Induction of vascularisation by an aqueous extract of the flowers of Calendula officinalis L. the European marigold.

Calendula officinalis L. (calendula) is a plant whose recorded history is indicative of intrinsic wound healing capabilities. The wound healing process involves several distinct phases in which the formation of new blood vessels plays an essential role. This report describes the angiogenic activity of a freeze-dried aqueous extract of the flowers of Calendula officinalis L. (the European marigold) utilizing the chick chorioallantoic membrane (CAM) assay. The CAM assay is a standard and well established method for assessing the angiogenic activity in impure and pure preparations and is suitable for studies requiring examination of large numbers of sample test materials. The angiogenic activity of calendula was measured by examination of CAMs using stereomicroscopy. Further histological investigation and quantification of neovascularization was performed utilizing microvascular counts. The histological sections of CAMs were also examined for the presence of hyaluronan (HA), a tissue glycosaminoglycan associated with neovascularization, by hyaluronidase digestion and staining of tissue sections by alcian blue. All calendula treated CAMs were positive for HA; no HA could be demonstrated in control CAMs. The numbers of microvessels in calendula-treated CAMs were statistically significantly higher than in the control CAMs (p < 0.0001). Thin layer chromatography indicated that the calendula extract contained water-soluble compounds such as flavonoids, but the exact nature of the active angiogenic component(s) has not yet been identified.

Separation and purification of oligonucleotides using a new bonded-phase packing material.

We describe a new bonded-phase packing material, based upon surface-stabilised microparticulate silica, suitable for the rapid separation and purification of oligonucleotides. Columns packed with this material were demonstrated to give rapid separations of individual oligonucleotide species of up to 44 base units with high purity; agarose gel electrophoresis showed that the products were essentially single bands, with only trace quantities of the (n-1)-mer present. Baseline resolution of the desired oligomer from (n +/- 1)-mer was achieved under preparative loading conditions, where up to 200-300 micrograms of oligonucleotide could be separated. The separation was essentially independent of structure or sequence of the oligonucleotides. The retention mechanism of the oligonucleotides was investigated, and the results used to determine the optimum column configuration and separation conditions.

The use of FT-IR for the determination of stratum corneum hydration in vitro and in vivo.

Measurement of the water content of stratum corneum plays an important role in physiological and therapeutic inquiries in dermatology. There are many techniques available for non-invasive determination of skin hydration such as measurement of electrical, mechanical, thermal and spectroscopic properties of the skin. Most techniques, however, suffer from the fact that they do not employ a direct measurement of water content rather a property caused by skin hydration. Recently, Potts et al., (Arch. Derm. Res. 277, 489-495, 1985) developed an FT-IR method for the determination of water content of the skin both in vitro and in vivo. The method employed attenuated total reflectance infrared (ATR-IR) to measure a weak O-H stretch formed by the presence of water at 2100 cm-1. This absorbance is distant from interferences due to skin and most topically applied substances and therefore may be used in the quantitation of skin water content (hydration). This report describes the use of this technique in an investigation into the effect of occlusion on the water content of the skin. Method development and validation employing an in vitro system is also discussed.

Application of a modified colorimetric enzyme assay to monitor plasma paracetamol levels following single oral doses to non-patient volunteers.

A modified enzyme-based colorimetric method has been used to determine plasma paracetamol profiles following single dose (2 x 500 mg) administration of three dosage forms to non-patient volunteers. The assay is linear over the concentration range 0.15-60 micrograms ml-1 with a coefficient of variation of 9.1% at the 1.5 micrograms ml-1 level. It is rapid, requiring small sample volumes; compares favourably with other techniques such as HPLC; and is not subject to interference from paracetamol metabolites and other drugs. Administration of paracetamol as two different dosage forms, as a solid tablet and as a dispersible tablet, resulted in no statistically significant difference in pharmacokinetic parameters between treatments.

Purification of homo- and hetero-oligonucleotides using high-performance charge-transfer chromatography.

Oligonucleotides employed in molecular biology have previously been purified by gel electrophoresis, gravity flow chromatography and more recently, high-performance liquid chromatography. However, these techniques have a number of problems and for this reason we investigated high-performance charge-transfer chromatography using the dye acriflavin coupled to silica as the stationary phase. Numerous oligonucleotides were purified using this technique and in this report we present data on four such oligonucleotides two homo-oligonucleotides and two hetero-oligonucleotides. Homogeneity of oligonucleotides eluted from the acriflavin matrices was determined by electrophoresis on 20% polyacrylamide gels and in each case they were greater than 90% pure.

A new rapid procedure for the preparation of plasmid DNA.

This report describes a simple and efficient procedure for the isolation of plasmid DNA free from chromosomal DNA, cellular RNA, and protein. The technique comprises a modified cleared lysate procedure of D.B. Clewell and D.R. Helinski (1969, Proc. Natl. Acad. Sci. USA, 62, 1159-1166) followed by high-performance liquid chromatography on a Dupont Bioseries GF250 surface stable diol-coated silica gel permeation column (Zorbax) for the final purification of the plasmid DNA. The use of HPLC facilitates rapid and high-resolution separations within 3-4 h. Plasmid DNA produced in this manner retains its biological activity and exhibits yields equal to those obtained by the conventional cesium chloride-ethidium bromide density centrifugation method.