Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Obtaining Bixin- and Tocotrienol-Rich Extracts from Peruvian Annatto Seeds Using Supercritical CO2 Extraction: Experimental and Economic Evaluation.

Currently, Bixa orellana L. extracts are used as a color source in the food, pharmaceutical, and cosmetic industries because they are important as a potential source of antioxidant activity. The extraction is carried out by conventional methods, using alkaline solutions or organic solvents. These extraction methods do not take advantage of the lipid fraction of annatto (Bixa orellana L.) seeds, and the process is not friendly to the environment. In this work, the objective was to obtain an extract rich in nutraceuticals (bixin and tocols) of high antioxidant power from Peruvian annatto seeds as a potential source for a functional food or additive in the industry using supercritical fluid extraction (SFE). Experiments related to extraction yield, bixin, tocotrienols, tocopherols, and antioxidant activity were carried out. The SFE was performed at 40 °C, 50 °C, and 60 °C, and 100, 150, and 250 bar with 0.256 kg/h carbon dioxide as the supercritical solvent (solvent-to-feed ratio of 10.2). Supercritical extraction at 60 °C and 250 bar presented the best results in terms of global extraction yield of 1.40 ± 0.01 g/100 g d.b., extract concentration of 0.564 ± 0.005 g bixin/g extract, 307.8 mg α-tocotrienol/g extract, 39.2 mg ß-tocotrienol/g extract, 2 mg γ-tocopherol/g extract, and IC50 of 989.96 µg extract/mL. Economical evaluation showed that 60 °C, 250 bar, and 45 min presented the lowest cost of manufacturing (2 × 2000 L, COM of USD 212.39/kg extract). This extract is a potential source for functional food production.

Potential use of bixin beyond function as a natural colourant in food and cosmetics.

Bixin is a carotenoid found in the covering of Bixa orellana seeds and widely used as a dye, mainly in food products and cosmetics. This compound is insoluble in water, but technologies have been developed for its use in aqueous preparations, as well as a component for new formulations to disperse other lipophilic ingredients. It has recently aroused the interest of researchers in areas such as Pharmacology, Endocrinology and Oncology, but also new applications in Food Technology. This work aimed to review the main studies in the period from 1911 to 2023, but we emphasised aspects in technologies related to the field of health such as Pharmacology and Pharmacy. We used 'bixin' as a keyword to search for articles on the Web of Science and obtained 488 results, most of which were original articles and 20 were reviews. The analysis demonstrated a continuing large number of studies in Food Technology, but a rapid growth in areas related to health aspects.

Annatto-Enriched Egg Improves the Perception of Satiety in Healthy Adults-Randomized Clinical Trial: EGGANT Study.

Diet is one of the factors that prevents the development and death from cardiovascular diseases (CVD). It has been proposed that diets high in protein, which increase satiety, and with a high content of antioxidants, help reduce cardiovascular risk factors. The egg is one of the foods that produces greater satiety and provides antioxidants. In addition, due to its lipophilic matrix, it could improve the bioavailability of other dietary antioxidants such as Annatto. OBJECTIVE: This study evaluated the effects of egg and annatto-enriched egg consumption on satiety markers and CVD risk factors in healthy adults from Colombia. METHODS: A parallel randomized clinical trial was conducted, where one hundred and five (n = 105) men and women, divided into three groups, consumed daily for 8 weeks: (a) two eggs (egg group), or (b) two eggs with annatto (egg + annatto group), or (c) two egg whites (placebo group). RESULTS: The three groups were similar in gender distribution. No significant changes were found over time (before vs. after) in any of the groups nor between the groups in anthropometric variables, physical activity, eating profile, and ghrelin as an objective marker of satiety. In the egg + annatto group, subjective satiety increased (effect size 0.431; p < 0.05) after consumption. CONCLUSIONS: In healthy adults, the intake of two eggs, or two eggs with annatto daily for 8 weeks, did not result in significant changes in ghrelin; but eggs with annatto tend to increase the perception of satiety.

Validation, Measurement Uncertainty, and Determination of Bixin and Norbixin in Processed Foods of Animal Resources Distributed in Korea.

This research aimed to validate a high-performance liquid chromatography method for the quantitative determination of bixin and norbixin in various foods. The Diode Array Detector (495 nm) technique was used. Method was validated for specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy, and the measurement uncertainty was assessed. The calibration curve showed excellent linearity (r2≥0.9999) over the tested concentration range of 0.2-25 mg/L. The LOD and LOQ were 0.03-0.11 and 0.02-0.05 mg/L for bixin and norbixin, respectively. The intra- and inter-day accuracies and precisions were 88.0±1.3-97.0±0.5% and 0.2%-2.6% relative SD (RSD) for bixin and 88.2±0.8-105.8±0.8% and 0.3%-2.7% RSD for norbixin, respectively. Inter-laboratory validation for accuracy and precision was conducted in three laboratories, and these results all met the AOAC guidelines. In addition, the relative expanded uncertainty (<22%) satisfied the CODEX recommendation. Furthermore, products distributed in Korea were monitored for annatto extracts using the proposed method to demonstrate its application. The developed analytical method is reliable for quantifying bixin and norbixin in various foods.

Application of different concentrations of the natural dye potassium norbixinate (annatto) in polyamide 6.6 fabrics.

Polyamide fabrics were dyed with concentrations ranging from 4% to 0.25% (o.w.f.) of the natural dye, potassium norbixinate (annatto). The exhaustion, chromatic coordinates, colouristic intensity (K/S), and fastness to washing and rubbing were evaluated. The natural dye was characterised, and its maximum absorption peaks were identified at 452 nm and 482 nm through UV-vis scanning. Its main chemical groups were identified by FTIR-ATR. All dyeings exhibited high exhaustion percentage, with a maximum of 98.4% for 1% dye concentration. The dyed samples displayed visually appealing orange hues, with a maximum K/S value of 6.9. Most of the fastness test results were rated between 5 and 4/5, remaining within the standards established by most textile industries. Potassium norbixinate exhibited a similar tinctorial behaviour to synthetic acid dyes for polyamide, suggesting ionic chemical reaction interaction between dye and polyamide, highlighting the potential use in the textile industry.

Therapeutic potential of bixin on inflammation: a mini review.

Chronic inflammation is the underlying mechanism for many diseases. Thus, inflammatory signaling pathways are valuable targets for new treatment modalities. Natural products have gained interest as a potential source of bioactive compounds which provide health benefits in combating inflammatory-related diseases. Recent reports have linked the medicinal values of Bixa orellana L. with its anti-inflammatory activities. Therefore, this review aims to examine the therapeutic potential of bixin, a major bioactive constituent found in the seeds of B. orellana, on inflammatory-related diseases based on existing in vitro and in vivo evidence. Additionally, the anti-inflammatory mechanism of bixin via signaling pathways is explored and possible toxic effects are addressed. The findings suggest that bixin may ameliorate inflammation via inhibition of toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) and thioredoxin-interacting protein/NOD-like receptor protein 3 (TXNIP/NLRP3) inflammasome mechanisms. More well-planned clinical studies should be performed to verify its effectiveness and safety profile.

Dyes Used in Processed Meat Products in the Polish Market, and Their Possible Risks and Benefits for Consumer Health.

Manufacturers are obliged to label processed meat products with information concerning the additives used and nutritional values. The aim of the study was to identify the dyes most frequently used in processed meat, evaluate their influence on specific food qualities, assess whether their use was correct and review their effect on health. The analysis was based on information on the labels and images of processed meat, and used a generalised linear model with a binary dependent variable. The risks and benefits for human health were defined based on the available literature. Twelve dyes were found to be used in the manufacture of processed meat. Carmine was found in 183 of 273 (67.03%) evaluated assortments containing dyes. The occurrence of water, flavourings and high fat and carbohydrate contents increased the chances that a dye would be present in a particular product. Unauthorised use of food additives was found in 20 products, with smoked meat products demonstrating the highest number of non-compliances. In general, the dyes used with food are considered safe; however, reservations are associated with the use of E150C and E150D caramels due to their potential carcinogenic effect, and carmine and annatto due to their allergic effects.

Bioactive cellulose acetate nanofiber loaded with annatto support skeletal muscle cell attachment and proliferation.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212 nm vs. 284 ± 130 nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Pharmacokinetics and bioavailability of tocotrienols in healthy human volunteers: a systematic review.

OBJECTIVE: To evaluate and compare the pharmacokinetic parameters, especially bioavailability, of annatto-based tocotrienol with palm tocotrienol-rich fraction in healthy human volunteers for better therapeutic outcome. METHODS: The systematic review was conducted between April and August 2021 in accordance with the Preferred Reporting Items for Systematic Review and Meta Analysis guidelines, and comprised search on PubMed, Google Scholar, Pakmedinet and Google search engines for open-label or double-blind randomised controlled trials involving healthy human volunteers published till January 2021. Key words used included annatto-based tocotrienol, palm tocotrienol-rich fraction, absorption and bioavailability. Boolean operators were also used, like tocotrienol AND bioavailability, annatto tocotrienol AND pharmacokinetics. RESULTS: Of the 230 articles identified, 50(21.7%) articles met the eligibility criteria. Of them, 7(14%) were selected for data extraction and detailed analysis. Pharmacokinetic parameters of annatto-based tocotrienol were better than palm-derived tocotrienol. Oral administration of all the isomers of annatto-based tocotrienols resulted in dose-dependent increase in area under curve and plasma levels. Amongst all the isomers of annatto-based and palm-derived tocotrienol, delta isomer of annatto-based tocotrienol had the highest bioavailability with area under curve 7450±89 ng/ml, time to reach peak plasma levels 4 hours, maximum plasma concentration 1591±43 ng/nl and elimination half-life 2. 68 ±0.29 hrs. Pharmacokinetic parameters of delta isomer of annatto-based tocotrienol was greater than palm tocotrienol-rich fraction. CONCLUSIONS: Bioavailability of annatto-based tocotrienol was better than that of palm-derived tocotrienol-rich fraction. Delta isomer of annatto-based tocotrienol had the highest bioavailability amongst all isomers of tocotrienol.

Introducing and assessing the efficacy of a novel method to reduce enamel damage after orthodontic bracket removal using two herbal-based resin colouring agents: An in vitro study.

PURPOSE: The differentiation of resin remnants from enamel is a critical factor to minimize enamel damage after bracket debonding. This study was conducted to produce, and ascertain the efficacy of two colouring agents in minimizing enamel loss, adhesive and bonding remnants, and surface roughness after debonding. METHODS: Two dyes containing annatto (orange colour) and curcumin (yellow colour) were produced. Seventy-two maxillary premolars were divided into three groups. After bracket bonding and debonding, the adhesive remnant was removed with a fine diamond bur. In groups 1 and 2, the orange and yellow dyes were utilized during the removal process, respectively. In group 3 (control) adhesive was removed with no colouring agent. The buccolingual dimension of the teeth was measured at the occlusal, middle, and apical areas, before bonding and after clean-up. The adhesive remnant index (ARI) and bonding remnant index (BRI) scores were recorded and the surface roughness parameters were measured. Data were analysed by ANOVA, Tukey, and Fisher's exact tests (α=0.05). RESULTS: Enamel loss was significantly lower in the groups cleaned by the use of colouring agents than that of the control group (P<0.05). No bonding agent was observed when the dyes were used, whereas 65% of teeth in the control group showed the remaining bonding material (P<0.001). There was no significant difference in ARI scores or surface roughness alterations among the study groups (P>0.05). CONCLUSION: The two dyes produced in this study were effective in enhancing the visibility of residual resin materials and minimizing enamel loss during the clean-up process.

Consumption of Eggs Alone or Enriched with Annatto (Bixa orellana L.) Does Not Increase Cardiovascular Risk in Healthy Adults-A Randomized Clinical Trial, the Eggant Study.

Most atherosclerotic cardiovascular diseases can be prevented by modifying lifestyles, including unhealthy diets. Eggs contain important carotenoids that may impact cardiovascular risk. The lipid nature of eggs can improve the bioavailability of other carotenoids, such as Annatto (Bixa orellana L.), with reported antioxidant properties. Although numerous studies have shown that there is no association between egg consumption and cardiovascular risk, there is still controversy. In addition, there is limited information about Annatto's effects on human health. This study evaluated the association between egg consumption and its enrichment with Annatto in lipid biomarkers of cardiovascular disease. In a parallel clinical trial, one hundred and five (n = 105) men and women were randomized by age, sex, and body mass index (BMI), and distributed into three groups. Subjects consumed daily, for 8 weeks, either two eggs, two eggs with Annatto, or two egg whites. Plasma lipids were measured by enzymatic colorimetric methods, plasma apolipoproteins and lipoprotein subfractions and size by nuclear magnetic resonance. There were no differences between groups in age, sex, and BMI. No significant changes were found over time or between groups in plasma triglycerides, LDL cholesterol, HDL cholesterol, apolipoprotein (apo) A1, apo B, or lipoprotein subfraction concentrations. In healthy adults, the intake of two eggs a day, or two eggs with Annatto for eight weeks, did not generate adverse changes in cardiovascular risk markers.

Green Extraction of Annatto Seed Oily Extract and Its Use as a Pharmaceutical Material for the Production of Lipid Nanoparticles.

This work developd nanomaterials formulated from annatto seed oily extract (ASE), myristic acid (tetradecanoic acid), and their fatty acid esters. The annatto seed oily extract was obtained using only soybean oil (ASE + SO) and Brazil nut oil (ASE + BNO). The UV/VIS analysis of the oily extracts showed three characteristic peaks of the bixin molecule at 430, 456 and 486 nm. The lipid nanoparticles obtained using myristic acid and ASE + BNO or only BNO showed better results than the oil soybean extract, i.e., the particle size was <200 nm, PDI value was in the range of 0.2−0.3, and had no visual physical instability as they kept stable for 28 days at 4 °C. Lipid nanoemulsions were also produced with esters of myristic acid and ASE + BNO. These fatty acid esters significantly influenced the particle size of nanoemulsions. For instance, methyl tetradecanoate led to the smallest particle size nanoemulsions (124 nm), homogeneous size distribution, and high physical stability under 4 and 32 °C for 28 days. This work demonstrates that the chemical composition of vegetable oils and myristic acid esters, the storage temperature, the chain length of fatty acid esters (FAE), and their use as co-lipids improve the physical stability of lipid nanoemulsions and nanoparticles from annatto seed oily extract.

Norbixin, a natural dye that improves serum lipid profile in rabbits and prevents LDL oxidation.

We hypothesized that norbixin, which is a carotenoid used as an orange/red natural food coloring additive, has anti-atherogenic properties. An in vitro oxidation assay with human LDL and a rabbit model of atherosclerosis were used to test this hypothesis. Norbixin inhibited the oxidation of isolated human LDL in a concentration-dependent manner. In the in vivo assay, rabbits were fed with a regular chow (control) or an atherogenic diet (0.5% cholesterol) alone or supplemented with norbixin (10, 30 or 100 mg/kg b.w.) for 60 days. Norbixin supplementation (30 and 100 mg/kg b.w.) increased HDL levels and reduced triglyceride levels and the atherogenic index of rabbits. This effect was associated with the decrease of serum levels of oxidized LDL, oxidized LDL antibodies and aortic tissue levels of lipid and protein oxidation in the atherogenic rabbits supplemented with norbixin. Atherogenic diet increased enzymatic (superoxide dismutase, catalase, glutathione reductase, and thioredoxin reductase-1) and non-enzymatic (non-protein thiol groups content) antioxidant defense systems in the aortic tissue but reduced the activity of paraoxonase-1 in the serum. All these changes were prevented by norbixin supplementation (10, 30 and 100 mg/kg b.w.). These results suggest that norbixin has atheroprotective potential by improving serum lipid profile and preventing oxidative modifications of circulating LDL and aortic tissue. Norbixin may, therefore, be beneficial in the control of atherosclerosis risk factors and can be further investigated as a candidate to be used not only as a functional food ingredient but also for therapeutic applications and in the nutraceutical industry.

Bixa orellana L. carotenoids: antiproliferative activity on human lung cancer, breast cancer, and cervical cancer cells in vitro.

Emerging evidence on the potential pro-oxidant effect of carotenoids provokes apoptosis of cancer cells. Bixa orellana L. is native to Central and South America, interestingly, is also cultivated worldwide. Apo-carotenoids present in B. orellana L. are mainly dominated by bixin and norbixin and demonstrate fundamental antioxidant activity. Anti-proliferative activity on human cancer cells is rarely investigated. We isolated bixin from B. orellana L. found in the island of Java using Ultra-Fast Liquid Chromatography and confirmed the isolated compound using Liquid Chromatography-MS/MS. Bixin and crude extract were examined on human lung cancer (A549), cervical cancer (HeLa), and breast cancer (MCF-7). Anti-proliferative activity revealed to be promising on both, the isolated pigment and crude extract. Further investigation on the mechanism of action and effect on other cell lines, both in vitro and in vivo, are required before clinical translation.

Liquid chromatography coupled to Venturi easy ambient sonic spray ionization mass spectrometry.

Venturi easy ambient sonic spray ionization (V-EASI) is a soft ambient ionization (AI) source that has the advantages of being suitable to the analysis of samples in solution (differently from the majority of AI sources), performing self-pumping, voltage- and heat-free ionization, and requiring minimum or no sample preparation. Since this ionization technique has not been fully explored, the present study provides a proof of principle of the coupling of liquid chromatography to mass spectrometry (LC-MS) using V-EASI as the interface. In order to test the performance of the developed LC-V-EASI-MS system, a quantification method for bixin, a natural dye from annatto (Bixa Orellana L.), which is known to be sensitive to the high voltage applied for electrospray ionization mass spectrometry (ESI-MS) analysis, was validated according to FDA criteria and tested in real samples. The analytical method was successfully applied and met the validation criteria, providing a detectability 10 times better than methods already reported to the quantification of bixin and no matrix effect was observed. Therefore, this proof of principle contributes to the continuous development of AI sources that represents the last great technological advance in MS towards becoming a miniaturized technique able to analyze samples closer to their actual state.

Annatto Oil Loaded Nanostructured Lipid Carriers: A Potential New Treatment for Cutaneous Leishmaniasis.

Annatto (Bixa orellana L.) is extensively used as food pigment worldwide. Recently, several studies have found it to have healing and antioxidant properties, as well as effective action against leishmaniasis. Therefore, the purpose of this study was to incorporate the oil obtained from annatto seeds into a nanostructured lipid carrier (NLC) and evaluate its physicochemical properties and biological activity against Leishmania major. Nanoparticles were prepared by the fusion-emulsification and ultrasonication method, with the components Synperonic™ PE (PL) as the surfactant, cetyl palmitate (CP) or myristyl myristate (MM) as solid lipids, annatto oil (AO) (2% and 4%, w/w) as liquid lipid and active ingredient, and ultra-pure water. Physicochemical and biological characterizations were carried out to describe the NLCs, including particle size, polydispersity index (PDI), and zeta potential (ZP) by dynamic light scattering (DLS), encapsulation efficiency (EE%), thermal behavior, X-ray diffraction (XRD), transmission electron microscopy (TEM), Electron Paramagnetic Resonance (EPR), cytotoxicity on BALB/c 3T3 fibroblasts and immortalized human keratinocyte cells, and anti-leishmaniasis activity in vitro. Nanoparticles presented an average diameter of ~200 nm (confirmed by TEM results), a PDI of less than 0.30, ZP between -12.6 and -31.2 mV, and more than 50% of AO encapsulated in NLCs. Thermal analyses demonstrated that the systems were stable at high temperatures with a decrease in crystalline structure due to the presence of AOs (confirmed by XRD). In vitro, the anti-leishmania test displayed good activity in encapsulating AO against L. major. The results indicate that the oily fraction of Bixa orellana L. in NLC systems should be evaluated as a potential therapeutic agent against leishmaniasis.

Assessment of the lipid oxidation, color and sensorial acceptance of fresh sausage formulated with potassium bixinate as a substitute for sodium nitrite and carmine.

This work aimed to evaluate the lipid oxidation, color, and sensory acceptance of sausages formulated with potassium bixinate (BP) as a substitute for carmine (Car) and sodium nitrite (SNi). Six different treatments (CT-control; CA-40 ppm Car; NIT-150 ppm SNi; C/N-40 ppm Car, and 150 ppm SNi; C/N/B-20 ppm Car, 75 ppm SNi, and 250 ppm BP; and BIX-500 ppm BP) were evaluated along 15 days. BP provided significantly lower levels of lipid oxidation to the samples (C/N/B and BIX) when compared to CT and significantly equal to CA, NIT, and C/N after 15 days (both p < 0.05). BP significantly increased the red (a*) and yellow (b*) intensity of sausages (p < 0.05), providing the highest values in both raw and roasted samples. Sausages formulated with BP did not differ in preference in visual analysis (p > 0.05), and both roasted were preferred over CT (p < 0.05). Although sausages formulated with carmine had better visual acceptance, BIX and C/N did not differ significantly (p > 0.05) after five days. BP was identified in taste analysis; however, it was preferred in the ordination analysis (both p < 0.01). Therefore, the replacement of SNi and Car by BP showed to be a viable alternative.

Cellulose acetate nanofibers loaded with crude annatto extract: Preparation, characterization, and in vivo evaluation for potential wound healing applications.

In this study we prepared annatto-loaded cellulose acetate nanofiber scaffolds and evaluated both in vitro cytotoxicity and potential for wound healing in a rat model. Annatto extract, which has been used to accelerate wound healing, was added to cellulose acetate polymer and the resulting material was used to produce nanofiber scaffolds via electrospinning. Physicochemical, and thermal evaluation of the resulting nanofiber mats showed that incorporating annatto did not significantly affect the thermal or chemical stability of the polymer. Annatto extract did not demonstrate cytotoxicity in the HET-CAM assay or MTT assay for fibroblast culture. Scanning electron microscopy of the fibroblasts confirmed that cells spread and penetrated into the nanofiber. In vivo experiments confirmed that cellulose acetate retained its biocompatibility when associated with crude annatto extract, and suggested that dose/response modulation occurs between the annatto-functionalized nanofibers and mast cells, indicating the potential of this material for wound healing applications.

Bixa orellana L. by-products' fractions from an industrial process: antiproliferative activity on tumor cells and chemical profile.

This study evaluated the phytochemical characterization of Bixa orellana (BO extract) unsaponifiable extract and resulting fractions (F fraction - FF, Geranyl fraction - GF and R fraction- RF) obtained as by-products of an industrial process investigating in vitro antiproliferative activities in human tumoral cells. The main compounds identified by GC-MS for BO extract were Geranylgeraniol (61.51%); for FF: Geranylgeraniol (70.23%); for GF: Geranylgeraniol (78.92%) and for RF: ß-cubebene (27.75%). Quantifications of geranylgeraniol by GC-FID presented the percentage content: BO 27.52%; FF 38.52%; GF 51.44% and RF 1.81%. BO extract showed a significant antiproliferative activity, with GI50 up to 4 µg/mL. All fractions had a remarkably similar antiproliferative activity profile (GI50 27-47 µg/mL). Data reported herein showed an important cytostatic effect for BO extract, nevertheless this activity is not attributed exclusively to geranylgeraniol. In conclusion, this by-product becomes of great value, being a potential candidate for development of new anti-tumor ingredients.