CN111087485B - Method for preparing noni polysaccharide with high anticancer and antioxidant activity by using gradient physical field - Google Patents
Method for preparing noni polysaccharide with high anticancer and antioxidant activity by using gradient physical field Download PDFInfo
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Abstract
The invention discloses a method for preparing noni polysaccharide with high anticancer and antioxidant activity by a gradient physical field, which comprises the following steps: (1) drying and powdering noni, degreasing and decoloring by using ethanol, and drying to obtain noni powder. (2) And preparing the polysaccharide extracting solution from the noni powder by adopting an intermittent pulsed electric field-ultrasonic wave-continuous pulsed electric field treatment mode. (3) Purifying the extractive solution, concentrating at low temperature, precipitating with ethanol, separating, and lyophilizing to obtain noni polysaccharide. The method has the advantages of high polysaccharide yield, low energy consumption and no need of adding acid and enzyme, and the prepared noni polysaccharide has smaller molecular weight, higher uronic acid content and better anticancer and antioxidant activity. Meanwhile, the noni polysaccharide and metallic zinc have higher chelating capacity, and the anticancer activity of the formed noni polysaccharide-zinc chelate is greatly increased.
Description
Technical Field
The invention relates to noni polysaccharide, in particular to a method for preparing noni polysaccharide with high anticancer and antioxidant activity by a gradient physical field.
Background
Noni fruit science, named Morinda citrifolia L, originally produced in southeast asia, oceania and australia, grows mainly in subtropical and tropical regions, and is mainly grown in the southern hai region in China. Noni fruit is known as the gift of god, the fruit is rich in polysaccharide, mineral substances, vitamins, polyphenol, flavonoid and other substances, and the noni fruit has been used as a traditional medical medicine in places such as Polynesia for more than two thousand years. In recent years, noni has been demonstrated to have antioxidant, anti-inflammatory, and anti-cancer health benefits, which are significantly related to its polysaccharide component.
The anti-cancer and anti-cancer activities of polysaccharides are significantly related to molecular weight and uronic acid content, and generally, the smaller the molecular weight, the higher the uronic acid content, the higher the anti-cancer and anti-cancer activities of the polysaccharides. Besides, the polysaccharide can be chelated with metal elements to form polysaccharide-metal element chelate so as to enhance the anticancer effect of the polysaccharide. For example, the dictyophora indusiata polysaccharide-zinc chelate disclosed in the patent publication No. CN104277134A and the prunella vulgaris polysaccharide-zinc chelate disclosed in the patent publication No. CN104031156A have stronger anticancer effects than the raw material polysaccharide. Wherein, the chelating ability of the polysaccharide and the metal elements has obvious influence on the anticancer ability of the polysaccharide. Therefore, the polysaccharide which has smaller molecular weight, higher uronic acid content and stronger metal element chelating ability can be obtained, and has important significance for the application of the polysaccharide in the aspect of anticancer.
The existing preparation methods of low molecular weight polysaccharide mainly comprise a high-temperature hydrolysis method, an acid hydrolysis method and an enzymolysis method. The high-temperature hydrolysis method is adopted to prepare the low-molecular-weight polysaccharide, so that the energy consumption is high, the time consumption is long, and the yield is low. When the acid hydrolysis method is adopted, acid waste liquid is generated, and certain pollution is caused to the environment. The enzymolysis method has high cost and high requirements on reaction conditions, and cannot be used for large-scale production. Therefore, the market urgently needs a method for preparing low molecular weight polysaccharide with high efficiency, green and safety. In addition, no relevant research on the preparation of low molecular weight polysaccharides by a gradient physical field is reported at present.
At present, the traditional hydrothermal extraction method is mainly adopted for extracting the noni polysaccharide, and the method has high energy consumption and long time, and has low noni polysaccharide yield, low purity and poor activity. Therefore, an extraction technology which is efficient and can protect the activity of the noni polysaccharide to the maximum extent is urgently needed in the field.
Disclosure of Invention
In order to overcome the defects and defects in the prior art, the invention aims to provide the method for preparing the noni polysaccharide by utilizing the multi-physical field synergy, low energy consumption, high efficiency and high yield are realized, the prepared product has small molecular weight and strong anticancer activity, and guidance is provided for the extraction and application of the noni polysaccharide.
The invention principle of the technology is as follows: the invention firstly uses the discontinuous pulse electric field to process the raw material, then immediately transfers the raw material into the ultrasonic field to process, and transfers the extracting solution into the continuous pulse electric field to process after the ultrasonic processing. Under the low-frequency discharge of a high-voltage pulse electric field, the noni powder breaks down the cell wall and the cell membrane to form a hole crack, so that the cavitation of ultrasonic waves can be more favorably exerted in cells and on the surface of the cells, and the high-efficiency extraction of polysaccharide is realized. The extracted macromolecular polysaccharide is decomposed into micromolecular polysaccharide under the action of a continuous high-frequency pulse electric field.
The object of the invention is achieved by the following steps:
a method for preparing noni polysaccharide with high anticancer and antioxidant activity by a gradient physical field comprises the following steps:
1) treating the noni raw material: drying noni, pulverizing, sieving, adding ethanol solution, refluxing, defatting, decolorizing, and drying to obtain noni powder;
2) preparing noni polysaccharide by a cascade physical field: adding distilled water into Morinda citrifolia powder, placing into intermittent pulsed electric field extraction equipment at room temperature, and treating for 30-40 times, with pulse frequency of 1-5Hz and treatment field intensity of 3-6 kV/cm; then transferring the mixture into ultrasonic equipment for treatment, wherein the ultrasonic power is 300-600W, the frequency is 20-40kHz, the temperature is maintained at 50-60 ℃, and the ultrasonic time is 20-30 min; then the extract is transferred into a continuous pulse electric field for treatment, the treatment field intensity is 10-25kV/cm, the frequency is 500-1300Hz, the pulse width is 60-80us, and the treatment time is 40-50 min;
3) and (3) purifying noni polysaccharide: and after treatment, performing centrifugal separation on the extracting solution to obtain polysaccharide crude extracting solution, adding a Sevage reagent, performing vortex oscillation to remove precipitates, repeating centrifugal separation, adding the Sevage reagent, performing vortex oscillation to remove the precipitates for 4-6 times until protein is completely removed and no precipitate is separated out, performing alcohol precipitation, performing centrifugal separation, performing precipitate freeze-drying, and thus obtaining noni polysaccharide.
To further achieve the object of the present invention, preferably, in the step 1), the drying of the noni is to dry the noni at 45-50 ℃ for 48-60 h.
Preferably, in the step 1), the sieving is 80-100 mesh sieving.
Preferably, in the step 1), the ethanol solution is 75-85% ethanol water solution by volume percentage.
Preferably, in the step 1), the reflux treatment refers to reflux treatment at 70-80 ℃ for 4 h.
Preferably, in step 1), the noni fruits are ripe noni fruits in white ripe stage.
Preferably, in the step 2), the feed-liquid ratio of the noni powder to the distilled water is 1:25-1:35, and the unit of the feed-liquid ratio of the noni powder to the unit of the distilled water is gram and milliliter.
Preferably, in the step 3), the rotation speed of the centrifugal separation is 4800-5000rpm, and the time of the centrifugal separation is 15-20 min.
Preferably, in step 3), the Sevage reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 3.5: 1-4.5: 1, wherein the volume ratio of the polysaccharide crude extract to the Sevage reagent is 1: 1-1: 5, the vortex oscillation time is 10-20 min.
Preferably, in the step 3), the alcohol precipitation is to add 3.5 to 4.5 times of volume of absolute ethyl alcohol into the crude extract to carry out alcohol precipitation for 12 to 20 hours at 1 to 4 ℃.
Compared with the prior art, the invention has the following advantages and beneficial effects.
1) Compared with the traditional water extraction method, the method has the advantages of low energy consumption, short extraction time, and obviously increased polysaccharide yield, uronic acid content and total sugar content by 98%, 20% and more than 150% respectively compared with the traditional hydrothermal method.
2) The invention can rapidly and efficiently prepare micromolecular noni polysaccharide by utilizing the cascade physical field technology, and compared with the noni polysaccharide extracted by the traditional hydrothermal method, the molecular weight is reduced by more than 40%. In addition, acid and enzyme are not required to be added in the extraction preparation process, and long-time heating is not required, so that the preparation process is green, energy-saving and low in cost.
3) Compared with the noni polysaccharide extracted by the traditional hydrothermal method, the noni polysaccharide extracted by utilizing the cascade physical field has obviously improved oxidation resistance and anticancer capability.
4) Compared with the noni polysaccharide extracted by the traditional hydrothermal method, the noni polysaccharide extracted by utilizing the gradient physical field has stronger chelating capacity to metal element zinc, and the noni polysaccharide-zinc chelate formed after chelating has better anticancer activity and larger amplification.
Drawings
FIG. 1 is a process flow chart of the method for preparing noni polysaccharide with high anticancer and antioxidant activity by using a cascade physical field.
FIG. 2 results of HPGPC molecular weight measurement of noni polysaccharide obtained by water extraction and gradient physical field method in example 1.
FIG. 3 shows the results of HPGPC measurement of the molecular weight of noni polysaccharide obtained by water extraction and step physical field method in example 2.
FIG. 4 shows the results of HPGPC measurement of the molecular weight of noni polysaccharide obtained by water extraction and step physical field method in example 3.
Detailed Description
For a better understanding of the present invention, the present invention will be further described with reference to the following drawings and examples, but the present invention is not limited thereto.
In the embodiment, the discontinuous pulse electric field equipment is a PEF-EX-500 type pulse electric field extractor of Xinan food science and technology Limited, Guangzhou city. The continuous pulse electric field equipment is SY-Z-500 type pulse electric field sterilization equipment of Xinan food science and technology limited company in Guangzhou city.
Example 1
As shown in fig. 1, a method for preparing noni polysaccharide with high anticancer and antioxidant activity by a gradient physical field comprises the following steps:
treating the noni raw material: drying noni fruit (the mature noni fruit in white ripe stage) at 50 deg.C, pulverizing, sieving (sieving with 80 mesh sieve), adding 4 volume percent 80% ethanol solution, reflux treating for 4 hr, defatting, decolorizing, and oven drying to obtain noni fruit powder.
The noni polysaccharide is extracted by the aid of a cascade physical field: adding 35g of noni powder into distilled water according to a material-liquid ratio of 1:35 (the mass of the noni powder and the unit of the distilled water are gram and milliliter respectively), placing the mixture into an intermittent pulsed electric field extraction device for treatment for 30 times, wherein the pulse frequency is 1Hz, the treatment field intensity is 3kV/cm, then carrying out ultrasonic treatment for 20min by adopting an ultrasonic device, the ultrasonic power is 300W, the ultrasonic frequency is 40kHz, and the temperature is maintained at 60 ℃. And after the ultrasonic treatment is finished, the mixture is transferred to continuous pulse electric field equipment for treatment for 40min, the field intensity is 20kV/cm, the pulse width is 60us, and the frequency is 500 Hz.
After the treatment, centrifuging the extracting solution at 4800rpm for 15min, centrifuging, taking supernatant, combining to obtain a polysaccharide crude extracting solution, and adding a Sevage reagent, wherein the preparation method of the Sevage reagent is that the volume ratio of chloroform to n-butyl alcohol is 4: 1, mixing the polysaccharide crude extract with a Sevage reagent in a volume ratio of 1: 1, carrying out vortex oscillation for 10min to remove precipitates, repeating centrifugal separation to obtain supernatant, merging the supernatant to obtain polysaccharide crude extract, adding a Sevage reagent, carrying out vortex oscillation to remove the precipitates for 6 times in total, keeping the preparation method of the Sevage reagent unchanged, completely removing proteins in the polysaccharide crude extract until no precipitates are separated out, adding 4 times of volume of absolute ethyl alcohol into the crude extract, carrying out alcohol precipitation for 12h at 4 ℃, then carrying out centrifugal separation, taking the precipitates, and freeze-drying to obtain noni polysaccharide.
Extracting noni polysaccharide by a traditional water extraction method: taking 35g of noni powder according to the material-liquid ratio of 1: distilled water was added to 35 ℃ and extracted in a water bath at 80 ℃ for 2 hours. After the treatment, centrifuging the extracting solution at 4800rpm for 15min, mixing the extracting solutions to obtain a polysaccharide crude extracting solution, and adding a Sevage reagent, wherein the preparation method of the Sevage reagent is that the volume ratio of chloroform to n-butanol is 4: 1, mixing the polysaccharide crude extract and a Sevage reagent in a volume ratio of 1: 1, carrying out vortex oscillation for 10min, removing precipitates, repeating for 6 times until no precipitate is separated out, adding 4 times of volume of absolute ethyl alcohol into the crude extract, carrying out alcohol precipitation for 12h at 4 ℃, then carrying out centrifugal separation, taking the precipitate, and freeze-drying to obtain noni polysaccharide.
Polysaccharide extraction ratio (%) ═ polysaccharide mass/noni powder mass × 100
The total sugar concentration was determined by the phenol-sulfuric acid method. And (3) determining the total sugar content of the noni polysaccharide by adopting a phenol-sulfuric acid method. And (3) making a standard curve: 1mg/ml glucose solution is prepared in a 1L volumetric flask, and 1, 2, 3, 4, 5 and 6ml mother liquor is respectively sucked into a 100ml volumetric flask for constant volume. Then respectively sucking 0.5ml of each gradient solution into a 10ml centrifuge tube, then adding 0.5ml of phenol solution (5%) and 2.5ml of concentrated sulfuric acid, fully mixing, standing for 10min at room temperature, bathing for 15min at 30 ℃, measuring the light absorption value at 490nm by using an enzyme-labeling instrument, and establishing a standard curve. Accurately weighing a certain amount of crude noni polysaccharide, adding deionized water for dissolving, measuring a light absorption value according to the operation, simultaneously ensuring that the light absorption value falls within the range of the standard curve, and obtaining the total sugar content of the crude noni polysaccharide through calculation.
And (4) measuring the content of uronic acid. The uronic acid content was determined by ion chromatography. The detection equipment is a Dionex ICS-3000 ion chromatograph, and the detection conditions are as follows: chromatographic column CarboPac PA20 analytical column, Carbo PA1 guard column; a detector for detecting the pulse ampere-times; the sample injection amount is 10 uL; the detection temperature is 30 ℃; the flow rate is 0.5 mL/min; the mobile phase A is ultrapure water, the B is 200 mM NaOH solution, the C is 500mM sodium acetate solution, and the D is 20mM NaOH solution. Weighing 1.0g of uronic acid standard, diluting to 1000mL, diluting to obtain 1.0, 2.0, 5.0, 8.0, 10.0ug/mL standard sample, detecting by sampling according to the above conditions, and drawing standard curve with peak area as abscissa and uronic acid content as abscissa. 10mg of the sample was hydrolyzed in 5mL of 4M trifluoroacetic acid at 105 ℃ for 6h, followed by diluting 1mL of the sample to 100mL with deionized water. Taking 1mL of the diluted sample, passing through a 0.22um water phase film, measuring, and substituting the peak area into a standard curve to calculate the content of uronic acid.
The molecular weight of the polysaccharide is determined by high performance gel chromatography (HPGPC). Using mobile phase (KH)2PO40.02M) prepared polysaccharide to a 1mg/mL solution and then subjected to sample detection. The detection conditions are as follows: column temperature 35 deg.C, flow rate 0.6mL/min, sample amount 20uL, detector Waters2414 differential detector, chromatographic column TSK-GEL5000K GEL column (7.8mm × 300mm PWXL) and TSK-GEL3000K GEL column (7.8mm × 300mm PWXL), 0.02mol/LKH2PO4(pH6.0) as a mobile phase.
The antioxidant activity of the noni polysaccharide is detected by three methods, namely a DPPH method, an ABTS method and a dehydroxylation free radical method.
The DPPH method: preparing noni polysaccharide into solutions of 1.0, 2.0, 3.0, 4.0 and 5.0mg/mL by using deionized water, accurately transferring 100uL of polysaccharide solution and 100uL of DPPH (200uM, prepared by absolute ethyl alcohol) into an ELISA plate, uniformly mixing, standing at room temperature for 30min, measuring a light absorption value at 517nm by using the ELISA plate, and taking ascorbic acid as a positive control. DPPH clearance was calculated according to the following formula:
DPPH radical scavenging ratio (%) [1- (As-Ab)/Ac ] x 100
Wherein, Ab is the light absorption value of the sample and absolute ethyl alcohol, and Ac is the light absorption value of deionized water replacing the sample and DPPH.
ABTS method: noni polysaccharide was formulated with deionized water into solutions of 1.0, 2.0, 3.0, 4.0, 5.0mg/mL for ready use. ABTS (7.00mM) solution and potassium persulfate solution (2.45mM) were mixed in equal volumes, and after 16h at room temperature in the absence of light, the mixture was diluted to an absorbance of 0.7 (+ -0.03) at 734 nm. After 20. mu.l of polysaccharide solution and 150. mu.l of ABTS solution are fully and uniformly mixed, the mixture is reacted for 30min in a dark place at room temperature, and the light absorption value of the mixture at 734nm is measured by a microplate reader. Ascorbic acid was used as a positive control. ABTS clearance was calculated according to the following formula:
ABTS free radical scavenging ratio (%) [1- (As/A) ]. times.100
Wherein As is the light absorption value of the sample group, and A is the light absorption value of the deionized water instead of the sample.
Dehydroxy radical method: the noni polysaccharide is prepared into solutions of 1.0, 2.0, 3.0, 4.0 and 5.0mg/mL by deionized water for later use. 50uL of ferrous sulfate solution (1.5mM), 35uL of hydrogen peroxide (6.7mM) and 15uL of sodium salicylate solution (20mM) were added to the plate, and then 100uL of the sample solution was added thereto, followed by mixing well, reaction at 37 ℃ for 1 hour, and absorbance at 562nm was measured. Ascorbic acid was used as a positive control. The dehydroxy radical clearance was calculated according to the following formula:
dehydroxylation radical clearance (%) [1- (As-Ab)/Ac ] x 100
Wherein As is the light absorption value of the sample group, Ab is the light absorption value of deionized water replacing sodium salicylate, and Ac is the light absorption value of deionized water replacing the sample.
Noni polysaccharide is chelated with Zn element. At room temperature, the zinc chloride solution (2mg/mL) was adjusted to pH to complete dissolution of zinc chloride, and then 5mg/mL of the noni polysaccharide solution extracted by the step physical field assisted extraction and the conventional water extraction method of the present example (10 mL) was added respectively and mixed, and the pH was adjusted to weak alkalinity (pH7-8) with sodium hydroxide. The two mixed solutions are respectively placed on a shaking table in an incubator at 50 ℃ and reacted for 2 days at constant temperature and humidity. After the reaction, the reaction solution was poured into a centrifuge tube, centrifuged at 8000 for 5min, and the supernatant was poured into a 2.5kDa dialysis bag, placed in a 4 ℃ refrigerator, and dialyzed with ultrapure water for 3 times. After the dialysis is finished, the mixed solution is frozen and dried in vacuum, and two dried noni polysaccharide-Zn chelate samples are placed in a dryer for storage and standby.
The content of chelate zinc element is determined by AAS element analyzer. Weighing a proper amount of sample in an erlenmeyer flask, adding a mixed acid (nitric acid: perchloric acid ═ 4: 1), digesting overnight, then digesting on a hot plate, and adding the mixed acid until the digestion solution is colorless and transparent after the digestion is completed. Transferring the digestive juice to a 25mL volumetric flask, fixing the volume with distilled water, using a Z-5000 element analyzer (AAS), drawing a standard curve by using a zinc standard solution, and measuring the content of the zinc element in the sample.
The anticancer effect of noni polysaccharide and noni polysaccharide zinc chelate is determined by adopting a CCK-8 method and taking human liver cancer HepG2 cells as experimental objects. 100 μ L of cell suspension was prepared in a 96-well plate at 4X 10 per well3The inoculation was carried out at each density. The plates were pre-incubated in an incubator for 24 hours (5% CO at 37 ℃)2Under the conditions of (a). To the plate, 10. mu.L of the test substance was added at different concentrations. After 48 hours incubation in the incubator, 10. mu.L of CCK-8 solution was added to each well (care was taken not to generate bubbles in the wells which would affect the OD reading). If the test substance is aerobic or reductive, the test substance can be removed by replacing the fresh medium (removing the medium and washing the cells twice with medium before adding CCK-8, and then adding new medium) before adding CCK-8. The plates were incubated in an incubator for 1-4 hours and absorbance at 450nm was measured using a microplate reader. Cell viability was calculated according to the following formula.
Cell survival (%) - (As-Ab)/(Ac-Ab) ]. times.100
As is the absorbance of the assay well (the absorbance of the well containing cells, medium, CCK-8 and test compound). Ab is absorbance of blank wells (absorbance of wells containing medium and CCK-8). Ac is the absorbance of the control wells (wells containing cells, medium and CCK-8).
The polysaccharide extraction yield, total sugar content and uronic acid content are shown in table 1. As can be seen from Table 1, the extraction rate of the gradient physical Favory polysaccharide is 13.42%, which is increased by 104% compared with the extraction rate of the traditional water extraction method (6.57%). Secondly, the total sugar content in the noni polysaccharide in the cascade physical site is up to 90.14 percent, which is increased by 27 percent compared with the total sugar content (74.14 percent) in the traditional water extraction method, and in addition, the uronic acid content in the noni polysaccharide in the cascade physical site is 3.21 percent, which is increased by 158 percent compared with the uronic acid content (1.24 percent) in the traditional water extraction method. The results show that the extraction rate of noni polysaccharide extracted by a cascade physical field is much higher than that of the traditional water extraction method, and the total sugar content and the uronic acid content of extracted polysaccharide are obviously higher than those of the polysaccharide extracted by the traditional water extraction method.
The molecular weight size of the noni polysaccharide is determined as shown in fig. 2, and the molecular weight of the noni polysaccharide extracted by the step physical field method is 34kDa and the molecular weight of the noni polysaccharide extracted by the hydrothermal method is 62kDa according to the high performance gel chromatography result. Compared with the noni polysaccharide extracted by a hydrothermal method, the molecular weight of the noni polysaccharide extracted by a cascade physical site is reduced by 45%.
TABLE 1 influence of different extraction methods on the extraction yield of noni polysaccharide, total sugar content and uronic acid content
The different letter representations are significantly different (p <0.05)
The results of the evaluation of the antioxidant activity of noni polysaccharide by the hydrothermal method and the step physical field method are shown in table 2. As shown in Table 2, the DPPH, ABTS and dehydroxylation free radical clearance of the polysaccharide in the cascade physical site at concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0mg/mL are higher than that of the polysaccharide in the hydrothermal method. The results show that the noni polysaccharide shows stronger antioxidant activity in the same concentration in the step physical site than the noni polysaccharide obtained by the hydrothermal method.
TABLE 2 antioxidant evaluation results of noni polysaccharide by hydrothermal method and cascade physical field method
After the noni polysaccharides were chelated with zinc, the zinc content in the two noni polysaccharide-zinc chelates was measured by ASS element analyzer, and the results are shown in fig. 4. The zinc content of the chelate formed by the noni polysaccharide extracted by the cascade physical field method is 6.44 percent, while the zinc content of the chelate formed by the noni polysaccharide extracted by the hydrothermal method is only 2.53 percent. Compared with the chelate formed by the noni polysaccharide by a hydrothermal method, the noni polysaccharide in the cascade physical site has stronger binding capacity to zinc element
The anticancer effect results of the polysaccharide and the polysaccharide-zinc chelate are shown in table 3.
The noni polysaccharide extracted by the two methods has certain inhibition effect on human liver cancer HepG2 cells and shows dose dependence. Wherein, under the selected concentration gradient, the HepG2 cell survival rate of the added gradient physical site noni polysaccharide is lower than that of the added gradient physical site noni polysaccharide prepared by a hydrothermal method. Specifically, under the concentration of 125, 250 and 500ug/mL, the survival rates of HepG2 cells added with the noni polysaccharide in the cascade physical site are respectively 61.2%, 42.3% and 24.5%, and the survival rates of HepG2 cells added with the noni polysaccharide extracted by the hydrothermal method are respectively 81.3%, 58.2% and 42.4%. The experimental results show that the morinda citrifolia polysaccharide in the ladder-grade physical site has stronger anticancer activity.
The anticancer effect of the two noni polysaccharides chelated with the zinc element is remarkably improved, and the noni polysaccharides show dose dependence. After the noni polysaccharide extracted by a hydrothermal method is chelated with zinc under three concentrations of 125, 250 and 500ug/mL, the survival rate of the human liver cancer HepG2 cells is reduced by 17.98%, 13.99% and 2.28%. After the noni polysaccharide is chelated with zinc in a cascade physical place, the survival rate of the human liver cancer HepG2 cells is reduced by 21.32%, 20.16% and 4.79%. The result shows that the anticancer activity of the morinda citrifolia polysaccharide in the cascade physical site is increased more than that of the morinda citrifolia polysaccharide prepared by a hydrothermal method after zinc chelation, and the reason is mainly that the morinda citrifolia polysaccharide in the cascade physical site has stronger chelating capacity for zinc elements and can be combined with more zinc elements to enhance the anticancer activity.
TABLE 3 Effect of two noni polysaccharides and their zinc chelates on human hepatoma HepG2 cell survival rate
Example 2
As shown in fig. 1, a method for preparing noni polysaccharide with high anticancer and antioxidant activity by a gradient physical field comprises the following steps:
treating the noni raw material: drying noni fruit (the noni fruit is mature noni fruit in white ripe stage), pulverizing at 48 deg.C, sieving (sieving with 80 mesh sieve), adding 4 volume percent of 85% ethanol solution, reflux treating for 4 hr, degreasing, decolorizing, and drying to obtain noni fruit powder.
The noni polysaccharide is extracted by the aid of a cascade physical field: taking 30g of noni powder according to the material-liquid ratio of 1: 30, adding distilled water, putting into an intermittent pulse electric field extraction device for treatment for 35 times, wherein the pulse frequency is 5Hz, the treatment field intensity is 5kV/cm, then performing ultrasonic treatment for 28min by adopting ultrasonic equipment, the ultrasonic power is 600W, the ultrasonic frequency is 40kHz, and the temperature is maintained at 55 ℃. And after the ultrasonic treatment is finished, the mixture is transferred to continuous pulse electric field equipment for treatment for 48min, wherein the field intensity is 20kV/cm, the pulse width is 70us, and the frequency is 1000 Hz.
After the treatment, centrifuging the extracting solution at 4900rpm for 18min, taking supernatant, combining the supernatant to obtain polysaccharide crude extracting solution, and adding Sevage reagent, wherein the preparation method of the Sevage reagent is that the volume ratio of chloroform to n-butyl alcohol is 3.5: 1, mixing the polysaccharide crude extract and a Sevage reagent in a volume ratio of 1: and 4, carrying out vortex oscillation for 15min to remove precipitates, repeating for 4 times until the proteins are completely removed and no precipitate is separated out, adding 3.5 times of volume of absolute ethyl alcohol into the crude extract, carrying out alcohol precipitation for 18h at 2 ℃, then carrying out centrifugal separation, taking the precipitate, and freeze-drying to obtain the noni polysaccharide.
Extracting noni polysaccharide by a traditional water extraction method: taking 30g of noni powder according to the material-liquid ratio of 1: distilled water was added to 30 ℃ and extracted in a water bath at 60 ℃ for 2 hours. After the treatment, centrifuging the extracting solution at 4900rpm for 18min, taking supernatant, combining the supernatant to obtain polysaccharide crude extracting solution, and adding Sevage reagent, wherein the preparation method of the Sevage reagent is that the volume ratio of chloroform to n-butyl alcohol is 3.5: 1, mixing the polysaccharide crude extract and a Sevage reagent in a volume ratio of 1: and 4, carrying out vortex oscillation for 15min to remove the precipitate, repeating for 4 times until the protein is completely removed and no precipitate is separated out, adding 3.5 times of volume of absolute ethyl alcohol into the crude extract, carrying out alcohol precipitation for 18h at the temperature of 2 ℃, then carrying out centrifugal separation, taking the precipitate, and freeze-drying to obtain the noni polysaccharide.
The obtained noni polysaccharide was tested for yield, total sugar content, uronic acid content, molecular weight, antioxidant activity, binding ability to zinc element, and anticancer activity according to the method of example 1.
The polysaccharide extraction yield, total sugar content and uronic acid content are shown in table 4. As can be seen from Table 4, the extraction rate of the gradient physical Favory polysaccharide is 12.45%, which is increased by 103% compared with the extraction rate (6.12%) of the traditional water extraction method. Secondly, the total sugar content in the noni polysaccharide in the cascade physical site is up to 88.43 percent, which is increased by 22 percent compared with the total sugar content (72.19 percent) in the traditional water extraction method, and in addition, the uronic acid content in the noni polysaccharide in the cascade physical site is 3.18 percent, which is increased by 165 percent compared with the uronic acid content (1.20 percent) in the traditional water extraction method. The results show that the extraction rate of noni polysaccharide extracted by a cascade physical field is much higher than that of the traditional water extraction method, and the total sugar content and the uronic acid content of extracted polysaccharide are obviously higher than those of the polysaccharide extracted by the traditional water extraction method.
TABLE 4 influence of different extraction methods on the extraction yield of noni polysaccharide, total sugar content and uronic acid content
The different letter representations are significantly different (p <0.05)
The molecular weight size of the noni polysaccharide is determined as shown in fig. 3, and the molecular weight of the noni polysaccharide extracted by the step physical field method is 37.6kDa and the molecular weight of the noni polysaccharide extracted by the hydrothermal method is 64.4kDa according to the high performance gel chromatography result. Compared with the noni polysaccharide extracted by a hydrothermal method, the molecular weight of the noni polysaccharide extracted by a cascade physical site is reduced by 41.46 percent. The results of the evaluation of the antioxidant activity of noni polysaccharide by the hydrothermal method and the step physical field method are shown in table 5. As can be seen from Table 5, the DPPH, ABTS and dehydroxylation free radical clearance of the polysaccharide in the cascade physical site at concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0mg/mL are all higher than that of the polysaccharide in the hydrothermal method. The results show that the noni polysaccharide shows stronger antioxidant activity in the same concentration in the step physical site than the noni polysaccharide obtained by the hydrothermal method.
After the noni polysaccharide is chelated with the zinc element, the zinc content in the two noni polysaccharide-zinc chelates is measured by an ASS element analyzer. The zinc content of the chelate formed by the noni polysaccharide extracted by the cascade physical field method is 5.98 percent, while the zinc content of the chelate formed by the noni polysaccharide extracted by the hydrothermal method is only 2.67 percent. Compared with a chelate formed by the noni polysaccharide extracted by a hydrothermal method, the noni polysaccharide extracted by a cascade physical site has stronger binding capacity to a zinc element.
TABLE 5 antioxidant evaluation results of noni polysaccharide by hydrothermal method and cascade physical field method
The anticancer effect results of the polysaccharide and the polysaccharide-zinc chelate are shown in table 6.
TABLE 6 influence of two noni polysaccharides and their zinc chelates on human liver cancer HepG2 cell survival rate
The noni polysaccharide extracted by the two methods has certain inhibition effect on human liver cancer HepG2 cells and shows dose dependence. Wherein, under the selected concentration gradient, the HepG2 cell survival rate of the added gradient physical site noni polysaccharide is lower than that of the added gradient physical site noni polysaccharide prepared by a hydrothermal method. Specifically, under the concentration of 125, 250 and 500ug/mL, the survival rates of HepG2 cells added with the noni polysaccharide in the cascade physical site are 61.32 percent, 45.23 percent and 26.32 percent respectively, and the survival rates of HepG2 cells added with the noni polysaccharide by a hydrothermal method are 82.12 percent, 53.21 percent and 45.13 percent respectively. According to the experimental result, the noni polysaccharide in the cascade physical site shows stronger anticancer activity.
The anticancer effect of the two noni polysaccharides chelated with the zinc element is remarkably improved, and the noni polysaccharides show dose dependence. After the noni polysaccharide extracted by a hydrothermal method is chelated with zinc under three concentrations of 125, 250 and 500ug/mL, the survival rate of the human liver cancer HepG2 cells is reduced by 17.98%, 13.99% and 2.28%. After the noni polysaccharide is chelated with zinc in a cascade physical place, the survival rate of the human liver cancer HepG2 cells is reduced by 21.32%, 20.16% and 4.79%. The result shows that the anticancer activity of the morinda citrifolia polysaccharide in the cascade physical site is increased more than that of the morinda citrifolia polysaccharide prepared by a hydrothermal method after zinc chelation, and the reason is mainly that the morinda citrifolia polysaccharide in the cascade physical site has stronger chelating capacity for zinc elements and can be combined with more zinc elements to enhance the anticancer activity.
Example 3
As shown in fig. 1, a method for preparing noni polysaccharide with high anticancer and antioxidant activity by a gradient physical field comprises the following steps:
treating the noni raw material: drying noni fruit (mature noni fruit in white ripeness stage) at 45 deg.C, pulverizing, sieving (sieving with 100 mesh sieve), adding 4 times of 75% ethanol solution, reflux treating for 4 hr, defatting, decolorizing, and drying to obtain noni fruit powder.
The noni polysaccharide is extracted by the aid of a cascade physical field: taking 25g of noni powder according to the material-liquid ratio of 1:25 adding distilled water, placing into intermittent pulsed electric field extraction equipment for treatment for 40 times, wherein the pulse frequency is 4Hz, the treatment field intensity is 6kV/cm, and then performing ultrasonic treatment for 20min by using ultrasonic equipment, the ultrasonic power is 600W, the ultrasonic frequency is 40kHz, and the temperature is maintained at 50 ℃. And after the ultrasonic treatment is finished, the mixture is transferred to continuous pulse electric field equipment for treatment for 45min, wherein the field intensity is 25kV/cm, the pulse width is 80us, and the frequency is 1300 Hz.
After the treatment, centrifuging the extracting solution at 4800rpm for 15min, taking supernatant, combining the supernatant to obtain polysaccharide crude extracting solution, and adding Sevage reagent, wherein the preparation method of the Sevage reagent is that the volume ratio of chloroform to n-butyl alcohol is 4.5: 1, mixing the polysaccharide crude extract and a Sevage reagent in a volume ratio of 1: and 5, carrying out vortex oscillation for 20min to remove precipitates, repeating for 4 times until the proteins are completely removed and no precipitate is separated out, adding 4.5 times of volume of absolute ethyl alcohol into the crude extract, carrying out alcohol precipitation for 20h at 1 ℃, then carrying out centrifugal separation, taking the precipitate, and freeze-drying to obtain the noni polysaccharide.
Extracting noni polysaccharide by a traditional water extraction method: taking 25g of noni powder according to the material-liquid ratio of 1: distilled water was added to 25 ℃ and extracted in a water bath at 60 ℃ for 2 hours. After the treatment, centrifuging the extracting solution at 4800rpm for 15min, taking supernatant, combining the supernatant to obtain polysaccharide crude extracting solution, and adding Sevage reagent, wherein the preparation method of the Sevage reagent is that the volume ratio of chloroform to n-butyl alcohol is 4.5: 1, mixing the polysaccharide crude extract and a Sevage reagent in a volume ratio of 1: and 5, carrying out vortex oscillation for 20min to remove precipitates, repeating for 4 times until protein is completely removed and no precipitate is separated out, adding 4.5 times of volume of absolute ethyl alcohol into the crude extract, carrying out alcohol precipitation for 20h at 1 ℃, then carrying out centrifugal separation, taking the precipitate, and freeze-drying to obtain the noni polysaccharide.
The obtained noni polysaccharide was tested for yield, total sugar content, uronic acid content, molecular weight, antioxidant activity, binding ability to zinc element, and anticancer activity according to the method of example 1.
The polysaccharide extraction yield, total sugar content and uronic acid content are shown in table 7. As can be seen from Table 7, the extraction rate of the ladder physical Favory polysaccharide is 13.24%, which is increased by 98.5% compared with the extraction rate (6.67%) of the conventional water extraction method. Secondly, the total sugar content in the noni polysaccharide in the cascade physical site is 87.13%, which is increased by 24% compared with the total sugar content (70.25%) in the traditional water extraction method, and in addition, the uronic acid content in the noni polysaccharide in the cascade physical site is 3.16%, which is increased by 165% compared with the uronic acid content (1.19%) in the traditional water extraction method. The results show that the extraction rate of noni polysaccharide extracted by a cascade physical field is much higher than that of the traditional water extraction method, and the total sugar content and the uronic acid content of extracted polysaccharide are obviously higher than those of the polysaccharide extracted by the traditional water extraction method.
TABLE 7 influence of different extraction methods on the extraction yield of noni polysaccharide, total sugar content and uronic acid content
The different letter representations are significantly different (p <0.05)
The molecular weight size of the noni polysaccharide is determined as shown in fig. 4, and the molecular weight of the noni polysaccharide extracted by the step physical field method is 33.45kDa and the molecular weight of the noni polysaccharide extracted by the hydrothermal method is 58.8kDa according to the high performance gel chromatography result. Compared with the noni polysaccharide extracted by a hydrothermal method, the molecular weight of the noni polysaccharide extracted by a cascade physical site is reduced by 43.11%.
The results of evaluation of antioxidant activity of noni polysaccharide by hydrothermal method and cascade physical field method are shown in Table 8. As can be seen from Table 8, the DPPH, ABTS, and dehydroxy radical clearance of the polysaccharide in the cascade physical location at concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0mg/mL were higher than that of the polysaccharide in the hydrothermal method. The results show that the noni polysaccharide shows stronger antioxidant activity in the same concentration in the step physical site than the noni polysaccharide obtained by the hydrothermal method.
TABLE 8 antioxidant evaluation results of noni polysaccharide by hydrothermal method and cascade physical field method
After the noni polysaccharide is chelated with the zinc element, the zinc content in the two noni polysaccharide-zinc chelates is measured by an ASS element analyzer. The zinc content of the chelate formed by the noni polysaccharide extracted by the cascade physical field method is 5.87 percent, while the zinc content of the chelate formed by the noni polysaccharide extracted by the hydrothermal method is only 2.59 percent. Compared with a chelate formed by the noni polysaccharide extracted by a hydrothermal method, the noni polysaccharide extracted by a cascade physical site has stronger binding capacity to a zinc element.
The anticancer effect results of the polysaccharide and the polysaccharide-zinc chelate are shown in Table 9.
TABLE 9 influence of two noni polysaccharides and their zinc chelates on human liver cancer HepG2 cell survival rate
The noni polysaccharide extracted by the two methods has certain inhibition effect on human liver cancer HepG2 cells and shows dose dependence. Wherein, under the selected concentration gradient, the HepG2 cell survival rate of the added gradient physical site noni polysaccharide is lower than that of the added gradient physical site noni polysaccharide prepared by a hydrothermal method. Specifically, under the concentration of 125, 250 and 500ug/mL, the survival rates of HepG2 cells added with the noni polysaccharide in the cascade physical site are 62.34%, 44.23% and 25.42% respectively, and the survival rates of HepG2 cells added with the noni polysaccharide by a hydrothermal method are 83.09%, 54.21% and 43.08% respectively. The experimental results show that the noni polysaccharide in the cascade physical site has stronger anticancer activity
The anticancer effect of the two noni polysaccharides chelated with the zinc element is remarkably improved, and the noni polysaccharides show dose dependence. After the noni polysaccharide extracted by a hydrothermal method is chelated with zinc under three concentrations of 125, 250 and 500ug/mL, the survival rate of the human liver cancer HepG2 cells is reduced by 20.28%, 9.97% and 2.85%. After the noni polysaccharide is chelated with zinc in a cascade physical place, the survival rate of the human liver cancer HepG2 cells is reduced by 21.23%, 24.15% and 7.28%. The result shows that the anticancer activity of the morinda citrifolia polysaccharide in the cascade physical site is increased more than that of the morinda citrifolia polysaccharide prepared by a hydrothermal method after zinc chelation, and the reason is mainly that the morinda citrifolia polysaccharide in the cascade physical site has stronger chelating capacity for zinc elements and can be combined with more zinc elements to enhance the anticancer activity.
The embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and they are included in the scope of the present invention.
Claims (10)
1. A method for preparing noni polysaccharide with high anticancer and antioxidant activity by a gradient physical field is characterized by comprising the following steps:
1) treating the noni raw material: drying noni, pulverizing, sieving, adding ethanol solution, refluxing, defatting, decolorizing, and oven drying to obtain noni powder;
2) preparing noni polysaccharide by a cascade physical field: adding distilled water into Morinda citrifolia powder, placing into intermittent pulsed electric field extraction equipment at room temperature, and treating for 30-40 times, with pulse frequency of 1-5Hz and treatment field intensity of 3-6 kV/cm; then transferring the mixture into ultrasonic equipment for treatment, wherein the ultrasonic power is 300-600W, the frequency is 20-40kHz, the temperature is maintained at 50-60 ℃, and the ultrasonic time is 20-30 min; then the extracting solution is transferred into a continuous pulse electric field for treatment, the treatment field intensity is 10-25kV/cm, the frequency is 500-1300Hz, the pulse width is 60-80us, and the treatment time is 40-50 min;
3) and (3) purifying noni polysaccharide: and after treatment, performing centrifugal separation on the extracting solution to obtain polysaccharide crude extracting solution, adding a Sevage reagent, performing vortex oscillation to remove precipitates, repeating centrifugal separation, adding the Sevage reagent, performing vortex oscillation to remove the precipitates for 4-6 times until protein is completely removed and no precipitate is separated out, performing alcohol precipitation, performing centrifugal separation, performing precipitate freeze-drying, and thus obtaining noni polysaccharide.
2. The method for preparing noni polysaccharide with high anticancer and antioxidant activity through the gradient physical field according to claim 1, wherein in the step 1), the noni fruit is dried at 45-50 ℃ for 48-60 h.
3. The method for preparing noni polysaccharide with high anticancer and antioxidant activity by using the stepped physical field according to claim 1, wherein in the step 1), the sieving is carried out by a sieve of 80-100 meshes.
4. The method for preparing noni polysaccharide with high anticancer and antioxidant activity through the gradient physical field according to claim 1, wherein in the step 1), the ethanol solution is 75-85% of ethanol water solution by volume percentage.
5. The method for preparing noni polysaccharide with high anticancer and antioxidant activity through the gradient physical field according to claim 1, wherein in the step 1), the reflux treatment is 70-80 ℃ for 4 hours.
6. The method for preparing noni polysaccharides with high anticancer and antioxidant activities through the gradient physical field according to claim 1, wherein in the step 1), the noni fruits are mature noni fruits in white ripe stage.
7. The method for preparing noni polysaccharide with high anticancer and antioxidant activity through the gradient physical field according to claim 1, wherein in the step 2), the feed-liquid ratio of the noni powder to the distilled water is 1:25-1:35, and the mass of the noni powder and the unit of the distilled water in the feed-liquid ratio are gram and milliliter respectively.
8. The method for preparing noni polysaccharide with high anticancer and antioxidant activity by using cascade physical field as claimed in claim 1, wherein in step 3), the rotation speed of the centrifugal separation is 4800-5000rpm, and the time of the centrifugal separation is 15-20 min.
9. The method for preparing noni polysaccharide with high anticancer and antioxidant activity by using gradient physical field as claimed in claim 1, wherein in step 3), the preparation method of Sevage reagent is that chloroform and n-butanol are mixed at a volume ratio of 3.5: 1-4.5: 1, wherein the volume ratio of the polysaccharide crude extract to the Sevage reagent is 1: 1-1: 5, the vortex oscillation time is 10-20 min.
10. The method for preparing noni polysaccharide with high anticancer and antioxidant activity through the gradient physical field according to claim 1, wherein in the step 3), the alcohol precipitation is that absolute ethyl alcohol with 3.5-4.5 times of volume is added into the crude extract to carry out alcohol precipitation for 12-20h at 1-4 ℃.
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