CN116942732A - Magnolia officinalis polyphenol extract and extraction method and application thereof - Google Patents

Magnolia officinalis polyphenol extract and extraction method and application thereof Download PDF

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CN116942732A
CN116942732A CN202310931655.XA CN202310931655A CN116942732A CN 116942732 A CN116942732 A CN 116942732A CN 202310931655 A CN202310931655 A CN 202310931655A CN 116942732 A CN116942732 A CN 116942732A
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magnolia bark
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李蓉
王劲松
缪园欣
徐艳
陈清婵
黄业传
张喜才
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Jingchu University of Technology
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    • 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
    • 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
    • A61K2236/55—Liquid-liquid separation; Phase separation

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Abstract

The application discloses a magnolia bark polyphenol extract and an extraction method and application thereof, and the magnolia bark polyphenol extract comprises the following steps: s1, obtaining magnolia bark powder; s2, adding an ethanol solution into the magnolia bark powder, and performing ultrasonic leaching and concentration to obtain a concentrated extract; s3, adding an extractant into the concentrated extract, extracting, concentrating, and freeze-drying to obtain a magnolia bark polyphenol extract; can inhibit pancreatic lipase activity, and has good in vitro and in vivo lipid lowering effect.

Description

Magnolia officinalis polyphenol extract and extraction method and application thereof
Technical Field
The application relates to the technical field of health-care products, in particular to a magnolia bark polyphenol extract, and an extraction method and application thereof.
Background
With the improvement of living standard and the change of people's eating habits, obesity is becoming an important problem affecting the health of a large population worldwide. Currently, the main prevention and treatment modes for obesity include surgery, medicines, sports, diet improvement and the like. A series of weight-losing medicaments including phentermine, fenframine and the like are forbidden due to the fact that the medicaments can cause side effects of cardiovascular diseases, hyperthyroidism and the like, and pancreatic lipase inhibitor medicaments, namely orlistat, are widely used as weight-losing medicaments in clinic, but can cause adverse reactions of diarrhea, flatulence, headache and the like after long-time administration. Thus, research is looking for new pancreatic lipase inhibitors that are natural and one of the hot spots for fat reduction and treatment of obesity.
The Magnolia officinalis is dried bark, root bark and branch bark of Magnolia officinalis or Magnolia officinalis of Magnolia of Magnoliaceae, is traditional Chinese medicinal material, and is widely distributed in multiple provinces of North and south. However, the extract of magnolia officinalis is mainly used for resisting oxidation, resisting inflammation, inhibiting bacteria, treating cardiovascular diseases and the like, and functional research on richer components in magnolia officinalis has not been performed yet.
Therefore, it is necessary to explore the application of magnolia bark extract in lipid lowering.
Disclosure of Invention
In view of the above, the application provides a magnolia bark polyphenol extract, an extraction method and application thereof, which can inhibit pancreatic lipase activity and has good in-vitro and in-vivo lipid-lowering effects.
In order to achieve the technical purpose, the application adopts the following technical scheme:
in a first aspect, the present application provides a method for extracting magnolia bark polyphenol extract, comprising the steps of:
s1, obtaining magnolia bark powder;
s2, adding an ethanol solution into the magnolia bark powder, and performing ultrasonic leaching and concentration to obtain a concentrated extract;
s3, adding an extractant into the concentrated extract, extracting, concentrating, and freeze-drying to obtain the magnolia bark polyphenol extract.
Preferably, the extractant is ethyl acetate and/or n-butanol.
Preferably, in the step S2, the concentration of the ethanol is 50-70%, and the ratio of the liquid to the solid in the ultrasonic leaching is 1:10-30.
Preferably, the temperature of ultrasonic leaching is 40-55 ℃, the frequency of ultrasonic leaching is 300-450W, and the time of ultrasonic leaching is 2-4h.
Preferably, the feed liquid ratio of the concentrated extract to the extractant is 1:10-20.
In a second aspect, the present application provides a magnolol extract.
In a third aspect, the present application provides an inhibitor of pancreatic lipase activity comprising an extract of magnolia bark polyphenol.
In a fourth aspect, the application provides an application of magnolia bark polyphenol extract as an active ingredient in preparing lipid-lowering medicaments and/or lipid-lowering health products.
In a fifth aspect, the present application provides a lipid-lowering drug and/or lipid-lowering health product comprising a magnolia bark polyphenol extract.
In a sixth aspect, the present application provides an application of magnolol extract in inhibiting pancreatic lipase.
The beneficial effects of the application are as follows: the application adopts ethanol ultrasonic leaching and combines ethyl acetate and n-butanol extraction to prepare the magnolia bark polyphenol extract, and the magnolia bark polyphenol extract has high total phenol content and rich types, and comprises magnolol, gallic acid, paeonol, epicatechin, catechin, apigenin, cinnamic acid and formononetin; the obtained magnolia bark polyphenol extract has the activity of inhibiting pancreatic lipase, has the equivalent activity of inhibiting pancreatic lipase compared with orlistat, can obviously reduce the fat content in caenorhabditis elegans, and has the prospect of developing health-care food or medicine with the lipid-lowering effect.
Drawings
FIG. 1 is a comparison of the results of inhibition of pancreatic lipase activity by different substances.
Detailed Description
The present application will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The application provides an extraction method of a magnolia bark polyphenol extract, which comprises the following steps:
s1, obtaining magnolia bark powder;
s2, adding an ethanol solution into the magnolia bark powder, and performing ultrasonic leaching and concentration to obtain a concentrated extract;
s3, adding an extractant into the concentrated extract, extracting, concentrating, and freeze-drying to obtain the magnolia bark polyphenol extract.
The extractant is ethyl acetate and/or n-butanol; more preferably, the extractant is ethyl acetate.
In the step S2, the concentration of the ethanol is 50-70%, and the ratio of the liquid to the solid in the ultrasonic leaching is 1:10-30; more preferably, the concentration of ethanol is 50%, the feed liquid ratio of ultrasonic leaching is 1:30, and the extraction is carried out by adopting low concentration and high feed liquid ratio, so that the extraction rate of polyphenol can be improved.
The ultrasonic leaching temperature is 40-55deg.C, ultrasonic leaching frequency is 300-450W, and ultrasonic leaching time is 2-4h.
The ratio of the magnolia bark powder to the extracting agent is 1:10-20; preferably, the feed liquid ratio of the concentrated extract to the extractant is 1:10, and under the condition, the extraction efficiency is higher.
The application provides a magnolia polyphenol extract which has high total phenol content and rich types, and comprises magnolol, gallic acid, paeonol, epicatechin, catechin, apigenin, cinnamic acid and formononetin.
The application provides a pancreatic lipase activity inhibitor containing a magnolia bark polyphenol extract, wherein pancreatic lipase is secreted into duodenum through a pancreatic bile duct system and works together with digestive juice in the duodenum, and the pancreatic lipase activity inhibitor is responsible for hydrolyzing 50% -70% of dietary fat in the total amount in a human body.
The application provides an application of magnolia bark polyphenol extract as an active ingredient in preparing a lipid-lowering medicament and/or a lipid-lowering health product, wherein the lipid-lowering medicament/health product is used for lowering the content of triglyceride.
The application provides a lipid-lowering medicament and/or lipid-lowering health care product containing magnolia bark polyphenol extract.
The application provides an application of a magnolia bark polyphenol extract in inhibiting pancreatic lipase.
The present application is further illustrated by the following specific examples.
Example 1
An extraction method of magnolia bark polyphenol extract comprises the following steps:
s1, drying magnolia bark raw materials in a baking oven at 60 ℃ for 24 hours, crushing and sieving with a 60-mesh sieve to obtain magnolia bark powder;
s2, weighing 500g of magnolia bark powder according to a feed liquid ratio of 1: adding 50% ethanol solution into 10g/mL, performing ultrasonic extraction at 40deg.C, preferably at 300W for 2 hr, mixing the obtained extractive solutions, and concentrating to obtain concentrated extract;
s3, adding the concentrated extract into a separating funnel, adding ethyl acetate for extraction, mixing the supernatant of the extract with the feed liquid ratio of the concentrated extract to the ethyl acetate of 1:10, performing rotary evaporation under reduced pressure, concentrating, recovering the solvent, and performing freeze drying to obtain the magnolia polyphenol extract.
Example 2
An extraction method of magnolia bark polyphenol extract comprises the following steps:
s1, drying magnolia bark raw materials in a baking oven at 60 ℃ for 24 hours, crushing and sieving with a 60-mesh sieve to obtain magnolia bark powder;
s2, weighing 500g of magnolia bark powder according to a feed liquid ratio of 1: adding 50% ethanol solution into 20g/mL, performing ultrasonic extraction at 45deg.C, preferably at 350W for 2 hr, mixing the obtained extractive solutions, and concentrating to obtain concentrated extract;
s3, adding the concentrated extract into a separating funnel, adding ethyl acetate for extraction, mixing the supernatant of the extract with the feed liquid ratio of the concentrated extract to the ethyl acetate is 1:20, performing rotary evaporation under reduced pressure, concentrating, recovering the solvent, and performing freeze drying to obtain the magnolia polyphenol extract.
Example 3
An extraction method of magnolia bark polyphenol extract comprises the following steps:
s1, drying magnolia bark raw materials in a baking oven at 60 ℃ for 24 hours, crushing and sieving with a 60-mesh sieve to obtain magnolia bark powder;
s2, weighing 500g of magnolia bark powder according to a feed liquid ratio of 1: adding 50% ethanol solution into 30g/mL, performing ultrasonic extraction at 45deg.C, preferably 400W for 4 hr, mixing the obtained extractive solutions, and concentrating to obtain concentrated extract;
s3, adding the concentrated extract into a separating funnel, adding ethyl acetate for extraction, mixing the supernatant of the extract with the feed liquid ratio of the concentrated extract to the ethyl acetate of 1:10, performing rotary evaporation under reduced pressure, concentrating, recovering the solvent, and performing freeze drying to obtain the magnolia polyphenol extract.
Example 4
An extraction method of magnolia bark polyphenol extract comprises the following steps:
s1, drying magnolia bark raw materials in a baking oven at 60 ℃ for 24 hours, crushing and sieving with a 60-mesh sieve to obtain magnolia bark powder;
s2, weighing 500g of magnolia bark powder according to a feed liquid ratio of 1: adding 60% ethanol solution into 10g/mL, performing ultrasonic extraction at 45deg.C, preferably 400W for 2 hr, mixing the obtained extractive solutions, and concentrating to obtain concentrated extract;
s3, adding the concentrated extract into a separating funnel, adding ethyl acetate for extraction, mixing the supernatant of the extract with the feed liquid ratio of the concentrated extract to the n-butanol is 1:10, performing rotary evaporation under reduced pressure, concentrating, recovering the solvent, and performing freeze drying to obtain the magnolia polyphenol extract.
Example 5
An extraction method of magnolia bark polyphenol extract comprises the following steps:
s1, drying magnolia bark raw materials in a baking oven at 60 ℃ for 24 hours, crushing and sieving with a 60-mesh sieve to obtain magnolia bark powder;
s2, weighing 500g of magnolia bark powder according to a feed liquid ratio of 1: adding 50% ethanol solution into 10g/mL, performing ultrasonic extraction at 40deg.C, preferably at 300W for 2 hr, mixing the obtained extractive solutions, and concentrating to obtain concentrated extract;
s3, adding the concentrated extract into a separating funnel, adding ethyl acetate for extraction, mixing the supernatant of the extract with the feed liquid ratio of the concentrated extract to the n-butanol is 1:20, performing rotary evaporation under reduced pressure, concentrating, recovering the solvent, and performing freeze drying to obtain the magnolia polyphenol extract.
Example 6
An extraction method of Magnolia bark polyphenol extract is the same as in example 3 except that the concentration of ethanol in step S2 is 70%.
Example 7
An extraction method of Magnolia officinalis polyphenol extract is the same as in example 3 except that the ratio of concentrated extract to ethyl acetate is 1:20.
Example 8
An extraction method of Magnolia bark polyphenol extract is the same as in example 3 except that ethyl acetate is replaced with n-butanol.
Comparative example 1
An extraction method of Magnolia bark polyphenol extract is the same as in example 3 except that the extractant is methyl acetate.
Comparative example 2
An extraction method of Magnolia bark polyphenol extract was the same as in example 3 except that the ethanol solution was replaced with water.
Evaluation test
The polyphenols of examples 1-7 and comparative examples 1-2 were tested for their content:
step 1: drawing a standard curve: gallic acid (gallic acid) is used as a standard substance, standard substance solutions with different concentrations are prepared by dissolving the gallic acid (gallic acid) in 50% methanol, 500 mu L of standard substance solution is mixed with 250 mu L of 50% Fu Lin Fen reagent and then reacted for 5min, 500 mu L of 5% sodium carbonate solution is added for light-proof reaction for 1h, absorbance is measured at 765nm, the concentration of gallic acid is taken as an abscissa, and absorbance is taken as an ordinate, so that a standard curve is drawn.
Step 2: determination of polyphenol content in magnolia bark polyphenol extract: after mixing 500. Mu.L of the sample with 250. Mu.L of 50% Fu Lin Fen reagent, reacting for 5min, adding 500. Mu.L of 5% sodium carbonate solution, reacting for 1h in a dark place, measuring absorbance at 765nm, and calculating total phenol content in the sample according to a standard curve, wherein the total phenol content is expressed as mg gallic acid per g of sample. The results are shown in Table 1:
TABLE 1 Total phenol content test results
As can be seen from Table 1, the concentration of ethanol, the leaching temperature, the feed-to-liquid ratio, the extraction solvent and the ratio of the extraction feed-to-liquid have a great influence on the total phenol content of the prepared Magnolia officinalis polyphenol extract, and in example 3, when the concentration of ethanol is 50%, the ratio of the feed-to-liquid of the ultrasonic leaching is 1:30, and the ratio of the feed-to-liquid of the concentrated extract to the extraction agent is 1:10, the obtained Magnolia officinalis total phenol content is the highest.
The results of testing the effect of the magnolol extract obtained in the application on the inhibition of pancreatic lipase activity in vitro by using example 3 and example 8 as test subjects and orlistat as comparison subjects are shown in fig. 1:
a proper amount of the magnolia bark polyphenol extract powder in the example 3 and the example 8 is accurately weighed, and dissolved by methanol to prepare a series of solutions with concentration.
A100U/mL pancrelipase solution was prepared using Tris-HCI buffer pH 7.0.
A10 mM solution of p-nitrophenyl palmitate was prepared using Tris-HCI buffer pH 7.0.
The whole reaction system was carried out in 96 microwell plates in a total volume of 200uL, including 20uL of orlistat solution or 20uL of Magnolia officinalis extract solution, 50uL of pancrelipase solution, 100uL of p-nitrophenyl palmitate solution, and the remaining volume was filled with Tris-HCI buffer (pH 7.0). The reaction was incubated at 37℃for 20min and then read at 405 nm. Pancreatic lipase inhibition was calculated as follows:
wherein: a is that 1 Absorbance at the time of enzyme addition to the sample; a is that 2 Absorbance values for the control group of samples without enzyme; a is that a Absorbance of control group without sample; a is that b Absorbance was measured for the control sample containing no enzyme.
As can be seen from fig. 1, the ethyl acetate fraction magnolol extract (example 3) and the n-butanol fraction magnolol extract (example 8) and orlistat have better pancreatic lipase inhibition activity, and the results show that the ethyl acetate fraction extract has higher pancreatic lipase inhibition activity than the n-butanol fraction extract, but slightly lower orlistat. Half inhibition concentration IC of ethyl acetate part on pancreatic lipase 50 IC of n-butanol fraction against pancreatic lipase with a value of 8.85mg/mL 50 IC of orlistat to pancreatic lipase with a value of 13.57mg/mL 50 The value was 7.21mg/mL.
The results of testing the lipid-lowering effect of the magnolia bark polyphenol extract obtained by the application in vivo by taking the example 3 and the example 8 as test subjects are shown in table 2:
the powder of Magnolia bark polyphenol extract in example 3 and example 8 was accurately weighed, dissolved in DMSO to prepare 50mg/mL of mother liquor, and stored at-80℃for use.
Enlarged culture of caenorhabditis elegans: and (3) coating a proper amount of E.coli OP50 bacterial suspension on the central position of the NGM flat plate, drying by a super clean bench, and placing in a 37 ℃ incubator for inversion culture for 24 hours. After the long bacterial NGM plate is cooled to normal temperature, the nematode needle is used to pick the needed nematode strain, and the nematode strain is inoculated on the NGM plate and cultured at 20 ℃. When a large number of nematodes grow on the NGM plate, the end of the metal spoon is burned by using an alcohol burner flame, the culture medium with the nematodes is cut and connected to a new NGM plate, and the culture is continued at 20 ℃.
Synchronization of caenorhabditis elegans: the nematodes in spawning period are washed from the NGM plate by using M9 buffer solution, put into a 1.5mL centrifuge tube, the supernatant is discarded after standing, and the bacteria adhered to the nematodes are removed by repeatedly washing with M9 three times. After the last time, adding 1mL of nematode lysate into a centrifuge tube, rapidly and severely oscillating for 15min until the nematode body is completely cracked, centrifuging at 10000rpm/min for 1min at normal temperature, discarding the supernatant, washing the obtained precipitate with M9 buffer solution for 4 times, finally dripping M9 buffer solution containing nematode eggs on an NGM plate coated with E.coli OP50, airing in an ultra-clean bench, and culturing in a culture box at 20 ℃ to obtain the nematode offspring at the same period.
Establishment of a high-fat dietary nematode model: culturing in 96-well plates, adding 100 mu L S-complete culture medium into each well, picking up wild nematodes synchronized to the L4 stage into the S-complete culture medium by using a picking needle, and picking up 10-15 nematodes per well, wherein the time for picking up the nematodes is set as day 0. 200 mu L M buffer solution is added to each of the outermost circles of the 96-well plates to prevent water evaporation, and the plates are sealed and then placed into 20 ℃ for culture. Wherein a blank group containing only DMSO, a high-fat group to which only 2% D-glucose was added, and a high-sugar treatment group to which a polyphenol extract of an ethyl acetate part of Magnolia officinalis 120. Mu.g/mL and a polyphenol extract of an n-butanol part of Magnolia officinalis 120. Mu.g/mL were added were set. After three days of nematode culture, nematodes of different treatment groups were collected, washed 3 times with M9 buffer, thoroughly ground on ice, centrifuged at 10000rpm/min for 5min at 4℃and the triglyceride and protein contents in the supernatant of the nematode homogenate were determined according to the procedure of the instructions of the Triglyceride (TG) and BCA assay kit of Nanjing, and each experiment was repeated three times, the results of which are shown in Table 2.
As can be seen from Table 2, compared with the blank control group, the triglyceride TG content in the caenorhabditis elegans in the high-fat group added with 2% of D-glucose in the diet is obviously improved by 2.73 times that of the blank control group, and after 120 mug/mL of the magnolia officinalis ethyl acetate part polyphenol extract (example 3) and 120 mug/mL of the magnolia officinalis n-butanol part polyphenol extract (example 8) are added, the TG content (mmol/g Protein) in the caenorhabditis elegans is obviously reduced, namely 67.9 percent (example 3) and 76.9 percent (example 8) of the TG content in the nematode in the high-fat control group respectively, so that the magnolia officinalis polyphenol extract has better lipid-lowering effect and can be applied to product development with lipid-lowering efficacy.
TABLE 2 Effect of Magnolia polyphenol extract on TG content in high fat caenorhabditis elegans
Group of TG(mmol/g Protein)
Blank control group 0.89±0.02 a
High fat control group 2.43±0.21 c
High sugar treatment group of ethyl acetate fraction extract (example 3) 1.65±0.18 b
High sugar treatment group of n-butanol fraction extract (example 8) 1.87±0.23 b
The results show that the magnolol extract obtained by ethanol ultrasonic leaching combined with the extractant has better pancreatic lipase inhibiting activity and in-vivo lipid lowering effect, and particularly has the potential of improving lipid metabolism disorder and losing weight when the extractant is ethyl acetate.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims (10)

1. The extraction method of the magnolia bark polyphenol extract is characterized by comprising the following steps of:
s1, obtaining magnolia bark powder;
s2, adding an ethanol solution into the magnolia bark powder, and performing ultrasonic leaching and concentration to obtain a concentrated extract;
s3, adding an extractant into the concentrated extract, extracting, concentrating, and freeze-drying to obtain the magnolia bark polyphenol extract.
2. The method for extracting magnolia bark polyphenol extract according to claim 1, wherein the extractant is ethyl acetate and/or n-butanol.
3. The method for extracting Magnolia bark polyphenol extract as claimed in claim 1, wherein in the step S2, the concentration of ethanol is 50-70%, and the ratio of the liquid to the solid of ultrasonic leaching is 1:10-30.
4. The method for extracting magnolol extract of claim 1, wherein the ultrasonic extraction temperature is 40-55deg.C, the ultrasonic extraction frequency is 300-450W, and the ultrasonic extraction time is 2-4h.
5. The method for extracting magnolia bark polyphenol extract according to claim 1, wherein the ratio of the concentrated extract to the extractant is 1:10-20.
6. An extract of Magnolia bark polyphenol obtained by the extraction method as claimed in any one of claims 1 to 5.
7. A pancreatic lipase activity inhibitor comprising the magnolia bark polyphenol extract of claim 6.
8. Use of the magnolol extract according to claim 6 as an active ingredient in the preparation of lipid-lowering drugs and/or lipid-lowering health products.
9. A lipid-lowering drug and/or lipid-lowering health product comprising the magnolia bark polyphenol extract of claim 6.
10. Use of the magnolol extract of claim 6 for inhibiting pancreatic lipase.
CN202310931655.XA 2023-07-25 2023-07-25 Magnolia officinalis polyphenol extract and extraction method and application thereof Pending CN116942732A (en)

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CN102240274A (en) * 2011-05-13 2011-11-16 吉林大学 Application of magnolol and honokiol in protein tyrosine phosphatase 1B inhibitor
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