Quality control of dentin biomodifiers based on Copaiba oil Controle de qualidade de biomodificadores dentinários à base de óleo de Copaíba

This study aimed to evaluate in vitro the pharmacological stability of the dentin biomodifiers emulsions based on copaiba oil X, at 0, 3, 6, 9, 12, 18, and 24 months, and copaiba oil Y, at 0, 6, and 12 months. The emulsions were stored in different environments and analyzed through centrifugation, pH, and density tests, as well as their organoleptic and microbiological characters were investigated. Emulsion X did not show phase separation at 0 and 6 months in the freezer and fridge and showed cleavage in all environments after 12 months. The Y emulsion did not exhibit phase separation at 0 and 6 months in the stove, fridge, room temperature protected, and exposed to light. Still, after 18 and 24 months, phase separation occurred in all analyzed environments. The emulsions X and Y did not show contamination at 12 months. In the organoleptic test, the main change observed was color. The emulsion X presented the slightest deviation of pH in the room temperature protected from light, air conditioning, and fridge while the Y emulsion at 6 months presented a smaller variation in the freezer and fridge, and at 12 months, freezer, stove, and air conditioning. Regarding density, emulsion X at 24 months showed a very slight deviation, and emulsion Y remained stable in all environments at 12 months. The emulsions tested showed stability for 12 months, with the best storage environment being the fridge. The analysis at 18 and 24 months reinforced the fridge as an ideal environment for storage.


INTRODUÇÃO
The technological development of herbal medicines from Brazilian biodiversity can be the alternative for access to safe, effective, and quality medicine, as it happens from the cultivation of plants, sustainable management, research, development, and innovation (RD&I), production, with the correct form of presentation and dosage, distribution and use (HASENCLEVER et al., 2017).
Quality control is an essential step for the product to meet the required standards, and its main advantages are the optimization of processes, from collection to product acquisition, the reduction of time and waste, the standardization of procedures and the quality of environments, inputs used and final products (ROCHA; GALENDE, 2014).
The stability study of herbal medicine directly interferes with the quality of pharmaceutical products. It aims to accelerate chemical degradation and/or physical changes in forced storage conditions, being carried out by packaging samples of the drug in its primary packaging. The stability study determines the shelf life and recommends the appropriate storage conditions after physical, chemical, biological, and micrological analyzes during the entire shelf life that has been defined, and, optionally, after that period, carried out within 24 months (BRAZIL, 2005;BRAZIL, 2019a).
Dental products produced from natural substances have good prospects in the market (FRANCISCO, 2010). The dentin biomodifier based on Copaifera multijuga oil developed to be used in the restorative process demonstrated antibacterial, antiinflammatory, and metalloproteinase inhibitory activity, which can assist in the adhesion of restorative materials to the dental structure, with perspectives of preventing the degradation of the adhesive interface, reducing or eliminating marginal nanoleakage, properties that researchers around the world have pursued for years, and still not achieved (DE BARI et al. , 2010;BANDEIRA et al., 2020;MEIRA et al., 2020;ARAÚJO et al., 2021, MOURA et al., 2021. Thus, this study aimed to characterize the physical-chemical, organoleptic, and microbiological aspects of copaiba (Copaifera multijuga Hayne) emulsion X in periods 0, 3, 6, 9, 12, 18, and 24 months and of emulsion Y in times 0, 6, 12 and 18 months, following the guidelines for standardization and norms of good manufacturing practices for emulsions from the Brazilian Pharmacopoeia and the National Health Surveillance Agency (ANVISA).

METODOLOGIA
This study was carried out at the Laboratory of Phytotherapy in Dentistry (LABFITO) of the Federal University of Amazonas. The quality and safety of medicines

Physical-chemical quality control tests
To carry out the quality control study of the emulsions based on copaiba oil, the emulsion X was analyzed at times 0, 3, 6, 9, 12, 18, and 24 months and the emulsion Y was analyzed at times 0, 6, and 12 months, being stored under different conditions of temperature and humidity, according to the environments described in Table 1. All bottles were coded and dated.

Centrifuge test
In this preliminary stability test, 5 mL of each prepared copaiba oil-based formulation were placed in previously sterilized Falcon tubes and subsequently centrifuged (5804R, Eppendorf, São Paulo, SP, Brazil) at 3000 rpm for 30 minutes, at room temperature, to observe a possible cremation or separation of the emulsion phases.
The test was performed in triplicate (BRAZIL, 2010b).

pH test
The pH was determined using a potentiometer (

Density determination
Density was obtained using an analytical balance (Shimadzu, Barueri, SP, Brazil) and a clean and dry glass pycnometer, with a capacity of 5 mL, previously calibrated, by determining the mass of the empty pycnometer and the mass of its content with distilled water. The sample was transferred to the pycnometer and weighed; the sample weight was determined through the mass difference of the full and empty pycnometer. Density was calculated by the ratio between the mass of the liquid sample and the mass of water.
The result was obtained through the average of three successive determinations (BRAZIL, 2010b).

Microbiological evaluation for research of contaminants -Microbiological control of the emulsion of Copaifera multijulga Hayne
The microbiological control of the Copaifera multijuga Hayne emulsion was determined by the total number of aerobic microorganisms and by researching yeast,

Evaluation of organoleptic characters
The evaluation of the organoleptic characters was based on the change in color, odor, brightness and consistency, verifying the most suitable place for the storage of the copaiba test emulsion, without changing its characteristics (BRAZIL, 2013). Color and brightness were analyzed in daylight. The consistency was assessed by touch, observing the presence or absence of granules.
A small sample of the emulsion was placed in the hand and inhaled slowly and repeatedly to determine the intensity of the odor: none, weak, distinct or strong; and then the sensation caused by that odor: aromatic, fruity, musty, rancid, or woody.

RESULTS
In the centrifugation test, the emulsion X did not show phase separation in 6 months in the refrigerator and freezer environments; in the other environments analyzed, phase separation was present. At 9 months, only the emulsions stored in the fridge and air conditioning did not undergo phase separation. At 12 months, the emulsion stored in the fridge did not undergo phase separation. After 18 and 24 months of analysis, all environments showed phase separation. The Y emulsion did not exhibit phase separation at times 0 and 6 months in the environments stove, fridge, room temperature protected from light and room temperature with exposure to light, showing changes in the environments freezer and air conditioning, and after 12 months of the analysis showed phase separation in all environments.
Regarding the pH test, emulsion X initially presented an average value of 4.23.
During the 24 months of analysis, there were changes in the average pH when compared to the initial value, with the stove and room temperature environment exposed to light being those that suffered the greatest pH variations; room temperature protected from light, air conditioning and fridge environments showed the lowest pH variation (Table 2 and Figure 1).

Time (months)
Environments    The pH test results for time 0 of the Y copaiba emulsion were 6.28. At the time of 6 months of storage, it was possible to notice that the environments that presented a lower initial pH variation were the freezer and fridge, while after 12 months, the freezer and stove (Table 3)     The density of a substance is characterized by the amount of mass of matter existing in a given volume. The results obtained at time 0 with the emulsion X was 1.00 g/cm. During 24 months of analysis, a very slight deviation in the density average was observed in some environments (Table 4).  At time zero, the Y emulsion of copaiba had a density of 0.99 g/cm³. The results found in the 6 and 12 months evaluation showed that the emulsion density remained stable in all environments (Table 5).

Time (months)
Environments  Table 5 -Average and Standard Deviation of the density of Emulsion Y, according to time and storage environment.ASno storage; A -stove; B -freezer; C -fridge; Droom temperature protected from light; Eair conditioning; Froom temperature with exposure to light.
The results for emulsion X in time up to 12 months showed the absence of contaminants in all environments tested. After 18 and 24 months, there was no contamination in storage at room temperature with exposure to light. There were no contaminants in the 12 months analyzed regarding the Y emulsion.
The organoleptic characters found in this study described the copaiba emulsion X and Y as having a milky white color, a strong woody odor, in addition to a liquid consistency without granules, with the presence of gloss at time 0. After evaluating 6, 9, 12, 18, and 24 months of emulsion X and evaluating 6 and 12 months of emulsion Y, changes occurred in all environments tested, except in the fridge environment. These changes were mainly related to color, as the emulsions took on more yellowish tones.

DISCUSSION
The microbiological test aimed to investigate the presence of contaminants, such as bacteria and fungi, in copaiba emulsions X and Y. The emulsion based on copaiba oil X was evaluated in the period of 0,3,6,9,12,18 and 24 months, while the emulsion Y Time (months) in the period of0, 6 and 12 months using centrifugation, density, pH, organoleptic, microbiological control for research of contaminants.
The emulsions were submitted to the centrifugation test, where initially they should not show phase separation, with the permanence of a homogeneous substance, which offers stability (BRASIL, 2008). The spin test is therefore performed to assess stability; phase separation occurs when there is not good stability and can be seen Another test performed to certify the physical-chemical stability is the pH test, which measures the acidity, alkalinity, or neutrality (BRAZIL, 2004). The pH scale ranges from 1 (acid) to 14 (alkaline) and can detect structural changes such as oxidation and incompatibility (BRAZIL, 2008). Both emulsions X and Y presented pH variation; however, room temperature protected from light, air conditioning and fridge environments showed the lowest pH variation for X after 24 months and the freezer, stove, and air conditioning for Y after 12 months.
Density analysis, used in quality control, may indicate the incorporation of air or the loss of volatile ingredients in the sample (BRAZIL, 2008). The measure for determining the density of a product depends on the characteristics of the components present in the formulation and if there was the incorporation of air during it (PEDRAZZI et al., 2012).
In this aspect, this study found favorable results with copaiba emulsions, as there was little value change on density.
The control of microbiological contaminants is essential to guarantee the quality of the product, as materials of plant origin may present the presence of fungi and bacteria, which are present in their microbiota, or which can be inserted at the time of handling. Quality control is one of the essential steps in manufacturing a drug, as it determines the quality, efficacy, and safety of a drug (CHAN, 2003). This study can assure that the two copaiba emulsions obtained satisfactory results, providing security to the final consumer (CHEN; HSU; LEE, 2019).

CONCLUSION
Based on the results of this research, the ideal shelf life for the Copaíba X and Y emulsions was 12 months, and the storage place, the fridge.