CN1149587A - Ginseng oligosaccharide element and ginseng monomer oligosaccharide and its preparing technology and use - Google Patents
Ginseng oligosaccharide element and ginseng monomer oligosaccharide and its preparing technology and use Download PDFInfo
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Abstract
The cells obtained through callus succeeeded geneneration culturing and suspension culturing of the ginseng primary wall cell system are conducted the pretreatment, the dried matter of cell wall is obtained through freeze drying, then the acid hydrolysis is conducted by using HCl of 0.7-1N to react for 1-1.5 hr under 90-98 deg.C. The ginseng oligosaccharide element composed of 3-12 monosaccharide degree of polymerization is obtained. Through active carbon column, ion exchange column and biological gel column separation, the segmental oligosaccharide components are obtained. By using HPLC separation, the ginseng trisaccharide to dodecasaccharide of monomer oligosaccharide are obtained. The mixture of ginseng monomer oligosaccharide and ginseng oligosaccharide element can be used to prepare the medicine for immunoenhancement and anticancer adjuvant drug and can also be used as revulsive of plant secondary metabolic substance.
Description
The present invention relates to biological technical field, relate in particular to the ginseng oligosaccharide element that obtains by genseng primary wall cell cultivation separation and purification, it is the genseng mixing oligosaccharides that the polymerization degree (DP) is made up of 3～12 monose, and further segmentation oligosaccharide compositions and the ginseng monomer oligosaccharide that obtains that separate, their preparation technology, and they are in the application aspect the elicitor of growth inducing of immunostimulant, anticancer adjuvant, food, plant cell culture, plant secondary metabolites.
Activated molecule is called oligosaccharide (Oligosacharin) in bar structure polysaccharide such as the U.S. Albershcim degraded fragment, the oligosaccharide of finding the earliest is activated Portugal seven sugar that obtain from the fungi Phytophythora f.sp.Glycinea cell walls that can infect soybean or mycelium in the world, thought structural polysaccharide in the plant cell wall that only plays protection in the past, be found to be adjusting molecule with special biological function, also can with protein, nucleic acid the same as informational molecule to growth and development of plant, morphogenesis and disease-resistant bacterium are infected etc. and to be of great importance, so U.S., state scientists such as Japan identify from separation and purification and structure so far, synthesizing of oligosaccharide, structure and function, mechanism of action aspect is studied the plant oligosaccharide, as document Augur C, et al, 1992 Pl.Physiol 99:180-185 have reported the xyloglucan that separation obtains from false prestige maple suspending nutrient solution cell walls, tool antiauxin activity; Document LerougeP, et al, 1990, Nuture, the 344:781-784 report, the DP that produces during infecting the clover process from root nodule bacterium is 4, concentration 10
-9The root nodule oligosaccharides of M can induce clover to form root nodule, document Priem Betal, and 1992, PlantPhysiol 98:399-401 report decomposes the DP-6 that obtains from ripe pectin, and the seminose oligosaccharides of consumption 1mg/g fruit can promote green tomato synthesizing ethylene and accelerate the ripening.At present there is no the report that obtains the high-quality ginseng oligosaccharide from the genseng culturing cell both at home and abroad, separation and Extraction oligosaccharides processing step is many, and solvent for use is more expensive, and instrument requires the precision height.In order to seek the fast and high new plant oligosaccharide resources of production of oligosaccharide content of a kind of cell growth, reach and promote oligosaccharide suitability for industrialized production schedule speed, it is material that the present invention adopts the higher genseng of economic worth (Panax-ginseng), reach the purpose of producing genseng and ginseng oligosaccharide element with these biotechnology means of cell cultures, the water soluble mixt that physico-chemical property is good, the polymerization degree (DP) is made up of 3-12 monose is provided, the genseng mixing oligosaccharides that provides the polymerization degree (DP) to be made up of 6-7 monose provides the panose to genseng ten disaccharides monomer oligosaccharides.
The present invention provides simultaneously from ginseng-cell and has cultivated the technology of producing above-mentioned ginseng oligosaccharide element and monomer oligosaccharide, and is used as growth inducing of immunostimulant, anticancer adjuvant, food, plant cell culture, the elicitor of plant secondary metabolism by the product of this explained hereafter.
To achieve these goals, the invention provides following technical scheme:
The ginseng oligosaccharide element mixture that the polymerization degree DP that is got by the ginseng-cell cultivation separation and purification is made up of 3-12 monose.
Get ginseng monomer oligosaccharide by the ginseng oligosaccharide element separation, above-mentioned ginseng oligosaccharide element mixture is passed through activated carbon, ion exchange column, the separation of xanthan gel post obtain the segmentation oligosaccharide compositions, then through HPLC separate the panose to the monomer oligosaccharide of genseng ten disaccharides, wherein genseng six sugared structural formulas are shown in (I):
The technology of preparation ginseng oligosaccharide element, monomer oligosaccharide, comprise that the cell that genseng primary wall cell system is obtained by callus succeeding transfer culture and suspension culture carries out pre-treatment, frost drying gets the cell walls dry-matter, carry out acid hydrolysis then, adopt 0.7-1N HCl under 90-98 ℃ of temperature, to react 1-1.5 hour, get ginseng oligosaccharide element; Again with above-mentioned ginseng oligosaccharide element liquid by active carbon column, ion exchange column, xanthan gel post separate the sectional oligosaccharide compositions, with HPLC separate the panose to the monomer oligosaccharide of genseng ten disaccharides.
In the above-mentioned technology, ginseng callus and suspension cell culture are cultivated with MS, additional 2mg/l 2, and 4-D, 0.1mg/l KT, 26 ± 1 ℃ of dark down cultivations, cultivating 20-25 days is one-period.
Acid hydrolysis condition optimization scope adopts 1N HCl to react 1 hour under 90 ℃ of temperature.
The invention provides a kind of immunostimulant, contain mixing oligosaccharides of the present invention and pharmacy acceptable carrier.
A kind of immunostimulant contains monomer oligosaccharide of the present invention and pharmacy acceptable carrier.
A kind of anticancer adjuvant contains mixing oligosaccharides of the present invention and pharmacy acceptable carrier.
A kind of anticancer adjuvant contains monomer oligosaccharide of the present invention and pharmacy acceptable carrier.
The elicitor of one kind of plant secondary metabolites contains mixing oligosaccharides of the present invention.
The elicitor of one kind of plant secondary metabolites contains monomer oligosaccharide of the present invention.
Technique scheme of the present invention is carried out according to following technological line:
Ginseng callus induce and succeeding transfer culture → suspension culture → cell pre-treatment (remove liposoluble part and protein part) → stem cell acid hydrolysis → neutralization, filtrations → active carbon column → ion exchange column → BiO-P-2 post → silica gel (G) post → HPLC (ODS semipreparative column) → FAB-MS and NMR → complete hydrolysis detects monose kind → make structure to identify → influence of plant tissue cell's cultivation is tested.
For flesh and blood of the present invention and beneficial effect are described better, will more specifically set forth technical scheme of the present invention and application test below.
Induce with the suspension cell culture method with the plant callus of routine and to obtain the genseng culturing cell, with cytoclasis homogenate after-filtration, go out liposoluble with 80% ethanol extracting and partly cast out, remove protein in the solution with the Se-vage method again, remove filtrate, acid hydrolysis (0.7-1NHCl will be carried out behind the remainder frost drying, 1-1.5 hour, 90-98 ℃) neutralization, leave standstill 4 ℃ of conditions, elimination precipitates, and obtains containing the ginseng oligosaccharide element mixture solution of 3-12 monose.
Above gained solution is removed pigment by active carbon column, Dowex50 (H
+) ion exchange column removes Na
+, separate with the xanthan gel post again, obtain four sections and have active ginseng oligosaccharide element fragment (DP1,2+DP3,4,5+DP6,7,8+DP9,10,11,12), separate the monomer oligosaccharide that obtains 3-12 with HPLC at last.Analyze the molecular structure of monose.
In order to reach better effect, the present invention adopts strict control hydrolysis condition in the acid hydrolysis process, and the little part of the polymerization degree accounts for significant proportion in the acid concentration oligosaccharide that obtains bigger than normal.Acid concentration is too small, and polysaccharide part content is bigger than normal again; To in time cool off after hydrolysis is finished with NaOH and interrupt reaction, prevent that hydrolysis reaction from proceeding.The solvent different with prior art of employing of the present invention simultaneously used 0.7-1N HCl, and reaction got final product in 1-1.5 hour under the boiling condition.The prior art majority adopts trifluoracetic acid or enzymolysis as solvent, the cost height, and consumption is big, is difficult to suitability for industrialized production.
Simultaneously, in order to remove the partial impurities in the ginseng oligosaccharide element first product, as salt, saponin and pigment, so before separating, adopt active carbon column and Dowex (H
+) column purification.In separating the oligosaccharide process, because it does not have absorption on ultraviolet, the application of HPLC is restricted, many both at home and abroad at present employings are reduced not at it, and termination one has ultraviolet absorption group (as ABBE, para-amino benzoic acid is ester derivative etc.) way separate, but this way has brought trouble for later activity identification.
The present invention adopts counter point, be about to 2,4-dinitrophenylhydrazine and oligosaccharides reaction, the reduction that is connected on oligosaccharides is not held, so that HPLC goes up ultraviolet detection, according to its peak type and retention time and oligose fragment size rule, as object of reference, repeat to do the separation of unreacted oligosaccharides under the same conditions, (forward slightly) fraction collection in corresponding retention time, after book was rolled over (TLC) detection layer by layer, same section merged.Make FAB-MS and NMR respectively and measure, analyze its structure and configuration, conformation, use sour complete hydrolysis subsequently after, the ply of paper folding is measured its monose and is formed.
Ginseng oligosaccharide element by the above-mentioned prepared of the present invention and monomer oligosaccharide to the safflower cell and among alpha-tocopherol, ginsenoside among pseudo-ginseng and ginseng-cell reach, the influence of content of taxol can be illustrated by following test-results among paniculatum cell and Shikonin and yew cell growth reached:
1, the ginseng oligosaccharide element mixture produced of the present invention can promote the growth of safflower (Carthamusiinctorius) culturing cell and improve the content of alpha-tocopherol in the cell, and wherein the effect with three kinds of oligosaccharides such as genseng disaccharides, panose, genseng pentasaccharides is comparatively remarkable; Added behind the oligosaccharides 1-3 days, the content of alpha-tocopherol promptly improves, and adds genseng one sugar, disaccharides, trisaccharide simultaneously, can make the safflower cell growth rate improve 18.11%, and the comparison of alpha-tocopherol content is according to improving 3.5 times, and its productivity ratio contrast improves 4.3 times.
2, the ginseng oligosaccharide element of the present invention's production makes the growth of Radix Notoginseng cell suspended culture cell improve 22.7%, makes the saponin productive rate improve nearly one times, and concrete test contains the mixture of 3-12 monose for the polymerization degree of the present invention of adding 15PPm.
3, monomer oligosaccharide genseng seven sugar and genseng eight sugar produced of the present invention can promote that in the Asian puccoon callus culture of Yunnan pigment synthetic optimal concentration is respectively 2.5ppm, when low concentration (1.0-2.5ppm) then slightly to suppress pigment synthetic.
4, monomer oligosaccharide genseng six sugar of the present invention's production are as shown in the table to the influence of growth of taxusyunnanensis suspended culture cell and content of taxol:
| Oligosaccharide concentration PPm | Growth velocity g/l.d | Content of taxol (%) |
| Total content in the substratum in the cell | ||
| 1.0 5.0 10.0 contrasts | ?????????0.322 ?????????0.350 ?????????0.271 ?????????0.256 | ????0.0164????0.0074????0.0238 ????0.0190????0.0205????0.0395 ????0.0108???????0??????0.0108 ????0.0098????0.0039????0.0137 |
As seen, genseng six sugared low concentration cell growth and content of taxol have significant inducing action, particularly 5PPm genseng six sugar make the taxol total content of Ramulus et folium taxi cuspidatae suspended culture cell and nutrient solution improve 188.32% to strengthening the synthetic of taxol and promoting that the effect of cell growth is the most obvious; Make Ramulus et folium taxi cuspidatae suspended culture cell growth velocity improve 36.72%.And descend on the contrary to the time effect of 10ppm concentration.
5, the ginseng oligosaccharide element of the present invention growth and monomer oligosaccharide The pharmacological results show and can significantly improve immune function of mice, to put, white corpuscle and blood platelet regenerating have remarkable effect after the chemotherapy; Vitro culture people alveolar cell carcinoma cell there is fine restraining effect.
6, the ginseng oligosaccharide element of the present invention's production can obviously increase the test tube knot potato number of potato, than generally acknowledging that effective tonka bean camphor and choline dichloride are good.
Show that by the invention described above technical scheme and active function result beneficial effect of the present invention is:
(1), the present invention is a purpose to produce ginseng oligosaccharide element, all inequality with other inventions both at home and abroad, fast and the oligosaccharide content height of genseng culturing cell growth, the ginseng-cell growth velocity reach the 15g dry weight/liter, the ginseng oligosaccharide cellulose content is on average more than 10%, and the oligosaccharide content of genseng crude drug is lower than dried/one.
(2), ginseng oligosaccharide element mixture provided by the invention, oligosaccharide compositions and monomer oligosaccharide physico-chemical property are good, soluble in water, stable to light, heat, PH2-12,121 ℃ of abilities, high pressure steam sterilization more than 15 pounds, storage period is more than 3 years, product is stable, tool various active function.The monomer oligosaccharide novel structure is beta configuration, and 1 → 4 and 1 → 3 mode of connection is made up of seminose, glucose and semi-lactosi, is the high structure of internationally recognized activity.
(3), stable preparation process of the present invention, simple being suitable for, the yield height, cost is low, is suitable for suitability for industrialized production, content assaying method accurately, good reproducibility, fast, simple being suitable for.
1. the immunocompetence test (seeing Table 1) of ginseng oligosaccharide element of the present invention (GOD):
The immunocompetence of table 1 ginseng oligosaccharide element (GOB) (ICR ♀ mouse 18-21 gram, administration is 5 days after the SRBC sensitization, dissection in the 6th day is measured)
| The sample title | Dosage mg/kg * d | Route of administration | The mouse number | Immune organ weight mg/10g body weight | |||
| Spleen | Thymus gland | Lymphoglandula | Liver | ||||
| The contrast ginseng oligosaccharide | ??N.S.×5 ??50×5 | ip ip | 9 9 | ??75±12 ??70±12 | ??33±11 ??29±9 | ??24±10 ??23±5 | ??545±85 ??562±38 |
| The sample title | ??PFC ??10 4Cell | Lymphopoiesis 3H-TdR cpm mixes | Complement C μ g/10 μ l serum | IgG μ g/10 μ l serum |
| The contrast ginseng oligosaccharide | ??598±27 ??596±22 | ????6585±1104 ????12183±1079 ↑ *** | ????14.2±2.4 ????16.3±1.7 ↑ * | ?237±60 ?208±45 |
With compare:
*P＜0.05,
* *P＜0.01. conclusion: ginseng oligosaccharide can make T cell proliferation quicken, and improves the serum complement C3 level, and immune-enhancing activity is arranged.
2. ginseng oligosaccharide element of the present invention (GOD) to put, the influence of chemotherapy blood picture test: table 2 ginseng oligosaccharide GOB+ radiotherapy is to B
16The observation of melanoma mouse blood picture variable effect
Project g/l x10
12/ l x10
9/ l x10
9The neutral lymph normal mice of/l % % group oxyphorase red corpuscle white corpuscle thrombocyte 113 5.65 8.10 123 27 73 physiological saline contrast 93 4.23 11.09 201 63 37GOB
150mg * 12 times84 4.38 11.47 211 65 3560CO
500CGY * 1 time84 3.29 5.97 108 54 46
60CO+GOB
500CGY 500mg * 12
96????3.89????6.56????108????48????52
60CO+GOB
600CGY 100mg×12
88????4.57????6.30????151????41????59
60CO+GOB
500CGY 150mg×12
76 3.56 7.00
*163
*41 59P＜0.05 table 3 ginseng oligosaccharide GOB+ chemotherapy is to the observation of Lewis lung cancer mouse blood picture variable effect
Project g/l x10
12/ l x10
9/ l x10
9The neutral lymph normal mice of/l % % group oxyphorase red corpuscle white corpuscle thrombocyte 113 5.65 8.10 123 27 73 physiological saline contrast 93 4.06 11.21 179 63 37GOB
50mg * 1282 4.38 11.47 211 65 35COB
100mg * 1276 4.26 12.63 198 71 29GOB
150mg * 1284 4.38 11.47 211 65 35GOB
200mg * 1267 4.76 9.43 117 71 29 endoxan
100mg * 2
89 4.77 7.90 137 45 55 endoxan+GOB
100mg * 2 100mg * 12
90 4.76 8.60 205 67 33 endoxan+GOB
100mg * 2 200mg * 12
86????4.90????8.70
*???213
*??61??38*P＜0.05
As shown in table 2, single all do not have obvious change, mouse warp with GOB to every index of small white mouse blood picture
60The Co500CGY irradiation once adds GOB150mg/kg and uses than single
60Co irradiation group white corpuscle raises 1.03 * 10
9/ L, thrombocyte improves 55 * 10
9/ L. endoxan 100mg/Kg adds the GOB200mg/Kg group and raises 0.8 * 10 than single with ring phosphamidon group white corpuscle
9/ L, thrombocyte improves 76 * 10
9/ L (table 3) finds out from the statistic data of each experimental group, when GOB concentration is 100-200mg/Kg with put, the chemotherapy coupling, white corpuscle and thrombocyte are had the certain protection effect, especially comparatively obvious to hematoblastic rising.Above-mentioned each group is got the marrow film-making simultaneously and is observed, and the result is consistent with peripheral hemogram.
3, genseng oligosaccharide GOB acute toxicity test result of the present invention:
Supply the reagent product: GOB of the present invention (ginseng oligosaccharide) 5% supplies to use with the physiological saline dilution.
Method and result press the provisions for new drugs approval technical requirements, 80 of Kunming mouses, and volume 18-23g, male and female half and half are measured ig and ivGOB acute toxicity respectively,
(1) after the administration of gastric infusion 5%GOB dosage n body weight is all mortality ratio
(g/kg) (g) time performance 0.35ml/10g 3.5 20 19 ± 1.5 creakily cries, tremble, dead 0.25ml/10g 1.25 20 19 ± 1.9 the same 2/20 (two) tail intravenously administrable ml/10g n body weight (g) all mortality ratio LD took place after not eating food 8/20 * 2 24 hour
50(g/Kg) 0.25 10 21 ± 1.2 8/100.213 10 21 ± 1.6 5,/10 1.04 (0.93--1.16) 0.18 10 19 ± 1.9 3/100.15 10 19 ± 1.2 1/10 conclusion: GOB5% test liquid ig or iv all show certain toxicity, after the life of ig death by poisoning is 24h, ivLD50 is 1.04/kg, death (10%) also takes place in ig1.25g/kg, 3.5g/kg mortality ratio reaches 40%, be limited to administration volume and concentration and can't obtain the LD50 that irritates stomach, but take temperature from absolute number, this product belongs to low toxicity material.
4, genseng oligosaccharide of the present invention (GOB) is tested the restraining effect of vitro culture human lung carcinoma cell:
Table 4GOB is to the effect of vitro culture human lung carcinoma cell
Conclusion: have when adding the GOB of 195 μ g in 95% the cancer cells endochylema a large amount of cavitys to occur, pyknosis appears in about 7% cancer cells nuclear, is 71.4% to the inhibiting rate of growth of cancer cells.Having 92% cancer cells endochylema cavity to occur when adding the GOB of 100 μ g, examine no pyknosis, is 22.1% to the inhibiting rate of growth of cancer cells.GOB has when being 10 μ g in 21% the cancer cells endochylema a small amount of cavity is arranged, and nucleus is normal, to the growth of cancer cells unrestraint.Endochylema, nuclear and cell growth to cancer cells when GOB is 1 μ g all do not have influence.
5, genseng oligosaccharide of the present invention (GOB) treatment leukopenia 44 routine clinical trials
The curative effect of table 5GOB treatment pancytopenia
| The sick kind | The example number | Curative effect |
| The produce effects enabledisable is efficient | ||
| Due to the width of cloth is penetrated due to the chronic idiopathic chemotherapy other | ????20 ????16 ????3 ????5 | ??4??????????10??????????6?????????70％ ??3??????????7???????????6?????????62.5％ ??/??????????3???????????/?????????100％ ??/??????????/???????????5?????????0％ |
| Add up to | ????44 | 7(15.9％)??20(45.5％)??17(38.6％)??61.4％ |
The GOB of conclusion: A, the clinical observation of this project by analysis, is to contain the oligosaccharide compound that β connects key.Toxicity trial finds no any toxicity, does not more have carcinogenic pathogenic effects, and clinical observation further proves nontoxic pair of effect, and is safe and reliable.
B, prove through 44 routine clinical observations that GOB is for oligoleukocythemia due to penetrating because of the width of cloth and after the chemotherapy, and the agnogenic chronic property leukopenia of waiting to send out, and the effect of leukocyte increasing is arranged, in the recent period efficient is 69.2% (27/39).
Oligoleukocythemia case after the chemotherapy that this group is observed mostly is leukemia and malignant lymphoma patient greatly, 10 examples effectively in, 7 examples clothes GOB2 in week white corpuscle obviously rise, and invalid cases of 6 examples, majority is the terminal stage of a disease, normal cell is counted severe inhibition in the marrow.
Part I in C, this group
9The case that G is on the low side or low, I behind the clothes GOB
9G rises to some extent.In conjunction with GOB promotion hepatitis B surface antigen(HBsAg) (HB is arranged
5A
9) the cloudy effect of changeing of positive person, point out this medicine may have the effect that improves humoral immune function.
Symptom had clear improvement after D, discovery under observation, part had the case of autonomic nervous dysfunction to obey GOB, may have the effect of regulating vegetative nerve function.
6, GOB treatment hepatitis B and hepatitis B surface antigen carrier clinical trial (see Table 6, table 7)
Table 6GOB treatment hepatitis B and hepatitis B surface antigen carrier 9 routine results
HBsAg eAg GTP TTT ZnT 1 1∶128 1∶64 20 5 3 4 7 82 1∶32 1∶16 90 25 8 7 14 123 1∶128 1∶32 6 8 4 6 8 ／4 1∶128 1∶256 60 10 14 8 14 125 1∶8 1∶8 ／ 2 3 5 4 11 96 1∶512 1∶128 ／ 5 15 5 6 10 107 1∶512 1∶512 90 10 10 8 11 108 1∶64 1∶128 ／ 30 10 4 6 9 99 1∶64 1∶8 ／ ／ ／ ／ ／ ／ ／1 （ 9 ） ； Treatment time short (15 days); Dosage is few, so do not find table
The face antigen person of turning out cloudy.
2 reagent GOB are in hepatitis b surface antigen positive person four examples, and four routine titres all have decline, its
In an example descend more obvious.
3 liver function deviants all transfer to normally, descend comparatively obvious with GTP especially.
Do not find any pair of effect during 4 treatments.Improve a poor appetite more, improve the reaction of sleep.
The case back case of taking medicine before table 7GOB examination is controlled hepatitis B 17 routine laboratory indexes observations projects and taken medicine
The positive feminine gender titre no change rising remarks of turning out cloudy
17 01934 pairs of three antigen projects of viral HBsAg descend
Unusual normal ///have that the transaminase 13 46115 of taking a turn for the better in various degree is efficient to reach 94.1%
Conclusion: through treatment, total effective rate is 94.1%.Most patient reaction's appetite increase, weak alleviating, and part patient's pain in the hepatic region disappears, and 2 routine hepatomegaly persons check normally; And find poison pair effect.According to symptom, sign, laboratory indexes evaluation curative effect, results of recent belongs to recovery from illness 1 example, produce effects 4 examples, progressive 8 examples, invalid 4 examples amount to effective 13 examples, account for 76.5%, as simple gauging surface antigen, turn out cloudy and titre totally 10 examples that descend, efficient is 58.8%, one routine model case, treat the anti-HBC in back and turn out cloudy earlier, twice cloudy commentaries on classics and normal of HBsAg, GTP subsequently.
Description of drawings:
Accompanying drawing 1 is with the oligosaccharide HPLC separation graph of the present invention of 2,4 one dinitrophenylhydrazine reactions;
Accompanying drawing 2, the FAB--MS that separates DP6 (7 peak) with the oligosaccharide HPLC of the present invention of 2,4 dinitrophenyl hydrazine reaction schemes;
Accompanying drawing 3 separates DP6 (7 peak) with the oligosaccharide HPLC of the present invention of 2,4 dinitrophenyl hydrazine reaction
13CNMR figure; Fig. 4, the FAB--MS figure of DP6 oligosaccharides of the present invention;
Accompanying drawing 5, the FAB--MS interlock figure of DP6 oligosaccharides of the present invention;
Accompanying drawing 6, DP6 oligosaccharides of the present invention
13C NMR figure
Accompanying drawing 7, DP6 oligosaccharide structure figure of the present invention
Accompanying drawing 8 is with the DP6 oligosaccharide structure figure of the present invention of 2,4 dinitrophenyl hydrazine reaction.
To essentiality content of the present invention be described by embodiment below, but technical scheme of the present invention, product are not limited thereto.
Adopt ginseng to induce callus, secretly be incubated at additional 2,4-D0.2PPm, on the MS substratum of KT0.1PPm, culture temperature is 26 ± 0.5 ℃, 25 days subcultures once, form clone, be (to use the 50ml triangular flask, 10ml MS solid medium, additional 2mg/l then with above-mentioned genseng primary wall cell by the callus succeeding transfer culture, 2.4-D with 0.1mg/l KT) and suspension cell culture (500ml and 1000ml triangular flask, 20% MS liquid nutrient medium), 26 ± 1 ℃, the dark cultivation, 120 commentaries on classics/min shaking culture, 25 days is one-period.
Behind the 500 gram ginseng oligosaccharide element cells adding aquae destillatas with frost drying, on refiner, centrifugal 15 minutes of 4000rpm uses filter paper filtering, distilled water flushing three times, discard filtrate, add 3 times of volume 80% ethanol and at room temperature soaked 24 hours, after the filtration, discard filtrate, filter residue is with the chloroform of 3 times of volumes: refluxed 6 hours under 60 ℃ of conditions of amylalcohol or butanols (4: 1), behind the elimination liquid with after acetone and the water washing drying cell walls dry-matter 480 grams.
Cell walls dry-matter 1N HCl carried out acid hydrolytic reaction 1.5 hours in 90 ℃ in rotary container.After hydrolysis finishes, cool off termination reaction immediately, the elimination precipitation, filtrate is neutralized to PH7.0 with NaOH, after 4 ℃ of following standing over night, elimination precipitate 14 liters in the ginseng oligosaccharide element mixture formed by 3-12 monose of polymerization degree DP, sugared assay contains sugared 50g approximately.
Embodiment 2 preparation DP3,4,5, DP6,7,8, DP9,10,11,12 oligosaccharide compositions:
Adopt earlier the method for embodiment 1 to obtain 14 liters in the ginseng oligosaccharide element mixture that the polymerization degree is made up of 3-12 monose, filtrate behind decolorizing with activated carbon, mistake Dowex50 (H
+) (1.0 * 15cm), the distilled water wash-out is removed sodium ion to ion exchange column, and effluent liquid concentrates.Use again Bio-Gel-P-2 (1.0 * 50cm) column chromatographies obtain DP3,4,5, DP6,7,8, DP9,10,11,12 oligosaccharide compositions.Carry out the oligosaccharides after the evaluation of oligosaccharide component: TLC detects the Bio-Gel-P-2 column chromatography then, solvent systems is a propyl carbinol: acetate; Water=2: 1: 1 or methyl alcohol: water=25: 1, some hangover of the latter.Positive phase-plate is efficient plate GF254 (Haiyang Chemical Plant, Qingdao's system, down together).The sucrose standard control, after the expansion, airing, spray naphthyl alcohol sulphuric acid soln (ethanol 40.5ml mixes mutually with water 4ml for 15%a-naphthols ethanolic soln 10.5ml, vitriol oil 6.5ml), baking 3-6min colour developing.General Bio-Gel-P-2 can obtain removing two (single's, two and polysaccharide) segmentation oligosaccharide fragment, is a purplish red long band after the colour developing, and wherein some point is blue look, and sucrose generally shows blue look.
Collect the component of Rf between initial point and sucrose standard substance.After concentrating this part solution, repeat the Bio-Gel-P-2 post and then can get the sectional oligosaccharide.Obtain 12g carbohydrate dry-matter.
Method with embodiment 1 obtains the ginseng oligosaccharide element mixture that the polymerization degree is a 3-12 monose earlier, method with embodiment 2 obtains the oligosaccharide compositions that the polymerization degree is a 6-8 monose again, about 2g crosses the silica gel G post with the concentrated back, II peak that Bio-Gel-P-2 assigns to, and collects MeoH: H
2O=25: 1 part, after concentrating 1.2g oligosaccharides dry-matter, separates as shown in Figure 1 with the HPLC (ODS post) of 2,4 dinitrophenyl hydrazine reaction oligosaccharides, T peak among the collection figure, do FAB=MS (accompanying drawing 2) and
13CNMR (accompanying drawing 3) measured the few peak of DP6.
The unreacted oligosaccharides divides three sections collections under the same conditions in retention time 4.5-5min, TLC detects, repeat twice and separate, obtain a 19mg component, this component is soluble in water, the flocks that is white in color in organic solvents such as methyl alcohol is white amorphous powder behind the frost drying.Detect through TLC, the sucrose standard substance in contrast, Rf value was 0.59 (as shown in Figure 4), handed over FAB--MS and NMS data to show that its structure is sugared identical with aforementioned 2,4 one dinitrophenylhydrazine reaction oligosaccharides HPLC separation DP6 six.
Get above-mentioned HPL divide after DP6 oligosaccharides sample and the complete acid hydrolysis of 5mg, through the ply of paper folding, the monose kind of measuring the DP6 oligosaccharides is Glc, Gal, three kinds of Man; Make FAB-MS and NMR collection of illustrative plates simultaneously, draw genseng six sugar and have structure as accompanying drawing 5, with the structure iron of 2,4 one dinitrobenzene hydrazine reactions as shown in Figure 6.
Claims (10)
1, ginseng oligosaccharide element is characterized in that being got by the ginseng-cell cultivation separation and purification, the mixture of being made up of 3-12 monose for polymerization degree DP.
2, the ginseng monomer oligosaccharide that gets by the described ginseng oligosaccharide element separation of claim 1, it is characterized in that above-mentioned ginseng oligosaccharide element mixture separated by activated carbon, ion exchange column, xanthan gel post obtaining the segmentation oligosaccharide compositions, then through HPLC separate the panose to the monomer oligosaccharide of genseng ten disaccharides.
3, monomer oligosaccharide as claimed in claim 2, its genseng six sugared structural formulas are shown in (I):
4, the technology of preparation claim 1 or 2 or 3 described ginseng oligosaccharide elements, monomer oligosaccharide, it is characterized in that comprising that the cell that genseng primary wall cell system is obtained by callus succeeding transfer culture and suspension culture carries out pre-treatment, frost drying gets the cell walls dry-matter, carry out acid hydrolysis then, adopt 0.7-1N HCl under 90-98 ℃ of temperature, to react 1-1.5 hour, get the described ginseng oligosaccharide element of claim 1; Again with above-mentioned ginseng oligosaccharide element liquid by active carbon column, ion exchange column, xanthan gel post separate the sectional oligosaccharide compositions, with HPLC separate the described panose of claim 2 to the monomer oligosaccharide of genseng ten disaccharides.
5, technology as claimed in claim 4, its spy is that said ginseng callus and suspension cell culture cultivate with MS, additional 2mg/l2,4-D, 0.1mg/lKT, 26 ± 1 ℃ of dark down cultivations, cultivating 20-25 days is one-period.
6, as claim 4 or 5 described technologies, its spy is that said acid hydrolysis condition optimization scope adopts 1N HCl to react 1 hour under 90 ℃ of temperature.
7, the application of ginseng oligosaccharide element as claimed in claim 1, the miscellany of forming by said monose add respectively corresponding pharmacy acceptable carrier get a kind of immunostimulant or a kind of anticancer adjuvant.
8, the application of ginseng monomer oligosaccharide as claimed in claim 2 adds corresponding pharmacy acceptable carrier respectively by said monomer oligosaccharide and gets a kind of immunostimulant or get a kind of anticancer adjuvant.
9, the application of ginseng oligosaccharide element as claimed in claim 1, the miscellany that said monose is formed is used to obtain the elicitor of a kind of plant secondary metabolites, and this elicitor contains the miscellany that right requires 1 said monose to form.
10, the application of ginseng monomer oligosaccharide as claimed in claim 2, said monomer oligosaccharide is used to obtain the elicitor of a kind of plant secondary metabolites, and this elicitor contains right and requires 2 said monomer oligosaccharides.
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| Application Number | Priority Date | Filing Date | Title |
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| CN95118079A CN1049221C (en) | 1995-10-30 | 1995-10-30 | Ginseng oligosaccharide element and ginseng monomer oligosaccharide and its preparing technology and use |
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| CN95118079A CN1049221C (en) | 1995-10-30 | 1995-10-30 | Ginseng oligosaccharide element and ginseng monomer oligosaccharide and its preparing technology and use |
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| CN1149587A true CN1149587A (en) | 1997-05-14 |
| CN1049221C CN1049221C (en) | 2000-02-09 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100594238C (en) * | 2005-12-31 | 2010-03-17 | 河北省生物研究所 | Production process for improving suspension cell saponin content of American ginseng |
| CN101502546B (en) * | 2009-02-24 | 2012-02-08 | 姜瑞芝 | Ginseng (root) oligosacchride preparation and novel use thereof |
| CN103385888A (en) * | 2013-07-24 | 2013-11-13 | 刘淑莹 | Application of ginseng oligosaccharide composition in preparation of anti-oxidation medicine or health-care food |
| CN105684782A (en) * | 2016-02-24 | 2016-06-22 | 丽江市农业科学研究所 | Regulation and control method for plants in potato breeder's seed production process |
-
1995
- 1995-10-30 CN CN95118079A patent/CN1049221C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100594238C (en) * | 2005-12-31 | 2010-03-17 | 河北省生物研究所 | Production process for improving suspension cell saponin content of American ginseng |
| CN101502546B (en) * | 2009-02-24 | 2012-02-08 | 姜瑞芝 | Ginseng (root) oligosacchride preparation and novel use thereof |
| CN103385888A (en) * | 2013-07-24 | 2013-11-13 | 刘淑莹 | Application of ginseng oligosaccharide composition in preparation of anti-oxidation medicine or health-care food |
| CN105684782A (en) * | 2016-02-24 | 2016-06-22 | 丽江市农业科学研究所 | Regulation and control method for plants in potato breeder's seed production process |
| CN105684782B (en) * | 2016-02-24 | 2018-10-19 | 丽江市农业科学研究所 | Plant in potato primary stock production process regulates and controls method |
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| Publication number | Publication date |
|---|---|
| CN1049221C (en) | 2000-02-09 |
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