PREPARATION AND CHARACTERIZATION OF GRAPEFRUIT OIL BASE MICROEMULSIONS OF CAFFEINE

Objective: The objective of the present work was to prepare and characterize grapefruit oil base microemulsions loaded with caffeine as a model hydrophilic compound. Methods: The formulation ingredients were selected based on surfactant efficiency and solubility studies. Ternary phase diagrams of grapefruit oil were constructed using the water titration method. Nine O/W microemulsions were constructed and prepared by mixing surfactant system, grapefruit oil, water and caffeine together. The resulting microemulsions were investigated for viscosity using Brookfield viscometer, for pH value using a digital pH meter, and for average particle size and polydispersity index (PDI) using a Zetasizer Nano. Ex vivo skin permeation through porcine ear skin was conducted using a side-by-side diffusion cell. The amount of caffeine was analyzed using HPLC-UV method. Results: Tween 20 yielded the highest emulsification ability for grapefruit oil and the highest caffeine solubility. It was selected as a major surfactant. Caffeine was slightly soluble in ethanol and isopropyl alcohol, but sparingly soluble in propylene glycol (PG). These ingredients were used as the cosurfactants. Nine grapefruit oil base microemulsions were prepared and characterized. The pH of microemulsions was within the range of 4.48-5.96. Particle size was in the range of 10.81±0.03 to 62.18±21.04 µm with the PDI of 0.13±0.02 to 0.64±0.11. Viscosity and particle size of microemulsions increased significantly with increasing grapefruit oil or tween 20 content. Addition of PG as cosurfactant resulted in the increases of viscosity, particle size and PDI. Depending on the formulation parameters, the permeation fluxes of caffeine from grapefruit oil base microemulsions were in the range of 28.4±3.4-361.4±15.2 µg/cm2/h. Conclusion: The grapefruit oil base microemulsions were successfully formulated. The physical properties and caffeine permeation of these microemulsions were found to be dependent on the grapefruit oil content, tween 20 content, cosurfactant type and content, as well as caffeine loading. The optimal formulation of grapefruit oil base microemulsion suggested composition of 5% grapefruit oil, 50% surfactant system (tween 20 and ethanol at the ratio of 9:1), and water

การใช้แป้งข้าวเหนียวดัดแปรด้วยเทคนิคไมโครเวฟเป็นสารยึดเกาะในยาเม็ด Microwave-treated Glutinous Rice Starch as A Tablet Binder

บทคัดย่อ วัตถุประสงค์: เพื่อศึกษาการใช้แป้งข้าวเหนียวที่ดัดแปรด้วยไมโครเวฟ (GRMi) เป็นสารยึดเกาะในการผลิตยาเม็ดที่เตรียมด้วยวิธีแกรนูลเปียก วิธีการศึกษา: เตรียมยาเม็ดด้วยวิธีแกรนูลเปียกที่ประกอบด้วยไมโครคริสทัลไลน์เซลลูโลส (MCC) หรือแลคโตสโมโนไฮเดรต (Lac) โดยใช้สารยึดเกาะในปริมาณร้อยละโดยน้ำหนักดังนี้ 1) ร้อยละ 0, 2) GRMi ร้อยละ 2.5 และ 3) แป้งข้าวเหนียวที่ให้ความร้อนโดยตรง (GRB) ร้อยละ 2.5 และใช้แมกนีเซียมสเตียเรตร้อยละ 1 โดยน้ำหนัก โดยตอกอัดยาเม็ดด้วยแรงที่ต่างกัน (40, 60 และ 80 kgf/cm2) แล้วประเมินสมบัติของยาเม็ด ผลการศึกษา: ยาเม็ดที่ใช้ MCC มีความต้านแรงอัดแตกในช่วง 9.52 - 17.72 kgf ซึ่งสูงกว่า Lac (1.10 - 5.98 kgf) เวลาในการแตกตัวและสภาพกร่อนของยาเม็ดที่ใช้ MCC อยู่ในช่วง 11.40 - 59.40 วินาที และร้อยละ 0.08 - 0.41 โดยน้ำหนัก ตามลำดับ ซึ่งต่ำกว่ายาเม็ดที่ใช้ Lac ที่อยู่ในช่วง 70.80 - 5,170.20 วินาที และร้อยละ 0.88 - 3.89 โดยน้ำหนัก ตามลำดับ และยังพบว่าการเพิ่มแรงตอกอัดทำให้ยาเม็ดมีค่าความต้านแรงอัดแตกและเวลาในการแตกตัวเพิ่มขึ้น แต่มีสภาพกร่อนลดลง เมื่อเพิ่มแรงอัดให้แก่ยาเม็ดที่ใช้สารยึดเกาะ GRMi และ GRB พบว่าสามารถทำให้ยาเม็ดมีค่าความแข็งแรง (CSFR) และดัชนีชี้วัดคุณภาพยาเม็ด (CSFR/DT index) เพิ่มมากขึ้นได้ แสดงถึงความสามารถในการยึดเกาะที่ดีและสมบัติการเป็นยาเม็ดที่ดีขึ้น สรุป: สามารถใช้แป้งข้าวเหนียวที่ดัดแปรด้วยไมโครเวฟเป็นสารยึดเกาะในการเตรียมยาเม็ดด้วยวิธีแกรนูลเปียกได้ คำสำคัญ: แป้งข้าวเหนียว, แป้งดัดแปรด้วยไมโครเวฟ, การเตรียมด้วยวิธีแกรนูลเปียก, สารยึดเกาะ Abstract Objective: To investigate microwave-treated glutinous rice starch (GRMi) used as a binder in wet granulation tablets. Methods: Being prepared with wet granulation method, each tablet consisted of filler (microcrystalline cellulose (MCC) or lactose monohydrate (Lac)), binder (0% w/w, 2.5% w/w of GRMi, or 2.5% w/w of heated glutinous rice starch (GRB)) and 1% w/w of magnesium stearate. All tablet formulations were compressed by various compression forces (40, 60, and 80 kgf/cm2) and tablet properties were evaluated. Results: The MCC-tablets had a crushing strength (CS) of 9.52-17.72 kgf. which was higher than that of Lac-tablets (1.10 - 5.98 kgf.). The disintegration time (DT) and friability (F) of MCC-tablets (11.40 - 59.40 sec and 0.08 - 0.41%, respectively) were lower than those of Lac-tablets (70.80 - 5,170.20 sec and 0.88 - 3.89%, respectively). The increase of compression force resulted in increased CS and DT and decreased F. When compression force was increased to tablets using GRMi and GRB as a binder, the higher crushing strength-friability ratio (CSFR) and crushing strength-friability/disintegration time ratio (CSFR/DT index) in both tablets were observed. These results indicated better binding capability with improved tablet properties. Conclusions: microwave-treated glutinous rice starch could be used as a binder in wet granulation tablets. Keywords: glutinous rice starch, microwave-treated starch, wet granulation method, binde

Preparation and Evaluation of Alcohol-Alkaline-Treated Rice Starch as a Tablet Disintegrant

Purpose: To prepare and characterize alcohol-alkaline modified rice starch (MRS) as a disintegrant for tablets.Methods: The preparation of MRS was carried out using 3 M NaOH and 40 % ethanol solution. Characterization carried out for MRS include morphology, swelling capacity, thermal and pasting properties. Direct-compressed tablets (DCT) containing either propranolol hydrochloride (PPNL) or hydrochlorothiazide (HCTZ) were evaluated for hardness, friability, disintegration time and drug release.Results: The microstructure of MRS was different in shape and dimension from that of rice starch (RS). The absence of gelatinization endotherm and FT-IR spectral peak for MRS correlated with change in MRS structure and arrangement. MRS showed significantly higher swelling capacity (p < 0.05) than RS, and also proved to be a disintegrant in DCT. The disintegration time of the tablets containing MRS was lower in the presence of large particles (3.55 ± 0.56 min); high content of MRS (1.03 ± 0.06 min); low content of lubricant (3.16 ± 0.44 min); water soluble filler (1.55 ± 0.16 min for Super-tab®); and model drug (0.84 ± 0.09 min for HCTZ) (p < 0.05).Conclusion: MRS exhibits improved water solubility and swelling capacity compared with RS, and is thus a good disintegrant for direct-compressed tablet formulations, especially in the presence of water insoluble fillers.Keywords: Rice starch, Alcohol-alkaline treatment, Disintegrant, Directly compressed tablet, Insoluble filler

Redox activity of melanin from the ink sac of Sepia officinalis by means of colorimetric oxidative assay

The redox properties of natural extract from cuttlefish ink sac (Sepia officinalis) and synthetic melanin used as a biomimetic in melanin structural investigation were determined by comparison of this phenol-based heterogeneous pigment with gallic acid used as a standard in Folinâ Ciocalteu colorimetric assay widely employed for characterisation of oxidative properties of biomaterials. Reactivity of sepia melanin reported here is much higher than previously indicated and this protocol should allow the redox characterisation of all melanins irrespective of their origin and composition.European Union’s Seventh Framework Programme (FP7/2007-2013) [grant agreement number REGPOT-CT2012-316331-POLARIS

Effect of melanomal proteins on sepia melanin assembly

Accepted manuscriptMelanins are phenol-based pigments with the potential for widespread applications including bioelectronics and tissue engineering. The concentration-dependent structural transition of sepia melanin in water is analyzed. This biopolymer at high concentration gives the well-known nanospheres, whereas sample dilution gives unforeseen nanofibres exhibiting the structural features of mature amyloid fibrils. We propose a mechanism of pigment self-assembly dependent on the interaction of residual melanosomal protein(s) with eumelanin heteropolymer. Our results contribute to understanding the peculiar physico-chemical properties of this ubiquitous pigmentEuropean Union's Seventh Framework Programme (FP7/2007–2013) under grant agreement No. REGPOT-CT2012–316331-POLARI

Exploitation of natural eumelanin and conductive polymers on the preparation of electroactive systems for skeletal muscle tissue engineering applications

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisSkeletal muscle tissues comprise about 40-45% of the total human body mass. The skeletal muscle functionality is commonly depreciated as a consequence of congenital abnormalities, traumatic injuries from accidents or sports activities, and neurological diseases. The defects in muscle structure can affect the physiological performance of the body as a whole, which further influences the psychological responses. It has been demonstrated that electrical stimulus can affect cell behavior, namely, adhesion, proliferation and differentiation and accelerate the healing of damaged tissues like bone and nerves. Moreover, electrical stimulation is one of the emerging tools being used in clinics with the purpose of inducing and recovering the physiological functionality in damaged muscle tissues. However, the clinical outcomes are limited to the specific local area, such as head, legs, arms and back. Despite the intrinsic capacity of skeletal muscle tissues to self-repair, in case of severe tissue damage or loss, such capacity is insufficient to restore the functionality. The conventional clinical treatment modalities include allocation of autologous muscle by surgical treatment, but clinical outcomes are not very satisfactory. Thus, by combining the advantages of electrical stimulus, including cell fate modulation, with electroactive materials and tissue engineering approaches, we expect to overcome some of the existing limitations. Indeed, skeletal muscle tissue engineering holds great potential to alleviate the increasing burden of defective muscle tissues by constructing viable substitutes for replacement therapies. Nevertheless, the reconstruction of tissue engineered skeletal muscle substitutes should mimic native skeletal muscle-like structural properties including myofibers uniformity and density throughout the functional muscle cells. This thesis is focus on the exploitation of novel biomaterials, in particular, natural and synthetic polymers, with electrical properties, for skeletal muscle tissue engineering applications. Eumelanin from Sepia ink (Sepia officinalis) was initially purified by a well-established protocol and analyzed by colorimetric oxidative assay for investigating the redox activity. To explore intrinsic properties of eumelanin nanoparticles and expand its applications, an innovative approach was developed, which consisted in the preparation of electrospun PVA nanofibers meshes containing eumelanin nanoparticles. The physical properties, in particular, electrical conductivity, and biological performance of the nanofibrous meshes were significantly improved upon eumelanin incorporation, indicating that EUNp-PVA nanofibrous matrices are excellent candidates to be used in skeletal muscle tissue engineering applications. In parallel, novel electroactive spongy-like hydrogels were developed by combining gellan gum (GG) with two different conductive synthetic polymers: polypyrrole (PPy) and polyaniline (PANi). GG is a linear anionic polysaccharide of natural origin that has been extensively studied for a wide range of biomedical applications and can be transformed in a hydrogel at body temperature by ionic crosslinking. When processed as spongy-like hydrogels, these materials retain the structural features of hydrogels relevant for tissue engineering applications and improved mechanical and cell adhesive properties. Thus, the rationale behind adding synthetic conducting polymers, PPy and PANi, was to improve the electroconductive properties of the final constructs, maintaining simultaneously the improved mechanical features and intrinsic cell-adhesive ability of GG spongy-like hydrogels. The physical, chemical and electrical properties were analyzed, and cytotoxicity, as well as, biocompatibility was assessed both in vitro and in vivo. Both PPy-GG and PANi-GG electroactive spongy-like hydrogels have shown high porosity and interconnected pores, showing enhanced cellular response during in vitro studies. Moreover, negligible inflammatory response was observed during in vivo analysis. The results demonstrate that the electroactive PPy-GG and PANi-GG spongy-like hydrogels meet all the functional requirements for mimicking the ECM microenvironment of muscle tissue, being interesting candidates to be used in skeletal muscle tissue regeneration strategies. The results reported in this thesis demonstrate the successful fabrication of eumelanin nanoparticlesincorporated PVA nanofibrous meshes by electrospinning, as well as, the production of electroactive PPy-GG and PANi-GG spongy-like hydrogels. Furthermore, physicochemical and biological assessment of these three different matrices prove that these biomaterials have beneficial properties to be employed in the development of tissue engineering strategies for the regeneration of skeletal muscle tissues or other tissues of interest that respond favorably to electrical stimulation.O tecido muscular esquelético constituí cerca de 40-45% da massa total do corpo humano. A funcionalidade do tecido muscular esquelético é frequentemente comprometida como consequência de anomalias congénitas, lesões traumáticas decorrentes de acidentes ou atividades desportivas e doenças neurológicas. Os defeitos no músculo-esquelético podem afetar o desempenho fisiológico do corpo, influenciando as suas respostas fisiológicas. Foi demonstrado que o estímulo elétrico pode afetar a comportamento celular, nomeadamente a adesão, proliferação e diferenciação, e acelerar a cura dos tecidos danificados como os ossos e os nervos. Apesar da estimulação elétrica ser uma ferramenta emergente usada na prática clínica com o objetivo de induzir e recuperar a funcionalidade fisiológica dos tecidos danificados, os resultados clínicos são limitados a uma área específica como cabeça, perna, braço e costas. Apesar da capacidade intrínseca do músculo-esquelético para auto-reparação, em casos de danos severos ou perda do tecido, essa capacidade é insuficiente para restaurar a funcionalidade do músculo. As modalidades de tratamento clínico convencionais incluem alocação de músculo autólogo por tratamento cirúrgico, no entanto, os resultados clínicos não são muito satisfatórios. Assim, através da combinação das vantagens do estímulo elétrico com materiais electrocondutores e engenharia de tecidos, pretendemos ultrapassar algumas das limitações existentes na regeneração do músculoesquelético. De facto, a engenharia de tecidos do músculo-esquelético apresenta grande potencial para reduzir a elevada percentagem de tecidos musculares defeituosos através da construção de substitutos viáveis para terapias de reposição. No entanto, a reconstrução do tecido fabricado pela engenharia de tecidos deve simular o tecido muscular nativo nas suas propriedades estruturais, incluindo a uniformidade e densidade das células musculares. Esta tese foca-se na exploração de novos biomateriais, em particular, polímeros materiais e sintéticos, com propriedades elétricas, para a engenharia de tecido muscular esquelético. A eumelaninn da Sepia ink (Sepia officinalis) foi inicialmente purificada por um protocolo estabelecido e analisada por um ensaio colorimétrico oxidativo para investigar as suas propriedades oxidativas/redutoras. Para explorar as propriedades intrínsecas das nanopartículas de eumelanina e alargar as suas aplicações, uma nova abordagem foi desenvolvida, que consistiu na preparação de nanofibras de PVA por “electrospun” contendo na malha das fibras nanopartículas de eumelanina. As propriedades físicas, em particular a condutividade e o desempenho biológico das malhas de nanofibras foram melhoradas significativamente após a incorporação de eumelanina, indicando que as matrizes de nanofibras de eumelanina nanopartícula-álcool polivinílico (EUNp-PVA) são excelentes para serem usadas em engenharia de tecidos do tecido muscular esquelético. Em paralelo, novos hidrogéis “spongy-like” foram desenvolvidos com gellan gum (GG) com dois polímeros condutores diferentes: polypyrrole (PPy) e polianilina (PANi). Gellan gum é um polissacarídeo aniónico linear cuja origem natural tem sido intensamente estudada para vasto número de aplicações biomédicas, que pode ser transformado num hidrogel à temperatura do corpo por reticulação iónica. Quando processado como um “spongy-like” hidrogel, retém as estruturas estruturais dos hidrogéis relevantes para engenharia de tecidos e melhoram as propriedades mecânicas e de adesão das células. Assim, o objetivo de adicionar polímeros condutores sintéticos, PPy and PANi, foi o de melhorar as propriedades electrocondutoras dos construtos finais, mantendo simultaneamente as características mecânicas e de adesão celular dos “spongy-like” GG hidrogéis. As propriedades físicas, químicas e elétricas foram analisadas, e a citoxicidade, assim como a biocompatibilidade foi avaliada in vitro e in vivo. Ambos “spongy-like” hidrogéis PPy-GG e PANi-GG apresentam grande porosidade e poros interconectados. Demonstrando assim melhor resposta celular durante os estudos in vitro. No entanto, inflamação celular não significativa foi detetada durante os estudos in vivo. Os resultados demonstram que os hidrogéis eletroactivos “spongy-like” PPy-GG e PANi-GG preenchem todos os requisitos funcionais para substituir a matriz extracelular (ECM) do tecido muscular, sendo candidatos interessantes para serem utilizados nas estratégias de regeneração do tecido muscular esquelético. Os resultados desta tese demostram a fabricação de nanopartículas de eumelanin e a sua incorparação em malhas de nanofibras à base de PVA produzidas por electrospinning, assim como a produção de hidrogéis “spongy-like” condutores. A avaliação físico-química e biológica destas 3 matrizes diferentes prova que estes biomateriais têm propriedades beneficiais para serem utilizados no desenvolvimento de estratégias de engenharia de tecidos para a regeneração do músculo esquelético ou outros tecidos de interesse que respondem favoravelmente à estimulação elétrica

Sodium Alginate-Quaternary Polymethacrylate Composites: Characterization of Dispersions and Calcium Ion Cross-Linked Gel Beads

The objective of this work was to examine the effect of quaternary polymethacrylate (QPM), a water-insoluble polymer with a positive charge, on the characteristics of the sodium alginate (SA) dispersions and the calcium alginate (CA) gel beads containing propranolol HCl (PPN). The SA-QPM composite dispersions presented the formation of flocculates with a negative charge due to the electrostatic interaction of both substances. The QPM addition did not affect the SA dispersions’ Newtonian flow, but the composite dispersions’ viscosity enhancement was found. The PPN-loaded CA-QPM gel beads had more spherical than the PPN-loaded CA gel beads. The incorporation of QPM caused a bigger particle size, higher drug entrapment efficiency, and greater particle strength of the gel beads. Despite the similar water uptake property, the PPN-loaded CA-QPM gel beads displayed lower burst release and slower drug release rate than the PPN-loaded CA gel beads. However, the drug release from the PPN-loaded CA-QPM gel beads involved drug diffusion and matrix swelling mechanisms. This study demonstrated that adding QPM into the SA dispersions leads to a viscosity synergism. The CA-QPM gel beads display a good potential for use as a bioactive compound delivery system

Synthesis and characterization of electroactive gellan gum spongy-like hydrogels for skeletal muscle tissue engineering applications

Advances on materials' research for tissue engineering (TE) applications have shown that animal cells respond directly to the material physical, chemical, mechanical, and electrical stimuli altering a variety of cell signaling cascades, which consequently result in phenotypic and genotypic alterations. Gellan gum (GG) spongy-like hydrogels (SLH) with open microstructure, mechanical properties, and cell performance have shown promising results for soft TE applications. Taking advantage of intrinsic properties of GG-SLH and polypyrrole (PPy) electroactivity, we developed electroactive PPy-GG-SLH envisaging their potential use for skeletal muscle TE. Three different methods of in situ chemical oxidative polymerization were developed based on the availability of pyrrole: freely dissolved in solution (method I and III) or immobilized into GG hydrogels (method II). PPy was homogeneously distributed within (method I and III) and on the surface (method II) of GG-SLH, as also confirmed by Fourier Transform infrared spectra. PPy-GG-SLH showed higher conductivity than GG-SLH (pâ <â 0.05) whereas PPy-GG-SLH (method I and II) showed the best conductivity among the 3 methods (â ¼1 to 2â à â 10-4 S/cm). The microarchitecture of PPy-GG-SLH (method I) was similar to GG-SLH but PPy-GG-SLH (method II and III) presented smaller pore sizes and lower porosity. PPy-GG-SLH (method I and II) compressive modulus (â ¼450-500 KPa) and recovering capacity (â ¼75-90%) was higher than GG-SLH, nevertheless the mechanical properties of PPy-GG-SLH (method III) were lower. The water uptake of PPy-GG-SLH was rapidly up to 2500% and were stable along 60 days of degradation being the maximum weight loss 20%. Mechanically stable and electroactive PPy-GG-SLH (method I and II) were analyzed regarding cellular performance. PPy-GG-SLH were not cytotoxic for L929 cells. In addition, L929 and C2C12 myoblast cells were able to adhere and spread within PPy-GG-SLH, showing improved spreading in comparison to GG-SLH performance. Overall, PPy-GG-SLH show promising features as an alternative electroactive platform to analyze the influence of electrical stimulation on skeletal muscle cells.The authors gratefully thank Elvira Fortunato, Daliana Muller and Guilherme Mariz de Oliveira Barra for performing the conductivity measurements of the samples produced using four probe methods equipment. The Researcher F.V.B. was supported by the grant of National Council for Scientific and Technological Development CNPq) of Brazil through the program Science without borders. P.S. was financially supported by Khon Kaen University, Thailand and L.P.d.S. was financially supported by Fundacao para a Ciencia e Tecnologia (FCT, SFRH/BD/565 78025/2011). The FCT fellowship distinction attributed to V.M.C. under the Investigator FCT program (IF/01214/2014) is also greatly acknowledged.info:eu-repo/semantics/publishedVersio

Electroactive gellan gum/polyaniline spongy-like hydrogels

he ability of electroactive materials to influence and modulate cell behavior has been revealing great potential, especially in the field of skeletal muscle tissue engineering. Herein, we propose PANi-GG electroactive spongy-like hydrogels as potential materials to modulate myoblast bioresponse. polyaniline (PANi) adds electroconductiviy to gellan gum (GG) spongy-like hydrogels that hold a high resemblance to the extracellular matrix (ECM), that is, water content, mechanical properties, and microarchitecture, and that can be further tuned to meet muscle tissue properties. For this purpose, PANi-GG spongy-like hydrogels were obtained by ionically cross-linking with calcium chloride (CaCl2) and further in situ aniline polymerization through oxidation with ammonium persulfate (APS) in a molar ratio of 1:1.05. The physicochemical characterization, surface morphology, electro-conductivity, and mechanical performance were assessed by FTIR, SEM, four-point probe technique, and compression testing, respectively. The viability and proliferation of L929 was not compromised after direct contact of PANi-GG spongy-like hydrogels with L929 cells, as determined by MTS assay and DNA quantification, respectively. C2C12 myoblasts were entrapped within the electroactive materials and cells adhered and spread. Moreover, cells proliferated along the cell culture period showing myosin expression after 7 days of culture. These results highlight that PANi-GG spongy like hydrogels are attractive candidates to be used in skeleton muscle tissue engineering.The authors gratefully thank Prof. Elvira Fortunato for the electrical conductivity measurement and Alessandra Zonari for confocal microscopy. Pathomthat Srisuk was financially supported by Khon Kaen University, Thailand. Fernanda V. Berti was supported by the grant of National Council for Scientific and Technological Development (CNPq) of Brazil through the program Science without borders and Lucília P. da Silva was financially supported by Fundação para a Ciência e Tecnologia (FCT, SFRH/BD/78025/2011). The FCT fellowship distinction attributed to Vitor M. Correlo under the Investigator FCT program (IF/01214/2014) is also greatly acknowledged. Pageinfo:eu-repo/semantics/publishedVersio

Redox activity of melanin from the ink sac of <i>Sepia officinalis</i> by means of colorimetric oxidative assay

<p>The redox properties of natural extract from cuttlefish ink sac (<i>Sepia officinalis</i>) and synthetic melanin used as a biomimetic in melanin structural investigation were determined by comparison of this phenol-based heterogeneous pigment with gallic acid used as a standard in Folin–Ciocalteu colorimetric assay widely employed for characterisation of oxidative properties of biomaterials. Reactivity of sepia melanin reported here is much higher than previously indicated and this protocol should allow the redox characterisation of all melanins irrespective of their origin and composition.</p