Abstract
Nicotinamide adenine dinucleotide (NAD+) depletion contributes to mitochondrial dysfunction, inflammation, cognitive and physical decline with aging, partly driven by upregulated cluster of differentiation 38 (CD38) activity. This 60-day randomized, double-blind, placebo-controlled trial evaluated LN22199, a polyphenol-rich blend of Punica granatum and Tagetes erecta, alone or with nicotinamide riboside (NR), in 140 healthy aging adults (age: 60.09 ± 4.20 years; male: female 59 %: 41 %). Participants received either a placebo, NR (500 mg), LN22199 (1000 mg), or LN22199 (500 mg) + NR (500 mg). Outcomes included NAD+ levels, CD38 activity, inflammatory biomarkers, cognitive function, physical performance, and quality-of-life measures. LN22199 increased blood NAD+ levels by 26.48 %, NR by 22.69 %, and their combination by 31.76 % (all p < 0.001 vs. baseline). CD38 activity decreased significantly with LN22199 alone (17.98 % reduction, p = 0.0025 vs. baseline; p = 0.0344 vs. placebo), while NR alone showed minimal change. Serum growth/differentiation factor 15 (GDF-15) levels declined by 15.07 % (LN22199; p = 0.0001), 11.36 % (NR; p = 0.0033), and 17.83 % (combination; p < 0.0001) vs. baseline. The combination group achieved the greatest improvements in functional outcomes, including a 9.94 % increase in six-minute walk distance and a 16.45 % improvement in Mini-Mental State Examination 2 cognitive scores. All treatments were well tolerated with no serious adverse events reported. LN22199, combined with NR, enhances NAD+ homeostasis, mitigates inflammation, and improves cognitive and physical function, representing a promising strategy for healthy aging.
Highlights
- LN22199, a polyphenol-rich formula of Punica granatum and Tagetes erecta extracts.
- LN22199 and NR increase NAD+ and reduce CD38 activity in aging adults.
- Combination therapy enhances cognition, physical performance, and quality of life.
- All treatments were well tolerated with no major adverse events.
Introduction
Nicotinamide adenine dinucleotide (NAD+) is a vital pyridine nucleotide coenzyme involved in fundamental cellular processes, including energy metabolism, redox reactions, DNA repair, and signal transduction (Covarrubias et al., 2021; Verdin, 2015). As an electron carrier, NAD+ facilitates glycolysis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation, and serves as an essential substrate for NAD+-consuming enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs), and cluster of differentiation 38 (CD38). These enzymes regulate vital biological functions such as genomic stability, mitochondrial function, and calcium signaling (Cantó et al., 2015; Verdin, 2015).
Advancing age and many age-related pathologies are associated with a steady reduction in NAD+ levels. This reduction contributes to mitochondrial dysfunction, elevated oxidative stress, and impaired activities of NAD+-dependent metabolic enzymes. (Mills et al., 2016; Rajman et al., 2018). Three primary biosynthetic pathways regulate the maintenance of intracellular NAD+ pools: (i) de novo synthesis from tryptophan, (ii) the Preiss–Handler pathway from nicotinic acid (NA), and (iii) the salvage pathway from nicotinamide (NAM) and nicotinamide riboside (NR). Among these, the salvage pathway predominates in mammalian cells and is critically dependent on nicotinamide phosphoribosyltransferase (NAMPT), the key rate-limiting enzyme that recycles NAM to NAD+ (Rajman et al., 2018; Trammell et al., 2016).
Numerous preclinical studies have demonstrated that supplementation with NR enhances NAD+ biosynthesis, improves mitochondrial biogenesis, and reduces inflammatory responses in models of aging and metabolic dysfunction (Mills et al., 2016; Zhang et al., 2016; Zhou et al., 2020). Human clinical trials further support the role of NR in restoring NAD+ levels and improving vascular function, skeletal muscle metabolism, and neuroprotection, thus offering a compelling strategy for promoting healthy aging and extending healthy life span (Martens et al., 2018; Elhassan et al., 2019; Remie et al., 2020; Xue et al., 2022; Sharma et al., 2023; Dölle & Tzoulis, 2025). However, declining NAD+ levels with advancing age are not solely due to impaired biosynthesis but are also driven by increased consumption of NAD+, particularly by CD38. CD38 is a multifunctional NADase upregulated in metabolic tissues, immune cells, and senescent cells during aging and chronic inflammation (Camacho-Pereira et al., 2016; Chini et al., 2020). It hydrolyses NAD+ into ADP-ribose and NAM, significantly contributing to the age-related depletion of NAD+. This poses a therapeutic limitation, as supplementation with NAD+ precursors alone may be inadequate to overcome accelerated NAD+ degradation that occurs with advancing age.
To overcome this limitation, combined strategies have been proposed, involving the use of NAD+ precursors (such as nicotinamide mononucleotide, NMN, and NR) in conjunction with the inhibition of NAD+-consuming enzymes, including CD38. Preclinical models suggest that pharmacological CD38 inhibition or genetic deletion, in combination with NAD+ precursors, more effectively restore intracellular NAD+ levels and improve mitochondrial and metabolic function (Peclat et al., 2022; Tarragó et al., 2018). Although synthetic NAD+ precursors offer promise, limited bioavailability, high costs, regulatory ambiguity, and potential long-term safety concerns impede their applications. As a result, attention is shifting toward natural and plant-derived compounds as safer and more sustainable alternatives for modulating NAD+ metabolism. Natural polyphenols such as apigenin and pomegranate-derived ellagitannins have been shown to inhibit CD38 and support mitochondrial health (Escande et al., 2013; Ogura et al., 2020), suggesting a potential complementary approach. However, clinical evidence supporting a dual-mode strategy that combines NAD+ precursor supplementation with targeted CD38 modulation to optimize and sustain intracellular NAD+ availability remains limited. (Chini et al., 2020).
In this context, LN22199, a standardized and proprietary polyphenol-rich herbal formulation composed of extracts from Punica granatum (pomegranate) whole fruit and Tagetes erecta (marigold) flowers, was developed. Preclinical studies demonstrated that LN22199 synergistically promoted NAD+ synthesis and inhibited CD38 activity in vitro. Immunoblot assays demonstrated that LN22199 increased the expression of Nicotinate phosphoribosyltransferase (NAPRT), NAMPT, and NMN adenylyltransferase (NMNAT) 1 and -3 in L6 rat skeletal myocytes (unpublished data). These observations suggested that LN22199 might help promote cellular NAD synthesis from NA and NAM, activating the Preiss-Handler and Salvage pathways. Moreover, it also helped reduce intracellular NAD breakdown via inhibiting CD38 enzyme activity. Subsequently, an in vivo proof-of-concept study demonstrated that LN22199 supplementation significantly elevated blood and tissue NAD+ levels in aged Sprague Dawley rats (unpublished data). Given LN22199’s dual mechanism—enhancing NAD+ biosynthesis and inhibiting CD38-mediated degradation, we hypothesized that LN22199 would produce greater improvements in NAD+ levels, anti-inflammatory responses, and functional health benefits as compared to NR alone. Moreover, combining LN22199 with NR was expected to deliver synergistic benefits, resulting in the highest gains in NAD+ restoration and age-related functional outcomes among all treatment groups.
Punica granatum has been used traditionally in Ayurveda and Unani medicine for cardiovascular, gastrointestinal, and general tonic purposes. It is rich in ellagitannins (notably punicalagin), ellagic acid, anthocyanins, and other polyphenols, which demonstrate potent antioxidant, anti-inflammatory, and anti-aging benefits (Jurenka, 2008). These phytochemicals have been shown to enhance mitochondrial function, modulate sirtuin pathways, and facilitate DNA repair (Zhao C et al., 2016; Guo et al., 2021). Tagetes erecta, widely used in traditional medicine in Latin America and Asia, is a rich source of lutein, zeaxanthin, and quercetagetin; these exhibit potent antioxidant and anti-inflammatory activities. (Escande et al., 2013). T. erecta has also demonstrated hepatoprotective, neuroprotective, and ocular benefits, particularly in age-related macular degeneration (Delgado-Vargas & Paredes-López, 2003).
This randomized, double-blind, placebo-controlled clinical trial was designed to evaluate the effects of LN22199, NR, and their combination (LN22199 + NR) on NAD+ and its metabolites in blood and urine over a 60-day duration in healthy aging individuals. This trial evaluated the efficacy of LN22199 alone and in combination with NR, while comprehensively assessing NAD+ metabolism, CD38 activity, and biomarkers of systemic and neurological well-being. The study assessed the impact of these interventions on a panel of molecular biomarkers including, growth differentiation factor 15 (GDF-15), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), CD38, neurofilament light chain (NFL), and functional health metrics that include endurance and cardiorespiratory fitness, cognitive health and quality of life, providing a comprehensive evaluation of cellular resilience, systemic inflammation, and functional capacity.
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Study Materials
LN22199 (BluNADBooster) is a patent-pending proprietary composition containing 90 % (w/w) blend of Punica granatum fruit water extract and Tagetes erecta flower aqueous-ethanol extract in a 3:1 ratio. It is combined with 10 % (w/w) neutral excipients, including rice maltodextrin, and syloid. The final product is standardized to contain not less than 1.5 % punicalagins, and 4.0 % quercetagetin, serving as phytochemical reference markers for P. granatum, and T. erecta, respectively. LN22199 was developed by Laila Nutra Private Limited, Vijayawada, Andhra Pradesh, India, and manufactured at its current Good Manufacturing Practices (cGMP)-certified production facility. This study utilized a batch of LN22199 (LPP22100194, produced in October 2022 with a minimum shelf life of two years). The major nutrient contents of this batch are presented in Supplementary Table S1.
Varun Kumar Bandi, Vittal Kumar Chundru, Aruna Yarasani, Ramesh Gajula, Vinay Kumar Pulipaka, Thabish Syed, Comparative efficacy of a standardized herbal composition LN22199, nicotinamide riboside, and their combination on NAD+ metabolism in healthy aging adults, Journal of Functional Foods, Volume 135, 2025, 107115, ISSN 1756-4646, https://doi.org/10.1016/j.jff.2025.107115.
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