WO2018058488A1

WO2018058488A1 - Method for preparing areca catechu extract from fresh areca catechu nuts

Info

Publication number: WO2018058488A1
Application number: PCT/CN2016/100977
Authority: WO
Inventors: 许启太
Original assignee: 许启太
Priority date: 2016-09-30
Filing date: 2016-09-30
Publication date: 2018-04-05
Also published as: CN108347983A

Abstract

Provided is a method for preparing an Areca catechu extract from fresh Areca catechu nuts, comprising the following steps: (1) pressing and extracting; (2) removing tannin; (3) removing a portion of alkaloid; (4) extracting residues, and (5) dehydrating to obtain the Areca catechu extract. The extraction method is a low-temperature, fast, efficient, and energy-saving industrialized extraction process which uses total polysaccharides and total polyphenols as quality control indexes and saves the effective ingredients of fresh Areca catechu, and solves the problem of fresh Areca catechu nuts being difficult to store. The process also removes tannin and a portion of alkaloid. Compared to conventional Areca catechu extracts prepared with common water extraction, the above-mentioned Areca catechu extract is safer, preserves the unique flavour of Areca catechu, can significantly reduce the cytotoxicity produced thereby and ensures that Areca catechu products are safe to consume.

Description

Method for preparing betel nut extract from betel nut fresh fruit

Technical field

The present invention relates to a method for preparing a betel nut extract from betel nut fresh fruit, and the obtained betel nut extract is applied to food development.

Background technique

Areca is a mature fruit of Areca catechu L., a palm tree belonging to the family Areca catechu L. Chewing betel nut is a traditional diet with more than 2,000 years of history. Chinese people are used to chewing betel nuts (Hainan, Taiwan) and dried fruit processed products (other parts of China), while betel nut chewing blocks are mainly in India and Southeast Asia. Betel nut has the effect of refreshing, euphoric and endurance. It is the world's fourth largest hobby after nicotine, ethanol and coffee. Currently, about 700 million people around the world consume betel nut. It is expected to form 20 in ten years. The annual output value of the billion-dollar global consumption of betel nut is expected to exceed RMB 2 trillion.

The existing betel nut processing method is simple, the technical content is low, and the food safety risk is high. In 2003, the World Health Organization (WHO) proposed a warning on the safety risk of chewing betel nuts. Scientific research shows that the unsafe factors of chewing betel nut are mainly related to traditional processing methods and betel nut coarse fiber. Excessive chewing of betel nut can cause submucosal fibrosis of the oral cavity, which may cause oral cancer; in addition, smoked fruit has strong surface smog. The carcinogen benzopyrene content also greatly exceeds the safety requirements of food hygiene. The safety of betel nut has attracted a lot of attention from the society. Therefore, it is urgent to develop a safe betel nut series.

Betel nut fruit contains a variety of chemical components, and 68 chemical compounds have been detected, including alkaloids, polyphenols and tannins. In the 2015 edition of the Chinese Pharmacopoeia, betel nut (dry mature seeds) has the effects of killing insects, eliminating product, qi, water, and malaria. Most of the above effects are positively related to arecoline, which is the main alkaloid in betel nut. It has fever, increases gastrointestinal motility, shrinks the bronchus, and causes physiological activity such as vasodilation. The research on the extraction process of arecoline has been relatively mature. Traditional betel nut extract contains a large amount of arecoline, which produces strong cytotoxicity and cannot be consumed in excess. Recent scientific studies have found that betel nut new effects include anti-fatigue, cholesterol lowering, anti-oxidation, anti-depression, anti-migraine, anti-age dementia The effect is related to betel nut polysaccharides and polyphenols. Most of these components are heat sensitive components, which decompose or lose activity at temperatures above 60 °C. At present, the betel nut extraction process and industrial production mostly use betel nut dried fruit as raw material. The processing temperature of betel nut dried fruit has far exceeded 60 °C, which causes some active ingredients to be destroyed during processing and lose their active effect. At the same time, dried fruit fiber is harder than fresh fruit and is not easy to be Destruction, so the dried fruit extracts the active ingredients without fresh fruit extraction completely. However, the fresh fruit of betel nut is not easy to preserve, and it needs to be processed quickly after picking, otherwise the fresh fruit is perishable and deteriorated. Therefore, the extraction process of betel nut needs to consider two factors: low temperature extraction and rapid processing.

Summary of the invention

The object of the present invention is to provide a method for preparing betel nut extract from betel nut fresh fruit. The present invention adopts betel nut fresh fruit pressing technology, and most likely retains all the nutrients ingested when chewing betel nut; and removes pressed juice by alcohol precipitation method. The enamel reduces the astringency brought to the subsequent foods, thereby improving the taste; at the same time, the extract residue is further extracted by the enzyme biolysis method, and the effective nutrients in the betel nut crude fiber cells are extracted in all directions.

In order to achieve the above object, the technical proposal of the present invention is to provide a method for preparing a betel nut extract from betel nut fresh fruit, comprising the following steps:

(1) Extracting and extracting: sorting the fresh betel nuts, cleaning them, crushing them, and pressing them by physical means to obtain juices. The juices are treated to obtain clear juice; the betel nut residue is combined and used;

(2) removing the enamel: adding edible ethanol to the clarified juice, mixing the final concentration of ethanol to 45-85%, standing overnight, taking the supernatant to obtain a juice solution;

(3) removing part of the alkaloid: the juice solution is concentrated under low pressure to remove ethanol, and the concentrated juice solution is passed through a solid phase column, and the solid column is rinsed with 1 to 10 volumes of water to collect all the liquid to obtain a juice extract; a solid phase column containing a beryllic acid molecularly imprinted polymer as a stationary phase;

(4) Residue extraction: the betel nut residue is added with 3 to 10 times its weight of water and cellulase for biolysis, the final concentration of cellulase after mixing is 0.01%-0.8%, the pH value of citric acid is adjusted to 3-6, ultrasonic extraction 1 to 6 hours, centrifugation 6000 ~ 12000 rpm, to obtain a residue extract;

(5) Dehydration: The juice extract is combined with the residue extract, and dehydrated by low-temperature decompression and concentration to obtain a solid matter, which is pulverized by 30 to 120 mesh, and tested to obtain a betel nut extract.

Further, the low temperature means that the temperature is 60 ° C or lower.

Further, the betel nut extract is applied to betel nut gum, betel nut, betel nut beverage, betel nut baked food.

Further, the solid phase column of the arecoline molecularly imprinted polymer is further eluted and concentrated, and is enriched and purified for application in drug development.

The method for preparing areca nut extract from betel nut fresh fruit of the present invention has the following beneficial effects:

1. The present invention adopts betel nut fresh fruit pressing technology, and most likely retains all the nutrients ingested when the betel nut is chewed; the alcohol deposition method is used to remove the tannin in the pressed juice, and the astringency in the subsequent food is reduced, thereby improving the taste; At the same time, the extract residue is further extracted by enzyme biolysis, and the effective nutrients in the crude fiber of betel nut are extracted in all directions.

2. The invention adopts molecular imprinting technology, aiming at removing some unsafe factors (becaline) in betel nut, and reducing the cytotoxicity produced by betel nut while retaining the functional site and characteristic flavor of betel nut.

3. The invention rapidly converts the betel nut fresh fruit into a betel nut extract dry powder, and solves the problem that the fresh fruit is difficult to preserve; the betel nut extract dry powder preparation process is carried out at a low temperature to maximize the protection of the functional ingredients.

4. The betel nut extract prepared by the invention can be used in food, and can develop betel nut series products such as betel nut gum, betel nut wine, betel nut beverage, betel nut baked food and the like, which are safe to eat and controllable in quality.

5. The arecoline removed by the molecular imprinting technique of the present invention can be further enriched and purified for use in subsequent drug development.

6. The extraction method of the invention has the advantages of low temperature, rapid, high efficiency and energy saving, and is an industrial extraction process for retaining fresh betel nut active ingredients by using total polysaccharides and total polyphenols as quality control indexes, and solving the problem that the betel nut fruits are difficult to preserve; The components that affect the quality of the subsequent foods (enamel and part of the alkaloids) are removed; at the same time, the in vitro toxicity test shows that the betel nut extract prepared by the invention is safer than the conventional betel nut extract prepared by ordinary water extraction, and the betel nut is unique in maintaining At the same time of flavor, it can significantly reduce the cytotoxicity it produces and ensure the safety of betel nut products.

detailed description

Example 1:

(1) Take 5kg of betel nut fresh fruit, sort, clean, crush, press, centrifuge 12000 rpm to obtain clarified juice; combine betel nut residue (squeezed after crushing and centrifugation), stand by;

(2) adding edible ethanol to the clarified juice, so that the final concentration of the ethanol after mixing is 65%, standing overnight, taking the supernatant to obtain a juice solution;

(3) The juice solution is concentrated under low pressure to remove ethanol, and the concentrated juice solution is passed through a solid phase column, and the solid column is rinsed with 8 volumes of water to collect all the liquid to obtain a juice extract; the solid phase column is fixed as a fixed liquid. Phase of arecoline molecularly imprinted polymer;

(4) Adding 5 times of water and cellulase to the betel nut residue for biolysis, the final concentration of cellulase is 0.25%, the pH value of citric acid is 4.8, the ultrasonic extraction is 3 hours, and the mixture is centrifuged at 6000 rpm to obtain a residue extract;

(5) The fruit juice extract and the residue extract are combined, and dehydrated by low-temperature decompression and concentration to obtain a solid matter, which is pulverized by 60 mesh and examined to obtain a betel nut extract.

The low temperature means that the temperature is 60 ° C or less.

The betel nut extract is applied to betel nut gum, betel nut, betel nut beverage, betel nut baked food.

The solid phase column of the arecoline molecularly imprinted polymer is further eluted and concentrated, and is enriched and purified for application in drug development.

Example 2:

(1) Take 5kg of betel nut fresh fruit, sort, clean, crush, squeeze, press 8000rpm, obtain clarified juice, combine betel nut residue, stand by;

(2) adding edible ethanol to the clarified juice, so that the final concentration of the ethanol after mixing is 75%, standing overnight, taking the supernatant to obtain a juice solution;

(3) The juice solution is concentrated under reduced pressure to remove ethanol, and the concentrated juice solution is passed through a solid phase column, and the solid column is rinsed with 10 volumes of water to collect all the liquid to obtain a juice extract; the solid phase column is fixed as a fixed liquid. Phase of arecoline molecularly imprinted polymer;

(4) Adding 7 times of water and cellulase to the betel nut residue for biolysis, the final concentration of cellulase after mixing is 0.5%, the pH value of citric acid is 4.5, the ultrasonic extraction is 6h, and the centrifugation is 12000 rpm. Residue extract;

(5) The juice extract and the residue extract are combined, and dehydrated by low-temperature decompression and concentration to obtain a solid matter, which is pulverized by 100 mesh and examined to obtain a betel nut extract.

The low temperature means that the temperature is 60 ° C or less.

Example 3:

(1) Take 5kg of betel nut fresh fruit, sort, clean, crush, squeeze, press 9000rpm, obtain clarified juice, and combine betel nut residue for use;

(2) adding edible ethanol to the clarified juice, so that the final concentration of the ethanol after mixing is 50%, standing overnight, taking the supernatant to obtain a juice solution;

(3) The juice solution is concentrated under low pressure to remove ethanol, and the concentrated juice solution is passed through a solid phase column, and the solid column is rinsed with 5 volumes of water to collect all the liquid to obtain a juice extract; the solid phase column is fixed as a fixed Phase of arecoline molecularly imprinted polymer;

(4) The betel nut residue was added with 8 times of water and cellulase for biolysis, so that the final concentration of cellulase after mixing was 0.4%, the pH value of citric acid was adjusted to 4.8, ultrasonic extraction was carried out for 3 hours, and centrifuged at 6000 rpm to obtain a residue extract. ;

The low temperature means that the temperature is 60 ° C or less.

Example 4 (without molecular imprinting techniques to remove some of the betel nut):

(1) 5kg of betel nut fresh fruit, sorted, cleaned, crushed, pressed, squeezed liquid 8000rpm, Obtain clarified juice; combine betel nut residue for use;

(2) adding edible ethanol to the clarified juice, so that the final concentration of the ethanol after mixing is 70%, standing overnight, taking the supernatant to obtain a juice solution;

(3) The juice solution is concentrated and removed by low temperature under reduced pressure to obtain a juice concentrate;

(4) The betel nut residue was added with 6 times of water and cellulase for biolysis, the final concentration of cellulase was 0.5%, the pH value of citric acid was 4.5, the ultrasonic extraction was carried out for 4 hours, and the mixture was centrifuged at 8000 rpm to obtain a residue extract;

(5) The fruit juice concentrate and the residue extract are combined, and dehydrated by low-temperature decompression and concentration to obtain a solid matter, which is pulverized by 100 mesh and examined to obtain a betel nut extract.

Experimental procedure:

(1) Determination of total polyphenols in the areca extract of the present invention by spectrophotometry:

Main reagents and instruments: gallic acid (biochemical reagent, China Food and Drug Administration Institute); methanol; 7.5% sodium carbonate solution; forinol test solution; DUV-2450 ultraviolet-visible spectrophotometer (Shimadzu, Japan).

1) Linear test The absorbance was measured by spectrophotometry at a wavelength of 765 nm without the addition of gallic acid. The concentration of gallic acid (mg/ml) was plotted on the abscissa, and the absorbance (A) was plotted on the ordinate. The standard curve was drawn and the linearity was calculated. Regression equation.

2) Calculation of results

Based on the linear regression equation of gallic acid, the polyphenol concentration C ₁ in the test solution to be tested was calculated. The polyphenols in the areca nut extract are expressed in mass fraction W ₁ and the values are expressed in %:

w ₁ - the mass fraction of the polyphenol component in the test article;

C ₁ - concentration of polyphenol component in the test solution, the unit is mg / mL;

V ₁ - the dilution volume of the test solution, in milliliters (mL);

m ₁ ^- —— the amount of sample to be tested, in milligrams (mg);

D - dilution factor.

The statistical results are shown in Table 1:

Table 1 Comparison of total polyphenol content in the betel nut extract of the present invention compared with conventional extraction data

(2) Determination of total polysaccharides in the areca extract of the present invention by spectrophotometry

Main reagents and instruments: glucose (biochemical reagent, China Food and Drug Administration Institute); 5% phenol solution; concentrated sulfuric acid solution; DUV-2450 UV-visible spectrophotometer (Shimadzu, Japan).

1) Linear test: The absorbance was measured by spectrophotometry at 490 nm without spectrophotometry. The glucose concentration (mg/ml) was used as the abscissa, the absorbance (A) was plotted on the ordinate, and the standard curve was drawn to calculate the linear regression equation. .

2) Calculation of results

Based on the linear regression equation of glucose, the concentration of polysaccharide C ₂ in the test solution to be tested was calculated. The polysaccharide in the areca nut extract is expressed in mass fraction W ₂ and the value is expressed in %:

w ₂ - the mass fraction of the polysaccharide component in the test article;

C ₂ - the concentration of the polysaccharide component in the test solution, the unit is mg / mL;

V ₂ - the dilution volume of the test solution, in milliliters (mL);

m ₂ —— the amount of sample to be tested, in milligrams (mg);

D - dilution factor;

The statistical results are shown in Table 2:

Table 2 Comparison of total polysaccharide content in betel nut extracts of the present invention with conventional extraction data

The data in Tables 1 and 2 show that the content of active ingredients (polyphenols and polysaccharides) in the betel nut extract obtained by the present invention is higher than that of the conventional areca nut extract.

(3) In vitro toxicity test (human oral mucosal keratinocyte model)

Main instruments and reagents: human oral mucosal keratinocytes (KC) serum-free medium (K-SFM), KC complete medium, DMEM medium, mouse anti-human keratin antibody and mouse anti-human spirulina monoclonal antibody, SABC immunohistochemistry kit, fluorescence microscope.

Experimental methods: KCs cultured in vitro were randomly divided into 3 groups: negative control group: K-SFM+KC; conventional areca nut extract treatment group (AW): AW+KC; betel nut extract treatment group of the present invention: Example (1) , 2, 3, 4) + KC.

Detection index: The number of cells was observed by a fluorescence microscope. Percent apoptosis = apoptotic cells / total cell number x 100%. The statistical results are shown in Table 3:

Table 3 Comparison of four embodiments of the present invention with conventionally extracted KC apoptotic rate data

According to the data in Table 3, the conventional areca nut extract (cut the betel nut fresh fruit, ethanol extraction) has the effect of significantly inducing KC apoptosis, and the apoptotic rate of KC in the betel nut extract of Example 4 (without molecular encapsulation to remove arecoline) The KC apoptotic rate of the conventional areca nut extract treatment group was close to that between the sample 1 and the conventional areca nut extract treatment group, and the betel nut extracts of Examples 1, 2, and 3 were removed by molecular imprinting technique. Arecoline) significantly reduced KC apoptotic rate, and there was no significant difference from the negative control group (no betel nut intervention). Therefore, it is proved that the method of using the molecular imprinting technique to remove some of the arecoline from the betel nut fresh fruit can reduce the cytotoxicity of the betel nut, reduce the risk of damage to the oral mucosa structure, and ensure the safety of the betel nut product.

(4) The content of arecoline in the areca extract of the present invention is determined by high performance liquid chromatography (HPLC).

Japanese high performance liquid chromatography system (Japan HITACHI series); with strong cation exchange bonded silica as a filler (SCX-strong cation exchange resin column); with acetonitrile-phosphoric acid solution (2 → 1000, concentrated ammonia test solution to adjust the pH value to 3.8) (55:45) is the mobile phase; the detection wavelength is 215 nm.

The statistical results are as follows:

Example 1: arecoline content 0.16%; Example 2: arecoline content 0.14%; Example 3: arecoline content 0.15%; Example 4: arecoline content 0.57%.

Experiments show that: Example 1, Example 2, and Example 3 have significantly different betelsine content than Example 4 which does not use molecular imprinting technology to remove arecoline. Combined with the results of the in vitro toxicity test, the method for preparing the betel nut extract from the betel nut fresh fruit by the invention can obviously reduce the cytotoxicity of the betel nut while maintaining the unique flavor of the betel nut, ensure the safe eating of the betel nut product, and guide the safety, civilization and health. Consumption of betel nut.

The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and thus equivalent changes made in the claims of the present invention are still within the scope of the present invention.

Claims

A method for preparing a betel nut extract from a betel nut fresh fruit, comprising the steps of:

(1) Squeeze extraction: sorting the betel nut fresh fruit, cleaning it, crushing it, and pressing it with physical method to obtain juice, the juice is processed to obtain clarified juice; the betel nut residue is combined and used;

(2) Removing the enamel: adding edible ethanol to the clarified juice, mixing the final concentration of ethanol to 45-85%, standing overnight, taking the supernatant to obtain a juice solution;

(3) removing part of alkaloids: the juice solution is concentrated under low pressure to remove ethanol, and the concentrated juice solution is passed through a solid phase column, and the solid phase column is rinsed with 1 to 10 volumes of water to collect all the liquid to obtain a juice extract; a solid phase column containing a arecoline molecularly imprinted polymer as a stationary phase;

(4) Residue extraction: the betel nut residue is added with 3 to 10 times its weight of water and cellulase for biolysis, the final concentration of cellulase after mixing is 0.01%-0.8%, the pH value of citric acid is adjusted to 3-6, ultrasonic extraction 1 to 6 hours, centrifugation 6000 ~ 12000 rpm, to obtain a residue extract;

(5) Dehydration: The juice extract is combined with the residue extract, and dehydrated by low-temperature decompression and concentration to obtain a solid matter, which is pulverized by 30 to 120 mesh, and tested to obtain a betel nut extract.
The method for preparing a betel nut extract from betel nut fresh fruit according to claim 1, wherein the low temperature means a temperature of 60 ° C or less.
The method for preparing a betel nut extract from betel nut fruit according to claim 1, wherein the betel nut extract is applied to betel nut gum, betel nut, betel nut beverage, betel nut baked food.
The method for preparing areca nut extract from betel nut fresh fruit according to claim 1, wherein the solid phase column of the arecoline molecularly imprinted polymer is further eluted and concentrated, and is enriched and purified for use in drug development.