# Antinociceptive Effect of an Aqueous Extract and Essential Oil from _Baccharis heterophylla_
## 3 Materials and Methods
### Method Validation  
The HPLC method was validated according to the International Conference on Harmonization Guidelines (ICH, 2005) and included a determination of selectivity, linearity, precision, accuracy, LOD, and LOQ. The linearity of the system was performed through the calibration curves of the standards (**1**, **3**, and **4**); compounds were accurately weighed (5 mg) and dissolved in 5 mL acetonitrile-water (5 mL, 1:5) to prepare stock solutions at a final concentration of 1 mg/mL. The solutions of the calibration curve were obtained by diluting the stock solution in water, ranging from 5\(-\)150 \(\upmu\)g/mL for **1**, 20\(-\)200 \(\upmu\)g/mL for **3** and 13.3\(-\)133.4 \(\upmu\)g/mL for **4**. The linearity was assessed estimating the slope, y-intercept, and the determination coefficient (\(R^{2}\)) using the least-squares analysis. LOD and LOQ were determined based on the standard deviation (\(\sigma\)) of the response and the slope (S) from calibration curve constructed with a series of appropriate concentrations for determination of the limits, using the following equations:  
\[LOD=\frac{3.3\ \sigma}{S} \tag{1}\]  
\[LOQ=\frac{10\ \sigma}{S} \tag{2}\]  
The precision was evaluated using repeatability (intraday) and intermediate precision (interday). Intraday and interday variations were established using six independent replicates of the standard reference of each compound on one and two different days, to determine intraday and interday precision, respectively. In order to study the accuracy of the method, recovery experiments were performed. The samples were spiked with known amounts of the standards, at different concentrations levels (low, medium and high):  
**1** (5, 50 and 150 \(\upmu\)g/mL), **3** (20, 100 and 200 \(\upmu\)g/mL) and **4** (13.3, 66.8 and 133.4 \(\upmu\)g/mL). The average recoveries were calculated according to the following formula:  
\[Recovery\ (\%)\ =\ \frac{amount\ found\ -\ original\ amount}{amount\ spiked}\ \times\ 100 \tag{3}\]  
The relative standard deviation (RSD) was calculated for each determination as a measure of precision and repeatability. **4. Conclusions**  
In summary, the aerial parts of _B. heterophylla_ exhibited antinociceptive activity when tested by the formalin assay. The main classes of compounds characterized in the traditional preparation were chlorogenic acid derivatives and flavonoids with known antinociceptive properties. In the case of the essential oil, the activity could be attributed to its high content of antinociceptive monoterpenes. In any case, the pharmacological effects might be achieved via pharmacology synergism and/or polypharmacology. A precise, reliable, and accurate HPLC method for quantifying chlorogenic acid derivatives' content in the infusion of the plant was developed and validated. This methodology will be useful for preparing standardized infusions or other formulations made up of this species. This method will also be proposed as composition test for the monograph of _B. heterophylla_ to be included in the new edition of the Mexican Herbal Pharmacopoeia. Altogether, our results tend to support the medicinal use of _B. heterophylla_ to treat painful complaints in Mexican folk medicine and contribute to the rational use of this valuable medicinal plant. **Supplementary Materials:** The following are available online at [[https://www.mdpi.com/2223-747/10/1/116/s1](https://www.mdpi.com/2223-747/10/1/116/s1)]([https://www.mdpi.com/2223-747/10/1/116/s1](https://www.mdpi.com/2223-747/10/1/116/s1)), **Figure S1**: Temporal course of the antinociceptive behavior (the liking time against time) and AUC from the time course curve for the Aqueous Extract (AE, 31.6-316 mg/kg). Each measurement represented as mean \(\pm\) SEM of \(n=6\).