Stepharine production in morphogenic cell cultures of <Emphasis Type="Italic">Stephania glabra</Emphasis> (ROXB.) Miers

Young leaves of Stephania glabra (ROXB.) Miers. (Menispermaceae) were used to establish callus cultures with the goal of studying their ability to produce the valuable alkaloid stepharine. The establishment of callus cultures from this plant is extremely difficult because primary calli are not viable and must be transferred to a liquid culture medium for subsequent subculturing. All obtained cultures possessed high morphogenic or rhizogenic potential. The methanolic extracts of two examined cell lines were analyzed by HPLC with high-resolution mass spectrometry to determine the alkaloid composition. Eleven alkaloids were detected in both cultures, including stepharine, magnoflorine, menisperine, roemerine, palmatine, corydalmine, N-methylcorydalmine, columbamine, tetrahydropalmatine, jatrorrhizine and tetrandrine. The predominant alkaloid was stepharine, the structure of which was identified through ¹H and ¹³C NMR, UV, HRMS and tandem MS. Quantitative HPLC-UV analysis showed that stepharine was produced at high levels (0.9?% dry cell weight) in the morphogenic callus culture S2-L cultivated in liquid media. To our knowledge, this is the highest level of stepharine production identified in plant cell cultures. The overall production parameters of the culture have remained stable over a long-term cultivation period for 3 years.     

Effects of water stress on berberine, jatrorrhizine and palmatine contents in amur corktree seedlings

Amur corktree (Phellodendron amurense) is one of the important associated species in broadleaved-Korean pine forests, and in the pharmaceutical resource plants of famous Chinese traditional medicine, named cortex phellodendri. Berberine, jatrorrhizine and palmatine are the main alkaloids to which high attention is paid by the researchers. In the present study, water stress treatments with four conditions, that is, mild drought, severe drought, waterlogging, and control (soil water potentials were controlled in the ranges of-40—-20 KPa,-80—-60 KPa, <-80 KPa and-20—0 KPa), were performed using the technique of root-sphere osmotic irrigation in a soil pond. The changes in the main medicinal compositions of berberine, jatrorrhizine and palmatine contents under different water conditions were discussed. As for the annual growing of amur corktree seedlings, mild drought was generally beneficial to the synthesis and accumulation of the three above-mentioned alkaloid contents. The three alkaloid contents did not show great changes under severe drought whereas those contents had significantly reduced under waterlogging compared with controls. Meanwhile, the growth of amur corktree seedlings was inhibited by the treatments of drought and waterlogging. The height, diameter and biomass of amur corktree seedlings were significantly lower than those of the control seedlings, which meant that the three alkaloid contents in a single seedling still kept the highest in control seedlings. Stem cortex was the medical part in Chinese traditional medicine and also kept the most abundant of the three alkaloid contents in amur corktree. Results indicated that short periods of mild drought could improve the berberine contents in the stem cortex, which might have reference value for the cultivation of amur corktree seedlings to obtain alkaloids.     

Screening the bioactive compounds in aqueous extract of Coptidis rhizoma which specifically bind to rabbit lung tissues β2-adrenoceptor using an affinity chromatographic selection method

A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to β₂-adrenoceptor (β₂-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a β₂-AR affinity column. The retention compounds eluted from the β₂-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the β₂-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized β₂-AR. Their association constants were (2.28 ± 0.11) × 10⁴/M and (3.00 ± 0.10) × 10⁴/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized β₂-AR, and the corresponding association constants were (2.20 ± 0.09) × 10⁻⁴/M and (6.78 ± 0.001) × 10⁵/M.     

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors

Suspension cultures of Berberis wilsonae produce 4 berberine-type alkaloids: berberine, palmatine, columbamine and jatrorrhizine. In particular the formation of the phenolic alkaloids columbamine and jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. With higher aeration rates, berberine and jatrorrhizine yields can be increased considerably. Thus we reached an alkaloid yield of more than 3 g × 1^–1 with 50% dissolved oxygen tension in the medium. As far as we know this is one of the best results in fermenting of alkaloid-producing cell cultures.Abbreviations pO₂ dissolved oxygen concentration in % saturation (using air) - HPLC high-performance liquid chromatography - vvm volume air × volume medium^–1 × minute^–1 - rpm revolutions per minute - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid     

Pyrrolizidine alkaloids in the antipodean genus Brachyglottis (Asteraceae)

The phylogeny of the genus Brachyglottis suggests that its constituent species should contain pyrrolizidine alkaloids. Consistent with this hypothesis, and the established occurrence of such alkaloids in Brachyglottis repanda, Brachyglottis kirkii, and Brachyglottis hectori, an investigation of Brachyglottis adamsii revealed the presence of senecionine and retrorsine; Brachyglottis huntii was found to contain senkirkine and retrorsine; 7-O-angelylheliotridine was the predominant alkaloid in Brachyglottis perdicioides, and the same alkaloid together with senecionine, senkirkine and intergerrimine was present in the Brachyglottis hectori × B. perdicioides “Alfred Atkinson” horticultural hybrid; Brachyglottis sciadophila contained clivorine and neopetasitenine (acetylfukinotoxin); the latter alkaloid was also present in B. kirkii together with the previously reported senkirkine and senkirkine 12-acetate.     

Alkaloid production in cultured cells of Dioscoreophyllum cumminsii

Jatrorrhizine and magnoflorine were isolated from cultured cells of Dioscoreophyllum cumminsii. The jatrorrhizine content in cultured cells was 40–100 times higher than that of the intact plant, but columbamine, which is a minor component in the original plant, was not detected. Moreover, it was observed that the addition of 1AA or NAA to the growth medium increases the alkaloid content as compared with 2,4-D.     

季节和地理差异对天然黄檗小檗碱和药根碱含量的影响

用高效液相色谱法测定了黄檗根皮、茎皮、多年生枝皮、1年生枝和叶片中小檗碱和药根碱的含量,研究黄檗不同部位两种生物碱的季节和地理差异。结果表明:不同季节,天然黄檗各部位的小檗碱、药根碱含量均表现为:根皮茎皮多年生枝皮1年生枝叶片。小檗碱含量随着季节变化在各部位呈现夏季升高,到秋季下降的趋势。而药根碱在不同部位中的含量随着季节变化的规律不同。根皮、茎皮中的药根碱含量随着季节推移逐渐增多,在夏季升高不明显,而到秋季大量积累。多年生枝皮、1年生枝、叶片中的药根碱在3个季节表现则为先降后升。从纬度水平上看,不同地域间黄檗各部位的生物碱差异显著,在调查的40°—50°N范围内,黄檗两种生物碱的含量普遍在纬度低的地区较高,随着纬度增大先逐渐降低,到N44°左右达到最低值,到了高纬度地区略有回升。     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Inhibitory effect of obestatin on glucose-induced insulin secretion in rats

Obestatin is a bioactive peptide encoded by the same gene that encodes ghrelin. Our aim was to investigate the effect of obestatin on insulin secretion. We evaluated the effects of obestatin on insulin secretion from rat islet cells which had been incubated overnight in the presence of 8.3, 11.1, and 22.2 mmol/l of glucose. In vivo, the serum levels of glucose and insulin were measured 0, 1, 5, 10, 20, 40, and 60 min after the intravenous administration of saline or glucose (1 g/kg), with or without obestatin, and the area under the 60 min curve of insulin concentration (AUC_insulin) was calculated. Obestatin (0.01-100 nmol/l) inhibited insulin secretion from rat islets in a dose-dependent fashion. In vivo, when administered intravenously to rats together with glucose, obestatin (10, 50, and 250 nmol/kg) inhibited both the rapid 1-min insulin response and the AUC_insulin in a dose-dependent fashion. Our data demonstrate that under glucose-stimulated conditions, exogenous obestatin acts as a potent inhibitor of insulin secretion in anaesthetized rats in vivo as well as in cultured islets in vitro.     

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n = 30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4 ± 0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2 ± 1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6 ± 15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4 ± 5.3) was higher (P < 0.05) than the MOET method (6.7 ± 3.7). However, pregnancy rates were lower (P < 0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P > 0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.     

Alkaloids of Zanthoxylum monophyllum and Z. punctatum

The known alkaloids N-methylcorydine and magnoflorine were major constituents of stems and branches of Zanthoxylum punctatum (Rutaceae). Berberine was the major alkaloid of Z. monophyllum, which also contained a new pyrano-2-quinolone alkaloid, zanthophylline, and its desmethyl analog. Zanthophylline has an —NCH₂OAc functional group which is readily hydrolyzed by dilute acid. Methyl vanillate and the furocoumarin columbianetin were also found in Z. monophyllum.     

Alkaloids and other constituents of Zanthoxylum williamsii,Z. monophyllum and Z. fagara

Zanthoxylum williamsii (Rutaceae) was found to contain (+)-asaranin, (+)-sesamin, esculetin dimethyl ether, nitidine, chelerythrine, magnoflorine, laurifoline, skimmianine and edulinine. The quaternary alkaloid fraction of Z. monophyllum contained berberine, magnoflorine, chelerythrine and a 1,2,9,10-substituted dihydroxydimethoxy-N,N-dimethylaporphinium salt. Leaves of Z. fagara were found to contain synephrine. Leaves of each species were examined for the presence of bishordeninyl terpene alkaloids, but none was found. Some chemotaxonomic relationships among Zanthoxylum species are discussed.     

Fraxinus excelsior seed extract FraxiPure™ limits weight gains and hyperglycemia in high-fat diet-induced obese mice

Purpose

The aim of this study was to determine whether a Fraxinus excelsior L. seed extract, FraxiPure™ (0.5% in the diet), limits weight gain and hyperglycemia in mice. In a previous report, we identified several secoiridoids in FraxiPure™, some of which activated peroxisome proliferator-activated receptor alpha (PPARα) in vitro and inhibited the differentiation of 3T3-L1 preadipocyte cells. In a separate study, FraxiPure™ reduced glycemia in healthy volunteers, following an oral glucose tolerance test. These findings suggest that FraxiPure™ has antiobesity and antihyperglycemia effects.

Materials and methods

FraxiPure™ was tested in mice that were fed a high-fat diet over 16 weeks and compared with low-fat and high-fat diet controls. Weight gain, omental and retroperitoneal fat, fasting blood glucose, and fasting blood insulin were measured.

Results

FraxiPure™ reduced gains in body weight by 32.30% (p < 0.05), omental fat by 17.92%, and retroperitoneal fat by 17.78%. FraxiPure™ also lowered fasting blood glucose levels by 76.52% (p < 0.001) and plasma insulin levels by 53.43% (p < 0.05) after 16 weeks. Moreover, FraxiPure™ lowered liver weight gains by 63.62% (p < 0.05) and the incidence of fatty livers by 66.67%.

Conclusions

Our novel results demonstrate the antiobesity effects of chronic administration of an F. excelsior seed extract and confirm its ability to regulate glycemia and insulinemia. In addition, this extract, which is rich in secoiridoid glucosides, protects against obesity-related liver steatosis.     

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve β-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve β-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC₅₀ value of 2.5 μM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic β-cell line MIN-6 only under high-glucose (16.8 mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8 mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.     

Hypoglycemic effect of aspalathin,a rooibos tea component from Aspalathus linearis,in type 2 diabetic model db/db mice

Effects of aspalathin, a green rooibos tea component, on glucose metabolism were studied in vitro and in vivo. We first examined the effect of aspalathin on glucose uptake by cultured L6 myotubes and on insulin secretion from cultured RIN-5F pancreatic β-cells in vitro, and then investigated the effect of dietary aspalathin on fasting blood glucose level and conducted an intraperitoneal glucose tolerance test (IPGTT) using type 2 diabetes model mice in vivo. Aspalathin dose-dependently and significantly increased glucose uptake by L6 myotubes at concentrations 1–100 μM. It also significantly increased insulin secretion from cultured RIN-5F cells at 100 μM. Dietary aspalathin (0.1–0.2%) suppressed the increase in fasting blood glucose levels of db/db mice for 5 weeks. In IPGTT, aspalathin improved impaired glucose tolerance at 30, 60, 90, and 120 min in db/db mice. These results suggest that aspalathin has beneficial effects on glucose homeostasis in type 2 diabetes through stimulating glucose uptake in muscle tissues and insulin secretion from pancreatic β-cells.     

Osteogenic effects of flavonoid aglycones from an osteoprotective fraction of Drynaria fortunei—An in vitro efficacy study

Drynaria fortunei (Kunze) J. Sm. is a traditional Chinese herb used for the treatment of osteoporosis and other bone metabolic disorders. Previous studies demonstrated that “small polar active fraction in Drynaria fortunei (SDF)”exerted osteoprotective effects in ovariectomized (OVX) mice. This study aims to investigate the constituents in SDF and systemically evaluate their osteogenic effects in vitro. Five flavonoid aglycones, naringenin, kurarinone, kushennol F, xanthogalenol, and sophoraflavanone G were identified in SDF. All the compounds did not show effects on proliferation of osteoblastic UMR 106 cells at the concentrations of 0.1-1000 nM, but significantly increased the ALP activity of the cells at most of the concentrations from 10 nm to 1000 nM. Xanthogalenol at the concentration of 100 nM significantly increased concentration of acid-solubilized calcium. ICI 182,780, antagonist of estrogen receptor (ER), diminished the effect of kushennol F on ALP activity and the effect of xanthogalenol on acid-solubilized calcium. In conclusion, flavonoid aglycones in SDF could promote differentiation and mineralization of osteoblastic UMR 106 cells in vitro, which was explained by activation of ER signaling pathway. This study provides scientific evidences for the conduction of in vivo experiments to confirm potential effects of flavonoid aglycones on preventing OVX-induced osteoporosis.     

Bishordeninyl terpene alkaloids and other constituents of Zanthoxylum culantrillo and Z. coriaceum

A new alkaloid, culantraramine, has been found in Zanthoxylum culantrillo, and the known alkaloid, alfileramine, has been identified from Z. coriaceum. Both alkaloids are members of a new structural type, bishordeninyl terpenes. Z. culantrillo was also found to contain eudesmin, epieudesmin, hordenine, N-methylisocorydine, magnoflorine, candicine, skimmianine, synephrine, tembetarine and a dihydroxy-dimethoxytetrahydroprotoberberine. Isolated from Z. coriaceum were N-methylisocorydine, dihydrochelerythrine, chelerythrine, N-methylcanadine and aegiline, in addition to alfileramine.     

Efficacy of an essential oil of Eugenia caryophyllata against Psoroptes cuniculi

The acaricidal activity of Eugenia caryophyllata essential oil was evaluated in vitro and in vivo on Psoroptes cuniculi, a mange mite. In vitro, different concentrations of the oil were tested and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R^®) controls. In vivo, six P. cuniculi infected rabbits were topically treated with the oil diluted at 2.5% and compared with untreated and treated control groups of six rabbits each. In vitro, up to the concentration of 0.10% the oil gave highly significant (P < 0.01) percentages of mite mortality respect to the untreated controls, but only up to 0.16% it showed the same efficacy of Acacerulen R^®. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed respect to the treated control group. The untreated rabbits remained infested.     

Elicitor enhanced production of protoberberine alkaloids from in vitro cell suspension cultures of <Emphasis Type="Italic">Tinospora cordifolia</Emphasis> (Willd.) Miers ex Hook. F. &amp; Thoms

The present research investigates the effect of Piriformospora indica, an endophytic fungus, on production of protoberberine alkaloids in in vitro cell suspension cultures of Tinospora cordifolia. Although T. cordifolia produces a number of protoberberine alkaloids, the simultaneous production of jatrorrhizine and palmatine in cell suspension cultures of T. cordifolia was observed for the first time with the use of P. indica as biotic elicitor. The cells in suspension cultures were elicitated with P. indica on 14th day of culture initiation and the production of the alkaloids on 16th day was monitored. The autoclaved as well as filter sterilized cultures of P. indica were used in addition to the use of fungal cell extract. The elicitor effect of P. indica was analyzed and compared with other abiotic elicitor (methyl jasmonate) and biotic elicitors (chitin and chitosan). The culture filtrate of P. indica in the filter sterilized (5.0% v/v) form gave better response with enhanced 4.2-fold production of jatrorrhizine (10.72 mg/g DW) and 4.0-fold production of palmatine (4.39 mg/g DW). The production of these compounds was at par with that achieved in methyl jasmonate (at 250 µM) treated cell suspension cultures.