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The efficacy of Isotretinoin-loaded solid lipid nanoparticles in comparison to Isotrex® on acne treatment

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Received: Mar. 15, 2013; Accepted: May. 12 , 2013 Vol. 1, No. 1, Autumn 2013, page 38-47 Online ISSN 2322-5904 http://nmj.mums.ac.ir Original Research The efficacy of isotretinoin-loaded solid lipid nanoparticles in comparison to Isotrex® on acne treatment Pouran Layegh1, Navid Mosallaei2, Danial Bagheri2, Mahmoud Reza Jaafari3, Shiva Golmohammadzadeh3* 1 Research Center for Cutaneous Leishmaniasis, Department of Dermatology, Mashhad University of Medical Sciences, Mashhad, Iran 2 School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran 3 Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Abstract Objective(s): Topical retinoids are considered as the first line therapy in the treatment of acne vulgaris, but they are associated with cutaneous irritation. Materials and Methods: In this study, isotretinoin-loaded solid lipid nanoparticles (IT-SLN) were prepared to treat the mild to moderate acne. Also using IT-SLN would minimize IT adverse effects in comparison to commercial product, Isotrex®. This study was conducted to prepare and characterize IT-SLN and assessing the efficiency of IT-SLN comparing to Isotrex® acne. IT-SLN was prepared using hot high pressure homogenization method. IT-SLN contained 0.05% IT in 5% of lipid phase (Glyceryl monostearate- GMS) and tween 80 (2.5 % w/v) was used as surfactant in the aqueous phase. IT-SLN was characterized by particle size analyzing, differential scanning calorimetry and transmission electron microscopy. Encapsulation efficacy was also obtained using spectrophotometry. The efficacy of IT-SLN was evaluated in a randomized, singleblind, parallel-group study and compared with Isotrex®. Forty patients encountered in the study and divided in two groups. Treatment regimen was once-nightly topical administration accompanied with topical administration of clindamycin 2% solution twice a day for 8 weeks. Results: The particle size of IT-SLN was around 60 nm with PDI of 0.4 and zeta potential was about -40 mV. Encapsulation efficacy of IT in SLN in crystalline form was 84±0.21%. IT-SLN produced significantly better treatment than Isotrex® in both non-inflammatory and inflammatory lesions according to its recovery percent after 8 weeks. Also IT-SLN gained better global assessment scores. Conclusion: Our results showed that IT-SLN had higher efficacy than Isotrex® to clear noninflammatory and inflammatory lesions. Keywords: Acne, Clinical trial, Isotretinoin, Solid lipid nanoparticles (SLN) *Corresponding author: Shiva Golmohammadzadeh, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Tel: +98511-8823255, Email: GolmohamadzadehSh@mums.ac.ir 38 Nanomed J, Vol. 1, No. 1, Autumn 2013 Effect of Isotretinoin-loaded SLN on acne Introduction Isotretinoin, as a derivative of retinoic acid (13-cis-retinoic acid), is included in the first generation of retinoids (1). It has been commonly used for the treatment of severe acne and the other dermatological diseases like neoplastic diseases (2). In recent years, dermatologists have used Isotretinoin to treat patients with less severe forms of acne which have the risk of scarring or excessive psychological distress. This regimen of therapy was preferred when; acne has demonstrated resistance to other conventional systemic treatments such as oral antibiotics (3). IT is also commonly used for treatment of some acne variants, such as acne conglobata, acne fulminans (in combination with corticosteroids) and acne inversa, as well as for acne with gram-negative folliculitis (4). IT adverse effects which may occur after oral administrations consist of mucosal dryness, visual disturbance, and skeletal hyperostosis with musculoskeletal symptoms. Elevation of serum triglycerides, blood glucose and hepatic enzymes are reported as well and should be closely monitored. One of the most important contraindications for IT is pregnancy (X) (5, 6, 7). Topical retinoids are highly effective in the treatment of both comedonal and inflammatory lesions of acne and are a vital part of almost any acne regimen. Topical IT should be primarily prescribed in the case of a relapse as a conventional treatment as well (1, 7). Hence a topical formulation of IT seems to be worthy. Topical preparations of IT gel and cream can be found on the market. Unfortunately, they showed systemic absorption, and skin irritation. Therefore, it is valuable to improve the skin uptake and acne treatment efficacy, while reducing irritation and systemic adverse effects of IT (6, 8, 9, 10). There are 4 major pathogenic steps in the production of acne: (1) increased sebum production; (2) abnormal keratinization of the follicular infundibulum; (3) Propionibacterium acnes-mediated responses; and (4) inflamemation. Topical retinoids show their efficacy in the treatment of acne through these pathways. Of course retinoids have no direct effect on decreasing sebum production but the other three factors are closely linked and modulation of one will often have a direct or indirect effect on another (1, 11). Adverse effects that patients usually complain about after topical administration of IT are erythema, dryness, peeling and skin photosensitivity. These adverse effects cause patient incompliance and the medication may be refused (1). In addition, IT is a photosensitive molecule which degrades while it is exposed to UV light. Thus, it was necessary to improve photostability, skin uptake and reduce systemic absorption of IT using a carrier which we showed in our previous study (12). Nanoparticulate carriers which are developed not only enhance percutaneous absorption but also target the active pharmaceutical ingredient to the skin or its substructure. Thus these systems like liposomes, nanoemulsions, niosomes and solid lipid nanoparticles (SLN) are potential for an improved benefit/risk ratio in topical drug therapy (10). SLN has been introduced as a novel drug delivery system for pharmaceutical drugs in various application routes of intravenous, intramuscular, oral and topical (13). SLN production by the aim of topical usage should be made up of lipids that remain in solid state at skin (32 °C) and room temperature. Its particle surface is covered by a surfactant which stabilizes the dispersion (10). As a skin drug delivery system, they are promising carriers for cosmetic active ingredients due to their numerous advantages over existing conventional formulations. SLN merits can be introduced as: (1) Protecting labile compounds against chemical degradation (14). (2) Producing controlled release of the active ingredients. This property depends on the way that active ingredient encapsulation. SLN with a drug-enriched shell shows burst release characteristics whereas SLN with a drugenriched core leads to sustained release (15, 16). (3) SLN has occlusive properties, i.e. they can be used in order to increase the water Nanomed J, Vol. 1, No. 1, Autumn 2013 39 Layegh P, et al. content of the skin (17, 18). (4) SLN shows a UV-blocking potential and they can act as physical sunscreens on their own by scattering sunlight (19, 20). (5) They have no biotoxicity of excipients. (6) They are produced in avoidance of using organic solvent and (7) ease of large scale production (21). Therefore, SLN has proven its ability as an effective skin drug delivery system and is used for many drugs like topical glucocorticoide, tretinoin, antimycotics (22, 23, 24, 25). In the current study we prepared and characterized SLN containing IT and evaluated its efficacy in patients suffering from mild to moderate acne. It is considered that in patients with mild-to-moderate mixed inflammatory and comedonal acne, a combination of a topical antimicrobial, either benzoyl peroxide or antibiotic, is optimal. Since it is well established that combination therapy works faster and more effective than monotherapy, we prepared and added topical solution of clindamycin 2% to the regimen (11). We compared the efficacy of our new nanoparticulate formulation with Isotrex® while they were used beside clindamycin 2% topical solution. in ethanol and poured on GMS. Ethanol was removed using rotary evaporator under vacuum condition. Then the isothermal aqueous phase containing Tween 80 (250 mg; 2.5 % w/v) was added to the melted lipid and homogenized using T 25 Ultra Turrax (Janke und Kunkel GmbH and Co KG Staufen, Germany) for 1 min at 20000 rpm and the preemulsion was produced. This pre-emulsion was processed at 1000 bar, at 80 °C, for 5 cycles using a high pressure homogenizer (EmulsiFlex-C5®, Avestin Inc., Canada) and IT-SLN suspension was obtained. Homogenized samples were then cooled to 4 °C and stored for 24 h before performing further characterizations (8, 12, 14). Particle size and zeta potential Dynamic light scattering (ZetaSizer Nano-ZS; Malvern Instruments Ltd., United Kingdom) was used to evaluate the mean particle size (Z-average (nm)), polydispersity index (PDI) and zeta potential (mV) of SLN dispersions. Samples were prepared by adding 990 µl deionized water to 10 µl SLN suspension. The mean diameters and zeta potential values during three months were calculated from the measurements performed at least in triplicates (8, 12). Materials and Methods Materials Isotretinoin (IT, Across, America), glyceryl monostearate (GMS, Gattefossé, France) and Tween 80 (Sigma, Germany) were used as received. Isotrex® 0.05% was manufactured in Stiefel, Ireland. Clindamycin phosphate powder was purchased from Sepidaj, Iran. Ethanol (Noor-e-Zakaria Razi, Iran) and double distilled water were used in experiments. Isopropyl alcohol, HCl and propylene glycol were obtained from Iranian companies. Transmission electron microscopy (TEM) TEM (CEM 902A; Zeiss, Germany) was performed to characterize the morphology of IT-SLN. IT-SLN suspension was diluted 50 times with double distilled water and placed on a carbon-coated copper grid and after 30 seconds, the excess water was wiped off with paper filter. Twenty μl of uranyl acetate 2 % in water was spread on SLN and after 30 seconds wiped off with paper filter. The grid was dried at room temperature and then observed through TEM (8, 12). Preparation of IT-SLN The best formulation of IT-SLN was investtigated in our previous study. Briefly, lipid phase contained GMS (500 mg; 5% w/v) melted at 80 °C in a pear-shaped vessel. IT (5 mg; 0.05% w/v of the formu-lation) dissolved Differential scanning calorimetry (DSC) DSC was performed to investigate the melting and recrystallization behavior of crystalline materials after solidification. DSC scans of IT, empty and IT-SLN was carried out in a Mettler DSC 821e (Mettler Toledo, 40 Nanomed J, Vol. 1, No. 1, Autumn 2013 Effect of Isotretinoin-loaded SLN on acne Germany). Approximately, 5 mg of samples were filled in aluminum oxide pans, sealed and analyzed. An empty aluminum pan served as reference. DSC was done at 25-250 °C temperature range by rate of 5°C/min under N2 flow and the melting point of SLN dispersions was compared to the bulk lipid (14, 26). Entrapment efficiency (EE) Calibration curve was resulted using UVspectroscopy technique. IT solutions in acidic isopropyl alcohol (HCl 10-5 N in isopropyl alcohol) were prepared at concentrations of 0.25, 0.5, 1, 2, 3, 4 and 5 mg/ml and its absorbance was detected at 349 nm wavelength. Calibration curve was drawn in triplicate, inter- and intra-daily during three days. Acidic isopropyl alcohol was served as blank. Encapsulation efficiency percent was determined indirectly by measuring the concentration of unentrapped IT (12, 27). A known dilution of the SLN dispersion was prepared and 500 μl was transferred to the upper chamber of centrifuge tubes fitted with an ultrafilter (Amicon Ultra-15, PLHK Ultracel-PL Membrane, 100 kDa, Millipore). Amicon tubes were centrifuged at 10000 rpm for 30 min. The filtrate was analyzed for unencapsulated IT at 349 nm using a validated UV-spectrophotometric method after suitable dilution and the entrapment efficacy was measured (27, 28). Preparation of clindamycin solution 2% To prepare 100 ml Clindamycin solution, its phosphate salt was dissolved in 27 ml double distilled water and added to 10 ml propylene glycol and mixed gently. This was then reached to the volume of 100 ml by ethanol 96 °. Evaluation of IT-SLN and Isotrex® efficacy on acne treatment in human The efficacy of IT-SLN (0.05%) was evaluated in a randomized, single-blind, parallelgroup study. The design included a screening and a baseline evaluation followed by randomization to the treatment with either IT- SLN, or commercial IT gel (Isotrex® 0.05%) at a once-nightly topical adminis-tration for 8 weeks. The regimen was accompanied by topical administration of clindamycin 2% solution twice a day. At weeks 0 (baseline), 4 and 8, numbers of noninflammatory (open and closed comedones) and inflammatory (papules and pustules) lesions were counted for each patient. Clinically significant improvement was defined as a between-group difference of 15% reduction in the percentage of total lesion count (sum of non-inflammatory and inflammatory lesions) from baseline to the end of the study. At the baseline, photographs were taken. Pictures were used to compare patients’ assessments of global response. Each patient’s global response to the treatment was assessed by a physician by counting the lesions at the first, fourth and eighth week of the treatment using a 7-point scale. The scale was as follows: 0 = completely cleared; 1= almost cleared ( ~90% improvement- very significant clearance in disease, with only traces of disease remaining); 2 = marked response (~75% improvement- significant improvement, with some disease remaining); 3 = moderate response (~50% improvement- intermediate between slight and marked response); 4 = slight response (~25% improvementsome improvement but significant disease remains); 5 = condition unchanged; and 6 = condition worsened. Treatment success was defined as a global assessment score of at least 3 points ( 50% global improvement) at the end of study (29). Before enrollment, written informed consent was obtained from each patient or from the patient’s parent or guardian if the patient was under the legal age of consent. The ethical committee code was 511/0363 (30). A dermatologist evaluated the result of treatment regimens and any side effects in weekly visits. Study population Patients for this study were recruited from dermatology department of Qaem Hospital, Mashhad University of Medical Sciences (MUMS). A total of 40 patients were scre- Nanomed J, Vol. 1, No. 1, Autumn 2013 41 Layegh P, et al. ened, enrolled treatment. and randomized to the Inclusion and exclusion criteria Male and female patients aged 13-30 years with mild to moderate facial acne vulgaris who were candidate of treatment with topical agents were enrolled. The following washout periods were required: 2 weeks for topical acne medications, 4 weeks for oral antibiotics, 12 weeks for hormone therapy (unless treatment had been used for >12 consecutive weeks immediately before study entry and was expected to continue throughout the study), and 6 months for oral retinoids. Patients were excluded if they had participated in any other study in the previous 30 days; had uncontrolled systemic disease; had any other skin condition that might interfere with assessment of the study medication like eczema; or were expected to undergo surgery, hospitalization, or excessive or prolonged exposure to the ultraviolet light (eg, sunlight, tanning bed) during the study. Volunteers were suggested to use the same oil-free sunscreen. Female patients who were pregnant, breastfeeding or planning to become pregnant were excluded (29). Recovery percent (treatment efficacy) was calculated using the following formula: Statistical analysis All the experiments of each preparation were repeated three times and data were expressed as Mean ± SD. Analysis of variance (ANOVA) was used to evaluate the statistical significance of differences among groups. Statistical data were analyzed using nonparametric techniques with a TukeyKramer test. Results with P < 0.05 were considered as statistically significant. Results Preparation of IT loaded SLN (IT-SLN) IT was dispersed in the pre-emulsion 42 homogenously, since we had dissolved it in ethanol and it was homogenized using a high speed stirrer (Ultra Turrax). Also we used the advantageous role of a hot water bath, in order to keep the pre-emulsion temperature over the melting point of lipids while it was forming. Hot high pressure homogenization method was performed to prepare our final product. The viscosity was enough to be stable and applicable on the skin without any excess excipient. The product had a milky color and good viscosity to apply. Particle size, polydispersity and zeta potential of IT-SLN Table 1 shows that particle size of IT-SLN was around 60 nm according to the number and PDI was 0.4. The results of particle size analyzing also showed an increase while IT was added to the formulation. This formulation was stable in view of particle size and zeta potential for 3 months, since no statistical significant change was occurred during the storage at 4 °C. Zeta potential was about -40 mV which can produce particle repulsion and would inhibit aggregations. Encapsulation efficacy of IT-SLN The maximum light absorbance of IT was observed at the wavelength of 349 nm using spectrophotometry technique. The resulted calibration curve equation of mentioned concentrations was and (A and C are absorbance and concentration (mg/ml), respectively). Encap-sulation efficacy of IT-SLN was 84±0.21 % (n=3). As a reason of high encapsulation efficacy, un-encapsulated IT was not washed out in the following clinical experiments. Thermal analysis of IT-SLN Figure 1 investigates crystalline behavior of IT-SLN through DSC. It can be seen that GMS formed foram contidos fisicamente em decúbito lateral direito, e as agulhas posicionadas sobre os acupontos (ACs) coração 7 (C-7 – Shenmen), localizado entre o processo estiloide lateral e o osso cárpico acessório, exatamente na dobra da articulação úmero-rádio-ulnar, e pericárdio 6 (PC-6 – Neiguan), localizado entre os tendões do músculo flexor carporradial e do músculo flexor digital superficial (Fig. 1A, 1B e 1C), conforme descrito por Draehmpaehl e Zohmann (1997). Já para a segunda seção, os animais foram novamente posicionados em decúbito lateral direito, e as agulhas inseridas em acupontos falsos (AF), localizados no oitavo e no nono espaço intercostal esquerdo (Figura 1D), onde não há interferência de nenhum outro meridiano, conforme descrito por (Draehmpaehl e Zohmann, 1994). Independentemente da seção, não foi realizada estimulação manual das agulhas, e os acupontos permaneceram estimulados por um período de 30 minutos, respeitando-se o período mínimo de 10 a 20 minutos sugerido por Scott (2001) para que o acuponto possa promover um efeito observável. Figura 1. Localização dos acupontos C-7 (seta em A) e PC-6 (seta em B) no cão (Adaptado de Draehmpaehl e Zohmann, 1997). Em C, acupuntura em cão do experimento acuponto C-7 (seta azul) e acuponto PC-6 (seta laranja). Em D, acupuntura em cão do experimento em acuponto falso. Arquivo pessoal, 2014.  Previamente à aplicação das agulhas nos ACs, foi realizado um registro eletrocardiográfico (ECG) com dois minutos de duração e, após a inserção das agulhas nos ACs, o registro eletrocardiográfico se manteve durante os 30 minutos de acupuntura. Para a realização do eletrocardiograma, foi utilizado um aparelho de eletrocardiografia computadorizada (ECGPC – TEB®), respeitando-se o padrão de fixação de eletrodos descrito por Tilley (1992), com registro das derivações bipolares e unipolares aumentadas e calibração de 25mm/s e 1mV (N). Os dados do ECG foram interpretados de forma comparativa pré e pós-estimulação dos acupontos, sendo os dados pós-estimulação coletados e interpretados em intervalos de cinco minutos até o final da gravação (30 minutos). Arq. Bras. Med. Vet. Zootec., v.68, n.1, p.252-256, 2016 253 Silva et al.  Para a análise da VFC, foi utilizada a fórmula disponível em Carareto et al. (2007). A normalidade dos dados obtidos foi verificada pelo teste de Shapiro-Wilk (P>0,05), seguida pelo teste ANOVA com pós-teste de Bonferroni para as variáveis paramétricas e pelo teste de Kruskal-Wallis com pós-teste de Dunn (P<0,05) para as variáveis não paramétricas. Foi realizada análise descritiva para as variáveis qualitativas. No presente estudo, 90% dos animais apresentaram arritmia sinusal respiratória, demonstrando maior influência do sistema nervoso simpático, em todos os momentos do grupo ACs, enquanto no grupo AF esse ritmo cardíaco esteve presente em 100% dos animais, em todos os momentos. O ritmo sinusal foi encontrado apenas em 10% dos animais do grupo ACs, sugestivamente por se tratar da primeira exposição dos animais ao experimento, resultando em maior influência parassimpática sobre o sistema cardiovascular. Conforme demonstrado na Tab. 1, houve pequena e irrelevante diferença quando comparada a FC de todos os momentos do grupo ACs com os momentos do grupo AF, o que evidencia, portanto, a ineficácia da estimulação dos acupontos sobre esse parâmetro. Tabela 1. Frequência cardíaca (FC) e variabilidade da frequência cardíaca (VFC) obtidas com a realização dos acupontos verdadeiros (AV) C-7 e PC-6 e do acuponto falso (AF) em diferentes momentos do ECG, expressos em mediana (percentil 25% - percentil 75%) para a FC e média±desvio-padrão para a VFC Momentos FC (bpm) VFC AV AF AV AF 0 minuto 107 (92-130)a 112 (93-129)a 12,79±0,46ab 12,71±0,47b 5 minutos 90 (79-121)a 93 (82-115)a 13,08± 0,48ab 13,09± 0,49ab 10 minutos 94 (79-118)a 88 (70-123)a 13,01± 0,48ab 13,12± 0,53ab 15 minutos 96 (76-120)a 99 (81-115)a 13,04± 0,52ab 13,17± 0,51a 20 minutos 95 (79-120)a 95 (81-112)a 13,13± 0,51ab 13,07± 0,54ab 25 minutos 104 (83-115)a 89 (76-108)a 13,15± 0,45ab 13,17± 0,53a 30 minutos 87 (82-107)a 95 (81-117)a 13,18± 0,46ab 13,11± 0,56ab P = 0,0890 P = 0,0051 Para a FC, valores seguidos por letras distintas diferem entre si (P<0,05) ao teste de Dunn. Para a VFC, valores seguidos por letras distintas diferem entre si (P<0,05) ao teste de Bonferroni. Não foi observada diferença na VFC (Tab. 1) entre os grupos do ACs e AF, exceto quando comparado apenas o momento basal com os momentos 15 e 25 minutos do AF, contudo sem relevância clínica para o estudo. Sabidamente, VFC constitui uma medida simples e não invasiva que avalia os efeitos do sistema nervoso autônomo sobre o coração. Variações nessa variabilidade são normais e esperadas, indicando habilidade do coração em responder a múltiplos estímulos fisiológicos e ambientais. Em síntese, uma alta VFC representa boa adaptação, caracterizando um indivíduo saudável, com mecanismos autonômicos eficientes, enquanto a baixa VFC representa adaptação anormal e insuficiente do SNA (Carareto et al., 2007). Dentre as opções terapêuticas para o tratamento de anormalidades dos mecanismos autonômicos, a acupuntura vem se destacando em vários estudos em animais, entretanto em nenhum desses estudos se objetivou a avaliação do efeito da acupuntura em indivíduos saudáveis, conforme ocorreu no presente estudo, fator limitante à discussão mais aprofundada. Observam-se, na literatura, diversos estudos em que foram observados os efeitos da acupuntura sobre pacientes com diferentes distúrbios cardiovasculares. Syuu et al. (2001) relataram efeitos benéficos da estimulação do acuponto PC-6 em cães anestesiados e sob toracotomia. Nesse estudo, os autores descrevem reduções na pressão arterial média, volume diastólico final, frequência cardíaca, débito cardíaco e pressão sistólica final mediante estimulação do acuponto. O efeito cardiovascular da acupuntura sobre o acuponto PC-6 em cães também foi observado por Jingbi (2004) em modelo de experimental de infarto agudo do miocárdio. Nesse estudo, foi observado que a acupuntura proporcionou redução do segmento ST do eletrocardiograma, além de reduzir as áreas de infarto induzidas pela ligadura coronariana. Entretanto, esses dois 254 Arq. Bras. Med. Vet. Zootec., v.68, n.1, p.252-256, 2016 Efeitos da acupuntura estudos diferem do presente pelo fato de as agulhas terem sido estimuladas eletricamente (eletroacupuntura), enquanto no presente estudo estas permaneceram em repouso. Cárdenas (2006) observou que a estimulação dos mesmos acupontos empregados no presente estudo em cavalos sob anestesia geral demonstrou efeitos positivos sobre a taquicardia ventricular induzida por infusão contínua de dopamina, nos quais a estimulação dos ACs C-7 e PC-6 promoveu diminuição do tempo da taquicardia ventricular, demonstrando, assim, a eficácia da estimulação desses acupontos em animais. Um estudo controlado em ratos demonstrou que a estimulação dos acupontos PC-5 e PC-6 reduziu significativamente a incidência de taquiarritmias induzidas experimentalmente pós-oclusão e reperfusão da artéria coronária esquerda (Lujan et al., 2007). Em humanos, a revisão de oito estudos utilizando acupuntura para o tratamento de arritmias cardíacas encontrou cerca de 87 a 100% que foram convertidos ao ritmo sinusal após a estimulação dos acupontos (Xie e Wedemeyer, 2012). estudo não se valeu da estimulação elétrica sobre os acupontos falsos ou verdadeiros (C-7 Shenmen e PC-6 Neiguan). Limitações a este estudo compreendem o fato de que a escolha dos acupontos explorados baseouse nas indicações terapêuticas descritas por Draehmpaehl e Zohmann (1994), em que o acuponto C-7 é indicado em casos de alterações funcionais do coração, e o acuponto PC-6 quando há dores miocárdicas. Ainda segundo os mesmos autores, essa forma de terapia alternativa objetiva normalizar funções orgânicas alteradas e, como no presente estudo foram utilizados animais hígidos, a acupuntura pode não ter promovido influência sobre a VFC nos cães que participaram do experimento. Limita-se, ainda, este estudo quando da observação das recomendações descritas por Scott (2001), quando ele destaca a necessidade de um ou dois tratamentos semanais durante quatro a seis semanas consecutivas, tempo em que se espera que possa haver a resposta desejada à terapia, fato não realizado no presente estudo. Finalmente, Kimura e Hara (2008) desenvolveram um estudo em que foram estudados os efeitos da eletroacupuntura sobre o ritmo do sistema nervoso autônomo em cães. Dentre outras análises, observaram o coeficiente de variação dos intervalos R-R (CVRR) do eletrocardiograma, o qual avalia o índice de atividade nervosa autônoma, de forma semelhante à VFC, a qual também se utiliza do intervalo R-R como unidade de medida. Seus até antes de 12 horas, 24 horas até antes de 72 horas aproximadamente de incubação, os valores foram superestimados. Subestimativas dos valores foram obtidas entre aproximadamente 12 horas às 24 horas e após 72 horas. Em nenhum momento os dados estimados foram iguais aos observados. Nas Fig. 2 e 3, apresentam-se a produção cumulativa de gases obtidos a partir dos dados observados e dos ajustados pelos dois modelos. Para estimativa da produção de gases, tanto o modelo logístico quanto o modelo de Gompertz podem ser utilizados, porém, de acordo com os parâmetros encontrados, o modelo logístico apresentou melhor valor de QME, critério que recomenda o seu uso. Com base nos gráficos de dispersões, pode-se verificar que tanto o modelo logístico como o Arq. Bras. Med. Vet. Zootec., v.63, n.1, p.136-142, 2011 139 Farias et al. Tempo de incubação (h) Figura 2. Curvas de produção cumulativa de gases da matéria seca do farelo e da torta de babaçu (Orbignya martiana) a partir da média dos dados observados e dos ajustados pelo modelo de Gompertz. Tempo de incubação (h) Figura 3. Curvas de produção cumulativa de gases da matéria seca do farelo e da torta de babaçu (Orbignya martiana) a partir da média dos dados observados e dos ajustados pelo modelo logístico bicompartimental. Barbosa (2007) obteve melhor ajuste para o modelo logístico em relação ao modelo de Gompertz, ao estudar a faveira e o pau-ferro. Azevedo (2007) encontrou, nos modelos logístico, exponencial e de France, ajuste adequado a todas as etapas do processo fermentativo, descrevendo as características de fermentação do material avaliado, tanto na fase inicial como na final. Na análise gráfica dos resíduos, o modelo logístico subestimou apenas no final da curva e superestimou no início. As curvas de produção de gases caracterizam-se por apresentar forma sigmoidal, podendo ser distinguidas três fases: inicial de baixa produção, fase exponencial de rápida produção e uma fase assintótica de lenta ou inexistente produção de gases (Noguera et al., 2004). Nas primeiras horas de fermentação, uma parte do substrato, geralmente os açúcares solúveis, é fermentada rapidamente, mas isto representa uma pequena porção do total a ser degradado. Na sequência, uma menor quantidade de alimento é hidratada e colonizada pela microbiota, dando origem a degradações distintas, dependendo do substrato, quanto aos constituintes solúveis e estruturais (Beuvink e Kogut, 1993). Giraldo et al. (2006), em estudo sobre a relação entre pressão e volume para a implantação da técnica in vitro de produção de gases, na Colômbia, obtiveram taxas de fermentação mais altas nas primeiras horas de incubação e atribuíram o fato à fermentação dos carboidratos solúveis, e também observaram grande diferença entre os alimentos estudados. Verificaram, ainda, que, entre seis e 12 horas de incubação, houve aumento na taxa de produção de gases, que foi atribuído à fermentação de carboidratos fibrosos ou estruturais. O QME, o R2 e o DMA dos modelos estudados para descrever a produção de gases oriundos da fermentação ruminal da FDN encontram-se na Tab. 3. Quanto ao QME, o modelo logístico bicompartimental foi o que apresentou menor valor. Os valores de R2 foram elevados (0,99) para os dois modelos. Os desvios médios absolutos diferiram entre os modelos, com o de Gompertz apresentando valor mais elevado. Azevedo (2007) obteve valores de R2 baixos para todos os modelos, sendo o menor valor para o de Gompertz. Schofield et al. (1994), ao compararem alguns modelos para descrever a cinética da digestão da fibra, obtiveram valores elevados de R2 para o logístico bicompartimental e o de Gompertz, com menor valor para o primeiro. 140 Arq. Bras. Med. Vet. Zootec., v.63, n.1, p.136-142, 2011 Avaliação dos modelos logístico. Tabela 3. Médias do quadrado médio do erro (QME), do coeficiente de determinação (R²) e do desvio médio absoluto (DMA) obtidas a partir dos ajustes dos dados de produção de gases da parede celular com os modelos logístico e de Gompertz Modelo QME R² DMA Logístico 6,39 0,99 1,98 de Gompertz 11,1 0,99 3,21 Nas Fig. 4 e 5, apresentam-se as produções cumulativas de gases obtidos a partir dos dados observados e dos ajustados pelos modelos de Gompertz e logístico bicompartimental para a FDN do farelo e da torta de babaçu. Figura 4. Modelo de Gompertz para o farelo e a torta de babaçu. Azevedo (2007) obteve melhor ajuste para o modelo logístico bicompartimental e exponencial do que para os de France e de Gompertz, no entanto utilizou o modelo de Gompertz, que, apesar de ter apresentado maior dispersão do resíduo, assim como valores mais elevados para QME e DMA, apresentou melhor estimativa para a curva de produção de gases. CONCLUSÕES O modelo logístico bicompartimental estimou os valores de produção de gases melhor do que o modelo de Gompertz. Desse modo, pode ser adotado na estimativa da cinética de fermentação ruminal da matéria seca e fibra em detergente neutro do farelo e da torta de babaçu. AGRADECIMENTOS Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pela bolsa de estudo concedida e pelo apoio financeiro necessário à condução da pesquisa. Figura 5. Modelo logístico para o farelo e a torta de babaçu. REFERÊNCIAS BIBLIOGRÁFICAS AZEVEDO, M.M.R. Parâmetros cinéticos da fermentação ruminal do pseudofruto de cinco clones de cajueiro pela técnica in vitro semiautomática de produção de gases. 2007. 44f. Dissertação (Mestrado) – Universidade Federal do Piauí, Teresina. BARBOSA, A.L. Valor nutritivo de vagens de faveira (Parkia platycephela Benth.) e pau-ferro (Caesalpinea ferrea Mart. Ex Tul.) através da técnica in vitro semiautomática de produção de gases. 2007. 44f. Dissertação (Mestrado) – Universidade Federal do Piauí, Teresina. BEUVINK, J.M.W.; KOGUT, J. Modeling gas production kinetics of grass silages incubated with buffered ruminal fluid. J. Anim. Sci., v.71, p.1041-1046, 1993. FRANCE, J.; DHANOA, M.S.; THEODOROU, M.K. et al. A model to interpret gas accumulation profiles associated with in vitro degradation of ruminant feeds. J. Theoret. Biol., v.163, p.99-111, 1993. Arq. Bras. Med. Vet. Zootec., v.63, n.1, p.136-142, 2011 141 Farias et al. GIRALDO, L.A.; GUTIÉRREZ, L.A.; SÁNCHEZ, J. et al. Relación entre presión y volumen para el montaje de la técnica in vitro de producción de gas em Colombia. Livestock Research for Rural Development, 2006. Disponível em: . Acessado em: 26 ago 2007. GONÇALVES, A.L.; LANA, R.P.; RODRIGUES, M.T. et al. Cinética de degradação de alguns volumosos usados na alimentação de cabras leiteiras por intermédio da técnica de produção de gases sob diferentes níveis de pH. Rev. Bras. Zootec., v.30, p.19041912, 2001. GROOT, J.C.J.; CONE, J.W.; WILLIAMS, B.A. et al. Multiphasic analysis of gas production kinetics for in vitro fermentation of ruminant feeds. Animal Feed Science and Technology, v.64, p.77-89, 1996. JAYME, D.G.; GONÇALVES, L.C.; MAURÍCIO, R.M. et al. Avaliação pela técnica de produção de gases das silagens de quarto genótipos de girassol (Helianthus annuus) (Rumbosol 91, Victoria 627, Victoria 807 e Mycogen 93338). Arq. Bras. Med. Vet. Zootec., v.61, p.1403-1410, 2009. LAVRENCIC, A.; STEFANON, B.; SUSMEL, P. An evaluation of the Gompertz model in degradability studies of forage chemical components. Anim. Sci., v.64, p.423-431, 1997. MAURICIO, R.M.; OWEN, E.; MOULD, F.L. et al. Comparison of bovine rumen liquor and bovine faeces as inoculum for an in vitro gas production technique for evaluating forages. Anim. Feed Sci. Technol., v.89, p.33-48, 2001. MAURÍCIO, R.M.; PEREIRA, L.G.R.; GONÇALVES, L.C. et al. Potencial da técnica in vitro semiautomática de produção de gases para avaliação de silagens de sorgo (Sorghum bicolor (L.) Moench). Rev. Bras. Zootec., v.32, p.10131020, 2003. MERTENS, D.R.; LOFTEN, J.R. The effect of starch on forage fiber digestion kinetics in vitro. J. Dairy Sci., v.63, p.1437-1446, 1980. NOGUERA, R.R.; SALIBA, E.O.; MAURICIO, R.M. Comparación de modelos matemáticos para estimar los parámetros de degradación obtenidos a través de la técnica de producción de gas. 2004. Disponível em: . Acessado em: 3 abr 2006. ØRSKOV, E.R.; McDONALD, I. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci., v.92, p.499-503, 1979. SARMENTO, J.L.R.; REGAZZI, A.J.; SOUSA, W.H. et al. Estudo da curva de crescimento de ovinos Santa Inês. Rev. Bras. Zootec., v.35, p.435-442, 2006. SCHOFIELD, P.; PITT, R.E.; PELL, A.N. Kinetics of fiber digestion from in vitro gas production. J. Anim. Sci., v.72, p.2980-2991, 1994. 142 Arq. Bras. Med. Vet. Zootec., v.63, n.1, p.136-142, 2011
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