CN103860598A - Hippocampus extract, efficacy verification method of same, preparation method, application and pharmaceutical composition thereof - Google Patents
Hippocampus extract, efficacy verification method of same, preparation method, application and pharmaceutical composition thereof Download PDFInfo
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Abstract
The invention provides a method for verifying the efficacy of a hippocampus extract, which comprises the following steps: a step of subjecting a hippocampus to breed identification to confirm the breed of the hippocampus, which comprises: extracting a DNA sample of the hippocampus; and performing origin identification (original identification) on a sequence fragment of a gene in the DNA sample to confirm the variety of the hippocampus; and (b) confirming the breed of the hippocampus, and then performing efficacy confirmation and/or quality confirmation on a hippocampus extract extracted from the hippocampus, wherein the step comprises: extracting the hippocampus with a solvent to obtain the hippocampus extract; and subjecting the hippocampal extract to a cancer cell survival test and/or confirming whether the hippocampal extract contains at least one indicator compound (indicator compound).
Description
Technical field
The present invention relates to a kind of effect verification method of Hippocampus extract, and be particularly related to a kind of effect verification method of Hippocampus extract, it is confirmed and/or quality determination in conjunction with Hippocampus cultivar identification and Hippocampus extract effect.
Background technology
Hippocampus is a kind of well-known marine pharmaceutical organism, it in Chinese medicine, is an important medical material, and the Hippocampus existing 600 years history of being used as medicine, it has the effect that comprises building body, kidney invigorating and YANG supporting, relaxing muscles and tendons and activating QI and blood in the collateral, anti-inflammatory analgetic, tranquillizing and allaying excitement, relieving cough and asthma etc., and it is also effective in cure to dermatosis, hypercholesterolemia, abundant expectoration, thyromegaly, heart disease and disease of lymph node etc., also there is defying age, anticancer effect, also therefore Hippocampus at home and abroad the market demand is very large, be the rare Chinese medicine that economic worth is higher.
The medicinal Hippocampus major part circulating in Chinese Medicinal Materials Markets at present is both at home and abroad wild fishing for, and drying processes that laggard pedlar sells, and kind is difficult for confirming.Again, Hippocampus complexity of a great variety, nearly more than 30 more than kind, and Hippocampus can change body colour appearance because of surrounding environment change, therefore commercially available Hippocampus Chinese crude drug, the difficult identification of kind, is difficult to confirm that the Hippocampus that uses has effect.According to Chinese Pharmacopoeia, only know that at present five kinds of Hippocampus have medicinal efficacy, therefore easily on market, buy the Chinese crude drug without the Hippocampus kind of medicinal efficacy.In addition, general Hippocampus Chinese crude drug is arranged in pairs or groups, and multiple medical material becomes compound recipe and medicated wine uses, it utilizes simple wine extraction and water extraction, Hippocampus extract is extracted, in the case, under multiple medical material effect, whether the Hippocampus Chinese crude drug that difficult confirmation is used has effect, or directly carries out clinical verification in particular hospital.Therefore, be badly in need of setting up at present the flow and method of Hippocampus extract effect checking.
Summary of the invention
Effect verification method that the invention provides a kind of Hippocampus extract, comprising: (a) Hippocampus is carried out to cultivar identification to confirm the step of kind of this Hippocampus, it comprises: a DNA sample that extracts this Hippocampus; And a sequence fragment of a gene of this DNA sample is carried out to the former qualification of base to confirm the kind of this Hippocampus; And (b) confirm after the kind of this Hippocampus, an extraction is carried out to the step of effect confirmation and/or quality determination from the Hippocampus extract of this Hippocampus, it comprises: extract this Hippocampus to obtain this Hippocampus extract with a solvent; And this Hippocampus extract is carried out to the test of cancerous cell cell survival rate and/or confirm whether this Hippocampus extract comprises at least one indication compound (indicator compound).
The present invention also provides a kind of method of preparing Hippocampus extract, comprising: (a) Hippocampus is carried out to cultivar identification to confirm the step of kind of this Hippocampus, it comprises: a DNA sample that extracts this Hippocampus; And a sequence fragment of a gene of this DNA sample is carried out to the former qualification of base to confirm the kind of this Hippocampus; And (b) confirm after the kind of this Hippocampus, extract this Hippocampus to obtain the step of this Hippocampus extract with a solvent.
The present invention provides again a kind of Hippocampus extract, and it has the effect that suppresses a growth of cancer cells, comprising: phylloxanthin and/or TYR are instruction composition.
The present invention also provides a kind of medical composition of anticancer growth, comprises that above-mentioned Hippocampus extract is effective ingredient.
The present invention more provides the purposes of a kind of above-mentioned Hippocampus extract for the preparation of the medicine of growth of cancer cells.
Brief description of the drawings
For above and other object of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and coordinate accompanying drawing, be described in detail below:
Figure 1A shows the step of an embodiment of effect verification method of Hippocampus extract of the present invention.
Figure 1B shows the thin portion of cultivar identification with the step of the kind of confirmation Hippocampus that a Hippocampus is carried out in Figure 1A.
Fig. 1 C shows the thin portion that an extraction is carried out to the step of effect confirmation and/or quality determination from the Hippocampus extract of above-mentioned Hippocampus in Figure 1A.
The impact of the survival rate of the hippocampus trimaculatus Leacs extract that Fig. 2 A shows variable concentrations on different carcinoma cell.
The impact of the survival rate of the sour jujube Hippocampus extract that Fig. 2 B shows variable concentrations on different carcinoma cell.
The storehouse that Fig. 2 C shows variable concentrations reaches the impact of the survival rate of Hippocampus extract on different carcinoma cell.
Fig. 3 A shows the result of the efficient liquid phase chromatographic analysis (condition A) of hippocampus trimaculatus Leacs extract.
Fig. 3 B shows the result of the efficient liquid phase chromatographic analysis (condition A) of sour jujube Hippocampus extract.
Fig. 3 C shows the result of the efficient liquid phase chromatographic analysis (condition B) that storehouse reaches Hippocampus extract.
Fig. 4 A shows the result of the efficient liquid phase chromatographic analysis (condition B) of hippocampus trimaculatus Leacs extract.
Fig. 4 B shows the result of the efficient liquid phase chromatographic analysis (condition A) of sour jujube Hippocampus extract.
Fig. 4 C shows the result of the efficient liquid phase chromatographic analysis (condition B) that storehouse reaches Hippocampus extract.
Fig. 5 A shows the result of the efficient liquid phase chromatographic analysis (condition B) of phylloxanthin.
Fig. 5 B shows the result of the efficient liquid phase chromatographic analysis (condition B) of TYR.
Detailed description of the invention
In an embodiment of the present invention, the present invention relates to a kind of effect verification method of Hippocampus extract.Effect verification method of Hippocampus extract of the present invention can comprise the steps, but be not limited to this.
Referring to Figure 1A, it illustrates the step of an embodiment of effect verification method of Hippocampus extract of the present invention.
First, carry out a Hippocampus is carried out to the step S1 of cultivar identification with the kind of confirmation Hippocampus.
In one embodiment, above-mentioned steps can comprise a DNA sample S1-1 who extracts this Hippocampus, and afterwards a sequence fragment of a gene of this DNA sample is carried out to the former qualification of base to confirm the kind S1-2 (referring to Figure 1B) of Hippocampus.In one embodiment, if when the sequence fragment of the sequence fragment of the said gene of the DNA sample of this Hippocampus and the said gene of the Hippocampus of a known kind is identical, confirm that this Hippocampus is above-mentioned known Hippocampus.
The example of the above-mentioned gene for the former qualification of base can comprise, (cytochrome is gene etc. b), but is not limited to this for such as cytochrome b.In one embodiment, a sequence fragment of a cytochrome b gene of the DNA sample of above-mentioned Hippocampus is carried out to the former qualification of base to confirm the kind of Hippocampus.In this embodiment, the above-mentioned sequence fragment of cytochrome b gene can be by being sequence identification number by the DNA sample of above-mentioned Hippocampus by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction and obtained.
Refer again to Figure 1A, then, confirming, after the kind of above-mentioned Hippocampus, to carry out the step of an extraction being carried out to effect confirmation and/or quality determination from the Hippocampus extract of above-mentioned Hippocampus.
In one embodiment, this step can comprise with a solvent and extract above-mentioned Hippocampus to obtain Hippocampus extract S2-1, and afterwards Hippocampus extract carried out to cancerous cell cell survival rate mensuration and/or confirms whether this Hippocampus extract comprises at least one indication compound S2-2 (referring to Fig. 1 C).In one embodiment, if cancerous cell cell survival rate is less than or equal to 90% and/or Hippocampus extract while comprising at least one indication compound, confirm that this Hippocampus extract possesses the effect that suppresses a growth of cancer cells.
The solvent that is suitable for extracting Hippocampus can include, but are not limited to ethanol, methanol, acetone, ethyl acetate etc.In one embodiment, be ethanol for extracting the solvent of Hippocampus.
And above-mentioned at least one indication compound can comprise phylloxanthin and/or TYR, but be not limited to this.
In an embodiment, can be comprised by the example of the cancerous cell that above-mentioned hippocampal cell suppressed again, such as hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell, breast cancer cell, ovarian cancer cell and colorectal cancer cells etc.
In a specific embodiment, when the above-mentioned sequence fragment of the above-mentioned sequence fragment of the said gene of above-mentioned Hippocampus DNA sample and the said gene of the Hippocampus of a known kind is identical, confirm that this Hippocampus is this known Hippocampus, and cancerous cell cell survival rate is less than or equal to 90% and/or above-mentioned Hippocampus extract while comprising this at least one indication compound, confirms that above-mentioned Hippocampus extract possesses effect.In addition, in this embodiment, one sequence fragment of one cytochrome b gene of above-mentioned DNA sample is carried out to the former qualification of base to confirm the kind of this Hippocampus, and the above-mentioned sequence fragment of cytochrome b gene is sequence identification number by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction to above-mentioned Hippocampus DNA sample and obtained, and above-mentioned at least one indication compound comprises phylloxanthin and TYR in addition.
In another embodiment of the present invention, the invention provides a kind of method of preparing Hippocampus extract.The method that the present invention prepares Hippocampus extract comprises the steps, but is not limited to this.
First, carry out a Hippocampus is carried out to the step of cultivar identification with the kind of confirmation Hippocampus.
In one embodiment, above-mentioned steps can comprise a DNA sample that extracts this Hippocampus, and afterwards a sequence fragment of a gene of this DNA sample is carried out to the former qualification of base to confirm to make the kind of Hippocampus.In one embodiment, if when sequence fragment Hippocampus or said gene of the sequence fragment DNA sample of this Hippocampus or said gene and a known kind is identical, confirm that this Hippocampus is above-mentioned known Hippocampus.
The above-mentioned gene for the former qualification of base can include, but are not limited to cytochrome b gene etc.In one embodiment, a sequence fragment of a cytochrome b gene of the DNA sample of above-mentioned Hippocampus is carried out to the former qualification of base to confirm the kind of Hippocampus.In this embodiment, the above-mentioned sequence fragment of cytochrome b gene can be by being sequence identification number by the DNA sample of above-mentioned Hippocampus by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction and obtained.
In one embodiment, above-mentioned Hippocampus may be confirmed to be, such as hippocampus trimaculatus Leacs (Hippocampus trimaculatus), sour jujube Hippocampus (Hippocampus spinosissimus) or storehouse reach Hippocampus (Hippocampus kuda) etc., but are not limited to this.
Then, confirming after the kind of above-mentioned Hippocampus, carrying out with a solvent and extract above-mentioned Hippocampus to obtain the step of Hippocampus extract.
The solvent that is suitable for extracting Hippocampus can include, but are not limited to ethanol, methanol, acetone, ethyl acetate etc.In one embodiment, extract Hippocampus with ethanol.
In a specific embodiment, one sequence fragment of one cytochrome b gene of above-mentioned Hippocampus DNA sample is carried out to the former qualification of base to confirm the kind of this Hippocampus, and the above-mentioned sequence fragment of cytochrome b gene can be sequence identification number by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction to above-mentioned Hippocampus DNA sample and obtained.Moreover in this embodiment, above-mentioned Hippocampus may be confirmed to be hippocampus trimaculatus Leacs, sour jujube Hippocampus or storehouse and reach Hippocampus, but is not limited to this, and extracts above-mentioned Hippocampus with ethanol.
In addition, in one embodiment, above-mentioned Hippocampus extract can comprise phylloxanthin and/or TYR is instruction composition.
In another embodiment, the invention still further relates to a Hippocampus extract, it is prepared the method preparation of Hippocampus extract by the invention described above and obtains.
Again, in another embodiment, the invention still further relates to a Hippocampus extract, it has the effect that suppresses a growth of cancer cells.The Hippocampus extract of the invention described above can comprise that phylloxanthin and/or TYR are instruction composition.Again, the example of the quenchable cancerous cell of Hippocampus extract of the present invention, can comprise hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell, breast cancer cell, ovarian cancer cell and colorectal cancer cells etc., but be not limited to this.
In one embodiment, Hippocampus extract of the present invention can be by a Hippocampus is extracted and obtained with a solvent.
Can be used to the example of the Hippocampus of preparing Hippocampus extract of the present invention, can comprise that such as hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse reach Hippocampus etc., but be not limited to this.
In addition the solvent that, is suitable for extracting Hippocampus extract of the present invention can include, but are not limited to ethanol, methanol, acetone, ethyl acetate etc.
In a specific embodiment, Hippocampus extract can by hippocampus trimaculatus Leacs, sour jujube Hippocampus or storehouse are reached Hippocampus with ethanol extract obtained.
In the present invention, again in another embodiment, the present invention is about a kind of medical composition of anticancer growth, and its Hippocampus extract with the effect that suppresses a growth of cancer cells that can comprise the invention described above is effective ingredient, but is not limited to this.
In one embodiment, the medical composition of above-mentioned anticancer growth also can more comprise pharmaceutically useful carrier or a salt.
In the medical composition of the invention described above anticancer growth, aforementioned pharmaceutically useful carrier can include, but are not limited to solvent, disperse medium (dispersion medium), coating (coating), antibacterial and antifungal agents and an isosmoticity and delayed absorption (absorption delaying) reagent etc. casts compatible person with pharmacy.For different administering modes, can utilize conventional method that pharmaceutical compositions is configured to dosage form (dosage form).
Again, above-mentioned pharmaceutically useful salt can include, but are not limited to salt and comprise inorganic cation, for example, alkaline metal salt, as sodium, potassium or ammonia salt, alkaline earth gold family salt, as magnesium, calcium salt, containing the salt of bivalence or quadrivalent cation, as zinc, aluminum or zirconates.In addition, be also organic salt, as hexanamine salt, methyl D-glycosamine, amidates, as arginine, from propylhomoserin, histidine, amide glutaminate.
The prepared drug administration of the present invention can be oral, parenteral, via sucking spraying (inhalation spray) or the mode by implanted reservoir (implanted reservoir).Parenterally comprise subcutaneous (subcutaneous), Intradermal (intracutaneous), intravenous (intravenous), intramuscular (intramuscular), intraarticular (intraarticular), intra-arterial (intraarterial), in synovial membrane (chamber) (intrasynovial), (intrasternal) subarachnoid space (intrathecal) in breastbone, (intralesional) injection and perfusion technique in disease location.
The form of oral composition can comprise, but be not limited to lozenge, capsule, Emulsion (emulsions), waterborne suspension (aqueous suspensions), dispersion liquid (dispersions) and solution.
Moreover the present invention also provides a kind of Hippocampus extract with the effect that suppresses a growth of cancer cells of the invention described above for the manufacture of the purposes of the medicine of anticancer growth.
Embodiment
Carry out Hippocampus cultivar identification
The former qualification of base
Commercially available hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse are reached to Hippocampus and extract its DNA with the Tissue & Cell Genomic DNA Purification Kit of BioKit label respectively.The DNA sample that the DNA sample of the DNA sample of obtained commercially available hippocampus trimaculatus Leacs, commercially available sour jujube Hippocampus and commercially available storehouse are reached to Hippocampus is that the introduction (forward) that introduction (forward) and the sequence of sequence identification number 1 is sequence identification number 2 carries out polymerase chain reaction to obtain the partial sequence of the cytochrome b gene in point other mitochondrion (mitochondrial) by sequence.
The reactant of above-mentioned polymerase chain reaction is as shown in table 1.
Table 1: the reactant of polymerase chain reaction
| Reactant composition | Volume (ul) | Ultimate density |
| 10X reacts |
5 | 1X |
| 10mM?dNTA | 1 | 0.25mM |
| 50mM? |
2 | |
| DNA | ||
| 4 | ? | |
| Forward (10pmol/ μ l) for introduction | 1 | 0.2pmol |
| (10pmol/ μ l) for reverse introduction | 1 | 0.2pmol |
| (5U/ μ l) for archaeal dna polymerase | 0.2-0.4 | 1-2U |
Visual response situation adds DMSO2-4%.
Above-mentioned reactant liquor is placed in to PCR reaction tube, and adding sterilized water to make final volume is 50 μ l.Fully, after mixed reactant, the reactor that the reaction tube of polymerase chain reaction is placed in to polymerase chain reaction reacts.PCR response procedures is as follows:
Step 1:94 DEG C, 10 minutes; Step 2:94 DEG C, 30 seconds; Step 3:52 DEG C, 30 seconds; Step 4:72 DEG C, 1 minute (35 circular response); Step 6:72 DEG C, 5 minutes; Step 7:25 DEG C.
After polymerase chain reaction finishes, the Polymerase Chain Reaction product that obtained commercially available hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse is reached to Hippocampus carries out respectively electrophoretic analysis.The condition of electrophoretic analysis is as follows:
Polymerase chain reaction product above-mentioned obtained 10 μ l is carried out to electrophoresis with 1.5% agaropectin (agarose gel), and voltage is made as 100V.Use DNA ladder as instruction.After electrophoresis completes, colloid is dyeed with ethidium bromide (ethidium bromide), and under UV light source, observe and take a picture.
Then, the polymerase chain reaction product that obtained commercially available hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse are reached to Hippocampus presents single fragment and the correct product of size entrusts sequence analysis company with dideoxy chain termination (dideoxy chain termination), carries out sequencing with ABI3700 or ABI3730DNA Sequencer.Institute's calling sequence is with DNA* (DNASTAR Inc.) computer software programs analysis and arrangement DNA sequence.Learnt by sequencing result, its sequence of polymerase chain reaction product that commercially available hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse reach Hippocampus is respectively sequence identification number: 3, sequence identification number: 4 with sequence identification number: 5.
Then by sequence identification number: 3, sequence identification number: 4 with sequence identification number: 5 compare with the login sequence of the GenBank of NCBI respectively, confirm that commercially available hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse reach Hippocampus and be really respectively hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse and reach Hippocampus.
The preparation of Hippocampus extract
Confirming that above-mentioned hippocampus trimaculatus Leacs, sour jujube Hippocampus and storehouse reach after the kind of Hippocampus, respectively above-mentioned three kinds of Hippocampus are being carried out to following extraction step to obtain point other Hippocampus extract.
(1) by weighing after Hippocampus chopping, add 10 times of volume ethanol (99%) to form a mixture;
(2) will under this mixture room temperature, cultivate 3 days with 50rpm concussion;
(3) take out upper strata liquid with filter paper filtering particle;
(4) residue adds and the isopyknic ethanol of step 1 (99%), and repeating step (2) is to (3);
(5) clear liquor of twice alcohol extraction is evaporated to respectively to surplus 5ml left and right;
(6) will concentrate extract and move on to centrifuge tube, overnight being kept at-70 DEG C, move to every other day freezer dryer and carry out lyophilization in 2 days.Weighing after dry, calculates alcohol extraction and for the second time response rate of alcohol extraction for the first time.
The impact of Hippocampus extract on cancerous cell survival
The hippocampus trimaculatus Leacs extract obtaining according to above-mentioned preparation method, sour jujube Hippocampus extract and storehouse are reached to Hippocampus extract processes different types of cancerous cell with variable concentrations respectively, and assessment is through the cell survival rate of different types of cancerous cell of the Hippocampus extract processing of variable concentrations, and its detailed experimental procedure is as follows.The cancerous cell not of the same race and its associated cancer that in this experiment, use are as shown in table 2.
Cell survival rate assessment through the cancerous cell of Hippocampus extract processing:
(1) with every hole 2.0 × 10
4cell seeding contain 100ul culture medium in every hole 96 porose discs in, and cultivate an evening at 37 DEG C;
(2) Hippocampus extract is dissolved in to DMSO, stock solution (stock solution) is 500mg/ml;
(3) remove culture medium, then add respectively the cultivation that 100ul contains various variable concentrations extracts to cultivate 2 days at 37 DEG C;
(4) remove the culture medium containing extract, then the cultivation that adds respectively 100ul to contain MTT (ultimate density is 10ug/m) is reacted 4 hours at 37 DEG C;
(5) remove the culture medium containing MTT, add 100ml DMSO to dissolve after MTT and measure OD with ELISAreader
550.
Survival rate (%)=cell is through the post-stimulatory OD of Hippocampus extract
550the OD that/cell stimulates without Hippocampus extract
550× 100%
Table 2: the cancerous cell not of the same race and its associated cancer that use in this experiment
And the impact of the survival rate of the hippocampus trimaculatus Leacs extract of variable concentrations on different carcinoma cell is if table 3 is with as shown in Fig. 2 A.
Table 3: the impact of the survival rate of hippocampus trimaculatus Leacs extract on different carcinoma cell
＊：p<0.05
Again, the impact of the survival rate of the sour jujube Hippocampus extract of variable concentrations on different carcinoma cell is if table 4 is with as shown in Fig. 2 B.
The impact of table 4, the survival rate of sour jujube Hippocampus extract on different carcinoma cell
＊：p<0.05
In addition the impact that, the storehouse of variable concentrations reaches the survival rate of Hippocampus extract on different carcinoma cell is if table 5 is with as shown in Fig. 2 C.
Table 5, storehouse reach the impact of the survival rate of Hippocampus extract on different carcinoma cell
＊：p<0.05
Can be learnt by table 3 and Fig. 2 A, table 4 and Fig. 2 B and table 5 and Fig. 2 C, it is below 90% to cancerous cell survival rate that hippocampus trimaculatus Leacs extract and storehouse reach Hippocampus extract in the time that concentration is 500 μ g/ml.Therefore whether Hippocampus extract can be made the survival rate of cancerous cell be equal to or less than 90% and be considered as an efficacy assessment benchmark.
The component analysis of Hippocampus extract
The hippocampus trimaculatus Leacs extract obtaining according to above-mentioned preparation method, sour jujube Hippocampus extract and storehouse are reached to Hippocampus extract and carry out efficient liquid phase chromatographic analysis.
A. efficient liquid phase chromatographic analysis (condition A)
Hippocampus trimaculatus Leacs extract, sour jujube Hippocampus extract and storehouse reach Hippocampus extract and carry out efficient liquid phase chromatographic analysis with following experimental technique:
(1), by Hippocampus samples weighing, add 0.5ml methanol again with ultrasonic vibrating 5 minutes.
(2) by the methanol solution of Hippocampus sample, with filter (filter) filtration, (0.45 μ m).
(3) draw in 20 μ l injection high performance liquid chromatographs (HPLC) and perform an analysis with 25 μ l syringe cylinders.
The condition A of efficient liquid phase chromatographic analysis is as follows:
Analytical tool: HITACHI Pump L-2130
HITACHI?Diode?L-7455
Eluting post (Elution column): Inertsil ODS3V4.6x250mm
Mobile phase: MeOH:H
2o (0.1%H
3pO
4) gradient (gradient).The condition of mobile phase is as shown in table 6.
Flow velocity: 1.0ml/ minute
Detect wavelength: 210nm
Analysis time: 65 minutes
The condition of table 6, mobile phase
| |
0 | 30 | 40 | 60 | 65 |
| CH 3OH | 5 | 70 | 100 | 100 | 5 |
| H 3PO 4(0.1%H 3PO 4) | 95 | 30 | 0 | 0 | 95 |
Hippocampus trimaculatus Leacs extract, sour jujube Hippocampus extract and storehouse reach Hippocampus extract analysis result respectively as shown in Fig. 3 A, Fig. 3 B and Fig. 3 C.
B. efficient liquid phase chromatographic analysis (condition B)
Hippocampus trimaculatus Leacs extract, sour jujube Hippocampus extract and storehouse reach Hippocampus extract and carry out efficient liquid phase chromatographic analysis with following experimental technique:
(1), by Hippocampus samples weighing, add 0.5ml methanol again with ultrasonic vibrating 5 minutes.
(2) by the methanol solution of Hippocampus sample, with filter (filter) filtration, (0.45 μ m).
(3) draw in 20 μ l injection high performance liquid chromatographs (HPLC) and perform an analysis with 25 μ l syringe cylinders.
The condition B of efficient liquid phase chromatographic analysis is as follows:
Analytical tool: HITACHI Pump L-2130
HITACHI?Diode?L-7455
Eluting post: Inertsil ODS SP4.6x250mm
Mobile phase: MeOH:H
2o (0.1%H
3pO
4)=2/98
Flow velocity: 1.0ml/ minute
Detecting wavelength: 210nm
Analysis time: 20 minutes
Hippocampus trimaculatus Leacs extract, sour jujube Hippocampus extract and storehouse reach Hippocampus extract analysis result respectively as Fig. 4 A, Fig. 4 B figure with Fig. 4 C as shown in.
Found that according to all efficient liquid phase chromatographic analysis above, hippocampus trimaculatus Leacs extract, sour jujube Hippocampus extract and storehouse reach Hippocampus extract and all have the crest of doubtful phylloxanthin and TYR.
Therefore further by the standard substance of phylloxanthin and TYR with condition B efficient liquid phase chromatographic analysis the crest to show that it is definite, result is respectively as shown in Fig. 5 A and 5B.Confirm that via comparison hippocampus trimaculatus Leacs extract, sour jujube Hippocampus extract and storehouse reach Hippocampus extract and all have phylloxanthin and TYR.Therefore, there is this to learn, phylloxanthin and/or TYR can be as the instruction composition of Hippocampus extract with the foundations as keyholed back plate Hippocampus extract.
Although the present invention discloses as above with preferred embodiment; so it is not in order to limit the present invention; anyly have the knack of this skill person; without departing from the spirit and scope of the present invention; when doing a little change and retouching, therefore protection scope of the present invention is when being as the criterion depending on the accompanying claim person of defining.
Symbol description
S1, S2, S1-1, S1-2, S2-1, S2-2～step.
Claims (31)
1. effect verification method of Hippocampus extract, comprising:
(a) Hippocampus is carried out to cultivar identification to confirm the step of kind of described Hippocampus, it comprises:
Extract a DNA sample for described Hippocampus; And
One sequence fragment of one gene of described DNA sample is carried out to the former qualification of base to confirm the kind of described Hippocampus; And
(b) confirm after the kind of described Hippocampus, an extraction is carried out to the step of effect confirmation and/or quality determination from the Hippocampus extract of described Hippocampus, it comprises:
Extract described Hippocampus to obtain described Hippocampus extract with a solvent; And
Described Hippocampus extract is carried out to the test of cancerous cell cell survival rate and/or confirm whether described Hippocampus extract comprises at least one indication compound.
2. effect verification method of Hippocampus extract claimed in claim 1, when this sequence fragment of the sequence fragment of the gene of wherein said DNA sample and this gene of the Hippocampus of a known kind is identical, confirms that described Hippocampus is this known Hippocampus.
3. effect verification method of Hippocampus extract claimed in claim 1, wherein said gene comprises cytochrome b gene.
4. effect verification method of Hippocampus extract claimed in claim 1 wherein carries out the former qualification of base to confirm the kind of this Hippocampus by a sequence fragment of a cytochrome b gene of described DNA sample in described step (a).
5. effect verification method of Hippocampus extract claimed in claim 4, this sequence fragment of wherein said cytochrome b gene is sequence identification number by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction to described DNA sample and obtained.
6. effect verification method of Hippocampus extract claimed in claim 1, wherein said cancerous cell cell survival rate is less than or equal to 90% and/or described Hippocampus extract while comprising described at least one indication compound, confirms that this Hippocampus extract possesses the effect that suppresses a growth of cancer cells.
7. effect verification method of Hippocampus extract claimed in claim 6, wherein said at least one indication compound comprises phylloxanthin and/or TYR.
8. effect verification method of Hippocampus extract claimed in claim 6, wherein said cancerous cell comprises hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell, breast cancer cell, ovarian cancer cell or colorectal cancer cells.
9. effect verification method of Hippocampus extract claimed in claim 1, wherein said solvent comprises ethanol, methanol, acetone or ethyl acetate.
10. effect verification method of Hippocampus extract claimed in claim 1, wherein said solvent is ethanol.
Effect verification method of 11. Hippocampus extracts claimed in claim 1, when this sequence fragment of the sequence fragment of the gene of wherein said DNA sample and this gene of the Hippocampus of a known kind is identical, confirm that described Hippocampus is this known Hippocampus, and described cancerous cell cell survival rate is less than or equal to 90% and/or described Hippocampus extract while comprising described at least one indication compound, confirms that described Hippocampus extract possesses effect.
Effect verification method of the Hippocampus extract described in 12. claim 11, wherein in described step (a), a sequence fragment of a cytochrome b gene of described DNA sample is carried out to the former qualification of base to confirm the kind of this Hippocampus, and this sequence fragment of described cytochrome b gene is sequence identification number by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction to described DNA sample and obtained, and wherein said at least one indication compound comprises phylloxanthin and TYR.
Prepare the method for Hippocampus extract, comprising for 13. 1 kinds:
(a) Hippocampus is carried out to cultivar identification to confirm the step of kind of this Hippocampus, it comprises:
Extract a DNA sample for described Hippocampus; And
One sequence fragment of one gene of described DNA sample is carried out to the former qualification of base to confirm the kind of described Hippocampus; And
(b) confirm after the kind of described Hippocampus, extract described Hippocampus to obtain the step of described Hippocampus extract with a solvent.
The method of preparing Hippocampus extract described in 14. claim 13, when this sequence fragment of the sequence fragment of the gene of wherein said DNA sample and this gene of the Hippocampus of a known kind is identical, confirms that described Hippocampus is this known Hippocampus.
The method of preparing Hippocampus extract described in 15. claim 13, wherein said gene comprises cytochrome b gene.
The method of preparing Hippocampus extract described in 16. claim 13 is wherein carried out the former qualification of base to confirm the kind of described Hippocampus by a sequence fragment of a cytochrome b gene of described DNA sample in described step (a).
The method of preparing Hippocampus extract described in 17. claim 16, this sequence fragment of wherein said cytochrome b gene is sequence identification number by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction to described DNA sample and obtained.
The method of preparing Hippocampus extract described in 18. claim 13, wherein confirms that described Hippocampus is that hippocampus trimaculatus Leacs, sour jujube Hippocampus or storehouse reach Hippocampus.
The method of preparing Hippocampus extract described in 19. claim 13, wherein said solvent comprises ethanol, methanol, acetone or ethyl acetate.
The method of preparing Hippocampus extract described in 20. claim 13, wherein in described step (a), a sequence fragment of a cytochrome b gene of described DNA sample is carried out to the former qualification of base to confirm the kind of described Hippocampus, and this sequence fragment of described cytochrome b gene is sequence identification number by sequence: 1 one first introduction and sequence are sequence identification number: 2 one second introduction is carried out a polymerase chain reaction to described DNA sample and obtained.
The method of preparing Hippocampus extract described in 21. claim 20, wherein confirm that described Hippocampus is that hippocampus trimaculatus Leacs, sour jujube Hippocampus or storehouse reach Hippocampus, and described solvent is ethanol.
The method of preparing Hippocampus extract described in 22. claim 13, wherein said Hippocampus extract comprises phylloxanthin and/or TYR is instruction composition.
23. 1 Hippocampus extracts, it has the effect that suppresses a growth of cancer cells, comprising:
Phylloxanthin and/or TYR are instruction composition.
Hippocampus extract described in 24. claim 23, wherein said Hippocampus extract is by a Hippocampus is extracted to acquisition with a solvent.
Hippocampus extract described in 25. claim 24, wherein said Hippocampus comprises that hippocampus trimaculatus Leacs, sour jujube Hippocampus or storehouse reach Hippocampus.
Hippocampus extract described in 26. claim 24, wherein said solvent comprises ethanol, methanol, acetone or ethyl acetate.
Hippocampus extract described in 27. claim 23, wherein said Hippocampus extract extracts acquisition by hippocampus trimaculatus Leacs, sour jujube Hippocampus or storehouse are reached to Hippocampus with ethanol.
Hippocampus extract described in 28. claim 23, wherein said cancerous cell comprises hepatoma carcinoma cell, lung carcinoma cell, prostate gland cancer cell, breast cancer cell, ovarian cancer cell or colorectal cancer cells.
The medical compositions of 29. 1 kinds of anticancer growth, comprise that the Hippocampus extract described in claim 23 is effective ingredient.
The medical composition of the anticancer growth described in 30. claim 29, also comprises pharmaceutically useful carrier or a salt.
Hippocampus extract described in 31. 1 kinds of claim 23 is for the preparation of the purposes of the medicine of anticancer growth.
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| KR20220078740A (en) * | 2020-12-03 | 2022-06-13 | 김경자 | Composition for preventing, ameliorating or treating prostate cancer comprising Hippocampus abdominalis and herbal medicine mixed extract as effective component |
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| 吴平等: "海马类药材的分子遗传标记鉴定研究", 《药学学报》 * |
| 张朝晖等: "海龙科药用动物的理化分析", 《中药材》 * |
| 王虹等: "小海马DPPH自由基清除作用及HPLC指纹图谱研究", 《中国药学杂志》 * |
| 胡嵘等: "海马、海龙基于COI条形码的DNA分子鉴定", 《杏林中医药》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20220078740A (en) * | 2020-12-03 | 2022-06-13 | 김경자 | Composition for preventing, ameliorating or treating prostate cancer comprising Hippocampus abdominalis and herbal medicine mixed extract as effective component |
| KR102558650B1 (en) * | 2020-12-03 | 2023-07-24 | 김경자 | Composition for preventing, ameliorating or treating prostate cancer comprising Hippocampus abdominalis and herbal medicine mixed extract as effective component |
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