Application of a Caulis Lonicerae Sichuanensis-Bark Pectin in Preparing Food with Anti-inflammatory Effect

Abstract

The application of a Clematis glauca pectin in the preparation of food with anti-inflammatory effect belongs to the technical field of food processing and health care. The Clematis glauca pectin is obtained by ultrasonic-assisted enzyme extraction and column chromatography purification with Clematis glauca pericarp as raw material. Experiments show that the pectin has significant DPPH free radical, ABTS free radical scavenging capacity and Fe 3+ reducing capacity, and good antioxidant activity. In a DSS-induced mouse colitis model, the pectin can significantly reduce DAI, improve colon shortening, reduce the levels of pro-inflammatory cytokines (TNF-alpha, IL-6, IL-17A) in serum and colon tissue, repair colon mucosal damage, and regulate intestinal flora structure, such as increasing the relative abundance of beneficial bacteria such as Bacteroides and Akkermansia. The present application provides a new way for developing new anti-inflammatory functional food or health care products for assisting the conditioning of colitis.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, specifically to the application of Akebia trifoliata pectin in the preparation of foods with anti-inflammatory effects. Background Technology

[0002] Inflammation is a defensive response of the body to stimuli. Moderate inflammation helps to eliminate pathogens and repair damaged tissues, but chronic inflammation can lead to a variety of diseases, such as cardiovascular disease, diabetes, arthritis, and intestinal inflammation, seriously threatening human health. With the increasing awareness of health, ingesting anti-inflammatory components through daily diet has become an important way to prevent and improve chronic inflammation. Therefore, developing functional foods rich in natural anti-inflammatory components has significant market value and social significance.

[0003] Pectin is a natural, water-soluble dietary fiber widely found in plant cell walls. It possesses gelling, thickening, and emulsifying properties, and also exhibits various physiological activities, such as regulating gut microbiota, lowering cholesterol, and enhancing immunity. It has been widely used in the food, pharmaceutical, and cosmetic industries. Currently, commercial pectin is mainly derived from citrus peels, apple pomace, and other raw materials. Its anti-inflammatory activity is relatively limited, and its sources are concentrated, making it difficult to meet market demand for highly active pectin and diversified raw materials.

[0004] *Akebia trifoliate* (Thunb.) Koidz. var. *australis* is a deciduous woody vine belonging to the genus *Akebia* in the family Lardizabalaceae. It is widely distributed in the Yangtze River basin and areas south of it in my country, and its fruits, stems, and leaves all possess certain medicinal value. Current research indicates that *Akebia trifoliate* contains abundant polysaccharides, flavonoids, saponins, and other active ingredients; however, there are no systematic reports on the extraction of pectin from *Akebia trifoliate*, its anti-inflammatory activity, and its application in functional foods. Fully exploring the utilization value of *Akebia trifoliate*, extracting its highly anti-inflammatory pectin, and applying it to functional foods can not only expand the sources of pectin raw materials but also provide new directions for the development of functional foods. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Akebia quinata pectin in the preparation of food with anti-inflammatory effects. Food or health food made using Akebia quinata pectin as an active ingredient has anti-inflammatory effects.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution.

[0007] Application of Akebia trifoliata pectin in the preparation of food with anti-inflammatory effects.

[0008] The food described in this invention is preferably a functional food.

[0009] The white akebia pectin described in this invention can be used alone or in combination with other active ingredients.

[0010] The functional food described in this invention is prepared using Akebia trifoliata pectin, which has anti-inflammatory properties, as the sole or main active ingredient, along with excipients acceptable for health foods.

[0011] The anti-inflammatory effect mentioned above refers to the relief and conditioning effect on inflammatory bowel diseases characterized by intestinal mucosal inflammation, barrier damage, and immune disorders.

[0012] The white akebia pectin described in this invention is prepared by the following method:

[0013] (1) Ultrasonic-assisted enzymatic extraction: Add deionized water at a material-to-liquid ratio of 1:10-1:30 (g / mL), add 0.03g-0.11g of cellulase, adjust the pH of the system to 4.1-4.9, place it in an ultrasonic device, and extract at 35-55℃ for 30-60 min, stirring once every 10 min during the process.

[0014] (2) Purification treatment: After extraction, the extract is centrifuged at 4000 r / min for 15 min, and the supernatant is filtered through a 0.45 μm filter membrane to remove solid impurities. The filtrate is passed through an anion exchange column to remove impurities, and finally eluted with 40-60% ethanol solution and the eluent is collected.

[0015] (3) Alcohol precipitation and drying: Concentrate the eluent to 1 / 5-1 / 3 of its original volume, slowly add 95% ethanol solution, let stand for 1.5h; collect the precipitate by centrifugation, wash with anhydrous ethanol 2-3 times, and freeze dry to obtain the finished product of white akebia pectin.

[0016] The present invention has the following advantages:

[0017] (1) The white akebia pectin in this invention exhibits certain DPPH and ABTS free radical scavenging rates and Fe in vitro. 3+ The reducing power results showed that Akebia quinata pectin has strong antioxidant activity. At the whole animal level, experiments using a colitis model revealed that it can significantly alleviate the intestinal mucosal inflammation response in colitis model animals, reduce the levels of pro-inflammatory cytokines such as TNF-α and IL-6 in intestinal tissue, and reduce the degree of intestinal mucosal damage and ulceration. It also confirmed that Akebia quinata pectin achieves its significant anti-inflammatory effect by regulating intestinal immune balance and repairing intestinal barrier function.

[0018] (2) The present invention found that white akebia pectin has an inhibitory effect on inflammatory factor-induced colitis at the whole animal level, and can alleviate the pathological damage of intestinal tissue and improve intestinal function in colitis. This indicates that it can be applied to health foods with anti-inflammatory and intestinal conditioning effects, thus providing a new direction for the modern application of natural active ingredients and the development of intestinal health products. It can also contribute to the auxiliary conditioning of colitis and the improvement of intestinal inflammation-related problems.

[0019] (3) The preparation method of Akebia quinata pectin in this invention has the characteristics of short cycle, low reagent consumption, simple process, safety and non-toxicity, stability and high efficiency. The ultrasonic-assisted enzymatic water extraction-anion exchange column purification combined process has high extraction efficiency, pectin yield of 7.1-7.6%, and the galacturonic acid content of purified Akebia quinata pectin is ≥75%. Attached Figure Description

[0020] Figure 1 The in vitro antioxidant capacity of Akebia trifoliata pectin. Among them, (A) is the ABTS antioxidant diagram, (B) is the DPPH antioxidant diagram, and (C) is the FRAP antioxidant diagram.

[0021] Figure 2 AP treatment trial flowchart.

[0022] Figure 3 DAI score of mice under AP treatment.

[0023] Figure 4 Figure 1 shows the weight change curve of mice treated with AP.

[0024] Figure 5 Changes in colon length in mice treated with AP; representative images of the colon are shown. In the image, A represents colon length, and B represents a representative colon image.

[0025] Figure 6 The concentrations of IL-6, IL-17A, and TNF-α in serum and colon were measured by ELISA. Where A represents the serum concentrations of IL-6, IL-17A, and TNF-α, and B represents the colon concentrations of IL-6, IL-17A, and TNF-α.

[0026] Figure 7 The concentrations of SOD, CAT, and T-AOC in serum were determined using a biochemical reagent kit.

[0027] Figure 8 H&E staining of colon tissue from mice treated with AP.

[0028] Figure 9 Quantitative analysis of H&E staining in colon tissue of mice treated with AP. In this study, A represents quantitative analysis of H&E-positive areas, and B represents tissue scoring.

[0029] Figure 10 PAS staining of colon tissue from mice treated with AP.

[0030] Figure 11 Quantitative analysis of PAS staining in colon tissue of mice treated with AP. In this study, A represents quantitative analysis of PAS-positive areas, and B represents quantitative analysis of goblet cell count.

[0031] Figure 12 Immunofluorescence staining of colon tissue in mice treated with AP.

[0032] Figure 13 The mRNA expression levels of tight junction proteins in the colon tissue of mice treated with AP. A represents Claudin-1, B represents Occludin, and C represents ZO-1 mRNA expression levels.

[0033] Figure 14 Venn diagram of gut microbiota analysis in mice treated with high doses of AP.

[0034] Figure 15 PCoA analysis of gut microbiota in mice treated with high doses of AP.

[0035] Figure 16 NMDS analysis of gut microbiota in mice treated with high doses of AP.

[0036] Figure 17 Alpha diversity index in mice treated with high doses of AP. A represents the Chao index analysis, B represents the Shannon index analysis, and C represents the Simpson index analysis.

[0037] Figure 18 Phylogenetic analysis of gut microbiota in mice treated with high doses of aspirin (AP).

[0038] Figure 19 Genus-level analysis of gut microbiota in mice treated with high doses of aspirin (AP).

[0039] Figure 20 Quantitative analysis of specific bacteria in the gut microbiota of mice treated with high doses of aspirin (AP). In this study, A represents quantitative analysis of Firmicutes, and B represents quantitative analysis of Akkermansia.

[0040] Figure 21 Evolutionary clade diagram of LEfSe analysis of gut microbiota in mice treated with high doses of AP. Detailed Implementation

[0041] The present invention will be further described in conjunction with the accompanying drawings and through the following embodiments.

[0042] Example 1

[0043] The Akebia quinata pectin provided in this experiment was prepared by the following method: Extraction was performed using an ultrasound-assisted enzymatic method. 10g of dried Akebia quinata powder was weighed and added to a specific material-to-liquid ratio (1g of Akebia quinata powder to 18.9mL of distilled water), followed by the addition of 0.084g of cellulase. After thorough mixing, the pH was adjusted to 4.565 using pure water, and the mixture was ultrasonically treated at 50℃ for 55min. Stirring was performed every 10min during this period.

[0044] Purification: The extract was centrifuged at 4000 r / min for 15 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was passed through an anion exchange column and eluted with 50% ethanol solution. The eluent was collected.

[0045] Alcohol precipitation and drying: Concentrate the eluent to 400 mL, slowly add 95% ethanol solution, and let stand at room temperature for 1.5 h; collect the precipitate by centrifugation, wash 3 times with anhydrous ethanol, and freeze dry to obtain the finished product of Akebia quinata pectin.

[0046] Example 2

[0047] The white akebia pectin provided in this experiment was characterized by the following galacturonic acid content: determined by gas chromatography, the white akebia pectin prepared in Example 1 had a galacturonic acid content of 75.53%.

[0048] Degree of esterification: The degree of esterification of this pectin was determined by titration to be 38.58%.

[0049] Table 1 shows the molecular weight: the weight-average molecular weight of the pectin was 61.670 kDa, as determined by high performance gel permeation chromatography.

[0050] Table 1. Molecular weight distribution of Akebia quinata pectin

[0051]

[0052] Wherein, Mn is the number-average molecular weight, Mp is the peak molecular weight, Mw is the weight-average molecular weight, and Mw / Mn is the molecular weight dispersion index. Its weight-average molecular weight is 61.670 kDa, indicating that Akebia quinata pectin is a diverse mixture composed of polysaccharide molecules of different sizes.

[0053] Example 3

[0054] In vitro antioxidant capacity analysis of Akebia trifoliata pectin

[0055] (1) DPPH free radical scavenging ability

[0056] Add 65 μmol·L to a 96-well microplate. -1280 μL of DPPH solution was added, followed by 20 μL of Trolox standard solution at different concentration gradients or 20 μL of sample solution at different concentrations. The mixture was shaken and incubated at room temperature in the dark for 30 min. The absorbance was then measured at 540 nm. The concentration gradients of the Trolox standards were set to 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mmol·L⁻¹. -1 Each sample was tested in triplicate. DPPH radical scavenging capacity was expressed as Trolox equivalents per gram of AP dry weight (μmol Trolox·g). -1 DW).

[0057] (2) ABTS free radical scavenging ability

[0058] The total antioxidant capacity of ABTS was determined using the Beyotime ABTS Total Antioxidant Capacity Assay Kit. ABTS working solution and Trolox standard solutions of various concentration gradients were prepared according to the instructions. 200 μL of ABTS working solution was added to a 96-well microplate, followed by 10 μL of each concentration gradient of Trolox standard solution or the sample solution to be tested. The mixture was gently shaken and incubated at room temperature and in the dark for 2–6 min. The absorbance was measured at 734 nm. Three replicates were set for each sample. The ABTS free radical scavenging capacity was expressed as Trolox equivalents per gram of AP dry weight (μmol Trolox·g). -1 DW).

[0059] (3) Fe 3+ Measurement of reducing power

[0060] The Beyotime FRAP assay kit was used. FRAP working solution and FeSO4 standard solutions of different concentrations were prepared according to the manufacturer's instructions. 180 μL of FRAP working solution was added to a 96-well microplate, followed by 5 μL of FeSO4 standard solutions or sample solutions of varying concentrations. The plates were incubated at 37 °C for 5 min, and absorbance was measured at 593 nm. Three replicates were performed for each sample. 3+ Reducing power is expressed as FeSO4 equivalents per gram of AP dry weight (μmol FeSO4·g) -1 DW).

[0061] Figure 1 The results showed differences among the concentrations. With increasing polysaccharide concentration, its DPPH radical scavenging ability significantly increased, a trend consistent with ABTS radical scavenging ability and Fe... 3+The results of the reducing power determination were consistent. In vitro antioxidant activity results indicated that Akebia quinata pectin possesses good bioactivity. This study, through multi-index in vitro experiments, confirmed the concentration-dependent antioxidant activity of Akebia quinata pectin, providing a scientific basis for its application in the development of functional components.

[0062] Experiment Example 4

[0063] Animal model construction: Thirty-two SPF-grade C57BL / 6 mice were used and fed acclimatized for one week. The mice were then randomly divided into four groups: normal group (NC), model group (DSS), low-dose pectin group (DSS-LAP), and high-dose pectin group (DSS-HAP). The experiment lasted for 14 days. Starting from day 8, except for the NC group mice, the other mice were given drinking water containing 2.5% DSS to establish an acute colitis model.

[0064] Grouping and administration: 32 mice were randomly divided into 4 groups of 8 mice each. The model control group was administered physiological saline by gavage, while the low-dose and high-dose groups were administered 50 mg / kg and 200 mg / kg of Akebia trifoliata pectin solution by gavage, respectively, once daily for 14 consecutive days.

[0065] Figure 2-7 The results showed that the average DAI score in the model control group was 7.75, while the scores in the low-dose and high-dose groups decreased to 6.5 and 7, respectively.

[0066] Colon morphology: The colon length in the model control group was 6.8 cm, while the low-dose and high-dose groups increased to 7.8 cm and 7.6 cm, respectively, which were significantly longer than those in the model control group (P < 0.05).

[0067] Inflammatory factors: In the model control group, the levels of TNF-α, IL-6, and IL-17A in the colon were 2517.44 pg / mL, 664.13 pg / mL, and 71.26 pg / mL, respectively. In the low-dose group, these levels decreased to 1068.97 pg / mL, 288.25 pg / mL, and 60.74 pg / mL, respectively. In the high-dose group, these levels decreased to 874.04 pg / mL, 319.29 pg / mL, and 57.45 pg / mL, respectively. Serum T... The levels of NF-α, IL-6, and IL-17A were 760.88 pg / mL, 740.29 pg / mL, and 180.13 pg / mL, respectively. In the low-dose group, these levels decreased to 629.88 pg / mL, 588.57 pg / mL, and 132.73 pg / mL, respectively, while in the high-dose group, they decreased to 600.88 pg / mL, 578.29 pg / mL, and 117.78 pg / mL, respectively. All differences were statistically significant (P < 0.05).

[0068] Figure 8-13The results showed that H&E staining revealed irregular arrangement of colonic mucosal epithelial cells and glands in the DSS group mice, with a small amount of inflammatory cell infiltration, while mice treated with Akebia trifoliata pectin showed significant improvement. Figure 8 PAS staining results showed that the colon of mice treated with Akebia quinata pectin had a more intact epithelial structure, more goblet cells, more uniform mucus distribution, and no abnormal cells or inflammatory infiltration compared to the model group. Figure 10 This is consistent with the RT-PCR results analysis. Figure 13 The lack of tight junction proteins increases the pericellular space and permeability, allowing intraluminal stimuli such as antigens, symbiotic bacteria, and toxins to enter the intestinal submucosa, stimulating the innate immune response and ultimately leading to intestinal inflammation. Therefore, the research results indicate that Akebia trifoliata pectin has a protective effect on the intestinal barrier.

[0069] Figure 14-21 The results show that Figure 14 The results showed overlap and differences in the number of microbial OTUs in the three groups of samples, indicating that the microbial community composition of different treatment groups has both unique and common components. Figure 15 (Principal coordinate analysis) and Figure 16 In (non-metric multidimensional scaling analysis), the clustering of sample points of different colors (NC, DSS, DSS+AP) intuitively reflects the differences in community structure between groups. Figure 18 The relative abundance of microorganisms at the phylum level differed among different treatment groups, suggesting that colitis and interventions affect the composition of the dominant phylum. Figure 19 Further analysis of community composition at the genus level revealed that the relative abundance of multiple genera differed among groups, indicating that changes in community composition were genus-specific. Figure 20 A shows that, compared to NC, Bacillot in DSS a The abundance of Firmicutes was significantly reduced (P<0.05); however, in the DSS+AP group after intervention, the abundance of Bacillota was significantly increased compared to the DSS group, suggesting that this phylum may be involved in the development of colitis, and that intervention can restore its abundance, potentially having a positive effect on intestinal homeostasis. Compared to the NC group, the abundance of Bacillota in the DSS group was significantly higher. Figure 20 B) The abundance of Akkermansia was significantly reduced (P<0.05); the abundance of Akkermansia in the DSS+AP group was significantly different from that in the DSS group (P<0.01), indicating that the abundance change of this genus is closely related to colitis and intervention measures, and may be one of the biomarkers of disease or intervention response.

Claims

1. The application of Akebia trifoliata pectin in the preparation of food with anti-inflammatory effects.

2. The application according to claim 1, characterized in that, The food mentioned is a functional food or a health food.

3. The application according to claim 1 or 2, characterized in that, The anti-inflammatory effect is to alleviate and regulate colon inflammation.

4. The application according to claim 3, characterized in that, The effects of alleviating and regulating colon inflammation include: reducing the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-17A in intestinal tissue or serum, repairing colonic mucosal damage, and / or regulating the intestinal flora structure.

5. The application according to claim 4, characterized in that, The regulation of gut microbiota structure includes increasing the relative abundance of Bacteroides and / or Akkermansia.

6. The application according to claim 1, characterized in that, The white akebia pectin is prepared by a method comprising the following steps: a) Take the fruit peel of Akebia trifoliata and perform impurity removal treatment; b) Mix the purified raw material powder with water, add cellulase, adjust the pH, and then perform ultrasonic-assisted extraction to obtain the extract; c) After centrifuging and filtering the extract, perform alcohol precipitation and collect the precipitate; d) The precipitate is purified to obtain purified Akebia quinata pectin.

7. The application according to claim 6, characterized in that, In step b), the material-to-liquid ratio is 1 g of raw material powder to 18.9 mL of water, the amount of cellulase added is 0.084 g, the pH is adjusted to 4.565, the ultrasonic temperature is 50℃, and the ultrasonic time is 55.31 minutes.

8. The application according to claim 6, characterized in that, The purification process in step d) includes: passing the crude pectin through a DEAE Sepharose Fast Flow anion exchange column, eluting it with different concentrations of NaCl solution, collecting the eluent, and then further purifying it through a Sepharose CL 6B gel column.

9. A food product with anti-inflammatory properties, characterized in that, It contains Akebia quinata pectin as described in claim 1 as an active ingredient.

10. The food product according to claim 9, characterized in that, The food product is a health food product in the form of tablets, capsules, granules, powders, or oral liquids.

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