Hypericum patulum (Guttiferae). Their structures were elucidated by extensive analysis of spectroscopic data , X-ray crystallography, electronic circular dichroism (ECD) spectroscopy and Rh2(OCOCF3)4-induced ECD. All compounds were tested for their cytotoxic activities on human HepG-2, HeLa, MCF-7, and A549 cell lines via 3--2,5-diphenyltetrazolium bromide (MTT) assay. Compound 5 exhibited significant cytotoxicities against HepG-2, HeLa and A549 cell lines with IC50 values of 9.52 ± 0.27, 11.87 ± 0.22 and 12.63 ± 0.12 μM, respectively.Six new compounds, hyperpatulones A–F (1–6), along with ten additional known related derivatives (7–16), were isolated from  Hypericum patulum and compound 5 exhibited significant cytotoxicities on various tumor cell lines.Six new PPAPs (1–6) were isolated from  Hypericum patulum (Guttiferae) is well known as “Jinsimei” in China, and is distributed mainly in southwest China, such as Guizhou, Sichuan and Yunnan Provinces.1 The herbs of H. patulum are used as a traditional medicine to clear heat, cool blood, relax tendons and activate collaterals, and to treat gonorrhea, hepatitis, colds, etc.2–6 Modern pharmacological investigations demonstrated that the plants of the genus Hypericum possessed anti-depression,7–11 anti-tumor,12–18 anti-bacterial,19,20 anti-viral,17,18,21–23 and liver protective activities.24 Previous phytochemical studies on these plants showed that derivatives of polycyclic polyprenylated acylphloroglucinols (PPAPs), which possessed a highly oxygenated bicyclo[3.3.1]nonane-2,4,9-trione or other related core decorated with C5H9 or C10H17 (prenyl or geranyl) side chains, were the main bioactive components.7,13–19,21,24,252(OCOCF3)4-induced ECD. Moreover, compounds 1–16 were evaluated for their cytotoxic activities on human HepG-2, HeLa, MCF-7, and A549 cell lines using the MTT assay. Among them, compound 5 shows significant cytotoxicities toward HepG-2, HeLa and A549 cell lines .In this paper, we report the isolation and structural elucidation of six new PPAPs (1–6) , togetheHypericum patulum was subjected to liquid–liquid fractionation to afford a petroleum ether (PE)-soluble fraction and an ethyl acetate (EtOAc)-soluble fraction. The PE fraction was separated by silica gel column chromatography, Sephadex LH-20 and preparative HPLC to obtain six new compounds (1–6) and ten known ones (7–16).The 95% EtOH extract of 3OH as colorless crystals with [α]25D +39.6 . Its molecular formula was deduced as C38H50O6 on the basis of 13C NMR and HRESIMS  data. IR spectroscopy suggested the presence of hydroxyl (3456 cm−1), carbonyl (1716 cm−1) and aromatic double bond  groups. The NMR data of 1 , an unconjugated carbonyl carbon , a methylene , a methine , and three quaternary carbons at δC 79.7 (C-2), 60.2 (C-6), and 49.7 (C-3), which suggested that 1 was a polycyclic polyprenylated acylphloroglucinol.7,26,27 Besides the above carbons, signals for eight methyls, six methylenes, nine methines and six quaternary carbons were observed. The NMR spectroscopic data of 1 resembled those of 32-epi-hyperforatin E.27 The main differences were that the absence of the 2-methylpropanoyl group , and the presence of a benzoyl group  in 1 (epi-hyperforatin E was replaced by a benzoyl group in 1. This was confirmed by the 1H–1H COSY cross-peaks between H-13/15 (δH 7.20) and H-12/16 (δH 7.41)/H-14 (δH 7.36), as well as the HMBC cross-peaks from H-12/16 to C-10 (δC 194.2)/C-14 (δC 132.3)  with H-5b (δH 1.63)/H-24, H-5b with H-34 and of H-5a (δH 2.10) with H-35 (δH 4.61) (in situ formed [Rh2(OCOCF3)4] complex.28,29 According to the bulkiness rule,28–30 the 20S configuration of 1 was confirmed by the Cotton effect (positive E band) of the Rh complex  and ECD calculations ] in 1 , which iC 132.3) . The relδH 4.61) . The abs20S, 35S . Based oα]25D +41.7 . The HRESIMS of compound 2 showed an [M + Na]+ ion peak at m/z 625.3506 , consistent with the molecular formula of C38H50O6. Compounds 2 and 1 were separated by using chiral HPLC over a CHIRALPAK IC column. And the NMR spectroscopic data of 2 4] complex-induced ECD spectrum . According to its 1D NMR spectra  between C-21 and C-22, but the presence of one additional oxygenated quaternary carbon (δC 73.1) and one additional methyl group (δC 26.4) in 3, and the chemical shifts of C-18, 19, 20, 23 shifted from δC 32.7, 32.0, 76.5, 17.8 in 1 to δC 34.2, 28.2, 79.5, 23.8 in 3, which indicated the olefinic carbons  in 1 were replaced by a tertiary alcohol hydroxy group and a methyl group in 3. This was confirmed by the HMBC cross-peaks from H-20 (δH 3.26)/H-22 (δH 1.18)/H-23 (δH 1.12) to C-21 (δC 73.1) (δH 1.18) with H-5b (δH 1.63)/H-24, of H-5b with H-34 and of H-5a (δH 2.10) with H-35 (δH 4.62) indicated that the relative configuration of 3 was identical to that of 1 (in situ [Rh2(OCOCF3)4] complex-induced ECD spectrum of 3 exhibited a positive E band for a 20S configuration  . NOESY chat of 1 . The in 33H42O6 by its HRESIMS m/z 643.3626 [M + Na]+ . The NMR data 4] complex-induced ECD spectrum of 4 . The 1D NMR data , with a 6/6/5 tricyclic spiro ring system, were a special subgroup of PPAPs. Detailed comparison of the NMR spectra of 5 with those of hyperascyrone G indicated the absence of a 3-methylbutanoyl group  in hyperascyrone G, but the presence of a benzoyl group  in 5. Thus, it could be deduced that the 3-methylbutanoyl group in hyperascyrone G was replaced by a benzoyl group in 5. This was confirmed by the 1H–1H COSY cross-peaks between H-30/32 (δH 7.37) and H-29/33 (δH 7.44)/H-31 (δH 7.43), as well as the HMBC cross-peaks from H-29/33 to C-27 (δC 191.5)/C-31 (δC 131.3) (δH 4.55) and H-22a (δH 2.66), between H-23 (δH 5.13) and H-17a (δH 2.15), between H-8 (δH 1.83) and Me-15 (δH 1.71)/Me-16 (δH 1.18)/H-14a (δH 4.78)  . The relδH 4.78) . The ECD25H36O6 by HRESIMS . The 1D NMR data , 1.78 and 1.44 (CH2), 1.22 (CH3), 0.83 (CH3); δC 205.1 (CO), 41.9 (CH), 25.3 (CH2), 19.5 (CH3), 12.3 (CH3)] in chipericumin D, but the presence of a propanoyl group  in 6. Thus, it could be deduced that the 2-methylbutanoyl group in chipericumin D was replaced by a propanoyl group in 6. The structure was supported by the 1H–1H COSY correlations between H-24 (δH 2.98) and Me-25 (δH 0.97) together with the HMBC correlations between Me-25 and C-23 (δC 201.0) (δH 1.91)/H-14a (δH 1.45), H-12 (δH 1.78)/H-14a, H-8 (δH 1.67)/Me-15 (δH 0.95), H-7b (δH 1.76)/Me-16, H-8/Me-16 (δH 1.40), Me-15/H-24 (δH 2.98) and H-7b/H-18(δH 4.55) (Compound 6 was assigned the molecular formula CC 201.0) . The relδH 4.55) . In addiδH 4.55) . Thus, s32 uralodin B (8),13 attenuatumione H (9),26 uralione D (10),7 uralione I (11),7 tomoeone A (12),15 tomoeone B (13),15 chipericumin D (14),31 hyperascyrone F (15),18 hypercohone G (16),33 by comparison of their spectroscopic and physical data with those of related literature.Ten known compounds were identified as uralodin A (7),50 values of 9.52 ± 0.27 to 42.33 ± 1.91 μM. Especially, compound 5 shows significant cytotoxicities toward HepG-2, HeLa and A549 cell lines .The isolates 1–16 were tested for their cytotoxic activities by MTT assay on human HepG-2, HeLa, MCF-7 and A549 cell lines. Cisplatin was used as the positive control. As shown in 18 preparative column  equipped with a G1311C pump and a G1315D photodiode array detector . All chemical reagents were purchased from Tianjin Damao Chemical Company .Optical rotations were obtained on a JASCO P-1020 polarimeter. UV spectra were recorded using a JASCO V-550 UV/VIS spectrophotometer. CD spectra were measured on a JASCO J-810 spectrometer. 1D and 2D NMR spectra were recorded on Bruker AV-500 NMR spectrometers with TMS as an internal standard. HRESIMS analyses were recorded on an Agilent 6210 ESI/TOF mass spectrometer. Column chromatography (CC) was performed with Silica gel , ODS  and Sephadex LH-20 . Preparative HPLC was conducted on a Cosmosil CHypericum patulum was collected in Guizhou Province of China, in August of 2016 and authenticated by Zhenqiu Mai, the senior engineer of Guangdong Province. A voucher specimen (no. 20160817) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, China.The whole plant of Hypericum patulum (12 kg) were extracted under reflux with 95% EtOH (30 L × 3) at room temperature. The combined ethanol extract was concentrated to afford a residue (654 g), which was suspended in water (4 L) and then extracted with petroleum ether (PE) (4 L × 3) and ethyl acetate (EtOAc) (4 L × 3). The PE extract (217 g) was subjected to silica gel column chromatography, eluting with PE-EtOAc  to yield seven fractions (Fr. A–F). Fr. C (18.7 g) was further applied to a silica gel CC with PE/EtOAc  to afford five subfractions (Fr. C1–C5). Fr. C2 (1.5 g) was purified by Sephadex LH-20  and further separated by preparative HPLC  to yield compounds 1 (21.2 mg), 2 (18.5 mg), 7 (11.7 mg) and 8 (16.9 mg). Fr. C3 (7.5 g) was purified by ODS CC and Sephadex LH-20 to obtain compounds 3 (12.7 mg), 4 (13.9 mg), 9 (19.7 mg), 10 (15.7 mg) and 11 (13.2 mg). Fr. D (21.9 g) was applied to ODS CC using a MeOH/H2O gradient  to afford five subfractions (Fr. D.1–D.5). Fr. D.3 (3.6 g) was further purified by Sephadex LH-20 CC  and preparative HPLC  and to yield compounds 5 (9.5 mg), 12 (21.3 mg) and 13 (25.1 mg). Fr. D.4 (5.8 g) was separated by preparative HPLC  to yield compounds 6 (15.8 mg) and 14 (11.9 mg). Fr. D.5 (4.9 g) was purified by preparative HPLC  to achieve compounds 15 (9.2 mg) and 16 (15.2 mg).The dried and powdered herbs of α]25D +39.6 ; UV (MeOH) λmax 203, 250 and 277 nm; IR (KBr) νmax 3456, 2981, 2931, 1716, 1693, 1624, 1450, 1369, 1227 cm−1; 1H and 13C NMR spectroscopic data, see Tables S1 and S2 .Colorless needle crystals (MeOH); mp 116–117 °C; 25D +56.6 ; UV (MeOH) λmax 204, 247 and 274 nm; IR (KBr) νmax 3425, 2977, 2931, 1705, 1600, 1442, 1389, 1273, 1215, 1119 cm−1; 1H and 13C NMR spectroscopic data, see Tables S1 and S2 .Colorless oil; 25D +51.8 ; UV (MeOH) λmax 203, 248 and 275 nm; IR (KBr) νmax 3413, 2974, 2927, 1709, 1604, 1446, 1381, 1281, 1219, 1122 cm−1; 1H and 13C NMR spectroscopic data, see Tables S1 and S2 .Colorless oil; 25D −37.5 ; UV (MeOH) λmax 208, 242 and 353 nm; IR (KBr) νmax 3413, 2965, 2927, 2877, 1716, 1612, 1454, 1376, 1269, 1153 cm−1; 1H and 13C NMR spectroscopic data, see Tables S1 and S3 .Colorless oil; 25D +22.8 ; UV (MeOH) λmax 203, 216, 249 and 279 nm; IR (KBr) νmax 3410, 2970, 2935, 2877, 1720, 1662, 1612, 1454, 1385, 1319, 1273, 1211, 1153, 1107 cm−1; 1H and 13C NMR spectroscopic data, see Tables S1 and S3 .Colorless oil;  × 100.Four selected human cancer cell lines at the logarithmic phase were seeded in 96-well plates at 5 × 10Hypericum patulum. Their structures were determined by spectroscopic data, X-ray crystallography, ECD spectrum and Rh2(OCOCF3)4-induced ECD. Moreover, compounds 1–16 were evaluated for their cytotoxic activities on human HepG-2, HeLa, MCF-7, and A549 cell lines using the MTT assay. Compound 5 shows significant cytotoxicity toward HepG-2, HeLa and A549 cell lines .In summary, six new PPAPs derivatives, hyperpatulone A–F (1–6), together with ten known analogs, were obtained from the dried herbs of The authors declare no competing financial interest.RA-009-C9RA00277D-s001RA-009-C9RA00277D-s002


Lonicera japonica Thunb., possesses antiviral and hepatoprotective activities, and is widely used as a health food and in cosmetics. However, its major constituents, caffeoylquinic acid derivatives, and their anti-HBV activity were lacking systematic research. In this study, four novel caffeoylquinic acids, five simple caffeic acids and fourteen known caffeoylquinic acids are isolated and identified from L. japonica. Most caffeoylquinic acids inhibited HBsAg and HBeAg secretion, and HBV DNA replication. In particular, 100 μg ml−1 monocaffeoylquinic acid 9 inhibits HBsAg and HBeAg secretion, and HBV DNA replication by 83.82, 70.76 and 39.36% compared to the control. Unfortunately, 50 μg ml−1 tricaffeoylquinic acid 23 promotes HBsAg and HBeAg secretion, and HBV DNA replication by 172.39, 9.92 and 55.40%. Finally, structure–activity relationships reveal that caffeoylquinic acids containing a caffeoyl group have better inhibitory activities. The results indicate that caffeoylquinic acids from L. japonica could serve as anti-HBV agents for functional food or medicinal use. Lonicera japonica Thunb., possesses antiviral and hepatoprotective activities, and is widely used as a health food and in cosmetics. Caffeoylquinic acids, which exist widely in the plant kingdom, especially in Asteraceae,2 Umbelliferae,3 and Caprifoliaceae,4 are a type of polyphenol compound formed by esterification and condensation of a molecule of quinic acid and a multimolecule caffeic acid. Caffeoylquinic acids and their derivatives are reported to possess anti-HBV activities.5,6The hepatitis B virus (HBV) is a major global health problem, due to its worldwide distribution and potential adverse consequences. Current therapeutic strategies for HBV, however, are far from satisfactory.Lonicera japonica Thunb. (Caprifoliaceae) is native to East Asia. In China, L. japonica is widely cultivated in Shandong and Henan Provinces. Jin Yin Hua, also known as Ren Dong, the flower buds of L. japonica, are famous in traditional Chinese medicine.7 During the recent decades, they have been used for treating influenza, cold, fever, and infections.8 Moreover, they have also been used in indigenous beverages, such as tea, in Korea and China for many years.9 Previous phytochemical investigations have been reported for different types of chemical constituents, including flavonoids, caffeoylquinic acids, iridoids, saponins, and other compounds.9–12 Furthermore, caffeoylquinic acids are major constituents of L. japonica.13,14 However, the caffeoylquinic acid derivatives form L. japonica and their anti-HBV activities are lacking in systematic research.L. japonica Thunb., as well as four new caffeoylquinic acids (1–4), together with five simple caffeic acids , and fourteen known caffeoylquinic acids  are systematically isolated and identified  as an internal standard. Electrospray ionization mass spectrometry (ESI-MS) data was obtained on a Bruker Esquire LC 200-Ion trap mass spectrometer , and high-resolution ESI-MS (HR-ESIMS) were measured on a Bruker microTOF-QII mass spectrometer . Optical rotations were measured using a JASCO P-1020 automatic digital polarimeter . Sephadex LH-20 , silica gel , and ODS  were used for column chromatography. Thin-layer chromatography (TLC) was carried out on silica gel 60 F254 , and spots were visualized by spraying the plates with 10% H2SO4 in EtOH and heating them at 105 °C. Preparative high-performance liquid chromatography (HPLC) was carried out on a EasyChrom 3.2.8.0 system  and an octadecyl silica (ODS) column . Diagnostic kits for HBsAg and HBeAg were purchased from Abbott Trading Shanghai Co. Ltd. , and a diagnostic kit for HBV DNA was purchased from Hunan Shengxiang Jiancheng Biotechnology Co. Ltd. . HPLC-grade methanol was purchased from Sigma-Aldrich Co. . All other analytical chemicals were obtained from Shanghai Chemical Reagents Co., Ltd .Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX-400 spectrometer using standard Bruker pulse programs . Chemical shifts are shown as L. japonica were purchased in September 2017 from Fukangwanjia Pharmaceuticals, Haozhou, Anhui Province, People's Republic of China. The plants were identified by Dr XB Zeng of Shenzhen People's Hospital and a voucher specimen (no. 20170930) was deposited at Center Lab of Longhua Branch, Shenzhen People's Hospital, Second Clinical Medical College of Jinan University, Shenzhen, China.The flower buds of L. japonica flower buds (6.5 kg) were extracted three times with 75% ethanol  under room temperature and concentrated by evaporation in vacuo. Then the EtOH extract (1.5 kg) was suspended in distilled water and successively partitioned three times with cyclohexane, ethyl acetate (EtOAc), and n-butyl alcohol, respectively. The EtOAc fraction (83 g) was separated by silica gel column chromatography  eluted with a gradient of dichloromethane-methanol  to afford 20 fractions (fractions 1–20) by TLC plate analysis.The air-dried and powdered 2Cl2–MeOH  to afford 6 subfractions (Fr. 4–1 to Fr. 4–6). Next, compound 5 (50.0 mg) was obtained through recrystallization in methanol from Fr. 4–6.Fr. 4  was subjected to Sephadex LH-20 column chromatography (2.3 × 75 cm) isocratic eluted with CH2Cl2–MeOH, 80 : 20, v/v) to yield 12 subfractions (Fr. 6–1 to Fr. 6–12). Fr. 6–9 was subjected to preparative HPLC  yielding compound 6 . Fr. 6–11 was also purified by preparative HPLC. HPLC  to afford compound 7 .Fr. 6  was separated by Sephadex LH-20 column chromatography  to afford 4 subfractions (Fr. 10–1 to Fr. 10–4). Fr. 10–3 was applied to preparative HPLC  and obtain compound 8 .Fr. 10  was submitted to Sephadex LH-20 column chromatography (2.3 × 75 cm) isocratic eluted with CH2Cl2–MeOH, 80 : 20, v/v) to afford subfractions Fr. 13–1 to 13–8 via TLC, and Fr. 13–4 was subjected to preparative HPLC  yielding compound 9 , compound 10  and compound 11 .Fr. 13  was chromatographed on Sephadex LH-20 column  to obtain 9 subfractions (Fr. 16–1 to Fr. 16–9). Compound 12 (150 mg) was obtained by recrystallizing in methanol from Fr. 16–2. Fr. 16–3 was subjected to preparative HPLC  yielding compound 13  and compound 14 . Fr. 16–4 was purified by preparative HPLC  using MeOH–H2O  as the eluent to give compound 15 , compound 16 , compound 1 , compound 2 , and compound 17 . Fr. 16–5 was separated by preparative HPLC  to afford compound 18 , compound 19 , compound 3  and compound 20 . Next, Fr. 16–6 was subjected to preparative HPLC  yielding compound 21 , compound 22 , compound 4  and compound 23 . L. japonica.Fr. 16 , was chromatographed on an ODS column (5.5 × 28 cm) gradually eluted with MeOH–Hm/z 547.1627 [M − H]−; 1H NMR  and 13C NMR  spectrum information, see Pale yellow, amorphous powder; HR-ESI-MS m/z 561.1877 [M − H]−; 1H NMR  and 13C NMR  spectrum information, see Pale yellow, amorphous powder; HR-ESI-MS m/z 529. 1506 [M − H]−; HR-ESI-MS2m/z 367.1129 [M–caffeoyl]−, 161.0289 [caffeoyl]−; 1H NMR  and 13C NMR  spectrum information, see Pale yellow, amorphous powder; HR-ESI-MS m/z 529.1573 [M − H]−; HR-ESI-MS m/z 529. 1506 [M − H]−; HR-ESI-MS2m/z 367.1185 [M–caffeoyl]−, 161.0295 [caffeoyl]−; 1H NMR  and 13C NMR  spectrum information, see Pale yellow, amorphous powder; HR-ESI-MS 1H NMR  δH: 7.43 , 6.82 , 3.79 ; 13C NMR  δC: 121.68 (C-1), 115.06 (C-2), 151.13 (C-3), 147.25 (C-4), 112.82 (C-5), 123.49 (C-6), 167.22 (C-7), 55.60 (–OCH3).Pale yellow, amorphous powder; 1H NMR  δH: 7.47 , 7.05 , 7.00 , 6.76 , 6.25 , 4.15 , 1.24 ; 13C NMR  δC: 125.47 (C-1), 114.77 (C-2), 148.34 (C-3), 145.53 (C-4), 115.69 (C-5), 121.29 (C-6), 144.95 (C-7), 114.01 (C-8), 166.50 (C-9), 59.64 (C-10), 14.22 (C-11).Pale yellow, amorphous powder; 1H NMR  δH: 9.70 , 7.27 , 7.23 , 6.90 ; 13C NMR  δC: 124.33 (C-1), 115.45 (C-2), 152.10 (C-3), 145.82 (C-4), 114.33 (C-5), 128.78 (C-6), 190.93 (C-7).Pale yellow, amorphous powder; 1H NMR  δH: 7.54 , 7.15 , 7.04 , 6.86 , 6.29 ; 13C NMR  δC: 127.12 (C-1), 117.13 (C-2), 149.38 (C-3), 146.83 (C-4), 115.99 (C-5), 122.55 (C-6), 145.98 (C-7), 116.51 (C-8), 169.34 (C-9).Pale yellow, amorphous powder; 1H NMR  δH: 7.39 , 7.02 , 6.96 , 6.77 , 6.11 , 5.02 , 4.00 , 3.89 , 3.58 , 2.11 , 1.93 , 1.77 , 1.12 ; 13C NMR  δC: 73.08 (C-1), 35.16 (C-2), 67.00 (C-3), 71.04 (C-4), 69.50 (C-5), 37.16 (C-6), 173.06 (C-7), 60.27 (C-8), 13.79 (C-9), 125.37 (C-1′), 115.84 (C-2′), 148.47 (C-3′), 145.62 (C-4′), 114.55 (C-5′), 121.28 (C-6′), 145.04 (C-7′), 113.85 (C-8′), 165.40 (C-9′).Yellow, amorphous powder; 1H NMR  δH: 7.50 , 7.43 , 7.04 , 6.99 , 6.78 , 6.26 , 6.13 , 5.19 , 5.09 , 4.05 , 3.85 , 2.20 , 2.01 , 1.15 ; 13C NMR  δC: 72.43 (C-1), 34.40 (C-2), 66.70 (C-3), 70.96 (C-4), 69.95 (C-5), 33.25 (C-6), 174.30 (C-7), 60.41 (C-8), 13.79 (C-9), 125.61 (C-1′), 125.33 (C-1′′), 115.89 (C-2′), 115.81 (C-2′′), 148.56 (C-3′), 148.31 (C-3′′), 145.31 (C-4′), 144.93 (C-4′′), 113.65 , 121.32 (C-6′), 121.20 (C-6′′), 145.65 (C-7′), 145.58 (C-7′′), 114.77 (C-8′), 114.62 (C-8′′), 165.99 (C-9′), 165.29 (C-9′′).Yellow, amorphous powder; 1H NMR  δH: 7.62 , 7.47 , 7.10 , 7.06 , 7.02 , 6.97 , 6.78 , 6.32 , 6.13 , 5.15 , 4.99 , 4.90 , 2.34 , 2.01 , 1.24 , 1.17 ; 13C NMR  δC: 72.95 (C-1), 38.56 (C-2), 66.67 (C-3), 70.68 (C-4), 69.17 (C-5), 37.78 (C-6), 177.55 (C-7), 125.33 (C-1′), 125.22 (C-1′′), 115.88 (C-2′), 115.77 (C-2′′), 148.88 (C-3′), 148.77 (C-3′′), 146.82 (C-4′), 146.32 (C-4′′), 113.06 (C-5′), 112.67 (C-5′′), 121.80 (C-6′), 121.49 (C-6′′), 145.67 (C-7′), 145.64 (C-7′′), 115.05 (C-8′), 114.78 (C-8′′), 164.88 (C-9′), 164.68 (C-9′′).Yellow, amorphous powder; 1H NMR  δH: 7.45 , 7.07 , 7.01 , 6.80 , 6.18 , 5.10 , 3.96 , 3.60 , 2.02 , 1.82 ; 13C NMR  δC: 74.85 (C-1), 37.63 (C-2), 71.75 (C-3), 72.20 (C-4), 69.47 (C-5), 38.52 (C-6), 176.35 (C-7), 126.92 (C-1′), 117.09 (C-2′), 149.66 (C-3′), 146.87 (C-4′), 116.08 (C-5′), 122.72 (C-6′), 146.29 (C-7′), 115.61 (C-8′), 167.09 (C-9′).Pale yellow, amorphous powder; 1H NMR  δH: 7.49 , 7.04 , 7.00 , 6.77 , 6.27 , 5.01 , 4.86 , 4.72 , 3.61 , 2.09 , 1.98 , 1.83 ; 13C NMR  δC: 75.98 (C-1), 38.20 (C-2), 64.58 (C-3), 73.82 (C-4), 65.74 (C-5), 38.20 (C-6), 174.01 (C-7), 51.87 (–OCH3), 125.83 (C-1′), 116.04 (C-2′), 148.54 (C-3′), 145.82 (C-4′), 114.97 (C-5′), 121.42 (C-6′), 145.05 (C-7′), 114.81 (C-8′), 166.43 (C-9′).Pale yellow, amorphous powder; 1H NMR  δH: 7.40 , 7.04 , 6.96 , 6.78 , 6.13 , 5.05 , 3.92 , 3.61 , 3.56 , 2.10 , 1.97 , 1.81 ; 13C NMR  δC: 73.47 (C-1), 35.61 (C-2), 67.44 (C-3), 71.14 (C-4), 69.84 (C-5), 37.38 (C-6), 173.82 (C-7), 52.00 (C–OCH3), 125.66 (C-1′), 116.08 (C-2′), 148.63 (C-3′), 145.79 (C-4′), 114.76 (C-5′), 121.58 (C-6′), 145.32 (C-7′), 114.17 (C-8′), 165.68 (C-9′).Pale yellow, amorphous powder; 1H NMR  δH: 7.42 , 7.06 , 6.95 , 6.79 , 6.16 , 5.11 , 3.98 , 3.64 , 3.57 , 2.06 , 1.86 ; 13C NMR  δC: 74.14 (C-1), 36.34 (C-2), 70.62 (C-3), 71.34 (C-4), 68.31 (C-5), 37.60 (C-6), 174.17 (C-7), 52.37 (–OCH3), 126.15 (C-1′), 116.42 (C-2′), 148.86 (C-3′), 146.05 (C-4′), 115.01 (C-5′), 122.00 (C-6′), 145.66 (C-7′), 114.61 (C-8′), 166.24 (C-9′).Pale yellow, amorphous powder; 1H NMR  δH: 7.56 , 7.52 ,7.12 , 6.97 , 6.82 , 6.32 , 6.24 , 5.34 , 5.29 , 3.96 , 2.25 , 2.11 ; 13C NMR  δC: 73.63 (C-1), 36.66 (C-2), 68.95 (C-3), 71.57 (C-4), 71.19 (C-5), 35.40 (C-6), 176.25 (C-7), 126.74 (C-1′), 126.67 (C-1′′), 116.65 , 148.95 (C-3′), 148.84 (C-3′′), 146.18 , 115.61 (C-5′), 115.43 (C-5′′), 122.49 (C-6), 122.32 (C-6′′), 146.07 (C-7′), 145.76 (C-7′′), 115.27 (C-8′), 115.07 (C-8′′), 167.23 (C-9′), 166.75 (C-9′′).Pale yellow, amorphous powder; m/z 193.0540 [M − H]−; 1H NMR  δH: 7.49 , 7.07 , 7.00 , 6.78 , 6.27 , 3.68 .Pale yellow, amorphous powder; HR-ESI-MS 1H NMR  δH: 7.48 , 7.03 , 6.99 , 6.76 , 6.23 , 5.13 , 4.33 , 3.76 , 3.67 , 2.18 , 2.09 , 1.97 .Pale yellow, amorphous powder; m/z 515.1276 [M − H]−; 1H NMR  δH: 7.52 , 7.46 , 7.05 , 6.98 , 6.77 , 6.27 , 6.18 , 5.42 , 5.02 , 4.23 , 2.20 , 2.09 , 1.96 ; 13C NMR  δC: 74.03 (C-1), 37.82 (C-2), 68.00 (C-3), 73.77 (C-4), 66.83 (C-5), 37.58 (C-6), 175.09 (C-7), 125.87 , 116.22 (C-2′), 116.16 (C-2′′), 148.80 , 145.92 , 115.18 (C-5′), 115.15 (C-5′′), 121.90 (C-6′), 121.82 (C-6′′), 145.89 , 114.28 (C-8′), 114.05 (C-8′′), 166.38 (C-9′), 165.98 (C-9′′).Pale yellow, amorphous powder; HR-ESI-MS 1H NMR  δH: 7.56 , 7.49 , 7.10 , 6.98 , 6.81 , 6.31 , 6.20 , 5.28 , 5.21 , 3.94 , 3.61 , 2.25 , 2.10 ; 13C NMR  δC: 73.27 (C-1), 35.77 (C-2), 67.91 (C-3), 71.33 (C-4), 70.74 (C-5), 35.03 (C-6), 174.30 (C-7), 52.38 (C–OCH3), 126.34 (C-1′), 126.08 (C-1′′), 116.45 (C-2′), 116.38 (C-2′′), 148.89 (C-3′), 148.67 (C-3′′), 146.04 (C-4′), 145.98 (C-4′′), 115.09 , 121.98 (C-6′), 121.89 (C-6′′), 145.85 (C-7′), 145.46 (C-7′′), 115.21 (C-8′), 114.41 (C-8′′), 166.69 (C-9′), 166.01 (C-9′′).Pale yellow, amorphous powder; 1H NMR  δH: 7.50 , 7.43 ,7.05 , 7.10 , 6.77 , 6.26 , 6.14 , 5.80 , 5.41 , 3.86 , 3.59 , 2.21 , 2.01 .Pale yellow, amorphous powder; 1H NMR  δH: 7.56 , 7.47 ,7.09 , 6.97 , 6.80 , 6.31 , 6.19 , 5.42 , 5.06 , 4.25 , 3.61 , 2.20 , 2.14 , 2.00 ; 13C NMR  δC: 74.16 (C-1), 37.93 (C-2), 68.10 (C-3), 73.32 (C-4), 66.60 (C-5), 37.23 (C-6), 173.86 (C-7), 52.50 (–OCH3), 126.09 (C-1′), 125.95 (C-1′′), 116.35 , 148.94 (C-3′), 148.84 (C-3′′), 146.15 (C-4′), 146.07 (C-4′′), 115.11 , 122.06 , 145.98 , 114.39 (C-8′), 113.99 (C-8′′), 166.55 (C-9′), 165.99 (C-9′′).Pale yellow, amorphous powder; 1H NMR  δH: 7.48 , 7.45 ,7.04 , 6.97 , 6.75 , 6.25 , 6.23 , 6.16 , 5.44 , 5.42 , 5.25 , 3.64 , 2.35 , 2.06 ; 13C NMR  δC: 72.52 (C-1), 38.89 (C-2), 67.68 (C-3), 69.45 (C-4), 67.75 (C-5), 39.10 (C-6), 173.61 (C-7), 52.04 (C–OCH3), 125.43 (C-1′), 125.29 (C-1′′), 125.22 (C-1′′′), 115.82 (C-2′), 115.77 (C-2′′), 115.71 (C-2′′′), 148.64 (C-3′), 148.58 (C-3′′), 148.43 (C-3′′′), 145.98 (C-4′), 145.87 (C-4′′), 145.48 (C-4′′′), 114.91 (C-5′), 114.83 , 121.56 (C-6), 121.44 (C-6′′), 121.36 (C-6′′′), 145.61 (C-7′), 145.56 , 113.86 (C-8′), 113.20 (C-8′′), 113.14 (C-8′′′), 165.74 (C-9′), 165.51 (C-9′′), 165.16 (C-9′′′).Yellow, amorphous powder; 18 analytical column , which was eluted isocratically with 25% acetonitrile in water (containing 0.1% formic acid) at a flow rate of 1 ml min−1. The temperature of the column oven was 25 °C, and 20 μl was injected into the system each time. The UV spectra were collected at 327 nm. Peaks of the ester hydrolysate of compounds 1–4 were identified by comparing the retention times of authentic samples of 4-O-caffeoylquinic acid methyl ester  after simultaneous treatment under the same conditions.Ester hydrolysates of compounds 1–4 were carried out according to the following method. In brief, compounds 1–4 (each 2.0 mg) were hydrolysed by reaction in 1 N NaOH (4 ml) and tetrahydrofuran (4 ml) at 0 °C for 4 h. The reaction mixture was extracted by adjusting pH to 4.5. The extraction product was directly analysed by reversed-phase HPLC using a Diamonsil C2, 95% relative humidity). The cytotoxicity assay was performed, according to the MTT method in 96-well microplates. Briefly, 200 μl of adherent cells was seeded into each well of the 96-well cell culture plates and allowed to adhere for 24 hours before drug addiction with an initial density of 1.0 × 105 cells per ml. Each tumour cell line was exposed to the test compounds at concentrations of 100, 50, and 20 μg ml−1 (DMEM with 0.1% DMSO) for three times within 48 hours. The control group was treated in the same solvent. After 48 hours, the media and test samples were replaced with MTT (5 mg ml−1) and incubated in the dark for 4 h. The formazan crystals formed were dissolved in DMSO. Absorbance was measured using a microplate reader at 520 nm. Cell viability (%) was calculated as Asample/Acontrol × 100%.The HepG 2 cells and HepG 2.2.15 cells were from the American Type Culture Collection . All the cells were maintained in DMEM containing 10% FBS  and cultured at 37 °C . The anti-HBV assay was performed according to previous research.15 Briefly, 500 μl of adherent cells was seeded into each well of the 24-well cell culture plates and allowed to adhere for 24 hours before drug addiction with an initial density of 3 × 105 cells per mL. The cell supernatants were collected every three days and fresh cell culture medium contain corresponding test compounds were added. The experiment lasted nine days. The collected cell supernatants were used to analyse the levels of HBsAg, HBeAg and HBV DNA with their corresponding reagent kit. The levels of HBsAg and HBeAg in the supernatants were measured with an ELISA method. Real-time PCR assay was used to detect the HBV DNA. The relative level was calculated using the equation: relative level (%) = [(Atest − Acontrol)/Acontrol] × 100 (where A is the levels of secreted HBsAg or HBeAg or HBV DNA), and the subscript words ‘test’ and ‘control’ represent that the test sample and control group, respectively.HepG 2.2.15 cells were maintained in DMEM containing 10% FBS  and cultured at 37 °C  test. Statistical significance is determined at 26H28O13 as deduced from the [M − H]− peak at m/z 547.1627  via HR-ESI-MS and supported by the 13C NMR spectral data. The 1H NMR and 13C NMR spectrum , three oxygen proton signals at δH 3.76 , 5.03 , 5.14 , four proton signals at δH 1.98  and 2.16  and eight carbon resonances at δC 72.90 (C-1), 35.46 (C-2), 67.47 (C-3), 71.07 (C-4), 70.33 (C-5), 34.82 (C-6), 174.10 (C-7), 52.11 (7–OCH3) by HSQC correlations analysis. An aromatic ABX system , together with a pair of coupled doublets [δH 7.49  and 6.24 ], reveal the presence of a trans-caffeoyl moiety. Furthermore, another aromatic ABX system  and three proton signals at δH 4.76 , 2.50 , 2.47  indicated the presence of a 3-hydroxy-3--propionyl moiety, which was confirmed according to the observed NOESY correlations of δH 4.76 (H-7′′) with H-2′′ (δH 6.75), H-6′′ (δH 6.58) and H-8′′ , and HMBC correlations of δH 2.47, 2.50 (H-8′′) to δC 69.35 (C-7′′), 169.87 (C-9′′) in O-trans-caffeoylquinic acid methyl ester (14), which was identified by HPLC analysis. Therefore, we concluded that the trans-caffeoyl moiety was attached to C-4 of quinic acid methyl ester. Moreover, the other part of the ester hydrolysates 3-hydroxy-3--propionic acid was measured to have an optical rotation value of [α]22D = −4.6°, which was nearly equal to [α]22D = −4.5° of β-d-glucopyranoside, 1′′-O-(7S)-7-(3-methoxyl-4-hydroxyphenyl)-7-methoxyethyl-3′′-α-l-rhamnopyranosyl-4′′-[(8E)-7-(3-methoxyl-4-hydroxyphenyl)-8-propenoate].16 Therefore, the C-7′′ configuration of compound 1 was identified as 7′′S. Consequently, compound 1 was determined as 4-O-trans-caffeoyl-5-O-[3S-hydroxy-3--propionyl] quinic acid methyl ester.Compound 1 was isolated as a yellow amorphous powder, with the molecular formula of Cspectrum  showed a27H30O13, based on HR-ESIMS at m/z 561.1877 . The 1H and 13C NMR spectroscopic features . The HMBC correlation from δH 3.07 to C-7′′ (δC 79.24) indicated that the extra methoxyl group was attached to C-7′′ of the propionyl moiety -propionyl] quinic acid methyl ester.Compound 2 was isolated as a yellow amorphous powder, and possessed the molecular formula of Cfeatures  were conl moiety . The 10 26H26O12 by a negative HR-ESIMS molecule at m/z 529.1506 [M − H]− and 529.1573 [M − H]− , respectively. The NMR data of compound 3 (O-caffeoylquinic acid methyl ester (20), except that the second pair of coupled doublet  was different. This suggested compound 3 was also a 4,5-disubstituted quinic acid methyl ester but contains a trans-caffeoyl group and a cis-caffeoyl group. At the same time, a comparison of the 1H NMR and 13C NMR spectra of compound 4 (O-caffeoylquinic acid methyl ester (22) proved that compound 4 was a 3,4-disubstituted quinic acid methyl ester contains a trans-caffeoyl group  and a cis-caffeoyl group . Ester hydrolysates of compounds 3 and 4 were all 4-O-trans-caffeoylquinic acid methyl ester (14), which were identified by HPLC analysis. The above results confirmed that the cis-caffeoyl group was linked to C-5 of compound 3 and C-3 of compound 4, respectively. The key HMBC and NOESY  correlations of compounds 3 and 4 were shown in O-trans-caffeoyl-5-O-cis-caffeoyl quinic acid methyl ester and 3-O-cis-caffeoyl-4-O-trans-caffeoyl quinic acid methyl ester, respectively.Compounds 3 and 4 were isolated as yellow amorphous powder. The molecular formula of compounds 3 and 4 were determined to be Cmpound 3  was simimpound 4  with tho17 caffeic acid ethyl ester (6),18 3,4-dihydroxybenzaldehyde (7),19 caffeic acid (8),20 4-O-caffeoylquinic acid ethyl ester (9),21 4,5-di-O-caffeoylquinic acid ethyl ester (10),22 1,4-di-O-caffeoylquinic acid (11),23 3-O-caffeoylquinic acid (12),20 1-O-caffeoylquinic acid methyl ester (13),24 4-O-caffeoylquinic acid methyl ester (14),25 3-O-caffeoylquinic acid methyl ester (15),25 4,5-di-O-caffeoylquinic acid (16),24,26 caffeic acid methyl ester (17),25 5-O-caffeoylquinic acid methyl ester (18) ,24 3,4-di-O-caffeoylquinic acid (19),24,26 4,5-di-O-caffeoylquinic acid methyl ester (20),27 3,5-di-O-caffeoylquinic acid (21),28 3,4-di-O-caffeoylquinic acid methyl ester (22),29 3,4,5-tri-O-caffeoylquinic acid methyl ester (23).30The other compounds were identified as isovanillic acid (5),−1 showed no significant cytotoxicity against HepG 2 and HepG 2.2.15 cells. Therefore, the above compounds were selected 100 μg ml−1 for test drug concentration in the subsequent anti-HBV assay. In addition, compounds 11, 18 and 23 exhibit weak cytotoxicity against HepG 2 and HepG 2.2.15 cells at concentration of 100 μg ml−1 and selected 50 μg ml−1 for the next anti-HBV activities. At the same time, compounds 6–8 and 17 were selected 20 μg ml−1 due to their significantly cytotoxicity against HepG 2 and HepG 2.2.15 cells at 50 μg ml−1 concentration.All the compounds (1–23) isolated were tested for their potential cytotoxicity against HepG 2 and HepG 2.2.15 cells. The purpose of this design was to determine their appropriate drug concentration in anti-HBV assay to eliminate the cytotoxicity interference. As shown in in vitro, as reported previously.15 The experiment results are summarized in 31,32 As shown in p < 0.001). HBeAg is a major structural protein in the core of HBV. Its level indicates the different stages of the HBV infection, which is related to the replication rate of HBV.31,33 The HBeAg results of p < 0.001). While HBV DNA reflects the extents of HBV replication . As seen in p < 0.05). Compound 8 showed the most significant inhibitory activity and its relative HBV DNA level was decreased by 83.11 ± 2.75%. However, several compounds  showed no significantly inhibitory activity and some compounds  showed strangely promoting activities. Unfortunately, we found that compound 23 promoted HBsAg and HBeAg secretion, and HBV DNA replication by 172.39 ± 2.59, 9.92 ± 6.91, and 55.40 ± 17.81%. Contrary to the study on anti-HBV activity of caffeoylquinic acids, there is presently no study that reports the HBV promoting activity of caffeoylquinic acids.To evaluate their anti-HBV activities, namely the inhibiting the secretion of Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg), as well as HBV DNA replication, compounds 1–23 were assayed on HepG 2.2.15 cell line stably transfected with the HBV genome 34 The two adjacent phenolic structure and α, β-conjugated unsaturated ester structure of caffeoyl groups increased benzene ring plane conjugation and resulted in the promotion of anti-HBV activity. For simple caffeic acid alone, compounds 6–8, 17 have significantly anti-HBV activity (p < 0.001). However, compound 5 expressed inferior anti-HBV activity when compared to the other simple caffeic acid because its relative HBV DNA level (68.35 ± 15.26%) was significantly increased compared to the control group (p < 0.001). This is because compound 5  could not form a benzene ring plane conjugation without a two adjacent phenolic structure, so it exhibited poor activity. For monocaffeoylquinic acid, only compound 12 (3-O-caffeoylquinic acid) showed excellent anti-HBV activities (p < 0.001). Other isolated monocaffeoylquinic acids, namely 13 (1-O-caffeoylquinic acid methyl ester), 14 (4-O-caffeoylquinic acid methyl ester), 15 (3-O-caffeoylquinic acid methyl ester), and 18 (5-O-caffeoylquinic acid methyl ester), did not significantly inhibited the secretion of HBsAg, HBeAg, and HBV DNA replication. These results suggest that carboxyl group in quinic acid was important for the accelerating effect on anti-HBV activities in monocaffeoylquinic acids. However, the caffeoylquinic acid ethyl ester derivatives (9) showed the best anti-HBV activity (p < 0.001). Perhaps further study on the structure–activity relationship of monocaffeoylquinic acids is worth discussing. For dicaffeoylquinic acids, compared to their structural analog (20), new compounds 1 and 2 have only one caffeoyl group in the molecule, so they exhibited weaker activities than that of compound 20. Moreover, the anti-HBV activity of new compound 3 was far better than that of compound 20, which could be due to the cis-caffeoyl group in compound 3. The above results may suggest that the existence and configuration of the double bond moiety in caffeoyl group is important for anti-HBV activity. Tricaffeoylquinic acid, compound 23 , had a steric hindrance because it contained three caffeoyl groups. The increased steric hindrance effect inhibited the anti-HBV activity of compound 23. Unfortunately, the relative HBsAg level (172.39 ± 2.59%) and HBV DNA level (55.40 ± 17.81%) of compound 23 was significantly promoted (p < 0.001). Therefore, more studies are necessary to further understand the anti-HBV action mechanism of caffeoylquinic acid derivatives.Structure–activity relationship analysis of these caffeoylquinic acids in anti-HBV activity suggested that the presence of caffeoyl groups in the molecule resulted in stronger anti-HBV activity. This conclusion resembles previous study on the structure–activity relationship of these compounds.L. japonica flower buds, which are widely used in traditional Chinese medicine, healthy food, and cosmetics. Four new caffeoylquinic acids (1–4), five simple caffeic acids  and fourteen known caffeoylquinic acids  were isolated from L. japonica, of which caffeoylquinic acids exhibited potent HBV inhibitory activities. The results indicated that the HBV inhibitory activity of L. japonica flower buds may occur primarily because of the amount of caffeoylquinic acids present and this could serve as anti-HBV agents for functional food or medical use. Also, we found that compound 23 appeared to significantly promote HBsAg and HBeAg secretion, and HBV DNA replication. Unlike the study on anti-HBV activity of caffeoylquinic acids, there is no current manuscript that reports caffeoylquinic acids can promote HBV activity.This study focuses on seeking anti-HBV agents from The authors declare no competing financial interest.RA-008-C8RA07549B-s001


Their structures were identified by a combination of spectroscopic analysis. The absolute configuration of compound 1 was elucidated on the basis of experimental and electronic circular dichroism calculation, and compounds 8 and 9 were determined by Mo2(OAc)4-induced circular dichroism experiments. Compounds 7–16 showed weak antibacterial activities against Stenotrophomonas maltophilia with MIC values ranging from 128 to 512 μg/mL .Three new compounds including a meroterpenoid ( Talaromyces was initially established by Benjamin and over 71 species have been reported − ion peak at m/z 471.2025 , implying 11 degrees of unsaturation. The 1H NMR spectrum of 1 exhibited five singlet methyl signals , a methoxyl peak , and one doublet methyl peak ). With the aid of HSQC, 13C NMR data , four methylenes , three methines , four sp3 quaternary carbons , three ester carbonyls , one keto carbonyl (δC 209.4), four olefinic carbons . The aforementioned data suggested that compound 1 was a meroterpenoid −. Analysis of 1H and 13C NMR spectra suggested that compound 8 was an isocoumarin. The 1H NMR spectrum exhibited signals for three aromatic protons at δH 7.42 , 6.81 , and 6.76 , suggesting a 1,2,3-trisubstituted benzene motif in the structure, three oxygenated methines , one methyl , and three methylenes , 3.01 ; 2.05 , 1.77 , 1.57 ). With the aid of HSQC, the 13C NMR spectrum revealed 14 carbon signals for six aromatic carbons , one ester carboxyl (δC 171.5), one methyl (δC 18.8), three methylenes , and three sp3 methines . All these spectroscopic data were very similar to those of aspergillumarin B [H/δC 3.35/76.2 or 3.55/71.8) in 8. The isochroman fragment was further confirmed by HMBC 4]-induced CD (ICD) spectra were further applied to determine the absolute configurations for 8 and 9. The ICD spectrum of 8 with [Mo2(OAc)4] in DMSO showed a positive Cotton effect at 317 nm  and B (3) 3OH); UV (MeOH) λmax (log ε): 245 (3.67) nm; IR (KBr) υmax: 2962, 2923, 2805, 1671, 1619, 1459, 1382, 1234, 1112, and 790 cm−1; HR ESIMS (neg.) m/z 471.2025 [M − H]− . CD  λmax nm (Δε) 244 (−9.98).Amestolkolide E (8): White amorphous powder; [α]3OH); UV (MeOH) λmax (log ε): 210 (3.95), 247 (3.44), 315 (2.70) nm; IR (KBr) υmax: 3310, 2896, 2857, 1629, 1581, 1425, 1355, 1201, 1089, and 790 cm−1; HR ESIMS (neg.) m/z 265.1081 [M − H]− ; CD  λmax nm (Δε) 216 (−5.46), 239 (+3.19), 257 (−4.94).(R)-3--3,4-dihydroxypentyl)-8-hydroxyisochroman-1-one (9): White amorphous powder; [α]3OH); UV (MeOH) λmax (log ε): 208 (3.98), 245 (3.40), 315 (3.22) nm; IR (KBr) υmax: 3232, 2911, 2865, 1633, 1585, 1428, 1303, 1201, 1085, 960, and 786 cm−1; HR ESIMS (neg.) m/z 265.1084 [M − H]− ; CD  λmax nm (Δε) 217 (−1.56), 239 (+0.88), 258 (−1.48).(R)-3--3,4-dihydroxypentyl)-8-hydroxyisochroman-1-one  at 30 °C and prepared for its suspension with a final concentration of 5 × 105 CFU/mL. Samples (1–16) were severally added into 96-well plates employing serial two-fold dilution from concentrations of 512 to 2 μg/mL. Whereafter, 100 μL bacterial suspension was transferred to the wells to incubate for 16 h at 30 °C. The final concentration of 0.25% TTC  in wells as microorganism growth indicator was added and the microorganism was continuously incubated for 3 h, and the concentration in well that showed no red color indicating complete growth inhibition of bacteria was determined as MIC. Equivalent amounts of DMSO (2.5%) and medium were successively used as negative and blank controls. Ceftriaxone sodium and levofloxacin were used as positive controls. Each experiment was performed in triplicate.The minimum inhibitory concentrations (MICs) were evaluated according to the reported procedure ,30 with 8 and 9 (3.0 mg) in 200 μL DMF was added 2,2-dimethoxypropane (2 equiv) and p-TsOH (0.5 equiv), respectively −.Compound 9a: 1H NMR  δ 7.43 , 6.82 , 6.77 , 4.62 , 3.73 , 3.55 , 3.02 , 2.95 , 1.95 , 1.87 , 1.60 , 1.33 , 1.32 , 1.23 . 13C NMR  δ 171.4 (C-1), 163.2 (C-8), 141.6 (C-10), 137.4 (C-6), 119.4 (C-5), 116.8 (C-7), 109.5 (C-9), 109.1 (C-6′), 83.6 (C-3′), 81.4 (C-3), 78.2 (C-4′), 33.7 (C-4), 32.8 (C-1′), 28.9 (C-2′), 27.6 (CH3-7′), 27.5 (CH3-8′), 17.7 (C-5′). ESI MS (neg.) m/z 305.0 [M − H]−.Compound Talaromyces amestolkiae MST1-15 collected from coal areas led to the isolation and identification of seven meroterpenoids (1–7) and eleven isocoumarins (8–16), including three new compounds named amestolkolide E (1), (R)-3--3,4-dihydroxypentyl)-8-hydroxyisochroman-1-one (8), and (R)-3--3,4-dihydroxypentyl)-8-hydroxyisochroman-1-one (9). Compounds 7–16 showed weak antibacterial activities against S. maltophilia with MIC values ranging from 256 to 512 μg/mL. This study enriched the chemical structure and biological activity from the secondary metabolites of T. amestolkiae.A chemical study of


Dianella ensifolia is a perennial herb with thickened rhizome and is widely distributed in tropical and subtropical regions of Asia, Australia, and the Pacific islands. This plant has the potential to be used as a source of herbal medicine. This study investigated further phytochemistry and tyrosinase inhibitory effect of some constituents isolated from D. ensifolia. Four new flavans, (2S)-4’-hydroxy-6,7-dimethoxyflavan (1), (2S)-3’,4’-dihydroxy-7-methoxy-8-methylflavan (2), (2S)-2’-hydroxy-7-methoxyflavan (3), and -4-hydroxy-4-(7-methoxy-8-methylchroman-2-yl)-cyclohex-2-enone (4), together with 67 known compounds, including 10 flavans (5–14), 5 flavanones (15–19), 3 flavone (20–22), 5 chalcones (23–27), 3 chromones (28–30), 15 aromatics (31–45), 7 phenylpropanoids (46–52), one lignan (53), 7 steroids (54–60), one monoterpene (61), one diterpene (62), 4 triterpenes (63–66), a carotenoid (67), 2 alkaloids (68 and 69), and 2 fatty acids (70 and 71) were isolated from D. ensifolia. Their structures were elucidated on the basis of physical and spectroscopic data analyses. Moreover, compounds 1–4, 8, 10–15, 20, 21, and 41 were evaluated for their mushroom tyrosinase inhibitory effect. Compounds 11 and 14 strongly inhibited mushroom tyrosinase activity with IC50 values of 8.6 and 14.5 μM, respectively. Dianella Lam. ex Juss. genus includes perennial herbs (or subshrubs), glabrous, and rhizomatous with aerial stems. It comprises about 40 species with accepted names, mainly distributed in tropical Asia, Australia, and the southern Pacific islands , two singlet aromatic protons , two methoxy singlet at δ 3.80 and 3.82, and one hydroxy group at δ 5.05. The NOESY spectrum of 1 }, C-2 possesses as S-configuration , two ortho coupled doublets (J = 8.0 Hz) at δ 6.44 and 6.85, a methoxy singlet at δ 3.80, and one methyl singlet at δ 2.20. The NOESY experiment of 2 }, which means C-2 has the S-configuration , four aromatic protons at δ 6.90, 6.91, 7.14, and 7.20, a methoxy singlet at δ 3.75. Since the NOESY experiment of 3  suggesting a hydroxyl group on B-ring. The NOESY correlations }  provided evidence of a cyclohexenone non-aromatic B-ring. Moreover, an additional carbonyl group at δ 198.2 (C-4’) and conjugated vinylic carbons at δ 129.5 (C-3’) and 148.7 (C-2’) in the 13C-NMR spectrum (4 provided strong support of this cyclohexenone moiety. HMBC correlations (4 was elucidated as 4-hydroxy-4-(7-methoxy-8-methylchroman-2-yl)-cyclohex-2-enone, which was further confirmed by COSY -4 -4-hydroxy-4-(7-methoxy-8-methylchroman-2-yl)-cyclohex-2-enone.Compound spectrum  of 4 wasspectrum  of 4 proelations  between  by COSY , NOESY -3,4’-dihydroxy-7-methoxy-8-methylflavan (tupichinol A) (5) D, and mass spectrometry data) with values found in the literature.The known compounds including 10 flavans, (2l A) (5) , (2S)-7,avan (6) , (2S)-4’avan (7) , (2S)-7,avan 8) , (2S)-4’avan 9) , (2S)-7,van (10) , (2S)-2’ , (2S)-7van (12) , (2S)-7,van (13) , (2S)-2’ , (2S)-4nin (15) , (2S)-farol (16) , (2S)-liher (17) , (2S)-5,one (18) , cyrtomi18) , seven aromatics , two alkaloids , one lignan [53 (17.8 mg)], and one monoterpene [61 (79.8 mg)].The EtOAc-soluble layer (185.0 g) was subjected to silica gel (5.0 kg) column chromatography with a gradient of c 0.01, MeOH); UV (MeOH) λmax (log ε): 286 (4.12), 225 (4.66) nm; IR (neat) νmax: 3424, 3010, 2935, 2859, 1605, 1512, 1214, 1116 cm−1; EIMS: m/z (rel. int.): 286 , 180 (8), 167 (41), 166 (13), 120 (7); HREIMS: 286.1209 ; 1H-NMR: see 13C-NMR: see Yellow oil; c 0.01, MeOH); UV (MeOH) λmax (log ε): 282 (3.66), 227 (4.08) nm; IR (neat) νmax: 3403, 3090, 2952, 2853, 1619, 1510, 1454, 1275, 1162 cm−1; EIMS: m/z (rel. int.): 286 , 269 (13), 164 (29), 151 (100), 136 (34); HREIMS: 286.1207 ; 1H-NMR: see 13C-NMR: see Brown oil; c 0.01, MeOH); UV (MeOH) λmax (log ε): 280 (3.84), 221 (4.25) nm; IR (neat) νmax: 3368, 3012, 2927, 2853, 1607, 1513 cm−1; EIMS: m/z (rel. int.): 256 , 239 (50), 150 (28), 137 (58), 73 (26), 59 (100); HREIMS: 256.1104 ; 1H-NMR: see 13C-NMR: see Brown oil; c 0.01, MeOH); UV (MeOH) λmax (log ε): 281 (3.51), 227 (4.48) nm; IR (neat) νmax: 3435, 3044, 2935, 1676, 1601, 1493, 1434, 1113 cm−1; EIMS: m/z (rel. int.): 288 , 270 (5), 178 (13), 177 (100), 112 (33); HREIMS: 288.1363 ; CD (MeOH): Δε392 0.47, Δε334 1.10 (dm3 mol−1 cm−1); 1H-NMR: see 13C-NMR: see Colorless oil; l-tyrosine  were mixed in 96-well plates. 20 μL of mushroom tyrosinase  was added to initiate the assay. The absorbance of the mixture was measured at 490 nm using a microplate reader . The percentage inhibition of mushroom tyrosinase was calculated according to the following equation: Inhibition (%) = [1 − (S − SB)/(C − CB)] × 100%, where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively.The mushroom tyrosinase inhibitory assay has been described in detail previously . BrieflyS)-4’-hydroxy-6,7-dimethoxyflavan (1), (2S)-3’,4’-dihydroxy-7-methoxy-8-methylflavan (2), (2S)-2’-hydroxy-7-methoxyflavan (3), and -4-hydroxy-4-(7-methoxy-8-methylchroman-2-yl)-cyclohex-2-enone (4), along with 67 known compounds were isolated from whole plants  of D. ensifolia. Selected compounds of D. ensifolia display anti-tyrosinase properties. The presence of 2′,4′-resorcinol (11 and 14) or catechol (2 and 20) moiety in the ring B of flavonoids appears to be required for the anti-tyrosinase activity. Although the detailed mechanism of action of these compounds remains to be determined, the results confirmed that D. ensifolia is a valuable source of herbal medicine from which natural-based whitening agents can be derived.Four new flavans, (2


Abutilon indicum L. , more often referred to as Peeli booti, Kanghi, and Kakhi, is a perennial shrub found in many countries of Asia. Traditionally, this plant is used as a diuretic to treat inflammation, discomfort, urethral infections, and gout. Inflammation and pain are key topics of interest for researchers throughout the globe, since they are linked to almost every illness that could affect humans or animals. The present study was conducted to isolate the phytoconstituents from the methanolic extract of Abutilon indicum collected from the Bihar state Koshi river belt in India, and to evaluate the isolated phytoconstituents’ ability to reduce nociception and inflammation. Furthermore, molecular docking was performed to investigate the molecular interaction profile, with possible therapeutic targets for anti-inflammatory medicines. A. indicum methanolic extract yielded two novel phytocompounds identified as 5′-hydroxyhexyl n-hexadecanoate (AB-01) and n-octanoyl-β-D-glucopyranosyl-(2′-1′′)-β-D-glucopyranosyl-(2′′-1′′′)-β-D-glucopyranosyl-(2′′′-1′′′′)-β-D-glucopyranoside (AB-05), together with three previously recognized phytocompounds such as ester glucoside. All isolated molecules were tested for the efficacy of analgesic and anti-inflammatory characteristics at doses of 5 and 10 mg/kg body weight. The isolated compound’s molecular interaction profile with anti-inflammatory drug targets cyclooxygenase-2 and tumor necrosis factor-alpha possessed high potential energy in molecular docking. These findings may aid in developing anti-inflammatory and analgesic drugs from A. indicum. Abutilon indicum L. , known locally as Peeli booti, Kanghi and Kakhi, is a perennial shrub found in many countries of Asia +(C22H44O3) (2.1), 311 (2.3), 239 (3.8), 117 (4.6).A colorless crystalline mass of AB-01 was obtained when the column was eluted with a mixture of chloroform and methanol in the proportion of 19:1. This was recrystallized with chloroform: methanol in a ratio of 1:1 that yielded 95 mg mass of yield 0.12%. The Rmax (MeOH): 210 nm; Rf 0.42 (MeOH: CHCl3-1:19); m.p. 115–118 °C; (KBr) IR νmax: 779, 867, 1059, 1231, 1403, 1721, 2852, 2936, 3265, 3389, 3450 cm−1. 1H NMR (MeOD)- 0.88 ; 1.29 , 1.39 , 2.56 , 3.10 , 3.34 , 3.48, 3.57 , 3.64 , 3.67 , 3.70 , 3.74 , 3.78 , 3.83 , 3.99 , 4.04 . 13C NMR (MeOD): 14.21(ME-8); 22.63 (CH2), 30.12 (4 × CH2), 34.82 (CH2), 62.74 (C-6′′), 64.55 (C-6′), 66.04 (C-4′′), 69.53 (C-4′), 71.39 (C-3′′),71.97 (C-3′), 71.97 (C-3′), 73.99 (C-2′′), 76.92(C-5′′), 77.64 (C-5′), 83.41(C-2′), 99.33(C-1′′), 103.29 (C-1′), 172.06 (C-1). +ESI MS m/z (rel.int): 468[M]+ (C20H36O12), 326 (12.5), (5.3), 163 (47.8), 127 (100), 143 (32.1).The column was then eluted using a mixture of methanol and chloroform (1:19) that yielded a colorless mass of AB-02. This was recrystallised with methanol, and a 103 mg mass of 0.11% yield was obtained. UV λHydrolysis of AB-02: Compound 2 (15 mg) was dissolved in ethanol (5 mL). Concentrated hydrochloric acid (2 mL) was then added, and the reaction mixture was heated for 1 h in a steam bath. Following this, it was chilled and extracted using petroleum ether, equivalent to a reference capric acid sample, on a TLC plate developed with petroleum ether to separate the capric acid. After separating the fatty acids using n-butanol-acetic acid water and a reference sugar sample, the residue was concentrated and chromatographed on a silica gel TLC plate . The sugar was revealed to be D-glucose, according to FR. 0.12.3 was eluted from the column with a mixture of chloroform and methanol (9:1), resulting in a colorless mass. It was then recrystallised with methanol, which eventually yielded 118 mg (0.16% yield). The Rf 0.51  and m.p. 120–122 °C. The UV λmax (MeOH) was noted at 270 nm. Using KBr- IR νmax was recorded: 779, 1059, 1240, 1402, 1638, 1721, 2930, 3261, 3389, 3425 cm−1. 1H NMR (MeOD)-δ 0.86 ; 1.52 , 2.23 , 3.30 , 3.32 , 3.35 , 3.46 , 3.50 , 3.58 , 3.62 , 3.68 , 3.71 , 3.78 , 6.5 Hz, H-2, 3.83 , 4.01 ,4.06 . 13C NMR (MeOD)-δ 22.61 (CH2), 14.23 (Me-8); 29.48 (5 × CH2), 30.94 (CH2), 34.91 (CH2), 64.33 (C-6′′), 64.66 (C-6′), 66.01 (C-4′′), 69.51 (C-4′), 71.38 (C-3′′), 71.96 (C-3′), 73.97 (C-2′′), 76.89 (C-5′), 77.65 (C-5′), 83.39 (C-2′), 99.33 (C-1′′), 103.27 (C-1′), 170.16 (C-1). + Ve ESI MS m/z (rel.int)- 497 [M+H]+ (C22H41O12), (7.25), 155 (16.5), 163 (20.8), 325 (18.3), 341 (12.2).Compound AB-0Hydrolysis of AB-03: Hydrochloric acid (conc.) was added to the reaction mixture with Compound 3 (10 mg), and the reaction took place for one hour in a hot steam bath. Caprylic acid was isolated by using petroleum ether as a solvent. To chromatograph the fatty acid residue over the developing solvent system of n-butanol, acetic acid, and water (4:1:5), silica gel TLC plates with a standard sample of sugars were used. D-glucose, FR 0.12, was identified as the sugar.f 0.72  and m.p. were reported to be 105–110 °C. UV λmax (MeOH) was shown at 217 nm. IR νmax (KBr)- 779, 1059, 1228, 1402, 1654, 1725, 2845, 2930, 3256, 3397, 3448 cm−1. 1H NMR (MeOD)- 0.88 ; 1.29 , 1.60 , 2.31 , 3.30 , 3.32, 3.35 , 3.49 , 3.58 , 3.61 , 3.64 , 3.74 , 3.78 , 3.83 , 3.96 , 4.01 , 4.04 . 13C NMR (MeOD)- 14.61 (Me-16); 22.55 (CH2), 30.03 (11 × CH2), 30.85 (CH2), 33.88 (CH2), 62.90 (C-6′′), 64.32 (C-6′), 64.66 (C-4′′), 66.02 (C-4′), 71.37 (C-3′), 71.95 (C-3′), 73.97 (C-2′′), 76.89 (C-5′′), 77.63(C-5′), 83.39 (C-2′), 99.32 (C-1′′), 103.27 (C-1′), 171.52 (C-1). +Ve ESI MS m/z (rel.int)-581 [M+H]+ (C28H53O12) (8.4), 163 (24.1), 239 (14.8), 255 (33.2).Crystals of AB-04, recrystallized from chloroform: methanol (l:1), were produced by further eluting the column with chloroform: methanol solvents (9:1). The yield was found to be 206 mg in terms of % yield 0.28. The RHydrolysis of AB-04: The reaction mixture containing Compound 4 (10 mg) was treated with ethanol (5 mL) and concentrated HCI (2 mL) for one hour in a hot steam bath. Palmitic acid was separated from the reaction mixture using chloroform extraction, and the co-TLC findings were then compared to these. After separating the fatty acid, it was concentrated and chromatographed on silica gel TLC. A reference sample of the sugar utilizing the top layer of n-BuOH-AcOH-H2O, 4:1:5 was used. The fatty acid was then separated. D-glucose was identified as the sugar (Rf 0.12) and was separated.f 0.63  and m.p. was found to be 125–126 °C. UV λmax (MeOH) ranged at 206 nm. The KBr- IR νmax values were 779, 1059, 1240, 1402, 1639, 1723, 2931, 3265, 3397, 3425 cm−1. 1H NMR (MeOD): δ 0.86 ; 1.25 , 1.52 , 2.51 , 3.29 , 3.31 , 3.34 , 3.46 , 3.48 , 3.52 , 3.57 , 3.61 , 3.64 , 3.69 , 3.78 , 3.78 , 3.82 , 3.84 , 3.87 , 3.93 , 3.96 , 4.00 , 4.02 , 4.04 , 4.06 . 13C NMR (MeOD): δ 14.27 (Me-8), 22.48 (CH2), 29.86 (4 × CH2), 33.13 (CH2), 62.62 (C-6′′′′), 62.63 (C-6′′′), 62.65 (C-6′′), 63.81 (C-6′), 64.62 (C-4′′′′), 65.13 (C-4′′′), 65.90 (C-4′′), 69.41 (C-4′), 71.28 (C-3′′′′), 71.87 (C-3′′′), 73.04 (C-3′′), 73.84 (C-3′), 74.89 (C-2′′′), 76.31 (C-5′′′′), 76.74 (C-5′′′), 77.51 (C-5′′), 77.99 (C-5′), 83.18 , 84.24 (C-2′), 98.16 (C-1′′′′), 99.33 (C-1′′′), 103.20 (C1′′), 105.89 ((C-1′), 171.13 (C-1). +ve ESI MS m/z (rel.int): 792[M]+ (C32H56O22) (6.2), 629 (7.0), 503 (14.8), 487 (6.2), 467(4.3), 305 (15.1), 289 (6.7), 179 (13.6), 163 (9.2), 143 (100), 127 (24.3).Column elution with methanol: chloroform in a ratio of (1:9) gave a crystalline mass of a light brown color AB-05, which was recrystallised again from methanol that yielded a 145 mg mass of 0.23% yield. The RHydrolysis of AB-05: A steam bath was used to heat a combination of 15 mg of Compound 5 for 1 h, using conc. HCL 2 mL and ethanol 5 mL. Petroleum ether was employed to extract capric acid from the reaction mixture, and co-TLC results were equivalent. The reaction mixture was concentrated, and the standard sugar was chromatographed on silica gel TLC with n-butanol-AcOH-H2O in a ratio of 4:1:5 as the developing solvent system. D-glucose, RF 0.12, was identified as the sugar.It was seen that the dosing of isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 up to 150 mg/kg orally to the mice did not incite drug-related harmfulness (toxicity) and mortality. The results revealed the mice endured the drug well and showed normal behavior up to 150 mg/kg orally. All animals were vigilant, with normal spruce, touch, and pain response, and there was no indication of accommodation or vocalization.p < 0.05) decrease in paw volume of rats from 2 h. Treatment with Indomethacin and AB-01, AB-02, AB-03, AB-04 and AB-05 at 10 mg/kg showed a significant reduction (p < 0.05) of paw volume in rats incited by carrageenan. The impact of AB-01 and AB-05 at a portion of 10 mg/kg was favored in lessening the swollen paw volume, rather than a higher portion of Indomethacin (10 mg/kg).The anti-inflammatory effect of isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 at doses of 5 & 10 mg/kg and Indomethacin at a dose of 10 mg/kg on carrageenan-disturbed rat paw is displayed in p < 0.01) augmented the response time of the animals towards the thermal source at various periods (AB-01 and AB-05 at a dose of 10 mg/kg and Tramadol significantly ( periods . The hotp < 0.001) reduced this response . The injection of acetic acid caused abdominal constriction and a contraction of the hind limbs, although AB-01, AB-05, and Diclofenac all substantially  and tumor necrosis factor-alpha (PDB id: 2AZ5). The docking methodology developed in AutoDock Vina was validated by re-docking all the co-crystallized ligands with their corresponding proteins. In addition, the docked poses of each ligand were compared with their crystal conformations, which exhibited a root-mean-square deviation (RMSD) of 2.0 A°, and validated the dependability of the docking approach used ,32,33. TA. indicum is an extensively utilized and accepted traditional medicinal plant. Early findings on the leaf extracts suggested antibacterial, hepatoprotective and larvicidal capabilities, and anti-inflammatory activity + and 117 [M-239]+. One hydroxyl group present at C-5′ (m/z 311 [M-CH(OH) Me]. Compound 1 1H NMR spectrum , ester function (1721 cm−1), and aliphatic chain (779 cm−1) and produced a positive Fehling solution for glucoside. The molecular ion peak of chemical AB-02 6CO] + and 143 [CH3(CH2)6 COO]+ indicated that aglycone capric acid was esterified with the diglycosidic glycone unit. The ion fragments generating at m/z 163 [C6H11O5]+ and 326  suggested that the aglycone unit was connected to the C12 sugar unit (1H NMR spectra (13C NMR spectra (13C NMR spectra. Capric acid D-glucose was produced via the compound AB-02’s acid hydrolysis (TLC comparable). The structure of component AB-02 was determined as n-octanoyl-β-D-glucopyranosyl-2′-β-D-glucopyranoside (From chloroform: methanol (19:1) eluants, the crystalline mass of chemical AB-02 capryloyl diglucoside was synthesized. It demonstrated IR absorption bands  for hydral AB-02  was idengar unit . Compoun spectra  revealed spectra  showed sranoside  based on−1) and ester groups (1721 cm−1), as well as aliphatic chains (779 cm−1). The compound 3 molecular ion peak 8CO]+ and 341, 325[C6H11O5-C6H11O5]+ and 163 [C6H11O5]- showed the existence of a caproyl group as an aglycone unit connected to a disaccharide chain -β-D-glucopyranoside . It accepted the glycoside Fehling solution test and displayed IR absorption bands  for hydrion peak  was idende chain . Two onempound 3  were att spectra  showed iranoside .−1), ester (1725 cm−1), and aliphatic chain (779 cm−1). According to its molecular formula C28H53O12, compound AB-04’s molecular ion peak was discovered at m/z 581[M+H], based on mass (13C NMR spectra. The ion peaks appearing at m/z 239 [CO(CH2)14CH3]+, 255[OCO(CH2)14-CH3]+, 325 [C6H10O5-C6H11O5]+ and 163 [C6H11O5]+ showed that the diglycoside unit had a palmityl chain connected to it , and three proton triplets at δ 0.88 (J = 6.2 Hz), were responsible for the C-16 main methyl protons. Signals for ester carbons at 171.52 (C-1), anomeric carbons at 103.27 (C-1) and 93.32 (C-1), other sugar carbons within δ 83.39 to 62.90, methylene carbons from δ 33.88 to δ 22.55, and methyl carbon at δ 14.61 were seen in compound AB 04 13C NMR spectra (C-16). The attachment of the second sugar unit at C-2′ was suggested by the existence of the C-2′ carbon signal in the deshielded area at δ 83.39. No signal was seen after δ 4.04 in the 1H NMR spectrum or from δ 171.52 to 103.27 in the 13C NMR spectrum (Chloroform and methanol (9:1) eluants were used to separate compound AB-04, palmityl diglucoside, a colorless crystalline mass. After carrying out a Fehling solution test, glycosides were revealed to be present, as well as IR absorption bands  for hydr on mass  and 13C ed to it . Two one spectra  were attspectrum , indicatspectrum .−1), IR absorption bands (−1), and aliphatic chains were visible (779 cm−1). Based on the mass 6CH3]+ and 143 [OCO(CH2)6CH3]+. The m/z 163-generated ion fragments 467 [M-C12H21O10]+, 305 [M-487]+, 289 [M-503]+, 487 [C6H11O5-C6H10O5-C6H11O5]+ 503 [C6H11O5-C6H10O5-C6H11O5], 179 [C6H11O6]+, [C6H11O5]+. The binding of a tetraglycosidic unit to a capryloyl group was facilitated by 629 [M-163]+ -β-D-glucopyranosyl-(2′′-1′′′)-β-D-glucopyranosyl- (2′′′-1′′′′)- β-D-glucopyranoside . It showed positive glycoside tests. The hydroxyl groups  in the volume of their paws. Compared to a higher dose of Indomethacin (10 mg/kg), the effects of AB-01 and AB-05 at 10 mg/kg were shown to be more effective in lowering the volume of the swollen paw. The aggravation is linked to the pathophysiology of different diseases, such as joint inflammation, cancerous growths, gout, and vascular diseases. In traditional clinical frameworks, various medicinal plants treat pain and inflammation signs. We found that at 5.0 and 10.0 mg/kg dosages, the AB-01, AB-02, AB-03, AB-04, and AB-05 extracted from A. indicum extract had a calming and analgesic effect, respectively. Carrageenan acted as a phlogistic factor, increasing the synthesis of prostaglandins and bradykinins at certain periods [ periods .All the drugs that were examined demonstrated a reduction in paw edema volume from 1 h to 5 h, and their anti-inflammatory effect was continued beyond 3 h. This analysis unequivocally showed that all examined medications may have had an effect that cooperated with the prostaglandins surge. Curiously, the effects of AB-05 on carrageenan-induced paw edema were consistent throughout the 1 h and 5 h time points. Inflammatory mediators such as prostaglandins and platelet-activating factors (PAF) were triggered by carrageenan, leading to inflammation. Histamine, 5-hydroxytryptamine, and kinin all arrived during the first stage (0–1 h), whereas the second stage (3–5 h) was linked to the production of prostaglandin and bradykinin. Even though the 10 mg/kg dose had a greater calming effect than the 0.05 mg/kg dose, this finding was directly connected to the effects shown in carrageenan-induced paw edema, where both dosages of isolated compounds markedly changed the inflammatory response triggered by carrageenan . The abdIn the ADMET studies, QPlogHERG values for compounds AB 01 and AB 04 were less than the specified value, indicating they possess some cardiotoxic potential. Predictions of the Qikprop  tool suggested that the non-active transport of these compounds across the gut-blood barrier was poor except for compound AB 01, with complete oral absorption. This was also the only compound capable of crossing the blood-brain barrier, as suggested by its QPlogBB and QPlogMDCK values. All compounds had some degree of skin permeability. As per Lipinski’s rule of five, compound AB 01 exhibited only one violation; compounds AB 02 and AB 03 showed two violations, while compounds AB 04 and AB 05 displayed three out of five . Hence, A. indicum, collected from the banks of the Koshi River in India, were depicted in this study. As a result, this study can significantly advance our understanding of the phytoconstituents in this herb and pave the way for the creation of novel medications with enormous promise for use in traditional Indian medicine. The current investigation suggests that AB-01, AB-03, and AB-05 show considerable analgesic and anti-inflammatory effects at 10 mg/kg. The main trigger for the release of platelet initiating factors (PAF), other inflammatory mediators and prostaglandins was carrageenan-induced edema. In conclusion, 5′-hydroxyhexyl n-hexadecanoate as AB-01 and n-octanoyl-β-D-glucopyranosyl-(2′-1′′)- β-D-glucopyranoside as AB-05 were isolated from A. indicum extract. These compounds showed anti-inflammatory and pain-relieving properties and may help to treat systemic inflammation caused by endotoxin. Since the precise purity of the extracted compounds has not yet been established, a potential contribution of minor compounds to the reported bioactivities cannot be completely ruled out. To the best of our knowledge, this work is the first to examine the presence of capryloyl tetra-glucoside and aliphatic ester in a methanolic extract of A. indicum. The study confirms that these strong isolated chemicals can be a useful diagnostic tool for the genus A. indicum. These results are thought to be crucial in nurturing the powerful compounds AB-01 and AB-05 for treating disorders caused by inflammation.The phytochemical components of


Although this push–pull dye absorbs intensively in the visible range and shows emission with large Stokes shifts in all organic solvents, it is strongly quenched in water. This Achilles’ heel prompted us to implement a new strategy to obtain a series of dyes that retain all the photophysical features of dEAF in water, conjugate readily with oligonucleotides, and furthermore demonstrate sensitivity to hydration, thus paving the way for a high-performance fluorogenic DNA hybridization probe.The intensive research for hybridization probes based on organic molecules with fluorogenic properties is currently attracting particular attention due to their potential to efficiently recognize different DNA conformations and the local environment. However, most established organic chromophores do not meet the requirements of this task, as they do not exhibit good brightness in aqueous buffer media, develop aggregation and/or are not easily conjugated to oligodeoxynucleotides (ODNs) while keeping their photophysics intact. Herein, an important modification strategy was employed for a well-known fluorophore, 2-(4-(diethylamino)phenyl)-3-hydroxychromone ( The use of hybridization probes composed of organic fluorophores attached to single strands (ss) of nucleic acids has found broad applications in nanotechnology, diagnostics, biology, and medicinal chemistry ,2,3. AmodEAF, π-Acceptor (D-π-A) relationship. The fluorophore dEAF incorporates in its structure a strong donor substituent—the diethylamino group—electronically coupled to the carbonyl acceptor group. It absorbs intensively in the visible range, exhibits large Stokes shifts in polar solvents, and is emissive in all screened solvents except water where it is strongly quenched. Consequently, shielding the dye from bulk water results in a turn-on of its emission (fluorogenicity), and thus a substantial increase of the signal-to-noise ratio (SNR). This fluorogenic behavior is expected when the labeled DNA single strand anneals to its target, as the environment of the formed duplex is hydrophobic on the inside and its grooves are less hydrated than the bulk water .,15.dEAF,lk water ,17.dEAF . For ss-DNA 2–3, the dye will be connected to a central or terminal position via a flexible tether. In the case of ds-DNA 2–3, the fluorescent reporter will be free to interact with the duplex either by intercalation/π-stacking or by groove binding. For our research program, we considered all options . Our objective was to synthesize labeled oligodeoxynucleotides (ODNs) with turn-on emission after annealing with their complementary strand. As a convenient and straightforward access, we selected an ODN labeling through a post-synthetic CuAAC coupling. For this purpose, we had to synthesize the corresponding azido and ethynyl derivatives of the reference 3-hydroxychromone dEAF . PositiodEAF . In ss-DdEAF derivatives with a significant increase in the water solubility and photostability while keeping intact, the rest of the photophysics. These modified dyes were also designed to be rapidly conjugated to ODNs using standard CuAAC click-chemistry protocol +; found: 388.0550, 390.0529 [M + H]+.6-Bromo-2-(4-diethylamino)phenyl-3-methoxy-4H-chromen-4-one (2): To a stirred suspension of 1  in DCM , were sequentially added 18-crown-6 , an aq. KOH solution  and dimethyl sulfate . The reaction mixture was stirred 30 min at rt. After addition of H2O (8 mL), the organic layer was extracted with DCM (3×), dried over MgSO4, filtered and the volatiles were removed in vacuo. The residue was purified by flash chromatography on silica gel eluted with DCM/DCM–1% MeOH mixture  to provide the desired compound 2 as a yellow powder . Rf = 0.29 (DCM/MeOH 99.5:0.5). 1H-NMR : δ 1.16 , 3.38 , 3.81 , 6.67 , 7.32 , 7.62 , 8.00 , 8.30 . 13C-NMR : δ 12.6 (NCH2-CH3), 44.5 (NCH2-CH3), 56.7 (OCH3), 110.8 (Cmeta), 116.4 (C6), 117.6 (Ci), 119.6 (C8), 127.7 (C10), 128.2 (C5), 130.3 (Cortho), 135.7 (C7), 140.0 (C3), 149.6 (Cpara), 153.8 (C2), 157.0 (C9), 173.2 (C4). HRMS (ESI+): m/z calcd for C20H20BrNO3H+: 402.0699, 404.0679 [M + H]+; found: 402.0698, 404.0679 [M + H]+.2-(4-(Diethylamino)phenyl)-3-methoxy-6-((trimethylsilyl)ethynyl)-4H-chromen-4-one (AlMF-TMS): To a stirred solution of 2  in dry DMF (3 mL) under argon, were sequentially added TMS-acetylene , triethylamine  and a mixture of CuI /PdCl2(PPh3)2 . The reaction mixture was warmed to 70 °C overnight. The volatiles were removed in vacuo and the residue was directly engaged in the next step.2-(4-(Diethylamino)phenyl)-6-ethynyl-3-methoxy-4H-chromen-4-one (AlMF): To a 10-mL vial containing AlMF-TMS , was added a minimum of MeOH (2 mL) to solubilize the compound. K2CO3  was then poured, and the resulting solution was stirred overnight at room temperature. The reaction mixture was quenched by acetic acid and reduced in vacuo to give the crude product. The residue was purified by preparative TLC (SiO2) eluted with DCM/MeOH  to provide the desired product AlMF as a yellow powder . Rf = 0.24 (DCM/MeOH 99.5:0.5). 1H-NMR : δ 1.16 , 3.04 , 3.38 , 3.82 , 6.67 , 7.38 , 7.62 , 8.00 , 8.31 . 13C-NMR : δ 12.6 (NCH2-CH3), 44.5 (NCH2-CH3), 59.7 (OCH3), 77.8 (HC≡C), 82.3 (HC≡C), 110.8 (Cmeta), 116.5 (C6), 118.0 (C8), 118.4 (Ci), 124.2 (C10), 129.9 (C5), 130.2 (Cortho), 136.0 (C7), 140.0 (C3), 149.5 (Cpara), 154.7 (C2), 156.8 (C9), 173.7 (C4). HRMS (ESI+): m/z calcd for C22H21NO3H+: 348.1594 [M + H]+; found: 348.1600.1-(5-(Chloromethyl)-2-hydroxyphenyl)ethan-1-one (3): Adapted from the protocol described in +; found: 379.1777.6-(Bromomethyl)-2-(4-(diethylamino)phenyl)-3-methoxy-4H-chromen-4-one (7): To a stirred solution of 5  in DCM (12 mL), were sequentially added 18-crown-6 , an aq. KOH solution  and dimethyl sulfate . The reaction mixture was stirred for 30 min at room temperature. The organic layer was extracted with DCM (3×), dried over MgSO4, filtered, and concentrated in vacuo. The residue was then purified by flash chromatography on silica gel eluted with cyclohexane/ethyl acetate  to give the desired compound 7 as a red oil . Rf = 0.58 (Cyclohexane/EtOAc 3:1). 1H-NMR : δ 1.23 , 3.45 , 3.88 , 4.59 , 6.74 , 7.49 , 7.67 , 8.08 , 8.24 . 13C-NMR : δ 12.6 (NCH2-CH3), 32.4 (BrCH2Ph), 44.5 (NCH2-CH3), 59.7 (OCH3), 110.8 (Cmeta), 116.6 (C6), 118.6 (C8), 124.2 (Ci), 125.8 (C5), 130.2 (Cortho), 133.7 (C7), 134.1 (C10), 140.1 (C3), 149.5 (Cpara), 154.7 (C2), 156.8 (C9), 174.1 (C4). HRMS (ESI+): m/z calcd for C21H22BrNO3H+: 416.0856, 418.0835 [M + H]+; found: 416.0870, 418.0848.Acid 3-(prop-2-yn-1-ylamino)propane-1-sulfonic (PYAPS): To a solution of propargylamine  in CH3CN (5 mL) was added dropwise 1,3-propane sultone 8 . The reaction mixture was stirred at room temperature for 2 d. The resulting precipitate was filtered and washed with CH3CN to afford the desired compound PYAPS as a pinkish solid . Rf = 0.25 (DCM/MeOH 4:1). Green staining by ninhydrin. 1H-NMR : δ 1.93 , 2.60 , 3.09 , 3.68 , 3.90 . 13C-NMR : δ 21.7 (C5), 35.6 (C3), 46.0 (C4), 48.8 (C6), 75.1 (C2), 79.4 (C1). HRMS (ESI+): m/z calcd for C6H11NO3SH+: 178.0532; [M + H]+; found: 178.0534.Sodium 3-(methyl(prop-2-yn-1-yl)amino)propane-1-sulfonate (Me-PYAPS): PYAPS , formol  (900 µL) and formic acid (903 µL) were heated overnight at 70 °C. Then, the solution was concentrated in vacuo until a paste was obtained. NaHCO3 (1 g) was added to the solid until bubbling ceased to provide the sodium sulfonate salt Me-PYAPS as a white solid . Bicarbonate salts contaminate the product due to the excess used. 1H-NMR : δ 1.60–1.73 , 2.15 , 2.32–2.42 , 3.07 , 3.25 . 13C-NMR : δ 22.7 (C5), 40.7 (C3), 44.3 (C6), 48.9 (C7), 53.7 (C4), 75.1 (C2), 81.5 (C1). HRMS (ESI+): m/z calcd for C7H12NO3SH+−: 192.0689; [M + H]+; found: 192.0690.2-Azido-N,N-dimethylethanamine (DMAZ): 3-Chloro-1,1-dimethylpropylamine 9  and NaN3  were sequentially dissolved in H2O . The solution was stirred at 80 °C for 24 h. After cooling to room temperature, the reaction mixture was adjusted to pH 10 by addition of an aq. 0.5 M NaOH solution. The organic layer was then extracted with diethyl ether (3×). The combined extracts were dried over MgSO4, filtered, and concentrated under vacuum to yield the product DMAZ as a yellowish liquid . 1H-NMR : δ 2.24 , 2.47 , 3.31 . 13C-NMR : δ 45.5 (C3 and C3′), 49.1 (C1), 58.0 (C2). HRMS (ESI+): m/z calcd for C4H10N4H+−: 115.0978; [M − H]+; found: 115.0982.Potassium N(3-(((2-(4-(diethylamino)phenyl)-3-methoxy-4H-chromen-6-yl)methyl), N(prop-2-yn-1-yl)amino)propane-1-sulfonate (AlMF−): To a solution of 7  in DMF (0.5 mL), were added PYAPS , H2O (0.4 mL), and K2CO3 . The solution was stirred at room temperature for 24 h. Then, DMF and water were evaporated in vacuo and the crude (solubilized with DCM/MeOH) was purified by flash chromatography on silica gel eluted with i-PrOH/NH3/H2O  to afford the desired product AlMF− as an orange solid . 1H-NMR : δ 1.22 , 1.97–2.08 , 2.73 , 2.90 , 3.49 , 3.78 , 3.79 , 3.94 , 4.84 , 6.80 , 7.60 , 7.77 , 8.07 , 8.08 . HRMS (ESI+): m/z calcd for C27H32N2O6SH+: 513.2054; [M − H]+; found: 513.2064.3-(((2-(4-(diethylamino)phenyl)-3-methoxy-4-oxo-4H-chromen-6-yl)methyl)(methyl)(prop-2-yn-1-yl)ammonio)propane-1-sulfonate (AlMF+−): To a solution of 7  in DMF (4.5 mL), were added Me-PYAPS and water (2 mL). The reaction mixture was stirred at room temperature for 24 h before the volatiles were removed under vacuum. The residue (diluted with DCM/MeOH) was purified by flash chromatography on silica gel eluted with i-PrOH/NH3/H2O  to give the desired product AlMF+− as a yellow oil . 1H-NMR : δ 1.23 , 2.07–2.17 , 2.86 , 2.88 , 3.07–3.14 , 3.50 , 3.68–3.69 , 4.30 , 4.79 , 6.81 , 7.72 , 7.96 , 8.07 , 8.35 . 13C-NMR : δ 11.5, 21.0, 44.0, 44.7, 53.8, 58.7, 60.9, 64.4, 71.1, 77.6, 82.6, 110.7, 115.4, 119.2, 123.5, 123.9, 130.0, 130.2, 137.1, 139.5, 150.1, 156.0, 158.0, 173.9. HRMS (ESI+): m/z calcd for C28H34N2O6SH+: 527.2210; [M + H]+; found: 527.2215.2-Azidoethyl-N-((2-(4-(diethylamino)phenyl)-3-methoxy-4-oxo-4H-chromen-6-yl)methyl)-N,N-dimethylethanammonium bromide (AzMF+): To a solution of 7  in anhydrous THF , was added DMAZ . The reaction mixture was stirred at room temperature for 6 h. The formed precipitate was centrifuged, and the upper layer was discarded. The compound was washed twice more after suspension in dry THF, centrifugation and removal of the supernatant. The recovered solid was dried under reduced pressure to provide AzMF+ as a yellow powder . 1H-NMR : δ 1.24 , 3.16 , 3.52 , 3.62 , 3.83 , 4.08 , 4.76 , 6.84 , 7.82 , 7.93 , 8.13 , 8.35 . 13C-NMR : δ 11.5 (NCH2-CH3), 44.0 (NCH2-CH3), 44.6 , 49.5 (N3CH2), 54.2 (N+CH2Ph), 58.7 (OCH3), 62.6 (CH2N+), 67.6 (N+CH2Ph), 110.8 (Cmeta), 115.4 (C6), 119.1 (C8), 123.8 (Ci), 123.9 (C10), 130.2 (C5), 130.2 (Cortho), 137.2 (C7), 137.2 (C3), 139.6 (Cpara), 150.2 (C2), 156.1 (C9), 174.1 (C4). HRMS (ESI+): m/z calcd for C25H32N5O3+: 450.2500 [M − H]+; found: 450.2500.1-Azido-1,2-dideoxy-3,5-di-O-p-toluoyl-d-ribofuranoseβ- (8): Adapted from the procedure described in +; found: 507.2240.dEAF were synthesized with different kinds of linkers  bearing appropriate functional groups, allowing the development of a toolbox of dyes, easy to conjugate with biomolecules. Compared to the parent chromone, these original fluorophores retained all photophysical properties intact, while improving solubility and photostability in aqueous media. They were then used to post-synthetically label ODNs of different compositions via Cu(I)-mediated click chemistry. The resulting ODN probes showed bright turn-on emission after annealing with the complementary strand. Position 6 of the chromone scaffold was chosen as the site of chemical modification because at this location, the impact on the photophysics of the dye is minimal. The methoxy group was preferred over the hydroxyl group to drastically enhance the photostability of the 3-OH chromone. A variety of tethers in terms of length and functionality were engineered and connected to different sites on the ODN . Conjugation of the dye at the 5′-terminus of the single strand generally demonstrated no change in its fluorescence emission upon hybridization due to its constant exposure to bulk water. However, when the dye was placed in the middle of the ODN, the quantum yield increased 13-fold compared to the single-stranded context. More importantly, this result—obtained after selective excitation at 488 nm —still revealed a prospective Stokes shift of 56 nm. In this regard, our fluorogenic AlMF conjugate compares favorably with the TO/JO FIT-probes, which were synthesized to generate a FRET pair and a larger Stokes shift for easier and more sensitive detection [In summary, novel fluorogenic derivatives of etection . We beli


The structures of new compounds were elucidated by comprehensive spectroscopic techniques, the planar structures of new compounds were determined by comprehensive spectroscopic techniques, and their absolute configurations were confirmed via gauge-independent atomic orbital calculations (GIAO), calculation of the electronic circular dichroism (ECD), and optical rotation (OR). These were determined by spectroscopic data analysis.Albocimea B-E ( Antrodiella albocinnamomea is a kind of wood-decay higher fungus, which belongs to the family Polyporaceae and is widely distributed in northeast China. The main characteristics are flat fruiting body, two-line hyphae, locked union of reproductive hyphae and saccular body in the fruiting layer + , corresponding to five degrees of unsaturation. The IR absorption bands at 3400, 1706, and 1677 cm−1 revealed the presence of hydroxyl, carbonyl, and C=C double bond groups. The 1H NMR spectrum , and revealed that those methyl groups were located at quaternary carbons. The 13C NMR and DEPT spectra (δC 121.3), and six quaternary carbons . These spectroscopic data revealed that 1 should be a dicyclic norsesquiterpenoid. The 1H-1H COSY spectrum  to C-13, C-11, C-10, and C-6, indicated C-12 and C-13 were located on C-11, and C-10 and C-6 were connected by C-11. The HMBC correlations (3-14 (δH 2.20) to C-7, C-8, and C-6, and H2-9 to C-7 indicated C-8 and C-6 were connected by C-7. Beside this, C-5 and C-1 were connected by C-6 from the 1H-1H COSY correlation of H1-4/H2-5, and HMBC correlations from H2-5 and H2-1 to C-6, C-7, and C-11, and so established a 6,6-bicylic skeleton via a C-6 spiro carbon. These data of 1 are closely related to those of antroalbol H +  via HR-ESI-MS, the molecular formula of 2 was carried out as C14H20O3, suggesting five degrees of unsaturation. The IR absorption band of 2 at 3440 cm−1 suggested the presence of a hydroxyl group, while the absorption band at 1712 cm−1 suggested the presence of a carbonyl group. The 1H NMR spectrum  suggested the existence of three methyl groups attached to quaternary carbons and an aldehyde group (δH 9.67). The 13C NMR and DEPT spectra , four methylenes , five quaternary carbons (including one ketone carbonyl at δC 213.4 and one oxygenated carbon at δC 80.0), one aldehyde group (δC 189.5), and a C=C double bond group . The 1H-1H COSY spectrum  to C-10; H1-3 (δH 6.75) to C-1, C-5; H2-4 (δH 2.83) to C-2, C-6, C-10; H2-8 (δH 2.76) to C-10; H2-9 (δH 1.66) to C-11; H3-11 (δH 0.88) to C-12; and H3-13 (δH 1.46) to C-5, C-7. These data are related to those of spiro[4.5]dec-6-en-1-ol, 2,6,10,10-tetramethyl [2 and spiro[4.5]dec-6-en-1-ol, 2,6,10,10-tetramethyl have the same mother nucleus structure and connect different functional groups.Compound spectrum  of 2 at  spectra  of 2 shospectrum  of 2 estspectrum , there w3-13 and H2-4 is very weak in the ROESY spectrum  level using the GIAO method. By comparison of 13C chemical shifts, the relative configurations of 2 can be determined. In order to determine its absolute configuration, the optical rotation (OR) value of configuration -2 was calculated; this value is −9.75. Compared with the experimental OR value of compound 2 (−10.2), the absolute configuration of 2 was finally determined as 5S,6S. Thus, the structure of albocimea C (2) was assigned as depicted.The correlation of Hspectrum . In orde3 was obtained as a white amorphous powder with the molecular formula C16H22O3, based on HR-ESI-MS at m/z 285.1462 [M + Na]+ , which corresponds to six degrees of unsaturation. The IR spectrum absorption band was at 3424 cm−1, indicating the presence of a hydroxyl group. The 1H NMR spectrum  showed the presence of four methyls. The 13C NMR and DEPT spectra . The 1H-1H COSY spectrum  to C-9, C-11; H2-3 (δH 2.64) to C-8, C-11; H1-4 (δH 6.88) to C-3, C-6, C-8; H2-10 (δH 3.52) to C-3, C-11; H3-12 (δH 2.29) to C-6; H2-13 (δH 3.68) to C-5, C-7; H3-15 (δH 2.19) to C-6, C-8. These data are closely related to those of compound M16 in the literature + , corresponding to four degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl group (3430 cm−1) and carbonyl group (1644 cm−1). The 1H NMR spectrum . The 13C NMR and DEPT spectra , three methylenes , one methyne (δC 75.5), and six quaternary carbons (including one carbonyl group at δC 207.4). The 1H-1H COSY spectrum (1-13/H3-14. The HMBC spectrum (2-1 (δH 2.62) to C-3, C-7, C-9, and C-10; H2-3 (δH 1.96) to C-8, C-11; H2-4 (δH 1.93) to C-6, C-8; H3-10 (δH 1.05) to C-11; H3-12 (δH 1.19) to C-6; H1-13 (δH 4.04) to C-4; H3-14 (δH 1.07) to C-5; H3-15 (δH 1.71) to C-6, C-8. These data are related to those of 6,8,8-trimethyl-bicyclonon-1-en-3-one non-1-en-3-one have the same mother nucleus structure and connect different functional groups.Compound spectrum  showed t spectra  showed 1spectrum  establisspectrum  displaye4 were insufficient for determining its relative configuration. Because compound 4 has three chiral centers, it has eight possible configurations. 13C-NMR calculations of eight of these possible configurations were carried out at the mPW1PW91/6-31 G (d) level using the GIAO method. The comparison of the 13C chemical shifts obtained revealed that the calculated chemical shifts of an enantiomer pair configuration 4a and 4e  and four known compounds (5–8) were acquired from the rice fermentation of the fungus A.albocinnamomea. The structures of these compounds were characterized using spectroscopic data. The antibacterial activity test of compounds 2 and 6 showed that they have no significant antibacterial activity.In conclusion, four previously undescribed sesquiterpenoids (18 were used for column chromatography (CC). Thin-layer chromatography (TLC) experiments were performed on a silica gel GF254 pre-coated plate. Fractions were monitored by TLC, and spots were visualized by spraying with 15% H2SO4 in ethanol.Optical rotations were taken on a JASCO P-1020 polarimeter. IR spectra were obtained on a Bruker Tensor 27 spectrometer with KBr pellets. NMR spectra were measured on a Bruker Avance III 600 MHz spectrometer with TMS as the internal standard. Mass spectra were recorded with an APIQSTAR time-of-flight spectrometer. CD spectra were recorded on an Applied Photophysics spectrometer. Silica gel (200–300 mesh), Sephadex LH-20, and Rp-CA. albocinnamomea was purchased from the China Institute of Microbiology. A voucher specimen (No. Yang20181012) was deposited at the Faculty of Life Science and Technology, Kunming University of Science and Technology. A rice medium was used to ferment the strain. The culture of the strain was divided into two steps. Firstly, the fungal strain was cultured in potato dextrose agar (PDA) medium at 24 °C, and the seed solution was obtained after 7 days of culture. Next, a rice medium was used for large-scale fermentation. The culture medium consisted of rice and water at a ratio of 1:1.4. When preparing the culture medium, 71 g of rice and 100 mL of water were put into 480 mL fermentation bottles. A total of 300 bottles were prepared. They were put into a high-pressure steam sterilization pot and sterilized at 121 °C for 30 min. The seed solution obtained before was divided into small parts, put into the prepared rice medium and incubated at room temperature for 45 days.2Cl2/MeOH (50:1–10:1) to afford fractions A–F. Fraction C (46.29 g) was subjected to CC over silica gel eluted with CH2Cl2/MeOH (50:1–10:1) to afford fractions C1–C12. Subfraction C4 (5.32 g) was further purified by silica gel CC , Sephadex LH-20 CC , silica gel CC , and analytical chromatography to afford 1 (1 mg) and 6 (3.2 mg).The fungus was cultured for 45 d, cut into small pieces, and then extracted three times with ethyl acetate (60 L × 72 h each time) at room temperature. The ethyl acetate solution was evaporated under vacuum to yield 137.33 g of crude extract. The extract was subjected to CC over silica gel and eluted with CH2Cl2/MeOH (50:1–0:1) to afford fractions D1–D10. Subfraction D5 (3.42 g) was further purified by silica gel CC , Sephadex LH-20 CC , and analytical chromatography to afford 2 (3.2 mg) and 5 (2.7 mg). D7 (237 mg) was further purified by Sephadex LH-20 CC  to afford 7 (4.7 mg) and 8 (3.6 mg).Fraction D (23.24 g) was subjected to CC over silica gel eluted with CH2O/MeOH (50–100%) to afford fractions E1–E8. Subfraction E6 (2.5 g) was further purified by silica gel CC , MPLC, and silica gel CC  to afford 3 (3.5 mg).Fraction E (40.00 g) was subjected to CC over Rp-18 with H2O/MeOH (20–100%) to afford fractions F1–F10. Subfraction F6 (4.2 g) was further purified by HPLC using a Sephadex LH-20 CC  to afford 4 (2.1 mg).Fraction F (30.00 g) was subjected to column chromatography (CC) over Rp-18 with HPeseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Monilia albican were purchased from the Nanjing Bianzhen Biotechnology Co., Ltd  and deposited at the Faculty of Life Science and Technology, Kunming University of Science and Technology.The strain of 8 colony froming units (CFU)/mL). A 0.5 mL amount of bacterial liquid was injected into the nutrient broth culture medium that had been cooled to about 50 °C; this was mixed evenly, poured into the plate (about 20 mL/plate), stood horizontally, and set aside after solidification. The filter paper soaked with the sample was put on the plate with sterile tweezers. Then, the plates were incubated in an incubator at 37 °C for 18 h, and the zones of inhibition were discussed.A suspension of the organism to be tested was prepared in a saline solution and measured equal to 0.5 McFarland standard (1 × 101): white amorphous powder; max 3401, 2958, 1705, 1676, 1626, 1226 cm−1; 1H  and 13C NMR  data, see m/z 275.1255 [M + Na]+ .Albocimea B (2): white amorphous powder; max 3440, 2957, 2923, 2853, 1703, 1630, 1384, 1272, 1161, 1103, 1063 cm−1; 1H  and 13C NMR  data, see m/z 236.1408 [M + Na]+ .Albocimea C (3): colorless oil; max 3436, 2962, 2930, 1733, 1646, 1605, 1436, 1261, 1165, 1033, 803 cm−1; 1H  and 13C NMR  data, see m/z 285.1462 [M + Na]+ .Albocimea D (4): colorless gum; max 3424, 2952, 2928, 2868, 1644, 1545, 1509, 1453, 1383, 1284, 1259, 1128, 1104 cm−1; 1H  and 13C NMR  data, see m/z 251.1653 [M − H]− .Albocimea E (