Background/Objectives: Microalgae are recognized as prolific producers of bioactive metabolites with pharmaceutical potential. This study aimed to isolate and characterize cytotoxic constituents from selected cytotoxic microalgae, collected in Hue city, Vietnam. Methods: Microalgal samples were collected from freshwater bodies, morphologically identified, and maintained in laboratory culture. Thirteen strains were successfully isolated and cultivated in BG11, Z8, and BBM media to determine optimal growth conditions. Cytotoxic effects of extracts/compounds were determined using the sulforhodamine B assay on human lung cancer (SK-LU-1) and human liver cancer (HepG2) cell lines. The methanol extract was partitioned with
n-hexane and CH
2Cl
2, followed by extensive chromatographic separation and HPLC purification to afford twelve compounds, including two new and ten known compounds. The structures were elucidated by HR-ESI-MS and NMR spectra, chemical methods, and comparing compounds in the literature. Results: From the phytoplankton samples collected across six freshwater bodies in Hue city, Vietnam, thirteen microalgal strains were successfully isolated and purified under laboratory conditions. These strains were morphologically and taxonomically identified to be
Microcystis aeruginosa HU05,
Microcystis viridis HU13,
Anabaena circinalis HU08,
Aphanizomenon flos-aquae HU02,
Dolichospermum smithii HU04,
Calothrix braunii HU14,
Nostoc muscorum HU12,
Nostoc punctiforme HU11,
Raphidiopsis raciborskii HU03,
Lyngbya spiralis HU15,
Planktothrix stagnina HU16,
Phormidium subtilis HU06, and
Scenedesmus quadricauda HU07. All methanol extracts of those microalgae were evaluated for cytotoxic activity. The MeOH extracts of
M. viridis (HU13) and
D. smithii (HU04) exhibited significant cytotoxic effects, with IC
50 values of 6.19 ± 0.80 and 4.89 ± 0.76 µg/mL for
M. viridis, and 9.51 ± 0.84 and 8.32 ± 0.94 µg/mL for
D. smithii against SK-LU-1 and HepG2 cell lines, respectively. Furthermore, chemical studies of
D. smithii HU04 led to the isolation of two new compounds, smithioside A (
1) and smithioside B (
2) and ten known ones, 3,4,5-trimethoxyphenyl-1-
O-β-D-glucopyranoside (
3), 4′-hydroxy-3′-methoxyphenol-β-D-[6-
O-(4″-hydroxy-3″,5″-dimethoxylbenzoate)]-glucopyranoside (
4), 4′-hydroxy-2′,6′-dimethoxyphenol 1-
O-β-D-(6-
O-syringoyl)glucopyranoside (
5), mallophenol B (
6), pisoninol II (
7), guaiacylglycerol (
8), (
E)-asarone (
9), deacetylsarmentamide B (
10), (
E)-2-hexenyl-β-D-glucopyranoside (
11), and 5,6-dihydropyridin-2(1
H)-one (
12). The cytotoxic activity of all isolated compounds was also evaluated against SK-LU-1 and HepG2 cancer cell lines. Compound
12 showed the strongest activity, with IC
50 values of 9.13 ± 0.89 µM (SK-LU-1) and 7.64 ± 0.46 µM (HepG2). Compounds
5 and
6 exhibited moderate cytotoxic activity on both human cancer cell lines with IC
50 values ranging from 25.99 to 51.47 µM. Conclusions: These results highlight the potential of
Dolichospermum smithii HU04 as a source of bioactive compounds, particularly in anticancer applications. These findings suggest that
D. smithii HU04 extracts could be developed for therapeutic purposes targeting cancer.
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