Summary of the invention
The object of the present invention is to provide a kind of compound that from shinyleaf yellowhorn fruit shell and carpopodium, extracts and extracting method and application, adopt the present invention not only can make depleted shinyleaf yellowhorn fruit shell and carpopodium obtain development and use, turn waste into wealth, and in field of medicaments, will develop a kind of treatment disease of brain and antineoplastic new drug.
The objective of the invention is to be achieved through the following technical solutions:
A kind of compound that extracts from shinyleaf yellowhorn fruit shell and carpopodium, this compound are named to Xanthoceraside (Xanthoceraside), are triterpene saponin componds, and its chemical structure is:
3-O-(α-L-arabinofuranosyl (1 → 3)-β-D-galactopyranosyl (1 → 2))-β-D-glucuronopyranosyl-21,22-diangeloyl-R
1-barrigenol, i.e. 3-O-(α-L-arabinofuranosyl (1 → 3)-β-D-galactopyranose base (1 → 2))-beta d glucopyranosiduronic acid base-21,22-two angeloyl groups-R
1-barrigenol.
This compound is white needle, and molten point is 267-268 ℃, is the light red blackening under the 254nm wavelength, and the sulfuric acid colour developing is purple, and the Molish reaction is positive.
Its extracting method is: shinyleaf yellowhorn fruit shell and/or carpopodium are pulverized, used the solvent lixiviate, filter, reclaim solvent and get concentrated solution, concentrated solution is crossed macroporous resin, uses solvent elution, with active constituent-enriched, removes impurity, reclaim solvent and get enriched material, evaporate to dryness obtains brown solid, i.e. total saponins; With the total saponins water dissolution, use n-butanol extraction, dry brown powder through silica gel column chromatography repeatedly, is carried out gradient elution with chloroform/methanol=100: 35-60, reclaims elutriant, and is refining, obtains white needle, is purpose compound Xanthoceraside;
Described solvent is water, methyl alcohol, ethanol, propyl alcohol, butanols and/or acetone, and the concentration expressed in percentage by volume of solvent methanol, ethanol, propyl alcohol, butanols or acetone is 35%-85%;
In the described solvent leaching process, adopt at interval and stir, promptly every 10-20 minute, stirred 1-5 minute, extraction temperature is 60-100 ℃, extraction time 1-3 hour.
Application of compound: be used for preparation treatment disease of brain and the medicine of tumour (solid tumor) and the functional health care food of prevention, treatment disease of brain and tumour.
The present invention has following advantage:
The present invention extracts a kind of brand-new compound from depleted shinyleaf yellowhorn fruit shell and carpopodium, through CA-CS (chemical substance index) and SCIFINDER retrieval, this compound does not appear in the newspapers, for triterpene saponin componds has increased a newcomer; The experimentation on animals of this compound shows: it has the brain function of raising and promptly promotes to remember, alleviate cerebral ischemia and anoxybiotic effect, and the cancer cell in vitro experiment of this compound shows: it has the effect that suppresses cancer cell in vitro; Thereby, the present invention has not only improved the biological value of exploiting and utilizing of depleted shinyleaf yellowhorn fruit shell and carpopodium greatly, the natural resources of saves valuable, and be expected in field of medicaments from now on, develop a kind of treatment disease of brain and anti-tumor drug and functional health care product thereof, its application prospect is very wide.
Description of drawings
Fig. 1 extracts the process flow sheet of new compound from shinyleaf yellowhorn shell and handle for the present invention.
Fig. 2 is Xanthoceraside ultraviolet spectrogram of the present invention (wavelength is 211.6nm, and its optical density is 0.4554, and peak heights is 0.1920).
Fig. 3 is Xanthoceraside hydrogen spectrogram (BMU-500-of the present invention
1H, δ are 0-12ppm (chemical shift)).
Fig. 4 is one of Xanthoceraside hydrogen spectrum enlarged view of the present invention (BMU-500-
1H, δ are 3.0-7.0ppm (chemical shift)).
Fig. 5 is two (BMU-500-of Xanthoceraside hydrogen spectrum enlarged view of the present invention
1H, δ are 0.4-2.4ppm (chemical shift)).
Fig. 6 is Xanthoceraside carbon spectrogram (BMU-500-of the present invention
13C, δ are 10-180ppm (chemical shift)).
Fig. 7 is one of Xanthoceraside carbon spectrum enlarged view of the present invention (BMU-500-
13C, δ are 10-50ppm (chemical shift)).
Fig. 8 is two (BMU-500-of Xanthoceraside carbon spectrum enlarged view of the present invention
13C, δ are 51-94ppm (chemical shift)).
Fig. 9 is three (BMU-500-of Xanthoceraside carbon spectrum enlarged view of the present invention
13C, δ are 100-155ppm (chemical shift)).
Figure 10 for the relevant collection of illustrative plates of Xanthoceraside carbon-hydrogen of the present invention (ARX-300,
1H-: δ is 0-10ppm,
13C-: δ is 0-180ppm).
Figure 11 amplify for Xanthoceraside carbon-hydrogen of the present invention is relevant one of collection of illustrative plates (ARX-300,
1H-: δ is 0.7-2.3ppm,
13C-: δ is 10-40ppm).
Figure 12 amplify for Xanthoceraside carbon-hydrogen of the present invention is relevant collection of illustrative plates two (ARX-300,
1H-: δ is 0.2-3.4ppm,
13C-: δ is 5-60ppm).
Figure 13 amplify for Xanthoceraside carbon-hydrogen of the present invention is relevant collection of illustrative plates three (ARX-300,
1H-: δ is 3-7ppm,
13C-: δ is 60-140ppm).
Figure 14 be the long-range relevant collection of illustrative plates of Xanthoceraside carbon-hydrogen of the present invention (BMU-500,
1H-:F
2Be 0.6-9ppm,
13C-:F
1Be 10-180ppm).
Figure 15 for one of long-range relevant amplification collection of illustrative plates of Xanthoceraside carbon-hydrogen of the present invention (BMU-500,
1H-:F
2Be 0.8-5.1ppm,
13C-:F
1Be 100-180ppm).
Figure 16 for the long-range relevant amplification collection of illustrative plates of Xanthoceraside carbon-hydrogen of the present invention two (BMU-500,
1H-:F
2Be 0.54-2ppm,
13C-:F
1Be 15-90ppm).
Figure 17 for the long-range relevant amplification collection of illustrative plates of Xanthoceraside carbon-hydrogen of the present invention three (BMU-500,
1H-:F
2Be 2.8-6.8ppm,
13C-:F
1Be 10-94ppm).
Figure 18 for the long-range relevant amplification collection of illustrative plates of Xanthoceraside carbon-hydrogen of the present invention four (BMU-500,
1H-:F
2Be 3.4-6.4ppm,
13C-:F
1Be 10-94ppm).
Figure 19 is the relevant collection of illustrative plates (ARX-300, δ are 0-10ppm) of Xanthoceraside hydrogen-hydrogen of the present invention.
Figure 20 is the relevant collection of illustrative plates (ARX-300, δ 0.5-7ppm) of Xanthoceraside hydrogen-hydrogen of the present invention.
Figure 21-1 is an Xanthoceraside one-level mass spectrum of the present invention (MS).
Figure 21-2 is Xanthoceraside second order ms figure of the present invention (MS).
Embodiment
Embodiment 1
As shown in Figure 1, it is 30 orders that the 2000g shinyleaf yellowhorn fruit shell is crushed to granularity, is the ethanol lixiviate of 35% (V/V) with the concentration of 10 times of shell volumes, extraction temperature is 60 ℃, and extraction time is 3 hours, stirs 1 minute every 10 minutes in the leaching process, filter then, reclaim ethanol and get concentrated solution, concentrated solution is crossed 101 macroporous resins, use 35% ethanol elution,, remove impurity with active constituent-enriched, reclaim ethanol and get enriched material, evaporate to dryness obtains brown solid, i.e. total saponins; With the total saponins water dissolution, use n-butanol extraction, reclaim propyl carbinol, the dry brown powder that gets, through silica gel column chromatography repeatedly, carry out gradient elution with chloroform/methanol=100: 35-60, reclaim elutriant, recrystallization obtains the white needle of about 50mg, is purpose compound Xanthoceraside.
Get the purpose compound Xanthoceraside of said extracted, do experimentation on animals.
20 of mouse, the male and female dual-purpose, body weight 18-23g divides two groups, the administration group is irritated stomach and is given Xanthoceraside by the dosage of mouse body weight 9mg/kg, and control group is irritated the physiological saline that stomach waits capacity, after the administration 40 minutes, two groups of mouse are put into Y type labyrinth respectively, experimentize.The study of record mouse reaches the number of times of continuous 10 these standards of correct response wrong reaction before, surpasses 30 persons in 30 times, and experimental result sees Table 1:
Table 1 Xanthoceraside has the experimental data table that promotes the learning and memory of little mouse effect
Group | Number of animals (only) | Dosage (mg/kg) | Wrong reaction number of times x ± SD | P |
Physiological saline | 10 | Deng dosage | 28.2±4.4 | - |
Xanthoceraside | 10 | 9 | 6.9±2.3 | <0.01 |
By in the table as seen, Xanthoceraside group mouse wrong reaction times obviously is less than physiological saline group (P<0.01), shows that Xanthoceraside has promotion learning and memory of little mouse effect.
Embodiment 2
As shown in Figure 1,2000g Wood of Shinyleaf Yellowhorn carpopodium being pulverized, is the propyl carbinol lixiviate of 85% (V/V) with the concentration of 8 times of carpopodium volumes, extraction temperature is 75 ℃, and extraction time is 1 hour, stirs 5 minutes every 20 minutes in the leaching process, filter then, reclaim propyl carbinol and get concentrated solution, concentrated solution is crossed 101 macroporous resins, with 85% propyl carbinol wash-out,, remove impurity with active constituent-enriched, reclaim propyl carbinol and get enriched material, the enriched material evaporate to dryness obtains brown solid, i.e. total saponins; With the total saponins water dissolution, use n-butanol extraction, reclaim propyl carbinol, the dry brown powder that gets, through silica gel column chromatography repeatedly, carry out gradient elution with chloroform/methanol=100: 35-60, reclaim elutriant, recrystallization obtains the white needle of about 45mg, is purpose compound Xanthoceraside.
Get the Xanthoceraside of said extracted, do experimentation on animals.
Press Y type labyrinth three arm random approachs training mouse, preliminary election reaches before continuous 10 correct responses wrong reaction and is less than 30 persons and is used for experiment, after the preliminary election mouse had a rest 24 hours, be divided into two groups at random, equal abdominal injection Scopolamine 2.0mg/kg, cause the mouse memory obstacle, after 15 minutes, the administration group is by the dosage of mouse body weight 6mg/kg, abdominal injection Xanthoceraside, capacity physiological saline such as control group abdominal injection, after the administration 20 minutes, two groups of mouse are put into Y type labyrinth respectively experimentize, record reaches continuous 10 correct responses wrong reaction number of times before, and experimental result sees Table 2:
Table 2 Xanthoceraside has the experimental data table that promotes the memory represents effect to Scopolamine induced mice dysmnesia
Group | Number of animals (only) | Dosage (mg/kg) | Wrong reaction number of times x ± SD | P |
Physiological saline | 8 | Deng dosage | 17.9±3.6 | - |
Xanthoceraside | 8 | 6 | 4.0±1.5 | <0.01 |
Xanthoceraside has the effect of the memory represents of promotion to Scopolamine induced mice dysmnesia as can be seen from the table.
Embodiment 3
1000g shinyleaf yellowhorn fruit shell and 1000g carpopodium are pulverized, and are the ethanol lixiviate of 50% (V/V) with the concentration of 10 times of shells and carpopodium volume, and extraction temperature is 90 ℃, extraction time is 2 hours, stirs 3 minutes every 15 minutes in the leaching process, filters then, reclaim ethanol and get concentrated solution, concentrated solution is crossed 101 macroporous resins, uses 50% ethanol elution, with active constituent-enriched, remove impurity, reclaim ethanol and get enriched material, evaporate to dryness, obtain brown solid, i.e. total saponins; With the total saponins water dissolution, use n-butanol extraction, reclaim propyl carbinol, the dry brown powder that gets, through silica gel column chromatography repeatedly, carry out gradient elution with chloroform/methanol=100: 35-60, reclaim elutriant, recrystallization obtains the white needle of about 50mg, is purpose compound Xanthoceraside.
Get the Xanthoceraside of said extracted, do the static anoxic experiment of conventional airtight bottle, observe the influence of Xanthoceraside the death time.
Control group and Xanthoceraside administration group are respectively established in experiment in two batches, with three index evaluation death times.Oral Xanthoceraside, every day secondary, 2mg/ time, totally 6 days.
1. organize mean time to death (xt) entirely;
2. the super average death time (xp): with control group mean time to death (xc) is standard, calculates greater than death time (xp) of the part animal of xc and averages;
3. prolong the death time (xp-xc): the promptly super average death time (xp) deducts xc and averages.
Experimental result sees Table 3.
Table 3 Xanthoceraside is to the influence of small white mouse hypoxia death time
Index | Control group | The administration group | P |
First xt (min) xp (min) xp-xc (min) second batch xt (min) xp (min) xp-xc (min) | 34.5±8.89(30※) 41.42±4.48(14) 5.54±4.46(14) 25.38±4.24(40) 27.54±2.87(24) 2.64±2.87(24) | 41.84±15.35(30※) 56.51±14.59(17) 14.59±14.83(17) 31.55±6.68(41) 33.40±4.94(33) 8.40±4.94(33) | <0.025 <0.05 <0.05 <0.001 <0.001 <0.001 |
※ is a number of animals
All there is tangible difference three death times of control group and two batches of experiments of administration group as can be seen from Table 3, and the death time of administration group is longer than control group, prove that Xanthoceraside has the animal of raising to the anoxybiotic tolerance.Brain is very responsive to anoxic, because at first involve brain during anoxic, Xanthoceraside has improved the anoxybiotic tolerance, thereby has prolonged the death time of small white mouse.
Embodiment 4
As shown in Figure 1, the 2000g shinyleaf yellowhorn fruit shell is pulverized, boiled lixiviate with the water of 15 times of shell volumes, extraction time is 3 hours, stirred 2 minutes every 10 minutes in the leaching process, filter then, filtrate is crossed 101 macroporous resins, the water wash-out, with active constituent-enriched, remove impurity, evaporation concentration, concentrated solution n-butanol extraction, reclaim propyl carbinol, dry brown powder through silica gel column chromatography repeatedly, is carried out gradient elution with chloroform/methanol=100: 35-60, reclaim elutriant, recrystallization obtains the white needle of about 40mg, is purpose compound Xanthoceraside.
In addition, extraction solvent of the present invention also can use methyl alcohol, propyl alcohol or acetone.
Embodiment 5
Get the Xanthoceraside that embodiment 3 extracts, do the tumor cell in vitro experiment, experimental technique and the results are shown in Table 4.
Table 4 Xanthoceraside is to the screening active ingredients result of external six types of tumour cells
Sequence number | Test model | Dosage (ug/ml) | Observation index | The test effect | The result | IC
50 (ug/ml)
|
1 2 3 4 5 6 | Mtt assay (HL-60 human leukemia) srb assay (PC-3M1E8 human prostata cancer) srb assay (BGC-823 people's cancer of the stomach) srb assay (MDA-MB-435-human breast carcinoma) srb assay (Bel-7402 people's liver cancer) srb assay (Hela human cervical carcinoma) | 0.5 5.0 50.0 0.5 5.0 50.0 0.5 5.0 50.0 0.5 5.0 50.0 0.5 5.0 50.0 0.5 5.0 50.0 | Inhibiting rate (%) inhibiting rate (%) inhibiting rate (%) inhibiting rate (%) inhibiting rate (%) inhibiting rate (%) | 9.34 17.68 56.90 -13.90 -7.63 92.03 -11.71 -5.92 92.45 -4.50 2.92 93.14 -9.78 -1.24 92.96 -12.72 -4.05 95.49 | + + + + + + | 41.29 40.98 40.24 18.58 39.32 34.23 |
Annotate: MTT: tetramethyl-azo azoles salt (dyeing), SRB: sulphur cyanamide B (dyeing)
IC
50The cell half-inhibition concentration.
As can be seen from the table: Xanthoceraside is remarkable to external six types of tumour cell restraining effect effects.From the IC that calculates
50(half-inhibition concentration) as can be seen, when the concentration of Xanthoceraside only is 18.58ug/ml, breast cancer cell promptly being had restraining effect, secondly is to cervical cancer, liver cancer, cancer of the stomach, prostate cancer and leukemia cell's inhibition effect, its half-inhibition concentration is respectively 34.23ug/ml, 39.32ug/ml, 40.24ug/ml, 40.98ug/ml, 41.92ug/ml.Hence one can see that, and Xanthoceraside promptly has the good restraining effect to solid tumor cell under lower concentration, through further investigation, can develop the new drug of treatment tumour.
To the embodiment of the invention 1,2,3 and the 4 purpose compounds that extract, applied chemistry method and spectroscopic analysis (comprising: UV, ID-NMR, 2D-DMR, EI-MS, ESI-MS etc.), carried out structure and identified and attribution data: according to its UV, ESI-MS,
1H-NMR,
13C-NMR, HMQC, HMBC, TOCSY spectrum data (seeing Figure of description 2-21 for details), and consult document [1] Chen Y J, Takeda T, Ogihara Y, et al.Studies on the constituents of Xanthocerassorbifolia Bunge.III.Minor prosapogenins from the fruits ofXanthoceras sorbifolia Bunge.[J] .Chem.Pharm.Bull, 1985,33 (1): 127-134; [2] Yoshikawa M, Harada E, Matsuda H, et a ElatosidesA and B, potent inhibitors of ethanol absorption in rats from thebark of Aralia elata Seem.:The structure-activity relationships ofoleanolic acid oligoglycosid[J] .Chem.Pharm.Bull.1993,41 (11): 2069-2071.[3] Yoshikawa M, Harada E, Murakami T, et al.Camelliasaponins B1, B2, C1 and C2, new type inhibitors of ethanolabsorption in rats from the seeds of Camellia japonica L.[J] .Chem.Pharm.Bull.1994,42 (3): it is 3-O-(α-L-arabinofuranosyl (1 → 3)-β-D-galactopyranosyl (1 → 2))-β-D-glucuronopyranosyl-21 that 742-744. identifies this compound structure, 22-diangeloyl-R
1-barrigenol, i.e. 3-O-(α-L-arabinofuranosyl (1 → 3)-β-D-galactopyranose base (1 → 2))-beta d glucopyranosiduronic acid base-21,22-two angeloyl groups-R
1-barrigenol, its chemical structure is as follows:
The detailed data ownership of purpose structure that compound carries out being identified through chemical method and spectroscopic analysis sees Table 5:
Table 5 Xanthoceraside nuclear magnetic data ownership table
No | 13C NMR
| 1H NMR
| No | 13C NMR
| 1H NMR
|
Aglycone moiety | Sugar moiety |
3-O-glucuronopyranosyl moiety |
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 | 39.7 26.7 89.9 39.0 55.7 18.9 36.4 41.1 47.2 37.0 24.0 125.5 143.7 47.8 67.6 73.4 48.4 41.5 47.0 36.8 78.6 73.6 28.0 16.8 15.8 17.6 21.3 63.2 29.6 | 3.21(1H,m) 5.48(1H,brs) 6.70(1H,d,J=10.2Hz) 6.32(1H,d,J=10.2Hz) 1.26(3H,s) 1.16(3H,s) 0.81(3H,s) 0.98(3H,s) 1.84(3H,s) 3.73,3.50(1H,d,J=10.2Hz) 1.09(3H,s) | 1′ 2′ 3′ 4′ 5′ 6′ | 105.2 78.9 86.4 71.7 77.3 172.0 | 4.89(1H,d,J=10.2Hz) |
2′-O-β-D-galactopyranosyl moiety |
1 2 3 4 5 6 | 104.9 73.5 75.2 69.8 76.7 61.9 | 5.32(1H,d,J=7.5Hz) |
3′-O-α-L-arabinofuranosyl moiety |
1 2 3 4 5 | 111.2 83.6 77.7 85.5 62.4 | 6.03(1H,brs) |
Angeloyl moiety |
1 2 3 4 5 1 2 3 4 | 167.8 129.0 137.4 15.9 21.0 168.2 129.2 136.6 15.7 | 5.96(1H,q,J=7.0Hz) 2.09(3H,dq,J=7.0Hz) 2.00(3H,m) 5.76(1H,q,J=6.6Hz) 1.93(3H,dq,J=6.6Hz) |
In sum, the triterpene saponin componds that is found to be of new compound has increased a newcomer, thereby the utility value of depleted shinyleaf yellowhorn fruit shell and carpopodium is improved greatly, is expected to develop in field of medicaments from now on the new drug of treatment disease of brain and tumour.