CHARACTERIZATION AND STANDARDIZATION OF THE HERBAL DRUG BACCHARIS TRIMERA (LESS.) DC AND ITS LYOPHILIZED EXTRACT

CHARACTERIZATION AND STANDARDIZATION OF THE HERBAL DRUG BACCHARIS TRIMERA (LESS.)DC AND ITS LYOPHILIZED EXTRACT

P. A. Ferreira ¹, F. L. A. Santos ¹, L. D. S. Alves ¹, L. R. M. Ferraz ¹, T. A. Rosa ¹, R. M. F. Silva ¹, G. M. A. Presmich ¹, Rolim, L. A. ², M. S. Silva ³, M. B. S. Maia ⁴ and P. J. Rolim-Neto ^*1

Department of Pharmaceutical Sciences ¹, Department of Physiology and Pharmacology ⁴, Federal University of Pernambuco, Brazil.

Pharmaceutical Sciences Collegiate ², Federal University of Vale do Sao Francisco, Brazil.

Department of Pharmaceutical Sciences ³, Federal University of Paraiba, Brazil.

ABSTRACT:Baccharis trimera is used in traditional medicine in South American countries for treatment of diseases, such as digestive disturbances, rheumatism, diabetes and inflammatory processes. Given its pharmacological potential, the aim was to characterize the vegetal drug B. trimera and attain and standardize its lyophilized extract to use as raw vegetal material in the development of phytotherapeutic medicine. Chemical identification and prospecting, granulometric determination, loss by desiccation, total ash and caffeic acid through High-Performance Liquid Chromatography were carried out. The lyophilized extract obtained was submitted to assays for determining caffeic acids, rheology, hygroscopicity, solubility, superficial area, porosity, particle size through laser granulometry and the thermogravimetric curve. The assays demonstrated that the sample is within the described specifications in literature and, therefore, is adequate to obtain the liquid extract and later dry and standardized in relation to the content of caffeic acids. The lyophilized extract was fine powder, demonstrating the necessity for addition of excipients that make possible the use of the input for the attainment of solid pharmaceutical forms. It was possible to infer characteristics of the vegetal drug and the dry extract obtained through the lyophilization process that will have to be considered in later studies of formularization.

Asteraceae, Baccharis trimera, caffeic acids, physicochemical characterization

INTRODUCTION: Medicinal plants are used as home remedies, over-the-counter medicines and as raw materials for the pharmaceutical industry. They possess the advantage of being well accepted by patients due to their traditional use and for being acquired at a lesser cost in relation to allopathic medicines, which make them more accessible to the needy population.

Moreover, it is a lucrative sector that moves billions of dollars, representing a substantial proportion of the global medicine market ¹.

In this overview, diverse organizations have stimulated the development of phytotherapeutics, through the achievements of research that comprise all aspects of technological development from botanical identification up to the assurance of the efficacy, safety and quality of the final pharmaceutical form. Thus, the development of a standardized phytotherapeutical adds a technological value in the final pharmaceutical forms that contain medicinal plants, such as Baccharis trimera ^{2, 3}.

Baccharis trimera (Less) DC (Asteraceae) is a medicinal plant found mainly in Brazil, Argentina, Uruguay and Paraguay. It is used in traditional medicine in the treatment of digestive disturbances, liver and renal illnesses, rheumatism, diabetes and inflammatory processes ^{4, 5}. In literature, its hepatoprotective potential ⁶, antiulcerogenic ⁷, analgesic, anti-inflammatory ⁸, immunomodulator ⁹, antioxidant ¹⁰, hypoglycemic ¹¹ and smooth musculature relaxant is described ¹². Additionally, the antiarthritic effect of the B. trimera aqueous extract prepared by infusion was demonstrated in vivo collagen-induced arthritis model. The results demonstrated a drastic reduction in the development of the illness with reduction in the migration, activation and proliferation of lymphocytes at an injury site, attributed to flavonoids present in the extract ¹³.

The variety of therapeutical actions in B. trimera extracts can be justified by the presence of composites of different polarities, amongst them mainly diterpene lactones, sesquiterpenes, flavonoids, saponins, polyphenols and cafeoilquinic acid ¹⁴.

Given the above, the present work had as an objective to carry out the characterization of the vegetal drug, as well as the attainment and standardization of lyophilized extract for its later use as vegetal raw material in the development of phytotherapeutic medicine based on B. trimera.

EXPERIMENTAL:

Vegetal Material

The aerial parts of B. trimera were collected on the morning of January 20th, 2011, in the city of Campinas, São Paulo, Brazil, at the following geographic coordinates: latitude 22° 47' 29.29''S and longitude 47° 6' 31.80''W. The fresh material was sprinkled with 70% ethyl alcohol, and after that dried in a circulating air kiln for five days at the temperature of 40ºC. After drying, the material was triturated in a cutting mill, with a 30 mesh.

The identification of the vegetal material was carried out by the Pluridisciplinary Center for Chemical, Biological and Agricultural Research of the University of Campinas (UNICAMP) and therefore the deposited exsiccate was found in the herbarium of the same institution, located in the city of Paulinea-SP, under number 1286.

 Chemical identification of B. trimera

The assay for the identification of the chemical marker (3-O-methylquercetine) was based on High-Performance Liquid Chromatography (HPLC) method ¹⁵. The sample used was a B. trimera aqueous extract obtained by infusion of 3.3% (w/v) of vegetal drug in distilled boiling water for 45 min, with later filtration and storage in an amber glass bottle ¹³.

Analysis was performed in a Shimadzu^® high efficiency chromatograph with a controlling system (Shimadzu SCL - 20VP). The following analytical conditions were used: Shimadzu^® reverse column phase C18 (250 x 4.6 mm) 5 μm particle size; mobile phase system A (water: ascetic acid 95.5:4.5, v/v) and B (100% acetonitrile), with a flow of 1.5 mL.min^-1 and injection volume of 10 µL; λ=325 nm. The elution gradient system used for analyses of the samples was: 0.01-15 min (15-60% B); 15.01-18 min (60-100% B); 18.01-21 min (100% B); 21.01-24 min (100-15% B).

Phytochemical Prospecting

The sample was prepared as described at chemical identification test. The infusion was partitioned without interruption in ethyl acetate and n-butanol. Aliquots of these fractions (about 15μL) underwent phytochemical prospecting via thin layer chromatography (TLC) using silica gel plates for chromatography (Macherey-Nagel^®). The reagents used in the characterization of the main secondary metabolite groups and representative standards of these groups are described in Table 1.

Additionally, for the identification of saponines the froth test was undertaken which consists of vigorous agitation of the solutions obtained for 1 min, followed by stasis. The presence of abundant foam and its persistent presence after 15 min of stasis was the criterion used to confirm the presence of saponosides ²⁰.

Determination of granulometric distribution of the vegetal drug

The dry vegetal raw material sample (25g) were submitted to vibration in agitator (Bertel^®) for 30 min in sieves with mesh openings of 850, 600, 425, 250, 150 and 90 µm ²¹. The fractions retained in the sieves and the collector was weighed. The results were expressed by the average of three determinations and the data were analyzed by graphical method, creating a histogram distribution and retention curves and going on to the determination of the average diameter of particles.

TABLE 1. CHROMATOGRAPHIC AND REAGENT SYSTEMS USED IN THE PHYTOCHEMICAL PROSPECTION OF DRY BACCHARIS TRIMERA.

Legend: ¹ 100:11:11:27 v/v; ² 50:50:50 v/v; ³ 40:50:10 v/v; ⁴ 90:10 v/v; AcOEt= Ethyl acetate, HCOOH = Formic Acid, H₂O= water, UV= ultraviolet, NEU = NEU Reagent (methanolic solution of diphenylboriloxietilamine 1%), n-BuOH = n-butanol, LB = Lieberman and Burchard Reagent.

Determination of the loss by desiccation and the total ash content of the vegetal drug

The analysis was carried out in triplicate using three weighing bottles, previously desiccated for 30 min in the same test conditions. One gram (1 g) of the vegetal drug was added to them. The weighing bottles with the samples were taken to the kiln at a temperature of 105ºC for a period of 2h and later cooled to room temperature in a desiccator and weighed. The procedure was repeated until reaching a constant weight.

The quantification of the non-volatile B. trimera residue powder was obtained in triplicate through determination of total ash ²¹, and the crucibles previously calcinated in a muffle furnace and then cooled and weighed. It were added 3 g of the dry drug powder to them, and the samples carbonized on a Bunsen burner and later incinerated in a muffle furnace at 500°C until constant weight.

Determination of the vegetal drug caffeic acids

The determination of caffeic acids was carried out according to a monograph of the same sort ²¹, ascertained by the quantification of chlorogenic acid in the sample via HLPC (Shimadzu^®). B. trimera sample solution was prepared at a concentration of 2.4 mg.mL^-1. For determination of the caffeic acid concentration, a calibration curve with standard chlorogenic acid (Sigma Aldrich, 99% purity) was constructed from a stock solution of 1.12 mg.mL^-1 to concentrations of 336, 224, 112, 84, 56, 28 and 11.2µg.mL^-1. The result of the

determination was expressed in grams of chlorogenic acid per 100 g of the drug (%), considering the loss by desiccation.

Attainment and control of the fluid extract

The attainment of B. trimera extract was carried out in accordance with Coelho et al. (2004) ¹³, aiming at evident antiarthritic activity, associated with anti-inflammatory and analgesic activities also described by other authors ^{8, 9}.  An aqueous infusion of the vegetal drug was made at 3.3% (w/v). As a quality control of this stage of the process the pH and the density of the fluid extract was evaluated.

 Attainment of lyophilized extract

The attainment of B. trimera dry extract was attained from the fluid extract, previously described, which was frozen in an ultrafreezer at -90° ± 5°C for 24 h, and later lyophilized (Liotop^®) under a pressure of 24 µmm of Hg; 220v ± 2 VCA vacuum for 96 h. The product was placed in a hermetically sealed vial and stored in a glass vacuum desiccator.

 Thermal characterization of vegetal drug and lyophilized extract

The drug thermogravimetry curves (TG) were obtained by means of a Shimadzu^® thermobalance, model DTG 60H, in a nitrogen atmosphere at 50 mL.min^-1 flow, with sample masses around 6 mg (± 0.5), packed in an alumina crucible, at the heating rate of 10 ^oC.min^-1, up to 700^oC. The thermoanalytical data were analyzed by means of Shimadzu^® TA-60WS^® (Thermal Analysis) version 2.20 software.

 Determination of caffeic acids via HPLC in the lyophilized extract

The determination of the lyophilized extract proceeded with the same adapted chromatographic conditions for the vegetal drug, with modification in the preparation of the sample. This solution, prepared from the lyophilized extract, was obtained in the concentration of 1 mg.mL^-1 of water.

The HPLC method was validated for the quantitative analysis of 5-o-caffeoylquinic acid (5-CQA) and the dicaffeoylquinic acids (3, 4-diCQA, 3,5-diCQA and 4,5-diCQA) in agreement with ICH guidelines, using the following analytical parameters: accuracy, linearity, precision (repeatability), limit of detection (LD) and limit of quantitation (LQ), robustness. Linearity was evaluated by the calculation of a regression line using the Least Squares Method. Precision was assessed by analyzing the average point of the extractive solution six times in the same day and by analyzing the same extract two times on two different days, performed by two different analysts (inter-day or intermediate precision).

The comparison between the means and the differences between them was evaluated by ANOVA one-way treatment. Accuracy was tested by determining the recovery of 5-CQA, 3, 4-diCQA, 3, 5-diCQA and 4, 5-diCQA at three different concentrations and by the calculation of the relative standard deviation (%RSD) of the recovery. The same was also performed for the extract contaminated with the standard substance of the vegetal material.

The values ​​were considered acceptable when the RSD values ​​were lower than 15%, in the case of raw vegetal material ²¹. The selectivity was determined by checking peak purity of all the peaks, using a DAD detector. LD e LQ were calculated dividing the standard deviation of the linear coefficients of the three calibration curves for testing the linearity, by means of obtained slopes of the curves multiplied by three and ten, respectively.Robustness was evaluated by the kind of agitation upon obtainment of the extract solution. Three different types of agitation were applied: manual shaking, magnetic stirring and sonication. To assess whether there are differences between the types of agitation, the comparison between means was performed using ANOVA One-Way. All treatments were performed using the confidence interval of 95%.

Determination of rheology of B. trimera lyophilized extract

Determinaion of superficial area and porosity of lyophilized extract

The lyophilized extract was weighed (200 mg) and previously treated at 100ºC for 5 h, in a kiln, for optimization of the adsorption process. Later, the sample was degasified for 48 h at 110°C to remove any adsorbed material in the interior of the pores and at the surface of the material.  The analyses were carried out thereby obtaining adsorption and desorption isotherms, applying the appropriate models for the adjustment of the experimental points.

The adsorption/desorption isotherm was obtained by gradual physical adsorption of nitrogen at 77 K on the material, and subsequent desorption. The application of the Brunauer-Emmett-Teller (BET) (1938) ²³ model on the appropriate portion of the curve supplied the superficial area value (SBET).

For the determination of porosity (size of pore and total volume of pores), the Barret-Joyner-Halenda (BJH) method was used. For the accomplishment of these assays, a Micromeritics^® ASAP 2440 Superficial Area and Pore Size Analyzer was used, loaded with its own software to determine the superficial area and porosity ²⁴.

 Determination of particle size by laser granulometry of lyophilized extract

The samples were dispersed in isopropyl alcohol in a 1:2 (mg.mL^-1) ratio. This dispersion was agitated in the ultrasonic bath (Unique^®, model USC-1400A), for 3 min before being analyzed. Microtac^® S3500 particle size analyzer was used. Each analysis was carried out in triplicate.

 Determination of solubility of lyophilized extract

For evaluation of solubility, about 10 mg of lyophilized extract was weighed, transferring it to an erlenmeyer. Slowly, the tested solvents (distilled water, methanol, ethanol, HCl and NaOH sodium sulphate 0.1 M and 1% sodium lauryl sulphate solution) were added until complete visual solubilization.

When there was no solubilization, even after the final 50 mL volume of solvent, the samples were submitted to sonication for 15 min. After persistent non-solubilization, a 25 mL quantity was added until complete solubilization, thereafter submitting the samples to sonication. At the end of adding a total of 100 mL, the assay was finalized ²¹.

 Determination of hygroscopicity of lyophilized extract

Samples of 0.5 g were placed in weighing bottles and kept in a desiccator, in triplicate, which ensures the RH saturation constant at room temperature. Then the samples were subjected to exposure to different conditions of relative humidity: 28, 74 and 95% using silica gel and saline solutions of sodium chloride and zinc sulphate, respectively, for the environment saturation. Such conditions have been confirmed through a J. Prolab^® model HS 122 digital thermohygrometer.

Samples were analyzed at 0, 4, 6, 8, 10, 12, 14 days and the percentage of water absorbed (U%) was calculated in regards to the weight in grams of dry weight (dw) and wet weight (ww) by the expression: U%= [(ww-dw)/dw] x100  ^{25, 26}.

RESULTS AND DISCUSSION:

Chemical identification of B. trimera

Beginning from the analysis performed, the chromatogram (Figure 1), in reference to the aqueous extract, was obtained. From this it is possible to visualize the peaks referring to the quercetin and 3-O-methylquercetine, previously analyzed, in retention times (T_R) of 14.7 and 15.1 min, respectively.

The scanning spectra obtained by DAD detector of the markers indicated that3-O-methylquercetine has a characteristic absorption at 355 nm, whereas quercetine has it at 369 nm (Figure 1).

FIGURE 1. CHROMATOGRAM AND ABSORPTION SPECTRA OF THE AQUEOUS EXTRACT OF B. TRIMERA INDICATED THE COMPOUNDS 3-O-METHYLQUERCETINE (A) AND QUERCETINE (B).

 The results obtained disclosed the presence of flavonoids, saponins, triterpenes, mono, di and sesquiterpenes and cinnamic derivatives. Were negative for coumarins, leucoanthocyanidins, proanthocyanidins, gallic tannins, and alkaloids. Taschetto (2010) ²⁷, using an aqueous infusion, did not observe the presence of alkaloids. A fact justified by the time of the collection. As to the extraction method some authors observed the presence of such a metabolyte only in organic solvents of low polarity like chloroform, n-hexane and n-butanol²⁸.

The analyses that presented discrepancy, as compared to the literature, in relation to the absence of tannins and coumarins, it can be justified by the extraction method and by the solvent used%