*4.2. Isolation and Identification of Compounds 1–***6**

Compounds **1**–**3**, **5**, and **6** (Figure 1) were isolated and characterized by 1H and 13C-NMR spectra from *Senecio roseiflorus* (**1**), *Polygonum senegalense* (**2** and **3**), *Gardenia ternifolia* (**5**), and *Psiadia punctulata* (**6**) plant species collected from Kenya [24,25,27,28] (*vide supra*). In addition, compound **4** was isolated from the leaves of *B. macrocalyx*, collected from Southern Tanzania (Mikindaniya Leo village). A voucher specimen (FMM 3579) was deposited at the Herbarium of the Department of Botany, University of Dar es Salaam, Tanzania. The dried pulverized plant material (5 kg) underwent sequential cold solvent extraction with hexane, EtOAc, and finally MeOH, each soaked in each solvent for 24 h, then the solvents were evaporated with a rotavap yielding hexane, EtOAc, and MeOH extracts (100, 300 and 200 g, respectively). Column chromatography of EtOAc extract (70 g) on silica gel (Merck silica gel 60; 0.40–0.63 mm) eluting with a hexane-DCM gradient, from 100% hexane to 100% DCM and finally in methanol afforded 10 fractions. The fractions eluting with hexane: DCM (3:7) afforded compounds **4** (8-desmethylsideroxylin, 13.2 mg); obtained as yellow needles, mp 275–277 ◦C (Lit. 275–277 ◦C; ESI MS (TOF +ve, Finnigan Mat SSQ 7000): *m*/*z* 299.1 ([M+H]<sup>+</sup>, C17H14O5+H) 1H-NMR (600 MHz, Avance Bruker): 6.82, s, 1H (H-3), 6.82, s, 1H (H-8), 7.95, *dd*, 2H, J = 2.4, 9.0 Hz (H-2- /6- ), 6.93, *dd*, 2H, J = 2.4, 9.0 Hz (H-3- /5- ), 1.98, *s*, 3H (C-6-Me), 3.90, *s*, 3H (C-7-OMe), 13.08, *s*, 1H (C-5-OH), 1H-NMR spectra was in agreement with those reported in the literature); 13C-NMR: 161.3 (C-2), 103.1 (C-3), 182.0 (C-4), 104.4 (C-4a), 157.5 (C-5), 107.6 (C-6), 163.1 (C-7), 90.4 (C-8), 155.5 (C-8a), 121.2 (C-1- ), 128.6 (C-2- /6- ), 116.2 (C-3- /5- ),163.9 (C-4- ), 7.4 (CH3 at C-6), 56.3 (OCH3 at C-7). All NMR spectra of **1**–**6** are provided in Supplementary Information.

### *4.3. MAO Inhibition Assay*

In this study, we have investigated the effect of the isolated constituents (**1**–**6**) on human recombinant MAO-A and -B. The kynuramine oxidation deamination assay was performed in 96-well plates as previously reported, with modification [18,35]. A fixed concentration of kynuramine substrate and varying concentrations of test compounds or inhibitor were used to determine the IC50 values. Kynuramine concentrations for MAO-A and -B were 80 μM and 50 μM, respectively. The concentrations of compounds **1**–**6** varied from 0.001 μM to 100 μM for the rhMAO-A and -B enzyme activity inhibition. The test compounds **1**–**6** were dissolved in DMSO, diluted in the buffer solution just before the assay, and pre-incubated with the enzyme for 10 min at 37 ◦C. The final concentration of DMSO in the enzyme-assay reaction mixtures did not exceed 1%. The enzymatic reactions were initiated by the addition of MAO-A (5 μg/mL) or -B (12.5 μg/mL), and incubated for 20 min at 37 ◦C. The enzyme reactions were terminated by the addition of 78 μL of 2N NaOH. The formation of 4-hydroxyquinoline (the enzyme reaction end product) was recorded fluorometrically on a SpectraMax M5 fluorescence plate

reader (Molecular Devices, Sunnyvale, CA, USA) with an excitation (320 nm) and emission (380 nm) wavelength, using the Soft MaxPro-6 program. The inhibition effects of enzyme activity were calculated as the percent of product formation compared to the corresponding controls (enzyme–substrate reaction) without inhibitors. The assay controls, to define the interference of the test compounds with the fluorescence measurements, were set up simultaneously, and the enzyme or the substrate was added after stopping the reaction.
