CN1594341A - Fatty acid ester compounds of ginsenoside secondary glycoside compound K and method for preparing same - Google Patents

Abstract

The invention discloses a fatty acid ester compounds of ginsenoside secondary glycoside compound K and method for preparing same, wherein the compound includes two derivatized compounds OM1 and SM1 having the structural formula disclosed in the specification, wherein R is oleoyl or stearoyl, and they are all esters formed by compound K with aliphatic acid. The invention also provides the method for synthesizing compound K and aliphatic acid, the pharmaceutical composition using the compositions as active composition, and the use of the compounds and pharmaceutical composition in preparing medicament for treating tumor.

Description

Fatty acid ester compound of ginsenoside secondary glycoside compound K and preparation method thereof

Technical field:

The invention relates to a fatty acid ester compound of ginsenoside secondary glycoside compound K, discloses the molecular structure of the compound, a synthesis method and a pharmaceutical composition using the compound as an active ingredient, and belongs to the technical field of semi-synthesis of effective ingredients of traditional Chinese medicine.

Background technique:

The ginsenoside secondary glycoside compound K (hereinafter referred to as M1) involved in the present invention is a metabolic product of intestinal bacteria of ginsenoside, and M1 has strong anticancer activity. M1 forms fatty acid esters (EM1) with fatty acids in the liver, which takes longer than M1 to accumulate in the liver. Moreover, through the pharmacokinetic study of M1 and EM1, it was found that esterification of M1 fatty acid to form EM1 improved its anti-tumor activity in vivo and reduced cytotoxicity. Therefore, the anticancer activity of ginseng originates from the metabolite M1 metabolized by intestinal bacteria after oral administration of ginseng and the fatty acid ester EM1 formed by fatty acid esterification in the liver. The intestinal bacterial metabolite M1 can directly exert anticancer effects.

After searching, there is no literature report on the above-mentioned artificially synthesized products.

Invention content:

The invention provides a fatty acid ester compound of ginsenoside secondary glycoside compound K with medicinal value, including two derivative compounds: OM1 and SM1.

The invention also provides a method for preparing OM1 and SM1 by synthesizing M1 and fatty acid chloride, which is suitable for industrial production.

The present invention further provides a pharmaceutical composition using the compound as an active ingredient for anticancer.

The ester compound synthesized by using M1 and fatty acid chloride in the present invention is effective for various cancers after screening for pharmacological activity.

Said compound of the present invention has following structural general formula:

The preparation method of the present invention comprises the following steps:

Take 30-50g of M1 and dissolve it in 1000-1500ml of ethyl acetate, add 1000-1500ml of water-saturated sodium bicarbonate under stirring condition, add 430-720g of oleic acid chloride under the condition of ice-water bath, stir overnight at room temperature; Separate the ethyl acetate layer and the water layer with a liquid funnel, and repeatedly extract the water layer with ethyl acetate, combine the ethyl acetate, centrifuge, rinse the supernatant with water repeatedly, recover the ethyl acetate under reduced pressure, dissolve the dry matter with methanol, and filter , the methanol solution was concentrated under reduced pressure to obtain dry matter;

The resulting material is subjected to HPLC, and C-18 column 100% methanol is eluted to obtain 20-(S)-protopanaxadiol-20-O-β-D-glucopyranose-6-O-oleate (hereinafter Abbreviation: OM1); 20-(S)-protopanaxadiol-20-O-β-D-glucopyranose-6-O-stearate (hereinafter abbreviated: SM1).

The obtained monomer compound is recrystallized with ethyl acetate to obtain the pure product of the present invention.

The above-mentioned compounds have the following physical and chemical properties respectively through spectral analysis:

SM1 is a colorless transparent oil, soluble in chloroform and methanol. Liebermann-Burchard reaction was positive; Molish reaction was positive.

In the TOF-MS spectrum, m/z[M+H]+ is 889.5, and its molecular weight is 888.

^{The 1} H NMR assignment of SM1 is as follows: ¹ H-NMR (500MHz, py-d ₅ ) δ: 5.33 1H, m, H-24), 5.14 (1H, d, J=7.5Hz, glc-H-1) , 5.05 (1H, d, J=10.1Hz, glc-Ha-6), 4.65 (1H, dd, J=7.0, 11.0Hz, glc-Hb-6), 4.19 (2H, m, glc-H-3 , glc-H-4), 3.98 (3H, H-12α glc-H-2, glc-H-5), 3.40 (1H, m, H-3α), 2.58 (2H, m, H-1α, Hb- 23), 2.41 (1H, m, Hb-22), 2.32 (1H, m, Hα-23), 2.01 (2H, m, Hb-11, H-13α), 1.88 (1H, m, Hb-2) , 1.88(1H, m, Hb-2), 1.85(1H, m, Hb-16), 1.82(1H, m, Ha-22), 1.80(1Hm, Ha-2), 1.69(1H, m, Hb -1), 1.67(3H, s, Me-21), 1.64(3H, s, Me-26), 1.63(3H, s, Me-27), 1.58(1H, m, Hb-6), 1.55( 1H, m, Ha-11), 1.52 (1H, m, Hb-15), 1.51 (1H, m, Hb-7), 1.45 (1H, m, Ha-6), 1.42 (1H, m, H- 9), 1.39(1H, Ha-16), 1.31(1H, m, Ha-7), 1.20(3H, s, Me-28α), 1.03(1H, m, Ha-15), 1.03(3H, s , Me-29β), 0.98 (3H, s, 19β), 0.95 (3H, s, Me-α), 0.89 (1H, m, Ha-1), 0.87 (3H, s, Me-1β) 0.85 (3H , t, J=7.5 Hz, fatty acid terminal Me), 0.79 (1H, d, J=11.0 Hz, H-5). ¹³ C-NMR (125MHz, py-d ₅ ) δ:

Its assignment is shown in Table 1, and its carbon assignment is compared with that of M1. On the ¹³ C-NMR spectrum, the C ₆ signal of the glucose residue shifts from δ62.7 to δ64.5 (Δ1.8); at ¹ On the H-NMR spectrum, the -CH ₂ OH signal of the glucose residue is from δ4.44 (1H, d, J=11.0Hz, glc-Ha-6) and 4.27 (1H, m, glc-Hb-6) to Downfield shifts to δ 5.05 (1H, d, J = 10.1 Hz, glc-Ha-6) and 4.65 (1H, dd, J = 7.0, 11.0 Hz, glc-Hb-6). The C3 signal (δ77.9) is exactly the same as the H-3α signal [δ3.40(1H, m)]. In the HMBC spectrum, δ5.05 (1H, d, J=10.1Hz, glc-Ha-6) and 4.65 (1H, dd, J=7.0, 11.0Hz, glc-Hb-6) are all related to fatty acyl δ173.3 (CO) has an HMBC relationship. Thus, the fatty acid residue is ester-bonded with the _-CH2OH of the glucose residue. Since the molecular weight of M1 is 622 and the molecular weight of stearic acid is 284 (C ₁₈ H ₃₆ O ₂ ), the molecular weight of the condensation product of the two is 888, which is consistent with the measured molecular weight. Compound 6 was determined to be the stearate of M1, and its name was determined to be: 20-(S)-protopanaxadiol-20-O-β-D-glucopyranose-6-O-stearate.

OM1 is a colorless transparent oil, soluble in chloroform and methanol. Spraying 10% H2SO4 reagent on the TLC plate after development showed purple; Liebermann-Burchard reaction was positive; Molish reaction was positive. Prompt the presence of triterpenoid saponins.

In the TOF-MS spectrum, m/z[M+H]+ is 887.5, so its molecular weight is 886.

¹³ C-NMR (125MHz, py-d ₅ ) δ: See Table 1 for its assignment. Compared with M1's carbon assignment, on the ¹³ C-NMR spectrum, the C ₆ signal of the glucose residue is from δ62.7 to low The field shifted to δ64.7 (Δ2.0); on the ¹ H-NMR spectrum, the -CH ₂ OH signal of the glucose residue was determined by δ4.44 (1H, d, J=11.0Hz, glc-Ha-6) and 4.27 (1H, m, glc-Hb-6) shifted downfield to δ5.02 (1H, d, J=11.0Hz, glc-Ha-6) and 4.63 (1H, d, J=7.0, 11.0Hz) respectively , glc-Hb-6). However, the C3 signal (δ78.0) and the H-3α signal [δ3.39 (1H, dd, J=5.0, 11.0Hz)] were completely consistent. Therefore, _CH2OH dehydration condensation of fatty acid residues and glucose residues. Since the molecular weight of M1 is 622 and the molecular weight of oleic acid is 282 (C ₁₈ H ₃₄ O ₂ ), the molecular weight of the condensation product of the two is 860, which is consistent with the measured molecular weight. Confirmed name: 20-(S)-protopanaxadiol-20-O-β-D-glucopyranose-6-O-oleate.

The above two substances have similar physiological activities, and both belong to the protection scope of the present invention.

¹³ C-NMR data of compounds SM1 and OM1 in Table 1

Compound

C

                                                                                                          

1 39.2 39.3

2 28.1 28.1

3 77.9 77.9

4 39.3 39.4

5 56.1 56.2

6 18.5 18.6

7 34.9 35.0

8 39.9 39.9

9 50.1 50.2

10 37.1 37.2

11 30.8 30.7

12 69.9 70.1

13 49.3 49.3

14 51.3 51.3

15 30.5 30.7

16 26.5 26.5

17 51.2 51.5

18 16.1 16.2

19 15.8 15.9

20 83.2 83.2

21 22.7 22.3

22 35.9 36.0

23 22.8 23.1

24 125.8 125.8

25 130.7 130.8

26 25.6 25.7

27 17.6 17.7

28 28.5 28.6

29 16.1 16.2

30 17.2 17.3

1' 97.8 98.1

2' 74.8 75.0

3' 79.0 79.2

4' 71.4 71.5

5' 77.8 78.2

6' 64.5 64.7

1” 173.3 173.0

2” 34.2 33.8

3” 25.1 25.0

4” 30.8 29.9

5” 29.3 28.6

6” 29.4 29.8

7” 29.4 29.3

8” 29.8 40.9

9” 29.8 129.2

10” 29.8 130.1

11” 29.8 41.2

12” 29.8 31.0

13” 29.6 29.5

14” 30.5 30.3

15” 29.8 30.1

16” 31.9 32.1

17” 22.7 22.6

18” 14.1 14.2

Through pharmacological experiment screening, the compound has anticancer physiological activity.

The pharmaceutical composition of the present invention contains the above-mentioned compound as an active ingredient, and one or more pharmaceutically acceptable carriers.

The compounds and compositions of the invention are useful in the treatment of cancer.

The carrier mentioned above refers to conventional drug carriers in the field of pharmacy, including diluents, excipients, fillers, binders, disintegrants, absorption promoters, surfactants, and adsorption carriers.

The present invention may be administered to cancer treatment patients in the form of compositions by oral, rectal, intravenous, intramuscular or parenteral administration. Various dosage forms such as tablets, granules, capsules, suppositories, sprays, sustained-release preparations and injections are prepared according to conventional production methods in the pharmaceutical field. The active ingredient can also be mixed with one or more carriers or drugs to make the desired dosage form.

The pharmaceutical composition of the present invention contains 1%-99.5% active ingredient, preferably 5%-85%, most preferably more than 90% of the compound of the present invention.

The dosage of the present invention can be varied according to the route of administration, patient's age, body weight, disease type and severity, etc., and the daily dosage is 0.1-10 mg/kg.

The synthesis, separation and structural identification of fatty acid ester compounds OM1 and SM1 of ginsenoside secondary glycoside compound K clarified the material basis of their functions and indications, and they have anticancer effects.

The following pharmacological experiments confirm the anticancer pharmacological activity of the compounds of the present invention.

Experimental example 1 Anti-cancer (melanoma) effect of OM1

1 Experimental materials and methods

1.1 Liver transplantation for melanoma

Experimental animals: ICR mice, half and half ♀♂, weight 18-22g, 12-16 weeks old, 30 mice were selected, randomly divided into 3 groups, 12-hour black and white cycle, and food and water were provided at any time.

Methods: Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg), and B16F-10 tumor cells (2×10 ⁵ cells) were injected intrahepatically as a control; mice were intrahepatically injected with B16F-10 tumor cells (2× 10 ⁵ cells) and M1 (5 mg/kg); mice were intrahepatically injected with B16F-10 tumor cells (2×10 ⁵ cells) and OM1 (5 mg/kg). After 10 days, the mice were sacrificed to remove the liver, and the weight of the cancerous tumor was weighed. Results: As shown in Figure 1-1, each column in the figure represents the mean value (EM±SD) of the tumor weights of 6 mice. #, p<0.002 compared with control; ^* p<0.02 compared with M1.

2 Statistical processing of data

The statistics of the experimental data adopt the t-test method; the results are expressed as mean plus or minus standard deviation ( x±S) said.

Referring to Figure 1-1, the effects of M1 and OM1 on tumor growth after intrahepatic transplantation of B16F-10 melanoma. Mice were injected intrahepatically with B16F-10 tumor cells (control); mice were intrahepatically injected with B16F-10 tumor cells and M1 (5 mg/kg); mice were intrahepatically injected with B16F-10 tumor cells and OM1 (5 mg/kg) . After 10 days, the mice were sacrificed to remove the liver, and the weight of the cancerous tumor was weighed. Each column in the figure represents the mean value (EM±SD) of the tumor weight of 6 mice. #, p<0.002 compared with control; ^* p<0.02 compared with M1.

The results of the study showed that the anticancer activity of M1 was significantly enhanced after the fatty acid ester was formed (see Figure 1).

Experimental Example 1-2 Anticancer (melanoma) effect of SM1

1 Experimental materials and methods

1.1 Liver transplantation for melanoma

Experimental animals: ICR mice, half and half ♀♂, weight 18-22g, 12-16 weeks old, 30 mice were selected, randomly divided into 3 groups, 12-hour black and white cycle, and food and water were provided at any time.

Methods: Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg), and B16F-10 tumor cells (2×10 ⁵ cells) were injected intrahepatically as a control; mice were intrahepatically injected with B16F-10 tumor cells (2× 10 ⁵ cells) and M1 (5 mg/kg); mice were intrahepatically injected with B16F-10 tumor cells (2×10 ⁵ cells) and SM1 (5 mg/kg). After 10 days, the mice were sacrificed to remove the liver, and the weight of the cancerous tumor was weighed. Results: As shown in Figures 1-2, each column in the figure represents the mean value (EM±SD) of the tumor weights of 6 mice. #, p<0.002 compared with control; ^* p<0.02 compared with M1.

2 Statistical processing of data

The statistics of the experimental data adopt the t-test method; the results are expressed as mean plus or minus standard deviation ( x±S) said.

Mice were injected intrahepatically with B16F-10 tumor cells (control); mice were intrahepatically injected with B16F-10 tumor cells and M1 (5mg/kg); mice were intrahepatically injected with B16F-10 tumor cells and SM1 (5mg/kg) . After 10 days, the mice were sacrificed to remove the liver, and the weight of the cancerous tumor was weighed. Each column in the figure represents the mean value (EM±SD) of the tumor weight of 6 mice. #, p<0.002 compared with control; ^* p<0.01 compared with M1.

The results of the study showed that the anticancer activity of M1 was significantly enhanced after the fatty acid ester was formed (Fig. 1-2).

Experimental Example 2-1 Retention time in the liver after intravenous administration of M1 and OM1

Experimental materials and methods

Experimental animals: ICR mice, ♀♂♂ in half, body weight 18-22g, 12-16 weeks old.

Methods: Mice were intravenously injected with M1 or OM1 (30mg/kg). The mice were slaughtered at the specified time after administration, and the liver was immediately taken out, homogenized with 6 ml of normal saline, extracted twice with 40 ml of ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. For HPLC analysis.

Result: as shown in Figure 2-1. M1 selectively enters the liver shortly after administration, reaches the peak within 10 minutes, and is rapidly cleared from the liver; while OM1 rapidly accumulates in the liver, and OM1 gradually decreases over time, exceeding 25% of the dose OM1 can remain in the liver for 24 hours.

Experimental Example 2-2 Retention time in the liver after intravenous administration of M1 and SM1

Experimental materials and methods

Experimental animals: ICR mice, ♀♂♂ in half, body weight 18-22g, 12-16 weeks old. Methods: Mice were intravenously injected with M1 or SM1 (30mg/kg). The mice were slaughtered at the specified time after administration, and the liver was immediately taken out, homogenized with 6 ml of normal saline, extracted twice with 40 ml of ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. For HPLC analysis.

Result: as shown in Figure 2-2. M1 selectively enters the liver shortly after administration, reaches the peak within 10 minutes, and is rapidly cleared from the liver; while SM1 rapidly accumulates in the liver, and SM1 gradually decreases over time, exceeding 27% of the dose SM1 can remain in the liver for 24 hours.

Experimental example 3-1 Effect of OM1 on liver metastasis caused by intrasplenic injection of B16F-10 cells in mice

Experimental animals: ICR mice, ♀♂♂ in half, body weight 18-22g, 12-16 weeks old.

Experimental method: Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg), and mice were intrasplenic injected with B16F-10 cells (2×10 ⁵ cells/mouse) to cause liver metastasis. OM1 (30 mg/kg) was injected intravenously on days 0, 2, and 4 after cancer inoculation. The spleen with early canceration was removed on the 7th day of inoculation. After 17 days, the mice were sacrificed to count the number of cancer metastases in the liver.

Experimental results: To evaluate the anti-metastasis effect of OM1, B16F-10 cells were injected into the spleen of mice to cause liver metastases. Three consecutive intravenous injections of OM1 (30mg/kg) had no significant effect on the body weight of mice. Show no toxic reaction. As shown in Figure 3-1A, OM1 has no inhibitory effect on spleen cancer tumors, but can significantly inhibit liver metastasis (Figure 3-1C). In addition, the spleen weight of mice treated with OM1 was significantly increased (Fig. 3-1A).

OM1 (30 mg/kg) was injected intravenously on days 0, 2, and 4 after cancer inoculation. The spleen with early canceration was removed 7 days after inoculation. After 17 days, the mice were sacrificed to count the number of cancer metastases in the liver. Each bar in the figure represents the mean value (EM±SD) of 5 mice. ^* , p<0.02;#,p<0.002 vs. control. A: Solid bars tumor weight, open bars indicate cancer-free spleen weight (at day 7). B: Number of cancer colonies in the liver at day 7. C: Photograph of liver metastases.

Experimental example 3-2 Effect of SM1 on liver metastasis caused by intrasplenic injection of B16F-10 cells in mice

Experimental animals: ICR mice, ♀♂♂ in half, body weight 18-22g, 12-16 weeks old.

Experimental method: Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg), and mice were intrasplenic injected with B16F-10 cells (2×10 ⁵ cells/mouse) to cause liver metastasis. SM1 (30 mg/kg) was injected intravenously on days 0, 2, and 4 after cancer inoculation. The spleen with early canceration was removed on the 7th day of inoculation. After 17 days, the mice were sacrificed to count the number of cancer metastases in the liver.

Experimental results: To evaluate the anti-metastasis effect of SM1, B16F-10 cells were injected into the spleen of mice to cause liver metastases. Three consecutive intravenous injections of SM1 (30mg/kg) had no significant effect on the body weight of mice. Show no toxic reaction. As shown in Figure 3-2A, SM1 has no inhibitory effect on spleen cancer tumors, but can significantly inhibit liver metastasis (Figure 3-2C). In addition, the spleen weight of mice treated with SM1 was significantly increased (Fig. 3-2A).

Experimental example 4-1 Spleen lymphocytes treated with OM1 have cytotoxic effect on B16-F10

1 Experimental method:

1.1 Preparation of lymphocytes Suspend liver or spleen in erythrocyte lysate [0.17 M NH ₄ Cl, 0.01 mM EDTA, 0.1 M Tris (pH 7.3)] through a stainless steel mesh, and centrifuge at room temperature at 100 x g for 6'. Cell pellets were washed twice with full medium, and liver cultured lymphocytes and 20 μM 2-mercaptoethanol were added to full medium.

1.2 Determination of cancer cell toxicity: cancer cells (1×10 ⁶ /well) and liver or spleen lymphocytes (3×10 ⁶ /well) were cultured in full medium, and OM1 (0.1-10μM) was present or absent , 5% CO ₂ atmosphere, cultured at 37°C. After 3 days, the adherent cells were harvested with trypsin and observed under a microscope to determine the number of cancer cells. Another group of splenic lymphocytes (1×10 ⁶ ) was previously treated with OM1 (0-240 μM). After 2 days, splenic lymphocytes were divided into adherent and non-adherent cells and immediately cultured with B16-F10 cells (2×10 ⁴ ) separately. Parallel experiment: Cancer cells were cultured with OM1 alone. One day later, the adherent cells were harvested with trypsin and the number of cancer cells was determined. The ability of lymphocytes to dissolve cancer cells is calculated by the following formula: dissolving power (%)=(1-test group/control group)×100, where test group=cancer cells and lymphocytes exist or do not exist under the condition of OM1, Number of cancer cells cultured; control group = number of cancer cells cultured without lymphocytes.

2 Statistical processing of data

The statistics of the experimental data adopt the t-test method; the results are expressed as mean plus or minus standard deviation ( x±S) said.

3 Results: As shown in Figure 4-1, when cancer cells were cultured with non-adherent cells treated with OM1 in advance, the inhibition rate of cancer cell growth increased with the change of concentration. Adhesive cells have no inhibitory effect on the growth of cancer cells. M1 also has no effect. These results suggest that the inhibitory effect of OM1 on cancer cell growth and metastasis may be related to the stimulation and activation of non-adherent cells to the disrupted cancer cell state.

Splenic lymphocytes pretreated with OM1 for 2 days were divided into two groups: adherent cells and non-adherent cells and cultured with B16-F10 cells, respectively. Parallel experiment: Cancer cells were cultured with OM1 alone. One day later, adherent cells were harvested with trypsin and the number of cancer cells was determined. Each point represents the mean of 3 plates (EM±SD). □, adherent cells; □, non-adherent cells; □, OM1 alone. ^* , p<0.01; ^** p<0.02 vs. control.

Experimental Example 4-2 Spleen lymphocytes treated with SM1 have toxic effect on B16-F10 cells

1 Experimental method:

1.1 Preparation of lymphocytes Suspend liver or spleen in erythrocyte lysate [0.17M NH ₄ Cl, 0.01mM EDTA, 0.1M Tris (pH 7.3)] through a stainless steel mesh, and centrifuge at room temperature at 100xg for 6'. Cell pellets were washed twice with full medium, and liver cultured lymphocytes and 20 μM 2-mercaptoethanol were added to full medium.

1.2 Determination of cancer cell toxicity: cancer cells (1×10 ⁶ /well) and liver or spleen lymphocytes (3×10 ⁶ /well) were cultured in full medium, and SM1 (0.1-10μM) was present or absent , 5% CO ₂ atmosphere, cultured at 37°C. After 3 days, the adherent cells were harvested with trypsin and observed under a microscope to determine the number of cancer cells. Another group of splenic lymphocytes (1×10 ⁶ ) was previously treated with SM1 (0-240 μM). After 2 days, splenic lymphocytes were divided into adherent and non-adherent cells and immediately cultured with B16-F10 cells (2×10 ⁴ ) separately. Parallel experiment: Cancer cells were cultured with SM1 alone. One day later, the adherent cells were harvested with trypsin and the number of cancer cells was determined. The ability of lymphocytes to dissolve cancer cells is calculated by the following formula: dissolving power (%)=(1-test group/control group)×100, wherein test group=cancer cells and lymphocytes exist or do not exist under the condition of SM1, Number of cancer cells cultured; control group = number of cancer cells cultured without lymphocytes.

2 Statistical processing of data

The statistics of the experimental data adopt the t-test method; the results are expressed as mean plus or minus standard deviation ( x±S) said.

3 Results: As shown in Figure 4-2, when cancer cells were cultured with non-adhesive cells treated with SM1 in advance, the inhibition rate of cancer cell growth increased with the change of concentration. Adhesive cells have no inhibitory effect on the growth of cancer cells. M1 also has no effect. These results suggest that the inhibitory effect of SM1 on cancer cell growth and metastasis may be related to the stimulation and activation of non-adherent cells to the disrupted cancer cell state.

Splenic lymphocytes pretreated with SM1 for 2 days were divided into two groups: adherent cells and non-adherent cells and cultured with B16-F10 cells, respectively. Parallel experiment: Cancer cells were cultured with SM1 alone. One day later, adherent cells were harvested with trypsin and the number of cancer cells was determined. Each point represents the mean of 3 plates (EM±SD). □, adherent cells; □, non-adherent cells; □, SM1 alone. ^* , p<0.01; ^** p<0.02 vs. control.

4 discussions

Studies have shown that fatty acid esters of M1 significantly enhanced the anticancer activity of M1. Through the pharmacokinetic study of M1 and OM1, it was found that the esterification of M1 fatty acid to form OM1 improved its anti-tumor activity in vivo. When the mice were inoculated with liver tumor cells, M1 or OM1 was administered at the same time, the inhibition rate of the M1 treatment group was 23%, but the difference was not significant compared with the control group; the same dose of OM1 treatment significantly inhibited tumor growth, and the difference was extremely significant compared with the control group , and the difference was also significant compared with the M1 treatment group. M1 selectively enters the liver shortly after administration, reaches the peak within 10 minutes, and is rapidly excreted from the liver; OM1 rapidly accumulates in the liver, and OM1 gradually decreases over time, exceeding 25% of the dose OM1 (wet tissue μg/g) can persist in the liver for 24 hours. It has been reported that fatty acid esterified cytarabine is more effective in treating hematological malignancies than cytarabine itself. Therefore, the enhanced antitumor activity of OM1 may be related to its prolonged residence time in the liver.

After oral administration, ginsenosides are metabolized by intestinal bacteria to form secondary glycosides and aglycones. Oral administration of Rb2 to mice found that Rb2 was only slightly oxidized in gastric juice and could not be decomposed. Rb1 was cultured with the contents of the rat gastrointestinal tract, and it was found that only when Rb1 was co-cultured with the contents of the large intestine, Rb1 was metabolized into M1 (also known as Compound K). This phenomenon was also observed in mice after oral administration of Rb1. This result indicated that enterobacteria metabolized Rb1 into M1 (Compound K).

When administered orally to rats, both ginsenosides and saponin metabolites had significant anti-metastatic effects; however, when administered intravenously, the enterobacterial metabolites of ginsenosides significantly inhibited tumor metastasis, but ginsenosides itself does not. Therefore, after oral administration of ginsenosides, not saponins but their intestinal bacterial metabolites are absorbed in the intestine and exert their effects.

OM1 promotes the lysis and apoptosis of tumor cells through the regulation of lymphocytes, especially non-adhesive splenocytes. OM1 induces host cell immune regulation more strongly than M1.

Therefore, the anticancer activity of ginseng originates from the metabolite M1 formed by the metabolism of intestinal bacteria after oral administration of ginseng and the fatty acid ester OM1 formed by the esterification of fatty acids in the liver. The metabolite M1 of intestinal bacteria has anticancer effects; the metabolites Fatty acid ester OM1 has stronger anticancer activity.

Evaluation of Experimental Example 5 Stearate SM1 Antitumor Activity

5.1 Materials and methods

5.1.1 Materials

Chemicals and test samples: SM1 was synthesized in this experiment with a purity of 97%. Tween was purchased from Shanghai Hualian Pharmaceutical Co., Ltd., batch number 020803, and cyclophosphamide was purchased from China National Pharmaceutical (Group) Shanghai Chemical Reagent Company, batch number 20001110.

Experimental animals: ICR mice, half ♀♂ and half ♀♂, weighing 18-22 g, provided by the Experimental Animal Center of Jilin University School of Basic Medicine. Experimental animals were housed in plastic cages with wire fences on top, sawdust on the bottom, 12-hour black-and-white cycle, and food and water available at any time.

5.1.2 Method

Animal xenograft tumor models were used, and normal saline group and positive drug cyclophosphamide group were used as controls to screen and evaluate the anticancer activity of SM1. The specific operations were as follows:

A. The inhibitory effect of SM1 on the growth of mouse liver cancer ascites type (HepA) cells: the tumor cells (HepA) of the mouse intraperitoneal passage for 7 days were taken out under sterile conditions, washed twice with normal saline, and diluted with normal saline , count the number of tumor cells per milliliter, adjust the cell concentration to 1×107·ml-1, inject intraperitoneally into the recipient mice, and subcutaneously inoculate 0.2ml of tumor fluid in the right armpit of each mouse. The survival rate of mice is 100%, and the effect on the host is similar, with little difference among individuals. Choose 30 mice, be divided into 3 groups at random, (inject normal saline group, positive drug cyclophosphamide group, SM1 administration group), intraperitoneal administration next day, positive drug cyclophosphamide group mice administration dose 30mg/ The dosage of the mice in the kg-daily administration and SM1 administration groups was 20 mg/kg/day. For 10 consecutive days, the mice were killed by dislocation of the neck 24 hours after drug withdrawal, the tumors were stripped out and electronically weighed, and the percentage of tumor inhibition was calculated.

B. The inhibitory effect of SM1 on the growth of mouse gastric cancer (MFC) cells: the tumor cells (MFC) of the mouse intraperitoneal passage for 7 days were taken out under sterile conditions, washed twice with normal saline, diluted with normal saline, and recorded The number of tumor cells per milliliter was counted, the cell concentration was adjusted to 1×107·ml-1, intraperitoneal injection was given to recipient mice, and 0.2ml of tumor fluid was inoculated subcutaneously in the right armpit of each mouse. The survival rate of mice is 100%, and the effect on the host is similar, with little difference among individuals. Choose 30 mice, be divided into 3 groups at random, (inject normal saline group, positive drug cyclophosphamide group, SM1 administration group), intraperitoneal administration next day, positive drug cyclophosphamide group mice administration dose 30mg/ The dosage of the mice in the kg-daily administration and SM1 administration groups was 20 mg/kg/day. For 10 consecutive days, the mice were killed by dislocation of the neck 24 hours after drug withdrawal, the tumors were stripped out and electronically weighed, and the percentage of tumor inhibition was calculated.

5.1.3 Statistical analysis of data

The statistics of the experimental data adopt the t-test method; the results are expressed as mean plus or minus standard deviation ( x±S) said.

Tumor inhibition rate (%)=(1-T/C)×100%

T: Average tumor weight of the experimental group C: Average tumor weight of the control group

5.1.4 Results

A. The inhibitory effect of SM1 on the growth of mouse hepatocarcinoma (HepA) cells

The tumor weight of the SM1 administration group was significantly lower than that of the control group treated with normal saline, and the difference was extremely significant compared with the control group (p<0.001), indicating that SM1 significantly inhibited the growth of liver cancer cells in mice. The comparative effect of phosphoramide group was inferior to that of cyclophosphamide, the tumor inhibition rate of SM1 administration group was 51.66%, and the tumor inhibition rate of positive drug cyclophosphamide group was 71.15%. The results are shown in Table 1, Figure 1, and Figure 3.

     Table 5-1 Effects of different treatments on mouse tumor cells (HepA)

Group N Tumor weight(g) Inhibitory rate(%)

Control 10 2.97±0.54 -

CTX 30mg/kg 10 0.86±0.35*** 71.15

SM1 20mg/kg 10 1.44±0.57*** 51.66

**P＜0.001 as compared with control group

5.2 Discussion

According to the experimental method of anti-tumor effect of traditional Chinese medicine in pharmacology, if the tumor weight inhibition rate of traditional Chinese medicine is more than 30%, and there is a significant difference after statistical processing, the anti-cancer effect of this medicine can be evaluated. The inhibitory effect of SM1 on mouse liver tumor (HepA) and gastric tumor (MFC) is much better than the evaluation standard of anti-tumor of traditional Chinese medicine, and it can be confirmed that SM1 has obvious anti-tumor effect. In addition, the pharmacological observation of SM1-administered mice showed no death, no adverse reactions, and no significant changes in physiological characteristics and body weight compared with the control group. It was preliminarily concluded that SM1 had no toxic and side effects. It can be seen that SM1 has a clear structure, significant effect, safety and stability. After further pharmacological and clinical experiments, a new class of anti-cancer drugs has a bright prospect.

When evaluating the antitumor activity of SM1, cyclophosphamide was introduced as a positive control. Cyclophosphamide is a chemical anti-tumor drug, which mainly interferes with the functions of DNA and RNA and inhibits the synthesis of DNA. It has a very significant anti-tumor effect, but its side effects are very strong. Although the anti-tumor effect of SM1 is not as good as cyclophosphamide, its anti-cancer effect is achieved by anti-metastasis, inhibition of new blood vessel formation, and enhancement of immune regulation, without toxic and side effects. The new drug developed by SM1 can achieve the effect of low toxicity, high efficiency, safety and reliability.

In the pharmacological experiments of SM1, when preparing the administration solution of SM1, SM1 was dissolved well after adding Tween, but after adding normal saline, the dissolution was not good and a small amount of crystals precipitated out, which indirectly affected the anticancer effect of SM1. Therefore, preparations, routes of administration and methods need to be further studied. Breakthroughs in this field favor the expression of SM1 anticancer activity.

The latest pharmacokinetic study of ginsenosides shows that fatty acid esters of M1 are a series of fatty acids (including stearic acid, oleic acid, palmitic acid or Palmitic acid) M1 monoester mixture. The esterification process of M1 represents a detoxification process, just as the esterification of cholesterol prevents cytotoxicity caused by excessive cholesterol intake. In addition, the esterification of M1 delays the excretion of M1 in the liver and enhances the immune regulation of host cells. Thus, esterification of M1 to EM1 reduces cytotoxicity and enhances anticancer activity. Studies have found that the fatty acids in the body fat of terrestrial higher animals are mainly stearic acid, palmitic acid and oleic acid, and 80% of the triterpene esters in the human liver are stearate and 20% are palmitate.

Description of drawings:

Figure 1-1: Effects of M1 and OM1 on tumor growth after intrahepatic transplantation of B16F-10 melanoma.

Figure 1-2 Effects of M1 and SM1 on tumor growth after intrahepatic transplantation of B16F-10 melanoma.

Figure 2-1: Retention time in the liver after intravenous administration of M1 and OM1.

Figure 2-2: Retention time in the liver after intravenous administration of M1 and SM1.

Figure 3-1A, B, C: Effects of OM1 on liver metastasis induced by intrasplenic injection of B16F-10 cells in mice.

Figure 3-2 A, B, C: The effect of SM1 on liver metastasis induced by intrasplenic injection of B16F-10 cells in mice.

Figure 4-1: The cytotoxic effect of OM1-treated spleen lymphocytes on B16-F10 cells.

Figure 4-2 The cytotoxic effect of SM1-treated splenic lymphocytes on B16-F10 cells.

Figure 5-1: The inhibitory effect of SM1 on liver cancer tumors.

Figure 5-2: Inhibitory effect of OM1 on liver cancer tumors.

Detailed ways:

Example 1

Take 30g of M1 and dissolve it in 1000ml of ethyl acetate, add 1000ml of water-saturated sodium bicarbonate under stirring condition, add 720g of oleic acid chloride under ice-water bath condition, and stir overnight at room temperature. Then use a separatory funnel to separate the ethyl acetate layer from the water layer, and repeatedly extract the water layer with ethyl acetate, combine the ethyl acetate, centrifuge (3000rpm), and wash the supernatant with water repeatedly, and the ethyl acetate is at low temperature (20 -30°C) for recovery under reduced pressure, the residue was dissolved in methanol, filtered, and the methanol solution was concentrated under reduced pressure to obtain dry matter.

Example 2

Take M140g and dissolve in 1200ml ethyl acetate, add 1000ml water-saturated sodium bicarbonate under stirring condition, add oleic acid chloride 700g under ice-water bath condition, stir overnight at room temperature. Then use a separatory funnel to separate the ethyl acetate layer from the water layer, and repeatedly extract the water layer with ethyl acetate, combine the ethyl acetate, centrifuge (3000rpm), wash the supernatant with water repeatedly, and store the ethyl acetate at a low temperature of 20°C ) under reduced pressure recovery, the residue was dissolved in methanol, filtered, and the methanol solution was concentrated under reduced pressure to obtain dry matter.

Example 3

Dissolve 50g of M1 in 1500ml of ethyl acetate, add 1500ml of water-saturated sodium bicarbonate under stirring conditions, add 720g of oleic acid chloride under ice-water bath conditions, and stir overnight at room temperature. Then use a separatory funnel to separate the ethyl acetate layer from the water layer, and repeatedly extract the water layer with ethyl acetate, combine the ethyl acetate, centrifuge (3000rpm), and wash the supernatant with water repeatedly. Recover under reduced pressure, dissolve the residue with methanol, filter, and concentrate the methanol solution under reduced pressure to obtain dry matter.

Example 4

The substances obtained in Examples 1, 2, and 3 were eluted by HPLC and C-18 column 100% methanol to obtain SM1 (20-(S)-protopanaxadiol-20-O-β-D-glucopyranose- 6-O-oleate 8g, 10g, 12g.

Example 5

The substances obtained in Examples 1, 2, and 3 were eluted by HPLC and C-18 column with 100% methanol to obtain OM1[20-(S)-protopanaxadiol-20-O-β-D-glucopyranose-6 -O-stearate] 4g, 6g, 8g.

Example 6

Preparation of anticancer tablets

Take 10 g of the compound prepared in Example 4 or Example 5, add 30 g of excipient pharmaceutical cyclodextrin, mix well, granulate and press into tablets to obtain tablets, each containing 10 mg of M1 fatty acid ester.

Example 4

Preparation of Anticancer Capsules

Take 10 g of the compound prepared in Example 4 or Example 5, add 10 g of excipient medicinal cyclodextrin, mix evenly, granulate, put into capsules, and each capsule contains 10 mg of fatty acid ester of M1.

Example 5

Preparation of anticancer injection

Get 10g of the compound prepared in Example 4 or Example 5, add 200ml of medicinal propylene glycol, 800ml of water for injection, dissolve, and membrane filter; membrane filter (0.2 μm), subpackage, 2ml per bottle, containing 20mg of fatty acid ester of M1. All operations should be performed under sterile conditions.

Claims (4)

1 one kinds of fatty acid ester compounds with ginsenoside secondary glycoside compound K of pharmaceutical use have following general structure:

Wherein, R is oleoyl or stearyl.

2, the preparation method of claim 1 compound may further comprise the steps:

1) gets M1 30-50g and be dissolved in the 1000-1500ml ethyl acetate, under agitation condition, add 1000-1500ml water saturation sodium bicarbonate, under the ice-water bath condition, add oleic acid acyl chlorides 430～720g, stir under the room temperature and spend the night; With separating funnel ethyl acetate layer is separated with water layer then, and, ethyl acetate is merged with ethyl acetate aqueous layer extracted repeatedly, centrifugal, the supernatant liquor water washes repeatedly, the reclaim under reduced pressure ethyl acetate, dry-matter dissolve with methanol, filtration, the methanol solution concentrating under reduced pressure gets dry-matter;

2) with the gained material through HPLC, C-18 post methanol-eluted fractions obtains 20-(S)-protopanoxadiol-20-O-B-D-Glucopyranose-6-O-oleic acid ester respectively; 20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-stearate.

3, according to the application of compound any in the claim 1 in the medicine of preparation antitumous effect.

4, be used to prepare anticancer pharmaceutical composition, wherein contain claim 1 compound and the pharmaceutically acceptable carrier for the treatment of significant quantity.

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