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A new quinazolinone alkaloid named peniquinazolinone A (<b>1</b>), as well as eleven known compounds, 2-(2-hydroxy-3-phenylpropionamido)-<i>N</i>-methylbenzamide (<b>2</b>), viridicatin (<b>3</b>), viridicatol (<b>4</b>), (±)-cyclopeptin (<b>5a</b>/<b>5b</b>), dehydrocyclopeptin (<b>6</b>), cyclopenin (<b>7</b>), cyclopenol (<b>8</b>), methyl-indole-3-carboxylate (<b>9</b>), 2,5-dihydroxyphenyl acetate (<b>10</b>), methyl <i>m</i>-hydroxyphenylacetate (<b>11</b>), and conidiogenone B (<b>12</b>), were isolated from the endophytic <i>Penicillium</i> sp. HJT-A-6. The chemical structures of all the compounds were elucidated by comprehensive spectroscopic analysis, including 1D and 2D NMR and HRESIMS. The absolute configuration at C-13 of peniquinazolinone A (<b>1</b>) was established by applying the modified Mosher’s method. Compounds <b>2</b>, <b>3</b>, and <b>7</b> exhibited an optimal promoting effect on the seed germination of <i>Rhodiola tibetica</i> at a concentration of 0.01 mg/mL, while the optimal concentration for compounds <b>4</b> and <b>9</b> to promote <i>Rhodiola tibetica</i> seed germination was 0.001 mg/mL. Compound <b>12</b> showed optimal seed-germination-promoting activity at a concentration of 0.1 mg/mL. Compared with the positive drug 6-benzyladenine (6-BA), compounds <b>2</b>, <b>3</b>, <b>4</b>, <b>7</b>, <b>9</b>, and <b>12</b> could extend the seed germination period of <i>Rhodiola tibetica</i> up to the 11th day.