Chemical Composition and Cytotoxic Activity of the Essential Oil and Oleoresins of In Vitro Micropropagated Ansellia africana Lindl: A Vulnerable Medicinal Orchid of Africa.

Orchids are rich treasure troves of various important phytomolecules. Among the various medicinal orchids, Ansellia africana stands out prominently in the preparing of various herbal medicines due to its high therapeutic importance. The nodal explants of A. africana were sampled from asymbiotically germinated seedlings on basal Murashige and Skoog (MS) medium and were micropropagated in MS medium supplemented with 3% sucrose and 10 µM meta topolin (mT) + 5 µM naphthalene acetic acid (NAA) +15 µM indole butyric acid (IBA) + 30 µM phloroglucinol (PG). In the present study, the essential oil was extracted by hydrodistillation and the oleoresins by the solvent extraction method from the micropropagated A. africana. The essential oil and the oleoresins were analysed by Gas Chromatography (GC) and GC/MS (Mass spectrometry). A total of 84 compounds were identified. The most predominant components among them were linoleic acid (18.42%), l-ascorbyl 2,6-dipalmitate (11.50%), linolenic acid (10.98%) and p-cresol (9.99%) in the essential oil; and eicosane (26.34%), n-butyl acetate (21.13%), heptadecane (16.48%) and 2-pentanone, 4-hydroxy-4-methyl (11.13%) were detected in the acetone extract; heptadecane (9.40%), heneicosane (9.45%), eicosane (6.40%), n-butyl acetate (14.34%) and styrene (22.20%) were identified and quantified in the ethyl acetate extract. The cytotoxic activity of essential oil and oleoresins of micropropagated A. africana was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) assay on Vero cells compared to the standard drug doxorubicin chloride. The present research contains primary information about the therapeutic utility of the essential oil and oleoresins of A. africana with a promising future research potential of qualitative and quantitative improvement through synchronised use of biotechnological techniques.

Wolfberry-derived zeaxanthin dipalmitate delays retinal degeneration in a mouse model of retinitis pigmentosa through modulating STAT3, CCL2 and MAPK pathways.

Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes blindness without effective treatment. The pathogenesis of retinal degeneration involves mainly oxidative stress and inflammatory responses. Zeaxanthin dipalmitate (ZD), a wolfberry-derived carotenoid, has anti-inflammatory and anti-oxidative stress effects. Here we investigated whether these properties of ZD can delay the retinal degeneration in rd10 mice, a model of RP, and explored its underlying mechanism. One shot of ZD or control vehicle was intravitreally injected into rd10 mice on postnatal day 16 (P16). Retinal function and structure of rd10 mice were assessed at P25, when rods degenerate substantially, using a visual behavior test, multi-electrode-array recordings and immunostaining. Retinal pathogenic gene expression and regulation of signaling pathways by ZD were explored using transcriptome sequencing and western blotting. Our results showed that ZD treatment improved the visual behavior of rd10 mice and delayed the degeneration of retinal photoreceptors. It also improved the light responses of photoreceptors, bipolar cells and retinal ganglion cells. The expression of genes that are involved in inflammation, apoptosis and oxidative stress were up-regulated in rd10 mice, and were reduced by ZD. ZD further reduced the activation of two key factors, signal transducer and activator of transcription 3 and chemokine (C-C motif) ligand 2, down-regulated the expression of the inflammatory factor GFAP, and inhibited extracellular signal regulated protein kinases and P38, but not the JNK pathways. In conclusion, ZD delays the degeneration of the rd10 retina both morphologically and functionally. Its anti-inflammatory function is mediated primarily through the signal transducer and activator of transcription 3, chemokine (C-C motif) ligand 2 and MAPK pathways. Thus, ZD may serve as a potential clinical candidate to treat RP.

Toxic metabolite profiling of Inocybe virosa.

Wild mushroom foraging involves a high risk of unintentional consumption of poisonous mushrooms which is a serious health concern. This problem arises due to the close morphological resemblances of toxic mushrooms with edible ones. The genus Inocybe comprises both edible and poisonous species and it is therefore important to differentiate them. Knowledge about their chemical nature will unambiguously determine their edibility and aid in an effective treatment in case of poisonings. In the present study, the presence of volatile toxic metabolites was verified in Inocybe virosa by gas chromatography. Methyl palmitate, phenol, 3,5-bis (1,1-dimethyl ethyl) and phytol were the identified compounds with suspected toxicity. The presence of the toxin muscarine was confirmed by liquid chromatography. The in vitro study showed that there was negligible effect of the digestion process on muscarine content or its toxicity. Therefore, the role of muscarine in the toxicity of Inocybe virosa was studied using a bioassay wherein metameters such as hypersalivation, immobility, excessive defecation, heart rate and micturition were measured. Administration of muscarine resulted in an earlier onset of symptoms and the extract showed a slightly stronger muscarinic effect in comparison to an equivalent dose of muscarine estimated in it. Further, the biological fate of muscarine was studied by pharmacokinetics and gamma scintigraphy in New Zealand white rabbits. Significant amount of the toxin was rapidly and effectively concentrated in the thorax and head region. This study closely explains the early muscarinic response such as miosis and salivation in mice. By the end of 24 h, a relatively major proportion of muscarine administered was accumulated in the liver which stands as an explanation to the hepatotoxicity of Inocybe virosa. This is one of the rare studies that has attempted to understand the toxic potential of muscarine which has previously been explored extensively for its pharmaceutical applications.

Ultrasonic-assisted extraction and high-speed counter-current chromatography purification of zeaxanthin dipalmitate from the fruits of Lycium barbarum L.

Zeaxanthin dipalmitate (ZDP) is a major non-saponified carotenoid in fully ripe fruits of Lycium barbarum L. In the present study, response surface methodology was used to optimize the ultrasonic-assisted extraction (UAE) conditions of carotenoids from the fruits of L. barbarum, and the optimal extraction conditions were determined as follows: ultrasonic power of 360 W, ultrasonic time of 40 min and the ratio of extraction solvent to sample of 30 mL/g. An actual value of ZDP content of 5.40 mg/g and short extraction time indicated the efficiency of UAE. Furthermore, a promising high-speed counter-current chromatography (HSCCC) method was established for the purification of ZDP from the fruits of L. barbarum. With a developed two-phase solvent system composed of n-hexane/dichloromethane/acetonitrile (10/3/7, v/v/v), ZDP with a purity of higher than 95% was successfully isolated from the crude extract. This is the first report on the purification of ZDP by using HSCCC.

Quantitating fatty acids in dried blood spots on a common collection card versus a novel wicking sampling device.

Blood biomarkers of n - 3 polyunsaturated fatty acids can serve as indicators of dietary intake and benefits and/or disease risk. The use of dried blood spots for fatty acid analyses is increasing but most of the reported data is qualitative (relative percentages of total fatty acids). The ability to quantitate concentrations of fatty acids on a common blood spot collection card and a novel wicking device designed to collect 10 µL of blood was compared with a wet blood sample in ten young adult participants. Prior to this comparison, the collection materials were screened for contaminants by gas chromatography with flame ionization and liquid chromatography/tandem mass spectrometry, and the blood volume and blood spot area relationship of the collection card was confirmed using technical replicates. Palmitate and stearate were detected as free fatty acids on both collection materials and as lysophosphatidylcholines on the wicking device. The low amounts (<1.0 µg) did not affect the quantitation of these fatty acids in either material. The relationship between blood volume and blood spot area was linear (r = 0.99, p < 0.001) and it was determined that a 6 mm hole punch contained 9.6 µL of blood. When compared with wet blood, the fatty acid determinations from the dried blood spots were largely similar although there were some minor differences in low abundant fatty acids. Quantitative fatty acid determinations of dried blood spots are possible and should be reported along with relative percentage data to improve interpretation in the future.

Efficient Separation and Analysis of Triacylglycerols: Quantitation of ß-Palmitate (OPO) in Oils and Infant Formulas.

A high-efficiency, convenient, and reliable method for the separation of structurally similar triacylglycerols is detailed and applied in the quantitative analysis of 1,3-dioleoyl-2-palmitoylglycerol (OPO) in infant formulas and OPO oils. OPO is an important lipid component in "humanized" infant formula. A fast preparative isolation of an OPO-containing fraction from the crude complex mixture, by nonaqueous reversed phase HPLC, followed by Ag(+)-HPLC with detection at 205 nm allowed fine separation and detection of the desired fraction. OPO was quantitated independently of its regioisomer 1,2-dioleoyl-3-palmitoylglycerol (OOP) and isomers of stearoyl-linoleoyl-palmitoyl glycerol that might be present in infant formulas. For samples with low OPO content, an evaporative light-scattering detector (ELSD) was more preferable than UV detection, with a calculated LOD of 0.1 µg of OPO injected and LOQ of 0.3 µg. The method, which showed high reproducibility (RSD < 5%), was suitable for both high OPO content oils and low OPO products such as unenriched infant formula. A number of possible interference issues were considered and dealt with.

A new highly oxygenated daphnane diterpene esters from the flower buds of Daphne genkwa.

The phytochemical investigation of the flower buds of Daphne genkwa yielded eight highly oxygenated daphnane-type diterpene esters, including a new one, genkwanine N 20-palmitate (1) and seven known ones (2-8). The molecular structures of the isolated compounds were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, NMR, and MS, and comparison with literature data. Furthermore, compound 1 showed potential cytotoxic activity in vitro, against A549 and Hep G2 tumour cell lines.

Chemical constituents of Hericium erinaceum associated with the inhibitory activity against cellular senescence in human umbilical vascular endothelial cells.

Hericium erinaceum is an edible and medicinal mushroom widely used in Korea, Japan, and China. On the search for biologically active compounds supporting the medicinal usage, the MeOH extract of the fruiting bodies of H. erinaceum was investigated for its chemical constituents. Six compounds were isolated and identified as hericenone D (1), (22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3ß-ol (2), erinacerin B (3), hericenone E (4), hericenone F (5) and isohericerin (6) by comparing their spectroscopic data with previously reported values. The inhibitory effects on adriamycin-induced cellular senescence in human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) of the isolates (1-6) were studied. Among the isolated compounds, ergosterol peroxide (2) reduced senescence associated ß-galactosidase (SA-ß-gal) activity increased in HUVECs treated with adriamycin. According to experimental data obtained, the active compound may inspire the development of a new pharmacologically useful substance to be used in the treatment and prevention of age-related diseases.

Characterization of electron ionization mass spectral (EIMS) fragmentation patterns of chloropropanol esters of palmitic acid using isotope labeling technique.

Chloropropanol (CP) esters are a class of thermally-induced toxicants that are mainly formed in refined edible oils. The structural diversity of these esters presents significant analytical challenges which have often been overcome through analysis of their corresponding free alcohols after a hydrolysis step. Mass spectrometry-based methodologies incorporating characteristic fragmentation patterns of particular isomers of CP esters greatly facilitates their identification. The electron ionization mass spectra (EIMS) of various isomers of synthetic and commercially available (13)C- and (2)H-labeled CP ester standards of palmitic (C16) and other short chain fatty acids (C3 to C10) were generated and analyzed using GC/MS. Short chain CP esters were synthesized by reacting their respective acid anhydrides with the corresponding 3-chloro- and 2-chloro- propanediols in addition to 1,3-dichloro- and 1,2-dichloropropanols. Five fragmentation pathways were identified. Four of the five pathways, such as α-cleavage, McLafferty rearrangement, α-H rearrangement and cyclic acyloxonium ion formation, were characteristic of CP mono- and diesters. The remaining pathway generating chloronium ion was found only in dichlorinated isomers. The proposed fragmentation pathways for the palmitic acid esters were confirmed through the use of (13)C- and (2)H-labeled CP ester standards of palmitic acid, and the generality of identified fragmentation patterns was confirmed through the identification of equivalent ions in the mass spectra of short chain fatty acids (C3 to C16). Characteristic ions that were identified in this study retaining the chlorine atom in their structures can be considered as potential markers for the presence of CP esters.

Androgenic effect of honeybee drone milk in castrated rats: roles of methyl palmitate and methyl oleate.

ETHNOPHARMACOLOGICAL RELEVANCE: Numerous honeybee (Apis mellifera) products have been used in traditional medicine to treat infertility and to increase vitality in both men and women. Drone milk (DM) is a relatively little-known honeybee product with a putative sexual hormone effect. The oestrogenic effect of a fraction of DM has recently been reported in rats. However, no information is available on the androgenic effects of DM. The purpose of the present study was to determine the androgen-like effect of DM in male rats and to identify effective compounds. MATERIALS AND METHODS: A modified Hershberger assay was used to investigate the androgenic effect of crude DM, and the plasma level of testosterone was measured. The prostatic mRNA and protein expression of Spot14-like androgen-inducible protein (SLAP) were also examined with real-time PCR and Western blot techniques. GC-MS and NMR spectroscopic investigations were performed to identify the active components gained by bioactivity-guided fractionation. RESULTS: The crude DM increased the relative weights of the androgen-dependent organs and the plasma testosterone level in castrated rats and these actions were flutamide-sensitive. DM increased the tissue mRNA and protein level of SLAP, providing further evidence of its androgen-like character. After bioactivity-guided fractionation, two fatty acid esters, methyl palmitate (MP) and methyl oleate (MO), were identified as active compounds. MP alone showed an androgenic effect, whereas MO increased the weight of androgen-sensitive tissues and the plasma testosterone level only in combination. CONCLUSION: The experimental data of DM and its active compounds (MO and MP) show androgenic activity confirming the traditional usage of DM. DM or MP or/and MO treatments may project a natural mode for the therapy of male infertility.

Cytotoxic steroids from the trunk of Berberis koreana.

A new steroid, itesmol 3-O-palmitate (1), along with two known steroids were isolated from the trunk of Berberis koreana. The structure of 1 was determined on the basis of spectroscopic analyses involving 2D NMR and chemical reactions. Compound 1 exhibited potential antiproliferative activity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines (respective IC(50) values of 7.41, 9.20, 4.53, and 12.91 µM).

Elimination of glycidyl palmitate in diolein by treatment with activated bleaching earth.

In this study, activated bleaching earth (ABE) was used to eliminate glycidyl esters from both triacyl- and diacylglycerol oils. To investigate the mechanism, glycerol dioleate containing glycidyl palmitate (GP) was treated with ABE and the fate of the GP was monitored by analyzing the feed, treated, and ABE-absorbed oils using a gas-liquid chromatograph equipped with a flame-ionized detector. GP was completely removed from both the treated and absorbed oils. This indicates that this treatment is useful for GE removal from diacylglycerol oil, although it was not achieved by absorption of GE on ABE but rather by modification of GP. The results of composition analysis demonstrate that GP is transformed to glycerol monopalmitate, glycerol palmitate oleate, and glycerol dipalmitate at a recovery rate of 99.1 ± 1.3 %. An increase in glycerol monooleate and trace amounts of free glycerol and fatty acids were also observed after treatment. The transformation is proposed to involve a ring-opening reaction of GP with water contained in the ABE and in the bulk oil followed by an interesterification reaction among the resultant monopalmitate and the glycerol dioleate of the bulk oil. All the generated compounds were simple acylglycerols and glycerol. Therefore, ABE treatment could be useful for GE removal during the manufacture of edible oils.

Isolation of a novel carotenoid, OH-chlorobactene glucoside hexadecanoate, and related rare carotenoids from Rhodococcus sp. CIP and their antioxidative activities.

In the course of screening for antioxidative carotenoids from bacteria, we isolated and identified a novel carotenoid, OH-chlorobactene glucoside hexadecanoate (4), and rare carotenoids, OH-chlorobactene glucoside (1), OH-γ-carotene glucoside (2) and OH-4-keto-γ-carotene glucoside hexadecanoate (3) from Rhodococcus sp. CIP. The singlet oxygen ((1)O(2)) quenching model of these carotenoids showed potent antioxidative activities IC(50) 14.6 µM for OH-chlorobactene glucoside hexadecanoate (4), 6.5 µM for OH-chlorobactene glucoside (1), 9.9 µM for OH-γ-carotene glucoside (2) and 7.3 µM for OH-4-keto-γ-carotene glucoside hexadecanoate (3).

Chemical composition and antioxidant activity of the essential oil of endemic Viola tianshanica.

The composition and in vitro antioxidant activities of the essential oil and methanol extract of the aerial parts of Viola tianshanica were evaluated in this research. GC-MS analysis of the essential oil resulted in the identification of 15 constituents, representing 89.67% of the oil. The major compounds detected in the essential oil were dibutyl phthalate (15.19%), hexadecanoate methyl (8.65%), n-hexadecanoic acid (3.07%) and 2,3-pentanedione (2.62%). Essential oil and methanol extract were tested for their antioxidant activities using 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging and ß-carotene linoleic acid assay. In addition, the total phenol of essential oil, polar subfraction and non-polar subfraction were determined.

Triterpenoids from Viburnum betulifolium.

Two new triterpenoids, ursa-12-sene-3ß,11ß-diol 3-O-palmitate (1) and ursa-12-sene-1ß,3ß,11α-triol 3-O-palmitate (2), were isolated from the 70% aqueous acetone extract of the aerial parts of Viburnum betulifolium, together with the artificial diene derivative of 2, ursa-12-dien-1ß,3ß-diol 3-O-palmitate (2a). Their structures were characterized by various spectroscopic methods, including 1D NMR, 2D NMR, and HR-ESI-MS.

Cinnamyl alcohols and methyl esters of fatty acids from Wedelia prostrata callus cultures.

Two methyl esters of fatty acids, namely octadecanoic acid methyl ester (methyl stearate) and hexadecanoic acid methyl ester (methyl palmitate), in addition to four cinnamyl alcohol derivatives, sinapyl alcohol, coniferyl alcohol, p-coumaryl alcohol and coniferyl alcohol 4-O-glucoside (coniferin), were isolated from callus cultures of Wedelia prostrata. The structure of coniferin was established by spectroscopic and chemical methods, while the other compounds were identified by gas chromatography-mass spectrometry and thin layer chromatography in comparison with standards.

Methyl palmitate: a potent vasodilator released in the retina.

PURPOSE: To determine whether palmitic acid methyl ester (PAME) or methyl palmitate is the retina-derived relaxing factor (RRF). METHODS: A superfusion bioassay cascade technique was used with rat isolated retina as donor tissue and rat aortic ring as detector tissue. The superfusate was analyzed with gas chromatography/mass spectrometry (GC/MS). The biochemical and pharmacologic characteristics of RRF and PAME were compared. RESULTS: The authors demonstrated that the retina on superfusion with Krebs solution spontaneously released RRF (indicated by aortic ring relaxation) and PAME (measured by GC/MS). The release of RRF and PAME was calcium dependent because the release was abolished when the retinas were superfused with calcium-free Krebs solution. Furthermore, aortic relaxations induced by RRF and PAME were not affected after heating their solutions at 70 degrees C for 1 hour, suggesting that both are heat stable. Exogenous PAME concentration dependently induced aortic relaxation with EC50 of 0.82+/-0.75 pM. The aortic relaxations induced by RRF and exogenous PAME were inhibited by 4-aminopyridine (2 mM) and tetraethylammonium (TEA, 10 mM) but were not affected by TEA at 1 mM or 3 mM, glibenclamide (3 microM), or iberiotoxin (100 nM). The vasodilator activity of Krebs solution containing RRF or exogenous PAME was greatly attenuated after hexane extraction. CONCLUSIONS: RRF and PAME share similar biochemical properties and react similarly to all pharmacologic inhibitors examined. Both act primarily on the voltage-dependent K+ (Kv) channel of aortic smooth muscle cells, causing aortic relaxation. These results suggest that PAME is the hydrophobic RRF.

Secondary metabolites from the red mould rice of Monascus purpureus BCRC 38113.

One new tetralone, monaspurpurone (1), was isolated from the EtOH extract of a yellow mutant of the fungus Monascus purpureus BCRC 38113 (Eurotiaceae) grown on rice, along with five known compounds, ß-sitosteryl palmitate (2), ergosterol (3), ankaflavin (4), monascin (5) and p-nitrophenol (6). They were characterised on the basis of spectral analysis and comparison with literature data. All the isolates were also evaluated for the scavenging properties towards the DPPH in TLC autographic and spectroscopic assays.

Methyl palmitate, an acaricidal compound occurring in green walnut husks.

Walnut, Juglans regia L., is known for its insecticidal activities to a range of herbivores and microbes. Isolation and identification of bioactive compounds from walnut is a potential approach for the development of new pesticides. Laboratory experiments were carried out to investigate the acaricidal activities of green husk extracts of walnut. Bioassay-guided fractionation of petroleum-ether extracts of walnut led to the identification of a common plant-borne fatty acid ester, methyl palmitate (MP), which produced strong acaricidal activity (62.8% mortality) at 1 mg/ml at 24 h. The structure of MP was characterized with infrared spectrum and NMR, and the identification of MP confirmed using an authentic standard on high-performance liquid chromatography. Based on a slide dip bioassay, 10 mg/ml MP provided 97.9% mortality against adults of Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae), whereas mortality against eggs was much lower (57.2%).

[Studies on chemical constituents from Grewia biloba].

OBJECTIVE: To investigate the chemical constituents of Grewia biloba and find its bioactive compounds. METHODS: Compounds were isolated by silica gel, MCI, Sephadex LH-20 and Rp-18 column chromatography, and purified by recrystallization. Their structures were elucidated by spectral analysis and other methods. RESULTS: Six compounds were isolated and identified as friedelin (1) epi-friedelan-3-ol (2), heneicosanoic acid (3), beta-sitosterol (4), Propyl palmitate (5), eatechin (6), respectively. CONCLUSION: Compounds 1-3, 5, 6 are isolated from Grewia genus for the first time and compounds 4 is isolated from Grewia biloba for the first time.