CHCl3 MeOH Research Articles

LC-ESI-MS/MS Chemical Characterization, Antioxidant and Antidiabetic Properties of Propolis Extracted with Organic Solvents from Eastern Anatolia Region.

Geographic conditions (altitude, climate, and local flora) lead to significant differences in the chemical composition of propolis. Therefore, more research is needed for propolis in different geographical regions. So, the aim of this study was to evaluate the phenolic profile, total phenolic content, antioxidant, and antidiabetic properties of Pülümür propolis from Turkey. Methanol (MeOH), chloroform (CHCl3 ), and hexane extracts of propolis were analyzed. LC-ESI-MS/MS analysis of the extracts showed that the most abundant phenolic compound is caffeic acid in the MeOH extract (2943.12±11.12 μg phenolics/g extract), while on the other hand, CHCl3 extract had the highest total phenolic content (125.75±1.02 mg GAE/g extract). Antioxidant activity was measured using ABTS and DPPH assays, whereas CHCl3 extract (IC50 =6.35±0.11 and 28.84±0.10 μg/mL, respectively) and MeOH extracts (IC50 =5.04±0.07 and 28.80±0.09 μg/mL, respectively) showed relatively high antioxidant activity. The MeOH extract showed better antidiabetic activity than the standard compound, acarbose (IC50 =0.544 and 0.805 mg/mL, respectively).

Phenolics from the heartwood of Tecoma mollis as potential inhibitors of COVID-19 virus main protease and spike proteins: An In silico study

Pharmacognosy Magazine,2021,17,06,s278-s286.DOI:10.4103/pm.pm_35_21Published:September 2021Type:Original ArticleAuthors:Lamya H Al-Wahaibi, Md Tabish Rehman, Muneera S M. Al-Saleem, Omer A Basudan, Ali A El-Gamal, Mohamed F AlAjmi, Enaam Y Backheet, Azza A Khalifa, and Wael Mostafa Abdel-Mageed Author(s) affiliations:Lamya H Al-Wahaibi1, Md Tabish Rehman2, Muneera S M. Al-Saleem1, Omer A Basudan2, Ali A El-Gamal2, Mohamed F AlAjmi2, Enaam Y Backheet3, Azza A Khalifa3, Wael Mostafa Abdel-Mageed4 1 Department of Chemistry, Science College, Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia 2 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia 3 Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt 4 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt Abstract:Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging novel coronavirus responsible for the viral pneumonia outbreak (coronavirus disease-19 [COVID-19]) that has impacted millions of people, causing a tremendous global public health concern and number of fatalities. The development of novel antiviral agents is considered an urgent research subject. Objectives: The objective of the study is to discover the phenolic constituents of the methanolic extract of Tecoma mollis Humb. and Bonpl. heartwood and to investigate their potential inhibitory action against SARS-CoV-2 protease and/or entry proteins. Materials and Methods: The heartwood of T. mollis was extracted by maceration with 70% EtOH until complete exhaustion. The extract was concentrated under reduced pressure, mixed with distilled H2O and defatted with CHCl3 to produce a CHCl3 fraction, and then subjected to solvent fractionation with n-butanol to produce an n-butanol fraction. The n-butanol fraction was subjected to a silica gel column using CHCl3–MeOH gradient mixtures followed by reversed-phase high-performance liquid chromatography. The isolated compounds were identified using one- and two-dimensional nuclear magnetic resonance as well as mass spectroscopy. Molecular docking studies have been implemented to identify the binding pattern between ligands and target enzymes, i.e. main protease (Mpro) and spike protein receptor-binding domain (RBD), and compared with the currently used COVID-19 inhibitors. Molecular dynamic simulations have been performed to evaluate the dynamics and stability of protein–ligand complexes. The obtained information is then correlated with the essential structural features, and finally the structure–activity relationship is suggested. Results: Fourteen phenolic glycosides were isolated from the methanolic extract of T. mollis Humb. and Bonpl. heartwood in addition to an iridoid, ixoside. The molecular docking study exhibited that the isolated compounds have a higher binding affinity toward the active site of Mpro and the angiotensin-converting enzyme-2 binding site of spike protein RBD. The phenylpropanoids have higher inhibitory action with higher binding energy toward SARS-CoV-2 Mpro protease as compared to spike protein RBD. Among all the isolated compounds, isoverbascoside (10) exhibited the most potent dual interaction with SARS-CoV-2 Mpro protease and spike protein RBD with high binding energy of − 8.8 and − 7.2 kcal/mol, respectively. This showed better potency than the currently used Mpro and spike–protein inhibitors. Conclusion: Our study is the first report on the potential inhibitory action of phenylpropanoids for SARS-CoV-2 protease and spike protein. It also correlates between the reported antiviral activities of some isolated compounds with their potential inhibitory action for COVID-19 viral proteins. Our results on T. mollis extract constituents could help in the discovery of a promising repurposable drug candidate that could contribute to the development of an effective therapy for COVID-19. Keywords:In silico study, isoverbascoside, phenylpropanoids, protease inhibition, severe acute respiratory syndrome coronavirus 2, spike protein, Tecoma mollisView:PDF (3.33 MB) Full Text

Quantification of herbal drug hypoxoside from the roots of South African Hypoxis hemerocallidea using cost-effective high-performance thin-layer chromatography–densitometry validated method

Hypoxis (Hypoxidaceae) consists of about 90 species of plants reported worldwide, of which 76 occur in Africa. As many as 41 species are indigenous to countries belonging to the Southern African Development Community (SADC), including South Africa. Of all the Hypoxis species, Hypoxis hemerocallidea has versatile application in traditional health care system of over 85% of South Africans and is regarded as one of the most ethnomedicinally important and most marketed species in South Africa. H. hemerocallidea corm’s water or alcoholic extract is widely used as traditional medicine for the treatment of benign hypertrophy and urinary tract infections as well as for boosting the immune system of people living with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) among others. However, the use of other parts of Hypoxis plant as medicine is vital for conservation purposes. The roots attached on the corm of H. hemerocallidea contain hypoxoside, but the roots are usually ripped off during the preparation of Hypoxis-containing traditional medicines and other herbal products. A developed and validated, affordable but reliable high-performance thin-layer chromatography (HPTLC) densitometry for the rapid and repeatable visualization and quantitative determination of hypoxoside from the roots of H. hemerocallidea was performed. After thin-layer chromatography analysis, the hypoxoside resolved and was visualized at RF of 0.30 in CHCl3–MeOH–H20 (70:30:2 v/v). The method was linear with R2 of 0.9876 over a calibration range of 0.20 × 10−4–1.80 × 10−3 mg mL−1. The limits of detection (LOD) and quantification (LOQ) were 5.08 × 10−4 and 1.65 × 10−3 mg mL−1, respectively, while the percentage recovery and the method repeatability (%RSD) were 84.10 and 4.98, respectively. The roots of H. hemerocallidea were found to contain 4.101 × 10−4 mg mL−1 of hypoxoside.

Structure-Directing Weak Interactions with 1,4-Diiodotetrafluorobenzene Convert One-Dimensional Arrays of [MII(acac)2] Species into Three-Dimensional Networks

The complexes [MII(acac)2] (M = Cu 1, Pd 2, Pt 3; Hacac = acetylacetone) and 1,4-diiodotetrafluorobenzene (FIB) were cocrystallized in CHCl3–MeOH solutions to form adducts (1–3)·FIB, whose structures were studied by X-ray diffraction. The association leads to unification of the three structures, thus demonstrating the potential of the isostructural Cu/Pd/Pt exchange for construction of supramolecular systems involving [MII(acac)2] complexes. In the crystal structures of (1–3)·FIB, the intermolecular bifurcated halogen bonding I···μ2-(O,O) and noncovalent interactions M···C were identified and then studied by density functional theory calculations and topological analysis of the electron density distribution within the framework of the QTAIM method at the M06/DZP-DKH level of theory. Apart from these unconventional interactions, two types of classic hydrogen bonding, viz. the C–H···I–C and C–H···F–C contacts between Me groups and halogen atoms of FIB, were detected. Collectively all these noncovalent struc...

A New N-Acyl Taurine from the South China Sea Marine Sponge Callyspongia sp.

The genus Callyspongia (order Haplosclerida, family Callyspongiidae) is widely distributed and contains various bioactive constituents [1]. In the course of our investigation on the marine sponge Callyspongia species, we have reported some metabolites [2, 3]. Further investigation on the marine sponge Callyspongia species has led to the isolation and characterization of a new N-acyl taurine (1). The sponge Callyspongia sp. was collected off the coast of Hainan Island, South China Sea, in January, 2007. The specimen was identified by Dr. Kyung Jin Lee. A voucher specimen (No. 20070101) was deposited in Natural History Museum, Hannam University, Daejon, Korea and Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, China. The wet marine sponge Callyspongia sp. (10 kg) was extracted with ethanol (90%). The ethanol was evaporated in vacuo to afford a syrupy residue that was suspended in distilled water and fractionated successively with petroleum ether, ethyl acetate, and n-butanol. The n-butanol soluble portion (80.1 g) was subjected to RP chromatography eluted with H2O–MeOH (9:1, fr. A), H2O–MeOH (8:2, fr. B), H2O–MeOH (7:3, fr. C), H2O–MeOH (6:4, fr. D), H2O–MeOH (4:6, fr. E), and H2O–MeOH (0:10, fr. F), to give six fractions. Fraction C (4.12 g) was subjected to silica gel chromatography with CHCl3–MeOH (8:2 to 6:4, gradient) to give three fractions (C1–C3). Fraction C2 (435 mg) was subjected to silica gel chromatography with CHCl3–MeOH (8:2), then purified by Sephadex LH-20 (CHCl3–MeOH, 1:1) to afford 1 (5.0 mg). Compound 1 was obtained as a yellow oil, and its molecular formula was deduced to be C13H25NO4S on the basis of the HR-ESI-MS spectrum at m/z 290.1422 [M – H]– (calcd 290.1426 for C13H24NO4S) and NMR data. The gross structure was determined by the aid of COSY, HMQC, and HMBC experiments. The 1H NMR, 13C NMR, and DEPT data for 1 indicated the presence of two methyls, eight sp3 methylenes, one sp2 methine, and two sp2 quaternary carbons. The sulfur and remaining three oxygens in the molecular formula suggested a sulfonic acid group (SO3H). The 1H NMR signals at 3.63 (2H, t, J = 7.0 Hz, H-1 ) and 3.00 (2H, t, J = 7.0 Hz, H-2 ), and 13C NMR signals at C 174.6 (C-1), 36.6 (C-1 ) and 51.5 (C-2 ), together with the HMBC correlations (Fig. 1) of H-2 with C-1 and H-2 with C-1 and C-1 , indicated the two methylenes C-1 and C-2 to be located between the amide and the sulfonic acid, which revealed a taurine moiety in the structure of 1 [4, 5]. The position of the double bond was ascertained at C-3 and C-4 on the basis of the HMBC correlations of H-4 with C-2 and H-5 with C-3. The methyl group (C-11) was determined to be located at C-3 by the HMBC correlations of H-11 with C-2 and C-4. The geometry of the double bond was deduced to be cis based on the chemical shifts of the allylic carbon C-5 at 29.2, which agreed well with the chemical shift for the allylic carbons of cis-olefins (approximately 29) [6]. Thus, the structure of compound 1 was elucidated as 2-(3-methyl-dec-3-enamido)ethanesulfonic acid. 2-(3-Methyl-dec-3-enamido)ethanesulfonic Acid (1). Yellow oil. 1H NMR (500 MHz, CDCl3, , ppm, J/Hz): 5.36 (1H, t, J = 6.5, H-4), 3.63 (2H, t, J = 7.0, H-1 ), 3.00 (2H, t, J = 7.0, H-2 ), 2.91 (2H, s, H-2), 2.07 (2H, m, H-5), 1.77 (3H, s, H-11), 1.18–1.27 (8H, overlap, H-6–9), 0.93 (3H, t, J = 6.5, H-10). 13C NMR (125 MHz, CDCl3, , ppm): 174.6 (C-1), 131.2 (C-4), 130.2 (C-3), 51.5 (C-2 ), 48.8 (C-2), 36.6 (C-1 ), 29.2 (C-5), 23.7–30.8 (C-6–9), 16.3 (C-11), 14.6 (C-10). HR-ESI-MS m/z 290.1422 [M – H]– (calcd 290.1426 for C13H24NO4S).

Chemical Constituents of Saxifraga umbellulata

Saxifraga umbellulata grows mainly in high, cold areas such as southwest of China and Qinghai and Gansu Provinces [1]. S. umbellulata is an important herbal medicine, which has been used as a traditional folk medicine for the treatment of hepatitis, cholecystitis, infuenza, fever, sore, and so on [2]. As part of our continuous research on S. umbellulata, five compounds were isolated from S. umbellulata for the first time. The air-dried whole plants of S. umbellulata were bought from Kumbum Monastery Tibetan Hospital in Qinghai Province of China in October of 2009 and was identified by Prof. Guo-Liang Zhang, Lanzhou University. The powdered S. umbellulata (3.0 kg) was extracted by ethanol refluxing (12 L 2, 2 h for the first time, 1 h for the second time). The EtOH extract was concentrated under reduced pressure to give a residue (320 g), which was sequentially partitioned with PE (90–100 C) (0.5 L 4), EtOAc (0.5 L 4), and n-BuOH (0.5 L 4). The EtOAc fraction (45 g) was chromatographed on silica gel column (4.0 120 cm, 450 g) and eluted with PE–acetone gradiently to yield 10 fractions (Fr.1–Fr.10) according to TLC analysis. Fraction 1 (2.0 g) was re-treated on a silica gel (300–400 mesh, 60 g) column and eluted with PE–EtOAc (20:1, v/v) to obtain 5 (7 mg). Fraction 2 (2.0 g) was re-treated on a silica gel (300–400 mesh, 60 g) column and eluted with PE–EtOAc (10:1, v/v) to obtain 1 (10 mg). Fraction 3 (3.0 g) was re-treated on a silica gel (300–400 mesh, 90 g) column eluted with CHCl3–MeOH (30:1, v/v) to obtain 2 (10 mg). Fraction 4 (2.5 g) was re-treated on a silica gel (300–400 mesh, 80 g) column eluted with PE–acetone (1:1, v/v) to obtain 3 (12 mg) and 4 (13 mg). p-Coumaric Acid (1). Colorless needles, C9H8O3. ESI-MS m/z: 165 [M + H]+, 187 [M + Na]+. IR (KBr, max, cm–1): 1310, 1640, 1705. 1H NMR (400 MHz, TMS, DMSO-d6, , ppm, J/Hz): 7.53 (2H, d, J = 8.8, H-2, 6), 6.81 (2H, d, J = 8.8, H-3, 5), 11.05 (1H, s, COOH). 13C NMR (100 MHz, TMS, DMSO-d6, , ppm): 159.7 (C-1), 115.8 (C-2, 6), 130.1 (C-3, 5), 125.4 (C-4), 144.2 (C-7), 115.5 (C-8), 168.1 (C-9) [3, 4]. Hirsutenone (2). Brown oil, C19H20O5. FAB-MS m/z 345 [M – H]+. 1H NMR (400 MHz, TMS, DMSO-d6, , ppm, J/Hz): 2.53–2.68 (4H, m, H-1, 2, 6, 7), 6.10 (1H, d, J = 15.6, H-4), 6.68–6.73 (4H, m, H-2 , 2 , 5 , 5 ), 6.48–6.52 (2H, m, H-6 , 6 ). 13C NMR (100 MHz, TMS, DMSO-d6, , ppm): 198.7 (C-3), 146.2 (C-3 ), 144.5 (C-3 ), 144.4 (C-5), 142.8 (C-4 ), 142.7 (C-4 ), 132.6 (C-1 ), 132.3 (C-1 ), 130.0 (C-4), 119.1 (C-6 ), 119.0 (C-6 ), 115.0 (C-5 ), 115.0 (C-5 ), 114.7 (C-2 ), 114.7 (C-2 ), 41.1 (C-2), 33.9 (C-6), 28.9 (C-1) [5]. 4-O-Caffeoylquinic Acid (3). Colorless needles, C16H18O9. FAB-MS m/z 353 [M + H] +. 1H NMR (300 MHz, TMS, DMSO-d6, , ppm, J/Hz): 7.53 (1H, d, J = 16.0, H-3 ), 7.04 (1H, dd, J = 2.0, H-5 ), 6.98 (1H, dd, J = 2.0, 8.0, H-9 ), 6.76 (1H, d, J = 8.0, H-8 ), 6.27 (1H, d, J = 16.0, H-2 ), 4.70–4.73 (2H, m, H-3, 5), 4.13 (1H, m, H-4), 2.34 (2H, m, H-6), 1.95 (2H, m, H-2). 13C NMR (75 MHz, TMS, DMSO-d6, , ppm): 177.3 (C-7), 166.0 (C-1 ), 148.7 (C-7 ), 145.8 (C-3 ), 145.7 (C-6 ), 125.6 (C-4 ), 121.8 (C-9 ), 116.0 (C-8 ), 114.7 (C-2 ), 113.9 (C-5 ), 75.9 (C-1), 71.6 (C-5), 68.7 (C-4), 62.9 (C-3), 36.6 (C-2), 35.7 (C-6) 6 . (5R)-5-Hydroxy-7-(3-methoxy-4-hydroxyphenyl)-1-(3,4-dihydroxyphenyl)-3-heptanone (4). Yellow oil, C20H24O6. EI-MS m/z 360 [M]+ (20). 1H NMR (400 MHz, TMS, CDCl3, , ppm, J/Hz): 6.88 (1H, m, H-2 ), 6.85 (1H, d, J = 8.0, H-5 ), 6.71 (1H, d, J = 8.0, H-5 ), 6.68 (1H, d, J = 1.8, H-2 ), 6.50 (1H, dd, J = 1.8, 7.8, H-6 ), 6.48 (1H, dd, J = 1.8, 7.8, H-6 ), 4.04 (1H, m, H-5), 3.64 (3H, m, OCH3), 2.77 (4H, m, H-1, 2), 2.68 (1H, m, H-7a), 2.60 (2H, m, H-4), 2.51 (1H, m, H-7b),

Chemical Constituents of Pteris fauriei

The group of ferns from the genus Pteris (Pteridaceae) contains 300 cosmopolitan species, with 66 species being endemic to China [1]. Several plants in the genus are widely used in traditional Chinese medicine for relieving internal heat or fever, swelling and diarrhea, and removing toxicity [2, 3]. Previously, phytochemical investigations on the Pteris genus had led to the isolation of various phenolic compounds, flavonol glycosides, kauranes, and pterosin sesquiterpenes [4–8]. Some extracts and compounds of plants in the genus showed various bioactivities such as antitumor, antibacterial, cytotoxic, and free radical-scavenging activities [9–11]. P. fauriei Hieron. is distributed in North Vietnam, Japan, and southern China. There have been no reports on the isolation of compounds from it. During the search for bioactive compounds from medicinal plants in Yunnan of China, we investigated the plant. P. fauriei was collected from Kunming of Yunnan, China in April, 2006. The air-dried whole plants (0.8 kg) were chopped and exhaustively extracted with 95% EtOH. Water (1.5 L) was added to the EtOH extract (100 g), and the resulting solution was extracted with petroleum ether and EtOAc successively (four times, each 0.8 L). The EtOAc extract (27 g) was separated on a silica gel column and eluted with petroleum ether containing increasing amounts of EtOAc to obtain nine fractions. Fraction B (4.2 g) was subjected to repeated column chromatography (silica gel, petroleum ether–EtOAc, 20:1) to afford 6 (47 mg). Fraction C (7.1 g) was isolated on a Sephadex LH-20 column (CHCl3–MeOH, 3:2) to give 5 (13 mg). Fraction D (1.3 g) was chromatographed over preparative TLC (petroleum ether–EtOAc, 3:1) to furnish 1 (8 mg). Fraction E (1.8 g) was purified on a Sephadex LH-20 column (MeOH) to obtain 2 (3 mg) and 3 (4 mg). Fraction F (1.5 g) was subjected to Sephadex LH-20 column chromatography (MeOH) to afford 4 (15 mg). Fraction G (4.7 g) was chromatographed over silica gel (CHCl3–Me2CO, 20:1) and then purified by chromatography over Sephadex LH-20 (MeOH–H2O, 9:1) to furnish 7 (43 mg). Fraction H (5.2 g) was isolated on MCI gel and eluted with a gradient of 0–100% MeOH in H2O to obtain 8 (16 mg). Aristolone (1), C15H22O, colorless amorphous powder. The mass spectrum exhibited peaks for ions at m/z 218 (M+, 43), 203, 191, 177, 161, 147, 91. PMR spectrum (CDCl3, , ppm, J/Hz): 1.19, 1.20, 1.30 (each 3H, s), 1.06 (3H, d, J = 6.7), 5.75 (1H, s). 13C NMR and DEPT spectra (CDCl3, , ppm): 196.6, 167.9, 124.7, 40.0, 39.6, 39.1, 35.9, 33.5, 31.0, 30.1, 26.5, 24.7, 22.9, 16.9 and 16.6 [12]. Kaempferol (2), C15H10O6, yellow amorphous powder 13 . Luteolin (3), C15H10O6, yellow amorphous powder [14].

Piperazine-based new sensor: selective ratiometric sensing of Fe3+, logic gate construction and cell imaging

Newly designed and synthesised chemosensor 1 selectively recognises Fe3+ ions in CHCl3–MeOH (1:1, v/v) by showing ratiometric change in emission and green colouration of the solution under the exposure of UV light. The ensemble 1·Fe3+ selectively detects F− ions over other halides and the phenomenon is useful to construct combinatorial logic gate. Furthermore, the probe 1 can be used for in vitro detection of Fe3+ in human cervical cancer (HeLa) cells.

Assembling coordination ladders with 4′-(4-methoxyphenyl)-4,2′:6′,4″-terpyridine as rails and rungs

The divergent ligand 4′-(4-methoxyphenyl)-4,2′:6′,4″-terpyridine (1) reacts with Cd(NO3)2·4H2O under room temperature crystallization conditions (MeOH/CHCl3) to give the coordination polymer [{Cd2(1)3(NO3)4} CHCl3 MeOH]n. The polymer adopts a ladder structure with 1 bridging pairs of cadmium(II) centres along both the edges (rails) and rungs of the ladder.

Phenolic Constituents of Pulicaria gnaphaloides

In continuation of our research on the chemical constituents of Pulicaria gnaphaloides [1], we isolated eight phenolic compounds (1–8) using column chromatography of EtOAc fractions and elution by CHCl3 and a CHCl3–MeOH gradient (9:1 and 4:1). The structures of the isolated compounds were elucidated using spectral data (PMR, 13C NMR, DEPT, HSQC, HMBC, COSY) and comparisons with the literature in addition to direct comparison with authentic samples. They were identified as p-hydroxybenzoic acid (1), 3,4-dihydroxybenzoic acid methyl ester (2), luteolin (3), quercetin (4), isoquercitrin (5), hyperoside (6), rutin (7), and quercetin 3-rhamnosyl-(1 6)-galactoside (8). p-Hydroxybenzoic acid (1), C7H6O3, white crystals, mp 159.5°C. Compound 1 was identified as p-hydroxybenzoic acid using spectral data [2]. 3,4-Dihydroxybenzoic Acid Methyl Ester (2). C8H8O4, white crystals, mp 137–138°C. Its spectral data agreed with those published [2]. Luteolin (3). C15H10O6, light-yellow powder, mp 328–330°C. PMR and 13C NMR spectra agreed with those published [3]. Quercetin (4). C15H10O7, light-yellow crystals, mp 305–307°C. Spectral data and a comparison with the literature identified 4 as quercetin [2]. Isoquercitrin (5). C21H20O12, yellow powder, mp 248–250 C. 1H NMR spectrum (600 MHz, DMSO-d6, , ppm, J/Hz): 7.58 (1I, d, J = 1.8, H-2 ), 7.57 (1I, d, J = 9.0, I-5 ), 6.85 (1I, dd, J = 1.8; 9.0, H-6 ), 6.39 (1I, d, J = 2.4, I-8), 6.19 (1I, d, J = 2.4, I-6), 5.46 (1I, d, J = 7.8, I-1 ), 12.63 (1I, 5-II), 10.92 (1I, 7-II), 9.67 (1I, 4 -II), 9.41 (1I, 3 -II). 13C NMR spectrum (150 MHz, DMSO-d6, , ppm): 156.29 (N-2), 133.28 (N-3), 177.38 (N-4), 161.21 (N-5), 98.65 (N-6), 162.21 (N-7), 93.47 (N-8), 156.11 (N-9), 103.90 (N-10), 121.56 (N-1 ), 116.15 (N-2 ), 144.78 (N-3 ), 148.44 (N-4 ), 113.41 (N-5 ), 121.12 (N-6 ), 104.84 (N-1 ), 74.07 (N-2 ), 76.48 (N-3 ), 70.91 (N-4 ), 77.55 (N-5 ), 60.95 (N-6 ) [4]. Acid hydrolysis of 5 produced quercetin and D-glucose. Hyperoside (6). C21H20O12, yellow powder, mp 231–232°C. Acid hydrolysis detected quercetin and D-galactose [1]. Rutin (7). C27H30O16, yellow powder, mp 193–195°C. Acid hydrolysis of 7 produced quercetin, D-glucose, and L-rhamnose [5]. Quercetin 3-rhamnosyl-(1 6)-galactoside (8). C27H30O16, yellow powder, mp 186–188 C. 1H NMR spectrum (400 MHz, DMSO-d6, , ppm, J/Hz): 7.55 (2H, dd, J = 1.8, 8.5, H-2 , 6 ), 6.84 (1H, d, J = 8.5, H-5 ), 6.33 (1H, d, J = 2.0, H-8), 6.14 (1H, d, J = 2.0, H-6), 5.33 (1H, d, J = 7.5, H-1 ), 4.39 (1H, s, H-1 ), 1.01 (3H, d, J = 6.0, CH3-6 ), 3.04–3.40 galactose and rhamnose protons, 12.56 (1I, s, 5-II). 13C NMR spectrum (100 MHz, DMSO-d6, , ppm): 156.94 (N-2), 133.62 (N-3), 177.52 (N-4), 161.55 (N-5), 99.48 (N-6), 165.21 (N-7), 94.20 (N-8), 156.74 (N-9), 103.81 (N-10), 121.99 (N-1 ), 116.52 (N-2 ), 145.24 (N-3 ), 149.07 (N-4 ), 115.63 (N-5 ), 121.43 (N-6 ), 101.79 (N-1 ), 74.51 (N-2 ), 76.29 (N-3 ), 70.97 (N-4 ), 76.87 (N-5 ), 67.42 (N-6 ), 101.17 (N-1 ), 70.77 (N-2 ), 70.39 (N-3 ), 72.26 (N-4 ), 68.66 (N-5 ), 18.17 (N-6 ) [6]. Acid hydrolysis of 8 produced quercetin, D-galactose, and L-rhamnose. p-Hydroxybenzoic acid, 3,4-dihydroxybenzoic acid methyl ester, luteolin, quercetin, and quercetin 3-rhamnosyl(1 6)-galactoside were isolated for the first time from the genus Pulicaria; isoquercitrin and rutin, for the first time from Pulicaria gnaphaloides.

Carbohydrates and Proteins from Helianthus tuberosus

Helianthus tuberosus has unlimited potential for multi-faceted application to food, feed, technical, medicinal, and ecological problems. Lipids [1], carbohydrates, and protein constituents of H. tuberosus variety Faiz Baraka that was developed in Uzbekistan were studied earlier [2]. In continuation of this research, we studied carbohydrates and proteins of H. tuberosus variety Muzhiza that was developed at the Uzbekistan Research Institute of Horticulture by individual and mass selection from voucher samples. The starting material of air-dried finely ground tubers was worked up with CHCl3–MeOH (2:1) in order to remove lipophilic substances. Successive extraction of the remaining raw material isolated water-soluble polysaccharides (WSPS) and fructooligosaccharides (FOS) using H2O at room temperature; inulin, hot H2O; pectinic substances (PS), a mixture of oxalic acid and ammonium oxalate solutions; and hemicellulose (HMC), base solution (5%). A light-brown powder was obtained after evaporation of the alcoholic mother liquor and work up with Me2CO. It contained glucose, fructose, and a series of FOS. The homologous series of FOS began with a saccharose of the lowest homolog (for n = 0) that was followed by trisaccharides differing from the previous one by a single fructose residue. The highest homolog of this series was inulin [3]. The contents of FOS and inulin were 35% and 9.1%, respectively. The polysaccharide contents in H. tuberosus tubers of the two studied varieties were compared. The results are given below:

Secondary Metabolites of Endophytic Fungus Trichoderma sp. YM 311505 of Azadirachta indica

Endophytic fungi colonize internal tissues of plants without resulting in overt damage while the tissue is alive. They are important sources to provide a wide variety of structurally unique and biologically potent natural products [1]. In our previous search for secondary metabolites of endophytic fungi from Azadirachta indica, some bioactive and/or new compounds were isolated [2–4]. Trichoderma species have long been studied as biological control agents of phytopathogens and are well known for their ability to produce a wide range of antibiotic substances and for their ability to parasitize other fungi [5]. In our ongoing work, we have studied the secondary metabolites of Trichoderma sp. YM 311505 from A. indica and obtained nine compounds. The fungal strain Trichoderma sp. YM 311505 was isolated from the fruit of A. indica collected in Yuanjiang Country, Yunnan Province, P. R. China. It was classified as a Trichoderma species by its morphological characteristics and ITS rDNA sequence analysis. The strain was deposited in Yunnan Institute of Microbiology, Kunming, P. R. China. The fungus was cultured in 500 mL Erlenmeyer flasks ( 750) containing 200 mL of PDB medium at 200 rpm at 28 C for 6 days on a rotary shaker. The culture broth was filtered to remove mycelia. The filtrate was concentrated in vacuum to 30 L and then extracted with EtOAc (5 30 L). The combined extracts were evaporated to yield 164.5 g of the crude extract, which was subjected to column chromatography on silica gel eluting with petroleum–acetone (9:1–3:7) to afford nine fractions (I–IX). Repeated chromatography of fraction II on silica gel with petroleum ether–EtOAc (5:1) and CHCl3–acetone (95:5) afforded compounds 1 (30 mg), 5 (50 mg), and 6 (4 mg). Fraction III was then subjected to column chromatography on RP-18 silica gel with acetone–H2O (8:2) to give compound 2 (28 mg). Fraction IV was subjected to repeated column chromatography on silica gel with petroleum ether–EtOAc (2:1) and RP-18 silica gel with acetone–H2O (8:2) to afford compound 9 (14 mg). Fraction V was submitted to column chromatography on silica gel with CHCl3–acetone (3:1) to give compound 3 (18 mg) and 8 (11 mg). Fraction VI was subjected to column chromatography on silica gel with petroleum ether–acetone (3:1) and RP-18 silica gel with acetone–H2O (1:1) to yeild compound 4 (10 mg). Fraction VIII was chromatographed on silica gel with CHCl3–MeOH (9:1) to give compound 7 (5 mg).

Flavonoids, Antioxidant and Antibacterial Activities of Eryngium triquetrum

The genus Eryngium belongs to the Apiaceae family (Umbelliferae), subfamily Saniculoideae, which consists of about 300 species [1]. Plants of this genus have shown various activities such as anti-inflammatory [2], antimalaria and antifungal [3], antioxidant and antimicrobial [4], antidiabetic [5], and analgesic properties [6]. Air-dried and powdered aerial parts (1.5 kg) of Eryngium triquetrum Vahl. were macerated at room temperature with MeOH–H2O (70:30, v/v) for 24 h three times. After filtration and concentration, the residue was dissolved in water (600 mL). The resulting solution was extracted successively with CHCl3, EtOAc, and n-butanol. Concentration in vacuo led to the following extracts: CHCl3 (0.8 g), EtOAc (1.3 g), and n-butanol (11 g). The butanolic extract of Eryngium triquetrum (11 g) was column chromatographed on polyamide SC6 with a gradient of toluene–MeOH with increasing polarity; Fr. 4 (70 mg), obtained from 85% toluene, was subjected to silica gel column chromatography eluted with EtOAc–MeOH–H2O (10:1:0.5), leading to two subfractions: Fr. 1 and Fr. 2. Subfraction 1 was separated by silica gel column chromatography eluted with CH2Cl2–MeOH (8:2) to afford compound 1 (10 mg). Subfraction 2 yielded compound 2 (60 mg), which was obtained as a yellow precipitate. Fraction 6 (70 mg), obtained from 80% toluene, was further subjected to silica gel column chromatography eluted with EtOAc–MeOH–H2O (10:1:0.5) to give compound 3 (40 mg). The ethyl acetate extract (1.3 g) was column chromatographed on silica gel with a gradient of CHCl3–MeOH with increasing polarity. Fraction 2 (30 mg) and Fr. 3 (35 mg), obtained from 95% and 90% CHCl3 successively, were separated by silica gel column chromatography eluted with EtOAc–MeOH–H2O (10:1:0.5) to afford compounds 4 (9 mg) and 5 (8.5 mg). The structures of compounds 1–5 were established by chemical and spectral analysis, UV, NMR, acid hydrolysis, and HPLC-UVDAD-MS, as well as by comparing their spectroscopic data with those reported in the literature. Acid Hydrolysis. The pure compounds were treated with 2M HCl at 100 C for 1 h. The hydrolysates were extracted with EtOAc, and the aglycons were identified by their UV spectra in methanol and by comparison of their Rf with authentic samples. Sugars were identified in the aqueous residue by comparison with authentic samples on silica gel TLC impregnated with 0.2 M NaH2PO4, solvent Me2CO–H2O (9:1), revealed with aniline malonate. Compound 1. C21H20O11. UV (MeOH, max, nm): 267, 347; +NaOH: 275, 325, 401; +AlCl3: 273, 398; +AlCl3/HCl: 273, 397; +NaOAc: 274, 377; +H3BO3: 268, 355. 1H NMR (250 MHz, CD3OD, , ppm, J/Hz): 8.09 (2H, d, J = 8.9, H-6 , 2 ), 6.91 (2H, d, J = 8.9, H-5 , 3 ), 6.42 (1H, d, J = 1.9, H-8), 6.22 (1H, d, J = 1.9, H-6), 5.28 (1H, d, J = 7.3, Glc H-1 ), 3.26–4.14 (sugar protons). This compound was characterized as kaempferol 3-O-D-glucoside [7]. Compound 2. C30H26O13. UV (MeOH, max, nm): 267, 315; +NaOH: 274, 368; +AlCl3: 274, 403; +AlCl3/HCl: 273, 403; +NaOAc: 274, 372; +H3BO3: 272, 371. 1H NMR (400 MHz, CD3OD, , ppm, J/Hz): 7.97 (2H, d, J = 8.9, H-6 , 2 ), 7.39 (1H, d, J = 15.9, H-7 ), 7.28 (2H, d, J = 8.6, H-2 , 6 ), 6.79 (2H, d, J = 8.9, H-5 , 3 ), 6.77 (2H, d, J = 8.6, H-3 , 5 ), 6.27 (1H, d, J = 1.7, H-8), 6.11 (1H, d, J = 1.7, H-6), 5.24 (1H, d, J = 7.2, Glc H-1 ), 4.32 (1H, d, J = 11.5, H-6 a), 4.19 (1H, dd, J = 12.5, 5.6, H-6 b), 3.20–4.40 (sugar protons). In addition, HPLC-UV-DAD permitted the characterization of the compound as kaempferol 3-O-[6 -O-E-p-coumaroyl]-D-glucopyranoside [8]. Compound 3. C27H30O16. UV (MeOH, max, nm): 269, 314; +NaOH: 276, 321, 365; +AlCl3: 276, 399; +AlCl3/HCl: 277, 399; +NaOAc: 275, 374; +H3BO3: 275, 372. 1H NMR (400 MHz, CD3OD, , ppm, J/Hz): 7.95 (2H, d, J = 8.9, H-6 , 2 ),

Chemical Components of the Tuber of Alocasia cucullata

Alocasia cucullata Schott is mainly distributed in Guangxi, Guangdong, Sichuan, Guizhou, and Fujian Provinces in China (Wu, 1990). Its tuber (Chinese name “jianweiyu”) is a well-known ethnic medicine of the Zhuang nationality in China and is used to treat cancer, reduce swelling, for detoxification, and to ease pain. The antitumor effects of this herb have been preliminarily confirmed because of its widespread clinical application in China for centuries. Nevertheless, up to now, no systematic study has been done on the antitumor constituents of A. cucullata, and the occurrence of an N-acetyl-D-lactosamine (LacNAc)-specific lectin has been reported in a previous investigation of A. cucullata [1]. Our previous pharmacological study further showed the presence of cytotoxic activity in a 50% ethanol extract [2]. In this article we report the isolation of a total of 12 substances: steroid (1), alkaloids (2–4), and phenolic substances (5–12) for the first time from the genus Alocasia. Plant Material. The tubers of A. cucullata were collected in Sichuang Province, China, in May 2011 and identified by Prof. Bao-Kang Huang (Second Military Medical University, Shanghai, China). A voucher specimen (Zheng 5580) has been deposited in the Herbarium of the Shanghai University of Traditional Chinese Medicine. Processing of Plant Material. The tubers of A. cucullata (5 kg) were powdered and extracted three times with 50% EtOH. The extract was concentrated under reduced pressure to give a crude residue (200 g), which was extracted successively with petroleum ether, ethyl acetate, n-butanol. The n-butanol fraction, concentrated to dryness (30 g), was further fractionated on a silica gel chromatography column (600 g of silica gel 60, 70–230 mesh; CHCl3–MeOH–H2O [(15:0.4:1,10:0.4:1, and 5:0.4:1) gradient)] to give four fractions, Frs. A, B, C, and D which were further fractionated on a silica gel chromatographic column (RP-18): CC of Fr. A (2.2 g) (RP-18, 200 g; CH3OH–H2O 4:1) gave 1 (30 mg), 2 (36 mg), 3 (30 mg), and 4 (28 mg); CC of Fr. B (6.2 g) (RP-18, 400 g; CH3OH–H2O 1:1) gave 3 (20 mg), 4 (100 mg), 5 (46 mg), 6 (30 mg), 7 (58 mg), and Fr. B (0.9 g), which were pooled. Fraction B was chromatographed on Sephadex LH-20 (CH3OH–H2O 1:1) yielding 8 (32 mg). CC of Fr. C (5.2 g) (RP-18, 400 g; CH3OH–H2O 2:1) gave 9 (30 mg) and 10 (34 mg). CC of Fr. D (2.1 g) (RP-18, 400 g; CH3OH–H2O 4:1) gave 11 (21 mg) and 12 (25 mg). Compound 1, C35H60O6, white amorphous powder (MeOH), mp 134–135 C. ESI-MS m/z 577 [M + H]+. Its physical constants and spectral data were in agreement with those of -daucosterol [3]. Compound 2, C10H13N5O4, white amorphous powder (MeOH), mp 234.4–235.4 C. ESI-MS m/z 268 [M + H] +. Its physical constants and spectral data were in agreement with those of -adenosine [4]. Compound 3, C6H6N2O, white amorphous powder (MeOH), mp 129–130 C. ESI-MS m/z 121 [M – H]–. Its physical constants and spectral data were in agreement with those of niacinamide [5]. Compound 4, C9H7NO, white gum (MeOH). ESI-MS m/z 146 [M + H] +. Its physical constants and spectral data were in agreement with those of 1H-indole-3-carboxaldehyde [5]. Compound 5, C9H10O3, yellow oil, mp 49–50 C. ESI-MS m/z 167 [M + H]+. Its physical constants and spectral data were in agreement with those of paeonol [6]. Compound 6, C7H6O5, yellow powder. ESI-MS m/z 171 [M + H] +. Its physical constants and spectral data were in agreement with those of gallic acid [6].

Chemical Constituents and Biological Activities of Dicranopteris linearis

The genus Dicranopteris (Gleicheniaceae) consists of about 10 species, distributed mainly in Asia. Several species of this genus are used as traditional medicines throughout the world [1]. The whole plant of Dicranopteris linearis is used commonly for the treatment of internal heat or fever, asthma, external wound, ulcers, and broils as a folk medicine in China [2]. Previously, the extracts of D. linearis have been reported to possess antinociceptive, anti-inflammatory, antipyretic, and cytotoxic activities [3, 4]. Aiming to find potentially bioactive secondary metabolites from this species, we investigated the whole plant of D. linearis and isolated 10 known compounds (1–10), in which compounds 1–6 were isolated from it for the first time. All these compounds were tested for antibacterial activities against five bacteria and cytotoxic activities against two selected cancer cells. Within the series of compounds tested, compounds 7–10 exhibited moderate activities against Escherichia coli, Bacillus cereus, Staphyloccocus aureus, Erwinia carotovora, and Bacillus subtilis with MICs ranging from 15.5 to 62.5 g/mL. Moreover, compound 7 showed the strongest activity against HL-60 and SMMC-7721 with IC50 values of 4.5 and 9.0 g/mL, respectively. To the best of our knowledge, this is the first report that the isolated compounds from D. linearis show antibacterial and cytotoxic activities. Plant Material. The whole plants of D. linearis were collected from Jianfengling of Hainan Province, China, in August 2007 and were identified by Senior Engineer Qiong-Xin Zhong at the Department of Biology, Hainan Normal University, China. A voucher specimen (No. 2007806) was deposited at the Natural Organic Academy of Lanzhou University. Extraction and Isolation. Air-dried whole plants (8.6 kg) of D. linearis were extracted three times (each for 7 days) with 90% ethanol (20 L) at room temperature. The ethanol filtrates were collected and concentrated under reduced pressure by a rotary evaporator at 40 C to provide 415 g of ethanol extract. The extract was suspended in water and partitioned with petroleum ether (PE), EtOAc, and n-BuOH, followed by concentration to yield 55 g of the petroleum ether extract, 80 g of EtOAc extract, 95 g of the n-BuOH extract, and 185 g of the water extract. The EtOAc extract (80 g) was subjected to silica gel column chromatography (200–300 mesh, 400 g) with a gradient of PE–EtOAc (30:1, 15:1, 8:1, 4:1, 2:1, 1:1) as eluent, and five fractions (Fr.1.1–Fr.1.5) were collected according to TLC analysis. Fraction 1.2 (1.2 g) was further separated on a silica gel column (200–300 mesh, 3 g) eluting again with PE–EtOAc (15:1) to provide compound 3 (8 mg). Fraction 1.3 (3.1 g) on the silica gel column eluting with PE–EtOAc (8:1) gave compounds 1 (6 mg) and 2 (5 mg). The n-BuOH extract (95 g) was applied to an HP20 column (1000 mL) with a gradient of H2O–EtOH (5:0, 4:1, 3:2, 2:3, 1:4, 0:5) as eluent, and six fractions (Fr.2.1–Fr.2.6) were collected according to TLC analysis. Fraction 2.3 (3.0 g) was chromatographed through a column of reversed-phase C18 silica gel eluting with MeOH–H2O (2:1) to afford compounds 4 (3 mg), 5 (2 mg), and 6 (3 mg). The separation of fraction 2.5 (8.7 g) was done on a silica gel column (200–300 mesh, 87 g) with a gradient of CHCl3–MeOH (8:1, 4:1, 2:1, 1:1) as the eluent, and three fractions were collected according to TLC analysis. Then the CHCl3–MeOH elution fraction (4:1, 100 mg) was further separated on a silica gel column (200–300 mesh, 3 g) eluting again with CHCl3–MeOH (8:1) to give compound 7 (20 mg). The CHCl3–MeOH fraction (2:1, 200 mg) was separated on a silica gel column (200–300 mesh, 1.5 g) eluting with CHCl3–MeOH (4:1) to afford compound 8 (7 mg). Further separation of the CHCl3–MeOH fraction (4:1, 100 mg) on a reversed-phase C18 silica gel column eluting with MeOH–H2O (1:2) gave compounds 9 (10 mg) and 10 (14 mg).

Chemical constituents of Penicillium chrysogenum, an endophytic fungus from Strychnos toxifera

Strychnos toxifera is a poisonous plant used by Amazon indians in the manufacture of curare poison [1]. Its toxicity is clue to indole alkaloids, which are the major constituents of this poison [2]. Endophytes are considered outstanding and underexplored sources of novel chemically diverse and bioactive compounds. These microorganisms can be detected at a particular moment within the tissues of apparently healthy plant hosts, and they have been found in all plant species examined to date [3]. As they occupy unique biological niches, the complex web of interactions with other endophytes and with the host might give rise to new chemical diversity and bioactive compounds [4]. In the course of screening for biologically active agents from Amazonian endophytic fungi [5], Penicillium chrysogenum, an endophytic fungus from the trunk of S. toxifera, was chosen for further phytochemical investigation. The MeOH extract of the mycelial mass from P. chrysogenum was subjected to chromatographic separation to afford four steroids [6] [ergosterol (1), brassicasterol (2), ergosterol peroxide (3), and cerevisterol (4)], two aromatic acids [7] [cinnamic acid (5) and p-hydroxybenzoic acid (6)], and three mycotoxins [8] [kojic acid (7), penicillic acid (8), and patulin (9)]. These compounds were obtained and characterized by a comparison of their physical and spectral data (UV, IR, NMR and MS), with values in the literature. S. toxifera was collected from Manaus, Brazil in July, 2008. The strain of P. chrysogenum was obtained, purified, and identified from the healthy tissues of the trunk using a previous methodology [9]. After cultivation in liquid media [5], the mycelial mass (1.1 kg) was extracted with MeOH (3 500 mL) at room temperature, and a MeOH extract (30.4 g) was obtained upon concentration under reduced pressure. The MeOH extract was chromatographed over silica gel (600 g, 70–230 mesh) using n-hexane containing increasing amounts of EtOAc to obtain nine fractions. Fraction 1 (321.7 mg) was purified on a silica gel column with n-hexane–CHCl3 with increasing gradient, yielding 1 (22 mg), 2 (7 mg), 3 (12 mg), and 4 (9 mg). Fraction 5 (507 mg) was subjected to silica gel chromatography with CHCl3–MeOH with increasing gradient, yielding 5 (4 mg), 6 (11 mg), 7 (89 mg), 8 (9 mg), and 9 (5 mg).

Chemical Constituents of Gentiana algida

There are about 240 species of the Gentiana genus grown in China, Gentiana algida Pall. is broadly distributed in the alpine zone of the world. In China, it is a little-investigated medicinal plant, that grows mainly in high cold areas such as Tibet, Sichuan, Xinjiang, Qinghai, and Jilin Provinces [1]. To the best of our knowledge, no work has been reported on the chemical constituents of Gentiana algida. As part of our continuing research on Gentiana algida Pall. [2], we have isolated six compounds from G. algida for the first time. The air-dried plants of G. algida were bought from Kumbum Monastery Tibetan Hospital in Qinghai Province of China in October of 2009 and was identified by Prof. Guo-Liang Zhang, Lanzhou University. The powdered G. algida (4.5 kg) was extracted by ethanol (30 L 4, each extraction lasting 7 days) at room temperature. The EtOH extract was concentrated under reduced pressure to give a residue (798 g), which was sequentially partitioned with petroleum ether (90–100 C) (1.0 L 4), EtOAc (1.0 L 4), and n-BuOH (1.0 L 4). The n-BuOH fraction (238 g) was chromatographed on a macroporous resin column (6.5 150 cm) and eluted with H2O–EtOH (90:10, 75:25, 50:50, 25:75, 5:95, 0:100, v/v) gradientwise and acetone to yield seven fractions (Fr. 1–Fr. 7) according to TLC analysis. Fraction 2 (3 g) was re-treated on a polyamide (60–100 mesh, 100 g) column and eluted with H2O–MeOH (5:1, v/v) to obtain 1 (25 mg), then futher eluted with H2O–MeOH (1:1, v/v) to obtain 2 (10 mg). Fraction 4 (0.5 g) was re-treated on a LH-20 (20–150 m, 30 g) column and eluted with H2O–MeOH (1:1, v/v) to obtain 3 (15 mg). Fraction 5 (1.5 g) was chromatographed on a silica gel (200–300 mesh, 50 g) column and eluted with CHCl3–MeOH (10:1, v/v) to obtain 4 (15 mg). Fraction 7 (1 g) was re-treated on a silica gel (200–300 mesh, 30 g) column and eluted with PE–EtOAc (10:1, v/v) to obtain coarse crystals of compounds 5 (30 mg) and 6 (20 mg), then recrystalled with petroleum ether to obtain 5 (15 mg) and 6 (10 mg). Isoscoparin (1), yellow powder, C22H22O11. ESI-MS m/z 461 [M – 1] +. 13C NMR (100 MHz, DMSO-d6, TMS, , ppm): 163.8 (C-2), 103.2 (C-3), 182.0 (C-4), 160.7 (C-5), 108.9 (C-6), 163.4 (C-7), 93.8 (C-8), 156.3 (C-9), 103.3 (C-10), 121.4 (C-1 ), 120.4 (C-2 ), 148.1 (C-3 ), 150.8 (C-4 ), 115.8 (C-5 ), 110.2 (C-6 ), 73.1 (C-1 ), 70.3 (C-2 ), 79.0 (C-3 ), 70.6 (C-4 ), 81.6 (C-5 ), 61.5 (C-6 ), 56.0 (OCH3-3 ) [3]. Gentiopicroside (2), white powder, C16H20O9. ESI-MS m/z 379 [M + Na] +. 1H NMR (400 MHz, DMSO-d6, TMS, , ppm, J/Hz): 7.38 (1H, s, H-3), 5.62 (1H, m, H-6), 5.60 (1H, d, J = 3.0, H-1), 5.19 (1H, m, H-10), 5.15 (1H, m, H-10), 5.04 (1H, m, H-7), 4.96 (1H, m, H-7), 4.48 (1H, d, J = 8.0, H-1 ), 3.14 (1H, t, J = 8.8, H-9). 13C NMR (100 MHz, DMSO-d6, TMS, , ppm): 96.7 (C-1), 149.1 (C-3), 103.5 (C-4), 125.1 (C-5), 116.4 (C-6), 70.1 (C-7), 134.1 (C-8), 44.5 (C-9), 116.2 (C-10), 163.1 (C-11), 98.9 (C-1 ), 73.0 (C-2 ), 77.4 (C-3 ), 69.4 (C-4 ), 76.7 (C-5 ), 61.3 (C-6 ) [4]. 3,6,4 -Trimethoxy-5,7-dihydroxyflavone (3), yellow powder, C18H16O7 [5]. -Sitosterol (4), white needles identical to an authentic sample. 10 -Hydroxy-7(11)-eremophilen-12,8 -olide (5), white needles, C15H22O3. EI-MS m/z 250 [M] +, 232, 126, 125, 109. 1H NMR (400 MHz, CDCl3, TMS, , ppm, J/Hz): 2.6 (1H, d, J = 14.0, H-6 ), 2.43 (1H, d, J = 14.0, H-6 ), 5.01 (1H, m, H-8), 2.15 (1H, dd, J = 7.2, 12.8, H-9 ), 1.96 (1H, dd, J = 11.2, 12.8, H-9 ), 1.81 (3H, s Me-13), 1.06 (s, Me-14), 0.84 (3H, d, J = 6.4, Me-15). 13C NMR (100 MHz, CDCl3, TMS, , ppm): 36.3 (C-1), 22.3 (C-2), 29.7 (C-3), 33.5 (C-4), 44.9 (C-5), 31.7 (C-6), 161.4 (C-7), 78.7 (C-8), 40.9 (C-9), 74.9 (C-10), 120.5 (C-11), 175.2 (C-12), 8.2 (C-13), 14.7 (C-14), 16.0 (C-15) [6].

A new ergostane from Monascus purpureus

A new ergostane, named 3,16,20,22,23,25-hexahydroxyergostane, was isolated by means of chromatographic techniques from Monascus purpureus Went., which is the main component of xuezhikang capsule, a Chinese traditional patent medicine. In China, it is used to treat hyperlipidemia, atherosclerosis, cancer, and osteoporosis [1]. Because this leavening agents showed good bioactivities, a remarkable anti-hyperlipidemia drug, lovastatin, was obtained from it [2]. Previous phytochemical studies of M. purpureus led to the isolation of polyketides, monacolins, terpenes, etc. [3–6]. At current, its analog is still widely studied in the world. In our study of the chemical constituents of this leavening, its chloroform extract was investigated. A new ergostane, 3,16,20,22,23,25-hexahydroxyergostane (1), was isolated, and its structure was elucidated on the basis of spectroscopic methods. The powders of Monascus purpureus Went. (2.0 kg) were exhaustively extracted ultrasonically with petroleum ether, chloroform, and methanol at room temperature. The chloroform-soluble fraction (250 g) was chromatographed on a column of silica gel eluting with CHCl3–MeOH (gradientwise) to give four fractions (Fr.1–Fr.4). Fraction 3 (CHCl3–MeOH 90:10) was rechromatographed on silica gel eluting with CHCl3–MeOH (gradientwise) to afford a mixture containing compound 1, then the pure compound 1 (35 mg) was obtained by preparative HPLC. 3,16,20,22,23,25-Hexahydroxyergostane (1) was obtained as a colorless crystals. The molecular formula of 1 was determined to be C28H46O6 on the basis of the negative HR-ESI-MS (m/z 479.3396 [M + H] +, calcd 479.3373 for C28H47O6). The IR spectrum showed the presence of hydroxy (3531cm–1) and double-bond (1636 cm–1) groups. The 1H NMR spectrum (Table 1) of 1 in CDCl3 showed six methyls at 1.12 (3H, s, Me-18), 0.96 (3H, s, Me-19), 1.43 (3H, s, Me-21) 1.20 (3H, s, MeO-26), 1.23 (3H, s, MeO-27), and 0.85 (3H, d, J = 7.0 Hz, MeO-28), four O-bearing methines at 3.63 (1H, m, H-3), 4.66 (1H, m, H-16), 4.19 (1H, m, H-22), and 3.66 (1H, m, H-23), and two olefinic H-atom signals at 5.56 (1H, d, J = 5.5, H-6) and 5.39 (1H, d, J = 5.5, H-7). In addition, overlapped hydrogen signals at high field were observed. The above data suggest that compound 1 is similar to ergosterol [7, 8], the differences between them being the side chain and the D-ring, in which one of H-C(16) was replaced by a hydroxyl group. This is also confirmed by the 13C NMR spectrum (Table 1), in which the chemical shift of the carbon signal at position 16 takes place at 23.4 of ergosterol to 83.6 of compound 1. The 13C NMR spectrum showed 28 carbon signals, including four C=C bond C-atoms ( 140.4, 119.4, 117.1, and 138.9) and six O-bearing C-atoms (84.3, 83.6, 80.3, 74.1, 72.4, and 70.4), etc. DEPT experiments differentiated them to be 6 CH3, 6 CH2, 10 CH, 6 C. The linkage positions of substituted groups were deduced by HMQC and HMBC experiments. In the HMBC spectrum (Fig. 1), correlations from H 1.91 (H-17) to C 84.3 (C-22), from 1.43 (Me-21) to 66.8 (C-17), and from 1.12 (Me-18) to 66.8 (C-17) indicated that the side chain was attached at the C-17 position. Furthermore, correlations from H 3.66 (H-23) to C 80.3 (C-20), 74.1 (C-25), and 13.9 (C-28) and from 1.75 (H-24) to 30.2 (C-26) and 23.9 (C-27) showed their arrangement. In addition, correlations from H 5.56 (H-6) to C 40.8 (C-4) and 37.2 (C-10) and from 5.39 (H-7) to 45.9 (C-9) and 53.9 (C-14) verify the position of C=C. The correlations from H 4.66 (H-16) to C 42.9 (C-13) and from 1.87 (H-14) to 83.6 (C-16) clarified that the linkage position of the hydroxyl group of D ring was C-16. Combining all the information above, we determined the structure of 1 as 3,16,20,22,23,25-hexahydroxyergostane (1). Subsequently, the cytotoxic activity of compound 1 was evaluated against HCT-8, A549, Bel-7402, BGC823, and A2780 using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The results showed that the compound was inactive (IC50 > 10 M).

Chemical constituents from the stems of Machilus philippinensis

Machilus philippinensis Merr. (Lauraceae) is a medium-sized evergreen tree distributed at an altitude of 500 to 1600 m in Taiwan and the Philippines [1]. In continuation of some studies of chemotaxonomy and biologically active metabolites from Formosan Lauraceous plants [2–10], a methanol extraction of the stems of M. philippinensis afforded 10 known compounds, including three lignans: cinnamophilin (1) [11], erythro-austrobailignan (2) [12], (+)-syringaresinol (3) [13]; one amide: cinnaretamine (4) [4]; four benzenoids: p-hydroxybenzoic acid (5) [14], p-hydroxybenzadehyde (6) [14], vanillic acid (7) [14], p-anisaldehyde (8) [15]; and two steroids: -sitosterol (9) [16] and -sitostenone (10) [16]. In addition to cinnamophilin [11], all of these compounds were found for the first time from this plant. The stems of M. philippinensis were collected from Taiwan County in May 2008. Plant material was identified by Dr. Yen-Ray Hsui (Chungpu Research Center, Taiwan Forestry Research Institute). A voucher specimen has been deposited at the Department of Medical Technology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan. The stems (6.14 kg) of M. philippinensis were extracted repeatedly with MeOH at room temperature for 24–48 h. The MeOH extract was dried and evaporated to leave a viscous residue (425.8 g). The residue was placed on a silica gel column and eluted with CH2Cl2 gradually enriched with MeOH to afford 9 fractions. Part of fraction 1 (13.7 g) was subjected to silica gel chromatography by eluting with n-hexane–acetone (40:1) and enriched gradually with acetone to furnish three fractions (1-1–1-3). Fraction 1-1 (4.3 g) was further purified on a silica gel column using n-hexane–acetone mixtures to obtain -sitostenone (10, 12 mg). Fraction 1-2 (4.3 g) was further purified on a silica gel column using n-hexane–acetone mixtures to obtain -sitosterol (9, 10 mg) and p-hydroxybenzaldehyde (6, 5 mg). Fraction 1-3 (3.2 g) was further purified on a silica gel column using n-hexane–acetone mixtures to obtain erythro-austrobailignan (2, 13 mg). Part of fraction 4 (8.8 g) was subjected to silica gel chromatography by eluting with n-hexane–acetone (20:1) and enriched with acetone to furnish two further fractions (4-1–4-2). Fraction 4-1 (6.3 g) was further purified on a silica gel column using n-hexane–acetone mixtures to obtain cinnamophilin (1, 30 mg). Part of fraction 4-2 (2.3 g) was further purified on a silica gel column using n-hexane–acetone mixtures to obtain p-hydroxybenzoic acid (5, 4 mg). Part of fraction 7 (12.4 g) was subjected to silica gel chromatography by eluting with n-hexane–acetone (10:1) and enriched with acetone to furnish two further fractions (7-1–7-2). Fraction 7-1 (6.4 g) was further purified on a silica gel column using n-hexane–acetone mixtures to obtain vanillic acid (7, 18 mg). Part of fraction 8 (24.6 g) was subjected to silica gel chromatography by eluting with CH2Cl2–MeOH (100:1) and enriched with MeOH to furnish three fractions (8-1–8-3). Fraction 8-2 (10.3 g) was further purified on a silica gel column using CHCl3–MeOH mixtures to obtain (+)-syringaresinol (3, 10 mg). Fraction 8-3 (7.3 g), eluted with CHCl3–MeOH (60:1), was further separated using silica gel column chromatography and preparative TLC (CHCl3–MeOH 100:1) and gave cinnaretamine (4, 5 mg). Fraction 9 (6.4 g) eluted with CHCl3–MeOH (50:1) was further separated using silica gel column chromatography and preparative TLC (CHCl3–MeOH 15:1) to give p-anisaldehyde (8, 22 mg).

Chemical Constituents from an Endophyte, Cercosporella sp.

Cercosporella sp., an endophyte, was isolated from Schisandra chinensis Baill., a medicinal plant. Extracts of Cercosporella sp. are nontoxic to human lymphocytic MT-4 cells and exhibited potent anti-HIV activity in an in vitro MTT assay [1]. The selectivity index (SI) value was 5.6. The fungi were cultured in wheat bran culture medium (containing dextrose, monopotassium phosphate, magnesium sulfate) on a rocking bed without light. The cultured fungus was filtered to separate mycelium from fermentation liquor. Mycelia were rinsed to eliminate fermentation liquor before desiccation, and dry mycelia, about 832 g, were extracted twice with 95% ethanol for 1 h by reflux. The solvents were removed under reduced pressure to give a crude extract (120 g). The aqueous suspension of the extract was partitioned successively with light petroleum, EtOAc, and n-BuOH, resulting in light petroleum (56 g), ethyl acetate (13 g), and n-BuOH (12 g) extracts. The ethyl acetate-soluble fraction was concentrated and subjected to 200–300 mesh silica gel column chromatography eluting with a gradient of CHCl3–MeOH (from 0:1 to 1:1 ) to yield thirteen fractions. The subfractions were then purified by successive column chromatography on 300–400 mesh silica gel using light petroleum–EtOAc and CHCl3–MeOH as elution systems, monitored by TLC, which led to the isolation of compounds 1–6. The structures were elucidated by 1H NMR, 13C NMR, HSQC, HMBC, and MS analysis. All these data were in good agreement with the literature data. All compounds were isolated from Cercosporella sp. for the first time. Compounds 1 and 4 exhibited potent anti-HIV activity, and the SI values were respectively 3.22 and 2.53 in the in vitro MTT assay. 5,6,7-Trihydroxyflavone (1) [2], 19 mg; yellow needles; C15H10O5, mp 223–226 C. Mass spectrum (EI, 70 eV), m/z: 270 (M+, 100%), 242 (M – CO), 168, 140, 112, 102, 77, 69. 5,7-Dihydroxy-6-methoxyflavone (2) [3], 28 mg; saffron yellow needles; C16H12O5, mp 231–232 C. PMR spectrum (500 MHz, CD3OD, , ppm, J/Hz): 8.02 (2H, dd, J = 9.0, 2.0, H-2 , H-6 ), 7.57 (3H, m, H-3 , H-4 , H-5 ), 6.76 (1H, s, H-8), 6.28 (1H, s, H-3), 3.93 (3H, s, 6-OCH3). 13C NMR spectrum data (125 MHz, CD3OD, , ppm): 184.0 (C-4), 165.4 (C-7), 159.0 (C-9), 158.3 (C-2), 151.3 (C-5), 133.1 (C-4 ), 132.5 (C-6), 130.3 (C-1 ), 129.3 (C-3 , C-5 ), 127.4 (C-2 , C-6 ), 105.9 (C-10), 105.4 (C-3), 100.4 (C-8), 62.0 (6-OCH3). Mass spectrum (EI, 70 eV), m/z: 284 (M+), 269 (M – 15, 100%), 241, 167, 139, 103, 77, 69. Physcion (3) [4], 38 mg; red needles; C16H12O5, mp 203–205 C. PMR spectrum (600 MHz, CD3OD, , ppm, J/Hz): 12.31 (1H, s, OH), 12.11 (1H, s, OH), 7.63 (1H, dd, J = 1.5, 0.5, H-14), 7.37 (1H, d, J = 2.5, H-10), 7.08 (1H, dd, J = 2.5, 1.0, H-9), 6.69 (1H, d, J = 2.5, H-13), 3.94 (3H, s, H-16), 2.45 (3H, s, H-15). 13C NMR spectrum data (150 MHz, CD3OD, , ppm): 190.8 (C-1), 182.0 (C-2), 162.5 (C-3), 166.6 (C-4), 148.4 (C-5), 165.2 (C-6), 133.2 (C-7), 113.7 (C-8), 124.5 (C-9), 110.3 (C-10), 135.3 (C-11), 108.2 (C-12), 106.8 (C-13), 121.3 (C-14), 22.1 (C-15), 56.1 (C-16). Indole-3-carboxylic Acid (4) [5], 10 mg; flavo-green crystal; C9H7O2N, mp 232–234 C. Mass spectrum (EI, 70 eV), m/z: 161 (M+, 100%), 144 (M – OH), 132 (M – CHO), 116 (M – COOH). 4-Hydroxyphenylacetic Acid (5) [5], 5 mg; colorless needles; C8H8O3, mp 148–150 C. 2-(4 -Hydroxyphenyl)ethanol (6) [5], 60 mg; colorless needles; C8H10O2, mp 89–92 C. Mass spectrum (EI, 70 eV), m/z: 138 (M+), 120 (M – H2O), 107 (M – CH2OH, 100%).