Moringa oleifera Lam.-Enriched Diet Boosts Serum Protein Levels Independently of Dietary Protein Intake

Moringa oleifera Lam.-Enriched Diet Boosts Serum Protein Levels Independently of Dietary Protein Intake | 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 Moringa oleifera Lam. -Enriched Diet Boosts Serum Protein Levels Independently of Dietary Protein Intake Pradeep B. Patil, Parimala Alduri, Gudepu Purushottam, Ashok Devarasetti, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5905107/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 Moringa oleifera Lam. leaves (MOL) have been a staple food source in India for centuries. Recent reports have highlighted their potential as immunomodulators, hepatoprotectants, and more. To verify these claims, we conducted a study aimed at shedding light on the nutritional benefits of MOL consumption. This six-week study employed an isocaloric and isonitrogenous feeding trial, using a pelleted diet with 20% protein, supplemented with either 2% or 4% MOL. Healthy adult male rats were subjected to a unique forced exercise regimen. We analyzed the biochemical parameters of 30 rats distributed across five groups. The results showed that the test groups had significantly lower levels of serum urea and liver enzymes (AST, ALT, and ALP) compared to the control group. Total protein levels increased significantly (14–19%) in all test groups, with no significant difference in creatinine levels. This analysis concludes that administering MOL powder orally at doses of 2% and 4% is biochemically safe and exhibits liver-protective and nephroprotective properties. In conclusion, our study suggests that MOL powder can be safely incorporated into both human and animal diets in a dose-dependent manner. Additionally, the synergistic effect of exercise enhances these benefits. Notably, our unique model provides long-term results without altering animal behavior, making MOL a promising option for combating protein-energy malnutrition and malnutrition in India. Animal Science Nutrition & Dietetics Food Science & Technology Moringa oleifera Lam. swimming forced-exercise model hepato-protective nephroprotective Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Moringa oleifera Lam. (MO) hails from the Moringaceae family [ 1 – 4 ] and originates from the Indian subcontinent. Its uses span from traditional herbal medicine [ 5 ] to a vegetable [ 6 ] and staple food in various countries, including India. Recent scientific reports emphasize the nutritional richness of dried Moringa oleifera Lam. leaf (MOL) powder [ 2 ], which falls into the superfood category due to its protein (19–35%), metabolizable energy (2273–2978 kcal/kg), fat (2.3–10%), crude fiber (9.14%), vitamins (A, B, C, and E), minerals (0.6–11.2%) - calcium (3.65%), phosphorus (0.3%), magnesium (0.5%), potassium (1.5%), sodium (0.164%), Sulphur (0.63%), zinc (13.03 mg/kg), copper (8.25%), manganese (86.8 mg/kg), iron (490 mg/kg) and selenium (363 mg/kg)[ 7 ], and essential amino acids content [ 8 ]. Furthermore, MOL contains various secondary plant compounds [ 1 , 2 ] and bioactive substances (such as beta-sitosterol, caffeoylquinic acid, kaempferol, quercetin, and zeatin, as well as alkaloids, reducing sugars, steroidal aglycones, tannins, and terpenoids) known for their anti-inflammatory, antibacterial, antioxidant, anti-cancer, hepatoprotective, and neuroprotective properties [ 2 , 4 ]. Protein energy malnutrition (PEM) is a pressing concern, particularly in India, affecting a significant portion of the population in terms of disease susceptibility, severity and mortality [ 9 ], especially children under five [ 10 ]. PEM contributes to both noncommunicable [ 11 , 12 ] and communicable [ 13 , 14 ] diseases, making it a priority for researchers and policymakers. MOL has been traditionally used as a medicinal plant [ 15 – 18 ] to address various health issues [ 19 – 21 ], but validation is an ongoing process. The phytoconstituents of Moringa oleifera (Shigru in Sanskrit) are rich and diverse, contributing to its extensive therapeutic benefits. According to Rasa-Panchaka, the plant is characterized by a Katu (pungent) and Tikta (bitter) Rasa (taste), with Guna (qualities) described as Laghu (light), Ruksha (dry), and Teekshna (sharp). It possesses Ushna (hot) Veerya (potency) and Katu Vipaka (pungent post-digestive effect). Its Doshaghnata (dosha-balancing properties) primarily pacifies Vaata and Kapha doshas. The plant exhibits various therapeutic actions (Karma), including Deepana (digestive stimulant), Hrudya (heart tonic), Vidahakruta (causing mild heat), Vishgna (detoxifying), Shukrala (enhancing reproductive health), Chakushya (improving vision), and Vaataghna (relieving Vata-related disorders) [ 17 ]. Moringa oleifera is a versatile tree with numerous medicinal applications, derived from various parts of the plant. The roots are used as an antilithic, rubefacient, vesicant, and stimulant, offering benefits such as anti-inflammatory and antifertility effects and treating conditions like rheumatism, kidney pain, and constipation. The leaves serve as a purgative and are applied for ailments like headaches, bronchitis, and sore throats, while the stem bark helps with eye diseases, spleen enlargement, tumors, and ulcers. The gum is employed in treating dental caries, fevers, and intestinal issues and is mixed with sesame oil for various therapeutic uses. Overall, Moringa oleifera exhibits diverse pharmacological activities, including antihypertensive, hepatoprotective, and antitumor properties, making it a valuable resource in traditional and modern medicine[ 18 ]. Shigru is recognized in Ayurvedic literature as one of the few herbs that exhibit both Balya (nourishing) and Medohara (anti-obesity) qualities [ 22 ] Shigru is also widely available and therapeutically potent remedy in diverse external therapeutic applications, a comprehensive literature survey revealed 149 formulations of Shigru, presented in 145 dosage forms, recommended for the treatment of 24 different diseases [ 23 ]. Shigru (Moringa oleifera) is a powerful antioxidant herb that protects the body from free radicals and helps combat various infections. Renowned for its exceptional nutritional value, it also boasts a broad spectrum of therapeutic applications [ 17 ]. Therefore, our experiment aimed to explore the hepatoprotective and nephroprotective properties of MOL while developing lean mass. Additionally, we investigated through review of literature for the potential of vegetarian diets using moringa leaves to use as alternative protein source where we found adequate evidences supporting its use for protein built up in fish study [ 24 ] and cattle study [ 25 ]. In addition it also provides evidence that it has low amount of anti-nutritional factors which helps in meat quality[ 26 ]. Therefore, in this manuscript we looked at its capacity to enhance the serum protein levels, independent of their protein content. Our study used a unique forced exercised rodent model with an isocaloric and isonitrogenous diet. Procedure The animal experiment was conducted at ICMR-NIN, Hyderabad, and was approved by IAEC (ICMR-NIN/IAEC-IV/02/001/2020). Male SD/NIN rats (n = 30), 3 months (12 weeks) old, were used in the study. The rats were acclimatized to a reversed day-night cycle (12hr:12hr) and forced swimming by housing them in an experimental room for 7 days prior to the feeding trial. Randomization of the rats was based on their body weight, and they were housed individually in open typed cages with netted floors. Environment was controlled as per CCSEA guidelines (Temperature 20 ± 2 0 C and relative humidity (45–55%). Ad libitum food and water were provided to the rats, and their food intake and body weight were measured weekly. Daily inspections of the cages were conducted, and blood samples were taken three times (‘0’, ‘21’ and ‘42’ days) during the experimental phase. Plant material collection and Preparation of Diet The dried MOL powder was procured from a local supplier [Medikonda Nutrients, Hyderabad, Telangana (Head Office: 17275 Old Tobacco Rd, Lutz, Florida 33558 USA)] which was shade-dried. The admixture of MOL in the feed was determined based on the proximate analysis of the powder. A standard protocol was followed to prepare pelleted feed, where raw materials were weighed and mechanically mixed for 15 minutes using a Hardcore Mixer™. The pellets were prepared using a pellet machine (Model No. 120 - Sanjeevani Agro-Machinery, Nagpur) with a mesh size of 6 mm and were then placed on sterilisation trays. The pellets were sterilised in a double-door autoclave (Steri-Horizontal Rectangular Steam Autoclave, Yorco Steriliser, Gaziabad) at 121ºC for 20 minutes and subsequently dried at 100ºC for 3 hours in a pellet dryer (IDS-48 Trays-Drier, Industrial Drying Systems, Madras). A study examining extraction temperatures (25–200°C) on Moringa leaf powder found that myricetin and kaempferol peaked at 100°C (2699 mg/kg and 3440 mg/kg, respectively) but decreased at 150°C. Quercetin levels, however, remained stable (1429–1488 mg/kg) across all temperatures [ 27 ]. The dried pellets were aseptically weighed, packed, and stored in a cold room until further use. In this study, to check the quality of feed both autoclaved and unautoclaved feed samples from three diet groups were sent for analysis about protein using Kjeldahl method (IS7219), quercetin and chlorogenic acid quantification using GCLC/MS and UV methods through NABL accredited lab. The protein levels in a conventional rodent diet (20%) remained unchanged when crafting a customized diet by incorporating 2% and 4% MOL powder. This adjustment was made without any alterations to the calorie content (355Kcal/100gm), fiber content (4%), or protein content (20%). The formulation of this custom diet was guided by the proximate analysis of MOL powder, as depicted in Table No. 1. A comprehensive breakdown of the custom feed composition (Standard maintenance diet and Moringa-enriched pelleted diet composition for rodents) can be found in Table No. 2. Diet Regimen The animals were housed individually in open type grilled bottom cage where diet was fed (standard and pelleted diet) to animals of respective groups during complete duration of the study. Daily 30 grams of diet was provided and remaining diet was measured to calculate feed intake. Exercise interventions Exercise interventions Based on rationale that exercise may have synergistic positive impact during feeding trial on body, two groups out of the five in the study design (Table No. 3) were required to undergo forced exercise. Therefore, twelve animals (2% and 4% MOL) from Group-3 and Group-5 were subjected to forced (intensive free-swimming) exercise for 30 minutes each day, six days a week, at a water temperature of 30 ± 5C. Determination of Biochemical Parameters After 6 weeks, the rats, including both the control and MOL-fed groups, were fasted for 16 hours. Blood was collected from retro-orbital plexus for serum separation, which was performed by centrifugation, and aliquots were stored at -20°C temperature until biochemical profiling. The serum samples were used to determine kidney function tests, including serum creatinine and serum urea, as well as liver function tests, such as serum alkaline phosphatase (ALP), serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and serum total protein (TP), using an automated biochemical analyser (COBAS, model C-311 analyser, Roche, Switzerland) at the National Institute of Nutrition, Hyderabad − 500007, India (ICMR-NIN). Then animals were euthanised using CO 2 asphyxiation and vital tissues were collected along with muscles for histology (data not published) and caecum of gut microbes ( In press ). All analytical kits were obtained from Roche, Switzerland, to ensure consistency in testing. Statistical methods The analysis of the experimental data was performed using SPSS statistical software (version-20th ). A one-way analysis of variance (ANOVA test) with a general linear model was used to analyze the results. Tukey's test was used to classify the means of the groups at a significance level of (P < 0.05). Results The proximate analysis of MOL indicated high levels of dietary fiber (31.4%) and protein (26.62%), as presented in Table 1. The chemical profile of Moringa oliefera Lam . was assessed through GCLC/MS and UV methods for Chlorogenic acid and Quercetin. Chlorogenic acid is a polyphenol compound found in coffee, fruits, and vegetables, known for its antioxidant, anti-inflammatory, blood sugar regulation, weight management, and potential anti-cancer properties. Quercetin is a potent antioxidant, anti-inflammatory, cardiovascular support, immune booster, anti-cancer agent, natural antihistamine, and exercise enhancer. Using this information, we formulated customized diets for the study. During the experimental period, there were no notable variations in feed intake or weight gain across all groups, as outlined in Tables 4 and 5. Nevertheless, a distinctive trend was observed, as depicted in Figs. 1 and 2. The Moringa-fed groups (Groups 2–5) exhibited an increase in feed intake (9.0%-18.2%) compared to the normal group (Group 1). Additionally, the forced exercise groups (Group 3 and Group 5) showed higher feed intake (18.2% and 17.5% respectively) compared to the non-exercise Moringa diet groups (Group 2: 9.0% and Group 4: 11.0%). Notably, significant changes in other biochemical parameters were observed and are explained below. The tables (Table no 6–11) show the results of evaluating the effect of the MOL diet on certain biochemical parameters of the rats at day '0', '21' and '42'. At day '0', there was no significant (P < 0.05) difference in the mean serum urea, AST, ALT, ALP, and serum total protein levels. However, a significant (P < 0.05) difference was observed between the levels on days ‘21’ and ‘42’. The MOL-based groups (Gr. 2–5) showed a significant decrease in serum urea levels compared to the control group (Gr. 1), with the rats in 4% Moringa enriched pelleted diet with forced exercise (4M-FE) group (Gr.5) having the lowest levels. The mean serum creatinine levels did not show a significant (P > 0.05) difference between all groups in different time periods. The mean values of liver enzymes AST, ALT, and ALP were significantly (P < 0.05) lower in all MOL-fortified test groups (Gr. 2–5) than in the control group. The rats in the MOL with exercise group had the lowest AST, ALT, and ALP levels. Mean serum total protein levels were highest in all MOL-enriched groups compared to the control group, with the rats in the 4% MOL with the exercise group having the highest serum total protein levels. All MOL-enriched diet groups (Gr. 2–5) exhibited a significant and consistent decrease in serum urea by 29–36%, whereas the control group showed an increase of 2.16% over a 6-week period (Fig. 3). The MOL-fed groups demonstrated a uniform decrease in AST (by 8–17%) and ALT (by 19.5–25.8%), while the control group showed a slight change in AST (2.1%) and ALT (1.5%) over a 6-week period (Figs. 4 & 5). The MOL-enriched diet groups also showed a significant and consistent decrease in serum ALP (by 22–35%), whereas the control group showed an increase of 7.4% over a 6-week period (Fig. 6). Conversely, serum total protein levels increased (by 14–19%) in all MOL groups, regardless of exercise, while water consumption was not measured in the study. These findings validate the results of a previous study [ 28 ], but in this case, we are presenting the dose-dependent outcomes of MOL powder instead of extracts. These findings suggest that liver function is normal and water consumption during exercise did not influence the results of the study, as two MOL groups without exercise (2M-WOE and 4M-WOE) yielded similar outcomes (Fig. 7). The impact of autoclaving was also checked on diet formulated through analyzing the protein, quercetin, and chlorogenic acid content (Fig. 8) in diet before and after autoclaving. The observed changes in the nutritional and bioactive profiles due to autoclaving can be attributed to its impact on moisture content and compound stability. Autoclaving significantly reduces the moisture content in the diets, as evidenced by the drop from 14% in the unautoclaved samples to 4% in the autoclaved ones. This reduction in moisture leads to a relative concentration of certain compounds, such as chlorogenic acid and quercetin, in the autoclaved Moringa-enriched diets (MED). The increase in chlorogenic acid content could be due to the release of bound forms or the conversion of precursor compounds during high-temperature processing. The protein content showed minimal changes across all diets and conditions. In the 2% MED, autoclaving resulted in a slight increase in protein content (from 13.29–15.86%), potentially attributed to moisture reduction concentrating the protein. A similar trend was observed in the standard control diet, where protein increased slightly from 13.98–14.06% post-autoclaving. Interestingly, the 4% MED displayed a decrease in protein content post-autoclaving (from 14.8–13.82%), suggesting that other factors, such as thermal denaturation or interaction with bioactive compounds, might influence protein stability at higher Moringa concentrations. Quercetin, a sensitive bioactive compound, displayed significant degradation due to autoclaving. In the 2% MED, quercetin levels dropped from 232.83 mg/g in the unautoclaved diet to 82.27 mg/g in the autoclaved diet, indicating the susceptibility of quercetin to thermal degradation despite the moisture-driven concentration effect. In the 4% MED, quercetin levels followed an unexpected trend, increasing from 27.33 mg/g in the unautoclaved diet to 42.91 mg/g in the autoclaved diet. This increase might be attributed to the enhanced release of bound quercetin forms at higher Moringa concentrations, which partially counteracted the degradation caused by autoclaving. The standard control diet exhibited low quercetin levels, with substantial reductions observed post-autoclaving (39.47 mg/g to 12.86 mg/g), emphasizing the protective effects of Moringa bioactives against thermal stress. Chlorogenic acid, another key bioactive compound, showed a remarkable increase in the autoclaved Moringa-enriched diets. In the 2% MED, chlorogenic acid content rose from 8.18 mg/g in the unautoclaved diet to 22.84 mg/g in the autoclaved diet, while in the 4% MED, it increased from 62.36 mg/g to 76.28 mg/g. These increases suggest that autoclaving might facilitate the release of bound chlorogenic acid or enhance its formation from precursor compounds during high-temperature processing. In contrast, the standard control diet exhibited negligible chlorogenic acid levels, with complete degradation observed post-autoclaving (2.03 mg/g to 0 mg/g), further emphasizing the importance of Moringa enrichment in preserving or enhancing bioactives during processing. The consistent reduction in moisture content from 14–4% across all diets due to autoclaving highlights its impact on the concentration and stability of bioactive compounds. While moisture loss concentrates certain compounds, thermal stress induces degradation, particularly for sensitive components like quercetin. Discussion Our study not only reaffirms the medicinal benefits of MOL but also highlights its potential to enhance serum protein levels. This increase falls within the normal range, indicating untapped potential for healthy individuals and potential benefits for those with protein-energy malnutrition (PEM). Previous research has shown that MOL supplementation can improve various aspects of animal health, such as meat quality, body weight gain, feed conversion ratio, antioxidant levels, and more in various animal models [ 29 , 30 ]. Overall, it positively affects anthropometric growth [ 31 ], as well as improves gut microbe [ 32 ]. Our study supports these findings and suggests that MOL can enhance food intake and assimilation efficiency, particularly when combined with exercise. The study also investigated the effects of MOL on liver and kidney function, demonstrating significant differences in biomarkers. These findings along with other data on makers for kidney damage (unpublished) suggest that MOL may protect against kidney and liver diseases and promote overall health. In contrast, groups with 2% MOL had a 7.7-fold increase in feed intake, and those with 4% MOL had a 9.2-fold increase. Forced exercise resulted in a 6-8-fold increase in feed intake in both MOL-enriched and control groups. While weight gain was not significant across all groups, it was 52–56% lower in MOL groups without exercise and 33 − 28% lower in MOL groups with forced exercise. The decrease in serum urea was significant and consistent in MOL-enriched groups by 29–36% but increased by 2.16% in the control group over a 6-week period (Fig. 1–2). These findings suggest that incorporating MOL in the diet, whether 2% or 4%, improves food intake and assimilation efficiency, with exercise providing a positive synergistic effect. This could be attributed to the antioxidative and gut microbiota-improving properties of MOL (unpublished data), as well as the release of vital organ potentials, such as those in the liver, gut, and kidneys. The objective of this experiment was to investigate the effects of MOL powder on liver and kidney function in rats by evaluating the activities of liver enzymes and kidney function tests, which serve as biomarkers for liver and kidney function. The results indicated significant differences in the markers for liver and kidney function. The available literature also demonstrated a mechanism by which MOL may protect against kidney disease by mitigating various pathological factors associated with the condition, such as inflammation and oxidative stress [ 33 ]. Additionally, it is evident that as the body adapts to exercise, as there is a noticeable decrease in urea levels. The lower mean serum urea levels (as shown in Table 6) were observed in MOL-enriched group with forced exercise (Gr. 4–5), followed by MOL-enriched group without forced exercise (Gr.2–3), indicating of reduced protein catabolism, which implies enhanced endurance. The mean serum creatinine levels did not show any significant differences between the groups at different time points. Literature suggested that the protective properties of MOL against kidney and liver tissue damage, such as anti-apoptotic, antioxidant, and anti-oxidative stress effects [ 34 ], may mitigate or reverse the effects of increased serum levels of ALT, AST, creatinine, and urea, as well as significantly decreased levels of total proteins, albumin, and globulin. Additionally, literature suggest that the hepatoprotective and antidiabetic effects of MO have been attributed to the phenolic bioactive compounds present in the methanolic leaf extracts of MO [ 35 ]. The animals from 4M-FE group exhibited the lowest mean values of AST (Table: 8), ALT (Table: 9), and ALP (Table: 10), followed by 2M-FE group, indicating the anti-apoptotic and antioxidant properties of MO. These properties are associated with increased endurance performance and restoration of liver enzymes and liver tissue to normal levels, which is consistent with previous research [ 36 ]. ALP plays a critical role in calcium homeostasis, and elevated calcium levels are associated with various dental and skeletal disorders. However, the present study demonstrated that MO has a significant effect on ALP levels, reducing it significantly (by 27–50%). The reduction in ALP not only increases bone mineral content but also decreases the risk of kidney stones. Although there was no significant change in body weight gain, an increase in lean mass, bone density, and bone content was observed in the MOL groups, which could be attributed to the reduced ALP activities. In addition to our unpublished findings demonstrating a positive alteration in gut microorganisms, several other studies have indicated that shifts in gut flora diversity and gene expression, both upregulation and downregulation, may be associated with the favorable increase in lean muscle mass [ 37 – 41 ]. The 4M-FE group (Gr.5) exhibited high serum total protein content on average (Table No. 11), followed by 2M-FE group (Gr. 3), indicating that the consumption of 4% MOL as a protein diet with 30minutes forced exercise significantly increased total protein content. The same trend was observed for 2% MOL with forced exercise, suggesting that trained muscles' ability to utilize protein as an energy source was enhanced, leading to an increase in lean muscle mass (unpublished data). This significant increase in total protein content is a crucial factor in enhancing swimming performance without fatigue. This finding is consistent with other studies [ 35 , 36 , 42 ]. The 4M-FE and 4M-WOE had 19% and 17% increase in serum total protein respectively. Similar pattern was observed in 2M-FE and 2M-WOE by 14% increase in serum total protein compared to standard diet (control group). These results suggest that forced exercise can only lead to better outcomes than an additional group if the diet includes a sufficient dose of MOL, which is dose-dependent. However, 2M-FE did not improve better than 2M-WOE, which may be due to inadequate MOL causing anabolism com-catabolism of proteins. The protein digestibility usually increases after autoclaving [ 43 ]. Based on this results study on PEM animal model will be performed. Interestingly, the significant increase in total serum protein without an increase in dietary protein suggests that a high-protein diet may not be necessary for improving anthropometric indices. Instead, measures to promote protein absorption, assimilation, and homeostasis should be emphasized. This positive balance of serum proteins can help address issues related to protein-energy malnutrition, anthropometric failure, and communicable diseases such as tuberculosis. Additionally, the decrease in urea and ALP enzymes observed in the study can benefit individuals with kidney and liver diseases, as well as iatrogenic hyperphosphatemia in childhood, dental caries, and postmenopausal mineral deficiency. Mass awareness campaigns, such as the Moringa Festival Week, could promote its use through the media. MOL-Ladoo made from dried MOL and dates could provide a simple solution to address malnutrition in all age groups. In the end as a quality check on diet the analysis on diet samples of unautoclaved and autoclaved feed samples were analyzed showed the contrasting trends in quercetin and chlorogenic acid levels between Moringa-enriched and standard diets highlight the dual effects of autoclaving. Moringa supplementation provides a protective and enhancing effect, reducing the extent of thermal degradation for some bioactives while facilitating the release or formation of others. However, the sensitivity of specific compounds to thermal processing underscores the need for optimizing autoclaving conditions to preserve the functional benefits of Moringa-enriched diets. These findings suggest that Moringa's bioactive compounds not only enrich the diet but also mitigate the adverse effects of processing, making it a promising component in nutritionally enhanced diets. Conclusion Our study highlights a critical insight: a 2% Moringa oleifera (MOL) diet is adequate to induce significant physiological changes in a sedentary lifestyle, effectively meeting dietary needs without the need for higher supplementation. However, when paired with physical exercise, the 4% MOL diet demonstrates a remarkable synergy, enhancing serum protein levels by 2%, showcasing its superior benefits in active individuals. This underscores the dynamic role of MOL in optimizing endurance, protecting liver and kidney health, and promoting muscle strength, making it an unparalleled choice for vegetarians seeking to build muscle while maintaining dietary thresholds. Contrary to preconceived notions about taste and utility, MOL emerges as a dietary powerhouse deserving widespread adoption. With strategic mass awareness campaigns, its potential to combat malnutrition across various demographics, especially in vulnerable age groups, becomes undeniable. This study reinforces the call for MOL's resurgence as a cornerstone in both human and animal nutrition. Abbreviations MOL - Moringa oleifera Lam. dried leaves powder PEM – Protein Energy Malnutrition 2M-WOE – 2% Moringa oleifera Lam. dried leaves in diet without forced exercise 4M-WOE – 4% Moringa oleifera Lam. dried leaves in diet without forced exercise 2M-FE - 2% Moringa oleifera Lam. dried leaves in diet with forced exercise 4M-FE - 4% Moringa oleifera Lam. dried leaves in diet with forced exercise ** P>0.05, no significant difference. *** P<0.05, a b Means with different superscripts in a column differ significantly. Declarations Conflict of interest: All authors in this manuscript are not having any conflict of interest in publishing the data. References Martínez-González CL et al (2017) Moringa oleifera, a species with potential analgesic and anti-inflammatory activities. Biomed Pharmacother 87:482–488 Paikra BK, Dhongade HKJ, Gidwani B (2017) Phytochemistry and Pharmacology of Moringa oleifera Lam. J Pharmacopunct 20(3):194–200 Sadek KM et al (2017) The chemo-prophylactic efficacy of an ethanol Moringa oleifera leaf extract against hepatocellular carcinoma in rats. Pharm Biol 55(1):1458–1466 Nwidu LL et al (2018) Vitro Anti-Cholinesterase and Antioxidant Activity of Extracts of Moringa oleifera Plants from Rivers State, Niger Delta, Nigeria, vol 5. Medicines (Basel), 3 López M et al (2018) Effects of Moringa oleifera leaf powder on metabolic syndrome induced in male Wistar rats: a preliminary study. J Int Med Res 46(8):3327–3336 Atawodi SE et al (2010) Evaluation of the polyphenol content and antioxidant properties of methanol extracts of the leaves, stem, and root barks of Moringa oleifera Lam. J Med Food 13(3):710–716 Moyo B et al (2011) Nutritional characterization of Moringa (Moringa oleifera Lam.) leaves. 10(60):12925–12933 Lamou B et al (2016) Antioxidant and Antifatigue Properties of the Aqueous Extract of Moringa oleifera in Rats Subjected to Forced Swimming Endurance Test. Oxid Med Cell Longev, 2016: p. 3517824 Gonakoti S, Osifo IF (2021) Protein-Energy Malnutrition Increases Mortality in Patients Hospitalized With Bacterial Pneumonia: A Retrospective Nationwide Database Analysis. Cureus 13(1):e12645 Bhutia DT (2014) Protein energy malnutrition in India: the plight of our under five children. J Family Med Prim Care 3(1):63–67 Brahmbhatt SR, Brahmbhatt RM, Boyages SC (2001) Impact of protein energy malnutrition on thyroid size in an iodine deficient population of Gujarat (India): Is it an aetiological factor for goiter? Eur J Endocrinol 145(1):11–17 Mohammed S et al (2023) Gestational low dietary protein induces intrauterine inflammation and alters the programming of adiposity and insulin sensitivity in the adult offspring. J Nutr Biochem 116:109330 Taylor AK et al (2013) Protein energy malnutrition decreases immunity and increases susceptibility to influenza infection in mice. J Infect Dis 207(3):501–510 Schaible UE, Kaufmann SH (2007) Malnutrition and infection: complex mechanisms and global impacts. PLoS Med 4(5):e115 Sonewane K et al (2022) Pharmacological, ethnomedicinal, and evidence-based comparative review of Moringa oleifera Lam.(Shigru) and its potential role. Manage malnutrition tribal Reg India especially Chhattisgarh 8(3):314–338 Gavadiya SK, Sharma T (2022) J.J.o.I.S.o.M. Bapna, External applications of Shigru (Moringa oleifera Lam): A comprehensive review . 10(4):241–250 Kumar M et al (2023) A Review on Nutritive and Medicinal Importance of Shigru (Moringa Oleifera Lam). 6(7):132–143 Maurya B et al (2021) Moringa oleifera (Shigru): A Miraculous Medicinal Plant with Many Therapeutic Benefits. 3(1): pp. 1–7 Azad SB et al (2017) Anti-hyperglycaemic activity of Moringa oleifera is partly mediated by carbohydrase inhibition and glucose-fibre binding. Biosci Rep, 37(3) Zeng B et al (2018) Effects of Moringa oleifera silage on milk yield, nutrient digestibility and serum biochemical indexes of lactating dairy cows. J Anim Physiol Anim Nutr (Berl) 102(1):75–81 Aremu A et al (2018) Methanolic leaf extract of Moringa oleifera improves the survivability rate, weight gain and histopathological changes of Wister rats infected with Trypanosoma brucei. Int J Vet Sci Med 6(1):39–44 Sonewane K et al (2022) Pharmacological, Ethnomedicinal, and Evidence-Based Comparative Review of Moringa oleifera Lam. (Shigru) and Its Potential Role in the Management of Malnutrition in Tribal Regions of India, Especially Chhattisgarh. World J Traditional Chin Med, 8(3) Gavadiya SK, Sharma T, Bapna VM (2022) External applications of Shigru (Moringa oleifera Lam): A comprehensive review. J Indian Syst Med, 10(4) Richter N, Siddhuraju P, Becker K (2003) Evaluation of nutritional quality of moringa (Moringa oleifera Lam.) leaves as an alternative protein source for Nile tilapia (Oreochromis niloticus L). Aquaculture 217(1):599–611 Mendieta-Araica B et al (2011) Moringa (Moringa oleifera) leaf meal as a source of protein in locally produced concentrates for dairy cows fed low protein diets in tropical areas. Livest Sci 137(1):10–17 Su B (2020) X.J.F.i.v.s. Chen. Curr status potential Moringa oleifera leaf as Altern protein source Anim feeds 7:53 Matshediso PG, Cukrowska E, Chimuka L (2015) Development of pressurised hot water extraction (PHWE) for essential compounds from Moringa oleifera leaf extracts. Food Chem 172:423–427 Ghasi S, Nwobodo E, Ofili JO (2000) Hypocholesterolemic effects of crude extract of leaf of Moringa oleifera Lam in high-fat diet fed wistar rats. J Ethnopharmacol 69(1):21–25 Selim S et al (2021) Impact of Dietary Supplementation with Moringa oleifera Leaves on Performance, Meat Characteristics, Oxidative Stability, and Fatty Acid Profile in Growing Rabbits. Anim (Basel), 11(2) Cui YM et al (2018) Effect of dietary supplementation with Moringa oleifera leaf on performance, meat quality, and oxidative stability of meat in broilers. Poult Sci 97(8):2836–2844 Sun B et al (2018) Effects of Moringa oleifera leaves as a substitute for alfalfa meal on nutrient digestibility, growth performance, carcass trait, meat quality, antioxidant capacity and biochemical parameters of rabbits. J Anim Physiol Anim Nutr (Berl) 102(1):194–203 Abu Hafsa SH et al (2020) Effect of dietary Moringa oleifera leaves on the performance, ileal microbiota and antioxidative status of broiler chickens. J Anim Physiol Anim Nutr (Berl) 104(2):529–538 Tsopanakis C, Tsopanakis A (1998) Stress hormonal factors, fatigue, and antioxidant responses to prolonged speed driving. Pharmacol Biochem Behav 60(3):747–751 Soliman MM et al (2020) The ameliorative impacts of Moringa oleifera leaf extract against oxidative stress and methotrexate-induced hepato-renal dysfunction. Biomed Pharmacother 128:110259 Muzumbukilwa WT, Nlooto M, Owira PMO (2019) Hepatoprotective effects of Moringa oleifera Lam (Moringaceae) leaf extracts in streptozotocin-induced diabetes in rats. J Funct Foods 57:75–82 Saleh SS, E.R.J.I.J.o.F M, Sarhat, Toxicology (2019) Effects of ethanolic Moringa oleifera extract on melatonin, liver and kidney function tests in alloxan-induced diabetic rats. 13(4):1015–1019 Moyo B et al (2012) Effect of supplementing crossbred Xhosa lop-eared goat castrates with Moringa oleifera leaves on growth performance, carcass and non-carcass characteristics. 44:801–809 Nkukwana T et al (2014) The effect of Moringa oleifera leaf meal supplementation on tibia strength, morphology and inorganic content of broiler chickens. 44(3):228–239 Cohen-Zinder M et al (2017) Dietary supplementation of Moringa oleifera silage increases meat tenderness of Assaf lambs. 151: pp. 110–116 Sebola N et al (2018) Comparison of meat quality parameters in three chicken strains fed Moringa oleifera leaf meal-based diets. 27(3):332–340 El-Rahman A et al (2019) Effect of Moringa leaves (Moringa oleifera Lam.) extract addition on luncheon meat quality. 46(6):2307–2316 Omara M et al (2018) Effects of supplementing rabbit diets with Moringa oleifera dry leaves at different levels on their productive performance. 21(2):443–453 Fadhilatunnur H, R.T.K.J.C.J.o.F S, Dewi, Technology (2021) Evaluation vitro protein digestibility moringa oleifera leaves Var Domest Cook 13(1) Tables Tables 1 to 11 are available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files tablerevisd.docx 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-5905107","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":407200231,"identity":"ed11e56f-e2e7-429b-a89d-35d1d822bcda","order_by":0,"name":"Pradeep B. Patil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYJACCTB5mIHxAQPDAWI0MMO1MBuQqOUAA5sEUVoMjvcfvF25454833HeY9U8NXfk+BmYHz66gU/LmcPMlmfPFBvOPMyXdpvn2DNjyQY2Y+McPFokZySzSTa2JTBuOMxjdpuH7XDihgM8bNJ4tcx/DNZiD9JSzPOPCC38EsxgLYkgLcy8bcRo4Uk2tmw8k5AM9Euy5Ny+w8aSzQT8wsZ+8OHNxh0Jtn3nzx788ObbYTl+9uaHj/FpAQPGBhDJw8DEA6KZCSlH1sL4gxjVo2AUjIJRMOIAAEmvTfjwl+h9AAAAAElFTkSuQmCC","orcid":"","institution":"Scientist D, ICMR-NIN, Hyderabad","correspondingAuthor":true,"prefix":"","firstName":"Pradeep","middleName":"B.","lastName":"Patil","suffix":""},{"id":407200232,"identity":"d1bdf0fd-cc31-42d4-a1b7-5d809414d5f2","order_by":1,"name":"Parimala Alduri","email":"","orcid":"","institution":"PG Student, Department of Veterinary Anatomy, College of Veterinary Science, Hyderabad, TS, India - 007","correspondingAuthor":false,"prefix":"","firstName":"Parimala","middleName":"","lastName":"Alduri","suffix":""},{"id":407200233,"identity":"f9cc139f-6b06-46a8-a8ef-ebdfe466a826","order_by":2,"name":"Gudepu Purushottam","email":"","orcid":"","institution":"Department of Veterinary Anatomy, College of Veterinary Science, Hyderabad, TS, India - 007","correspondingAuthor":false,"prefix":"","firstName":"Gudepu","middleName":"","lastName":"Purushottam","suffix":""},{"id":407200234,"identity":"8badf55d-a5c6-4818-a046-349113a0bac5","order_by":3,"name":"Ashok Devarasetti","email":"","orcid":"","institution":"Department of Veterinary Biochemistry, College of Veterinary Science, Hyderabad, TS, India - 007","correspondingAuthor":false,"prefix":"","firstName":"Ashok","middleName":"","lastName":"Devarasetti","suffix":""},{"id":407200235,"identity":"1f65df64-4d04-4ce8-b5c9-ccf30c8956ae","order_by":4,"name":"Kumar reddy","email":"","orcid":"","institution":"ICMR-NIN, Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"Kumar","middleName":"","lastName":"reddy","suffix":""},{"id":407200236,"identity":"0b105b9a-c86e-4c47-a99c-9c02d71e9077","order_by":5,"name":"Swapnil Deshmukh","email":"","orcid":"","institution":"ICMR-NIN, Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"Swapnil","middleName":"","lastName":"Deshmukh","suffix":""},{"id":407200237,"identity":"f65e92cf-e3f6-439d-9b32-95cf74063e02","order_by":6,"name":"Sreenivasa Rao J","email":"","orcid":"","institution":"ICMR-NIN, Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"Sreenivasa","middleName":"Rao","lastName":"J","suffix":""},{"id":407200238,"identity":"e486a4d8-ec72-4a79-a0fe-e6fb90bd3c29","order_by":7,"name":"SSYH Qadri","email":"","orcid":"","institution":"ICMR-NIN, Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"SSYH","middleName":"","lastName":"Qadri","suffix":""},{"id":407200239,"identity":"7ee4caf8-c911-4b5e-8432-2be23cbd319c","order_by":8,"name":"B. Tulja","email":"","orcid":"","institution":"ICMR-NIN, Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"B.","middleName":"","lastName":"Tulja","suffix":""}],"badges":[],"createdAt":"2025-01-26 07:50:42","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5905107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5905107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75688181,"identity":"42cede2e-cd06-467b-a42e-1280d598146d","added_by":"auto","created_at":"2025-02-07 06:46:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1016440,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0001.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/aa1bbd2ba30f373429835882.png"},{"id":75688179,"identity":"028dd398-e4e7-40c6-82a6-2aac6f8bfbb4","added_by":"auto","created_at":"2025-02-07 06:46:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":899200,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0002.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/b92cedf3163bd53cdb7df02b.png"},{"id":75688187,"identity":"9c0cbed2-6117-4f10-884e-4c2b79046aee","added_by":"auto","created_at":"2025-02-07 06:46:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1045641,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0003.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/fcf81cb1069c54b9ea8fb6e1.png"},{"id":75688185,"identity":"48d73563-db05-4f18-a031-373ca464edf0","added_by":"auto","created_at":"2025-02-07 06:46:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":867251,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0004.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/71face81cd3a0c6ac06efd72.png"},{"id":75689451,"identity":"1190c66b-762c-47c8-b7e8-28a80150e480","added_by":"auto","created_at":"2025-02-07 07:02:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":942595,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0005.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/59af3c2a640a18a530c8e021.png"},{"id":75689167,"identity":"98419d4b-db81-4b49-b9d0-855cafbc469d","added_by":"auto","created_at":"2025-02-07 06:54:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1039658,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0006.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/d9750e38db07939df68397b4.png"},{"id":75688189,"identity":"90b0c5de-0bc0-4eff-bf45-db2d766e650e","added_by":"auto","created_at":"2025-02-07 06:46:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":810069,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0007.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/6ba62a645bc9118086486672.png"},{"id":75688186,"identity":"19207eb7-3fd5-4686-bc0a-9dc5c46e0ff5","added_by":"auto","created_at":"2025-02-07 06:46:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1020506,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figuresrevised19122024page0008.png","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/03144c95955aad08626d580d.png"},{"id":75689452,"identity":"528696dc-eab3-4615-ac04-c1d128bd4686","added_by":"auto","created_at":"2025-02-07 07:02:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9138145,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/c1f3fe09-8de9-4c1c-9033-9086e2cc8bef.pdf"},{"id":75688176,"identity":"3d22414c-1e16-4316-a4ba-3379536ee145","added_by":"auto","created_at":"2025-02-07 06:46:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":82067,"visible":true,"origin":"","legend":"","description":"","filename":"tablerevisd.docx","url":"https://assets-eu.researchsquare.com/files/rs-5905107/v1/aa282458a7640b750cc43a8b.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMoringa oleifera Lam.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Enriched Diet Boosts Serum Protein Levels Independently of Dietary Protein Intake\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eMoringa oleifera Lam.\u003c/em\u003e (MO) hails from the Moringaceae family [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and originates from the Indian subcontinent. Its uses span from traditional herbal medicine [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] to a vegetable [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and staple food in various countries, including India.\u003c/p\u003e \u003cp\u003eRecent scientific reports emphasize the nutritional richness of dried \u003cem\u003eMoringa oleifera Lam.\u003c/em\u003e leaf (MOL) powder [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], which falls into the superfood category due to its protein (19\u0026ndash;35%), metabolizable energy (2273\u0026ndash;2978 kcal/kg), fat (2.3\u0026ndash;10%), crude fiber (9.14%), vitamins (A, B, C, and E), minerals (0.6\u0026ndash;11.2%) - calcium (3.65%), phosphorus (0.3%), magnesium (0.5%), potassium (1.5%), sodium (0.164%), Sulphur (0.63%), zinc (13.03 mg/kg), copper (8.25%), manganese (86.8 mg/kg), iron (490 mg/kg) and selenium (363 mg/kg)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and essential amino acids content [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, MOL contains various secondary plant compounds [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and bioactive substances (such as beta-sitosterol, caffeoylquinic acid, kaempferol, quercetin, and zeatin, as well as alkaloids, reducing sugars, steroidal aglycones, tannins, and terpenoids) known for their anti-inflammatory, antibacterial, antioxidant, anti-cancer, hepatoprotective, and neuroprotective properties [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein energy malnutrition (PEM) is a pressing concern, particularly in India, affecting a significant portion of the population in terms of disease susceptibility, severity and mortality [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], especially children under five [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. PEM contributes to both noncommunicable [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and communicable [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] diseases, making it a priority for researchers and policymakers. MOL has been traditionally used as a medicinal plant [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] to address various health issues [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], but validation is an ongoing process. The phytoconstituents of Moringa oleifera (Shigru in Sanskrit) are rich and diverse, contributing to its extensive therapeutic benefits. According to Rasa-Panchaka, the plant is characterized by a Katu (pungent) and Tikta (bitter) Rasa (taste), with Guna (qualities) described as Laghu (light), Ruksha (dry), and Teekshna (sharp). It possesses Ushna (hot) Veerya (potency) and Katu Vipaka (pungent post-digestive effect). Its Doshaghnata (dosha-balancing properties) primarily pacifies Vaata and Kapha doshas. The plant exhibits various therapeutic actions (Karma), including Deepana (digestive stimulant), Hrudya (heart tonic), Vidahakruta (causing mild heat), Vishgna (detoxifying), Shukrala (enhancing reproductive health), Chakushya (improving vision), and Vaataghna (relieving Vata-related disorders) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moringa oleifera is a versatile tree with numerous medicinal applications, derived from various parts of the plant. The roots are used as an antilithic, rubefacient, vesicant, and stimulant, offering benefits such as anti-inflammatory and antifertility effects and treating conditions like rheumatism, kidney pain, and constipation. The leaves serve as a purgative and are applied for ailments like headaches, bronchitis, and sore throats, while the stem bark helps with eye diseases, spleen enlargement, tumors, and ulcers. The gum is employed in treating dental caries, fevers, and intestinal issues and is mixed with sesame oil for various therapeutic uses. Overall, Moringa oleifera exhibits diverse pharmacological activities, including antihypertensive, hepatoprotective, and antitumor properties, making it a valuable resource in traditional and modern medicine[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eShigru is recognized in Ayurvedic literature as one of the few herbs that exhibit both Balya (nourishing) and Medohara (anti-obesity) qualities [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Shigru is also widely available and therapeutically potent remedy in diverse external therapeutic applications, a comprehensive literature survey revealed 149 formulations of Shigru, presented in 145 dosage forms, recommended for the treatment of 24 different diseases [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Shigru (Moringa oleifera) is a powerful antioxidant herb that protects the body from free radicals and helps combat various infections. Renowned for its exceptional nutritional value, it also boasts a broad spectrum of therapeutic applications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, our experiment aimed to explore the hepatoprotective and nephroprotective properties of MOL while developing lean mass.\u003c/p\u003e \u003cp\u003eAdditionally, we investigated through review of literature for the potential of vegetarian diets using moringa leaves to use as alternative protein source where we found adequate evidences supporting its use for protein built up in fish study [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and cattle study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition it also provides evidence that it has low amount of anti-nutritional factors which helps in meat quality[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, in this manuscript we looked at its capacity to enhance the serum protein levels, independent of their protein content. Our study used a unique forced exercised rodent model with an isocaloric and isonitrogenous diet.\u003c/p\u003e"},{"header":"Procedure","content":"\u003cp\u003e The animal experiment was conducted at ICMR-NIN, Hyderabad, and was approved by IAEC (ICMR-NIN/IAEC-IV/02/001/2020). Male SD/NIN rats (n\u0026thinsp;=\u0026thinsp;30), 3 months (12 weeks) old, were used in the study. The rats were acclimatized to a reversed day-night cycle (12hr:12hr) and forced swimming by housing them in an experimental room for 7 days prior to the feeding trial. Randomization of the rats was based on their body weight, and they were housed individually in open typed cages with netted floors. Environment was controlled as per CCSEA guidelines (Temperature 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003e0\u003c/sup\u003eC and relative humidity (45\u0026ndash;55%). \u003cem\u003eAd libitum\u003c/em\u003e food and water were provided to the rats, and their food intake and body weight were measured weekly. Daily inspections of the cages were conducted, and blood samples were taken three times (\u0026lsquo;0\u0026rsquo;, \u0026lsquo;21\u0026rsquo; and \u0026lsquo;42\u0026rsquo; days) during the experimental phase.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material collection and Preparation of Diet\u003c/h2\u003e \u003cp\u003eThe dried MOL powder was procured from a local supplier [Medikonda Nutrients, Hyderabad, Telangana (Head Office: 17275 Old Tobacco Rd, Lutz, Florida 33558 USA)] which was shade-dried. The admixture of MOL in the feed was determined based on the proximate analysis of the powder. A standard protocol was followed to prepare pelleted feed, where raw materials were weighed and mechanically mixed for 15 minutes using a Hardcore Mixer\u0026trade;. The pellets were prepared using a pellet machine (Model No. 120 - Sanjeevani Agro-Machinery, Nagpur) with a mesh size of 6 mm and were then placed on sterilisation trays. The pellets were sterilised in a double-door autoclave (Steri-Horizontal Rectangular Steam Autoclave, Yorco Steriliser, Gaziabad) at 121\u0026ordm;C for 20 minutes and subsequently dried at 100\u0026ordm;C for 3 hours in a pellet dryer (IDS-48 Trays-Drier, Industrial Drying Systems, Madras). A study examining extraction temperatures (25\u0026ndash;200\u0026deg;C) on Moringa leaf powder found that myricetin and kaempferol peaked at 100\u0026deg;C (2699 mg/kg and 3440 mg/kg, respectively) but decreased at 150\u0026deg;C. Quercetin levels, however, remained stable (1429\u0026ndash;1488 mg/kg) across all temperatures [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The dried pellets were aseptically weighed, packed, and stored in a cold room until further use. In this study, to check the quality of feed both autoclaved and unautoclaved feed samples from three diet groups were sent for analysis about protein using Kjeldahl method (IS7219), quercetin and chlorogenic acid quantification using GCLC/MS and UV methods through NABL accredited lab.\u003c/p\u003e \u003cp\u003eThe protein levels in a conventional rodent diet (20%) remained unchanged when crafting a customized diet by incorporating 2% and 4% MOL powder. This adjustment was made without any alterations to the calorie content (355Kcal/100gm), fiber content (4%), or protein content (20%). The formulation of this custom diet was guided by the proximate analysis of MOL powder, as depicted in Table No. 1. A comprehensive breakdown of the custom feed composition (Standard maintenance diet and Moringa-enriched pelleted diet composition for rodents) can be found in Table No. 2.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDiet Regimen\u003c/h3\u003e\n\u003cp\u003eThe animals were housed individually in open type grilled bottom cage where diet was fed (standard and pelleted diet) to animals of respective groups during complete duration of the study. Daily 30 grams of diet was provided and remaining diet was measured to calculate feed intake.\u003c/p\u003e\n\u003ch3\u003eExercise interventions\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eExercise interventions\u003c/div\u003e \u003cp\u003eBased on rationale that exercise may have synergistic positive impact during feeding trial on body, two groups out of the five in the study design (Table No. 3) were required to undergo forced exercise. Therefore, twelve animals (2% and 4% MOL) from Group-3 and Group-5 were subjected to forced (intensive free-swimming) exercise for 30 minutes each day, six days a week, at a water temperature of 30\u0026thinsp;\u0026plusmn;\u0026thinsp;5C.\u003c/p\u003e\n\u003ch3\u003eDetermination of Biochemical Parameters\u003c/h3\u003e\n\u003cp\u003eAfter 6 weeks, the rats, including both the control and MOL-fed groups, were fasted for 16 hours. Blood was collected from retro-orbital plexus for serum separation, which was performed by centrifugation, and aliquots were stored at -20\u0026deg;C temperature until biochemical profiling. The serum samples were used to determine kidney function tests, including serum creatinine and serum urea, as well as liver function tests, such as serum alkaline phosphatase (ALP), serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and serum total protein (TP), using an automated biochemical analyser (COBAS, model C-311 analyser, Roche, Switzerland) at the National Institute of Nutrition, Hyderabad \u0026minus;\u0026thinsp;500007, India (ICMR-NIN). Then animals were euthanised using CO\u003csub\u003e2\u003c/sub\u003e asphyxiation and vital tissues were collected along with muscles for histology (data not published) and caecum of gut microbes (\u003cem\u003eIn press\u003c/em\u003e). All analytical kits were obtained from Roche, Switzerland, to ensure consistency in testing.\u003c/p\u003e\n\u003ch3\u003eStatistical methods\u003c/h3\u003e\n\u003cp\u003eThe analysis of the experimental data was performed using SPSS statistical software (version-20th ). A one-way analysis of variance (ANOVA test) with a general linear model was used to analyze the results. Tukey's test was used to classify the means of the groups at a significance level of (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe proximate analysis of MOL indicated high levels of dietary fiber (31.4%) and protein (26.62%), as presented in Table\u0026nbsp;1. The chemical profile of \u003cem\u003eMoringa oliefera Lam\u003c/em\u003e. was assessed through GCLC/MS and UV methods for Chlorogenic acid and Quercetin. Chlorogenic acid is a polyphenol compound found in coffee, fruits, and vegetables, known for its antioxidant, anti-inflammatory, blood sugar regulation, weight management, and potential anti-cancer properties. Quercetin is a potent antioxidant, anti-inflammatory, cardiovascular support, immune booster, anti-cancer agent, natural antihistamine, and exercise enhancer.\u003c/p\u003e \u003cp\u003eUsing this information, we formulated customized diets for the study. During the experimental period, there were no notable variations in feed intake or weight gain across all groups, as outlined in Tables\u0026nbsp;4 and 5. Nevertheless, a distinctive trend was observed, as depicted in Figs.\u0026nbsp;1 and 2. The Moringa-fed groups (Groups 2\u0026ndash;5) exhibited an increase in feed intake (9.0%-18.2%) compared to the normal group (Group 1). Additionally, the forced exercise groups (Group 3 and Group 5) showed higher feed intake (18.2% and 17.5% respectively) compared to the non-exercise Moringa diet groups (Group 2: 9.0% and Group 4: 11.0%). Notably, significant changes in other biochemical parameters were observed and are explained below.\u003c/p\u003e \u003cp\u003eThe tables (Table no 6\u0026ndash;11) show the results of evaluating the effect of the MOL diet on certain biochemical parameters of the rats at day '0', '21' and '42'. At day '0', there was no significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) difference in the mean serum urea, AST, ALT, ALP, and serum total protein levels. However, a significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) difference was observed between the levels on days \u0026lsquo;21\u0026rsquo; and \u0026lsquo;42\u0026rsquo;. The MOL-based groups (Gr. 2\u0026ndash;5) showed a significant decrease in serum urea levels compared to the control group (Gr. 1), with the rats in 4% Moringa enriched pelleted diet with forced exercise (4M-FE) group (Gr.5) having the lowest levels. The mean serum creatinine levels did not show a significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) difference between all groups in different time periods.\u003c/p\u003e \u003cp\u003eThe mean values of liver enzymes AST, ALT, and ALP were significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower in all MOL-fortified test groups (Gr. 2\u0026ndash;5) than in the control group. The rats in the MOL with exercise group had the lowest AST, ALT, and ALP levels. Mean serum total protein levels were highest in all MOL-enriched groups compared to the control group, with the rats in the 4% MOL with the exercise group having the highest serum total protein levels.\u003c/p\u003e \u003cp\u003eAll MOL-enriched diet groups (Gr. 2\u0026ndash;5) exhibited a significant and consistent decrease in serum urea by 29\u0026ndash;36%, whereas the control group showed an increase of 2.16% over a 6-week period (Fig.\u0026nbsp;3). The MOL-fed groups demonstrated a uniform decrease in AST (by 8\u0026ndash;17%) and ALT (by 19.5\u0026ndash;25.8%), while the control group showed a slight change in AST (2.1%) and ALT (1.5%) over a 6-week period (Figs.\u0026nbsp;4 \u0026amp; 5). The MOL-enriched diet groups also showed a significant and consistent decrease in serum ALP (by 22\u0026ndash;35%), whereas the control group showed an increase of 7.4% over a 6-week period (Fig.\u0026nbsp;6). Conversely, serum total protein levels increased (by 14\u0026ndash;19%) in all MOL groups, regardless of exercise, while water consumption was not measured in the study. These findings validate the results of a previous study [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but in this case, we are presenting the dose-dependent outcomes of MOL powder instead of extracts. These findings suggest that liver function is normal and water consumption during exercise did not influence the results of the study, as two MOL groups without exercise (2M-WOE and 4M-WOE) yielded similar outcomes (Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eThe impact of autoclaving was also checked on diet formulated through analyzing the protein, quercetin, and chlorogenic acid content (Fig.\u0026nbsp;8) in diet before and after autoclaving. The observed changes in the nutritional and bioactive profiles due to autoclaving can be attributed to its impact on moisture content and compound stability. Autoclaving significantly reduces the moisture content in the diets, as evidenced by the drop from 14% in the unautoclaved samples to 4% in the autoclaved ones. This reduction in moisture leads to a relative concentration of certain compounds, such as chlorogenic acid and quercetin, in the autoclaved Moringa-enriched diets (MED). The increase in chlorogenic acid content could be due to the release of bound forms or the conversion of precursor compounds during high-temperature processing.\u003c/p\u003e \u003cp\u003eThe protein content showed minimal changes across all diets and conditions. In the 2% MED, autoclaving resulted in a slight increase in protein content (from 13.29\u0026ndash;15.86%), potentially attributed to moisture reduction concentrating the protein. A similar trend was observed in the standard control diet, where protein increased slightly from 13.98\u0026ndash;14.06% post-autoclaving. Interestingly, the 4% MED displayed a decrease in protein content post-autoclaving (from 14.8\u0026ndash;13.82%), suggesting that other factors, such as thermal denaturation or interaction with bioactive compounds, might influence protein stability at higher Moringa concentrations.\u003c/p\u003e \u003cp\u003eQuercetin, a sensitive bioactive compound, displayed significant degradation due to autoclaving. In the 2% MED, quercetin levels dropped from 232.83 mg/g in the unautoclaved diet to 82.27 mg/g in the autoclaved diet, indicating the susceptibility of quercetin to thermal degradation despite the moisture-driven concentration effect. In the 4% MED, quercetin levels followed an unexpected trend, increasing from 27.33 mg/g in the unautoclaved diet to 42.91 mg/g in the autoclaved diet. This increase might be attributed to the enhanced release of bound quercetin forms at higher Moringa concentrations, which partially counteracted the degradation caused by autoclaving. The standard control diet exhibited low quercetin levels, with substantial reductions observed post-autoclaving (39.47 mg/g to 12.86 mg/g), emphasizing the protective effects of Moringa bioactives against thermal stress.\u003c/p\u003e \u003cp\u003eChlorogenic acid, another key bioactive compound, showed a remarkable increase in the autoclaved Moringa-enriched diets. In the 2% MED, chlorogenic acid content rose from 8.18 mg/g in the unautoclaved diet to 22.84 mg/g in the autoclaved diet, while in the 4% MED, it increased from 62.36 mg/g to 76.28 mg/g. These increases suggest that autoclaving might facilitate the release of bound chlorogenic acid or enhance its formation from precursor compounds during high-temperature processing. In contrast, the standard control diet exhibited negligible chlorogenic acid levels, with complete degradation observed post-autoclaving (2.03 mg/g to 0 mg/g), further emphasizing the importance of Moringa enrichment in preserving or enhancing bioactives during processing.\u003c/p\u003e \u003cp\u003eThe consistent reduction in moisture content from 14\u0026ndash;4% across all diets due to autoclaving highlights its impact on the concentration and stability of bioactive compounds. While moisture loss concentrates certain compounds, thermal stress induces degradation, particularly for sensitive components like quercetin.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study not only reaffirms the medicinal benefits of MOL but also highlights its potential to enhance serum protein levels. This increase falls within the normal range, indicating untapped potential for healthy individuals and potential benefits for those with protein-energy malnutrition (PEM).\u003c/p\u003e \u003cp\u003ePrevious research has shown that MOL supplementation can improve various aspects of animal health, such as meat quality, body weight gain, feed conversion ratio, antioxidant levels, and more in various animal models [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Overall, it positively affects anthropometric growth [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], as well as improves gut microbe [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our study supports these findings and suggests that MOL can enhance food intake and assimilation efficiency, particularly when combined with exercise. The study also investigated the effects of MOL on liver and kidney function, demonstrating significant differences in biomarkers. These findings along with other data on makers for kidney damage (unpublished) suggest that MOL may protect against kidney and liver diseases and promote overall health.\u003c/p\u003e \u003cp\u003eIn contrast, groups with 2% MOL had a 7.7-fold increase in feed intake, and those with 4% MOL had a 9.2-fold increase. Forced exercise resulted in a 6-8-fold increase in feed intake in both MOL-enriched and control groups. While weight gain was not significant across all groups, it was 52\u0026ndash;56% lower in MOL groups without exercise and 33\u0026thinsp;\u0026minus;\u0026thinsp;28% lower in MOL groups with forced exercise. The decrease in serum urea was significant and consistent in MOL-enriched groups by 29\u0026ndash;36% but increased by 2.16% in the control group over a 6-week period (Fig.\u0026nbsp;1\u0026ndash;2). These findings suggest that incorporating MOL in the diet, whether 2% or 4%, improves food intake and assimilation efficiency, with exercise providing a positive synergistic effect. This could be attributed to the antioxidative and gut microbiota-improving properties of MOL (unpublished data), as well as the release of vital organ potentials, such as those in the liver, gut, and kidneys.\u003c/p\u003e \u003cp\u003eThe objective of this experiment was to investigate the effects of MOL powder on liver and kidney function in rats by evaluating the activities of liver enzymes and kidney function tests, which serve as biomarkers for liver and kidney function. The results indicated significant differences in the markers for liver and kidney function. The available literature also demonstrated a mechanism by which MOL may protect against kidney disease by mitigating various pathological factors associated with the condition, such as inflammation and oxidative stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, it is evident that as the body adapts to exercise, as there is a noticeable decrease in urea levels. The lower mean serum urea levels (as shown in Table\u0026nbsp;6) were observed in MOL-enriched group with forced exercise (Gr. 4\u0026ndash;5), followed by MOL-enriched group without forced exercise (Gr.2\u0026ndash;3), indicating of reduced protein catabolism, which implies enhanced endurance.\u003c/p\u003e \u003cp\u003eThe mean serum creatinine levels did not show any significant differences between the groups at different time points. Literature suggested that the protective properties of MOL against kidney and liver tissue damage, such as anti-apoptotic, antioxidant, and anti-oxidative stress effects [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], may mitigate or reverse the effects of increased serum levels of ALT, AST, creatinine, and urea, as well as significantly decreased levels of total proteins, albumin, and globulin. Additionally, literature suggest that the hepatoprotective and antidiabetic effects of MO have been attributed to the phenolic bioactive compounds present in the methanolic leaf extracts of MO [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e The animals from 4M-FE group exhibited the lowest mean values of AST (Table: 8), ALT (Table: 9), and ALP (Table: 10), followed by 2M-FE group, indicating the anti-apoptotic and antioxidant properties of MO. These properties are associated with increased endurance performance and restoration of liver enzymes and liver tissue to normal levels, which is consistent with previous research [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. ALP plays a critical role in calcium homeostasis, and elevated calcium levels are associated with various dental and skeletal disorders. However, the present study demonstrated that MO has a significant effect on ALP levels, reducing it significantly (by 27\u0026ndash;50%). The reduction in ALP not only increases bone mineral content but also decreases the risk of kidney stones. Although there was no significant change in body weight gain, an increase in lean mass, bone density, and bone content was observed in the MOL groups, which could be attributed to the reduced ALP activities. In addition to our unpublished findings demonstrating a positive alteration in gut microorganisms, several other studies have indicated that shifts in gut flora diversity and gene expression, both upregulation and downregulation, may be associated with the favorable increase in lean muscle mass [\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 4M-FE group (Gr.5) exhibited high serum total protein content on average (Table No. 11), followed by 2M-FE group (Gr. 3), indicating that the consumption of 4% MOL as a protein diet with 30minutes forced exercise significantly increased total protein content. The same trend was observed for 2% MOL with forced exercise, suggesting that trained muscles' ability to utilize protein as an energy source was enhanced, leading to an increase in lean muscle mass (unpublished data). This significant increase in total protein content is a crucial factor in enhancing swimming performance without fatigue. This finding is consistent with other studies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The 4M-FE and 4M-WOE had 19% and 17% increase in serum total protein respectively. Similar pattern was observed in 2M-FE and 2M-WOE by 14% increase in serum total protein compared to standard diet (control group). These results suggest that forced exercise can only lead to better outcomes than an additional group if the diet includes a sufficient dose of MOL, which is dose-dependent. However, 2M-FE did not improve better than 2M-WOE, which may be due to inadequate MOL causing anabolism com-catabolism of proteins.\u003c/p\u003e \u003cp\u003eThe protein digestibility usually increases after autoclaving [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Based on this results study on PEM animal model will be performed. Interestingly, the significant increase in total serum protein without an increase in dietary protein suggests that a high-protein diet may not be necessary for improving anthropometric indices. Instead, measures to promote protein absorption, assimilation, and homeostasis should be emphasized. This positive balance of serum proteins can help address issues related to protein-energy malnutrition, anthropometric failure, and communicable diseases such as tuberculosis. Additionally, the decrease in urea and ALP enzymes observed in the study can benefit individuals with kidney and liver diseases, as well as iatrogenic hyperphosphatemia in childhood, dental caries, and postmenopausal mineral deficiency. Mass awareness campaigns, such as the Moringa Festival Week, could promote its use through the media. MOL-Ladoo made from dried MOL and dates could provide a simple solution to address malnutrition in all age groups.\u003c/p\u003e \u003cp\u003eIn the end as a quality check on diet the analysis on diet samples of unautoclaved and autoclaved feed samples were analyzed showed the contrasting trends in quercetin and chlorogenic acid levels between Moringa-enriched and standard diets highlight the dual effects of autoclaving. Moringa supplementation provides a protective and enhancing effect, reducing the extent of thermal degradation for some bioactives while facilitating the release or formation of others. However, the sensitivity of specific compounds to thermal processing underscores the need for optimizing autoclaving conditions to preserve the functional benefits of Moringa-enriched diets. These findings suggest that Moringa's bioactive compounds not only enrich the diet but also mitigate the adverse effects of processing, making it a promising component in nutritionally enhanced diets.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study highlights a critical insight: a 2% Moringa oleifera (MOL) diet is adequate to induce significant physiological changes in a sedentary lifestyle, effectively meeting dietary needs without the need for higher supplementation. However, when paired with physical exercise, the 4% MOL diet demonstrates a remarkable synergy, enhancing serum protein levels by 2%, showcasing its superior benefits in active individuals. This underscores the dynamic role of MOL in optimizing endurance, protecting liver and kidney health, and promoting muscle strength, making it an unparalleled choice for vegetarians seeking to build muscle while maintaining dietary thresholds.\u003c/p\u003e \u003cp\u003eContrary to preconceived notions about taste and utility, MOL emerges as a dietary powerhouse deserving widespread adoption. With strategic mass awareness campaigns, its potential to combat malnutrition across various demographics, especially in vulnerable age groups, becomes undeniable. This study reinforces the call for MOL's resurgence as a cornerstone in both human and animal nutrition.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMOL - \u003cem\u003eMoringa oleifera Lam.\u0026nbsp;\u003c/em\u003edried leaves powder\u003c/p\u003e\n\u003cp\u003ePEM \u0026ndash; Protein Energy Malnutrition\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2M-WOE \u0026ndash; 2% \u003cem\u003eMoringa oleifera Lam.\u0026nbsp;\u003c/em\u003edried leaves in diet without forced exercise\u003c/p\u003e\n\u003cp\u003e4M-WOE \u0026ndash; 4% \u003cem\u003eMoringa oleifera Lam.\u0026nbsp;\u003c/em\u003edried leaves in diet without forced exercise\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2M-FE - 2% \u003cem\u003eMoringa oleifera Lam.\u0026nbsp;\u003c/em\u003edried leaves in diet with forced exercise\u003c/p\u003e\n\u003cp\u003e4M-FE - 4% \u003cem\u003eMoringa oleifera Lam.\u0026nbsp;\u003c/em\u003edried leaves in diet with forced exercise\u003c/p\u003e\n\u003cp\u003e** P\u0026gt;0.05, no significant difference.\u003c/p\u003e\n\u003cp\u003e*** P\u0026lt;0.05, a b Means with different superscripts in a column differ significantly.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eAll authors in this manuscript are not having any conflict of interest in publishing the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Gonz\u0026aacute;lez CL et al (2017) Moringa oleifera, a species with potential analgesic and anti-inflammatory activities. Biomed Pharmacother 87:482\u0026ndash;488\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaikra BK, Dhongade HKJ, Gidwani B (2017) Phytochemistry and Pharmacology of Moringa oleifera Lam. J Pharmacopunct 20(3):194\u0026ndash;200\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadek KM et al (2017) The chemo-prophylactic efficacy of an ethanol Moringa oleifera leaf extract against hepatocellular carcinoma in rats. Pharm Biol 55(1):1458\u0026ndash;1466\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNwidu LL et al (2018) Vitro Anti-Cholinesterase and Antioxidant Activity of Extracts of Moringa oleifera Plants from Rivers State, Niger Delta, Nigeria, vol 5. Medicines (Basel), 3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oacute;pez M et al (2018) Effects of Moringa oleifera leaf powder on metabolic syndrome induced in male Wistar rats: a preliminary study. J Int Med Res 46(8):3327\u0026ndash;3336\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtawodi SE et al (2010) Evaluation of the polyphenol content and antioxidant properties of methanol extracts of the leaves, stem, and root barks of Moringa oleifera Lam. J Med Food 13(3):710\u0026ndash;716\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoyo B et al (2011) Nutritional characterization of Moringa (Moringa oleifera Lam.) leaves. 10(60):12925\u0026ndash;12933\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamou B et al (2016) \u003cem\u003eAntioxidant and Antifatigue Properties of the Aqueous Extract of Moringa oleifera in Rats Subjected to Forced Swimming Endurance Test.\u003c/em\u003e Oxid Med Cell Longev, 2016: p. 3517824\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonakoti S, Osifo IF (2021) Protein-Energy Malnutrition Increases Mortality in Patients Hospitalized With Bacterial Pneumonia: A Retrospective Nationwide Database Analysis. Cureus 13(1):e12645\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhutia DT (2014) Protein energy malnutrition in India: the plight of our under five children. J Family Med Prim Care 3(1):63\u0026ndash;67\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrahmbhatt SR, Brahmbhatt RM, Boyages SC (2001) Impact of protein energy malnutrition on thyroid size in an iodine deficient population of Gujarat (India): Is it an aetiological factor for goiter? Eur J Endocrinol 145(1):11\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed S et al (2023) Gestational low dietary protein induces intrauterine inflammation and alters the programming of adiposity and insulin sensitivity in the adult offspring. J Nutr Biochem 116:109330\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor AK et al (2013) Protein energy malnutrition decreases immunity and increases susceptibility to influenza infection in mice. J Infect Dis 207(3):501\u0026ndash;510\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaible UE, Kaufmann SH (2007) Malnutrition and infection: complex mechanisms and global impacts. PLoS Med 4(5):e115\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonewane K et al (2022) Pharmacological, ethnomedicinal, and evidence-based comparative review of Moringa oleifera Lam.(Shigru) and its potential role. Manage malnutrition tribal Reg India especially Chhattisgarh 8(3):314\u0026ndash;338\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGavadiya SK, Sharma T (2022) J.J.o.I.S.o.M. Bapna, \u003cem\u003eExternal applications of Shigru (Moringa oleifera Lam): A comprehensive review\u003c/em\u003e. 10(4):241\u0026ndash;250\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar M et al (2023) A Review on Nutritive and Medicinal Importance of Shigru (Moringa Oleifera Lam). 6(7):132\u0026ndash;143\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaurya B et al (2021) \u003cem\u003eMoringa oleifera (Shigru): A Miraculous Medicinal Plant with Many Therapeutic Benefits.\u003c/em\u003e 3(1): pp. 1\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzad SB et al (2017) Anti-hyperglycaemic activity of Moringa oleifera is partly mediated by carbohydrase inhibition and glucose-fibre binding. Biosci Rep, 37(3)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng B et al (2018) Effects of Moringa oleifera silage on milk yield, nutrient digestibility and serum biochemical indexes of lactating dairy cows. J Anim Physiol Anim Nutr (Berl) 102(1):75\u0026ndash;81\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAremu A et al (2018) Methanolic leaf extract of Moringa oleifera improves the survivability rate, weight gain and histopathological changes of Wister rats infected with Trypanosoma brucei. Int J Vet Sci Med 6(1):39\u0026ndash;44\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonewane K et al (2022) Pharmacological, Ethnomedicinal, and Evidence-Based Comparative Review of Moringa oleifera Lam. (Shigru) and Its Potential Role in the Management of Malnutrition in Tribal Regions of India, Especially Chhattisgarh. World J Traditional Chin Med, 8(3)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGavadiya SK, Sharma T, Bapna VM (2022) External applications of Shigru (Moringa oleifera Lam): A comprehensive review. J Indian Syst Med, 10(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichter N, Siddhuraju P, Becker K (2003) Evaluation of nutritional quality of moringa (Moringa oleifera Lam.) leaves as an alternative protein source for Nile tilapia (Oreochromis niloticus L). Aquaculture 217(1):599\u0026ndash;611\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendieta-Araica B et al (2011) Moringa (Moringa oleifera) leaf meal as a source of protein in locally produced concentrates for dairy cows fed low protein diets in tropical areas. Livest Sci 137(1):10\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu B (2020) X.J.F.i.v.s. Chen. Curr status potential Moringa oleifera leaf as Altern protein source Anim feeds 7:53\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatshediso PG, Cukrowska E, Chimuka L (2015) Development of pressurised hot water extraction (PHWE) for essential compounds from Moringa oleifera leaf extracts. Food Chem 172:423\u0026ndash;427\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhasi S, Nwobodo E, Ofili JO (2000) Hypocholesterolemic effects of crude extract of leaf of Moringa oleifera Lam in high-fat diet fed wistar rats. J Ethnopharmacol 69(1):21\u0026ndash;25\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelim S et al (2021) Impact of Dietary Supplementation with Moringa oleifera Leaves on Performance, Meat Characteristics, Oxidative Stability, and Fatty Acid Profile in Growing Rabbits. Anim (Basel), 11(2)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui YM et al (2018) Effect of dietary supplementation with Moringa oleifera leaf on performance, meat quality, and oxidative stability of meat in broilers. Poult Sci 97(8):2836\u0026ndash;2844\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun B et al (2018) Effects of Moringa oleifera leaves as a substitute for alfalfa meal on nutrient digestibility, growth performance, carcass trait, meat quality, antioxidant capacity and biochemical parameters of rabbits. J Anim Physiol Anim Nutr (Berl) 102(1):194\u0026ndash;203\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbu Hafsa SH et al (2020) Effect of dietary Moringa oleifera leaves on the performance, ileal microbiota and antioxidative status of broiler chickens. J Anim Physiol Anim Nutr (Berl) 104(2):529\u0026ndash;538\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsopanakis C, Tsopanakis A (1998) Stress hormonal factors, fatigue, and antioxidant responses to prolonged speed driving. Pharmacol Biochem Behav 60(3):747\u0026ndash;751\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoliman MM et al (2020) The ameliorative impacts of Moringa oleifera leaf extract against oxidative stress and methotrexate-induced hepato-renal dysfunction. Biomed Pharmacother 128:110259\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuzumbukilwa WT, Nlooto M, Owira PMO (2019) Hepatoprotective effects of Moringa oleifera Lam (Moringaceae) leaf extracts in streptozotocin-induced diabetes in rats. J Funct Foods 57:75\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaleh SS, E.R.J.I.J.o.F M, Sarhat, Toxicology (2019) Effects of ethanolic Moringa oleifera extract on melatonin, liver and kidney function tests in alloxan-induced diabetic rats. 13(4):1015\u0026ndash;1019\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoyo B et al (2012) Effect of supplementing crossbred Xhosa lop-eared goat castrates with Moringa oleifera leaves on growth performance, carcass and non-carcass characteristics. 44:801\u0026ndash;809\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNkukwana T et al (2014) The effect of Moringa oleifera leaf meal supplementation on tibia strength, morphology and inorganic content of broiler chickens. 44(3):228\u0026ndash;239\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen-Zinder M et al (2017) \u003cem\u003eDietary supplementation of Moringa oleifera silage increases meat tenderness of Assaf lambs.\u003c/em\u003e 151: pp. 110\u0026ndash;116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSebola N et al (2018) Comparison of meat quality parameters in three chicken strains fed Moringa oleifera leaf meal-based diets. 27(3):332\u0026ndash;340\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Rahman A et al (2019) Effect of Moringa leaves (Moringa oleifera Lam.) extract addition on luncheon meat quality. 46(6):2307\u0026ndash;2316\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmara M et al (2018) Effects of supplementing rabbit diets with Moringa oleifera dry leaves at different levels on their productive performance. 21(2):443\u0026ndash;453\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFadhilatunnur H, R.T.K.J.C.J.o.F S, Dewi, Technology (2021) Evaluation vitro protein digestibility moringa oleifera leaves Var Domest Cook 13(1)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 11 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"ICMR-National Institute of Nutrition, Hyderabad","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":"Moringa oleifera Lam., swimming, forced-exercise model, hepato-protective, nephroprotective","lastPublishedDoi":"10.21203/rs.3.rs-5905107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5905107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMoringa oleifera Lam.\u003c/em\u003e leaves (MOL) have been a staple food source in India for centuries. Recent reports have highlighted their potential as immunomodulators, hepatoprotectants, and more. To verify these claims, we conducted a study aimed at shedding light on the nutritional benefits of MOL consumption.\u003c/p\u003e \u003cp\u003eThis six-week study employed an isocaloric and isonitrogenous feeding trial, using a pelleted diet with 20% protein, supplemented with either 2% or 4% MOL. Healthy adult male rats were subjected to a unique forced exercise regimen. We analyzed the biochemical parameters of 30 rats distributed across five groups.\u003c/p\u003e \u003cp\u003eThe results showed that the test groups had significantly lower levels of serum urea and liver enzymes (AST, ALT, and ALP) compared to the control group. Total protein levels increased significantly (14\u0026ndash;19%) in all test groups, with no significant difference in creatinine levels. This analysis concludes that administering MOL powder orally at doses of 2% and 4% is biochemically safe and exhibits liver-protective and nephroprotective properties.\u003c/p\u003e \u003cp\u003eIn conclusion, our study suggests that MOL powder can be safely incorporated into both human and animal diets in a dose-dependent manner. Additionally, the synergistic effect of exercise enhances these benefits. Notably, our unique model provides long-term results without altering animal behavior, making MOL a promising option for combating protein-energy malnutrition and malnutrition in India.\u003c/p\u003e","manuscriptTitle":"Moringa oleifera Lam.-Enriched Diet Boosts Serum Protein Levels Independently of Dietary Protein Intake","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 06:46:13","doi":"10.21203/rs.3.rs-5905107/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":"eaa69493-7897-403e-9431-f61b2178ff06","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43423447,"name":"Animal Science"},{"id":43423448,"name":"Nutrition \u0026 Dietetics"},{"id":43423449,"name":"Food Science \u0026 Technology"}],"tags":[],"updatedAt":"2025-02-07T06:46:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-07 06:46:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5905107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5905107","identity":"rs-5905107","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}