Pharmacokinetic study and phytochemical analysis of beetroot powder as an initial stage of the development of an NO-boosting formulation as a food supplement with cardioprotective properties and potential donor of nitric oxide

Introduction

Discovery of endogenous nitric oxide (NO) synthesis in the body in 1980s and subsequent identification of various signaling pathways linked to NO represented one of the main achievements in cardiovascular medicine [1]. NO regulates important functions of the cardiovascular, immune, and nervous systems and is involved in blood coagulation and inflammation. Imbalance in NO levels is associated with multiple diseases linked to excessive or insufficient synthesis or bioavailability of NO.

Human body has a functional NO cycle starting from NO synthesis and followed by oxidation of NO by oxygen to form nitrite (NO₂^-) and nitrate (NO₃^-). Then, NO₃^- may be reduced to NO₂^– by various enzyme, and NO₂^- may be further reduced to form bioactive NO [1]. These molecules have specific biological and physiological activities, some of which are associated with the regulation of the cardiovascular system [1, 2].

Biomedical studies demonstrated that NO production and levels are modulated by changes in endogenous synthesis of NO by NO-synthases from L-arginine and by exogenous consumption of NO metabolites or precursors [1, 2]. The levels of bioactive NO are influenced by diet containing various plant-based products that have high levels of nitrate or nitrite (Table 1). Regular consumption of NOx has been shown to normalize the levels of NO in the blood and tissues and thus compensate for alterations in endogenous synthesis of NO [1, 2].

Table 1. Classification of vegetables based on the NO3- ion contents [3]

Healthy nutrition that compensates for pathological changes in NO synthesis is important for prophylaxis of cardiovascular diseases. The data shown in Table 1 indicate that beetroot has the highest level of NO₃^- [3] and is considered a suitable source for exogenous nutritional boosting of NO levels.

The goal of the present study was to facilitate the development of an NO-boosting nutritional supplement formulated from beetroot. Thus, we aimed to assay the contents of NOx in finely dispersed dried beetroot powder to determine NOx shelf life as a potential NO precursor and to determine pharmacokinetic properties of NOx in the blood within 24 h. 

Material and Methods

Pharmacokinetic study

The study enrolled 10 healthy volunteers who signed an informed consent and agreed to use the nutritional supplement at the dose of 11 g dissolved in water orally. The Local Ethics Committee of Saratov State Medical University, Saratov, Russia approved the protocol of the study according to Helsinki Declaration. All participants were warned to avoid consumption of vegetables (beetroot, green salad, and cabbage) and processed foods within 12 h prior to the consumption of the beetroot powder solution. Prior to the ingestion of the solution, a catheter was placed in the cubital vein, and the blood was collected at six time points: before ingestion of the solution and in 0.5, 2.5, 5.5, 8.5, and 24 h after the ingestion. The pharmacokinetic curve was constructed by plotting the mean NOx concentration in the serum for all 10 volunteers versus time.

Serum preparation

The serum was prepared by centrifugation of the blood at 1,000 g for 20 min at room temperature and was aliquoted and stored at -24С until NOx assay.

Beetroot powder formulation used for NOx assay

Finely dispersed beetroot powder was prepared by infrared dehydration using an infrared drying chamber IKS-70, Russia. The chamber was supplied by the Research Center of Healthy Nutrition Technology of Razumovsky Medical University, Saratov, Russia. The powder was dark-pink colored and had characteristic beetroot odor. A total of 100 mg of the powder was dissolved in 10 ml of deionized water to prepare 10 g/l solution that was filtered through a paper filter and used to prepare dilutions for the NOx assay.

Assay of NOx in biological media and fluids.

The method is based on diazotization reaction after reduction of nitrate to nitrite by vanadium chloride (III) in deproteinized medium. The method has been developed by us previously and registered by the Ministry of Health Care of Russian Federation in 2008 under the name “Express method for the assay of nitric oxide metabolites in biological media as markers of vascular endothelial dysfunction” (certificate AA 0001634 for the use of novel medical technology number 2008/229; October 23, 2008).

NOx was assayed in the beetroot samples twice, including immediately after receipt of the packaging from the manufacturing facility and in 12 months after storage at room temperature in a dry place.

NOx was measured by the Griess reaction after reduction with VCl₃ solution in 1 M HCl as described previously [4, 5]. Optical density was measured at 540 nm using a plate reader (Tecan Infinite 200 Pro, Switzerland). NOx concentrations were calculated using Magellan software (Tecan) based on a calibration curve constructed using variable nitrate concentrations from 5 to 320 µM. Serum samples were deproteinized by ultrafiltration through SPIN-X UF columns (Corning, UK)

Results

Assay of NOx in beetroot powder solutions

NOx concentration in 10 g/l aqueous beetroot powder solution was 2.43±0.25 mM. Assuming molecular weight of KNO₃ of 101.1 g/mol, the mean concentration of NOx in the solution was 0.25 g/l. Thus, the mean content of NOx was estimated to be 25.2 g/kg dry weight of the powder. The second assay was performed after 12 months of storage. In this case, the concentration of NOx was 2.40±0.32 mM. The two values were compared using Mann-Whitney test, and the P value was >0.05, indicating that the storage of the powder for 12 months did not result in significant changes in the content of NOx. Thus, the dry powder can be stored for at least 12 months.

Based on previously published studies [7-9], we have selected the dose of 400 mg of NOx for pharmacokinetic experiments in volunteers, and this dose corresponded to 16 g of the beetroot powder.

Pharmacokinetics of the beetroot powder

The pharmacokinetic curve after the ingestion of a single dose of the beetroot powder corresponding to 400 mg of NOx was based on the mean concentrations of NOx in the serum of 10 healthy volunteers (Figure 1).

Figure 1. Dynamics of NOx concentrations in the serum of healthy volunteers after a single ingestion of beetroot powder solution (N=10).

The data indicated that Tmax was 30 min after the ingestion of the formulation. This Tmax corresponded to Cmax of 209 µM (approximately 52% of the total ingested dose of 400 mg). Half-life of the active ingredient NOx (Т1/2) was estimated to be 10 h. The rate of absorption appeared to be relatively fast and was approximately equal to 7 µM NOx/min, reaching the Cmax after 30 min.

Discussion

The data of Table 1 [3] and our measurements indicated that the content of NO₃^- was 2.4 g/kg of wet beetroot weight, corresponding to a very high level. These results are in agreement with the data of another study [6] that used ionometry to measure content NO₃^- to be 1.90 g/kg in beetroot according to the State Standard of Russian Federation number 4329-77.4. Thus, infrared dehydration increased the content of NOx in the product by an order of magnitude. Use of dry powder simplifies storage and consumption of NOx as NO precursor and is expected to facilitate further development of beetroot-based nutritional supplements.

Previous studies estimate effective doses of nitrate based on tolerance to physical load, effect on blood pressure, and other cardiovascular parameters. On average, the dose of nitrate in clinical studies was 400 mg [6-10]. This dose corresponded to approximately 16-20 g of beetroot powder used in the present study.

Beetroot-based formulations have been shown to be beneficial for physical fitness in athletes [11-13]. Positive effects of beetroot nitrate on high-load exercise tolerance were confirmed in numerous studies [14-17].

Recent DASH study (Dietary Approaches to Stop Hypertension) demonstrated that consumption of large volumes of fruits and vegetables while limiting the consumption of total fat and saturated fat lowered blood pressure compared to that in subjects fed a diet with low volume of vegetables and fruits and excessive consumption of saturated fat [18-22]. Moreover, Mediterranean diet is linked to low cardiovascular and cancer mortality compared to standard Western diet, which contains only 6% of NOx content of a typical Mediterranean diet [23-30]. These findings suggest that beneficial effects of Mediterranean diet may be due to high levels of NOx and polyphenols [25-30]. Thus, NOx supplementation in the form of a beetroot powder formulation is expected provide considerable cardiovascular health benefits.

Conclusion

The results of the present study indicated that the content of NOx was 25.2 g/kg of dried beetroot powder. The powder used in the present study was stored for 12 months without a detectable decrease in the content of NOx. The estimated oral dose of the powder was 16-20 g corresponding to 400 mg of nitrate. The data confirm feasibility of the development of a nutritional supplement using formulation used in the present study to boost the levels of NO in the body and benefit the cardiovascular system.

Funding

The study was supported by the Ministry of Health Care of Russian Federation, project number 124013100892-7 (2024-2026).

Conflicts of interest

The authors declare no conflicts of interest.

Author participation

Generation and collection of the data: N.L. Bogdanova; statistical analysis, conceptualization, design, editing, and writing: N. G. Gumanova; administration: A. R. Kiselev, O.M. Drapkina.