## 2 Materials and Methods
### Isolation and Purification of the Growth Inhibitory Substances  
The powdered _Albizia richardiana_ leaves (3000 g) were (dissolved in 14 L of 70% aqueous methanol and 14 L of methanol) extracted as mentioned in the extract preparation and were concentrated (at 40 \({}^{\circ}\)C) using a rotary evaporator to make aqueous residues. The concentrated residues were then adjusted to a pH of 7.0 with 1 molar of phosphate buffer solution. Partitioning was done with the same amount of the ethyl acetate (seven times, 150 mL/time) and separated into aqueous fraction and ethyl acetate fraction. The growth suppression effects of the aqueous fraction and the ethyl acetate fraction were measured with a cress assay as mentioned previously. The ethyl acetate fraction displayed the highest activity, so this fraction was then evaporated until complete dryness after using anhydrous sodium sulphate (Na\({}_{2}\)SO\({}_{4}\)) to remove water for overnight. Firstly, the fraction of ethyl acetate was separated through a column of silica gel (60 g of silica gel 60, spherical, 70-230 mesh; Nacalai Tesque, Inc.) and eluted stepwise with n-hexane (150 mL/step), holding increasing quantities of ethyl acetate (10%/step, \(v/v\)) from 20% to 80%, ethyl acetate (150 mL), and two times cold methanol (300 mL) to produce 9 fractions. The biological effects of these nine fractions were measured using a cress assay. From the assay results, it was observed that fraction 8 (80% ethyl acetate in n-hexane) showed thehighest biological activity. The residues were evaporated to dryness and separated through a column of Sephadex LH-20 (GE Healthcare, 50 g; Bio-Sciences AB, SE-751 84 Uppsala, Sweden). The Sephadex column was loaded with different percentages (fractions 1 to 5) of aqueous methanol (e.g., 20, 40, 60, and 80% (\(v/v\))) (150 mL/step), and two times methanol (300 mL). After collection, these fractions were evaporated until dry and, to check the biological effects of these fractions, a cress bioassay was set. Assay chromatography showed that the 40% aqueous methanol fraction (fraction 2) was the most active. The residues were evaporated until dry, diluted with aqueous methanol 20% (\(v/v\)), and loaded onto a reverse-phase C\({}_{18}\) cartridge (YMC Co. Ltd., Kyoto, Japan). The C\({}_{18}\) cartridge was eluted with 20%, 30%, 40%, 50%, 60%, and 80% (\(v/v\)) aqueous methanol (15 mL per step) and two times methanol (30 mL per step). The aqueous methanol of 30% (fraction 2) showed the most activity against cress. The highest active fraction was purified using reverse-phase high-performance liquid chromatography (HPLC, 500 mm \(\times\) 10 mm I.D. ODS AQ-325; YMC Co. Ltd.) at a flow rate of 1.5 mL/min with 40% aqueous methanol and was detected at a wavelength of 220 nm and an oven temperature of 40 \({}^{\circ}\)C. Two active peaks were found at retention times 65-70 min (compound **1**) and 76-80 min (compound **2**). These two active peak fractions were checked again using reverse-phase HPLC (5 \(\upmu\)m, 4.6 mm \(\times\) 250 mm I.D., Inertsil(r) ODS-3; GL Science Inc., Tokyo, Japan). The column was eluted at a flow rate of 0.8 mL/min with 20% (\(v/v\)) aqueous methanol for compound **1** and 25% (\(v/v\)) aqueous methanol for compound **2**. The active peaks of these two compounds were detected at 220 nm, 40 \({}^{\circ}\)C, and at a retention time of 68-74 min as a colorless substance (compound **1**) and at a retention time of 56-63 min as a whitish substance (compound **2**). These two active compounds were then characterized through HRESIMS. ESIMS spectra were obtained on an LCT Premier XE time-of-flight (TOF) mass spectrometer using positive ion mode; \({}^{1}\)H-NMR, NMR spectral data were recorded on a JEOL JNM-ECX400 spectrometer at 400 MHz. \({}^{1}\)H NMR chemical shifts were referenced to residual CHD2 OD observed at 8H 3.31 and a specific rotation.