Abstract
Declining testosterone levels are associated with reduced muscle mass, fatigue, and reproductive challenges in men. Although testosterone replacement therapy is available, it is often associated with risks such as cardiovascular complications and endocrine imbalance, highlighting the need for safer alternatives. Nutraceutical approaches offer a promising strategy for supporting endogenous testosterone production. In this study, Testolift, a novel testosterone-supporting nutraceutical formulation, was developed under the Natural Ingredients for Mental and Performance (NIMP) Platform using InSitu360 molecular complexation technology.
The formulation integrates fenugreek saponins, black ginger (Kaempferia parviflora) polymethoxyflavones, and zinc methionine to achieve synergistic enhancement of testosterone. The formulation was characterized using FT-IR, SEM, TEM, DSC, EDS, and UPLC, confirming the successful integration of bioactives and favorable physicochemical properties. Quantitative analysis verified the presence of 20% saponins, 0.93% polymethoxyflavones, and 1.4% elemental zinc. Morphological studies revealed uniformly dispersed spherical nano- and microspheres with sub-200 nm particles, supporting structural stability and efficient incorporation of bioactives within the matrix. Dynamic light scattering analysis confirmed a mean particle size of 193 nm with a distribution range of 120–260 nm, while the zeta potential of −32.24 mV indicated good colloidal stability.
A 180 days stability study demonstrated >95% retention of methoxyflavones, saponins, and zinc across refrigerated (4 ± 2 °C), ambient (25 ± 2 °C), and accelerated (45 ± 2 °C) conditions, confirming excellent compositional and physicochemical stability. In a 42 days in vivo experimental study in male rats, Testolift significantly increased serum testosterone by 22.7% compared to 4.1% in controls (p < 0.05) without adverse effects. The InSitu360 technology enhanced the physicochemical and compositional stability of the formulation, supporting its robustness and suggesting potential for improved bioavailability, which warrants confirmation in future clinical studies. These results position Testolift as a promising natural testosterone-support formulation with potential applications in male health and muscle endurance.
Introduction
Testosterone is a principal androgen hormone essential for male reproductive function, muscle mass, bone density, skeletal integrity, erythropoiesis, physical stamina, mood regulation, and overall vitality. Declining testosterone levels, whether due to advancing age or lifestyle-related factors such as obesity, chronic disease, and stress, are increasingly recognized as a significant health concern in both older and younger male populations. Symptoms of testosterone deficiency (TD) include diminished libido, fatigue, loss of muscle mass, depression, and cognitive impairment. Global prevalence estimates for TD range from 10% to 40% among adult men, and even mild reductions can adversely affect quality of life and elevate risks for metabolic and cardiovascular disorders. Despite growing awareness, the diagnosis and management of TD remain inconsistent.1,2 Although testosterone replacement therapies (TRT) are available, they are associated with drawbacks such as cardiovascular risks, infertility, hepatotoxicity, mood disturbances, and suppression of endogenous testosterone synthesis, potentially leading to dependency and testicular atrophy.2–4 These limitations highlight the need for safe and effective alternatives that support natural testosterone production.
Nutraceuticals, defined as products that provide health benefits beyond basic nutrition, have emerged as promising candidates for long-term testosterone support due to their natural origin and favorable safety profile. Unlike synthetic hormones, nutraceutical interventions typically act by supporting the hypothalamic-pituitary-gonadal (HPG) axis, enhancing luteinizing hormone (LH) secretion, reducing oxidative stress, and upregulating steroidogenic enzymes.2–4 Current testosterone-supporting nutraceuticals include herbal extracts such as ashwagandha, tongkat ali, tribulus terrestris, and fenugreek, as well as trace minerals like zinc. However, many single-ingredient products suffer from limited clinical validation, poor bioavailability, or insufficient synergy to achieve meaningful improvements in testosterone status and muscle performance.5–13 Advanced formulation technologies that combine multiple bioactives and enhance absorption may therefore offer significant advantages.
Testolift is a novel testosterone-boosting nutraceutical formulation that integrates saponin-rich fenugreek (Trigonella foenum-graecum) extract, polymethoxyflavone-rich black ginger (Kaempferia parviflora) extract, and zinc methionine. Fenugreek saponins support testosterone biosynthesis through modulation of 5α-reductase and stimulation of luteinizing hormone (LH) secretion; black ginger polymethoxyflavones enhance nitric oxide synthesis, mitochondrial activity, and steroidogenic enzyme expression; and zinc methionine, a bioavailable chelated form of zinc, serves as a critical cofactor in testosterone biosynthesis and provides antioxidant protection.11–18 The combination offers a multifaceted approach to improving testosterone status, muscle strength, and reproductive health. The formulation was developed under the Natural Ingredients for Mental and Performance (NIMP) Platform, a research and development initiative focused on optimizing the efficacy and delivery of phytoactive compounds for human performance enhancement. Within this framework, InSitu360 molecular complexation technology employed a proprietary encapsulation platform that enables real-time interaction between fenugreek saponins, black ginger polymethoxyflavones, and zinc methionine during processing. This three-stage approach (i) targeted extraction and purification of bioactives, (ii) enrichment of fenugreek saponins, and (iii) in situ molecular complexation under controlled shear, pressure, and thermal conditions creates a stable nanostructured matrix. The process enhances solubility, stability, and intestinal absorption while promoting synergistic physiological effects. By improving molecular alignment and sustained release, InSitu360 overcomes common challenges of poor bioavailability in plant-based nutraceuticals and provides a next-generation delivery strategy for testosterone support and muscle endurance, consistent with principles established in spray-drying and nano-encapsulation literature.19–23
Specifically, fenugreek-derived steroidal saponins have been reported to influence androgen-associated pathways and sex hormone metabolism, with human studies demonstrating improvements in testosterone-related parameters, strength, body composition, and symptoms associated with androgen deficiency.24,25 Black ginger (Kaempferia parviflora) polymethoxyflavones have been associated with improvements in mitochondrial bioenergetics, endothelial nitric oxide signaling, physical performance, and male sexual health parameters.26–28 Zinc is an essential trace element that serves as a cofactor in numerous enzymatic processes involved in steroidogenesis and reproductive physiology, and zinc deficiency has been consistently associated with reduced testosterone concentrations, while zinc supplementation has been shown to support normal testosterone status in deficient individuals.29,30 Accordingly, the formulation strategy was designed to target multiple biological determinants of testosterone status simultaneously rather than relying on a single mechanistic pathway. Given the substantial body of evidence supporting the individual efficacy of these ingredients, our intention was to investigate a novel formulation combining these well-characterized bioactives and to evaluate whether their concurrent administration could provide enhanced benefits compared with existing single-ingredient approaches. The objective of the present study was to formulate and characterize Testolift using InSitu360 technology and to evaluate its physicochemical attributes and efficacy in enhancing serum testosterone levels in male Sprague Dawley rats.
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2.1. Materials
Rhizomes of Kaempferia parviflora were sourced from Ava Corporation, Thailand, and seeds of Trigonella foenum-graecum (fenugreek) were obtained from Kuber Impex Limited, Indore, India. Botanical authentication of both plant materials was performed by a qualified local botanist, although no voucher specimens were archived. Zinc methionine was procured from Wilincare, China, and phosphatidylcholine was obtained from Shankar Nutricon, India. Gum arabic used in the formulation was purchased from Nexira, France. Reference standards of polymethoxyflavones such as 5,7-dimethoxyflavone (≥95% purity; CAS No. 21392-57-4), 5,7,4′-trimethoxyflavone (≥90% purity; CAS No. 5631-70-9), and 3,5,7,3′,4′-pentamethoxyflavone (≥90% purity; CAS no. 1247-97-8) were purchased from Sigma-Aldrich (India). All organic solvents used in the study, including ultra-performance liquid chromatography (UPLC), were of analytical grade and were procured from Merck India. Ultrapure water generated by a Millipore Milli-Q system was employed throughout the experimental procedures.
Amalraj A, Ramachandran Reshna K, Varma K, Jogy AM, Balakrishnan P, Gopi S. Formulation, characterization, and efficacy evaluation of testolift: a novel testosterone-enhancing nutraceutical featuring synergistic black ginger methoxyflavones, fenugreek saponins, and zinc methionine via InSitu360 technology. RSC Adv. 2026 Jul 2. doi: 10.1039/d6ra02748b. Epub ahead of print. PMID: 42395749; PMCID: PMC13326673.
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Black Ginger as a Functional Ingredient for Performance, Skin Vitality, and Muscle Preservation
