CN103483410B - Xanthoceraside preparation method - Google Patents
Xanthoceraside preparation method Download PDFInfo
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- CN103483410B CN103483410B CN201310419295.1A CN201310419295A CN103483410B CN 103483410 B CN103483410 B CN 103483410B CN 201310419295 A CN201310419295 A CN 201310419295A CN 103483410 B CN103483410 B CN 103483410B
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Abstract
The invention belongs to the field of medical technology and relates to a new method for preparing xanthoceraside in shinyleaf yellowhorns. The highly purified xanthoceraside can be prepared efficiently with the preparation method. The preparation method is easy and convenient to use and reliable and reduces redundant intermediate operation steps, thus, product loss is reduced, and the yield of the preparation method is obviously increased compared with that of a former method. Crude extract is directly processed with a silica gel column, eluted with the chloroform/dichloromethane-methyl alcohol eluent with the volume ratio of 10:1-7:1, the ethyl acetate-methyl alcohol eluent with the volume ratio of 10:1-5:1 or the acetone-methyl alcohol eluent with the volume ratio of 10:1-6:1, and then eluted with the chloroform/dichloromethane-methyl alcohol eluent with the volume ratio of 6:1-2:1, the ethyl acetate-methyl alcohol eluent with the volume ratio of 4:1-1:1 or the acetone-methyl alcohol eluent with the volume ratio of 5:1-3:1, solvent recovery, purification and recrystallization are performed, and finally the xanthoceraside is obtained.
Description
Technical field
The invention belongs to medical art, relate to the novel preparation method of Xanthoceraside in Wood of Shinyleaf Yellowhorn.
Background technology
Wood of Shinyleaf Yellowhorn (Xanthocerassorbifolia Bunge) for Sapindaceae (
sapindaceae) Wood of Shinyleaf Yellowhorn platymiscium, one belongs to a kind of, and shrub or arbor, be distributed in the provinces such as China Liaoning, Hebei, Shaanxi.Its heartwood, stem branch and fruit etc. all can enter medicinal.Have and dispel rheumatism, swelling and pain relieving, hold back the effects such as dry yellow water, once list 2005 editions Pharmacopoeias of the People's Republic of China in.
Research in recent years shows, Xanthoceraside (also known as bunkanka saponins E in bibliographical information) is the principle active component of Wood of Shinyleaf Yellowhorn, have anti-inflammatory, antitumor, suppress hiv protease, improve learning and memory and improve the effects such as human body anti-glycosuria.Can research and develop and become a kind of medicine or healthcare products for the treatment of disease of brain.
Through retrieval domestic and foreign literature, the research report about Xanthoceraside is little.Not yet there is the enriching method of good Xanthoceraside.
Current result of study show its saponin fraction be its main efficient part (Lee l. accounts for woods, Li Xi, Li Ning, Li Wen, Sha Yi. the chemical composition of shinyleaf yellowhorn fruit shell. Shenyang Pharmaceutical University's journal 2005,22 (4): 271 one 272; 2. discipline snow flies, Liu Xinxia, and Wu clamors, Yang Baizhen, Wang Lihua, Zou Libo. and shinyleaf yellowhorn shell extract activates the improvement result of thing learning memory disorder to amyloid-beta. Shenyang Pharmaceutical University's journal 2007,24 (4): 232 one 237.).Oneself is through there being multiple development and utilization patent (l.UnitedstatesPatent:7,189,420 about shinyleaf yellowhorn fruit shell at present; 2. Chinese patent: brain active function material preventing and treating encephalopathic and intelligence development and preparation method thereof, Authorization Notice No.: CN1236792C; 3. Chinese patent: brain active function material preventing and treating encephalopathic and intelligence development and preparation method thereof, publication number: CN1416852A; 4. Chinese patent: shinyleaf yellowhorn fruit shell extract improves in brain function food and medicine in preparation to be applied, publication number: CN134982OA; 5 one kinds are extracted compound and extracting method thereof and application, publication number: CN1626545, Authorization Notice No.: CN1281616C from shinyleaf yellowhorn fruit shell).
Substantially macroporous resin is all adopted to carry out enrichment to Wood of Shinyleaf Yellowhorn total saponins in these patents, and then utilize other chromatographic process, comprise solvent extraction, silica gel chromatography, high-speed countercurrent chromatography etc. and carry out further separation and purification, the good compound of purity can be obtained.These methods, step is more, and therefore compound loses excessive in preparation process, causes ultimate yield too low; In addition, because adopt macroporous adsorbing resin for purification more, or solvent-extracted operation, not only owing to easily producing residue in building-up process, contaminated samples causes residual macroporous adsorbent resin itself, and consumption of organic solvent is comparatively large, cost is high, inflammable and explosive, be unfavorable for environmental protection, also larger to the infringement of workman's health, security is poor; In addition, the costs of equipment such as high-speed countercurrent chromatography are higher, are unfavorable for carrying out of enterprise's scale operation.
summary of the invention:
The object of the present invention is to provide a kind of preparation method that is novel, Xanthoceraside fast, the preparation of high-level efficiency, highly purified Xanthoceraside can be realized.This preparation method is reliably easy, reduces over many intermediary operations link, thus reduces product loss, and yield is comparatively previous methods raising obviously; Also therefore effectively reduce organic residue, reduce reagent and instrument expense, workable, economize in raw materials cost, is easy to realize industrialization scale operation.
In the inventive method for the preparation of Xanthoceraside, also known as bunkanka saponins E (bunkanka saponins E, 3-O-(3-O-
α-L-arabinofuranosyl-2-O-
β-D-galactopyranosyl)-
β-D-glucuronopyranosyl-21,22-di-O-angeloyl-R
1-barrienol) its structure is as follows:
Its structured data is as follows:
ESI-MS：[M+Na]
+ m/
z1163.3，
m/
z1008.4 [M-132]
+，846.8 [M-132-162]
+，652.9 [M-132-162-176-H
2O]
+。
1h NMR(300 MHz, pyridine-
d 5):
δ5.48 (1H, brs), 6.70(1H, d,
j=10.2 Hz, H-21), 6.32(1H, d,
j=10.2 Hz, H-22), 1.26(3H, s, CH
3-23), 1.16(3H, s, CH
3-24), 0.81(3H, s, CH
3-25), 0.98(3H, s, CH
3-26), 1.84(3H, s, CH
3-27), 3.73,3.50 (1H each, d,
j=10.2 Hz, H-28), 1.09(3H, s, CH
3-29), 1.31(3H, s, CH
3-30), 4.89(1H, q,
j=7.3 Hz, H-1 '), 5.32(1H, q,
j=7.5 Hz, H-1 ' '), 6.03(1H, s, H-1 ' ' '); 21-
o-angeloyl groups:
δ5.96(1H, q,
j=7.0 Hz, H-3), 2.09(3H, d,
j=7.2 Hz, H-4), 2.00(3H, s, H-5); 22-
o-angeloyl groups:
δ5.76(1H, q,
j=6.6 Hz, H-3), 1.93(3H, d,
j=6.6 Hz, H-4), 1.72(3H, s, H-5).
The present invention adopts following technical scheme:
(1) choose drying, without the shinyleaf yellowhorn fruit shell, carpopodium, leaf, the flower that go mouldy, be crushed into 20-40 object powdery medicinal material;
(2) by powdery medicinal material, by the volume percent 50%-85% ethanol of 6-10 times of volume, preferred 65%-70% ethanol, temperature 65 DEG C-80 DEG C, heating and refluxing extraction, filter, merging filtrate, decompression and solvent recovery lower than 5%, obtains crude extract to determining alcohol; Extracting mode comprises one or more modes such as leaching, seepage flow, decoction, temperature leaching, backflow and combines, and extract 2-3 time, the consumption of alcohol is volume/weight multiple is 6-10 times of Wood of Shinyleaf Yellowhorn medicinal material coarse powder weight, each 1.5-3h, filters, merging filtrate, recycling design, obtains crude extract.
(3) above-mentioned crude extract is directly through silica gel column chromatography separation and purification, take volume ratio as chloroform-methanol or the methylene chloride-methanol of 10:1-7:1 be eluent, preferred proportion is 8:1-7:1, afterwards further with 6:1-2:1 chloroform-methanol or methylene chloride-methanol for eluent, preferred proportion is 3:1-2:1; Or volume ratio be the acetate-methanol of 10:1-5:1 is eluent, preferred proportion is 6:1-5:1, afterwards further with the acetate-methanol of 4:1-1:1 for eluent, preferred proportion is 3:1-2:1; Or volume ratio be 10:1-6:1 acetone-methanol is eluent, preferred proportion is 7:1-6:1, be 5:1-3:1 acetone-methanol is afterwards further eluent with volume ratio, and preferred proportion is 5:1-4:1.Elutriant atmospheric distillation or distillation under vacuum recycling design, room temperature (18 DEG C-28 DEG C) is placed, and after crystallization is separated out completely, obtains rough Xanthoceraside;
(4) the rough saponin(e described in above-mentioned steps (3) utilizes recrystallization method to refine, and selects suitable solvent to carry out recrystallization, obtains the Xanthoceraside of the high purity (more than 98%) of refining;
When crude drug source is unstable, when Xanthoceraside content is lower, high performance liquid chromatography can be adopted to be further purified sample.By dissolving the Xanthoceraside of gained in above-mentioned steps (4) through half preparation or preparative high performance liquid chromatography (HPLC) separation and purification further, can be moving phase wash-out with methanol-water, obtaining target compound-Xanthoceraside.
(5) to the Xanthoceraside that above-mentioned steps obtains, nuclear-magnetism method can be adopted, comprise
1h NMR and
13c NMR carries out Structural Identification.
In the preparation method of Xanthoceraside provided by the invention, the alcohol of step (2) is ethanol, and its volumetric concentration is 50%-85%, preferred 65%-70%;
In the preparation method of Xanthoceraside provided by the invention, step (3) the normal phase silica gel column chromatography method that uses carries out separation and purification, and its filler is refined type 100-200,200-300,300-400 order column chromatography silica gel.Silica gel column chromatography solvent system is the chloroform-methanol of 10:1-7:1 or methylene chloride-methanol first by volume, or volume ratio is the acetate-methanol of 10:1-5:1, or volume ratio is the acetone-methanol of 10:1-6:1, remove other compositions of non-Xanthoceraside (as sterol, triterpenoid sapogenin, and polarity is less than the compound etc. of Xanthoceraside), the chloroform-methanol of preferred 8:1-7:1 or methylene chloride-methanol, the acetate-methanol of 6:1-5:1, the acetone-methanol of 7:1-6:1; Again further with the chloroform-methanol of volume ratio 6:1-2:1 or methylene chloride-methanol, or the acetate-methanol of 4:1-1:1, or volume ratio is that the acetone-methanol of 5:1-3:1 elutes target component, preferred proportion is chloroform-methanol or the methylene chloride-methanol of 3:1-2:1, or the acetate-methanol of 3:1-2:1, or the acetone-methanol of 5:1-4:1; Enrichment obtains rough Xanthoceraside.
In the preparation method of Xanthoceraside provided by the invention, the solvent that step (4) is selected is acetone, methyl alcohol, water, or acetone-water, methanol-water, its ratio is 0:1-1:0, or acetone-methanol-water, its ratio is 1:1:1-1:1:0.5, obtains the Xanthoceraside of the high purity (more than 98%) of refining after recrystallization.
In the preparation method of Xanthoceraside provided by the invention, the methanol-water of the solvent that high performance liquid chromatography adopts to be volume ratio be 50-85:15-50, flow velocity is 1.0-3.0 mL.min
-1, determined wavelength is 210nm, and column temperature is 34 DEG C.
Xanthoceraside provided by the invention, can adopt nuclear-magnetism method, comprise
1h NMR and
13c NMR carries out Structural Identification.
Present invention employs an one-step preparation method, the extraction process of Xanthoceraside is optimized, not only simplify technique, reagent and instrument cost are saved, and decrease sample and the loss that cause and organic residue more due to step in preparation process, thus increase substantially the yield of sample.Utilize the inventive method can realize the preparation of high-level efficiency, highly purified Xanthoceraside, this preparation method's step is reliably easy, and extraction and isolation efficiency is high, workable, economizes in raw materials and plant and instrument cost, is easy to realize industrialization scale operation.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment 1
Getting pulverizing is 20 object shinyleaf yellowhorn fruit shell 100 g, with concentration of volume percent 50% ethanol of 10 times of volumes, temperature 65 DEG C, heating and refluxing extraction 3 times, time is 3 hours/time, recycling design, concentrate and obtain crude extract, this crude extract is the separation and purification of 100-200 object normal phase silica gel column chromatography method through order number, be chloroform-methanol or the methylene chloride-methanol of 7:1 and 2:1 respectively with volume ratio be eluting solvent wash-out, reflux, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, room temperature is placed, adopt methanol-water solvent system recrystallization, repeatedly obtain the highly purified Xanthoceraside refined after recrystallization, purity is 99.5%, and yield is 0.047%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 20 object shinyleaf yellowhorn fruit shell 100 g, after pulverizing, with concentration of volume percent 50% ethanol of 10 times of volumes, temperature 65 DEG C, heating and refluxing extraction 3 times, time is 3 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 50% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.0%, and yield is 0.021%
0.
Embodiment 2
Getting pulverizing is 30 object shinyleaf yellowhorn fruit shell 150 g, with concentration of volume percent 70% ethanol of 8 times of volumes, temperature 70 C, heating and refluxing extraction, time 2 h/time, extract 2 times, reflux, decompression and solvent recovery, the medicinal extract obtained after concentrated is the separation and purification of 200-300 object normal phase silica gel column chromatography method through order number, is that chloroform-methanol or the methylene chloride-methanol of 8:1 and 3:1 is eluting solvent wash-out respectively with volume ratio, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, obtains rough saponin(e; Adopt recrystallizing methanol, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.9%, and yield is 0.048%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 30 object shinyleaf yellowhorn fruit shell 150 g, after pulverizing, with concentration of volume percent 70% ethanol of 8 times of volumes, temperature 70 C, heating and refluxing extraction 2 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 65% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.1%, and yield is 0.026%
0.
Embodiment 3
Getting pulverizing is 30 object shinyleaf yellowhorn fruit shell 150 g, with concentration of volume percent 65% ethanol of 8 times of volumes, temperature 70 C, heating and refluxing extraction, 2.5 hours/time time, extract 3 times, reflux, decompression and solvent recovery, the medicinal extract obtained after concentrated is the separation and purification of 200-300 object normal phase silica gel column chromatography method through order number, is that chloroform-methanol or the methylene chloride-methanol of 7:1 and 3:1 is eluting solvent wash-out respectively with volume ratio, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, obtains rough saponin(e; Adopt recrystallizing methanol, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.9%, and yield is 0.051%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 30 object shinyleaf yellowhorn fruit shell 150 g, after pulverizing, with concentration of volume percent 70% ethanol of 8 times of volumes, temperature 70 C, heating and refluxing extraction 3 times, time is 2.5 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 65% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.1%, and yield is 0.026%
0.
Embodiment 4
Getting pulverizing is 40 object shinyleaf yellowhorn shell 200 g, with concentration of volume percent 80% ethanol of 7 times of volumes, temperature 75 DEG C, heating and refluxing extraction, 1.5 hours/time time, extract 2 times, recycling design, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, is that chloroform-methanol or the methylene chloride-methanol of 10:1 and 6:1 is eluting solvent with volume ratio, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-methanol-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.6%, and yield is 0.045%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 40 object shinyleaf yellowhorn fruit shell 200 g, after pulverizing, with concentration of volume percent 80% ethanol of 7 times of volumes, temperature 75 DEG C, heating and refluxing extraction 2 times, time is 1.5 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 70% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain target compound Xanthoceraside, its purity is 98.8%, and yield is 0.029%
0.
Embodiment 5
Getting pulverizing is 40 object shinyleaf yellowhorn shell 200 g, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction, time 2 h/time, extract 3 times, recycling design, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, is that chloroform-methanol or the methylene chloride-methanol of 7:1 and 3:1 is eluting solvent with volume ratio, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-methanol-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.3%, and yield is 0.042%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 40 object shinyleaf yellowhorn fruit shell 200 g, after pulverizing, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction 3 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 80% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.6%, and yield is 0.026%
0.
Embodiment 6
Getting pulverizing is 20 object shinyleaf yellowhorn fruit shell 100 g, with concentration of volume percent 50% ethanol of 10 times of volumes, temperature 65 DEG C, heating and refluxing extraction 3 times, time is 3 hours/time, recycling design, concentrate and obtain crude extract, this crude extract is the separation and purification of 100-200 object normal phase silica gel column chromatography method through order number, be the acetate-methanol of 10:1 and 4:1 respectively with volume ratio be eluting solvent wash-out, reflux, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, room temperature is placed, adopt methanol-water solvent system recrystallization, repeatedly obtain the highly purified Xanthoceraside refined after recrystallization, purity is 98.3%, and yield is 0.037%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 20 object shinyleaf yellowhorn fruit shell 100 g, after pulverizing, with concentration of volume percent 50% ethanol of 10 times of volumes, temperature 65 DEG C, heating and refluxing extraction 3 times, time is 3 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 50% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.0%, and yield is 0.021%
0.
Embodiment 7
Getting pulverizing is 30 object shinyleaf yellowhorn fruit shell 150 g, with concentration of volume percent 70% ethanol of 8 times of volumes, and temperature 70 C, heating and refluxing extraction, time 2 h/time, extract 2 times, reflux, decompression and solvent recovery, the medicinal extract obtained after concentrated is the separation and purification of 200-300 object normal phase silica gel column chromatography method through order number, be the acetate-methanol of 5:1 and 1:1 is respectively eluting solvent wash-out with volume ratio, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, obtains rough saponin(e; Adopt recrystallizing methanol, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.2%, and yield is 0.040%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 30 object shinyleaf yellowhorn fruit shell 150 g, after pulverizing, with concentration of volume percent 70% ethanol of 8 times of volumes, temperature 70 C, heating and refluxing extraction 2 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 65% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.1%, and yield is 0.026%
0.
Embodiment 8
Getting pulverizing is 30 object shinyleaf yellowhorn fruit shell 150 g, with concentration of volume percent 65% ethanol of 8 times of volumes, and temperature 70 C, heating and refluxing extraction, 2.5 hours/time time, extract 3 times, reflux, decompression and solvent recovery, the medicinal extract obtained after concentrated is the separation and purification of 200-300 object normal phase silica gel column chromatography method through order number, be the acetate-methanol of 6:1 and 3:1 is respectively eluting solvent wash-out with volume ratio, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, obtains rough saponin(e; Adopt recrystallizing methanol, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.9%, and yield is 0.041%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 30 object shinyleaf yellowhorn fruit shell 150 g, after pulverizing, with concentration of volume percent 70% ethanol of 8 times of volumes, temperature 70 C, heating and refluxing extraction 3 times, time is 2.5 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 65% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.1%, and yield is 0.026%
0.
Embodiment 9
Getting pulverizing is 40 object shinyleaf yellowhorn shell 200 g, with concentration of volume percent 80% ethanol of 7 times of volumes, temperature 75 DEG C, heating and refluxing extraction, 1.5 hours/time time, extract 2 times, recycling design, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, take volume ratio as the acetate-methanol of 5:1 and 2:1 is eluting solvent, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-methanol-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.6%, and yield is 0.042%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 40 object shinyleaf yellowhorn fruit shell 200 g, after pulverizing, with concentration of volume percent 80% ethanol of 7 times of volumes, temperature 75 DEG C, heating and refluxing extraction 2 times, time is 1.5 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 70% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain target compound Xanthoceraside, its purity is 98.8%, and yield is 0.029%
0.
Embodiment 10
Getting pulverizing is 40 object shinyleaf yellowhorn shell 200 g, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction, time 2 h/time, extract 3 times, recycling design, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, take volume ratio as the acetone-methanol of 10:1 and 5:1 is eluting solvent, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-methanol-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.3%, and yield is 0.040%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 40 object shinyleaf yellowhorn fruit shell 200 g, after pulverizing, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction 3 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 80% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.6%, and yield is 0.026%
0.
Embodiment 11
Getting pulverizing is 40 object shinyleaf yellowhorn shell 200 g, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction, time 2 h/time, extract 3 times, recycling design, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, take volume ratio as the acetone-methanol of 6:1 and 3:1 is eluting solvent, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-methanol-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.3%, and yield is 0.040%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 40 object shinyleaf yellowhorn fruit shell 200 g, after pulverizing, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction 3 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 80% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.6%, and yield is 0.026%
0.
Embodiment 12
Getting pulverizing is 40 object shinyleaf yellowhorn shell 200 g, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction, time 2 h/time, extract 3 times, recycling design, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, take volume ratio as the acetone-methanol of 7:1 and 5:1 is eluting solvent, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-methanol-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.3%, and yield is 0.038%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 40 object shinyleaf yellowhorn fruit shell 200 g, after pulverizing, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction 3 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 80% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.6%, and yield is 0.026%
0.
Embodiment 13
Getting pulverizing is 40 object shinyleaf yellowhorn shell 200 g, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction, time 2 h/time, extract 3 times, recycling design, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, take volume ratio as the acetone-methanol of 6:1 and 4:1 is eluting solvent, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-methanol-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 98.3%, and yield is 0.042%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 40 object shinyleaf yellowhorn fruit shell 200 g, after pulverizing, with concentration of volume percent 85% ethanol of 10 times of volumes, temperature 75 DEG C, heating and refluxing extraction 3 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 80% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.6%, and yield is 0.026%
0.
Embodiment 14
Getting pulverizing is that 25 object Wood of Shinyleaf Yellowhorn spend 125 g, with concentration of volume percent 75% ethanol of 7 times of volumes, temperature 75 DEG C, heating and refluxing extraction 2 times, the time is 2.5 hours/time, decompression and solvent recovery, concentrate and obtain crude extract, the medicinal extract obtained after concentrated is the separation and purification of 300-400 order normal phase silica gel column chromatography method through order number, is that chloroform-methanol or the methylene chloride-methanol of 8:1 and 6:1 is eluting solvent with volume ratio, recycling design, enrichment obtains Xanthoceraside stream part; Adopt acetone-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Refining Xanthoceraside E is further through the separation and purification of HPLC method, and with methyl alcohol: water (85:15, V:V) is moving phase, flow velocity is 3.0mL/min, and determined wavelength is 210 nm, obtains subject monomers composition-Xanthoceraside, and purity is 99.5%, and yield is 0.031%
0.
Embodiment 15
Getting pulverizing is 35 object Wood of Shinyleaf Yellowhorn leaf 200 g, with concentration of volume percent 60% ethanol of 6 times of volumes, temperature 75 DEG C, heating and refluxing extraction 4 times, time is 2 hours/time, decompression and solvent recovery, concentrate and obtain crude extract, this crude extract is the separation and purification of 100-200 object normal phase silica gel column chromatography method through order number, be chloroform-methanol or the methylene chloride-methanol of 7:1 and 3:1 respectively with volume ratio be eluting solvent wash-out, reflux, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, room temperature is placed, after crystallization is separated out completely, adopt acetone-methanol-water solvent system recrystallization, repeatedly obtain the highly purified Xanthoceraside refined after recrystallization, refining Xanthoceraside E is further through the separation and purification of HPLC method, and with methyl alcohol: water (80:20, V:V) is moving phase, flow velocity is 2.5mL/min, and determined wavelength is 210 nm, obtains subject monomers composition-Xanthoceraside, and purity is 98.9%, and yield is 0.028%
0.
Embodiment 16
Getting pulverizing is 40 object Wood of Shinyleaf Yellowhorn leaf 400 g, with concentration of volume percent 80% ethanol of 7 times of volumes, temperature 65 DEG C, heating and refluxing extraction 3 times, time is 1.5 hours/time, decompression and solvent recovery, concentrate and obtain crude extract, this crude extract is the separation and purification of 100-200 object normal phase silica gel column chromatography method through order number, be chloroform-methanol or the methylene chloride-methanol of 8:1 and 4:1 respectively with volume ratio be eluting solvent wash-out, reflux, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part; Adopt acetone-water solvent system, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Refining Xanthoceraside E is further through the separation and purification of HPLC method, and with methyl alcohol: water (70:30, V:V) is moving phase, flow velocity is 3.0mL/min, and determined wavelength is 210 nm, obtains subject monomers composition-Xanthoceraside, and purity is 98.6%, and yield is 0.032%
0.
Embodiment 17
Getting pulverizing is 35 object Wood of Shinyleaf Yellowhorn handle 250 g, with concentration of volume percent 65% ethanol of 6 times of volumes, temperature 80 DEG C, heating and refluxing extraction 4 times, time is 1.5 hours/time, decompression and solvent recovery, concentrate and obtain crude extract, this crude extract is the separation and purification of 100-200 object normal phase silica gel column chromatography method through order number, be chloroform-methanol or the methylene chloride-methanol of 7:1 and 2:1 respectively with volume ratio be eluting solvent wash-out, reflux, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, room temperature is placed, adopt methanol-water solvent system recrystallization, repeatedly obtain the highly purified Xanthoceraside refined after recrystallization, purity is 99.3%, and yield is 0.047%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 35 object Wood of Shinyleaf Yellowhorn carpopodium 250 g, after pulverizing, with concentration of volume percent 65% ethanol of 6 times of volumes, temperature 80 DEG C, heating and refluxing extraction 4 times, time is 1.5 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 70% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, through silica gel column chromatography, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 98.1%, and yield is 0.022%
0.
Embodiment 18
Getting pulverizing is 20 object Wood of Shinyleaf Yellowhorn handle 125 g, with concentration of volume percent 70% ethanol of 8 times of volumes, temperature 75 DEG C, heating and refluxing extraction 2 times, time is 2 hours/time, decompression and solvent recovery, concentrate and obtain crude extract, the medicinal extract obtained after concentrated is the separation and purification of 200-300 object normal phase silica gel column chromatography method through order number, be chloroform-methanol or the methylene chloride-methanol of 8:1 and 4:1 respectively with volume ratio be eluting solvent wash-out, decompression and solvent recovery, enrichment obtains Xanthoceraside stream part, obtains rough saponin(e; Adopt recrystallizing methanol, repeatedly after recrystallization, obtain the highly purified Xanthoceraside refined; Purity is 99.2%, and yield is 0.042%
0.
According to patent (publication number: CN1626545 before, Authorization Notice No.: CN1281616C) method, getting pulverizing is equally 15 object Wood of Shinyleaf Yellowhorn carpopodium 125 g, after pulverizing, with concentration of volume percent 70% ethanol of 8 times of volumes, temperature 75 DEG C, heating and refluxing extraction 2 times, time is 2 hours/time, recycling design, concentrate and obtain concentrated solution, macroporous adsorbent resin crossed by concentrated solution, with 75% ethanolic soln wash-out, recycling design obtains enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins, by total saponins water dissolution, with n-butanol extraction, dry brown powder, gradient elution is carried out with chloroform-methanol 100: 35 ~ 60 through silicagel column, reclaim elutriant, refining, obtain object compound Xanthoceraside, its purity is 99.1%, and yield is 0.025%
0.
Embodiment 19
Gained Xanthoceraside is carried out physics and chemistry and spectral data test, to identify structure:
White needles (methyl alcohol), mp 267-268 DEG C.10% ethanol solution of sulfuric acid shows purple, Liebermann-Burchard reacting positive, and Molish reacting positive shows for triterpene saponin componds.ESI-MS provides quasi-molecular ion peak [M+Na]
+ m/
z1163.3, in conjunction with its NMR data, supposition molecular formula is C
57h
88o
23, in mass spectrum, provide fragmention
m/
z1008.4([M-132]
+),
m/
z846.8([M-132-162]
+),
m/
z652.9([M-132-162-176-H
2o]
+), show in molecule containing a pentose fragment, a hexose fragment and a hexuronic acid fragment.
1during H NMR composes, high field region provides R
1seven methyl singlets signals of-barrigenol feature:
δ(1.26 3H, s), 1.16 (3H, s), 0.81 (3H, s), 0.98 (3H, s), 1.84 (3H, s), 1.09 (3H, s), 1.31 (3H, s) and 12 alkene Hydrogen Proton signals:
δ5.48 (1H, brs); Give the proton signal of two groups of angeloyl groups features, A group simultaneously:
δ2.00(3H, s), 2.09(3H, d,
j=7.0Hz), 5.97(1H, q,
j=7.0Hz), B group:
δ1.72(3H, s), 1.93(3H, d,
j=6.6 Hz), 5.77(1H, q,
j=6.6 Hz).
13c NMR provides R in composing
1-barrigenol parent nucleus 30 carbon signals and two groups of angeloyl groups feature carbon signals, A group:
δ21.0,15.9,167.8,137.4,129.0, B group:
δ20.7,15.7,168.2,136.6,129.2.Thus infer that the mother nucleus structure of this compound is 21,22-di-
o-angeloyl-R
1-barrigenol, and only there is glycosyl replacement on 3, A ring.
1h NMR spectrum provides three sugared anomeric proton signals:
δ4.89(1H, d,
j=7.3 Hz), 5.32(1H, d,
j=7.5 Hz), 6.30(1H, br s).
13c NMR Pu Zhongtang district provides 17 carbon signals, wherein
δ105.2 104.9 and 111.2 is 3 sugared end group carbon signals.In conjunction with ESI-MS sugar chain cracking rule, determine the order of connection of glycosyl, wherein
β-D-glucopyranosiduronic acid is inner side sugar, is directly connected with parent nucleus 3,
β-D-galactopyranose and
α-L-arabinofuranose is outside sugar, and is connected to
β-
d2 of-glucopyranosiduronic acid and 3.
By the spectral data of gained compound and data in literature (Li Z. L., Yang B. Z., Li X.,
et al.. Triterpenoids from the husks of
xanthoceras sorbifoliabunge. [J]. Journal of Asian Natural Products Research) compare, completely the same, therefore identify that this compound is 3-
o-(3-
o-
α-L-arabinofuranosyl-2-
o-
β-D-galactopyranosyl)-
β-D-glucuronopyranosyl-21,22-di-
o-angeloyl-R
1-barrigenol, i.e. Xanthoceraside.
Claims (10)
1. a preparation method for Xanthoceraside, is characterized in that: get drying, without the shinyleaf yellowhorn fruit shell, carpopodium, leaf or the flower that go mouldy, pulverize, adopt alcohol water extraction, united extraction liquid, filter and concentrated filtrate, obtain crude extract, above-mentioned crude extract is through purification on normal-phase silica gel column chromatography for separation purifying, take volume ratio as the chloroform/methylene chloride-methanol of 10:1-7:1, volume ratio is the acetate-methanol of 10:1-5:1 or volume ratio is 10:1-6:1 acetone-methanol eluent, be the chloroform/methylene chloride-methanol of 6:1-2:1 again through volume ratio, the acetone-methanol of volume ratio to be the acetate-methanol of 4:1-1:1 or volume ratio be 5:1-3:1 is eluent, select suitable solvent through recrystallization operation repeatedly afterwards, if desired and in conjunction with high performance liquid chromatography purifying, to obtain highly purified Xanthoceraside.
2. the preparation method of a kind of Xanthoceraside according to claim 1, is characterized in that: pulverizing medicinal materials is 20-40 object powdery medicinal material.
3. the preparation method of a kind of Xanthoceraside according to claim 1, it is characterized in that: Wood of Shinyleaf Yellowhorn medicinal material adopts alcohol water extraction, its volumetric concentration is the alcohol-water of 50%-85%, and one or more modes that extracting mode comprises in leaching, seepage flow, decoction, temperature leaching, backflow combine, and extract 2-3 time, the consumption of alcohol is volume/weight multiple is 6-10 times of Wood of Shinyleaf Yellowhorn medicinal material coarse powder weight, each 1.5-3h, filters, merging filtrate, recycling design, obtains crude extract.
4. the preparation method of a kind of Xanthoceraside according to claim 3, is characterized in that: Wood of Shinyleaf Yellowhorn medicinal material adopts alcohol water extraction, and its volumetric concentration is 65%-70% alcohol-water.
5. the preparation method of a kind of Xanthoceraside according to claim 1, is characterized in that: take volume ratio as 8:1-7:1 chloroform/methylene chloride-methanol, the volume ratio acetate-methanol that is 6:1-5:1 or the volume ratio acetone-methanol wash-out that is 7:1-6:1.
6. the preparation method of Xanthoceraside according to claim 5, is characterized in that: again through volume ratio be 3:1-2:1 chloroform/methylene chloride-methanol, the acetone-methanol of volume ratio to be 3:1-2:1 acetate-methanol or volume ratio be 5:1-4:1 is eluent.
7. the preparation method of Xanthoceraside according to claim 3, is characterized in that: what the method for recycling design adopted is atmospheric distillation, distillation under vacuum.
8. the preparation method of Xanthoceraside according to claim 1, is characterized in that: the silicagel column filler of described normal phase silica gel column chromatography method is 100-200,200-300,300-400 object column chromatography silica gel.
9. the preparation method of Xanthoceraside according to claim 1, is characterized in that: utilize recrystallization method to refine, and the solvent selected is acetone, methyl alcohol, water, or acetone-water, methanol-water, its ratio is 0:1-1:0, or acetone-methanol-water, and its ratio is 1:1:1-1:1:0.5.
10. the preparation method of Xanthoceraside according to claim 1, it is characterized in that: when crude drug source is unstable, Xanthoceraside is when in medicinal material, content is lower, can high performance liquid chromatography be adopted to be further purified sample after recrystallization, the methanol-water of the solvent adopted to be volume ratio be 50-85:15-50, flow velocity is 1.0-3.0 mL.min
-1, determined wavelength is 210nm, and column temperature is 34 DEG C.
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