| Summary: | In our continuing research for bioactive constituents from natural resources, a new methyl threonolactone glucopyranoside (<b>1</b>), a new methyl threonolactone fructofuranoside (<b>2</b>), 2 new pyroglutamates (<b>3</b> and <b>4</b>), and 10 known compounds (<b>5</b>–<b>14</b>) were isolated from the whole plant of <i>Spilanthes acmella</i> (L.) L. The structures of these compounds were determined based on various spectroscopic and chemical analyses. All of the isolated compounds were evaluated on bone formation parameters, such as ALP (alkaline phosphatase) and mineralization stimulatory activities of MC3T3-E1 cell lines. The results showed that the new compound, 1,3-butanediol 3-pyroglutamate (<b>4</b>), 2-deoxy-<span style="font-variant: small-caps;">d</span>-ribono-1,4-lactone (<b>6</b>), methyl pyroglutamate (<b>7</b>), ampelopsisionoside (<b>10</b>), icariside B<sub>1</sub> (<b>11</b>), and benzyl α-<span style="font-variant: small-caps;">l</span>-arabinopyranosyl-(1→6)-β-<span style="font-variant: small-caps;">d</span>-glucopyranoside (<b>12</b>) stimulated both ALP and mineralization activities.
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