Introduction

Natural color pigments are found in plant tissues, animal cells (e.g., carminic acid and kermesic acid), the metabolism of microorganisms, or mineral sources (e.g., calcium carbonate, titanium dioxide) [1]. Betalains are a group of naturally occurring water-soluble pigments and specialized (secondary) metabolites that contain nitrogen (chromoalkaloids) [2]. Compared to anthocyanins, these pigments are water-soluble and have three times the tinctorial strength and stability at lower pH levels [3]. Plants in the order Caryophyllales are the main producers of betalains [4], which are found in the leaves, roots, and fruits of certain species within the order, including the genera Beta, Amaranthus, Opuntia, and Hylocereus [5]. Additionally, betalains are reported to be potent antioxidants [6, 7].

Betalain pigments can be categorized into two main structural groups: betaxanthins and betacyanins, which are distinguished by their yellow-orange and reddish-violet colorations, respectively [2, 8, 9]. The amounts of betalain pigments, such as betaxanthins and betacyanins, differ from plant to plant [10]. Both betacyanins and betaxanthins have betalamic acid as the core compound. Specifically, betacyanins are conjugated with cyclo-dihydroxyphenylalanine (DOPA) and undergo O-glycosylation or acetylation [11], whereas betaxanthins are formed by the condensation of betalamic acid with various amino acids, such as proline in indicaxanthin and glutamine in vulgaxanthin I [12] (Figure 1).

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Figure 1. Structure of (A) betacyanin parent molecule and (B) betaxanthin parent molecule. R1 and R2: hydrogen or sugar moieties; R3 and R4: amino acids, amines, or their derivatives. Adapted from [13]. Copyright © 2023 Ornelas García, Guerrero Barrera, Avelar González, Chávez Vela and Gutiérrez Montiel. CC BY.

Betanidin-5-O-β-glucoside (betanidin) is the primary structure of all betacyanins, by which acylation and glycosylation of the 5-O- or 6-O-glucosides yields various betacyanin structures [14]. Examples of betacyanins include betanin, isobetanin, probetanin, neobetanin, amaranthin, and isoamaranthin [5, 15]. Betacyanins are classified into four distinct types based on attachment of glucosyl groups to oxygen atoms in the O-position on the cyclo-dopa moiety [14], including (i) betanin-type, (ii) gomphrenin-type, (iii) amaranthine-type, and (iv) bougainvillein-type pigments [16]. The betanin-type group has a glucosyl or derivative linked to the C5 carbon and a hydroxyl attached to C6; the amaranthin-type group contains a glucuronyl-glucosyl moiety or derivative linked to C5; the gomphrenin-type group has a hydroxyl at C5, and glucosyl or derivative at C6; and the bougainvillein-type group may harbor a diglucosyl moiety or derivative at C5 or C6 of carboxylated or decarboxylated betacyanins [14, 16].

This review discusses the natural sources, biosynthesis, bioavailability, extraction methods, and industrial applications of betacyanins. Building on this, we examine their health benefits, particularly in relation to cardiometabolic disease, and consider their potential roles as anticancer and antimicrobial agents. Furthermore, we expand on previous publications by discussing their molecular docking properties and exploring their emerging applications in food, natural products, and health.

Natural sources of betacyanins

Betanin (betanidin-5-O-β-glucoside) is the most prevalent betacyanin found in plants [17]. In particular, betanin is mainly found in red beetroots (Beta vulgaris L.), one of the best sources of betacyanins [5, 18]. Furthermore, betacyanin traces are also visible in all tested varieties of B. vulgaris [19]. The natural red food colorant betanin, approved under food additive regulations (E162), is also permitted in small quantities and used in pharmaceuticals and cosmetics [20].

The fruits of Opuntia species contain several betacyanins, including betanidin, isobetanin, isobetanidine, and neobetanin [21], with examples like Opuntia stricta, O. joconostle, and O. dillenii [22–24]. Betacyanins, mainly betanidin 5-O-β-sophoroside, were also recovered from the edible fruits of four Melocactus species: M. violaceus, M. bahiensis, M. amoenus, and M. curvispinus [25]. Betacyanin levels are generally higher than betaxanthin levels in pear cactus and bougainvillea [10]. The peel of red dragon fruit or pitaya (Hylocereus polyrhizus) contains high betacyanin concentration, giving the peel its reddish-violet hue [26, 27]. Higher concentrations of betacyanins are recovered in the peel, which is usually discarded as food waste [28]. Interestingly, although earlier reports suggested that betacyanins and anthocyanins are mutually exclusive in plants [29, 30], studies have found both in red pitaya [27, 31], a finding that is still under discussion [32, 33]. Despite this, distinctions exist between these two compounds; for instance, anthocyanins are more stable in acidic conditions [34], whereas betacyanins are more stable from pH 3–7 [11]. The different types of plants and their betacyanin contents, such as B. vulgaris [19, 35, 36], Opuntia ficus-indica [37], Amaranth [37], H. polyrhizus [38], and Melocactus spp. [25] are summarized in Table 1.

Plants of the Amaranthaceae family contain betacyanins, consisting of amaranthine-type, gomphrenin-type, and betanin-type pigments [39]. For instance, Alternanthera, Amaranthus, Beta, Chenopodium, Celosia, and Gomphrena are several genera in Amaranthaceae containing betalains [40]. Amaranthus gangeticus is rich in betacyanins, including amaranthine, iso-amaranthine, betanin, and iso-betanin [41]. Gomphrenin-I, a specific betacyanin, has been identified in the flowers of Gomphrena globosa [39, 42, 43]. Betanin monoglucoside, and its 4-coumaroyl and feruloyl derivatives, are the main betacyanins present in the fresh juice of Basella rubra fruits [44]. The fruits of nine Mammillaria species contain betanidin 5-O-(6′-O-malonyl)-β-sophoroside, identified via spectroscopic techniques [45]. The fruits of Basella alba L. and Basella alba L. var. “Rubra”, and mushrooms of genera Amanita, Hygrocybe, and Hygrophorus also contain betalains [11]. Additional sources of betacyanins include Talinum triangulare [46], some Bougainvillea species [47], and wild pokeweed (Phytolacca americana) [48].

Identifying new natural sources of betacyanin can increase available pigment alternatives beyond Caryophyllales plants. For example, Garcinia, with over 250 species, should be explored for its betacyanin content due to its variety and colorful nature [49]. In addition, extracting pigments from underutilized sources or food waste like fruit peel is promising for its environmental and practical benefits [50, 51].

Biosynthesis of betacyanins

Several steps are needed for the biosynthesis of betacyanins (Figure 2). Betanin goes through additional glycosylation and acylation reactions, leading to more complex betacyanins [52]. The formation of beta cyclins is a complex process involving many enzymes. Studies in plants have shown a positive correlation between betacyanin levels and a bifunctional tyrosinase enzyme (cytochrome P450 enzyme CYP76AD1). This enzyme is necessary for the hydroxylation and oxidation activities needed for the biosynthesis of betalains, which are further modified to give betacyanins. This correlation was discovered by Wang et al. [53] when they observed tyrosinase synthesis together with betacyanin production in the cotyledons of Suaeda salsa seedlings grown in the dark. When exposed to light, the levels of tyrosinase and betacyanin decreased, suggesting a link between exposure to light and the degradation of both molecules. Other studies also show an inverse relationship between betacyanin synthesis and temperature. Lower temperatures favor synthesis, and higher temperatures reduce synthesis in B. vulgaris root samples [54, 55]. Apart from temperature, other ecological factors play roles in the synthesis and accumulation of betacyanin. These include salinity, pH (4–6), and the presence of metals in the environment [56].

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Figure 2. The biosynthesis of betacyanin pigments. In a series of reactions, tyrosine is converted to L-DOPA by tyrosine hydroxylase, which is then used by 4,5-DOPA-extradiol-dioxygenase (DOD) to produce 4,5-seco-DOPA, which is transformed into betalamic acid, which serves as a building block for either betacyanins or betaxanthins synthesis. This figure was drawn following pathway information synthesized from other papers referenced in this review [52–68]. L-DOPA: L-dihydroxyphenylalanine.

Apart from the tyrosinase enzyme, other enzymes are instrumental in the biosynthesis of betacyanins. These include 4,5-DOPA-extradiol-dioxygenase (DOD) and 5-O-glucosyl transferase (5GT) [57]. Their heterologous expression has been shown to lead to de novo production of betacyanin pigments in transgenic Eustoma plants [58]. DOD is a critical enzyme in this pathway. It catalyzes the formation of a betalamic acid, a structural intermediate for all betalains [59]. Researchers can make either beta-cyclins or betaxanthins using betalamic acid as a starting point [60]. The reaction begins with a tyrosine amino acid converted to L-DOPA by tyrosine hydroxylase. From DOPA, several pathways can branch off to give betanidin. In one pathway, DOD uses DOPA as a substrate for this reaction and cleaves it via an extradiol cleavage reaction, producing 4,5-seco-DOPA, which spontaneously undergoes a recyclization reaction, forming betalamic acid, which spontaneously becomes betanidin.

In another pathway, L-DOPA is metabolized by various enzymes like tyrosinase to give DOPA-quinone. DOPA-quinone undergoes an intramolecular cyclization, forming a molecule called aminochrome, which spontaneously converts to cyclo-DOPA. Cyclo-DOPA is glycosylated at the 5-O position by cyclo-DOPA 5-O-glucosyltransferase to form cyclo-DOPA 5-O-glucoside. Finally, cyclo-DOPA 5-O-glucoside spontaneously condenses with betalamic acid to form betanidin [11, 61–63]. Betanidin is further glycosylated by betanidin 5-O-glucosyl-transferase encoded by B5GT to form betanin, the most common betacyanin [64, 65].

Recent studies show that betalamic acid can be engineered chemically [60, 66]. Other researchers are looking for ways to increase yield by optimizing the pathway from tyrosine to the final betalain product by selecting the best-performing isozymes from betalain-producing species and the optimal combination of regulatory elements that drive their expression [64]. Timoneda et al. [64] reported a seven-fold increase in betalain production in Nicotiana benthamiana. Other studies are exploring whether betalain production could be engineered in various organisms to have new potential sources for betalains. These developments and other current biochemical metabolic engineering studies have been discussed by Polturak and Aharoni [52].

Further, metabolic engineering for betalain production in crops like vegetables, fruits, and cereals may offer new food resources beneficial for healthcare, since betalains are primarily found naturally in plants of the order Caryophyllales. Several advancements have been made, for example, with the help of appropriate specific promoters, the transgenic tomato fruits and potato tubers with co-expression of the genes involved in betacyanin biosynthesis CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase), and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis Jalapa, have dark red tissues with an enriched accumulation of betacyanins (betanin and isobetanin) [67].

Current developments in betanin production include combinatorial engineering of plant P450 and UDP-glycosyltransferases (UGTs) enzymes and precursor metabolisms to enhance the de novo betanin production in Saccharomyces cerevisiae [68]. Li et al. [68] employed a multifaceted strategy focusing on improving the activity of the key cytochrome P450 enzyme, CYP76AD. Targeted mutagenesis of the CYP76AD enzyme led to the discovery of a mutant that exhibited a ~7-fold increase in betanin titer compared to the wild-type enzyme, underscoring the effectiveness of targeted mutagenesis in fine-tuning P450 activity. Notably, upregulation of the bio-synthetic pathway and optimizing the UDP-glucose levels supported efficient glycosylation, a critical step in betanin formation [68]. Glitz et al. [69], critically assessed which of the UGTs increased the production of betanin in yeast.

The authors’ comprehensive in vivo screening of 27 plant-derived UGTs in Saccharomyces cerevisiae revealed two previously uncharacterized UGTs—CqGT2 (UGT73A37) from Chenopodium quinoa and BgGT2 (UGT92X1) from Bougainvillea glabra—which the authors suggest are likely involved in betanin synthesis in their native plants [69]. Functional validation of these UGTs in Yarrowia lipolytica revealed that CqGT2 was the most effective UGT for betanin production in this non-conventional yeast host. Li et al. [68] and Glitz et al. [69] highlight how protein engineering and pathway optimization can enable high-level production of complex plant-derived natural products in microbial hosts.

Green extraction methods of betacyanins

Extraction processes for obtaining phytochemicals such as betacyanins from plant sources need to be carefully chosen [70]. Conventional methods, such as Soxhlet and decoction, are often inefficient and environmentally unfavorable. This occurs due to the use of large amounts of organic solvents and the need for continuous heating, sometimes lasting several hours, raising environmental and safety concerns [71, 72]. This has driven the development of green extraction techniques, aiming to minimize solvent use, reduce labor, and improve sustainability. This section explores various green extraction methods for betacyanin extraction, including ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), enzyme-assisted extraction (EAE), and pulsed electric field (PEF). These methods offer improved automation, enhanced selectivity, higher extraction efficiency, and reduced solvent consumption [70, 71].

Pulsed electric field (PEF)

This method induces electroporation, in which an intense electric field permeabilizes the cell membrane through dielectric breakdown [87]. Here, two electrodes are used to apply short-duration pulses of moderate electric voltage (0.5–20 kV/cm) to a substrate [88]. The PEF method has been utilized to extract betacyanin. Koubaa et al. [89] tested PEF to extract betacyanins from purple Opuntia fruits. They adjusted the electric fields and number of pulses using water as the extraction solvent, with constant agitation at room temperature for 1 h [89]. They found that applying 50 pulses at 20 kV/cm produced a yield of 50 mg betacyanins per 100 g fresh weight (fw), which was a significant two-fold improvement compared to the conventional method of constant agitation with water at room temperature for 1 h, which yielded only 20 mg per 100 g fw [89].

Among the methods discussed, UAE is generally the most effective for preserving bioactivity, as its cavitation mechanism enhances mass transfer and compound release while operating at relatively low temperatures. Compared with MAE, UAE better maintains pigment color and antioxidant potential under mild conditions, especially when optimized or combined with enzymatic treatments [73, 75]. MAE provides rapid extraction and high yields of betalains and phenolics, often resulting in elevated antioxidant activity in assays such as DPPH, ABTS, and FRAP. However, because MAE is a thermal technique, prolonged exposure or high power can degrade pigments and reduce color stability, making it less suitable for bioactivity preservation unless parameters are carefully optimized [79].

SFE, which uses CO₂ with a polar co-solvent, also excels at retaining pigment integrity and antioxidant activity because it operates at low temperatures and limits oxidative degradation. Nevertheless, its total pigment recovery is typically lower than that of MAE or UAE, meaning it better preserves bioactivity per compound extracted but yields less extract overall [83, 84]. Low-temperature EAE has demonstrated superior pigment integrity and color stability compared to conventional solvent extraction, owing to enzymatic hydrolysis under mild conditions that prevent thermal degradation [85]. Combining EAE with UAE further enhances both pigment release and antioxidant capacity while maintaining gentle extraction conditions [87]. Finally, PEF is recognized as one of the most promising non-thermal pretreatments for preserving bioactivity, as it disrupts cell membranes through electroporation and enables subsequent extractions under mild conditions. When applied before UAE or solid-liquid extraction, PEF improves both yield and the retention of heat-sensitive compounds [87].

Selecting the optimal betacyanin extraction method requires careful consideration of factors like sustainability, yield, cost, and future applications. While methods like microwave or ultrasound excel in resource efficiency, factors like extraction time might favor techniques such as supercritical fluid or MAE. The green extraction methods are illustrated in Figure 3 and Table 2.

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Figure 3. Green methods used in betacyanin extractions. (a) Ultrasound-assisted extraction (UAE); (b) microwave-assisted extraction (MAE); (c) supercritical fluid extraction (SFE); (d) enzyme-assisted extraction (EAE); and (e) pulsed electric field (PEF). Adapted with permission from [90]. © 2023 Elsevier B.V.

Table 2. Green extraction methods for betacyanin extraction.

Methods
Ultrasound-assisted extraction
Source	Solvent	Temperature (°C)	Time duration (min); ultrasound frequency (kHz/W)	Solid:liquid ratio (g/mL)	Betacyanin content	References
Hylocereus polyrhizus (F.A.C.Weber) Britton & Rose	Methanol in water	10–60	2–30; 200/24	0.1:10–0.3:10	0.84–1.31 mg/g dw	[73]
Hylocereus polyrhizus	n.d.	30–70	2–32; n.d.	n.d.	134.87–154.86 mg/L	[75]
Beta vulgaris L.	Water	30–60	30–60; 25	25–75 mg/mL	3.36–4.48 mg/g	[76]
Microwave-assisted extraction
Source	Solvent	Power (W)	Time; cycles (s)	Solid:liquid ratio (g/mL)	Betacyanin content	References
Beta vulgaris L.	Ethanol	360–900	2–6 s; 140–420 s	1:20	130 mg/L	[78]
Amaranthus tricolour	Water	200–700	5–15 min; 0.5–1 s	1:80	71.95 mg/g dw	[79]
Beta vulgaris L.	Water	100–800	30–50 s; n.d.	0.1–0.2 w/v	115.89 mg/100 g fw	[80]
Supercritical fluid extraction
Source	Solvent	Temperature (°C); pressure (psi/MPa)	Time (min)	Solid:liquid ratio (g/mL)	Betacyanin content	References
Beta vulgaris L.	n.d.	45–85; 10–60 and 200–500	5–10	n.d.	10–23%	[82]
Hylocereus polyrhizus	Ethanol in water	50; 25	90	1:10	28.44–120.28 mg/100 mL	[83]
Hylocereus polyrhizus	Ethanol in water	40–60; 20–30	90	1:10	25.49 mg/100 mL	[84]
Enzyme-assisted extraction
Source	Solvent	Enzyme (%)	Temperature (°C); time (min)	Solid:liquid ratio (g/mL)	Betacyanin content	References
Beta vulgaris ssp.	Acetate buffer	CL: 37, PG and PL: 28, and XL: 35	25–45; 20–300	1:15	6.03–12.15 mg/L/U	[85]
Hylocereus polyrhizus	Water	AA: 0.25–2.75	20–50; 5–30	1:10	0.21–0.30 mg/g	[86]
Beta vulgaris ssp.	Water	AN: 0.1	30–70; 30–180	n.d.	135.23–154.42 mg/L	[75]

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CL: cellulase; PG: polygalacturonase; PL: pectin-liasic; XL: xylanase; AA: Aspergillus aculeatus; AN: Aspergillus niger; n.d.: not described.

Industrial applications of betacyanins

Bioavailability of betacyanins

Bioavailability studies are important to measure the potential bioactive effects of phytocompounds in living subjects; this can be further translated into humans for health assessments and applications [140]. Currently, the mechanisms of how betacyanins are absorbed, metabolized, and excreted are yet to be fully described. Betacyanins begin to degrade immediately upon ingestion in the stomach. A study administering betacyanin solution into Wistar rat stomachs demonstrated that while continuous exposure to gastric secretions degrades these pigments, a portion is still absorbed directly through the gastric wall. The dose and exposure time significantly influence the rate of degradation and the intensity of absorption [141]. This is supported by earlier work calculating that 24–29% of betalain pigments are degraded in the stomach [142], a figure consistent with in vitro models showing a 21–35% loss in the gastric phase [143, 144].

The intestinal phase is where the most significant degradation occurs, drastically reducing bioaccessibility. Bioaccessibility refers to the proportion of a compound that is released from the food matrix in the gastrointestinal tract and becomes available for intestinal absorption. In vitro models show losses of 46–55% in the intestinal phase [143, 144], leaving only 29–38% of ingested betalains remaining [145]. The food matrix plays a critical role; liquid matrices like juices show better intestinal absorption and bioavailability than solid (fiber-rich) foods [146]. This is due to several factors. For example, solid foods must first undergo mechanical and enzymatic breakdown before pigments can be solubilized. These extra steps tend to delay release so that molecules face more extensive intestinal/microbial degradation before release. Pigments in liquid matrices are already solubilized, less trapped by fiber, and more exposed to the absorptive environment [145]. Fermentation can increase bioaccessibility, as shown by fermented red dragon fruit juice having higher betanin levels (46.22%) than raw juice (43.76%) after digestion [26]. This is because the fermentation process partially breaks down the plant cell walls, increasing the release of betacyanin pigments into the solution phase and increasing the bioaccessible fraction during digestion.

Despite significant digestion, betacyanins and their metabolites are absorbed into the systemic circulation. They have been detected in human and animal blood plasma and urine, both in their native form [147, 148] and as deglucosylated, decarboxylated, and dehydrogenated metabolites [149, 150]. Absorption occurs via paracellular transport and can be hindered by specific gut transporters like multidrug resistance-associated protein 2 (MRP2) and competition with glucose transporters due to its glycosylated structure [151, 152]. The liver contributes to metabolism, with first-pass extraction reducing systemic concentrations by approximately 23% [141].

Elimination occurs primarily through renal and fecal excretion, with overall bioavailability being very low. Human studies show a renal recovery of only 0.13% and rapid elimination [145]. Fecal excretion shows a higher degree of decomposition, indicating extensive fragmentation and interactions with gut microbiota [145]. A strong positive correlation between urinary and fecal metabolite profiles underscores the gut’s profound impact on systemic bioavailability and is a key source of inter-individual variability. Excretion rates are also influenced by the food matrix, with betacyanins from beet juice being excreted faster than those from solid beet slices [150].

Several critical mechanisms remain unresolved. The specific role of the liver is not fully explained; for example, biliary excretion of betacyanins appears limited [153–155]. This suggests that the pigments may be rapidly metabolized into uncharacterized or polar secondary compounds that are not detected by current analytical methods or that the presence of alternative hepatic clearance pathways. The precise nature of the interaction with gut microbiota and the identity of all resulting metabolites require further elucidation [141, 145]. As mentioned earlier, decarboxylation and dehydrogenation reactions convert betacyanins into multiple decarboxylated and oxidized pigments (e.g., C‑2/C‑17 decarboxylates, 2,15‑bidecarboxy forms, neobetanin derivatives) [27], but there is a dearth of data on their direct bioactivity. Other similar types of pigments show that metabolites have some activity. Decarboxylated gomphrenin-type metabolites, a subset of betacyanins, have been shown to have strong anti-inflammatory effects in vitro, modulating cytokine release and immune cell activity more effectively than native or parent molecules [156, 157]. Hence, a major future direction is to characterize these unknown metabolites to fully understand the pharmacokinetic pathway and the compounds responsible for the observed health benefits.

The absorption and bioavailability of betacyanins are affected by a complex interplay of numerous factors. These include dosage, gastric conditions, metabolic processes in different organs, the presence of aglycones, specific transport mechanisms (e.g., MRP2), biotransformation pathways, and excretion processes. Crucially, the food matrix (liquid vs. solid, fermented vs. raw) and the structural form of the betacyanin (e.g., glycosylated) are significant determinants of its absorption efficiency [26, 150–152]. Ultimately, intestinal uptake and systemic metabolism are closely related to biotransformation by gut microbiota [141, 145].

Together, these studies show that the absorption of betacyanins is affected by numerous factors and potentially is degraded mainly by the gastrointestinal tract. Several factors that influence the absorption of betacyanins have been identified, among them are dosage, gastric conditions, metabolic processes in different organs, the presence of aglycones, transport mechanisms, biotransformation pathways, food matrices, fermentation status, excretion processes, intestinal biotransformation, and gut microbiota interaction. The bioavailability of betacyanins is summarized in Figure 4.

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Figure 4. The absorption and excretion of betacyanin pigments in various body systems and excretions. Orally given betacyanins showed low urinary recoveries, suggesting rapid metabolism or enhanced bodily elimination. Betacyanins are highly degraded in the stomach and intestines. Absorption of betacyanins is affected by the food matrix.

Betacyanins and metabolic syndrome

Metabolic syndrome is a constellation of conditions, including central obesity, hypertension, type 2 diabetes or impaired glucose metabolism, and dyslipidemia. This syndrome is associated with an increased risk of cardiovascular disease and other complications such as chronic inflammation, chronic kidney disease, and non-alcoholic fatty liver disease [158, 159]. Among natural compounds showing promise for metabolic syndrome management, betacyanins—vibrant plant pigments found in red beetroot, prickly pear, and dragon fruit—have emerged as particularly interesting candidates due to their multi-target biological activities. This section aims to discuss recent reports on the effects of betacyanins on cardiometabolic diseases and proposes their mechanisms of action on both animal and human models.

The findings from preclinical data on betacyanins show consistent demonstration of their dual capacity to combat both oxidative stress and inflammation, two processes intricately linked in metabolic syndrome. Reactive oxygen species (ROS) can activate pro-inflammatory signaling pathways, while inflammatory cells themselves generate further ROS. Betacyanins appear to disrupt this vicious cycle at multiple points. In animal models of diet-induced obesity, streptozotocin-induced diabetes, and diclofenac (DF)-induced acute liver injury model, administration of betanin (the most studied betacyanin) at doses of 10–40 mg/kg consistently acts as a direct redox regulator. It significantly reduces the concentrations of toxic lipid peroxidation end-products like malondialdehyde (MDA) and protein carbonyls (PC), key markers of oxidative damage to cellular structures [160–162].

Apart from acting as free-radical scavengers, betacyanins also enhance the body’s endogenous antioxidant defense system. Studies report a significant upregulation in the activity and gene expression of fundamental antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [160–163]. The master regulator of this coordinated defense is the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) [164]. Research by Mousavi et al. [161] and Villa-Jaimes et al. [162] demonstrates that betacyanins activate the Nrf2 pathway, leading to the increased transcription of a battery of cytoprotective genes, including hepatic enzymes. This Nrf2 activation represents a sophisticated, indirect mechanism by which betacyanins boost the cell’s long-term resilience to oxidative attack.

The reduction in oxidative stress translates into the corresponding anti-inflammatory effects. A reduced oxidative load inherently dampens the activation of redox-sensitive inflammatory pathways. The most critical of these is the NF-κB pathway, a primary regulator of the inflammatory response. Abedimanesh et al. [163] showed that betanin treatment in diabetic rats directly reduced the mRNA expression of NF-κB in liver and blood tissues. Further qPCR analysis increased the expression level of adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin-1 (SIRT1) in blood and liver [163]. Another study also noted a decrease in inflammatory markers (IL-6, IL-1β, and TNF-α) while increasing anti-inflammatory markers (IL-10) in diet-induced obese mice [128]. These findings showed that betanin can reduce inflammation and oxidative stress symptoms associated with diabetes and may produce the same effects in humans.

This molecular finding is corroborated by functional outcomes. In vitro, betanin demonstrated a marked ability to inhibit the expression of key NF-κB-driven pro-inflammatory mediators in macrophage cells, including cytokines like IL-6 and IL-1β, as well as enzymes like iNOS and COX-2 [165]. Simultaneously, it promoted the expression of the anti-inflammatory cytokine IL-10. In vivo, these mechanisms manifest as reduced hepatic inflammation and pancreatic lesions, as observed in histological analyses [128, 163]. The study by Fernando et al. [165] further illustrates the synergy between antioxidant and anti-inflammatory actions by showing that betanin’s radical-scavenging capacity counteracted hydrogen peroxide-induced ROS generation, thereby proactively suppressing the trigger for inflammation.

The convergence of these antioxidant and anti-inflammatory mechanisms may explain the significant improvements seen in key organs associated with metabolic syndrome pathogenesis. Betacyanins demonstrate remarkable hepatoprotective effects across multiple models. In obese Wistar rats, betanin administration reversed hepatic tissue damage, evidenced by reduced aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, decreased lipid accumulation, and improved liver structure [160]. Similarly, in DF-induced acute liver injury, Opuntia robusta fruit extract rich in betacyanins reduced oxidative biomarkers while restoring glutathione levels and activating the Nrf2 pathway [162]. The administration of betacyanins from red and white pitaya also improved liver steatosis in high-fat diet-induced obesity in mice [8, 128]. These findings suggest betacyanins protect against both metabolic and toxic liver injury through complementary antioxidant and anti-inflammatory mechanisms.

Human studies provide promising evidence for cardiovascular benefits. A pilot randomized crossover trial by Rahimi et al. [166] demonstrated that two-week supplementation with betalain and betacyanin-rich (50 mg betacyanins/betalains daily) extracts (O. stricta and red beetroot) improved several cardiovascular markers in male patients, with clinically meaningful reductions in homocysteine, LDL, and non-HDL cholesterol concentrations. Additionally, Cheok et al. [167] reported that red dragon fruit powder (24 g) consumption (providing 33 mg betalains daily for 14 days) improved endothelial function and arterial stiffness in healthy adults, though it did not significantly reduce blood pressure as shown by Rahimi et al. [166]. These findings suggest betacyanins may primarily benefit vascular function and atherogenic lipid profiles. However, the short intervention period, small sample size, and specific age group limit the application of this study to the wider population. Hence, further studies with a longer intervention period, a wider age group, and a larger sample size are suggested to increase the validity of this study.

The renal protective effects of betacyanins are particularly relevant given the high risk of diabetic nephropathy in metabolic syndrome. Sutariya and Saraf [168] demonstrated that betanin treatment from Opuntia elatior (50–100 mg/kg/day) in diabetic rats inhibited proteinuria, reduced urine output, reduced blood glucose concentration, and restored antioxidant enzyme activities in kidney tissue. Importantly, betanin modulated the expression of genes involved in epithelial-mesenchymal transition [transforming growth factor beta (TGF-β), type IV collagen, alpha-smooth muscle actin (α-SMA), E-cadherin] which are the markers of myofibroblasts, and cell-cell adhesion molecule, suggesting it can counteract diabetic nephropathy progression by inhibiting the TGF-β signaling pathway and increasing antioxidant defense systems [168].

Betacyanins demonstrate significant effects on glucose homeostasis and insulin sensitivity, one of the major defects in metabolic syndrome. In streptozotocin-induced diabetic rats, betanin treatment (10–40 mg/kg) produced comprehensive anti-diabetic effects: reduced fasting blood glucose, improved glucose tolerance, and increased serum insulin levels [163]. Histological analyses revealed pancreatic regeneration, suggesting potential for β-cell protection and restoration [163]. The mechanisms underlying these improvements extend beyond antioxidant and anti-inflammatory effects to include modulation of key metabolic sensors. The upregulation of AMPK and SIRT1 [163] is particularly significant, as these enzymes enhance insulin sensitivity, promote glucose uptake in muscles, and improve mitochondrial function. Additionally, betacyanins decrease fibroblast growth factor 21 (FGF21) resistance, further contributing to improved metabolic regulation [8]. These multi-target effects on glucose metabolism make betacyanins particularly promising for addressing the complex pathophysiology of insulin resistance in metabolic syndrome.

Additionally, betacyanins demonstrate significant effects on adipose tissue biology and lipid metabolism. In obesity models, betacyanin supplementation reduced body weight gain, visceral adipose tissue size, and serum lipid levels [8, 128]. In a study published by Khoo et al. [27], the addition of red pitaya extracts containing betacyanins and anthocyanins for 48 h effectively reduced the H₂O₂-induced oxidative stress in 3T3-L1 cells. Additionally, the peel extract showed better antioxidant properties than the pulp due to the higher concentration of betacyanin and anthocyanin in the peel. The peel extract also showed better lipid reduction effects on 3T3-L1 cells compared to the pulp extract [27]. However, as both betacyanins and anthocyanins were reported in this study, it is difficult to elucidate the exact effects of these two phytochemicals, as both may have synergistic effects on cellular physiology. Thus, further studies should aim to assess the effects of combined phytochemicals, e.g., betacyanins and anthocyanins starting from in vitro to in vivo studies.

Mechanistically, in vitro betacyanin treatment in 3T3L adipocytes inhibits adipogenesis through downregulation of key transcriptional regulators, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c), and reduction in PPARγ protein expression involved in adipogenesis [169]. While simultaneously promoting fatty acid oxidation in vivo, through increased expression of lipid metabolism-related genes (AdipoR2, Cpt1a, Cpt1b, Acox1, PPARγ, Insig1, and Insig2) [8]. These dual actions on lipid storage and utilization make betacyanins particularly promising for addressing adipose tissue dysfunction in metabolic syndrome.

Recent evidence suggests betacyanin’s metabolic benefits may be partially mediated through gut microbiota modulation. The consumption of red beetroot juice containing betacyanins in humans correlated with increased beneficial bacteria, including Akkermansia muciniphila, Bifidobacterium, and Coprococcus, while decreasing less desirable species [170]. This microbial shift was associated with increased production of short-chain fatty acids (SCFAs), particularly butyric and isobutyric acid, with specific betacyanin metabolites showing strong correlations with SCFA production [170]. The results showed the ability of betacyanin-containing juice to increase beneficial gut bacteria populations. For instance, the increase in Akkermansia population has been correlated with a healthy diet and decreased risk of obesity and other metabolic disorders [170]. Although the increase of E. coli is thought to be harmful, a recent report showed that commensal E. coli may be beneficial for iron transport [171]. Highlighting the influence of strain specificity of this bacterium and its functions in the human gut [172].

In an animal study, supplementation of 200 mg/kg of betacyanins for 14 days also improved (Firmicutes:Bacteroides) F/B ratio and increased Anaerotruncus, Mucispirillum, and Akkermansia populations at the genus level [128]. An increase in Akkermansia population was also seen after supplementation of Davidson’s plums in Wistar rats’ diet, which is associated with better metabolic and physiological parameters after being given a high-carbohydrate, high-fat diet [173]. The consistent increase in Akkermansia muciniphila across studies [128, 170] is particularly noteworthy given its association with improved metabolic parameters. The ability of betacyanins to modulate the gut-microbiota-liver axis represents a novel mechanism through which they may influence systemic metabolism in metabolic syndrome.

Overall, betacyanins were shown to have beneficial effects against cardiometabolic symptoms in various models (Table 3). Their effects are mediated through several mechanisms such as antioxidant [27, 160–162, 165], anti-inflammatory [163, 165], hepatoprotective [160, 162, 163], pancreatoprotective [163], renal-protective [168], antidiabetic [8, 128, 160, 163], antilipidemic [8, 128, 160, 163], antiobesity [8, 27, 128] and cardioprotective [166, 167] mechanisms, potentially through modulation of gut microbiota population [128, 170] with correlative changes at gene expression levels (Figure 5).

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Figure 5. The potential health-promoting effects of betacyanin pigments in cardiometabolic disorders, cancer, and as antimicrobial agents. Betacyanin acts on these disorders through various mechanisms, including physiological, biochemical, and gene alterations.

Despite promising evidence, several limitations must be acknowledged. First, effective animal doses (10–100 mg/kg) may not be easily achieved through human dietary intake. Second, human studies remain limited and often use combination extracts, making it difficult to attribute effects solely to betacyanins. The bioavailability issue is particularly important, as human studies show minimal increases in plasma polyphenol levels despite functional benefits [167], suggesting metabolites rather than parent compounds may mediate effects.

Future research should focus on well-designed human randomized controlled trials in populations using standardized betacyanin preparations. Research identifying the most bioactive metabolites and understanding how inter-individual variability in gut microbiota affects response to betacyanin intervention will be crucial for developing personalized nutrition approaches. Additionally, more studies are needed to elucidate potential synergistic effects between betacyanins and other phytochemicals, as well as their interactions with conventional pharmacological treatments for metabolic syndrome.

However, translation to clinical practice requires addressing significant challenges, including bioavailability issues, standardization of effective doses, and understanding individual variability in response. While betacyanin-rich foods can be recommended as part of a healthy diet for metabolic syndrome management, their specific therapeutic application requires further rigorous clinical investigation to establish efficacy, optimal dosing, and potential for personalized nutrition approaches based on individual metabolic and microbial profiles.

Betacyanin and cancer

Numerous studies were conducted over the years to investigate the anticancer properties of betacyanins. Betacyanin-rich plants such as B. vulgaris (red beetroot), Basella rubra (Malabar spinach), Amaranth cruentus (sprouts), Opuntia ficus-indica (cactus pear), and H. polyrhizus (red pitaya) demonstrated anticancer potentials against various human cancer cell lines such as HepG2, HeLa, MCF-7, and Caco-2, elucidating several fundamental mechanisms through which betacyanins exert their anticancer effects.

Sreekanth et al. [174] (2007) investigated the anticancer potential of betanin isolated from cactus pear. There was a 50% decrease in K562 cell proliferation after treatment with 40 µM betanin for 24 h. Betanin caused apoptotic cell death, as morphological and structural changes such as a condensed nucleus and membrane blebbing were observed in K562 cells. DNA fragmentation in K562 cells was detected in a progressively increasing manner after treatment with betanin at different concentrations for 24 h [174]. This corresponded to inter-nucleosomal cleavage, which is one of the characteristics of apoptosis. Furthermore, decreased mitochondrial membrane potential was also observed with leakage of cytochrome c into the cytosolic fractions of the K562 cells after treatment with 40 µM betanin at different time points [174], showing the effect of betanin in intrinsic mitochondrial apoptosis.

The anticancer potentials of Pachycereus weberi (Chico) and Escontria chiotilla (Jiotilla) showed that both Chico and Jiotilla possessed high phytochemical contents of betalains and other phenolic compounds. Chico fruit juice was able to inhibit the growth of Caco-2 and MCF-7 cell lines, whereas Jiotilla fruit extract decreased the cell viability of HepG2 and PC-3 cell lines [175]. Extracted betanin/isobetanin concentrate from fresh beetroot was found to upregulate apoptosis-related proteins, in MCF-7 cells treated with 30 µM betanin/isobetanin concentrate for 24 h, such as B-cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad), TNF-related apoptosis-inducing ligand receptor 4 (TRAILR4), FAS, and phosphorylated p53, besides altering the mitochondrial membrane. Hence, it indicated involvement of this extract in both mitochondrial and death-receptor pathways. Furthermore, autophagosome vesicles were observed in the MCF-7 cells, suggesting that there was autophagic cell death as well when cells were treated with betanin/isobetanin [176]. This effect was also demonstrated in T21 bladder cancer cells, where a combination of purified betacyanin and vitexin-2-O-xyloside (XVX) from B. vulgaris var. cicla L. inhibits the proliferation of T24 cancer cells at 24 h and 48 h of incubation. Apoptosis was induced through activation of caspase 8 activity [177].

The anticancer properties of the beetroot hydro-alcoholic extract (BHE) of the red beetroots (B. vulgaris) were investigated previously [178]. Both BHE and its constituent betanin inhibited the growth of Caco-2 and HT-29 cell lines in a time and dose-dependent manner. The IC₅₀ values of betanin were 64 μg/mL (HT-29), 90 μg/mL (Caco-2), and the IC₅₀ values of BHE were 92 μg/mL (HT-29), 107 μg/mL (Caco-2) after 48 h treatment. Apoptosis was confirmed by 4’,6-diamidino-2-phenylindole (DAPI) staining, where both cell lines showed apoptotic characteristics. Flow cytometry indicated an increase in the proportion of both Caco-2 and HT-29 cells in early and late apoptosis after treatment with BHE and betanin for 48 h. This pro-apoptotic effect was further supported by the upregulation of genes including Bad, Fas-R, caspases-3, -8, and -9. Notably, the expression level of the Bad gene was found to be higher than the positive control group, 5-fluorouracil (5-FU), after both BHE and betanin treatment. Conversely, BHE treatment downregulated the expression of anti-apoptotic genes such as Bcl-2 [178]. Looking at an alternative view, as Polygonum minus phytochemicals enhance 5-FU efficacy, betacyanins could similarly synergize with 5-FU by targeting complementary pathways [179]. Additionally, given that combinations of phytochemicals have been shown to enhance colorectal cancer treatment efficacy, future research could explore the co-administration of betacyanins with complementary agents such as isothiocyanates and carotenoids or drugs to modulate intersecting signaling pathways [e.g., phosphoinositide 3-kinase (PI3K)-Akt and NF-κB] [180].

Chenopodium formosanum (djulis) was investigated for its anticancer effects on hepatoma cells, both in vitro and in vivo. Betanin was one of the bioactive compounds identified in the extract using high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-MS analysis, and it was selected to undergo further evaluation. Betanin was able to induce apoptosis in HepG2 cells by increasing apoptotic bodies, decreasing mitochondrial membrane potential, enhancing the ratio of Bcl-2-associated X protein (Bax)/Bcl-2, and activating caspase-3 and cleaved Poly (ADP-ribose) polymerase (PARP) levels. At the concentrations between 50 and 200 μM, betanin increased ROS generation in the HepG2 cells, which may contribute to the cellular antiproliferation due to oxidative stress and eventually apoptosis [181].

In parallel, nanotechnology approaches have leveraged betacyanins for enhanced efficacy using betacyanin-modified selenium nanoparticles for anticancer treatment [182]. The presence of betacyanin on the surface of these nanoparticles enhanced their anticancer properties compared to the non-modified selenium nanoparticles. These modified nanoparticles inhibited the growth of HepG2 cell lines in a dose-dependent manner [182]. Another research group synthesized Ag nanoparticle-integrated ZnO nanoflakes (ZnO/Ag nanocomposite) using red beetroot juice. The HeLa and SKOV-3 cell lines showed a significant decrease in viability after treatment with ZnO/Ag nanocomposites in a dose-dependent manner [183]. There was a high ROS generation in both HeLa and SKOV-3 cells after treatment with the nanocomposites at 2.0 mg/mL, leading to oxidative stress and ultimately cell death. In addition to this, a cell migration assay was performed, which reported a significant reduction in migration for both cell lines after 48 h incubation with the nanocomposites [183].

The anticancer effect of betanin towards human osteosarcoma (MG-63) cells was recently reported [184]. This phytocompound induced apoptosis and inhibited migration as well as adhesion of MG-63 cells in a dose-dependent manner. Morphological changes were also observed, such as DNA strand breaks and a large nucleoid appearance. Besides, apoptosis was also triggered by betanin as ROS generation was induced and subsequently decreased the mitochondrial membrane potential. This study also revealed that betanin inhibited osteosarcoma via the PI3K/Akt/mechanistic target of rapamycin (mTOR) signaling pathway [184], indicating betacyanin’s effects in modulating the oncogenic signaling pathway.

Basella rubra, containing betalain, demonstrated good antioxidant activity besides exhibiting cytotoxicity towards human cervical cancer cells (SiHa). Cellular viability of SiHa cells decreased significantly by approximately 80% after treatment with 50 mg/mL of the fruit extract for 24 h [185]. The treated cells showed morphological changes, such as cell shrinkage, blebbing, and detachment of the cells from the substratum, exhibiting classical features of apoptotic cell death. These changes became visible after 24 h of extract treatment but were absent in control cells, and the changes became more remarkable with the increasing extract concentrations. A dose-dependent decrease in growth kinetics was observed [185].

It was reported that the red pitaya peel extract possessed higher total betacyanin content (35.12 ± 0.01 mg/g sample) than the pulp extract (30.15 ± 0.03 mg/g sample) [27]. The phytochemical results also revealed that the red pitaya peel extract had a higher betanin content (9.44 ± 0.01 mg/g sample) than the pulp extract (7.44 ± 0.03 mg/g sample). While both extracts showed very low cancer-killing potential, IC₅₀ against liver cancer cells (HepG2), the peel was slightly more effective, likely due to its higher content of these compounds. The study found that pure betanin was much more potent at killing cancer cells than either the pulp or peel extracts, as evidenced by the lower IC₅₀ value [27].

Wang and Wang [186] (2023) fermented red beetroot juices using water kefir grains, and it was found that the fermentation process increased the betacyanin content in the juices. The juices were then tested on various cancer cell lines, such as HepG2 cells. Results clearly indicated that the red beetroot juice possessed stronger cellular inhibitory potentials after fermentation by water kefir grains, as the inhibition rate of the fermented red beetroot juice increased by 32.08% after 24 h of fermentation as compared with the unfermented red beetroot juice [186]. The fermented juices exerted growth-inhibitory effects towards HepG2 cells by increasing cellular ROS production, leading to cell apoptosis [186].

As reported by Kapadia et al. [187] (2011), B. vulgaris extract showed cytotoxicity towards human prostate (PC-3) and breast (MCF-7) cancer cell lines. However, the beetroot extract was less potent than doxorubicin. Its cytotoxic effect was most likely due to its high betacyanin content, specifically betanin [187]. Lee et al. [188] (2014) also reported that betacyanins like betanin and betaine decreased the HepG2 cell viability in a dose-dependent manner, and betanin was shown to be more effective than betaine. A study conducted by Krajka-Kuźniak et al. [189] (2013) reported that the growth of HepG2 cells showed a dose-dependent decrease after betanin treatment, with a decrease of 50% in cell proliferation at the concentration of 200 µM betanin. A notable contradiction exists in colon cancer models, where Caco-2 cells remained viable after 24 h treatment with the highest dose of betanin [190], while others reported significant apoptosis at lower concentrations [178], potentially reflecting differences in experimental conditions or cell characteristics.

In terms of cell cycle arrest, the treatment of betanin in K562 cells was found to have increased hypodiploid apoptotic DNA content and a reduced number of cells in the S and G2 phases, as shown through flow cytometry analysis [174]. Fermented juice containing betacyanins was also shown to arrest the growth of cancer cells at the G1 phase, and the population of cells entering the next cell cycle was also significantly reduced [186].

Based on the studies conducted so far, we conclude that betacyanins, specifically betanin and betalains, indeed possess promising potential as anti-cancer agents since they inhibit growth in various cancer cell lines in vitro. These mechanisms demonstrate consistent dose-dependent and time-dependent responses across multiple cancer cell lines, while revealing important structure-activity relationships and cell-type-specific variations. The findings are summarized in Table 4.

They were found to be effective against human breast (MCF-7), human bladder (T24), human colon (Caco-2, HT-29), human liver (HepG2), human cervical (SiHa), and human chronic myeloid leukemia (K562) cell lines. These phytocompounds were able to induce apoptotic cell death towards cancer cells via both intrinsic and extrinsic pathways [174, 176–178, 181, 184–186], and also affect cell migration [184]. Nevertheless, more in-depth investigations using in vivo model systems are vital to validate betacyanins’ therapeutic abilities against cancer for clinical application. Furthermore, employing proteomics and metabolomics analyses can facilitate clarifying the specific pathways (e.g., 2-oxocarbodylic acid metabolism) that are potentially affected by betacyanin, as demonstrated by the 5-FU nanoparticle combination for colon cancer [191].

Antimicrobial properties of betacyanins

Antimicrobial resistance (AMR) is of significant concern worldwide and impacts human, animal, and food health and safety [192]. AMR is projected to worsen due to the lack of innovation, a decline in funding, and an emphasis on antimicrobial research over the last several decades, antibiotic overuse, inappropriate prescribing, extensive use in agricultural production, and the lack of new antibiotics [192–195]. As such, it is imperative that new, effective, and accessible antimicrobials are developed.

Many new antibiotics are synthetic derivatives of older drugs such as tetracycline, fluoroquinolones, and aminoglycosides, for example [196], but these may not meet the needs for clinical practice, especially in hard-to-reach and low-resource communities. In fact, many micro-organisms are resistant to these drugs. Alternative and supplementary treatments will help bridge this gap in treating infectious diseases and recent trends in research and a surge in interest in natural therapies is driving a re-evaluation of the health benefits of natural products. For example, drimenol, a sesquiterpene alcohol from mainly Polygonum spp. and Drimys spp. has been reported to have significant antimicrobial activities [197]. The antimicrobial activity of betalains is well documented, and recent studies show antimicrobial activity of betacyanins and betanin.

Biofilms are of significant concern due to their impact on healthcare, water systems, and the food industry [198]. These bacterial communities are difficult to remove, and where removal is possible, harsh physical or chemical methods may be required [198]. Preventing biofilm formation is a legitimate and preferable goal [199]. A study by Yong et al. [200], showed that extracts from Amaranthus dubius and H. polyrhizus inhibited biofilm formation by approximately 30–50% when in combined action. Yong et al. [200], also separately showed that H. polyrhizus alone had antibacterial activity against 10 pathogenic Gram-positive and 6 Gram-negative bacteria and showed that refrigerating the extract at 4 degrees for 6 days increased the antibacterial activity by reducing the MIC by 8–16 fold.

Vulić et al. [201] reported that commercial beetroot pomace had antibacterial activity against 6 Gram-negative bacteria and 5 Gram-positive bacteria, comparing quite favorably to conventional controls (ceforaxime/claulanic acid), with the pomace showing reduced growth in an area that was at least half to as large as the control. This study showed that the pomace was not as effective as the conventional drugs they were compared with [201]. Gong et al. [202] reported that beetroot extract could inhibit the growth of L. monocytogenes and maintain cooked pork for a longer period of time without spoiling. Using a C. elegans model, Choo et al. [203] showed that beetroot extract could completely inhibit the growth of MRSA at 76 h. Furthermore, at 18 h post-infection, beetroot extract completely inhibited further MRSA growth [203]. Betacyanins from H. polyrhizus and A. dubius were shown to decrease the proliferation of Dengue virus type two in a Vero cell culture. The A. dubius extract exhibited significantly greater inhibition of dengue virus [204].

The extraction method has been shown to significantly impact the concentration and antimicrobial efficacy of the betacyanins from fruits and vegetables [205]. Tenore et al. [205] showed that different extract fractions from peel and fruit flesh of red pitaya fruits had differing effects on the antimicrobial properties. Betacyanins extracted from the flesh of red pitaya showed higher efficacy than whole flesh or peel extract [205]. The antibacterial activity of the flesh and peel extracts of betacyanin-rich extracts is dependent on the quantity and quality of betacyanin in the extract fraction. The fraction extracted from the flesh was more efficacious with a lower minimum inhibitory concentration [205]. Tenore et al. [205] study and others indicate the important considerations affecting the antimicrobial capacity include the extraction method, method and length of storage [101, 206, 207], environmental growth conditions [53], and the state of ripeness [19]. These considerations affect the test results reported, but can also affect the efficacy of any prescribed usage for therapeutic or preventative purposes.

The review by Wijesinghe and Choo [208] provides a broad look at the betalains and addresses some of the problems with comparing studies. There are different standards for reporting concentrations, the different sources of the active betacyanins, and the extraction, storage, and application methods. A review of studies indicates that the minimum inhibitory concentration of conventional drugs is much lower than that of the betacyanin extracts from different sources when comparing broth dilution and agar well diffusion methods [208].

Sutor and Wybraniec [25] report the quantitation of betacyanins from M. violaceus, M. bahiensis, M. amoenus, and M. curvispinus using chromatographic and mass spectrometric methods. The wide range in quality and quantity of betacyanin suggests that more work needs to be done to describe and understand the possibility of using all plants that produce betacyanins for antimicrobial purposes [25]. The available research suggests that betacyanins or betanin may play an important role in the control of infectious diseases as research provides important information on the best extraction, characterization, and concentration of the active phytochemicals, location of the most efficacious betacyanins or betanin fractions (i.e., pulp, peel, etc.), shelf-life, and other information necessary for use in therapeutics. Some of the antimicrobial effects of betacyanins are summarized in Table 5.

Molecular docking properties of betacyanins

Molecular docking of natural products is increasingly used to predict phytochemical interactions with new biological targets, explaining traditional uses and uncovering novel medicinal applications [209]. This approach accelerates drug discovery by saving time and costs and enabling the introduction of new molecular scaffolds for further testing and optimization [210].

The well-documented radical scavenging activity of betacyanins is computationally rationalized by their strong predicted interactions with key enzymatic sources of oxidative stress. Enzymatically, xanthine oxidase (XO) catalyzes the conversion of xanthine and hypoxanthine into uric acid, generating ROS implicated in oxidative stress-related pathologies [211]. Molecular docking studies by Ramirez-Velasquez et al. [17] revealed that betanin, isobetanin, betanidin, and isobetanidin exhibit strong interactions with XO’s active site, with binding affinities exceeding that of febuxostat, a known XO inhibitor [212]. Betanidin, isobetanidin, and betanin formed stable complexes via electrostatic, hydrogen bonding, and hydrophobic interactions, supporting their potential as effective XO inhibitors and antioxidants [17].

In the inflammation pathway, NF-κB activation involves its nuclear translocation and induction of pro-inflammatory pathways [213]. ElSayed et al. [214] (2023) demonstrated via molecular docking and CASTp analysis that betanin binds the DNA-binding domain of NF-κB. Its central indole ring anchors via π-stacking with TYR36, stabilized by polar interactions with Arg33, Lys122, Arg124, and Lys218 [214]. This binding site overlap with DNA suggests betanin competitively inhibits NF-κB activation, corroborated by in vivo data showing reduced NF-κB expression post-treatment. Furthermore, lipoxygenase (LOX) enzymes mediate inflammation via eicosanoid production and numerous phytochemicals and natural products induce the desired anti-inflammatory effects through inhibition of these enzymes [215]. Betanidin significantly inhibits LOX activity in a dose-dependent manner. Molecular docking indicates that indoline-derived betacyanin occupies a pocket near the loop preceding helix 2. However, glucosylation of betanidin to betanin markedly diminishes its LOX-inhibitory capacity [216]. However, a high docking score is necessary but not sufficient for biological activity, as it fails to account for crucial pharmacokinetic factors like bioavailability and metabolism.

When considering its antimicrobial activity, Chaari et al. [217] (2023) investigated the interactions of betacyanin and betaxanthin—constituting 75–95% and 5–25% of betalains, respectively—with bacterial DNA/RNA polymerases and cell membranes of S. aureus and Salmonella enterica. Molecular docking revealed betacyanin formed stable complexes with DNA and RNA polymerases via van der Waals, hydrogen bonding, and various π-interactions. Both pigments also disrupted membrane integrity by inhibiting efflux pumps (AcrAB-TolC in S. enterica; MepR/MepA in S. aureus), indicating promising antibacterial activity against foodborne pathogens. Molecular docking studies have demonstrated strong interactions between betacyanin and critical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins, particularly the spike glycoprotein in its closed state (PDB ID: 6VXX) and the receptor-binding domain (PDB ID: 6YLA). These interactions exhibited binding affinities comparable to established antiviral agents such as nelfinavir and hydroxychloroquine sulfate, indicating potential for therapeutic intervention in COVID-19 [218].

In cancer-related targets, betanin has shown promising binding behavior with enzymes involved in DNA replication and cell cycle regulation. Notably, it formed more favorable interactions with human DNA topoisomerase I than topoisomerase II, and demonstrated robust affinity with cyclin-dependent kinase (CDK)-6, an essential cell cycle protein. Additionally, betanin exhibited moderate interactions with apoptosis-associated proteins, Bcl-2 and caspase-3, with docking scores of −4.522 and −6.108 kcal/mol, respectively. Supporting the role of betanin in inhibiting DNA replication, suppressing cell cycle progression, and promoting apoptotic activity, paralleling the effects of the chemotherapeutic agent doxorubicin, to which it bears structural similarity [219].

Molecular docking provides a valuable framework for connecting the dots between betalain chemistry and observed biology. It translates empirical findings into structural hypotheses, suggesting that betalains may act as multi-target agents by directly modulating key enzymes and receptors. However, critical interpretation is important as the consistent theme of high binding affinity across diverse targets may point to both their therapeutic potential and a limitation of docking algorithms. The true mechanism of action likely involves a combination of direct target engagement and indirect effects stemming from their potent redox activity and modulation of core signaling pathways like Nrf2 and NF-κB. Therefore, docking predictions must be viewed as the starting point for rigorous laboratory experimental validation, not as conclusive proof of mechanisms [220].

Overall, these molecular docking studies, despite being computational, reveal that betacyanins can target key enzymes and proteins involved in inflammation, antioxidant defense, antimicrobial, antiviral, and anticancer pathways. This supports their promising role as nutraceuticals with diverse therapeutic applications (Figure 6).

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Figure 6. The molecular docking targets of betacyanin pigments on enzymes and/or proteins involved in various cellular pathways. Betacyanins demonstrated inhibitory activities in LOX, xanthine oxidase, in bacterial DNA and RNA polymerases, in human DNA topoisomerase I and IIa, in NF-κB protein, in genes involved in cell cycle and apoptosis, and in SARS-CoV-2 virus spike protein and receptor binding domain. These observations likely explain the molecular aspects of betacyanins’ activities observed in biological studies. LOX: lipoxygenase; NF-κB: nuclear factor-kappa B.

Conclusion

This review discusses the natural sources, biosynthesis, extraction methods, applications, antimicrobial, anticancer, and the positive effects of betacyanins against cardiometabolic diseases, with novel reports of the effects of this compound group on gut microbiome and molecular docking properties. Despite the favorable effects reported in the article, it is important to note that the results reported in these studies should be interpreted with some limitations. For instance, the number of studies reporting the effects of specific betacyanins from plants on disease models is still limited. Many studies used products that have a combination of different phytochemicals and pigments, thus complicating the interpretation. Betalain-rich food also comprises several other potentially relevant compounds, such as polyphenols and nitrates, and their interactions in biological systems should be considered. To answer this, further studies could consider separating individual components and comparing their effects with complete or combined betacyanins from whole fruits or plants.

To enhance bioavailability, diversifying food sources, food processing, and food matrices could potentially increase betacyanin concentration and thus enhance absorption, overcoming the challenges of their limited absorption. Moreover, more studies should be done to increase the extraction of betacyanins from plant waste such as fruit peels. This will greatly reduce the environmental impact through food waste and generate income for growers. More rigorous clinical trials should be implemented soon to provide strong evidence-based information for clinical practice. Considering the current information available in the literature, existing evidence is pointing towards the application of betacyanins as a promising natural food pigment having multiple health benefits, acting through various biological mechanisms, including their interactions with the gut microbiome and binding of key cellular components.