Safety of a formulation containing chitosan microparticles with chamomile: blind controlled clinical trial

ABSTRACT Objective: to evaluate the safety of a topical formulation containing chamomile microparticles coated with chitosan in the skin of healthy participants. Method: phase I blind, controlled, non-randomized, single-dose clinical trial with control for skin, base formulation, and formulation with microparticles. The variables analyzed were irritation and hydration by the Wilcoxon and Kruskall-Wallis tests. Results: the study started with 35 participants with a mean age of 26.3 years. Of these, 30 (85.71%) were female, 29 (82.90%) were white skinned and 32 (91.40%) had no previous pathologies. One participant was removed from the study reporting erythema at the site of application, and four other participants for not attending the last evaluation. In the 30 participants who completed the study, the tested formulation did not cause erythema, peeling, burning, pruritus or pain; there was an improvement in cutaneous hydration in the site of application of the formulation with microparticles. In the evaluation of the barrier function, there was an increase in transepidermal water loss in all sites. Conclusion: the formulation with chamomile microparticles is safe for topical use, not causing irritation and improving skin hydration over four weeks of use. Its effects on barrier function need further investigation. No. RBR-3h78kz in the Brazilian Registry of Clinical Trials (ReBEC).


Introduction
The skin, as the interface of the human body with the external environment, carries part of our identity: it provides information about our age, genetics, health status, lifestyle and even our emotional state (1) . The skin has different roles, among them, the function of barrier, thermoregulation, vitamin D synthesis and also protection of the body against harmful agents (2)(3) .
Currently, there is a growing interest on skin care products and their protective and healing properties, especially those with botanical extracts (4) . Among the latter, Chamomilla recutita (L.) rauschert (chamomile) is a popular plant (5) which has its use as a phytotherapic released by the National Agency of Sanitary Surveillance (ANVISA) (6) . Chamomile has flavonoids, among which apigenin and apigenin-7-glycoside are the most abundant (7) . In different studies, these substances have proved to have antimicrobial (8) , analgesic (9) , antiinflammatory (6,8,(10)(11) , cicatrizant (12)(13) , antitumor (14)(15) and immunomodulator (16) potential. Moreover, the antioxidant blend present in chamomile extract is effective in reducing free radicals and brings potential benefits when used in skin formulations by reducing water loss, improving hydration, and aiding the maintenance of the barrier function (4) .
The risks associated with the use of this plant are small and are related to the reduction of platelet aggregation (17) and anaphylactic reactions to people sensitive to its components (18) . However, a recent study that evaluated allergic reactions to herbal compounds over the last 27 years found no reports related to Chamomilla recutita (L.) rauschert (19) .
Despite the potential of apigenin and apigenin-7glycoside, they have low stability (15,(20)(21)(22) . An alternative to improve this issue is the use of controlled release systems. The pharmaceutical sector, in line with technological developments, has gradually improved the processes of obtaining different products and invested in the forms of producing and applying them. To this end, microencapsulation is a widely used technology aimed at optimizing industrial processes as well as increasing the bioavailability and stability of the formulations. This process can be accomplished by different methods, using various coatings. In this study, the selected coating was chitosan and the method of production was spray drying (21) .
This polymer was also used to coat microparticles containing endothelial and epidermal growth factors, with positive results in improving the cicatrization process (29) , and also as a coating of microparticles capable of capturing and expanding specific cells in order to accelerate the anti-inflammatory and cicatrization processes (30) .
Despite all the potentialities of chitosan-coated microparticles and the therapeutic properties of chamomile, no studies with these compounds were identified in the literature. These microparticles are incorporated in lanolin-based formulation, a safe substance for topical applications that incorporates various bioactive agents (31) .
In view of the above, a study was carried out to evaluate the safety of the topical formulation containing chitosan-coated Chamomilla recutita (L.) Rauschert microparticles for application on the skin of healthy volunteers evaluating the following variables: erythema, variation in the amount of melanin, desquamation, burning, pruritus, pain and alterations in cutaneous hydration. The hypothesis was that the use of this formulation would be safe for cutaneous application over four weeks of use.

Method
Blind, controlled, non-randomized, single-dose Phase I clinical trial in which a low dose with biological activity of the active ingredient was administered (32) .
Extraction and microencapsulation methodologies developed and validated in a previous study were applied for the development of the microparticles used in this study (21) . Quality tests of the plant acquired according to the guidelines of the Brazilian Pharmacopoeia (34) were carried out in another study developed by the main author, as well as preliminary permeation and stability tests of the formulation in an ex vivo model. The criteria for inclusion of participants were: age (18 years and over); healthy skin in the site of application of the product (forearms); absence of history of hypersensitivity to fish, seafood or any component of the formulation (chamomile, chitosan or lanolin); non-use of heparin, oral anticoagulants, and antiplatelet agents. The exclusion criteria were: injury at the sites of application, express intention to stop participation, and non-application of the product for more than four consecutive days. National (35) and international (32) recommendations about studies aiming at the initial evaluations of tolerance and safety in healthy humans were considered; the participation of 20 to 100 individuals is recommended.
The study was carried out in partnership with the  in the proximal portion of both, no product was applied to allow assessment of skin conditions (right negative control -C1; left negative control -C2). The dose was applied once a day, daily, always at the same time, and the participants were instructed not to use any other product at the application and evaluation sites during the 28 days of study.

Results
A total of 52 participants were evaluated for eligibility and recruited by the principal investigator. Of these, 17 did not attend the appointment; thus, 35 participants were allocated to the study. The study was completed after four weeks, according to the initial schedule plan.      Table 2.     Concerning sex, 30 (85.7%) of the participants were female (Table 1). Skin characteristics such as erythema, melanin, elasticity, thickness, transepidermal water loss and pH vary in the different anatomical sites between men and women and also in the different age groups (39)(40)(41)(42) . Authors point out the importance of considering variations in the biophysical properties of the skin at different ages, genders and anatomical locations because these differences are related to individual susceptibility to skin diseases; they should be considered in studies and in the production of skin products (41) .

Discussion
The same occurs with BMI. This variable influences skin quality and, therefore, selecting a sample with a wide variation of BMI is important to understand the action of the product in a broader way. In this study, BMI also varied considerably, with a mean of 23.2 (Standard  (41) . Besides anatomical issues, this fact can be explained by difference in sun exposure (41) , corroborating with the findings of this study. Personal factors such as age, sex, race, anatomical site and skin surface properties, as well as environmental factors such as light conditions, temperature, humidity and climatic variations can influence the color of the skin (41,44) .
The function of melanin is the protection of the DNA of the keratinocytes against radiation (45) ; it is known that its concentration, its type and its location represent important factors in the evaluation of skin color, as well as in the evaluation of blood flow, thickness, softness and degradation of skin proteins (46) . A study that evaluated the effects of a tamarindcontaining emulsion on melanin identified a reduction in the amount of melanin at the application sites and attributed this result to the presence of phenolic compounds present in the extract (47) . It is known that chamomile contains several phenolic compounds in its composition, a fact that suggests the need for future studies to better investigate this property.
In the pH evaluation, there was no difference between the sites in D0 (p = 0.0819), but there was a pH is also a variable that varies in the different body regions (42) . In this study, it is believed that the difference is the result of the application of the formulation with chamomile, which caused an increase in its mean value, despite remaining within the physiological limits. Chamomile extract was evaluated for toxicity, presenting safety at the dosages recommended for humans, without cytotoxic, genotoxic or mutagenic effects (48) . The use of chitosan in nanocapsules with alginate, for the treatment of infectious or inflammatory conditions of the skin showed antibacterial, antiinflammatory and controlled release activity, without causing skin irritation (49) . A study on the toxicity of lanolin and its effect on animal cicatrization concluded that it has no toxic effect on monocytes, important cells of the cicatrization process (50) .
As for the participant who was removed from the study in D1 due to a report of local reaction, the event is attributed to a possible unknown personal sensitivity to the components of the formulation, since this was an However, these products do not present chitosan microparticles with chamomile; microparticles that promote an increase in the stability of the encapsulated botanical extract and also the slow and controlled release of their actives that can be a differential.
The study had as possible limitations the nonrandomization of the application sites and the nonmeasurement of the exact amount of the product to be applied.
It is, therefore, a technological product that uses in its composition an active principle with proven biological activities with the advantage of presenting a

Conclusion
The hypothesis that the lanolin formulation,