Characterization of Betalains-Enriched Nutraceutical Tablets from Beta vulgaris L. Pomace Powder

doi:10.21203/rs.3.rs-4732104/v1

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Research Article

Characterization of Betalains-Enriched Nutraceutical Tablets from Beta vulgaris L. Pomace Powder

https://doi.org/10.21203/rs.3.rs-4732104/v1

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The objective of this research was to develop nutraceutical tablets enriched with phytonutrients and antioxidants by dehydrating beetroot pomace, which is abundant in bioactive compounds, and integrating it with pomace extract obtained using ultrasonication. The research involved evaluating the packaging, microstructure, and physical and chemical properties of freeze-dried powder. The freeze-dried powder exhibited favorable flow properties, reduced Hausner ratio, and low hygroscopicity. Analysis confirmed higher phytochemical content, supported by FTIR. SEM and particle size distribution analysis indicated consistent particle generation. The freeze-dried powder was mixed with pomace extract in a 1:1 weight-to-volume ratio to augment phytonutrient levels before compression into tablets. The tablets exhibited rapid disintegration and in vitro release profiles of betalains. Storage at 25 ± 2°C over five months revealed significant humidity and water movement, retaining approximately 81% of phytochemicals and 78% antioxidant activity. This approach not only effectively utilizes beetroot waste but also produces bioavailable nutraceutical tablets, offering significant health benefits.

Beetroot pomace

Characterization

Freeze drying

Nutraceutical

Phytochemicals

The agricultural sector produces substantial amounts of by-products including pomace, seeds, pulp, pods, stems, husks, and peels [26]. These by-products are abundant in bioactive compounds such as vitamins, dietary fibers, oils, carotenoids, polyphenols, and enzymes, associated with health benefits against chronic conditions including heart disease, obesity, arthritis, and diabetes [20, 14]. Consequently, there has been growing interest among researchers and manufacturers worldwide in utilizing agricultural waste for producing nutraceutical, functional, and fortified foods, aiming to reduce waste production [17].

Beta vulgaris L., commonly known as red beetroot and belonging to the Chenopodiaceae family, is distinguished by its elevated levels of betalains, which are water-soluble nitrogenous pigments encompassing yellow betaxanthins and reddish-purple betacyanins [1]. This plant, deeply rooted in Ayurveda and traditional medicine, is valued for its rich array of phytochemicals, offering diverse health benefits including diuretic, antiviral, stomachic, antioxidant, antiproliferative, antidiabetic, and antiallergic effects [29].

During industrial processing, beetroot is predominantly utilized for juice extraction, resulting in approximately 45% of the raw material being discarded as waste in the form of seeds and pomace [30]. This waste, considered low in worth and susceptible to microbial contamination, raises concerns regarding environmental pollution. Despite its rich nutritional profile, pomace is typically underutilized, often relegated to animal fodder due to its high moisture content, which accelerates degradation [12]. Therefore, there is a compelling need to explore efficient strategies for utilizing beetroot pomace in the production of nutraceutical food products.

Drying is widely recognized as the optimal and economically feasible method for preserving perishable commodities, reducing moisture content to below 10% to extend shelf life and inhibit microbial growth [32]. Various drying techniques have been employed for fruits and vegetables, each potentially impact phytonutrient content differently. However, dried powders offer advantages such as extended shelf life, reduced transportation and storage costs, and enhanced nutritional potential in diverse food applications [9]. Thus, identifying appropriate and cost-effective drying methods that preserve the quality of fresh produce is critical.

In today's market, there is increasing promotion and consumption of dehydrated nutraceutical products including powders, tablets, and encapsulated formulations. Nutraceutical tablets, often produced through direct compression, require powders that are free-flowing and compactable [14]. The objective was to assess the effects of freeze-drying on the physical properties and concentrations of bioactive compounds in beetroot pomace, and to formulate nutraceutical tablets using direct compression to augment phytochemical content and antioxidant capacities.

Materials

Fresh beetroot (Beta vulgaris L.) sourced from a local market in Chandigarh procured and after extraction of juice, beetroot pomace immediately transported to the laboratory under controlled environmental conditions.

Pomace drying and tablet preparation

Freeze drying

The pomace underwent freezing at -40°C (Coldlab, CL model 120 − 40, Brazil), followed by lyophilization (SCANVAC Coolsafe 55 − 4), grinding, and storage in high-density polyethylene bags and sealed plastic containers for analysis.

Analysis

Physicochemical Properties of Powder

Moisture & water activity

The moisture level was assessed utilizing a hot air oven. Following the initial weighing, a 2.0 gram sample underwent heating in an oven at 130°C for 3hrs. After a 3 hrs drying, the petri dishes were removed, covered, transferred to desiccators, and left to cool. After the samples cool down, their weights are measured again, and the moisture content is determined using the following formula.

Moisture content (%) = $\:\frac{weight\:of\:fresh\:sample-weight\:of\:dried\:sample}{weight\:of\:fresh\:sample}\times\:100$

The water activity of 1 gram of powder was measured using a water activity meter (Aqualab PAWKIT, DECAGON Devices, Inc., USA).

Color analysis

The powder's color was assessed using a Color Flex meter (Hunter Lab Color Flex 150, Hunter Associates Inc., USA), utilizing a 45°/0° geometry and standard illuminant C, where L* represents brightness (with 0 indicating black), a* signifies green (-) or red (+), and b* indicates blue (-) or yellow (+) in a scientific color model.

Hygroscopicity

Tao et al. [31] reported a slight adjustment to the method for measuring hygroscopicity.

One gram of powder was exposed to a sodium chloride solution saturated at 75% relative humidity in a desiccator for 24 hours, and the hygroscopicity was quantified as grams of absorbed moisture per 100 grams of dry powder. The measurements were taken in triplicates.

Particle size distribution and glass transition temperature (Tg)

The laser light diffraction instrument (LA-950 V2, Horiba, Kyoto, Japan) was used to measure D10, D50, and D90, representing diameters at which 10%, 50%, and 90% of the powder mass was accumulated, respectively. Furthermore, span, representing particle size distribution dispersion, was calculated using the method outlined by Kaur et al. [15] following their provided equation.

$\:\text{S}\text{p}\text{a}\text{n}=\frac{\text{D}90-\text{D}10}{\text{D}50}$

The glass transition temperature of the powders was assessed using differential scanning calorimetry (DSC) according to the procedure outlined by Kaur et al. [15] with the Pyris 1 instrument (Perkin Elmer, Massachusetts, USA). The Tg value, determined by heating a 10 mg sample in an aluminum container to 150°C followed by controlled cooling to 25°C, marks the temperature where a half change in heat capacity (ΔCp) occurs.

Flowability parameters

The flow properties of pomace powder were assessed through measurements of angle of repose, Hausner ratio, and Carr's Index. A funnel with a constant height of 3 cm was used on a flat white surface to facilitate powder flow, with the angle of repose determined by the arctangent of the ratio of pile height to radius. The Hausner ratio, derived from the ratio of tapping density to packing density, assesses cohesion, while Carr's index evaluates powder flow and compressibility. Correia et al. [3] determined the bulk and tapped densities of the powders and applied Carr's compressibility index formula for analysis.

$$\:\text{C}\text{I}=\frac{\text{T}\text{a}\text{p}\text{p}\text{e}\text{d}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}-\text{B}\text{u}\text{l}\text{k}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}}{\text{T}\text{a}\text{p}\text{p}\text{e}\text{d}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}}\text{X}\:100$$

Determination of the phytochemicals

A 0.2 gram sample was dissolved in 10 ml of distilled water and agitated for 20 minutes at 250 rpm using a magnetic stirrer, followed by centrifugation (Hitachi) at 4000 rpm for 10 minutes, for the analysis of total betalains, total phenol content, and total flavonoid content.

The total betalains concentration (mg/g) was calculated by summing spectrophotometric measurements of betacyanin at 535 nm and betaxanthin at 480 nm.

$$\:\text{B}\text{e}\text{t}\text{a}\text{l}\text{a}\text{i}\text{n}\text{s}\:\left(\text{m}\text{g}\:\text{o}\text{f}\:\text{B}\text{X}\:\text{o}\text{r}\:\text{B}\text{C}/\text{g}\right)=\frac{\text{A}\times\:\text{D}\text{F}\times\:\text{V}\times\:\text{M}\text{W}}{\text{E}\times\:\text{L}\times\:\text{M}}$$

Where E represents the molar extinction coefficient (Lmol − 1 cm − 1) with values of 60,000 for betacyanins and 48,000 for betaxanthins, A corresponds to A538 for betacyanins (BCs) and A480 for betaxanthins (BXs), DF denotes the dilution factor, MW stands for Molecular Weight (550 g/mol for betacyanin and 308 g/mol for betaxanthin), V represents the extract volume, L is the quartz cuvette path length in centimeters, and M signifies the mass of dry material used in extraction [25].

The total phenolic content (TPC) of both powder and tablet extracts was quantified using the Folin-Ciocalteu (FC) method [15], involving mixing 1 mL of extract with 1 mL of FC reagent, 1 mL of 10% sodium carbonate solution, adjusting to 10 mL with double-distilled water, and measuring absorbance at 760 nm after one hour of incubation, with results expressed in milligrams of gallic acid equivalent (GAE) per gram of dry weight (dw). Total flavonoid content was determined according to the method outlined by Kaur et al. [14], involving the combination of a 2% AlCl3 solution with 1.5 mL of the extract in equal proportions, followed by measuring absorbance at 430 nm after 15 minutes at room temperature in darkness, with calculations based on quercetin equivalent (mg/g dw) presented as QE mg/g (dw) or mg/g.

Assessment of antioxidant properties

The DPPH free radical scavenging activity of tablet and pomace powder extracts was evaluated following the method outlined by Ibrahim et al. [11]. A 0.2 gram sample was dissolved in 10 mL of distilled water, stirred for 20 minutes at 250 rpm using a magnetic stirrer, centrifuged at 4000 rpm for 10 minutes (Hitachi), and mixed with various concentrations of samples in 3 mL of methanol-soluble DPPH, followed by dilution to 10 mL with distilled water, incubated in the dark at room temperature for 30 minutes, and absorbance measurements were recorded at 517 nm. By employing this formula, calculate the inhibition percentage:

$$\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:\left(\text{%}\right)=\frac{\text{A}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\:\text{A}\:\text{t}\text{e}\text{s}\text{t}}{\text{A}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\text{x}\:100$$

A _control = absorbance of control

A _test = absorbance of test

Ravichandran et al. [27] utilized the DPPH assay, diluting 0.011 grams of DPPH in 25 mL methanol to achieve an absorbance range of 0.80 − 0.05 at 515 nm. A mixture of 3.8 mL DPPH solution and 0.20 mL BRP extract was incubated for 30 minutes at room temperature, followed by spectrophotometric analysis at 515 nm wavelengths.

Mineral profiling

The mineral content of pomace powder was analyzed using inductively coupled plasma atomic emission spectroscopy (X-Series2, Thermo Scientific, USA). Following digestion with a solution of perchloric and nitric acid in 1:3 ratios, the sample (0.5 mg) was diluted to 25 mL with deionized water for mineral assessment, with results reported in mg/L.

Fourier Transform Infrared

FTIR analysis was conducted to examine structural changes using a Fourier transform spectrophotometer (PerkinElmer, UK) with a resolution of 0.5 cm − ¹, scanning wavelengths from 400 to 4000 cm − 1.

X-Ray diffraction

Structural analysis of the samples was conducted using a Panalytical-X'pert PRO diffractometer (Almelo, Netherlands) with CuK (A⁰) = 1.54056 radiation, scanning from 5 to 80 degrees (2θ) following the method outlined by Ma et al. [18].

Scanning electron microscopy (SEM)

The composition and effects of tablet and pomace powder on structures were investigated. SEM analysis utilized an XL30 scanning electron microscope (FEI Philips, France), offering magnifications from 10 to 300,000.

Tablet formulation

Preparation of extract

Ultrasonic assisted extraction (UAE) involved sonicating freeze-dried pomace in a distilled water solution with 1% citric acid (1:40, w/v ratio) using an ultrasonic instrument (MODEL-Q500, Qsonica, LLC, Newtown, CT, USA) set at 500 W power, 20 kHz frequency, and 5 sec pulse duration. The slurry underwent ultrasonic treatment for 30 minutes at 40% amplitude, followed by filtration using Whatman filter paper 1. The extracted liquid was stored in amber-colored containers at 4 ± 2°C and subsequently used in tablet formulation.

Tablet preparation

The freeze-dried powder, optimized for flowability and phytochemical content, was mixed with beetroot pomace extract obtained via ultrasonic extraction in a 1:1 (w/v) ratio to enhance bioactive compounds and antioxidant capacity, followed by subsequent freeze-drying. Tablets were then prepared by compressing the pomace powder alone using a hydraulic press (Delfabro, Cordoba, Argentina) at 3 kN for 10 seconds, using a 10 mm diameter flat die to standardize each tablet to a weight of 500 ± 10 mg.

Compact disintegration time

The disintegration time, referring to the duration for complete tablet disintegration, was assessed using a disintegration apparatus as per the United States Pharmacopeia guidelines established by the United States Pharmacopeial Convention [34]. The experiment was conducted at 30 ± 0.5°C using 800 ml of distilled water and six tablets.

In vitro bioavailability of phytochemicals

Total betalains released from the tablets were quantified using a dissolution apparatus (708-DS, Agilent Technologies, USA), where each tablet immersed in 500 ml of distilled water at 32 ± 0.5°C and 100 rpm, following a method adapted from Garrido Makinistian et al. [8]. After the designated time interval, 5 mL of the solution was withdrawn and replaced with an equivalent volume of fresh distilled water for identification of betalains using the previously described method.

Storage studies

Amber plastic bottles containing 100g tablets were stored at room temperature (25 ± 2°C) for 5 months, with monthly assessments conducted to monitor changes in moisture levels, color, total betalains, phytochemical composition, and antioxidant properties.

Statistical analysis

Mean values, standard deviations, and analysis of variance (ANOVA) were computed using SPSS 18.0 software, followed by Duncan's multiple range tests at a significance level of p ≤ 0.05 for comparisons, with the mean and standard deviation (S.D.) of the entire dataset calculated thrice.

Physicochemical attributes of Beetroot pomace powder and tablet

Table 1 summarizes the characteristics of beetroot powder, including its moisture content, Carr's index, Hausner ratio, water activity, water retention capacity, water holding capacity, angle of repose, swelling capacity, hygroscopicity, and color, relevant to nutraceutical applications. Water has a profound impact on the characteristics of powdered materials, including their physical, chemical, and biological properties. Water activity and moisture content are critical indicators influencing water presence and storage stability of products over time [36]. The moisture levels of the powder, indicating the residual water content, showed satisfactory results. In industrial level, it is usually preferable for storage stability to be < 5% [28]. The observation indicated a moisture content of 2.55% in the pomace powder and 2.56% in the tablet (Table 1). The quantity of available water within the product is directly linked to its water activity. Elevated water activity indicates a greater presence of free water, facilitating microbial and chemical interactions, consequently leading to increased degradation. The suggested water activity level for powder is < 0.6 [10]. The pomace powder that underwent freeze-drying exhibited the lowest water activity, measuring at 0.32 as indicated in Table 1. The low moisture levels and water activity of the powder create an environment unsuitable for microbial growth or deteriorative reactions. Color plays a crucial role in determining acceptability as it enhances the visual appeal of products (Fig. 1). The freeze drying process notably enhanced the color values of the produced powder. The freeze-dried powder exhibited decreased L* value and increased a* and b* values (Table 1). The vibrant red color of beetroot comes from betalains, which break down more at higher drying temperatures. This degradation possibly happened during conventional hot air drying methods [6]. The study revealed that the color of the freeze-dried powder closely matched that of fresh pomace, with powder flow properties linked to resting angle, Hausner ratio, and Carr's index. Powders characterized by a Hausner ratio greater than 1.30% and a Carr's index exceeding 15% were recognized as cohesive, typically hindering flow. Powder particles with a high Hausner ratio tend to clump together, resulting in reduced powder flowability [15]. Table 1 demonstrated the notable flow properties of the freeze-dried powder, including a 31.22-degree angle of repose, 14.99% Carr's Index, and a 1.19 Hausner ratio. The hygroscopic nature of any food material refers to its ability to absorb moisture from the environment. The physicochemical parameters of freeze-dried powder and tablet powder did not show significant differences (p < 0.05).

Table 1

Physico and phyto-chemical Charateristics of Beta Vulgaris L. pomace powder and Tablet
Physico-chemical analysis	Moisture (%)	Water activity (a_w)	Angle of respose	Carr index (%)	Hausner’s Ratio	Hygrocopicity (g/100g)	D_{50 (µm)}	Span	Glass transition temperature (T_g)	pH
BPP	2.55^a ± 0.03	0.32^b ± 0.01	31.22^a ± 1.31	14.99^a ± 0.02	1.19^a ± 0.01	15.04^a ± 0.44	275.74^a ± 0.15	1.22^a ± 0.02	52.38^a ± 1.77	5.74^a ± 0.05
TB	2.56^a ± 0.02	0.31^b ± 0.01	31.19^b ± 1.30	14.97^b ± 0.01	1.17^b ± 0.02	15.01^b ± 0.37	269.12^b ± 0.11	1.23^a ± 0.02	51.98^b ± 1.31	5.65^b ± 0.02
Mineral content (mg/100g)	Calcium	Copper	Iron	Potassium	Magnesium	Manganese	Sodium	Phosphorous	Sulphur	Zinc
BPP	276.5^b ± 0.01	44.8^b ± 0.15	5.8^b ± 0.88	1230.7^b ± 0.14	153.7^b ± 0.11	2.5^b ± 0.15	291.5^b ± 0.01	160.6^b ± 0.15	171.8^b ± 0.02	2.8^b ± 0.05
TB	306.1^a ± 0.02	49.1^a ± 0.11	5.9^a ± 0.71	1306.2^a ± 0.12	167.1^a ± 0.21	3.4^a ± 0.09	316.1^a ± 0.04	167.1^a ± 0.07	178.3^a ± 0.01	2.9^a ± 0.01
Phytochemical analysis	Total phenols (mg GAE/g)	Total betalains (mg/g)	Betacyanins (mg/g)	Betaxanthins (mg/g)	Flavonoids (mg QE/g)	DPPH (%)	DPPH (mg AAE/g)	Color profiling
Phytochemical analysis	Total phenols (mg GAE/g)	Total betalains (mg/g)	Betacyanins (mg/g)	Betaxanthins (mg/g)	Flavonoids (mg QE/g)	DPPH (%)	DPPH (mg AAE/g)	L*	a*	b*
BPP	193.26^b ± 0.04	5.19^b ± 0.11	3.33^b ± 0.15	1.86^b ± 0.13	49.32^b ± 0.03	80.96^b ± 0.08	26.36^b ± 3.25	41.98^a ± 0.13	3.50^b ± 0.03	1.40^b ± 0.01
TB	253.26^a ± 0.03	11.18^a ± 0.08	6.33^a ± 0.09	4.85^a ± 0.04	98.32^a ± 0.02	87.34^a ± 0.11	46.36^a ± 2.97	40.88^b ± 0.07	3.67^a ± 0.01	1.46^a ± 0.02
Values are expressed as means of three replications ± standard deviation. Values with different letters in superscript differ significantly (p ≤ 0.05).

This characteristic significantly affects the stability during handling and storage. As depicted in Table 1, the powders exhibited hygroscopicity levels that fell comfortably within the optimal range (< 20%), implying the necessity for careful handling and storage [22]. Particle size significantly influenced the physical properties of powdered materials, encompassing handling, storage, and stability considerations.

The freeze-dried powder particles had an average size of 275.74 µm, indicating that they were relatively smaller. The span values of the powder indicated a narrow particle distribution, with value being less than 2 (Table 1). The flowability characteristics of the beetroot pomace powder were consistent with the results. As per the data presented in Table 1, the glass transition temperatures (Tg) of the powders, measuring 52.38°C, suggest their stability under ambient conditions. The results of this research align with the outcomes reported by Jovanović et al. [13].

Phytochemical analysis

Beetroots were acknowledged for their abundant reservoirs of phytochemicals, encompassing phenolic compounds and a wide range of betalains, presenting manifold health advantages. Food processing industries encountered challenges in preserving food items due to the loss of nutrients during the processing stage. The evaluation of drying methods on antioxidant potential and bioactive components revealed significant enhancement through freeze-drying (P < 0.05). The freeze-drying technique widely acknowledged for its capacity to preserve a significant quantity of phytochemicals while minimizing degradation, as indicated by Sęczyk et al. [29] and demonstrated in Table 1. The amount of phytochemicals present in a food item was correlated with its antioxidant properties [4]. The pomace extract obtained through ultrasonic extraction exhibited concentrations of 5.19 mg/g of total betalains, 193.26 mg GAE/g of total phenolics, 49.32 mg QE/g of total flavonoids, and 26.36 mg AAE/g of DPPH on a dry weight basis.

Mineral profiling

Farhan et al. [5] reported that beetroot was believed to contain diverse minerals including calcium, magnesium, sodium, potassium, iron, and zinc. These elements are thought to potentially address deficiencies in micronutrients among vulnerable populations. The mineral profile of tablet powder was significantly (p < 0.05) increased calcium, iron, copper, potassium, magnesium, manganese, sodium, phosphorus, sulfur, and zinc concentrations i.e., 306.1, 49.1, 5.9, 1306.2, 167.1, 3.4, 316.1, 167.1, 178.3 and 2.9 mg 100 g^− 1, respectively than freeze dried pomace powder.

Scanning Electron Microscopy

SEM analysis was conducted on the powder and tablet powder to investigate the morphological alterations arising from the drying procedure. The SEM images distinctly illustrate that freeze drying resulted in particles of relatively uniform size (Fig. 2). The improved flow characteristics of the freeze-dried powder were attributed to previous mentions, while the tablet surface became smooth due to compression during the manufacturing process.

Fourier transform infrared spectra

The FT-IR spectra of both the powder and tablets showed comparable bands (Fig. 3), indicating the presence of a variety of bioactive compounds. Peaks observed between 3928 to 2032 cm^− 1 in the spectrum indicated the presence of O–H functional groups in sugars and phenolic components, suggesting the presence of betalain pigments. The appearance of a stretching band around 1739 to 1617 cm^− 1 indicates the likely presence of flavonoid compounds, suggesting the existence of a pyran ring structure. Phenolic compounds are identified through absorption peaks at specific wavelengths: 1383 − 1380 cm^− 1, 1246–1233 cm^− 1, and 1071–917 cm^− 1. The peaks were representative of stretching vibrations involving CH3, C–O–C, CH–CH, C–CH, and C–OH bonds, with the presence of C–O stretching indicating the presence of phenols, characterized by a distinct band in the 1300–1000 cm^− 1 spectrum. The stretching of C-Br is responsible for the absorption bands that emerged at 692 cm^− 1 [21].

X-ray diffraction

The X-ray diffraction (XRD) technique was employed to analyze the crystallinity, orientation, and chemical composition of both powder and tablet samples, depicted in Fig. 4, with sharper peaks indicating crystallinity and wider peaks suggesting an amorphous form. The analysis of these characteristics assessed the crystallinity level in powder particles, showing that amorphous particles dissolved more readily in water compared to crystalline ones, as indicated by XRD analysis showing peak broadening as a feature of the semi-crystalline structure of the powder particles. Amorphous particles lacked crystallinity, resulting in peak broadening due to weakened molecular bonding within and between chains, thereby reducing crystallinity. Peak broadening served as an indicator of particle size, as smaller particles generally resulted in wider diffraction peaks [24]. The particle size results obtained from the freeze-dried powder were validated through XRD patterns of both the tablet and powder.

Tablet Disintegration Time and Dissolution Characteristics

The process of tablet preparation involved using freeze-dried powder, with each tablet weighing approximately 500 ± 20 mg. This weight was determined based on considerations of flowability, phytochemical composition, and antioxidant capacity (Fig. 1). The findings indicated that each tablet contains 3.75 mg of betacyanins, 2.28 mg of betaxanthins, 5.59 mg of betalains, 126.63 mg of total phenols, and 49.16 mg of total flavonoids. It also demonstrated significant antioxidant properties, showing DPPH free radical scavenging activity equivalent to 23.18 mg AAE. Fu et al. [6] & Menezes et al. [19] suggested that consuming two tablets daily can provide around 30–50% of the suggested daily betalains (35–50 mg) and phenols (497–1143 mg) intake, respectively. According to the United States Pharmacopeia [35], an oral tablet should dissolve within an hour. The beetroot pomace tablet fulfilled the requirement, disintegrating in approximately 30 minutes without the need for any additional substances to aid its breakdown. It also demonstrated significant antioxidant properties, showing DPPH free radical scavenging activity equivalent to 23.18 mg AAE. The dissolution test conducted on the beetroot pomace tablet showed a rapid release of betalains from the tablet. In the initial 15 minutes, about half of the phytochemicals were dissolved; approximately 85% of the chemicals were discharged within 50 minutes. The dissolving assay results matched those for several other herbal medications published by Gallo et al. [7] The formulation designed for in vitro release exhibited satisfactory dissolution performance. The color measurements of the tablet matched closely with those of the pomace powder, showing L* 43.33, a* 3.12, and b* 1.41.

Storage studies

Effects of Storage Conditions on the Physical and Chemical Properties of Tablets

The research investigated the influence of storage conditions on the chemical, physical, and compound characteristics of plant-derived tablets. The tablet characteristics were evaluated longitudinally, taking into account variables including temperature, humidity, and storage duration. The findings provide insights into the stability and quality maintenance of tablets during storage, crucial for ensuring their efficacy and safety in pharmaceutical applications. Ensuring the stability of any food item throughout its storage period is crucial for assessing the preservation of bioactive compounds [23]. Over a five-month period, we periodically assessed the levels of various plant compounds and antioxidant properties to evaluate the tablets' stability under room temperature storage (25 ± 2°C). At the end of storage, a statistically significant increase (p < 0.05) in moisture content occurred, rising from 3.23–4.04%, with moisture content and water activity identified as key factors affecting food storage quality. The increase was attributed to moisture absorption, influenced by storage conditions, including temperature and humidity, as depicted in Fig. 5.

During storage, there was a slight rise in water activity, noticeable at a significance level < 0.05. At the conclusion of the 5-month storage period, the water activity reached 0.27, as depicted in Fig. 5. During the storage experiments, both moisture levels and water activity remained within acceptable limits. Storage did not cause any notable change in the water activity of the tablets.

Betalains, among phytochemicals, are recognized as highly prone to degradation during storage [2]. Throughout the entire 5-month storage period, a noticeable linear decline in total betalains was observed statistical significant (P < 0.05). These compounds decreased from an initial value of 11.18 to 7.94 mg/g (Fig. 5). This decline can be attributed to the sensitivity exhibited by these compounds to storage conditions. One significant factor is the exposure to oxygen in the air, which can lead to oxidation reactions, thereby breaking down the betalain molecules. Additionally, moisture present in the surrounding environment can facilitate enzymatic reactions that degrade betalains. Furthermore, fluctuations in temperature and exposure to light can also accelerate the degradation process. Overall, the combined effects of oxygen exposure, moisture, temperature fluctuations, and light exposure contribute to the degradation of betalains during storage at ambient temperature [16].

Improvements in storage techniques revealed a decrease in overall phenolic content.

This finding suggests that the decrease in total phenol content from 253.26 mg GAE/g to 225.56 mg GAE/g over 5 months reflects an approximate retention of 89% of total phenols, possibly linked to phenolic compound degradation during storage or processing. Kaur et al. [15] similarly found a reduction in phenolic compounds during storage in jamun pomace powder. Despite a notable decrease in total flavonoid content, with a statistical significance of (P < 0.05), following a storage period of 5 months, approximately 76% of the initial total flavonoids remained detectable. Specifically, the quantity diminished from 98.32 to 74.52 mg QE/g, as illustrated in Fig. 5. The reduction in flavonoids could possibly be attributed to the oxidative breakdown and hydrolysis of flavonols [15]. The tablets exhibited a notable decrease in antioxidant activities from 46.36 mg GAE/g to 36.46 mg AAE/g as storage time progressed, correlating with the levels of phytochemical concentration (Fig. 5).

Although there was a notable reduction, an average of 78% of antioxidant properties was preserved.

The freeze-dried powder demonstrated enhanced physical attributes and a more advantageous phytochemical profile. FTIR, SEM, and XRD examinations confirmed this, leading to its subsequent utilization in tablet formulation. To enhance the level of bioactive compounds and antioxidant power, a blend of pomace powder and pomace extract obtained through ultrasonic extraction was combined in equal proportions. After the storage period, about 71% of betalains, 89% of phenols, 76% of flavonoids, and 78% of antioxidant capacity were still preserved at 25°C. Drying pomace can assist in reducing the environmental impact of waste, while tablet formulation offers a nutraceutical functional product for health-conscious consumers.

Acknowledgement: Ms. Akashdeep Kaur expressed gratitude to Panjab University, Chandigarh, for awarding her a Ph.D. fellowship.

Funding Declaration: The study did not receive any funding.

Conflict of Interest Declaration: None of the authors had conflicts of interest.

Human and Animal Studies: No research involving human or animal subjects was conducted in this study.

Ethics Approval: Ethics approval was unnecessary for this research.

Author Contribution Declaration: Ms. Akashdeep conducted practical experiments, authored the initial manuscript, and received conceptual guidance and editing from Dr. Gargi.

Novelty

This research innovatively utilizes beetroot pomace, a bioactive compound-rich waste, to develop nutraceutical tablets enriched with phytonutrients and antioxidants. Advanced techniques like ultrasonication and freeze-drying were employed to enhance the phytochemical content and flow properties of the powder. Comprehensive analyses, including FTIR, SEM, and particle size distribution, confirmed the high-quality and consistency of the freeze-dried powder. The resultant tablets demonstrated rapid disintegration and high stability, retaining significant phytochemical and antioxidant activity after five months of storage.

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Characterization of Betalains-Enriched Nutraceutical Tablets from Beta vulgaris L. Pomace Powder

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Abstract

Figures

Introduction

Materials and methods

Materials

Pomace drying and tablet preparation

Freeze drying

Analysis

Physicochemical Properties of Powder

Moisture & water activity

Color analysis

Hygroscopicity

Particle size distribution and glass transition temperature (Tg)

Flowability parameters

Determination of the phytochemicals

Assessment of antioxidant properties

Mineral profiling

Fourier Transform Infrared

X-Ray diffraction

Scanning electron microscopy (SEM)

Tablet formulation

Preparation of extract

Tablet preparation

Compact disintegration time

Storage studies

Statistical analysis

Results and discussions

Physicochemical attributes of Beetroot pomace powder and tablet

Phytochemical analysis

Mineral profiling

Scanning Electron Microscopy

Fourier transform infrared spectra

X-ray diffraction

Tablet Disintegration Time and Dissolution Characteristics

Storage studies

Effects of Storage Conditions on the Physical and Chemical Properties of Tablets

Conclusion

Declarations

References

Supplementary Files

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