Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3749

Full Length Research Paper

Phyllanthin and hypophyllanthin determination by gas chromatography-mass spectrometry of six stonebreaker species from different regions of Brazil

Kátia Viviane Alves Novellino
  • Kátia Viviane Alves Novellino
  • Departamento de Produtos Naturais, Instituto de Tecnologia em Fármacos, Farmanguinhos, Fundação Oswaldo Cruz. Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ - 21040-900, Brasil.
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Suellen Rodrigues Bernardo
  • Suellen Rodrigues Bernardo
  • Departamento de Métodos Analíticos, Instituto de Tecnologia em Fármacos, Farmanguinhos, Fundação Oswaldo Cruz. Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ - 21040-900, Brasil.
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Marcelo Raul Romero Tappin
  • Marcelo Raul Romero Tappin
  • Departamento de Métodos Analíticos, Instituto de Tecnologia em Fármacos, Farmanguinhos, Fundação Oswaldo Cruz. Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ - 21040-900, Brasil.
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Rosa de Belém das Neves Alves
  • Rosa de Belém das Neves Alves
  • Recursos Genéticos e Biotecnologia, Empresa Brasileira de Pesquisa Agropecuária, Embrapa, Parque Estação Biológica - PqEB s/n°, Brasília, DF - 70770-901, Brasil.
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Dijalma Barbosa da Silva
  • Dijalma Barbosa da Silva
  • Recursos Genéticos e Biotecnologia, Empresa Brasileira de Pesquisa Agropecuária, Embrapa, Parque Estação Biológica - PqEB s/n°, Brasília, DF - 70770-901, Brasil.
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Marcos José da Silva
  • Marcos José da Silva
  • Departamento de Botânica,Instituto de Ciências Biológicas I, Universidade Federal de Goiás, UFG, Campus Samambaia II, Sala 204. Setor Itatiaia, Goiânia, GO - 74001-970, Brasil.
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Roberto Fontes Vieira
  • Roberto Fontes Vieira
  • Recursos Genéticos e Biotecnologia, Empresa Brasileira de Pesquisa Agropecuária, Embrapa, Parque Estação Biológica - PqEB s/n°, Brasília, DF - 70770-901, Brasil.
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Maria das Dores Dutra Behrens
  • Maria das Dores Dutra Behrens
  • Departamento de Produtos Naturais, Instituto de Tecnologia em Fármacos, Farmanguinhos, Fundação Oswaldo Cruz. Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ - 21040-900, Brasil.
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Davyson de Lima Moreira
  • Davyson de Lima Moreira
  • Departamento de Produtos Naturais, Instituto de Tecnologia em Fármacos, Farmanguinhos, Fundação Oswaldo Cruz. Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ - 21040-900, Brasil.
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  •  Received: 17 January 2020
  •  Accepted: 13 March 2020
  •  Published: 30 April 2020

 ABSTRACT

Stonebreaker is a medicinal plant traditionally used for the treatment of urolithiasis among other illnesses. However, several species of the genus Phyllanthus L. are called “quebra-pedra” (stonebreaker) by the general population in Brazil, due to taxonomic similarities, although the use of a species with a low content of active compounds may lead to a less effective treatment. P. niruri and P. tenellus are recognized as stonebreaker in the Brazilian Pharmacopoeia, and the Brazilian Ministry of Health included Phyllanthus spp in a list of medicinal plants recommended for research and development, indicating the need for further studies. Quantitative determination of the active compounds of different Phyllanthus species from Brazil is important because it allows to assess the impact of their role in public health and so contribute to the goal of RENISUS, the official list of species of interest to the national health service. Lignans comprise a group of compounds found in Phyllanthus species that have diverse pharmacological properties. Some of the most studied lignans are phyllanthin and hypophyllanthin, which have antihyperuricemic activity. The aim of this study was to develop an analytical method for the quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by GC-MS and to assess the content of these lignans in six species of the genus from four different regions of Brazil. The phyllanthin and hypophyllanthin contents were found to be very variable in the analyzed species, ranging from not detectable or a mere trace to 0.6% (m/m), the highest value, found in P. amarus.

Key words: Traditional medicine, urolithiasis, stonebreaker, quebra-pedra, Phyllanthus, lignans, gas chromatography-mass spectrometry (GC-MS)

 


 INTRODUCTION

Stonebreaker is a  medicinal  plant  traditionally  used  for the   treatment  of  urolithiasis,   diabetes   and   hepatitis. Several species of the genus Phyllanthus L. (Phyllanthaceae) are commonly recognized as “quebra-pedra” (stonebreaker) and due to their taxonomic similarities, all of them are collected as such by a population unable to differentiate them scientifically, which is liable to result in a less effective treatment when a species with low content of the active compounds is used (Ingliset al., 2018). Officially in Brazil, Phyllanthusniruri and Phyllanthustenellus both have monographs in the Brazilian Pharmacopoeia (Brazilian Health Surveillance Agency, 2019) and both the infusion and the tincture from P. niruriare indicated for the treatment of urolithiasis, as specified in the National Phytotherapy Formulary of the Brazilian Pharmacopoeia (Brazilian Health Surveillance Agency, 2011). The Brazilian Ministry of Health also included Phyllanthus spp in a list of species recommended for research and development of herbal medicines for public health indicating the need for further studies. The list included four species of the genus found in Brazil, to be compared in order to define scientifically which is best suited for production and therapeutic use (Brazilian Ministry of Health, 2009).

Several pharmacological properties are described for species of the genus Phyllanthus, justifying the interest on these species. They include the activities: anti-inflammatory and analgesic (Santos et al., 1994; Calixtoet al., 1998; Zhang et al., 2014; Chen and Chen, 2011; Chopade and Sayyad, 2013), antihyperuricemic (Murugaiyah and Chan, 2006), urolithiasis (Boim et al., 2010; Woottisinet al., 2011; Barros et al., 2003, 2006), hepatoprotective (Huang et al., 2003; Sriramaet al., 2012; Lee et al., 2006; Jain and Singhai, 2011), hypoglycemic (Hnatyszynet al., 2002) and antibacterial (Oliveira et al., 2007; Silva et al., 2010; Windayaniet al., 2015; Cesariet al., 2015). Different classes of compounds present in Phyllanthusextracts are believed to be responsible for the pharmacological properties, among them tannins, alkaloids, lignans and flavonoids (Calixtoet al., 1998). Variability in the content of these active compounds among different species of the genus Phyllanthuswas already known in India (Ravikanthet al., 2012) and poses a significant challenge also in Brazil because of the diversity of environments the country has. Therefore, the quantitative determination of these compounds in different species of this genus, and of their differences in content, would help assess the impact of their use in health (Nahar et al., 2012) and so contribute to the goalof RENISUS, the official list of species of interest to the national health service (Brazilian Ministry of Health, 2009).

Lignans   are   among   the    most    important     activein Phyllanthusspecies, as they have many described pharmacological properties such as antihyperuricemic (Murugaiyah and Chan, 2006) and hepatoprotective activities (Huang et al., 2003; Sriramaet al., 2012; Lee et al., 2006; Jain and Singhai, 2011). These are phenylpropanoid dimers with a broad range of structural diversity widely distributed in higher plants and identified in species of some 70 families. Many of these compounds have been used in traditional medicine and isolated from different plant parts such as bark, wood, resin, roots, leaves, flowers, fruits and seeds (Konuklugil, 1995). Antinociceptive studies using a murine model byChopade and Sayyad (2013) showed that the tannin corilagin and the lignans phyllanthin and hypophyllanthin were responsible for the analgesic effect of Phyllanthus amarus and Phyllanthus fraternus extracts. Murugaiyah and Chan (2006) demonstrated, also in a murine model, that the methanolic extract obtained from P. niruriand its lignans were able to reverse the high plasmatic uric acid levels in hyperuricemic animals. The antihyperuricemic effect was attributed to the lignansphyllanthin, hypophyllanthin and phyltetralin.

Tripathiet al. (2006) have quantitatively determined phyllanthin and hypophyllanthin (Figure 1) in several Phyllanthus species from India and found significant variation in the contents of these two lignans, which raises even more concern given the taxonomic similarities among these species. However, the monographs of P. niruri and P. tenellusin Brazilian Pharmacopoeia do not include the analysis of the lignan content, as though these species do not have lignans. Therefore, the aim of this study is to determine the contents of phyllanthin and hypophyllanthin in the four species of the genus Phyllanthus of the RENISUS list plus two other species commonly found in Brazil. To achieve this goal, Gas Chromatography-Mass Spectrometry (GC-MS) was used, three different GC columns were tested and a central composite design was performed to develop a method in the selected column.

 

 


 MATERIALS AND METHODS

Plant sample collection

Samples were obtained from collection sites in South, Southeast, Central-West, Northeast and North regions of Brazil.Phyllanthus niruri L. and Phyllanthus stipulates (Raf.) G.L. Webster were obtained from the Collection of Bioactive Plants of theAgricultural Research and Rural Extension Company of Santa Catarina Epagri, in Itajaí, Santa Catarina, Brazil (GPS 26º57'10''S; 48º45'47''W, South region); P. amarusSchum. &Thonn.was collected in the Multidisciplinary Center for Chemical, Biological and Agricultural    Research    in    Campinas,    São   Paulo,  Brazil  (GPS22º47'43''S; 47º06'46''W, Southeast region); P. tenellusRoxb. was collected in the State University of Santa Cruz in Ilhéus, Bahia, Brazil (GPS 14º10'26''S; 47º04'28''W, Northeast region); Phyllanthus caroliniensis Walter was collected from the Brazilian Agricultural Research Corporation, Embrapa, Genetic Resources and Biotechnology, Brasília, Brazil (GPS 15º43'50.1''S; 47º54'06.2''W, Central-West region) and Phyllanthus urinariaL. was collected in the Embrapa Eastern Amazon Research Institute in Belém, Pará, Brazil (GPS 01º27'21''S; 48º30'14''W, North region). The vouchers were deposited and identified at the Embrapa CEN Herbarium.

Plant sample preparation

Samples were processed and storage according to the Good Practices for medicinal plants (Brazilian Health Surveillance Agency, 2013). Aerial parts were dried in a Marconi laboratory stove model MA 035/5 (Piracicaba, Brazil) and ground in a Tecnal mill model TE-650 (Piracicaba, Brazil). The resulting material was sieved to particle size between 32-60 mesh. Approximately 100 mg of each sample was weighed on an analytical balance (Sartorius model CP225D) in a glass tube. To each sample was added 1 ml of n- hexane 95% (HPLC grade/ Spectro, Tedia, Brazil) and then sonicated (USC 1850 A, UNIQUE) for 45 min at 25°C, 25 KHZ frequency and power of 154W. The samples were filtered with hydrophobic polytetrafluoroethylene filters (FilterPro) with 0.22 μm pore size and 4-mm diameter into pore size and 4-mm diameter into 1-ml volumetric flasks. Samples were transferred to vials with an insert and analyzed.

Development of the analytical method

The chromatographic method was developed using a sample of P. niruriL. The development of the method for the lignans analysis was carried out in an Agilent GC 6890N gas chromatograph (GC) equipped with a GC Sampler 120 automatic liquid sampler and coupled with a mass spectrometer (MS) model 5973N. Helium was used as carrier gas. Initially three capillary columns were tested, all had polymethylphenylsiloxane stationary phase with different proportions of phenyl and methyl groups: DB-5ms (5% phenyl), DB-35 (35% phenyl) and DB-17HT (50% phenyl). All columns were from Agilent Technologies and had the following dimensions: 30 m length, 0.25 mm inner diameter and 0.250 µm film thickness. For the comparison between columns, a method published by Moslkaet al.(2014) was used. The chromatographic method was further developed with the DB-17HT column. The development was performed by central composite design, with the following parameters: Initial oven temperature, oven heating rate and carrier gas flow. Table 1 shows the performed experiments. All calculations were performed in JMP Statistical Discovery Software version 8.0 (SAS, Cary, USA). The otherchromatographic parameters were set to: Injection volume of 1 μm pore size and 4 mm diameter, injector temperature at 300°C in splitless mode, transfer line temperature at 300°C, ion source temperature at 300°C and quadrupole analyzer at 200°C. These parameters were maintained in all analysis. Injection of the samples in the different experiments was randomly performed. The responses used were four resolutions between major fragments of different peaks of lignans and their contaminants. The resolutions with the respective fragments and the substances involved are presented in Table 2.

 

 

 

Quantitative determination of phyllanthin and hypophyllanthin

Identification of phyllanthin and hypophyllanthin

Phyllanthin and hypophyllanthin were identified by comparison withstandard mass spectra and retention times (Rt). The  standards were purchased from Chromadex (Irvine, USA) and both have 98.9% purity.

Preparation of internal standard solution

Alpha-humulene (Sigma-Aldrich, Brazil) was weighed (37.90 mg) into a 5-ml volumetric flask to prepare the internal standard stock solution. This compound was dissolved in 95% n-hexane (HPLC grade/Spectro, Tedia, Brazil), in an ultrasonic bath, and after dissolution the flask volume was completed with n-hexane. The internal standard working solution was prepared by diluting 187.5 μm pore size and 4-mm diameter into the stock solution in a 5-ml volumetric flask, with a final concentration of 284.25 μm pore size and 4 mm diameter in g/ml.

Method validation

Validation was performed based on the International Council for Harmonisation (ICH) guidelines (2005). Standard calibration curves of phyllanthin and hypophyllanthinlignans were prepared. The stock solution of each lignan was prepared with 1 mg of each standard, weighed in a 2-ml volumetric flask. Subsequently, the compounds were dissolved with n-hexane (HPLC grade/ Spectro, Tedia, Brazil) using an ultrasonic bath, after dissolution the flask volume was completed with n-hexane. Solutions of different concentrations of the calibration curve were prepared from this solution (ranging from about 10 to 500 µg/ml) with the addition of 50 μm  pore  size  and  4 mm diameter into the internal standard working solution. For quantitative determination of the samples, the fragments m/z 151 of phyllanthin and hypophyllanthin were divided by the area of fragments m/z 93 of the internal standard. With this value, the linear regression was calculated. Precision was calculated as the coefficient of variation between different preparations of the P. nirurisample. Limit of quantification (LOQ) was considered the lowest concentration of the calibration curve and limit of detection (LOD) was calculated from the analytical noise of the chromatographic method.

Sample preparation method for the quantitative determination of lignans

Sample preparation followed the same method used for the standard stock solutions with the addition of 50 µl of internal standard working solution before completing the volume.

Lignans content

The phyllanthin and hypophyllanthin contents (% m/m) were calculated using the following expression:

Where ASais the area of fragment m/z 151 of each lignan, AIS is the area of fragment m/z 93 of the internal standard, a is the linear coefficient of the curve, b is the angular coefficient of the curve and MSais the sample weight.

 

 


 RESULTS AND DISCUSSION

Chromatographic method development

The choice of Gas Chromatography-Mass Spectrometry as analytical technique  rather  than  the  more  frequently used liquid chromatography for the analysis of phyllanthin and hypophyllanthin has  the advantage of GC-MS being less expensive to acquire and to maintain, using minimum amounts of organic solvents and able to identify several lignans as demonstrated by Molskaet al. (2014).

Analysis of the P. nirurisample in different columns showed that the DB-35 and DB-17HT columns have a greater discrimination capacity for lignans than the DB-5ms column, similar to that used by Molskaet al. (2014) (Figure 2). The higher content of phenyl groups in these columns allows for a better interaction of the lignans  with the stationary phase, due to the higher availability of groups able to perform dipole interactions, which results in better separations. Between the columns DB-35 and DB-17HT, the latter showed a slightly better separation and therefore was chosen for optimization of the analytical method. Before the optimization, several lignans present in the P. nirurisample were identified by CG-MS and are presented in Table 3.

 

 

 

As can be observed in Figure 2, although phyllanthin and hypophyllanthin peaks showed good chromatographic resolution, the chromatogram of the sample P. niruriin DB-17HT column shows some peaks coeluting with other lignans, indicating the need for further optimization of the chromatographic method. An experimental design was therefore, performed to optimize the analytical method. The three factors studied, initial oven temperature, oven heating rate and carrier gas flowrate were chosen because they were the most significant factors in previous studies. The responses used were four resolutions, indicated as R1, R2, R3 and R4, between two lignans or a lignan and another substance. These are detailed in Table 2 which presents the two substances involved in each resolution, along with their characteristic fragments used for resolution calculation. The study intervals of each factor were based on the method of Molskaet al. (2014), using  the  author’s initial oven temperature (60°C) as the lowest value (-1), their oven heating rate (6°C/min) used as the lowest value (-1) and their carrier gas flow rate (1.5 ml/min) used as the central value (0). The three factors were significant for the responses studied as shown in Figure 3. The best chromatographic condition was established by contour analysis. Figure 4 presents a contour graph between carrier gas flow and oven heating rate as the two most significant factors. For this contour graph, initial oven temperature is fixed at 120°C. In the graph, the regions where the resolutions were below 1.5 were shaded. Only a small region was not shaded, with high carrier gas flow and low heating rate. Within this region, the best chromatographic condition comprises a 120°C initial oven temperature, 6°C/min. heating rate and 2 ml/min carrier gas flow rate. A chromatogram of P. nirurisample in this condition is presented in Figure 5.

 

 

 

 

Method validation

The calibration curves of phyllanthin and hypophyllanthin were considered adequate with a coefficient of correlation r2= 0.996. A duplicate analysis of this sample showed a coefficient of variation below 4% and was therefore considered adequate.  The  LOQ  of  both  the   lignans    was considered the lowest concentration of the calibration curve, at 12.2 µg/ml for phyllanthin and at 11.1 µg/ml for hypophyllanthin. The LOD was obtained for phyllanthinat 1.4 µg/ml and for hypophyllanthin at 0.8 µg/ml.

Phyllanthin and hypophyllanthin quantitative determination

The contents of phyllanthin and hypophyllanthindetermined are shown in Table 4. Phyllanthin and hypophyllanthin were detected in three of six samples: P. niruri, P. amarusand P. urinaria. Nevertheless, only P. amarushad minimal quantifiable contents of both lignans. P. amarushad about 0.6% (m/m) of phyllanthin, very similar to the contents found by Tripathiet al. (2006) although the content of hypophyllanthin was about ten times smaller. The same authors did not detect these lignans in P. urinariaalthough this study found them in small amounts in this species. Nahar et al. (2012) claimed that P. niruriand P. urinariaare the major lignan- producing species, but the results here obtained indicate that although they do produce lignans, P. amarusis the major producing species among the samples analyzed.

The results also contradict the P. niruri  monograph  in the Brazilian Pharmacopoeia whichstates that this species does not have phyllanthin. At the same time, P. amaruswas the only species that produced substantial amounts of phyllanthin and hypophyllanthin. The overall results demonstrate the high variability of phyllanthin and hypophyllanthin production among different species of the genus Phyllanthus and even the variability in the production of these lignans by the same species from different countries, implying  that  a  broader study encompassing several species from different regions of Brazil is necessary, in order to establish the best conditions for cultivation and to confirm P. amarusas the most suitable. The RENISUS list, as already stated, includes Phyllanthusspp, without defining which ones but indicating four promising species to be studied (P. amarus, P. niruri, P. tenellusand P. urinaria). This study contributes  to  this effort, at least in relation to phyllanthin and hypophyllanthin content, by revealing P. amarusas the most promising species.

 

 

Lignan content in the different Phyllanthus species

Eleven lignans were identified in the samples analyzed (Table 3), with P. amarusbeing the only sample in which all eleven were detected and had been all previously detected in other studies (Nahar et al., 2012; Molskaet al., 2014; Qi et al., 2014), confirming that it was the major producer of lignans among the studied species. Seven lignans were detected in P. niruribut, only niranthin, hypophyllanthin and dextrobursehernin were detected for the first time (Nahar et al., 2012; Qi et al., 2014). Five lignans were detected in P. urinaria, andof these only hypophyllanthin  was  detected for the first time (Nahar et al., 2012; Molskaet al., 2014). No lignans were detected in the remaining three species analyzed in agreement with previously published literature. These results demonstrate again the high variability in the occurrence of lignans among Phyllanthusspecies and in the same species from different locations. The authors reaffirm the necessity of a broader study in Brazil of the occurrence of lignans in Phyllanthus species.

 

 

 

 


 CONCLUSION

These results confirmed the high variability of lignan content among species of Phyllanthus. Furthermore, they demonstrate that P. amarus species is the most promising species to be studied in Brazil due to its high lignan content. The results also contribute to the RENISUS goal for developing a herbal medicine from Phyllanthus spp. Additionally, the method developed by GC-MS offers a precise identification and quantitation of lignans found in Phyllanthusspp is less expensive and complies with the principles of green chemistry.

 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.

 


 ACKNOWLEDGMENTS

The authors would like to thank all personnel involved in the Brazilian Living Pharmacies Program – Brazilian Ministry of Health, who generously provided samples for this study. The study received a Scientific Research Access Authorization by the Brazilian Institute of Environment and Renewable Natural Resources – IBAMA under register number 0201.000663/2012-91 and was registered at the Council for the Management of Genetic Resources under number A670412. The authors would like to thank Dr. Benjamin  Gilbert  for  revising  the English text. This work was funded by the Brazilian Agricultural Research Corporation – Embrapa.

 



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