BR102020010354A2 - PROCESS FOR OBTAINING PHYTOTHERAPY INPUT BASED ON A DRY AND STANDARDIZED EXTRACT OF DYSPHANIA AMBROSIOIDES (L.) MOSYAKIN - CLEMANTS (SIN: CHENOPODIUM AMBROSIOIDES L.) WITH APPLICATIONS IN HUMAN AND VETERINARY MEDICINE - Google Patents

Abstract

process of obtaining phytotherapeutic input based on dry and standardized extract of dysphania ambrosioides(l.) mosyakin& clemants(syn: chenopodium ambrosioides l.) with applications in human and veterinary medicine. The present invention consists of obtaining a natural product of plant origin (pharmaceutical raw material in the form of a standardized dry extract) made from the extraction, characterization, standardization and drying by nebulization/atomization (spray drying) of the extract obtained from parts aerials of St. Mary's weed (dysphania ambrosioides (l.) mosyakin & clemants).

Description

PROCESS FOR OBTAINING PHYTOTHERAPY INPUT BASED ON A DRY AND STANDARDIZED EXTRACT OF DYSPHANIA AMBROSIOIDES (L.) MOSYAKIN - CLEMANTS (SIN: CHENOPODIUM AMBROSIOIDES L.) WITH APPLICATIONS IN HUMAN AND VETERINARY MEDICINE Descriptive Report: I. Field of Invention:

[001] The present invention consists in obtaining the natural product of plant origin (pharmaceutical raw material in the form of standardized dry extract) prepared from the extraction, characterization, standardization and drying by nebulization/atomization (spray drying) of the extract obtained from the aerial parts of Santa Maria herb (Dysphania ambrosioides (L.) Mosyakin & Clemants)

II. Description of the State of the Technique:

[002] Among the patents requested for Dysphania ambrosioides at the INPI are BR 10 2013 000137 6 A2 referring to pharmaceutical compositions based on the extract and its use as an anti-inflammatory and healing agent, also PI 1101651-5 A2 which includes processes for obtaining a lyophilized aqueous extract of the aerial parts of the plant, formulating a pharmaceutical composition and using it as a bone graft. In the search for international patent banks, there are several claims related to the properties of D. ambrosioides extract in the treatment of gastritis and ulcer caused by H. pylori (US20090130203), of uterine fibrosis (US20030082250). As well as methods to inhibit, prevent, kill, repel insects and mites using mixtures of the plant under the deposit of (EP2440061). A patent deposit was also found on the method of obtaining ascaridol, from the extraction of the essential oil of D. ambrosioides and purification through a chromatographic method on positive silica gel or reverse silica gel (CN101676289).

[003] After analyzing the filed patents, it was concluded that no innovative document was found on the development of a phytotherapic input from the dry and standardized extract of Dysphania ambrosioides obtained by the nebulization/sprinkling technique.

[004] Considering that no reports were found in the literature of standardized dry extract of this plant species, the present invention has an innovative character and fits in the area of research, development and innovation of processes and products, with applications in human and veterinary medicine.

[005] The utility of this invention is based on marketing, technological and public health issues, since the growing use of phytotherapy as an integrative practice is being observed in several countries around the world (WHO, 2014). Phytotherapy has always been present in the health therapies of the Brazilian population and presents a growth trend with an effective contribution to people's health care, mainly through public health programs. Global drug spending could reach US$1.5 trillion by 2021 (IMS, 2016).

[006] New strategies to promote health and reduce inequalities are therefore necessary. The United Nations (UN) has defined as one of the goals of the “Sustainable Development Goals” until 2030: ensuring access to safe, effective and affordable medicines (UN, 2015). In this context, herbal medicines represent an opportunity.

[007] Consequently, the standardization of herbal medicines, including their manufacturing process, is a prerequisite for quality assurance, as well as for the constancy of their therapeutic effects and patient safety (KLEIN, 2012).

[008] In 2009, in Brazil, the Ministry of Health, from the National Program of Medicinal Plants and Phytotherapics, released the National List of Medicinal Plants of Interest to SUS (RENISUS) which presents 71 medicinal plants with the potential to generate products . The objective of this list was to direct studies and research that support the elaboration of the list of herbal medicines available for use by the population (BRASIL, 2009) and the species Dysphania ambrosioides is part of this list.

[009] The technological development of dry extracts has provided a growing expansion of solid herbal forms. According to Carvalho et al. (2008), in Brazil there are more than 500 registrations of herbal medicines and around 70% of these are marketed in solid pharmaceutical form. The vegetable raw material almost always consists of dry extracts, as these have the advantage of greater chemical, microbiological and physicochemical stability. In addition to facilitating standardization, they allow an increase in the concentration of actives and have the ability to differentiate from other pharmaceutical forms (OLIVEIRA; PETROVICK, 2010).

[010] One of the processes used for drying extracts is spray drying. It is one of the most versatile techniques in the transformation of powdered products, allowing the drying of compounds in a flow from millimeters per hour to tens of tons per hour. Initially used in the drying of eggs, in the middle of the 18th century, its use was only widespread around 1920 in the milk and soap drying industry. It is currently present in the most diverse industrial segments, such as the chemical, pharmaceutical and food industries (MASTERS, 1985; CAL; SOLLOHUB, 2010).

[011] Drying in spray dryer allows an adequate choice of cycle according to the final product to be developed, with the control of particle size and uniformity. The process can run continuously without interruption if the cycle needs to be changed, in addition to being fast and with satisfactory performance. Another positive point is the low aggressiveness to the manipulated product, although the extract is exposed to heat, the contact time in the drying chamber is fast. Finally, spray dryer equipment requires considerable initial investment, which is offset by low maintenance and production costs (WALTON, 2002; MEZHERICHER; LEVY; BORDE, 2010; GONG et al., 2014).

[012] By using this technique, the results can be optimized, as the spray dryer allows the adjustment of some process parameters, such as drying air temperature (°C), extract feed flow rate at the sprinkler nozzle (mL /min) and proportion of drying adjuvant on yield (%, m/m), moisture (%), water activity and contents (%, m/m) of active vegetable raw material in the dry extracts ( WALTON, 2002).

[013] Spray drying has been used quite successfully in plant extracts. The use of additives in the drying of extracts from plants is responsible for its stability and quality, even affecting its bioavailability and flowability. In addition to increasing the yield, as plant extracts have molecules such as sugars and organic acids, which can remain adhered in the drying chamber and the adjuvants reduce this adhesion. Among the most used additives are colloidal silicon dioxide, maltodextrin, gelatin, gum arabic, cellulose derivatives, proteins, among others (CAL; SOLLOHUB, 2010; OLIVEIRA; PETROVICK, 2010).

[014] In order for the best physicochemical properties to be achieved in the dry extract, some care must be taken in spray drying. In the production of the dry extract, the fluid is taken to high temperatures, although the contact is fast, it is necessary to evaluate the impact of this parameter on the quality of the dry extract (OLIVEIRA; PETROVICK, 2010).

[015] The standardized dry extract of this invention constitutes an innovation and an important pharmaceutical and technological alternative in the manufacture of herbal medicines based on Dysphania ambrsioides, in classical pharmaceutical forms (solid, semi-solid, liquid, aerosols, etc.) (microcapsules, nanostructured systems, phytosomes, matrix systems, pellets, beads, etc.), with a high standard of quality, efficiency and therapeutic safety.

[016] The species Dysphania ambrosioides L. known in Brazil as yerba-de-santamaria, mastruz, belonging to the Amaranthaceae family, has great acceptance in traditional and popular medicine in several countries and in some of these it is consumed as a condiment. The plant has antibacterial, antifungal, anti-inflammatory, antioxidant, antiparasitic, antitumor properties (JARDIM et al., 2010; BARROS et al., 2013; MONZOTE et al., 2014a,b; TRIVELLATO-GRASSI et al., 2013; JESUS et al., 2014a,b; TRIVELLATO-GRASSI et al., 2013; JESUS et al., 2014a,b; al., 2018). It is a native species of Tropical America, with probable origin in Mexico. It is widely distributed in tropical, subtropical and temperate regions (KOKANOVA-NEDIALKOVA; NEDIALKOV; NIKOLOV, 2009). D. ambrosioides is a herbaceous plant, annual or perennial, very branched, with an unpleasant odor. It has an annual life cycle of easy cultivation and its propagation happens through seeds (LORENZI, 1982).

[017] From Dysphania ambrosioides the whole plant is used. Although the species has several pharmacological activities, one of its active principles present in the essential oil in high concentrations, ascaridol, is also responsible for its toxicity (GADANO et al., 2002). Thus, studies with liquid extracts of D. ambrosioides demonstrate that flavonoids are the main components of the plant and relate their presence to plant activities, making them possible markers (BARROS et al., 2013; JESUS et al., 2018) . Flavonoids have antioxidant, anti-inflammatory, antitumor and antimicrobial properties (LAY et al., 2014; PARHIZ et al., 2015; YAKOUB et al., 2018). Barros et al. (2013) found rutin, quercetin and kaempferol derivatives in greater amounts in the methanolic extract of aerial parts of D. ambrosioides. Jesus et al. (2018) identified the presence of the flavonoids rutin, quercetin and chrysin in the ethanol extract of leaves of the species and in the chloroform, ethyl acetate and n-butanol fractions.

[018] Antioxidant and anti-inflammatory properties were recorded, both in the ethanol extract and in the aqueous extract of aerial parts of D. ambrosioides. Barros et al. (2013) and Song et al. (2015) verified antioxidant properties for the aqueous extract obtained from the infusion of aerial parts of D. ambrosioides. For the methanolic extract, antitumor properties were observed against colon, cervical and hepatocellular carcinoma cell lines. There is still research demonstrating antibacterial activities for Helicobacter pylori, both in vivo and in vitro (LIU et al., 2013; YE et al., 2015). Jesus et al. (2018) were able to relate the presence of rutin, quercetin and chrysin in the extracts of leaves of the species to antimicrobial activity, with promising results for the chloroform fraction in the fight against Mycobacterium tuberculosis. The study by Calado et al. (2015) found that the ethanolic extract of D. ambrosioides leaves is able to reduce pain and synovial inflammation in an experimental model with animal guinea pigs. Some studies have demonstrated hypotensive and anti-diabetic activities (EDDOUKS et al., 2002; TAHRAOUI et al., 2007; ASSAIDI et al., 2014). In addition, the research by Jardim et al. (2010) evaluated the antifungal activity of hexane extract obtained from leaves of D. ambrosioides. The hexane fraction purified at a concentration of 0.1% inhibited the growth of the fungi Aspergillus flavus, Aspergillus glaucus, Aspergillus ochraceous, Fusarium semitectum, Fusarium oxysporum, Colletotrichum gloeosporioides, Aspergillus niger, Colletotrichum musae. The work by Shah (2014) found promising antifungal activity for the ethyl acetate fraction obtained from extracts of roots of D. ambrosiodes, which reached an inhibitory concentration of 40 and 35% for the fungi A. niger and Rhizopus tolenapur, respectively. . Due to the toxicity of compounds present in the essential oil, its therapeutic use is discouraged. However, with the increase in zoonoses, the essential oil of D. ambrosioides has gained a new focus due to its repellent and insecticidal action (PAVELA et al., 2017). There are also promising results for the treatment with essential oil of aerial parts of D. ambrosioides for cutaneous leishmaniasis (MONZOTE et al., 2014a). With respective proof of activity for amastigote and promastigote forms. In addition to demonstrating effect for Plasmodium falciparum and Trypanosoma brucei (MONZOTE et al., 2014b).

[019] Given the medicinal potential of the plant D. ambrosioides and the absence of high quality herbal products of this plant species, certified by searches of patent databases, such as the National Institute of Industrial Property (INPI), World Intellectual Property Organization (WIPO) and in the Google Patents search base, the need for technological development of the process of extracting the aerial parts of this species is identified, as well as the development of an efficient drying process to obtain a standardized dry extract and the evaluation of its toxicity. The aim is to contribute to the development of technologically elaborated inputs with high added value, enabling the development, for example, of pharmaceutical formulas with greater stability, uniformity of dosage and therapeutic adherence.

III. Summary of the invention:

[020] Different techniques can be used to obtain standardized extracts. The nebulization/atomization drying technique makes it possible to obtain a solid particulate form from a dispersed system (solution, suspension or emulsion) sprayed, in the form of small droplets, in a drying chamber at varying temperatures, where the liquid is quickly eliminated by evaporation (mass transfer) (COUTO et al., 2011). This type of technique favors the rapid evaporation of the solvent contained in the extract, which reduces the processing time, and thus, changes in heat-sensitive products (OLIVEIRA and PETROVICK, 2010), with the obtaining of pulverized solid products with well defined characteristics. defined, such as particle size and shape. The low costs of spray drying production are also evident, mainly because it is a process by which dry powders are obtained from a liquid dispersion, through a single operation that presents high yield (AULTON, 2005).

[021] In this sense, the present inventors have been researching ways to advance in studies related to the pharmacological and therapeutic aspects of this medicinal plant, as well as producing standardized dry extracts with high quality and stability, with levels of known active principles, in order to insert an innovative pharmaceutical product on the market in the near future.

[022] In this way, the standardized dry extract of Dysphania ambrosioides, obtained by nebulization/atomization (spray drying) represents a perspective of technological advancement for the future development of herbal medicines for use in public and private health systems, due to its analgesic properties and anti-inflammatory.

[023] The dry extract standardized in flavonoids, expressed as rutin equivalents, from the aerial parts of D. ambrosioides, was obtained considering the traceability of the entire production process, feasibility of processes and the final product. Briefly, the aerial parts of D. ambrosioides were collected, dried and pulverized. The pulverized material was characterized by pharmacopoeial methods. Part of the material was macerated, percolated in 70% (w/w) ethanol, in a drug/solvent ratio of 10% (w/w). The obtained extract was concentrated, characterized and added with colloidal silicon dioxide for spray drying. A spectrophotometric analytical method was adapted and validated for the quantification of total flavonoids, expressed as rutin, in the dry extract samples. The dry extract standardized in flavonoids and characterized was submitted to toxicological tests in vitro in Artemia salina at concentrations from 62.5 to 1000 µg/mL and also in vivo, of acute oral toxicity in Wistar rats, approved by the ethics committee on the use of animals, at doses of 300 and 2000 mg/kg. The spectrophotometric analytical method met the criteria of selectivity, linearity, precision, accuracy and robustness, being fast, cheap and easy to perform in the determination of flavonoids expressed as rutin. To obtain the standardized dry extract, the best drying conditions in spray dryer, in this study, were: concentration of drying adjuvant of 50% (w/w), inlet temperature of 148°C, feed flow of 0.5 L /H. For toxicological tests, the standardized dry extract was considered non-toxic in the Artemia salina test and classified as low toxicity in the protocol of acute oral toxicity in rodents, with LD50 between 2000 and 5000 mg/Kg. The results obtained contribute to the development of high quality and low toxicity raw materials from the aerial parts of D. ambrosioides.

IV. Detailed description of the invention:

b) etapa 2: Foi realizado estudo de caracterização morfoanatômica, com amostras de folhas do material vegetal, como um dos parâmetros para o controle de qualidade.
c) etapa 3: O material coletado foi submetido à secagem em estufa com circulação forçada de ar à 37±5°C, até atingir o teor de umidade entre 8 a 14%. Em seguida, o material seco foi pulverizado em moinho de facas.
d) etapa 4: O pó obtido foi analisado segundo os critérios da Farmacopeia Brasileira, a saber: identificação das estruturas anatômicas presentes (observou-se fragmentos da epiderme, de tricomas tectores e glandulares); determinação do teor de voláteis (8,45% ±0,03); determinação de cinzas totais (11,01% ±0,03); determinação de cinzas insolúveis em ácido clorídrico (0,54% ±0,03); determinação da granulometria do pó (grosso, conforme parâmetros da Farmacopeia Brasileira); determinação do índice de intumescência (0,2 mL ±0,26) e triagem fitoquímica (flavonoides, compostos fenólicos, heterosídeos antraquinônicos, taninos e terpenos). Foi realizada a análise do óleo essencial extraído do material vegetal e os principais compostos estão demonstrados na Tabela 2.
Tabela 2: Composição química do óleo essencial de partes aéreas de Dysphania ambrosioides

e) etapa 5: Para a quantificação de flavonoides totais, expressos como rutina, na droga vegetal (material pulverizado) de D. ambrosioides foi utilizado o método proposto por Rolim et al. (2005) com adaptações. Resumidamente, para a extração dos flavonoides da amostra, a 1g da droga vegetal foi adicionada quantidade suficiente para completar 10mL de etanol (70% p/p), o material foi mantido em banho de ultrassom (UNIQUE mod. USC – 2800A, frequência 40kHz e potência 154W) por 30 minutos a temperatura ambiente. O extrato foi filtrado em papel de filtro e 100µl do filtrado foi diluído em 3900 µl de uma solução composta por metanol e ácido acético 0,02M (99:1), e em seguida foi realizada a leitura em espectrofotômetro (modelo SP22 Biospectro) a 364 nm, utilizando como branco a solução de metanol em ácido acético 0,02M (99:1). O cálculo do teor de flavonoides totais, expressos como rutina, foi realizado com o auxílio da equação da reta, obtida por meio da análise de regressão linear, da curva de calibração do padrão rutina (Sigma Aldrich). A validação do método espectrofotométrico foi realizada conforme parâmetros estabelecidos pela legislação brasileira (BRASIL, 2017a). O teor de flavonoides totais, equivalentes a rutina, na droga vegetal foi de 0,015% (m/m).
f) etapa 6: Para a produção do extrato líquido das partes aéreas de Dysphania ambrosioides, a ser utilizado na produção do extrato seco em spray dryer, foram utilizados 3 kg de droga vegetal pulverizada, colocados em maceração por 24h em etanol 70% (p/p). Em seguida, o macerado foi transferido para percoladores de aço inox, completados com solução extrativa, e deixados em repouso por 4h. Então a percolação propriamente dita foi iniciada e mantida até que o líquido extrator saturasse e fosse substituído por um novo. Cada 1kg de droga vegetal consumiu 10 L de solvente, num total de 30 L. O extrato coletado foi rotulado, acondicionado em frascos plásticos e armazenado a -20°C. O rendimento da percolação foi de 27 litros de extrato, que foi concentrado em rotaevaporador Ika 10 (temperatura 40°C, rotação de 25 rpm e pressão de 70 bar) até cerca de 10 L. O concentrado foi homogeneizado e armazenado em frasco plástico a -20°C, até sua utilização nos ensaios de caracterização e na secagem.
g) etapa 7: O extrato líquido obtido conforme item anterior foi caracterizado quanto aos seguintes parâmetros: teor de sólidos totais; pH; densidade relativa; e viscosidade. A determinação de sólidos totais do percolado foi obtida com auxílio de analisador de umidade com aquecimento por lâmpada de halogênio Shimadzu modelo Unibloc. A análise foi feita em triplicata, em cada experimento foi pesado cerca de 1g de extrato líquido e aquecido a 105°C por aproximadamente15 minutos. A porcentagem de sólidos totais foi expressa pela Equação 1:
%sólidos totais = 100 – U (Eq.1)
Em que:
U = Leitura do teor de voláteis indicada no display do analisador para a amostra.
A leitura do pH do extrato líquido foi realizada em triplicata pelo método potenciométrico, de acordo com a Farmacopeia Brasileira 5° edição (BRASIL, 2010). O equipamento utilizado foi da marca Marconi modelo MA-552 previamente calibrado com tampões pH 7,0 e 4,0. A densidade relativa do extrato líquido foi determinada em triplicata, conforme Farmacopeia Brasileira 5° edição (BRASIL, 2010) por metodologia gravimétrica, com utilização de picnômetro de 10 ml, limpo, seco, previamente calibrado e pesado. A densidade relativa foi calculada através da razão entre massa da amostra e massa da água purificada expressa na Equação 2:
Densidade Relativa = ((Pe- Pv))/((Pa- Pv)) (Eq.2)
Em que:
Pe – massa do picnômetro com o extrato líquido
Pv – massa do picnômetro vazio
Pa – massa do picnômetro com água purificada
As medidas de viscosidade foram realizadas segundo metodologia descrita na Farmacopeia Brasileira 5° edição (BRASIL, 2010) em viscosímetro de Brookfield – DV-II + Viscosimeter, com conjunto de spindles tipo LV, operado nas condições de 100 rpm, torque 4%, temperatura ambiente.
Os resultados obtidos são apresentados na Tabela 3.
Tabela 3: Caracterização do extrato líquido concentrado obtido de partes aéreas de D. ambrosioides

Legenda: DP: Desvio padrão; DPR: Desvio padrão relativo
h) etapa 8: Antes da realização dos procedimentos de secagem do extrato líquido de D. ambrosioides em spray dryer foi avaliado o efeito da temperatura sobre a degradação da rutina, principal marcador do extrato. Para tanto, foram obtidos dados consolidados da literatura sobre a análise termogravimétrica do padrão analítico rutina (COSTA et al., 2002; QI et al., 2015). O estudo de Costa et al. (2002) demonstra que, na curva de decaimento da rutina com ar atmosférico, da temperatura de 50 a 261°C ocorre perda de massa de 4,3%, entre 261 a 609°C a decomposição da rutina chega a 69,5%. Quando a ATG é realizada com gás nitrogênio, a perda de massa entre as temperaturas de 25 a 139°C é de 5%, mas na faixa de 139 a 304°C a decomposição da rutina chega a 15,8% e a curva de decaimento se mantem estável até 892°C. Em outro estudo que realizou a análise com nitrogênio gasoso, o processo de fusão e decomposição da rutina ocorreu na temperatura de 274,41°C, com perda de massa próxima aos 10% (QI et al., 2015). Tendo em vista que a secagem do extrato por aspersão ocorre na câmera de secagem em presença de ar atmosférico aquecido, para preservação dos flavonoides estabeleceu-se a faixa de temperatura entre 120 e 160°C para os experimentos de secagem.
i) etapa 9: A secagem do extrato líquido concentrado foi realizada em um equipamento tipo “mini” Spray Dryer LABMAQ® - modelo LM-MSD 1.0, com fluxo co-corrente. Foram realizados experimentos de secagem no spray dryer utilizando dois adjuvantes de secagem: maltodextrina e dióxido de silício coloidal (Aerosil®). Os adjuvantes foram testados nas concentrações de 20, 40 e 60% (p/p) em relação à massa de sólidos totais do extrato líquido concentrado. A incorporação dos adjuvantes de secagem às amostras de extrato líquido foi realizada com o auxílio de homogeneizador do tipo Ultra Turrax. Os parâmetros de secagem utilizados estão descritos na Tabela 4:
Tabela 4: Parâmetros de secagem no spray dryer utilizados na avaliação preliminar dos adjuvantes maltodextrina e Aerosil®

A secagem do extrato líquido utilizando maltodextrina como adjuvante de secagem não melhorou o rendimento de secagem, nem mesmo auxiliou na atomização do extrato. Já os extratos secos com Aerosil® nas mesmas concentrações contribuíram para a formação dos mesmos e melhora de rendimento, por isso este último foi escolhido como adjuvante de secagem nos experimentos de otimização.
j) etapa 10: Para o processo de otimização da secagem do extrato líquido obtido das partes aéreas de D. ambrosioides foi empregada a metodologia de superfície de resposta (MSR) em um modelo Box Behnken (33 ), com as variáveis independentes selecionadas com base na literatura (Tabela 5).
Tabela 5: Variáveis independentes e seus níveis avaliados pela metodologia de superfície de resposta para otimização do processo de secagem por aspersão (Spray Drying) do extrato líquido de D. ambrosioides

Legenda: *porcentagem em relação à massa de sólidos totais
O modelo estatístico gerou 15 experimentos, com auxílio do software Statistica® versão 12.0 (STATSOFT, 2010) cuja execução foi realizada de forma aleatória. Para avaliação do erro puro três experimentos eram repetições do ponto central. Os coeficientes foram interpretados pelo teste F. A análise dos dados foi executada por meio de três ferramentas: análise de variância (ANOVA), análise de regressão e plotagem de superfície de resposta. Foram considerados significativos valores de p < 0,05. As variáveis respostas foram: rendimento de secagem (%); perda por dessecação (%); atividade de água (Aw); e teor de flavonoides, expressos como rutina (%). O rendimento da secagem foi calculado pela relação entre o peso do extrato seco obtido na secagem pela massa de teor de sólidos totais presentes no extrato líquido utilizado no processo, expressa em porcentagem, conforme a Equação 3:

Em que:
U – rendimento (%); P1 – peso do extrato seco obtido (g); P2 – massa de sólidos totais (incluindo a massa de adjuvante acrescentada) presentes na amostra (g)
A perda por dessecação dos extratos secos foi obtida com auxílio de analisador de umidade com aquecimento por lâmpada de halogênio Shimadzu modelo Unibloc. A análise foi feita em triplicata, sendo que em cada experimento foram utilizados cerca de 200 mg de extrato seco, que foram aquecidos a 105°C por aproximadamente 5 minutos. A atividade de água de cada extrato seco foi determinada em triplicata no equipamento PRE AQUA LAB modelo 450 com aproximadamente 200 mg da amostra, a 25°C.
O doseamento de flavonoides totais, expressos como rutina, em cada amostra de extrato seco foi realizada de acordo com a etapa 5. Posteriormente convertido em teor (%, m/m) pela Equação 4.

Em que:
C – concentração de flavonoides totais expressos como rutina (µg/mL); Ca – concentração da amostra (µg/mL)
Os resultados dos 15 experimentos estão descritos na Tabela 6. As análises de variância (ANOVA) e regressão linear múltipla foram verificadas em conjunto com a metodologia de superfície de resposta. A ANOVA permite comparar a variação das repostas aos erros randômicos inerentes à mensuração dessas respostas e mensurar a significância da regressão usada para prever respostas, de acordo com a variabilidade experimental (BEZERRA et al., 2008). Na análise estatística foi verificada que dos parâmetros analisados, a atividade de água demonstrou falta de ajuste (p=0,04), de modo que na Tabela 7 estão descritos os efeitos mais relevantes e respectivas significâncias. As variáveis, concentração de Aerosil® (1), temperatura de entrada (2) e fluxo de alimentação (3) exerceram efeito linear significativo sobre o rendimento de secagem. O modelo foi significante (p = 0,000133) e não demonstrou falta de ajuste (p = 0,112957), apontou um coeficiente de determinação (r2 ) de 0,90884 e r2 ajustado de 0,74476. O efeito linear dos parâmetros foi observado na análise de superfície de resposta, o rendimento é aumentado à medida que a concentração de adjuvante (Desenho 1) é elevada, o mesmo é observado para o fluxo de alimentação (Desenho 1). Já na temperatura de entrada (Desenho 1) é observado um efeito linear negativo nas temperaturas mais baixas apresentaram menor rendimento. Na análise estatística foi observada uma relação significativa entre a temperatura de entrada e fluxo de alimentação (Desenho 1), de modo que os maiores rendimentos foram alcançados em temperaturas inferiores a 125°C e fluxo de alimentação superiores a 0,44 L/h.
Tabela 6: Matriz experimental do modelo Box Behnken (33 ), rendimento e ensaios de caracterização dos extratos secos de partes aéreas de D. ambrosioides obtidos por aspersão

Legenda: CA- concentração do adjuvante; TE- temperatura de entrada; FA- fluxo de alimentação; Rrendimento; TV- teor de voláteis; AW- atividade de água.*porcentagem de Aerosil® em relação a massa de sólidos totais. Condições das secagens: diâmetro do bico aspersor de 0,7 mm, fluxo do ar comprimido 40L/min, pressão do ar comprimido de 4,0 Kg/F.
Tabela 7: Sumário dos efeitos das variáveis independentes e suas significâncias (ANOVA) sobre as respostas analisadas no planejamento Box-Benhken 33 para obtenção de extrato seco de D. ambrosioides por aspersão

Legenda: *Nível de significância p < 0,05, ** p < 0,1. 1- concentração de adjuvante, 2- temperatura de entrada, 3- fluxo de alimentação, L- linear, Q- quadrática.
A concentração de Aerosil® exerceu efeitos lineares e quadráticos negativos sobre o teor de voláteis. O modelo foi significativo (p = 0,000042) e não demonstrou falta de ajuste (p = 0,082978), apontou um coeficiente de determinação (r2) de 0,91515 e r2 ajustado de 0,76242. Os gráficos de superfície de resposta, descritos no Desenho 2, sugerem que as condições adequadas do teor de voláteis foram alcançadas em concentrações de adjuvante superiores a 30% da massa do teor de sólidos do extrato, independente do valor do parâmetro analisado dentro do intervalo estabelecido. Os melhores teores de flavonoides foram alcançados nos intervalos com fluxo de alimentação superior a 0,4 L/h e temperatura inferior a 120°C e fluxo de alimentação inferior a 0,48 L/h e temperatura superior a 155°C, não evidenciando nenhum efeito significativo entre as variáveis individuais ou interação entre elas conforme dados estatísticos da Tabela 7.
O teor de flavonoides, expressos como equivalentes de rutina, não apresentou relações significativas para as variáveis, concentração de Aerosil® (1), temperatura de entrada (2) e fluxo de alimentação (3). Entretanto, a interação dos parâmetros Aerosil® e fluxo de alimentação foi significativa (p=0,07) quando se considerou o valor de p menor que 0,1. O modelo foi significativo (p = 0,001437) e não demonstrou falta de ajuste (p = 0,189142), apontou um coeficiente de determinação (r2 ) de 0,73323. Na análise de superfície de resposta, conforme Desenho 3, foi observada uma relação significativa entre concentração de adjuvante e fluxo de alimentação, indicando que os maiores teores foram alcançados em concentração de adjuvante superior a 30% e fluxo de alimentação superiores a 0,38 L/h. Embora altas temperaturas de entrada possam gerar a degradação dos componentes do extrato, o parâmetro não apresentou efeito significativo em relação às variáveis observadas. No entanto, nota-se que os melhores teores de flavonoides foram alcançados em temperaturas superiores a 130°C (Desenho 3), o que está de acordo com a análise termogravimétrica descrita na literatura, cuja decomposição do marcador escolhido ocorre significativamente em temperaturas superiores a 261°C (COSTA et al., 2002).
As melhores condições de secagem do extrato de D. ambrosioides, para as variáveis concentração de adjuvante (Aerosil®), temperatura de entrada (°C) e fluxo de alimentação (L/h) foram determinadas a partir dos dados das superfícies de resposta e da função geral de otimização e estão descritos na Tabela 8, a uma desejabilidade de 0,739467. Os valores preditos pelo modelo estatístico para as respostas, rendimento de secagem, teor de voláteis e teor de flavonoides totais, nas condições otimizadas, também são apresentados na Tabela 8.
As condições previstas pelo modelo foram validadas por meio da análise do rendimento e caracterização de 3 secagens conduzidas nas condições otimizadas para que a reprodutibilidade fosse observada. Os dados da triplicata são apresentados na Tabela 9.
Tabela 8: Melhores condições de secagem do extrato de D. ambrosioides para as variáveis, concentração de adjuvante (Aerosil®), temperatura de entrada (°C) e fluxo de alimentação (L/h) e valores preditos pelo modelo para as variáveis dependentes

Legenda: R- rendimento, TV- teor de voláteis; TF- teor de flavonoides.*porcentagem de Aerosil® em relação a massa de sólidos totais.
Tabela 9: Valores observados para o rendimento, teor de voláteis e teor de flavonoides totais expressos como rutina do extrato seco obtido de partes aéreas de D. ambrosioides por aspersão

Legenda: DPR: desvio padrão relativo; %* porcentagem em relação ao valor predito. Condições das secagens: diâmetro do bico aspersor de 0,7 mm, fluxo do ar comprimido 40L/min, pressão do ar comprimido de 4,0 Kg/F; concentração de adjuvante: 50% (p/p), temperatura de entrada de 148°C, fluxo de alimentação: 0,5 L/h.
Os resultados observados na triplicata demonstraram que o modelo foi eficiente na predição das respostas. O experimento mostrou valores com boa concordância aos resultados preditos, demonstrando a adequabilidade do modelo escolhido em demonstrar a influência destes parâmetros na otimização das variáveis respostas para a secagem dos extratos obtidos das partes aéreas de D. ambrosioides.
k) etapa 11: Após a obtenção do extrato seco nas condições otimizadas, a morfologia das partículas foi analisada por microscopia eletrônica de varredura (MEV). As amostras do extrato seco foram metalizadas em ouro e a caracterização morfológica foi obtida em Microscópio Eletrônico de Varredura modelo Jeol, JSM – 6610, equipado com EDS, Thermo scientific NSS Spectral Imaging, no Laboratório Multiusuário de Microscopia de Alta Resolução (LabMic) da Universidade Federal de Goiás. Verificaram-se a presença de estruturas aglomeradas compostas de grãos individuais de superfície porosa unidos por poeira submicrométrica do mesmo material, de tamanhos e formas variados (Desenho 4). Os formatos dos grãos variavam de irregulares a esféricos e o tamanho de 1,4 µm a partículas com dimensões superiores a 22,7 µm de diâmetro. No extrato seco também foi observado que as esferas maiores eram preenchidas por esferas menores. Característica que pode ser explicada pela permeabilidade e elasticidade da superfície da esfera, gerando grãos com a superfície imperfeita ou fragmentada, sólidas ou ocas (OLIVEIRA; PETROVICK et al., 2010).
l) etapa 12: O bioensaio em Artemia salina Leach foi usado para estimar a bioatividade do extrato seco de D. ambrosioides. O bioensaio, utilizando concentrações previamente definidas do extrato seco, foi realizado em microplacas de poliestireno de 96 poços com o microcustáceo, de acordo com a metodologia proposta por Molina-Salinas e SaidFernandez (2006), com adaptações. Neste trabalho, 60 mg de cistos de Artemia salina foram incubados em um artemilheiro com metade de água marinha sintética, preparada com a dissolução de sal marinho em água destilada (36 g/L), acrescida de extrato de leveduras (6 mg/L) e esterilizada em autoclave. Esta solução foi utilizada para eclosão dos ovos de A. salina e no preparo das demais diluições. Durante um período de 36 horas o meio foi preservado sobre saturação constante de oxigênio à temperatura ambiente e iluminação natural para a eclosão dos ovos. As amostras (extrato seco de D. ambrosioides e Aerosil®) foram diluídos em 5% de propilenoglicol e salina para obter as concentrações de 2000, 1000, 500, 250 e 125 μg/mL. Para o experimento foram utilizadas 8 colunas das microplacas, das quais 3 colunas foram reservadas para o extrato seco em concentrações decrescentes, 3 colunas para o Aerosil® com as concentrações distribuídas de forma semelhante a do extrato, e 1 coluna para o controle positivo (K2CrO7) e 1 coluna para o controle negativo (água salina). Cada poço foi preenchido com 10 ± 1 náuplios suspensos em 100 μL de solução salina e completados com 100 μL do respectivo tratamento. Posteriormente, após 24 horas de incubação, realizou-se a contagem de microcrustáceos mortos ou imóveis por mais de 10 segundos. Todos os ensaios foram feitos em triplicata. A toxicidade foi expressa através da concentração letal a 50% da população do microcrustáceo por meio do cálculo da CL50 estimada a partir da regressão linear obtida da correlação entre a porcentagem de indivíduos mortos e a concentração da substância teste, usando o método Probitos. A classificação do nível de toxidade foi estabelecida de acordo com o critério Nguta et al. (2012). O estudo divide como toxicidade forte valores de CL50 até 100 µg/mL, toxicidade moderada para CL50 entre 100 e 500 µg/ml e toxicidade baixa para CL50 entre 500 µg/mL e 1000 µg/mL, e não tóxico acima de 1000 µg/mL. O extrato seco de D. ambrosioides e o Aerosil® não ocasionaram mortes de artemias nas concentrações testadas (2000, 1000, 500, 250 e 125 μg/mL). Em decorrência da ausência de mortes não foi possível estimar a CL50, sugere-se portanto que está acima das concentrações analisadas. O estudo de Nguta et al. (2012) classifica que extratos vegetais com CL50 superior a 1000 µg/ml são atóxicos corroborando para classificação do extrato seco de D. ambrosioides como atóxico.
m) etapa 13: Para avaliar a toxicidade oral aguda do extrato seco de D. ambrosioides em roedores foi utilizado o protocolo n° 423 da Organização para Cooperação Econômica e Desenvolvimento (Organization for Economic Co-operation and Development – OEDC, 2001). Os experimentos foram conduzidos de acordo com as normas do Conselho de Experimentação Animal (CONCEA). Foram aprovados pela Comissão de Ética no Uso de animais (CEUA) da Universidade Estadual de Goiás (UEG), conforme protocolo nº 006/2017. Foram utilizados ratos fêmeas da linhagem Wistar (Rattus norvegicus), nulíparas, com idade entre 68 e 81 dias, pesando entre 144,22 – 210,68 g. Os animais foram mantidos durante todo o período experimental no Biotério do Laboratório de Farmacologia e Toxicologia de Produtos Naturais e Sintéticos da Universidade Estadual de Goiás (UEG). As condições de temperatura e umidade foram controladas, a luminosidade seguiu um ciclo claro/escuro de 12 horas (luzes acesas das 7:00 às 19:00 horas) e os animais foram tratados com água e ração a vontade. Neste estudo os animais foram divididos em dois grupos experimentais (grupo 1: controle (Aerosil®) grupo 2: Teste (dose de 300 e 2000 mg/kg) com três ratas em cada grupo. As amostras foram administradas via gavagem. Os animais foram observados por 8 horas seguidas no primeiro dia de experimento. Nos 14 dias restantes foram observados 1 hora no período da manhã. Os sinais de toxicidade investigados foram: mudanças ou alterações na pele ou no pêlo; alterações nos olhos e nas membranas mucosas; nos sistemas circulatório, respiratório, nervoso central e autônomo. Além disso, também foram investigadas possíveis alterações no padrão comportamental e somatomotor (tremores, prostração, paralisia, respostas exageradas ao barulho ou ao toque – hiperreflexia). Ao final do período experimental todos os animais foram submetidos à necropsia. Os órgãos foram analisados quanto ao seu aspecto geral, e determinou-se a massa dos rins, fígado. O extrato seco das partes aéreas de D. ambrosioides foi dissolvido em 10% de propilenoglicol e água, nas concentrações de acordo com o peso dos animais, a fim de se obter as doses de 300 e 2000 mg/kg. O volume de administração utilizado foi de 0,5 mL para cada 100g de massa corporal para as doses de 300 e 2000mg/kg. No grupo controle foi utilizado o Aerosil® na dose de 667 mg/Kg para verificar se o adjuvante influenciava na toxicidade do extrato, e este foi preparado em 10% de propilenoglicol e água. Utilizou-se a análise de variância ANOVA seguida pelo teste Student-Newman-Keuls para identificar as diferenças entre os grupos controle e experimental, através do software Statistica® versão 12.0 (STATSOFT, 2010). Para a avaliação do ganho de massa corporal e massa relativa dos órgãos foi utilizada ANOVA unifatorial e para avaliação do consumo de água e ração ANOVA para medidas repetidas. As diferenças estatisticamente significativas foram indicadas quando p<0,05. Os sinais clínicos comportamentais apresentados pelos animais durante as primeiras horas, evidenciados pelo screening hipocrático, após a administração do extrato foram: no grupo controle os animais apresentaram hiperatividade, diminuição de resposta a pressão da cauda, e comportamento compulsivo. Na dose de 300mg/kg observou-se dificuldade na respiração (hiperventilação), e redução de resposta à pressão da cauda. Na dose de 2000mg/kg observou-se nos primeiros 15 minutos que a respiração das mesmas estava ofegante, e persistiu a redução de resposta à pressão da cauda. A persistência da analgesia da cauda em especial nas doses maiores do extrato é explicada em parte pelas propriedades antinoniceptiva e anti-inflamatória da planta (IBIRONKE; AJIBOYE, 2007; TRIVELLATO-GRASSI et al., 2013; CALADO et al., 2015). Durante o ensaio não foram verificadas mortes dos animais nos grupos estudados, nem mesmo durante a etapa de repetição do ensaio. De forma que o extrato seco padronizado para flavonoides obtido de partes aéreas de D. ambrosioides é classificado na categoria “5”, com DL50 estimada entre 2000 a 5000 mg/Kg, de acordo com o guia n° 423 da OECD. Este guia considera DL50 superiores a 2000 mg/Kg como praticamente atóxicas, e não recomenda a continuidade do experimento para a dose de 5000 mg/Kg. Uma vez que a dose é de difícil solubilidade e administração, além da necessidade de proteção da vida animal utilizada em laboratório (OECD, 2001). Na análise estatística ANOVA one way para o ganho de massa corporal não foi demonstrada interferência do tratamento sobre os grupos analisados (F2,15 = 0,28; p = 0,76). Para a ANOVA de medidas repetidas, quanto a massa corporal relativa, também não foi demonstrado efeito do tratamento sobre os grupos (F2,15 = 2,16; p = 0,15) e não apresentou interação significativa quando foi analisado o efeito do tratamento sobre os dias (F26,195 = 0,71; p = 0,85). No entanto o fator dias demonstrou significativo no decorrer do ensaio (F13,195 = 79,44; p < 0,01). Ao analisar o efeito dos tratamentos sobre o consumo de ração, na ANOVA unifatorial não foi constatada interação (F2,15 = 0,80; p = 0,47). Ao avaliar o consumo de ração relativo a cada 3 dias, a ANOVA para medidas repetidas não demonstrou efeito do tratamento (F2,15 = 0,34; p = 0,71) e nem interação entre dias e tratamento (F8,60 = 0,79; p = 0,61). Entretanto o fator dias, analisado separadamente, demonstrou ser significativo (F4,60 = 11,03; p < 0,01). Na análise do consumo relativo de água a cada 3 dias, em detrimento do tratamento, a ANOVA unifatorial não foi significativa (F2,15 = 1,05; p = 0,37). Na ANOVA para medidas repetidas não foi demonstrado efeito do tratamento (F2,15 = 0,85; p = 0,45), também não houve efeito para o fator dias (F4,60 = 1,91; p = 0,12), e nem interação entre dias e tratamento (F8,60 = 0,33; p = 0,95). Na análise de massa relativa dos órgãos, a ANOVA unifatorial não apontou diferenças entre os grupos para a massa dos rins (F2,15 = 0,71; p = 0,50) e fígado (F2,15 = 2,36; p = 0,13).[024] The process for obtaining phytotherapic input based on dry and standardized extract of Dysphania ambrosioides comprises the following steps:
a) step 1: To obtain a sufficient amount of plant material, specimens of Dysphania ambrosioides were collected at the Horto de Plantas Medicinais at the Campus Anápolis de Ciências Exatas e Tecnológicas (CCET), at the Universidade Estadual de Goiás and at the Horto de Plantas Medicinais do Jardim Botanical of Goiânia, city of Goiânia, Goiás. The specimens were also collected in residential backyards in the regions of Anápolis, Goiânia, Itapuranga and Pirenópolis, in the state of Goiás. The pool collected included the aerial parts of the plant species with the presence of stem, leaves , inflorescences and seeds. The exsiccates were deposited in the Herbarium of the State University of Goiás, as shown in Table 1. The collections were carried out between December 2016 and March 2017.
Table 1: Data from the exsiccates deposited in the herbarium of the State University of Goiás

b) step 2: A morphoanatomical characterization study was carried out, with leaf samples of plant material, as one of the parameters for quality control.
c) step 3: The material collected was subjected to drying in an oven with forced air circulation at 37±5°C, until reaching a moisture content between 8 and 14%. Then, the dried material was pulverized in a knife mill.
d) step 4: The powder obtained was analyzed according to the criteria of the Brazilian Pharmacopoeia, namely: identification of the anatomical structures present (fragments of the epidermis, tector and glandular trichomes were observed); determination of volatile content (8.45% ±0.03); determination of total ash (11.01% ±0.03); determination of ash insoluble in hydrochloric acid (0.54% ±0.03); determination of powder granulometry (coarse, according to Brazilian Pharmacopoeia parameters); determination of the swelling index (0.2 mL ±0.26) and phytochemical screening (flavonoids, phenolic compounds, anthraquinone heterosides, tannins and terpenes). The analysis of essential oil extracted from plant material was performed and the main compounds are shown in Table 2.
Table 2: Chemical composition of essential oil from aerial parts of Dysphania ambrosioides

e) step 5: The method proposed by Rolim et al. (2005) with adaptations. Briefly, to extract the flavonoids from the sample, 1g of the plant drug was added to a sufficient amount to complete 10mL of ethanol (70% w/w), the material was kept in an ultrasound bath (UNIQUE mod. USC – 2800A, frequency 40kHz and power 154W) for 30 minutes at room temperature. The extract was filtered through filter paper and 100µl of the filtrate was diluted in 3900 µl of a solution composed of methanol and 0.02M acetic acid (99:1), and then the reading was performed in a spectrophotometer (model SP22 Biospectro) at 364 nm, using a solution of methanol in 0.02M acetic acid (99:1) as a blank. The calculation of the total flavonoid content, expressed as rutin, was performed with the aid of the straight line equation, obtained through linear regression analysis, of the calibration curve of the rutin standard (Sigma Aldrich). The validation of the spectrophotometric method was performed according to parameters established by Brazilian legislation (BRASIL, 2017a). The total flavonoid content, equivalent to rutin, in the plant drug was 0.015% (m/m).
f) step 6: For the production of the liquid extract of the aerial parts of Dysphania ambrosioides, to be used in the production of the dry extract in spray dryer, 3 kg of pulverized plant drug were used, placed in maceration for 24 hours in 70% ethanol (p. /P). Then, the macerate was transferred to stainless steel percolators, completed with extractive solution, and left to rest for 4 h. Then the percolation itself was started and maintained until the extractor liquid was saturated and it was replaced by a new one. Each 1kg of plant drug consumed 10 L of solvent, totaling 30 L. The collected extract was labeled, placed in plastic bottles and stored at -20°C. The percolation yield was 27 liters of extract, which was concentrated in an Ika 10 rotary evaporator (temperature 40°C, rotation of 25 rpm and pressure of 70 bar) to about 10 L. The concentrate was homogenized and stored in a plastic bottle at -20°C, until its use in the characterization tests and drying.
g) step 7: The liquid extract obtained according to the previous item was characterized in terms of the following parameters: total solids content; pH; relative density; and viscosity. The determination of total solids of the leachate was obtained with the aid of a moisture analyzer heated by a Shimadzu halogen lamp, Unibloc model. The analysis was performed in triplicate, in each experiment, about 1g of liquid extract was weighed and heated at 105°C for approximately 15 minutes. The percentage of total solids was expressed by Equation 1:
%total solids = 100 - U (Eq.1)
On what:
U = Volatile content reading indicated on the analyzer display for the sample.
The pH reading of the liquid extract was performed in triplicate by the potentiometric method, according to the Brazilian Pharmacopoeia 5th edition (BRASIL, 2010). The equipment used was a Marconi brand, model MA-552, previously calibrated with pH 7.0 and 4.0 buffers. The relative density of the liquid extract was determined in triplicate, according to the Brazilian Pharmacopoeia 5th edition (BRASIL, 2010) by gravimetric methodology, using a 10 ml pycnometer, clean, dry, previously calibrated and weighed. The relative density was calculated through the ratio between the mass of the sample and the mass of purified water expressed in Equation 2:
Relative Density = ((Pe- Pv))/((Pa- Pv)) (Eq.2)
On what:
Pe – mass of the pycnometer with the liquid extract
Pv – mass of the empty pycnometer
Pa – mass of the pycnometer with purified water
Viscosity measurements were performed according to the methodology described in the Brazilian Pharmacopoeia 5th edition (BRASIL, 2010) in a Brookfield viscometer – DV-II + Viscosimeter, with a set of LV-type spindles, operated at 100 rpm, 4% torque, temperature environment.
The results obtained are shown in Table 3.
Table 3: Characterization of concentrated liquid extract obtained from aerial parts of D. ambrosioides

Caption: DP: Standard Deviation; DPR: Relative Standard Deviation
h) step 8: Before carrying out the drying procedures of the liquid extract of D. ambrosioides in spray dryer, the effect of temperature on the degradation of rutin, the main marker of the extract, was evaluated. For that, consolidated data were obtained from the literature on the thermogravimetric analysis of the rutin analytical standard (COSTA et al., 2002; QI et al., 2015). The study by Costa et al. (2002) shows that, in the decay curve of rutin with atmospheric air, from 50 to 261°C there is a mass loss of 4.3%, between 261 to 609°C the rutin decomposition reaches 69.5% . When ATG is carried out with nitrogen gas, the mass loss between temperatures from 25 to 139°C is 5%, but in the range from 139 to 304°C the rutin decomposition reaches 15.8% and the decay remains stable up to 892°C. In another study that performed the analysis with nitrogen gas, the rutin melting and decomposition process occurred at a temperature of 274.41°C, with a mass loss close to 10% (QI et al., 2015). Considering that the drying of the extract by spraying occurs in the drying chamber in the presence of heated atmospheric air, for the preservation of flavonoids, a temperature range between 120 and 160°C was established for the drying experiments.
i) step 9: The drying of the concentrated liquid extract was performed in a “mini” Spray Dryer LABMAQ® equipment - model LM-MSD 1.0, with co-current flow. Drying experiments were carried out in the spray dryer using two drying adjuvants: maltodextrin and colloidal silicon dioxide (Aerosil®). Adjuvants were tested at concentrations of 20, 40 and 60% (w/w) in relation to the total solids mass of the concentrated liquid extract. The incorporation of drying aids to the liquid extract samples was carried out with the aid of an Ultra Turrax homogenizer. The drying parameters used are described in Table 4:
Table 4: Drying parameters in the spray dryer used in the preliminary evaluation of maltodextrin and Aerosil® adjuvants

Drying the liquid extract using maltodextrin as a drying aid did not improve the drying performance, nor did it help in the atomization of the extract. The dry extracts with Aerosil® at the same concentrations contributed to their formation and yield improvement, so the latter was chosen as a drying aid in the optimization experiments.
j) step 10: For the process of optimizing the drying of the liquid extract obtained from aerial parts of D. ambrosioides, the response surface methodology (MSR) was used in a Box Behnken model (33 ), with the independent variables selected based on in the literature (Table 5).
Table 5: Independent variables and their levels evaluated by the response surface methodology for optimization of the spray drying process (Spray Drying) of the liquid extract of D. ambrosioides

Caption: *percentage in relation to the mass of total solids
The statistical model generated 15 experiments, with the help of the Statistica® software version 12.0 (STATSOFT, 2010) whose execution was performed randomly. For pure error evaluation, three experiments were repetitions of the central point. The coefficients were interpreted by the F test. Data analysis was performed using three tools: analysis of variance (ANOVA), regression analysis and response surface plotting. Values of p < 0.05 were considered significant. The response variables were: drying yield (%); desiccation loss (%); water activity (Aw); and flavonoid content, expressed as rutin (%). The drying yield was calculated by the ratio between the weight of the dry extract obtained in the drying by the mass of total solids content present in the liquid extract used in the process, expressed as a percentage, according to Equation 3:

On what:
U – yield (%); P1 – weight of the dry extract obtained (g); P2 - mass of total solids (including the mass of adjuvant added) present in the sample (g)
The loss by desiccation of the dry extracts was obtained with the aid of a moisture analyzer heated by a Shimadzu halogen lamp, Unibloc model. The analysis was performed in triplicate, and in each experiment about 200 mg of dry extract were used, which were heated at 105°C for approximately 5 minutes. The water activity of each dry extract was determined in triplicate in the PRE AQUA LAB model 450 equipment with approximately 200 mg of the sample, at 25°C.
The determination of total flavonoids, expressed as rutin, in each dry extract sample was performed according to step 5. Subsequently converted into content (%, m/m) by Equation 4.

On what:
C – concentration of total flavonoids expressed as rutin (µg/mL); Ca – sample concentration (µg/mL)
The results of the 15 experiments are described in Table 6. Analysis of variance (ANOVA) and multiple linear regression were verified in conjunction with the response surface methodology. ANOVA allows comparing the variation of responses to the random errors inherent in the measurement of these responses and measuring the significance of the regression used to predict responses, according to the experimental variability (BEZERRA et al., 2008). In the statistical analysis, it was verified that of the analyzed parameters, the water activity showed a lack of adjustment (p=0.04), so that Table 7 describes the most relevant effects and their respective significance. The variables, Aerosil® concentration (1), inlet temperature (2) and feed flow (3) exerted a significant linear effect on the drying yield. The model was significant (p = 0.000133) and showed no lack of fit (p = 0.112957), it showed a coefficient of determination (r2 ) of 0.90884 and an adjusted r2 of 0.74476. The linear effect of the parameters was observed in the response surface analysis, the yield is increased as the adjuvant concentration (Drawing 1) is high, the same is observed for the feed flow (Drawing 1). Already at the inlet temperature (Drawing 1) a negative linear effect is observed at the lower temperatures showed lower yield. In the statistical analysis, a significant relationship between inlet temperature and feed flow was observed (Drawing 1), so that the highest yields were achieved at temperatures below 125°C and feed flow above 0.44 L/h.
Table 6: Experimental matrix of the Box Behnken model (33 ), yield and characterization assays of dry extracts of aerial parts of D. ambrosioides obtained by spraying

Legend: CA- adjuvant concentration; TE- inlet temperature; FA- feed flow; Yield; TV- volatile content; AW- water activity.*percentage of Aerosil® in relation to the mass of total solids. Drying conditions: sprinkler nozzle diameter of 0.7 mm, compressed air flow of 40L/min, compressed air pressure of 4.0 Kg/F.
Table 7: Summary of the effects of independent variables and their significance (ANOVA) on the responses analyzed in the Box-Benhken 33 design for obtaining dry extract of D. ambrosioides by spraying

Caption: * Significance level p < 0.05, ** p < 0.1. 1- adjuvant concentration, 2- inlet temperature, 3- feed flow, L- linear, Q- quadratic.
The concentration of Aerosil® exerted negative linear and quadratic effects on the volatile content. The model was significant (p = 0.000042) and showed no lack of fit (p = 0.082978), it showed a coefficient of determination (r2) of 0.91515 and an adjusted r2 of 0.76242. The response surface graphs, described in Drawing 2, suggest that adequate conditions for the volatile content were achieved at adjuvant concentrations greater than 30% of the mass of the solids content of the extract, regardless of the value of the analyzed parameter within the established range . The best levels of flavonoids were achieved in the intervals with feed flow above 0.4 L/h and temperature below 120°C and feed flow below 0.48 L/h and temperature above 155°C, showing no effect. significant between individual variables or interaction between them according to statistical data in Table 7.
The flavonoid content, expressed as rutin equivalents, did not show significant relationships for the variables, Aerosil® concentration (1), inlet temperature (2) and feed flow (3). However, the interaction of Aerosil® parameters and feed flow was significant (p=0.07) when p-value lower than 0.1 was considered. The model was significant (p = 0.001437) and showed no lack of fit (p = 0.189142), indicating a coefficient of determination (r2 ) of 0.73323. In the response surface analysis, according to Drawing 3, a significant relationship between adjuvant concentration and feed flow was observed, indicating that the highest levels were achieved in adjuvant concentration greater than 30% and feed flow greater than 0.38 L /H. Although high inlet temperatures can cause the degradation of extract components, the parameter did not present a significant effect in relation to the observed variables. However, it is noted that the best levels of flavonoids were achieved at temperatures above 130°C (Drawing 3), which is in agreement with the thermogravimetric analysis described in the literature, whose decomposition of the chosen marker occurs significantly at temperatures above 261°C (COSTA et al., 2002).
The best drying conditions of the D. ambrosioides extract, for the variables adjuvant concentration (Aerosil®), inlet temperature (°C) and feed flow (L/h) were determined from the response surface data and of the general optimization function and are described in Table 8, at a desirability of 0.739467. The values predicted by the statistical model for the responses, drying yield, volatile content and total flavonoid content, under optimized conditions, are also presented in Table 8.
The conditions predicted by the model were validated through the analysis of the yield and characterization of 3 dryings carried out in the optimized conditions so that the reproducibility was observed. The triplicate data are presented in Table 9.
Table 8: Best drying conditions of D. ambrosioides extract for the variables, adjuvant concentration (Aerosil®), inlet temperature (°C) and feed flow (L/h) and values predicted by the model for the dependent variables

Legend: R- yield, TV- volatile content; TF- content of flavonoids.*percentage of Aerosil® in relation to the mass of total solids.
Table 9: Values observed for yield, volatile content and total flavonoid content expressed as rutin in the dry extract obtained from aerial parts of D. ambrosioides by spraying

Legend: RSD: relative standard deviation; %* percentage over predicted value. Drying conditions: sprinkler nozzle diameter of 0.7 mm, compressed air flow of 40L/min, compressed air pressure of 4.0 Kg/F; adjuvant concentration: 50% (w/w), inlet temperature 148°C, feed flow: 0.5 L/h.
The results observed in triplicate showed that the model was efficient in predicting responses. The experiment showed values in good agreement with the predicted results, demonstrating the suitability of the chosen model in demonstrating the influence of these parameters in the optimization of the response variables for the drying of extracts obtained from the aerial parts of D. ambrosioides.
k) step 11: After obtaining the dry extract under optimized conditions, the morphology of the particles was analyzed by scanning electron microscopy (SEM). The dry extract samples were gold plated and the morphological characterization was obtained in a Jeol Scanning Electron Microscope, JSM - 6610, equipped with EDS, Thermo scientific NSS Spectral Imaging, at the Multiuser Laboratory of High Resolution Microscopy (LabMic) of the University Federal de Goiás. It was verified the presence of agglomerated structures composed of individual grains of porous surface joined by submicron dust of the same material, of different sizes and shapes (Drawing 4). The shapes of the grains varied from irregular to spherical and the size of 1.4 µm to particles with dimensions greater than 22.7 µm in diameter. In the dry extract, it was also observed that the larger spheres were filled by smaller spheres. This characteristic can be explained by the permeability and elasticity of the surface of the sphere, generating grains with an imperfect or fragmented surface, solid or hollow (OLIVEIRA; PETROVICK et al., 2010).
l) step 12: The Artemia salina Leach bioassay was used to estimate the bioactivity of the dry extract of D. ambrosioides. The bioassay, using previously defined concentrations of the dry extract, was performed in 96-well polystyrene microplates with the microcustaceous, according to the methodology proposed by Molina-Salinas and SaidFernandez (2006), with adaptations. In this work, 60 mg of Artemia salina cysts were incubated in a pot with half of synthetic marine water, prepared by dissolving sea salt in distilled water (36 g/L), plus yeast extract (6 mg/L). and sterilized in an autoclave. This solution was used to hatch A. salina eggs and to prepare the other dilutions. During a period of 36 hours, the medium was preserved under constant oxygen saturation at room temperature and natural lighting for the eggs to hatch. The samples (dry extract of D. ambrosioides and Aerosil®) were diluted in 5% propylene glycol and saline to obtain concentrations of 2000, 1000, 500, 250 and 125 μg/mL. For the experiment, 8 columns of microplates were used, of which 3 columns were reserved for the dry extract in decreasing concentrations, 3 columns for Aerosil® with concentrations distributed similarly to the extract, and 1 column for the positive control (K2CrO7 ) and 1 column for the negative control (saline water). Each well was filled with 10 ± 1 nauplii suspended in 100 μL saline and topped up with 100 μL of the respective treatment. Subsequently, after 24 hours of incubation, dead or immobile microcrustaceans were counted for more than 10 seconds. All assays were performed in triplicate. Toxicity was expressed as the lethal concentration at 50% of the microcrustacean population by calculating the LC50 estimated from the linear regression obtained from the correlation between the percentage of dead individuals and the concentration of the test substance, using the Probitos method. The classification of the level of toxicity was established according to the criterion Nguta et al. (2012). The study divides as strong toxicity values of LC50 up to 100 µg/mL, moderate toxicity for LC50 between 100 and 500 µg/ml and low toxicity for LC50 between 500 µg/mL and 1000 µg/mL, and non-toxic above 1000 µg/mL. ml. The dry extract of D. ambrosioides and Aerosil® did not cause deaths of brine shrimp at the concentrations tested (2000, 1000, 500, 250 and 125 μg/mL). Due to the absence of deaths, it was not possible to estimate the LC50, therefore, it is suggested that it is above the analyzed concentrations. The study by Nguta et al. (2012) classifies that plant extracts with LC50 higher than 1000 µg/ml are non-toxic, corroborating the classification of the dry extract of D. ambrosioides as non-toxic.
m) step 13: To evaluate the acute oral toxicity of the dry extract of D. ambrosioides in rodents, protocol No. 423 of the Organization for Economic Co-operation and Development (Organization for Economic Co-operation and Development – OEDC, 2001) was used. The experiments were conducted in accordance with the norms of the Council for Animal Experimentation (CONCEA). They were approved by the Ethics Committee on the Use of Animals (CEUA) of the State University of Goiás (UEG), according to protocol No. 006/2017. Nulliparous female Wistar rats (Rattus norvegicus), aged between 68 and 81 days, weighing between 144.22 - 210.68 g were used. The animals were kept throughout the experimental period in the vivarium of the Laboratory of Pharmacology and Toxicology of Natural and Synthetic Products of the State University of Goiás (UEG). Temperature and humidity conditions were controlled, light followed a 12-hour light/dark cycle (lights on from 7:00 am to 7:00 pm) and animals were treated with water and feed ad libitum. In this study the animals were divided into two experimental groups (group 1: control (Aerosil®) group 2: Test (dose of 300 and 2000 mg/kg) with three rats in each group. The samples were administered via gavage. observed for 8 consecutive hours on the first day of the experiment. In the remaining 14 days, 1 hour was observed in the morning. The signs of toxicity investigated were: changes or alterations in the skin or fur; alterations in the eyes and mucous membranes; in the circulatory, respiratory, central nervous and autonomic. In addition, possible alterations in the behavioral and somatomotor pattern (tremors, prostration, paralysis, exaggerated responses to noise or touch - hyperreflexia) were also investigated. at necropsy. The organs were analyzed regarding their general appearance, and the mass of the kidneys, liver was determined. The dry extract of the aerial parts of D. ambrosioides was dissolved 10% propylene glycol and water, in concentrations according to the weight of the animals, in order to obtain doses of 300 and 2000 mg/kg. The administration volume used was 0.5 mL for each 100g of body mass for doses of 300 and 2000mg/kg. In the control group, Aerosil® was used at a dose of 667 mg/Kg to verify if the adjuvant influenced the toxicity of the extract, and this was prepared in 10% propylene glycol and water. ANOVA analysis of variance followed by the Student-Newman-Keuls test was used to identify the differences between the control and experimental groups, using the Statistica® software version 12.0 (STATSOFT, 2010). One-way ANOVA was used to evaluate body mass gain and relative organ mass, and ANOVA for repeated measures was used to evaluate food and water consumption. Statistically significant differences were indicated when p<0.05. The behavioral clinical signs presented by the animals during the first hours, evidenced by the Hippocratic screening, after the administration of the extract were: in the control group the animals showed hyperactivity, decreased response to tail pressure, and compulsive behavior. At the dose of 300mg/kg, difficulty in breathing (hyperventilation) and reduced response to tail pressure were observed. At the dose of 2000mg/kg, it was observed in the first 15 minutes that the animals were breathing heavily, and the reduction in response to tail pressure persisted. The persistence of tail analgesia, especially at higher doses of the extract, is partly explained by the antinociceptive and anti-inflammatory properties of the plant (IBIRONKE; AJIBOYE, 2007; TRIVELLATO-GRASSI et al., 2013; CALADO et al., 2015). During the test, there were no deaths of animals in the studied groups, not even during the test repetition stage. Thus, the standardized dry extract for flavonoids obtained from aerial parts of D. ambrosioides is classified in category “5”, with an estimated LD50 between 2000 and 5000 mg/Kg, according to OECD guide n° 423. This guide considers LD50 higher than 2000 mg/Kg as practically non-toxic, and does not recommend the continuation of the experiment for the dose of 5000 mg/Kg. Since the dose is difficult to dissolve and administer, in addition to the need to protect animal life used in the laboratory (OECD, 2001). In the one-way ANOVA statistical analysis for body mass gain, no interference of the treatment on the analyzed groups was demonstrated (F2.15 = 0.28; p = 0.76). For the repeated measures ANOVA, regarding the relative body mass, there was also no effect of the treatment on the groups (F2.15 = 2.16; p = 0.15) and there was no significant interaction when the treatment effect was analyzed. on days (F26.195 = 0.71; p = 0.85). However, the days factor proved to be significant during the trial (F13.195 = 79.44; p < 0.01). When analyzing the effect of treatments on feed intake, in the one-way ANOVA, no interaction was observed (F2.15 = 0.80; p = 0.47). When evaluating the feed consumption relative to every 3 days, the ANOVA for repeated measures showed no treatment effect (F2.15 = 0.34; p = 0.71) and no interaction between days and treatment (F8.60 = 0 .79; p = 0.61). However, the days factor, analyzed separately, proved to be significant (F4.60 = 11.03; p < 0.01). In the analysis of the relative consumption of water every 3 days, to the detriment of treatment, the one-way ANOVA was not significant (F2.15 = 1.05; p = 0.37). In the ANOVA for repeated measures, no treatment effect was demonstrated (F2.15 = 0.85; p = 0.45), there was also no effect for the days factor (F4.60 = 1.91; p = 0.12) , and no interaction between days and treatment (F8.60 = 0.33; p = 0.95). In the analysis of the relative mass of the organs, the one-way ANOVA did not show differences between the groups for the mass of the kidneys (F2.15 = 0.71; p = 0.50) and liver (F2.15 = 2.36; p = 0.13).

[25] The result was the dry extract, standardized in total flavonoids expressed as rutin, from the aerial parts of D. ambrosioides, with a high level of quality. As for the toxicity tests, in the Artemia salina toxicity test, the dry extract was considered non-toxic. In the experiment of acute oral toxicity in rats, the dry extract falls into category “5”, of low toxicity with LD50 between 2000 and 5000 mg/kg. This extract presents technological innovations applied to the processes of obtaining standardized extracts. It is raw material to be used in the development of technologically elaborated medicines, with high added value, enabling the development of different pharmaceutical formulas, which present greater stability and dosage uniformity.

V. Brief Description of the Drawings

[26] Drawing 1: Graphs of response surface for yield (A – C) as a function of independent variables in obtaining dry extract of aerial parts of D. ambrosioides by spraying. Legend: (A) adjuvant concentration (%, w/w)* and inlet temperature (°C); (B) adjuvant concentration (%, w/w)* and feed flow (L/h); (C) inlet temperature (°C) and feed flow (L/h).* percentage of Aerosil® in relation to the mass of total solids.

[27] Drawing 2: Graphs of response surface for volatile content (A – C) as a function of independent variables in obtaining dry extract of aerial parts of D. ambrosioides by spraying. Legend: (A) adjuvant concentration (%, w/w)* and inlet temperature (°C); (B) adjuvant concentration (%, w/w)* and feed flow (L/h); (C) inlet temperature (°C) and feed flow (L/h).* percentage of Aerosil® in relation to the mass of total solids.

[28] Drawing 3: Graphs of response surface for total flavonoids (A – C), expressed as rutin equivalents, as a function of independent variables in obtaining dry extract of aerial parts of D. ambrosioides by spraying. Legend: (A) adjuvant concentration (%, w/w)* and inlet temperature (°C); (B) adjuvant concentration (%, w/w)* and feed flow (L/h); (C) inlet temperature (°C) and feed flow (L/h).* percentage of Aerosil® in relation to the mass of total solids.

[29] Drawing 4: Scanning electron microscopy of dry extract of D. ambrosioides A/B – photomicrograph of the 1st replicate; C/D - photomicrograph of the 2nd replicate; E/F - photomicrograph of the 3rd replicate.

Claims (22)

Standardized dry extract characterized by comprising flavonoids, expressed in rutin equivalents, obtained by the nebulization/atomization technique (spray drying), from the aerial parts of Dysphania ambrosioides with therapeutic applications. Process for obtaining the standardized dry extract of claim "1" characterized in that it comprises the following steps;

• a) Submit the plant matrix (aerial parts) of the plant Dysphania ambrosioides (santa-maria herb) to pre-treatment, comprising drying and grinding;
• b) Characterize the powder obtained: granulometric analysis, volatile content, investigation of flavonoids by spectrophotometry;
• c) Standardize extractive methods;
• d) Extract the active ingredients by the percolation extractive method, using a hydroethanolic solution;
• e) Concentrate the macerated/percolated extract in a concentrator;
• f) Characterize the crude extract: determine the pH, relative density, viscosity, alcohol content, solids content, investigation of flavonoids, expressed in rutin equivalents;
• g) Dry the crude extract, using the nebulization process (spray drying);
• h) Evaluate the drying process: inlet and outlet temperatures of drying air, drying air flow, drying air pressure, compressed air flow, compressed air pressure, dispersion flow to nebulize/atomize, yield of the powder obtained, percentage of degradation of the markers;
• i) Characterize the dry extract: water content, volatile content, scanning electron microscopy, investigation and quantification of flavonoids, expressed in rutin equivalents, by spectrophotometric method;
• j) Define the pharmacotechnical properties of the dry extract according to the application.

Use of the standardized dry extract obtained by the nebulization/atomization technique (spray dryong) of claims "1 and 2", characterized by being used in the preparation of a drug, analgesic, anti-inflammatory, antimicrobial, for human, veterinary, dental, cosmetic or dermocosmetic. Liquid pharmaceutical composition, oral administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Liquid pharmaceutical composition, topical administration route, characterized by containing flavonoids, expressed in rutin equivalents, process of obtaining and applications according to claims “1, 2 and 3”; Semi-solid pharmaceutical composition, topical administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Solid pharmaceutical composition, oral administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Liquid pharmaceutical composition, nasal administration route, characterized by containing flavonoids, expressed in rutin equivalents, process of obtaining and applications according to claims “1, 2 and 3”; Solid pharmaceutical composition, nasal administration route, characterized by containing flavonoids, expressed in rutin equivalents, process of obtaining and applications according to claims “1, 2 and 3”; Pharmaceutical composition of microcapsules, oral administration route, characterized by containing flavonoids, expressed in rutin equivalents, process of obtaining and applications according to claims "1, 2 and 3" Pharmaceutical composition in the form of microcapsules, topical administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of microcapsules, nasal administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of nanocapsules, oral administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of nanocapsules, nasal administration route, characterized by containing flavonoids, expressed in rutin equivalents, process of obtaining and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of nanocapsules, topical administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of liposomes, topical administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of liposomes, oral administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of liposomes, nasal administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of nanoemulsions, topical administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of nanoemulsions, oral administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of a release system composed of liquid crystals, characterized by containing flavonoids, expressed in rutin equivalents, process of obtaining and applications according to claims “1, 2 and 3”; Pharmaceutical composition in the form of beads, oral administration route, characterized by containing flavonoids, expressed in rutin equivalents, obtaining process and applications according to claims “1, 2 and 3”;

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