Development and validation of an analytical method by HPLC-DAD for determination of caffeine in products based on guarana extracts (Paullinia cupana)

Abstract Guarana (Paullinia cupana) is a native plant from the Amazon whose seeds contain a high concentration of caffeine. Aqueous extract of guarana is widely used in the world. In this study, the objective was to develop and validate a High-Performance Liquid Chromatography method for the determination of caffeine in extracts and commercial beverages based on guarana. A sensitive, simple, and viable high performance liquid chromatographic method without the need of an analyte extraction procedure was developed and validated according to Brazilian and international requirements. The method presented high performance, fulfilling Brazilian and international requirements, in addition to allowing product compliance tests. Results confirmed high selectivity and linearity (>0.999) between 5 to 135 ug/mL, with no significant matrix effect. Detection and quantification limits were 0.02 µg/mL and 2 µg/mL, respectively. Precision was less than 4 %, and accuracy varied from 99.9-120 %. Applicability of the method was demonstrated by conducting a limited evaluation in products containing caffeine. Commercial extracts showed quite different caffeine levels, while carbonated drinks follow Brazilian and American recommendations. Our results indicate that the developed method can be used to evaluate the quality of the guarana extract and of products containing caffeine

Analysis of Chemical Constituents in Paullinia cupana Dried Seeds by UPLC-Q-TOF-MS / 中国实验方剂学杂志

Objective:The chemical constituents in guarana （<italic>Paullinia cupana</italic> dried seeds） were systematically analyzed to provide a basis for further research， development and utilization of this plant. Method:The contents of crude protein， crude fat， crude polysaccharide and crude fiber in guarana were determined according to national standards and related documents， and the chemical constituents of guarana was qualitatively analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF-MS）， ACQUITY UPLC-HSS-T3 column （2.1 mm×100 mm， 1.8 μm） was used with 0.1% formic acid aqueous solution （A）-0.1% formic acid acetonitrile solution （B） as mobile phase for gradient elution （0-5 min， 2%-10%B； 5-6 min， 10%-20%B； 6-9 min， 20%-30%B； 9-9.5 min， 30%-35%B； 9.5-10.5 min， 35%-45%B； 10.5~13 min， 45%-55%B； 13-15 min， 55%-80%B； 15-19 min， 80%-98%B； 19-20 min， 98%B； 20-20.3 min， 98%-2%B； 20.3-23 min， 2%B）， the electrospray ionization （ESI） was used for detection in positive and negative ion modes， the scanning range was <italic>m</italic>/<italic>z</italic> 50-1 500， and the structure was identified according to the relative molecular weight and fragment information combined with database matching and comparison of reference substances. Result:The contents crude protein， crude fat， crude polysaccharide and crude fiber in guarana were （0.63±0.03）%， （2.73±0.09）%， （3.23±0.12）% and （8.89±0.59）%， respectively. A total of 42 chemical constituents in guarana were identified by UPLC-Q-TOF-MS， including 3 methylxanthines， 2 nucleosides， 1 amino acid， 3 organic acids， 33 flavonoids， 3 （<italic>L</italic>-tryptophan， epigallocatechin gallate， daidzein） of which were first discovered in guarana. Conclusion:Guarana is rich in nutrients and has good potential to be developed as a functional food. UPLC-Q-TOF-MS technique provides a simple， rapid and accurate method for the identification of chemical constituents in guarana. Methylxanthines and proanthocyanidins are the main chemical constituents of guarana， which is meaningful for quality evaluation and material basis of guarana.

Colletotrichum siamense, a Mycovirus-Carrying Endophyte, as a Biological Control Strategy for Anthracnose in Guarana Plants

Abstract Guarana, the fruit of Paullinia cupana, is known for its stimulating and medicinal properties by the Amazonian indigenous population and communities. However, it presents serious phytopathological problems, such as anthracnose disease caused by Colletotrichum spp. The objective of this study was to verify if C. siamense, a mycovirus-carrying endophytic fungus, could protect guarana seedlings, by reducing or by eliminating characteristic disease symptoms. Other physiological changes in the plant caused by the presence of this endophyte were also evaluated. The cuttings of the Cereçaporanga cultivar were dipped in a biological control suspension and planted in a specific substrate. After four months in the greenhouse, the seedlings were sprayed with a suspension of phytopathogen conidia, and a portion of these seedlings received the fungicide indicated for the crop to be compared with the control seedlings. After 28 days, the number of lesions, morphophysiological and macro characteristics, and leaf micronutrients were evaluated. The seedlings treated with C. siamense showed a lower percentage of lesions and an increased aerial part and root system compared to the other treatments. There were no significant differences between treatments regarding the percentage of macronutrients and micronutrients.

Microparticles containing guaraná extract obtained by spray-drying technique: development and characterization

AbstractGuaraná (Paullinia cupana Kunth, Sapindaceae) is well known for its dietary and pharmaceutical potential, and the semipurified extract of guaraná shows antidepressant and panicolytic effects. However, the low solubility, bioavailability and stability of the semipurified extract limit its use as a component of pharmaceutical agents. Delivery of the semipurified extract in a microparticle form could help to optimize its stability. In this study, microparticles containing semipurified extract of guaraná were obtained by the spray-drying technique, using a combination of maltodextrin and gum arabic. The raw materials and microparticles produced were characterized by particle size analysis, differential scanning calorimetry, thermogravimetric analysis, and scanning electron microscopy. The drug content and antioxidant capacity were also evaluated. In vitrodissolution tests using flow cell dissolution apparatus, were carried out to investigate the influence of formulation parameters on the release of semipurified extract of guaraná from the microparticles. The spray-drying technique and the processing conditions selected gave satisfactory encapsulation efficiency (80–110%) and product yield (55–60%). The mean diameter of microparticles was around 4.5 µm. The DPPH radical scavenging capacity demonstrated that microparticles can protect the semipurified extract of guaraná from the effect of high temperatures during the process maintained the antioxidant capacity. Differential scanning calorimetry results indicated an interaction between semipurified extract of guaraná and gum arabic: maltodextrin in the microparticles, and thermogravimetric analysis indicate that the profile curves of the microparticles are similar to the adjuvants used in drying, probably due to the higher proportion of adjuvants compared to semipurified extract of guaraná. In vitro dissolution tests demonstrate that all formulations complete dissolution within 60 min. Microencapsulation improved the technological characteristics of the powders and preserved the antioxidant properties. The study demonstrated the feasibility of producing these microparticles for a one-step process using spray drying. The composition of each formulation influenced the physical and chemical characteristics. This spray-drying technique can be used as an efficient and economical approach to produce semipurified extract of guaraná microparticles.

Evaluation of neurotransmitters involved in the anxiolytic and panicolytic effect of the aqueous fraction of Paullinia cupana (guaraná) in elevated T maze

This study investigated the effects of repeatedly administration of an aqueous fraction of Paullinia cupana Kunth, Sapindaceae (guaraná) seeds (8 mg/kg) on rats submitted to the elevated T-maze, model of generalized anxiety and panic disorders. The selective serotonin reuptake inhibitor paroxetine (3 mg/kg), was used as a positive control. To evaluate possible neurotransmissions involvement, ineffective doses of metergoline (3 mg/kg - non-selective serotonin receptor antagonist), sulpiride (20 mg/kg - non-selective dopaminergic receptor antagonist) or ketamine (0.125 mg/kg - non-selective glutamate receptor antagonist) were acutely administered in association with the aqueous fraction of P. cupana. Both aqueous fraction and paroxetine decrease the inhibitory avoidance latencies of the elevated T-maze, indicating anxiolytic effect and increased one-way escape latencies from the open arm of the elevated T-maze, indicating a panicolytic effect. The pre-treatment with metergoline, sulpiride and ketamine blocked the anxiolytic effect of aqueous fraction. The panicolytic effect of aqueous fraction was blocked by both metergoline and sulpiride. These results show that the serotonergic, dopaminergic and glutamatergic neurotransmission systems are involved in anxiolytic effect promoted by aqueous fraction, whereas only the serotonergic and the dopaminergic neurotransmission systems are involved in the panicolytic effect promoted by aqueous fraction of P. cupana. The effects produced by paroxetine, were blocked only by metergoline, validating this experimental procedure.

Development of tablets containing semipurified extract of guaraná (Paullinia cupana)

This study evaluated the technological feasibility of producing a semipurified extract of guaraná (Paullinia cupana Kunth, Sapindaceae) in tablet form, using a direct-compression process. Maltodextrin and gum arabic were used to produce the extract microparticles, in order to protect the microparticles against such factors as temperature, oxidation, and humidity. Using pharmacopoeial methodologies, technological and physicochemistry tests (determination of residual moisture, of bulk and tapped density, Hausner ratio, compressibility and compactibility index, appearance, mean weight, hardness, friability, disintegration time, determination of EPA amount in tablets and in vitro release profile) were conducted. The formulation containing 200 mg of microparticles, 170 mg microcrystalline cellulose, and 10 mg lactose gave the best results in terms of hardness (116 N), friabilility (0.28%), mean weight (0.3821 g), and disintegration time (25 min) for a tablet designed for oral administration. The results met pharmacopoeial specifications, and the tablets are suitable for oral administration.

Associação entre características da parte aérea e do sistema radicular em mudas de guaranazeiro por correlações canônicas / Canonical correlations among characteristics of shoots and roots system in guarana seedlings

Este trabalho teve como objetivo quantificar o grau de asociação entre variáveis de parte aérea e de raízes em mudas clonadas de guaranazeiro, utilizando-se correlações canônicas, a fim de aperfeiçoar o procedimento de seleção de mudas para garantir o aumento da porcentagem de sobrevivência das mudas após o plantio. Foram avaliados dois grupos de variáveis em mudas aptas ao plantio definitivo de 36 clones de guaranazeiro. O delineamento usado foi o aleatorizado em blocos com cinco repetições e 10 plantas por parcela, sob condições de viveiro. Os caracteres avaliados foram submetidos à análise de correlações canônicas. Utilizou-se a análise de correlações canônicas. O grupo de variáveis da parte aérea não se mostrou independente do grupo de variáveis do sistema radicular. Através de seleção baseada em variáveis da parte aérea pode-se melhorar o sistema radicular, principalmente através do maior comprimento do ramo (CRA). A seleção de clones de guaraná para maior peso de raiz pode ser efetuada de forma indireta, realizando-se mensurações do comprimento dos ramos, o que evita a necessidade de se destruir as mudas.

This study aimed to quantify the degree of association between variables of shoots and roots system of seedlings cloned from guarana, using canonical correlations, in order to improve the procedure of selection of seedlings to ensure increased survival percentage of seedlings after planting. Two groups of variables suitable for final planting seedlings in 36 guarana clones. The experimental design was a randomized complete block with five replications and 10 plants per plot, under nursery conditions. We used the canonical correlation analysis. The group of variables of shoot is not independent of variable group of root system. Through selection based on variables from the air, you can improve the root system, mainly through the greater length of the branch (CRA). Can practice the selection of clones of guarana for increased root weight without the need to destroy the seedlings carrying only measurements of the length of the branches.

Dissolution test of herbal medicines containing Paullinia cupana: validation of methods for quantification and assessment of dissolution / Ensaio de dissolução de medicamentos fitoterápicos contendo Paullinia cupana: validação de métodos de quantificação e avaliação de dissolução

"Guaraná" (Paullinia cupana) is used as a physical activity enhancer and stimulator due to its methylxanthines and condensed tannins. The aim of this work was to evaluate the dissolution behavior of five herbal medicines in the form of capsules and tablets containing guaraná. Assay and dissolution methods were validated and results obtained allowed simultaneous marker quantification with precision, accuracy, selectivity and robustness. Findings showed that 100 percent of the herbal medicinal products analyzed did not provide satisfactory results concerning the presence of four markers, 60 percent had three markers (caffeine, catechin and epicatechin), while 40 percent had only caffeine at tested dosage forms. In addition, after 30 minutes, only capsule A showed at least 80 percent of the dissolved markers. In other capsules, marker dissolution did not exceed 60 percent whereas 60 percent of the samples had some characteristic pharmacotechnical problems. These results evidence the need for rigorous quality control to help ensure the therapeutic action of these drugs. To this end, dissolution studies are an essential tool for quality assurance of herbal medicines.

Guaraná (Paullinia cupana) é utilizado como revigorante e estimulante devido à presença de metilxantinas e taninos condensados. Este trabalho visou avaliar o comportamento de dissolução de cinco fitoterápicos, na forma de cápsulas e comprimidos, contendo guaraná. O método de quantificação e de dissolução foram validados e os resultados obtidos permitiram a quantificação dos marcadores simultaneamente, com precisão, exatidão, seletividade e robustez. Foi verificado que 100 por cento dos fitoterápicos analisados encontravam em desacordo quanto à presença dos quatro marcadores, sendo que 60 por cento apresentaram três marcadores (cafeína, catequina e epicatequina) e 40 por cento apresentaram somente a cafeína. Além disso, após o tempo de 30 minutos de ensaio foi possível observar que somente a cápsula A apresentou pelo menos 80 por cento dos marcadores dissolvidos. Nas demais cápsulas o comportamento de dissolução apresentado pelos marcadores não ultrapassou 60 por cento e, além disso, 60 por cento das amostras apresentaram alguns problemas farmacotécnicos característicos. Frente aos resultados obtidos torna-se evidente a necessidade de um rigoroso controle de qualidade que contribua para assegurar a ação terapêutica desses medicamentos e, nesse sentido, o estudo de dissolução constitui uma ferramenta essencial para a garantia de qualidade dos fitoterápicos.

Produção e distribuição de fitomassa no guaranazeiro (Paullinia cupana H.B.K. var. sorbilis (Mart.) Ducke) / Production and distribution of phytomass in guarana plants

Apesar da importância econômica do guaranazeiro (Paullinia cupana H.B.K. var. sorbilis (Mart.) Ducke) em algumas regiões do Brasil, existem poucas informações sobre sua caracterização fenológica. Assim, realizou-se um estudo cujo objetivo foi de quantificar a produção de fitomassa e sua distribuição, em uma população de guaranazeiros, originadas de propagação sexuada. Foram avaliadas cinco plantas adultas, que lançaram mais de 100 ramos por planta ano-1, dos quais apenas 50 por cento produziram frutos. As plantas lançaram entre 445 e 1294 inflorescências por ano, porém, de 67 por cento a 98 por cento produziram apenas um fruto. A produção de matéria seca total foi estimada em 15,8 kg por planta ao ano, o que corresponde 6350 kg ha ano-1. Nas plantas, o maior acúmulo de matéria seca apresentou a seguinte ordem: folhas>ramos>sementes>ráquis>casca dos frutos+arilo> pecíolos.

The commerce of guarana seeds (Paullinia cupana H.B.K. var. sorbilis (Mart.) Ducke) is economically important in some regions of Brazil. However, few studies are found about its phenological characteristics. The aim of this paper was to study the production and distribution of organic matter in adult plants of guarana. Five individuals, which had more than 100 branches per plant/year, were evaluated. One found out that only 50 percent of the branches had fruits. The guarana plants had between 445 and 1294 inflorescences but of these, 67 percent to 98 percent had only one fruit. The total organic matter was evaluated in 15.8 kg per plant year-1 which means around 6350 kg ha year-1. The organic matter storage in guarana plant had the following sequence: leaves>branches>seeds>rachis> fruit peel+aril> petiole.

Spectrophotometric determination of tannins and caffeine in preparations from Paullinia cupana var. sorbilis

The present work was designed to quantify the caffeine and total polyphenols in the extractive solution and the granulated form from the seeds of Paullinia cupana var. sorbilis, by the spectrophotometric method. The method showed linearity for the caffeine and polyphenols in the range of 5-25 µg/ml and 2.4-5.6 µg/ml respectively. The solutions of the semipurified fraction (EPA) and granulated form (GRA) showed linear responses in the range of 0.288-0.672 and 0.4-1.2 µg/ml, respectively. The precision and accuracy were determined for the EPA solution at a concentration of 100 µg/ml. The spectrophotometric method performed well in quantifying the caffeine and total polyphenols.

A qualidade de preparações fitofarmacêuticas deve ser avaliada de acordo com os requisitos estabelecidos pela Agência Nacional de Vigilancia Sanitária na RDC n. 48 (ANVISA-BRASIL). As análises para avaliar a integridade dos produtos da droga vegetal incluem a quantificação de substâncias marcadoras através de métodos validados. O trabalho objetivou quantificar cafeína e polifenóis totais em soluções extrativas e no granulado obtidos das sementes de P. cupana var. sorbilis através de método espectrofotométrico. O método apresentou linearidade para a cafeína e polifenóis no intervalo de 5-25 µg/ml e 2,4-5,6 µg/ml, respectivamente. Soluções da fração semipurificada (EPA) e do granulado (GRA) mostraram resposta linear no intervalo de 0,288-0,672 µg/ml e 0,4-1,2 µg/ml respectivamente. A precisão e exatidão foram determinadas para a solução de EPA na concentração de 100 µg/ml. O método espectrofotométrico obteve um bom desempenho na quantificação de cafeína e polifenóis totais, uma vez que a presença de interferentes foi previamente avaliada.

Determinação do teor de princípios ativos em matérias-primas vegetais / Determination of active principles in vegetable raw materials

Castanha-da-índia (Aesculus hippocastanum L.), hipérico (Hypericum perforatum L.), guaraná (Paullinia cupana H.B.K.) e ginkgo (Ginkgo biloba L.) fazem parte de formulações de medicamentos fitoterápicos muito comercializados no Brasil. Neste trabalho foi avaliada a qualidade de amostras de pós e extratos destas plantas. Para a determinação do teor de princípios ativos foram aplicados métodos farmacopéicos ou citados na literatura científica e os resultados demonstraram serem adequados para a análise destes produtos farmacêuticos. Verificou-se que 60 por cento das amostras analisadas de guaraná e ginkgo estavam em desacordo com as especificações farmacopéicas ou as declaradas pelos laudos dos fabricantes, em relação ao teor de princípios ativos. Além disso, 100 por cento e 20 por cento, respectivamente, das amostras de hipérico e de castanha-da-índia apresentaram teores abaixo das especificações. Os dados obtidos mostraram ser de grande importância e necessidade a implementação de técnicas quantitativas no controle de qualidade físico-químico de matérias-primas vegetais.

Pharmaceutical preparations containing Aesculus hippocastanum L., Hypericum perforatum L., Paullinia cupana H.B.K. and Ginkgo biloba L. have large market. In this work the quality of the powder or extracts of these plants were evaluated. Pharmacopoeia methods or those cited on cientific literature for quantification of active substances were employed and the data showed them to be adequate for these analyses. The analytical results demonstrated that 60 percent of the samples of P. cupana and G. biloba, and 100 percent and 20 percent, respectively of samples of H. perforatum and A. hippocastanum were not in agreement with the specifications. This analysis showed to be valuable for herbal medicines quality control.

Avaliação físico-química de sementes de guaraná secas por diferentes métodos / Physico-chemical evaluation of guarana seeds dried by different methods

Amostras de sementes de guaraná provenientes da região amazônica e secas por diferentes métodos, foram analisadas por técnicas farmacopéicas e outras, e o extrato liofilizado foi analisado por cromatografia líquida de alta eficiência (CLAE). Realizou-se o doseamento de metilxantinas (MX) e dos taninos totais (TT), através de métodos espectrofotométricos, para a comparação das amostras. O maior teor de MX foi obtido com as sementes secas em tacho metálico por 4 h com adição de água, enquanto que o maior teor de TI foi obtido com a amostra torrada em tacho metálico por 4 h sem água. A análise cromatográfica por CLAE apresentou tempos de retenção para catequina, epicatequina e cafeína de 6,17; 8,85 e 11,91 min, respectivamente.

Contrai quality of the vegetable drug "guaraná", with samples of seeds obtained from Amazonian area, were carried out by pharmacopoeial assays, and the extract were analysed by HPLC. The determination of methylxantines (MX) and total tannins (TI) was performed by spectrophotometric methods. The most yield of MX were showed by the sample GTIB and for TI the best result were obtained by the sample GTIM4s. The analysis by HPLC showed retention times for catechin, epicatechin and cafteine of 6.17,8.85 and 11.91 min, respectively.

On the occurrence of cyanolipids In Paullinia carpopodea cambess And P. cupana Kunth seed oils

Paullinia carpopodea seed oil contains 70% type Icyanolipids with cyanogenetic properties, as proven by chemical and spectrometric techniques. P. cupana seed oil also contains cyanogenic type I cyanolipids, according to its 1-NMR spectrum. The existing controversy in the literature about the presence and/or type of cyanolipids in P. cupana seed oil is probably due to the low amount of these compounds (0.2%) in the seeds.

O óleo das sementes de Paullinia carpopodea contem 70% de cianolipidios cianogenéticos, do tipo I, como mostrado através de métodos químicos e espectrométricos. O óleo de P. cupana contém o mesmo tipo de cianolipídios, como evidenciado através de seu espectro de 1-NMR. A controvérsia existente na literatura sobre a presença e/ou tipo de cianolipídios no óleo de P. cupana foi, provavelmente, causada pelo baixo teor com que estes componentes ocorrem nas suas sementes (0,2%).

Introdução da técnica espectrofotométrica na detecção da cafeína em guaraná / Use of the spectrophotometric method in the caffein determination in guarana

Foram comparados os resultados da determinação quantitativa da cafeína em guaraná de acordo com o método gravimétrico descrito na Farmacopeia Brasileira, 3a. edição, com os da determinação espectrofotométrica dos resíduos obtidos dissolvidos em ácido clorídrico 0,1 N, com leitura a 273nm. A presença de outras substâncias de natureza não xantílica no resíduo foi mostrada através de cromatografia em camada delgada, usando-se fases móveis de diferentes polaridades. Os valores obtidos por espectrofotometria foram sempre menores do que os obtidos por gravimetria (AU).