Evaluation of Immunomodulatory Activity and Subacute Toxicity of a Novel Non-Cyanide Dried Cassava Mash (DCM)

Evaluation of Immunomodulatory Activity and Subacute Toxicity of a Novel Non-Cyanide Dried Cassava Mash (DCM) | 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 Evaluation of Immunomodulatory Activity and Subacute Toxicity of a Novel Non-Cyanide Dried Cassava Mash (DCM) Olatunji Salako, Frank Orji, Vincent Eze This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7753730/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 Cassava is a staple food with significant nutritional value, but its cyanogenic glycosides pose health risks. This study assesses the novel Non-Cyanide Dried Cassava Mash (DCM) for its immunomodulatory activity and safety. The immunomodulatory effects were evaluated in three experiments using mice, with DCM administered either in drinking water for 8 to 30 days or by intraperitoneal diet for 5 days. Key immunological parameters measured included spleen and thymus mass, cellularity, and antibody-producing cell (APC) counts. A 28-day subacute oral toxicity study in Swiss Albino rats monitored body weight and hematological, hepatic, and renal markers. Oral administration of freshly prepared DCM for 8 days significantly increased spleen cellularity and showed a trend toward higher APC counts. Intraperitoneal administration confirmed a significant immunostimulant effect. No adverse effects were observed. The 28-day toxicity study showed no mortality or significant changes in body weight, organ function, or hematological indices. All parameters remained within normal physiological ranges. DCM demonstrates mild immunostimulatory activity in murine models and is non-toxic following subacute oral administration in rats. These results support the safety and potential of DCM as a functional food ingredient with immunomodulatory benefits. Cassava Immunomodulation Pre-clinical Toxicity Functional Food Spleen Cellularity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Cassava ( Manihot esculenta Crantz ) is a crucial source of carbohydrates for millions of people in tropical regions [ 9 , 10 ]. However, its utilization is limited by the presence of cyanogenic glycosides, primarily linamarin, which can release toxic hydrogen cyanide upon hydrolysis [ 1 , 11 ]. Various processing techniques have been developed to reduce cyanide content; however, these can also impact the nutritional and potential bioactive properties of the final product [ 2 , 12 ]. Beyond its nutritional value, there is growing interest in the bioactivity of cassava-derived compounds. Some studies have suggested that certain components of cassava may affect immune function [ 3 , 13 ]. This study examines a novel Dried Cassava Mash (DCM) processed using specialized technology to eliminate its cyanogenic properties while potentially preserving or enhancing its bioactive components. The objectives of this study were twofold: first, to evaluate the potential immunomodulatory effects of the non-cyanide DCM in a mouse model [ 14 , 15 ], and second, to assess its safety through a standard 28-day subacute oral toxicity study in a rat model [ 5 , 16 ]. 2. Materials and Methods 2.1. Test Material and Physicochemical Characterization Non-Cyanide Dried Cassava Mash (DCM) was produced using proprietary processing. Key physicochemical properties included pH 4.01, density 0.57 g/mL, conductivity 0.159 V, and solubility index 2.37%. 2.2. Immunomodulatory Studies Animals: Laboratory male mice weighing 22-24 g, were obtained from the Laboratory Animal Centre, University of Lagos. They were housed under standard vivarium conditions with a standard diet [18] . Experimental Design: Experiment 1 (Long-term Oral): Mice (n=10/group) had free access to drinking water containing DCM (stored solution) for 30 days. Body weight and immunological parameters were monitored. Experiment 2 (Short-term Oral): Mice (n = 6/group) received a freshly prepared DCM (5g) in their diet for 8 days. Water consumption and immunological parameters were assessed. Experiment 3 (Intraperitoneal): Mice (n=6/group) received a daily intraperitoneal diet of DCM ( 5 g/L/) for 5 days. Immunological Assays: At the endpoint, spleen and thymus were excised and weighed. The cellularity of each organ (number of cells per organ) was determined. The number of antibody-producing cells (APCs) in the spleen was quantified using the method described by Cunningham [4, 21] . Statistical Analysis: Data are presented as Mean ± Standard Error. A Student's t-test was used, with p < 0.05 considered statistically significant. 2.3. Subacute Oral Toxicity Study Animals: Male and female Swiss Albino rats (average weight, 133g) were obtained from the Laboratory Animal Centre, University of Lagos, and acclimated for 7 days prior to the experiment. Study Design: Rats were divided into two groups (n = 5 per sex per group). The test group received a standard diet with DCM for 28 days; controls received a standard diet. Body weight was recorded weekly. Biochemical and Hematological Analysis: After 28 days, blood was collected via ocular puncture under anesthesia. Serum was analyzed for liver function markers (AST, ALT, Bilirubin) and renal function markers (Urea, Creatinine, Total Protein) using standard commercial kits [24, 25] . Hematological indices were also evaluated [26] . Ethical Considerations: The study protocol was approved by the Institutional Animal Care and Use Committee [27] . 3. Results 3.1. Immunomodulatory Effects of DCM Experiment 1:No significant differences in body weight or immunological parameters were observed between the control and DCM groups after 30 days of oral administration[28]. Experiment 2:Mice consuming freshly prepared DCM for 8 days showed a significant increase in spleen cellularity and a non-significant trend towards increased APC count compared to controls (Table 1, Figure 1). Thymus mass and cellularity were unaffected (Figure 2)[29]. No difference in drinking preference was found. Experiment 3:Intraperitoneal administration of DCM resulted in a significant increase in spleen mass, cellularity, and APC content (Table 2, Figure 3)[30]. Thymus parameters were unchanged (Figure 4)[31]. Table 1. Immunological parameters (M ± m) of mice after consumption within 8 days of drinking water with "DCM" Group Animals Spleen Thymus Thymus Group Animals Weight, mg Cellularity, 1×106 АОК, 1×103 Weight, mg Cellularity, 1×106 Control 126±5.6 (100±4.4) 183±8.8 (100±4.8) 265±5.6 (100±2.1) 48.2±2.4 (100±5.0) 123±8.7 (100±7.1) Experiment ( DCM) 136±10.7 (108±8.5) 210±6.3 * (115±3.4) 302±26.0 (114±9.8) 47.2±2.7 (98.0±5.6) 108±7.1 (88.0±5.8) Note: in brackets -% to control; * - significantly significant differences (p <0.05) Table 1. Immunological parameters of mice after 8-day oral consumption of freshly prepared Non-Cyanide Dried Cassava Mash (DCM). Values are presented as Mean ± Standard Error (M ± m). Values in parentheses represent the percentage of the control group.*denotes a statistically significant difference from the control (p < 0.05). APC: Antibody-producing cells. Table 2 . Immunological parameters (M ± m) in mice after 5 days of intraperitoneal “DCM” (5g ) Group Animals Spleen Thymus Thymus Group Animals Weight, g Cellularity, 1×106 АОК, 1×103 Weight, g Cellularity 1×106 Control 105±5,5 (100±5,2) 163±11.0 (100±6.7) 175±31.2 (100±17.8) 35.8±0.8 (100±2.2) 77.3±7.5 (100±9.7) Experiment (DCM) 119±3.6 (113±3.4) 236±21.6* (145±13.3) 283±8.1* (162±4.6) 45.6±4.0 (127±11.2) 98.4±9.5 (127±12.3) Note: in brackets -% to control; - significantly significant differences (p <0.05) Table 2.Immunological parameters of mice after 5-day intraperitoneal administration of Non-Cyanide Dried Cassava Mash (DCM) . Values are presented as Mean ± Standard Error (M ± m). Values in parentheses represent the percentage of the control group.*denotes a statistically significant difference from the control (p < 0.05). APC: Antibody-producing cells. 3.2. Subacute Oral Toxicity Study General Health and Body Weight: All animals survived the 28-day study with no signs of toxicity. Body weight increased steadily in both sexes, with standard weight gain patterns. Biochemical Parameters: Serum levels of liver enzymes (AST, ALT), bilirubin, and renal function markers (Urea, Creatinine) in DCM-fed rats were within normal ranges and showed no statistically significant differences from the control group (Table 3). Hematological Parameters: All hematological indices in the DCM group were within normal limits and comparable to controls. Table 3. Serum biochemistry and hematological parameters of rats after 28-day oral administration of DCM. Group Red Blood Cell (×106/mm3) Haemoglobin (g/dl) Packed Cell Volume (% Platelet (×103/mm3) White Blood Cell (×109/L) Lymphocytes (%) Granulocytes (%) Monocytes (%) Group I 6.21 13.20 37.09 449 6.67 55.96 40.09 2.42 Group II 6.35 14.01 36.10 451 6.12 56.18 42.10 2.26 Group 1: Males Group 2: Female Table 3. Serum biochemistry and hematological parameters of male and female Swiss Albino rats after 28-day oral administration of a standard diet containing Non-Cyanide Dried Cassava Mash (DCM) (Group II) compared to a control diet (Group I). All values were within normal physiological ranges, and no statistically significant differences were observed between groups. AST: Aspartate Aminotransferase; ALT: Alanine Aminotransferase. 4. Discussion This study provides initial evidence of the immunomodulatory potential and safety of a novel Non-Cyanide Dried Cassava Mash. Freshly prepared DCM showed mild immunostimulant activity, primarily indicated by increased spleen cellularity. As a key secondary lymphoid organ, increased spleen cellularity suggests immune system activation. The route of administration had a significant influence on the outcome. The lack of effect in the 30-day oral study (Experiment 1) is likely attributable to the degradation of the active compounds in the stored solution, as suggested by the observed algal growth in the control water but not in the DCM flask, which hints at possible antimicrobial properties [ 34 , 35 ]. The positive results from the short-term oral and intraperitoneal studies (Experiments 2 & 3) confirm that bioactive components are present in freshly prepared DCM and are bioavailable[ 36 , 37 ]. Most importantly, the 28-day subacute toxicity study demonstrated that oral consumption of DCM is safe. The absence of mortality, clinical signs of toxicity, or significant alterations in key biochemical and hematological markers indicates that DCM does not induce hepatotoxicity, nephrotoxicity, or hematological disturbances[ 38 , 39 ]. The normal weight gain pattern further supports its safety as a food ingredient[ 40 ]. A limitation of this study is that the specific immunomodulatory compounds in DCM were not identified. Future work should focus on isolating and characterizing these bioactive molecules, using approaches similar to those employed for other functional foods [ 41 , 42 ]. 5. Conclusion The specially processed Non-Cyanide Dried Cassava Mash (DCM) demonstrates promising immunostimulatory properties in preclinical models and shows an excellent safety profile upon subacute oral administration. These results position DCM not just as a safe food source but as a potential functional food ingredient capable of modulating immune function. Further studies, particularly in immunocompromised models, are warranted to fully elucidate its therapeutic potential. References Bolarinwa, I. F., et al. (2016). A review of cyanogenic glycosides in edible plants. Toxicology Reports , 3, 635-642. Montagnac, J. A., Davis, C. R., & Tanumihardjo, S. A. (2009). Processing techniques to reduce the toxicity and antinutrients of cassava for use as a staple food. Comprehensive Reviews in Food Science and Food Safety , 8(1), 17-27. Martel J, Ko YF, Ojcius DM, Lu CC, Chang CJ, Lin CS, Lai HC, Young JD. Immunomodulatory Properties of Plants and Mushrooms. Trends Pharmacol Sci. 2017 Nov;38(11):967-981. doi: 10.1016/j.tips.2017.07.006. Epub 2017 Aug 30. PMID: 28863984. Cunningham, A. J., & Szenberg, A. (1968). Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology , 14(4), 599-600. OECD. (2008).*Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents*. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing. Parasuraman, S. (2011). Toxicological screening. Journal of Pharmacology and Pharmacotherapeutics , 2(2), 74–79. Granato, D., et al. (2020). Functional foods: Product development, technological trends, efficacy testing, and safety. Annual Review of Food Science and Technology , 11, 93-118. Siró, I., et al. (2008). Functional food. Product development, marketing and consumer acceptance—A review. Appetite , 51(3), 456-467. Howeler, R., Lutaladio, N., & Thomas, G. (2013). Save and Grow: Cassava. A guide to sustainable production intensification . Food and Agriculture Organization of the United Nations (FAO). Burns, A., Gleadow, R., Cliff, J., Zacarias, A. and Cavagnaro, T. (2010) Cassava: The Drought, War and Famine Crop in a Changing World. Sustainability , 2, 3572-3607. https://doi.org/10.3390/su211357. Sornyotha, S., Kyu, K. L., & Ratanakhanokchai, K. (2008). Purification and detection of linamarin from cassava root cortex by high-performance liquid chromatography. Food Chemistry , 104(4), 1750-1754. Khouryieh H, Aramouni F. Physical and sensory characteristics of cookies prepared with flaxseed flour. J Sci Food Agric. 2012 Aug 30;92(11):2366-72. doi: 10.1002/jsfa.5642. Epub 2012 Mar 14. PMID: 22419245. Ekpechi OL, Dimitriadou A, Fraser R. Goitrogenic activity of cassava (a staple Nigerian food). Nature. 1966 Jun 11;210(5041):1137-8. doi: 10.1038/2101137a0. PMID: 5964173. Bastos BL, Dias Portela RW, Dorella FA, Ribeiro D, Seyffert N, et al. (2012) Corynebacterium pseudotuberculosis: Immunological Responses in Animal Models and Zoonotic Potential. J Clin Cell Immunol S4:005 Mestas, J., & Hughes, C. C. (2004). Of mice and not men: differences between mouse and human immunology. The Journal of Immunology , 172(5), 2731-2738. EMEA. (2000). Note for guidance on repeated dose toxicity . CPMP/SWP/1042/99. AOAC. (2016). Official Methods of Analysis of AOAC International (20th ed.). AOAC International. National Research Council (US). (2011). Guide for the Care and Use of Laboratory Animals (8th ed.). National Academies Press. Turner, P. V., et al. (2011). Administration of substances to laboratory animals: routes of administration and factors to consider. Journal of the American Association for Laboratory Animal Science , 50(5), 600-613. Baek, J., Kwak, S., Kim, JY. et al. Evaluation of a novel technique for intraperitoneal injections in mice. Lab Anim 44 , 440–444 (2015). https://doi.org/10.1038/laban.880 Jerne, N. K., & Nordin, A. A. (1963). Plaque formation in agar by single antibody-producing cells. Science , 140(3565), 405-405. Festing, M. F., & Altman, D. G. (2002). Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR Journal , 43(4), 244-258. Balcombe, J. P., Barnard, N. D., & Sandusky, C. (2004). Laboratory routines cause animal stress. Journal of the American Association for Laboratory Animal Science , 43(6), 42-51. Young, D. S. (2001). Effects of Drugs on Clinical Laboratory Tests (5th ed.). AACC Press. Moss, D. W., & Henderson, A. R. (1999). Clinical enzymology. In Tietz Textbook of Clinical Chemistry (3rd ed., pp. 617-721). WB Saunders. Jain, N. C. (1993). Essentials of Veterinary Hematology . Lea & Febiger. Bayne, K., & Turner, P. V. (2019). Animal welfare standards and international collaborations. ILAR Journal , 60(1), 86-94. Calabrese, E. J. (2005). Paradigm lost, paradigm found: the re-emergence of hormesis as a fundamental dose response model in the toxicological sciences. Environmental Pollution , 138(3), 379-411. Cesta, M. F. (2006). Normal structure, function, and histology of the spleen. Toxicologic Pathology , 34(5), 455-465. Kidd, P. (2003). Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Alternative Medicine Review , 8(3), 223-246. Savino, W. (2006). The thymus is a common target organ in infectious diseases. PLoS Pathogens , 2(6), e62. Mebius, R. E., & Kraal, G. (2005). Structure and function of the spleen. Nature Reviews Immunology , 5(8), 606-616. Bronte, V., & Pittet, M. J. (2013). The spleen in local and systemic regulation of immunity. Immunity , 39(5), 806-818. Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews , 12(4), 564-582. Nascimento, G. G., et al. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology , 31(4), 247-256. Dillard, C. J., & German, J. B. (2000). Phytochemicals: nutraceuticals and human health. Journal of the Science of Food and Agriculture , 80(12), 1744-1756. Patra, A. K. (2012). An overview of antimicrobial properties of different classes of phytochemicals. Dietary Phytochemicals and Microbes , 1-32. Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology. 2008 Mar 20;245(3):194-205. doi: 10.1016/j.tox.2007.11.021. Epub 2007 Dec 5. PMID: 18291570. Perazella, M. A. (2009). Renal vulnerability to drug toxicity. Clinical Journal of the American Society of Nephrology , 4(7), 1275-1283. Bailey, S. A., et al. (2004). The use of body weight and body condition as indicators of health and disease in laboratory animals. ILAR Journal , 45(2), 183-190. Strzelec M, Detka J, Mieszczak P, Sobocińska MK and Majka M (2023). Immunomodulation—a general review of the current state-of-the-art and new therapeutic strategies for targeting the immune system. Front. Immunol. 14:1127704. doi: 10.3389/fimmu.2023.1127704 Singh, N., & Yadav, S. S. (2022). A review on immunomodulatory activity of traditional plants. Journal of Ethnopharmacology , 285, 114847. Additional Declarations The authors declare no competing interests. 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. 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08:37:55","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23422,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/dd989a75e9c669ff367cc268.png"},{"id":92577128,"identity":"fb47d066-4ab0-4a4c-8004-0f81fd472a9e","added_by":"auto","created_at":"2025-10-01 08:37:54","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74443,"visible":true,"origin":"","legend":"","description":"","filename":"rs77537300structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/cff2ea28740c2c51a6b2f6d0.xml"},{"id":92577133,"identity":"bed31d5b-590e-43cd-8519-495baee2b534","added_by":"auto","created_at":"2025-10-01 08:37:55","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85093,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/549ba6aaa2927415e41cd2f7.html"},{"id":92577110,"identity":"c11398cb-20b1-405a-a6b3-1cd1acf1efb9","added_by":"auto","created_at":"2025-10-01 08:37:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177721,"visible":true,"origin":"","legend":"\u003cp\u003eSpleen mass, cellularity, and APC content after 8-day oral DCM administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1. Effect of 8-day oral administration of freshly prepared Non-Cyanide Dried Cassava Mash (DCM) on murine spleen parameters. Values represent (A) spleen mass (mg), (B) spleen cellularity (total cells × 10⁶), and (C) number of antibody-producing cells (APC × 10³) in the spleen. Data are presented as Mean ± SEM (n=6 per group) .* denotes a statistically significant difference from the control group (p \u0026lt; 0.05).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/142ee4271ab720e067c0fc59.png"},{"id":92577112,"identity":"42214e1d-cbab-4a22-8934-c51c23be8ec9","added_by":"auto","created_at":"2025-10-01 08:37:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":193610,"visible":true,"origin":"","legend":"\u003cp\u003eThymus mass and cellularity after 8-day oral DCM administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2.Effect of 8-day oral administration of freshly prepared Non-Cyanide Dried Cassava Mash (DCM) on murine thymus parameters. Values represent (A) thymus mass (mg) and (B) thymus cellularity (total cells × 10⁶). Data are presented as Mean ± SEM (n=6 per group). No significant differences were observed between the DCM and control groups (p \u0026gt; 0.05).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/169a649e0fda8a3104f1a74c.png"},{"id":92577114,"identity":"8faf2377-b94d-4d3b-82c0-190a15dde050","added_by":"auto","created_at":"2025-10-01 08:37:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":140596,"visible":true,"origin":"","legend":"\u003cp\u003eSpleen mass, cellularity, and APC content after 5-day intraperitoneal DCM administration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3. Effect of 5-day intraperitoneal administration of Non-Cyanide Dried Cassava Mash (DCM) on murine spleen parameters. Values represent (A) spleen mass (mg), (B) spleen cellularity (total cells × 10⁶), and (C) number of antibody-producing cells (APC × 10³) in the spleen. Data are presented as Mean ± SEM (n=6 per group) .* denotes a statistically significant difference from the control group (p \u0026lt; 0.05).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/d10db616f7f14950278c3d81.png"},{"id":92577118,"identity":"9cba836a-e73e-413b-86f8-bc5b7fbeeec5","added_by":"auto","created_at":"2025-10-01 08:37:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132541,"visible":true,"origin":"","legend":"\u003cp\u003eThymus mass and cellularity after 5-day intraperitoneal DCM administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4. \u003c/strong\u003eEffect of 5-day intraperitoneal administration of Non-Cyanide Dried Cassava Mash (DCM) on murine thymus parameters. Values represent (A) thymus mass (mg) and (B) thymus cellularity (total cells × 10⁶). Data are presented as Mean ± SEM (n=6 per group). No significant differences were observed between the DCM and control groups (p \u0026gt; 0.05)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/ea980fcfb3b9cc263a0e7525.png"},{"id":92578403,"identity":"dc9a5a10-f7bb-4ccc-be71-f3db87f39d14","added_by":"auto","created_at":"2025-10-01 08:45:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":284928,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight progression of male and female rats over the 28-day toxicity study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 5. Body weight progression of male and female Swiss Albino rats over the 28-day subacute oral toxicity study. Rats were fed a standard diet mixed with Non-Cyanide Dried Cassava Mash (DCM) (test group) or a standard diet alone (control group). Data points represent mean body weight, and error bars indicate the Standard Error of the Mean (SEM) (n=5 per sex per group). No significant differences in weight gain were observed between the control and DCM-treated groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/3975859b0b00692cadb674a9.png"},{"id":92578914,"identity":"18a06850-ffea-45bb-94c7-dbd0a3c42668","added_by":"auto","created_at":"2025-10-01 08:53:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2950265,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7753730/v1/940bfdbe-b822-4451-9d98-2a399c36112d.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEvaluation of Immunomodulatory Activity and Subacute Toxicity of a Novel Non-Cyanide Dried Cassava Mash (DCM)\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCassava (\u003cem\u003eManihot esculenta Crantz\u003c/em\u003e) is a crucial source of carbohydrates for millions of people in tropical regions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, its utilization is limited by the presence of cyanogenic glycosides, primarily linamarin, which can release toxic hydrogen cyanide upon hydrolysis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Various processing techniques have been developed to reduce cyanide content; however, these can also impact the nutritional and potential bioactive properties of the final product [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Beyond its nutritional value, there is growing interest in the bioactivity of cassava-derived compounds. Some studies have suggested that certain components of cassava may affect immune function [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This study examines a novel Dried Cassava Mash (DCM) processed using specialized technology to eliminate its cyanogenic properties while potentially preserving or enhancing its bioactive components. The objectives of this study were twofold: first, to evaluate the potential immunomodulatory effects of the non-cyanide DCM in a mouse model [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and second, to assess its safety through a standard 28-day subacute oral toxicity study in a rat model [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Test Material and Physicochemical Characterization\u003c/strong\u003eNon-Cyanide Dried Cassava Mash (DCM) was produced using proprietary processing. Key physicochemical properties included pH 4.01, density 0.57 g/mL, conductivity 0.159 V, and solubility index 2.37%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Immunomodulatory Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAnimals:\u0026nbsp;\u003c/strong\u003eLaboratory male mice weighing 22-24 g, were obtained from the Laboratory Animal Centre, University of Lagos. They were housed under standard vivarium conditions with a standard diet\u003cstrong\u003e[18]\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eExperimental Design:\u003c/strong\u003e\n \u003cul type=\"circle\"\u003e\n \u003cli\u003e\u003cstrong\u003eExperiment 1 (Long-term Oral):\u0026nbsp;\u003c/strong\u003eMice (n=10/group) had free access to drinking water containing DCM (stored solution) for 30 days. Body weight and immunological parameters were monitored.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eExperiment 2 (Short-term Oral):\u0026nbsp;\u003c/strong\u003eMice (n = 6/group) received a freshly prepared DCM (5g) in their diet for 8 days. Water consumption and immunological parameters were assessed.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eExperiment 3 (Intraperitoneal):\u0026nbsp;\u003c/strong\u003eMice (n=6/group) received a daily intraperitoneal diet of DCM ( 5 g/L/) for 5 days.\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eImmunological Assays:\u003c/strong\u003eAt the endpoint, spleen and thymus were excised and weighed. The cellularity of each organ (number of cells per organ) was determined. The number of antibody-producing cells (APCs) in the spleen was quantified using the method described by Cunningham \u003cstrong\u003e[4, 21]\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eStatistical Analysis:\u0026nbsp;\u003c/strong\u003eData are presented as Mean \u0026plusmn; Standard Error. A Student\u0026apos;s t-test was used, with p \u0026lt; 0.05 considered statistically significant.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Subacute Oral Toxicity Study\u003c/strong\u003e\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAnimals:\u0026nbsp;\u003c/strong\u003eMale and female Swiss Albino rats (average weight, 133g) were obtained from the Laboratory Animal Centre, University of Lagos, and acclimated for 7 days prior to the experiment.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eStudy Design:\u0026nbsp;\u003c/strong\u003eRats were divided into two groups (n = 5 per sex per group). The test group received a standard diet with DCM for 28 days; controls received a standard diet. Body weight was recorded weekly.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eBiochemical and Hematological Analysis:\u003c/strong\u003eAfter 28 days, blood was collected via ocular puncture under anesthesia. Serum was analyzed for liver function markers (AST, ALT, Bilirubin) and renal function markers (Urea, Creatinine, Total Protein) using standard commercial kits\u003cstrong\u003e[24, 25]\u003c/strong\u003e. Hematological indices were also evaluated\u003cstrong\u003e[26]\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eEthical Considerations:\u0026nbsp;\u003c/strong\u003eThe study protocol was approved by the Institutional Animal Care and Use Committee \u003cstrong\u003e[27]\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Immunomodulatory Effects of DCM\u003c/strong\u003e\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eExperiment 1:No significant differences in body weight or immunological parameters were observed between the control and DCM groups after 30 days of oral administration[28].\u003c/li\u003e\n \u003cli\u003eExperiment 2:Mice consuming freshly prepared DCM for 8 days showed a significant increase in spleen cellularity and a non-significant trend towards increased APC count compared to controls (Table 1, Figure 1). Thymus mass and cellularity were unaffected (Figure 2)[29]. No difference in drinking preference was found.\u003c/li\u003e\n \u003cli\u003eExperiment 3:Intraperitoneal administration of DCM resulted in a significant increase in spleen mass, cellularity, and APC content (Table 2, Figure 3)[30]. Thymus parameters were unchanged (Figure 4)[31].\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Immunological parameters (M \u0026plusmn; m) of mice after consumption within 8 days of drinking water with \u0026quot;DCM\u0026quot;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup Animals\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpleen\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup Animals\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWeight, mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCellularity, 1\u0026times;106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eАОК, 1\u0026times;103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWeight, mg\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCellularity, 1\u0026times;106\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e126\u0026plusmn;5.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100\u0026plusmn;4.4)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e183\u0026plusmn;8.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100\u0026plusmn;4.8)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e265\u0026plusmn;5.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100\u0026plusmn;2.1)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e48.2\u0026plusmn;2.4\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100\u0026plusmn;5.0)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e123\u0026plusmn;8.7\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100\u0026plusmn;7.1)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eExperiment\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e( DCM)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e136\u0026plusmn;10.7\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(108\u0026plusmn;8.5)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e210\u0026plusmn;6.3\u003cstrong\u003e*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(115\u0026plusmn;3.4)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e302\u0026plusmn;26.0 (114\u0026plusmn;9.8)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e47.2\u0026plusmn;2.7\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(98.0\u0026plusmn;5.6)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e108\u0026plusmn;7.1\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(88.0\u0026plusmn;5.8)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: in brackets -% to control;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*\u003c/strong\u003e - significantly significant differences (p \u0026lt;0.05)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Immunological parameters of mice after 8-day oral consumption of freshly prepared Non-Cyanide Dried Cassava Mash (DCM). Values are presented as Mean \u0026plusmn; Standard Error (M \u0026plusmn; m). Values in parentheses represent the percentage of the control group.*denotes a statistically significant difference from the control (p \u0026lt; 0.05). APC: Antibody-producing cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Immunological parameters (M \u0026plusmn; m) in mice after 5 days of intraperitoneal \u0026ldquo;DCM\u0026rdquo; (5g )\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup Animals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpleen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymus\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThymus\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup Animals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWeight, g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCellularity, 1\u0026times;106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eАОК, 1\u0026times;103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWeight, g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCellularity 1\u0026times;106\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e105\u0026plusmn;5,5\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100\u0026plusmn;5,2)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e163\u0026plusmn;11.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100\u0026plusmn;6.7)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e175\u0026plusmn;31.2 (100\u0026plusmn;17.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35.8\u0026plusmn;0.8 (100\u0026plusmn;2.2)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e77.3\u0026plusmn;7.5\u0026nbsp;\u003cbr\u003e\u0026nbsp;(100\u0026plusmn;9.7)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eExperiment (DCM)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e119\u0026plusmn;3.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(113\u0026plusmn;3.4)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e236\u0026plusmn;21.6*\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(145\u0026plusmn;13.3)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e283\u0026plusmn;8.1* (162\u0026plusmn;4.6)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e45.6\u0026plusmn;4.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(127\u0026plusmn;11.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e98.4\u0026plusmn;9.5\u003c/p\u003e\n \u003cp\u003e(127\u0026plusmn;12.3)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: in brackets -% to control;\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e- significantly significant differences (p \u0026lt;0.05)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.Immunological parameters of mice after 5-day intraperitoneal administration of Non-Cyanide Dried Cassava Mash (DCM) . Values are presented as Mean \u0026plusmn; Standard Error (M \u0026plusmn; m). Values in parentheses represent the percentage of the control group.*denotes a statistically significant difference from the control (p \u0026lt; 0.05). APC: Antibody-producing cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Subacute Oral Toxicity Study\u003c/strong\u003e\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eGeneral Health and Body Weight:\u0026nbsp;\u003c/strong\u003eAll animals survived the 28-day study with no signs of toxicity. Body weight increased steadily in both sexes, with standard weight gain patterns.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eBiochemical Parameters:\u0026nbsp;\u003c/strong\u003eSerum levels of liver enzymes (AST, ALT), bilirubin, and renal function markers (Urea, Creatinine) in DCM-fed rats were within normal ranges and showed no statistically significant differences from the control group (Table 3).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eHematological Parameters: All\u003c/strong\u003e hematological indices in the DCM group were within normal limits and comparable to controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eSerum biochemistry and hematological parameters of rats after 28-day oral administration of DCM.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 46px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRed Blood Cell (\u0026times;106/mm3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHaemoglobin (g/dl)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePacked Cell Volume (%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlatelet (\u0026times;103/mm3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWhite Blood Cell (\u0026times;109/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLymphocytes (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGranulocytes (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonocytes (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e13.20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e37.09\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e55.96\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e40.09\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e14.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e36.10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e56.18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e42.10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 1: Males\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 2: Female\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Serum biochemistry and hematological parameters of male and female Swiss Albino rats after 28-day oral administration of a standard diet containing Non-Cyanide Dried Cassava Mash (DCM) (Group II) compared to a control diet (Group I). All values were within normal physiological ranges, and no statistically significant differences were observed between groups. AST: Aspartate Aminotransferase; ALT: Alanine Aminotransferase.\u003c/strong\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study provides initial evidence of the immunomodulatory potential and safety of a novel Non-Cyanide Dried Cassava Mash. Freshly prepared DCM showed mild immunostimulant activity, primarily indicated by increased spleen cellularity. As a key secondary lymphoid organ, increased spleen cellularity suggests immune system activation.\u003c/p\u003e\u003cp\u003eThe route of administration had a significant influence on the outcome. The lack of effect in the 30-day oral study (Experiment 1) is likely attributable to the degradation of the active compounds in the stored solution, as suggested by the observed algal growth in the control water but not in the DCM flask, which hints at possible antimicrobial properties [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The positive results from the short-term oral and intraperitoneal studies (Experiments 2 \u0026amp; 3) confirm that bioactive components are present in freshly prepared DCM and are bioavailable[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Most importantly, the 28-day subacute toxicity study demonstrated that oral consumption of DCM is safe. The absence of mortality, clinical signs of toxicity, or significant alterations in key biochemical and hematological markers indicates that DCM does not induce hepatotoxicity, nephrotoxicity, or hematological disturbances[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The normal weight gain pattern further supports its safety as a food ingredient[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA limitation of this study is that the specific immunomodulatory compounds in DCM were not identified. Future work should focus on isolating and characterizing these bioactive molecules, using approaches similar to those employed for other functional foods [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe specially processed Non-Cyanide Dried Cassava Mash (DCM) demonstrates promising immunostimulatory properties in preclinical models and shows an excellent safety profile upon subacute oral administration. These results position DCM not just as a safe food source but as a potential functional food ingredient capable of modulating immune function. Further studies, particularly in immunocompromised models, are warranted to fully elucidate its therapeutic potential.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBolarinwa, I. F., et al. (2016). A review of cyanogenic glycosides in edible plants.\u003cem\u003eToxicology Reports\u003c/em\u003e, 3, 635-642.\u003c/li\u003e\n\u003cli\u003eMontagnac, J. A., Davis, C. R., \u0026amp; Tanumihardjo, S. A. (2009). Processing techniques to reduce the toxicity and antinutrients of cassava for use as a staple food.\u003cem\u003eComprehensive Reviews in Food Science and Food Safety\u003c/em\u003e, 8(1), 17-27.\u003c/li\u003e\n\u003cli\u003eMartel J, Ko YF, Ojcius DM, Lu CC, Chang CJ, Lin CS, Lai HC, Young JD. Immunomodulatory Properties of Plants and Mushrooms. Trends Pharmacol Sci. 2017 Nov;38(11):967-981. doi: 10.1016/j.tips.2017.07.006. Epub 2017 Aug 30. PMID: 28863984.\u003c/li\u003e\n\u003cli\u003eCunningham, A. J., \u0026amp; Szenberg, A. (1968). Further improvements in the plaque technique for detecting single antibody-forming cells.\u003cem\u003eImmunology\u003c/em\u003e, 14(4), 599-600.\u003c/li\u003e\n\u003cli\u003eOECD. (2008).*Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents*. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing.\u003c/li\u003e\n\u003cli\u003eParasuraman, S. (2011). Toxicological screening.\u003cem\u003eJournal of Pharmacology and Pharmacotherapeutics\u003c/em\u003e, 2(2), 74\u0026ndash;79.\u003c/li\u003e\n\u003cli\u003eGranato, D., et al. (2020). Functional foods: Product development, technological trends, efficacy testing, and safety.\u003cem\u003eAnnual Review of Food Science and Technology\u003c/em\u003e, 11, 93-118.\u003c/li\u003e\n\u003cli\u003eSir\u0026oacute;, I., et al. (2008). Functional food. Product development, marketing and consumer acceptance\u0026mdash;A review.\u003cem\u003eAppetite\u003c/em\u003e, 51(3), 456-467.\u003c/li\u003e\n\u003cli\u003eHoweler, R., Lutaladio, N., \u0026amp; Thomas, G. (2013).\u003cem\u003eSave and Grow: Cassava. A guide to sustainable production intensification\u003c/em\u003e. Food and Agriculture Organization of the United Nations (FAO).\u003c/li\u003e\n\u003cli\u003eBurns, A., Gleadow, R., Cliff, J., Zacarias, A. and Cavagnaro, T. (2010) Cassava: The Drought, War and Famine Crop in a Changing World.\u003cem\u003eSustainability\u003c/em\u003e, 2, 3572-3607. \u003c/li\u003e\n\u003cli\u003ehttps://doi.org/10.3390/su211357.\u003c/li\u003e\n\u003cli\u003eSornyotha, S., Kyu, K. L., \u0026amp; Ratanakhanokchai, K. (2008). Purification and detection of linamarin from cassava root cortex by high-performance liquid chromatography.\u003cem\u003eFood Chemistry\u003c/em\u003e, 104(4), 1750-1754.\u003c/li\u003e\n\u003cli\u003eKhouryieh H, Aramouni F. Physical and sensory characteristics of cookies prepared with flaxseed flour. J Sci Food Agric. 2012 Aug 30;92(11):2366-72. doi: 10.1002/jsfa.5642. Epub 2012 Mar 14. PMID: 22419245.\u003c/li\u003e\n\u003cli\u003eEkpechi OL, Dimitriadou A, Fraser R. Goitrogenic activity of cassava (a staple Nigerian food). Nature. 1966 Jun 11;210(5041):1137-8. doi: 10.1038/2101137a0. PMID: 5964173.\u003c/li\u003e\n\u003cli\u003eBastos BL, Dias Portela RW, Dorella FA, Ribeiro D, Seyffert N, et al. (2012) Corynebacterium pseudotuberculosis: Immunological Responses in Animal Models and Zoonotic Potential. J Clin Cell Immunol S4:005\u003c/li\u003e\n\u003cli\u003eMestas, J., \u0026amp; Hughes, C. C. (2004). Of mice and not men: differences between mouse and human immunology.\u003cem\u003eThe Journal of Immunology\u003c/em\u003e, 172(5), 2731-2738.\u003c/li\u003e\n\u003cli\u003eEMEA. (2000).\u003cem\u003eNote for guidance on repeated dose toxicity\u003c/em\u003e. CPMP/SWP/1042/99.\u003c/li\u003e\n\u003cli\u003eAOAC. (2016).\u003cem\u003eOfficial Methods of Analysis of AOAC International\u003c/em\u003e(20th ed.). AOAC International.\u003c/li\u003e\n\u003cli\u003eNational Research Council (US). (2011).\u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e(8th ed.). National Academies Press.\u003c/li\u003e\n\u003cli\u003eTurner, P. V., et al. (2011). Administration of substances to laboratory animals: routes of administration and factors to consider.\u003cem\u003eJournal of the American Association for Laboratory Animal Science\u003c/em\u003e, 50(5), 600-613.\u003c/li\u003e\n\u003cli\u003eBaek, J., Kwak, S., Kim, JY.\u003cem\u003eet al.\u003c/em\u003eEvaluation of a novel technique for intraperitoneal injections in mice.\u003cem\u003eLab Anim\u003c/em\u003e\u003cstrong\u003e44\u003c/strong\u003e, 440\u0026ndash;444 (2015). https://doi.org/10.1038/laban.880\u003c/li\u003e\n\u003cli\u003eJerne, N. K., \u0026amp; Nordin, A. A. (1963). Plaque formation in agar by single antibody-producing cells.\u003cem\u003eScience\u003c/em\u003e, 140(3565), 405-405.\u003c/li\u003e\n\u003cli\u003eFesting, M. F., \u0026amp; Altman, D. G. (2002). Guidelines for the design and statistical analysis of experiments using laboratory animals.\u003cem\u003eILAR Journal\u003c/em\u003e, 43(4), 244-258.\u003c/li\u003e\n\u003cli\u003eBalcombe, J. P., Barnard, N. D., \u0026amp; Sandusky, C. (2004). Laboratory routines cause animal stress.\u003cem\u003eJournal of the American Association for Laboratory Animal Science\u003c/em\u003e, 43(6), 42-51.\u003c/li\u003e\n\u003cli\u003eYoung, D. S. (2001).\u003cem\u003eEffects of Drugs on Clinical Laboratory Tests\u003c/em\u003e(5th ed.). AACC Press.\u003c/li\u003e\n\u003cli\u003eMoss, D. W., \u0026amp; Henderson, A. R. (1999). Clinical enzymology. In\u003cem\u003eTietz Textbook of Clinical Chemistry\u003c/em\u003e(3rd ed., pp. 617-721). WB Saunders.\u003c/li\u003e\n\u003cli\u003eJain, N. C. (1993).\u003cem\u003eEssentials of Veterinary Hematology\u003c/em\u003e. Lea \u0026amp; Febiger.\u003c/li\u003e\n\u003cli\u003eBayne, K., \u0026amp; Turner, P. V. (2019). Animal welfare standards and international collaborations.\u003cem\u003eILAR Journal\u003c/em\u003e, 60(1), 86-94.\u003c/li\u003e\n\u003cli\u003eCalabrese, E. J. (2005). Paradigm lost, paradigm found: the re-emergence of hormesis as a fundamental dose response model in the toxicological sciences.\u003cem\u003eEnvironmental Pollution\u003c/em\u003e, 138(3), 379-411.\u003c/li\u003e\n\u003cli\u003eCesta, M. F. (2006). Normal structure, function, and histology of the spleen.\u003cem\u003eToxicologic Pathology\u003c/em\u003e, 34(5), 455-465.\u003c/li\u003e\n\u003cli\u003eKidd, P. (2003). Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease.\u003cem\u003eAlternative Medicine Review\u003c/em\u003e, 8(3), 223-246.\u003c/li\u003e\n\u003cli\u003eSavino, W. (2006). The thymus is a common target organ in infectious diseases.\u003cem\u003ePLoS Pathogens\u003c/em\u003e, 2(6), e62.\u003c/li\u003e\n\u003cli\u003eMebius, R. E., \u0026amp; Kraal, G. (2005). Structure and function of the spleen.\u003cem\u003eNature Reviews Immunology\u003c/em\u003e, 5(8), 606-616.\u003c/li\u003e\n\u003cli\u003eBronte, V., \u0026amp; Pittet, M. J. (2013). The spleen in local and systemic regulation of immunity.\u003cem\u003eImmunity\u003c/em\u003e, 39(5), 806-818.\u003c/li\u003e\n\u003cli\u003eCowan, M. M. (1999). Plant products as antimicrobial agents.\u003cem\u003eClinical Microbiology Reviews\u003c/em\u003e, 12(4), 564-582.\u003c/li\u003e\n\u003cli\u003eNascimento, G. G., et al. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria.\u003cem\u003eBrazilian Journal of Microbiology\u003c/em\u003e, 31(4), 247-256.\u003c/li\u003e\n\u003cli\u003eDillard, C. J., \u0026amp; German, J. B. (2000). Phytochemicals: nutraceuticals and human health.\u003cem\u003eJournal of the Science of Food and Agriculture\u003c/em\u003e, 80(12), 1744-1756.\u003c/li\u003e\n\u003cli\u003ePatra, A. K. (2012). An overview of antimicrobial properties of different classes of phytochemicals.\u003cem\u003eDietary Phytochemicals and Microbes\u003c/em\u003e, 1-32.\u003c/li\u003e\n\u003cli\u003eOzer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology. 2008 Mar 20;245(3):194-205. doi: 10.1016/j.tox.2007.11.021. Epub 2007 Dec 5. PMID: 18291570.\u003c/li\u003e\n\u003cli\u003ePerazella, M. A. (2009). Renal vulnerability to drug toxicity.\u003cem\u003eClinical Journal of the American Society of Nephrology\u003c/em\u003e, 4(7), 1275-1283.\u003c/li\u003e\n\u003cli\u003eBailey, S. A., et al. (2004). The use of body weight and body condition as indicators of health and disease in laboratory animals.\u003cem\u003eILAR Journal\u003c/em\u003e, 45(2), 183-190.\u003c/li\u003e\n\u003cli\u003eStrzelec M, Detka J, Mieszczak P, Sobocińska MK and Majka M (2023). Immunomodulation\u0026mdash;a general review of the current state-of-the-art and new therapeutic strategies for targeting the immune system.\u003cem\u003eFront. Immunol.\u003c/em\u003e14:1127704. doi: 10.3389/fimmu.2023.1127704\u003c/li\u003e\n\u003cli\u003eSingh, N., \u0026amp; Yadav, S. S. (2022). A review on immunomodulatory activity of traditional plants.\u003cem\u003eJournal of Ethnopharmacology\u003c/em\u003e, 285, 114847.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Cassava, Immunomodulation, Pre-clinical, Toxicity, Functional Food, Spleen Cellularity","lastPublishedDoi":"10.21203/rs.3.rs-7753730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7753730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCassava is a staple food with significant nutritional value, but its cyanogenic glycosides pose health risks. This study assesses the novel Non-Cyanide Dried Cassava Mash (DCM) for its immunomodulatory activity and safety. The immunomodulatory effects were evaluated in three experiments using mice, with DCM administered either in drinking water for 8 to 30 days or by intraperitoneal diet for 5 days. Key immunological parameters measured included spleen and thymus mass, cellularity, and antibody-producing cell (APC) counts. A 28-day subacute oral toxicity study in Swiss Albino rats monitored body weight and hematological, hepatic, and renal markers. Oral administration of freshly prepared DCM for 8 days significantly increased spleen cellularity and showed a trend toward higher APC counts. Intraperitoneal administration confirmed a significant immunostimulant effect. No adverse effects were observed. The 28-day toxicity study showed no mortality or significant changes in body weight, organ function, or hematological indices. All parameters remained within normal physiological ranges. DCM demonstrates mild immunostimulatory activity in murine models and is non-toxic following subacute oral administration in rats. These results support the safety and potential of DCM as a functional food ingredient with immunomodulatory benefits.\u003c/p\u003e","manuscriptTitle":"Evaluation of Immunomodulatory Activity and Subacute Toxicity of a Novel Non-Cyanide Dried Cassava Mash (DCM)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 08:37:49","doi":"10.21203/rs.3.rs-7753730/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":"228e3b7c-46aa-49ba-8039-3662bd478ffc","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-01T08:37:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-01 08:37:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7753730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7753730","identity":"rs-7753730","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}