CN113694155B - Kaempferia galanga total flavone extract and preparation method and application thereof - Google Patents
Kaempferia galanga total flavone extract and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a small flower kaempferia galangal total flavone extract and a preparation method and application thereof. The invention takes dried rhizome of kaempferia parviflora as raw material, after crushing and extraction, AB-8 or HPD300 macroporous resin column is adopted for adsorption, separation and purification, and the kaempferia parviflora total flavone extract with the purity of more than 90 percent is obtained after drying. The invention prepares the kaempferia galanga total flavone extract into a solid dispersion of the kaempferia galanga total flavone, and combines the kaempferia galanga total flavone extract with wheat germ oil to obtain a preparation of soft capsules, micro-emulsion and self-micro-emulsion, thereby achieving the purpose of improving the solubility and bioavailability of the kaempferia galanga total flavone. The composition of the kaempferia galangal total flavone extract and the wheat germ oil prepared by the invention has a synergistic interaction effect, can obviously enhance the reproductive function of fruit flies, has an anti-aging effect, and has high nutritional and medicinal values.
Description
Technical Field
The invention relates to a kaempferia galanga total flavone extract as well as a preparation method and application thereof, belonging to the technical field of medicines.
Background
Kaempferia parviflora (Kaempferia parviflora) is a plant belonging to the family Zingiberaceae for many years and is mainly produced in northern Thailand, northeast, laos and southeast Asia. The rhizome of kaempferia galanga, also called as black ginger, has long application history, is used for treating various diseases, such as inflammation, ulcer, gout, abscess, allergy, osteoarthritis, fatigue, gastrointestinal dysfunction, sexual dysfunction, cancer, hypertension and the like, is mainly related to active ingredient methoxy flavonoid compounds contained in the rhizome of kaempferia galanga, and needs to be systematically researched and developed in order to better develop and utilize the resource of kaempferia galanga due to lower total content of methoxy flavonoid in medicinal materials.
Chinese patent application (application No. 201580026991.3) discloses a black ginger oil extract and a manufacturing method thereof, wherein water, a hydrophilic solvent or a mixture thereof is firstly contacted with black ginger to extract methoxyflavone to obtain an intermediate extract, and then oil such as soybean oil is contacted with the intermediate extract to achieve the purpose of extracting methoxyflavone. However, this method has the disadvantage of a complex composition of the fat, which leads to the extraction of methoxyflavonoids and the introduction of other impurities which are difficult to remove. Chinese patent application (201810869975.6) discloses an anti-aging black ginger extract and a preparation method thereof, wherein the black ginger extract is prepared by freezing an effective part of black ginger with liquid nitrogen, and crushing into powder to obtain black ginger powder; then extracting the black ginger powder by using a pyridine bromide saline solution as an extraction solvent to obtain an anti-aging black ginger extract; then, ethyl acetate and n-hexane-ethanol are respectively adopted as extracting agents to carry out extraction twice, so as to achieve the purposes of purifying and enriching the methoxy flavonoid compounds. However, this method has the disadvantages of harsh conditions for the disruption step and complex extraction reagents, which may lead to the potential toxicity risk of organic reagent residues.
Disclosure of Invention
The invention aims to provide a kaempferia galanga total flavone extract, which is prepared into a solid dispersion form and is compounded with wheat germ oil to form a preparation form of microemulsion and self-microemulsion, so that the solubility and bioavailability of the kaempferia galanga total flavone are improved, and the kaempferia galanga total flavone extract can be used in the fields of nutrition, health care and pharmacy.
The kaempferia galanga total flavone extract and wheat germ oil composition have a synergistic effect, have an anti-aging effect while remarkably enhancing the reproductive function of fruit flies, and have high nutritional and medicinal values.
The preparation method of the kaempferia galanga total flavone extract provided by the invention comprises the following steps:
s1, crushing rhizome of kaempferia galanga to obtain rhizome powder of kaempferia galanga;
s2, extracting the rhizome powder of the rhizoma kaempferiae by adopting an ethanol water solution to obtain an extracting solution;
s3, concentrating the extracting solution, separating by using macroporous adsorption resin, collecting eluent, and drying to obtain a small flower kaempferia galangal total flavone extract;
in the kaempferia galanga total flavone extract obtained by the method, the purity (calculated by the total of six components, namely 5,7,3',4' -tetramethoxyflavone, 3,5,7,3',4' -pentamethoxyflavone, 5,7-dimethoxyflavone, 5,7,4 '-trimethoxyflavone, 3,5,7-trimethoxyflavone and 3,5,7,4' -tetramethoxyflavone) detected by HPLC (high performance liquid chromatography) reaches over 90 percent.
In the preparation method, in the step S1, the rhizome of kaempferia galanga is dried and then crushed, and the powder of the rhizome of kaempferia galanga is obtained by sieving the dried rhizome with a 60-mesh sieve.
In the above preparation method, in step S2, the volume content of ethanol in the ethanol aqueous solution is 50 to 90%, preferably 60%;
the proportion of the kaempferia galanga rhizome powder to the ethanol water solution is as follows: 1;
extracting under the condition of ultrasound;
the extraction time is 20-30 min, preferably 25min, and the extraction time is 1-3 times, preferably 2 times.
In the preparation method, in the step S3, the extracting solution is concentrated under reduced pressure until the density of the concentrated solution is 4-6 g/mL;
the macroporous adsorption resin is AB-8 macroporous adsorption resin or HPD300 macroporous adsorption resin;
eluting with 95% ethanol solution, collecting eluate, concentrating under reduced pressure by evaporation to obtain soft extract, and vacuum drying.
The kaempferia galanga total flavone extract prepared by the method also belongs to the protection scope of the invention.
The invention also provides a solid dispersion of the kaempferia galanga total flavone extract, which is prepared from the kaempferia galanga total flavone extract and polyethylene glycol;
in the solid dispersion of the kaempferia galanga total flavone extract, the content of the kaempferia galanga total flavone extract is 10-20% by mass;
the polyethylene glycol is PEG6000 or PEG4000, preferably PEG6000;
the solid dispersion of the kaempferia galanga total flavonoid extract can obviously improve the solubility of the kaempferia galanga total flavonoid extract and provide a usable preparation for improving the bioavailability of the kaempferia galanga total flavonoid extract.
The solid dispersion of the kaempferia galanga total flavonoid extract can be prepared by the following steps:
adding the polyethylene glycol into the kaempferia galanga total flavone extract, heating and melting to mix, then placing in an ice-water bath for solidification, and drying and grinding the cooled solid to obtain the kaempferia galanga total flavone extract;
the cooling time can be 5-30 min;
drying under reduced pressure at 40 deg.C for 1h, grinding, and sieving with 60 mesh sieve.
The invention also compounds the kaempferia galangal total flavone extract and wheat germ oil to form a composition, and concretely forms a soft capsule form;
the mass ratio of the kaempferia galangal total flavone extract to the wheat germ oil can be 1:0.5 to 10, such as 1:1 to 5 or 1:2;
the soft capsule is prepared from the kaempferia galangal total flavone extract, wheat germ oil, gelatin, glycerol, titanium dioxide and water, and can be prepared by the following steps:
mixing the kaempferia galanga total flavone extract and the wheat germ oil to obtain soft capsule contents;
mixing the gelatin, the glycerol, the titanium dioxide and the water for swelling, and uniformly stirring to obtain a soft capsule shell glue solution;
pressing and forming the soft capsule content and the soft capsule shell glue solution to obtain the soft capsule;
the titanium dioxide, the glycerol, the gelatin and the water can be prepared according to a conventional proportion;
the soft capsule content and the soft capsule shell glue solution can be prepared according to the conventional proportion.
The invention also compounds the kaempferia galanga total flavone extract and wheat germ oil to form a composition, and particularly forms a micro-emulsion or self-micro-emulsion form;
the mass ratio of the kaempferia galangal total flavone extract to the wheat germ oil can be 1:0.5 to 10, such as 1:1 to 5 or 1:2;
the microemulsion is prepared from polyoxyethylene hydrogenated castor oil, propylene glycol, the kaempferia galangal total flavone extract, the wheat germ oil and water, and can be prepared by the following steps:
mixing the polyoxyethylene hydrogenated castor oil, the propylene glycol and the wheat germ oil as an oil phase;
adding the kaempferia galanga total flavone extract into the oil phase, uniformly stirring, dropwise adding the water, and uniformly stirring to obtain the kaempferia galanga total flavone extract;
the mass ratio of the kaempferia galanga total flavone extract to the polyoxyethylene hydrogenated castor oil, the propylene glycol and the water can be 1:5 to 15:3 to 6:25 to 35, such as 1:13.5:4.5:28.5.
the self-microemulsion is prepared from glyceryl triacetate, polyoxyethylene hydrogenated castor oil, diethylene glycol monoethyl ether, the kaempferia galanga total flavone extract and the wheat germ oil, and can be prepared by the following steps:
mixing the polyoxyethylene hydrogenated castor oil and the diethylene glycol monoethyl ether to serve as a mixed emulsifier;
mixing the mixed emulsifier with the glycerol triacetate and the wheat germ oil, magnetically stirring in a constant-temperature water bath at 37 ℃ for 10min, and uniformly mixing to obtain the wheat germ oil-containing water-based emulsifier;
the mass ratio of the kaempferia galanga total flavone extract to the polyoxyethylene hydrogenated castor oil, the glyceryl triacetate and the diethylene glycol monoethyl ether can be 1:5 to 15:2 to 5:8 to 15, such as 1:8.75:3.75:11.5.
the microemulsion and the self-microemulsion can improve the solubility and bioavailability of the kaempferia galanga total flavonoids, and the kaempferia galanga total flavonoid extract and the wheat germ oil composition have a synergistic effect, so that the reproductive function of the fruit flies is obviously enhanced, and the fruit flies have an anti-aging effect and high nutritional and medicinal values.
The invention has the following beneficial technical effects:
1. the preparation method of the kaempferia galanga total flavone extract provided by the invention has the advantages of simple preparation process and high extract purity, and provides a technical basis for industrial production and application in the fields of nutrition and pharmacy of the kaempferia galanga total flavone;
2. the composition of the kaempferia galanga total flavone extract and the wheat germ oil provided by the invention can obviously improve the reproductive activity of fruit flies and prolong the life of the fruit flies.
3. The solid dispersion preparation of the kaempferia galanga total flavone extract provided by the invention can obviously improve the solubility of the kaempferia galanga total flavone extract and provides a usable preparation for improving the bioavailability of the kaempferia galanga total flavone extract.
4. The micro-emulsion and self-micro-emulsion preparation of the composition of the kaempferia galanga total flavone extract and the wheat germ oil can obviously improve the release of the kaempferia galanga total flavone, thereby improving the bioavailability of the medicine in vivo.
Drawings
FIG. 1 is a response surface diagram showing the influence of interaction between two factors on composite score in example 1 of the present invention.
FIG. 2 shows the extraction rate of 6 methoxy flavonoids in Kaempferia galanga when ethanol with different concentrations is used for extraction.
Fig. 3 shows the sexual activity (mating latency (left histogram), mating duration (right histogram) of drosophila flies in different test groups within 30min and 60min, P <0.05 compared to normal control group.
Fig. 4 shows the experimental results of the fruit fly fertility in each test group, compared to the normal control group, P <0.05.
Fig. 5 shows the experimental results of the survival lives of the drosophila flies in each test group (half the death time, the average life and the average maximum life from left to right), wherein P is less than 0.05 compared with the normal control group.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The rhizome of Kaempferia parviflora used in the following examples was purchased from Guangxi Shuitai Biotech Co., ltd. (batch: 201805) and identified as a dried rhizome of Kaempferia parviflora of Zingiberaceae.
Example 1: optimization of extraction method of kaempferia galanga general flavone
The instrument comprises: LC-20AT high performance liquid chromatography (Shimadzu corporation, japan); KQ-500DE model digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); RT-12SF Mill (Tester instruments, inc., tianjin); MS104S analytical balance (ningbo xinzhi biotechnology ltd); 78100-11 acid-proof refrigerated desktop concentrators (Labconco, usa); LD5-2B centrifuge (Beijing King-Li centrifuge, inc.), milli-Q ultrapure water meter (Milipore, USA); eppendorf centrifuge 5417R small high-speed refrigerated centrifuge (Germany professional Life sciences Co.).
Liquid chromatography detection conditions: lichrospher RP-18 endblocked column (4.6 mm. Times.250mm, 5 μm); mobile phase: 0.5% formic acid aqueous solution (A) and 0.5% formic acid acetonitrile solution (B), gradient elution (0-10min, 38-20min, 38-40% B, 20-35min, 40% B, 35-50min, 40-44% B, 50-55min, 44-38% B, v/v); flow rate 0.8min mL -1 (ii) a The column temperature is 30 ℃; the detection wavelength is 350nm, and the sample injection amount is 10 mu L.
Preparation of mixed control solution: taking a proper amount of reference substances of 5,7,3',4' -tetramethoxyflavone, 3,5,7,3',4' -pentamethoxyflavone, 5, 7-dimethoxyflavone, 5,7,4 '-trimethoxyflavone, 3,5, 7-trimethoxyflavone and 3,5,7,4' -tetramethoxyflavone, precisely weighing, placing in a 10mL measuring flask, adding ethanol for dissolving and fixing the volume to prepare mixed reference substances with the mass concentrations of 102.00, 296.25, 213.00, 262.50, 102.50 and 86.80 mu g/mL respectively for later use.
On the basis of single-factor optimization, a response surface method is adopted to investigate and optimize the preparation method of the kaempferia galanga total flavonoids. Taking comprehensive scores (M) obtained by calculating the percentage content of 6 methoxy flavonoid components in medicinal materials as evaluation indexes, adopting a three-factor three-level response surface method to carry out optimization test design, carrying out 17 extraction experiments in total, and specifically comprising the following steps:
the dried rhizome of kaempferia galanga is crushed, sieved by a No. four sieve, precisely weighed to obtain 1g of powder, placed in a conical flask with a plug, added with ethanol (40, 50 and 60%) with a certain concentration according to a certain material-liquid ratio (1. Taking a proper amount of supernatant, diluting by a certain multiple, filtering by a 0.45 mu m microporous filter membrane, and carrying out HPLC detection analysis.
Taking the percentage content of 6 main methoxy flavonoid components in kaempferia galanga as an index, solving the weight coefficient of each index by adopting an information entropy method, calculating a comprehensive score M, and establishing an original evaluation index matrix (X) ij ) mn, processing the detection data of the methoxy flavonoid components in the 17 times extraction solution, and establishing a probability matrix (P) ij ) mn, converting the original evaluation matrix into a probability matrix.
And analyzing the experimental result by using Design-Expert10.0.7 software, and obtaining a binomial regression fitting equation of the ethanol concentration (A), the feed-liquid ratio (B) and the ultrasonic time (C) by using the comprehensive score (M) as a response value.
M＝0.96+0.06A+0.089B-0.012C-0.016AB-9.325×10 -3 AC-8.45×10 -3 BC-0.013A 2 -0.1B 2 -0.019C 2
Type F value was 62.19 and significance level of the overall model was P<0.0001, which shows that the regression model is extremely remarkable, and shows that the relation between each index and M can be functionalized by using the model; the distortion term F =6.85, p =0.0470<0.05, the equation can be used for replacing a real test point for analysis, and the R of the model can be obtained from the analysis value of software 2 Is 0.9876 of R 2 (Adj) is 0.9718, which indicates good fitness of the regression equation; C.V% is 1.79, the test accuracy is good, and the data show that the model can be well used for predicting the optimization of the extraction process of the main flavonoid components in the kaempferia galanga. Factors A, B 2 And C 2 The effect on the composite score M is very significant (P)<0.0001 And factors C, A) 2 And the interaction items AB, AC and BC have no significant difference on the influence of the comprehensive score M (P)>0.05)。
And (3) fitting the experimental result data by using Design-Expert10.0.7 software to obtain a response surface graph (figure 1) of the interaction of every two factors on the influence of the comprehensive score M, and analyzing and evaluating the interaction effect of any two factors influencing the M value by using the response surface graph. The optimization process through simulation analysis comprises the following steps: the ethanol concentration is 60%, and the feed-liquid ratio is 1 (g/mL), and the extraction is carried out for 2 times, each time for 25min.
Furthermore, experimental verification is carried out on the optimized extraction process, and the specific steps are as follows:
precisely weighing 3 parts of medicinal materials, each part of the medicinal materials is 1.0g, extracting according to an optimized process, wherein the ethanol concentration is 60%, and the material-liquid ratio is 1 (g/mL), and extracting for 2 times, each time for 25min. The comprehensive score M of the verification result is 0.9939 +/-0.340, the RSD is 0.340%, under the condition, the extraction rates of 6 methoxy flavone components are 0.094%, 0.712%, 2.891%, 1.215%, 0.398% and 0.487%, the total extraction rate is 5.797%, the comprehensive score of the verification experiment is highly approximate to the predicted value (1.024) of the model, the model is accurate and credible, and the method can be used for extracting the flavonoid components in the kaempferia galanga.
Example 2: comparative analysis of extraction rate of methoxyl total flavonoids by using different ethanol concentrations
Fixing the feed-liquid ratio of 1. Taking a proper amount of extract to prepare a sample for HPLC detection and analysis. The percentage content of 6 methoxy flavones is plotted against the concentration of ethanol, as shown in figure 2. When the concentration of the ethanol is 60 percent, the extraction rate of 6 methoxy flavones is the highest; when the concentration of the ethanol is 90%, the extraction rate of 6 methoxy flavones is 5.44%, which is slightly lower than that when the concentration of the ethanol is 60%; when the concentration of the ethanol is 30%, the extraction rate of the 6 methoxy flavones is only 3.15%. When the concentration of the ethanol is lower, the extraction yield of the methoxy total flavonoids is increased along with the increase of the concentration of the ethanol, the extraction yield is the highest when the concentration of the ethanol is 60 percent, and the concentration of the ethanol is increased to 90 percent, so that the yield of the methoxy total flavonoids is not further improved.
Example 3: purification and enrichment method of total flavone components of kaempferia galanga
Purifying and enriching the total flavone components of the kaempferia galanga by column chromatography. Firstly, three different types of macroporous resin AB-8, HPD300 and NKA-2 are selected to examine the adsorption performance of the macroporous resin AB-8, the HPD300 and the NKA-2 on flavonoid components. The method comprises the following specific steps:
(1) And performing reduced pressure rotary evaporation concentration on the kaempferia galanga extract by using a rotary evaporator to ensure that the density of the concentrated extract is 5g of medicinal material powder/mL.
(2) Placing 5.0g of each of the three macroporous resins in a conical flask, adding 1mL of the kaempferia galanga concentrated extract and 50mL of distilled water, and carrying out static adsorption for 24h. After the solution is observed, the appearance of the solution is observed, and it can be seen that the liquid is still cloudy after the NKA-2 resin is statically adsorbed for 24 hours and has no obvious change from the appearance of the solution before adsorption; after static adsorption of the two resins AB-8 and HPD300 for 24 hours, the solution was essentially clear.
(3) And separating the three adsorbed macroporous resins and the solution by adopting a suction filtration method. And (3) respectively taking a proper amount of prepared liquid phase detection samples from the separated solution, detecting the content of 6 kaempferia galanga methoxy flavone components in the solution by using an established HPLC method, and investigating the adsorption effect of the three macroporous resins.
HPLC detection results show that after NKA-2 is statically adsorbed for 24 hours, representative methoxy flavonoid components can still be detected; and the solutions after static adsorption of AB-8 and HPD300 do not detect representative methoxy flavonoid components, which shows that the two macroporous resins have good adsorption effect on the flavonoid components.
(4) And collecting the three macroporous resins after adsorption, respectively transferring the three macroporous resins into a new conical flask, adding 50mL of 95% ethanol, and standing for 24h. After the reaction is finished, a proper amount of solution is taken to prepare a liquid phase detection sample, the content of 6 kaempferia galangal methoxy flavone components in the sample is detected by an established HPLC method, and the desorption effect of the three macroporous resins in 95% ethanol is inspected.
HPLC detection results show that the content of 6 kaempferiylmethoxy flavone components in the solution after the NKA-2 macroporous resin is statically desorbed for 24 hours is the lowest; the content of methoxyl flavone in the solution after desorption of AB-8 and HPD300 macroporous resin is relatively high, and AB-8 is slightly higher than HPD300.
From the analysis, the macroporous resin AB-8 and HPD300 are suitable for purifying and enriching the total flavone components of the kaempferia galanga, and the AB-8 effect is optimal.
Example 4: preparation of small flower kaempferia galanga total flavone solid dispersion
The water solubility of the methoxyflavone is low, and the invention provides a small flower kaempferia galangal total flavone solid dispersion, and the solubility of the small flower kaempferia galangal total flavone solid dispersion in water is measured.
The formula is as follows: kaempferia galanga extract solid powder 0.5g (60 mesh), PEG 6000.0 g.
The preparation method comprises the following steps: preparing the kaempferia galanga extract-PEG 6000 solid dispersion by a melting method. Weighing the solid powder of the kaempferia galanga extract and PEG6000, putting the solid powder and the PEG6000 into a 250mL beaker, heating and melting the mixture, and uniformly mixing the mixture. Placing the beaker in ice water bath, rapidly solidifying, cooling for 10min, drying at 40 deg.C under reduced pressure, grinding, and sieving (60 mesh).
And (3) respectively taking appropriate amounts of the prepared solid powder of the kaempferia galanga extract and the solid dispersion of the kaempferia galanga extract to ensure that the contained kaempferia galanga extract is equal, adding 20mL of pure water, magnetically stirring for 2h, taking the upper solution to prepare a sample, and carrying out HPLC (high performance liquid chromatography) detection, wherein the detection results of 6 main methoxy flavonoids show that the water solubility of the solid dispersion to the kaempferia galanga extract is remarkably improved and is about 2 times of the physical mixture of the solid powder.
The pharmacokinetic results of oral administration of the solid dispersion in rats show that the bioavailability of the solid dispersion is improved by more than 50 percent compared with that of a physical mixture.
TABLE 1 solubility contrast of kaempferia parviflora extract solid dispersions and solid powders
Example 5: preparation method of micro emulsion of composition of kaempferia galangal total flavone extract and wheat germ oil
The formula is as follows: RH40 g, propylene glycol 18g, wheat germ oil 8g, kaempferia galanga general flavone extract 4g and distilled water 114g.
The preparation method comprises the following steps: weighing RH40, propylene glycol and wheat germ oil according to the prescription amount, and uniformly stirring the mixture to obtain an oil phase of the kaempferia galanga total flavone extract; adding 2g of rhizoma Kaempferiae total flavone extract into the oil phase, stirring for 2h, and mixing well; and dropwise adding 120g of distilled water, and uniformly stirring to obtain the water-soluble organic silicon dioxide.
The particle diameter of the prepared microemulsion of the medicine-carrying kaempferia galanga total flavone extract and wheat germ oil composition is 41.79 +/-0.35nm, POI is 0.12 +/-0.03, zeta potential is-18.98 +/-1.87 mV, encapsulation rate is 90.23 +/-1.05%, and the medicine-carrying amount of the kaempferia galanga total flavone is 4.71 +/-0.02%. The prepared drug-loaded microemulsion does not generate turbidity after being placed at room temperature for 1 month, and the particle size does not have obvious change, which shows that the prepared kaempferia galanga total flavone extract and wheat germ oil composition microemulsion has good stability.
Example 6: preparation of self-microemulsion of kaempferia galanga total flavone extract and wheat germ oil composition
The formula is as follows: 15g of glycerol triacetate, 35g of RHF 40, 46g of diethylene glycol monoethyl ether, 8g of wheat germ oil and 4g of kaempferia galanga total flavone extract.
The preparation method comprises the following steps: weighing RH40 and diethylene glycol monoethyl ether according to the prescription amount, uniformly mixing to obtain a mixed emulsifier, mixing the mixed emulsifier with oil-phase glycerol triacetate and wheat germ oil, magnetically stirring in a constant-temperature water bath at 37 ℃ for 10min, and uniformly mixing to obtain the compound emulsifier.
The quality of the prepared drug-loaded kaempferia galanga total flavone extract and wheat germ oil composition self-microemulsion is evaluated, the self-emulsifying time, the self-microemulsion particle size and the saturated drug-loading amount are measured, and the measurement is repeated for 3 times. The prepared self-microemulsion is a light yellow oily liquid, the self-emulsifying time is 14.32 +/-0.45 s, the particle size of the self-microemulsion is 27.88 +/-0.72 nm, and the drug-loading rate of the kaempferia galanga total flavone is 3.98 +/-0.08%.
Referring to the solubility determination method of '0931 dissolution and release degree determination method' of the general rule of the four divisions in the year 2020 edition of Chinese pharmacopoeia, an appropriate amount of kaempferia galangal total flavone extract and wheat germ oil composition self-microemulsion and kaempferia galangal total flavone extract are respectively weighed, 900mL of distilled water is used as a dissolution medium, the temperature is (37 +/-0.5) DEG C, the rotating speed is 50r/min, sample liquid is taken at 5min, 10min, 20min, 30min, 45 min, 60min, 90 min and 120min respectively, the content of 6 main methoxy flavone components in kaempferia galangal is detected according to an HPLC method, and the accumulated dissolution rate (n = 6) is calculated, more than 75% of the drug of the self-microemulsion preparation is dissolved into the medium in 10min, and the accumulated drug release rate of the kaempferia galangal total flavone extract reaches 25% in 30 min. Therefore, the self-microemulsion prepared by the method can effectively improve the dissolution rate of the medicine, thereby being expected to improve the bioavailability of the oral medicine.
Example 7: preparation of soft capsule of composition of kaempferia galanga total flavone extract and wheat germ oil
The formula of the contents is as follows: 4g of kaempferia galangal total flavone extract and 8g of wheat germ oil;
the capsule shell comprises the following components: 30g of gelatin, 30g of distilled water, 10g of glycerol and 0.15g of titanium dioxide.
The preparation method comprises the following steps: weighing the kaempferia galanga total flavone extract and wheat germ oil according to the prescription amount, and uniformly stirring to obtain a capsule content which is uniformly mixed, easy to disperse and viscous; weighing gelatin, glycerol, titanium dioxide and distilled water according to the prescription amount, mixing and swelling, and uniformly stirring to obtain capsule shell glue solution with good elasticity and moderate bubbles generated in the swelling process; and pressing and forming the prepared proper amount of contents and the glue solution to obtain the soft capsule of the composition of the kaempferia galanga total flavone extract and the wheat germ oil.
The capsule surface of the soft capsule of the composition of the kaempferia galangal total flavone extract and the wheat germ oil is smooth, and the conditions of adhesion, leakage and the like do not exist. The obtained soft capsule is subjected to disintegration time limit inspection according to requirements of 0921 disintegration time limit inspection method of the four ministry of general regulation of China pharmacopoeia 2020 edition, and 6 soft capsules are completely disintegrated within 20min, and meet the requirement of complete disintegration within 60min specified by the pharmacopoeia. The kaempferia galanga total flavone extract and wheat germ oil composition are prepared into the soft capsule, so that the soft capsule has accurate dosage and high precision, can be absorbed by a human body more quickly, can prevent the medicine from being oxidized and decomposed, and has the characteristics of good taste, easiness in carrying and convenience in use.
Example 8: effect of Kaempferia galanga Total Flavonoids and wheat germ oil composition on reproductive function
The fruit fly model of the invention inspects the influence of the composition of the kaempferia galangal total flavonoids and the wheat germ oil on the reproductive function and the life span of the fruit flies.
1. Preparation of fruit fly culture medium
(1) Preparing a blank culture medium: placing 5000mL of purified water into an electric pan, heating, adding yeast, corn flour, agar, white sugar and brown sugar according to the formula (shown in table 2) while stirring until the mixture is uniformly stirred; stopping heating when the culture solution is boiled, adding propionic acid according to the prescription amount after 3min, uniformly stirring, standing at room temperature for 30min, adding antibiotic according to the prescription amount, and uniformly stirring to obtain the drosophila culture solution. And filling the fruit fly culture solution into a fruit fly tube, and cooling to solidify to obtain a blank fruit fly culture medium.
TABLE 2 blank Medium formulation
(2) Preparation of a test article culture medium: adopting a formula shown in Table 3 to prepare a conditioned medium of a test sample of a kaempferia galanga total flavone extract group, a wheat germ oil group, a kaempferia galanga total flavone extract and wheat germ oil composition group, wherein the mass ratio of the two test samples of the kaempferia galanga total flavone extract to the wheat germ oil composition group is 1:2. the normal control group was given a blank medium for normal culture.
TABLE 3 culture Medium formulation containing test article
2. Experimental method
(1) And (3) testing the sexual activity: collecting the emerged fruit fly and CO within 8 hours 2 After anesthesia, male and female parts are distinguished and transferred into a conditioned medium respectively for culture. A normal control group, a small flower kaempferia galangal total flavone group, a wheat germ oil group,The composition group of the kaempferia galangal total flavonoids and the wheat germ oil is provided with 3 male flies and 3 female flies, and 20 flies per tube. The culture conditions are as follows: the temperature is 25 +/-1) DEG C, the relative humidity is 45-75%, the culture medium is replaced once every 3 days, and the continuous breeding is carried out for 15 days. Sexual activity assay was performed on the last day: the female and male fruit flies in each group were paired at a ratio of 1 to 1 (12 tubes/group, 5 pairs/tube, 60 pairs total), and the number of pairs of copulating fruit flies, the mating latency and the duration within 30 minutes and 60 minutes were recorded.
(2) And (3) fertility experiment: collecting the emerged fruit fly and CO within 8 hours 2 After anesthesia, male and female parts are distinguished and transferred into a conditioned medium respectively for culture. The normal control group, the small flower rhizoma Kaempferiae total flavone group, the wheat germ oil group, the small flower rhizoma Kaempferiae total flavone and wheat germ oil composition group are respectively provided with 1 tube of male flies and 1 tube of female flies, and 30 flies per tube. The culture conditions are as follows: the temperature is 25 +/-1 ℃, and the relative humidity is 45-75 percent. The medium was changed every 3 days. After 2 weeks of feeding, randomized cohort trials were performed. And 5 pairs of male and female fruit flies in each group of 6 fruit flies in each group are inoculated for 7 days, parents are removed, the number of the fruit flies eclosion every day is counted, and the number of the imagoes hatched within 8 days after the 1 st imagoes are hatched is taken as the number of the F1 fruit flies.
(3) And (3) determining the life: collecting the emerged fruit fly and CO within 8 hours 2 After anesthesia, male and female are distinguished, and the male and female are respectively transferred into a conditioned medium for culture. The normal control group, the small flower rhizoma Kaempferiae total flavone group, the wheat germ oil group, the small flower rhizoma Kaempferiae total flavone and wheat germ oil composition group are respectively provided with 1 tube of male flies and 1 tube of female flies, and 30 flies per tube. The culture conditions are as follows: the temperature is 25 +/-1 ℃ and the relative humidity is 45-75 percent. The medium was changed every 3 days. The number of dead fruit flies was counted up to all natural deaths by observing every day from day 20, and half of the death time, the average life and the maximum life of each group were calculated. Half of the days required for death of the drosophila in each group was half of the death time, the average of the total days for survival of all the drosophila was the average life span, and the average of the last 10 death of drosophila in each group was the average maximum life span of the drosophila in the group.
3. Results of the experiment
(1) And (4) measuring the sexual activity: within 30 minutes, the normal control group and the kaempferia galangal total flavone group are mated without fruit flies, the mating latency period is more than 30 minutes, and the wheat germ oil group, the kaempferia galangal total flavone and wheat germ oil composition group are mated with fruit flies, the mating latency period is obviously shortened (P is less than 0.01), wherein the mating rate within 30 minutes of the composition group reaches 30%, and the mating rate within 30 minutes of the wheat germ oil group reaches 17%.
Within 60 minutes, the mating logarithm of the fruit flies in the small flower galangal total flavone group, the wheat germ oil group, the small flower galangal total flavone and wheat germ oil composition group is higher than that in the normal control group. Wherein the mating rate of the wheat germ oil, the kaempferia galangal and wheat germ oil composition group reaches 20 percent and 45 percent within 60 minutes, which are respectively 4 times and 9 times of that of the normal group. The composition of the wheat germ oil, the kaempferia galangal total flavonoids and the wheat germ oil can obviously improve the sexual activity of the fruit flies, and the effect is obviously better than the single action of the wheat germ oil. The results of the sexual activity test of the fruit flies in the different test groups within 30 minutes and 60 minutes are shown in fig. 3.
(2) The result of the fertility experiment: taking the number of the imagoes hatched out of each culture tube in 8 days as the total number of the drosophila melanogaster generation F1 as an evaluation index, the reproductive capacity of the drosophila melanogaster in the kaempferia galangal total flavone group, the wheat germ oil group, the kaempferia galangal total flavone group and the wheat germ oil composition group is obviously increased compared with that in the normal group. The amount of the imagoes in the composition of the kaempferia galanga total flavonoids and the wheat germ oil is 2.9 times of that in the normal control group, and the imagoes in the composition of the kaempferia galanga total flavonoids and the wheat germ oil is 1.3 times and 1.9 times of that in the normal control group respectively. The Drosophila fecundity results for each test group are shown in FIG. 4.
(3) The experimental result of the life span is as follows: the kaempferia galanga total flavone group, the wheat germ oil group, the kaempferia galanga total flavone and wheat germ oil composition group have different degrees of prolonging effects on the life span of the fruit flies, as shown in fig. 5. The wheat germ oil has a certain life prolonging effect, but is not obvious compared with normal fruit flies; the kaempferia galanga total flavone group, the kaempferia galanga total flavone and wheat germ oil composition group can obviously prolong the life of the fruit flies, and the effect of the kaempferia galanga total flavone and wheat germ oil composition is most obvious.
The experimental results show that the kaempferia galanga total flavone group, the wheat germ oil group, the kaempferia galanga total flavone and wheat germ oil composition have different degrees of influence on the sexual activity and the reproductive capacity of the fruit flies. The composition of the kaempferia galanga total flavonoids and the wheat germ oil has the most obvious effects of improving the sexual activity and reproductive capacity of fruit flies and prolonging the life of the fruit flies, and the combined action of the kaempferia galanga total flavonoids and the wheat germ oil shows that the composition not only can reflect the influence of the kaempferia galanga total flavonoids and the wheat germ oil on the reproductive function and the life of the fruit flies, but also has a synergistic effect, and compared with the composition which is used singly, the composition has the remarkable effects of improving the reproductive function and the life of the fruit flies.
Claims (4)
1. A composition for increasing reproductive activity and/or anti-aging comprises Kaempferia galanga total flavone extract and wheat germ oil;
the mass ratio of the kaempferia galanga total flavone extract to the wheat germ oil is 1:1 to 5;
the preparation method of the kaempferia galanga total flavone extract comprises the following steps:
s1, crushing rhizome of kaempferia galanga to obtain rhizome powder of kaempferia galanga;
s2, extracting the rhizome powder of the rhizoma kaempferiae by adopting an ethanol water solution to obtain an extracting solution;
s3, concentrating the extracting solution, separating by using macroporous adsorption resin, collecting eluent, and drying to obtain a small flower kaempferia galangal total flavone extract;
in the step S1, the rhizome of kaempferia galanga is dried and then crushed, and the crushed rhizome is sieved by a 60-mesh sieve to obtain powder of the rhizome of kaempferia galanga;
in the step S2, the volume content of the ethanol in the ethanol water solution is 50-90%;
the proportion of the rhizome powder of the kaempferia galanga to the ethanol water solution is as follows: 1: 10-15 g/mL;
extracting under the condition of ultrasound;
the extraction time is 20-30 min, and the extraction is carried out for 1-3 times;
in the step S3, decompressing and concentrating the extracting solution until the density of the concentrated solution is 4-6 g/mL;
the macroporous adsorption resin is AB-8 macroporous adsorption resin or HPD300 macroporous adsorption resin;
eluting with 95% ethanol solution, collecting eluate, concentrating under reduced pressure by evaporation to obtain soft extract, and vacuum drying.
2. The composition of claim 1, wherein: the composition is a soft capsule;
the content of the soft capsule is prepared from kaempferia galanga total flavone extract and wheat germ oil.
3. The composition of claim 1, wherein: the composition is a microemulsion or a self-microemulsion;
the microemulsion is prepared from polyoxyethylene hydrogenated castor oil, propylene glycol, the kaempferia galangal total flavone extract, the wheat germ oil and water;
the self-microemulsion is prepared from glyceryl triacetate, polyoxyethylene hydrogenated castor oil, diethylene glycol monoethyl ether, the kaempferia galanga total flavone extract and the wheat germ oil.
4. Use of a composition according to any of claims 1-3 for the manufacture of a product for increasing reproductive activity and/or anti-ageing.
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| WO2013172681A1 (en) * | 2012-05-16 | 2013-11-21 | 연세대학교 산학협력단 | Use of kaempferia parviflora wall. ex. baker extracts or flavone compound for preventing or treating muscle diseases, or improving muscle function |
| CN104257715B (en) * | 2014-10-17 | 2018-12-25 | 朴光春 | Herba Artemisiae extract and its preparation method and application |
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