Use of botanicals in food supplements. Regulatory scope, scientific risk assessment and claim substantiation. 2005.

BACKGROUND/AIMS: In the European Union, an elaborate legal framework regulates botanical products both under food and medicinal law. The decision as to which legal framework applies to an individual product may differ between the Member States. In the case of botanical food supplements, all food law provisions apply to their manufacturing, composition and marketing, including the new claims legislation. METHODS: Elements from EU and national law, scientific and other publications are brought together to investigate how to clarify the differentiation between the use of botanicals for medicinal and health-promoting purposes on a scientific basis. RESULTS: Guidance on the safety assessment and quality evaluation of botanicals is proposed in light of the different approaches described in the scientific literature with particular attention to the concept of long-term use as an integral part of safety evaluation. Guidance on claims substantiation is also included, taking into consideration the proposed legislation, the concept of long-term experience and grading of evidence. CONCLUSIONS: A model for safety and efficacy assessment of botanical food supplements in the EU is proposed, and should be taken into consideration in the development of legislation and scientific research on botanicals.

Zinopin--the rationale for its use as a food supplement in Traveller's thrombosis and motion sickness.

Venous thrombo-embolism (VTE) has been associated with periods of prolonged immobility during air, sea and road travel. Motion sickness (MS) has also been reported during both long and short journeys. Current prophylactic therapies for both these indications are generally associated with side effects. Physiological profiles of Pycnogenol and Standardized Ginger Root Extract (SGRE) representing active constituents of Zinopin have been described and reviewed in relation to their activities involved in the patho-physiology of VTE (Traveller's Sickness) and MS and their safe use as food supplement, in traveller's thrombosis and motion sickness. The pathophysiology of VTE and MS is discussed in light of epidemiological data and risk factors associated with these conditions. Rationale of development of Zinopin and its mechanism of action are discussed based on physiological synergy of Pycnogenol and SGRE. Conclusions are made in light of preliminary clinical findings obtained in an open controlled clinical trial. Further clinical study on Zinopin on these lines is suggested.

The effect of Pycnogenol on fluoride induced rat kidney lysosomal damage in vitro.

Pycnogenol (PYC) is a procyanidin-rich extract of the bark of French maritime pine (Pinus pinaster) with a potent ability to scavenge free radicals. Lysosome-rich fractions from rat renal cortices were incubated with varying amounts of PYC and challenged with noxious doses of fl uoride. Controls were also included. The release of N-acetyl-beta-D-glucosaminidase (NAG) isozymes in the supernatant was estimated by spectrophotometric methods. The protein content of the renal cortex was also determined. Our results show that fluoride in unhealthy doses can cause a concentration dependent release of N-acetyl-beta-D-glucosaminidase (NAG) isozymes from the renal lysosomes. This may be related to its known ability to initiate free radical formation or direct damaging effects on the lysosomal membrane. As a blend of bio flavonoids pycnogenol has a potent ability to scavenge free radicals. In our study PYC was effective in preventing fluoride induced release of NAG isozymes from the renal lysosomes.

Pycnogenol prevents haemolytic injury in G6PD deficient human erythrocytes.

Glucose6 phosphate dehydrogenase (G6PD) deficiency is the most common X-linked disorder of human erythrocytes where cells have inadequate capacity to destroy peroxides and high susceptibility towards haemolytic changes. Pycnogenol is a proprietary dry extract of the French Maritime pine (Pinus pinaster) bark with high ability to scavenge free radicals. In the present study we have investigated if Pycnogenol can protect G6PD deficient erythrocytes against haemolytic cell damage. Venous blood samples were obtained from six subject of Mediterranean origin with known G6PD deficiency which was also confirmed with standard techniques. Erythrocyte haemolysis in the presence and absence of Pycnogenol was induced either with tert-butylhydroperoxide (t-BHP) or quinine and the haemoglobin release in the supernatant was determined by recording the optical density at 540 nm in a Shimadzu spectrophotometer. Our results have shown that Pycnogenol has protective action against a Xenobiotic chemical induced haemolysis in G6PD deficient human erythrocytes.

Effect of fruits of Moringa oleifera on the lipid profile of normal and hypercholesterolaemic rabbits.

Rabbits were fed Moringa oleifera (200mg/kg/day, p.o.) or lovastatin (6mg/kg/day, p.o.) in banana pulp along with standard laboratory diet and hypercholesterolaemic diet for 120 days. Moringa oleifera and lovastatin were found to lower the serum cholesterol, phospholipid, triglyceride, VLDL, LDL, cholesterol to phospholipid ratio and atherogenic index, but were found to increase the HDL ratio (HDL/HDL-total cholesterol) as compared to the corresponding control groups. Treatment with M. oleifera or lovastatin in normal rabbits decreased the HDL levels. However, HDL levels were significantly increased or decreased in M. oleifera- or lovastatin-treated hypercholesterolaemic rabbits, respectively. Lovastatin- or M. oleifera-treated hypercholesterolaemic rabbits showed decrease in lipid profile of liver, heart and aorta while similar treatment of normal animals did not produce significant reduction in heart. Moringa oleifera was found to increase the excretion of faecal cholesterol. Thus, the study demonstrates that M. oleifera possesses a hypolipidaemic effect.

Pycnogenol inhibits the release of histamine from mast cells.

Oxygen derived free radicals are now increasingly regarded as a primary force of tissue destruction and also have the ability to release histamine from mast cells. Pycnogenol is an extract of the bark of French maritime pine (Pinus pinaster) containing bioflavonoids with a potent ability to scavenge free radicals. Therefore Pycnogenol was investigated for inhibition of histamine release from rat peritoneal mast cells. In addition, its effects were compared with sodium cromoglycate, a known inhibitor of histamine release from the mast cell. Rat peritoneal mast cells were isolated and purified by differential centrifugation and cells pooled from 3-4 animals were suspended at approximately 10(6) cells/mL buffered salt solution. Histamine release was induced by compound 48/80 or the calcium ionophore A-23187 and estimated from supernatant following extraction and by fluorimetric methods. Pycnogenol produced a concentration dependent inhibition of histamine release induced by the two secretagogues. Its inhibitory effect on mast cell histamine release was favourably comparable to sodium cromoglycate.

Treatment of melasma with Pycnogenol.

Melasma (or chloasma) is a common disorder of cutaneous hyperpigmentation predominantly affecting sun-exposed areas in women. The pathogenesis of melasma is not fully understood and treatments are frequently disappointing and often associated with side effects. Pycnogenol is a standardized extract of the bark of the French maritime pine (Pinus pinaster), a well-known, potent antioxidant. Studies in vitro show that Pycnogenol is several times more powerful than vitamin E and vitamin C. In addition, it recycles vitamin C, regenerates vitamin E and increases the endogenous antioxidant enzyme system. Pycnogenol protects against ultraviolet (UV) radiation. Therefore its efficacy in the treatment of melasma was investigated. Thirty women with melasma completed a 30-day clinical trial in which they took one 25 mg tablet of Pycnogenol with meals three times daily, i.e. 75 mg Pycnogenol per day. These patients were evaluated clinically by parameters such as the melasma area index, pigmentary intensity index and by routine blood and urine tests. After a 30-day treatment, the average melasma area of the patients decreased by 25.86 +/- 20.39 mm(2) (p < 0.001) and the average pigmentary intensity decreased by 0.47 +/- 0.51 unit (p < 0.001). The general effective rate was 80%. No side effect was observed. The results of the blood and urine test parameters at baseline and at day 30 were within the normal range. Moreover, several other associated symptoms such as fatigue, constipation, pains in the body and anxiety were also improved. To conclude, Pycnogenol was shown to be therapeutically effective and safe in patients suffering from melasma.

Influence of chronic treatment of rats with isoprenaline and calcium channel blockers on response of isolated right ventricle to noradrenaline.

Influence of chronic treatment of rats with and calcium channel blockers (CCBs) and isoprenaline (ISP) on responses to noradrenaline (NA) was investigated on electrically--driven isolated right ventricle preparations. The ventricles were obtained from animals treated with chronic ISP or CCBs alone and chronic nifedipine, verapamil, diltiazem or nimodipine plus chronic ISP. A decreased response to NA as evidenced by an increase in EC50 for contraction which was observed in chronic ISP- treated preparations may be due to development of desensitisation (down-regulation) of beta-adrenoceptors. In chronic CCB-treated preparations there was a significant decrease in the EC50 of NA and decreased contractile response suggesting an increase in the beta-adrenoceptors and decreased availability of calcium, respectively. In chronic CCBs + ISP treated preparations further decreases in the EC50 values were observed suggesting that the voltage gated L-type Ca2+ channels may be affected directly or indirectly by change in beta-adrenoceptor activity. By the above results a proposed mechanism of interrelationship of beta-adrenoceptors with voltage gated L-type calcium channels in cardiac muscle is supported.

Interaction of calcium channel blockers with different agonists in aorta from normal and diseased rats.

The interactions of calcium channel blockers (CCBs) with noradrenaline (NA), phenylephrine (PE), dopamine (DA) and KCl have been investigated in rat isolated aortic strip. In preparations from control and hypertensive (DOCA-saline) rats chronically treated with verapamil, nifedipine and diltiazem, there was partial inhibition of contractions to NA, PE and DA. However, with nimodipine, there was potentiation of responses. This could be related to the occurrence of different isoforms of L-type calcium channels. In preparations obtained from hyperthyroid rats the concentration-response curves of NA, PE and KCl were shifted to the right with depressed maximal response which could be secondary to the primary effect exerted on the heart. In preparations from L-thyroxine + nimodipine/nifedipine treated rats the concentration-response curves of NA, PE and KCl were shifted to the right and the maxima was depressed suggesting that this may be due to decreased alpha receptor density (NA and PE) and down-regulation of voltage operated channels (KCl).

Interaction of calcium channel blockers (CCBs) with histamine and 5-hydroxytryptamine in aorta from normal and diseased rats.

The present study attempts to investigate the interaction of calcium channel blockers (CCBs) with histamine (H) and 5-hydroxytryptamine (5-HT) in rat isolated aortic strip preparations. In preparations obtained from rats chronically treated with various CCBs the contractile responses to H were completely blocked suggesting that this may be due to inhibition of the voltage-dependent channels and inositol 1,4,5-triphosphate induced release of calcium from intracellular stores. The decreased contractions of the aortic strip preparations with 5-HT obtained from rats chronically treated with various CCBs implies a decrease in 5-HT receptor density. DOCA-saline hypertensive rats chronically treated with various CCBs showed variable responses to H and 5-HT suggesting that these changes may be due to different isoforms of L-type calcium channels. In L-thyroxine-treated preparations or those simultaneously treated with L-thyroxine and CCBs the responses to H were abolished and those to 5-HT were partially blocked with decrease in maxima which could be secondary to the primary effect on the heart and to generalised reduced senstivity of the rat aorta.

Evaluation of xanthotoxol for central nervous system activity.

Xanthotoxol (XT), 8-hydroxypsoralen, exhibited dose-graded sedative activity in dogs, cats, rats, mice and hamsters. At doses of 5-20 mg/kg intraperitoneally (i.p.) in cats and 3-100 mg/kg orally (p.o.) in dogs, XT blocked predatory mouse/rat killing behavior. In mice, XT (10-300 mg/kg i.p.) exhibited a dose-dependent reduction in locomotor activity but was less potent in this regard than reference diazepam (10-100 mg/kg i.p.). XT in mice (0.1-10.0 mg/kg i.p.) and in hamsters (0.1-10.0 mg/kg p.o.) antagonized amphetamine-induced hypermobility but was less potent than diazepam. XT elevated the electrical threshold in foot-shock-induced fighting behavior in rats. XT (0.1-30.0 mg/kg p.o.) potentiated pentobarbital-induced narcosis in hamsters at otherwise subeffective doses of pentobarbital. Conditioned avoidance responses in rats were not significantly altered with 1-3 mg/kg i.p. and 30-100 mg/kg p.o. doses of XT but 300 mg/kg p.o. blocked both conditioned and unconditioned response. Doses of 100-1000 mg/kg i.p. of XT in mice were used to study 48-h acute toxicity of XT and its LD50 was estimated to be 468 mg/kg. Doses of 10, 40 and 80 mg/kg p.o. were used to study the chronic toxicity of XT in rats for 6 months and no side effects or abnormalities in reproductive activity or endocrine integrity were noted. The F1 generation of rats from 6-month XT-treated parents were free of teratogenic effects.

Effect of cadmium on contractile response to spasmogens in vascular and nonvascular tissues.

In rat isolated aorta low concentration of CdCl2 (4.8 x 10(-8) M) produced a significant increase in pD2 value of KCl and noradrenaline (NA) with an increase in the maxima, while higher concentration of CdCl2 (1.44 x 10(-5) M) produced a significant rightward shift of the dose-response curve with a depression of maxima. In rat isolated portal vein 4.8 x 10(-7) M CdCl2 produced a significant increase in the pD2 value of KCl with an increase in the maxima, while higher concentration of CdCl2 (4.8 x 10(-5) M) produced a significant rightward shift of the dose-response curve of KCl and NA with a depression of maxima. In rat isolated vas deferens and anococcygeus muscle 4.8 x 10(-8) M CdCl2 produced a significant increase in pD2 value of KCl with an increase in the maxima, while higher concentrations of CdCl2 (4.8 x 10(-6) M and 1.44 x 10(-5) M) produced a significant rightward shift of the dose-response curve of KCl and NA. It is suggested that enhancement and reduction of response to KCl and NA, in presence of different concentrations of CdCl2 might be due to the alteration in the fluxes of calcium ion since these spasmogens produce their action by increasing the availability of calcium ions for the contractile machinery.

The acute pressor response to cadmium in rats.

In rats, acute i.v. administration of CdCl2 (0.5 and 1 mg/kg) produced a depressor response followed by a pressor response, while the acute i.p. administration of CdCl2 (0.5 and 1 mg/kg) produced only a pressor response. Phentolamine (5 mg/kg, i.v.), hexamethonium (10 mg/kg, i.v.), propranolol (2 mg/kg, i.v.) and indomethacin (20 mg/kg, i.p.) as well as bilateral adrenalectomy, acute reserpinization and chemical sympathectomy by guanethidine did not modify the acute pressor response to CdCl2. Verapamil (1 mg and 2 mg/kg, i.v.) and nifedipine (0.25 and 0.5 mg/kg, i.v.) prevented the acute pressor response to CdCl2 administered by either i.v. or i.p. route. Phentolamine (10 micrograms/ml) could not prevent the rise in perfusion pressure of the rat hindquarter due to intra-arterial CdCl2 administration. However, verapamil (50 and 100 micrograms/ml) prevented rise in perfusion pressure. It is concluded that cadmium ion might mimic calcium ion and produce a direct contractile effect on the vascular smooth muscle.

Modification of the antihypertensive effect of indapamide by indomethacin.

Oral treatment with indapamide was found to reduce blood pressure of hypertensive rats but not of normotensive rats. Chronic indomethacin treatment had no effect on blood pressure of untreated normotensive and hypertensive rats. Also indomethacin did not modify the antihypertensive effect of indapamide excluding the direct involvement of PGs in the antihypertensive effect of indapamide. Vascular reactivity to pressor agents NA, ADR and ANG was significantly increased after indomethacin treatment. This may be due to the blockade of the actions of PG in modifying vascular reactivity to vasoconstrictor agents or may be a direct effect of indomethacin on calcium fluxes. Indapamide reduced the reactivity to NA and ANG in the presence of indomethacin suggesting that the antihypertensive effect of indapamide may be through a decrease in reactivity to pressor agents which is independent of increase in the synthesis of vasodilator PGs.

Cadmium-induced hypertension in rats.

Chronic cadmium chloride (CdCl2, 0.5 and 1.0 mg/kg, i.p.) treatment in female albino rats for 2 weeks resulted in elevation of blood pressure. In chronic CdCl2-treated rats the pressor responses to different doses of noradrenaline, angiotensin II and depressor responses to acetylcholine and isoprenaline were unaltered. In rat hindquarter preparation there was elevation of perfusion pressure and the sensitivity of vascular bed to noradrenaline was increased in the CdCl2-induced hypertensive rats. Complete bilateral adrenalectomy or chemical sympathectomy or treatment with captopril did not prevent the development of CdCl2-induced hypertension. Treatment with verapamil (15 mg/kg/day, p.o.) or nifedipine (10 mg/kg/day, p.o.) for 2 weeks prevented the development of hypertension with chronic CdCl2 treatment. It is suggested that chronic treatment of rats with CdCl2 induces hypertension. It is possible that cadmium mimics the calcium ion for the induction of hypertension in rats.

Effects of Abana, an Ayurvedic preparation, on rabbit atrium and intestine.

The effects of Abana, an Ayurvedic remedy, administered orally to rabbits was studied for its effects on isolated atria and intestine. Administration of Abana for 3 days increased the basal amplitude and reduced the responses of atria to isoprenaline and norepinephrine. Combined treatment with Abana and isoprenaline reduced this effect. It is possible that Abana treatment for 3 days has an action similar to that of chronic administration of isoprenaline (down regulation of beta receptors). A similar down regulation of beta receptors of smooth muscle of rabbit intestine also seems to occur. Abana pretreatment potentiated the inotropic responses of histamine and CaCl2. These effects may be due to a specific depressant effect of Abana on the adrenergic receptors and a direct sensitization of the atrium manifested by an increased response to CaCl2.

Evidence for multiple prejunctional receptor sites in rat isolated anococcygeus muscle.

In rat isolated anococcygeus muscle, dopamine (2.6 x 10(-11) M to 8.3 x 10(-10) M) produced a concentration-dependent inhibitory effect on field stimulated contractions. The effect of dopamine was blocked by pimozide (2.2 x 10(-8) M) but not by yohimbine (2.6 x 10(-10) M) or propranolol (1.0 x 10(-7) M), suggesting a specific prejunctional inhibitory effect. Higher concentrations of dopamine (1.4 x 10(-7) M to 1.4 x 10(-4) M) elicited concentration-dependent contractions which were blocked competitively with higher concentrations of pimozide (2.2 x 10(-7) M to 1.4 x 10(-4) M) and 2.2 x 10(-6) M), suggesting a postjunctional activity. ACh in very low concentrations (2.8 x 10(-11) M to 4.4 x 10(-10) M) blocked the field stimulated contractions. Atropine (2.6 x 10(-9) M) per se augmented them and also antagonized the inhibitory effects of ACh, suggesting a prejunctional activity of ACh. Higher concentrations of ACh (5.5 x 10(-7) M to 7.0 x 10(-5) M) produced contractions which were not altered by atropine in a concentration (2.6 x 10(-9) M) which antagonized the prejunctional activity of ACh. Histamine, in a wide range (3.1 x 10(-12) M to 2.6 x 10(-8) M), did not modify field stimulated contractions. Very low concentrations of 5-HT (1.2 x 10(-11) M to 3.8 x 10(-10) M) had an inhibitory effect on field stimulated contractions. Methysergide (2.8 x 10(-9) M) enhanced the responses to electrical stimulation and antagonized the 5-HT-induced inhibitory effect. Still higher concentrations of 5-HT (1.9 x 10(-6) M to 1.0 x 10(-3) M) produced concentration-dependent contractions. Methysergide (8.5 x 10(-7) M) failed to antagonize, whereas phentolamine (1.0 x 10(-6) M) antagonized 5-HT competitively. Dopamine (8.3 x 10(-10) M), ACh (4.4 x 10(-10) M) or 5-HT (3.9 x 10(-10) M), in concentrations which produced a maximal prejunctional inhibitory effect, did not alter the EC50 value of NA, ruling out a post-junctional effect. Moreover, the concentration ratios of these agents for EC50 pre to EC50 post were less than 1, suggesting their preferential prejunctional site of action. It is concluded that multiple prejunctional site of action. It is concluded that multiple prejunctional receptor activities for DA, ACh (muscarinic) and 5-HT, which modify the release of neurotransmitter, may be operative in this preparation.

Action of some amides of substituted ethylenediamines on central nervous system.

Five of the substituted ethylenediamine amides (LMG I to V) were tested for various CNS attributes and for acute toxicity (24 hr mortality). All compounds were potent analgesics in various animal tests, LMG V being most potent. All reduced spontaneous activity of mice and potentiated ether anaesthesia. However, CAR was not altered and anti-MES were not pronounced in rats. Compounds appear to have a wide safety margin considering ED50 and LD50 in mice.

The role of the sympathetic nervous system in oestrogen-induced hypertension in rats.

Albino rats of either sex received chronic ethinyl oestradiol (EO) treatment (1.5 mg kg-1 daily, i.m.) for 3 weeks. Untreated control rats received arachis oil vehicle alone. Chronic EO treatment resulted in elevation of blood pressure in both sexes. Female rats exhibited significantly greater elevation in blood pressure than males. In chronic EO-treated rats pressor responses to low doses (0.5 micrograms kg-1) of noradrenaline were significantly increased, while those to angiotensin II, acetylcholine and isoprenaline were unaltered. Chronic EO treatment also sensitized the vascular bed of the rats' hindquarters to noradrenaline. EO-induced hypertension was associated with significant increase in dopamine-beta-hydroxylase activity of adrenal glands. Complete bilateral adrenalectomy or chemical sympathectomy prevented the development of EO-induced hypertension. It is suggested that chronic treatment of rats with EO induces and maintains hypertension. The peripheral sympathetic system plays an important role in this phenomenon.