*3.3. Sample Preparation*

The dried samples (rhizomes, stems, and leaves) were ground and then passed through a 100 mesh sieves. Each sample powder (25 mg) was accurately weighed and extracted while using 1.5 mL 80% methanol-water solution, at 25 ◦C. The samples were extracted while using an Ultrasonic extractor for 40 min. The final extract was filtered with a 0.22 μm syringe filter into an HPLC vial and then subjected to HPLC analysis [16,58].

#### *3.4. Instrumentation and HPLC Analysis*

Chromatographic analyses were performed with an Agilent 1260 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA), which was equipped with a G1315D diode-array detector, a G1329B ALS autosampler, and a thermostated column compartment. The HPLC fingerprint was recorded by Chemstation software (Agilent Technologies, Waldbron, Germany).

The analytical separation was adopted from a published method for chemical fingerprinting analysis [16]. The separation was achieved on a reversed phase C18 (Agilent Intersil, 5 μm, 4.6 × 150 mm) column (Agilent, Santa Clara, CA, USA). The composition of the mobile phase was: (A) 0.1% phosphoric acid in water and (B) 100% acetonitrile. The separation was as follows: 0.00–2.50 min: 7–10% B, 2.50–20.00 min: 10–26% B, 20.00–29.02 min: 26–58.3% B, 29.02–30.00 min: 58.3–90% B. The column was subsequently washed with 90% B and re-equilibrated with 7% B prior to injection of the next sample. The flow rate was 1.0 mL/min and the column temperature was 30 ◦C. The injection volume was 5 μL and the detective wavelength of UV spectra was set at 241 nm. Chromatographic data was processed while using OpenLab software (Agilent Technologies) [16,58].
