Anti-Obesity and Anti-Steatotic Effects of Bixin (Apocarotenoid from Bixa orellana L. Seeds): Dose-Dependent Correlations with Bioactivity ^†

Abstract

Annatto, a tropical shrub from Central and South America and parts of India, contains Bixin, an apocarotenoid pigment. Bixin is conventionally used as a natural food colorant and is now receiving attention for its health-promoting nutraceutical properties, particularly in chronic diseases (metabolic syndrome—MetS—and cancers). This study investigates the dose-dependent anti-obesity and anti-steatotic effects of Bixin in in vitro cell culture models. The anti-adipogenic and anti-steatotic effects of Bixin were examined in well-established in vitro models of obesity and non-alcoholic fatty liver disease (NAFLD/steatosis) using 3T3-L1 preadipocytes (by a differentiation protocol) and HepG2cells (steatosis-induced with oleic acid), respectively. Bixin was administered in the following concentration range: 1 μg mL⁻¹–20 μg mL⁻¹ (obesity model) and 2.5 μg mL⁻¹–10 μg mL⁻¹ (NAFLD/steatosis model). The neutral lipid content was estimated by Oil Red O staining; ROS/RNS were quantified by 2′,7′-dichlorofluorescein diacetate (DCFDA) and nitrite assays; and malondialdehyde (MDA), the biochemical marker of lipid peroxidation, was assessed by TBARS assay. At lower concentrations, 5 μg mL⁻¹ in steatotic cells and 10 μg mL⁻¹ in matured adipocytes, Bixin significantly reduced lipid accumulation in both hepatocytes and adipocytes (p < 0.05), demonstrating its potential as an anti-steatotic and anti-obesity agent. This beneficial effect was correlated with a reduction in oxidative stress levels (decreased MDA and ROS/RNS levels). Strikingly, at higher concentrations (>10 μg mL⁻¹), Bixin showed increased lipid accumulation and oxidative stress. Bixin exhibits anti-obesity and anti-steatotic effects at lower doses, which correlates with its antioxidant properties. However, its bioactivity is dose-dependent, meaning that at higher concentrations, it ceases to inhibit adipogenesis. This opposing response is accompanied by elevated oxidative stress levels, indicating a pro-oxidant effect at higher doses, which suggests its anti-cancer potential. The present study highlights the significance of dosage optimization of nutraceuticals and dietary ingredients with respect to their intended biological applications, such as MetS and cancer treatment.

1. Introduction

Metabolic syndrome comprises a cluster of metabolic abnormalities, including obesity, insulin resistance, hypertension, and dyslipidemia, with non-alcoholic fatty liver disease (NAFLD) as a key manifestation [1]. Both NAFLD and obesity are characterized by excessive lipid accumulation in the liver and adipose tissue, contributing to its pathophysiological features [2]. The prevalence of NAFLD is high, ranging from 22.5% to 44.0% in overweight individuals and reaching up to 90% in those with obesity [3]. Current pharmacological interventions for these conditions face significant limitations, including side effects and limited efficacy, highlighting the urgent need for alternative approaches. While obesity is commonly managed with anti-obesity drugs, their potential adverse effects remain a concern. Furthermore, effective and targeted pharmacological treatment for NAFLD has not been established, largely due to its complex and poorly understood pathogenesis. As a result, lifestyle modifications, including dietary interventions, physical activity, and management of associated comorbidities, remain the cornerstone of NAFLD management. These challenges underscore the importance of exploring natural compounds with therapeutic potential as safer and more effective strategies for managing metabolic disorders [4,5].

Bixa orellana L., commonly known as annatto, is a tropical plant that is widely recognized for its seeds, which are rich in Bixin, an apocarotenoid pigment [6]. Out of all the carotenoids, only Bixin presents a 9-cis configuration [7]. Traditionally used as a natural food colorant, Bixin has gained scientific interest due to its potential health-promoting properties, including antioxidant, anti-inflammatory, and lipid-lowering effects [8,9].

Dose optimization is crucial when using phytochemicals, as their biological effects can vary depending on concentration. A study on HL-60 cells demonstrated that curcumin exhibits a biphasic effect on ROS generation. At low doses (10 μM), it reduces ROS levels and acts as an antioxidant, while at high doses (50 μM), it promotes ROS production, functioning as a pro-oxidant and leading to cell death [10]. Similarly, carotenoids such as Bixin, lycopene, and β-carotene are renowned for their antioxidant properties at physiological levels, where they neutralize reactive oxygen species and protect against oxidative damage. However, studies have shown that carotenoids can act as pro-oxidants at higher concentrations, increasing oxidative stress and potential cytotoxicity [11]. These biphasic effects highlight the importance of determining the optimal dosages for therapeutic and dietary applications with different biological effects (anti-cancer, antioxidant, anti-inflammatory, and anti-lipemic). In this study, we investigated the dose-dependent effects of an apocarotenoid, Bixin, in adipogenic and steatotic cell culture models.

2. Materials and Methods

2.1. Chemicals and Reagents

Pure Bixin was obtained from HPC Molecules Analytical Lab Solutions Pvt Ltd. (Hyderabad, India). Chemicals such as DMSO, potassium hydroxide, hydrochloric acid, monopotassium phosphate, sodium hydroxide, tris buffer, TCA (Trichloroacetic acid), and TBA (Thiobarbituric acid) were sourced from Sigma Aldrich (St. Louis, MO, USA).

2.2. In Vitro Disease Models

• Adipogenesis model: The 3T3-L1 preadipocytes were obtained from NCCS (National Centre for Cell Science, Pune, India) and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ atmosphere. Adipogenesis was initiated 48 h post-confluence (day 0) using a differentiation cocktail of insulin (10 μg mL⁻¹), dexamethasone (0.25 µM), and (IBMX) 3-isobutyl-1-methylxanthine (500 µM), followed by insulin-only medium from day 2. Different dosages of Bixin (2.5–20 μg mL⁻¹) were administered on day 4 of differentiation [12].
• Hepatic steatosis model: HepG2 cells from NCCS (Pune, India) were cultured in MEM with 10% FBS and 1% penicillin-streptomycin at 37 °C, 5% CO₂. For steatotic experiments, cells were seeded in 96-well plates (5 × 10⁵ cells/well), serum-starved for 24 h, and treated with 1 mM oleic acid in MEM with 1% BSA for 48 h to induce steatosis. This was followed by treatment with multiple concentrations of Bixin (2.5–10 μg mL⁻¹) for 24 h [13].

2.3. Assessment of Lipid Accumulation Through Lipid Staining

Oil Red O (ORO) staining was used to assess the dose-dependent impact of Bixin on lipid accumulation in cells following adipogenic and steatotic induction. Cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and stained with 0.3% ORO for 20 min. After rinsing with water, lipid droplets were visualized under an inverted microscope. The lipid-bound dye was dissolved in 100% isopropanol, and absorbance was measured at 490 nm to evaluate the effect of varying Bixin concentrations on lipid accumulation.

2.4. TBARS–Lipid Peroxidation Assay

The TBARS assay was used to assess the dose-dependent impact of Bixin on the antioxidant activity in adipogenic and steatotic models by measuring the malondialdehyde (MDA) levels. Cells were treated with varying Bixin concentrations for 24 h, washed, and lysed. After homogenization and centrifugation, the protein content was normalized. The lysate was incubated with Tris buffer and monopotassium phosphate, followed by the addition of TCA and TBA to generate a pink adduct. Absorbance was measured spectrophotometrically at 530 nm and compared to a standard MDA curve.

2.5. Griess Assay

Griess assay was used to measure the nitrate and nitrite concentrations, indicating the Nitric Oxide (NO) levels, in the adipogenic and steatotic models, as well as in the Bixin-treated groups. Nitrate was enzymatically converted to nitrite, and the Griess reagent was added to the cell culture medium. After a 15 min incubation, the optical density (OD) was measured at 540 nm, and the nitrite concentration was determined by comparison to a standard nitrite curve (µM).

2.6. Quantification of Intracellular ROS/RNS Levels

DCFDA assay was used to measure intracellular ROS/RNS levels in steatotic and adipogenic models treated with varying concentrations of Bixin for 24 h. After treatment, cells were incubated with 10 μM DCFDA for 60 min in the dark. The fluorescence intensity (Excitation/Emission = 485/535 nm) was measured, showing a dose-dependent response to Bixin.

2.7. Statistical Analysis

The in vitro experiments were performed at least thrice, in triplicate sets. Statistical analysis was performed using one-way ANOVA to compare the experimental groups, and their statistically significant differences in values were expressed as follows: p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

3. Results and Discussion

The effects of Bixin concentrations on controlling adipogenesis and steatosis, and their associated biochemical sequel such as oxidative stress, were assessed using lipid staining and oxidative stress markers. The outcomes are discussed below.

3.1. Assessment of Lipid Accumulation by Oil Red O Staining

Our previous study identified the non-cytotoxic doses of Bixin for 3T3-L1 preadipocytes and HepG2 cells before optimizing these concentrations for their anti-adipogenic and anti-steatotic effects. We reported that Bixin at concentrations greater than 10 μg mL⁻¹ and 5 μg mL⁻¹ resulted in decreased cell viability in adipocyte and steatotic models [14]. This was a pointer that Bixin could exert cytoprotective and cytotoxic effects at different concentrations. Therefore, multiple dosages below 20 μg mL⁻¹ and 10 μg mL⁻¹ were tested as safe dosages for anti-adipogenic and anti-steatotic effects.

The potential of pure Bixin to inhibit lipid accumulation was assessed in both mature adipocytes and oleic acid-induced HepG2 steatotic cells. Preadipocytes were treated with varying concentrations of Bixin (2.5, 5, 10, 15, and 20 μg mL⁻¹) starting on day 4 of differentiation. For the steatotic model, HepG2 cells were treated with Bixin (2.5, 5, 7.5, and 10 μg mL⁻¹) 24 h after steatosis induction. Both mature adipocytes and steatotic cells showed a significant reduction in lipid accumulation, with a noticeable decrease up to certain concentrations: in adipocytes, this occurred up to 10 μg mL⁻¹, and in steatotic cells, up to 5 μg mL⁻¹. Interestingly, as the concentration increased beyond these values, the lipid accumulation increased, as evidenced by Oil Red O staining (Figure 1). This clearly suggests that there is a dosage threshold, which determines the biphasic nature of Bixin, that is, whether it promotes or supresses adipogenesis and steatosis. In other words, it is important to identify the dosages which would result in reduction in lipid accumulation in order to realize the anti-obesity and anti-NAFLD potential of Bixin. There are mixed reports of the adipogenic property of Bixin. For instance, while certain doses of Bixin (70 µM) promoted adipogenesis in 3T3-L1 adipocytes [15], another study reported that a lower dose of Bixin (20 µM) inhibited the differentiation of 3T3-L1 preadipocytes [16]. Our study characterized this dual nature of Bixin using a low to high dosage range. The present outcomes clearly explain these mixed reports on the basis of the concentration of apocarotenoid.

Along similar lines with respect to another phytochemical, curcumin, it was shown that low doses of curcumin (15 μM) attenuate adipocyte differentiation, whereas higher concentrations (30 μM) induce apoptotic signaling [17], implying dose-dependent changes in the mechanism. The present study extended the investigation of Bixin’s anti-lipid accumulation efficacy in yet another model, which was steatotic NAFLD. The outcomes in the NAFLD model were in line with the adipogenic model, wherein there was a dosage threshold wherein concentrations of 2.5 μg mL⁻¹ and 5 μg mL⁻¹ led to reductions in steatosis, while a dosage above 5 μg mL⁻¹ resulted in a gradual increase in steatosis.

3.2. Assessment of Oxidative Stress

The assessment of oxidative stress in Bixin-treated cells was carried out using multiple biochemical assays. Through the TBARS assay, DCFDA, and nitrite assays, we observed significantly elevated levels of lipid peroxidation, total intracellular ROS/RNS, and nitrite levels in both the adipogenic and steatotic models compared to the control group (### p < 0.001) (Figure 2). Interestingly, at lower concentrations, Bixin treatment (up to 10 μg mL⁻¹ in adipogenic cells and 5 μg mL⁻¹ in steatotic cells) significantly reduced the total ROS/RNS production and lipid peroxidation (p < 0.01), suggesting its potential antioxidant effects. This supports the potential multifaceted protection that Bixin can exert in obesity and NAFLD by alleviating lipid accumulation and associated oxidative damage. However, oxidative stress was elevated at higher concentrations (>10 μg mL⁻¹ in adipogenic cells and >5 μg mL⁻¹ in steatotic cells), indicating a shift towards a pro-oxidant effect of Bixin. This dose-dependent effect may be partly explained by the increase in lipid accumulation, as shown above, with higher dosages. These results show the importance of dosage optimization in the clinical application of Bixin, particularly in the context of MetS, NAFLD, and cancer, where oxidative stress plays a crucial role in disease progression.

The antioxidant properties of carotenoids are attributed to their ability to scavenge free radicals [18]. Bixin, with its conjugated double bonds, acts as an antioxidant by donating electrons to neutralize free radicals, thereby preventing oxidative damage [19]. However, studies have demonstrated that at higher concentrations, carotenoids such as β-carotene, lycopene, and lutein lose their antioxidant activity and exhibit pro-oxidant effects [20,21]. Another study investigated the effects of β-carotene supplementation in HT29 cells. It demonstrated that at low doses (1–3 μM), β-carotene effectively scavenged radicals within the membrane, thereby preventing lipid peroxidation. In contrast, it increased the membrane fluidity at higher concentrations (4–10 μM), potentially facilitating greater oxidant damage [22].

4. Conclusions

The biphasic behavior of Bixin concentrations underscores the necessity of dose optimization in its anti-obesity and anti-NAFLD applications. The present outcomes, taken together with other related reports, clearly implicate that the efficacy and safety of nutraceuticals are closely dependent on their concentrations. Therefore, it is imperative to understand the specific biological context and intended health outcomes before standardizing the dosage of the phytochemicals. Further, the nature of administration, such as a single phytochemical or combinations, pure chemical or botanical extracts, and different delivery systems such as encapsulations, would all need corresponding dosage optimization with respect to the intended biological effect and actual bioavailability of the phytochemical.