Chapter

Synergy in Whole Plant Medicine: Crataegus spp.: An Example

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  • Irish College of Traditional & Integrative Medicine
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Abstract

Extracts of Crataegus spp. have been the subject of extensive in vivo and in vitro investigations, primarily for the treatment of cardiovascular diseases. Effects include antioxidant, hypolipidemic, hypotensive, positive inotropic activity, increased blood vessel wall integrity, improved coronary blood flow, and positive oxygen utilization, among others. Flavonoids and procyanidins are the principle constituents. This chapter reviews published English language scientific literature for evidence of indications of interactive combination effects including synergistic effects and/or multiple target effects within the body. Synergistic effects have only been reported using effect‐based measurement approaches. There was considerable diversity among the studies regarding their methodology, substrate type and their analysis, which made direct comparisons challenging. The authors suggest the need for universal use of consistent and standardized methods, using activity‐led, systems‐based reverse analysis or “omic” research, owing to the multifactorial requirements of plant‐based studies.

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S ummary A quantitative analysis of the toxicity of drugs or poisons applied jointly requires that they be administered at several dosages in mixtures containing fixed proportions of the ingredients. From a study of the dosage‐mortality curves for several such mixtures, preferably in comparison with equivalent curves for the isolated active ingredients, most cases of combined action can be classified into one of three types: (1) The first type is that in which the constituents act independently and diversely, so that the toxicity of any combination can be predicted from that of the isolated components and from the association of susceptibilities to the two components. The coefficient of association can be measured experimentally and should be constant at all proportions of the ingredients. When high, the toxicity of the mixture is reduced. The form of the dosage‐mortality curve has been examined for several hypothetical mixtures. Whenever the curves for the two constituents were assumed to differ in slope, there was a relatively abrupt bend in the curve for the mixture, the rectilinear segments above and below the break approaching in slope the values for the original constituents. This observation indicates that in homogeneous populations the slope of a dosage‐mortality curve is of toxicological significance. Since the same numerical relations would be expected if a single poison were to have two independent lethal effects within the animal, there is theoretical basis for fitting the linear segments of a dosage‐mortality curve separately when a break occurs after transformation to probits and logarithms. This argument has been extended to time‐mortality experiments to explain the smoothly concave curves characteristic of natural mortality. (2) The second type of joint action is that in which the constituents act independently but similarly, so that one ingredient can be substituted at a constant ratio for any proportion of a second without altering the toxicity of the mixture. With homogeneous populations, dosage‐mortality curves for the separate ingredients and for all mixtures should be parallel. Although by hypothesis the susceptibility to one ingredient is completely correlated with that to the other, mixtures in this category are more toxic than in the preceding class where association may vary from 0 to 1. The numerical relations have been illustrated by an experiment on the toxicity to the house‐fly of solutions containing pyrethrin and rotenone. A mixture with a little less than four equitoxic units of pyrethrin to one of rotenone agreed closely with the definition but one in which the ingredients were about equally balanced showed a significantly greater toxicity than expected on the hypothesis of independent action, indicating the presence of synergism. (3) Synergism forms the third type of joint action, characterized by a toxicity greater than that predicted from studies on the isolated constituents. It is the reverse of antagonism, which has not been considered directly. Two methods are proposed for the analysis of synergism. The more direct is to relate equitoxic dosages of mixture to its percentage composition in terms of the more active ingredient. When both are in logarithms the relation is linear over a useful range of compositions. This procedure preserves the original structure of the experiment, can be extended readily to three or more ingredients and leads to a convenient practical result. 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None of the samples contained a very small proportion of one ingredient, so that several equations were equally applicable, one of them being (1+0–714 A ) B = 56·1, from which the intensity of synergism was 40. The problem of measuring synergism in fumigants has been discussed briefly.
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The significance of antioxidants in preventive medicine is well known. Increasing interest has been devoted to naturally occurring compounds – polyphenols – because of their beneficial health effects. The subject of this study was to examine the antioxidative activity of polyphenolic preparations containing oligomeric procyanidin from the bark of common pine (Pinus sylvestris L.) and hawthorn (Crataegus oxyacantha L.) and flavones of skullcap (Scutellaria baicalensis Georgi) roots. Multi-constituent mixtures were fractionated, and the antioxidative activity of fractions was tested in vitro with linoleic acid oxidation by AAPH-generated radicals. All preparations at 6 and 12 ppm concentrations exhibited protective activity, from 45% to 95% in relation to the control sample. The average activity of preparations was higher than those of their fractions used at the same concentrations, and it was similar to trolox and BHT activity.
Article
Extracts from hawthorn leafs and flowers (Crataegus sp., Rosaceae) are widely used as a rational based phytomedicine for declining cardiac performance. According to present literature C-glycosylated flavones and oligomeric procyanidins are considered to be the active ingredients, despite the fact that no systematic data are available on systemic bioavailability of proanthocyanidins after oral intake. The present study aims to review the actual state of literature in this field and to investigate the intestinal absorption mechanisms of defined hawthorn PAs with different degrees of polymerization by validated in vitro Caco-2 monolayer permeation system. Hawthorn OPCs with DP 2 to 6 were isolated as defined clusters. Procyanidin B2 and the procyanidin clusters DP 4, 5 and 6 had very low P(app) values between 0.6 and 6×10⁻⁷ cm/s for apical to basolateral permeation. The higher the molecular weight the lower permeation coefficients were calculated. The observed low-level transport was mainly due to passive paracellular permeation. Additionally cellular uptake of OPCs by transcellular permeation was possible; on the other side procyanidins were shown to be p-glycoprotein substrates, which leads to subsequent excretion of PAs by the efflux pump to the apical side. Mixtures of the different OPCs did not have an increased permeation. Transport experiments of complex OPC mixtures together with hawthorn flavonoids did not indicate any improved permeation or synergistic effects. In principle this raises the question if systemic pharmacological activities of hawthorn extracts, can really be attributed to OPCs with very low systemic bioavailability.
Article
Two or more drugs that individually produce overtly similar effects will sometimes display greatly enhanced effects when given in combination. When the combined effect is greater than that predicted by their individual potencies, the combination is said to be synergistic. A synergistic interaction allows the use of lower doses of the combination constituents, a situation that may reduce adverse reactions. Drug combinations are quite common in the treatment of cancers, infections, pain, and many other diseases and situations. The determination of synergism is a quantitative pursuit that involves a rigorous demonstration that the combination effect is greater than that which is expected from the individual drug's potencies. The basis of that demonstration is the concept of dose equivalence, which is discussed here and applied to an experimental design and data analysis known as isobolographic analysis. That method, and a related method of analysis that also uses dose equivalence, are presented in this brief review, which provides the mathematical basis for assessing synergy and an optimization strategy for determining the dose combination.
Article
Since the 1800s, natural health products that contain hawthorn (Crataegus spp.) have been used in North America for the treatment of heart problems such as hypertension, angina, arrhythmia, and congestive heart failure. Traditionally, Native American tribes used hawthorn (Crataegus spp.) to treat gastrointestinal ailments and heart problems, and consumed the fruit as food. Hawthorn also has a long history of use in Europe and China for food, and in traditional medicine. Investigations of Crataegus spp. typically focus on the identification and quantification of flavonoids and anthocyanins, which have been shown to have pharmacological activity. The main flavonoids found in Crataegus spp. are hyperoside, vitexin, and additional glycosylated derivatives of these compounds. Reviewed herein are the botany, ethnobotany, and traditional use of hawthorn while focusing on the phytochemicals that have been reported in Crataegus species, and the variation in the described chemistry between individual species.
Article
The isobole is well established and commonly used in the quantitative study of agonist drug combinations. This article reviews the isobole, its derivation from the concept of dose equivalence, and its usefulness in providing the predicted effect of an agonist drug combination, a topic not discussed in pharmacology textbooks. This review addresses that topic and also shows that an alternate method, called "Bliss independence," is inconsistent with the isobolar approach and also has a less clear conceptual basis. In its simplest application the isobole is the familiar linear plot in cartesian coordinates with intercepts representing the individual drug potencies. It is also shown that the isobole can be nonlinear, a fact recognized by its founder (Loewe) but neglected or rejected by virtually all other users. Whether its shape is linear or nonlinear the isobole is equally useful in detecting synergism and antagonism for drug combinations, and its theoretical basis leads to calculations of the expected effect of a drug combination. Numerous applications of isoboles in preclinical testing have shown that synergism or antagonism is not only a property of the two agonist drugs; the dose ratio is also important, a fact of potential importance to the design and testing of drug combinations in clinical trials.
Article
Methanol extracts prepared from five plant materials native to the Mediterranean area, namely olive tree (Olea europaea) leaf, St. John's wort (Hypericum perforatum), hawthorn (Crataegus laevigata), oregano (Origanum vulgare) and laurel leaf (Lauris nobilis), were examined for their phenolic components. Total phenolic content was determined by the Folin–Ciocalteu method. The content of proanthocyanidins in acid-hydrolysed extracts was determined spectrophotometrically. The contents of free flavones (apigenin and luteolin) and flavonols (kaempferol, myricetin and quercetin) were determined by HPLC analysis. The time of hydrolysis of flavones, flavonols and proanthocyanidins was optimised.Antioxidant activities of apigenin, luteolin, kaempferol, myricetin, quercetin and of plant extracts were examined. Antioxidative activities were studied in sunflower oil at 98 °C, by measuring peroxide value, and in an aqueous emulsion system of β-carotene and linoleic acid by measuring the absorbance of the sample. Among flavones and flavonols investigated, only myricetin inhibited oxidation of sunflower oil. All other flavones and flavonols showed pro-oxidative activity. Oppositely, in the emulsion system, only apigenin showed pro-oxidative activity while other flavones and flavonols and plant extracts inhibited oxidation of β-carotene.
Article
Many studies have been carried out on bioactivities of individual herbs, however, no collective study on their comparative antioxidant and cytoprotective activities against oxidative damage has been reported. We selected 17 common commercial herbs and studied their relative phenolic contents, antioxidant activities, and cytoprotective activities on gap–junction intercellular communication and antioxidative enzymes in vitro under the same conditions. Total polyphenol content ranged from 464 to 870 gallic acid equivalents (GAE) mg/100 g and total flavonoid content from 212 to 494 catechin equivalents (CE) mg/100 g. Among the samples, chamomile, rosehip, hawthorn, lemon verbena, and green tea contained relatively high total phenolics (769–844 mg GAE/100 g) and flavonoids (400–4 mg CE/100 g). Chamomile also showed the highest antioxidant activity with 960 mg/100 g of vitamin C equivalent (VCE), followed by hawthorn (929 mg VCE/100 g) and black tea (916 mg VCE/100 g). Total phenolic and total flavonoids showed a higher correlation with antioxidant activity. Most of herbs enhanced cell viability and showed protective effects against oxidative stress induced by hydrogen peroxide in Chinese hamster lung fibroblast (V79-4) cells. Furthermore, herbs used in this study showed higher protective effect on gap–junction intercellular communication (GJIC) as compared to gallic acid and catechin, and also enhanced activity of the antioxidative enzymes such as superoxide dismutase (SOD) and catalase (CAT) in a dose-dependent manner.
Article
Despite the extensive traditional use of Croton gratissimus Burch. var. gratissimus for medicinal purposes, scientific studies validating the therapeutic properties of this indigenous plant are lacking. As the bark, roots and leaves of C. gratissimus are used separately as well as in combination, this study focused on determining antimicrobial efficacies of the plant parts independently and in combination to assess possible pharmacological interactions (e.g. synergy, antagonism). The hydro-distilled leaf essential oil and extracts of bark, root and leaf were comparatively assessed for antimicrobial activity by means of microdilution minimum inhibitory concentration (MIC). The fractional inhibitory concentrations (FIC) were determined for the leaf and root (1:1), bark and root (1:1), leaf and bark (1:1) combination. Isobolograms were plotted to demonstrate interactions between various ratios of the roots and leaves. The MIC and FIC results indicated variable efficacies for the various plant part combinations, the greatest of which was noted for Cryptococcus neoformans in the root and leaf combination (MIC 0.4 mg/ml and FIC of 0.4). Isobolograms indicated the greatest synergy for Bacillus cereus, Candida albicans and Cryptococcus neoformans. The observed synergistic interactions clearly indicate that the reductionist approach may often be short-sighted and that biological activity may be improved through combination therapy, where different complex metabolic pools collectively contribute to the enhanced effect.
Article
Inhaled anesthetics have been postulated to act at multiple receptors, with modest action at each site summing to produce immobility to noxious stimulation. Recent experimental results affirm prior findings that inhaled anesthetics interact additively. Synergy implies multiple sites of action by definition. In this essay, we explore the converse: does additivity imply a single site of action? The interaction of one versus two ligands competing for the same binding site at a receptor was explored using the law of mass action. Circuits were then constructed to investigate how the potency of drugs and the steepness of the concentration versus response relationship is amplified by the arrangement of suppressors into serial circuits, and enhancers into parallel circuits. Assemblies of suppressor and enhancer circuits into signal processing units were then explored to investigate the constraints signal processing units impose on additive interactions. Lastly, the relationship between synergy, additivity, and fractional receptor occupancy was explored to understand the constraints imposed by additivity. Drugs that compete for a single receptor, and that similarly affect the receptor, must be additive in their effects. Receptors that bind suppressors in serial circuits, or enhancers in parallel circuits, increase the apparent potency of the drugs and the steepness of the concentration versus response relationship. When assemblies of suppressor and enhancer circuits are arranged into signal processing units, the interactions may be additive or synergistic. The primary determinant is the relationship between the concentration of drug associated with the effect of interest and the concentration associated with 50% receptor occupancy, k(d). Effects mediated by very low concentrations are more likely to be additive. Similarly, inhaled anesthetics that act at separate sites are unlikely to exhibit additive interactions if anesthetic drug effect occurs at concentrations at or above 50% receptor occupancy. However, if anesthetic drug effect occurs at very low levels of receptor occupancy, then additivity is expected even among anesthetics acting on different receptors. Additivity among drugs acting on different receptors is only likely if the concentrations responsible for the drug effect of interest are well below the concentration associated with 50% receptor occupancy.
Article
To study the hypolipidemic active compounds from Crataegus pinnatifida and mechanism of action of those. Guided by the inhibitory activity to HMG-CoA reductase, the active compounds were separated and purified with macroporous resin and silica gel. Four active compounds were obtained, which were quercetin, hyperoside, rutin and chlorogenic acid, the sum of their inhibitory rate was 50.01%, and the total inhibitory rate of the mixture of four active compounds matched was 79.48%. Quercetin and hyperoside were the principle active components inhibiting HMG-CoA reductase in Hawthorn fruit, and there were synergistic action among them.
Article
Hawthorn medicinal extract has long been a favored herbal remedy in Europe. The active components of this slow-acting cardiotonic agent are thought to be flavonoids and oligomeric procyanidins. The most studied hawthorn extracts are WS 1442 and LI 132. Reviews of placebo- controlled trials have reported both subjective and objective improvement in patients with mild forms of heart failure (New York Heart Association classes I through III). Other studies of hawthorn in patients with heart failure have revealed improvement in clinical symptoms, pressure-heart rate product, left ventricular ejection fraction, and patients' subjective sense of well-being. However, there is no evidence of a notable reduction in mortality or sudden death. Hawthorn is well tolerated; the most common adverse effects are vertigo and dizziness. Theoretic interactions exist with antiarrhythmics, antihypertensives, digoxin, and antihyperlipidemic agents. Proven conventional therapies for heart failure are still recommended until the safety and effectiveness of hawthorn has been proven in long-term studies.
Article
The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors from hawthorn fruit ( Crataegus pinnatifida Bge.) were isolated and evaluated for their antihyperlipidemic effect induced by high-fat diet in mice. After being further purified with silica and polyamide column chromatography from the fractions (fractions A, F, H, and G) with a high inhibitory rate (IR) to HMGR, 24 chromatographic fractions were obtained, including 8 active fractions with a high IR to HMGR. However, the total inhibitory activity of 24 fractions was decreased by about 70%. From eight active fractions, four compounds were obtained by recrystallization and identified as quercetin (a), hyperoside (b), rutin (c), and chlorogenic acid (d), the contents of which in hawthorn EtOH extract were 0.16, 0.32, 1.45, and 0.95%, respectively. The IR values of compounds a-d to HMGR were 6.28, 9.64, 23.53, and 10.56% at the corresponding concentrations of 0.16, 0.32, 1.45, and 0.95 mg/mL, respectively. It was discovered that the IR of a mixture (2.85 mg/mL) matching the original percentage of compounds a-d in hawthorn EtOH extract was up to 79.5%, much higher than that of the single compound and the total IR of these four compounds (50.01%). The in vivo results also revealed that the mixture had a more significant lipid-lowering efficacy than the monomers. Structure-activity relationship revealed the inhibitory activity and lowering-lipid ability of compounds a-c decreased with increasing glycoside numbers. It was concluded that there were synergetic effects on inhibiting HMGR and lowering lipid among compounds a-d, and the weak hydrophilic ability benefits the inhibition to HMGR and lowering-lipid efficacy.
Article
Unlabelled: In our present investigation the neuroprotective effect of alcoholic extract of Hawthorn (Crataegus oxycantha) was evaluated against middle cerebral artery occlusion induced ischemia/reperfusion injury in rats. Male Sprague-Dawley rats were pretreated with 100 mg/kg body weight of the extract by oral gavage for 15 days. The middle cerebral artery was then occluded for 75 min followed by 24 h of reperfusion. The pretreated rats showed significantly improved neurological behavior with reduced brain infarct when compared to vehicle control rats. The glutathione level in brain was found to be significantly (p<0.05) low in vehicle control rats after 24 h of reperfusion when compared to sham operated animals. However, in Hawthorn extract pretreated rats the levels were found to be close to that of sham. Malondialdehyde levels in brain of sham and pretreated group were found to be significantly lower than the non-treated vehicle group (p<0.05). The nitric oxide levels in brain were measured and found to be significantly (p<0.05) higher in vehicle than in sham or extract treated rats. Conclusion: Our results suggest that Hawthorn extract which is a well known prophylactic for cardiac conditions may very well protect the brain against ischemia-reperfusion. The reduced brain damage and improved neurological behavior after 24 h of reperfusion in Hawthorn extract pretreated group may be attributed to its antioxidant property which restores glutathione levels, circumvents the increase in lipid peroxidation and nitric oxide levels thereby reducing peroxynitrite formation and free radical induced brain damage.
Article
Opinions about the therapeutic efficacy of medicinal herbs differ significantly. Some reported herbal efficacies at low doses of active ingredients suggest a need for investigating whether these are because of placebo or multi-ingredient synergistic effects. This review discusses the opinions, methods and outcomes of herbal synergism investigations and analyzes indications from 48 in vivo tests and 106 rigorous clinical trials. Analyses of ingredient-mediated interactions at molecular and pathway levels indicate multi-ingredient synergism in 27 of the 39 reported cases of herbal synergism with available ingredient information. Synergistic actions may be responsible for the therapeutic efficacy of a substantial number of herbal products and their mechanisms may be studied by analyzing ingredient-mediated molecular interactions and network regulation.
Article
The longstanding, successful use of herbal drug combinations in traditional medicine makes it necessary to find a rationale for the pharmacological and therapeutic superiority of many of them in comparison to isolated single constituents. This review describes many examples of how modern molecular-biological methods (including new genomic technologies) can enable us to understand the various synergistic mechanisms underlying these effects. Synergistic effects can be produced if the constituents of an extract affect different targets or interact with one another in order to improve the solubility and thereby enhance the bioavailability of one or several substances of an extract. A special synergy effect can occur when antibiotics are combined with an agent that antagonizes bacterial resistance mechanisms. The verification of real synergy effects can be achieved through detailed pharmacological investigations and by means of controlled clinical studies performed in comparison with synthetic reference drugs. All the new ongoing projects aim at the development of a new generation of phytopharmaceuticals which can be used alone or in combination with synthetic drugs or antibiotics. This new generation of phytopharmaceuticals could lend phytotherapy a new legitimacy and enable their use to treat diseases which have hitherto been treated using synthetic drugs alone.
Article
To explore the physiological role of endogenous gaseous sulfur dioxide (SO(2)) on vascular contractility and its underlying cellular and molecular mechanisms, vasodilation experiment of isolated rat thoracic aortic rings by gaseous SO(2) was carried out and the signal transduction pathways involved in the vascular effects of SO(2) were investigated. In the present study, SO(2) gas and SO(2) gas-bubbled solution (SO(2) stock solution) were first used to relax vascular tissues. The results show: (1) Gaseous SO(2) relaxed rat thoracic aortic rings in a dose-dependent manner (from 1 to 2000microM). The vasorelaxant effect of SO(2) at physiological relevant and low concentrations (<450microM) was endothelium-dependent, and at high concentrations (>500microM) was endothelium-independent. (2) The vasorelaxation by addition of SO(2) stock solution (final concentrations 2mM) was actually caused by SO(2) molecules, not by sulfite or bisulfite, and the characteristic of vasorelaxation by SO(2) was different from that of sulfite and bisulfite. (3) The vasorelaxant effect of SO(2) was not due to the altered neurotransmitter release from the autonomous or nonadrenergic and noncholinergic (NANC) nerve endings, also not due to superoxide and hydrogen peroxide produced in the vascular tissues, also disapproving the involvement of prostaglandin, PKC, beta-adrenoceptor and cAMP pathways. (4) The vasorelaxant effect of SO(2) at the physiological relevant and low concentrations was mediated by the cGMP pathway. (5) There was the synergistic effect on smooth muscle relaxation between much lower concentrations of SO(2) (3microM) and NO (3 or 5nM). These findings led to the conclusions: endogenous gaseous SO(2) was a vasoactive factor, which might regulate vascular smooth muscle tone in synergy with NO.
Article
There are a number of different mechanisms that can lead to an improved therapeutic result by the combination of radiotherapy and chemotherapy. Some require interaction between the effects of the two modalities; others require independence of effect. Enhanced killing of tumor cells is only one possible mechanism. There are serious conceptual problems in demonstrating greater-than-additive cell kill whenever dose-response curves are nonlinear. An approach to this problem is suggested, based on an envelope of additivity in an iso-effect plot.