Quantitative HPLC Analysis of some Marker Compounds of Hydroalcoholic Extracts of Piper aduncum L .

Plants have always played an important role for the mankind, especially as food and medicine. Some ancients as Assyrians, Arabs, Egyptians and Greeks left a legacy of over a thousand years of medicinal plants use. They have discovered not only their medicinal properties, but also some dose-related effects of the plants and their extracts. Despite the major advances in the modern medicine, the development of new drugs from natural products is still considered important. This seems to be even more relevant for the developing countries, where the costs to develop a drug are prohibitive. Since 1980, the World Health Organization has been encouraging countries to identify and exploit traditional medicine and phytotherapy. In Brazil, phytotherapy is a promissing alternative due to its biodiversity and many studies devoted to the description of plant chemistry and their biological activities. However the development of phytotherapeutical products is dependent on their standardization, to guarantee not only the authenticity of the plant extract, but also to enable a doserelated use. Chromatographic methods have been recommended to the quality control of phytotherapeutical products with many advantages over other methods. The genus Piper is widely spread and many species present important biological activities. In the present study, we have studied some hydroalcohol extracts from the aerial parts of Piper aduncum L., popularly known as “aperta-ruão”. P. aduncum was registered in the First Edition of Brazilian Pharmacopeia and many studies in the literature describe its phytochemicals and its antimicrobial effects. This work describes part of the efforts for the standardization and quality control of hydroalcohol extracts of P. aduncum, based on the HPLC quantification of some marker compounds. This was a colaborative work with Siema Eco Essências da Amazônia, a company that develops an antimicotic product trademarked as Dermodilapiol from aerial parts of Piper aduncum.


Introduction
Plants have always played an important role for the mankind, especially as food and medicine.Some ancients as Assyrians, Arabs, Egyptians and Greeks left a legacy of over a thousand years of medicinal plants use.They have discovered not only their medicinal properties, but also some dose-related effects of the plants and their extracts. 1espite the major advances in the modern medicine, the development of new drugs from natural products is still considered important.This seems to be even more relevant for the developing countries, where the costs to develop a drug are prohibitive.Since 1980, the World Health Organization has been encouraging countries to identify and exploit traditional medicine and phytotherapy. 2In Brazil, phytotherapy is a promissing alternative due to its biodiversity and many studies devoted to the description of plant chemistry and their biological activities.
However the development of phytotherapeutical products is dependent on their standardization, to guarantee not only the authenticity of the plant extract, but also to enable a doserelated use.][5] The genus Piper is widely spread and many species present important biological activities. 6,7In the present study, we have studied some hydroalcohol extracts from the aerial parts of Piper aduncum L., popularly known as "aperta-ruão".P. aduncum was registered in the First Edition of Brazilian Pharmacopeia 8 and many studies in the literature describe its phytochemicals and its antimicrobial effects. 9,10This work describes part of the efforts for the standardization and quality control of hydroalcohol extracts of P. aduncum, based on the HPLC quantification of some marker compounds.This was a colaborative work with Siema Eco Essências da Amazônia, a company that develops an antimicotic product trademarked as Dermodilapiol from aerial parts of Piper aduncum.

Plant material and extracts
Four different batches of hydroalcohol extracts of aerial parts of P. aduncum were prepared by the company Siema Eco Essências da Amazônia.The first three batches were prepared from wild specimens located in Manaus, whereas the fourth batch from cultivated specimens collected at Itacoatiara City.Voucher specimens are deposited in the INPA herbarium under the numbers 214711 and 21334.The extracts were prepared by maceration of ca.4.0 kg of dried and powdered aerial parts with ca.40 L of ethanol:water (96:4) for seven days at room temperature.The material was then filtered and the concentration of the final product was adjusted to reach a final concentration of 20 g L -1 of extractives.

Isolation of the marker compounds
Previous HPLC analysis of the extracts had showed that all four batches had two major components (marker compounds).After several chromatographic steps (liquidliquid partition, normal and reversed-phase column chromatography, preparative thin-layer chromatography), 94 mg of marker compound 1 and 18 mg of the marker compound 2 have been isolated.

Quantitative analysis
For the quantification, the external standard method was employed, using the purified marker compounds as standards.Stock solutions of 10, 20, 40 e 80 μg mL -1 of dillapiol and 1, 2, 4 e 8 μg mL -1 of the benzoic acid derivative were prepared in methanol.Calibration curves of dillapiol and benzoic acid derivative were obtained from the analysis at 280 and 254 nm, respectively.
Samples of each batch of the hydroalcohol extracts have been previously submitted to SPE treatment to enhance the detectability and resolution.Initially, 100 μL of the extract were diluted with 900 μL of water, eluted into a SPE cartridge (Seppak C-18 Waters) and was washed with up to 5 mL of water to elute the polar components.The cartridge was then washed with up to 10 mL of acetonitrile (ACN) to obtain a second fraction to be quantified (with the chemical markers).Before the injection, this sample was diluted with 10 mL of water to prevent undesirable separations.A third fraction eluted with additional 10 mL of ACN was obtained (apolar components).All fractions have been analysed to ensure that all components, including the marker compounds, were completely removed from the cartridge.

Results and Discussion
The sample treatment with SPE was very efficient to concentrate the interested compounds solely in the first fraction eluted with ACN.The chromatographic profiles of all batches were very similar, showing two major components (Figure 2) with good separation.The peak of each marker compound has been identified with the help of coinjection of standards.
For the quantification of dillapiol and the benzoic acid derivative, calibration curves of each marker compound have been obtained in triplicate.All calibration curves have shown good linearity in the tested range.The regression equations were y = 21004x + 2257.2 (r = 0.9939) for dillapiol and y = 42676 x 2921.5 (r = 0.9955) for the benzoic acid derivative.The relative standard deviations (RSD) for the peak areas were inferior to 2 %.
The four batches have been also analysed in triplicate and the results of both marker compounds (Table 1) have shown good linearity, precision of injection and repeatability (RSD < 3 %).
Very similar chromatographic profiles were observed for all analysed hydroalcoholic extracts of aerial parts of Piper aduncum, indicating a common composition of their metabolites.The quantitative results have also shown small differences for the analysed marker compounds 1 and 2 in the four batches, indicating also that the product has a good stability, since the batches were produced along an entire year.

Conclusions
The established HPLC method has shown good linearity, precision and repeatability.The analysis time is not long and achieved good resolution for both marker compounds.These results indicate that this method may be applied to quality control this product.

Figure 2 .
Figure 2. HPLC chromatogram obtained at 254 nm with the identification of the corresponding peaks of dillapiol and the benzoic acid derivative.

Table 1 .
Marker compounds contents in four different batches of hydroalcohol extracts of aerial parts of Piper aduncum