Mistletoe extracts standardized to mistletoe lectins in oncology: review on current status of preclinical research.

Since the identification and characterization of mistletoe lectins as pharmacologically active constituents at the end of the 1980s, research on mistletoe has made substantial advances. Mistletoe extracts are now available that are standardized in terms of the active mistletoe lectins (measured as mistletoe lectin I, ML I). This constitutes an indispensable precondition for reproducible investigations. Preclinical studies have shown that mistletoe extracts standardized in terms of ML I or pure ML I itself have highly potent cytotoxic and immunostimulating effects, predominantly on the cellular immune system. The immunostimulating effect is correlated with the apoptosis of immunologically active cells at low concentrations. Cytotoxic effects on tumor cells are likewise apoptosis-related, but at higher levels necrotic cell death predominates. Due to these properties, mistletoe extracts or pure ML I showed antitumoral activities in different animal models. The objective of this review is to present the current state of preclinical research on standardized mistletoe extracts which hence may be included in the category of rationalphytotherapy.

Effects of long-term treatment with anthranoids and sodium picosulphate on the contents of vasoactive intestinal polypeptide, somatostatin and substance P in the rat colon.

OBJECTIVE: To examine the effects of chronic treatment and a single high-dose application of anthranoids and sodium picosulphate on the neuropeptide content of the rat colon. DESIGN AND METHODS: Over a 6-month period, eight groups of rats were each given one of the following: sennosides or sodium picosulphate in low daily doses (10 and 2.5 mg/kg/day, respectively), in high daily doses (40 and 10 mg/kg/day, respectively), and in high twice-weekly doses (30 and 7.5 mg/kg/day, respectively); high daily doses of danthron (500 mg/kg/day); and vehicle (tragacanth 0.5%) only. Four further groups of rats each received a single dose of vehicle or a high dose of one of the three laxatives. All rats were killed 48 h after the last dose. The ascending and descending colon were removed and separated into mucosa, submucosa, and muscularis externa. Vasoactive intestinal polypeptide (VIP), somatostatin, and substance P were extracted by boiling and homogenizing the tissue in acetic acid, and their levels were determined using validated radioimmunoassays. RESULTS: After long-term treatment with high doses of sennosides and danthron, but not after a single high-dose administration, there was a significant reduction in mucosal levels of VIP and somatostatin and in submucosal levels of somatostatin of both colonic segments, as well as in the level of VIP in the muscularis externa of the descending colon. Substance P levels remained unaffected. Sodium picosulphate had no effect. CONCLUSIONS: Chronic treatment with anthranoids in high doses, but not with sodium picosulphate, reduces VIP and somatostatin levels in the rat colon. This may represent damage to the enteric nervous tissue or a pharmacological effect of the anthranoids, causing decreased synthesis or increased breakdown of these peptides.

Sennoside-induced secretion is not caused by changes in mucosal permeability or Na+,K(+)-ATPase activity.

The effect of sennosides (50 mg kg-1) on the rat colon in-situ was studied 6 h after oral treatment when the laxative effect was maximal. In a second experiment, rhein (4 x 10(-3) M), an active sennoside metabolite, was administered into the lumen of the colon for 1 h. Both sennosides and rhein reduced net H2O and Na+ absorption or reversed it to net secretion. Paracellular permeability, as measured using erythritol as a small marker molecule, was increased 2- to 3-fold; permeability to a large molecule, PEG 1000, was unchanged. The activity of Na+,K(+)-ATPase in the colon mucosa was not affected. There was no damage of the epithelial cells as determined by lactic acid dehydrogenase release. These results indicate that neither inhibition of Na+,K(+)-ATPase nor damage of the colon epithelium are involved in the secretory effect of sennosides or rhein. The increased paracellular permeability of small molecules fits into the concept of stimulation of active chloride secretion by sennosides, which is electrochemically and osmotically balanced by an increase in Na+ and H2O flow via the paracellular pathway.

Sennoside-induced secretion and its relevance for the laxative effect.

The effect of oral treatment with sennosides (50 mg/kg) on the time-course of net H2O and electrolyte transport rates was studied in 1-hour incubation experiments in the rat colon in vivo. Net H2O, Na+ and Cl- absorption rates did not change during the first 4 h after treatment, but were reversed to net secretion after 6 h and partly recovered during the next 18 h. K+ and Ca2+ were secreted in controls, and net secretion increased from 6 to 24 h after treatment. Paracellular permeability of [14C]erythritol was 3-fold 6 h after treatment but unchanged at other times after treatment (2, 4, 12 or 24 h). LDH leakage into the lumen was not enhanced by treatment. Neither mucosal Na+, K(+)-ATPase activity nor cAMP or phosphodiesterase activity was affected by sennosides. As stool consistency and acceleration of transit by sennosides has entirely normalized 24 h after treatment but not net absorption of H2O and electrolytes, it is concluded that there may be regional differences in the absorptive behavior of the colon induced by sennosides. Slow transit and increased absorption in some parts of the colon may overcome secretion in other parts.

Effects of long-term sennoside treatment on in vitro motility of rat colon.

Colonic motility of rats chronically pretreated with either 10 or 40 mg sennosides/kg body weight daily, 30 mg sennosides/kg twice a week or 500 mg danthron/kg daily for 6 months has been evaluated in ex vivo preparations. Registration of colonic contractions 48 h after the last application of drugs or placebo revealed striking differences between the ascending, transverse and descending parts of the colon as well as between corresponding circularly or longitudinally orientated segments. Treatment did not influence motility in the various parts of the colon except some reduction in spontaneous motility in the descending colon. Response to electrical stimulation and acetylcholine was independent of treatment in most segments, but a decrease in maximal contraction was observed by the high daily sennoside dose and by danthron in the ascending colon and descending colon, respectively. Sensitivity to rhein, an active metabolite of sennosides, was similar in all groups. Thus, chronic treatment with anthranoids reveals no abnormal motility pattern but a residual pharmacological activity 48 h after the last administration of high doses.

Chronic sennoside treatment does not cause habituation and secondary hyperaldosteronism in rats.

Rats were treated with sennosides (6 x 10, 6 x 40 or 2 x 30 mg/kg weekly) or with danthron (6 x 500 mg/kg weekly) for 6 months. The laxative effect as measured by faecal wet weight during the first 10 h after treatment increased 3- to 4-fold by the higher sennoside doses (daily or intermittently) and 1- to 3-fold by danthron. The low sennoside dose had no measurable effect except on the 1st day (2 fold) compared with the control group. Mean faecal water content increased from 53% (controls) to 66-79% in rats treated with high sennoside doses and to 57 (1st day) -69% in danthron-treated rats. Serum aldosterone levels and mucosal Na(+)-K(+)-ATPase activities in the small intestine and colon did not change with treatment. There were no signs of habituation or secondary hyperaldosteronism due to sennosides or danthron in spite of chronic diarrhoea over 6 months.

Plantago ovata seeds as dietary fibre supplement: physiological and metabolic effects in rats.

In rats, the effects of a 4-week supplementation of a fibre-free elemental diet with 100 or 200 g Plantago ovata seeds/kg was compared with that of the husks and wheat bran. The seeds increased faecal fresh weight up to 100%, faecal dry weight up to 50% and faecal water content up to 50%. The husks, at the high concentration only, were more effective and wheat bran less effective. Length and weight of the small intestine were not greatly affected by the seeds, but both variables increased significantly in the large intestine. The husks had more pronounced effects, especially in the small intestine, and wheat bran almost no effect. Faecal bacterial mass as estimated from the 2,6-diaminopimelic acid output was increased to the greatest extent by the seed-containing diet and by the high concentration of husks, but to a lesser extent by wheat bran. Faecal and caecal protein content was enhanced by the seeds and wheat bran, but to a lesser extent by the husks. Total acetate in caecal contents or faeces was highest on the seeds and husks diet and not elevated by wheat bran. Total faecal bile acid excretion was stimulated and beta-glucuronidase (EC 3.2.1.31) activity reduced by both Plantago ovata preparations, but not by wheat bran. Mucosal digestive enzyme activities were inhibited to different degrees by all dietary fibres in the jejunum, and sometimes activated in the ileum. These results suggest that Plantago ovata seeds are a partly-fermentable dietary fibre supplement which increases stool bulk; metabolic and mucosa-protective effects are also probable.

Interference of dietary fibres with gastrointestinal enzymes in vitro.

Dietary fibres (Plantago ovata seeds, P. ovata husks, wheat bran, alfalfa, pectin, xylan) were incubated in vitro with gastrointestinal enzymes (pepsin, trypsin, chymotrypsin, lipase, alpha-amylase, maltase, lactase) in buffer solutions at concentrations of 1-5% for 10-30 min at 37 degrees C. All fibres induced sometimes pronounced changes in enzyme activity, but the effect of the different fibres on the various enzymes varied individually and was not predictable. Both P. ovata preparations had no (pepsin, trypsin, alpha-amylase) or only stimulating (chymotrypsin, lipase, lactase) actions whereas all other fibres showed inhibiting as well as stimulating influences. Wheat bran induced the most pronounced alterations increasing lipase, maltase and lactase activity and inhibiting alpha-amylase activity. Pectin and xylan were comparable in decreasing lipase and pepsin activity and in increasing chymotrypsin activity but had opposite effects on maltase activity. Alfalfa was able to stimulate lactase and lipase activity but depressed trypsin and alpha-amylase activity. The inactivation of enzymes by dietary fibres can, at least partly, be explained by adsorption to the fibre or by the presence of enzyme inhibitors especially in natural compounds. The reasons for activation processes are unknown. As enzyme activities are decisive for food digestion, the properties of the individual fibres should be carefully considered when used as dietary supplement in physiological or pathological conditions.

Effects of sennosides A + B and bisacodyl on rat large intestine.

Oral and intracecal administration of bisacodyl or sennosides A + B (10-100 mg/kg each) to rats induced a similar quantity of soft feces within 24 h and a similar acceleration of large intestinal transit time, but in each case bisacodyl had a prolonged action. Net fluid absorption in the perfused rat colon was reduced to a comparable extent by local bisacodyl in 1/10 of the molar concentration of rhein, an active metabolite of sennosides; recovery was delayed after bisacodyl. These results show that the time course of the laxative action of both drugs is different, probably attributable to their different pharmacokinetics. Both drugs influence colon motility as well as colonic secretion, but fluid secretion may contribute more to the laxative effect of bisacodyl than to that of sennosides.

Effect of rhein on electrogenic chloride secretion in rabbit distal colon.

The effect of the laxative component rhein on electrical properties and Cl transport was investigated in partially stripped epithelial sheets of rabbit distal colon under short-circuit conditions. Whereas mucosal rhein had no effect, serosally applied rhein stimulated electrogenic Cl secretion in a dose-dependent manner with a half-maximal stimulation at 0.05 mmol/l. The stimulation was partially reversible by serosal bumetanide. Indomethacin preincubation inhibited the effect of rhein, but did not prevent theophylline-induced Cl secretion. The results suggest a prostaglandin-mediated effect of rhein on the apical Cl conductance of the colonic cells.

Instability of rhein-9-anthrone as a problem in pharmacological and analytical use.

The stability of rhein-9-anthrone, one of the main and most active metabolites of the sennosides A + B, was studied under physiological conditions. A high-pressure liquid chromatographic method for the determination of rhein-9-anthrone and its oxidation products, rhein and sennidins A + B, was developed. Rhein-9-anthrone, dissolved in Tyrode buffer at pH 6.5 or 7.5 at a concentration of 10(-4) mol/l, completely disappeared from a solution warmed to 37 degrees C within 30 min. More than 90% were transformed into rhein and sennidins A + B. Pharmacological experiments in rats showed that net water absorption in the small intestine was reversed to net secretion by rhein-9-anthrone, whereas rhein and sennidins in corresponding concentrations were ineffective. As at least rhein is known to stimulate secretion under different experimental conditions, all pharmacological results with rhein-9-anthrone must be interpreted with caution and should be checked whether they essentially differ from those of its main oxidation products.

Effect of sennosides and related compounds on intestinal transit in the rat.

The effect of pure sennosides A + B on large intestinal transit (LIT) was investigated in the rat. LIT was defined as the time from intracecal administration of a color marker through a chronically implanted catheter until first appearance of colored feces. Sennosides (50 mg/kg, administered orally 2-24 h before the marker) reduced LIT from greater than 6 h in controls to a minimum of 30-20 min after a 4- or 6-hour pretreatment. Longer pretreatment times increased LIT again reaching normal values after 24 h. Intracecal administration of sennosides and their natural metabolites (sennidins A + B, rhein-9-anthrone, rhein) simultaneously with the marker accelerated LIT to approximately 50-70 min. The laxative effect was less pronounced after rhein compared with the other compounds. Indometacin, loperamide, and calcium-channel antagonists (verapamil, nifedipine) partially antagonized the effect of intracecal sennosides on LIT and delayed, but did not suppress, appearance of soft feces.

Dual effect of orally administered sennosides on large intestine transit and fluid absorption in the rat.

Quantity and consistency of the faecal output, large intestine transit time, and colonic net fluid absorption were investigated in rats after oral administration of sennosides A + B (12.5-200 mg kg-1). The release of normal faecal pellets was accelerated 3-4 h after drug administration; excretion of soft faeces was evident within 4-5 h and reached its maximum 5-7 h after administration. Large intestine transit time was dose- and time-dependently influenced by sennoside treatment. A highly significant reduction in transit time from more than 6 h in controls to 90 min for a 2 h pretreatment and a nearly maximal reduction to 30 min for a 4 h pretreatment was induced by a dose of 50 mg kg-1. Inhibition of net fluid absorption in the colon was maximal with the same dose, but clearly more pronounced after a 6 h pretreatment period than after a 4 h period. Since the increase in fluid volume due to net fluid secretion is delayed compared with the acceleration of large intestine transit, the early motility effect seems to be largely independent of the changes in absorption mechanisms. Therefore, the laxative effect of the sennosides consists of changes in colon motility as well as in colonic fluid absorption, but motility may be an earlier and more sensitive parameter than net absorption.

Acceleration of large intestine transit time in rats by sennosides and related compounds.

Sennosides A + B and their natural metabolites, sennidins A + B, rheinanthrone and rhein, as well as the synthetic laxative danthron, were investigated for their influence on small and large intestine transit time in rats. Carmine red, as a marker, was administered through a gastric tube for small intestine transit or intracaecally by a chronically implanted catheter for colon transit. High doses of sennosides (250-500 mg kg-1) given orally from 20 min or up to 6 h before marker administration had no effect on small intestine transit time. The metabolites and danthron (10-100 mg kg-1 p.o.) also did not accelerate upper gastrointestinal passage. Intracaecal administration at the same time as carmine red, however, reduced the time for the appearance of the first coloured faeces from more than 8 h in the controls to 46 +/- 9 min after sennosides, 34 +/- 11 min after sennidins, 53 +/- 83 min after rhein and 16 +/- 4 min after rheinanthrone (50 mg kg-1 of each). Danthron was ineffective. Thus, sennosides and their natural metabolites specifically influence large intestinal motility. Acceleration of colonic transport seems to be a major component of the laxative action whereas for danthron motility changes are not responsible for its laxative action. Indomethacin partly inhibited the acceleration of large intestine transit induced by sennosides. An involvement of endogenous prostaglandins may therefore be possible, although a local bolus administration of PGF2 alpha or PGE2 into the caecal lumen neither influenced transit time nor induced diarrhoea.

Spasmolytische wirkung des isoasaronfreien kalmus1.

ACORUS CALAMUS yields different drug types with varying content of beta-asarone, a substance which proved to be carcinogetic in rats. We tested essential oils with different beta-asarone content for their activity against histatnine-spasms in the isolated guinea pig ileum. At a dose level of 10 microg/cm (3), the beta-asarone-free oil (type I) had a pronounced, spasmolytic activity which was comparable to that of the antihistaminic drug pyrilaminemaleate used as a standard antagonist (4 microg/cm (3). Each of these drugs changed the EC (50) for histamine from 29 microg/cm (3) to 118 microg/cm (3) approximately. At a dose level of 10 microg/cm (3) the beta-asarone-rich oil (type IV) showed no spasmolytic activity at all. The essential oil of the european calamus with low content of beta-asarone (type II) had also a good spasmolytic effect, however inferior to that of the beta-asarone-free oil, so that the above cited decrease of the EC (50) value of histamine could be seen in a concentration of 32 microg/cm (3). For this reasons and for better drug-safety, only the rhizomas of the beta-asarone-free diploid or of the triploid calamus with low content of beta-asarone should be used.

Effect of anthraquinone derivatives on canine and rat intestinal motility.

The effects on gastrointestinal motility of 3 senna preparations containing 18% oxidized Ca-sennosides, 60% Ca-sennosides, or pure sennosides A + B were tested in dogs and rats as measured by electromyography. Oral administration of the oxidized products in the fasted animal increased the activity of the small intestine within 2 h and reduced both caecal and colonic contractions for 24 h. Severe diarrhoea was present 4-6 h after administration and lasted for at least 1 day. Ca-sennosides had a similar, but weaker effect while pure sennosides affected motility only 6-10 h after oral administration. The intracolonic administration of the oxidized products resulted in an immediate reduction of colon motility for 7-8 h and diarrhoea was present within 40 min. Intracolonic Ca-sennosides and sennosides A + B induced only small changes in the intestinal motility, but diarrhoea also appeared. The results confirm that pure sennosides act predominantly on large intestine motility after their degradation by colonic microorganisms. Oxidized products are already effective in the upper gastrointestinal tract. The early action of Ca-sennosides requires further investigation. Side effects after oral senna treatment such as griping or nausea may be caused by motility changes induced by the presence of small amounts of oxidized products in the drug.