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Guayusa is the common name for Ilex guayusa which is a wild tree that belongs to the family Aquifoliaceae (Radice et al., 2017). It is a holly species found in Colombia, Ecuador and Perú (Dueñas et al., 2016). Decoctions of the leaves are consumed as a stimulating morning drink (Tene et al., 2007). Amazonian Kichwa people prepare infusions every day to promote conviviality, for festivals and rituals (Overing et al., 2000). On the other hand, mestizos and white populations in Ecuador consume the infusion as an additive to spirits (Dueñas et al., 2016).
The main compounds reported in the leaves and tea of I. guayusa are caffeine, theobromine and polyphenols (Radice et al., 2017); chlorogenic acid, lutein and quercetin-3-O-hexose (Villacís-Chiriboga et al., 2017); phenolic mono- and dicaffeoylquinic acid derivatives (Pardau et al., 2017). In addition, these authors report guayusa leaves have antioxidant and anti-inflammatory activity. Caffeine (1,3,7-trimethylxanthine) is an alkaloid of great interest because of the beneficial effects that brings to individuals. For instance, it increases alertness, improves concentration and exhibits antioxidant properties (Turnbull et al., 2017; Metro et al., 2017). Thus, to quantify the content of caffeine in the leaves of I. guayusa, it is important to determinate the quality of the final product.
The Ecuadorian guayusa has a great demand in the international market for its good sensory attributes assigned to the preparations obtained from the leaves of the species. Ecuador exports almost 100% of the harvest which is reduced 75% to dry of the total fresh leaves collected (Proecuador, 2017)1.
The producers commercialize aromatic and medicinal herbs by putting them through a drying processes. This is a complementary way of selling and inserting guayusa into new markets. The drying method is a critical step in the process because it extends the shelf life of herbal products (Figiel et al., 2016). This process inhibits microbial growth and prevents biochemical changes. Thus, it minimizes microbial contamination and possible modifications from the physical and chemical point of view and increases the shelf life of the product (García, 2014).
The most common methods are air-drying (AD), convection oven (CO) and solar drying (SOD). AD is very simple and economical, but it takes long periods to reach the adequate moisture content and may increase the probability of microbial spoilage (Kim et al., 2012). CO is considered as a cost - effective method but some compounds can get lost as a consequence of the high temperature (García, 2014). However, we have not found studies about the drying methods used to improve the stability of the leaves of I. guayusa.
The objective of this research was to determine the effect of air-drying, convection oven and solar drying on the physical and chemical properties of the leaves of Ilex guayusa.
MATERIALS AND METHODS
Plant materials and reagents
Fresh leaves of I. guayusa in vegetative phenological state were collected in Sucumbíos, Ecuador. A sample of the plant material was taken for botanical identification at National Herbarium of Ecuador, keeping an herbal witness (CIBE 020) at Centro de Investigaciones Biotecnológicas del Ecuador (Guayaquil - Ecuador). The plant material was previously washed and selected before drying process. After that, the dried material was grinded to homogeneous powder (800 µm) previous to physical and chemical analysis. All the chemicals were of analytical grade. The water was purified in a Milli-Q water purification system (Millipore, Bedford, MA, USA).
Drying methods
Air Drying (AD). Leaves of Ilex guayusa were air-dried at ambient temperature in a ventilated room without exposure to solar light. The highest temperature recorded was 31.83 °C and the lowest was 21.94 °C. The loss weight was measured every 24 h for 13 days until it reached a constant weight.
Convection Oven (CO). The experiments were performed at 46 °C in a convection oven (VWR Scientific Products, Atlanta, USA). Leaves were distributed uniformly on perforated stainless-steel trays (0.76 x 0.51 m). The time required to reach a constant weight of dried leaves was 13 h (Díaz-Maroto et al., 2003).
Solar Drying (SOD). The plant material was dried in a handmade chamber solar dryer equipped with a forced air circulation system through an average temperature of 33.54 °C. Samples were placed uniformly on perforated stainless-steel trays (0.50 x 0.50 m) into the system. The time required to reach a constant weight of dried leaves was 36 h.
Physical and chemical properties
Weight loss for fresh samples were obtained by the three different drying methods until constant mass was reached. Moisture was determined from sample weight loss after oven drying at 105 °C for 3 h (AOAC, 2005; method 930.15). Total ash, acid-insoluble ash and water-soluble ash were determinate for all samples (WHO, 1998).
Phytochemical analysis
Qualitative phytochemical analysis was performed using ethyl ether, ethanol and water solvents according to the procedure described by Miranda and Cuellar (2001). Briefly, 30 g of sample was weighted and 300 mL of solvent of increasing polarity was added, each one after 24 h of successive maceration extraction at temperature room. The presence or not of the following chemical compounds was determined: oils - fats; alkaloids; flavonoids; quinones; saponins; lactones; phenol and tannins; reducing sugars; mucilages; bitter principles; steroid and triterpenoid; aminoacids; resins.
Determination of caffeine
Grinded guayusa (500 mg) was added into 25 mL of distilled water at boiling temperature. Then, it remained without further heating for 10 min. The extracts passed through a filter paper (Whatman number 43), and then filtered through 0.22 µm disposable filters before analysis (Cai et al., 2004; Lima et al., 2004; Venditti et al., 2010). Five working solutions of standard caffeine were prepared in the range of 10 to 100 mg L-1 to determine the percentage of caffeine in aqueous solution. From the calibration curve, the correlation coefficient was r 2=0.998.
Chromatographic analysis was performed in a high-performance liquid chromatography (HPLC) with diode array detector system (Perkin Elmer, Norwalk, CA, USA) equipped with a C18 250 mm x 4 mm (5 μm). The mobile phase consisted of a mixture of acetonitrile and water (10:90, v/v) at a flow rate of 1.0 mL min-1. Chromatograms were measured at 254 nm with a run time of 5 min (Rojo et al., 1999).
RESULTS AND DISCUSSION
Physical and chemical properties
The observed values of total ash, ash insoluble in acid and soluble in water, showed no significant differences depending on the drying methods used (Table 1). This result was expected since mineral elements have a high stability. Further, minerals in the soil represent the ashes. The plant absorbs them through the roots. Hence, depending on the place of collection and the type of soil, ashes can vary, both in concentration and in qualitative composition.
The total content of ashes found (6.8%) was higher than that reported by Kapp et al. (2016) (4.9%) for guayusa leaves. It is high for the ranges established in the pharmacopoeias and the norms that determine a total index of ashes up to 5%. However, this value might relate to the concentration of minerals in the soil. As observed in the percentage of water-soluble ash, they are basically constituted by alkaline and alkaline-earth minerals. Furthermore, the percentage of ash insoluble in acid, representative of the presence of heavy metals, is within the established limits (Zhi-cen, 1980; WHO, 2011).
Moisture is the water content that remains inside the cell, once the extracellular water clears away by drying. Thus, moisture is of great importance because, in high amounts, it indicates a poor drying process and can cause deterioration of the material plant growth by microorganisms and biotransformation of secondary metabolites. The pharmacopoeias refer values of 8 and 14% depending on the vegetable organ studied.
The results of the physical analysis showed moisture values close to 8%, without significant differences between the different types of drying, which means that regardless of the method and the time used, a good drying of the plant material was achieved.
Another aspect analyzed was the concentration of caffeine (expressed in percentage). Hence, a small increase in concentration was observed in the CO method (3.71% ± 0.11). This may be related to a shorter drying time (13 h CO) compared to air drying (13 d AD) and solar drying (36 h SOD), avoiding the fermentation or degradation of the active ingredients (Christensen and Kaufmann, 1974; Reis et al., 2003). The content of caffeine found in guayusa is comparable to values reported for Camellia sinensis (Nishitani and Sagesaka, 2004) and higher than the content described for Ilex paraguariensis (1.8%) (Isolabella et al., 2010).
Phytochemical analysis
Phytochemical screening is an important test for the identification of a new source of therapeutic compounds from medicinal plants (Ambasta et al., 1986). In this study, the identification of metabolites of Ilex guayusa leaves dried by different methods were performed at ethyl ether (EEE), ethanol (EE) and aqueous extracts (AE). Plant constituents such as alkaloids, flavonoids, phenols, tannin, reducing sugars were identified at EE and AE. Meanwhile steroid, triterpenoid and quinones were only found at EE and saponins at AE (Table 2).
These results differ in the diversity of groups with those reported by Pacha (2012), which only indicated the presence of alkaloids and reducing compounds in the AE. However, they coincide in diversity with the work of Kothiyal et al. (2012), who report the presence in the genus of different pentacyclic triterpenoids derived from α and β-amyrins, saponins, flavonoids and purine alkaloids, among others. In addition, Villacís-Chiriboga et al. (2017) identified and quantified a total of 14 phenolic compounds in the leaves where chlorogenic acid and quercetin-3-O hexose were the major. Also, seven carotenoids were detected and lutein was the main component. The presence of alkaloids in the extracts could be explained by the caffeine content (purine alkaloid) of the leaves which has been reported by Verpoorte et al. (2007). Moreover, the different drying methods did not affect the presence of the compound in the different extracts.
CONCLUSIONS
The sample dried in a conventional oven showed a slight increase in the concentration of caffeine in comparison with the other drying methods (AD and SOD). Although, AD and SOD methods did not affect the caffeine concentration. The physical properties such as moisture, total ash, water soluble ash and acid insoluble ash, did not differ significantly for any method. The chemical groups found in all the samples were flavonoids, phenols, tannins, reducing compounds, saponins, triterpenes-steroids, quinones, fats and alkaloids.
