CN102406670A - Extraction method of total alkaloids from tripterygium hypoglaucum and application of extract - Google Patents

Extraction method of total alkaloids from tripterygium hypoglaucum and application of extract Download PDF

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CN102406670A
CN102406670A CN2010102891958A CN201010289195A CN102406670A CN 102406670 A CN102406670 A CN 102406670A CN 2010102891958 A CN2010102891958 A CN 2010102891958A CN 201010289195 A CN201010289195 A CN 201010289195A CN 102406670 A CN102406670 A CN 102406670A
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extraction
total alkaloids
tripterygium hypoglaucum
distilling
value
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刘晋祎
曹佳
敖琳
杨录军
黄晓春
姜晓
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Third Military Medical University TMMU
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Third Military Medical University TMMU
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Abstract

The invention discloses an extraction method of total alkaloids from tripterygium hypoglaucum and an application of the extract. The extraction method comprises the following steps of: taking a certain amount of tripterygium hypoglaucum, pulverizing after peeling and removing the root, adding a certain amount of ethanol solution of a finite concentration for performing multiple extraction assisted by ultrasonic equipment, combining the extracted solutions and concentrating, adding concentrated hydrochloric acid of a finite concentration to the concentrated solution for regulating the pH value, filtering after fully mixing for a period of time, extracting the filtrate by chloroform many times, reserving the raffinate phase, regulating the pH value of the raffinate phase to be alkaline, extracting the alkaline solution by chloroform many times, combining the chloroform phases, and evaporating for recovering the chloroform to obtain total alkaloids, or performing solid phase extraction on the alkaline solution through C18 columns to obtain total alkaloids. The extract obtained according to the invention can be used for treating tumor and autoimmune disease.

Description

The method for distilling of Tripterygium hypoglaucum total alkaloids and the application of this extract
Technical field
The invention belongs to plant extract technology and drug research field, be specially the method and the extract obtained application of this method for distilling of extract total alkaloids in the Tripterygium hypoglaucum plant.
Background technology
Cancer constitutes significant threat to human beings'health and existence, and antitumor drug is still one of main means of treatment of cancer in quite long at present and in the future period.There is the shortcoming that selectivity is low, toxicity is big in existing most tumor chemotherapeutic drug.Find that the new type antineoplastic medicine of high selectivity, low toxicity, high specificity has become the important directions of current antitumor drug research and development.China's population the first in the world, tumor incidence height, the anti-cancer agent exploitation also is the emphasis of China's new drug research and exploitation.
The discovery of new type antineoplastic medicine is faced with the chemical entities source and how screens two key issues.Come aspect the source problem at anti-cancer agent, natural product is the precious resources of China's new drug research and exploitation and good point of penetration.Tripterygium hypoglaucum (Tripterygium Hypoglaucum Hutch (THH)) is the Celastraceae tripterygium plant, mainly is distributed in each provinces and regions on the south China the Changjiang river, southwest district and Taiwan Province, and main product is in Yunnan Province.Be used for treating rheumatoid arthritis, lupus erythematosus, tumor etc. mainly as immunosuppressant clinically, and have analgesia, antiinflammatory, anti-male fertility and mutagenic action.The toxic action of experiment proof THH is much smaller than the congener Radix Tripterygii Wilfordii.The Main Ingredients and Appearance of THH has alkaloid, terpenoid, glycoside, saccharide, organic acid, polysaccharide etc., and wherein alkaloid curative effect clinically is best and toxic and side effects is minimum.
Traditional Tripterygium hypoglaucum total alkaloids (Total alkaloids of THH (THHta)) method for distilling drug immersion overlong time (adds the organic solvent soak time and reaches 6 days; Like document [Ao Lin; Cao Jia, Xu Ying etc., Tripterygium hypoglaucum total alkaloids bring out the apoptotic observation of HL60. the journal .2001 of Third Military Medical University; 23 (11): 1273-1275]) and the total alkaloids impurity that obtains more, not easily separated purification.The present invention has overcome the problem of method for distilling existence in the past, and the one, utilize ultrasound assisted extraction technique, improved the yield of THHta, improved extraction efficiency thereby shortened soak time; The 2nd, on the basis of ultrasonic extraction, utilize the lipotropy of C18 to carry out SPE, when reducing the chloroform consumption, improved the purity of total alkaloids.
Summary of the invention
The object of the present invention is to provide a kind of from Tripterygium hypoglaucum the method for extract total alkaloids, and be applied to the treatment of tumor and autoimmune disease.
The present invention utilizes ultrasound assisted extraction technique; Supersound extraction, solvent extraction and SPE are combined; Confirmed that concrete extraction conditions extracts the total alkaloids in the Tripterygium hypoglaucum; The anti-tumor activity experiment shows that THHta can suppress tumor cell proliferations activity such as MOLT-4, HCT116 and PC3 to this extract through the inside and outside, and growth also has the obvious suppression effect to HCT116 cell transplanted tumor in nude mice, has significant anti-tumor in vivo activity.
The present invention realizes goal of the invention through following method.
The method for distilling step of mountain, Kunming total alkaloids is following:
(1) Tripterygium hypoglaucum peeling root is pulverized the back and is crossed the 5-15 mesh sieve;
(2) adding Tripterygium hypoglaucum peeling root 10-40, doubly to measure 61-69% ethanol multiple extraction under ultrasound condition;
(3) merge extractive liquid,, the concentrated hydrochloric acid that concentrates back adding 30-45% is regulated pH value to acid, fully mixes after-filtration;
(4) filtrating is with chloroform extraction 3-5 time, and the gained raffinate phase is used Na 2CO 3Regulate pH value to 9-11, reuse chloroform extraction 3-5 time merges extraction phase;
(5) under 40-50 ℃ of condition, the rotary evaporation extraction phase reclaim behind the chloroform total alkaloids;
Or
(1) Tripterygium hypoglaucum peeling root is pulverized the back and is crossed the 5-15 mesh sieve;
(2) adding Tripterygium hypoglaucum peeling root 10-40, doubly to measure 61-69% ethanol multiple extraction under ultrasound condition;
(3) merge extractive liquid,, the concentrated hydrochloric acid that concentrates back adding 30-45% is regulated pH value to acid, fully mixes after-filtration;
(4) filtrating is with chloroform extraction 3-5 time, and the gained raffinate phase is used Na 2CO 3Regulate pH value to 9-11, with this raffinate phase through containing the solid-phase extraction device of C18, earlier with distilled water flushing 30 minutes, reuse 30-45% alcohol flushing, collecting this flushing liquor must total alkaloids after rotary evaporation reclaims ethanol.
Wherein the supersound extraction of step (2) adopts the continuous multifunction ultrasonic extractor of Based Intelligent Control of Jining Sinobest Electronics Co., Ltd.'s production to accomplish.
The specific embodiment
1. experiment purpose
Detect this patent method gained Tripterygium hypoglaucum total alkaloids (THHta) inside and outside anti-tumor activity, for the antitumor drug exploitation provides experimental basis.
2. experiment material
2.1 confession test agent: THHta obtains via the said method of this patent.
2.2 experimental cell strain: tumor cell line HL60, MOLT-4, HCT116 and PC3 (available from Shanghai cell biological institute cell bank).
2.3 laboratory animal:
18 of BALB/c-nu nude mouses, 4 ages in week, body weight 14.2 ± 0.9g, female Mus is provided by Chinese Academy of Medical Sciences's laboratory animal, SPF level, laboratory animal production licence number: (capital) 2009-0004.
3. experimental technique and result
3.1THHta inhibited proliferation to tumor cell
THHta with variable concentrations (5,10,25,50,100 μ g/ml) handles 4 kinds of tumor cells such as HL60, MOLT-4, HCT116 and PC3, and 48h carries out the MTT experiment after drug treating.
The result shows: THHta can suppress the proliferation activity of HL60, MOLT-4, HCT116 and four kinds of tumor cells of PC3, and is obvious dependence with dosage, and the result sees table 1.The IC that THHta effect HL60, MOLT-4, HCT116 and PC3 cell 48h are corresponding 50Value is 1.59,8.77,18.86,49.34 μ g/ml.
3.2THHta inhibitory action to the growth of HCT116 transplanted tumor in nude mice
The HCT116 cell inoculation is subcutaneous in nude mice, when tumor forms and the long-pending 100mm that reaches of tumor block 3During the left and right sides, nude mice is divided into matched group, THHta treatment group (50mg/kg group, 100mg/kg group) at random by gross tumor volume.Dosage regimen: intraperitoneal injection, administration is 5 days weekly, and intermittently two days, successive administration 24 days.The heavy corresponding equal-volume solvent (normal saline that contains 0.2%DMSO) of matched group donor.Experimental session was measured a transplantable tumor volume parameter in per three days and was calculated the knurl volume, and each is organized average knurl volume and sees Table 2.According to the relative tumor volume of the transplanted tumor volume calculation of each measurement (RTV), and further calculate corresponding tumor proliferation rate (T/C%) relatively, see table 3.
The result shows: the average external volume of treatment group HCT116 transplanted tumor in nude mice is compared with matched group, and after the 6th day, 100mg/kg group tumor volume has significant significant difference (P＜0.01) less than matched group in administration; In administration extremely experiment end in the 9th day, THHta two treatment group tumor volumes are all significantly less than matched group (P＜0.01).When experiment finishes, when the average external volume of matched group, THHta low dosage, high dose group transplanted tumor is the grouping administration respectively 9.39 times of gross tumor volume, 1.93 times and 0.83 times.THHta50mg/kg group began relative tumor proliferation rate (T/C%) less than 40% on the 12nd day after administration; THHta100mg/kg group after administration the 9th day beginning T/C value less than 40%.Each time point T/C value explanation THHta of experimental session can suppress HCT116 transplanted tumor in the intravital proliferate of nude mice, and is dependence with time and dosage.
Table 1.MTT method method is measured the suppression ratio (% of THHta effect 48h to following 4 kinds of tumor cells; N=8,
Figure BSA00000279871600031
)
Figure BSA00000279871600032
Annotate: compare * P＜0.05, * * P＜0.01 with normal group
Table 2.HCT116 transplanted tumor in nude mice volume (mm 3)
Annotate: compare * P＜0.05, * * P＜0.01 with matched group
The RTV and the T/C value of table 3.THHta treatment HCT116 transplanted tumor
Figure BSA00000279871600042
Figure BSA00000279871600043
annotates: it is invalid that T/C＞40% is regarded as; T/C of≤40%, and be regarded as effectively through statistical procedures P＜0.05.Relative tumour volume (RTV); Relative tumor appreciation rate (T/C)
3.3 date processing
Record index and all use mean ± standard deviation
Figure BSA00000279871600044
expression; Adopt the SPSS12.0 software kit to carry out statistical analysis; Relatively check between group with F; Relatively with the q check, there is statistical significance P＜0.05 for difference in twos.
4. conclude the speech
It is active that THHta can significantly suppress MOLT-4, HL60, HCT116 and PC3 tumor cell proliferation, and be tangible dose-effect relationship.THHta can suppress the growth of HCT116 cell transplanted tumor in nude mice, has significant anti-tumor in vivo activity.
Embodiment 1:
Get Tripterygium hypoglaucum peeling root 2kg; Pulverize the back and cross 10 mesh sieves, placed ultrasonic instrument simultaneously 3 hours, leach medicinal liquid with 10 times of amount 61% soak with ethanol; Repeat to add twice of 10 times of amount 61% ethanol; Soak and to place ultrasonic instrument simultaneously respectively 2 hours and 1 hour, leach behind the medicinal liquid and the combining medicine first time, concentrated.Add 30%HCl and regulate pH between the 2-3, with the chloroform extraction of a great deal of 3 times, the reservation water.Add Na 2CO 3, regulate pH to 9-11, reuse a great deal of chloroform extraction 3 times, combining extraction liquid get total alkaloids 19.8g after 40-50 ℃ of rotary evaporation reclaims chloroform;
Embodiment 2:
Get Tripterygium hypoglaucum peeling root 100g; Pulverize the back and cross 10 mesh sieves, placed ultrasonic instrument simultaneously 3 hours, leach medicinal liquid with 10 times of amount 69% soak with ethanol; Repeat to add twice of 10 times of amount 69% ethanol; Soak and to place ultrasonic instrument simultaneously respectively 2 hours and 1 hour, leach behind the medicinal liquid and the combining medicine first time, concentrated.Add 45%HC1 and regulate pH between the 2-3, with the chloroform extraction of a great deal of 3 times, the reservation water.Add people Na 2CO 3, regulate pH to 9-11, through the C18 solid-phase extraction device, water flushing solid-phase extraction device 30 minutes is used 45% alcohol flushing, collects flushing liquor after rotary evaporation reclaims ethanol and gets total alkaloids 0.97g with this alkalies;
Embodiment 3:
Get Tripterygium hypoglaucum peeling root 100g; Pulverize the back and cross 10 mesh sieves, placed ultrasonic instrument simultaneously 3 hours, leach medicinal liquid with 10 times of amount 65% soak with ethanol; Repeat to add twice of 10 times of amount 65% ethanol; Soak and to place ultrasonic instrument simultaneously respectively 2 hours and 1 hour, leach behind the medicinal liquid and the combining medicine first time, concentrated.Add 40%HCl and regulate pH between the 2-3, with the chloroform extraction of appropriate amount 3 times, the reservation water.Add Na 2CO 3, regulate pH to 9-11, through the C18 solid-phase extraction device, water flushing solid-phase extraction device 30 minutes is used 50% alcohol flushing, collects flushing liquor after rotary evaporation reclaims ethanol and gets total alkaloids 0.95g with this alkalies.

Claims (8)

1. the method for distilling of a Tripterygium hypoglaucum total alkaloids is characterized in that leaching process comprises following steps:
(1) Tripterygium hypoglaucum peeling root is pulverized the back and is crossed the 5-15 mesh sieve;
(2) adding Tripterygium hypoglaucum peeling root 10-40, doubly to measure 61-69% ethanol multiple extraction under ultrasound condition;
(3) merge extractive liquid,, the concentrated hydrochloric acid that concentrates back adding 30-45% is regulated pH value to acid, fully mixes after-filtration;
(4) filtrating is with chloroform extraction 3-5 time, and the gained raffinate phase is used Na 2CO 3Regulate pH value to 9-11, reuse chloroform extraction 3-5 time merges extraction phase;
(5) under 40-50 ℃ of condition, the rotary evaporation extraction phase reclaim behind the chloroform total alkaloids;
Or
(1) Tripterygium hypoglaucum peeling root is pulverized the back and is crossed the 5-15 mesh sieve;
(2) adding Tripterygium hypoglaucum peeling root 10-40, doubly to measure 61-69% ethanol multiple extraction under ultrasound condition;
(3) merge extractive liquid,, the concentrated hydrochloric acid that concentrates back adding 30-45% is regulated pH value to acid, fully mixes after-filtration;
(4) filtrating is with chloroform extraction 3-5 time, and the gained raffinate phase is used Na 2CO 3Regulate pH value to 9-11; With the SPE of this raffinate phase through containing C18 (Solid-Phase Extraction, SPE) device is earlier with distilled water flushing 30 minutes; Reuse 30-45% alcohol flushing is collected this flushing liquor and after rotary evaporation reclaims ethanol, is got total alkaloids.
2. method for distilling according to claim 1 is characterized in that amount of ethanol in the said step 2 is 15 times of Tripterygium hypoglaucum grain weight amount.
3. method for distilling according to claim 1 is characterized in that the concentration of alcohol in the said step 2 is 65%.
4. method for distilling according to claim 1 is characterized in that multiple extraction in the said step 2 is 3 times, is 3 hours for the first time, is 2 hours for the second time, is 1 hour for the third time.
5. method for distilling according to claim 1 is characterized in that the pH value in the said step 3 is 2-3.
6. method for distilling according to claim 1 is characterized in that the pH value in the said step 4 is 10.
7. according to the extract obtained application in antitumor and anti-autoimmune disease of the said method for distilling of claim 1, it is characterized in that adding in a certain amount of total alkaloids an amount of pharmaceutic adjuvant and process various common dosage forms, comprise tablet, capsule, injection.
8. according to the extract obtained application in antitumor and anti-autoimmune disease of the said method for distilling of claim 7, it is characterized in that the gained preparation is a tablet, writing out a prescription is total alkaloids, starch, 7%-15% starch slurry, magnesium stearate.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105213463A (en) * 2015-11-18 2016-01-06 重庆市中药研究院 A kind of colguhoumia root external preparation for eczema, preparation method and application thereof
CN105250349A (en) * 2015-11-18 2016-01-20 重庆市中药研究院 Colquhoumia root cataplasma for rheumatoid arthritis as well as preparation method and application of colquhoumia root cataplasma
CN107691485A (en) * 2017-08-21 2018-02-16 长沙新气象自动化技术有限公司 A kind of pond Chinese herbal medicinal bactericide and its manufacture craft
CN109394844A (en) * 2018-12-21 2019-03-01 昆明市中医医院 Compound tripterygium hypoglaucum hutcs chewing gum
CN109806291A (en) * 2019-03-22 2019-05-28 楚雄医药高等专科学校 The method for extraction and purification of effective component in a kind of Yi nationality's medicine tripterygium hypoglaucum hutcs
CN112110818A (en) * 2020-09-29 2020-12-22 天津师范大学 Malus spectabilis ketone 3-position modified derivative and application thereof in preparation of antitumor drugs

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105213463A (en) * 2015-11-18 2016-01-06 重庆市中药研究院 A kind of colguhoumia root external preparation for eczema, preparation method and application thereof
CN105250349A (en) * 2015-11-18 2016-01-20 重庆市中药研究院 Colquhoumia root cataplasma for rheumatoid arthritis as well as preparation method and application of colquhoumia root cataplasma
CN107691485A (en) * 2017-08-21 2018-02-16 长沙新气象自动化技术有限公司 A kind of pond Chinese herbal medicinal bactericide and its manufacture craft
CN109394844A (en) * 2018-12-21 2019-03-01 昆明市中医医院 Compound tripterygium hypoglaucum hutcs chewing gum
CN109806291A (en) * 2019-03-22 2019-05-28 楚雄医药高等专科学校 The method for extraction and purification of effective component in a kind of Yi nationality's medicine tripterygium hypoglaucum hutcs
CN112110818A (en) * 2020-09-29 2020-12-22 天津师范大学 Malus spectabilis ketone 3-position modified derivative and application thereof in preparation of antitumor drugs
CN112110818B (en) * 2020-09-29 2023-06-06 天津师范大学 Malus spectabilis ketone 3-position modified derivative and application thereof in preparation of antitumor drugs

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