Aqueous extract from urucum (Bixa orellana L.): antimicrobial, antioxidant, and healing activity

Abstract

Background: Annatto was obtained from seed B orellana (urucum) and is commonly used in food and cosmetic industries. The objective of this study was to identify the antimicrobial and antioxidant activity of the aqueous extract from urucum seeds and its skin healing potential in exposed cutaneous lesions in rats treated with the gel containing the extract.

Methods: Three types of extracts from seeds were made using chloroform, sodium hydroxide, water, and estimated bixin and norbixin. In the presence of antioxidants, antibacterial was observed and then evaluated the skin healing in rats using aqueous extract.

Results: Annatto dyes have been evaluated in all three extracts. When the seeds were extracted with chloroform, bixin was detected. If extraction was performed by sodium hydroxide or water, norbixin was detected. For healing use, 10% of aqueous extract was mixed in a gel base. The finding obtained from the antioxidant assay revealed that the activities of the water extract could be used as a source of polyphenolic compounds. In chloroform extract, the antioxidant was not effective because it has weak radical scavengers. With respect to antimicrobial activity, it has been observed that aqueous extract has more effect. For skin healing assay, a total of 3 study groups were tested: negative control group (gel base), positive control group (fibrinase), and test group (gel with urucum aqueous extract). After 7 days of treatment, animals treated with fibrinase had an improvement of 4.7% in total wound area when compared with the negative control while those treated with urucum aqueous extract presented an improvement of 51.55% in comparison. After 14 days, the total wound area of animals within the test group had a decrease of 94.97% when compared with the negative control (gel base) results while the control group presented an improvement of 56.58% in total wound area. These results indicate that wounds treated with urucum aqueous extract were 38.39% more efficient than fibrinase, a cream used for skin healing.

Conclusions: It is possible to conclude that gel with aqueous extract is effective in skin healing in rats, being used as a phytotherapic, besides possessing antioxidant and antimicrobial activity.

Introduction

B orellana L. is a plant originating in South America, more specifically in the Amazon region. Its popular name comes from the Tupi word “Uru-ku” and denominated Urucum, which means red. Fruits grown from that plant are small hedgehogs covered by malleable spines. When they mature, red seeds can be seen, which are traditionally used by indigenous people of different ethnicities for body painting and protection of the skin against insect bites. Pigments present in those seeds are commercially known as annatto dyes. It is also widely used by food, pharmaceutical, and cosmetics industries.^1,2

Annatto color is a carotenoid pigment obtained by several methodologies, such as immersion of seeds in hot vegetable oil, dilute alkaline aqueous solutions, solvents, and water, for efficient extraction of bixin and/or norbixin.^3–10 For fat-soluble dye, bixin is the major constituent and can be extracted with organic solvent such as chloroform (1:5) or chloroform and 0.5% acetic acid.⁷ As for water-soluble norbixin, the extraction is performed with an aqueous alkaline solution (NaOH) under controlled conditions, with the hydrolysis of the ester group of the main carotenoid bixin and norbixin salt (norbixinate), soluble in the medium.¹¹ Norbixin water-based dye can be obtained by homogenization of seeds in water, generating a solution with norbixin as its main constituent, and owing to this compound reaction with casein, it allows for its use as a dairy products coloring agent.¹¹

Pigments can be identified by visible spectrophotometry, thin-layer chromatography, and liquid chromatography (hight pressure liquid chromatography). Most carotenoids present absorption in the visible region of the spectrum between 400 and 500 nm.^12–14 Reith and Gielen⁷ extracted cis-bixin in chloroform where the absorbance of the diluted solution was measured at 470 and 501 nm, and the content was calculated using the absorption coefficients of 2880 (501 nm) and 3230 (470 nm). According to Rodriguez-Amaya,¹¹ norbixin was extracted with sodium hydroxide where the absorbance of the diluted solution was measured at 453 and 482 nm. Norbixin content was calculated using the absorption coefficients of 2850 (453 nm) and 2550 (482 nm).

Among the natural carotenoids, bixin and norbixin stand out for being capable of intercepting and deactivating reactive singlet oxygen molecules, acting in antioxidant defense,¹⁵ and protecting against mutagenic and genotoxic effects induced.¹⁶ According to Lima et al,¹⁷ the antioxidant action of norbixin and bixin was important in the prevention of atherosclerosis.

Based on in vitro studies and using various parts of the plant, it was possible to note that B orellana showed antibacterial characteristics with inhibitory behavior against the following bacteria: Clostridium perfringens, Lactococcus lactis, Streptococcus thermophilus, Bacillus cereus, Lactobacillus casei subsp. casei, Paenibacillus polymyxa, Listeria monocytogenes, and Enterococcus durans.¹⁸ Fleischer et al¹⁹ demonstrated that the ethanolic extract showed a good antimicrobial activity against gram-positive and gram-negative bacteria and the yeast-like fungus Colletotrichum albicans.

The objective of this study was to identify the antimicrobial and antioxidant activity of the aqueous extract from urucum seeds and its skin healing potential in exposed cutaneous lesions treated with the gel containing the extract.

Materials and methods

Extraction

One kilogram of seeds was collected from Ibiúna, São Paulo, Brazil, dried in a closed circulation oven for 72 hours at 55°C, and crushed. The seeds were submitted for three types of extraction: (1) extracted with aqueous NaOH solution, (2) extracted with water, and (3) extracted with organic solvents (chloroform).

Extracts with aqueous NaOH solution and organic solvent (chloroform) were based on the work from Bhalkar and Dubash¹ using 2 g of seeds per 40 mL of solutions or solvents (chloroform).

To prepare the aqueous extract, 20 g of seeds were soaked overnight in 50 mL of water and the obtained extract was stored at 2°C–8°C until being used.^19,20

Estimation of bixin and norbixin

Ten milligram seed powder from alkaline extract and chloroform was dissolved in 100 mL of NaOH solution or chloroform. With aqueous extract, 1 mL was diluted to 30 mL of water. The three diluted extracts were scanned in a spectrophotometer from 400 to 600 nm.

The content of bixin was determined in sample diluted with chloroform using a wavelength of 470 nm and an absorption coefficient of 3230 according to Reith and Gielen.⁷ The norbixin content, extracted with an alkaline solution, used a wavelength of 453 nm with an absorption coefficient of 2850.¹¹

Antioxidants

As an antioxidant, the 2,2′-azinobis (3-etilbenzotiazolina-6-ácido sulfônico) (ABTS)²¹ method was used with the reaction involving 7 mM ABTS (5 mL) and 2.45 mM of potassium persulfate (88 mL) after incubation at room temperature and dark for 16 hours. After that period, it was diluted in 80% ethanol to present an absorbance of 0.700 ± 0.005 at 734 nm. Then, 2.7 mL of ABTS solution was mixed slowly with 0.3 mL of samples. After incubation for 30 minutes at 30°C, the spectrophotometer was measured at an absorbance of 734 nm and compared with the standard Trolox.

Antimicrobial assay

For antimicrobial testing, both Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were used. The minimum inhibitory concentration was determined using 4 mL of norbixin solution with 150, 75, 37.5, and 18.75 mg, with the addition of 1 mL of inoculum (15 × 108 CFU/mL) in 3 tubes. After incubation, the growth of microorganisms was observed and also the disc halo was performed in the Petri plates.

Animals

For wound skin healing in rats (Ethics Committee AN 37/2014), a total of 18 adults, male Wistar rats (weight 200–250 g) (UNINOVE Vivarium), divided into three groups were used. Group 1 consisted of animals treated with 1 mL of 10% aqueous extract (norbixin) in gel, group 2 treated with 1 mL of distilled aqueous in gel (negative control), and group 3 used fibrinase (commercially available skin healing cream) (positive control) as treatment. Daily application of the different treatments was performed on a surgically inflicted wound with a total area of 4 cm² in the dorsal region of each animal. Wound evaluation was performed microscopically in days 0, 7, and 14, and the wound evolution and skin healing retraction measures were evaluated by using digital planimetry.

Results/discussion

Extraction and estimation of bixin and norbixin

The scanning spectrum of the 3 extracts is presented in Fig. 1. The chloroform extract showed peaks at 470 and 501 nm that correspond to bixin, being in accordance with the study of Reith and Gielen.⁷ The NaOH extract showed peaks at 453 and 482 nm corresponding to norbixin, matching what was found by Rodrigez-Amaya.¹¹

Figure 1.

Scanning spectrum from three types of extracts: water, chloroform, and NaOH.

The extract obtained using water showed a peak of 453 nm and another of 550–555 nm of unknown identity. This aqueous extract can be recommended as a plant derivative and be easily mixed with gel. Usually there is a problem when using extracts prepared with an apolar organic solvent because of the possibility of precipitate formation, besides the solvent presenting a higher toxicity potential when compared with water.

For the quantification of the aqueous extract, it was based on the calculation of norbixin involving reading at 453 nm and the specific extinction coefficients (2850) as follows:

$$\text{Norbixin content}=\frac{\text{reading in spectrophotometer} \times \text{dilution factor }}{\text{absorptivity coefficient} \times \text{seed mass}}.$$

For skin healing assessment, an annatto dye of 250 mg from 10% aqueous extract mixed in a gel base was used.

Antioxidant assay

Of all the three extracts from B orellana seeds, water extract showed significant radical scavenging activity (147.5 µMol Trolox) followed by sodium hydroxide extract (118 µMol Trolox), while the chloroform extract presented minimal activities (Table 1). The results obtained from the antioxidant assay indicated that the aqueous extract, because of its higher concentration of polyphenolic compounds, presented an improved kidnapping of free radicals when compared with the chloroform-based extract. This discrepancy between results occurred mostly because of the difference between polyphenolic compounds concentration within the samples, and while the chloroform-based extract displayed the presence of these compounds, the water-based had a higher concentration present.

Table 1.

Antioxidant activity (micromol Trolox) in extracts from urucum seeds

	Aqueous extract	Chloroform extract	NaOH extract
µMol Trolox	147.50*,†	60.10‡	118.00§

^*Average of three tests for each treatment. †, ‡, and § Means from three tests from treatments are different statistical at the level of 5% (t test).

Abayomi et al²² extracted dye from urucum seeds using potassium hydroxide and also observed high antioxidant activity.

Antimicrobial assay

For antimicrobial assay, four dilutions were used: 150, 75, 37.5, and 18.75 mg of annatto dye from aqueous extract. In the minimum inhibitory concentration test using liquid medium, it was observed that the extract in concentrations of 150 mg, 75 mg, and 37.5 mg annatto dye did not show visible growth in the media inoculated with S aureus, whereas in the tubes inoculated with E coli, the concentrations of the extract that led to no visible growth were 150 mg and 75 mg (Table 2). The results indicated that the aqueous annatto extract has an antimicrobial action in concentrations of 150 and 75 mg of annatto dye against S aureus and E coli.

Table 2.

Microbial growth in different concentrations of aqueous extract from urucum

Concentration of aqueous extract (equivalent a norbixin)
Microorganism	150 mg	75 mg	37.5 mg	18.75 mg	H2O	Hypochlorite 2%
E coli	−	−	+	+	+	−
S aureus	−	−	-	+	+	−

− did not show turbidity of the medium. + presented turbidity in the medium

In the agar diffusion test, it was observed that after 24 hours of incubation at 37°C, the inhibition zone for E coli was 18 mm with a concentration of 150 mg, 9 mm for 75 mg, 7 mm for 37.5 mg, and 6 mm for 18.75 mg.

For S aureus, the inhibition zone was 21 mm at a concentration of 150 mg, 17 mm at 75 mg, 11 mm at 37.5 mg, and 8 mm at 18.75 mg.

The disks used as a positive control (antibiotic) showed an 8-mm inhibition halo in the E coli plate and 11 mm in the S aureus plate. The disks used as negative controls (saline 0.9%) did not present an inhibition halo (Table 3). The results indicated that aqueous extract promotes an inhibition of growth action in 150 and 75 mg of annatto dye against S aureus and E coli.

Table 3.

Inhibition halo at different concentrations of aqueous extract from urucum

Concentration of aqueous extract (equivalent a norbixin)
Microorganism	150 mg	75 mg	37.5 mg	18.75 mg	Antibiotic	Saline
E coli	18 mm	9 mm	7 mm	6 mm	8 mm	−
S aureus	21 mm	17 mm	11 mm	8 mm	11 mm	−

− did not show halo.

The results are in agreement with those of Galindo-Cuspinera et al¹⁸ where norbixin-based fractions were responsible for the antibacterial activity mainly against S aureus. Many authors cite the importance of annatto extracts as an antimicrobial action in maintaining the quality of foods such as cheese, butter, meat, and others.^23,24

Skin wound healing

For the skin healing assay, the group used as negative control had as treatment, the gel base alone, aiming to evaluate whether the gel base alone wound not present a potential skin healing effect, thus causing a possible interference in the results. Regarding fibrinase, which is a commercially available cream used as a skin healing agent, was observed to have an improvement of 4.7% in total wound area after 7 days of treatment and 56.58% after 14 days. These results are based on the comparison with the gel base group (negative control). When evaluating the test group, with use of annatto aqueous extract gel after 7 days, an improvement of 51.55% in total wound area was observed, and after 14 days, the improvement was 94.97% when compared with the negative control.

When compared with the fibrinase, the group treated with annatto aqueous extract gel presented an improved skin healing effect, with a decrease of 38.39% in total wound area when using the aqueous extract–based gel. These results are presented in Fig. 2.

Figure 2.

Graphic involved time of wound healing from rats treated with aqueous extract of urucum seeds + gel, gel–water, and fibrinase. Percentage of healing wound included on table. Photographs from treatments (300× magnification photographs).

Santos et al²⁵ evaluated the healing process of open dermal wounds of rats treated with an aqueous solution of annatto containing 2.5% of norbixin, acquired commercially, which was obtained with sodium hydroxide or organic solvent. The results that group obtained proved that the solution was not innocuous to skin tissues and has proinflammatory and proangiogenic effects during the process of skin wound healing in rats, interfering in the physiological healing process.

Results obtained in this work prove the opposite observed by Santos et al²⁵ because the aqueous extract presents norbixin with excellent healing. This proves that for healing to occur, there cannot have residue of organic solvent and also alkaline substance. Thus, aqueous extract has an antioxidant factor, antimicrobial properties, and acted as a healing agent.

Conclusion

This study indicates that the gel with 10% aqueous annatto extract is effective at skin healing in rats, being can be used as a phytoterapic, besides possessing antioxidant and antimicrobial activity and is a natural product non-toxic.

Conflicts of interest

The authors declare no conflicts of interest.