CN107879920A - A kind of bittersweet sesquialter terpenoid extract and its preparation method and application - Google Patents
A kind of bittersweet sesquialter terpenoid extract and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of bittersweet sesquialter terpenoid extract and its preparation method and application, including step：1) extract and effectively eliminate chlorophyll, take the bittersweet original plant of 18~22 parts by weight, add 95% ethanol, ethanol liquid level is higher by medicinal material 2.5~3.5cm of plane, heating and refluxing extraction 3 times, and extract solution merges, in 0.08~0.12MPa, it is concentrated under reduced pressure at 48~52 DEG C to remove the impurity such as fat-soluble chlorophyll, then extracted repeatedly using chloroform, at 33~37 DEG C, chloroform extract merges under 0.05~0.07MPa, be concentrated under reduced pressure to obtain concentrate；2) flavone compound is effectively eliminated；3) steroid alkaloid is effectively dispelled；4) effective acquisition of sequiterpene chemical fraction, anti-tumor biological is obvious, has crucial antitumor drug effect.
Description
Technical field
The present invention relates to biomedicine technical field, more particularly to a kind of bittersweet sesquialter terpenoid extract and preparation method thereof
And application.
Background technology
Bittersweet, also known as solanum lyratum, it is the drying herb of Solanaceae activity of Solanum Lyratum Thunb (Solanum lyratum Thunb),
The effect of with clearing heat and detoxicating, dispelling wind and eliminating dampness, for clinical conventional antineoplastic.The relevant antitumor effective substance of bittersweet is ground
Study carefully, domestic and international scientific and technical personnel have been focused into steroid alkaloid, steroid saponin and flavone compound, although these compounds
Certain anti-tumor biological is shown, but clinical effectiveness is unsatisfactory.
The content of the invention
It is obvious that the technical problems to be solved by the invention are to provide a kind of anti-tumor biological, has crucial antitumor
The bittersweet sesquialter terpenoid extract of drug effect.
In order to solve the above technical problems, the technical scheme is that：
A kind of bittersweet sesquialter terpenoid extract, the extract include adhering to 32 sequiterpenes of 6 structure types separately, wherein
18 sequiterpenes are noval chemical compound；
6 sequiterpene structure types are respectively：
As preferable technical scheme, described 18 new sesquiterpenoids are respectively：
1) A of type of compounds, Type B：
2) C, D, E, F type of type of compounds：
As preferable technical scheme, the chemical constitution of known sequiterpene is in 32 sequiterpenes：
The present invention also provides a kind of method for preparing bittersweet sesquialter terpenoid extract, including step：
1) extract and effectively eliminate chlorophyll
The bittersweet medicinal material original plant of 18~22 parts by weight is taken, adds 95% ethanol, ethanol liquid level is higher by medicinal material plane 2.5
~3.5cm, heating and refluxing extraction 3 times, extract solution merge, are concentrated under reduced pressure at 0.08~0.12MPa, 48~52 DEG C to remove
The impurity such as fat-soluble chlorophyll, then extracted repeatedly using chloroform, at 33~37 DEG C, chloroform extract under 0.05~0.07MPa
Merge, be concentrated under reduced pressure to obtain concentrate；
2) flavone compound is effectively eliminated
Chloroform extract is adsorbed by polyamide column chromatography, is eluted first with the deionized water of 13~17 parts by volume
With except impurity such as depigmentatons, then with 45% ethanol eluate of 18~22 parts by volume, and flavone compound continues absorption and existed
On polyamide chromatography post, thus effectively remove flavone compound；
3) steroid alkaloid is effectively dispelled
45% ethanol eluate of polyamide column chromatography, it is first after being concentrated under reduced pressure at 0.09~0.11MPa, 53~57 DEG C
First dissolved with the ethyl acetate of 1.3~1.7 parts by volume, then go out steroid alkaloid with 3% watery hydrochloric acid continuous extraction, it is described dilute
The 1st~2 dosage of hydrochloric acid is 1.5 parts by volume, and the 3rd~4 time dosage is 1.0 parts by volume, and sesquiterpenoids then stays in second
Acetoacetic ester position；
4) effective acquisition of sequiterpene chemical fraction
Ethyl acetate extract after extraction, is eluted first with ammoniacal liquor, and the ammoniacal liquor PH is that the 10, the 1st dosage is
1.5 parts by volume, the 2nd~3 dosage are 1.0 parts by volume, then by ethyl acetate extract in 0.07~0.09MPa, 43~47 DEG C
Under be concentrated under reduced pressure, that is, obtain sequiterpene chemical fraction.
As preferable technical scheme, 3 specific Extracting temperatures of heating and refluxing extraction are 80 DEG C in the step 1), the 1st
Secondary extraction 1 hour, the 2nd~3 time is respectively 40 minutes.
As preferable technical scheme, it is 60 DEG C that step 1) the petrochina ether extracts specifically petroleum ether boiling range repeatedly
~90 DEG C, the 1st~2 dosage is 2.0 times of the volume of concentrate, and the 3rd~4 dosage is 1.5 times of the volume of concentrate, the 5th~
6 dosages are 1.0 times of the volume of concentrate.
As preferable technical scheme, the chloroform of the step 1) extracts repeatedly specifically extracts 6 times, wherein chloroform the 1st
~2 dosages are 2.0 times of extract volume, and the 3rd~4 dosage is 1.5 times of extract volume, the 5th~6 use
Measure as 1.0 times of extract volume.
As preferable technical scheme, ethanol eluate is also containing sequiterpene, steroid alkaloid in the step 2).
Bittersweet sesquialter terpenoid extract is used to make antineoplastic by the present invention.
The anti-tumor biological research of bittersweet sequiterpene
(1), antitumor activity screening：
1st, experimental method：With RPMI-1640 medium culture lymph cancer P388 cells, people's nasopharynx containing 5% hyclone
Cancer HONE-1 cells, oral epithelium cancer KB cells and colon cancer HT29 cells, tumour cell are transferred to 24 after being in exponential phase
Well culture plate, cell concentration are 5000/ml/ holes.The decoction to be measured of various concentrations is added in culture hole, is cultivated 72 hours；Should
With the micro enzyme reaction colorimetric method of tetramethyl azo azoles salt, using Podophyllum emodi var chinense ethylidene and cis-platinum as positive control, evaluation sequiterpene monomer
Suppress tumor cell growth activity, diagrammatically calculate IC50Value.
2nd, experiment conclusion：Using mtt assay, 18 English-Chinese new sequiterpene monomers of dialogue have carried out antitumor activity first
Screening, as a result shows：18 sequiterpenes are to thin to lymph cancer P388 cells, human nasopharyngeal carcinoma HONE-1 cells, oral epithelium cancer KB
Born of the same parents and colon cancer HT29 cells have good growth inhibition effect, its half-inhibition concentration (IC50) it is 2.6-8.3 μ Μ, carefully
Born of the same parents' cytotoxic activity is much better than the steroid alkaloid, steroid saponin and flavone compound of document report, thereby determines that sesquiterpenoids
Compound is the critical active material that bittersweet shows good antitumor drug effect.
(2) antitumor mechanism research：
1st, experimental method：
(1) to the influence in human lung carcinoma cell cycle：Inoculating cell normal culture 24 hours, adds different in six orifice plates
The compound of concentration, continue culture 24 hours, pancreas enzyme -EDTA vitellophag, take 1 × 106Cell PBS is washed twice, and 70% ethanol-
20 DEG C are fixed more than 2 hours, and centrifugation PBS is washed twice, are added the μ l of 200 μ g/ml RNaseA 50 and are digested at room temperature 0.5 hour, add
Enter the 50 μ g/ml μ l of PI 200,4 DEG C are incubated 0.5 hour, flow cytometer detection, analyze the ratio of different cell cycles, and with compareing
Group is compared.
(2) to the influence of human lung carcinoma cell apoptosis：The cell pancreas enzyme -EDTA digestion of compound processing, takes 1 × 106Cell
PBS is washed twice, is then dyed using Annexin-V FITC apoptosis kits, specific steps are according to kit specification
Operation, after dyeing terminates, flow cytomery Annexin-V FITC and PI positive ratio, calculate the hundred of Apoptosis
Divide ratio, and compared with control group.
2nd, experiment conclusion：On the basis of preliminary antitumor structure-effect relationship analysis, bittersweet alcohol D (Lyratol D) is selected
Structural modification is carried out for target compound, and carries out antitumor mechanism research.Experiment display, after bittersweet alcohol D is acted on 24 hours,
Drugs block can be significantly reduced in G1 phases, S phase cell proportions, meanwhile, can obvious inducing apoptosis of tumour cell.
By adopting the above-described technical solution, a kind of bittersweet sesquialter terpenoid extract and its preparation method and application, including step
Suddenly：1) extract and effectively eliminate chlorophyll, take the bittersweet original plant of 18~22 parts by weight, add 95% ethanol, ethanol liquid level is high
Go out medicinal material 2.5~3.5cm of plane, heating and refluxing extraction 3 times, extract solution merges, subtracted at 0.08~0.12MPa, 48~52 DEG C
Then pressure concentration is extracted with to remove the impurity such as fat-soluble chlorophyll repeatedly using chloroform, at 33~37 DEG C, 0.05~0.07MPa
Lower chloroform extract merges, be concentrated under reduced pressure to obtain concentrate；2) flavone compound is effectively eliminated, chloroform extract passes through polyamides
Amine column chromatography is adsorbed, and is eluted first with the deionized waters of 13~17 parts by volume to remove the impurity such as depigmentaton, then with 18~
45% ethanol eluate of 22 parts by volume, and flavone compound continues absorption on polyamide chromatography post, thus effectively remove
Remove flavone compound；3) steroid alkaloid is effectively dispelled, 45% ethanol eluate of polyamide column chromatography, 0.09~
0.11MPa, after being concentrated under reduced pressure at 53~57 DEG C, dissolved first with the ethyl acetate of 1.3~1.7 parts by volume, it is then dilute with 3%
Hydrochloric acid continuous extraction goes out steroid alkaloid, and described the 1st~2 dosage of watery hydrochloric acid is 1.5 parts by volume, and the 3rd~4 dosage is 1.0
Parts by volume, and sesquiterpenoids then stays in ethyl acetate extract；4) effective acquisition of sequiterpene chemical fraction, extracts it
Ethyl acetate extract afterwards, is eluted first with ammoniacal liquor, and the ammoniacal liquor PH is that the 10, the 1st dosage is 1.5 parts by volume, and the 2nd~3
Secondary dosage is 1.0 parts by volume, and then ethyl acetate extract is concentrated under reduced pressure at 0.07~0.09MPa, 43~47 DEG C, produced
To sequiterpene chemical fraction, antitumor or anti-inflammatory bioactivity is obvious, has crucial antitumor drug effect.
The present invention is directed to the obvious progress of prior art：
1st, in bittersweet sequiterpene chemical research：Based on the extraction and separation process flow of novelty, steroidal life is effectively being eliminated
While the type compounds such as alkaloids, steroid saponin and flavones disturb, it is successfully separated to obtain sequiterpene chemical fraction first；It is logical
Modern chromatography technology is crossed, finds to adhere to 32 sequiterpenes of 6 structure types separately altogether from bittersweet, wherein 18 sequiterpenes
For noval chemical compound.Chemical structural type B, D contain 2, the 6- dimethyl benzyl structure fragments of novelty, for China nightshade institute
It is peculiar, 5 sequiterpenes with such a structure fragment are successively found from bittersweet.
2nd, the anti-tumor biological of sequiterpene is studied in bittersweet：1. antitumor activity screening：It is right first using mtt assay
18 new sequiterpene monomers in bittersweet have carried out antitumor activity screening, as a result show：18 sequiterpenes are to lymph cancer
P388 cells, human nasopharyngeal carcinoma HONE-1 cells, oral epithelium cancer KB cells and colon cancer HT29 cells have good growth suppression
Make and use, its half-inhibition concentration (IC50) it is 2.6-8.3 μ Μ.2. antitumor mechanism research：Based on preliminary structure-effect relation
Analysis, the bittersweet alcohol D that content is high and structure is novel is selected, carries out structural modification, while carry out anti-human lung cancer Mechanism Study.It is real
Display is tested, after bittersweet alcohol D is acted on 24 hours, drugs block can be significantly reduced, can substantially induced in G1 phases, S phase cell proportions
Apoptosis.
Embodiment
With reference to embodiment, the present invention is expanded on further.In the following detailed description, only by way of explanation
Describe some one exemplary embodiments of the present invention.Undoubtedly, one of ordinary skill in the art will recognize, not
In the case of deviateing the spirit and scope of the present invention, the described embodiments may be modified in various different ways.
Therefore, description is inherently illustrative, is not intended to limit the scope of the claims.
Embodiment 1
A kind of method for preparing bittersweet sesquialter terpenoid extract, including step：
1) extract and effectively eliminate chlorophyll
18kg bittersweet medicinal material original plant is taken, adds 95% ethanol, ethanol liquid level is higher by medicinal material plane 2.5cm, heated back
Stream extraction 3 times, extract solution merges, is concentrated under reduced pressure at 0.08MPa, 48 DEG C to remove the impurity such as fat-soluble chlorophyll, Ran Houli
Extracted repeatedly with chloroform, at 33 DEG C, chloroform extract merges under 0.05MPa, be concentrated under reduced pressure to obtain concentrate；
2) flavone compound is effectively eliminated
Chloroform extract is adsorbed by polyamide column chromatography, is eluted first with 13L deionized water to remove depigmentaton
Deng impurity, then with 18L 45% ethanol eluate, and flavone compound continues absorption on polyamide chromatography post, thus
Effectively remove flavone compound；
3) steroid alkaloid is effectively dispelled
45% ethanol eluate of polyamide column chromatography, after being concentrated under reduced pressure at 0.09MPa, 53 DEG C, first with 1.3L's
Ethyl acetate dissolves, and then goes out steroid alkaloid with 3% watery hydrochloric acid continuous extraction, described the 1st~2 dosage of watery hydrochloric acid is
1.5L, the 3rd~4 time dosage is 1.0L, and sesquiterpenoids then stays in ethyl acetate extract；
4) effective acquisition of sequiterpene chemical fraction
Ethyl acetate extract after extraction, is eluted first with ammoniacal liquor, and the ammoniacal liquor PH is that the 10, the 1st dosage is
1.5L, the 2nd~3 dosage are 1.0L, and then ethyl acetate extract is concentrated under reduced pressure at 0.07MPa, 43 DEG C, that is, are obtained again
Hemiterpene chemical fraction 100g.
3 specific Extracting temperatures of heating and refluxing extraction are 80 DEG C in the step 1), the 1st extraction 1 hour, the 2nd~3
Secondary is respectively 40 minutes.
It is 60 DEG C~90 DEG C that step 1) the petrochina ether extracts specifically petroleum ether boiling range repeatedly, the 1st~2 dosage
For 2.0 times of the volume of concentrate, the 3rd~4 dosage is 1.5 times of the volume of concentrate, and the 5th~6 dosage is the volume of concentrate
1.0 times.
The chloroform of the step 1) extracts repeatedly specifically to be extracted 6 times, and wherein the 1st~2 dosage of chloroform is extract
2.0 times of volume, the 3rd~4 dosage are 1.5 times of extract volume, and the 5th~6 dosage is extract volume
1.0 again.
Ethanol eluate is also containing sequiterpene, steroid alkaloid in the step 2).
Bittersweet sesquialter terpenoid extract is used to make antineoplastic.
Embodiment 2
A kind of method for preparing bittersweet sesquialter terpenoid extract, including step：
1) extract and effectively eliminate chlorophyll
20kg bittersweet medicinal material original plant is taken, adds 95% ethanol, ethanol liquid level is higher by medicinal material plane 3cm, is heated to reflux
Extraction 3 times, extract solution merge, are concentrated under reduced pressure at 0.1MPa, 50 DEG C to remove the impurity such as fat-soluble chlorophyll, then utilized
Chloroform extracts repeatedly, and at 35 DEG C, chloroform extract merges under 0.06MPa, be concentrated under reduced pressure to obtain concentrate；
2) flavone compound is effectively eliminated
Chloroform extract is adsorbed by polyamide column chromatography, is eluted first with 15L deionized water to remove depigmentaton
Deng impurity, then with 20L 45% ethanol eluate, and flavone compound continues absorption on polyamide chromatography post, thus
Effectively remove flavone compound；
3) steroid alkaloid is effectively dispelled
45% ethanol eluate of polyamide column chromatography, after being concentrated under reduced pressure at 0.1MPa, 55 DEG C, first with 15 volumes
The ethyl acetate dissolving of part, then goes out steroid alkaloid, described the 1st~2 dosage of watery hydrochloric acid with 3% watery hydrochloric acid continuous extraction
For 1.5L, the 3rd~4 time dosage is 1.0L, and sesquiterpenoids then stays in ethyl acetate extract；
4) effective acquisition of sequiterpene chemical fraction
Ethyl acetate extract after extraction, is eluted first with ammoniacal liquor, and the ammoniacal liquor PH is that the 10, the 1st dosage is
1.5L, the 2nd~3 dosage are 1.0L, and then ethyl acetate extract is concentrated under reduced pressure at 0.08MPa, 45 DEG C, that is, are obtained again
Hemiterpene chemical fraction 102g.
3 specific Extracting temperatures of heating and refluxing extraction are 80 DEG C in the step 1), the 1st extraction 1 hour, the 2nd~3
Secondary is respectively 40 minutes.
It is 60 DEG C~90 DEG C that step 1) the petrochina ether extracts specifically petroleum ether boiling range repeatedly, the 1st~2 dosage
For 2.0 times of the volume of concentrate, the 3rd~4 dosage is 1.5 times of the volume of concentrate, and the 5th~6 dosage is the volume of concentrate
1.0 times.
The chloroform of the step 1) extracts repeatedly specifically to be extracted 6 times, and wherein the 1st~2 dosage of chloroform is extract
2.0 times of volume, the 3rd~4 dosage are 1.5 times of extract volume, and the 5th~6 dosage is extract volume
1.0 again.
Ethanol eluate is also containing sequiterpene, steroid alkaloid in the step 2).
Bittersweet sesquialter terpenoid extract is used to make antineoplastic.
Embodiment 3
A kind of method for preparing bittersweet sesquialter terpenoid extract, including step：
1) extract and effectively eliminate chlorophyll
22kg bittersweet medicinal material original plant is taken, adds 95% ethanol, ethanol liquid level is higher by medicinal material plane 3.5cm, heated back
Stream extraction 3 times, extract solution merges, is concentrated under reduced pressure to remove the impurity such as fat-soluble chlorophyll, then at 0.12MPa, 52 DEG C DEG C
Extracted repeatedly using chloroform, at 37 DEG C, chloroform extract merges under 0.07MPa, be concentrated under reduced pressure to obtain concentrate；
2) flavone compound is effectively eliminated
Chloroform extract is adsorbed by polyamide column chromatography, is eluted first with 17L deionized water to remove depigmentaton
Deng impurity, then with 22L 45% ethanol eluate, and flavone compound continues absorption on polyamide chromatography post, thus
Effectively remove flavone compound；
3) steroid alkaloid is effectively dispelled
45% ethanol eluate of polyamide column chromatography, after being concentrated under reduced pressure at 0.11MPa, 57 DEG C, first with 1.7L's
Ethyl acetate dissolves, and then goes out steroid alkaloid with 3% watery hydrochloric acid continuous extraction, and described the 1st~2 dosage of watery hydrochloric acid is 1.5
Parts by volume, the 3rd~4 time dosage is 1.0 parts by volume, and sesquiterpenoids then stays in ethyl acetate extract；
4) effective acquisition of sequiterpene chemical fraction
Ethyl acetate extract after extraction, is eluted first with ammoniacal liquor, and the ammoniacal liquor PH is that the 10, the 1st dosage is
1.5L, the 2nd~3 dosage are 1.0L, and then ethyl acetate extract is concentrated under reduced pressure at 0.09MPa, 47 DEG C, that is, are obtained again
Hemiterpene chemical fraction 104g.
3 specific Extracting temperatures of heating and refluxing extraction are 80 DEG C in the step 1), the 1st extraction 1 hour, the 2nd~3
Secondary is respectively 40 minutes.
It is 60 DEG C~90 DEG C that step 1) the petrochina ether extracts specifically petroleum ether boiling range repeatedly, the 1st~2 dosage
For 2.0 times of the volume of concentrate, the 3rd~4 dosage is 1.5 times of the volume of concentrate, and the 5th~6 dosage is the volume of concentrate
1.0 times.
The chloroform of the step 1) extracts repeatedly specifically to be extracted 6 times, and wherein the 1st~2 dosage of chloroform is extract
2.0 times of volume, the 3rd~4 dosage are 1.5 times of extract volume, and the 5th~6 dosage is extract volume
1.0 again.
Ethanol eluate is also containing sequiterpene, steroid alkaloid in the step 2).
Bittersweet sesquialter terpenoid extract is used to make antineoplastic.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these
Changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims
And its equivalent thereof.
Claims (9)
- A kind of 1. bittersweet sesquialter terpenoid extract, it is characterised in that：The extract includes adhering to 32 sesquialters of 6 structure types separately Terpene, wherein 18 sequiterpenes are noval chemical compound；6 structure types are respectively：
- 2. bittersweet sesquialter terpenoid extract as claimed in claim 1, it is characterised in that described 18 new sesquiterpenoid difference For：1) A of type of compounds, Type B：2) C, D, E, F type of type of compounds：
- 3. bittersweet sesquialter terpenoid extract as claimed in claim 1, it is characterised in that known sequiterpene in 32 sequiterpenes Chemical constitution be：
- A kind of 4. method for preparing the bittersweet sesquialter terpenoid extract as described in claim 1,2 or 3, it is characterised in that including step Suddenly：1) extract and effectively eliminate chlorophyllTake the bittersweet medicinal material original plant of 18~22 parts by weight, add 95% ethanol, ethanol liquid level be higher by medicinal material plane 2.5~ 3.5cm, heating and refluxing extraction 3 times, extract solution merge, are concentrated under reduced pressure at 0.08~0.12MPa, 48~52 DEG C to remove liposoluble Property the impurity such as chlorophyll, then extracted repeatedly using chloroform, at 33~37 DEG C, under 0.05~0.07MPa chloroform extract merge, Be concentrated under reduced pressure to obtain concentrate；2) flavone compound is effectively eliminatedChloroform extract is adsorbed by polyamide column chromatography, is eluted first with the deionized water of 13~17 parts by volume to remove The impurity such as pigment, then with 45% ethanol eluate of 18~22 parts by volume, and flavone compound continues absorption in polyamide On chromatographic column, thus effectively remove flavone compound；3) steroid alkaloid is effectively dispelled45% ethanol eluate of polyamide column chromatography, after being concentrated under reduced pressure at 0.09~0.11MPa, 53~57 DEG C, is used first The ethyl acetate dissolving of 1.3~1.7 parts by volume, then goes out steroid alkaloid with 3% watery hydrochloric acid continuous extraction, the watery hydrochloric acid the 1~2 dosage is 1.5 parts by volume, and the 3rd~4 time dosage is 1.0 parts by volume, and sesquiterpenoids then stays in ethyl acetate Position；4) effective acquisition of sequiterpene chemical fractionEthyl acetate extract after extraction, is eluted first with ammoniacal liquor, and ammoniacal liquor PH used is that the 10, the 1st dosage is 1.5 volumes Part, the 2nd~3 dosage is 1.0 parts by volume, is then depressurized ethyl acetate extract at 0.07~0.09MPa, 43~47 DEG C dense Contracting, that is, obtain sequiterpene chemical fraction.
- 5. the method as claimed in claim 4 for preparing bittersweet sesquialter terpenoid extract, it is characterised in that：Heated in the step 1) 3 specific Extracting temperatures of refluxing extraction are 80 DEG C, the 1st extraction 1 hour, and the 2nd~3 time is respectively 40 minutes.
- 6. the method as claimed in claim 4 for preparing bittersweet sesquialter terpenoid extract, it is characterised in that：Step 1) the petrochina It is 60 DEG C~90 DEG C that ether extracts specifically petroleum ether boiling range repeatedly, and the 1st~2 dosage is 2.0 times of the volume of concentrate, the 3rd~4 Secondary dosage is 1.5 times of the volume of concentrate, and the 5th~6 dosage is 1.0 times of the volume of concentrate.
- 7. the method as claimed in claim 4 for preparing bittersweet sesquialter terpenoid extract, it is characterised in that：The chloroform of the step 1) Extraction is specifically extraction 6 times repeatedly, and wherein the 1st~2 dosage of chloroform is 2.0 times of extract volume, the 3rd~4 dosage For 1.5 times of extract volume, the 5th~6 dosage is 1.0 times of extract volume.
- 8. the method as claimed in claim 4 for preparing bittersweet sesquialter terpenoid extract, it is characterised in that：Ethanol in the step 2) Eluent is also containing sequiterpene, steroid alkaloid.
- 9. the bittersweet sesquialter terpenoid extract described in claim 1 to 8 any claim is used to make antineoplastic.
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