Comparison of physicochemical characteristics and biological activities of polysaccharides from Lycium barbarum L. at different tree ages

Comparison of physicochemical characteristics and biological activities of polysaccharides from Lycium barbarum L. at different tree ages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of physicochemical characteristics and biological activities of polysaccharides from Lycium barbarum L. at different tree ages Ningli Wang, Han Wang, Hao Meng, Xinyu Guo, Dong Pei, Yingli Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2334172/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The biological activity is related to the content of active substances, and the accumulation of active substances is closely related to the tree age. The relationship between the physicochemical characteristics and biological activities of Lycium barbarum polysaccharides (LBPs) and tree ages was studied in this study. The physicochemical characteristics of LBPs at second (Y2), fourth (Y4), sixth (Y6), eighth (Y8), tenth (Y10) and fifteenth (Y15) tree ages, such as total carbohydrate content (TCC), protein content (PC), uronic acid content (UAC), monosaccharide composition, molecular weight distribution, infrared characteristics were determined. And effects of LBPs of different tree ages on PC12 cells and APRE-19 cells function were evaluated in vitro. The results indicated that there was no significant difference in PC of LBPs at different tree ages. The UAC and TCC of LBPs from Y4 to Y10 were not significantly different, but were significantly higher than those in Y2 and Y15. LBPs of different tree ages showed similar preliminary structural characteristics. Compared with the LBPs of other tree ages, the LBPs of Y4, Y6, Y8 and Y10 had a good role in resisting oxidative stress. These results indicated that molecular weight, TCC and UAC of LBPs had important effects on their antioxidant activity in vitro. The accumulation of UAC and TCC in LBPs was closely related to tree age. The physicochemical characteristics and biological activities of LBPs fromY4 to Y10 were better than Y2 and Y15. Therefore, Lycium barbarum L. from Y4 to Y10 should be selected when preparing LBPs. Lycium barbarum polysaccharides (LBPs) Tree age Physicochemical characteristics Resistance to oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Lycium barbarum L. belongs to Solanaceae, Solaneae, Lyciinae, Lycium L. The traditional Chinese medicine Lycii Fructus refers to the mature and dry fruit of Lycium barbarum L.. Modern pharmacological studies have demonstrated that Lycium barbarum L. presents multiple biological activities, for instance regulating immunity, anti-aging, anti-oxidation, anti-tumor, anti-inflammatory, relieving physical fatigue, anti Alzheimer's disease, lowering blood lipid, lowering blood sugar [ 1 – 3 ]. The main physiological active components isolated from Lycium barbarum L. are polysaccharides, carotenoids, flavonoids, alkaloids, organic acids, etc. [ 4 , 5 ]. These active ingredients are widely applied in food and medicine. Lycium barbarum L., as a raw material with potential nutritional and medicinal value, has attracted much attention. Studies have shown that Lycium barbarum L. played a variety of activities mainly related to its resistance to oxidative stress. Wang et al. investigated the protective effect and mechanism of Lycium barbarum polyphenols on ethanol-induced cognitive impairment in mice. They found that Lycium barbarum polyphenols reduced intracellular oxidative stress level by increasing the expression levels of key proteins and related factors, and ultimately improved ethanol-induced cognitive impairment [ 6 ]. Gong et al. compared the inhibitory effects of Lycium barbarum polysaccharides (LBPs) fractions on a variety of cancer cells, including breast cancer cells, cervical cancer cells, liver cancer cells and gastric cancer cells. Then the mechanism of LBPs was studied from cell cycle, mitochondrial function, oxidative stress pathway, apoptosis and other aspects [ 7 ]. Therefore, the evaluation of the ability of the main active components of Lycium barbarum L. to resist oxidative stress is of guiding significance to reveal the mechanism of the active components. In addition, the physicochemical characteristics and biological activities of Lycium barbarum L. are affected by many factors [ 8 ]. In the research of LBPs, Xie et al. showed that the similar effects of polysaccharides in different regions of China should be related to their similar chemical properties. However, the extent to which they promote macrophage function was different, which might be due to their different active polysaccharide content [ 9 ]. Thus, the content of polysaccharide in Lycium barbarum L. from different regions was different, which made the biological activity of LBPs different. Similarly, the content of LBPs may be different in different tree ages, which makes the biological activity of LBPs different, because tree age is a key factor affecting the accumulation of nutrients and active ingredients. Tree age could affect the content of ascorbic acid, total phenol and other active components in fruit juice and the antioxidant activity of fruit juice [ 10 ]. Meena et al. studied the effects of three different tree ages of 6, 18 and 30 years on mineral elements, functional components, etc. in Amrapali Mango ( Mangifera indica ) Fruits, so as to provide mangoes with the most appropriate tree age for people to eat and product development [ 11 ]. However, the effect of tree age on the main active components and biological activities of Lycium barbarum L. has not been reported in the same way. In this paper, as one of the main active ingredients in Lycium barbarum L., LBPs were obtained by the same preparation method from different tree ages, and their total carbohydrate content (TCC), protein content (PC), uronic acid content (UAC), monosaccharide composition, molecular weight distribution were investigated. Their structural characteristics were preliminarily explored, and the role of LBPs of different tree ages in preventing H 2 O 2 induced APRE-19 and CoCl 2 induced PC12 oxidative stress was evaluated. It was expected to reveal the correlation between the physicochemical properties of LBPs of different tree ages and their antioxidant effects in order to provide scientific basis for selecting the best tree age of Lycium barbarum L. to separate active LBPs, and thus provide the best source of raw materials for food and medicine with LBPs as the main component. 2. Materials And Methods 2.1 Chemicals and reagents The reference substances with purity greater than 98% of mannose (Man), galactose (Gal), glucose (Glc), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucosamine (GlcN), xylose (Xyl) and arabinose (Ara) were obtained from China National Institute for Drug and Food Control. Chloroform, n-butanol and sulfuric acid were obtained from Sinopharm Chemical Reagent Co., Ltd., China and were analytical grade. Methanol and acetonitrile were chromatographic grade and were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd., China. 2.2 Materials The Lycium barbarum fruits of different tree ages were collected by researcher Ying Wang from South China Botanical Garden of the Chinese Academy of Sciences in 2017 and identified as the dried fruits of Lycium barbarum L. [ 12 ]. They were protected from light and moisture. The storage temperature was − 20°C. The Y2, Y4, Y6, Y8, Y10 and Y15 represented Lycium barbarum L. of 2, 4, 6, 8, 10 and 15 tree ages respectively. 2.3 Preparation of LBPS About 5.0 g of Lycium barbarum powder dried to constant weight at 105°C and 125 mL of distilled water were put into a 250 mL conical flask. The Lycium barbarum powder was extracted under the conditions of ultrasonic power 200 W and temperature 60°C for 30 min. After the whole system was cooled to room temperature, the supernatant was collected by centrifugation 4500 r·min − 1 for 10 min. Then the supernatant was concentrated under reduced pressure and made up to 20 mL. Under the stirring condition of the rotating speed of 120 r·min − 1 , 80 mL of absolute ethanol was added to the concentrated solution at a rate of 4 mL·min − 1 with a separatory funnel, and it was allowed to stand for 10 h. Then the supernatant was removed. The collected precipitate was added with 50 mL of distilled water, centrifuged at 4500 r·min − 1 for 10 min, and the supernatant was collected. Sevage reagent was added to the supernatant and the mixed solution was vigorously shaken to remove agglomerated flocs. The above steps were repeated several times until no floccules were precipitated at the interface between the Sevage reagent and the polysaccharide solution, which indicated that the protein had been completely removed. The polysaccharide solution was added to the dialysis bag with the interception molecular weight of 1000 Da. The dialysis bag was dialyzed with tap water for 2 days and replaced with distilled water for 1 day. Under the condition that the temperature of the cold trap was − 50°C and the degree of vacuum was 8 Pa, the polysaccharide solution after dialysis was dried in a freeze dryer to obtain LBPs. 2.4 Determination of chemical composition content in LBPS The method of phenol–sulfuric acid was utilized to measure the TCC of LBPs [ 13 ]. The TCC was calculated as the D-anhydroglucose calibration curve. Coomassie Brilliant Blue G-250 method was used to determine the PC with BSA as reference [ 14 ]. With GalA as reference, carbazole-sulfate method was used to determine UAC [ 15 ]. 2.5 Determination of molecular weight The molecular weight was determined by the size exclusion chromatography coupled with multi-angel laser light scattering (SEC-MALLs, Wyatt Technologies Corporation, USA) with reference to reported method [ 16 , 17 ]. OHpak SB-803 HQ (8.0 mm I.D.×300 mm, 6 µm) and OHpak SB-804 HQ (8.0 mm I.D.×300 mm, 6 µm) were connected in series to be used at 25°C. The injection volume was 250 µL and the injection concentration was 1 mg·mL − 1 at a flow rate of 1.0 mL·min − 1 . 2.6 Monosaccharide-composition analysis Hydrolysis of LBPs (5.0 mg) was hydrolyzed using 0.5 mL of 2.0 mol/L TFA at 110°C for 6 h. The solution was blown dry by nitrogen and dissolved in 0.5 mL of water to obtain a hydrolyzed solution of LBPs. 400 µL hydrolyzed solution of LBPs, 400 µL 0.5 mol·L − 1 PMP-methanol solution and 400 µL 0.3 mol·L − 1 NaOH solution were added to a 5 mL snap-top bottle, mixed well, and reacted in a water bath at 70°C for 100 min. After the water bath reaction, 500 µL of 0.3 mol·L − 1 hydrochloric acid solution was added to the snap-top bottle, mixed well, washed 3 times with 2 mL of chloroform each time, and the chloroform solution was discarded. The supernatant was filtered through a 0.45 µm membrane filter and analyzed by A LC-20A high performance liquid chromatography (HPLC, Shimadzu Corporation, Japan) system with a Inertsil ODS-3 column (4.6 mm I.D. × 250 mm, 5 µm) at 245 nm and 30°C. The flow rate was 1.2 mL·min − 1 . The loading volume was 10 µL. The procedure for monosaccharide standards was the same as above. 2.7 Fourier transform-infrared spectroscopy (FT-IR) analysis The FT-IR spectra of LBPs (1.0 mg) were detected on a Bruker tensor 27 FT-IR spectrometer (Bruker, Germany) and the detection range was 4000 cm − 1 to 500 cm − 1 with a resolution of 4 cm − 1 . 2.8 The inhibition of oxidative stress activity PC12 cells and APRE-19 cells in logarithmic growth phase were taken and cultured in 96-well plates. After the cells adhered, samples of different concentrations (1, 10, 50, 100, 200 and 500 µg·mL − 1 ) were added. After 6 h of treatment, CoCl 2 solution and H 2 O 2 solution was respectively added for modeling treatment. And after 24 h, the cell viability was measured by MTT assay to evaluate the inhibition of CoCl 2 -induced oxidative stress activity of PC12 cells and the inhibition of H 2 O 2 -induced oxidative stress activity of ARPE-19 cells by LBPs. 2.9 Statistical analysis Results are expressed as mean ± standard deviation (M ± SD). SPSS 22.0 statistical software, Origin 2021 and GraphPad Prism were used for data processing and graph analysis, and one-way analysis of variance (ANOVA) was used for comparison between groups. 3. Results And Discussion 3.1 Effect of tree age on the content of chemical constituents for LBPs During the whole development process of Lycium barbarum L. trees, the accumulation of secondary metabolites in the fruits is not consistent. There are regular changes in morphology and physiology, which in turn affect their ability to absorb nutrients from the outside [ 18 ]. The content of chemical constituents in LBPs of different tree ages was shown in Fig. 1 . The results indicated that there was no significant difference in the content of protein for LBPs at different tree ages. The UAC and TCC of LBPs in the trees from Y4 to Y10 were not significantly different but were significantly higher than those in the trees of Y2 and Y15. This indicated that the Y2 was in the growing stage with a lot of the intake of nutrients, but the vast majority was used for their own metabolism and nutrition supply. The trees of Lycium barbarum L. have developed and matured from the Y4 to Y10, which belongs to the fruit bearing period. At this stage, the contents of all chemical constituents in LBPs were relatively stable and had no significant difference. However, the TCC and UAC decreased at Y15 and the quality of LBPs decreased, which may be due to the slow metabolism of Lycium barbarum L. trees, leading to the decrease of the content for secondary metabolites. There was no data on the content of chemical constituents for LBPs after Y15 and the small sample size above results, so it was necessary to further increase the sample size to verify this conclusion. 3.2 Effect of tree age on molecular weight of LBPs The molecular weight distribution and molecular weight percentage of LBPs at different tree ages was shown in Fig. 2 . High molecular weight distribution was 2.099 × 10 6 ~ 3.644 × 10 6 Da, and low molecular weight distributed at 4.537 × 10 4 ~ 8.942 × 10 4 Da. The MW1 range and proportion of LBPs at Y8 were larger than those at other tree ages, and the molecular weights of MW1 and MW3 range of LBPs at Y15 were smaller than those at other tree ages. The MW1, MW2 and MW3 ranges of Y4 and Y10 are almost consistent with their proportions. 3.3 Effect of tree age on monosaccharide-composition of LBPs The monosaccharide composition of LBPs at different tree ages was shown in Fig. 3 . LBPs of different tree ages was composed of 8 kinds of monosaccharides, and glucose, galactose and arabinose were the main monosaccharides units of polysaccharide structure, which was the typical monosaccharide composition profile of LBPs [ 19 ]. At the second age, the molar ratio of arabinose was higher than that of glucose and galactose. However, from Y4 to the Y15, the monosaccharide composition of LBPs was similar, in the order of glucose > arabinose > galactose. 3.4 Effect of tree age on FT-IR of LBPs The main functional groups of polysaccharides can be determined qualitatively by FT-IR, which is usually used to clarify the preliminary structural information of polysaccharides [ 20 ]. As shown in Fig. 4 , the two characteristic absorption peaks near 3403 cm − 1 and 2925 cm − 1 were relevant to O-H and C-H tensile vibrations, respectively, which were typical characteristics of LBPs [ 18 , 21 ]. The characteristic absorption peaks around 1643 cm − 1 and 1420 cm − 1 indicated tensile vibrations of symmetric and asymmetric C = O groups, indicating there was uronic acid in LBPs [ 22 ], which was consistent with monosaccharide composition and chemical composition measurements. The absorption peak about 1078 cm − 1 belonged to the bending vibration of group C-H [ 18 ]. The results of FT-IR showed that tree age had little effect on the main functional groups of LBPs. 3.5 Effect of tree age on the inhibition of oxidative stress activity of LBPs The effect of LBPs of different tree ages on the proliferation of APRE-19 induced by H 2 O 2 was shown in Fig. 5 A. Compared with the control group, the LBPs at Y4, Y6, Y8, Y10 and Y15 had significant effects on promoting APRE-19 proliferation ( P ༜0.01). However, LBPs at Y2 has no obvious APRE-19 proliferation effect, indicating that the LBPs has a good anti-oxidative stress effect from Y4 to Y15. The correlation analysis between the APRE-19 proliferation promoting activity of LBPs at different tree ages and the TCC, PC and UAC showed that the TCC was significantly positively correlated with the proliferation promoting activity (Pearson correlation coefficient r = 0.920, P < 0.01). And the UAC was also positively correlated with the proliferation promoting activity (Pearson correlation coefficient r = 0.765), which indicated that the higher the TCC and UAC of LBPs in different tree ages, the stronger the proliferation activity of APRE-19 induced by H 2 O 2 . The TCC in Y2 was higher than that in Y15, but it did not promote APRE-19 proliferation, which may be related to the monosaccharide composition of polysaccharides [ 23 ]. It could be seen from Fig. 3 that although the main skeleton structure of LBPs at different tree ages was composed of glucose, galactose and arabinose, the composition molar ratio of Y2 was significantly different from that of other tree ages, and its glucose molar ratio was smaller than arabinose. However, the molar ratio of glucose was greater than arabinose in other tree ages, and the composition ratio of polysaccharides chain might be one of the main factors affecting its APRE-19 proliferation activity. Furthermore, different oligosaccharide fragments need to be obtained by means of partial hydrolysis of polysaccharides for further verification. The influence of LBPs of different tree ages on the proliferation of PC12 induced by CoCl 2 was shown in Fig. 5 B. Compared with the control group, the LBPs in Y4, Y6, Y8 and Y10 significantly promoted the proliferation of PC12 induced by CoCl 2 ( P < 0.01), while LBPs in Y2 and Y15 did not show similar activity. The results suggested that LBPs from Y4 to Y10 had a good anti-oxidative stress effect. Correlation analysis showed that the activity was positively correlated with TCC and UAC (Pearson correlation coefficient r = 0.835 and 0.855, P < 0.05), but not with the PC. 4. Conclusion In conclusion, this study confirmed that the tree age had an impact on the chemical composition, monosaccharide composition and molecular weight distribution of polysaccharides obtained from Lycium barbarum L., and LBPs of different tree ages showed similar preliminary structural characteristics. Compared with the LBPs of other tree ages, the LBPs of Y4, Y6, Y8 and Y10 had a good role in resisting oxidative stress. The molecular weight, TCC and UAC of LBPs had important effects on their antioxidant activity in vitro. In general, these results indicated that Lycium barbarum L. from Y4 to Y10 was more suitable for preparing LBPs with higher nutritional value and biological activity. And the LBPs could be used as the main functional components of food and medicine. The structure analysis and biological activity evaluation of LBPs of different tree ages in vivo will be carried out in the next step. Declarations Authors’ Contributions Ningli Wang: Conceptualization, methodology, experiments performing, data analysis, writing-original draft, review and editing; Han Wang: Research advice, data analysis, review and editing; Hao Meng: Research advice, investigation, review and editing; Xinyu Guo: Investigation, review and editing; Dong Pei: Funding acquisition, review, and editing; Yingli Yang: Supervision, conceptualization, funding acquisition, review, and editing, validation; Jianfei Liu: Supervision, conceptualization, funding acquisition, review, and editing, validation; Duolong Di: Review and editing, validation. Funding This research was supported by Gansu Science and Technology Major Project (No. 22ZD6FA021), LICP Cooperation Foundation for Young Scholars (No. HZJJ21-08), the Science Foundation of Gansu Province (No. 20JR10RA054), and Lanzhou Science and Technology Plan Project (No. 2022-2-2). Data Availability All data generated or analysed during this study are included in this published article. Competing Interests The authors declare no competing interests. Conflict of Interest The authors have declared no conflict of interest. Ethics Approval Not applicable. Consent to Participate Not applicable. Consent for Publication The authors hereby consent to the publication of this study in the journal of Plant Foods for Human Nutrition. The author warrants that the study has not been published before in any form and is not being concurrently submitted to and is not under consideration by another publisher. References Chang CC, So KF (2015) Lycium Barbarum and Human Health. Springer Netherlands. Tian XJ, Liang TS, Liu YL et al (2019) Extraction, Structural Characterization, and Biological Functions of Lycium Barbarum Polysaccharides: A Review. 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Ind Crop Prod 184:115036. https://doi.org/ 10.1016/j.indcrop.2022.115036 Liu JF, Li YC, Pu QS et al (2022) A polysaccharide from Lycium barbarum L.: Structure and protective effects against oxidative stress and high-glucose-induced apoptosis in ARPE-19 cells. Int. J. Biol. Macromol. 201:111-120. https://doi.org/ 10.1016/j.ijbiomac.2021.12.139 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Liu","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianfei","middleName":"","lastName":"Liu","suffix":""},{"id":157517699,"identity":"10d0c948-ac90-42c3-9f8b-86138e1ba82e","order_by":7,"name":"Duolong Di","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Duolong","middleName":"","lastName":"Di","suffix":""}],"badges":[],"createdAt":"2022-12-01 14:47:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2334172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2334172/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30019106,"identity":"5d774d47-37f8-4c44-bf7c-b8a056933ee8","added_by":"auto","created_at":"2022-12-07 16:09:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25249,"visible":true,"origin":"","legend":"\u003cp\u003eThe content of chemical constituents in LBPs of different tree ages\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2334172/v1/8994677cc49dda22742f219a.png"},{"id":30019109,"identity":"94332636-97c7-44c8-a8b6-8c295c29ffbd","added_by":"auto","created_at":"2022-12-07 16:09:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":438788,"visible":true,"origin":"","legend":"\u003cp\u003eThe molecular weight distribution (A) and molecular weight percentage (B) of LBPs at different tree ages\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2334172/v1/1bb1f8e8a479a9b9cb65887f.png"},{"id":30020493,"identity":"a438f3d8-1640-46d5-8efb-e18c3400d827","added_by":"auto","created_at":"2022-12-07 16:17:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":456898,"visible":true,"origin":"","legend":"\u003cp\u003eHigh performance liquid chromatography (A) and molar percentage (B) of monosaccharide composition for LBPs at different tree ages\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2334172/v1/15abbabf9b67e73997406816.png"},{"id":30020495,"identity":"c9ae1303-875e-4089-9dc1-3d1904df4df5","added_by":"auto","created_at":"2022-12-07 16:17:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":152621,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR of LBPs at different tree ages\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2334172/v1/bdc75ed0790cd8dfaa35eac9.png"},{"id":30021495,"identity":"dad28f5c-d17e-4116-b474-430e46ec1c51","added_by":"auto","created_at":"2022-12-07 16:25:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":197768,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of LBPs of different tree ages on the proliferation of APRE-19 induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (A) and PC12 induced by CoCl\u003csub\u003e2\u003c/sub\u003e (B)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2334172/v1/f1263486c27ae25f9783568a.png"},{"id":30047503,"identity":"56f8b148-3fbe-42da-a664-065ac8d8880c","added_by":"auto","created_at":"2022-12-08 05:59:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":936327,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2334172/v1/0cb758bd-487e-4449-bb16-de5abafe4ed5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of physicochemical characteristics and biological activities of polysaccharides from Lycium barbarum L. at different tree ages","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eLycium barbarum\u003c/em\u003e L. belongs to Solanaceae, Solaneae, Lyciinae, \u003cem\u003eLycium\u003c/em\u003e L. The traditional Chinese medicine \u003cem\u003eLycii Fructus\u003c/em\u003e refers to the mature and dry fruit of \u003cem\u003eLycium barbarum\u003c/em\u003e L.. Modern pharmacological studies have demonstrated that \u003cem\u003eLycium barbarum\u003c/em\u003e L. presents multiple biological activities, for instance regulating immunity, anti-aging, anti-oxidation, anti-tumor, anti-inflammatory, relieving physical fatigue, anti Alzheimer's disease, lowering blood lipid, lowering blood sugar [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The main physiological active components isolated from \u003cem\u003eLycium barbarum\u003c/em\u003e L. are polysaccharides, carotenoids, flavonoids, alkaloids, organic acids, etc. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These active ingredients are widely applied in food and medicine.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLycium barbarum\u003c/em\u003e L., as a raw material with potential nutritional and medicinal value, has attracted much attention. Studies have shown that \u003cem\u003eLycium barbarum\u003c/em\u003e L. played a variety of activities mainly related to its resistance to oxidative stress. Wang et al. investigated the protective effect and mechanism of \u003cem\u003eLycium barbarum\u003c/em\u003e polyphenols on ethanol-induced cognitive impairment in mice. They found that \u003cem\u003eLycium barbarum\u003c/em\u003e polyphenols reduced intracellular oxidative stress level by increasing the expression levels of key proteins and related factors, and ultimately improved ethanol-induced cognitive impairment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Gong et al. compared the inhibitory effects of \u003cem\u003eLycium barbarum\u003c/em\u003e polysaccharides (LBPs) fractions on a variety of cancer cells, including breast cancer cells, cervical cancer cells, liver cancer cells and gastric cancer cells. Then the mechanism of LBPs was studied from cell cycle, mitochondrial function, oxidative stress pathway, apoptosis and other aspects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, the evaluation of the ability of the main active components of \u003cem\u003eLycium barbarum\u003c/em\u003e L. to resist oxidative stress is of guiding significance to reveal the mechanism of the active components. In addition, the physicochemical characteristics and biological activities of \u003cem\u003eLycium barbarum\u003c/em\u003e L. are affected by many factors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the research of LBPs, Xie et al. showed that the similar effects of polysaccharides in different regions of China should be related to their similar chemical properties. However, the extent to which they promote macrophage function was different, which might be due to their different active polysaccharide content [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, the content of polysaccharide in \u003cem\u003eLycium barbarum\u003c/em\u003e L. from different regions was different, which made the biological activity of LBPs different. Similarly, the content of LBPs may be different in different tree ages, which makes the biological activity of LBPs different, because tree age is a key factor affecting the accumulation of nutrients and active ingredients. Tree age could affect the content of ascorbic acid, total phenol and other active components in fruit juice and the antioxidant activity of fruit juice [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Meena et al. studied the effects of three different tree ages of 6, 18 and 30 years on mineral elements, functional components, etc. in Amrapali Mango (\u003cem\u003eMangifera indica\u003c/em\u003e) Fruits, so as to provide mangoes with the most appropriate tree age for people to eat and product development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the effect of tree age on the main active components and biological activities of \u003cem\u003eLycium barbarum\u003c/em\u003e L. has not been reported in the same way.\u003c/p\u003e \u003cp\u003eIn this paper, as one of the main active ingredients in \u003cem\u003eLycium barbarum\u003c/em\u003e L., LBPs were obtained by the same preparation method from different tree ages, and their total carbohydrate content (TCC), protein content (PC), uronic acid content (UAC), monosaccharide composition, molecular weight distribution were investigated. Their structural characteristics were preliminarily explored, and the role of LBPs of different tree ages in preventing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induced APRE-19 and CoCl\u003csub\u003e2\u003c/sub\u003e induced PC12 oxidative stress was evaluated. It was expected to reveal the correlation between the physicochemical properties of LBPs of different tree ages and their antioxidant effects in order to provide scientific basis for selecting the best tree age of \u003cem\u003eLycium barbarum\u003c/em\u003e L. to separate active LBPs, and thus provide the best source of raw materials for food and medicine with LBPs as the main component.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eThe reference substances with purity greater than 98% of mannose (Man), galactose (Gal), glucose (Glc), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucosamine (GlcN), xylose (Xyl) and arabinose (Ara) were obtained from China National Institute for Drug and Food Control. Chloroform, n-butanol and sulfuric acid were obtained from Sinopharm Chemical Reagent Co., Ltd., China and were analytical grade. Methanol and acetonitrile were chromatographic grade and were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd., China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Materials\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eLycium barbarum\u003c/em\u003e fruits of different tree ages were collected by researcher Ying Wang from South China Botanical Garden of the Chinese Academy of Sciences in 2017 and identified as the dried fruits of \u003cem\u003eLycium barbarum\u003c/em\u003e L. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They were protected from light and moisture. The storage temperature was \u0026minus;\u0026thinsp;20\u0026deg;C. The Y2, Y4, Y6, Y8, Y10 and Y15 represented \u003cem\u003eLycium barbarum\u003c/em\u003e L. of 2, 4, 6, 8, 10 and 15 tree ages respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of LBPS\u003c/h2\u003e \u003cp\u003eAbout 5.0 g of \u003cem\u003eLycium barbarum\u003c/em\u003e powder dried to constant weight at 105\u0026deg;C and 125 mL of distilled water were put into a 250 mL conical flask. The \u003cem\u003eLycium barbarum\u003c/em\u003e powder was extracted under the conditions of ultrasonic power 200 W and temperature 60\u0026deg;C for 30 min. After the whole system was cooled to room temperature, the supernatant was collected by centrifugation 4500 r\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 10 min. Then the supernatant was concentrated under reduced pressure and made up to 20 mL. Under the stirring condition of the rotating speed of 120 r\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 80 mL of absolute ethanol was added to the concentrated solution at a rate of 4 mL\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a separatory funnel, and it was allowed to stand for 10 h. Then the supernatant was removed. The collected precipitate was added with 50 mL of distilled water, centrifuged at 4500 r\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 10 min, and the supernatant was collected. Sevage reagent was added to the supernatant and the mixed solution was vigorously shaken to remove agglomerated flocs. The above steps were repeated several times until no floccules were precipitated at the interface between the Sevage reagent and the polysaccharide solution, which indicated that the protein had been completely removed. The polysaccharide solution was added to the dialysis bag with the interception molecular weight of 1000 Da. The dialysis bag was dialyzed with tap water for 2 days and replaced with distilled water for 1 day. Under the condition that the temperature of the cold trap was \u0026minus;\u0026thinsp;50\u0026deg;C and the degree of vacuum was 8 Pa, the polysaccharide solution after dialysis was dried in a freeze dryer to obtain LBPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination of chemical composition content in LBPS\u003c/h2\u003e \u003cp\u003eThe method of phenol\u0026ndash;sulfuric acid was utilized to measure the TCC of LBPs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The TCC was calculated as the D-anhydroglucose calibration curve. Coomassie Brilliant Blue G-250 method was used to determine the PC with BSA as reference [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. With GalA as reference, carbazole-sulfate method was used to determine UAC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Determination of molecular weight\u003c/h2\u003e \u003cp\u003eThe molecular weight was determined by the size exclusion chromatography coupled with multi-angel laser light scattering (SEC-MALLs, Wyatt Technologies Corporation, USA) with reference to reported method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. OHpak SB-803 HQ (8.0 mm I.D.\u0026times;300 mm, 6 \u0026micro;m) and OHpak SB-804 HQ (8.0 mm I.D.\u0026times;300 mm, 6 \u0026micro;m) were connected in series to be used at 25\u0026deg;C. The injection volume was 250 \u0026micro;L and the injection concentration was 1 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a flow rate of 1.0 mL\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Monosaccharide-composition analysis\u003c/h2\u003e \u003cp\u003eHydrolysis of LBPs (5.0 mg) was hydrolyzed using 0.5 mL of 2.0 mol/L TFA at 110\u0026deg;C for 6 h. The solution was blown dry by nitrogen and dissolved in 0.5 mL of water to obtain a hydrolyzed solution of LBPs. 400 \u0026micro;L hydrolyzed solution of LBPs, 400 \u0026micro;L 0.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PMP-methanol solution and 400 \u0026micro;L 0.3 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOH solution were added to a 5 mL snap-top bottle, mixed well, and reacted in a water bath at 70\u0026deg;C for 100 min. After the water bath reaction, 500 \u0026micro;L of 0.3 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e hydrochloric acid solution was added to the snap-top bottle, mixed well, washed 3 times with 2 mL of chloroform each time, and the chloroform solution was discarded. The supernatant was filtered through a 0.45 \u0026micro;m membrane filter and analyzed by A LC-20A high performance liquid chromatography (HPLC, Shimadzu Corporation, Japan) system with a Inertsil ODS-3 column (4.6 mm I.D. \u0026times; 250 mm, 5 \u0026micro;m) at 245 nm and 30\u0026deg;C. The flow rate was 1.2 mL\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The loading volume was 10 \u0026micro;L. The procedure for monosaccharide standards was the same as above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Fourier transform-infrared spectroscopy (FT-IR) analysis\u003c/h2\u003e \u003cp\u003eThe FT-IR spectra of LBPs (1.0 mg) were detected on a Bruker tensor 27 FT-IR spectrometer (Bruker, Germany) and the detection range was 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 The inhibition of oxidative stress activity\u003c/h2\u003e \u003cp\u003ePC12 cells and APRE-19 cells in logarithmic growth phase were taken and cultured in 96-well plates. After the cells adhered, samples of different concentrations (1, 10, 50, 100, 200 and 500 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were added. After 6 h of treatment, CoCl\u003csub\u003e2\u003c/sub\u003e solution and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution was respectively added for modeling treatment. And after 24 h, the cell viability was measured by MTT assay to evaluate the inhibition of CoCl\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress activity of PC12 cells and the inhibition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress activity of ARPE-19 cells by LBPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (M\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). SPSS 22.0 statistical software, Origin 2021 and GraphPad Prism were used for data processing and graph analysis, and one-way analysis of variance (ANOVA) was used for comparison between groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of tree age on the content of chemical constituents for LBPs\u003c/h2\u003e \u003cp\u003eDuring the whole development process of \u003cem\u003eLycium barbarum\u003c/em\u003e L. trees, the accumulation of secondary metabolites in the fruits is not consistent. There are regular changes in morphology and physiology, which in turn affect their ability to absorb nutrients from the outside [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The content of chemical constituents in LBPs of different tree ages was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results indicated that there was no significant difference in the content of protein for LBPs at different tree ages. The UAC and TCC of LBPs in the trees from Y4 to Y10 were not significantly different but were significantly higher than those in the trees of Y2 and Y15. This indicated that the Y2 was in the growing stage with a lot of the intake of nutrients, but the vast majority was used for their own metabolism and nutrition supply. The trees of \u003cem\u003eLycium barbarum\u003c/em\u003e L. have developed and matured from the Y4 to Y10, which belongs to the fruit bearing period. At this stage, the contents of all chemical constituents in LBPs were relatively stable and had no significant difference. However, the TCC and UAC decreased at Y15 and the quality of LBPs decreased, which may be due to the slow metabolism of \u003cem\u003eLycium barbarum\u003c/em\u003e L. trees, leading to the decrease of the content for secondary metabolites. There was no data on the content of chemical constituents for LBPs after Y15 and the small sample size above results, so it was necessary to further increase the sample size to verify this conclusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of tree age on molecular weight of LBPs\u003c/h2\u003e \u003cp\u003eThe molecular weight distribution and molecular weight percentage of LBPs at different tree ages was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e. High molecular weight distribution was 2.099 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e ~ 3.644 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e Da, and low molecular weight distributed at 4.537 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e ~ 8.942 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e Da. The MW1 range and proportion of LBPs at Y8 were larger than those at other tree ages, and the molecular weights of MW1 and MW3 range of LBPs at Y15 were smaller than those at other tree ages. The MW1, MW2 and MW3 ranges of Y4 and Y10 are almost consistent with their proportions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of tree age on monosaccharide-composition of LBPs\u003c/h2\u003e \u003cp\u003eThe monosaccharide composition of LBPs at different tree ages was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e. LBPs of different tree ages was composed of 8 kinds of monosaccharides, and glucose, galactose and arabinose were the main monosaccharides units of polysaccharide structure, which was the typical monosaccharide composition profile of LBPs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. At the second age, the molar ratio of arabinose was higher than that of glucose and galactose. However, from Y4 to the Y15, the monosaccharide composition of LBPs was similar, in the order of glucose\u0026thinsp;\u0026gt;\u0026thinsp;arabinose\u0026thinsp;\u0026gt;\u0026thinsp;galactose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of tree age on FT-IR of LBPs\u003c/h2\u003e \u003cp\u003eThe main functional groups of polysaccharides can be determined qualitatively by FT-IR, which is usually used to clarify the preliminary structural information of polysaccharides [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the two characteristic absorption peaks near 3403 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2925 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were relevant to O-H and C-H tensile vibrations, respectively, which were typical characteristics of LBPs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The characteristic absorption peaks around 1643 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1420 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated tensile vibrations of symmetric and asymmetric C\u0026thinsp;=\u0026thinsp;O groups, indicating there was uronic acid in LBPs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which was consistent with monosaccharide composition and chemical composition measurements. The absorption peak about 1078 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e belonged to the bending vibration of group C-H [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The results of FT-IR showed that tree age had little effect on the main functional groups of LBPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of tree age on the inhibition of oxidative stress activity of LBPs\u003c/h2\u003e \u003cp\u003eThe effect of LBPs of different tree ages on the proliferation of APRE-19 induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. Compared with the control group, the LBPs at Y4, Y6, Y8, Y10 and Y15 had significant effects on promoting APRE-19 proliferation (\u003cem\u003eP\u003c/em\u003e༜0.01). However, LBPs at Y2 has no obvious APRE-19 proliferation effect, indicating that the LBPs has a good anti-oxidative stress effect from Y4 to Y15. The correlation analysis between the APRE-19 proliferation promoting activity of LBPs at different tree ages and the TCC, PC and UAC showed that the TCC was significantly positively correlated with the proliferation promoting activity (Pearson correlation coefficient \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.920, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). And the UAC was also positively correlated with the proliferation promoting activity (Pearson correlation coefficient \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.765), which indicated that the higher the TCC and UAC of LBPs in different tree ages, the stronger the proliferation activity of APRE-19 induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The TCC in Y2 was higher than that in Y15, but it did not promote APRE-19 proliferation, which may be related to the monosaccharide composition of polysaccharides [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It could be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e that although the main skeleton structure of LBPs at different tree ages was composed of glucose, galactose and arabinose, the composition molar ratio of Y2 was significantly different from that of other tree ages, and its glucose molar ratio was smaller than arabinose. However, the molar ratio of glucose was greater than arabinose in other tree ages, and the composition ratio of polysaccharides chain might be one of the main factors affecting its APRE-19 proliferation activity. Furthermore, different oligosaccharide fragments need to be obtained by means of partial hydrolysis of polysaccharides for further verification.\u003c/p\u003e \u003cp\u003eThe influence of LBPs of different tree ages on the proliferation of PC12 induced by CoCl\u003csub\u003e2\u003c/sub\u003e was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. Compared with the control group, the LBPs in Y4, Y6, Y8 and Y10 significantly promoted the proliferation of PC12 induced by CoCl\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while LBPs in Y2 and Y15 did not show similar activity. The results suggested that LBPs from Y4 to Y10 had a good anti-oxidative stress effect. Correlation analysis showed that the activity was positively correlated with TCC and UAC (Pearson correlation coefficient \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.835 and 0.855, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not with the PC.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, this study confirmed that the tree age had an impact on the chemical composition, monosaccharide composition and molecular weight distribution of polysaccharides obtained from \u003cem\u003eLycium barbarum\u003c/em\u003e L., and LBPs of different tree ages showed similar preliminary structural characteristics. Compared with the LBPs of other tree ages, the LBPs of Y4, Y6, Y8 and Y10 had a good role in resisting oxidative stress. The molecular weight, TCC and UAC of LBPs had important effects on their antioxidant activity in vitro. In general, these results indicated that \u003cem\u003eLycium barbarum\u003c/em\u003e L. from Y4 to Y10 was more suitable for preparing LBPs with higher nutritional value and biological activity. And the LBPs could be used as the main functional components of food and medicine. The structure analysis and biological activity evaluation of LBPs of different tree ages in vivo will be carried out in the next step.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u0026nbsp;\u003c/strong\u003eNingli Wang: Conceptualization, methodology, experiments performing, data analysis, writing-original draft, review and editing; Han Wang: Research advice, data analysis, review and editing; Hao Meng: Research advice, investigation, review and editing; Xinyu Guo: Investigation, review and editing; Dong Pei: Funding acquisition, review, and editing; Yingli Yang: Supervision, conceptualization, funding acquisition, review, and editing, validation; Jianfei Liu: Supervision, conceptualization, funding acquisition, review, and editing, validation; Duolong Di: Review and editing, validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis research was supported by Gansu Science and Technology Major Project (No. 22ZD6FA021), LICP Cooperation Foundation for Young Scholars (No. HZJJ21-08), the Science Foundation of Gansu Province (No. 20JR10RA054), and Lanzhou Science and Technology Plan Project (No. 2022-2-2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e All data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors have declared no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e The authors hereby consent to the publication of this study in the journal of Plant Foods for Human Nutrition. The author warrants that the study has not been published before in any form and is not being concurrently submitted to and is not under consideration by another publisher.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChang CC, So KF (2015)\u003cem\u003e Lycium Barbarum\u003c/em\u003e and Human Health. Springer Netherlands. \u003c/li\u003e\n\u003cli\u003eTian XJ, Liang TS, Liu YL et al (2019) Extraction, Structural Characterization, and Biological Functions of \u003cem\u003eLycium Barbarum\u003c/em\u003e Polysaccharides: A Review. Biomolecules 9:389. https://doi.org/10.3390/biom9090389\u003c/li\u003e\n\u003cli\u003eBai XL, Luo YJ, Fan WQ et al (2022) Neuroprotective Effects of \u003cem\u003eLycium Barbarum\u003c/em\u003e Fruit Extract on Pink1\u003csup\u003eB9\u003c/sup\u003eDrosophila Melanogaster Genetic Model of Parkinson\u0026rsquo;s Disease. 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Macromol. 201:111-120. https://doi.org/ 10.1016/j.ijbiomac.2021.12.139\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lycium barbarum polysaccharides (LBPs), Tree age, Physicochemical characteristics, Resistance to oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-2334172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2334172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe biological activity is related to the content of active substances, and the accumulation of active substances is closely related to the tree age. The relationship between the physicochemical characteristics and biological activities of \u003cem\u003eLycium barbarum\u003c/em\u003e polysaccharides (LBPs) and tree ages was studied in this study. The physicochemical characteristics of LBPs at second (Y2), fourth (Y4), sixth (Y6), eighth (Y8), tenth (Y10) and fifteenth (Y15) tree ages, such as total carbohydrate content (TCC), protein content (PC), uronic acid content (UAC), monosaccharide composition, molecular weight distribution, infrared characteristics were determined. And effects of LBPs of different tree ages on PC12 cells and APRE-19 cells function were evaluated in vitro. The results indicated that there was no significant difference in PC of LBPs at different tree ages. The UAC and TCC of LBPs from Y4 to Y10 were not significantly different, but were significantly higher than those in Y2 and Y15. LBPs of different tree ages showed similar preliminary structural characteristics. Compared with the LBPs of other tree ages, the LBPs of Y4, Y6, Y8 and Y10 had a good role in resisting oxidative stress. These results indicated that molecular weight, TCC and UAC of LBPs had important effects on their antioxidant activity in vitro. The accumulation of UAC and TCC in LBPs was closely related to tree age. The physicochemical characteristics and biological activities of LBPs fromY4 to Y10 were better than Y2 and Y15. Therefore, \u003cem\u003eLycium barbarum\u003c/em\u003e L. from Y4 to Y10 should be selected when preparing LBPs.\u003c/p\u003e","manuscriptTitle":"Comparison of physicochemical characteristics and biological activities of polysaccharides from Lycium barbarum L. at different tree ages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-07 16:09:12","doi":"10.21203/rs.3.rs-2334172/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9af132fe-f541-4fcc-a525-a75f85c324f3","owner":[],"postedDate":"December 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-08T05:59:31+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-07 16:09:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2334172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2334172","identity":"rs-2334172","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}