**2. Materials and Methods**

#### *2.1. Preparation of HLP and Detection of Polyphenolic Compounds*

One hundred grams of *H. sabdari*ff*a* L. (Malvaceae) dried leaves, obtained from Taitung City, Taitung Country, Taiwan, were extracted three times with methanol (300 mL) at 50 ◦C for 3 h, and the samples were filtered after each extraction. The methanol was evaporated under reduced pressure, and the residue was dissolved in 500 mL of distilled water at 50 ◦C and extracted with 200 mL of hexane to remove pigments. The aqueous phase was extracted three times with 180 mL of ethyl acetate, and the solvent was removed from the extract with a vacuum rotary evaporator. The residue was re-dissolved in 250 mL of distilled water and was lyophilized to obtain about 2.5 g of HLP. The polyphenolic components of HLP were further analyzed as follows. All reagents and pure compounds were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Total phenolic acid content was determined by the Folin-Ciocalteau method [20] using gallic acid (GA) as a standard. To start, 0.1 mg of HLP was first dissolved in a tube with 1 mL of distilled water, and 0.5 mL of Folin-Ciocalteu reagen<sup>t</sup> (2 N) was added and mixed thoroughly. After 3 min, 3 mL of Na2CO3 solution (2%) was added, and the mixture was allowed to stand for 15 min. The absorbance of the mixture at 750 nm was measured on a spectrophotometer (Beckman Coulter DU 730, Brea, CA, USA). The concentration of total flavonoid was assayed according to the Jia method [21]. A standard curve using rutin (Rut) was also prepared. Next, 0.5 mL of HLP (1 mg/mL) was diluted with 1.25 mL of distilled water. Afterwards, 75 μL of NaNO2 solution (5%) was added to the mixture. After an interval of 6 min, 150 μL of AlCl3·6H2O solution (10%) was added, and the mixture was allowed to stand for another 5 min. Then, 0.5 mL of NaOH (1 M) and 2.5 mL of distilled water were added. The solution was mixed, and the absorbance was immediately measured against the prepared control at 510 nm. The polyphenolic components of HLP were confirmed by high performance liquid chromatography (HPLC) system using a Hewlett-Packard Vectra 436/33 N system with a diode array detector (all from Waters Corp., Milford, MA, USA). The HLP was filtered through a 0.45 μm filter disc, and then 20 μL of HLP was injected onto a 5 μm RP-18 column (4.6 × 150 mm i.d.; Phenomenex, Inc., Torrance, CA, USA). The mobile phase contained two solvents, including solvent A (formic acid/water = 10:90) and solvent B (formic acid/acetonitrile/water = 10:30:60), run by a linear gradient method at room temperature as follows: From 10% solvent B to 40% solvent B (flow rate = 1.0 mL/min) over 25 min. The chromatography was monitored at 240 and 345 nm, and an ultraviolet (UV) spectrum (Beckman Coulter Inc., Brea, CA, USA) was collected to confirm peak purity. The HPLC analysis of 10 kinds of standard polyphenols showed the retention times (RT) as follows: GA (4.58 min), protocatechuic acid (PCA, 7.50 min), Cat (9.39 min), ECG (11.21 min), EA (13.29 min), Rut (14.01 min), ρ-coumaric acid (CA, 14.44 min), FA (15.28 min), Que (21.57 min), and naringenin (Nar, 24.48 min), respectively. Consistent with our previous study [16], the yield of HLP was approximately 25.0%, and polyphenols were indeed present in HLP (Table S2).
