Optimization of Ultrasonic Pretreatment and Structure Analysis of Chlorogenic Acid in Potato Leaves

Optimization of Ultrasonic Pretreatment and Structure Analysis of Chlorogenic Acid in Potato Leaves | 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 Article Optimization of Ultrasonic Pretreatment and Structure Analysis of Chlorogenic Acid in Potato Leaves Xin Wang, Xianyun Gong, Binbin Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3934142/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 May, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Chlorogenic Acid was extracted from potato leaves by ultrasonic pretreatment technology were optimized through orthogonal design experiment. The extraction conditions had been controlled according to the results of Gauss calculation, which consisted of acidity, coordination and hydrolysis in molecules. The results showed that the optimization extraction temperature was 60 ℃, the extraction time was 60 minutes, the ratio of material to liquid was 1:20, the concentration of ethanol was 40%, and the yield of chlorogenic acid was 6.35%. The purified chlorogenic acid was analyzed by HPLC, TLC, UV-Vis and molecular fluorescence. Biological sciences/Chemical biology Health sciences/Molecular medicine Physical sciences/Chemistry Physical sciences/Materials science Ultrasonic extraction Potato leaf Chlorogenic acid Structure analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction With the advent of the post epidemic era, traditional Chinese medicine has taken the stage in the protection and treatment of COVID-19. Honeysuckle has played an extremely important role in the prevention and control of the epidemic. Studies have shown that the chlorogenic acid contained in honeysuckle has a good effect on inhibiting bacteria and viruses [1-5] . Chlorogenic acid is well known as an important member of the "seventh nutrient type" and is widely used in medical care [6-9] . Liu Jun showed that chlorogenic acid inhibited the activity of hepatitis B virus significantly. The anti-inflammatory effect of chlorogenic acid is closed due to the activity of hyaluronidase, and is stronger than that of aspirin [10] . Chlorogenic acids can also be used as antioxidants in the processing and storage of foods. Pang Xiufen [11] confirmed that chlorogenic acids can effectively maintain th e appearance and nutritional values of fruits during storage, therefore the physiological activities and the storage period of fruits can be extended. At present, the cost of chlorogenic acid is high due to the limited resources of raw materials. Meanwhile the more production of Chinese patent medicine, the more demand of honeysuckle is obviously insufficient. So it is urgent to find a series of substitute for honeysuckle. In addition to honeysuckle, other traditional Chinese medicines such as eucommia, and foods such as potato leaves and coffee beans also contain a certain amount of chlorogenic acid. The leaves of sweet potato (perennial trailing herb) contain a large amount of chlorogenic acid and a variety of effective ingredients as flavonoids, polysaccharides and proteins that can strengthen the body's immunity, delay aging, reduce blood sugar, facilitate urination and prevent night blindness [12-15] . In this paper, chlorogenic acid in potato leaf was extracted by a device of our own design. The extraction conditions had been designed according to the results of Gauss calculation. The optimization extraction conditions had been verified through orthogonal experiment by HPLC(High performance liquid chromatography). The structure and properties of chlorogenic acid were analyzed by spectrum and chromatography. It is hopeful that the results could promote the further development and application of chlorogenic acid. 1 Materials and methods 1.1 Materials and instruments 1.1.1 Materials Sweet potato leaves: laboratory planting(Technical Regulations for Planting Agricultural Products of The Ministry of Agriculture of China); ethanol: analytical pure, Tianjin Yongda Chemical Reagent Co., Ltd; Chlorogenic acid: Standard sample, Shanghai Aladdin Biochemical Technology Co., Ltd; Acetonitrile: chromatographically pure, Thermo Fisher Scientific (China) Co., Ltd; Methanol: chromatographically pure, Thermo Fisher Scientific (China) Co., Ltd. 1.1.2 Instruments and Equipments High performance liquid chromatograph: LC-10AT, Shimadzu, Japan; C18 chromatographic column: LC-C18 150×4.6mm, Shimadzu, Japan; UV visible spectrophotometer: UV-255, Shimadzu Instrument Co., Ltd. Luminescence spectra were measured on a Perkin-Elmer LS55 Luminescence spectrometer at room temperature. 1.2 Methods 1.2.1 Preparation of potato leaf extract: weigh 0.5000g ground potato leaf powder into a round bottom flask, add absolute ethanol and distilled water as mixed solution. The research has been focused on finding a set of reaction conditions, which could yield higher active products in the sealed environment of fluid N2. As shown in Figure 1, the samples were prepared in a sealed chamber (part 1). The heater and ultrasonic effected the reaction with N2 (part 3) flowing into the chamber. Conditions for the thermal treatment of mixed solution were investigated by changing reaction temperature, time, and ratio. Regarding to simplify and reduce the number of variables, four remarkable factors are proposed as follows: material liquid ratio (1:20, 1:30, 1:40, the solvent is 55% ethanol solution), extraction time (0.5 h, 1.0 h, 1.5 h), extraction temperature (50℃, 60℃, 70℃), ethanol volume fraction (40%, 50%, 60%) . 1.2.2 Standard solution preparation: chlorogenic acid 2.0mg and 50% methanol dissolve in 50mL volumetric flask, forming 40 μg/mL concentration of standard solution. Then transfer the standard solution(1mL, 3mL, 5mL, 7mL and 9mL) to 10mL for a series of concentration 4 μg/mL, 12 μg/mL, 20 μg/mL, 28 μg/mL and 36 μg/mL chlorogenic acid standard solution. 1.2.3 Purification: The crude extraction was purified by AB-8 macroporous adsorption resin as carrier. Added ten times of deionized water for washing and centrifuge in a vacuum rotary evaporator, and repeated washing and precipitation twice. Then the precipitate dissolved with ethanol, centrifuged and spined to dryness for a purified sample of chlorogenic acid. 1.2.4 The chromatographic conditions for HPLC analysis were: LC-C18 (250mm×4.6mm) ; mobile phase: 0.2% phosphoric acid acetonitrile (85:15, V/V) ; Detection wavelength: 324nm; Column temperature: 30℃; Injection volume: 20 μL；Current Speed: 1.000mL/min. 2 Results and Analysis 2.1 Selection and optimization of extraction parameters from orthogonal experiments Molecular structure analysis of chlorogenic acid had been controlled by Gauss calculation, which consisted of acidity, coordination and hydrolysis in molecules. The calculation method used as well as the basis sets are included in the Gauss 03 package. The orbitals are labeled by the principal quantum number and the orbital quantum number of the atom. A valence basis set consists of all valence atomic orbital, occupied or unoccupied, up to the shell of the principal quantum number of highest occupied valence electrons. Chlorogenic acid was formed by the ester bond between o-diphenol acrylic acid and polyhydroxy saturated hexacyclic carboxylic acid. It can be seen from the stick model diagram of chlorogenic acid molecule in Fig. 2 (a). There were some functional groups with unstable chemical properties like unsaturated bonds, carboxyl groups, phenolic hydroxyl groups, ester bonds in the chemical structure of chlorogenic acid. The calculation of frontier orbital theory showed the polyhydroxy saturated six member rings in the molecular structure, which presented a stable chair configuration, and distorted as certain angle between its plane and the phenol acrylate conjugated system (shown in Fig. 2 (b) and (c)). The molecule was belonged to C1 symmetry. The contribution of molecular frontier orbitals came from the phenol acrylate conjugated system, and the chemical activity was mainly reflected in the hydroxyl phenol ring and acrylate group. The calculated results showed its acidity and coordination properties. The hydrogen in phenol hydroxyl was easy to lose and showed acidity, while carboxyl oxygen accepted metal cations and showed a good coordination ability. The cleavage of ester bond was helpful to promote the separation of saturated six membered ring due to its hydrolytic property. According to the theoretical calculation analysis, chlorogenic acid is easy to decompose, migrate and rearrange at high temperature. Therefore, extraction temperature and other conditions should be strictly controlled during extraction proceeding. According to Gauss calculation results, a group of L 9 (3 4 ) orthogonal experiments were designed for the extraction experiment in Table 1. Extraction temperature: 50, 60, 70℃; extraction time: 30, 60, 90min; material liquid ratio: 1:20, 1:30, 1:40; ethanol concentration: 40%, 50%, 60%. The purified production is quantitatively analyzed by HPLC (high performance liquid chromatography). According to the variance calculation results of orthogonal experiment, the optimal extraction conditions of chlorogenic acid were: extraction temperature 60℃, extraction time 60 min, material liquid ratio 1: 20, ethanol concentration 40%, then the yield of chlorogenic acid reached to 6.35%. Table 1 Orthogonal experiment of ethanol ultrasonic extraction and chlorogenic acid content Extraction temperature/℃ Extraction time/min Material liquid ratio/g∙mL -1 Ethanol concentration/% Chlorogenic acid content/mg∙g -1 1 2 3 4 5 6 7 8 9 50 60 70 50 60 70 50 60 70 30 30 30 60 60 60 90 90 90 1: 20 1: 30 1: 40 1: 40 1: 20 1: 30 1: 30 1: 40 1: 20 40% 50% 60% 60% 40% 50% 50% 60% 40% 0.1500 0.1000 0.1750 0.1750 3.1750 0.0500 0.0750 0.4250 0.6500 2.3 Spectroscopy analysis of chlorogenic acid 2.3.1 Infrared spectroscopy analysis Fig. 3 (a) showed the infrared spectrum of chlorogenic acid fitted by Gauss 03, and Fig. 3 (b) showed the experiment diagram of extraction. 1 is C-H stretching vibration on the benzene ring; Peak 2 is the stretching vibration of carbonyl group; The absorption peak 3 is the stretching vibration of C=C; The absorption peaks 4 and 5 are the skeleton vibration of benzene ring; The absorption peak 6 is the characteristic absorption of CH 2 shear bending; The absorption intensity of peak 7 is large, which should be the characteristic absorption peak of C-O bond (ester, alcohol). The test results are basically consistent with the fitting results, indicating that the extract is the target product. 2.3.2 Ultraviolet visible and molecular fluorescence spectrophotometry Take the diluted potato leaf extract and chlorogenic acid standard solution, take 50% methanol as the blank solution, and scan at the wavelength of 200-500nm. Fig. 4 (a) is the UV spectrogram, where the max of the extract is 324nm, which is consistent with the test results of chlorogenic acid standard. Fig. 4 (b) shows the fluorescence spectrum. It can be seen from the figure that the excitation wavelength and emission wavelength of the extract of sweet potato leaves are the same as those of the chlorogenic acid standard, both of which are 279nm and 445nm. The results of UV and fluorescence tests prove that the extract contains chlorogenic acid. 2.4 Determination of chlorogenic acid by chromatography 2.4.1 Thin layer chromatography Take a small amount of extract solution and standard solution to sample on the silica gel GF254 thin layer plate, place the silica gel thin layer plate in the liquid tank of developing agent ethyl acetate acetone formic acid water (7:3:2:1, V/V), ethyl acetate acetone formic acid (7:3:2, V/V), and ethyl acetate formic acid water (10:3:1, V/V), dry and observe at 254nm of the ultraviolet analyzer. Compared with Fig. 5, the spots of chlorogenic acid sample in (a) are clear, but not on the same horizontal line with those of the extract of ground melon leaves; (b) The sample spot and the extract spot clearly appeared on the same horizontal line, and there was no obvious tailing phenomenon; (c) Although the two spots in are on the same horizontal line, they are slightly blurred and appear trailing. The qualitative analysis of thin-layer chromatography showed that in order to make the experimental results clear and free of abnormal phenomena, the ultraviolet light source should be 254nm, and the developing agent was ethyl acetate acetone formic acid (7:3:2, V/V). 2.4.2 High performance liquid chromatography It can be seen from Figure 6 that the peak time of chlorogenic acid standard is the same as that of the 250 times diluted potato leaf extract, and it can be concluded that the chemical composition of the extract contains chlorogenic acid. The the linear regression equation of chlorogenic acid is y=3.24x×10 -8 , correlation coefficient R=0.9927, the results show that chlorogenic acid is between 4-36 μg/mL. The concentration range of g/mL shows a good linear relationship, which can be used for quantitative analysis. 3 Conclusion In this paper, the molecular properties of chlorogenic acid were calculated and predicted by using the frontier orbital theory of quantum chemistry, and its purified products were qualitatively analyzed by HPLC, thin-layer chromatography, UV visible spectrophotometry, and molecular fluorescence spectrophotometry. The calculation results showed that the chemical activity of the molecules was mainly reflected in the hydroxyl phenol ring and acrylate base sites, showing a certain degree of acidity, coordination, and hydrolysis, The extraction temperature should be strictly controlled when extracting from sweet potato leaves. The orthogonal experiment was designed to optimize the ultrasonic pretreatment process conditions of sweet potato leaves. The results showed that the optimal process conditions of ethanol ultrasonic method were: extraction temperature 60℃, extraction time 60 min, material liquid ratio 1:20, ethanol concentration 40%, under which the yield of chlorogenic acid was 6.35%. In view of the complex composition of sweet potato leaves, it is difficult to further improve the content of chlorogenic acid. Therefore, it is of guiding significance to effectively combine the theoretical prediction results and improve the content of chlorogenic acid by optimizing the experimental process. Declarations Author Contribution Xin wrote the main manuscript text and Xianyun and Binbin had taken the test. All authors reviewed the manuscript. References Gao X., He Z., Luo P., et al. Overview of research on medium and high frequency single drug of traditional Chinese medicine compound against novel coronavirus pneumonia (COVID-19) [J]. Foreign medicine (Antibiotics). 2020,4:283-289. Liu W., Gong P., Gu J. Discussion on the treatment of novel coronavirus pneumonia (COVID-19) with antipyretic, anti-inflammatory and immunomodulatory Chinese medicine [J]. Traditional Chinese medicine. 2020,8:2081-2088. Fan Q., Pan X., He Y. Research progress on the immunomodulatory effects of traditional Chinese medicine and its compound formulas on viral pneumonia [J] Chinese herbal medicine. 2020,51(8). Zhu W., Ren H., Zheng Y., et al. Research progress on functional components and biological activities of honeysuckle [J]. Food Industry Technology, 2020,11:26. Li H., Zhou Z., Chen Y. Study on the separation process of chlorogenic acid and total flavonoids from Lonicera japonica using macroporous resin [J]. Chemical World. 2020,11:760-766. National Pharmacopoeia Commission. Chinese Pharmacopoeia (Part 1) [M]. Beijing: China Medical Science and Technology Press, 2020:221. Gao X.,Yue C.,Tian R. Akkermansia muciniphila-directed polyphenol chlorogenic acid intervention for obesity in mice[J]. Food Science and Human Wellness. 2024,13(1): 90-100. HM Rawel,J Kroll,B Riese. Reactions of Chlorogenic Acid with Lysozyme: Physicochemical Characterization and Proteolytic Digestion of the Derivatives.Journal of Food Science[J]. 2001,72(1): 59-71. Miao Q. Study on the preparation process of high-purity chlorogenic acid in the crude extract of Eucommia ulmoides leaves [D]. Yantai: Yantai Institute of Coastal Zone, Chinese Academy of Sciences,2017. Zhang B.,Nan T.G.,Zhan Z.L.,et al. A monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of chlorogenic acid in honeysuckle[J]. Journal of Pharmaceutical and Biomedical Analysis,2018,148:1-5. Liu J., Huang Z., Wang X., Tian D., Cao W. Inhibitory effect of chlorogenic acid on hepatitis B virus HBsAg and HBeAg [J] Pharm J Chin PLA, 2010,1: 33-36. Pang X., Duan R., Zhao Q. Optimization of ultrasound assisted Soxhlet extraction of chlorogenic acid from Lonicera japonica fruit [J]. Biological resources,2017,39(2):125-129. Liang Y., Li J., Ma W. Optimization of ultrasonic assisted ethanol extraction of chlorogenic acid from peach blossoms [J]. Applied chemical engineering,2018,47(02):319-321. Liu Z. Ma W.Chen B. Recovery of chlorogenic acid from the DES-based extract of Eucommia ulmoides leaves by molecularly imprinted solid-phase extraction. Industrial Crops and Products. 2023,195:116406-116417. Bin T.,Yanmei H.,Xiangling M. Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to human serum albumin:Effects of esteryl position on affinity[J].Food Chemistry,2016,212:434-442. Corrigan H. Dunne A.Purcell N. Conceptual functional-by-design optimisation of the antioxidant capacity of trans-resveratrol, quercetin, and chlorogenic acid: Application in a functional tea. Food Chemistry. 2023,428:136764-136776. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 May, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Feb, 2024 Reviews received at journal 26 Feb, 2024 Reviews received at journal 26 Feb, 2024 Reviewers agreed at journal 18 Feb, 2024 Reviewers agreed at journal 16 Feb, 2024 Reviewers invited by journal 16 Feb, 2024 Editor assigned by journal 16 Feb, 2024 Editor invited by journal 16 Feb, 2024 Submission checks completed at journal 16 Feb, 2024 First submitted to journal 06 Feb, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3934142","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":273491726,"identity":"02e2f95b-e335-4c75-8679-aad5f6628ee1","order_by":0,"name":"Xin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYNACA4Z6+/bmgw8SKmqI15JgwHMs2eDBmWPE25NgIOFjJvmwhZkI84+fPfyap+BOnrkEj1lFYgMbA397dwJ+LWfy0ixnGDwrtpzdVnYjcYcMg8SZsxvwajE7kGNm8MHgMGPDncPbbiSeYWMwkMgloOX8GzODBJCWGwlmBYltzERouZFj/ABoS+KGGylmDERpsb/xxoxxhsFhY8meY8kSCWeO8RD0i2R/jvFnnj+H5fjZmw9+/FFRI8ff3otfCxCwSSDzeAgpBwHmD8SoGgWjYBSMghEMAMaiUHEivvLOAAAAAElFTkSuQmCC","orcid":"","institution":"Harbin University","correspondingAuthor":true,"prefix":"","firstName":"Xin","middleName":"","lastName":"Wang","suffix":""},{"id":273491727,"identity":"5e9ed5ad-b25a-4319-b92a-05aaa8f41b69","order_by":1,"name":"Xianyun Gong","email":"","orcid":"","institution":"Harbin University","correspondingAuthor":false,"prefix":"","firstName":"Xianyun","middleName":"","lastName":"Gong","suffix":""},{"id":273491728,"identity":"9d9514d0-4f29-4991-b8de-70cc7a41b38f","order_by":2,"name":"Binbin Lin","email":"","orcid":"","institution":"Harbin University","correspondingAuthor":false,"prefix":"","firstName":"Binbin","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2024-02-06 14:16:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3934142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3934142/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-61139-7","type":"published","date":"2024-05-09T21:18:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51311888,"identity":"52a7b7d7-39f7-4036-9862-15e2ebc7159d","added_by":"auto","created_at":"2024-02-19 11:22:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35446,"visible":true,"origin":"","legend":"\u003cp\u003eSelf-designed device for the extraction of chlorogenic acid in potato leaf powders\u003c/p\u003e\n\u003cp\u003ePart 1: Sealed chamber; Part 2: A device set heat and ultrasonic; Part 3: The compressed gas cylinder of N\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3934142/v1/b1d8829ecb138ed936018ed6.png"},{"id":51311894,"identity":"c87100d7-48aa-456d-9232-e2c777a9a712","added_by":"auto","created_at":"2024-02-19 11:22:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189569,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular theoretical calculation diagram of chlorogenic acid\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3934142/v1/bff98d4e002b21771b1fd952.png"},{"id":51311890,"identity":"466a8e30-c048-4d9b-ba65-ae812b909212","added_by":"auto","created_at":"2024-02-19 11:22:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86982,"visible":true,"origin":"","legend":"\u003cp\u003eIR spectrum of chlorogenic acid:(a) Fitting of Gauss 98; (b) Extraction\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3934142/v1/00dbbba83b1298147099b225.png"},{"id":51312025,"identity":"2072ad2e-b4fa-4177-9888-8fdefd66a686","added_by":"auto","created_at":"2024-02-19 11:30:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102117,"visible":true,"origin":"","legend":"\u003cp\u003eUV spectrum (a) and fluorescence spectrum (b) of chlorogenic acid: curve 1 is chlorogenic acid standard; curve 2 is extraction\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3934142/v1/dec0b5ceb8b71958f621164b.png"},{"id":51311892,"identity":"bf4c67e7-b613-4473-9b45-8cea381db570","added_by":"auto","created_at":"2024-02-19 11:22:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298478,"visible":true,"origin":"","legend":"\u003cp\u003eTLC (A: chlorogenic acid standard; B: potato leaf extract) 1: ethyl acetate acetone formic acid water (7:3:2:1, V/V); 2: ethyl acetate acetone formic acid (7:3:2, V/V); 3: ethyl acetate formic acid water (10:3:1, V/V)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3934142/v1/2c6cc7aab567d6384888c75b.png"},{"id":51311891,"identity":"750f6c58-c53e-4a5e-85d6-f66026c4277b","added_by":"auto","created_at":"2024-02-19 11:22:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13734,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC of chlorogenic acid 1: chlorogenic acid standard; 2: potato leaf extraction\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3934142/v1/ffb309e2c426e2a51051a244.png"},{"id":56488370,"identity":"b38f62a4-4ba2-4d16-81a9-8a03485e1191","added_by":"auto","created_at":"2024-05-14 21:32:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":873626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3934142/v1/262449f0-19e7-462a-bd9a-c2fe9fadcf0e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of Ultrasonic Pretreatment and Structure Analysis of Chlorogenic Acid in Potato Leaves","fulltext":[{"header":"Introduction","content":"\u003cp style=\"text-align: left;\"\u003eWith the advent of the post epidemic era, traditional Chinese medicine has taken the stage in the protection and treatment of COVID-19. Honeysuckle has played an extremely important role in the prevention and control of the epidemic. Studies have shown that the chlorogenic acid contained in honeysuckle has a good effect on inhibiting bacteria and viruses\u003cstrong\u003e[1-5]\u003c/strong\u003e. Chlorogenic acid is well known as an important member of the \u0026quot;seventh nutrient type\u0026quot; and is widely used in medical care\u003cstrong\u003e[6-9]\u003c/strong\u003e. Liu Jun showed that chlorogenic acid inhibited the activity of hepatitis B virus significantly. The anti-inflammatory effect of chlorogenic acid is closed due to the activity of hyaluronidase, and is stronger than that of aspirin \u003cstrong\u003e[10]\u003c/strong\u003e. Chlorogenic acids can also be used as antioxidants in the processing and storage of foods. Pang Xiufen\u003cstrong\u003e [11] \u003c/strong\u003econfirmed that chlorogenic acids can effectively maintain th e appearance and nutritional values of fruits during storage, therefore the physiological activities and the storage period of fruits can be extended. At present, the cost of chlorogenic acid is high due to the limited resources of raw materials. Meanwhile the more production of Chinese patent medicine, the more demand of honeysuckle is obviously insufficient. So it is urgent to find a series of substitute for honeysuckle.\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003eIn addition to honeysuckle, other traditional Chinese medicines such as eucommia, and foods such as potato leaves and coffee beans also contain a certain amount of chlorogenic acid. The leaves of sweet potato (perennial trailing herb) contain a large amount of chlorogenic acid and a variety of effective ingredients as flavonoids, polysaccharides and proteins that can strengthen the body\u0026apos;s immunity, delay aging, reduce blood sugar, facilitate urination and prevent night blindness\u003cstrong\u003e [12-15]\u003c/strong\u003e. \u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003eIn this paper, chlorogenic acid in potato leaf was extracted by a device of our own design. The extraction conditions had been designed according to the results of Gauss calculation. The optimization extraction conditions had been verified through orthogonal experiment by HPLC(High performance liquid chromatography). The structure and properties of chlorogenic acid were analyzed by spectrum and chromatography. It is hopeful that the results could promote the further development and application of chlorogenic acid. \u003c/p\u003e"},{"header":"1 Materials and methods","content":"\u003cp\u003e1.1\u0026nbsp;Materials and instruments\u003c/p\u003e\n\u003cp\u003e1.1.1\u0026nbsp;Materials\u003c/p\u003e\n\u003cp\u003eSweet potato leaves: laboratory planting(Technical Regulations for Planting Agricultural Products of The Ministry of Agriculture of China); ethanol: analytical pure, Tianjin Yongda Chemical Reagent Co., Ltd; Chlorogenic acid: Standard sample, Shanghai Aladdin Biochemical Technology Co., Ltd; Acetonitrile: chromatographically pure, Thermo Fisher Scientific (China) Co., Ltd; Methanol: chromatographically pure, Thermo Fisher Scientific (China) Co., Ltd.\u003c/p\u003e\n\u003cp\u003e1.1.2\u0026nbsp;Instruments and Equipments\u003c/p\u003e\n\u003cp\u003eHigh performance liquid chromatograph: LC-10AT, Shimadzu, Japan; C18 chromatographic column: LC-C18 150\u0026times;4.6mm, Shimadzu, Japan; UV visible spectrophotometer: UV-255, Shimadzu Instrument Co., Ltd. Luminescence spectra were measured on a Perkin-Elmer LS55 Luminescence spectrometer at room temperature.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1.2\u0026nbsp;Methods\u003c/p\u003e\n\u003cp\u003e1.2.1 Preparation of potato leaf extract: weigh 0.5000g ground potato leaf powder into a round bottom flask, add absolute ethanol and distilled water as mixed solution.\u0026nbsp;The research has been focused on finding a set of reaction conditions, which could yield higher active products in the sealed environment of fluid N2. As shown in Figure 1, the samples were prepared in a sealed chamber (part 1). The heater and ultrasonic effected the reaction with N2 (part 3) flowing into the chamber. Conditions for the thermal treatment of\u0026nbsp;mixed solution\u0026nbsp;were investigated by changing reaction temperature, time, and ratio. Regarding to simplify and reduce the number of variables, four remarkable factors are proposed as follows: material liquid ratio (1:20, 1:30, 1:40, the solvent is 55% ethanol solution), extraction time (0.5 h, 1.0 h, 1.5 h), extraction temperature (50℃, 60℃, 70℃), ethanol volume fraction (40%, 50%, 60%) .\u003c/p\u003e\n\u003cp\u003e1.2.2 Standard solution preparation: chlorogenic acid 2.0mg and 50% methanol dissolve in 50mL volumetric flask, forming 40 \u0026mu;g/mL concentration of standard solution. Then transfer the standard solution(1mL, 3mL, 5mL, 7mL and 9mL) to 10mL for a series of concentration 4 \u0026mu;g/mL, 12 \u0026mu;g/mL, 20 \u0026mu;g/mL, 28 \u0026mu;g/mL and 36 \u0026mu;g/mL chlorogenic acid standard solution.\u003c/p\u003e\n\u003cp\u003e1.2.3 Purification: The crude extraction was purified by AB-8 macroporous adsorption resin as carrier. Added ten times of deionized water for washing and centrifuge in a vacuum rotary evaporator, and repeated washing and precipitation twice. Then the precipitate dissolved with ethanol, centrifuged and spined to dryness for a purified sample of chlorogenic acid.\u003c/p\u003e\n\u003cp\u003e1.2.4\u0026nbsp;The chromatographic conditions for HPLC analysis were: LC-C18 (250mm\u0026times;4.6mm) ; mobile phase: 0.2% phosphoric acid acetonitrile (85:15, V/V) ; Detection wavelength: 324nm; Column temperature: 30℃; Injection volume: 20 \u0026mu;L；Current Speed: 1.000mL/min.\u003c/p\u003e"},{"header":"2 Results and Analysis","content":"\u003cp\u003e2.1 Selection and optimization of extraction parameters from orthogonal experiments\u003c/p\u003e\n\u003cp\u003eMolecular structure analysis of chlorogenic acid had been controlled by Gauss calculation, which consisted of acidity, coordination and hydrolysis in molecules. The calculation method used as well as the basis sets are included in the Gauss 03 package. The orbitals are labeled by the principal quantum number and the orbital quantum number of the atom. A valence basis set consists of all valence atomic orbital, occupied or unoccupied, up to the shell of the principal quantum number of highest occupied valence electrons.\u003c/p\u003e\n\u003cp\u003eChlorogenic acid was formed by the ester bond between o-diphenol acrylic acid and polyhydroxy saturated hexacyclic carboxylic acid. It can be seen from the stick model diagram of chlorogenic acid molecule in Fig. 2 (a). There were some functional groups with unstable chemical properties like unsaturated bonds, carboxyl groups, phenolic hydroxyl groups, ester bonds in the chemical structure of chlorogenic acid. The calculation of frontier orbital theory showed the polyhydroxy saturated six member rings in the molecular structure, which presented a stable chair configuration, and distorted as certain angle between its plane and the phenol acrylate conjugated system (shown in Fig. 2 (b) and (c)). The molecule was belonged to C1 symmetry. The contribution of molecular frontier orbitals came from the phenol acrylate conjugated system, and the chemical activity was mainly reflected in the hydroxyl phenol ring and acrylate group. The calculated results showed its acidity and coordination properties. The hydrogen in phenol hydroxyl was easy to lose and showed acidity, while carboxyl oxygen accepted metal cations and showed a good coordination ability. The cleavage of ester bond was helpful to promote the separation of saturated six membered ring due to its hydrolytic property. According to the theoretical calculation analysis, chlorogenic acid is easy to decompose, migrate and rearrange at high temperature. Therefore, extraction temperature and other conditions should be strictly controlled during extraction proceeding.\u003c/p\u003e\n\u003cp\u003eAccording to Gauss calculation results, a group of L\u003csup\u003e9\u003c/sup\u003e(3\u003csup\u003e4\u003c/sup\u003e) orthogonal experiments were designed for the extraction experiment\u0026nbsp;in Table 1. Extraction temperature: 50, 60, 70℃; extraction time: 30, 60, 90min; material liquid ratio: 1:20, 1:30, 1:40; ethanol concentration: 40%, 50%, 60%. The purified production is quantitatively analyzed by HPLC (high performance liquid chromatography). According to the variance calculation results of orthogonal experiment, the optimal extraction conditions of chlorogenic acid were: extraction temperature 60℃, extraction time 60 min, material liquid ratio 1: 20, ethanol concentration 40%, then the yield of chlorogenic acid reached to 6.35%.\u003c/p\u003e\n\u003cp\u003eTable 1\u0026nbsp;Orthogonal experiment of ethanol ultrasonic extraction and chlorogenic acid content\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.1231884057971%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.028985507246375%\" valign=\"top\"\u003e\n \u003cp\u003eExtraction temperature/℃\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1231884057971%\" valign=\"top\"\u003e\n \u003cp\u003eExtraction time/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1231884057971%\" valign=\"top\"\u003e\n \u003cp\u003eMaterial liquid ratio/g∙mL\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.028985507246375%\" valign=\"top\"\u003e\n \u003cp\u003eEthanol concentration/%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.57246376811594%\" valign=\"top\"\u003e\n \u003cp\u003eChlorogenic acid content/mg∙g\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.1231884057971%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.028985507246375%\" valign=\"top\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1231884057971%\" valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.1231884057971%\" valign=\"top\"\u003e\n \u003cp\u003e1: 20\u003c/p\u003e\n \u003cp\u003e1: 30\u003c/p\u003e\n \u003cp\u003e1: 40\u003c/p\u003e\n \u003cp\u003e1: 40\u003c/p\u003e\n \u003cp\u003e1: 20\u003c/p\u003e\n \u003cp\u003e1: 30\u003c/p\u003e\n \u003cp\u003e1: 30\u003c/p\u003e\n \u003cp\u003e1: 40\u003c/p\u003e\n \u003cp\u003e1: 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.028985507246375%\" valign=\"top\"\u003e\n \u003cp\u003e40%\u003c/p\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003cp\u003e40%\u003c/p\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003cp\u003e40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.57246376811594%\" valign=\"top\"\u003e\n \u003cp\u003e0.1500\u003c/p\u003e\n \u003cp\u003e0.1000\u003c/p\u003e\n \u003cp\u003e0.1750\u003c/p\u003e\n \u003cp\u003e0.1750\u003c/p\u003e\n \u003cp\u003e3.1750\u003c/p\u003e\n \u003cp\u003e0.0500\u003c/p\u003e\n \u003cp\u003e0.0750\u003c/p\u003e\n \u003cp\u003e0.4250\u003c/p\u003e\n \u003cp\u003e0.6500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e2.3 Spectroscopy analysis of chlorogenic acid\u003c/p\u003e\n\u003cp\u003e2.3.1 Infrared spectroscopy analysis\u003c/p\u003e\n\u003cp\u003eFig. 3 (a) showed the infrared spectrum of chlorogenic acid fitted by Gauss 03, and Fig. 3 (b) showed the experiment diagram of extraction. 1 is C-H stretching vibration on the benzene ring; Peak 2 is the stretching vibration of carbonyl group; The absorption peak 3 is the stretching vibration of C=C; The absorption peaks 4 and 5 are the skeleton vibration of benzene ring; The absorption peak 6 is the characteristic absorption of CH\u003csub\u003e2\u003c/sub\u003e shear bending; The absorption intensity of peak 7 is large, which should be the characteristic absorption peak of C-O bond (ester, alcohol). The test results are basically consistent with the fitting results, indicating that the extract is the target product.\u003c/p\u003e\n\u003cp\u003e2.3.2 Ultraviolet visible and molecular fluorescence spectrophotometry\u003c/p\u003e\n\u003cp\u003eTake the diluted potato leaf extract and chlorogenic acid standard solution, take 50% methanol as the blank solution, and scan at the wavelength of 200-500nm. Fig. 4 (a) is the UV spectrogram, where the max of the extract is 324nm, which is consistent with the test results of chlorogenic acid standard. Fig. 4 (b) shows the fluorescence spectrum. It can be seen from the figure that the excitation wavelength and emission wavelength of the extract of sweet potato leaves are the same as those of the chlorogenic acid standard, both of which are 279nm and 445nm. The results of UV and fluorescence tests prove that the extract contains chlorogenic acid.\u003c/p\u003e\n\u003cp\u003e2.4 Determination of chlorogenic acid by chromatography\u003c/p\u003e\n\u003cp\u003e2.4.1 Thin layer chromatography\u003c/p\u003e\n\u003cp\u003eTake a small amount of extract solution and standard solution to sample on the silica gel GF254 thin layer plate, place the silica gel thin layer plate in the liquid tank of developing agent ethyl acetate acetone formic acid water (7:3:2:1, V/V), ethyl acetate acetone formic acid (7:3:2, V/V), and ethyl acetate formic acid water (10:3:1, V/V), dry and observe at 254nm of the ultraviolet analyzer. Compared with Fig. 5, the spots of chlorogenic acid sample in (a) are clear, but not on the same horizontal line with those of the extract of ground melon leaves; (b) The sample spot and the extract spot clearly appeared on the same horizontal line, and there was no obvious tailing phenomenon; (c) Although the two spots in are on the same horizontal line, they are slightly blurred and appear trailing. The qualitative analysis of thin-layer chromatography showed that in order to make the experimental results clear and free of abnormal phenomena, the ultraviolet light source should be 254nm, and the developing agent was ethyl acetate acetone formic acid (7:3:2, V/V).\u003c/p\u003e\n\u003cp\u003e2.4.2 High performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eIt can be seen from Figure 6 that the peak time of chlorogenic acid standard is the same as that of the 250 times diluted potato leaf extract, and it can be concluded that the chemical composition of the extract contains chlorogenic acid. The the linear regression equation of chlorogenic acid is y=3.24x\u0026times;10\u003csup\u003e-8\u003c/sup\u003e, correlation coefficient R=0.9927, the results show that chlorogenic acid is between 4-36 \u0026mu;g/mL. The concentration range of g/mL shows a good linear relationship, which can be used for quantitative analysis.\u003c/p\u003e"},{"header":"3 Conclusion","content":"\u003cp\u003eIn this paper, the molecular properties of chlorogenic acid were calculated and predicted by using the frontier orbital theory of quantum chemistry, and its purified products were qualitatively analyzed by HPLC, thin-layer chromatography, UV visible spectrophotometry, and molecular fluorescence spectrophotometry. The calculation results showed that the chemical activity of the molecules was mainly reflected in the hydroxyl phenol ring and acrylate base sites, showing a certain degree of acidity, coordination, and hydrolysis, The extraction temperature should be strictly controlled when extracting from sweet potato leaves. The orthogonal experiment was designed to optimize the ultrasonic pretreatment process conditions of sweet potato leaves. The results showed that the optimal process conditions of ethanol ultrasonic method were: extraction temperature 60℃, extraction time 60 min, material liquid ratio 1:20, ethanol concentration 40%, under which the yield of chlorogenic acid was 6.35%. In view of the complex composition of sweet potato leaves, it is difficult to further improve the content of chlorogenic acid. Therefore, it is of guiding significance to effectively combine the theoretical prediction results and improve the content of chlorogenic acid by optimizing the experimental process.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXin wrote the main manuscript text and Xianyun and Binbin had taken the test. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGao X., He Z., Luo P., et al. Overview of research on medium and high frequency single drug of traditional Chinese medicine compound against novel coronavirus pneumonia (COVID-19) [J]. Foreign medicine (Antibiotics). 2020,4:283-289.\u003c/li\u003e\n\u003cli\u003eLiu W., Gong P., Gu J. Discussion on the treatment of novel coronavirus pneumonia (COVID-19) with antipyretic, anti-inflammatory and immunomodulatory Chinese medicine [J]. Traditional Chinese medicine. 2020,8:2081-2088.\u003c/li\u003e\n\u003cli\u003eFan Q., Pan X., He Y. Research progress on the immunomodulatory effects of traditional Chinese medicine and its compound formulas on viral pneumonia [J] Chinese herbal medicine. 2020,51(8).\u003c/li\u003e\n\u003cli\u003eZhu W., Ren H., Zheng Y., et al. Research progress on functional components and biological activities of honeysuckle [J]. Food Industry Technology, 2020,11:26.\u003c/li\u003e\n\u003cli\u003eLi H., Zhou Z., Chen Y. Study on the separation process of chlorogenic acid and total flavonoids from Lonicera japonica using macroporous resin [J]. Chemical World. 2020,11:760-766.\u003c/li\u003e\n\u003cli\u003eNational Pharmacopoeia Commission. Chinese Pharmacopoeia (Part 1) [M]. Beijing: China Medical Science and Technology Press, 2020:221.\u003c/li\u003e\n\u003cli\u003eGao X.,Yue C.,Tian R. Akkermansia muciniphila-directed polyphenol chlorogenic acid intervention for obesity in mice[J]. Food Science and Human Wellness. 2024,13(1): 90-100.\u003c/li\u003e\n\u003cli\u003eHM Rawel,J Kroll,B Riese. Reactions of Chlorogenic Acid with Lysozyme: Physicochemical Characterization and Proteolytic Digestion of the Derivatives.Journal of Food Science[J]. 2001,72(1): 59-71.\u003c/li\u003e\n\u003cli\u003eMiao Q. Study on the preparation process of high-purity chlorogenic acid in the crude extract of Eucommia ulmoides leaves [D]. Yantai: Yantai Institute of Coastal Zone, Chinese Academy of Sciences,2017.\u003c/li\u003e\n\u003cli\u003eZhang B.,Nan T.G.,Zhan Z.L.,et al. A monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of chlorogenic acid in honeysuckle[J]. Journal of Pharmaceutical and Biomedical Analysis,2018,148:1-5.\u003c/li\u003e\n\u003cli\u003eLiu J., Huang Z., Wang X., Tian D., Cao W. Inhibitory effect of chlorogenic acid on hepatitis B virus HBsAg and HBeAg [J] Pharm J Chin PLA, 2010,1: 33-36. \u003c/li\u003e\n\u003cli\u003ePang X., Duan R., Zhao Q. Optimization of ultrasound assisted Soxhlet extraction of chlorogenic acid from Lonicera japonica fruit [J]. Biological resources,2017,39(2):125-129.\u003c/li\u003e\n\u003cli\u003eLiang Y., Li J., Ma W. Optimization of ultrasonic assisted ethanol extraction of chlorogenic acid from peach blossoms [J]. Applied chemical engineering,2018,47(02):319-321.\u003c/li\u003e\n\u003cli\u003eLiu Z.\u0026ensp;Ma W.Chen B. Recovery of chlorogenic acid from the DES-based extract of Eucommia ulmoides leaves by molecularly imprinted solid-phase extraction. Industrial Crops and Products. 2023,195:116406-116417.\u003c/li\u003e\n\u003cli\u003eBin T.,Yanmei H.,Xiangling M. Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to human serum albumin:Effects of esteryl position on affinity[J].Food Chemistry,2016,212:434-442.\u003c/li\u003e\n\u003cli\u003eCorrigan H.\u0026ensp;Dunne A.Purcell N. Conceptual functional-by-design optimisation of the antioxidant capacity of trans-resveratrol, quercetin, and chlorogenic acid: Application in a functional tea. Food Chemistry. 2023,428:136764-136776.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ultrasonic extraction, Potato leaf, Chlorogenic acid, Structure analysis","lastPublishedDoi":"10.21203/rs.3.rs-3934142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3934142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChlorogenic Acid was extracted from potato leaves by ultrasonic pretreatment technology were optimized through orthogonal design experiment. The extraction conditions had been controlled according to the results of Gauss calculation, which consisted of acidity, coordination and hydrolysis in molecules. The results showed that the optimization extraction temperature was 60 ℃, the extraction time was 60 minutes, the ratio of material to liquid was 1:20, the concentration of ethanol was 40%, and the yield of chlorogenic acid was 6.35%. The purified chlorogenic acid was analyzed by HPLC, TLC, UV-Vis and molecular fluorescence.\u003c/p\u003e","manuscriptTitle":"Optimization of Ultrasonic Pretreatment and Structure Analysis of Chlorogenic Acid in Potato Leaves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-19 11:22:38","doi":"10.21203/rs.3.rs-3934142/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-27T04:06:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-26T17:23:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-26T13:59:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7ffa0269-9abe-4acc-ab74-1b96ba8f1cd0","date":"2024-02-18T09:59:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a775dc43-e78e-4ca4-aa5b-8892bf3eee19","date":"2024-02-17T00:40:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-16T17:58:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-16T17:55:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-16T11:17:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-16T11:10:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-06T14:14:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6ec0be4-90d4-4d2e-bd07-cc0593f870cd","owner":[],"postedDate":"February 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28822748,"name":"Biological sciences/Chemical biology"},{"id":28822749,"name":"Health sciences/Molecular medicine"},{"id":28822750,"name":"Physical sciences/Chemistry"},{"id":28822751,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2024-05-14T21:27:34+00:00","versionOfRecord":{"articleIdentity":"rs-3934142","link":"https://doi.org/10.1038/s41598-024-61139-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-05-09 21:18:12","publishedOnDateReadable":"May 9th, 2024"},"versionCreatedAt":"2024-02-19 11:22:38","video":"","vorDoi":"10.1038/s41598-024-61139-7","vorDoiUrl":"https://doi.org/10.1038/s41598-024-61139-7","workflowStages":[]},"version":"v1","identity":"rs-3934142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3934142","identity":"rs-3934142","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}