CN100586450C - Phenolic extract of duchesnea indica focke, preparation method and application thereof - Google Patents
Phenolic extract of duchesnea indica focke, preparation method and application thereof Download PDFInfo
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- CN100586450C CN100586450C CN200710063000A CN200710063000A CN100586450C CN 100586450 C CN100586450 C CN 100586450C CN 200710063000 A CN200710063000 A CN 200710063000A CN 200710063000 A CN200710063000 A CN 200710063000A CN 100586450 C CN100586450 C CN 100586450C
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Abstract
The present invention discloses an Indian strawberry phenol extraction extracted from an Indian strawberry whole grass, which also relates to the preparation method of the extraction and the functionof the extraction. The preparation method of the Indian strawberry phenol extraction is as following, which comprises soaking and extraction of the Indian strawberry by water, ethanol, or a mixed solution with water and ethanol in a certain ratio; vacuum concentration of the Indian strawberry extraction liquid; then the ethanol is removed from the extraction liquid and passed through a malodorousresin column; the liquid is eluted by the ethanol after elution of the non affection component with the water; the ethanol elution liquid and vacuum concentration are collected, therefore the Indian strawberry phenol extraction is obtained. The Indian strawberry phenol extraction of the present invention possesses functions of prohibiting the generation and growth of various kinds of tumor cells.Therefore the present invention is applicable for treatment of a plurality of cancer diseases comprising the lung cancer, the liver cancer, the nasopharynx cancer, the gastric cancer, the cervical cancer, the endometrium cancer and the ovarian cancer, etc. In addition, the Indian strawberry phenol extraction in the present invention has active function of improving the immune ability in the organism.
Description
Technical field
The present invention relates to a kind of Chinese medicine effective component extracts, relate in particular to a kind of phenol extract that obtains that from the plant Herba Duchesneae Indicae, extracts, the invention still further relates to the purposes of this this preparation method of extract and this extract, belong to the field of Chinese medicines.
Background technology
Herba Duchesneae Indicae [Drchesnea indica (Andr) Focke] is a Rosaceae Herba Duchesneae Indicae platymiscium, call three wind, the certain kind of berries, Herba Duchesneae Indicae, five-fingered jack, wild Fructus Myricae rubrae etc., be medical herbs commonly used among the people.Herba Duchesneae Indicae has heat-clearing and toxic substances removing, detumescence dissipating blood stasis, the effect of astringing to arrest bleeding, removing heat from blood with all herbal medicine.Existing bibliographical information Herba Duchesneae Indicae has notable antitumor activity, but what reported all is that the water of Herba Duchesneae Indicae is carried or the crude extract of alcohol extraction, up to now, does not obtain the antitumor component of Herba Duchesneae Indicae as yet.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and the antitumor component of a kind of Herba Duchesneae Indicae is provided, promptly Herba Duchesneae Indicae phenol extract (Drchesnea polyphenolic extract, DPE).
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method for preparing Herba Duchesneae Indicae phenol extract comprises:
(1) mixed liquor formed with arbitrary proportion of water, 50-80% ethanol or ethanol and water serves as to extract solvent soaking to extract Herba Duchesneae Indicae (extracting solvent did not have Herba Duchesneae Indicae to get final product), filter the Herba Duchesneae Indicae extracting solution;
(2) will pass through macroporous adsorptive resins behind the Herba Duchesneae Indicae extracting solution concentrating under reduced pressure removal ethanol, behind elder generation's water flush away non-active ingredients, reuse 50-95% ethanol (90-95% ethanol more preferably) eluting macroporous adsorptive resins, collect ethanol elution, be evaporated to dried Herba Duchesneae Indicae phenol extract (DPE).
In the above-mentioned preparation method, preferred, the mixed liquor that water, 50-80% ethanol or ethanol and water are formed with arbitrary proportion in the step (1) serves as to extract solvent soaking to extract Herba Duchesneae Indicae 3 times, extracts 1 hour at every turn; Be to extract solvent soaking to extract Herba Duchesneae Indicae 3 times more preferably, extracted 1 hour at every turn with 80 ℃ of hot water.
Non-active ingredients described in the step (2) is sugar, organic salt, aminoacid, protein, pigment etc.;
Macroporous adsorptive resins is preferably and uses AB-8, D3520, DM130, D8, D101 or D201 macroporous adsorptive resins in the step (2).
Herba Duchesneae Indicae phenol extract of the present invention has propagation and the growth effect that suppresses kinds of tumor cells, can be used for multiple treatment for cancer, comprises pulmonary carcinoma, hepatocarcinoma, nasopharyngeal carcinoma, gastric cancer, cervical cancer, carcinoma of endometrium, ovarian cancer etc.; In addition, Herba Duchesneae Indicae phenol extract of the present invention can improve cell and humoral immune function.Therefore, Herba Duchesneae Indicae phenol extract of the present invention can be used for being prepared into the medicine of antitumor or raising body immunity.
After Herba Duchesneae Indicae phenol extract extract of the present invention can add various adjuvants and pharmaceutically acceptable excipient or carrier required when preparing different dosage form, method of Chinese medicinal with routine is prepared into any suitable clinical preparation, for example can be injection (powder pin, freeze-dried powder, liquid drugs injection, transfusion etc.), tablet, oral liquid, granule, capsule, soft capsule or drop pill etc.; Wherein, described adjuvant can be antioxygen chelating agent, filler, framework material etc.;
Described pharmaceutically acceptable carrier is one or more in xylitol, mannitol, lactose, fructose, glucosan, glucose, polyvinylpyrrolidone, low molecular dextran, sodium chloride, calcium gluconate or the calcium phosphate.
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
The specific embodiment
Embodiment 1
Get Herba Duchesneae Indicae herb 200g, add 80 ℃ of hot water 3L, soak and extract 3 times, each 1 hour.Remove by filter medicinal residues, merge extractive liquid, slowly passes through AB-8 type macroporous adsorptive resins with extracting solution, behind non-active ingredients such as 10L washing desaccharide, organic salt, aminoacid, again with 6L 90% ethanol elution, collect ethanol elution, be evaporated to dried Herba Duchesneae Indicae phenol extract 11g.With spectrophotography (Scalbert A, Monties B, Jannin, G.Tanins in wood:comparison of different estimation methods.Journal of Agricultural andFood Chemistry 1989,37:1324-1329.) record, contain the phenol total amount in this extract and count 46.1% with epicatechin.
Embodiment 2
Get Herba Duchesneae Indicae herb 150g, add entry 2.5L, make and soaked powder, soak and extract 3 times, each 1 hour.Remove by filter medicinal residues, merge extractive liquid,, extracting solution is slowly passed through AB-8 type macroporous adsorptive resins, behind non-active ingredients such as 10L0.1% aqueous hydrochloric acid solution flush away sugar, organic salt, aminoacid, again with 6L 95% ethanol elution, collect ethanol elution, be evaporated to dried Herba Duchesneae Indicae phenol extract 9.6g.With spectrophotography (Scalbert A, Monties B, Jannin, G.Tanins in wood:comparison of different estimation methods.Journal of Agricultural and Food Chemistry 1989,37:1324-1329.) record, contain the phenol total amount in this extract and count 41.9% with epicatechin.
Embodiment 3
Get Herba Duchesneae Indicae herb 130g, add 80% ethanol 2L and soak extraction 3 times, each 1 hour.Remove by filter medicinal residues, merge extractive liquid,, after the extracting solution concentrating under reduced pressure removed ethanol, add the dilution of 5L water, filtrate is by D3520 type macroporous adsorptive resins, earlier with non-active ingredients such as 10L washing desaccharide, organic salt, aminoacid, again with 6L 90% ethanol elution, collect ethanol elution, be evaporated to dried Herba Duchesneae Indicae phenol extract 5.2g.With spectrophotography (Scalbert A, Monties B, Jannin, G.Tanins in wood:comparison of different estimationmethods.Journal of Agricultural and Food Chemistry 1989,37:1324-1329.) record, contain the phenol total amount in this extract and count 40.2% with epicatechin.
Test example 1 Herba Duchesneae Indicae phenol extract is to the inhibitory action of growth of human tumor cells
The take the logarithm tumor cell (available from China Concord Medical Science University basis institute cell centre) of trophophase is inoculated in 96 orifice plates with 5000/hole, in 37 ℃ of 5%CO
2Cultivate after 10-12 hour under the condition, the administration group adds DPE (embodiment of the invention 1,2 or 3 is prepared), making its final concentration is 20-240 μ g/mL, the blank group is 0.08% dimethyl sulfoxide, positive controls is the cisplatin of 20nmol/L, adopt the MTT colorimetry, measure the absorbance A value in wavelength 570nm/650nm place.Calculate the suppression ratio of medicine according to the A value to growth of tumour cell.
The growth inhibition ratio of tumor cell=(the A value of the A value/negative control group of 1-experimental group) * 100%
Result of the test shows that DPE has inhibition proliferation function (table 1) to kinds of tumor cells, especially pulmonary carcinoma, nasopharyngeal carcinoma and hepatoma carcinoma cell is had very strong inhibitory action, and the gynecological tumor cell is also had good inhibitory effect.
Table 1. Herba Duchesneae Indicae phenol extract is to the inhibited proliferation of tumor cell
Test example 2 Herba Duchesneae Indicae phenol extracts are to the inhibitory action of mice transplanted tumor U14
Get well-grown ascites cells (available from institute of Materia Medica,Chinese Academy of Medical Sciences), washing back makes with an amount of physiological saline solution that to contain cell number be 2.5 * 10
7The tumor cell suspension of individual/mL.0.2mL (contains oncocyte 5 * 10 respectively at right this tumor cell suspension of axillary fossa subcutaneous vaccination of mice (available from Chinese Academy of Medical Sciences's animal center)
6Individual).Inoculation is divided into 5 groups at random with mice with tumor next day, every group 10, wherein 3 groups are the administration group, and oral administration gavage gives DPE (embodiment of the invention 1,2 or 3 is prepared) with 3 various dose (0.25 respectively, 0.5 and 1g/kg), model group is that oral administration gavage gives the equal-volume distilled water.Administration group and model group all gavage 20 days continuously.Positive controls is intraperitoneal injection of cyclophosphamide (CTX) 60mg/kg, only gives once next day behind the inoculation oncocyte.Each treated animal is all conventional raises, drinking-water, and food is not limit.Mice all took off cervical vertebra in the time of 21 days puts to death, and strips subcutaneous solid tumor tumor piece, weighs, and calculates tumor control rate (%) by following formula.
Tumor control rate (%)=(the average tumor of the average tumor weight-experimental group of model group is heavy)/average tumor of model group heavy * 100%.
Result of the test (table 2) shows that the DPE of low dosage (0.25g/kg) has facilitation to the growth of mouse carcinoma of uterine cervix U14; And the DPE of high dose (0.5g/kg and 1g/kg) can obviously suppress the growth of U14, and tumour inhibiting rate is respectively 27% and 63%; Each dosage group of DPE there is no tangible toxic reaction, and mice all survived when treatment finished.
The effect of table 2.DPE anti-tumor in vivo (mean value SD, n=10)
| The experiment grouping | Dosage (g/kg) | Tumor heavy (g) |
| Contrast | - | 2.420±0.839 |
| CTX | 0.06 | 0.652±0.756 |
| DPE | 0.25 | 2.584±0.718 |
| DPE | 0.5 | 1.150±0.590 ** |
| DPE | 1 | 0.777±0.552 ** |
*, P＜0.01 is compared with matched group
The test of test example 3 Herba Duchesneae Indicae phenol extract immunologic functions
1. pair cell Immune Effects-T lymphocyte transformation test
In test example 2, after the execution of 20 days U14 tumor-bearing mice of successive administration, under aseptic, get spleen immediately, grind with cold aseptic PBS flushing back, the splenocyte after the grinding makes 5 * 10 with the RPMI-1640 that contains 10% hyclone after the PBS washing
6The cell suspension of/mL.Add this cell suspension in 96 orifice plates, 100 μ L/ holes.And adding ConA 10 μ L (final concentration 2.5 μ g/mL).Cultivate after 60 hours, every hole adds MTT 20 μ L (final concentration 0.5mg/mL), continues to cultivate after 4 hours, adds three liquid, 100 μ L[and contains SDS 10% (w/v), isobutanol 5% (v/v) and HCl 0.01mol/L (v/v)], 37 ℃ are spent the night.Microplate reader 570nm place surveys absorption value A.
2. to the influence-hemolysin spectrophotography of humoral immune function
Select Kunming female mice (available from Chinese Academy of Medical Sciences's animal center) for use, after the inoculation of U14 oncocyte, animal grouping and route of administration, method, dosage and time are all identical in the example 2 with test, and the 5%SRBC to mouse peritoneal injection 0.4mL on the 16th makes animal sensitization in the inoculation back.Put to death animal and get spleen in drug withdrawal on the 21st.Spleen channel is ground, after the washing, make with cold PBS and to contain 2 * 10
10The cell suspension of/L.With this splenocyte suspension 1mL and 0.2%SRBC 1mL and 1: 10 abundant mixing of dilution complement 1mL, behind 37 ℃ of water-bath 60min, the centrifugal 5min of 3000rpm.Get supernatant and survey absorption value A in the 413nm place.
Above T lymphocyte transformation experiment and hemolysin spectrophotometric experimental result (table 3) show that compare with normal mouse, the cell and the humoral immune function of tumor-bearing mice all obviously weaken.Herba Duchesneae Indicae phenol extract 1g/kg can obviously promote the lymphocytic propagation of T of tumor-bearing mice; But the DPE of low dosage (0.25g/kg) obviously suppresses lymphocytic propagation.The DPE of various dose all can make the humoral immune function of tumor-bearing mice strengthen, and the antibody amount that shows as dose dependent increases.
The influence that table 3DPE discharges T lymphopoiesis and bone-marrow-derived lymphocyte antibody (mean value SD, n=10)
| Group | Dosage (g/kg) | T lymphopoiesis (A 570value) | Antibody discharges (A 413value) |
| Normal | - | 0.449±0.144 | 0.367±0.077 |
| Model | - | 0.408±0.098 | 0.083±0.109## |
| CTX | 0.06 | 0.322±0.046 * | 0.105±0.06 |
| DPE | 0.25 | 0.270±0.072 ** | 0.153±0.119 |
| DPE | 0.5 | 0.385±0.096 | 0.464±0.385 ** |
| DPE | 1 | 0.588±0.112 ** | 0.512±0.337 ** |
##, P＜0.01 is compared with normal group
*, P＜0.05,
*, P＜0.01 is compared with model group
Claims (9)
1, a kind of method for preparing Herba Duchesneae Indicae phenol extract comprises:
(1) mixed liquor formed with arbitrary proportion of water, 50-80% ethanol or ethanol and water serves as to extract solvent soaking to extract Herba Duchesneae Indicae, filters to remove medicinal residues and get the Herba Duchesneae Indicae extracting solution;
(2) the Herba Duchesneae Indicae extracting solution behind Herba Duchesneae Indicae aqueous extract or the decompression removal ethanol is passed through macroporous adsorptive resins, behind elder generation's water or the 0.1% hydrochloric acid flush away non-active ingredients,, collect ethanol elution again with the 50-95% ethanol elution, be evaporated to dried, Herba Duchesneae Indicae phenol extract; Described macroporous adsorptive resins is selected from AB-8, D3520, DM130, D8, D101 or D201 macroporous adsorptive resins.
2, according to the method for claim 1, it is characterized in that: the mixed liquor that water, 50-80% ethanol or ethanol and water are formed with arbitrary proportion in the step (1) serves as when extracting solvent soaking extraction Herba Duchesneae Indicae, to extract 3 times, extracts 1 hour at every turn.
3, according to the method for claim 2, it is characterized in that: step (1) is middle to be to extract solvent soaking to extract Herba Duchesneae Indicae with 80 ℃ of hot water.
4, according to the method for claim 1, it is characterized in that: the non-active ingredients described in the step (2) comprises sugar, organic salt, aminoacid, protein or pigment.
5, a kind of Herba Duchesneae Indicae phenol extract is characterized in that: the product that is prepared by any one method of claim 1-4.
6, according to the Herba Duchesneae Indicae phenol extract of claim 5, it is characterized in that: be prepared into tablet, pill, granule, injection or oral liquid according to conventional method of Chinese medicinal.
7, the purposes of the Herba Duchesneae Indicae phenol extract of claim 5 in preparation medicine for treating tumor thing.
8, according to the purposes of claim 7, it is characterized in that: described tumor comprises pulmonary carcinoma, hepatocarcinoma, nasopharyngeal carcinoma, gastric cancer, cervical cancer, carcinoma of endometrium or ovarian cancer.
9, the purposes of the Herba Duchesneae Indicae phenol extract of claim 5 in preparation enhancing human body immunity power medicine.
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| CN101486771B (en) * | 2009-02-27 | 2011-08-24 | 中国药科大学 | Duchesnea polysaccharide, as well as preparation and use thereof |
| CN101701107A (en) * | 2009-10-29 | 2010-05-05 | 西北工业大学 | Preparation method of mock-strawberry fruit haematochrome |
| CN103768185A (en) * | 2012-10-25 | 2014-05-07 | 奇复康药物研发(苏州)有限公司 | Extraction method for Indian strawberry flavone |
| CN103263455B (en) * | 2013-03-20 | 2018-04-06 | 中国医学科学院药用植物研究所 | A kind of preparation and application of the medicine with neuroprotection |
| CN112500468B (en) * | 2020-12-14 | 2022-07-15 | 上海交通大学 | Bioactive peptide RLAFIAHPKLG, and preparation method and application thereof |
| CN116832088A (en) * | 2023-06-20 | 2023-10-03 | 重庆派佰乐生物科技有限公司 | Application of Indian mockstrawberry herb extract in preparation of constipation relieving medicine |
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| 蛇莓化学成分研究. 王强等.河南科学2006，第24卷第4期. 2006 |
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