Herman J. Woerdenbag

TRADITIONAL CHINESE HERBAL MEDICINE

Herbal medicine, acupuncture and moxibustion, and massage are the three major constituent parts of traditional Chinese medicine. Although acupuncture is well known in many Western countries, Chinese herbal medicine, the most important part of traditional Chinese medicine, is less well known in the West. This article gives a brief introduction to the written history, theory, and teaching of Chinese herbal medicine in China. It also describes modern scientific research into and the quality control of Chinese herbal medicines in China. Some examples of how new drugs derived from Chinese herbs have been developed on the basis of traditional therapeutic experience are presented. Finally, the situation of Chinese herbal medicine in the West is discussed.

PRODUCTION OF THE NEW ANTIMALARIAL DRUG ARTEMISININ IN SHOOT CULTURES OF ARTEMISIA-ANNUA L

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l-1 BAP, 0.05 mg l-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l-1 gibberellic acid, 0.5 g l-1 casein hydrolysate, 10 mg l-1 or 20 mg l-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.

PROGRESS IN THE RESEARCH OF ARTEMISININ-RELATED ANTIMALARIALS - AN UPDATE

Artemisinin, a sesquiterpene lactone endoperoxide isolated fromArtemisia annua L., and a number of its semisynthetic derivatives have shown to possess antimalarial properties. They are all eflective againstPlasmodium parasites that are resistant to the newest and commonly used antimalarial drugs. This article gives a survey of the literature dealing with artemisinin-relaled antimalarial issues that have appeared from the end of 1989 up to the beginning of 1994. A broad range of medical and pharmaceutical disciplines is covered, including phytochemical aspects like the selection of high-producing plants, analytical procedures, and plant biotechnology. Furthermore, the organic synthesis of artemisinin derivatives is discussed, as well as their mechanism of action and antimalarial activity, metabolism and pharmacokinetics, clinical studies, sideeffects and toxicology, and biological activities other than antimalarial activity.

Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis

In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis.

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

Abstract Endophytes, microorganisms that colonize internal tissues of all plant species, create a huge biodiversity with yet unknown novel natural products, presumed to push forward the frontiers of drug discovery. Next to the clinically acknowledged antineoplastic agent, paclitaxel, endophyte research has yielded potential drug lead compounds with antibacterial, antiviral, antioxidant, insulin mimetic, anti-neurodegenerative and immunosuppressant properties. Furthermore, while being implicated in livestock neurotoxicosis, some endophyte-produced alkaloids have been shown to display insecticidal activity. The endophyte-host relationship is postulated to be a ‘balanced antagonism’. Moreover, the plausibility of horizontal gene transfer (HGT) hypothesis is taken into account. Knowledge of the genetic background of endophytic natural product biosynthesis is discussed on the basis of loline alkaloids, ergopeptines, lolitrems and maytansinoids. The current dynamic progress in genomics will contribute to a better understanding of endophytic microbes and to further exploiting them as a source of pharmaceutically relevant compounds.

Artemisia annua L.: a source of novel antimalarial drugs

Artemisia annua L. contains artemisinin, an endoperoxide sesquiterpene lactone, mainly in its leaves and inflorescences. This compound and a series of derivatives have attracted attention because of their potential value as antimalarial drugs. In this review a survey of the currently available literature data is given. It includes phytochemical aspects, such as constituents ofA. annua, the artemisinin content during the development of the plant and its biosynthesis, isolation, analysis and stability. Total chemical synthesis of artemisinin is referred to, as well as structure—activity relationships of derivatives and simplified analogues. Pharmacological studies are summarized, including the mechanism of action, interaction of the antimalarial activity with other drugs, possible occurrence of resistance to artemisinin, clinical results, toxicological aspects, metabolism and pharmacokinetics. Finally, plant cell biotechnologyy is mentioned as a possible means to obtain plants and cell cultures with higher artemisinin contents, allowing an industrial production of pharmaceuticals containing this novel drug.

Analytical aspects of phytotherapeutic valerian preparations

A high performance liquid chromatographic method combined with diode array detection is described by which the valerian constituents valtrate, isovaltrate, acevaltrate, didrovaltrate, isovaleroxyhydroxydidrovaltrate, valerenic acid, hydroxyvalerenic acid and acetoxyvalerenic acid, as well as the valepotriate decomposition products baldrinal and homobaldrinal, can be separated and identified simultaneously. Using this procedure, roots of Valeriana officinalis, which are used for the production of phytomedicines, were analysed. The influence of different ethanol:water mixtures, used as extraction liquid, on the composition of extracts of V. officinalis is reported. The analytical procedure was also applied to a number of valerian-containing phytomedicines available on the Dutch market. In order to study the stability of the valepotriates and the formation of their decomposition product(s), samples of freshly prepared valerian tinctures were analysed after being stored at 4, 20, and 36 degrees C for up to one month.

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

SummaryCell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of β-cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with β-cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. β-Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble β-D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with β-cyclodextrin, but coniferin is not commercially available.

The essential oil of Tanacetum parthenium (L.) Schultz-Bip.

The composition of the essential oil of Tanacetum parthenium (L.) Schultz-Bip. (feverfew; Asteraceae) of various origins was investigated using GC and GC-MS. Camphor and chrysanthenyl acetate were the main constituents of the samples originating from England and The Netherlands. No infraspecific variation in the composition of the oil was found. Furthermore, the essential oil content and composition of Dutch feverfew during a vegetative period was studied. The young herb, before the formation of the stems, yielded a relatively high percentage of oil (0.53%, v/w), calculated on the dry weight. After a sharp decline at the beginning of the formation of the stems (0.30%, v/w), the percentage of the oil increased until full bloom (0.83%, v/w). During the development of the plant the percentage of camphor rose from 28% to 48%, whereas the amount of chrysanthenyl acetate decreased from 30% to 22%. In none of the oil samples investigated could the potentially toxic monoterpenes α- or β-thuojone be detected. This is important, because feverfew is used over long periods of time as a migraine prophylactic agent. The sesquiterpene lactone parthenolide is held responsible for this biological activity. In addition, chrysanthenyl acetate may display an analgesic effect by inhibiting the enzyme prostaglandin synthetase. Based on the results of our investigations and literature data, a number of recommendations are proposed with respect to the essential oil of T. parthenium.

Cytotoxic potential of valerian constituents and valerian tinctures.

Underground parts of three Valeriana species, namely V. officinalis L. s.l., V. wallichii DC. (V. jatamansi Jones), and V. edulis Nutt. ex Torr & Gray ssp. procera (H.B.K.) F. G. Meyer (V. mexicana DC.), are used in phytotherapy because of their mild sedative properties. Characteristic constituents of these species, which are regarded also as the active principles, were tested for cytotoxicity against GLC(4), a human small-cell lung cancer cell line, and against COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. Valepotriates of the diene type (valtrate, isovaltrate and acevaltrate) displayed the highest cytotoxicity, with IC50 values of 1-6 μM, following continuous incubation. The monoene type valepotriates (didrovaltrate and isovaleroxyhydroxydidrovaltrate) were 2- to 3-fold less toxic. Baldrinal and homobaldrinal, decomposition products of valepotriates, were 10- to 30-fold less toxic than their parent compounds. Isovaltral had a higher cytotoxicity than its parent compound isovaltrate. Valerenic acids (valerenic acid, acetoxyvalerenic acid, hydroxyvalerenic acid and methyl valerenate), which are characteristic for V. officinalis, had a low toxicity with IC(50) values between 100 and 200 μM. Freshly prepared and stored tinctures, prepared from roots and rhizomes of the three valerian species, were analysed for valepotriates, baldrinals and valerenic acids, and also tested for cytotoxicity. There was a clear relationship between the valepotriate contents of the freshly prepared tinctures and their toxicity. Upon storage, valepotriates decomposed, which was reflected in a significant reduction of the cytotoxic effect.