CN103040932A - Phlomis maximowiczii extract, as well as extraction method and application thereof - Google Patents
Phlomis maximowiczii extract, as well as extraction method and application thereof Download PDFInfo
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Abstract
The invention discloses a Phlomis maximowiczii extract, as well as an extraction method and application thereof in preparation of antioxidant drugs and hepatoprotective drugs, and belongs to the technical field of medicines. The Phlomis maximowiczii extract contains a petroleum ether extract, an ethyl acetate extract and an n-butanol extract. The extraction method comprises the following steps: (1) collecting whole plants of Phlomis maximowiczii, washing, drying in a dark place, grinding and then extracting by soaking in methanol, recovering methanol to obtain a total extract; (2) dispersing the total extract in an ethanol aqueous solution, adding petroleum ether for extraction, and recovering petroleum ether to obtain the petroleum ether extract; (3) adding ethyl acetate into the solution for extraction after petroleum ether extraction, and recovering ethyl acetate to obtain the ethyl acetate extract; and (4) adding n-butanol into the solution after ethyl acetate extraction and recovering n-butanol to obtain the n-butanol extract. The Phlomis maximowiczii extract has antioxidant and hepatoprotective effects and can be used for preparing antioxidant and hepatoprotective health foods or drugs.
Description
Technical field
The present invention relates to a kind of Da Ye Herba phlomidis extract, extracting method and the application aspect preparation antioxidation class medicine and hepatoprotective class medicine thereof, belong to medical technical field.
Background technology
Hepatitis is one of hepatic disease of common serious harm human health, China is as the district occurred frequently of hepatopathy, infected the number of hepatitis virus approximately more than 100,000,000, but all there is hepatic injury in various degree in major part, groups of people develop to chronic hepatitis, liver cirrhosis, hepatocarcinoma, block or delay it to worsen the challenge that has become the treatment hepatopathy.The general game of liver disease comprises protection, improves liver function at present, reverses liver histological and changes, and the blocking-up chronic hepatitis develops to liver cirrhosis, thereby prevents the pernicious differentiation of " hepatopathy trilogy ".
Acute liver damage is one of emergency treatment common disease, can be divided into violence nature, pathologic, chemical and the hepatic injury of feelings will type, wherein pathologic hepatic injury and chemical liver injury are the most common, and main manifestations is hepatic necrosis or apoptosis, hepatocellular degeneration, hepatic cell fattydegeneration, the infringement of silt gallbladder, inflammatory reaction etc.Cause clinically the main cause of acute liver damage to have: viral infection, drug toxicity, ethanol, pesticide or herbicide, industrial chemical, alimentary toxicosis, stay up late excessively, other diseases such as acute pancreatitis, cardiac insufficiency, diabetes, tumor etc.Modern medicine there is no the specific drug for the treatment of hepatic injury, diet, vitimin supplement are had a rest, regulated to adopt and by taking antiviral agents (such as interferon and lamivudine), immunomodulator (such as the thymus skin) and improving the symptomatic treatment such as function medicine (such as bifendate) more, although the various kinds of drug of symptomatic treatment respectively has clinically certain curative effect and is verified, but owing to have strict indication and serious toxic and side effects, so clinical practice has been subject to restriction to a certain degree.Along with the development of Chinese medicine and combination of Chinese and Western medicine research, use Chinese herbal medicine and preparation for treating hepatic disease thereof and more and more come into one's own in China.
At present, the pathogenetic research of various hepatic disease and the screening of medicine all rely on foundation and the application of the experimental animal model similar to human liver disease's pathomechanism to a great extent.The method of making the Experimental Hepatic Damage animal model is a lot, such as chemical liver injury (carbon tetrachloride, aminogalactose are induced hepatic injury etc.), drug induced hepatic injury (acetaminophen is induced hepatic injury), immunologic liver injury (bacillus calmette-guerin vaccine and lipopolysaccharide-induced hepatic injury, concanavalin A, Con A are induced hepatic injury) and alcoholic liver injury.Wherein the acute liver damage animal model of tetrachloro-methane induction is the most classical experimental liver injury model.
Oxidative stress is not only the part of hepatic insufficiency, also is the pathophysiological basis of all hepatic injury.Hepatocyte is interior in case responding property oxidation product ROS(reactive oxygen species, ROS) excessive generation and Antioxidation Mechanism are low, just easily cause oxidative stress status, Ca in Cell membrane lipids peroxidating, organelle dysfunction, the mitochondrion occurs
2+Overload, inflammatory reaction etc., even the oxidation of nuclear occurs further develop and can cause canceration.
Chinese medicine and effective ingredient thereof can cross expressions, free radical resisting damage by suppressing cytokine, reduce the generation that causes inflammation and apoptosis associated media, promote liver blood circulation and hepatocellular regeneration, regulate the aspects such as immunity, antiviral protects hepatocyte, prevents hepatic injury.Wherein, screening has anti-oxidation stress from Chinese herbal medicine, and the hepatic that suppresses lipid peroxidation also is the focus of studying at present.As: barbaloin, rhodioloside, chimonin, Radix Puerariae total flavones, willow tea, Radix Glycyrrhizae extract, Herba Polygoni Perfoliati, Fructus Canarii albi total flavones, Radix Helicteris, Herba Goldffussiae Psilostachydis polysaccharide, hyperin etc. can reduce glutamate pyruvate transaminase, glutamic oxaloacetic transaminase, GOT and Mda During in various degree; the approach such as increased SOD or GSH-PX activity; strengthen hepatic antioxidant active; suppress lipid peroxidation, thereby acute liver damage is played a protective role.
Da Ye Herba phlomidis (Phlomis maximowiczii) is Labiatae Paraphlomis plant, originate in Jilin, Liaoning and Hebei, be born in border or riverbank, fruit can be extracted oil, oil content is 20-34%, root is traditional herbal medicine, bitter in the mouth, suffering, cool in nature, energy clearing heat for detumescence, its root or herb all can be used as medicine, the energy clearing heat for detumescence dispels the wind, and cures mainly furuncle, innominate toxic swelling, have the effects such as treatment flu, invigorating the liver and kidney, reuniting the fractured tendons and bones, arresting bleeding and miscarriage prevention, cold expelling, granulation promoting, the local resident uses it as a kind of medical herbs that has no side effect.At present except Gu Haipeng etc. studies the liposoluble constituent of Da Ye Herba phlomidis, have no other to the report of Da Ye Herba phlomidis chemical composition and pharmacologically active.
Summary of the invention
The purpose of this invention is to provide a kind of Da Ye Herba phlomidis extract.
In order to realize above purpose, the technical solution adopted in the present invention is:
A kind of Da Ye Herba phlomidis extract comprises ligroin extraction, ethyl acetate extract and n-butanol extract; The extracting method step is as follows: (1) gets Da Ye Herba phlomidis herb, cleans, dries in the shade, and soaks extraction with methanol after pulverizing, and reclaims methanol, gets total extractum for subsequent use; (2) the total extractum with step (1) disperses to add petroleum ether extraction in the ethanol water, petroleum ether extraction liquid is reclaimed petroleum ether get ligroin extraction; (3) add ethyl acetate in the solution behind step (2) petroleum ether extraction and extract, acetic acid ethyl acetate extract is reclaimed ethyl acetate get ethyl acetate extract; (4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, butanol extraction liquid is reclaimed n-butyl alcohol get n-butanol extract.
Simultaneously, the present invention also provides a kind of extracting method of Da Ye Herba phlomidis extract, and concrete steps are as follows:
(1) gets Da Ye Herba phlomidis herb, clean, dry in the shade, soak extraction with methanol after pulverizing, reclaim methanol, get total extractum for subsequent use;
(2) the total extractum with step (1) disperses to add petroleum ether extraction in the ethanol water, petroleum ether extraction liquid is reclaimed petroleum ether get ligroin extraction;
(3) add ethyl acetate in the solution behind step (2) petroleum ether extraction and extract, acetic acid ethyl acetate extract is reclaimed ethyl acetate get ethyl acetate extract;
(4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, butanol extraction liquid is reclaimed n-butyl alcohol get n-butanol extract.
Preferably, the immersion number of times of methanol is 3 times in the described step (1), and soak time is 3 days.
The present invention also provides the application of a kind of Da Ye Herba phlomidis extract aspect preparation antioxidation class health food or medicine.
Further, ethyl acetate extract and the n-butanol extract application aspect preparation hepatoprotective food or medicine in the Da Ye Herba phlomidis extract.
Further, ethyl acetate extract and the n-butanol extract application aspect the anti-acute liver damage class health food of preparation or medicine in the Da Ye Herba phlomidis extract.
Further, ethyl acetate extract and n-butanol extract reduce the health food of serum GPT vigor in the acute liver damage or the application aspect the medicine in preparation in the Da Ye Herba phlomidis extract.
Further, ethyl acetate extract and n-butanol extract reduce the health food of serum GOT vigor in the acute liver damage or the application aspect the medicine in preparation in the Da Ye Herba phlomidis extract.
Further, the health food of ethyl acetate extract and n-butanol extract hepatic SOD in preparation rising acute liver damage or the application aspect the medicine in the Da Ye Herba phlomidis extract.
Further, ethyl acetate extract and n-butanol extract reduce the health food of liver MDA level in the acute liver damage or the application aspect the medicine in preparation in the Da Ye Herba phlomidis extract.
Beneficial effect of the present invention:
Da Ye Herba phlomidis extract of the present invention comprises ligroin extraction, ethyl acetate extract and n-butanol extract, get Da Ye Herba phlomidis herb and obtain to extract with petroleum ether, ethyl acetate, n-butyl alcohol successively behind the methanol extraction first again, this extracting method is simple.The Da Ye Herba phlomidis extract that the present invention obtains extraction has carried out antioxidation activity in vitro test and CCl
4The test of the acute liver of inducing; the result shows that the antioxidant activity of n-butanol extract in the Da Ye Herba phlomidis extract is the strongest; ethyl acetate extract takes second place; ligroin extraction is the most weak; n-butanol extract and ethyl acetate extract all can be by strengthening GPT vigor and GOT vigor in the serum in the acute liver test simultaneously; play the effect of hepatoprotective; simultaneously by the rising SOD in liver; reduce the antioxidative functional that the MDA contents level strengthens the acute hepatic injury mice body; suppress lipid peroxidation; the protection body is avoided the further injury of free radical, performance hepatoprotective effect.Da Ye Herba phlomidis extract of the present invention can for the preparation of antioxidation class health food and medicine, further, can be brought into play effect aspect preparation antioxidation class hepatoprotective food and medicine.
Description of drawings
Fig. 1 is that Da Ye Herba phlomidis extract is to the clearance rate of DPPH free radical;
Fig. 2 is that Da Ye Herba phlomidis extract is to the clearance rate of ABTS free radical.
The specific embodiment
Below in conjunction with embodiment the present invention is elaborated, but do not consist of any limitation of the invention.
Embodiment 1
A kind of Da Ye Herba phlomidis extract comprises ligroin extraction, ethyl acetate extract and n-butanol extract in the present embodiment; The extracting method step is as follows: (1) gets Da Ye Herba phlomidis herb, cleans, dries in the shade, and soaks, extracts with methanol after pulverizing, and reclaims methanol, gets total extractum for subsequent use; (2) the total extractum with step (1) disperses to add petroleum ether extraction in the ethanol water, petroleum ether extraction liquid is reclaimed petroleum ether get ligroin extraction; (3) add ethyl acetate in the solution behind step (2) petroleum ether extraction and extract, acetic acid ethyl acetate extract is reclaimed ethyl acetate get ethyl acetate extract; (4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, butanol extraction liquid is reclaimed n-butyl alcohol get n-butanol extract.Concrete extraction step is:
(1) get Da Ye Herba phlomidis herb 1000g, clean, dry in the shade, pulverize, add 2000mL methanol at every turn and soak, soak 2 times, soaked 4 days at every turn, decompression merges lixiviating solution, reclaims methanol, gets total extractum for subsequent use;
(2) total extractum of step (1) is scattered in 15% the ethanol water, adds the 1000mL petroleum ether at every turn and extract, extract 2 times, merge the petroleum ether extraction layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim petroleum ether, namely get ligroin extraction;
(3) add ethyl acetate in the solution behind step (2) petroleum ether extraction, add 1000mL at every turn, extract 2 times, the combined ethyl acetate extract layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim ethyl acetate, namely get ethyl acetate extract;
(4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, add 1000mL at every turn, extract 2 times, merge the n-butanol extraction layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim n-butyl alcohol, namely get n-butanol extract.
Embodiment 2
A kind of Da Ye Herba phlomidis extract comprises ligroin extraction, ethyl acetate extract and n-butanol extract in the present embodiment; The extracting method step is as follows: (1) gets Da Ye Herba phlomidis herb, cleans, dries in the shade, and soaks, extracts with methanol after pulverizing, and reclaims methanol, gets total extractum for subsequent use; (2) the total extractum with step (1) disperses to add petroleum ether extraction in the ethanol water, petroleum ether extraction liquid is reclaimed petroleum ether get ligroin extraction; (3) add ethyl acetate in the solution behind step (2) petroleum ether extraction and extract, acetic acid ethyl acetate extract is reclaimed ethyl acetate get ethyl acetate extract; (4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, butanol extraction liquid is reclaimed n-butyl alcohol get n-butanol extract.Concrete extraction step is:
(1) get Da Ye Herba phlomidis herb 1000g, clean, dry in the shade, pulverize, add 2000mL methanol at every turn and soak, soak 4 times, soaked 2 days at every turn, decompression merges lixiviating solution, reclaims methanol, gets total extractum for subsequent use;
(2) total extractum of step (1) is scattered in 10% the ethanol water, adds the 1000mL petroleum ether at every turn and extract, extract 3 times, merge the petroleum ether extraction layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim petroleum ether, namely get ligroin extraction;
(3) add ethyl acetate in the solution behind step (2) petroleum ether extraction, add 1000mL at every turn, extract 3 times, the combined ethyl acetate extract layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim ethyl acetate, namely get ethyl acetate extract;
(4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, add 1000mL at every turn, extract 3 times, merge the n-butanol extraction layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim n-butyl alcohol, namely get n-butanol extract.
Embodiment 3
A kind of Da Ye Herba phlomidis extract comprises ligroin extraction, ethyl acetate extract and n-butanol extract in the present embodiment; The extracting method step is as follows: (1) gets Da Ye Herba phlomidis herb, cleans, dries in the shade, and soaks, extracts with methanol after pulverizing, and reclaims methanol, gets total extractum for subsequent use; (2) the total extractum with step (1) disperses to add petroleum ether extraction in the ethanol water, petroleum ether extraction liquid is reclaimed petroleum ether get ligroin extraction; (3) add ethyl acetate in the solution behind step (2) petroleum ether extraction and extract, acetic acid ethyl acetate extract is reclaimed ethyl acetate get ethyl acetate extract; (4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, butanol extraction liquid is reclaimed n-butyl alcohol get n-butanol extract.Concrete extraction step is:
(1) get Da Ye Herba phlomidis herb 1000g, clean, dry in the shade, pulverize, add 1000mL methanol at every turn and soak, soak 3 times, soaked 3 days at every turn, decompression merges lixiviating solution, reclaims methanol, gets total extractum for subsequent use;
(2) total extractum of step (1) is scattered in 5% the ethanol water, adds the 1000mL petroleum ether at every turn and extract, extract 4 times, merge the petroleum ether extraction layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim petroleum ether, namely get ligroin extraction;
(3) add ethyl acetate in the solution behind step (2) petroleum ether extraction, add 1000mL at every turn, extract 4 times, the combined ethyl acetate extract layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim ethyl acetate, namely get ethyl acetate extract;
(4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, add 1000mL at every turn, extract 4 times, merge the n-butanol extraction layer, under the vacuum condition in Rotary Evaporators evaporate to dryness reclaim n-butyl alcohol, namely get n-butanol extract.
Test example 1
The antioxidation activity in vitro test of Da Ye Herba phlomidis extract
Test material and method
The Da Ye Herba phlomidis extract that material: embodiment 2 prepares is with the positive contrast of BHT, not add Da Ye Herba phlomidis extract as blank.
Reagent: the DPPH(Tokyo changes into Industrial Co., Ltd), ABTS(U.S. Fluka company), TPTZ(Belgium Acros organics company), Trolox(U.S. Aldrich company), dibenzylatiooluene (BHT, Belgium Acros organics company), methanol (AR level).
Instrument: UV-2000 type ultraviolet-uisible spectrophotometer (but You Ni Shanghai Instr Ltd.), electronic balance (prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit Instr Ltd.), ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), CS-H1 type blender (Beijing is won and encouraged positive scientific ﹠ technical corporation), various pipettors and rifle are first-class.
Method: (1) DPPH(hexichol is for bitterness acyl group free radical) method, first sample is mixed with a series of concentration with methanol, getting 0.1mL sample adding 3.5mL concentration is 0.06mmol/L DPPH methanol solution, and mixing is measured its absorbance at the 515nm place behind the 30min; Same method is with the absorbance of methanol replacement sample determination blank.Every duplicate samples operation repetitive 3 times is averaged, and the following formula of recycling calculates the DPPH free radical scavenging activity:
DPPH clearance rate (%)=[(A
Blank-A
Sample)/A
Blank] * 100
Then use Origin6.0 Software on Drawing concentration-clearance rate graph of a relation, calculation sample is removed the half suppression ratio IC of DPPH free radical
50Value.Principle is: DPPH is a kind of very stable free radical centered by nitrogen, and its lone pair electrons have strong absorption near 515nm.When the antioxidant that hydrogen supply capacity is arranged in the sample existed, DPPH free radical lone pair electrons were paired, and absorption disappears or weakens, and absorbance diminishes, and the degree that the degree that it diminishes and free radical are eliminated is quantitative relationship.
(2) ABTS(2,2 '-Lian ammonia-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) method, first sample is mixed with a series of concentration with methanol, get the 0.15mL sample and add 2.85mL ABTS free radical working solution, mixing is measured its absorbance at the 734nm place behind the 10min; The absorbance that replaces the sample determination blank with method with methanol.Every duplicate samples operation repetitive 3 times is averaged, and the following formula of recycling calculates the ABTS free radical scavenging activity:
ABTS clearance rate (%)=[(A
Blank-A
Sample)/A is blank] * 100
Then use Origin6.0 Software on Drawing concentration-clearance rate graph of a relation, calculation sample is removed the half suppression ratio IC50 value of ABTS free radical.Principle is: ABTS is a kind of water miscible radical initiator, generates stable aeruginous radical cation ABTS after the active oxygen oxidation
+, when containing polyphenoils, this material meeting and ABTS to the measured matter that wherein adds
+React and make reaction system color fade, ABTS
+At the 734nm place absorption maximum is arranged, adopt K herein
2S
2O
8Generate stable ABTS with the ABTS direct reaction
+Come the oxidation resistance of working sample.
(3) FRAP(iron ion reducing power) method, first sample is mixed with the solution that initial concentration is 2mg/mL with methanol, get the 0.2mL sample and add the freshly prepared TPTZ working solution of 3.8mL, mixing, measure it at 593nm place absorbance behind 37 ℃ of reaction 30min, every duplicate samples operation repetitive 3 times is averaged.If institute's working sample absorbance surpasses the range of linearity, then need further dilute sample.Trolox is configured to concentration range in a series of concentration of 25～400 μ mol/L with methanol, measures with method with sample, draw concentration-absorbance standard curve, as the foundation of sample treatment.
Principle: in the solution of low pH value, Fe
3+-three pyridines three mute piperazines (tripyridyl-triazine, TPTZ) can be reduced to Fe by antioxidant
2+Form presents obvious blueness, and at the 593nm place absorption maximum is arranged, and calculates the power of antioxidant activity according to the size of absorbance, and the result can be with Fe
2+Equivalent or represent with respect to the ability of standard anti-oxidant adopts Trolox to represent the antioxidant activity of sample herein.
The antioxidation activity in vitro result of the test of Da Ye Herba phlomidis extract sees the following form 1 and Fig. 1,2.
The antioxidant activity of table 1 Da Ye Herba phlomidis extract
Annotate :-expression does not detect activity.
Result of the test and discussion
Da Ye Herba phlomidis extract is to the result of the test of DPPH free radical: table 1 shows, n-butanol extract is to the removing ability (IC of DPPH free radical
50=25.15 μ g/mL) slightly be weaker than BHT(IC
50=18.71 μ g/mL).Ligroin extraction and ethyl acetate extract are because the primary dcreening operation suppression ratio is lower than 50%, IC
50The value undetermined, but ethyl acetate extract to the clearance rate (49.65%) of DPPH free radical greater than ligroin extraction (17.47%).As seen, ligroin extraction, ethyl acetate extract, n-butanol extract and positive control BHT to the removing ability of DPPH free radical sequentially are: BHT〉n-butanol extract〉ethyl acetate extract〉ligroin extraction〉blank.Fig. 1 shows, in the experimental concentration scope, antioxidant almost is to be linear to increase along with mass concentration increases to the clearance rate of DPPH free radical, when the antioxidant mass concentration is lower than 40.49 μ g/mL, n-butanol extract and BHT are all lower to the clearance rate of DPPH free radical, and n-butanol extract is lower than BHT, along with the increase of mass concentration; When mass concentration increased to 40.49 μ g/mL, n-butanol extract and BHT were equal to the clearance rate of DPPH free radical, and clearance rate is increased to 71.24%; Continue to increase mass concentration, also in gradually increase, but n-butanol extract is higher than BHT to the clearance rate of DPPH free radical for n-butanol extract and BHT.
Da Ye Herba phlomidis extract is to the result of the test of ABTS free radical: table 1 shows, ethyl acetate extract and n-butanol extract are to the removing ability (IC of ABTS free radical
50Be respectively 36.73 and 18.96 μ g/mL) all be weaker than BHT(IC
50=7.72 μ g/mL); Ligroin extraction is lower than 50%, IC because the primary dcreening operation suppression ratio is 38.27%
50The value undetermined.As seen, ligroin extraction, ethyl acetate extract, n-butanol extract and positive control BHT to the removing ability of DPPH free radical sequentially are: BHT〉n-butanol extract〉ethyl acetate extract〉ligroin extraction〉blank.Fig. 2 shows, in the quality of experiments concentration range, ethyl acetate extract and n-butanol extract to the clearance rate of ABTS free radical all less than positive control BHT, n-butanol extract to the clearance rate of ABTS free radical greater than ethyl acetate extract; And ethyl acetate extract and BHT almost are linear to the ABTS free radical scavenging activity with the mass concentration increase to be increased; When mass concentration is lower than 50 μ g/mL, n-butanol extract to the clearance rate of ABTS free radical along with the increase of mass concentration be linear and increase, but continue to increase concentration, clearance rate tends towards stability.
The result of the test of Da Ye Herba phlomidis extract reduced iron ion: table 1 shows, n-butanol extract reduction Fe
3+Ability the strongest (Trolox equivalent=2775.6 ± 144.18 μ mol/g), be better than positive control BHT(Trolox equivalent=1581.68 ± 97.41 μ mol/g), be about 1.75 times of BHT; Ligroin extraction and ethyl acetate extract reduction Fe
3+Ability (Trolox equivalent=502.4 ± 9.88 and 606.4 ± 5.64 μ mol/g) all be weaker than BHT.As seen, ligroin extraction, ethyl acetate extract, n-butanol extract and BHT reduction Fe
3+The order of ability is: n-butanol extract〉BHT〉ethyl acetate extract〉ligroin extraction〉blank.
Above 3 kinds of methods experiment results show, ligroin extraction, ethyl acetate extract and n-butanol extract all have the ability of certain removing DPPH and ABTS free radical and reduced iron ion.Wherein the n-butanol extract antioxidant activity is the strongest, is ethyl acetate extract secondly, and the ligroin extraction activity is the most weak.As seen, Da Ye Herba phlomidis oxidation-resistant active ingredient mainly concentrates on middle polarity or the large position of polarity.
Test example 2
Da Ye Herba phlomidis extract is used for the hepatoprotective test of acute liver damage
Test material and method
The Da Ye Herba phlomidis extract that material: embodiment 2 prepares is with the positive contrast of bifendate, not add Da Ye Herba phlomidis extract as model contrast, take not modeling, do not add Da Ye Herba phlomidis extract and be blank.
Experimental animal: 90 of kunming mices, sex is random, age in 4-6 week, body weight 22 ± 2g.
Reagent: carbon tetrachloride analytical pure (Kaifeng chemical reagent factory); olive oil (Weihui City, Shanghai chemical reagent factory); bifendate drop pill (Zhejiang Prov WanBang Pharmaceutical Co., Ltd; lot number: 10031); Coomassie brilliant blue G250 (packing factory of Solution on Chemical Reagents in Shanghai company; lot number: 20070867); bovine serum albumin (the extensive and profound in meaning star in Beijing Bioisystech Co., Ltd); glutamic oxaloacetic transaminase, GOT is measured test kit (GOT; lot number: 20120720); glutamate-pyruvate transaminase determination reagent kit (GPT; lot number: 20120717), the Mda test kit (MDA, lot number: 20120724) and superoxide dismutase measure test kit (SOD; lot number: 20111201), above test kit all is purchased from Nanjing and builds up bio-engineering research institute.
Instrument: UV-2000 type ultraviolet-uisible spectrophotometer (You Nike Shanghai Instr Ltd.), Multiskan MK3 microplate reader (U.S. Thermo Electron company), LRH-150 constant incubator (Shanghai one permanent Science and Technology Ltd.), electronic balance (prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit Instr Ltd.), Rotary Evaporators (German Heidolph company), CS-H1 type blender (Beijing is won and encouraged positive scientific ﹠ technical corporation), TGL-16 high speed centrifuge (in the Community of Jin Tan County city large instrument plant), and various pipettor and rifle are first-class.
Test method: the kunming mice random assortment is contrasted and the blank group in each dosetest group of embodiment 2 extracts and model, with the ethyl acetate extract of embodiment 2 according to high, medium and low dosage group (800,400 and 200mg/kg Mus heavy), n-butanol extract according to high, medium and low dosage group (600,300 and 150mg/kg Mus heavy) gavage mice every day, successive administration 8d, 2h(after the 8d administration is except blank), according to the heavy lumbar injection 0.4%CCl of 0.1mL/10g Mus
4Olive oil solution carries out modeling, water 12h is can't help in then fasting, pluck eyeball and get blood, the blood plasma room temperature leaves standstill the 30min separation of serum, serum places 0～3 ℃ to save backup, the sharp separation liver, preparation 10% and 1% liver homogenate liquid is measured SOD vigor and MDA level in the vigor, liver homogenate liquid of GOT and GPT in the serum according to the test kit description again.Measure serum albumin content with Coomassie brilliant blue G-250 method.
Ethyl acetate extract and n-butanol extract see the following form 2 to the impact of GPT in the acute hepatic injury mice serum and GOT vigor in the Da Ye Herba phlomidis extract, and the SOD vigor of polarity hepatic injury mouse liver and the impact of MDA level are seen the following form 3.
Table 2 Da Ye Herba phlomidis extract is on the impact of GPT in the acute hepatic injury mice serum and GOT vigor
Annotate: compare with the blank group:
△ △ △P＜0.001,
△ △P＜0.01,
△P＜0.05;
Compare with model group:
* *P＜0.001,
*P＜0.01,
*P＜0.05;
Compare with positive controls:
###P＜0.001,
##P＜0.01,
#P＜0.05.
Conclusion and discussion
Table 2 demonstration is compared with blank, and the GPT in the model control group mice serum and GOT vigor all are utmost point significance and reduce (P＜0.001), and the modeling success is described; Compare with model group, the ethyl acetate extract low dose group can reduce GPT and GOT vigor (P＜0.001) in the serum extremely significantly, high dose group can reduce GOT vigor in the serum (P＜0.001) extremely significantly, the high, medium and low dosage group of n-butanol extract all can reduce GPT vigor in the serum (P＜0.001) significantly, respectively can be extremely significantly, GOT level (P＜0.001, P＜0.001 and P＜0.01) extremely significantly and in the reduction serum of highly significant; Compare with positive controls, the vigor (P＜0.001) of dosage group and n-butanol extract low dose group energy significance rising GOT in the ethyl acetate extract, to GPT there was no significant difference (P〉0.05), other dosage groups are compared positive controls there are no significant difference (P〉0.05) to the impact of GPT and GOT, but ethyl acetate extract and n-butanol extract high dose group reduce the energetic for positive control of GOT; The ethyl acetate extract low dosage reduces the energetic for positive control of GPT, and GOT is a little more than positive control.Above result shows, ethyl acetate extract and n-butanol extract all can reduce CCl
4GPT and GOT vigor have certain liver protection function in the acute hepatic injury mice serum of inducing, and be wherein best with n-butanol extract senior middle school low dose group and ethyl acetate extract low dosage effect.
Table 3 Da Ye Herba phlomidis extract is on the impact of acute hepatic injury mice hepatic SOD and MDA level
Annotate: compare with the blank group:
△ △ △P＜0.001,
△ △P＜0.01,
△P＜0.05;
Compare with model group:
* *P＜0.001,
*P＜0.01,
*P＜0.05;
Compare with positive controls:
###P＜0.001,
##P＜0.01,
#P＜0.05.
Result and discussion
Table 3 shows, compares with blank, and SOD vigor utmost point significance reduces (P＜0.001) in the model control group mouse liver homogenate, and MDA content utmost point significance raises (P＜0.001, P＜0.001 and P＜0.05), and the modeling success is described; Compare with model control group, all can raise the very significantly vigor (P＜0.001) of SOD in the liver homogenate liquid of high, the middle dosage group of ethyl acetate extract, the vigor of the reduction SOD of low dose group significance (P＜0.05), the high, medium and low dosage group of the n-butanol extract SOD vigor (P＜0.001, P＜0.001, P＜0.05) in the liver homogenate liquid that raises extremely significantly, extremely significantly and significantly respectively; The high, medium and low dosage group of ethyl acetate extract high and low dose group and n-butanol extract all can reduce MDA content in the liver homogenate liquid (P＜0.001) by the utmost point significantly, the dosage group can reduce the content of MDA in the liver homogenate liquid in the ethyl acetate extract, but there was no significant difference (P〉0.05); Compare with positive controls, in the high, medium and low dosage group of ethyl acetate extract and the n-butanol extract, low dose group all can the utmost point increased SOD vigor (P＜0.001) significantly, n-butanol extract high dose group increased SOD vigor is similar to positive control, there was no significant difference (P〉0.05), the high, medium and low dosage group of ethyl acetate extract low dose group and n-butanol extract all can reduce MDA content, but there are no significant difference (P＞0.05).Above result shows; the ability of SOD vigor and reduction MDA content is better than ethyl acetate extract in the n-butanol extract rising liver homogenate liquid; both strengthen the anti-oxidation function of acute hepatic injury mice body by this approach, the protection body is avoided the further injury of free radical.
Claims (10)
1. a Da Ye Herba phlomidis extract is characterized in that: comprise ligroin extraction, ethyl acetate extract and n-butanol extract; The extracting method step is as follows: (1) gets Da Ye Herba phlomidis herb, cleans, dries in the shade, and soaks extraction with methanol after pulverizing, and reclaims methanol, gets total extractum for subsequent use; (2) the total extractum with step (1) disperses to add petroleum ether extraction in the ethanol water, petroleum ether extraction liquid is reclaimed petroleum ether get ligroin extraction; (3) add ethyl acetate in the solution behind step (2) petroleum ether extraction and extract, acetic acid ethyl acetate extract is reclaimed ethyl acetate get ethyl acetate extract; (4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, butanol extraction liquid is reclaimed n-butyl alcohol get n-butanol extract.
2. the extracting method of a Da Ye Herba phlomidis extract as claimed in claim 1, it is characterized in that: concrete steps are as follows:
(1) gets Da Ye Herba phlomidis herb, clean, dry in the shade, soak extraction with methanol after pulverizing, reclaim methanol, get total extractum for subsequent use;
(2) the total extractum with step (1) disperses to add petroleum ether extraction in the ethanol water, reclaims petroleum ether and gets ligroin extraction;
(3) add ethyl acetate in the solution behind step (2) petroleum ether extraction and extract, reclaim ethyl acetate and get ethyl acetate extract;
(4) add n-butyl alcohol in the solution behind step (3) ethyl acetate extraction, reclaim n-butyl alcohol and get n-butanol extract.
3. the extracting method of Da Ye Herba phlomidis extract according to claim 2 is characterized in that: the immersion number of times of methanol is 3 times in the step (1), and soak time is 3 days.
4. the application of a Da Ye Herba phlomidis extract as claimed in claim 1 is characterized in that: the application of described Da Ye Herba phlomidis extract aspect preparation antioxidation class health food or medicine.
5. the application of Da Ye Herba phlomidis extract according to claim 1 is characterized in that: ethyl acetate extract and the n-butanol extract application aspect preparation hepatoprotective food or medicine in the described Da Ye Herba phlomidis extract.
6. the application of Da Ye Herba phlomidis extract according to claim 5 is characterized in that: ethyl acetate extract and the n-butanol extract application aspect the anti-acute liver damage class health food of preparation or medicine in the described Da Ye Herba phlomidis extract.
7. the application of Da Ye Herba phlomidis extract according to claim 6 is characterized in that: ethyl acetate extract and n-butanol extract reduce the health food of serum GPT vigor in the acute liver damage or the application aspect the medicine in preparation in the described Da Ye Herba phlomidis extract.
8. the application of Da Ye Herba phlomidis extract according to claim 6 is characterized in that: ethyl acetate extract and n-butanol extract reduce the health food of serum GOT vigor in the acute liver damage or the application aspect the medicine in preparation in the described Da Ye Herba phlomidis extract.
9. the application of Da Ye Herba phlomidis extract according to claim 6 is characterized in that: the health food of ethyl acetate extract and n-butanol extract hepatic SOD in preparation rising acute liver damage or the application aspect the medicine in the described Da Ye Herba phlomidis extract.
10. the application of Da Ye Herba phlomidis extract according to claim 6 is characterized in that: ethyl acetate extract and n-butanol extract reduce the health food of liver MDA level in the acute liver damage or the application aspect the medicine in preparation in the described Da Ye Herba phlomidis extract.
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| CN104069348A (en) * | 2014-06-30 | 2014-10-01 | 天津中医药大学 | Sealwort extract as well as preparation method and use thereof |
| CN105434575A (en) * | 2014-09-24 | 2016-03-30 | 李敏 | Application of Eseye and extract thereof in preparation of medicines or health food for protecting liver |
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| CN1970049A (en) * | 2006-09-21 | 2007-05-30 | 彭美中 | Prescription of a health-preserving wine and preparation process thereof |
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| 20120228 顾海鹏，陈 龙，张一冰，康文艺＊ 18-19,29 1 第３１卷, 第１期 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069348A (en) * | 2014-06-30 | 2014-10-01 | 天津中医药大学 | Sealwort extract as well as preparation method and use thereof |
| CN105434575A (en) * | 2014-09-24 | 2016-03-30 | 李敏 | Application of Eseye and extract thereof in preparation of medicines or health food for protecting liver |
| CN105434575B (en) * | 2014-09-24 | 2020-04-21 | 李敏 | Application of Russian leaves and extract thereof in preparation of liver-protecting medicines or health-care foods |
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