CN101392015A - Triterpene saponin in camellia seeds, preparation method and medical use thereof - Google Patents
Triterpene saponin in camellia seeds, preparation method and medical use thereof Download PDFInfo
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Abstract
The invention pertains to the field of medical technology and provides triterpenoid saponin in camellia seeds, and a preparation method and medical application thereof. The triterpenoid saponin is shown in a general formula (1), wherein, R1 is hydrogen or oxhydryl or acyloxy; R2 is the hydrogen or the acyloxy; R3 is the hydrogen or the acyloxy; R4 is the hydrogen or the acyloxy; R5 is methyl or methylol or aldehyde group or carboxyl or metheyl carboxyl; R6 is the hydrogen or orglycosyl; the preparation method comprises the following steps: macroporous resins are degreased, extracted by ethanol, decocted in water, desugared and decolored to obtain crude total saponins and then go though an opened ODS chromatographic column and repeated HPLC method to obtain the total saponins of camellia seeds and monomer theasaponin. The triterpenoid saponin compounds in the camellia seeds have the functions of protecting gastric mucosa, being antineoplastic, reducing blood sugar and blood fat and the like and are used for preparing medicaments or health-care foods having the functions of protecting gastric mucosa, being antineoplastic and reducing blood sugar and blood fat.
Description
Technical field:
The invention belongs to medical technical field, relate to triterpenoid saponin in the camellia seeds and preparation method thereof and medicinal use.
Background technology:
Camellia Camellia sinensis is a Theaceae Camellia plant, in China's commerial growing, is a kind of good cash crop.The systematic research report of the chemical ingredients of its tender shoots---tealeaves and biological activity.Show that according to existing research data the chemical ingredients of tea has 600 kinds more than, organic compound wherein reaches more than 500 kinds.Their biochemical reaction approach synthetic and that transform have the relation that interknits, conditions each other.Its biological activity relates to: anti-inflammatory, antianaphylaxis, anti-oxidant, remove many-sided activity such as free radical, hypoglycemic, reducing blood-fat, stomach and intestine promote, protect the liver, premalignant lesion prevention.
In recent years, along with asexual cottage propagation is taked in tea place plant husbandry, a large amount of camellia seeds is demanded further rational exploitation and utilization urgently as by product.Compendium of Material Medica record: tea seed bitter cold, poisonous, control and breathe heavily anxious phlegm and cough, remove the phlegm dirt.In our early-stage Study, find, contain a large amount of saponin components in the tea seed.Tea kind total saponins is a kind of excellent surfactant, is the raw material of novel cosmetic and washing composition.There are better protecting stomach mucous membrane, hypoglycemic, reducing blood-fat, antitumor, antimycotic, anti-inflammatory, antianaphylaxis and anti-angiogenic dilating effect in the medical treatment aspect.The extraction of camellia seeds total saponins and utilize its effective constituent process for processing to become preparation that report is not arranged.
Summary of the invention:
The object of the present invention is to provide triterpenoid saponin constituents in the camellia seeds shown in general formula (1), and preparation method thereof and medicinal use.
The invention provides the triterpenoid saponin in the camellia seeds, it is a represented oleanane derivative in the following general formula (1):
General formula (1)
The cautious chromyl angeloyl groups of ethanoyl cinnamoyl
In the general formula (1), R1 is hydrogen or hydroxyl or acyloxy; R2 is hydrogen or acyloxy (the acyl group kind comprises ethanoyl, angeloyl groups, cautious chromyl, cinnamoyl); R3 is hydrogen or acyloxy (the acyl group kind comprises ethanoyl, angeloyl groups, cautious chromyl, cinnamoyl); R4 is hydrogen or acyloxy (the acyl group kind comprises ethanoyl, angeloyl groups, cautious chromyl, cinnamoyl); R5 is methyl or methylol or aldehyde radical or carboxyl or carboxyl ester; R6 is hydrogen or glycosyl (comprising glucal acidic group, Arabic glycosyl, galactosyl, glucosyl group, xylosyl and the oligonucleotide chain of being made up of them).
Wherein new compound is:
The present invention also provides the preparation method of triterpenoid saponin in the camellia seeds, and its preparation method is as follows:
(1) gets dry camellia seeds oil expression back gained camellia oleosa seed cake, add and extract solvent soaking, medicinal material is 1:5 to 1:12 with the ratio of extracting solvent, heats 0.5～3 hour under 50～100 ℃ of conditions, extract 1～4 time, merge 2～10 times of volumes that each extracting solution is concentrated into the medicinal material amount;
(2) extracting solution is through macroporous resin (HPD100 or resins for universal use such as D101 or AB-8) absorption, and the rare pure flush away impurity with 0～30% gets the thick total saponins of camellia seeds (content 50%～70%) with 40%～100% pure liquid wash-out again;
(3) the thick total saponins of gained camellia seeds adds the less water dissolving, through opening ODS column chromatography, with 0～30% rare pure flush away tea flavones impurity, gets camellia seeds total saponins (content 70%～95%) with 40%～90% pure liquid wash-out again;
(4) gained camellia seeds total saponins is through the further separation and purification of HPLC: reversed-phase bonded silica is stationary phase (C-18, C-30, C-8 etc.), with methanol-water (containing 0.05% Glacial acetic acid) volume ratio 30～80% or acetonitrile-water (containing 0.05 Glacial acetic acid) volume ratio 20%～70% or methyl alcohol-acetonitrile-water (containing 0.05% Glacial acetic acid) volume ratio 2:50:48～16:20:64 is moving phase, (190～220nm) detectors detect, and obtain all kinds triterpenoid saponin constituents shown in the general formula (1) for differential or ultraviolet.
The preparation method of triterpenoid saponin in the camellia seeds provided by the invention, the extraction solvent in the described step (1) is water, methyl alcohol, ethanol etc., methyl alcohol or concentration of ethanol are 5%～95%; Extracting method is decocting method or heating and refluxing extraction method.
The preparation method of triterpenoid saponin in the camellia seeds provided by the invention, pure liquid is the methyl alcohol or the ethanol of different concns in the described step (3).
The invention provides the medical use of triterpenoid saponin in the camellia seeds, the triterpene saponin componds in the camellia seeds as shown in general formula (1) has effects such as gastric mucosal protection, antitumor, hypoglycemic, reducing blood-fat; Can be used for preparing the medicine or the protective foods of protection stomach mucous membrane, antitumor, hypoglycemic, reducing blood-fat.
Advantage of the present invention is as follows:
(1) preparation method's gained camellia seeds total saponins yield height of the present invention, purity height;
(2) 32 compounds as implied above are new compound, are the endemic elements of camellia seeds, can be used as the index of quality control;
(3) gained monomer theasaponin yield height of the present invention, purity height, structure is clear and definite, can carry out the targeted structural modification, with further development and use;
(4) the present invention is raw materials used is industrial waste for the camellia oleosa seed cake after the camellia seeds oil expression, and cost is low, be easy to get, help environmental protection;
(5) easy, the easy row of preparation method pollutes and lacks, and is suitable for industrial production;
(6) camellia seeds total saponins and monomer theasaponin biological activity is various, effect is remarkable.
Description of drawings:
Fig. 1 is theasaponin T1's
1H NMR collection of illustrative plates.
Fig. 2 is theasaponin T1's
13The collection of illustrative plates of C NMR.
Fig. 3 is theasaponin T10's
1H NMR collection of illustrative plates.
Fig. 4 is theasaponin T10's
13The collection of illustrative plates of C NMR.
Fig. 5 is theasaponin T12's
1The collection of illustrative plates of H NMR.
Fig. 6 is theasaponin T12's
13The collection of illustrative plates of C NMR.
Fig. 7 is theasaponin T20's
1The collection of illustrative plates of H NMR.
Fig. 8 is theasaponin T20's
13The collection of illustrative plates of C NMR.
Fig. 9 is the collection of illustrative plates of the 1H NMR of theasaponin T33.
Figure 10 is theasaponin T33's
13The collection of illustrative plates of C NMR.
Figure 11 is the collection of illustrative plates of the 1H NMR of theasaponin T34.
Figure 12 is theasaponin T34's
13The collection of illustrative plates of C NMR.
Figure 13 is the collection of illustrative plates of the 1H NMR of theasaponin T36.
Figure 14 is theasaponin T36's
13The collection of illustrative plates of C NMR.
Embodiment:
The present invention is described in further detail for following examples, and the present invention is not limited.
The preparation of camellia total saponins
Get dry camellia seeds oil expression back gained camellia oleosa seed cake 10kg, remove impurity, dust.
In the traditional Chinese medicine extraction jar, add the water of medicinal material and 10 times of amounts, be heated to 80 ℃, extract twice, each 2 hours, merge 4 times of volumes that each extracting solution is concentrated into the medicinal material amount.
Extracting solution with 20% ethanol flush away impurity, gets thick total saponins 680 grams (content 60%) of camellia seeds with 90% ethanol elution through macroporous resin adsorption again.
The thick total saponins of gained camellia seeds adds 5 times of water gaging dissolvings, through opening ODS column chromatography (sample size: filler 1: 10), with 20% ethanol flush away tea flavones impurity, get camellia seeds total saponins 380 grams (content 85%) with 85% ethanol elution again.
Gained camellia seeds total saponins is through further separation and purification of reversed-phase HPLC (Tianjin, island L6A): with ODS and C-30 bonded silica gel is stationary phase, be moving phase with methanol-water (containing 0.05% Glacial acetic acid) volume ratio 70:30 respectively, acetonitrile-water (containing 0.05 Glacial acetic acid) volume ratio is moving phase at 40: 60, methyl alcohol-acetonitrile-water (containing 0.05% Glacial acetic acid) volume ratio 8:32:60 be the flowing opposite subdivision from, the differential detector detects, and obtains all kinds triterpenoid saponin constituents shown in the general formula (1).
The preparation of camellia total saponins
(1) gets dry camellia seeds oil expression back gained camellia oleosa seed cake 6kg, remove impurity, dust.
(2) 95% ethanol of adding medicinal material and 8 times of amounts in the traditional Chinese medicine extraction jar is heated to 80 ℃, extracts twice, each 1.5 hours, merges 2 times of volumes that each extracting solution is concentrated into the medicinal material amount.
(3) extracting solution with 30% ethanol flush away impurity, gets thick total saponins 770 grams (content 65%) of camellia seeds with 90% ethanol elution through macroporous resin adsorption again.
(4) the thick total saponins of gained camellia seeds adds 4 times of water gaging dissolvings, through opening ODS column chromatography (sample size: filler 1:12), with 20% ethanol flush away tea flavones impurity, get camellia seeds total saponins 430 grams (content 87%) with 90% ethanol elution again.
(5) gained camellia seeds total saponins is through further separation and purification of reversed-phase HPLC (Tianjin, island L6A): with ODS and C-30 bonded silica gel is stationary phase, be moving phase with methanol-water (containing 0.1% Glacial acetic acid) volume ratio 65:35 respectively, acetonitrile-water (containing 0.1 Glacial acetic acid) volume ratio 33:67 is a moving phase, methyl alcohol-acetonitrile-water (containing 0.05% Glacial acetic acid) volume ratio 4:34:62 be the flowing opposite subdivision from, the differential detector detects, and obtains all kinds triterpenoid saponin constituents shown in the general formula (1).
Investigate of the influence of camellia seeds total saponins to S180 tumor-bearing mice tumor growth
Laboratory animal: 8 age in week male ICR mouse, mean body weight 20 gram.Provide by Zhejiang University's Experimental Animal Center.
Medicine and knurl strain: the camellia seeds total saponins is according to the method preparation of describing in claims 2; Endoxan (CTX), 200mg/ props up, and Hualian Pharmaceutical Co., Ltd., Shanghai produces, and S180 ascitic tumor mouse provides for Zhejiang University experimentation on animals center.
The preparation of S180 ascitic tumor cell suspension: aseptic condition is operation down, get the S180 sarcoma ascitic type tumor-bearing mice of inoculation 10d, sterilization animal skin of abdomen, penetrate the abdominal cavity with the 5mL disposable sterilized injector, draw the good ascites of growth of tumour cell, institute taps the abdomen and puts into aseptic Erlenmeyer flask, is positioned on the ice chest.Other carries out cell counting at the ascites that takes a morsel: with 100 times of physiological saline dilutions, getting diluent 0.9mL adds platform and expects orchid (expecting blue normal saline solution for 0.2%) 0.1mL, mixing, with cell counting analyze tumour cell sum and dead cell number (purple or blue, viable count should 95%).Ascites is diluted with physiological saline, and adjusting oncocyte concentration is 1 * 107/mL (entire operation is carried out on ice chest, keeps the activity of tumour cell with this).
Kunming mouse is transplanted the foundation of S180 sarcoma model: get the Kunming mouse about body weight 20g, it is subcutaneous that every mouse 0.2mL (tumor cell number is about 2 * 106) is inoculated in the right fore armpit, weigh behind the 24h, be divided into 3 groups at random, the grouping situation is: lotus knurl control group, endoxan (CTX) group, camellia seeds saponin(e group group.Calculate knurl and heavily reach other indexs of correlation.
Lotus knurl control group: after the transplantation tumor, gavage 0.4mL physiological saline every day.Endoxan (CTX) group: every day 1 time, 100mg/kg body weight, abdominal injection, 3d continuously.Camellia seeds total saponins dosage group: gavage 1 time by the 100mg/kg body weight every day, continuously 10d.
The detection of index: stop administration behind the administration 10d, next day is put to death mouse in drug withdrawal, takes by weighing the mouse body weight, dissects the knurl body, weighs.
Experimental result: see Table 1
Table 1 camellia seeds total saponins is to the influence of S180 tumor-bearing mice tumor growth
Adopt mtt assay and srb assay, monomer theasaponin anti tumor activity in vitro is tested
Experimental technique is as follows:
Cell cultures: the human body tumour cell in the vegetative period of taking the logarithm, diluting with the RPMI-1640 nutrient solution that includes 10% (v/v) foetal calf serum (fetal bovine serum) is the cell suspending liquid of 5 * 104/L, be inoculated in 96 orifice plates, 100 μ L/ holes place 37 ℃, saturated humidity, 5%CO2 incubator to cultivate 24h.
Add reagent: after a small amount of DMSO sample dissolution, with the RPMI-1640 nutrient solution dilution that contains 10% (v/v) foetal calf serum is 10,20,30,40, the sample solution of 50 μ m/L concentration gradients adds above-mentioned pastille nutrient solution in 96 orifice plates, is positioned in the incubator with cell cultures the same terms and cultivates 48h.
The result measures:
Adopt mtt assay to measure MCF-7 and HepG2 knurl strain experimental result.
Adopt srb assay to measure K562 and HL-60 knurl strain experimental result.
Experimental result: see Table 2
Table 2 monomer theasaponin anti tumor activity in vitro test result
Investigate of the influence of camellia kind total saponins to the glucose load rat blood sugar
Laboratory animal: male Wistar rat, body weight 130～150g is available from Japanese KiwaLabora tory Animal Co.Ltd. (Wakayama).Animal constant temperature (23 ± 2 ℃) is raised, and feed is available from Japanese MF.Oriental Yeast Co.Ltd. (Tokyo).The animal fasting is 20～24 hours before the experiment, freely fetches water.
Experimental technique: get the male Wistar rat of 130～150g, fasting was irritated stomach and is given and camellia kind total saponins after 20～24 hours, 0.5 after hour, irritate stomach and give sucrose (1.0g/kg), after 0.5 hour, blood is got on the eyeground, adopts the determination of glucose oxidase glucose level behind the centrifugal separation plasma.
Experimental result: provided the influence of camellia kind total saponins to glucose load rat blood sugar level in the table 3, camellia kind total saponins can significantly reduce glucose load rat blood sugar level at 100～400mg/kg dosage.
Table 3 camellia kind total saponins is to the influence of glucose load rat blood sugar
Values?represent?the?means±S.E.M.
For?statistical?analysis：one-way?analysis?of?variance?followed?by?Dunnett’s?test
Significantly?different?from?the?control?group，
*p<0.05，
**p<0.01.
Investigate of the influence of camellia kind total saponins to triglyceride levels in the oil load mice plasma
Laboratory animal: male DDY mouse, body weight 25～30g is available from Japanese Kiwa LaboratoryAnimal Co.Ltd. (Wakayama).Animal constant temperature (23 ± 2 ℃) is raised, and feed is available from Japanese MF.Oriental Yeast Co.Ltd. (Tokyo).The animal fasting is 20～24 hours before the experiment, freely fetches water.
Experimental technique: get the male DDY mouse of 25～30g, fasting was irritated stomach and is given and camellia kind total saponins after 20～24 hours, 0.5 after hour, irritate stomach and give sweet oil (5mL/kg), after 2 hours, blood is got on the eyeground, adopts triglyceride levels in the WAKO kit measurement blood plasma behind the centrifugal separation plasma.
Experimental result: provided the influence of camellia kind total saponins to triglyceride levels in the oil load mice plasma in the table 4, camellia kind total saponins can significantly reduce triglyceride levels in the oil load mice plasma at 400mg/kg dosage.
Table 4 camellia kind total saponins is to the influence of triglyceride levels in the oil load mice plasma
Values?represent?the?means±S.E.M.
For?statistical?analysis：one-way?analysis?of?variance?followed?by?Dunnett’s?test
Significantly?different?from?the?control?group，
*p<0.05，
**p<0.01.
Investigate of the influence of camellia kind total saponins to alcohol induced gastric mucosa injury
Laboratory animal: male SD rat, body weight 230～250g is available from Japanese Kiwa LaboratoryAnimal Co.Ltd. (Wakayama).Animal constant temperature (23 ± 2 ℃) is raised, and feed is available from Japanese MF.Oriental Yeast Co.Ltd. (Tokyo).The animal fasting is 24～26 hours before the experiment, freely fetches water.
Experimental technique: get the male SD rat of 230～250g, fasting was irritated stomach and is given and tea kind total saponins (5% gum arabic suspendible) after 24～26 hours, and after 1 hour, the filling stomach causes gastric mucosa injury to 99.5% ethanol (1.5mL), and after 1 hour, etherization is put to death.Take out the rat stomach, behind the extraction content, the formalin solution of injection 10mL1.5% is coat of the stomach fixedly, and with flattening after the flushing with clean water, computer scanning calculates the gastric mucosa injury rate repeatedly.
Experimental result: the result is as shown in table 5, and camellia kind total saponins can significantly reduce the gastric mucosa injury that ethanol causes at 2.5～20mg/kg dosage.
Table 5 camellia kind total saponins is to the influence of alcohol induced gastric mucosa injury
Values?represent?the?means±S.E.M.
For?statistical?analysis：one-way?analysis?of?variance?followed?by?Dunnett’s?test
Significantly?different?from?the?control?group，
*p<0.05，
**p<0.01.
Embodiment 8
Investigate of the influence of camellia kind total saponins to indomethacin inductive gastric mucosa injury
Laboratory animal: male SD rat, body weight 230～250g is available from Japanese Kiwa LaboratoryAnimal Co.Ltd. (Wakayama).Animal constant temperature (23 ± 2 ℃) is raised, and feed is available from Japanese MF.Oriental Yeast Co.Ltd. (Tokyo).The animal fasting is 24～26 hours before the experiment, freely fetches water.
Experimental technique: the male SD rat of getting 230～250g, after the fasting 24～26 hours, irritating stomach gives and camellia kind total saponins (5% gum arabic suspendible), after 1 hour, (the 20mg/kg indomethacin is dissolved in 5% sodium hydrogen carbonate solution, behind the distilled water diluting to indomethacin to irritate stomach, add the neutralization of 0.2M hydrochloric acid, 1.5ml/rat) cause gastric mucosa injury, after 4 hours, etherization is put to death.Take out the rat stomach, behind the extraction content, the formalin solution of injection 10mL1.5% is coat of the stomach fixedly, and with flattening after the flushing with clean water, the gastric mucosa injury rate is calculated in scanning repeatedly.
Experimental result: experimental result is as shown in table 6, and camellia kind total saponins can significantly reduce the gastric mucosa injury that indomethacin causes at 50～200mg/kg dosage.
Table 6 camellia kind total saponins is to the influence of indomethacin inductive gastric mucosa injury
Values?represent?the?means±S.E.M.
For?staistical?analysis：one-way?analysis?of?variance?followed?by?Dunnett’s?test
Significantly?different?from?the?control?group，
*p<0.05，
**p<0.01
The monomer theasaponin that the investigation separation obtains is to the provide protection of alcohol induced gastric mucosa injury.
Laboratory animal and test method are with embodiment 7.
Experimental result: as shown in table 7, the partial monosomy theasaponin is the 5mg/kg level at dosage, can significantly suppress alcohol induced gastric mucosa injury, and its effect is better than positive control drug omeprazole and bent propionate salts hydrochlorate.
Table 7 monomer theasaponin is to the provide protection of alcohol induced gastric mucosa injury
Values?represent?the?means±S.E.M.
For?staistical?analysis：one-way?analysis?of?variance?followed?by?Dunnett’s?test
Significantly?different?from?the?contrql?group，
*p<0.05，
**p<0.01.
The structural identification of embodiment 10 monomer theasaponin T1
White needle (methyl alcohol), mp219～220 ℃, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and has existed; Acid hydrolysis, detecting has glucuronic acid, semi-lactosi, pectinose and wood sugar exist.IR(KBr?pellet)vmax?3453(OH)，1718(C＝O)，1649(C＝O)，1O78(C-O)cm-1。
Pos.FAB-MS?m/z：1213[M+Na]
+，Neg.FAB-MS?m/z：1189[M-H]
-，1057[M-H-132]
-，925[M-H-2×132]
-，895[M-H-132-162]
-。HR?FABMS?m/z：1213.5627(calcd.1213.5618?for?C
57H
90O
26Na)。
1H-NMR,
13The C-NMR data see Table 1, table 2.
1H-NMR,
13C-NMR sees Figure of description 1, Fig. 2.
The structural identification of embodiment 11 monomer theasaponin T10
White needle (methyl alcohol), mp220～221 ℃, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and acetate and has existed; Acid hydrolysis, detecting has glucuronic acid, semi-lactosi, pectinose and wood sugar exist.IR(KBr?pellet)v
max?3453(OH)，1731(C＝O)，1647(C＝O)，1080(C-O)cm
-1 Pos.FAB-MS?m/z：1281[M+Na]
+，Neg.FAB-MS?m/z：1257[M-H]
-，1125[M-H-132]
-，963[M-H-132-162]
-。HR?FABMS：1281.5886(calcd.1281.5880for?C
61H
94O
27Na)。
1H-NMR,
13The C-NMR data see Table 1, table 2.
1H-NMR,
13C-NMR sees Figure of description 3, Fig. 4.
The structural identification of embodiment 12 monomer theasaponin T12
White needle (methyl alcohol), mp223～224 ℃, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and has existed; Acid hydrolysis, detecting has glucuronic acid, semi-lactosi, pectinose and wood sugar exist.IR(KBr?pellet)v
max?3453(OH)，1741(C＝O)，1085(C-O)cm
-1。
Pos.FAB-MS?m/z：1197[M+Na]
+，Neg.FAB-MS?m/z：、1173[M-H]
-，1041[M-H-132]
-。HR?FABMS：1197.5657(calcd.1197.5669?for?C
57H
90O
25Na)。
1H-NMR,
13The C-NMR data see Table 1, table 2.
1H-NMR,
13C-NMR sees Figure of description 5, Fig. 6.
Table 1 theasaponin T1, T10, T12's
13The C-NMR data
Ac＝acetyl，Ang＝angeloyl；Xyl：β-D-xylose；Ara：α-L-arabinose；GlcA：β-D-glucuronic?acid；
Glc：β-D-glucose；Gal：β-D-galactose；
Table 2 theasaponin T1, T10, T12's
1The H-NMR data
Ac＝acetyl，Ang＝angeloyl，
Xyl：β-D-xylose；Ara：α-L-arabinose；GlcA：β-D-glucuronic?acid；Gal：β-D-glucose；Glc：β-D-galactose；
The structural identification of embodiment 13 monomer theasaponin T20
White powder (methyl alcohol), mp196～198 ℃, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected cautious chromic acid and acetate and has existed; Acid hydrolysis, detecting has glucuronic acid, semi-lactosi, pectinose and wood sugar exist.IR(KBr?pellet)v
max?3453(OH)，1743(C＝O)，1085(C-O)cm
-1。
Pos.FAB-MS?m/z：1253[M+Na]
+，Neg.FAB-MS?m/z：1229[M-H]
-，1097[M-H-132]
-，1067[M-H-162]
-，965[M-H-2×132]
-，803[M-H-2×132-162]
-。HR?FABMS：1253.5573(calcd.1253.5567?for?C
59H
90O
27Na)。
1H-NMR,
13The C-NMR data see Table 3, table 4.
1H-NMR,
13C-NMR sees Figure of description 7, Fig. 8.
The structural identification of embodiment 14 monomer theasaponin T33
White powder (methyl alcohol), mp217～218 ℃, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and acetate and has existed; Acid hydrolysis, detecting has glucuronic acid, semi-lactosi, pectinose and wood sugar exist.IR(KBr?pellet)v
max?3453(OH)，1718(C＝O)，1650(C＝O)，1080(C-O)cm
-1。
Pos.FAB-MS?m/z：1283[M+Na]
+，Neg.FAB-MS?m/z：1259[M-H]
-，1127[M-H-132]
-，1097[M-H-162]
-，995[M-H-2×132]
-，965[M-H-132-162]
-。HRFABMS：1283.5682(calcd.1283.5673?for?C
60H
92O
28Na)。
1H-NMR,
13The C-NMR data see Table 3, table 4.
1H-NMR,
13C-NMR sees Figure of description 9, Figure 10.
The structural identification of embodiment 15 monomer theasaponin T34
White powder (methyl alcohol), 218～219 ℃ of mp, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and has existed; Acid hydrolysis, detecting has glucuronic acid, semi-lactosi, pectinose and wood sugar exist.IR(KBr?pellet)v
max?3453(OH)，1717(C＝O)，1638(C＝O)，1080(C-O)cm
-1。
Pos.FAB-MS?m/z：1225[M+Na]
+，Neg.FAB-MS?m/z：1201[M-H]
-，1069[M-H-132]
-，937[M-H-2×132]
-，907[M-H-132-162]
-。HRFABMS：1225.5613(calcd.125.5618?for?C
58H
90O
26Na)。
1H-NMR,
13The C-NMR data see Table 3, table 4.
1H-NMR,
13C-NMR sees Figure of description 11, Figure 12.
The structural identification of embodiment 16 monomer theasaponin T36
White powder (methyl alcohol), mp225～226 ℃, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and has existed; Acid hydrolysis, detecting has glucuronic acid, semi-lactosi, pectinose and wood sugar exist.IR(KBr?pellet)v
max?3453(OH)，1717(C＝O)，1638(C＝O)，1080(C-O)cm
-1。
Pos.
FAB-MS?m/z：1195[M+Na]
+，Neg.FAB-MS?m/z：1171[M-H]
-，1039[M-H-132]
-，907[M-H-2×132]
-，877[M-H-132-162]
-。HR?FABMS：1195.5520(calcd.1195.5512?for?C
57H
88O
25Na)。
1H-NMR,
13The C-NMR data see Table 3, table 4.
1H-NMR,
13C-NMR sees Figure of description 13, Figure 14.
Table 3 theasaponin T20, T33, T34, T36's
13The C-NMR data
Ac＝acetyl，Ang＝angeloyl；Xyl：β-D-xylose；Ara：α-L-arabinose；GlcA：β-D-glucuronic?acid；
Glc：β-D-glucose；Gal：β-D-galactose；
Table 4 theasaponin T20, T33, T34, T36's
1The H-NMR data
Ac＝acetyl，Ang＝angeloyl，
Xyl：β-D?xylose；Ara：α-L-arabinose；GlcA：β-D-glucuronic?acid；Gal：β-D-glucose；Glc：β-D-galactose；
Claims (10)
1, the triterpenoid saponin in the camellia seeds is characterized in that: it is represented oleanane derivative in the following general formula (1):
General formula (1)
The cautious chromyl angeloyl groups of ethanoyl cinnamoyl
In the general formula (1): R1 is hydrogen or hydroxyl or acyloxy; R2 is hydrogen or acyloxy; R3 is hydrogen or acyloxy; R4 is hydrogen or acyloxy; R5 is methyl or methylol or aldehyde radical or carboxyl or carboxyl ester; R6 is hydrogen or glycosyl.
2, the triterpenoid saponin in the camellia seeds according to claim 1 is characterized in that: the acyl group in the described acyloxy is one or more in ethanoyl, angeloyl groups, cautious chromyl, the cinnamoyl.
3, the triterpenoid saponin in the camellia seeds according to claim 1 is characterized in that: described glycosyl is one or more in the following glycosyl: glucal acidic group, Arabic glycosyl, galactosyl, glucosyl group, xylosyl and the oligonucleotide chain base of being made up of them.
4, the preparation method of triterpenoid saponin in a kind of camellia seeds as claimed in claim 1, it is characterized in that: its preparation method is as follows:
(1) gets dry camellia seeds oil expression back gained camellia oleosa seed cake, add and extract solvent soaking, medicinal material is 1:5 to 1:12 with the ratio of extracting solvent, heats 0.5～3 hour under 50～100 ℃ of conditions, extract 1～4 time, merge 2～10 times of volumes that each extracting solution is concentrated into the medicinal material amount;
(2) extracting solution is through macroporous resin (HPD100 or resins for universal use such as D101 or AB-8) absorption, and the rare pure flush away impurity with 0～30% gets the thick total saponins of camellia seeds with 40%～100% pure liquid wash-out again;
(3) the thick total saponins of gained camellia seeds adds the less water dissolving, through opening ODS column chromatography, with 0～30% rare pure flush away tea flavones impurity, gets the camellia seeds total saponins with 40%～90% pure liquid wash-out again;
(4) gained camellia seeds total saponins is through the further separation and purification of HPLC: reversed-phase bonded silica is a stationary phase, is moving phase with methanol-water or acetonitrile water, and differential or UV-detector detect, and obtain all kinds triterpenoid saponin constituents shown in the general formula (1).
5, the preparation method of triterpenoid saponin in the camellia seeds according to claim 4 is characterized in that: the extraction solvent in the described step (1) is a kind of in water, methyl alcohol, the ethanol.
6, the preparation method of triterpenoid saponin in the camellia seeds according to claim 4, it is characterized in that: the extracting method in the described step (1) is decocting method or heating and refluxing extraction.
7, the preparation method of triterpenoid saponin in the camellia seeds according to claim 4 is characterized in that: pure liquid is methyl alcohol or alcoholic acid solution in the described step (3).
8, the preparation method of triterpenoid saponin in the camellia seeds according to claim 4 is characterized in that: reversed-phase bonded silica is a kind of in C-30～C-2 bonded silica gel in the described step (4).
9, the preparation method of triterpenoid saponin in the camellia seeds according to claim 4 is characterized in that: the moving phase in the described step (4) is 30%～80% methanol-water or acetonitrile water, and contains 0.01～0.5% Glacial acetic acid in methanol-water or the acetonitrile water.
10, the triterpenoid saponin in the camellia seeds is in preparation protection stomach mucous membrane, antitumor, hypoglycemic, the medicine of reducing blood-fat or the application in the protective foods.
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