Matthias Hamburger

7. Bioactivity in plants: the link between phytochemistry and medicine

Abstract The development of medicinal plant research over the last 30 years is reviewed with reference to the search for new active principles. Difficulties inherent to activity guided isolation and the specific requirements of bioassays are discussed. An overview is given on currently used systems for various bioactivities, with emphasis on simple bioassays for phytochemical laboratories. The progress in medicinal plant research is illustrated by selected examples of plant derived compounds of importance as drugs or pharmacological tools.

Pressurized liquid extraction of medicinal plants

The suitability of pressurized liquid extraction (PLE) in medicinal plant analysis was investigated. PLE extracts from a selection of representative herbs were compared with extracts obtained according to Pharmacopoeia monographs with respect to yield of relevant plant constituents, extraction time and solvent consumption. In all cases a significant economy in time and solvents was realized, while extraction yields of the analytes were equivalent or higher.

Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts

UNLABELLED PHARMACOLOGICAL RELEVANCE: Presentation of the scratch assay as a convenient and inexpensive in vitro tool to gain first insights in the wound healing potential of plant extracts and natural compounds. AIM OF THE STUDY The present study deals with the optimization of the scratch assay which can be used as an in vitro model for quantification of fibroblast migration to and proliferation into the wounded area. It is suitable for the first evaluation of the wound re-epithelialization potential of crude herbal extracts, isolated compounds and pharmaceutical preparations. As a proof of concept three preparations from traditional medicinal plants were investigated. MATERIALS AND METHODS Swiss 3T3 albino mouse fibroblasts were used in monolayers and platelet derived growth factor as positive control. Hexane and ethanolic extracts from Calendula officinalis and Matricaria recutita, Hypericum oil as well as the triterpenoids faradiol myristate and palmitate were studied. To differentiate between proliferation and migration antimitotic mitomycin C was added. RESULTS Both extracts of Calendula officinalis stimulated proliferation and migration of fibroblasts at low concentrations, e.g. 10 microg/ml enhanced cell numbers by 64.35% and 70.53%, respectively. Inhibition of proliferation showed that this effect is mainly due to stimulation of migration. Faradiol myristate and palmitate gave comparable stimulation rates at an almost 50 microg/ml concentration, indicating that they contribute partially, but not most significantly to the wound healing effects of Calendula preparations. Extracts from Matricaria recutita were only moderately active. Hypericum oil was cytotoxic at concentrations higher than 0.5 microg/ml. CONCLUSIONS The scratch assay in the present form can be used as a promising scientific approach and platform to differentiate between plant extracts known for their wound healing and their anti-inflammatory properties.

Medicinal Herbs for the Treatment of Rheumatic Disorders : a Survey of European Herbals from the 16th and 17th Century

ETHNOPHARMACOLOGICAL RELEVANCE From the 16th up into the 18th century botanical and medicinal knowledge in Europe was documented and spread in magnificently illustrated herbals. For the most part modern science has neglected this source of knowledge and old remedies have not been systematically evaluated pharmacologically. AIM OF THE STUDY Rheumatic disorders and chronic inflammatory and degenerative diseases of the musculoskeletal system were chosen in an attempt to discuss remedies described in the old herbals in the viewpoint of modern science. MATERIALS AND METHODS Five of the most important European herbals of the 16th and 17th century were searched for terms related to rheumatic diseases, and plants and recipes described for their treatment. An extensive search of the scientific data banks Medline and SciFinder scholar was done to find recent results concerning the phytochemistry and possible antiphlogistic activities of the plants. RESULTS Sixty-three plants were identified in the herbals for this indication. More than half of them have shown in vitro or in vivo antiphlogistic activities. CONCLUSIONS European herbals may be a valuable source of information for the selection of plants for focussed screening programmes. Information contained in these herbals should be explored in a systematic manner.

Antimicrobial flavonoids from Psiadia trinervia and their methylated and acetylated derivatives

Abstract From a dichloromethane extract and a hydrolysed methanolic extract from the leaves of Psiadia trinervia, 13 3-methylated flavonols have been isolated. Their structures were established by the usual spectroscopic methods (UV, EIMS, 1H and 13C NMR). Ayanin, casticin, chrysosplenol-D and 5,7,4′-trihydroxy-3,8-dimethoxyfiavone were responsible for the antifungal activity found in the preliminary screening. Chrysosplenol-D, isokaempferide, 5,7,4′- trihydroxy-3,3′-dimethoxyflavone and 5,7,4′-trihydroxy-3,8-dimethoxyflavone displayed antibacterial activity. Twenty-nine derivatives were prepared by permethylation and selective methylation of the free hydroxyl group at C-5. The antimicrobial activities of the isolates and derivatives were determined by bioautographic assays using C. cucumerinum and B. cereus as test organisms.

Preparative purification of the major anti-inflammatory triterpenoid esters from Marigold (Calendula officinalis)

A method for the efficient preparative purification of faradiol 3-O-laurate, palmitate and myristate, the major anti-inflammatory triterpenoid esters in the flower heads of the medicinal plant Calendula officinalis has been developed. Gram quantities of the individual compounds were obtained with 96 to 98% purity by a combination of supercritical fluid extraction (SFE), normal-phase and reversed-phase column chromatography. During the work-up of the faradiol esters, accompanying minor compounds of the triterpene ester fraction were purified and identified by spectroscopic means as maniladiol 3-O-laurate and myristate.

Concepts and technologies for tracking bioactive compounds in natural product extracts: generation of libraries, and hyphenation of analytical processes with bioassays

Since the advent of high-throughput screening (HTS) in the early 1990s, a wealth of innovative technologies have been proposed and implemented for the effective localization and characterization of bioactive constituents in complex matrices. The latest developments in this field are reviewed under the perspective of their applicability to natural product-based drug discovery. The approaches discussed here include TLC-based bioautography, HPLC-based assays with on-line, at-line and off-line detection, as well as affinity-based methods, such as frontal affinity chromatography, pulsed ultrafiltration mass spectrometry, imprinted polymers, and affinity capillary electrophoresis. Selected practical examples are given to illustrate the strengths and limitations of these approaches in contemporary natural product lead discovery. In addition, compatibility issues of natural product extracts and HTS are addressed, and selected protocols for the generation of high quality libraries are presented.

Isatis tinctoria - From the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical

An account on the reinvestigation of the old dye and medicinal plant Isatis tinctoria as an anti-inflammatory and current research on the active principles in woad is given. In a broad-based screening, a dichloromethane extract from the leaves displayed significant activities on several clinically relevant targets of inflammation. The cyclooxygenase-2 inhibitory principle was identified with the aid of HPLC-based activity profiling as the alkaloid tryptanthrin. In cell based assays, tryptanthrin strongly inhibited eicosanoid synthesis catalyzed by cyclooxygenase-2 and 5-lipoxygenase. A supercritical carbon dioxide extraction process was developed to replace the dichloromethane extract. Dichloromethane and carbon dioxide extracts showed in vivo anti-inflammatory activity in topical and oral application. With the aid of electrospray ionization liquid chromatography-mass spectrometry coupled skin microdialysis, tryptanthrin was found to penetrate the skin. The penetration from the extract was better than for the pure alkaloid. A screening of 67 woad samples of different geographic origin revealed up to 30-fold differences in tryptanthrin content in leaves.

Antiplasmodial lanostanes from the Ganoderma lucidum mushroom

In a screen of 880 extracts from plants and fungi for antiplasmodial, antitrypanosomal, and leishmanicidal activity, an ethyl acetate extract of the mushroom Ganoderma lucidum showed antiplasmodial activity with 79% inhibition at 4.9 microg/mL. HPLC-based activity profiling and subsequent isolation of the antiplasmodial compounds yielded seven lanostanes (1-7), of which three (2, 3, and 7) were new. A new benzofuran derivative (8) of the farnesyl hydroquinone ganomycin B was also identified. The structures and relative configurations of the new compounds were elucidated by comprehensive spectroscopic analysis and by comparison of their NMR data with those of related compounds. The lanostanes exhibited in vitro antiplasmodial activity with IC(50) values from 6 to greater than 20 microM.

Drug discovery and development with plant-derived compounds

An overview is given on current efforts in drug development based on plant-derived natural products. Emphasis is on projects which have advanced to clinical development. Therapeutic areas covered include cancer, viral infections including HIV, malaria, inflammatory diseases, nociception and vaccine adjuvants, metabolic disorders, and neurodegenerative diseases. Aspects which are specific to plant-based drug discovery and development are also addressed, such as supply issues in the commercial development, and the Convention on Biological Diversity.