CN101392015B - Triterpene saponin in camellia seeds, preparation method and medical use thereof - Google Patents

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 thelike 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

Triterpenoid saponin in camellia seeds and preparation method thereof and medicinal use

Technical field:

The invention belongs to medical technical field, relate to triterpenoid saponin in camellia seeds and preparation method thereof and medicinal use.

Background technology:

Camellia Camellia sinensis is the Theaceae Camellia Plants, in China's commerial growing, is a kind of good cash crop.The systematic research report of the chemical composition of its tender shoots---tealeaves and biological activity.Show that according to existing research data the chemical composition 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.Find in our early-stage Study, contain a large amount of saponin components in the tea seed.Tea kind total saponins is a kind of good tensio-active agent, 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 saponins in the camellia seeds as shown in general formula (1), and preparation method thereof and medicinal use.

The invention provides the triterpenoid saponin in camellia seeds, it is represented oleanane derivative in following general formula (1):

General formula (1)

The cautious chromyl angeloyl groups of ethanoyl cinnamoyl

In 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, aralino, galactosyl, glucosyl group, xylosyl and the oligonucleotide chain that is comprised 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) get the rear gained camellia oleosa seed cake of dry camellia seeds oil expression, add the extraction 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 adsorbs through macroporous resin (HPD100 or the resins for universal use such as D101 or AB-8), the rare alcohol wash decon with 0～30%, then get the thick total saponins of camellia seeds (content 50%～70%) with 40%～100% pure liquid wash-out;

(3) the thick total saponins of gained camellia seeds adds a small amount of water dissolution, through opening ODS column chromatography, washes away tea flavones impurity with 0～30% rare alcohol, then gets camellia seeds total saponins (content 70%～95%) with 40%～90% pure liquid wash-out;

(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.), take 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 as moving phase, (190～220nm) detectors detect, and obtain all kinds triterpenoid saponins shown in general formula (1) for differential or ultraviolet.

The preparation method of triterpenoid saponin in camellia seeds provided by the invention, the extraction solvent in described step (1) is water, methyl alcohol, ethanol etc., the concentration of methyl alcohol or ethanol is 5%～95%; Extracting method is decocting method or heating and refluxing extraction method.

The preparation method of triterpenoid saponin in camellia seeds provided by the invention, in described step (3), pure liquid is methyl alcohol or the ethanol of different concns.

The invention provides the medical use of triterpenoid saponin in camellia seeds, the triterpene saponin componds in the camellia seeds as shown in general formula (1) has the effects such as gastric mucosal protection, antitumor, hypoglycemic, reducing blood-fat; Can be used for preparing 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 of the present invention is high, purity is high;

(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 of the present invention is high, purity is high, and structure is clear and definite, can carry out the targeted structural modification, with further exploitation;

(4) the present invention is raw materials used is industrial waste for the camellia oleosa seed cake after camellia seeds oil expression, and cost is low, be easy to get, be conducive to environmental protection;

(5) easy, the easy row of preparation method pollutes less, 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.

Embodiment 1

The preparation of camellia total saponins

Get the rear gained camellia oleosa seed cake 10kg of dry camellia seeds oil expression, remove impurity, dust.

The water that adds medicinal material and 10 times of amounts in Chinese medicine extracting tank is heated to 80 ℃, extracts twice, each 2 hours, merges 4 times of volumes that each extracting solution is concentrated into the medicinal material amount.

Extracting solution washes away impurity through macroporous resin adsorption with 20% ethanol, then gets thick total saponins 680 grams of camellia seeds (content 60%) with 90% ethanol elution.

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), wash away tea flavones impurity with 20% ethanol, then get camellia seeds total saponins 380 grams (content 85%) with 85% ethanol elution.

Gained camellia seeds total saponins is through further separation and purification of reversed-phase HPLC (Shimadzu L6A): take ODS and C-30 bonded silica gel as stationary phase, respectively take methanol-water (containing 0.05% Glacial acetic acid) volume ratio 70: 30 as moving phase, 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 for the flowing opposite subdivision from, the differential detector detects, and obtains all kinds triterpenoid saponins shown in general formula (1).

Embodiment 2

The preparation of camellia total saponins

(1) get the rear gained camellia oleosa seed cake 6kg of dry camellia seeds oil expression, remove impurity, dust.

(2) add 95% ethanol of medicinal material and 8 times of amounts in Chinese medicine extracting tank, be heated to 80 ℃, extract twice, each 1.5 hours, merge 2 times of volumes that each extracting solution is concentrated into the medicinal material amount.

(3) extracting solution through macroporous resin adsorption, washes away impurity with 30% ethanol, then gets thick total saponins 770 grams of camellia seeds (content 65%) with 90% ethanol elution.

(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), wash away tea flavones impurity with 20% ethanol, then get camellia seeds total saponins 430 grams (content 87%) with 90% ethanol elution.

(5) gained camellia seeds total saponins is through further separation and purification of reversed-phase HPLC (Shimadzu L6A): take ODS and C-30 bonded silica gel as stationary phase, respectively take methanol-water (containing 0.1% Glacial acetic acid) volume ratio 65: 35 as moving phase, acetonitrile-water (containing 0.1 Glacial acetic acid) volume ratio is moving phase at 33: 67, methyl alcohol-acetonitrile-water (containing 0.05% Glacial acetic acid) volume ratio 4: 34: 62 for the flowing opposite subdivision from, the differential detector detects, and obtains all kinds triterpenoid saponins shown in general formula (1).

Embodiment 3

Investigate the camellia seeds total saponins to the impact of S180 tumor-bearing mice tumor growth

Laboratory animal: 8 the week age male ICR mouse, mean body weight 20 grams.Provided 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's experimentation on animals center.

The preparation of S180 Ascitic Tumor Cells suspension: operate under aseptic condition, 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 ice chest.The ascites that separately takes a morsel is carried out cell counting: with 100 times of normal saline dilutions, getting diluent 0.9mL adds platform and expects orchid (expecting blue normal saline solution for 0.2%) 0.1mL, mixing, analyze tumour cell sum and dead cell number (purple or blue, viable count answers＞95%) with cell counting.The ascites normal saline dilution, adjusting oncocyte concentration is 1 * 107/mL (whole operating on ice chest carried out, and keeps the activity of tumour cell with this).

Kunming mouse is transplanted the foundation of S180 sarcoma model: the Kunming mouse of getting body weight 20g left and right, it is subcutaneous that every mouse 0.2mL (tumor cell number is about 2 * 106) is inoculated in the right fore armpit, weigh after 24h, be divided at random 3 groups, 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 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 after administration 10d, next day is put to death mouse in drug withdrawal, takes Mouse Weight, dissects the knurl body, weighs.

Experimental result: see Table 1

The impact of table 1 camellia seeds total saponins on S180 tumor-bearing mice tumor growth

Embodiment 4

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, cell suspending liquid take the RPMI-1640 nutrient solution dilution that includes 10% (v/v) foetal calf serum (fetal bovine serum) as 5 * 104/L, be inoculated in 96 orifice plates, 100 μ L/ holes are put in 37 ℃, saturated humidity, 5%CO2 incubator and are cultivated 24h.

Add reagent: after a small amount of DMSO dissolution sample, be 10 with the RPMI-1640 nutrient solution dilution that contains 10% (v/v) foetal calf serum, 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.

Result is measured:

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

Embodiment 5

Investigate camellia kind total saponins to the impact of glucose load rat blood sugar

Laboratory animal: male Wistar rat, body weight 130～150g is available from Japanese KiwaLaboratory Animal Co.Ltd. (Wakayama).Animal constant temperature (23 ± 2 ℃) is raised, and feed is available from Japanese MF.Oriental Yeast Co.Ltd. (Tokyo).Before experiment, the animal fasting is 20～24 hours, freely fetches water.

Experimental technique: get the male Wistar rat of 130～150g, fasting is after 20～24 hours, and gavage is given and camellia kind total saponins, 0.5 after hour, gavage is to sucrose (1.0g/kg), after 0.5 hour, blood is got on the eyeground, adopts the determination of glucose oxidase glucose level after centrifugal separation plasma.

Experimental result: provided the impact of camellia kind total saponins on glucose load rat blood sugar level in table 3, camellia kind total saponins can significantly reduce glucose load rat blood sugar level at 100～400mg/kg dosage.

The impact of table 3 camellia kind total saponins on the 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.

Embodiment 6

Investigate camellia kind total saponins to the impact of triglyceride levels in 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).Before experiment, the animal fasting is 20～24 hours, freely fetches water.

Experimental technique: get the male DDY mouse of 25～30g, fasting is after 20～24 hours, and gavage is given and camellia kind total saponins, 0.5 after hour, gavage is to sweet oil (5mL/kg), after 2 hours, blood is got on the eyeground, adopts triglyceride levels in WAKO kit measurement blood plasma after centrifugal separation plasma.

Experimental result: provided the impact of camellia kind total saponins on triglyceride levels in oil load mice plasma in table 4, camellia kind total saponins can significantly reduce triglyceride levels in oil load mice plasma at 400mg/kg dosage.

The impact of table 4 camellia kind total saponins on triglyceride levels in 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.

Embodiment 7

Investigate camellia kind total saponins to the impact of 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).Before experiment, the animal fasting is 24～26 hours, freely fetches water.

Experimental technique: get the male SD rat of 230～250g, fasting is after 24～26 hours, and gavage is given and tea kind total saponins (5% gum arabic suspendible), after 1 hour, gavage causes gastric mucosa injury to 99.5% ethanol (1.5mL), and after 1 hour, etherization is put to death.Take out the rat stomach, after extracting content out, the formalin solution of injection 10mL 1.5% is coat of the stomach fixedly, and with flattening after flushing with clean water, computer scanning calculates the gastric mucosa injury rate repeatedly.

Experimental result: result is as shown in table 5, and camellia kind total saponins can significantly reduce at 2.5～20mg/kg dosage the gastric mucosa injury that ethanol causes.

The impact of table 5 camellia kind total saponins on 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 the impact of the gastric mucosa injury that camellia kind total saponins induces indomethacin

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).Before experiment, the animal fasting is 24～26 hours, freely fetches water.

Experimental technique: the male SD rat of getting 230～250g, after fasting 24～26 hours, gavage is given and camellia kind total saponins (5% gum arabic suspendible), after 1 hour, (the 20mg/kg indomethacin is dissolved in 5% sodium hydrogen carbonate solution to gavage, after distilled water diluting to indomethacin, 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, after extracting content out, the formalin solution of injection 10mL 1.5% is coat of the stomach fixedly, and with flattening after 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 at 50～200mg/kg dosage the gastric mucosa injury that indomethacin causes.

The impact of the gastric mucosa injury that table 6 camellia kind total saponins is induced indomethacin

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 9

The provide protection of the monomer theasaponin that the investigation separation obtains to 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.

The provide protection of table 7 monomer theasaponin to 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.

The structural identification of embodiment 10 monomer theasaponin T1

White needle (methyl alcohol), 219～220 ℃ 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)vmax?3453(OH)，1718(C＝O)，1649(C＝O)，1O78(C-O)cm-1。 ${\left[\mathrm{\α}\right]}_D^{20}=+6.5(c2.5,\mathrm{MeOH}).$ 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.1213.5618for?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), 220～221 ℃ of mp, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and acetic acid 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 ${\left[\mathrm{\α}\right]}_D^{20}=-10.0(c2.4,\mathrm{MeOH}).$ 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), 223～224 ℃ 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)，1741(C＝O)，1085(C-O)cm ^-1。 ${\left[\mathrm{\α}\right]}_D^{20}=+7.9(c2.0,\mathrm{MeOH}).$ 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), 196～198 ℃ of mp, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected cautious chromic acid and acetic acid 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。 ${\left[\mathrm{\α}\right]}_D^{20}=+10.9(c3.0,\mathrm{MeOH}).$ 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.1253.5567for?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), 217～218 ℃ of mp, Liebermann-Burchard reacting positive, Molish reacting positive.Basic hydrolysis has detected angelicic acid and acetic acid 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。 ${\left[\mathrm{\α}\right]}_D^{20}=+25.1(c2.0,\mathrm{MeOH}).$ 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。 ${\left[\mathrm{\α}\right]}_D^{20}=+10.0(c0.9,\mathrm{MeOH}).$ 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] ^-。HR?FABMS：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), 225～226 ℃ 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。 ${\left[\mathrm{\α}\right]}_D^{20}=-1.3(c1.0,\mathrm{MeOH}).$ 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 (2)

1. the preparation method of triterpenoid saponin in camellia seeds is characterized in that:

Get the rear gained camellia oleosa seed cake 10kg of dry camellia seeds oil expression, remove impurity, dust;

The water that adds medicinal material and 10 times of amounts in Chinese medicine extracting tank is heated to 80 ℃, extracts twice, each 2 hours, merges 4 times of volumes that each extracting solution is concentrated into the medicinal material amount;

Extracting solution washes away impurity through macroporous resin adsorption with 20% ethanol, then gets thick total saponins 680 grams of camellia seeds, content 60% with 90% ethanol elution;

The thick total saponins of gained camellia seeds adds 5 times of water gaging dissolvings, and through opening ODS column chromatography, sample size: filler=1:10 washes away tea flavones impurity with 20% ethanol, then gets camellia seeds total saponins 380 grams, content 85% with 85% ethanol elution;

gained camellia seeds total saponins is through reversed-phase HPLC, Shimadzu L6A, further separation and purification: take ODS and C-30 bonded silica gel as stationary phase, respectively take the methanol-water that contains 0.05% Glacial acetic acid of volume ratio 70:30 as moving phase, the acetonitrile-water that contains 0.05% Glacial acetic acid of volume ratio 40:60 is moving phase, methyl alcohol-the acetonitrile-water that contains 0.05% Glacial acetic acid of volume ratio 8:32:60 be the flowing opposite subdivision from, the differential detector detects, obtain all kinds triterpenoid saponins shown in general formula (1)

In general formula (1): R ₁Be hydrogen or hydroxyl or acyloxy; R ₂Be hydrogen or acyl group; R ₃Be hydrogen or acyl group; R ₄Be hydrogen or acyl group; R ₅Be methyl or methylol or aldehyde radical or carboxyl or carboxyl ester; R ₆Be hydrogen or glycosyl; Described acyl group is ethanoyl, angeloyl groups, cautious chromyl or cinnamoyl; Described glycosyl is the oligonucleotide chain base of glucal acidic group, aralino, galactosyl, glucosyl group, xylosyl or their compositions.

2. the preparation method of triterpenoid saponin in camellia seeds is characterized in that:

Get the rear gained camellia oleosa seed cake 6kg of dry camellia seeds oil expression, remove impurity, dust;

95% ethanol that adds medicinal material and 8 times of amounts in Chinese medicine extracting tank 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;

Extracting solution washes away impurity through macroporous resin adsorption with 30% ethanol, then gets thick total saponins 770 grams of camellia seeds, content 65% with 90% ethanol elution;

The thick total saponins of gained camellia seeds adds 4 times of water gaging dissolvings, and through opening ODS column chromatography, sample size: filler=1:12 washes away tea flavones impurity with 20% ethanol, then gets camellia seeds total saponins 430 grams, content 87% with 90% ethanol elution;

Gained camellia seeds total saponins is through reversed-phase HPLC, Shimadzu L6A, further separation and purification: take ODS and C-30 bonded silica gel as stationary phase, respectively take the methanol-water that contains 0.1% Glacial acetic acid of volume ratio 65:35 as moving phase, the acetonitrile-water that contains 0.1% Glacial acetic acid of volume ratio 33:67 is moving phase, the methyl alcohol-acetonitrile-water that contains 0.05% Glacial acetic acid of volume ratio 4:34:62 be the flowing opposite subdivision from, the differential detector detects, obtain all kinds triterpenoid saponins shown in general formula (1)

In general formula (1): R ₁Be hydrogen or hydroxyl or acyloxy; R ₂Be hydrogen or acyl group; R ₃Be hydrogen or acyl group; R ₄Be hydrogen or acyl group; R ₅Be methyl or methylol or aldehyde radical or carboxyl or carboxyl ester; R ₆Be hydrogen or glycosyl; Described acyl group is ethanoyl, angeloyl groups, cautious chromyl or cinnamoyl; Described glycosyl is the oligonucleotide chain base of glucal acidic group, aralino, galactosyl, glucosyl group, xylosyl or their compositions.

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