Isolation of Polysaccharides From Curcuma Longa and Its Interaction With Bovine Serum Albumin

Isolation of Polysaccharides From Curcuma Longa and Its Interaction With Bovine Serum Albumin | 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 Isolation of Polysaccharides From Curcuma Longa and Its Interaction With Bovine Serum Albumin Washim Raja This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6207332/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 traditional medicine most of the diseases have been treated by administration of plant or plant product. Curcuma longa L. (Zingiberaceae), is a useful traditional medicinal plant in India. There are several data in the literature indicating a great variety of pharmacological activities of C.longa which exhibit anti-inflammatory, anti-human immunodeficiency virus, anti-bacteria, antioxidant effects and nematocidal activities. It is now considered as a valuable source of unique natural products for development of medicines against various diseases and also for the development of industrial products.Our aim is to develop new potent drug candidate. For this we studied the interaction of the polysaccharide with BSA(bovine serum albumin). To study the interaction with polysaccharides and BSA present, I have isolated polysaccharides from depigmented plant materials of C. longa by extraction with cold water. The cold water extracted materials, which were isolated in 3.9 % yield, have been designated as WE. Ultraviolet spectrometric analyses showed that the studied water extracted carbohydrate polymer (WE) interact with BSA. Natural Product Chemistry Medicinal Plant Polysaccharides BSA Carbohydrate Polymer Figures Figure 1 Figure 2 Figure 3 Introduction Curcuma longa (CL, common name Turmeric), a plant species from the Curcuma genus (family Zingiberaceae ), is common ingredient in many health supplements in Asia.Turmeric ( Curcuma longa ) has been used for centuries in Ayurvedic medicine, which amalgamate the medicinal goods of herbs with food. This astonishing herb has established its way into the attention in the west because of its wide range of medicinal benefits [ 1 ]. In ayurveda , rhizome of turmeric is used as medicines against skin, gastrointestinal, respiratory, hepatic, and biliary disorders [ 2 ]. The active constituents of C. longa are the flavonoid curcumin (diferuloylmethane) and various volatile oils, including tumerone, atlantone, and zingiberene. Other constituents include sugars, proteins, and resins. The best researched active constituent is curcumin,which comprises 0.3–5.4 percent of raw turmeric [ 1 ]. Components of turmeric especially curcumin has been shown to have anti-inflammatory, antiviral, and anticancer properties [ 3 – 5 ]. Among the phytoconstituents of C. longa , curcuminoids are considered as an important active molecule and also exhibited wide range of pharmacological activities. The literature review confirmed plethora of information available on safety aspects of curcumin and essential oil fractions of C.longa [ 3 , 6 – 8 ].While curcuminoids based extract were well studied for their pharmacological and safety aspects, polysaccharide extract of C. longa is gaining importance since it showed to have various pharmacological activities, which include antidiabetic, antitumour, antidepressant, antioxidant, antimicrobial, antifertility, hepatoprotective, and immunomodulatory properties [ 9 – 16 ]. NR-INF-02, a polysaccharide extract prepared from rhizome of C. longa had shown clinical efficiency in a randomized placebo controlled study on 120 human patients (37 males and 83 females) affected with primary osteoarthritis [ 17 ].NR-INF-02 deserved as an effective option for the treatment of patients with primary painful knee and joint pains and also reduced the need of analgesics as a rescue medication [ 17 ]. Also, NR-INF-02 showed immunestimulatory and anti-inflammatory effects in in vitro models by influencing various cytokines involved in immune regulation [ 18 ]. Similarly, the immunostimulatory effects of C.longa polysaccharides on peripheral blood mononuclear cells was investigated and the findings revealed the potential use of C.longa polysaccharide extract as an adjuvant supplement for cancer patients, whose immune activities were suppressed during chemotherapies [ 16 ].Polysaccharide extract of C.longa needs evaluation for their safety due to its growing demand on reported preclinical therapeutic indications. Also, before setting of a clinical trial of an herbal product, its safety must be evaluated by toxicity test procedures. Also, this toxicity evaluation is helpful for the estimation of an initial safe starting dose and dose range for the human trials and the identification of parameters for clinical monitoring for potential adverse effects. Hence, the goal of the present investigation is to characterize the toxicological profile of polysaccharide extract from C.longa rhizome (NR-INF-02). In order to evaluate toxicological profile, the ability of NR-INF-02 to induce mutations was assessed in indicator microorganisms using bacterial reverse mutation test. The effect of NR-INF-02 on the genetic system was weighed up by analyzing induced chromosomal aberrations and micronucleus in mammalian cells. An acute oral toxicity study was performed after single oral dose of NR-INF-02 to determine immediate toxic effect. All these tests were conducted based on recommendation of the OECD guidelines [ 19 – 22 ]. Experimental Section Plant material. Powdered root of curcuma longa were collected from the ayurvedic medical store of Birbhum, West Bengal, India. General procedures. The chemicals used were of an analytical grade or the best available. All experiments were conducted at least in duplicate. Evaporations were carried out under reduced pressure at around 50°C (SB 1100 Rotary Evaporator; Eyela, Tokyo, Japan). Toluene was used to inhibit microbial growth. Small volumes of samples are freeze-dried (Cool Safe 55-F freeze drier;Scanvac, Lynge, Denmark). Total carbohydrate content were determined by the phenol–sulfuric acid using galactose as standard. UV−VIS spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan). GC performed with a Shimadzu GC-17A chromatograph (Shimadzu, Kyoto, Japan), fitted with a flame ionization detector, and a DB-225 column (30 m x 0.53 mm i.d.), using a program that maintained an isocratic temperature of 210°C for 18 min and helium as gas vector. GC-MS was performed with a Shimadzu QP 5050A GC-MS instrument (Shimadzu) at 70 eV. Conditions for GC-MS were as described previously. Isolation of carbohydrate polymer. The powder material of curcuma longa root ( plant material 10gm) was extracted with distilled water (3 × 100 mL, pH 5) at 4–8°C for 6 h. The insoluble residue was removed by centrifugation (10,000 g, 15 min) and filtration of the supernatant through a glass filter (G 2). The extract was then diluted with 4 volume cold acetone (× 3), evaporated to a small volume, and EtOH was added (x 3 volumes). The resulting precipitates were dissolved in H 2 O and lyophilized to yield the water extracted carbohydrate polymer (WE, ~ 0.55 mg). Monosaccharide Analysis Polysaccharide fraction were hydrolyzed either with 2M trifluoro acetic acid for 3 hr at 100 o C, centrifuged. Then TFA was removed on rotary evaporator. The presence of monosaccharide in hydrolysate in WE was also indicated by presence spots on TLC. Monosaccharides in the acid hydrolysate were also analyzed by thin layer chromatography on kieselgel 60F plate (Merck) using EtOH/ PhOH/pyridine/0.1 M H3PO4 (5/1/1/2 v/v) as eluent. Carbohydrates were then detected by heating at 100°C after treatment with a saturated solution of aniline phthalate. FT-IR Analysis IR spectra were obtained on a FT-IR spectrophotometer (JASCO FT-IR-420) using KBr disks containing finely ground samples. The analyses were conducted five times. Bovine serum albumin (BSA)-Carbohydrate Polymer interaction . UV−VIS Spectra. UV-VIS absorption spectra of 1mg/mL BSA alone and in presence of 0.2-2 mg/mL WE in a 10 mM phosphate buffer of pH 7.4 were recorded on a UV-VIS spectrophotometer at room temperature (298 K). The spectra of 0.2-2 mg/mL WE were also recorded under similar condition as blank in order to eliminate the spectral inferences on the BSA-WE system from WE. Results and discussion Chemical Characterization. C.longa Was extracted with H2O, and the extract was subjected to fractional precipitation with EtOH to obtain a precipitate of polysaccharide, precipitate was then lyophilized giving the water extracted carbohydrate polymer (WE). Chromatographic analyses indicate the presence of a heteropolysaccharide made up of arabinose and galactose in the water extracted polymer. FT-IR Analysis FT-IR spectrum of WE P fraction showed bands characteristic of polysaccharide. It consisted, inter alia, of (i) a broad band around 3403 cm − 1 (Fig. 1)originated from stretching vibrations of OH groups in sugar moieties, (ii) a band at 2933 cm − 1 derived from (C–H) stretching vibrations of methylene or methyl groups, and (iii) a band at 1371 cm − 1 related to the carbonyl stretching of the carboxylate anion. Effect of carbohydrate polymer on BSA. The UV-VIS spectra of BSA (1.0 mg/mL) alone and in the presence of CP (0.05-1 mg/mL) are indicated in Fig. 4 at pH7.4. BSA has two absorption peaks. The peak at 228 nm and 277.5 nm (Fig. 2) is attributed to the π-π* transition of characteristic polypeptide backbone structure and n-π* transition of aromatic amino acids. At pH 7.4 with gradual addition of CP to BSA solution, the intensity of the peak at 228 nm decreases and the λmax for the particles in the solution shifted towards longer wavelength feebly (7.5 nm). These spectral changes might arise from the disturbance of the microenvironment around the polypeptide caused by the binding of WE with BSA. Such complex formation occurs above the isoelectric point (Ip) of BSA (Ip = 4.7) may be attributed to the electrostatic interaction between locally positively charged patches on the protein [24] and negatively charged polysaccharide because of the presence of galacturonic acid. Conclusion In conclusion, this study has shown that about 0.63% of WE of C.longa could be obtained by simple water extraction method. Chromatographic analyses indicate the presence of a heteropolysaccharide made up of arabinose and galactose in the water extracted polymer. Further research will be directed towards a more detailed characterization of the purified polysaccharides and their biological activity study will be of interest. In addition, the interaction between bovine serum albumin (BSA) and carbohydrate polymer (WE) has been studied. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding No funding was received to assist with the preparation of this manuscript. Authors' contributions Dr Washim Raja contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dr Washim Raja. The first draft of the manuscript was written by Dr Washim Raja. Dr Raja read and approved the final manuscript. Acknowledgements Technical support from Turku Hansda Lapsa Hemram Mahavidyalay and the University of Burdwan to W. R. is gratefully acknowledged. Data availability statements The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. References M. Akram, Shahab-Uddin, A. Ahmed et al., “ Curcuma longa and curcumin: a review article,” Romanian Journal of Biology ,vol. 55, pp. 65–70, 2010. The Ayurvedic Pharmacopoeia of India, part 1 , vol. 8, The Controller of Publications, Civil Lines, Delhi, India, 1st edition,2011. N. Chainani-Wu, “Safety and anti-inflammatory activity of curcumin: a component of tumeric ( Curcuma longa ),” Journal of Alternative and Complementary Medicine , vol. 9, no. 1, pp. 161–168, 2003. M. H. Chen, M. Y. Lee, J. J. Chuang et al., “Curcumin inhibits HCV replication by induction of heme oxygenase-1 and suppression of AKT,” International Journal of Molecular Medicine , J.-L. Ji, X.-F. Huang, and H.-L. Zhu, “Curcumin and its formulations:potential anti-cancer agents,” Anti-Cancer Agents in Medicinal Chemistry , vol. 12, no. 3, pp. 210–218, 2012. V. B. Liju, K. Jeena, and R. Kuttan, “Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric ( Curcuma longa L),” Food and Chemical Toxicology , vol. 53, pp. 52–61, 2012. S. Balaji and B. Chempakam, “Toxicity prediction of compounds from turmeric ( Curcuma longa L),” Food and Chemical Toxicology , vol. 48, no. 10, pp. 2951–2959, 2010. J. Joshi, S. Ghaisas, A. Vaidya et al., “Early humansafety study of turmeric oil ( Curcuma longa oil) administered orally in healthy volunteers,” Journal of Association of Physicians of India , vol. 51,pp. 1055–1060, 2003. S. Mohankumar and J. R. McFarlane, “An aqueous extract of Curcuma longa (turmeric) rhizomes stimulates insulin release and mimics insulin action on tissues involved in glucose homeostasis in vitro,” Phytotherapy Research , vol. 25, no. 3, pp. 396–401, 2011. S. S.Deshpande, A.D. Ingle, andG. B.Maru, “Chemopreventive efficacy of curcumin-free aqueous turmeric extract in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumorigenesis,” Cancer Letters , vol. 123, no. 1, pp. 35–40, 1998. Z. F. Yu, L. D. Kong, and Y. Chen, “Antidepressant activity of aqueous extracts of Curcuma longa in mice,” Journal of Ethnopharmacology , vol. 83, no. 1-2, pp. 161–165, 2002. R. Selvam, L. Subramanian, R. Gayathri, and N. Angayarkanni, “The anti-oxidant activity of turmeric ( Curcuma longa ),” Journal of Ethnopharmacology , vol. 47, no. 2, pp. 59–67, 1995. J. Anbu Jeba Sunilson, R. Suraj, G. Rejitha, K. Anandarajagopal, A. V. Anita Gnana Kumari, and P. Promwichit, “In vitro antimicrobial evaluation of Zingiber officinale, Curcuma longa and Alpinia galanga extracts as natural food preservatives,” American Journal of Food Technology , vol. 4, no. 5, pp. 192–200,2009. R. K.Mishra and S. K. Singh, “Reversible antifertility effect of aqueous rhizome extract of Curcuma longa L. in male laboratory mice,” Contraception , vol. 79, no. 6, pp. 479–487, 2009. L. Subramanian and R. Selvam, “Prevention of CCl4—Induced hepatotoxicity by aqueous extract of turmeric,” NutritionResearch , vol. 19, no. 3, pp. 429–441, 1999. G. G. L. Yue, B. C. L. Chan, P.-M. Hon et al., “Immunostimulatoryactivities of polysaccharide extract isolated from Curcumalonga ,” International Journal of Biological Macromolecules , vol.47, no. 3, pp. 342–347, 2010. K.Madhu, K. Chanda, and Saji, “Safety and efficacy of Curcumalonga extract in the treatment of painful knee osteoarthritis: arandomized placebo controlled trial,” Inflammopharmacology ,vol. 21, pp. 129–136, 2013. C. V. Chandrasekaran, K. Sundarajan, R. J. Edwin et al., “Immune-stimulatory and anti-inflammatory activities of Curcuma longa extract and its polysaccharide fraction,” Pharmacognosy Research , vol. 5, pp. 71–79, 2013. OECD Guidelines for Testing of Chemicals, “Bacterial Reverse Mutation Test, No. 471,” Organisation for Economic Cooperation and Development: Paris, France, 1997. OECD Guidelines for Testing of Chemicals, “In vitro Mammalian Chromosomal Aberration Test, No. 473,” Organisation for Economic Co-operation and Development: Paris, France,1997. OECD Guidelines for Testing of Chemicals, “In vitroMicronucleus Test, No. 487,” Organisation for Economic Co-operation and Development: Paris, France, 2004. OECD Guidelines for Testing of Chemicals, “Acute Oral Toxicity-Fixed Dose Procedure, No 420,” Organisation for Economic Co-operation and Development: Paris, France, 2001. Park JM, Muhoberac BB, Dubin P, Xia, J Effects of Protein Charge Heterogeneity in Protein-Polyelectrolyte Complexation ,Macromolecules, 25:290,1992. Additional Declarations The authors declare no competing interests. Supplementary Files floatimage1.jpeg Graphical Abstract 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-6207332","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427513393,"identity":"6e270f41-31ac-4316-8b2d-d6188bd1ca96","order_by":0,"name":"Washim Raja","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYHACNoYEMH2A8cGHCiDNzNxAtBZmwxlnQFoYidACY0jztoFoAloMjh9/9uBhDkM+f+Phx8a882qj+duBWn5UbMOt5UyOuUHiNgbLGQeOGT6cu+147ozDjA2MPWdu49RidiCHTQKoxYDhwAFjg7fbjuU2ALUwM7bh0XL++TOwFvkDx79J8M45ljufoJYbCWZgLQYHzphJ8jbU5G4gpMX+xhuQFgkDwwNnig1nHDuQuxGo5SA+v0j2pz+T/LnNxkDuxvGNDz7U1OXOO3/44IMfFbi1QIEEEB0AMQ6DuQcIqYcA/gYQWUec4lEwCkbBKBhRAAAA72NAB2kbZQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0005-6062-1567","institution":"Turku Hansda Lapsa Hemram Mahavidyalay","correspondingAuthor":true,"prefix":"","firstName":"Washim","middleName":"","lastName":"Raja","suffix":""}],"badges":[],"createdAt":"2025-03-12 00:13:25","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6207332/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6207332/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78447146,"identity":"4cc13869-7e22-449a-9f42-54b9ae53687c","added_by":"auto","created_at":"2025-03-13 10:15:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82571,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectrum of WE fraction\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6207332/v1/c5c82321f3c5489c11daf0a5.png"},{"id":78446208,"identity":"a1abeb91-c2f5-4737-b098-6ba558886a4c","added_by":"auto","created_at":"2025-03-13 10:07:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":197139,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of WE fraction with BSA\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6207332/v1/12644105471aa534bdc6dd18.png"},{"id":78446207,"identity":"41d8e27e-53d8-465f-8f76-afe6e006e441","added_by":"auto","created_at":"2025-03-13 10:07:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101068,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimental Section section.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6207332/v1/8b4e7ab6d9c55816d8266403.png"},{"id":78448913,"identity":"de643680-09dd-47e1-bb2e-06635a19b349","added_by":"auto","created_at":"2025-03-13 10:39:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":773852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6207332/v1/d309dcf2-e3c3-445f-a284-84c419311b18.pdf"},{"id":78447150,"identity":"3e952292-b4c8-49d3-80cf-ab229337174a","added_by":"auto","created_at":"2025-03-13 10:15:44","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":309389,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6207332/v1/d594b751543bae29fbeb65af.jpeg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eIsolation of Polysaccharides From Curcuma Longa and Its Interaction With Bovine Serum Albumin\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eCurcuma longa\u003c/em\u003e (CL, common name Turmeric), a plant species from the \u003cem\u003eCurcuma\u003c/em\u003e genus (family \u003cem\u003eZingiberaceae\u003c/em\u003e), is common ingredient in many health supplements in Asia.Turmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e) has been used for centuries in Ayurvedic medicine, which amalgamate the medicinal goods of herbs with food. This astonishing herb has established its way into the attention in the west because of its wide range of medicinal benefits [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In \u003cem\u003eayurveda\u003c/em\u003e, rhizome of turmeric is used as medicines against skin, gastrointestinal, respiratory, hepatic, and biliary disorders [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The active constituents of \u003cem\u003eC. longa\u003c/em\u003e are the flavonoid curcumin (diferuloylmethane) and various volatile oils, including tumerone, atlantone, and zingiberene. Other constituents include sugars, proteins, and resins. The best researched active constituent is curcumin,which comprises 0.3\u0026ndash;5.4 percent of raw turmeric [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Components of turmeric especially curcumin has been shown to have anti-inflammatory, antiviral, and anticancer properties [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among the phytoconstituents of \u003cem\u003eC. longa\u003c/em\u003e, curcuminoids are considered as an important active molecule and also exhibited wide range of pharmacological activities. The literature review confirmed plethora of information available on safety aspects of curcumin and essential oil fractions of \u003cem\u003eC.longa\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].While curcuminoids based extract were well studied for their pharmacological and safety aspects, polysaccharide extract of \u003cem\u003eC. longa\u003c/em\u003e is gaining importance since it showed to have various pharmacological activities, which include antidiabetic, antitumour, antidepressant, antioxidant, antimicrobial, antifertility, hepatoprotective, and immunomodulatory properties [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. NR-INF-02, a polysaccharide extract prepared from rhizome of \u003cem\u003eC. longa\u003c/em\u003e had shown clinical efficiency in a randomized placebo controlled study on 120 human patients (37 males and 83 females) affected with primary osteoarthritis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].NR-INF-02 deserved as an effective option for the treatment of patients with primary painful knee and joint pains and also reduced the need of analgesics as a rescue medication [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Also, NR-INF-02 showed immunestimulatory and anti-inflammatory effects in \u003cem\u003ein vitro\u003c/em\u003e models by influencing various cytokines involved in immune regulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, the immunostimulatory effects of \u003cem\u003eC.longa\u003c/em\u003e polysaccharides on peripheral blood mononuclear cells was investigated and the findings revealed the potential use of \u003cem\u003eC.longa\u003c/em\u003e polysaccharide extract as an adjuvant supplement for cancer patients, whose immune activities were suppressed during chemotherapies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].Polysaccharide extract of \u003cem\u003eC.longa\u003c/em\u003e needs evaluation for their safety due to its growing demand on reported preclinical therapeutic indications. Also, before setting of a clinical trial of an herbal product, its safety must be evaluated by toxicity test procedures. Also, this toxicity evaluation is helpful for the estimation of an initial safe starting dose and dose range for the human trials and the identification of parameters for clinical monitoring for potential adverse effects. Hence, the goal of the present investigation is to characterize the toxicological profile of polysaccharide extract from \u003cem\u003eC.longa\u003c/em\u003e rhizome (NR-INF-02).\u003c/p\u003e \u003cp\u003eIn order to evaluate toxicological profile, the ability of NR-INF-02 to induce mutations was assessed in indicator microorganisms using bacterial reverse mutation test. The effect of NR-INF-02 on the genetic system was weighed up by analyzing induced chromosomal aberrations and micronucleus in mammalian cells. An acute oral toxicity study was performed after single oral dose of NR-INF-02 to determine immediate toxic effect. All these tests were conducted based on recommendation of the OECD guidelines [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cp\u003e\u003cstrong\u003ePlant material.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePowdered root of \u003cstrong\u003ecurcuma longa\u003c/strong\u003e were collected from the ayurvedic medical store of Birbhum, West Bengal, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemicals used were of an analytical grade or the best available. All experiments were conducted at least in duplicate. Evaporations were carried out under reduced pressure at around 50\u0026deg;C (SB 1100 Rotary Evaporator; Eyela, Tokyo, Japan). Toluene was used to inhibit microbial growth. Small volumes of samples are freeze-dried (Cool Safe 55-F freeze drier;Scanvac, Lynge, Denmark). Total carbohydrate content were determined by the phenol\u0026ndash;sulfuric acid using galactose as standard. UV\u0026minus;VIS spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan). GC performed with a Shimadzu GC-17A chromatograph (Shimadzu, Kyoto, Japan), fitted with a flame ionization detector, and a DB-225 column (30 m x 0.53 mm i.d.), using a program that maintained an isocratic temperature of 210\u0026deg;C for 18 min and helium as gas vector. GC-MS was performed with a Shimadzu QP 5050A GC-MS instrument (Shimadzu) at 70 eV. Conditions for GC-MS were as described previously.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of carbohydrate polymer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe powder material of \u003cstrong\u003ecurcuma longa\u003c/strong\u003e root \u003cem\u003e(\u003c/em\u003eplant material 10gm) was extracted with distilled water (3 \u0026times; 100 mL, pH 5) at 4\u0026ndash;8\u0026deg;C for 6 h. The insoluble residue was removed by centrifugation (10,000 g, 15 min) and filtration of the supernatant through a glass filter (G 2). The extract was then diluted with 4 volume cold acetone (\u0026times; 3), evaporated to a small volume, and EtOH was added (x 3 volumes). The resulting precipitates were dissolved in H\u003csub\u003e2\u003c/sub\u003eO and lyophilized to yield the water extracted carbohydrate polymer (WE, ~\u0026thinsp;0.55 mg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMonosaccharide Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolysaccharide fraction were hydrolyzed either with 2M trifluoro acetic acid for 3 hr at 100\u003csup\u003eo\u003c/sup\u003eC, centrifuged. Then TFA was removed on rotary evaporator. The presence of monosaccharide in hydrolysate in WE was also indicated by presence spots on TLC. Monosaccharides in the acid hydrolysate were also analyzed by thin layer chromatography on kieselgel 60F plate (Merck) using EtOH/ PhOH/pyridine/0.1 M H3PO4 (5/1/1/2 v/v) as eluent. Carbohydrates were then detected by heating at 100\u0026deg;C after treatment with a saturated solution of aniline phthalate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFT-IR Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIR spectra were obtained on a FT-IR spectrophotometer (JASCO FT-IR-420) using KBr disks containing finely ground samples. The analyses were conducted five times.\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003eBovine serum albumin (BSA)-Carbohydrate Polymer interaction\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eUV\u0026minus;VIS Spectra.\u003c/em\u003e UV-VIS absorption spectra of 1mg/mL BSA alone and in presence of 0.2-2 mg/mL WE in a 10 mM phosphate buffer of pH 7.4 were recorded on a UV-VIS spectrophotometer at room temperature (298 K). The spectra of 0.2-2 mg/mL WE were also recorded under similar condition as blank in order to eliminate the spectral inferences on the BSA-WE system from WE.\u003c/p\u003e\n\u003c/div\u003e\n"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eChemical Characterization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC.longa\u003c/em\u003e Was extracted with H2O, and the extract was subjected to fractional precipitation with EtOH to obtain a precipitate of polysaccharide, precipitate was then lyophilized giving the water extracted carbohydrate polymer (WE).\u003c/p\u003e\n\u003cp\u003eChromatographic analyses indicate the presence of a heteropolysaccharide made up of arabinose and galactose in the water extracted polymer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFT-IR Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFT-IR spectrum of WE\u003csub\u003eP\u003c/sub\u003e fraction showed bands characteristic of polysaccharide. It consisted, inter alia, of (i) a broad band around 3403 cm\u003csup\u003e− 1\u003c/sup\u003e (Fig. 1)originated from stretching vibrations of OH groups in sugar moieties, (ii) a band at 2933 cm\u003csup\u003e− 1\u003c/sup\u003e derived from (C–H) stretching vibrations of methylene or methyl groups, and (iii) a band at 1371 cm\u003csup\u003e− 1\u003c/sup\u003e related to the carbonyl stretching of the carboxylate anion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of carbohydrate polymer on BSA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UV-VIS spectra of BSA (1.0 mg/mL) alone and in the presence of CP (0.05-1 mg/mL) are indicated in Fig. 4 at pH7.4. BSA has two absorption peaks. The peak at 228 nm and 277.5 nm (Fig. 2) is attributed to the π-π* transition of characteristic polypeptide backbone structure and n-π* transition of aromatic amino acids. At pH 7.4 with gradual addition of CP to BSA solution, the intensity of the peak at 228 nm decreases and the λmax for the particles in the solution shifted towards longer wavelength feebly (7.5 nm). These spectral changes might arise from the disturbance of the microenvironment around the polypeptide caused by the binding of WE with BSA. Such complex formation occurs above the isoelectric point (Ip) of BSA (Ip = 4.7) may be attributed to the electrostatic interaction between locally positively charged patches on the protein [24] and negatively charged polysaccharide because of the presence of galacturonic acid.\u003c/p\u003e\n\n"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study has shown that about 0.63% of WE of \u003cem\u003eC.longa\u003c/em\u003e could be obtained by simple water extraction method. Chromatographic analyses indicate the presence of a heteropolysaccharide made up of arabinose and galactose in the water extracted polymer. Further research will be directed towards a more detailed characterization of the purified polysaccharides and their biological activity study will be of interest. In addition, the interaction between bovine serum albumin (BSA) and carbohydrate polymer (WE) has been studied.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;No funding was received to assist with the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDr Washim Raja contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dr Washim Raja. The first draft of the manuscript was written by Dr Washim Raja. Dr Raja read and approved the final manuscript.\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTechnical support from Turku Hansda Lapsa Hemram Mahavidyalay and the University of Burdwan to W. R. is gratefully acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eData availability statements\u003c/h4\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eM. Akram, Shahab-Uddin, A. Ahmed et al., “\u003cem\u003eCurcuma longa \u003c/em\u003eand curcumin: a review article,” \u003cem\u003eRomanian Journal of Biology\u003c/em\u003e,vol. 55, pp. 65–70, 2010.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eThe Ayurvedic Pharmacopoeia of India, part 1\u003c/em\u003e, vol. 8, The Controller of Publications, Civil Lines, Delhi, India, 1st edition,2011.\u003c/li\u003e\n \u003cli\u003eN. Chainani-Wu, “Safety and anti-inflammatory activity of curcumin: a component of tumeric (\u003cem\u003eCurcuma longa\u003c/em\u003e),” \u003cem\u003eJournal of Alternative and Complementary Medicine\u003c/em\u003e, vol. 9, no. 1, pp. 161–168, 2003.\u003c/li\u003e\n \u003cli\u003eM. H. Chen, M. Y. Lee, J. J. Chuang et al., “Curcumin inhibits HCV replication by induction of heme oxygenase-1 and suppression of AKT,” \u003cem\u003eInternational Journal of Molecular Medicine\u003c/em\u003e,\u003c/li\u003e\n \u003cli\u003eJ.-L. Ji, X.-F. Huang, and H.-L. Zhu, “Curcumin and its formulations:potential anti-cancer agents,” \u003cem\u003eAnti-Cancer Agents in Medicinal Chemistry\u003c/em\u003e, vol. 12, no. 3, pp. 210–218, 2012.\u003c/li\u003e\n \u003cli\u003eV. B. Liju, K. Jeena, and R. Kuttan, “Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (\u003cem\u003eCurcuma longa \u003c/em\u003eL),” \u003cem\u003eFood and Chemical Toxicology\u003c/em\u003e, vol. 53, pp. 52–61, 2012.\u003c/li\u003e\n \u003cli\u003eS. Balaji and B. Chempakam, “Toxicity prediction of compounds from turmeric (\u003cem\u003eCurcuma longa \u003c/em\u003eL),” \u003cem\u003eFood and Chemical Toxicology\u003c/em\u003e, vol. 48, no. 10, pp. 2951–2959, 2010.\u003c/li\u003e\n \u003cli\u003eJ. Joshi, S. Ghaisas, A. Vaidya et al., “Early humansafety study of turmeric oil (\u003cem\u003eCurcuma longa \u003c/em\u003eoil) administered orally in healthy volunteers,” \u003cem\u003eJournal of Association of Physicians of India\u003c/em\u003e, vol. 51,pp. 1055–1060, 2003.\u003c/li\u003e\n \u003cli\u003eS. Mohankumar and J. R. McFarlane, “An aqueous extract of \u003cem\u003eCurcuma longa \u003c/em\u003e(turmeric) rhizomes stimulates insulin release and mimics insulin action on tissues involved in glucose homeostasis in vitro,” \u003cem\u003ePhytotherapy Research\u003c/em\u003e, vol. 25, no. 3, pp. 396–401, 2011.\u003c/li\u003e\n \u003cli\u003eS. S.Deshpande, A.D. Ingle, andG. B.Maru, “Chemopreventive efficacy of curcumin-free aqueous turmeric extract in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumorigenesis,” \u003cem\u003eCancer Letters\u003c/em\u003e, vol. 123, no. 1, pp. 35–40, 1998.\u003c/li\u003e\n \u003cli\u003eZ. F. Yu, L. D. Kong, and Y. Chen, “Antidepressant activity of aqueous extracts of \u003cem\u003eCurcuma longa \u003c/em\u003ein mice,” \u003cem\u003eJournal of Ethnopharmacology\u003c/em\u003e, vol. 83, no. 1-2, pp. 161–165, 2002.\u003c/li\u003e\n \u003cli\u003eR. Selvam, L. Subramanian, R. Gayathri, and N. Angayarkanni, “The anti-oxidant activity of turmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e),” \u003cem\u003eJournal of Ethnopharmacology\u003c/em\u003e, vol. 47, no. 2, pp. 59–67, 1995.\u003c/li\u003e\n \u003cli\u003eJ. Anbu Jeba Sunilson, R. Suraj, G. Rejitha, K. Anandarajagopal, A. V. Anita Gnana Kumari, and P. Promwichit, “In vitro antimicrobial evaluation of Zingiber officinale, \u003cem\u003eCurcuma longa \u003c/em\u003eand Alpinia galanga extracts as natural food preservatives,” \u003cem\u003eAmerican Journal of Food Technology\u003c/em\u003e, vol. 4, no. 5, pp. 192–200,2009.\u003c/li\u003e\n \u003cli\u003eR. K.Mishra and S. K. Singh, “Reversible antifertility effect of aqueous rhizome extract of \u003cem\u003eCurcuma longa \u003c/em\u003eL. in male laboratory mice,” \u003cem\u003eContraception\u003c/em\u003e, vol. 79, no. 6, pp. 479–487, 2009.\u003c/li\u003e\n \u003cli\u003eL. Subramanian and R. Selvam, “Prevention of CCl4—Induced hepatotoxicity by aqueous extract of turmeric,” \u003cem\u003eNutritionResearch\u003c/em\u003e, vol. 19, no. 3, pp. 429–441, 1999.\u003c/li\u003e\n \u003cli\u003eG. G. L. Yue, B. C. L. Chan, P.-M. Hon et al., “Immunostimulatoryactivities of polysaccharide extract isolated from \u003cem\u003eCurcumalonga\u003c/em\u003e,” \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e, vol.47, no. 3, pp. 342–347, 2010.\u003c/li\u003e\n \u003cli\u003eK.Madhu, K. Chanda, and Saji, “Safety and efficacy of \u003cem\u003eCurcumalonga \u003c/em\u003eextract in the treatment of painful knee osteoarthritis: arandomized placebo controlled trial,” \u003cem\u003eInflammopharmacology\u003c/em\u003e,vol. 21, pp. 129–136, 2013.\u003c/li\u003e\n \u003cli\u003eC. V. Chandrasekaran, K. Sundarajan, R. J. Edwin et al., “Immune-stimulatory and anti-inflammatory activities of \u003cem\u003eCurcuma longa \u003c/em\u003eextract and its polysaccharide fraction,” \u003cem\u003ePharmacognosy \u003cem\u003eResearch\u003c/em\u003e, vol. 5, pp. 71–79, 2013.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003eOECD Guidelines for Testing of Chemicals, “Bacterial Reverse Mutation Test, No. 471,” Organisation for Economic Cooperation and Development: Paris, France, 1997.\u003c/li\u003e\n \u003cli\u003eOECD Guidelines for Testing of Chemicals, “In vitro Mammalian Chromosomal Aberration Test, No. 473,” Organisation for Economic Co-operation and Development: Paris, France,1997.\u003c/li\u003e\n \u003cli\u003eOECD Guidelines for Testing of Chemicals, “In vitroMicronucleus Test, No. 487,” Organisation for Economic Co-operation and Development: Paris, France, 2004.\u003c/li\u003e\n \u003cli\u003eOECD Guidelines for Testing of Chemicals, “Acute Oral Toxicity-Fixed Dose Procedure, No 420,” Organisation for Economic Co-operation and Development: Paris, France, 2001.\u003c/li\u003e\n \u003cli\u003ePark JM, Muhoberac BB, Dubin P, Xia, J Effects of Protein Charge Heterogeneity in Protein-Polyelectrolyte Complexation ,Macromolecules, 25:290,1992.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Turku Hansda Lapsa Hemram Mahavidyalay","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":"Medicinal Plant, Polysaccharides, BSA, Carbohydrate Polymer","lastPublishedDoi":"10.21203/rs.3.rs-6207332/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6207332/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn traditional medicine most of the diseases have been treated by administration of plant or plant product. \u003cem\u003eCurcuma longa\u003c/em\u003e \u003cem\u003eL. (Zingiberaceae), \u003c/em\u003eis a useful traditional medicinal plant in India. There are several data in the literature indicating a great variety of pharmacological activities of \u003cem\u003eC.longa\u003c/em\u003e \u0026nbsp;which exhibit anti-inflammatory, anti-human immunodeficiency virus, anti-bacteria, antioxidant effects and nematocidal activities.\u003c/p\u003e\n\u003cp\u003eIt is now considered as a valuable source of unique natural products for development of medicines against various diseases and also for the development of industrial products.Our aim is to develop new potent drug candidate. For this we studied the interaction of the polysaccharide with BSA(bovine serum albumin). To study the interaction with polysaccharides and BSA present, I have isolated polysaccharides from depigmented plant materials of \u0026nbsp;\u003cem\u003eC. longa\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eby extraction with cold water. The cold water extracted materials, which were isolated in 3.9 % yield, have been designated as WE. Ultraviolet spectrometric analyses showed that the studied water extracted carbohydrate polymer (WE) interact with BSA.\u003c/p\u003e","manuscriptTitle":"Isolation of Polysaccharides From Curcuma Longa and Its Interaction With Bovine Serum Albumin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-13 10:07:39","doi":"10.21203/rs.3.rs-6207332/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":"7375a5d6-7e85-4ec9-b016-327e27575dc1","owner":[],"postedDate":"March 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45626187,"name":"Natural Product Chemistry"}],"tags":[],"updatedAt":"2025-03-13T10:07:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-13 10:07:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6207332","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6207332","identity":"rs-6207332","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}