CN117820437A - Jerusalem artichoke peptide, preparation method thereof and application thereof in kidney-protecting auxiliary diabetes treatment antioxidant product - Google Patents

Abstract

The invention relates to the technical field of jerusalem artichoke, which has various effects of controlling blood sugar, regulating immunity, resisting tumor and the like. The invention relates to jerusalem artichoke peptide, a preparation method thereof and application thereof in kidney-protecting auxiliary diabetes treatment antioxidant products. The amino acid sequence of the Jerusalem artichoke peptide selected from Pep2 and Pep2 is sequentially shown as SEQ ID NO.2, and the Jerusalem artichoke peptide is derived from Jerusalem artichoke (Helianthus tuberosus (L.1753)). The jerusalem artichoke peptide has antioxidant and antibacterial effects in vitro, can effectively reduce the blood sugar of hereditary hyperglycemia mice in vivo, can reduce diabetic complications, and has obvious protective effect on kidneys in particular.

Description

Jerusalem artichoke peptide, preparation method thereof and application thereof in kidney-protecting auxiliary diabetes treatment antioxidant product

Technical Field

The invention relates to the technical field of jerusalem artichoke, in particular to jerusalem artichoke peptide, a preparation method thereof and application thereof in kidney-protecting auxiliary diabetes treatment antioxidant products.

Background

Jerusalem artichoke (Helianthus tuberosus) is also called Jerusalem artichoke, is a perennial herb plant of Helianthus of Compositae, is also called Jerusalem artichoke, rhizoma picrorhizae and rhizoma Zingiberis recens, has effects of relieving constipation, promoting bile flow, detumescence, removing dampness, regulating stomach, etc., and has potential value for treating diabetes and rheumatism. The aerial parts of Jerusalem artichoke are stem leaves and flowers, the underground parts are roots and tubers, and each part contains various bioactive components such as inulin, phenolic acid, terpenes and the like, and has various effects of controlling blood sugar, regulating immunity, resisting tumor and the like.

Inulin contained in Jerusalem artichoke has certain antidiabetic effect. A comparison of WANG Z Q, SEUNGHWAN H, SUNYOUB L, et al, fermentation of purple Jerusalem artichoke extract to improve the alpha-glucosidase in hibitory effect in vitro and ameliorate blood glucose in db/db mic [ J ]. Nutrition Research & Practice,2016,10 (3): 282-287 ] shows that Jerusalem artichoke (L.plantarum-fermented purple Jerusalem artichoke, LJA) fermented by lactobacillus plantarum has the best inhibitory activity on alpha-glucosidase, and the inhibition rate reaches 49.34%; further, the efficacy of LJA on non-insulin dependent diabetic animals was examined, and as a result, it was found that the oral administration of LJA mg/kg group decreased blood glucose level by 42.25% as compared with the control group; serum insulin, high density lipoprotein and high density total cholesterol in LJA group mice increased by 30%, 38.89% and 13.52%, respectively; glycosylated hemoglobin and triglycerides were reduced by 10.32% and 65.27%, respectively.

Jerusalem artichoke also contains abundant amino acids, coumarin and polyacetylene compounds. Kim et al, "KIM D, FAN J P, CHUNG H C, et al, changes in extractability and antioxidant activity of Jerusalem artichoke (Helianthus tuberosus L.) tubers by various high hydrostatic pressure treatments [ J ]. Food Science & Biotechnology,2010,19 (5): 1365-1371" research shows that Jerusalem artichoke is rich in 10 non-essential amino acids of lysine, methionine, threonine, isoleucine, phenylalanine, valine and leucine, aspartic acid, serine, glutamic acid, glycine, alanine, cystine, tyrosine, histidine, arginine and proline.

However, the prior art does not find that peptide substances from jerusalem artichoke have the effect of reducing blood sugar, and further research and development on peptide substances from jerusalem artichoke resources are necessary for further opening the jerusalem artichoke resources.

Disclosure of Invention

In view of the above, the invention discloses a hypoglycemic antioxidant jerusalem artichoke peptide, a preparation method and application thereof. The jerusalem artichoke peptide has antioxidant and antibacterial effects in vitro, can effectively reduce the blood sugar of hereditary hyperglycemia mice in vivo, can reduce diabetic complications, and has obvious protective effect on kidneys in particular.

The invention aims to provide a blood sugar-reducing and antioxidant jerusalem artichoke peptide, which is selected from any one of Pep1, pep2, pep3, pep4 or Pep5, wherein the amino acid sequences of Pep1, pep2, pep3, pep4 and Pep5 are sequentially shown as SEQ ID NO. 1-5, and the jerusalem artichoke peptide is derived from jerusalem artichoke (Helianthus tuberosus (L.1753)) leaves.

The aforementioned Pep1 had an IC50 of 237.9.+ -. 20.6. Mu.g/ml for DPPH radical scavenging rate, 328.1.+ -. 43.8. Mu.g/ml for ABTS radical scavenging rate, 183.5.+ -. 24.3. Mu.g/ml for hydroxyl radical scavenging rate, 436.8.+ -. 50.9. Mu.g/ml for superoxide anion scavenging rate, 169.3.+ -. 18.5. Mu.g/ml for iron ion chelating activity, 208.7.+ -. 21.6. Mu.g/ml for copper ion chelating activity, and 188.6.+ -. 19.9. Mu.g/ml for lipid peroxidation inhibiting activity.

The aforementioned Pep2 had an IC50 for DPPH radical scavenging rate of 562.2.+ -. 36.4. Mu.g/ml, an IC50 for ABTS radical scavenging rate of 486.3.+ -. 43.2. Mu.g/ml, an IC50 for hydroxyl radical scavenging rate of 359.7.+ -. 29.5. Mu.g/ml, an IC50 for superoxide anion scavenging rate of 836.2.+ -. 36.8. Mu.g/ml, an IC50 for iron ion chelating activity of 284.8.+ -. 28.1. Mu.g/ml, an IC50 for copper ion chelating activity of 245.2.+ -. 24.8. Mu.g/ml, and an IC50 for lipid peroxidation inhibiting activity of 235.2.+ -. 63.5. Mu.g/ml.

The aforementioned Pep3 had an IC50 of 202.3.+ -. 17.9. Mu.g/ml for DPPH radical scavenging rate, an IC50 of 286.9.+ -. 32.0. Mu.g/ml for ABTS radical scavenging rate, an IC50 of 179.3.+ -. 20.7. Mu.g/ml for hydroxyl radical scavenging rate, an IC50 of 336.9.+ -. 36.5. Mu.g/ml for superoxide anion scavenging rate, an IC50 of 187.2.+ -. 20.3. Mu.g/ml for iron ion chelating activity, an IC50 of 193.2.+ -. 18.4. Mu.g/ml for copper ion chelating activity, and an IC50 of 208.3.+ -. 21.3. Mu.g/ml for lipid peroxidation inhibiting activity.

The aforementioned Pep4 had an IC50 of 302.4.+ -. 29.1. Mu.g/ml for DPPH radical scavenging rate, 313.5.+ -. 36.7. Mu.g/ml for ABTS radical scavenging rate, 193.2.+ -. 29.2. Mu.g/ml for hydroxyl radical scavenging rate, 368.4.+ -. 30.3. Mu.g/ml for superoxide anion scavenging rate, 197.6.+ -. 25.4. Mu.g/ml for iron ion chelating activity, 212.3.+ -. 22.5. Mu.g/ml for copper ion chelating activity, and 213.1.+ -. 29.3. Mu.g/ml for lipid peroxidation inhibiting activity.

The aforementioned Pep5 had an IC50 for DPPH radical scavenging rate of 216.7.+ -. 26.5. Mu.g/ml, an IC50 for ABTS radical scavenging rate of 289.3.+ -. 37.9. Mu.g/ml, an IC50 for hydroxyl radical scavenging rate of 156.3.+ -. 22.8. Mu.g/ml, an IC50 for superoxide anion scavenging rate of 305.5.+ -. 28.7. Mu.g/ml, an IC50 for iron ion chelating activity of 148.6.+ -. 15.2. Mu.g/ml, an IC50 for copper ion chelating activity of 183.7.+ -. 16.9. Mu.g/ml, and an IC50 for lipid peroxidation inhibiting activity of 157.4.+ -. 17.2. Mu.g/ml.

The invention also provides a preparation method of the jerusalem artichoke peptide, which comprises the following steps:

extracting fresh jerusalem artichoke leaves with alkali liquor, juicing, filtering, regulating pH of the filtrate to 6.5 with acid liquor, flocculating, centrifuging, and drying to obtain protein product;

and (3) carrying out enzymolysis on the protein finished product by papain, pepsin and acyltransferase I.

In the foregoing method, the step of enzymolysis with papain comprises:

suspending the protein product in 50mM phosphate buffer with pH of 7.0 and final concentration of 2% (w/v), and adding papain with mass-volume ratio of 4%; after water bath at 65 ℃ for 1 hour, heating in boiling water for 10min, and stopping the reaction; after cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

In the foregoing method, the step of performing enzymolysis with pepsin includes:

suspending the freeze-dried product in 50mM KCl-HCl buffer solution with pH of 1.5 and final concentration of 2% (w/v), and adding pepsin with mass-volume ratio of 3%; after the water bath is carried out for 1 hour at 37 ℃, heating is carried out in boiling water for 10min, and the reaction is stopped; after cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

In the foregoing method, the step of performing enzymolysis with the acyltransferase I comprises:

suspending the freeze-dried product in 50mM Tris-HCl buffer solution with pH of 8.0 and final concentration of 2% (w/v), and adding 6% of acyltransferase I by mass-volume ratio; after carrying out ultrasonic reaction for 1h at 57 ℃ in water bath and 25kHZ, heating in boiling water for 10min, and stopping the reaction; after cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

The invention also provides an antioxidant peptide composition, which comprises at least one of Pep1, pep2, pep3, pep4 or Pep5, wherein the amino acid sequences of Pep1, pep2, pep3, pep4 and Pep5 are sequentially shown as SEQ ID NO. 1-5, and the jerusalem artichoke peptide is derived from jerusalem artichoke (Helianthus tuberosus (L.1753)) leaves.

The invention also provides a bacteriostatic peptide composition, which comprises at least one of Pep1, pep2, pep3, pep4 or Pep5, wherein the amino acid sequences of Pep1, pep2, pep3, pep4 and Pep5 are sequentially shown as SEQ ID NO. 1-5, and the jerusalem artichoke peptide is derived from jerusalem artichoke (Helianthus tuberosus (L.1753)) leaves.

The invention also provides application of the jerusalem artichoke peptide in preparing a medicine for reducing hereditary hyperglycemia.

The invention also provides application of the jerusalem artichoke peptide in preparing a medicament for reducing diabetic complications and protecting kidneys.

Compared with the prior art, the invention has at least one of the following beneficial effects:

according to the invention, protein is extracted from fresh jerusalem artichoke leaves, enzymolysis is carried out for a plurality of times, and enzymolysis liquid is separated and purified, so that five jerusalem artichoke peptides of Pep 1-5 are obtained. The five jerusalem artichoke peptides are found to have obvious antioxidation and antibacterial effects, can effectively reduce the blood sugar of hereditary hyperglycemia mice in vivo, can reduce diabetic complications, and particularly has obvious protective effect on kidneys.

Drawings

FIG. 1 shows LC-ESI-Q-TOF MS/MS spectra of Pep1 (A), pep2 (B), pep3 (C), pep4 (D).

FIG. 2 shows LC-ESI-Q-TOF MS/MS spectra of Pep5 (A), pep6 (B), pep7 (C), pep8 (D).

FIG. 3 is a graph of HE staining of kidney tissue sections of mice from each group of db/db diabetes experiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. 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 invention. The reagents not specifically and individually described in the present invention are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

1. Material and extraction

Protein extraction is carried out by taking jerusalem artichoke (Helianthus tuberosus (L.1753)) leaves as materials.

Cutting fresh jerusalem artichoke leaves to small sections of 1-2 cm, weighing 10g, adding into 500mL of 1M sodium hydroxide solution extractant, fully and uniformly mixing, adding into a juicer for juicing for 2min, and filtering with 200mm filter cloth to obtain dark green extract. The pH was adjusted to 6.5 with 1M hydrochloric acid and flocculated in a thermostatic water bath at the set temperature for 20min. Cooling with cold water to room temperature, centrifuging at 10000rpm for 5min to obtain leaf protein precipitate, oven drying at 60deg.C to obtain leaf protein product, weighing, measuring crude protein content and leaf protein yield, and calculating crude protein extraction rate. Wherein, according to GB/T5009.5-2010, the content of crude protein in jerusalem artichoke leaves is measured by adopting a Kjeldahl nitrogen determination method. As a result, the protein content in the dried protein names was 34.5%.

2. Enzymolysis

Example 1:

and (3) carrying out enzymolysis on the finished product of the protein by papain, pepsin and acyltransferase I.

Suspending the protein product in 50mM phosphate buffer (pH 7.0) at a final concentration of 2% (w/v), and adding papain (product No. 76220, sigma-Aldrich) at a mass/volume ratio of 4%; after reacting in a water bath at 65℃for 1 hour, heating in boiling water for 10min, and stopping the reaction. After cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

The lyophilized product was further suspended in 50mM KCl-HCl buffer (pH 1.5) at a final concentration of 2% (w/v), and pepsin (product No. 1.07185, sigma-Aldrich) was added in a mass-to-volume ratio of 3%; after reaction in a water bath at 37℃for 1h, the reaction was stopped by heating in boiling water for 10 min. After cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

The lyophilizate was suspended in 50mM Tris-HCl buffer (pH 8.0) at a final concentration of 2% (w/v), and 6% by mass/volume of acyltransferase I (product number 01818, sigma-Aldrich) was added; after ultrasonic reaction for 1h at 57℃in a water bath at 25kHZ, the reaction was stopped by heating in boiling water for 10 min. After cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

Comparative example 1:

and only papain is used for enzymolysis of the finished protein product.

Suspending the protein product in 50mM phosphate buffer (pH 7.0) at a final concentration of 2% (w/v), and adding papain (product No. 76220, sigma-Aldrich) at a mass/volume ratio of 15%; after 3h reaction in water bath at 65 ℃, heating in boiling water for 10min, and stopping the reaction. After cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

3. Separation and purification of enzymolysis product and detection of antioxidant activity

And sequentially passing the enzymolysis liquid obtained under the optimal enzymolysis parameters through an ultrafiltration membrane with the molecular weight of 30kDa and 10kDa to obtain filtrate with the molecular weight of less than 10 kDa. And then the filtrate is passed through an ultrafiltration cup (model UFSC 20001) with a Millipore ultrafiltration membrane of 3KD, and the obtained filtrate is subjected to vacuum freeze drying to obtain a jerusalem artichoke peptide crude product.

The crude Jerusalem artichoke peptide is subjected to chromatographic separation by using a gel chromatographic column (flow rate of 2.6X100 cm eluting distilled water is 1 mL/min) filled with Sephadex G-25 (source leaf, product number S14031), and then the components with absorption peaks at 280nm are respectively collected, and are subjected to vacuum freeze drying. The antioxidant activity of each component is measured separately, and a component having a better antioxidant activity is preferable.

The preferred components were structurally identified using liquid chromatography tandem triple quadrupole tandem time of flight mass spectrometry (UPLC-Q-TOF, waters, manchester, UK). The identification method was carried out according to the method of "ZHUANG H, TANG N, YUAN Y. Purification and identification of antioxidant peptides from corn gluten meal [ J ]. Journal of Functional Foods,2013,5 (4): 1810-1821 ], and the jerusalem artichoke peptides shown in Table 1 were obtained by comparing the relevant reference sequences provided by NCBI with respect to Helianthus tuberosus.

The results are shown in Table 1, and Jerusalem artichoke peptides Pep1 to Pep5 were obtained by the enzymolysis method provided in example 1, and Jerusalem artichoke peptides Pep6 to Pep8 were obtained by the enzymolysis method provided in comparative example 1. The mass spectrum is shown in figures 1 and 2.

TABLE 1 Jerusalem artichoke peptides and sequences

4. Jerusalem artichoke peptide antioxidant activity detection

DPPH radical scavenging Rate determination: the concentration IC50-1 of Jerusalem artichoke peptide at DPPH radical removal of 50% was calculated by measuring according to the method of "ZHANG T, LI Y, MIAO M, et al purification and characterisation of a new antioxidant peptide from chickpea (Cicer arietium L.) protein hydrolysates [ J ]. Food Chemistry,2011,128 (1): 28-33 ].

ABTS radical clearance assay: the concentration of Jerusalem artichoke peptide IC50-2 at AN ABTS radical clearance of 50% was calculated by measuring according to the method "TIRONI V A, AN M C.amaranth proteins as a source of antioxidant peptides: effect of proteolysis [ J ]. Food Research International,2010,43 (1): 315-322".

Determination of the hydroxyl radical removal rate: the concentration IC50-3 of the jerusalem artichoke peptide at a hydroxyl group removal rate of 50% was calculated by measuring according to the method of "WANG J, WANG Y, DANG X, et al, houseforce larvae hydrolysate: orthogonal optimization of hydrolysis, antioxidant activity, amino acid composition and functional properties [ J ]. BMC Research Notes,2013,6 (1): 1-10".

And (3) measuring the superoxide anion clearance rate: the concentration IC50-4 of the Jerusalem artichoke peptide at a superoxide anion clearance of 50% was calculated by measuring according to the method "LI Y, JIANG B, ZHANG T, et al, analytical and free radio-scavenging activities of Chickpea Protein Hydrolysate (CPH) [ J ]. Food Chemistry,2008,106 (2): 444-450".

Determination of iron ion chelating Activity: the concentration of Jerusalem artichoke peptide IC50-5 at 50% iron ion chelating activity was calculated by measuring according to the method of "SABEENA FARVIN K H, BARON C P, NIELSEN N S, et al, annexidant activity of yoghurt peptides, part 1-in vitro assays and evaluation in omega-3 enriched mill [ J ]. Food Chemistry,2010,123 (4): 1081-1089 et al.

Cu2+ chelation activity assay: the concentration of Jerusalem artichoke peptide IC50-6 at a Cu < 2+ > chelating activity of 50% was calculated by measuring according to the method of ZHU L J, CHEN J, TANG X Y, et al reducing, radical scavenging, and chelation properties of in vitro digests of alcalase-treated zein hydrolysate [ J ]. Journal of Agricultural and Food Chemistry,2008,56 (8): 2714-2721 ].

Lipid peroxidation inhibition activity assay: the concentration of Jerusalem artichoke peptide IC50-7 at a lipid peroxidation inhibition activity of 50% was calculated by measuring according to the method of "CHANDRASEKARA A, SHAHIDI F. Antioxidant phenolics of millet control lipid peroxidation in human LDL cholesterol and food systems [ J ]. Journal of the American Oil Chemists' Society,2012,89 (2): 275-285 ].

The IC50-1, IC50-2, IC50-3, IC50-4, IC50-5, IC50-6 and IC50-7 of the Jerusalem artichoke peptides Pep1, pep2, pep3, pep4 and Pep5 prepared in example 1 are shown in Table 2. Table 3 shows the IC50-1, IC50-2, IC50-3, IC50-4, IC50-5, IC50-6 and IC50-7, respectively, of the Jerusalem artichoke peptides Pep6, pep7 and Pep8 prepared in comparative example 1. In tables 2 and 3, "-" indicates undetected. Comparing tables 2 and 3, the jerusalem artichoke peptides Pep1, pep2, pep3, pep4 and Pep5 prepared in example 1 can remove DPPH free radical, ABTS free radical, hydroxyl and superoxide anion, and can also have activity of chelating iron ion and copper ion simultaneously, and can also inhibit lipid peroxidation. As shown in table 3, the jerusalem artichoke peptides Pep6, pep7, pep8 provided in comparative example 1 do not have the activity of simultaneously chelating iron ions and copper ions and inhibiting lipid peroxidation, and have less antioxidant effect than the jerusalem artichoke peptides Pep1, pep2, pep3, pep4, pep5 are comprehensive, and have weaker antioxidant ability.

Table 2 (μg/ml)

Jerusalem artichoke peptide	Pep1	Pep2	Pep3	Pep4	Pep5
						IC50-1	237.9±20.6	562.2±36.4	202.3±17.9	302.4±29.1	216.7±26.5
IC50-2	328.1±43.8	486.3±43.2	286.9±32.0	313.5±36.7	289.3±37.9
						IC50-3	183.5±24.3	359.7±29.5	179.3±20.7	193.2±29.2	156.3±22.8
IC50-4	436.8±50.9	836.2±36.8	336.9±36.5	368.4±30.3	305.5±28.7
						IC50-5	169.3±18.5	284.8±28.1	187.2±20.3	197.6±25.4	148.6±15.2
IC50-6	208.7±21.6	245.2±24.8	193.2±18.4	212.3±22.5	183.7±16.9
						IC50-7	188.6±19.9	235.2±63.5	208.3±21.3	213.1±29.3	157.4±17.2

Table 3 (μg/ml)

Jerusalem artichoke peptide	Pep6	Pep7	Pep8
				IC50-1	1559.2±125.2	3638.9±539.8	4963.8±832.1
IC50-2	1824.3±162.1	4293.5±835.4	7548.2±770.9
				IC50-3	2638.9±256.5	－	－
IC50-4	4195.5±350.4	－	－
				IC50-5	－	－	－
IC50-6	－	－	－
				IC50-7	－	－	－

5. Antioxidant stability assay

Jerusalem artichoke peptides Pep1, pep2, pep3, pep4, pep5, pep6, pep7 and Pep8 are respectively prepared into 10mg/mL, 5mg/mL, 1mg/mL, 500 mug/mL, 200 mug/mL and 100 mug/mL, and the Jerusalem artichoke peptides are treated in a water bath at 70 ℃ for 2 hours, and then IC50-1, IC50-2, IC50-3, IC50-4, IC50-5, IC50-6 and IC50-7 are respectively detected by the methods.

As shown in Table 4, after 2 hours of treatment in a water bath at 70 ℃, the Jerusalem artichoke peptides Pep1, pep2, pep3, pep4 and Pep5 prepared in example 1 still can remove DPPH free radicals, ABTS free radicals, hydroxyl and superoxide anions, have the activity of chelating iron ions and copper ions at the same time, and can inhibit lipid peroxidation. In table 5, "-" indicates undetected. As shown in Table 5, after 2 hours of treatment in a water bath at 70 ℃, the Jerusalem artichoke peptides Pep6, pep7, pep8 provided in comparative example 1 do not have activities of simultaneously chelating iron ions and copper ions and inhibiting lipid peroxidation, and have an antioxidant effect less than the Jerusalem artichoke peptides Pep1, pep2, pep3, pep4, pep5 in all aspects, and have a weaker antioxidant ability. After the treatment for 2 hours in a water bath at 70 ℃, pep6 loses the effect of scavenging hydroxyl and superoxide anions, and Pep7 and Pep8 almost lose the antioxidation.

TABLE 4 (μg/ml) 70℃water bath treatment for 2h

Jerusalem artichoke peptide	Pep1	Pep2	Pep3	Pep4	Pep5
						IC50-1	535.5±42.8	936.2±77.1	439.2±30.5	516.3±48.6	352.5±31.6
IC50-2	428.1±37.5	821.3±64.2	332.7±49.1	489.7±52.4	320.7±40.2
						IC50-3	352.6±31.9	562.2±40.5	367.8±32.9	363.8±34.5	193.3±26.4
IC50-4	802.5±62.4	1036.8±92.8	527.3±41.6	497.3±48.2	389.2±36.5
						IC50-5	322.2±40.3	497.0±53.7	259.6±35.7	352.6±32.0	186.9±22.7
IC50-6	343.8±32.1	426.3±37.5	326.7±31.2	423.2±37.2	213.5±20.8
						IC50-7	469.5±50.5	378.6±43.5	292.6±28.6	408.7±31.6	189.4±23.5

TABLE 5 (μg/ml) 70℃water bath treatment for 2h

Jerusalem artichoke peptide	Pep6	Pep7	Pep8
				IC50-1	9635.8±263.4	18425.7±2671.2	－
IC50-2	19367.5±649.7	－	－
				IC50-3	－	－	－
IC50-4	－	－	－
				IC50-5	－	－	－
IC50-6	－	－	－
				IC50-7	－	－	－

6. Bacteriostasis test

The strain used for the test was Salmonella (Salmonella enteritidis subspecies enteritidis ATCC14028, north Biotechnology Co., ltd.) Proteus (Proteus mirabilis CMCC49027, north Biotechnology Co., ltd.), escherichia coli (Escherichia coli ATCC 35218), yersinia (Yersinia enterocolitica)Yersinia enterocolitica->North Biotechnology Co., ltd.) of Shanghai. Wherein, escherichia coli is cultured in LB medium. Salmonella was cultured in SS (Salmonella-Shigella) medium. Proteus is cultured in nutrient agar medium. Yersinia is cultivated in CIN-1 medium.

And (3) selecting a plurality of test strains by using an inoculating loop, inoculating the test strains into sterile water with glass beads, fully dispersing, injecting the sterile water into the total yellow agar culture medium for testing, and fully cooling. The disc filter paper is immersed in an aqueous solution containing 3mg/mL jerusalem artichoke peptides Pep1, pep2, pep3, pep4, pep5, pep6, pep7 or Pep8 for 1h by using tweezers, taken out, placed in the center of a bacteria-carrying culture plate, covered with a cover, cultured at 30 ℃ for 24h, and the size of a bacteria inhibition zone is measured.

In table 6, "-" indicates undetected. Table 6 shows the inhibition zone sizes of 3mg/mL Jerusalem artichoke peptides Pep1, pep2, pep3, pep4, pep5, pep6, pep7 or Pep8 against Salmonella, proteus, E.coli and Yersinia. As a result, it was found that 3mg/mL of the Jerusalem artichoke peptides Pep1, pep2, pep3, pep4, pep5 had antibacterial effects on Salmonella, proteus, E.coli and Yersinia, while 3mg/mL of the Jerusalem artichoke peptides Pep6, pep7 or Pep8 had no antibacterial effects on Salmonella, proteus, E.coli and Yersinia.

Table 6 size of inhibition zone (mm)

Jerusalem artichoke peptide	Salmonella bacteria	Proteus (Proteus) and its preparation method	Escherichia coli	Yersinia genus
					Pep-1	5.23±0.52	5.72±0.73	8.30±0.42	4.08±0.34
Pep-2	5.09±0.49	5.58±0.64	8.16±0.66	4.27±0.41
					Pep-3	4.85±0.37	5.47±0.59	8.36±0.52	4.36±0.32
Pep-4	4.69±0.35	5.32±0.29	8.49±0.57	4.21±0.26
					Pep-5	5.86±0.67	6.14±0.58	9.16±0.83	4.41±0.39
Pep-6	－	－	－	－
					Pep-7	－	－	－	－
Pep-8	－	－	－	－

7. Alpha-glucosidase inhibitory Activity assay

Further, the alpha-glucosidase inhibitory activity of the jerusalem artichoke peptide provided by the invention is measured.

(1) Test article

Experimental group: jerusalem artichoke peptide solutions containing different concentrations were prepared with 0.01mol/LPBS buffer, and Pep1, pep2, pep3, pep4, pep5, pep6, pep7, pep8 were prepared as 10mg/mL, 5mg/mL, 1mg/mL, 500 μg/mL, 200 μg/mL, 100 μg/mL as experimental groups.

Positive group: taking fresh jerusalem artichoke tuber, cleaning, airing, slicing, drying at 60 ℃, crushing, and sieving with a 40-mesh sieve to obtain jerusalem artichoke dry powder. 200g of fresh jerusalem artichoke dry powder is repeatedly extracted twice by deionized water (solid-to-liquid ratio 1:20), and the extraction temperature is 90 ℃. The extracts are combined and filtered twice to obtain filtrate, and the filtrate is concentrated to 250mL under reduced pressure. Adding absolute ethanol with a certain volume to make the final concentration of ethanol 40%,60%,80% and 90% respectively, standing overnight at 4deg.C in refrigerator, collecting precipitate with various alcohol precipitation concentrations, redissolving, and centrifuging to remove impurities. Removing impurities from the extractive solutions with various alcohol precipitation concentrations respectively by using D101 macroporous adsorbent resin, decolorizing, concentrating the eluate under reduced pressure to 200mL, adding 1/3 volume of Sevage reagent (chloroform: n-butanol-4:1) respectively, stirring for 90 min, extracting in a separating funnel for 30min, detecting protein content by using BCA protein concentration measuring kit, and repeating the above steps until no protein exists. Adding water, concentrating under reduced pressure to remove organic reagent to 165mL, placing the solutions in dialysis bags with molecular weight cut-off of 1000Da respectively, dialyzing with flowing water in refrigerator at 4deg.C for 48h, and lyophilizing for 3 days to obtain Jerusalem artichoke crude polysaccharide. The Jerusalem artichoke crude polysaccharide was formulated as a positive group at 10mg/mL, 5mg/mL, 1mg/mL, 500 μg/mL, 200 μg/mL, 100 μg/mL.

The method comprises the following steps: to the ELISA plate, 0.4mL of test solution of the experimental group or the positive group was added. A blank group and a negative group are set,0.4mL of 0.01mol/LPBS buffer was added separately. 0.3U/mL of alpha-glucosidase (G3651, sigma-Aldrich) solution was added to each of the experimental group, the positive group and the negative group, the experimental group, the positive group, the blank group and the negative group were kept at a constant temperature of 37℃for 5 minutes after shaking uniformly, 0.4mL of PNPG solution was added to each of the experimental group, the positive group, the blank group and the negative group at 2.5mmol/L, and 1.6mL of PNPG solution was added after 30 minutes of reaction at 370C ₂ CO ₃ Stopping the reaction, taking PBS buffer solution as blank control, measuring absorbance at 405nm, repeating 3 times for each group, and calculating the inhibition rate of 8 jerusalem artichoke peptides and jerusalem artichoke polysaccharide on alpha-glucosidase. Wherein, the inhibition rate of alpha-glucosidase = [1- (OD experimental group/positive-OD blank group)]/(OD negative group-OD blank group) ×100%.

TABLE 7 inhibition of alpha-glucosidase%

Jerusalem artichoke peptide

10mg/mL

5mg/mL

1mg/mL

500μg/mL

200μg/mL

100μg/mL

Pep-1

49.36±2.37

36.57±3.05

24.72±1.36

18.35±1.15

11.12±1.41

2.35±0.16

Pep-2

48.32±2.18

34.29±3.26

22.49±1.82

17.62±1.63

10.26±1.23

2.09±0.28

Pep-3

47.59±2.44

35.26±2.82

25.18±2.15

16.49±1.35

9.34±0.79

2.34±0.31

Pep-4

50.13±2.59

37.29±4.12

28.37±2.32

19.57±1.68

12.36±1.16

3.27±0.48

Pep-5

51.32±2.72

39.21±3.05

29.53±2.51

20.35±1.71

13.53±1.25

5.08±0.55

Pep-6

－

－

－

－

－

－

Pep-7

－

－

－

－

－

－

Pep-8

－

－

－

－

－

－

Jerusalem artichoke polysaccharide

26.84±2.68

17.36±1.55

10.34±0.73

5.24±0.19

－

－

Table 7 shows the inhibition ratios of 8 jerusalem artichoke peptides and jerusalem artichoke polysaccharides provided by the invention to alpha-glucosidase at concentrations of 10mg/mL, 5mg/mL, 1mg/mL, 500 μg/mL, 200 μg/mL, 100 μg/mL, respectively, wherein "-" indicates undetected. From Table 7, it is clear that Pep1, pep2, pep3, pep4, pep5 and Jerusalem artichoke polysaccharide have inhibitory activity against alpha-glucosidase, and that Pep1, pep2, pep3, pep4, pep5 have higher inhibitory activity against alpha-glucosidase than Jerusalem artichoke polysaccharide, and still have inhibitory activity at a concentration as low as 100. Mu.g/mL. Therefore, the jerusalem artichoke peptides Pep1, pep2, pep3, pep4 and Pep5 provided by the invention can be used as alpha-glucosidase inhibitors to regulate in-vivo sugar metabolism disorder or be used as diabetes medicines.

8. Animal experiment

(1) Hereditary hyperglycemia experiment

Hereditary hyperglycemia mice: KK/Upj-Ay/J mice, 3 months old, 35-45 g body mass, male and female halves, have hyperglycemia, hyperinsulinemia, glucose intolerance characteristics, pass number 00080269, provided by Beijing Fukang Biotechnology Co., ltd., license number SCXK11-00-0006.

KK/Upj-Ay/J mice with fasting blood glucose above 11.0mmol/L are randomly divided into a model group, an experimental group and a positive group. The model group was perfused with distilled water 1 time per day. The positive group was perfused with 3mg/kg body weight of glucose per day (Tianjin Pacific pharmaceutical Co., ltd.). The experimental group was filled with 100mg/kg body weight of jerusalem artichoke peptide Pep1, pep2, pep3, pep4, pep5, pep6, pep7 or Pep8 per day. Blood glucose was measured by taking blood from the orbit 1.5h after the last administration, following 4 weeks of continuous experiments with 12h fasting. Blood glucose content was measured using a blood glucose measuring kit (glucose oxidase method, beijing Lidammann Biochemical technology Co., ltd.).

Table 8 fasting blood glucose levels (mM) in hereditary hyperglycemic mice

As can be seen from table 8, the body mass of the mice in the model group was significantly reduced (P < 0.01) compared to the control group after 4 weeks of administration; the mice in each administration group had a significantly increased mass (P < 0.05) compared to the model group, wherein the mice in the compound Betula platyphylla blood glucose-reducing tablet group had no significant difference but had a tendency to increase compared to the Betula platyphylla polysaccharide group and the Fagopyrum tataricum polysaccharide group. The compound betulin blood sugar reducing tablet can relieve emaciation symptoms of diabetic mice, and has slightly better effect than betulin polysaccharide and tartary buckwheat polysaccharide.

(2) Diabetic complications and kidney experiments

Male SPF-class m/m normal mice (5 weeks old) and male SPF-class db/db diabetes model mice (5 weeks old) were purchased from Kwangsi laboratory animal Co., ltd., animal license number 2018D046.

M/m mice were set as normal groups and were perfused with an equal amount of physiological saline daily.

The db/db diabetes model mice are diabetic mice which are tested for initial blood sugar after 7d of adaptive feeding, and compared with normal m/m mice, the db/db diabetes model mice are diabetic mice, so that the experimental requirements are met. Db/db mice were randomly divided into model groups and were perfused with an equivalent amount of physiological saline daily; the experimental group was filled with 100mg/kg body weight of jerusalem artichoke peptide Pep1, pep2, pep3, pep4, pep5, pep6, pep7 or Pep8 daily. Positive group, 300mg/kg metformin hydrochloride suspension per day.

Observation of mental state of mice and measurement of body weight and blood sugar the physiological state, hair and feed intake, water intake and excretion of the mice were observed, the body weight of the mice was measured weekly, and the initial body weight after 7d of adaptive feeding of the mice and the body weight data after 2 and 4 weeks of gastric lavage were selected for statistical analysis.

The normal group of mice were in an active state in their physiological spirit, had normal feed intake and water intake, and had normal excretion (data not shown here). The feed intake and water intake of the model group are obviously increased, and the excretion is large, so that constipation appears in the middle and later stages of the experiment. Slow and slow movement, rough hair, even partial hair falling, moist padding and need to be replaced once in 3 days. The physiological states of the positive group and the experimental group (Pep 1-Pep 5) are improved, and the hair is smooth and glossy.

The tail vein of the mice was bled, the blood glucose value of each week was measured by a glucometer and blood glucose test paper, and the initial random blood glucose of the mice, and fasting blood glucose values of 8 hours after 2 and 4 weeks of gastric lavage were selected for data analysis. The results are shown in Table 9. The fasting blood glucose levels were significantly elevated in both the model group at weeks 2 and 4 relative to the normal group. Compared with the model group, the fasting blood glucose content of the mice in the positive group and the experimental group (Pep 1-Pep 5) is obviously reduced, and the fasting blood glucose content of the mice in the experimental group (Pep 1-Pep 5) at the 2 nd week and the 4 th week is lower than that of the mice in the positive group. Therefore, the jerusalem artichoke peptides Pep 1-Pep 5 provided by the invention not only can reduce the fasting blood glucose content of db/db diabetic mice, but also has better effect than metformin hydrochloride.

Table 9db/db diabetes test groups mice fasting blood glucose levels (mM)

Table 10 shows the weight data of mice from each group of db/db diabetes experiments. The results showed that the mice in the model group had significantly higher body weights at weeks 2 and 4 than the normal group. The body weight of mice in the positive group and the experimental group (Pep 1 to Pep 5) was significantly reduced relative to the model group, and the body weight of mice in the experimental group (Pep 1 to Pep 5) was lower than that in the positive group at weeks 2 and 4, respectively. Therefore, the jerusalem artichoke peptides Pep 1-Pep 5 provided by the invention not only can relieve obesity caused by diabetes of db/db diabetic mice, but also have better effect than metformin hydrochloride.

Table 10db/db diabetes experiment groups mice weights (g)

Determining the organ index of the mice facilitates assessment of hypoglycemic activity of jerusalem artichoke peptides on diabetic mice. After 4 weeks of experiment, all animals were anesthetized by ether inhalation, blood was taken from the eyeballs and placed in a 2mL centrifuge tube, the mice were sacrificed after neck removal, and after 30min, the animals were centrifuged at 4℃and 3000 Xg for 10min to collect the supernatant serum. Weighing kidney, liver, spleen and heart, and quickly freezing viscera and serum with liquid nitrogen at-80deg.C for subsequent experimental analysis. Organ index = mouse organ weight/mouse weight x 100%.

As can be seen from Table 11, the indices of the organs of the mice in the normal group were significantly lower than those in the model group, indicating that diabetes caused different degrees of organ lesions in the db/db mice. The kidney index was significantly lower in both the positive and experimental groups (Pep 1-Pep 5) than in the model group. The jerusalem artichoke peptides Pep 1-Pep 5 provided by the invention can protect organs of db/db diabetic mice and reduce the harm effect of diabetic complications to the mice.

Table 11db/db diabetes experiments groups of mice organ indices

Renal malondialdehyde is lipid peroxide, free radicals in the body of diabetics are excessive, oxidation reaction is severe, and the content of malondialdehyde is high, so that the renal malondialdehyde is one of indexes of kidney injury. Measurement of renal function-related indicators 24h urine was collected after 4 weeks of treatment. Urine protein content was determined using Bradford method using Bovine Serum Albumin (BSA) as a standard. The content of malondialdehyde in blood urea nitrogen, serum creatinine, blood uric acid and kidney tissue homogenates was determined using ELISA kit (Roche diagnostics products (Shanghai Co.). The measurement method of each index is strictly operated according to the instruction of the kit.

Table 12db/db diabetes mellitus experiments groups of mice kidney serum indicators

Table 12 shows the kidney serum index for each group of mice in the db/db diabetes test. Compared with the normal group, the db/db diabetic mice in the model group have obviously raised indexes. While the kidney index of both the positive and experimental groups (Pep 1-Pep 5) was significantly lower than that of the model group. The jerusalem artichoke peptides Pep 1-Pep 5 provided by the invention can protect the kidney of a db/db diabetic mouse and reduce the harm effect of diabetic high glucose metabolism to the kidney of the mouse. The Jerusalem artichoke peptides Pep 6-Pep 8 do not obviously reduce various kidney serum indexes of db/db diabetic mice, and do not have obvious kidney protection effect on the mice.

As can be seen from FIG. 3, the histological analysis using HE shows that the normal mice of the normal group have uniform blood cell distribution, the glomerulus and the kidney capsule are normal without obvious pathological changes, the blood cell distribution of the model group is disordered, the kidney capsule is extruded and atrophic, the basement membrane is thickened, and the mesangial region is widened, which are typical pathological symptoms of diabetic nephropathy. The diabetic mice with db/db administered with Jerusalem artichoke peptides Pep 6-Pep 8 are not improved in substitution, and the diabetic mice with db/db administered with Jerusalem artichoke peptides Pep 1-Pep 5 are improved in pathological symptoms of diabetic nephropathy, which shows that the Jerusalem artichoke peptides Pep 1-Pep 5 provided by the invention have an improving effect on diabetic nephropathy of the diabetic mice with db/db, play a certain protection role on kidneys, presumably because of remarkable hypoglycemic activity, reduce ROS, thereby reducing basement membrane thickening and intercellular matrix deposition caused by collagen denaturation.

The present invention 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 invention are intended to be included in the scope of the present invention.

Claims (7)

1. The sequence of the jerusalem artichoke peptide for reducing blood sugar and resisting oxidation is shown as SEQ ID NO.2, and the jerusalem artichoke peptide is derived from jerusalem artichoke (Helianthus tuberosus (L.1753)).

2. The method for producing a jerusalem artichoke peptide of claim 1, comprising:

extracting fresh jerusalem artichoke leaves with alkali liquor, juicing, filtering, regulating pH of the filtrate to 6.5 with acid liquor, flocculating, centrifuging, and drying to obtain protein product;

carrying out enzymolysis on the protein finished product by papain, pepsin and acyltransferase I;

wherein, the step of enzymolysis by the papain comprises the following steps: suspending the protein product in 50mM phosphate buffer with pH of 7.0 and final concentration of 2% (w/v), and adding papain with mass-volume ratio of 4%; after water bath at 65 ℃ for 1 hour, heating in boiling water for 10min, and stopping the reaction; cooling, centrifuging for 20min at 10000g, collecting supernatant, and lyophilizing to obtain first lyophilized product;

wherein, the step of adopting pepsin enzymolysis comprises the following steps: suspending the first freeze-dried product in 50mM KCl-HCl buffer solution with pH of 1.5 and final concentration of 2% (w/v), and adding pepsin with mass-volume ratio of 3%; after the water bath is carried out for 1 hour at 37 ℃, heating is carried out in boiling water for 10min, and the reaction is stopped; cooling, centrifuging for 20min at 10000g, collecting supernatant, and lyophilizing to obtain second lyophilized product;

wherein, the step of adopting the acyltransferase I for enzymolysis comprises the following steps: suspending the second lyophilized product in 50mM Tris-HCl buffer solution with pH of 8.0 and final concentration of 2% (w/v), and adding 6% mass/volume of acyltransferase I; after carrying out ultrasonic reaction for 1h at 57 ℃ in water bath and 25kHZ, heating in boiling water for 10min, and stopping the reaction; after cooling, the supernatant was collected by centrifugation at 10000g for 20min and freeze-dried.

3. An antioxidant peptide composition comprises Pep2, pep3, pep4 or Pep5, wherein the amino acid sequences of the Pep2, the Pep3, the Pep4 and the Pep5 are sequentially shown as SEQ ID NO. 2-5, and the Pep2, the Pep3, the Pep4 and the Pep5 are derived from blades of jerusalem artichoke (Helianthus tuberosus) L.1753.

4. The antibacterial peptide composition comprises Pep2, pep3, pep4 or Pep5, wherein the amino acid sequences of the Pep2, the Pep3, the Pep4 and the Pep5 are sequentially shown as SEQ ID NO. 2-5, and the Pep2, the Pep3, the Pep4 and the Pep5 are derived from blades of jerusalem artichoke (Helianthus tuberosus) L.1753.

5. The use of the jerusalem artichoke peptide of claim 1 for the preparation of a medicament for the treatment of hereditary hyperglycemia.

6. The use of the jerusalem artichoke peptide of claim 1 for the preparation of a medicament for improving pathological symptoms of diabetic nephropathy.

7. Use of the jerusalem artichoke peptide of claim 1 for the preparation of a kidney protecting product.