Study on the ultrasonic-assisted deep eutectic solvent extraction of total flavonoids from flos sophorae immaturus

Study on the ultrasonic-assisted deep eutectic solvent extraction of total flavonoids from flos sophorae immaturus | 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 Study on the ultrasonic-assisted deep eutectic solvent extraction of total flavonoids from flos sophorae immaturus Lifen Meng, Ye Tan, Mengting Su, Yinying Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6275463/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 In this paper, four different kinds of deep eutectic solvents were prepared to carry out ultrasonic-assisted extraction of total flavonoids from flos sophorae immaturus . The content of total flavonoids was determined by ultraviolet spectrophotometry. The effects of the type, molar ratio, moisture of deep eutectic solvent, the solid-liquid ratio, the ultrasonic time and the ultrasonic temperature on the extraction of total flavonoids from flos sophorae immaturus were investigated by condition experiment. Meanwhile, the standard addition recovery rate experiment was performed to verify the feasibility of the method. The results showed that the optimal extraction results could be obtained when the moisture was 10%, the molar ratio of choline chloride/1,2-propylene glycol deep eutectic solvent 1:2, the solid-liquid ratio 1:35, the ultrasonic temperature 40℃, and the ultrasonic time 2.5h. Under the optimal conditions, the content of total flavonoids extraction was up to 4.97mg/g, and the standard addition recovery rate reached about 89%. deep eutectic solvent ultrasonic flos sophorae immaturus total flavonoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Flavonoids in natural products had been a research focus in recent years. Flos sophorae immaturus was the dried flower bud of Sophora japonica L. , which had the function of cooling blood and stopping bleeding, clearing liver and purging internal heat. It could be used to treat symptoms such as hematochezia, hematemesis, liver heat and red eyes, headache and vertigo [ 1 , 2 ] . Flos sophorae immaturus mainly contained flavonoids, saponins, fatty acids and other active ingredients. Flavonoids with a high content of 30%, were the main components, which could be used clinically for hemostasis, hypoglycemic therapy, antioxidant, anti-tumor, anti-inflammatory and so on [ 3 – 9 ] . So far, the methods for extracting flavonoids included soxhlet extraction [ 10 ] , ethanol reflux method [ 11 ] , ultrasonic method [ 12 – 14 ] , enzyme assisted extraction [ 15 ] , microwave extraction [ 16 ] , and supercritical fluid extraction [ 17 ] , which were effective, but can be quite time-consuming. Hu Q H et al. [ 12 ] studied the extraction amount and antioxidant activity of total flavonoids from Flammulina velutipes . The crude flavonoids were extracted three times with ethyl acetate, resulting in a maximum value of 3.88 mg of total flavonoids. The supercritical fluid extraction technology was susceptible to variety of factors, such as chemical composition, the state of supercritical fluid, and pressure, etc., limiting its functions in practical use. In general, most flavonoids were extracted using organic solvents, which were volatile and could cause environmental pollution during use [ 18 – 20 ] . Therefore, it was necessary to reduce the use of such solvents and replace with green solvents. Deep eutectic solvent (DES) was a new type of eutectic mixed solvent formed by hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD). It was similar to ionic liquids (ILS) in viscosity, refractive index, high conductivity and other physical properties. However, compared with ILS, DES had the advantages of simple preparation, low toxicity, biodegradability, biocompatibility and recyclability, and can be used as a new ionic liquid substitute [ 21 ] . Since the discovery of deep eutectic solvent, it had been used in various fields, such as the extraction of natural products, the organic reactions, and the food and environmental detection etc. In natural products, deep eutectic solvent replacing volatile organic compounds had become a hot issue [ 22 – 24 ] . It had been reported in literatures that flavonoids are extracted from fruits of hawthorn, leaves of ginkgo biloba, and flowers of honeysuckle by using the ultrasonic-assisted deep eutectic solvent [ 25 ] . However, there were few studies on the extraction of total flavonoids from flos sophorae immaturus by ultrasound-assisted deep eutectic solvent. In this paper, several important factors such as the type, molar ratio, moisture of deep eutectic solvent, the solid-liquid ratio, the ultrasonic time and the ultrasonic temperature were investigated, based on the content of total flavonoids from flos sophorae immaturus extracted by ultrasound-assisted deep eutectic solvent. The methods for detecting total flavonoids included UV-visible spectrophotometry (UV-vis) [ 26 ] , high performance liquid chromatography (HPLC) [ 27 , 28 ] , and Infrared spectrometry [ 29 ] , UHPLC-MS/MS [ 30 ] , HPCE [ 31 ] . This paper determined the optimal extraction process conditions by measuring the content of total flavonoids using UV-vis. This method provided a reference for the study of the extraction process of total flavonoids from flos sophorae immaturus . 2. Experiment 2.1 Experimental materials 2.1.1 Main instruments Electronic analytical balance (Shimadzu), Ultraviolet-visible spectrophotometer (Cary 50, Shimadzu), Fourier transform infrared spectrometer (Beijing Beifen-Ruili Analytical Instrument (Group) Co., Ltd), DF-101Z Collective constant temperature heating magnetic stirrer (Shanghai Yushen Instrument Co., Ltd.) and TDZ4-WS Table-type low-speed autobalancing centrifuge (Changsha Maijiasen Instrument Equipment Co., 0Ltd.) were used for this experiment. 2.1.2 Main reagents The flos sophorae immaturus was purchased from Anhui Province, rutin standard substance was from Guizhou University. The choline chloride, 1,2-Propylene, glycol, glycerol, lactic acid, glucose, ethanol, sodium nitrite, aluminium nitrate, sodium hydroxide were all analytical grade, which were from Aladdin Reagent Co., Ltd. 2.2 Experimental methods 2.2.1 Preparation of DES The four types of DES composed of choline chloride and 1, 2-propylene glycol, glycerol, lactic acid, glucose were weighed at a molar ratio of 1:2, and placed in a round-bottom flask of 50ml. After 4h of magnetic stirring in water bath at constant temperature of 60℃, the round-bottom flask was taken out and cooled to obtain DES, which was used as a solvent to extract total flavonoids from flos sophorae immaturus . 2.2.2 Preparation of standard solution According to the 2020 edition of Chinese Pharmacopoeia , rutin of 50mg as standard substance was accurately weighed and placed in a volumetric flask of 25 ml. Then, an appropriate amount of ethanol of 60% was added. After slightly heated and dissolved in water bath, the solution was cooled and added with 60% of ethanol to scale line. When shaken well, the solution of 10ml was accurately measured and placed in a volumetric flask of 100 ml. Water was then added to the scale line. By shaking, the standard solution of rutin was obtained, containing 0.2 mg/ml of rutin. 2.2.3 Preparation of sample solution Flos sophorae immaturus powder of 1g was precisely weighed and placed into a conical flask. The prepared DES was then added at solid-liquid ratio of 1:15. The solution was put in an ultrasonic instrument for ultrasonic extraction of 1h. After centrifugation, the supernatant was collected and its volume was recorded. Liquid of 1ml was then taken from the supernatant, added with 60% of ethanol, and put into a volumetric flask of 50 ml for constant volume. Thus, the sample solution was obtained for later use. 2.2.4 Production of standard curve According to the 2020 edition of Chinese Pharmacopoeia , standard solutions of 1-5ml were accurately measured and respectively placed into volumetric flasks of 25ml. Water was added into each flask to scale line of 6ml. A total of 1ml of sodium nitrite solution of 5% was added, mixed and placed for 6 minutes. Then, a total of 1ml of aluminum nitrate solution of 10% was added, shaken and placed for 6 min. Finally, test solution of sodium hydroxide of 4% was added and water was also added to the scale line. The mixture was shaken well and placed for 15 min. Taking the reagent without the standard solution as the blank control, the absorbance was determined by ultraviolet spectrophotometry at 200 ~ 800nm. The absorbance was regarded as the ordinate and the concentration as the abscissa to draw a standard curve. 2.2.5 Determination of total flavonoids in flos sophorae immaturus The sample solution of 3 ml was accurately measured and placed in a volumetric flask of 25 ml. As per the steps in article 2.2.4, the absorbance was measured, following the steps from ‘water was added into each flask to scale line of 6ml’. According to the standard curve, the concentration of total flavonoids in the extract (mg/ml) and the content of total flavonoids in flos sophorae immaturus (mg/g) were calculated. The formula was as follows: $$\:\text{W=}\frac{\text{C×V×n}}{\text{m×1000}}$$ Where: W- content of total flavonoids, mg/g C-solution mass concentration, mg/ml V-extraction volume, ml n-dilution multiple m-powder mass, g 3. Result and Discussion 3.1 Infrared spectrum analysis of DES The infrared spectrum analysis was performed on DES of choline chloride/1, 2-propylene glycol with moisture of 10% and molar ratio of 1:1. The infrared absorption spectrum of DES is shown in Fig. 1 . The wide and strong stretching vibration peak at 3200–3400 cm -1 is created by the associated O-H bond, indicating that there are a large number of hydrogen bonds in the DES structure. The stretching vibration peak at 2800–3000 cm -1 is created by C-H bond, and the bending vibration peak at 1600-1800cm -1 by N-H bond. The in-plane deformation vibration peak of C-H is at about 1400cm -1 , the stretching vibration peak of C-O at about 900-1200cm -1 , and the in-plane rocking vibration absorption band of CH 3 at about 800cm -1 . It can be seen that the molecular structure of DES is intact when using choline chloride and 1, 2-propylene glycol for preparation. 3.2 UV absorption spectroscopy characterization of standard samples The standard curve wavelength of rutin is as shown in Fig. 2 . The standard solution of 5 ml was accurately taken as the standard group, and prepared by following the steps from ‘water was added into each flask to scale line of 6ml’. The reagent without standard solution was taken as the blank control group, and prepared by following the steps from ‘water was added into each flask to scale line of 6ml’, as per article 2.2.4. Ultraviolet spectrophotometry was used for detection. The standard group had peaks at 505nm and 350nm, while the blank control group had peaks only at 350nm, which means that the peak wavelength of the standard solution was at 505 nm. 3.3 Standard curve of rutin The standard curve of rutin is shown in Fig. 3 . The regression equation is y = 0.46829x + 0.00149, R 2 = 0.9995 (y is absorbance, x is the concentration of rutin (mg/mL)), indicating that the standard substance of rutin has a good linear relationship with absorbance in the range of 0.2 ~ 1.0 mg/ml. 3.4 Investigation of DES 3.3.1 Analysis of the effect of DES types on the content of total flavonoids Different components of DES will affect its melting point, density, viscosity and other physical and chemical properties, thus affecting the extraction content of flavonoids. In this experiment, the effects of the following four different types of DES, ChCl/1, 2-propylene glycol (1:2), ChCl/lactic acid (1:2), ChCl/glucose (1:2), ChCl/glycerol (1:2) on the extraction content of total flavonoids from flos sophorae immaturus were compared. The results are shown in Fig. 4 . The highest extraction content was obtained by choline chloride/1, 2-propylene glycol, reaching 3.35 mg/g, followed by choline chloride/glucose and choline chloride/glycerol, totaling 2.30 mg/g and 2.24 mg/g, respectively. The choline chloride/lactic acid failed to make a peak at 505nm. Therefore, DES composed of choline chloride/1, 2-propylene glycol was used as the extraction solvent of total flavonoids from flos sophorae immaturus . 3.3.2 Analysis of the effect of DES molar ratio on the content of total flavonoids Different molar ratios of DES also play a decisive role in its physical and chemical properties, which in turn affects the extraction effect. In the experiment, the effects of different molar ratios of choline chloride/1, 2-propylene glycol on the extraction content of total flavonoids were studied. The results are shown in Fig. 5 . It can be seen from Fig. 5 that the content of total flavonoids from flos sophorae immaturus increased with the increase of the molar ratio of 1, 2-propylene glycol. When the molar ratio of choline chloride/1, 2-propylene glycol was 1:2, the content reached the maximum, that is, 3.55 mg/g. When the molar ratio was reduced to 1:3, the extraction content of total flavonoids decreased to 2.81 mg/g. The choline chloride/1, 2-propylene glycol with a molar ratio of 3:1 and 2:1 failed to completely dissolve into a homogeneous and transparent solution during preparation, and could not be used for the extraction of flavonoids from flos sophorae immaturus , so the flavonoid content of these two groups was 0. Therefore, the extraction effect was the best when the molar ratio of choline chloride/1, 2-propylene glycol was 1:2. 3.3.3 Analysis of the effect of DES moisture on the content of total flavonoids The effect of DES moisture on the content of total flavonoids from flos sophorae immaturus is shown in Fig. 6 . With the increase of DES moisture, the content of total flavonoids increased. When the moisture reached 10%, the content of flavonoids reached the peak, namely 3.69 mg/g. However, when the moisture of DES exceeded 10%, the extraction content of flavonoids decreased until the moisture reached 40%-50%. No flavonoids were detected in these two groups. The reason may be that the increase of moisture led to the decrease of DES viscosity, which was more conducive to the dissolution of flavonoids. However, when the moisture increased to a certain extent, it would result in the rise of solvent polarity, which had adverse effect on the extraction. Therefore, the best moisture of DES was 10% in the experiment. 3.4 Investigation of extraction process conditions 3.4.1 Analysis of the effect of solid-liquid ratio on the total content of flavonoids It can be seen from Fig. 7 that within a certain range, the content of flavonoids increased with the increase of the solid-liquid ratio. When the solid-liquid ratio achieved 1:35, the content of total flavonoids reached the maximum, namely, 4.68 mg/g. However, when the solid-liquid ratio continued to increase, the content of total flavonoids decreased. It was likely that with the increase of the solid-liquid ratio, the total flavonoids may have been mostly extracted from flos sophorae immaturus . Although the content of flavonoids decreased at liquid-solid ratio of 1:25 − 1: 30 and then increased at liquid-solid ratio of 1:30 ~ 1:35, it showed an overall upward trend. Therefore, the optimal solid-liquid ratio for extraction was 1:35. 3.4.2 Analysis of the effect of extraction time on the total content of flavonoids Time is also an important factor affecting the extraction content. As shown in Fig. 8 , the content of total flavonoids extracted from flos sophorae immaturus increased as time prolongs. At the time point of 2.5 h, the extraction content of total flavonoids culminated, reaching 3.80 mg/g. After 2.5 h, the content of flavonoids decreased. The long extraction time may possibly lead to the change of some structurally unstable compounds in flos sophorae immaturus , resulting in a decrease in flavonoid content. Therefore, the ideal time for extraction was 2.5 h. 3.4.3 Analysis of the effect of extraction temperature on the total content of flavonoids Usually, the rise of temperature can accelerate the movement between molecules and raise up the diffusion coefficient, thereby reducing the viscosity of the solvent, which is conducive to the dissolution of active ingredients. As shown in Fig. 9 , the extraction content of total flavonoids from flos sophorae immaturus reached the climax, namely 4.08 mg/g, at the temperature of 40℃. Subsequently, the flavonoid content decreased with the elevation of temperature. Although the flavonoid content saw a small rise from 50℃ to 60℃, it generally showed a downward trend after 40℃. The reason for the decrease may be that the gradual heating up damaged the structure of flavonoids. Hence, the ideal temperature for extraction was 40℃. 3.5 Standard addition recovery test A total of 12 portions of flos sophorae immaturus powder of 1g were accurately weighed, of which 6 portions were added with a certain volume of standard solution, and extracted under the optimal conditions. Among them, 6 portions without standard solution were used as blank control group, and the other 6 portions with standard solution were used as standard addition group. The extract supernatant of 3 ml was taken and treated by following the steps from ‘water was added into each flask to scale line of 6ml’, as per article 2.2.4. The absorbance was detected by ultraviolet spectrophotometry, and the flavonoid content and recovery rate were calculated. The flavonoid content of the blank control group was within the range of 4.97 ~ 4.81 mg/g, and the recovery rate of the standard addition group within the range of 88.3%~90.1%. See Table 1 . The results proved the feasibility of the extraction method of this experiment. Table 1 Results of standard addition recovery test Group Measured value of blank control group(mg/g) Standard Addition Value(mg) Standard addition measured value(mg/g) Recovery rate(%) 1 4.97 5 9.47 90.0 2 4.85 5 9.30 89.3 3 4.88 5 9.30 88.3 4 4.94 10 13.95 90.1 5 4.80 10 13.69 88.9 6 4.81 10 13.75 89.4 4. Conclusion In this paper, the optimal process of ultrasonic-assisted DES extraction of total flavonoids from flos sophorae immaturus was studied. The results of condition optimization experiment showed that for the extraction of total flavonoids from flos sophorae immaturus , it’s best to use DES made of choline chloride/1, 2-propylene glycol with a moisture of 10% at a molar ratio of 1:2. Meanwhile, the optimal process conditions for ultrasonic-assisted DES extraction of total flavonoids from flos sophorae immaturus were determined. The solid-liquid ratio was 1:35, and the extraction time 2.5h, the extraction temperature 40℃. Under the optimal conditions, the content of total flavonoids extracted from flos sophorae immaturus was up to 4.97 mg/g, and the recovery rate was about 89%, confirming that the method has good applicability. In this study, DES was used as the extration solvent to extract total flavonoids from flos sophorae immaturus by ultrasonic method, which provided a reference for the research on the extraction method of total flavonoids from flos sophorae immaturus . Compared with the traditional organic solvent extraction, the use of DES improved the extraction yield of total flavonoids, and reduced the environmental pollution caused by organic solvents. Therefore, this method is worthy of further study. Declarations CRediT authorship contribution statement Lifen Meng: Writing-original draft, Visualization, Software, Methodology. Ye Tan: Validation, Software, Methodology. Mengting Su, Yinying Zhang: Conceptualization, Methodology, Validation. Conflicts of Interest All authors declare no conflict of interest. Author Contribution Lifen Meng: Writing-original draft, Visualization, Software, Methodology. Ye Tan: Validation, Software, Methodology. Mengting Su, Yinying Zhang: Conceptualization, Methodology, Validation. Acknowledgements This work was supported by Bijie City Science and Technology Bureau Joint Fund Project ((2023)46). References Yuhong G, Liuping F, Li W et al (2023) Flos Sophorae Immaturus: Phytochemistry, bioactivities, and its potential applications. Food Reviews Int 39(6):3185–3203 Ping S, Jiajun L, Ting D et al (2023) Chemical composition and pharmacological properties of Flos sophorae immaturus, Flos sophorae and Fructus sophorae: a review. 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J Chromatogr A 1679:463395 Yan N, Wen DS, Zhao YR et al (2018) Epimedium sagittatum inhibits TLR4/MD-2 mediated NF-kappaB signaling pathway with anti-inflammatory activity. BMC Complement Altern Med 18(1):303 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6275463","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441923183,"identity":"3e349edb-df9a-43dc-8260-f78c0d09d461","order_by":0,"name":"Lifen Meng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACNmbm459/VNjUt7E3HyBOCx97Wxozw5k0xn6eYwnEaZHjOWPGzNh2mHHmjBwDIh0mkWD2uOBMGrPBgZyPN94w2MnpNhDWkm48o8KGzeDA2c2WcxiSjc0OENZyQILnTBqPwcHebdI8DAcStxHWktggwdt2WMLgMM8zIrXwHGaTBmoxkGzjYSNSC3sbs+GMM2kJ/DxsxpZzDIjwi3wz/8cHHypsEtjkHz+88abCTo6gFhQgwUNk1CBrIVXHKBgFo2AUjAgAAIiQQoI/typlAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou University of Engineering Science","correspondingAuthor":true,"prefix":"","firstName":"Lifen","middleName":"","lastName":"Meng","suffix":""},{"id":441923185,"identity":"0a063bfb-248c-45ef-9d4a-352a5726de5f","order_by":1,"name":"Ye Tan","email":"","orcid":"","institution":"Guizhou University of Engineering Science","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Tan","suffix":""},{"id":441923186,"identity":"a6c10e20-fae3-4c82-ba4c-43775168d47a","order_by":2,"name":"Mengting Su","email":"","orcid":"","institution":"Guizhou University of Engineering Science","correspondingAuthor":false,"prefix":"","firstName":"Mengting","middleName":"","lastName":"Su","suffix":""},{"id":441923188,"identity":"7e05142b-38e8-4d6e-a9b4-f975461bd6ca","order_by":3,"name":"Yinying Zhang","email":"","orcid":"","institution":"Guizhou University of Engineering Science","correspondingAuthor":false,"prefix":"","firstName":"Yinying","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-21 08:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6275463/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6275463/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80575681,"identity":"da9b3e36-a0ff-4b46-8e3f-2dd605d2510f","added_by":"auto","created_at":"2025-04-14 21:42:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38614,"visible":true,"origin":"","legend":"\u003cp\u003eInfrared absorption spectrum of DES\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/4b40c78b8cc985a5265f8dc9.jpg"},{"id":80575815,"identity":"fc0b9ebb-4b39-47b8-b8ad-76ab2b8cb944","added_by":"auto","created_at":"2025-04-14 21:50:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33051,"visible":true,"origin":"","legend":"\u003cp\u003eUV absorption spectrum of rutin\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/6b135626bfc62b19608db223.jpg"},{"id":80575682,"identity":"2188b9ac-ec42-4afd-b99a-c4ad77c362c5","added_by":"auto","created_at":"2025-04-14 21:42:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30236,"visible":true,"origin":"","legend":"\u003cp\u003eThe standard curve of rutin\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/27a73e26160b28fcaf780a9a.jpg"},{"id":80576259,"identity":"043e7edf-0490-42e0-a66c-6a663113f474","added_by":"auto","created_at":"2025-04-14 22:06:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29589,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of DES types on total flavonoid content\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/b97bc005099f858a47c0b362.jpg"},{"id":80576159,"identity":"8b76ac09-3b5b-4124-ba47-bc8bc9143ab1","added_by":"auto","created_at":"2025-04-14 21:58:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":25626,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of molar ratio of DES on total flavonoid content\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/1a167ff590cdf5fe7ecd480c.jpg"},{"id":80575687,"identity":"6141d7aa-b9f0-45c7-8e65-e4775b7ddf1f","added_by":"auto","created_at":"2025-04-14 21:42:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":29405,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of DES moisture on the content of total flavonoids\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/dcd07c8c0f6778171f2cd34d.jpg"},{"id":80575689,"identity":"365c057b-5683-4dbc-8b29-78dab97333d8","added_by":"auto","created_at":"2025-04-14 21:42:12","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37782,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of solid-liquid ratio on total flavonoid content\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/7389caae35fa59bab66d9731.jpg"},{"id":80575690,"identity":"bc9d9100-8369-4336-bea7-01b52e006cf6","added_by":"auto","created_at":"2025-04-14 21:42:12","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":37363,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of extraction time on total flavonoid content\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/98ebdcd4aa294feb42255cc8.jpg"},{"id":80575695,"identity":"7658e0d5-89b2-491d-9b7c-0e9b517701ea","added_by":"auto","created_at":"2025-04-14 21:42:12","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":33939,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of extraction temperature on total flavonoid content\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/5253d50e98988eb9513dd0a3.jpg"},{"id":86247694,"identity":"e57d93e3-3347-45ef-a7d6-8b30a9d78e07","added_by":"auto","created_at":"2025-07-08 12:03:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1242300,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6275463/v1/b0a9aa73-1e40-406a-a692-525ccba65f8c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on the ultrasonic-assisted deep eutectic solvent extraction of total flavonoids from flos sophorae immaturus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFlavonoids in natural products had been a research focus in recent years. \u003cem\u003eFlos sophorae immaturus\u003c/em\u003e was the dried flower bud of \u003cem\u003eSophora japonica L.\u003c/em\u003e, which had the function of cooling blood and stopping bleeding, clearing liver and purging internal heat. It could be used to treat symptoms such as hematochezia, hematemesis, liver heat and red eyes, headache and vertigo \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eFlos sophorae immaturus\u003c/em\u003e mainly contained flavonoids, saponins, fatty acids and other active ingredients. Flavonoids with a high content of 30%, were the main components, which could be used clinically for hemostasis, hypoglycemic therapy, antioxidant, anti-tumor, anti-inflammatory and so on \u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSo far, the methods for extracting flavonoids included soxhlet extraction \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, ethanol reflux method \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, ultrasonic method \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, enzyme assisted extraction \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, microwave extraction \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, and supercritical fluid extraction \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, which were effective, but can be quite time-consuming. Hu Q H et al. \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e studied the extraction amount and antioxidant activity of total flavonoids from \u003cem\u003eFlammulina velutipes\u003c/em\u003e. The crude flavonoids were extracted three times with ethyl acetate, resulting in a maximum value of 3.88 mg of total flavonoids. The supercritical fluid extraction technology was susceptible to variety of factors, such as chemical composition, the state of supercritical fluid, and pressure, etc., limiting its functions in practical use. In general, most flavonoids were extracted using organic solvents, which were volatile and could cause environmental pollution during use \u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Therefore, it was necessary to reduce the use of such solvents and replace with green solvents.\u003c/p\u003e \u003cp\u003eDeep eutectic solvent (DES) was a new type of eutectic mixed solvent formed by hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD). It was similar to ionic liquids (ILS) in viscosity, refractive index, high conductivity and other physical properties. However, compared with ILS, DES had the advantages of simple preparation, low toxicity, biodegradability, biocompatibility and recyclability, and can be used as a new ionic liquid substitute \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Since the discovery of deep eutectic solvent, it had been used in various fields, such as the extraction of natural products, the organic reactions, and the food and environmental detection etc. In natural products, deep eutectic solvent replacing volatile organic compounds had become a hot issue \u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt had been reported in literatures that flavonoids are extracted from fruits of hawthorn, leaves of ginkgo biloba, and flowers of honeysuckle by using the ultrasonic-assisted deep eutectic solvent \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. However, there were few studies on the extraction of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e by ultrasound-assisted deep eutectic solvent.\u003c/p\u003e \u003cp\u003eIn this paper, several important factors such as the type, molar ratio, moisture of deep eutectic solvent, the solid-liquid ratio, the ultrasonic time and the ultrasonic temperature were investigated, based on the content of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e extracted by ultrasound-assisted deep eutectic solvent. The methods for detecting total flavonoids included UV-visible spectrophotometry (UV-vis) \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, high performance liquid chromatography (HPLC) \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and Infrared spectrometry \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, UHPLC-MS/MS \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, HPCE \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. This paper determined the optimal extraction process conditions by measuring the content of total flavonoids using UV-vis. This method provided a reference for the study of the extraction process of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Experiment","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Main instruments\u003c/h2\u003e \u003cp\u003eElectronic analytical balance (Shimadzu), Ultraviolet-visible spectrophotometer (Cary 50, Shimadzu), Fourier transform infrared spectrometer (Beijing Beifen-Ruili Analytical Instrument (Group) Co., Ltd), DF-101Z Collective constant temperature heating magnetic stirrer (Shanghai Yushen Instrument Co., Ltd.) and TDZ4-WS Table-type low-speed autobalancing centrifuge (Changsha Maijiasen Instrument Equipment Co., 0Ltd.) were used for this experiment.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e2.1.2 Main reagents\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eflos sophorae immaturus\u003c/em\u003e was purchased from Anhui Province, rutin standard substance was from Guizhou University. The choline chloride, 1,2-Propylene, glycol, glycerol, lactic acid, glucose, ethanol, sodium nitrite, aluminium nitrate, sodium hydroxide were all analytical grade, which were from Aladdin Reagent Co., Ltd.\u003c/p\u003e\n\u003ch3\u003e2.2 Experimental methods\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2.1 Preparation of DES\u003c/h2\u003e \u003cp\u003eThe four types of DES composed of choline chloride and 1, 2-propylene glycol, glycerol, lactic acid, glucose were weighed at a molar ratio of 1:2, and placed in a round-bottom flask of 50ml. After 4h of magnetic stirring in water bath at constant temperature of 60℃, the round-bottom flask was taken out and cooled to obtain DES, which was used as a solvent to extract total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.2.2 Preparation of standard solution\u003c/h2\u003e \u003cp\u003eAccording to the 2020 edition of \u003cem\u003eChinese Pharmacopoeia\u003c/em\u003e, rutin of 50mg as standard substance was accurately weighed and placed in a volumetric flask of 25 ml. Then, an appropriate amount of ethanol of 60% was added. After slightly heated and dissolved in water bath, the solution was cooled and added with 60% of ethanol to scale line. When shaken well, the solution of 10ml was accurately measured and placed in a volumetric flask of 100 ml. Water was then added to the scale line. By shaking, the standard solution of rutin was obtained, containing 0.2 mg/ml of rutin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2.3 Preparation of sample solution\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eFlos sophorae immaturus\u003c/em\u003e powder of 1g was precisely weighed and placed into a conical flask. The prepared DES was then added at solid-liquid ratio of 1:15. The solution was put in an ultrasonic instrument for ultrasonic extraction of 1h. After centrifugation, the supernatant was collected and its volume was recorded. Liquid of 1ml was then taken from the supernatant, added with 60% of ethanol, and put into a volumetric flask of 50 ml for constant volume. Thus, the sample solution was obtained for later use.\u003c/p\u003e\n\u003ch3\u003e2.2.4 Production of standard curve\u003c/h3\u003e\n\u003cp\u003eAccording to the 2020 edition of \u003cem\u003eChinese Pharmacopoeia\u003c/em\u003e, standard solutions of 1-5ml were accurately measured and respectively placed into volumetric flasks of 25ml. Water was added into each flask to scale line of 6ml. A total of 1ml of sodium nitrite solution of 5% was added, mixed and placed for 6 minutes. Then, a total of 1ml of aluminum nitrate solution of 10% was added, shaken and placed for 6 min. Finally, test solution of sodium hydroxide of 4% was added and water was also added to the scale line. The mixture was shaken well and placed for 15 min. Taking the reagent without the standard solution as the blank control, the absorbance was determined by ultraviolet spectrophotometry at 200\u0026thinsp;~\u0026thinsp;800nm. The absorbance was regarded as the ordinate and the concentration as the abscissa to draw a standard curve.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2.5 Determination of total flavonoids in\u003c/b\u003e \u003cb\u003eflos sophorae immaturus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe sample solution of 3 ml was accurately measured and placed in a volumetric flask of 25 ml. As per the steps in article 2.2.4, the absorbance was measured, following the steps from \u0026lsquo;water was added into each flask to scale line of 6ml\u0026rsquo;. According to the standard curve, the concentration of total flavonoids in the extract (mg/ml) and the content of total flavonoids in \u003cem\u003eflos sophorae immaturus\u003c/em\u003e (mg/g) were calculated. The formula was as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{W=}\\frac{\\text{C\u0026times;V\u0026times;n}}{\\text{m\u0026times;1000}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere: W- content of total flavonoids, mg/g\u003c/p\u003e \u003cp\u003eC-solution mass concentration, mg/ml\u003c/p\u003e \u003cp\u003eV-extraction volume, ml\u003c/p\u003e \u003cp\u003en-dilution multiple\u003c/p\u003e \u003cp\u003em-powder mass, g\u003c/p\u003e "},{"header":"3. Result and Discussion","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Infrared spectrum analysis of DES\u003c/h2\u003e \u003cp\u003eThe infrared spectrum analysis was performed on DES of choline chloride/1, 2-propylene glycol with moisture of 10% and molar ratio of 1:1. The infrared absorption spectrum of DES is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The wide and strong stretching vibration peak at 3200\u0026ndash;3400 cm\u003csup\u003e-1\u003c/sup\u003e is created by the associated O-H bond, indicating that there are a large number of hydrogen bonds in the DES structure. The stretching vibration peak at 2800\u0026ndash;3000 cm\u003csup\u003e-1\u003c/sup\u003e is created by C-H bond, and the bending vibration peak at 1600-1800cm\u003csup\u003e-1\u003c/sup\u003e by N-H bond. The in-plane deformation vibration peak of C-H is at about 1400cm\u003csup\u003e-1\u003c/sup\u003e, the stretching vibration peak of C-O at about 900-1200cm\u003csup\u003e-1\u003c/sup\u003e, and the in-plane rocking vibration absorption band of CH\u003csub\u003e3\u003c/sub\u003e at about 800cm\u003csup\u003e-1\u003c/sup\u003e. It can be seen that the molecular structure of DES is intact when using choline chloride and 1, 2-propylene glycol for preparation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 UV absorption spectroscopy characterization of standard samples\u003c/h2\u003e \u003cp\u003eThe standard curve wavelength of rutin is as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The standard solution of 5 ml was accurately taken as the standard group, and prepared by following the steps from \u0026lsquo;water was added into each flask to scale line of 6ml\u0026rsquo;. The reagent without standard solution was taken as the blank control group, and prepared by following the steps from \u0026lsquo;water was added into each flask to scale line of 6ml\u0026rsquo;, as per article 2.2.4. Ultraviolet spectrophotometry was used for detection. The standard group had peaks at 505nm and 350nm, while the blank control group had peaks only at 350nm, which means that the peak wavelength of the standard solution was at 505 nm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Standard curve of rutin\u003c/h2\u003e \u003cp\u003eThe standard curve of rutin is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The regression equation is y\u0026thinsp;=\u0026thinsp;0.46829x\u0026thinsp;+\u0026thinsp;0.00149, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9995 (y is absorbance, x is the concentration of rutin (mg/mL)), indicating that the standard substance of rutin has a good linear relationship with absorbance in the range of 0.2\u0026thinsp;~\u0026thinsp;1.0 mg/ml.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Investigation of DES\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Analysis of the effect of DES types on the content of total flavonoids\u003c/h2\u003e \u003cp\u003eDifferent components of DES will affect its melting point, density, viscosity and other physical and chemical properties, thus affecting the extraction content of flavonoids. In this experiment, the effects of the following four different types of DES, ChCl/1, 2-propylene glycol (1:2), ChCl/lactic acid (1:2), ChCl/glucose (1:2), ChCl/glycerol (1:2) on the extraction content of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e were compared. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The highest extraction content was obtained by choline chloride/1, 2-propylene glycol, reaching 3.35 mg/g, followed by choline chloride/glucose and choline chloride/glycerol, totaling 2.30 mg/g and 2.24 mg/g, respectively. The choline chloride/lactic acid failed to make a peak at 505nm. Therefore, DES composed of choline chloride/1, 2-propylene glycol was used as the extraction solvent of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3.2 Analysis of the effect of DES molar ratio on the content of total flavonoids\u003c/h2\u003e \u003cp\u003eDifferent molar ratios of DES also play a decisive role in its physical and chemical properties, which in turn affects the extraction effect. In the experiment, the effects of different molar ratios of choline chloride/1, 2-propylene glycol on the extraction content of total flavonoids were studied. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e that the content of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e increased with the increase of the molar ratio of 1, 2-propylene glycol. When the molar ratio of choline chloride/1, 2-propylene glycol was 1:2, the content reached the maximum, that is, 3.55 mg/g. When the molar ratio was reduced to 1:3, the extraction content of total flavonoids decreased to 2.81 mg/g. The choline chloride/1, 2-propylene glycol with a molar ratio of 3:1 and 2:1 failed to completely dissolve into a homogeneous and transparent solution during preparation, and could not be used for the extraction of flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e, so the flavonoid content of these two groups was 0. Therefore, the extraction effect was the best when the molar ratio of choline chloride/1, 2-propylene glycol was 1:2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3.3 Analysis of the effect of DES moisture on the content of total flavonoids\u003c/h2\u003e \u003cp\u003eThe effect of DES moisture on the content of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. With the increase of DES moisture, the content of total flavonoids increased. When the moisture reached 10%, the content of flavonoids reached the peak, namely 3.69 mg/g. However, when the moisture of DES exceeded 10%, the extraction content of flavonoids decreased until the moisture reached 40%-50%. No flavonoids were detected in these two groups. The reason may be that the increase of moisture led to the decrease of DES viscosity, which was more conducive to the dissolution of flavonoids. However, when the moisture increased to a certain extent, it would result in the rise of solvent polarity, which had adverse effect on the extraction. Therefore, the best moisture of DES was 10% in the experiment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Investigation of extraction process conditions\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Analysis of the effect of solid-liquid ratio on the total content of flavonoids\u003c/h2\u003e \u003cp\u003eIt can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e that within a certain range, the content of flavonoids increased with the increase of the solid-liquid ratio. When the solid-liquid ratio achieved 1:35, the content of total flavonoids reached the maximum, namely, 4.68 mg/g. However, when the solid-liquid ratio continued to increase, the content of total flavonoids decreased. It was likely that with the increase of the solid-liquid ratio, the total flavonoids may have been mostly extracted from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e. Although the content of flavonoids decreased at liquid-solid ratio of 1:25\u0026thinsp;\u0026minus;\u0026thinsp;1: 30 and then increased at liquid-solid ratio of 1:30\u0026thinsp;~\u0026thinsp;1:35, it showed an overall upward trend. Therefore, the optimal solid-liquid ratio for extraction was 1:35.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4.2 Analysis of the effect of extraction time on the total content of flavonoids\u003c/h2\u003e \u003cp\u003eTime is also an important factor affecting the extraction content. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the content of total flavonoids extracted from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e increased as time prolongs. At the time point of 2.5 h, the extraction content of total flavonoids culminated, reaching 3.80 mg/g. After 2.5 h, the content of flavonoids decreased. The long extraction time may possibly lead to the change of some structurally unstable compounds in \u003cem\u003eflos sophorae immaturus\u003c/em\u003e, resulting in a decrease in flavonoid content. Therefore, the ideal time for extraction was 2.5 h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4.3 Analysis of the effect of extraction temperature on the total content of flavonoids\u003c/h2\u003e \u003cp\u003eUsually, the rise of temperature can accelerate the movement between molecules and raise up the diffusion coefficient, thereby reducing the viscosity of the solvent, which is conducive to the dissolution of active ingredients. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the extraction content of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e reached the climax, namely 4.08 mg/g, at the temperature of 40℃. Subsequently, the flavonoid content decreased with the elevation of temperature. Although the flavonoid content saw a small rise from 50℃ to 60℃, it generally showed a downward trend after 40℃. The reason for the decrease may be that the gradual heating up damaged the structure of flavonoids. Hence, the ideal temperature for extraction was 40℃.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Standard addition recovery test\u003c/h2\u003e \u003cp\u003eA total of 12 portions of \u003cem\u003eflos sophorae immaturus\u003c/em\u003e powder of 1g were accurately weighed, of which 6 portions were added with a certain volume of standard solution, and extracted under the optimal conditions. Among them, 6 portions without standard solution were used as blank control group, and the other 6 portions with standard solution were used as standard addition group. The extract supernatant of 3 ml was taken and treated by following the steps from \u0026lsquo;water was added into each flask to scale line of 6ml\u0026rsquo;, as per article 2.2.4. The absorbance was detected by ultraviolet spectrophotometry, and the flavonoid content and recovery rate were calculated. The flavonoid content of the blank control group was within the range of 4.97\u0026thinsp;~\u0026thinsp;4.81 mg/g, and the recovery rate of the standard addition group within the range of 88.3%~90.1%. See Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results proved the feasibility of the extraction method of this experiment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of standard addition recovery test\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeasured value of blank control group(mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard Addition Value(mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStandard addition measured value(mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRecovery rate(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e89.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e88.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e88.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e89.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this paper, the optimal process of ultrasonic-assisted DES extraction of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e was studied. The results of condition optimization experiment showed that for the extraction of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e, it\u0026rsquo;s best to use DES made of choline chloride/1, 2-propylene glycol with a moisture of 10% at a molar ratio of 1:2. Meanwhile, the optimal process conditions for ultrasonic-assisted DES extraction of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e were determined. The solid-liquid ratio was 1:35, and the extraction time 2.5h, the extraction temperature 40℃. Under the optimal conditions, the content of total flavonoids extracted from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e was up to 4.97 mg/g, and the recovery rate was about 89%, confirming that the method has good applicability.\u003c/p\u003e \u003cp\u003eIn this study, DES was used as the extration solvent to extract total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e by ultrasonic method, which provided a reference for the research on the extraction method of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e. Compared with the traditional organic solvent extraction, the use of DES improved the extraction yield of total flavonoids, and reduced the environmental pollution caused by organic solvents. Therefore, this method is worthy of further study.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLifen Meng: Writing-original draft, Visualization, Software, Methodology. Ye Tan: Validation, Software, Methodology. Mengting Su, Yinying Zhang: Conceptualization, Methodology, Validation.\u0026nbsp;\u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eAll authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLifen Meng: Writing-original draft, Visualization, Software, Methodology. Ye Tan: Validation, Software, Methodology. Mengting Su, Yinying Zhang: Conceptualization, Methodology, Validation.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by Bijie City Science and Technology Bureau Joint Fund Project ((2023)46).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYuhong G, Liuping F, Li W et al (2023) Flos Sophorae Immaturus: Phytochemistry, bioactivities, and its potential applications. Food Reviews Int 39(6):3185\u0026ndash;3203\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePing S, Jiajun L, Ting D et al (2023) Chemical composition and pharmacological properties of Flos sophorae immaturus, Flos sophorae and Fructus sophorae: a review. 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BMC Complement Altern Med 18(1):303\u003c/span\u003e\u003c/li\u003e\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":"deep eutectic solvent, ultrasonic, flos sophorae immaturus, total flavonoids","lastPublishedDoi":"10.21203/rs.3.rs-6275463/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6275463/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this paper, four different kinds of deep eutectic solvents were prepared to carry out ultrasonic-assisted extraction of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e. The content of total flavonoids was determined by ultraviolet spectrophotometry. The effects of the type, molar ratio, moisture of deep eutectic solvent, the solid-liquid ratio, the ultrasonic time and the ultrasonic temperature on the extraction of total flavonoids from \u003cem\u003eflos sophorae immaturus\u003c/em\u003e were investigated by condition experiment. Meanwhile, the standard addition recovery rate experiment was performed to verify the feasibility of the method. The results showed that the optimal extraction results could be obtained when the moisture was 10%, the molar ratio of choline chloride/1,2-propylene glycol deep eutectic solvent 1:2, the solid-liquid ratio 1:35, the ultrasonic temperature 40℃, and the ultrasonic time 2.5h. Under the optimal conditions, the content of total flavonoids extraction was up to 4.97mg/g, and the standard addition recovery rate reached about 89%.\u003c/p\u003e","manuscriptTitle":"Study on the ultrasonic-assisted deep eutectic solvent extraction of total flavonoids from flos sophorae immaturus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-14 21:42:07","doi":"10.21203/rs.3.rs-6275463/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":"9330cd19-7da7-4213-9f79-db762a3d0258","owner":[],"postedDate":"April 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-08T11:55:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-14 21:42:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6275463","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6275463","identity":"rs-6275463","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}