CN1651452A - Panaxcoside secondary glucoside fatty acid ester compound, its preparation method and medicinal composition using said compound as active ingredient - Google Patents
Panaxcoside secondary glucoside fatty acid ester compound, its preparation method and medicinal composition using said compound as active ingredient Download PDFInfo
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Abstract
A secondary ginsenosides ester of fatty acid, its preparing process, its medicinal composition and the application of said compound and its medicinal composition in preparing medicine for treating cancer are disclosed.
Description
Technical field:
The present invention relates to ginsenoside secondary glycoside (being called for short M1) fatty acid ester compound (being called for short FM1), and disclose the molecular structure, synthetic method of this compound and be the pharmaceutical composition of activeconstituents, the semi-synthetic technical field of pharmaceutically active ingredient in belonging to this compound.
Background technology:
Ginsenoside secondary glycoside involved in the present invention (being called for short M1) is the ginsenoside secondary glycoside that the bacterium metabolism produces in intestines, and M1 can obtain by enzymolysis or fermentation process.Find that after deliberation M1 has very strong antitumour activity.M1 forms fatty acid ester (being called for short EM1) with lipid acid in liver, it is longer the integration time in liver than M1.And the pharmacokinetic that passes through M1, EM1 finds that the M1 fatty acid ester that the M1 fatty acid esterification forms (being called for short EM1) has improved its anti-tumor in vivo activity, and has reduced cytotoxicity.Therefore, the antitumour activity of genseng comes from oral genseng after the metabolin M 1 that the bacterium metabolism forms in the intestines and the EM1 of intrahepatic fat acid esters formation, and the bacterium metabolin M 1 can directly be brought into play antitumous effect in the intestines.
Before I am engaged in this research, do not see the bibliographical information of above-mentioned synthetic product by retrieval.
Summary of the invention:
The object of the present invention is to provide a class to have ginsenoside secondary glycoside fatty acid ester (the being called for short FM1) compound of pharmaceutical use.
Another object of the present invention has provided the synthetic and separation method of ginsenoside secondary glycoside fatty acid ester (being called for short FM1) compound, is suitable for industrialized mass.
It is the pharmaceutical composition that is used for the treatment of cancer of activeconstituents with this compound that further aim of the present invention has provided a kind of.
Another purpose of the present invention is above-claimed cpd and the purposes of composition aspect the medicine of preparation treatment cancer.
The present invention M1 and C
8~ C
16Fat acyl chloride synthetic ester compound, through the pharmacologically active screening, effective for multiple cancer.
The compounds of this invention has following general structural formula (I):
Wherein: R is C
8~ C
16Fatty acyl group, this compound abbreviates FM1 as.
Above-mentioned C
8~ C
16Fatty acyl group is the acyl group with straight or branched carboxylic acid of 8-16 carbon atom, and aliphatic carboxylic acid wherein comprises naturally occurring saturated or unsaturated straight chain fatty acid, reaches the saturated or unsaturated side chain lipid acid of synthetic.The preferred compound of the present invention is that wherein R is C
8~ C
16The formula of straight chain fatty acyl group (I) compound; Preferably wherein R is C
8~ C
16The formula of saturated straight chain fatty acyl group (I) compound; Particularly preferably being wherein, R is C
8~ C
16Formula (I) compound of saturated straight chain even carbon fatty acyl group; Most preferably wherein R is C
16The formula of saturated straight chain fatty acyl group (I) compound.
Wherein R is C
16The formula of saturated straight chain fatty acyl group (I) compounds process for production thereof may further comprise the steps:
A. with the ginsenoside secondary glycoside of genseng through the preparation of microbial fermentation or enzymolysis, by the ODS reversed-phase column, or silica gel column chromatography separates, with the methanol solution wash-out;
B. collect and be rich in ginsenoside secondary glycoside elutriant part, reclaim methyl alcohol, be concentrated into dried, the ginsenoside secondary glycoside, be called for short M1;
C. get M1 and be dissolved in the ethyl acetate, under the agitator condition of stirring, add saturated sodium bicarbonate aqueous solution, under the ice-water bath condition, add mole number 2-20 saturated straight chain palmitoyl chlorine doubly, stir under the room temperature and spend the night.With separating funnel ethyl acetate layer is separated with water layer then, and with ethyl acetate aqueous layer extracted repeatedly, ethyl acetate is merged, centrifugal, the supernatant liquor water washes repeatedly, ethyl acetate is reclaim under reduced pressure under 0-20 ℃ of cold condition, product dissolve with methanol, filtration, and the methanol solution concentrating under reduced pressure gets dry-matter;
D. with the gained dry-matter through HPLC, C-18 post, 100% methanol-eluted fractions, obtain 20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-16 carbonic ethers [20-O-β-D-glucopyranosyl (6-O-palmitoyl)-20 (S)-protopanaxadi], be called for short PM1; Yield is counted 35-60% with M1.
The gained monomeric compound can get the pure product of product of the present invention with re-crystallizing in ethyl acetate.
R is other C
8~ C
16The formula of fatty acyl group (I) compound can make according to above-mentioned preparation method with the acyl chlorides of corresponding carbon atom.And the inventor finds that employing the inventive method is utilized C
8~ C
16The yield of the formula of fatty acyl group (I) compound apparently higher than other yields of the formula of high-carbon fatty acyl group (I) compound more, is C as R
18The yield of the formula of saturated or unsaturated straight chain fatty acyl group (I) compound has only 5-20%.
The present invention studies show that the fatty acid ester of M1 has obviously strengthened the antitumour activity of M1, especially FM1, particularly PM1.Pharmacokinetic by M1, PM1 finds that the M1 fatty acid esterification forms FM1, particularly PM1, has improved its anti-tumor in vivo activity.Administration M1 or PM1 in the mouse inoculation tumor cell of liver, M1 treatment group inhibiting rate is 23%, but not obvious with the control group comparing difference; PM1 processing with same dose then obviously suppresses tumor growth, and is remarkable with the poor heteropole of control group, also remarkable with M1 treatment group comparing difference.Soon optionally enter liver after the M1 administration, in 10 minutes, reach the climax, and from liver, discharge rapidly; And PM1 promptly accumulates in liver, though As time goes on PM1 also reduces gradually, the PM1 (the μ g/g of wet tissue) that surpasses dosage 25% can retain in liver 24 hours.This with fatty acid esterification after cytosine arabinoside treatment hematologic malignancies have an identical reason than cytosine arabinoside itself is more effective.Therefore, the enhancing of PM1 anti-tumor activity may prolong relevant with its residence time in liver.
The contriver is through also discovery of pharmacological experiment screening, though The compounds of this invention FM1 and R are C
18The formula of saturated or unsaturated straight chain fatty acyl group (I) compound (SM1 or OM1) all has anticancer physiologically active; various cancers had inhibition, alleviation and therapeutic action; but obviously the anticancer physiologically active of The compounds of this invention FM1 is stronger; inhibition, alleviation and result of treatment to various cancers are better; the PM1 among the FM1 particularly; the ability that anti-lung carcinoma cell shifts obviously is better than SM1, OM1, and toxicity is lower.
It is activeconstituents that pharmaceutical composition of the present invention contains above-mentioned general formula (I) the compound F 17-hydroxy-corticosterone M1 that treats effective dose, and contains one or more pharmaceutically acceptable carriers.
Compound of the present invention and pharmaceutical compositions can be used for preparing the medicine for the treatment of various cancers.
Above-mentioned described pharmaceutically acceptable carrier is meant the pharmaceutical carrier of pharmaceutical field routine, comprises thinner, vehicle such as water etc.; Weighting agent such as starch, sucrose etc.; Tamanori such as Mierocrystalline cellulose and derivative, gelatin; Wetting agent such as glycerine; Disintegrating agent such as sodium bicarbonate, lime carbonate etc.; Absorption enhancer such as quaternary ammonium salt; Tensio-active agent such as high-carbon fatty alcohol; Absorption carrier such as kaolin, soap clay; Lubricant such as talcum powder, calcium stearate and poly-hexylene glycol etc.; Can also in composition, add other assistant agent such as sweeting agent, flavouring agent etc.
The compounds of this invention can be applied to cancer patients's treatment with the form of composition by oral, rectum, vein, muscle or parenteral admin mode.Also can be according to the conventional production method of pharmaceutical field, as make its activeconstituents and one or more carriers or medicament mixed, prepare various formulations such as tablet, electuary, capsule, suppository, sprays etc., preferred form is tablet, electuary, capsule, suppository, sprays, sustained release dosage and injection; Particularly preferably be preparation in the direct administration of lesions position.Pharmaceutical composition of the present invention contains the activeconstituents FM1 that weight ratio is 1%-99.5%, and preferably containing weight ratio is the activeconstituents FM1 of 45%-99.5%, and preferably containing weight ratio is the activeconstituents FM1 of 90%-99.5%.
Formulation rate of the present invention can be according to variations such as route of administration, patient age, body weight, disease type and severity, and per daily dose is the 0.01-10mg/kg body weight, preferred 0.1-8mg/kg body weight.Can use by one or many.
Embodiment
The following examples can help those skilled in the art more fully to understand the present invention, but do not limit the present invention in any way.
Embodiment 1.20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-16 carbonic ethers [20-O-β-D-glucopyranosyl (6-O-palmitoyl)-20 (S)-protopanaxadi] (being called for short PM1) preparation: with the M1 of genseng through the glucolase fermentative preparation, by the ODS reversed-phase column, or silica gel column chromatography separates, behind the methanol solution wash-out, M1 elutriant part is rich in collection, reclaim methyl alcohol, be concentrated into dried, purity is 98% M1; Get M1 50g and be dissolved in the 1000ml ethyl acetate, under the agitator condition of stirring, add 1000ml water saturation sodium bicarbonate, under the ice-water bath condition, add saturated straight chain palmitoyl chlorine 420g respectively, stir under the room temperature and spend the night.With separating funnel ethyl acetate layer is separated with water layer then, and with ethyl acetate aqueous layer extracted repeatedly, ethyl acetate is merged, centrifugal (3000rpm), the supernatant liquor water washes repeatedly, ethyl acetate is reclaim under reduced pressure under (20 ℃) condition at low temperatures, and product is with dissolve with methanol, filtration, and the methanol solution concentrating under reduced pressure gets dry-matter.The gained dry-matter through HPLC, C-18 post, 100% methanol-eluted fractions, is obtained 40g 20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-16 carbonic ethers (PM1), and yield counts 58.1% with M1.
Product is a water white transparency oily thing, is dissolved in chloroform, methyl alcohol.
Liebermann---the Burchard reaction is positive,
In the TOF-MS spectrum, m/z[M+H]
+861.6 its molecular weight is 860.
1H-NMR(500MHz，py-d
5)δ：5.32(1H，t，J＝6.5Hz，H-24)，5.11(1H，d，J＝7.5Hz，glc-H-1)，
5.02(1H，d，J＝11.0Hz，glc-Ha-6)，4.63(1H，dd，J＝7.0，11.0Hz，glc-Hb-6)，4.18(1H，t，J＝8.5Hz，glc-H-4)，4.16(1H，t，J＝8.5Hz，glc-H-4)，3.98(1H，t，J＝9.5Hz，H-12α)，3.95(1H，t，J＝8.0Hz，glc-H-2)，3.92(1H，t，J＝9.0Hz，glc-H-5)，3.39(1H，dd，J＝5.0，11.0Hz，H-3α)，2.59(1H，m，H-
17α)，2.55(1H，m，Hb-23)，2.43(1H，m，Hb-22)，2.31(1H，m，Ha-23)，2.02(1H，m，Hb-11)，2.00(1H，m，H-13)，1.88(1H，m，Hb-2)，1.85(1H，m，Hb-16)，1.82(1H，m，Ha-22)，1.80(1H，m，Ha-2)，
1.69(1H，m，Hb-1)，1.67(3H，s，Meα)，1.64(3H，s，Me-26)，1.62(3H，s，Me-27)，1.59(1H，m，
Hb-6)，1.55(1H，m，Ha-1)，1.53(1H，m，Hb-15)，1.50(1H，m，Hb-7)，1.45(1H，m，Ha-6)，1.42(1H，
m，H-9α)，1.39(1H，Ha-10)，1.31(1H，Ha-7)，1.20(3H，s，Me-28α)，1.03(1H，m，Ha-15)，1.02
(3H，s，Me-29β)，0.98(3H，s，19β)，0.94(3H，s，Me-30α)，0.89(1H，m，Ha-1)，0.87(3H，s，
Me-18 β) 0.85 (3H, t, J=7.5Hz, lipid acid terminal M e), 0.79 (1H, d, J=11.0Hz, H-5).
13C-NMR (125MHz, py-d
5) δ: its ownership sees table 1 for details, and its carbon ownership and M1 compare,
13On the C-NMR wave spectrum, the C of glucosyl residue
6Signal by δ 62.7 to low field displacement to δ 64.7 (Δ 2.0);
1On the H-NMR wave spectrum, glucosyl residue-CH
2The OH signal by δ 4.44 (1H, d, J=11.0Hz, glc-Ha-6) and 4.27 (1H, m, glc-Hb-6) respectively to low field displacement to δ 5.02 (1H, d, J=11.0Hz, glc-Ha-6) with 4.63 (1H, d, J=7.0,11.0Hz, glc-Hb-6).And C3 signal (δ 78.0) and H-3 signal [δ 3.39
(1H, dd, J=5.0,11.0Hz)] in full accord.Therefore, the CH of fatty acid residue and glucosyl residue
2The OH dehydrating condensation.Because the molecular weight of M1 is that the molecular weight of 622, ten six carbonic acid is 256 (C
16H
32O
2), both condensation product molecular weight are 860, this is identical with the institute molecular weight of surveying.Determine that this compound is M1 16 carbonic acid acid esters, promptly 20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-cetylate abbreviates PM1 as.
Table 1. Compound P M1 and M1's
13The C-NMR data
| C | Compound | C | Compound | |||
| PM1 | M1 | PM1 | M1 | |||
| 1 | 39.6 | 39.3 | 27 | 17.8 | 17.7 | |
| 2 | 28.3 | 28.1 | 28 | 28.7 | 28.6 | |
| 3 | 78.1 | 77.9 | 29 | 16.4 | 16.2 | |
| 4 | 39.4 | 39.4 | 30 | 17.5 | 17.3 | |
| 5 | 56.4 | 56.2 | 1’ | 98.1 | 98.1 | |
| 6 | 18.8 | 18.6 | 2’ | 75.1 | 75.0 | |
| 7 | 35.2 | 35.0 | 3’ | 79.2 | 79.2 | |
| 8 | 39.1 | 39.9 | 4’ | 71.5 | 71.5 | |
| 9 | 50.3 | 50.2 | 5’ | 78.2 | 78.2 | |
| 10 | 37.4 | 37.2 | 6’ | 64.7 | 62.7 | |
| 11 | 30.8 | 30.7 | 1 | 173.2 | ||
| 12 | 70.1 | 70.1 | 2 | 34.5 | ||
| 13 | 49.5 | 49.3 | 3 | 25.3 | ||
| 14 | 51.5 | 51.3 | 4 | 29.4 | ||
| 15 | 31.0 | 30.7 | 5 | 28.3 | ||
| 16 | 26.7 | 26.5 | 6 | 29.6 | ||
| 17 | 51.6 | 51.5 | 7 | 30.0 | ||
| 18 | 16.3 | 16.2 | 8 | 30.0 | ||
| 19 | 16.0 | 15.9 | 9 | 30.0 | ||
| 20 | 83.2 | 83.2 | 10 | 30.0 | ||
| 21 | 22.9 | 22.3 | 11 | 30.0 | ||
| 22 | 36.1 | 36.0 | 12 | 29.6 | ||
| 23 | 23.0 | 23.1 | 13 | 29.8 | ||
| 24 | 126.1 | 125.8 | 14 | 32.1 | ||
| 25 | 131.0 | 130.8 | 15 | 22.1 | ||
| 26 | 25.8 | 25.7 | 16 | 14.3 |
The preparation of embodiment 2.20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-lauric acid ester (being called for short DM1): as embodiment 1 preparation M1; Get M1 50g and be dissolved in the 1000ml ethyl acetate, under the agitator condition of stirring, add 1000ml water saturation sodium bicarbonate, under the ice-water bath condition, add saturated straight chain 12 carbonyl chloride 70g respectively, 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 centrifugal (3000rpm), supernatant liquor with ethyl acetate aqueous layer extracted repeatedly
Water washes repeatedly, and ethyl acetate is reclaim under reduced pressure under (20 ℃) condition at low temperatures, and product is with dissolve with methanol, filtration, and the methanol solution concentrating under reduced pressure gets dry-matter.The gained dry-matter is washed through HPLC, C-18 post, 100% methyl alcohol
Take off, obtain 23g 20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-lauric acid ester (DM1), yield counts 35.7% with M1.Product is a water white transparency oily thing, is dissolved in chloroform, methyl alcohol.
The preparation of embodiment 3.20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-octanoate (being called for short OM1): as embodiment 1 preparation M1; Get M1 50g and be dissolved in the 1000ml ethyl acetate, under the agitator condition of stirring, add 1000ml water saturation sodium bicarbonate, under the ice-water bath condition, add saturated straight chain capryl(yl)chloride 156g respectively, stir under the room temperature and spend the night.With separating funnel ethyl acetate layer is separated with water layer then, and with ethyl acetate aqueous layer extracted repeatedly, ethyl acetate is merged, centrifugal (3000rpm), the supernatant liquor water washes repeatedly, ethyl acetate is reclaim under reduced pressure under (20 ℃) condition at low temperatures, and product is with dissolve with methanol, filtration, and the methanol solution concentrating under reduced pressure gets dry-matter.The gained dry-matter through HPLC, C-18 post, 100% methanol-eluted fractions, is obtained 34.7g 20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-octanoate (OM1), and yield counts 58% with M1.Product is a water white transparency oily thing, is dissolved in chloroform, methyl alcohol.
Following pharmacological evaluation has confirmed the anticancer pharmacologically active of The compounds of this invention.
Experimental example 4. ten six carbonic ethers (PM1) are to the restraining effect of liver tumor, gastric tumor growth
1. experiment material
Pharmaceutical chemicals and confession test agent: PM1: synthetic by this experiment, purity 97%, tween: available from Hualian Pharmaceutical Co., Ltd., Shanghai, lot number 020803, endoxan: available from China Medicine (Group) Shanghai Chemical Reagent Co.,, lot number 20001110.
Laboratory animal: the ICR mouse, ♀ ♂ half and half, body weight 18～22g is provided by preclinical medicine institute of Jilin University Experimental Animal Center.Experimental animal feeding is that the bottom is covered with sawdust in the plastics cage of iron wire fence at the top, and food and water are supplied in black and white circulation in 12 hours at any time.
2. experimental technique
Adopt the animal-transplanted tumor model, compare, the PM1 antitumour activity is carried out screening and assessment with physiological saline group, positive drug endoxan group, as follows with concrete operations:
The restraining effect that A.PM1 grows to rat liver cancer ascitic type (HepA) cell: the oncocyte (HepA) that went down to posterity in the mouse peritoneal 7 days is taken out under aseptic condition, with physiological saline washing 2 times, dilute with physiological saline, oncocyte number in every milliliter counts, adjust cell concn to 1? 07ml-1, make abdominal injection for the acceptor mouse, every the right armpit subcutaneous vaccination of mouse 0.2ml knurl liquid.Mouse surviving rate 100%, similar to host's influence, interindividual variation is very little.Choose 30 of mouse, be divided into 3 groups at random, (injecting normal saline group, positive drug endoxan group, PM1 administration group), next day intraperitoneal administration, positive drug endoxan group mouse dosage 30mg/kg every other day administration, PM1 administration group mouse dosage 20mg/kg/day.Continuous 10 days, took off neck execution mouse in 24 hours after the drug withdrawal, separate the tumour electronic scale and weigh, calculate the tumor suppression percentage.
The restraining effect that B.PM1 grows to Mouse Gastric Cancer (MFC) cell: the oncocyte (MFC) that went down to posterity in the mouse peritoneal 7 days is taken out under aseptic condition, with physiological saline washing 2 times, dilute with physiological saline, oncocyte number in every milliliter counts, adjust cell concn to 1 * 107ml-1, make abdominal injection for the acceptor mouse, every the right armpit subcutaneous vaccination of mouse 0.2ml knurl liquid.Mouse surviving rate 100%, similar to host's influence, interindividual variation is very little.Choose 30 of mouse, be divided into 3 groups at random, (injecting normal saline group, positive drug endoxan group, PM1 administration group), next day intraperitoneal administration, positive drug endoxan group mouse dosage 30mg/kg every other day administration, PM1 administration group mouse dosage 20mg/kg/day.Continuous 10 days, took off neck execution mouse in 24 hours after the drug withdrawal, separate the tumour electronic scale and weigh, calculate the tumor suppression percentage.
3. the statistical study of experimental data
The statistics of experimental data adopts the t-method of inspection; The result adds and subtracts standard deviation (X ± S) expression with mean.
Tumor control rate (%)=(1-T/C) * 100%
T: the heavy C of the average knurl of experimental group: the average knurl of control group is heavy
4. experimental result and conclusion
A.PM1 is to the restraining effect of rat liver cancer (HepA) cell growth
PM1 administration group knurl heavily is starkly lower than the control group that physiological saline is handled, with the poor heteropole of control group remarkable (p＜0.001), illustrate that PM1 has significantly suppressed the growth of liver cancer cell in the mouse body, PM1 administration group and positive drug endoxan group relatively act on not as good as endoxan, PM1 administration group tumor control rate is 53.66%, and positive drug endoxan group tumor control rate is 73.15%.As table 2.
Table 2. different treatment is to the influence of mouse tumor cell (HepA)
| Handle group | Number of animals | Tumour heavy (g) | Inhibiting rate (%) |
| Contrast | 10 | 2.97±0.54 | - |
| CTX?30mg/kg | 10 | 0.86±0.35*** | 73.15 |
| PM1?20mg/kg | 10 | 1.44±0.57*** | 53.66 |
Data are 3 test averages in * * P＜0.001 table.
B.PM1 is to the restraining effect of Mouse Gastric Cancer (MFC) growth
PM1 administration group knurl heavily is starkly lower than the control group that physiological saline is handled, with control group comparing difference remarkable (p＜0.05), illustrate that PM1 has significantly suppressed vitro growth of gastric cancer cell in the mouse body, PM1 administration group and positive drug endoxan group relatively act on not as good as endoxan, PM1 administration group tumor control rate is 54%, and positive drug endoxan group tumor control rate is 72.11%.As table 3.
Table 3. different treatment is to the influence of Mouse Gastric Cancer cell (MFC) growth
| Handle group | Number of animals | Tumour heavy (g) | Inhibiting rate (%) |
| Contrast | 10 | 3.32±1.39 | - |
| CTX?30mg/kg | 10 | 0.99±0.51*** | 72.11 |
| PM1?20mg/kg | 10 | 1.60±0.68** | 54.00 |
Data are 3 test averages in * P＜0.01 * * * P＜0.001 table.
Experimental example 5.PM1 is to the restraining effect of melanoma growth
1. liver transplantation melanoma
Laboratory animal: the ICR mouse, ♀ ♂ half and half, body weight 18～22g chooses 30 of mouse age in 12-16 week, is divided into 3 groups at random, and food and water are supplied in black and white circulation in 12 hours at any time.
2. experimental technique
Mouse peritoneal is injected Sodital (50mg/kg) anesthesia, with injection into liver inoculation B16F-10 tumour cell (2 * 10
5Individual cell) in contrast; Inject B16F-10 tumour cell (2 * 10 in the Mouse Liver simultaneously
5Individual cell) and M1 (5mg/kg); Inject B16F-10 tumour cell (2 * 10 in the Mouse Liver simultaneously
5Individual cell) and PM1 (5mg/kg) respectively as handling.After 10 days, slaughter mouse and extract liver, weighing tumor weight.
3. the statistical procedures of data
The statistics of experimental data adopts the t-method of inspection; The result adds and subtracts standard deviation (X ± S) expression with mean.
4. experimental result and conclusion
The mean value of 10 mouse tumor weights of calculating (EM ± SD), analyse by statistics, result such as table 4 by credit.
Result of study shows that M1 forms after the fatty acid ester PM1, and antitumour activity obviously strengthens.
Table 4.PM1 is to the restraining effect of melanoma growth
| Handle group | Number of animals | Tumour heavy (g) | Inhibiting rate (%) |
| Contrast | 10 | 2.4±0.56 | - |
| M1?5mg/kg | 10 | 1.85±0.47* | 22.9 |
| PM1?5mg/kg | 10 | 0.95±0.35# | 60.4 |
#, p＜0.002 with compare; *, compare with M1 p＜0.02.
The splenic lymphocyte that experimental example 6 PM1 handle is to the B16-F10 cytotoxic effect
1. experiment material and method
Lymphocytic preparation: liver or spleen are suspended in blood cytolysate [0.17MNH by stainless steel mesh
4Cl, 0.01mM EDTA, 0.1M Tris (pH7.3)] in, centrifugal 6 minutes of 100xg room temperature.Cell granulations is washed 2 times with full medium, and lymphocyte and 20 μ M 2 mercapto ethanols that liver is cultivated are added in the full medium.
2. the cancer cells toxic action is measured
Cancer cells (1 * 10
6/ hole) with liver or splenic lymphocyte (1-3 * 10
6/ hole) in full medium, cultivate, and under PM1 (0.1-10 μ M) existence or the non-existent condition, 5%CO
2In the gas, 37 ℃ of cultivations.After 3 days, collect AC and examine under a microscope mensuration cancer cells number with trypsinase.Another organizes splenic lymphocyte (1 * 10
6) use PM1 (0-240 μ M) to handle in advance.After 2 days, splenic lymphocyte be divided into AC and non-AC and immediately respectively with B16-F10 cell (2 * 10
4) cultivate.Parallel laboratory test: cancer cells and PM1 single culture.After 1 day, collect AC and measure the cancer cells number with trypsinase.The ability of lymphocytolysis cancer cells is used following formula and is calculated: solvency power (%)=(1-test group/control group) * 100, wherein test group=cancer cells with lymphocyte PM1 exist or non-existent condition under, the cancer cells number of cultivation; The cancer cells number that control group=do not have lymphocyte is cultivated.
Be divided into two groups with 2 days splenic lymphocyte of PM1 pre-treatment: AC and non-AC and cultivate with thin 103 born of the same parents of B16-F10 respectively.Parallel laboratory test: cancer cells and PM1 single culture.After 1 day, obtain AC and measure the cancer cells number with trypsinase.
3. the statistical procedures of data
The statistics of experimental data adopts the t-method of inspection; The result adds and subtracts standard deviation (X ± S) expression with mean.
4. experimental result and conclusion
Measure PM1 different concns cancer cells number down, get following 3 the dish mean numbers of each concentration of the PM1 (growth rate of calculating cancer cells of EM ± SD).Result such as table 5.
The splenic lymphocyte that table 5.PM1 handles is to the B16-F10 cytotoxic effect
| PM1 concentration (μ m) is handled group | 0 | 60 | 120 | 240 |
| Cancer cells+PM1 | 100 | 100 | 100 | 100 |
| Cancer cells+AC+PM1 | 100 | 100 | 100 | 100 |
| Cancer cells+non-AC+PM1 | 100 | 85 | 60 | 30 |
The result shows: cancer cells is cultivated with the non-AC of handling with PM1 in advance, and along with the variation of concentration, the growth of cancer cells inhibiting rate increases.And AC does not have restraining effect to the growth of cancer cells.Also not effect of M1 separately.These results show the restraining effect of PM1 to growth of cancer cells and transfer, possible right and wrong AC to the stimulation of destructive cancer cells state with activate relevant.
The comparative studies of embodiment 7.DM1, PM1, SM1, OM1 anti-tumor activity
1. experiment material
The Lewis lung tumor cell
Laboratory animal: the ICR mouse, ♀ ♂ half and half, body weight 19～23g chooses 50 of mouse age in 12-16 week, is divided into 5 groups at random, and food and water are supplied in black and white circulation in 12 hours at any time.
2. experimental technique
Mouse peritoneal is injected Sodital (50mg/kg) anesthesia, and behind the intravenous inoculation cancer cells, respectively to different treatment group oral administration, control group is given corresponding suspensoid with the dosage of 3mg/Kg, after 10 days, slaughters mouse and takes out lungs and weigh.
3. the statistical procedures of data
Statistics such as table 6.The statistics of experimental data adopts the t-method of inspection; The result is with (X ± SD) expression.
Table 6.DM1, PM1, SM1, OM1 oral administration are to the spontaneous useless curative effect that shifts of Lewis lung cancer
| Group | Dosage (mg/kg) | Dosage regimen | Number of animals (beginning/end) | The weight of animals (g, beginning/end) | X clone number (g, ± SD) | Inhibiting rate (%) |
| DM1 PM1 SM1 OM1 contrast | 3.00 3.00 3.00 3.00 corresponding suspensions | po×10qod po×10qod po×10qod po×10qod po×10qod | 10/10 10/10 10/10 10/10 10/10 | 19.1±21.6 19.2±21.7 19.5±21.4 19.0±22.3 19.3±22.7 | 1.67±0.23 1.70±0.23 4.60±5.17 4.30±5.10 7.60±6.60 | 77.48** 77.63** 39.47** 43.42** |
* P＜0.05 * * P＜0.01, data are 3 experiment averages in the table.
4. experimental result
DM1, PM1, SM1, the OM1 oral administration is to the spontaneous useless curative effect that shifts of Lewis lung cancer, DM1, PM1, SM1, OM1 oral administration group knurl heavily is starkly lower than the control treatment group, with the poor heteropole of control group remarkable (p＜0.01), DM1 is described, PM1, SM1, OM1 has significantly suppressed the growth of lung carcinoma cell in the mouse body to be shifted, DM1, PM1 administration group and SM1, OM1 administration group relatively acts on and obviously is better than the latter, DM1, PM1 administration group tumor control rate is respectively 77.48%, 77.63%, and SM1 administration group tumor control rate is 39.42%, and OM1 administration group tumor control rate is 43.42%.The result shows: the ability that the anti-lung carcinoma cell of DM1, PM1 shifts obviously is better than SM1, OM1.
The preparation of embodiment 8. anticancer tablets: get the compound 10g of embodiment 1 preparation, add the medicinal cyclodextrin 30g of vehicle, mix, the granulation compressing tablet makes tablet, and every contains PM1 10mg.
The preparation of embodiment 9. anti-cancer capsules: get the compound 10g of embodiment 1 preparation, add the medicinal cyclodextrin 10g of vehicle, mix, granulation incapsulates, and every contains PM1 10mg.
The preparation of embodiment 10. anticancer injections: get the compound 10g of embodiment 3 preparations, add medicinal propylene glycol 200ml, water for injection 800ml, dissolving, membrane filtration; Membrane filtration (0.2 μ m), packing, every bottle of 2ml contains PM1 20mg.All operations all should carry out under aseptic condition.
The preparation of embodiment 11. anti-cancer capsules: get the compound 10g of embodiment 2 preparations, add the medicinal cyclodextrin 10g of vehicle, mix, granulation incapsulates, and every contains PM1 10mg.
Claims (8)
1. the panaxcoside secondary glucoside fatty acid ester compound that has antitumous effect is characterized by and has following structure:
Wherein R is C8 ~ C16 fatty acyl group.
2. according to the compound of claim 1, it is characterized by R is C8 ~ C16 straight chain fatty acyl group.
3. according to the compound of claim 2, it is characterized by R is C8 ~ C16 saturated straight chain fatty acyl group.
4. according to the compound of claim 3, it is characterized by C8 ~ C16 saturated straight chain even carbon fatty acyl group.
5. according to the compound of claim 4, it is characterized by R is C16 saturated straight chain fatty acyl group.
6. the preparation method of the compound of claim 5 is characterized by and may further comprise the steps:
A. with the ginsenoside secondary glycoside of genseng through the preparation of microbial fermentation or enzymolysis, by the ODS reversed-phase column, or silica gel column chromatography separates, with the methanol solution wash-out;
B. collect and be rich in ginsenoside secondary glycoside elutriant part, reclaim methyl alcohol, be concentrated into dried, the ginsenoside secondary glycoside, be called for short M1;
C. get M1 and be dissolved in the ethyl acetate, under the agitator condition of stirring, add saturated sodium bicarbonate aqueous solution, under the ice-water bath condition, add the saturated straight chain palmitoyl chlorine of mole number 2-20 multiple, stir under the room temperature and spend the night; With separating funnel ethyl acetate layer is separated with water layer then, and with ethyl acetate aqueous layer extracted repeatedly, ethyl acetate is merged, centrifugal, the supernatant liquor water washes repeatedly, ethyl acetate is reclaim under reduced pressure under 0-20 ℃ of cold condition, product dissolve with methanol, filtration, and the methanol solution concentrating under reduced pressure gets dry-matter;
D. with the gained dry-matter through HPLC, C-18 post methanol-eluted fractions, obtain 20-(S)-protopanoxadiol-20-O-β-D-Glucopyranose-6-O-16 carbonic ethers, be called for short PM1.
7. any one compound is used in the medicine of preparation treatment cancer among the claim 1-5.
8. the pharmaceutical composition that is used for the treatment of cancer is characterized by the compound and the pharmaceutically acceptable carrier that wherein contain the claim 1 for the treatment of significant quantity.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921305A (en) * | 2010-06-23 | 2010-12-22 | 大连大学 | Method for preparing ginsenoside metabolite M1 fatty acid monoester compound |
| CN102827233A (en) * | 2011-06-14 | 2012-12-19 | 李晓辉 | Ester derivatives of ginsenoside Compound K and application thereof in preparing drugs of preventing and treating atherosclerosis |
| CN102942610A (en) * | 2012-11-26 | 2013-02-27 | 吉林农业大学 | Preparation of ginsenoside M1 n-butyrate and application of antidiabetic medicines of ginsenoside M1 n-butyrate |
| WO2017057851A1 (en) * | 2015-09-30 | 2017-04-06 | (주)아모레퍼시픽 | Ginsenoside fatty acid ester compound, method for preparing same, and cosmetic composition comprising same |
| CN107619847A (en) * | 2017-11-09 | 2018-01-23 | 宁波润沃生物科技有限公司 | A kind of method that biofermentation prepares panaxoside metabolin |
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2004
- 2004-12-01 CN CNB2004101554932A patent/CN100484951C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921305A (en) * | 2010-06-23 | 2010-12-22 | 大连大学 | Method for preparing ginsenoside metabolite M1 fatty acid monoester compound |
| CN101921305B (en) * | 2010-06-23 | 2012-09-05 | 大连大学 | Method for preparing ginsenoside metabolite M1 fatty acid monoester compound |
| CN102827233A (en) * | 2011-06-14 | 2012-12-19 | 李晓辉 | Ester derivatives of ginsenoside Compound K and application thereof in preparing drugs of preventing and treating atherosclerosis |
| CN102827233B (en) * | 2011-06-14 | 2016-04-13 | 李晓辉 | Ginsenoside Compound K ester derivative and the application in the medicine of preparation control arteriosclerosis thereof |
| CN102942610A (en) * | 2012-11-26 | 2013-02-27 | 吉林农业大学 | Preparation of ginsenoside M1 n-butyrate and application of antidiabetic medicines of ginsenoside M1 n-butyrate |
| WO2017057851A1 (en) * | 2015-09-30 | 2017-04-06 | (주)아모레퍼시픽 | Ginsenoside fatty acid ester compound, method for preparing same, and cosmetic composition comprising same |
| CN108137640A (en) * | 2015-09-30 | 2018-06-08 | 爱茉莉太平洋股份有限公司 | Ginsenoside fatty acid ester compound, preparation method and the cosmetic composition containing it |
| CN108137640B (en) * | 2015-09-30 | 2021-09-07 | 爱茉莉太平洋股份有限公司 | Ginsenoside fatty acid ester compound, preparation method thereof and cosmetic composition containing same |
| CN107619847A (en) * | 2017-11-09 | 2018-01-23 | 宁波润沃生物科技有限公司 | A kind of method that biofermentation prepares panaxoside metabolin |
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