Teijo Yrjönen

Formulation and Stability of Cytokine Therapeutics

Cytokines are messenger proteins that regulate the proliferation and differentiation of cells and control immune responses. Interferons, interleukins, and growth factors have applications in cancer, autoimmune, and viral disease treatment. The cytokines are susceptible to chemical and physical instability. This article reviews the structure and stability issues of clinically used cytokines, as well as formulation strategies for improved stability. Some general aspects for identifying most probable stability concerns, selecting excipients, and developing stable cytokine formulations are presented. The vast group of cytokines offers possibilities for new biopharmaceuticals. The formulation approaches of the current cytokine products could facilitate development of new biopharmaceuticals.

Dibenzocyclooctadiene lignans from Schisandra spp. selectively inhibit the growth of the intracellular bacteria Chlamydia pneumoniae and Chlamydia trachomatis

Lignans from Schisandra chinensis berries show various pharmacological activities, of which their antioxidative and cytoprotective properties are among the most studied ones. Here, the first report on antibacterial properties of six dibenzocyclooctadiene lignans found in Schisandra spp. is presented. The activity was shown on two related intracellular Gram-negative bacteria Chlamydia pneumoniae and Chlamydia trachomatis upon their infection in human epithelial cells. All six lignans inhibited C. pneumoniae inclusion formation and infectious progeny production. Schisandrin B inhibited C. pneumoniae inclusion formation even when administered 8 h post infection, indicating a target that occurs relatively late within the infection cycle. Upon infection, lignan-pretreated C. pneumoniae elementary bodies had impaired inclusion formation capacity. The presence and substitution pattern of methylenedioxy, methoxy and hydroxyl groups of the lignans had a profound impact on the antichlamydial activity. In addition our data suggest that the antichlamydial activity is not caused only by the antioxidative properties of the lignans. None of the compounds showed inhibition on seven other bacteria, suggesting a degree of selectivity of the antibacterial effect. Taken together, the data presented support a role of the studied lignans as interesting antichlamydial lead compounds.

Determination of terpenoid indole alkaloids in hairy roots of Rhazya stricta (Apocynaceae) by GC-MS

INTRODUCTION Rhazya stricta Decne. (Apocynaceae) is a medicinal plant rich in terpenoid indole alkaloids (TIAs), some of which possess important pharmacological properties. The study material including transgenic hairy root cultures have been developed and their potential for alkaloid production are being investigated. OBJECTIVE In this study, a comprehensive GC-MS method for qualitative and quantitative analysis of alkaloids from Rhazya hairy roots was developed. METHODS The composition of alkaloids was determined by using GC-MS. In quantification, the ratio between alkaloid and internal standard was based on extracted ion from total ion current (TIC) analyses. RESULTS The developed method was validated. An acceptable precision with RSD ≤ 8% over a linear range of 1 to 100 µg/mL was achieved. The accuracy of the method was within 94-107%. Analysis of hairy root extracts indicated the occurrence of a total of 20 TIAs. Six of them, pleiocarpamine, fluorocarpamine, vincamine, ajmalicine and two yohimbine isomers are reported here for the first time in Rhazya. Trimethylsilyl (TMS) derivatisation of the extracts resulted in the separation of two isomers for yohimbine and also for vallesiachotamine. Clearly improved chromatographic profiles of TMS-derivatives were observed for vincanine and for minor compounds vincamine and rhazine. CONCLUSION The results show that the present GC-MS method is reliable and well applicable for studying the variation of indole alkaloids in Rhazya samples.

Rotation planar extraction and medium-pressure solid-liquid extraction of onion (Allium cepa)

Extraction of onion (Allium cepa L.) with 80:20 (v/v) methanol—water in water by rotation planar extraction (RPE) and medium- pressure solid—liquid extraction (MPSLE) has been studied systematically. Rotation planar extraction was studied by use of an ExtraChrom separation instrument prototype. Only rotation planar extraction performed by use of the ExtraChrom enabled efficient extraction of dried onion. The equilibration time proved to be the most important variable in the extraction. The particle size of the sample had a variable effect on extraction efficiency, mainly because of the carbohydrates predominating in the extracts. It was possible to extract oligofructans with a degree of polymerization of up to 12, and these were the main components of the extract. Fructose, glucose, and saccharose, in total, accounted for 10% of the mass of the extract; their mass ratio in the extract was 3:1:6. After mild hydrolysis of the extract with oxalic acid only fructose and glucose were obtained. Although TLC screening of the extract before acid hydrolysis detected no flavonoids, one phenolic acid was observed. After acid hydrolysis with hydrochloric acid, quercetin could be detected.

Effects of microcrystalline plant sterol suspension and a powdered plant sterol supplement on hypercholesterolemia in genetically obese Zucker rats

Because dietary fat appears to be an effective vehicle for dispensing plant sterols into the diet, a special plant‐sterol‐containing ingredient has recently been developed. This ingredient is a plant sterol suspension in oil in which the sterols are in microcrystalline form. The objective of the present study was to analyse the cholesterol‐lowering effects and safety of two different plant sterol preparations, an orally administered microcrystalline plant sterol suspension (MPS) in rapeseed oil and a powdered plant sterol supplement, in obese Zucker rats. Dietary plant sterol supplements (0.5%, w/w) were given concurrently with a high cholesterol diet (HCD, 1% cholesterol and 18% fat, w/w). No significant changes in serum triglyceride, blood glucose, serum glutamate oxaloacetic transaminase and glutamic pyruvic transaminase values or body and liver weights were observed. The powdered plant sterol supplement lowered the serum cholesterol by 25% (P< 0.05) and the MPS diet by 35% (P< 0.001) compared with HCD by the end of the 12‐week experiment. Interestingly, the plant sterol supplements also produced a marked reduction in serum ubiquinone levels, suggesting a possible effect on isoprene synthesis. Unlike the powdered plant sterol, both MPS and plain rape‐seed oil decreased the serum baseline diene conjugation values, suggesting that they protect against oxidative stress‐induced lipid peroxidation in rats. This lipid peroxidation diminishing effect is probably due to some antioxidative components in rapeseed oil. These findings indicate that an unesterified plant sterol, such as the microcrystalline suspension in oil, effectively prevents cholesterol absorption in obese Zucker rats.

Analysis of Indole Alkaloids from Rhazya stricta Hairy Roots by Ultra-Performance Liquid Chromatography-Mass Spectrometry.

Rhazya stricta Decne. (Apocynaceae) contains a large number of terpenoid indole alkaloids (TIAs). This study focused on the composition of alkaloids obtained from transformed hairy root cultures of R. stricta employing ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). In the UPLC-MS analyses, a total of 20 TIAs were identified from crude extracts. Eburenine and vincanine were the main alkaloids followed by polar glucoalkaloids, strictosidine lactam and strictosidine. Secodine-type alkaloids, tetrahydrosecodinol, tetrahydro- and dihydrosecodine were detected too. The occurrence of tetrahydrosecodinol was confirmed for the first time for R. stricta. Furthermore, two isomers of yohimbine, serpentine and vallesiachotamine were identified. The study shows that a characteristic pattern of biosynthetically related TIAs can be monitored in Rhazya hairy root crude extract by this chromatographic method.

Profiling of Coumarins in Peucedanum palustre (L.) Moench Populations Growing in Finland

The coumarin composition of Peucedanum palustre (L.) Moench populations growing in Finland was investigated. A total of 132 flowering P. palustre specimens from 43 locations in southern and central Finland were collected, divided into root, stem, leaf, and umbel samples, and analyzed by HPLC. HPLC coupled to high‐resolution mass spectrometry was used to aid the identification of coumarins. A total of 13 coumarin‐structured compounds were quantitatively analyzed from the samples. The coumarin profile of root samples was found to differ from the aerial plant parts. The main coumarins in roots were oxypeucedanin and columbianadin. In aerial parts, peulustrin isomers were the most abundant coumarin components. Umbels and leaves also contained a considerable amount of umbelliprenin, which was only found in traces in roots. Based on hierarchical cluster analysis of the coumarin profiles, some populations shared common characteristics. The most distinct property connecting certain populations was their high peulustrin content. Another notable common property between some populations was the high umbelliprenin content in aerial plant parts. Some populations were clustered together due to their low overall coumarin content.

Comparison of Medium Pressure Solid–Liquid Extraction and Rotation Planar Extraction of Ficus Leaves with Reference to Optimum Operating Parameters

Abstract The effect of three operating variables on the extraction efficiency of medium pressure solid–liquid extraction (MPSLE) was studied using a factorial experimental design with three levels. The operating variables tested were medium particle size of the plant material, volume of extractant, and the equilibrium time between consecutive extractions of the same material. The possibility of scaling up the MPSLE extraction process on larger columns was also confirmed. The extraction efficiency did not markedly decrease, and a systematic increase in the extraction yields was observed on scaling up the MPSLE procedure. The results of the MPSLE experiments were compared with those obtained by the rotation planar extraction (RPE) method using a prototype ExtraChrom® separation instrument with particle size of the plant material and volume of extractant as the operating variables. Both methods proved to be suitable for the efficient extraction of the leaves of various Ficus species. The strong points of RPE were the ease of operation of the ExtraChrom® separation instrument and significant time savings achieved, whereas the possibility of scaling up the extraction process made MPSLE a suitable method for even larger scale preparative extractions.

Bioluminescent whole-cell reporter gene assays as screening tools in the identification of antimicrobial natural product extracts.

We describe novel tools, bioluminescent whole-cell reporter gene assays, for facilitating the use of natural products in antimicrobial drug discovery. As proof-of-concept, a plant extract library was screened and follow-up experiments were carried out. Primary results can be obtained in 2-4h with high sensitivity, leading to significant improvements of the process.