LC Characterisation of Guaco Medicinal Extracts, Mikania laevigata and M. glomerata, and their Effects on Allergic Pneumonitis

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Extract preparation
• LC analysis
• Method validation
• Animals
• Induction of the allergic pneumonitis
• Bronchoalveolar lavage
• Total and differential cell counts
• Histopathological analysis
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

The leaves of guaco (Mikania glomerata and M. laevigata) are widely used for the treatment of asthma and bronchitis. An LC method for the quantification of coumarin and o-coumaric acid in medicinal extracts was developed and validated for linearity, limit of detection, accuracy, precision, as well as intra- and inter-day variations. Extracts and isolated markers were tested in the mice allergic pneumonitis model and the histopathological profile of the lung tissue was analysed. The values found for coumarin and o-coumaric acid in a fluid extract were 1.53 and 1.69 mg/mL, respectively, for M. glomerata, and 0.96 and 0.38 mg/mL for M. laevigata. The values found for the lyophilised aqueous extract were 0.22 and 0.11 mg/mL of coumarin and o-coumaric acid in M. glomerata and 0.05 and 0.02 mg/mL in M. laevigata, respectively. The analysed samples from the species M. glomerata presented more coumarin and o-coumaric acid than the analogous M. laevigata species. Both coumarin and o-coumaric acid are part of the phytocomplex which is responsible for the therapeutic activity of the guaco species. The lyophilisation process generated some alterations in the extract, in comparison with the fresh aqueous extract, and these extracts did not present anti-inflammatory activity. Comparing the histopathological images of the groups tested, a haemorrhagic profile of lung tissue of animals treated with lyophilised extract, o-coumaric acid and coumarin is observed, but not for the group treated with hydroalcoholic extract. It is probable that some protective effect of the whole extract (lost during the lyophilisation process) blocks the harmful effects of the isolated markers.

Introduction

Popularly known as ‘guaco’, Mikania laevigata Schultz Bip. ex Baker and M. glomerata Sprengel, (Asteraceae) are morphologically related plants and are traditionally used to treat respiratory affections. The ‘guaco’ leaves are traditionally used as an extract, syrup or infusion to treat bronchitis, asthma and coughs [1].

The species M. glomerata is considered an official drug in the Brazilian Pharmacopoeia (1^st edn., 1929). In southern Brazil, M. laevigata is mostly harvested, rather than M. glomerata, due to its local abundance. The main difference between the two is the flowering period, with that of the former occurring in September, and that of the latter occurring in January [2]. The leaves are slightly different, the lobes being more prominent in the former species, and both present the characteristic odour of coumarin.

Phytochemical studies of the leaves from these species indicate a similar composition; presenting diterpene acids (ent-kaurane derivatives), triterpenes and steroids (friedelin, stigmasterol and lupeol) and cinnamic acid derivatives [3], [4].

M. glomerata has been subjected to more frequent pharmacological investigations than M. laevigata, and has recently been described for its antiophidian [5], antiallergic [6] and bronchodilatador activities [7], and its influence on the rat spermogenic cycle was observed [8]. For M. laevigata, antiulcerogenic and anti-inflammatory activities have recently been investigated [9], [10], and no mutagenic effect was observed [9]. Both species show antimicrobial activity [4].

In this paper, the coumarin and o-coumaric acid contents were determined, through a validated HPLC methodology, in hydroalcoholic and aqueous extracts (lyophilised and freshly prepared), in both ”guaco” species. The antiallergic properties of M. laevigata extracts were investigated in an allergic pneumonitis model in isogenic mice, by determining the total number of cells and eosinophils in the bronchoalveolar lavage, and histopathological analysis of the lung tissue.

Materials and Methods

Plant material

Leaves of cultivated specimens obtained from vegetative propagations of authentic Mikania glomerata Sprengel and M. laevigata Sch ex Baker (identified by Pedro M. Magalhães, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil) were collected in Telemaco Borba (Paraná, Brazil) and Itajaí (Santa Catarina, Brazil). Voucher specimens of M. glomerata are deposited at the Klabin Herbarium [1, (23/07/02)] and specimens of M. laevigata are deposited at the Herbario Barbosa Rodrigues [HBR Biavatti 30 (30/08/01)] Itajaí, Santa Catarina, Brazil

Extract preparation

The leaves were air dried (40 °C, forced ventilation), powdered and sieved. Only particles between 0.85 - 1.4 mm were used to prepare the extracts. Hydroalcoholic extracts were prepared by percolation [12] with ethanol-water 1 : 2 (v/v) and concentrated to obtain a 1 : 2 (w/v) extract. For the LC analysis, the extracts were diluted in ultrapure water 1 : 5 (M. laevigata) and 1 : 10 (M. glomerata). For the preparation of the aqueous extract, boiling mineral water (100 mL) was added to the leaves (1 g), the mixtures were covered and allowed to reach room temperature, then filtered and analysed by HPLC. These extracts were also evaluated after lyophilisation.

LC analysis

The HPLC system consisted of a Waters 600 pump and 2996 PDA detector, and a manual injector Rheodyne 7725i (loop 20 μL). The analyses were carried out on an RPC18 Nova-Pak Waters column (150 × 3.9 mm, 4 μm). The mobile phase was MeCN:0.01 % aqueous acetic acid (20 : 80 v/v), flow rate 1.2 mL/min, the analysis was monitored at 275 nm, and the column oven set to 30 °C.

All the solvents were HPLC grade, and were degassed using an ultrasonic bath before use. The water was purified using a Milli-Q system (Millipore; Milford, MA, USA). All the solutions were filtered through 0.45 μm membranes (Schleicher & Schuell; Dassel, Germany).

Method validation

The HPLC method was validated for linearity, limit of detection, limit of quantification, peak purity, accuracy, precision and repeatability. Each sample solution was injected in triplicate.

Coumarin (1,2-benzopyrone, Sigma-Aldrich; St. Louis, MS, USA) and o-coumaric acid (Spectrum; Gardena, CA, USA) were used as external standards, dissolved in the mobile phase (500 μg/mL) and diluted in triplicate to 0.013, 0.05, 0.10, 0.20 and 0.35 μg/mL. The software Millenium Empower (Waters; Milford, MA, USA) was used to fit the regression curve and calculate the corresponding correlation coefficients. The LOD and LOQ were calculated through the RMSE (root mean square error) method [13]. The peak purity was confirmed by studying the PDA and GC-MS data of all the relevant peaks The accuracy of the method was determined through an analyte recovery test [14], adding known standard concentrations from 50, 150 and 250 μg/mL (hydroalcoholic extracts) and from 10, 30 and 70 μg/mL (lyophilised extracts) to the appropriately diluted matrix sample in triplicate, regarding the linearity of the method. The analyte recovery in the presence and absence of the extract matrix was compared, in order to analyse the specificity of the method. To verify the precision, intra- and inter-day precision tests were performed on 3 consecutive days. Repeatability was confirmed by evaluating the consistency of retention times and standard deviations.

Animals

Male BALB/c mice weighing 28 - 32 g, 6 - 8 weeks old, from our own animal facilities, were housed in a room with a 12 h light-dark cycle and water and food ad libitum. Animal care and research protocols were used, in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the Committee for Animal Research (UNIVALI).

Induction of the allergic pneumonitis

The mice were sensitised on days 0 and 7, by intraperitoneal injection of a mixture containing 50 μg of ovalbumin and 1 mg of Al₂O₃ in saline (a total volume of 0.6 mL). At 14 and 21 days after the first immunisation, the animals were challenged by exposure to an ovalbumin aerosol (50 : 50 grade II and III, Sigma; St. Louis, MS, USA) generated by a nebuliser (INALAR; São Paulo, Brazil) delivering particles of 0.5 - 10 μm in diameter at approximately 0.75 mL/min for 20 min. the concentration of ovalbumin in the nebuliser was 2.5 % wt/vol. The control group consisted of animals immunised as described and challenged with saline solution.

Bronchoalveolar lavage

The animals were sacrificed by an intraperitoneal injection of ketamine/xylasine (50 μL of a 100 mg/mL solution) 24 h after exposure to the second aerosol challenge. A tracheal cannula was inserted via a mid-cervical incision, and the airways were lavaged three times with 1 mL of phosphate-buffered saline (PBS, pH 7.4 at 4 °C).

Total and differential cell counts

The bronchoalveolar lavage fluid was centrifuged at 170 g for 10 min at 4 °C, the supernatant was removed, and the cell pellet was resuspended in 1 mL of PBS. One part of a solution containing 0.5 % crystal violet dissolved in 30 % acetic acid was added to nine parts of the cell suspension. The total number of cells was determined by counting in a Neubauer chamber. Differential cell counts were performed after cytocentrifugation and staining with haematoxylin-eosin.

Histopathological analysis

The lungs were removed after bronchoalveolar lavage collection, perfused via the right ventricle with 10 mL PBS to remove residual blood, immersed in 10 % phosphate-buffered formalin for 24 h and then in 70 % ethanol, whereafter they were embedded in paraffin. Tissues were sliced in 5 micron sections and stained with periodic acid-Schiff (PAS)/haematoxylin for evaluation of morphological parameters (presence of mucus, bronchial epithelium desquamation, bronchoconstriction).

Statistical analysis

The results obtained are presented as mean ± standard error, using the Instat software. For comparison of the two groups with normal distribution, the Student t test was used, and the Mann-Whitney U test for abnormal distribution. Values of p < 0.05 were considered significant.

Results and Discussion

Various solvent systems based on several mixtures of acetonitrile or methanol with water or 0.01 % acetic acid were tested, in order to achieve optimal separation in a relatively short time. The chromatographic parameters and analytical features obtained for the optimal conditions adopted in this work are shown in Table S1 in the Supporting Information. The selectivity and resolution obtained were α = 1.43 and Rs = 2.428, respectively. The chromatographic profiles of the standard and extracts are presented in Figure S1 in the Supporting Information. The interval tested for linearity and the accuracy shown CV below 2 %, standards recovery values are presented in Table S2 in the Supporting Information. Although many validation protocols are internationally accepted, there is a lack of specificity for phytopharmaceuticals products, which are complex matrices. Acceptable recovery values could vary between 70 - 120 % [15]. The recovery values of coumarin and o-coumaric acid were in between 102.05 ± 1.27 % and 97.69 ± 1.65 % for M. glomerata and 99.51 ± 1.77 % and 96.71 ± 1.50 % for M. laevigata, respectively, in the hydroalcoholic extract samples (CV < 2 %). No relevant matrix effect was verified for the analytes, but a major influence on the lyophilised extracts was observed, which presented recovery values of 73.59 ± 4.93 % (o-coumaric acid) and 93.73 ± 4.37 % (coumarin). Similar results have been obtained and published for other plant extracts [16], [17].

The results of the intra-day and inter-day validation assays presented in Table S3 of the Supporting Information indicate lower CV values (0.5 - 2 %) for hydroalcoholic extracts, compared with the lyophilised extracts (2.1 - 4.5 %). The repeatability is in accordance with Brazilian norm RE nº 899 [18], which considers values of up to 5 % as acceptable. Peak purity was investigated by studying the PDA and MS data of the peaks of interest; no indications for impurities could be found. All standards and samples were injected in triplicate. A maximum relative standard deviation of 1.5 % for the retention time of 2 compounds was found for o-coumaric acid (Rt = 4.63 min) and coumarin (Rt = 6.50 min) and very stable retention times over the whole study period (approximately 300 injections) confirmed the precision of the method. The method of analysis developed complies with international standards required for analysis of pharmaceuticals for human use.

The concentration found for the selected markers was largest for M. glomerata, the official species (Table [1]). The species M. laevigata presented considerable variations in concentration in terms of geographical origin, when collected during the same season and period and processed in the same way. The lyophilised extracts presented a diminished quantitative of the selected markers (ca. 60 % for o-coumaric acid and 50 % for coumarin) in relation to the freshly analysed aqueous extract. Observing the corresponding chromatograms of the lyophilised extracts, a new peak can be detected at 5.2 min, showing that some alteration has occurred during the lyophilisation process. The fresh aqueous extract of M. laevigata presented 13 and 34.33 μg/mL of o-coumaric acid and coumarin, respectively, representing approximately 1.95 and 5.15 mg in one tea cup. For the hydroalcoholic extract, (1 : 2) 0.57 and 0.84 mg/mL were found, equivalent to one teaspoon of a 10 % syrup. For the species M. glomerata, double this concentration could be expected.

The animals were treated by gavage with M. laevigata extracts and the dosage was established proportionally to the traditional usage of guaco by adult subjects (multiplied by ten, due to the metabolic differences).

The 7-day treatment with lyophilised aqueous extract did not significantly alter the total and differential influx of cells to the bronchoalveolar space. However, a tendency to reduce the total leukocytes (p = 0.194) can be observed in Fig. [1] A. The mice treated with hydroalcoholic extract showed a significant influx inhibition of leukocytes (p = 0.0384) and eosinophiles (p = 0.0317) (Fig. [1] B). Similarly, animals treated with o-coumaric acid and coumarin also presented a reduced influx of total cells when compared with the control group (Figs. [1] C and D), mainly due to the reduced number of eosinophils, since a significant difference in the number of neutrophils and mononuclear cells was observed in relation to the control group.

Asthma is characterised by oedema and hyperaemia of the mucosa, and infiltration of leukocytes, mainly lymphocytes Th₂ and eosinophils [19]. Since the positive control in the in vivo model evaluated in this work presented recognised alterations in the cellular pattern (Fig. S2 in the Supporting Information), the treatment with hydroalcoholic extract and the markers showed important anti-inflammatory activity in the allergic inflammation.

As previously described, the lyophilisation process generated some alterations in the extract, in comparison with the fresh aqueous extract. Since the concentration of the markers was drastically diminished, and these extracts did not present anti-inflammatory activity, it can be concluded that the lyophilised extracts do not present the same pharmacological profile as the drug.

Coumarin (5 mg/kg), when intraperitoneally and orally administered, inhibited carragenin-induced paw oedema in rats by 82.4 % and 34.8 %, respectively [20]. In several reports, coumarin was also found to possess smooth muscle relaxant activity and an antioedematous profile [21], [7], [22]. A recent paper described the bronchodilator activity of 1,2-benzopyrone and its probable mechanism of action, verifying that coumarin probably stimulates the restoration of Ca²⁺ into sarcoplasmic reticulum and provokes bronchodilation by internal calcium redistribution [23]. For o-coumaric acid, few pharmacological investigations have been reported. One paper demonstrated that this acid did not decrease the inflammatory mediator production by human whole blood cultures [24].

The treatment of mice with M. laevigata extracts was also evaluated through histopathological analysis of lungs (Fig. [2]). Despite the encouraging results obtained with the diminution of inflammatory cells, the effect of the treatment with extracts and markers on the lung tissue can be visualised. The frame observed is suggestive of accentuated vasodilation, causing blood stasis as verified by erythrocyte accumulation inside lung vessels in all the groups treated. Vasodilation can lead to an increase in the hydrostatic pressure in the vessels with outflow of erythrocytes, proteins and plasma characterising haemorrhagic exudates. The activated inflammatory cells may have caused endothelial injury, and could not be observed in the analysed histological sections. The simple coumarin is an anticoagulant, and according to [25], there is confusion between coumarins and the anticoagulant effect. There are more than 3400 naturally occurring coumarins in around 160 plant families. Of these, only few were investigated in terms of their anticoagulant activity in vitro or in vivo, and just a dozen are clinically available. From 13 natural coumarins tested as antithrombotics and anticoagulants, 7 were active, including 1,2-benzopyrone. The term coumarin is clinically used to refer to warfarin (Coumadin; Bristol-Myers Squibb, Princeton, NJ, USA) or other synthetic coumarins, specifically anticoagulants. Comparing the histopathological images of the groups tested (Fig. [2]), is possible to observe a haemorrhagic profile in the lung tissue of animals treated with lyophilised extract, o-coumaric acid and coumarin (Figs. [2] C, D and F, with decreasing intensity, respectively), which was not observed in the group treated with hydroalcoholic extract. This probably means that some protective effect of the whole extract (lost during the lyophilisation process) is blocking the harmful effects of the isolated markers.

From the results observed, caution is recommended in patients who use anticoagulant drugs, when using guaco extracts. Also, lyophilised extracts of guaco are not the same as the fresh variety. Both coumarin and o-coumaric acid are part of the phytocomplex responsible for the therapeutic activity of the guaco species and M. laevigata is active in allergic inflammation. The haemorrhagic feature observed in the histopathological analysis suggests that detailed studies are needed to verify the result of this anticoagulant effect.

Acknowledgements

Funbio (Fundo Brasileiro para a Biodiversidade) and Projeto Fazenda Monte Alegre (Klabin do Paraná) are gratefully acknowledged.

References

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Prof. Dr. Maique Weber Biavatti

Curso de Farmácia

CCS

University of Vale do Itajaí (UNIVALI)

Rua: Uruguai 458

Caixa postal 360

Itajaí

Santa Catarina (SC) 88302-202

Brazil

Phone: +55-47-3341-7601

Fax: +55-47-3341-7601

Email: maique@univali.br

References

• 1 Oliveira F, Alvarenga M A, Akisue G, Akisue M K. Isolamento e identificação de compostos químicos de Mikania glomerata Spreng e Mikania laevigata Schultz Bip. ex Baker.  Rev Farm Bioquím. 1984;  20 169-83
  PubMedSearch in Google Scholar
• 2 Moraes M D. A família Asteraceae na planície litorânea de Picinguaba - Município de Ubatuba [master thesis]. São Paulo, Brazil; Universidade Estadual de Campinas 1997
  Search in Google Scholar
• 3 Veneziani R CS, Camilo D, Oliveira R. Constituents of Mikania glomerata Sprengel.  Biochem Syst Ecol. 1999;  27 99-102
  CrossrefPubMedSearch in Google Scholar
• 4 Yatsuda R, Rosalen P L, Cury J A, Murata R M, Rehder V LG, Melo L V. et al . Effects of Mikania genus plants on growth and cell adherence of mutans streptococci.  J Ethnopharmacol. 2005;  97 83-9
  CrossrefPubMedSearch in Google Scholar
• 5 Maiorano V A, Marcussi S, Daher M AF, Oliveira C Z, Couto L B, Gomes O A. et al . Antiophidian properties of the aqueous extract of Mikania glomerata.  J Ethnopharmacol. 2005;  102 364-70
  CrossrefPubMedSearch in Google Scholar
• 6 Fierro I M, Silva A CB, Lopes C S, Moura R S, Barja-Fidalgo C. Studies on the anti-allergic activity of Mikania glomerata .  J Ethnopharmacol. 1999;  66 19-24
  CrossrefPubMedSearch in Google Scholar
• 7 Soares de Moura R, Costa S S, Jansen J M, Silva C A, Lopes C S, Bernardo-Filho M. et al . Bronchodilator activity of Mikania glomerata Sprengel on human bronchi and guinea-pig trachea.  J Pharm Pharmacol. 2002;  54 249-56
  CrossrefPubMedSearch in Google Scholar
• 8 Silveira e Sá R C, Leite M N, Reporedo M M, Almeida R N. Evaluation of long-term exposure to Mikania glomerata (Sprengel) extract on male Wistar rats’ reproductive organs, sperm production and testosterone level.  Contraception. 2003;  67 327-31
  CrossrefPubMedSearch in Google Scholar
• 9 Bighetti A E, Antônio M A, Kohn L K, Rehder V LG, Foglio M A, Possenti A. et al . Antiulcerogenic activity of a crude hydroalcoholic extract and coumarin isolated from Mikania laevigata Schultz Bip.  Phytomedicine. 2005;  12 72-7
  CrossrefPubMedSearch in Google Scholar
• 10 Suyenaga E S, Reche E, Farias F M, Schapoval E E, Chaves C G, Henriques A T. Antiinflammatory investigation of some species of Mikania .  Phytother Res. 2002;  16 519-23
  CrossrefPubMedSearch in Google Scholar
• 11 Fernandes J B, Vargas V M. Mutagenic and antimutagenic potential of the medicinal plants M. laevigata and C. xanthocarpa .  Phytother Res. 2003;  17 269-73
  CrossrefPubMedSearch in Google Scholar
• 12 British P harmacopoeia. Vol. II. London; The Stationery Office 2000: p 1661
  Search in Google Scholar
• 13 Corley J. Best practices in establishing detection and quantification limits for pesticide residues in foods. In: Lee PW, editor Handbook of residue analytical methods for agrochemicals. Chichester; Wiley & Sons 2002: p 1-18
  Search in Google Scholar
• 14 IC H. Q2B Validation of Analytical Procedures: Methodology. Available at http://www.fda.gov/cder/guidance/1320fnl.pdf. Accessed in 2005
  Search in Google Scholar
• 15 Green M J. A practical guide to analytical method validation.  Anal Chem. 1996;  68 305A-9A
  PubMedSearch in Google Scholar
• 16 Lin-Chin Y R, Huang C Y, Wen K C. Evaluation of quantitative analysis of flavonoid aglycones in Ginkgo biloba extract and its products.  J Food Drug Anal. 2000;  8 289-6
  PubMedSearch in Google Scholar
• 17 Deng F, Zito W. Development and validation of a gas chromatographic-mass spectrometric method for simultaneous identification and quantification of marker compounds including bilobalide, ginkgolides and flavonoids in Ginkgo biloba L. extract and pharmaceutical preparations.  J Chromatogr A. 2003;  986 121-7
  CrossrefPubMedSearch in Google Scholar
• 18 BRASI L. Resolução RE 899. Guia para validação de métodos analíticos e bioanalíticos. Brasília; Ministério da Saúde, Agência Nacional de Vigilância Sanitária, Diário Oficial da União 2003
  Search in Google Scholar
• 19 Frieri M. Inflammatory issues in allergic rhinitis and asthma.  Allergy Asthma Proc. 2005;  26 163-9
  PubMedSearch in Google Scholar
• 20 Leite M GR, Souza C L, Silva M AM, Moreira L KA, Matos F JA, Viana G SB. Estudo farmacológico comparativo de Mikania glomerata Spreng (guaco), Justicia pectoralis Jacq (anador) e Torresea cearensis Fr. All (cumarú).  Rev Bras Farm. 1993;  74 12-5
  PubMedSearch in Google Scholar
• 21 Leal L KAM, Ferreira A AG, Bezerra G A, Matos F JA, Viana G SB. Antinociceptive, anti-inflammatory and bronchodilator activities of Brazilian medicinal plants containing coumarin: a comparative study.  J Ethnopharmacol. 2000;  70 151-9
  CrossrefPubMedSearch in Google Scholar
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Prof. Dr. Maique Weber Biavatti

Curso de Farmácia

CCS

University of Vale do Itajaí (UNIVALI)

Rua: Uruguai 458

Caixa postal 360

Itajaí

Santa Catarina (SC) 88302-202

Brazil

Phone: +55-47-3341-7601

Fax: +55-47-3341-7601

Email: maique@univali.br

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