CN116549516A - Paeonia lactiflora seed meal extract and preparation method and application thereof - Google Patents
Paeonia lactiflora seed meal extract and preparation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
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- Public Health (AREA)
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- Microbiology (AREA)
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- Alternative & Traditional Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Heart & Thoracic Surgery (AREA)
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Abstract
The invention belongs to the technical field of plant component extraction, and provides a paeonia lactiflora seed meal extract, a preparation method and application thereof, wherein the preparation method comprises the following steps: pulverizing radix Paeoniae seed cake, mixing with ethanol solution, extracting, filtering to obtain filtrate, and concentrating the filtrate to obtain radix Paeoniae seed cake extract. The paeonia lactiflora seed meal extract prepared by the preparation method has obvious blood pressure reducing effect, is simple in preparation method and low in cost, and is safe in extracted components, thereby providing a new idea for developing blood pressure reducing products.
Description
Technical Field
The invention relates to the technical field of plant component extraction, in particular to a paeonia lactiflora seed meal extract and a preparation method and application thereof.
Background
Paeonia lactiflora (Paeonia lactiflora pall.) is a perennial herb of the genus Paeonia of the family Paeoniaceae. Paeonia lactiflora is one of economic plants which can be used for both medicine and ornamental, and the root tuber of Paeonia lactiflora can be used as a medicine, and has the functions of regulating liver, spleen and nutrient and blood. The paeonia lactiflora seed can be used for extracting natural oil, and the obtained paeonia lactiflora seed oil has anti-inflammatory and antibacterial effects. The residual paeonia lactiflora seed meal after the paeonia lactiflora seed oil is squeezed is generally directly discarded, but researches show that the paeonia lactiflora seed meal contains abundant monoterpene glycoside active ingredients, and the paeonia lactiflora seed meal can cause environmental pollution and waste of resources after being directly discarded.
With the improvement of the living standard of people, the incidence probability of some chronic diseases is gradually increased. Hypertension is the most common chronic disease, the incidence rate of which tends to increase with the increase of age, and for patients suffering from hypertension, long-term administration of antihypertensive drugs is required, and side effects such as effects on blood lipid, blood sugar and blood uric acid metabolism are brought about after long-term administration of antihypertensive drugs, and also damage to other organs is brought about to different extents. Therefore, there is a need to develop a new blood pressure lowering product.
Disclosure of Invention
The invention aims to provide the paeonia lactiflora seed meal extract, the preparation method and the application thereof, wherein the paeonia lactiflora seed meal extract is prepared by enriching the effective components in waste paeonia lactiflora seed meal, and has a simple preparation method, and the obtained paeonia lactiflora seed meal extract has an obvious blood pressure reducing effect and provides a new idea for developing blood pressure reducing products.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a paeonia lactiflora seed meal extract, which comprises the following steps:
pulverizing radix Paeoniae seed cake, mixing with ethanol solution, extracting, filtering to obtain filtrate, and concentrating the filtrate to obtain radix Paeoniae seed cake extract.
Preferably, the volume percentage concentration of the ethanol solution is 40-60%.
Preferably, the ratio of the paeonia lactiflora seed meal to the ethanol solution is 1g: 5-15 mL.
Preferably, the times of extraction are 1-3 times, the time of each extraction is 5-10 hours, and the temperature of each extraction is 20-30 ℃.
Preferably, the concentration is 20-40% of the original volume.
Preferably, the concentration is preceded by a step of volatilizing ethanol.
The invention also provides the paeonia lactiflora seed meal extract prepared by the preparation method.
The invention also provides application of the paeonia lactiflora seed meal extract in preparation of a reduced pressure product.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes waste paeonia lactiflora seed meal, and after active ingredients in the paeonia lactiflora seed meal are enriched, the paeonia lactiflora seed meal extract with the blood pressure reducing effect is obtained. The preparation method provided by the invention is simple, not only reduces the environmental pollution caused by random discarding of waste paeonia lactiflora seed meal, but also provides a new idea for developing blood pressure lowering products.
Drawings
FIG. 1 shows the weight and food intake changes of spontaneously hypertensive rats during administration;
FIG. 2 shows changes in systolic (A), mean arterial (B) and diastolic (C) pressures in spontaneously hypertensive rats during dosing;
FIG. 3 shows the results of measurement of red blood cells (A), white blood cells (B) and platelets (C) in each group of rats;
FIG. 4 is a comparison of liver and kidney function related indicators for each group of rats;
FIG. 5 shows Alpha diversity sparse curve (A) versus abundance level curve (B);
FIG. 6 is a graph of Alpha diversity index;
FIG. 7 is PCA of the beta diversity of the sample flora of each group of rats;
FIG. 8 shows the results of the changes in intestinal flora at portal level for each group of rats;
FIG. 9 shows the results of the changes in intestinal flora at the genus level for each group of rats.
Detailed Description
The invention provides a preparation method of a paeonia lactiflora seed meal extract, which comprises the following steps:
pulverizing radix Paeoniae seed cake, mixing with ethanol solution, extracting, filtering to obtain filtrate, and concentrating the filtrate to obtain radix Paeoniae seed cake extract.
In the present invention, the peony seed meal is first crushed by a conventional crushing method in the art, and the crushed peony seed meal is then sieved by a 20-50 mesh sieve, preferably 25-45 mesh, and more preferably 30-40 mesh.
In the present invention, the volume percentage concentration of the ethanol solution is 40 to 60%, preferably 45 to 55%, and more preferably 48 to 52%.
In the invention, the ratio of the paeonia lactiflora seed meal to the ethanol solution is 1g: 5-15 mL, preferably 1g:7 to 13mL, more preferably 1g: 9-11 mL.
In the present invention, the number of times of the extraction is 1 to 3, preferably 2; the time of each extraction is 5-10 hours, preferably 6-9 hours, and more preferably 7-8 hours; the temperature of each extraction is 20 to 30 ℃, preferably 22 to 28 ℃, and more preferably 24 to 26 ℃.
In the present invention, the concentration is 20 to 40%, preferably 25 to 35%, more preferably 28 to 32% of the original volume; the concentration method can be a conventional concentration method in the field.
In the invention, the concentration is preceded by a step of volatilizing ethanol.
The invention also provides the paeonia lactiflora seed meal extract prepared by the preparation method.
The invention also provides application of the paeonia lactiflora seed meal extract in preparation of a reduced pressure product.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The embodiment provides a preparation method of a paeonia lactiflora seed meal extract, which comprises the following steps:
pulverizing radix Paeoniae seed meal by a pulverizer, sieving with a 20-mesh sieve, and taking 100g of radix Paeoniae seed meal and 40% ethanol solution according to a feed-liquid ratio of 1g: mixing 10mL, extracting at 25deg.C for 8h, separating filtrate and residue, and mixing residue with 40% ethanol solution according to a feed-liquid ratio of 1g: mixing at a ratio of 10mL, extracting at 25deg.C for 8 hr, and mixing the filtrates. Volatilizing ethanol in the filtrate, and concentrating under reduced pressure to 40% of the original filtrate volume to obtain radix Paeoniae seed cake extract.
Example 2
The embodiment provides a preparation method of a paeonia lactiflora seed meal extract, which comprises the following steps:
pulverizing radix Paeoniae seed meal by a pulverizer, sieving with a 30-mesh sieve, and taking 100g of radix Paeoniae seed meal and 50% ethanol solution according to a feed-liquid ratio of 1g: mixing 15mL, extracting at 20deg.C for 10h, separating filtrate and residue, and mixing residue with 50% ethanol solution according to a feed-liquid ratio of 1g: mixing at a ratio of 15mL, extracting at 25deg.C for 10 hr, and mixing the filtrates. Volatilizing ethanol in the filtrate, and concentrating under reduced pressure to 30% of the original filtrate volume to obtain radix Paeoniae seed cake extract.
Example 3
The embodiment provides a preparation method of a paeonia lactiflora seed meal extract, which comprises the following steps:
pulverizing radix Paeoniae seed meal by a pulverizer, sieving with a 50-mesh sieve, and taking 100g of radix Paeoniae seed meal and 60% ethanol solution according to a feed-liquid ratio of 1g: mixing 8mL, extracting at 30deg.C for 5h, separating filtrate and residue, and mixing residue with 60% ethanol solution according to a feed-liquid ratio of 1g: mixing at a ratio of 8mL, extracting at 30deg.C for 5h, and mixing the filtrates. Volatilizing ethanol in the filtrate, and concentrating under reduced pressure to 20% of the original filtrate volume to obtain radix Paeoniae seed cake extract.
Experimental example 1
The experimental example explores the improvement effect of the paeonia lactiflora seed meal extract on high-pressure rats, and the specific steps are as follows:
45 male spontaneous hypertensive rats (tail artery systolic blood pressure >140mm Hg), SPF grade, 11 week old, purchased from si Bei Fu (beijing) biotechnology limited, license number: SCXK (Beijing) 2019-0010 was randomly divided into model group, positive group, low dose group, medium dose group, and high dose group, 9 each. 9 healthy male Wistar-Kyoto (WKY) rats, SPF grade, 11 week old, purchased from si Bei Fu (beijing) biotechnology limited, license number: SCXK (Beijing) 2019-0010, set as blank group. All rats were housed in separate cages for 10d with no more than 5 rats per cage. The experimental animals are fed to SPF-class animal experiment centers of pharmaceutical inspection institute in Gansu province, and the use license number of the experimental animals is as follows: SYXK 2021-0003, room temperature 20+ -2deg.C, humidity 40-60%, day and night light and shade alternation time of 12h/12h, and free intake of drinking water. Blank group, model group rats: the stomach was irrigated with physiological saline, 10mL/kg/d daily, for 6 weeks. Positive control group: captopril, 2.25mg/kg/d was infused daily for 6 weeks continuously. The low dose group, the medium dose group and the high dose group were respectively and daily lavaged for 6 weeks with 0.45g/kg/d, 0.90g/kg/d, 1.80g/kg/d of the peony seed meal extract prepared in example 1.
The blood pressure of the rat in the awake state was measured using a BP-2010A intelligent noninvasive sphygmomanometer (available from Beijing soft-tipped biotechnology Co., ltd., model: BP-2010A) and kept quiet indoors. The pressurizing sensor is fixed at 1/3 position of the tail near the root for pressurizing, and after the pulse wave is stable, the systolic pressure and the diastolic pressure are automatically measured. Each rat was measured 3 times in succession, averaged, and the measurement was recorded, once every two weeks, with each rat blood pressure measurement being made during the same time period. The general status of spontaneous hypertensive rats was observed daily, and the general status of mental state, ingestion of drinking water, urination and defecation, respiration, hair shape, posture, etc. was recorded. Feed was weighed for rats 1 time a week in the morning and the remaining amount was weighed the same time the next day in the morning, and daily food intake was calculated. Rat body weight was recorded at the same time every week. After the experiment was performed for 6 weeks, the rats of each group were fasted without water for 12 hours, and the rats were anesthetized by intraperitoneal injection of chloral hydrate at 10% and blood was taken from femoral artery. Part of the mixture is gently mixed in an anticoagulation tube and used for blood routine (red blood cells, white blood cells and platelets) detection. The other part is not anticoagulated, and after standing for 1h at room temperature, the serum is separated by centrifugation at 3000r/min for 15min, and the serum Albumin (ALB), globulin (GLB), plasma albumin/globulin (white ball ratio, A/G), glutamic oxaloacetic transaminase/glutamic pyruvic transaminase (AST/ALT) and bile acid (TBA) levels are measured by a full-automatic biochemical analyzer. The experimental results were all statistically analyzed using SPSS 25.0 software.
Experimental results:
1. general condition, body mass, and food intake of rats
All rats were well-conditioned during the experiment, and only 1 rat was dead, and the dissection was found to be due to the lavage technique, and the death caused by the drug was not observed. The initial weight difference of each group of rats has no statistical significance, daily average food intake and weight are in an increasing state during the administration period, and the food intake and weight difference of each group in the same period have no statistical significance (P > 0.05), and the results are shown in tables 1-2 and figure 1.
TABLE 1 spontaneous hypertensive rats weight change during dosing [ (]n＝9，g)
Table 2. Spontaneous hypertensive rats fed changes during dosing (x+ -s, n= 9,g)
2. Blood pressure
The blood pressure of the blank group is stable at different time points, has no obvious rising or falling trend, and eliminates the change of the blood pressure caused by the factors of external environment, diet and the like. The blood pressure of the model group continuously rises, and the difference is obvious compared with the normal control group at different times, and the difference is statistically significant (P < 0.05). The systolic pressure, the mean arterial pressure and the diastolic blood pressure of the high-dose group and the positive group are obviously reduced compared with the model group after two weeks of administration, the effect of the high-dose group is slightly better than that of the positive group, the low-dose group is obviously different from that of the positive group, and the difference is statistically significant (P < 0.05). The blood pressure of each administration group was significantly reduced compared with the model group after four weeks of administration, and there was no significant difference from the positive drug, and the results are shown in tables 3 to 5. The trend of the high-dose group is the same as that of the positive group, the early-stage depressurization is rapid and then is slow, the medium-dose group shows a uniform descent process, and the administration groups have no obvious difference after 6 weeks of administration, and the result is shown in figure 2.
TABLE 3 spontaneous hypertensive rats systolic pressure changes during dosingn＝9，mmHg)
| Group of | Before administration of the drug | Administration is carried out for 2 weeks | Administration is carried out for 4 weeks | Administration is carried out for 6 weeks |
| Blank group | 120.97±6.44 | 126.17±3.09 | 123.10±3.27 | 120.47±2.19 |
| Model group | 189.11±14.09 ### | 192.26±12.11 ### | 195.26±14.59 ### | 196.33±13.95 ### |
| Low dose group | 186.19±12.31 | 182.22±23.12 Δ | 166.22±16.80 ** | 158.19±11.77 *** |
| Medium dose group | 191.15±10.95 | 177.56±19.17 | 163.31±14.73 *** | 148.41±18.43 *** |
| High dose group | 185.93±9.82 | 158.70±10.35 *** | 145.33±12.85 *** | 141.48±8.26 *** |
| Positive group | 186.19±9.66 | 160.33±19.74 ** | 151.20±17.50 *** | 142.78±14.6 *** |
Note that the blank set was compared to the model set, ### P<0.001; the dosing group was compared to the model group, ** P<0.01， *** P<0.001; each dose group was compared with the positive group, Δ P<0.05。
TABLE 4 mean arterial pressure changes in spontaneously hypertensive rats during dosingn＝9，mmHg)
Note that the blank set was compared to the model set, ### P<0.001; the dosing group was compared to the model group, ** P<0.01， *** P<0.001; each dose group was compared to the positive group, ΔP<0.05。
TABLE 5 spontaneous hypertensive rat diastolic pressure variation during dosingn＝9，mmHg)
Note that the blank set was compared to the model set, ### P<0.001; the dosing group was compared to the model group, ** P<0.01， *** P<0.001; each dose group was compared with the positive group, Δ P<0.05。
3. comparison of conventional correlation index of blood of rats of each group
Compared with the blank group, the red blood cells and platelets of the model group are obviously increased, the white blood cells are obviously decreased, and the difference is statistically significant (P < 0.001). The red blood cells are obviously improved after administration, and the effects of medium dose and high dose are better than those of positive groups (P < 0.05). After administration, the white blood cells of the positive group further decreased, and the other administration groups of each dose were improved, and the high dose variability was remarkable (P < 0.01). After administration, there was no significant difference between the platelet administration groups, and the results are shown in fig. 3.
4. Comparison of liver and kidney function related indicators of rats of each group
The model group showed significantly elevated albumin compared to the blank group, the difference was statistically significant (P < 0.01), and the remaining dosing groups were not significantly different from the model group. After administration, the AST (glutamic oxaloacetic transaminase)/ALT (glutamic oxaloacetic transaminase) of each administration group is improved compared with the model group, and the effect of the low and medium dose groups is better than that of the positive group, but no obvious difference exists. After administration, there was no significant difference (P > 0.05) in globulin, white ball ratio, bile acid compared to model group, see figure 4.
The results show that the blood pressure of the model group continuously rises in the whole treatment period, the blood pressure reducing trend of the high-dose group and the positive group is the same, the early blood pressure reduction is rapid and then is slower, and the medium-low dose group shows a uniform speed descending process. Compared with the model, the high dose and the positive group have obvious antihypertensive effect after two weeks of administration, the medium dose and the high dose have no obvious difference from the positive drug, the blood pressure of each administration group is obviously reduced after 6 weeks of administration, and each administration group has no obvious difference. In general, the extract prepared by the invention has better antihypertensive effect. The blood routine examination is one of the most basic clinical laboratory examinations, mainly comprises a red blood cell system, a white blood cell system and a platelet system, has important significance for diagnosing early signs of systemic diseases, anemia, blood system diseases and the like, and has the defects of increased white blood cells and inflammation, and is mostly bacterial infection. Compared with the model group, the red blood cells and the white blood cells are significantly improved after the administration group. Blood biochemistry detects liver and kidney function related indexes, rats in each group have no obvious liver and kidney injury, and have certain improvement after administration compared with a model group, but the rats in each group have no obvious difference, and the total rats are in a normal range.
Experimental example 2
The experimental example explores the effect of the paeonia lactiflora seed meal extract on improving intestinal flora of a hypertensive rat, and the method comprises the following specific steps:
after the grouping administration of different groups of rats is finished, 2-3 particles of fresh feces of each group of rats are collected in a sterilization centrifuge tube, and frozen at-80 ℃ for standby;
DNA extraction and detection: DNA was quantified using Nanodrop and DNA extraction quality was checked by 1.2% agarose gel electrophoresis.
PCR amplification of target fragment: amplification conditions: 95 ℃ for 5min;95 ℃ for 1min;50 ℃ for 1min;72 ℃ for 1min; 7min to 15cycles at 72 ℃;1.8% agarose gel, 120V40 min electrophoresis, cut the fragment of interest, and recovery.
And (3) purifying and recovering amplified product magnetic beads: (1) to 25. Mu. LPCR product was added 0.8 volume of magnetic beads (Vazyme VAHTSTM DNACleanBeads), shaken, fully suspended, adsorbed on a magnetic rack for 5min, and the supernatant was aspirated with a pipette. (2) Adding 20 mu L of 0.8 times magnetic bead washing liquid, vibrating, fully suspending, placing on a magnetic rack, adsorbing for 5min, and carefully sucking out the supernatant. (3) 200 mu L of 80% ethanol is added, and the mixture is reversely placed on a magnetic rack, and is adsorbed on the other surface of the PCR tube by using magnetic beads, and the supernatant is sucked out after full adsorption. (4) Standing at room temperature for 5min until alcohol volatilizes completely, and the magnetic beads are cracked. (5) Add 25. Mu.L of the Elution Buffer for Elution. (6) The PCR tube was placed on an adsorption rack for 5min, fully adsorbed, and the supernatant was removed to a clean 1.5mL centrifuge tube for storage.
Fluorescent quantification of amplified products: the PCR amplification recovery product was subjected to fluorescent quantitation with a quantitive reagent Quant-iTPicoGreen dsDNAAssayKit and a quantitation instrument Microplate reader (BioTek, FLx 800).
Sequencing library preparation: sequencing libraries were prepared using TruSeqNano DNALT LibraryPrep Kit from Illumina corporation. (1) Firstly, repairing the sequence End of an amplification product, cutting off a protruding base at the 5 'End of a DNA sequence through End Repair mix 2 in a kit, and simultaneously adding a phosphate group and supplementing a missing base at the 3' End; (2) adding an A base at the 3' end of the DNA sequence to prevent the DNA fragment from self-linking, and simultaneously ensuring that the target sequence can be connected with a sequencing joint; (3) adding a sequencing linker containing a library-specific tag (i.e., index sequence) at the 5' end of the sequence to allow the DNA molecule to be immobilized on the Flow Cell; (4) adopting BECKMANAMPure XP Beads, removing the self-connecting segment of the joint through magnetic bead screening, and purifying the library system added with the joint; (5) carrying out PCR amplification on the DNA fragments connected with the connectors so as to enrich the template of the sequencing library, and purifying the enriched product of the library again by adopting BECKMANAMPure XP Beads; (6) the final fragment selection and purification was performed on the library by 2% agarose gel electrophoresis. Prior to on-machine sequencing, the library was inspected for quality on an AgilentBioanalyzer using Agilent High Sensitivity DNAKit. The qualified library had and only a single peak and was linker-free. The library was then quantitated using Quant-iT PicoGreen dsDNAAssayKit on a Promega QuantiFluor fluorescent quantitation system, and the qualified library concentration should be above 2 nM. After the qualified sequencing libraries (Index sequence is not repeatable) are diluted in a gradient manner, the sequencing libraries are mixed according to the required sequencing amount according to the corresponding proportion, and are denatured into single strands by NaOH for sequencing on the machine.
NovaSeq on-machine sequencing: double-ended sequencing was performed using a NovaSeq sequencer, with the corresponding reagent being NovaSeq 6000SP reagent kit (500 cycles).
High throughput sequencing: the library was first quality checked at Agilent Bioanalyzer using Agilent High Sensitivity DNAKit and then quantified using Quant-iT PicoGreen dsDNA Assay Kit on Promega QuantiFluor fluorescent quantitation system (library concentration should be above 2 nM). After the qualified sequencing libraries of each machine are diluted in a gradient manner, the sequencing libraries are mixed according to the required sequencing amount according to the corresponding proportion, and are denatured into single strands by NaOH to carry out machine-on sequencing (Novageq sequencer is used for double-end sequencing, and the corresponding Reagent is Novageq 6000SP Reagent Kit (500 cycles)).
Experimental results:
alpha diversity analysis
The sparse curve (Rarefaction Curve) can directly reflect the rationality of the amount of sequencing data, with a flatter curve indicating that the sequencing result is sufficient to reflect the diversity contained in the current sample. As shown in a in fig. 5, the number of OTUs tends to be gentle as the amount of sequencing data extracted increases, indicating that the amount of sequencing data is progressive. The abundance ranking curve (Rank abundance curve) can visually reflect the abundance and uniformity of species in the sample. As shown in B in fig. 5, the abundance level graph eventually flattens, which indicates that the sample size used in the sequencing of the experiment is sufficient, and the species in the sample are uniformly distributed and have wide coverage. Both figures demonstrate that this sequencing depth and sample size are sufficient and that all data meet the analysis requirements.
Alpha diversity refers to diversity within a particular environment or ecosystem, primarily reflecting species richness and uniformity and sequencing depth. The overt-patterns index (also called Richness index) and the Chao1 index were used to evaluate the abundance of the flora; simpson index and Shannon index were used to evaluate microbial diversity; the Pielou index reflects only uniformity, the larger the value, the more uniform; the goods_coverage index is used to evaluate whether the sequencing result represents the true status of the microorganisms in the sample. As can be seen from fig. 6, compared with the normal group, the model group has significant differences (P < 0.05) in the indexes of the bacterial population diversity of rats, and has a certain improvement effect on the bacterial population diversity and the bacterial population diversity after each administration group is compared with the model group, and the improvement effect of each administration group of the extract prepared by the invention is better than that of the positive control group.
Beta diversity analysis
Principal Component Analysis (PCA) was used to investigate the differences in beta diversity of the intestinal flora of rats. As can be seen from fig. 7, the model group was far from the rest of the groups, and the rest of the five groups were gathered together, indicating that the intestinal flora of spontaneous hypertensive rats was significantly changed. In the blank group and each administration group, the aggregation of the positive group, the medium dose and the normal group was more remarkable, which means that the medium dose and the positive group were better in improvement effect and more approximate to the blank group, but the medium dose was larger in individual difference relative to the positive group. It can be seen in general that the intestinal flora of the rats tends to be normal after administration of each group.
3. Changes in flora abundance
The abundance results of the pre-20 species of the intestinal flora of each group of rats at the portal level indicate (fig. 8) that the model group has an increased abundance of Firmicutes (Firmicutes) compared to the normal group, and that the bacteroides relative abundance (bacterioides) has decreased, and accordingly the F/B ratio of the model group has increased. In addition to the two dominant mycoplasmas, the relative abundance of TM7 mycoplasmas, amycolates (tendrils), proteasomes (Proteobacteria) of the model group was increased. After administration, the bacteroides abundance of each administration group is increased, the bacteroides abundance is reduced, and the F/B ratio of each group is increased to a certain extent correspondingly. In addition, the administration groups of TM7 phylum, proteus were all somewhat lower than the model group. All the changes show that the improving effect of the preparation on the hypertension is closely related to intestinal flora, and the occurrence and development of the hypertension are highly related to the abundance changes of bacteroides, proteus, TM7 bacteria and thick-walled bacteria.
As shown in FIG. 9, the abundance of the species 20 before the excretion of the intestinal flora of each group of rats at the genus level indicates that the genus Lactobacillus, prevotella, ruminococcus, oscillospira, coprococcus, clostridium, blautia, allobaculum, bifidobacterium is dominant. The intestinal bacteria of the spontaneous hypertension rat are greatly changed before and after administration. The abundance of the genus Lactobacillus, prevotella, ruminococcus, oscillospira, coprococcus, clostridium, blautia, allobaculum, bifidobacterium, turicibacter, parabacteroides, bacteroides, etc. in the model group was varied to some extent as compared to the normal group. Wherein the bacteria with increased abundance in the model group are mainly Lactobacillus, clostridium, allobaculum; the fungus genus with reduced abundance of the model group mainly comprises Prevotella, oscillospira, coprococcus, blautia;
under normal state, the abundance of each intestinal microorganism is in dynamic balance, and under the participation of multiple ways of intestinal flora, each life activity of human body is orderly carried out. The development of many diseases results in a disruption of the balance of intestinal flora, resulting in a reduction in beneficial bacterial species, an increase in harmful bacterial species, and a change in the proportion of various bacteria, which in turn can further exacerbate the disease or cause other complications, and thus, in the course of disease treatment, attention is paid to changes in intestinal flora in addition to phenotype. Compared with the model group rats, the model group rats have reduced flora diversity, richness and uniformity, obvious change of flora at the portal level and the genus level, and great improvement after administration.
According to the embodiment, the invention provides the paeonia lactiflora seed meal extract, the preparation method and the application thereof, the effective components in the waste paeonia lactiflora seed meal are enriched, the preparation method is simple, and the obtained paeonia lactiflora seed meal extract has obvious blood pressure reducing effect and provides a new idea for developing blood pressure reducing products.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The preparation method of the paeonia lactiflora seed meal extract is characterized by comprising the following steps of:
pulverizing radix Paeoniae seed cake, mixing with ethanol solution, extracting, filtering to obtain filtrate, and concentrating the filtrate to obtain radix Paeoniae seed cake extract.
2. The preparation method of the paeonia lactiflora seed meal extract according to claim 1, wherein the volume percentage concentration of the ethanol solution is 40-60%.
3. The method for preparing the paeonia lactiflora seed meal extract according to claim 1 or 2, wherein the proportion of the paeonia lactiflora seed meal to the ethanol solution is 1g: 5-15 mL.
4. The method for preparing paeonia lactiflora seed meal extract according to claim 1, wherein the extraction times are 1-3, the time of each extraction is 5-10 h, and the temperature of each extraction is 20-30 ℃.
5. The method for preparing paeonia lactiflora seed meal extract according to claim 1, wherein the concentration is 20-40% of the original volume.
6. The method for preparing paeonia lactiflora seed meal extract according to claim 1 or 5, wherein the step of volatilizing ethanol is further included before the concentration.
7. The peony seed meal extract prepared by the preparation method of any one of claims 1 to 6.
8. The use of paeonia lactiflora seed meal extract according to claim 7 for preparing a reduced pressure product.
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