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Crataegus laevigata decreases neutrophil elastase and has hypolipidemic effect: a randomized, double-blind, placebo-controlled trial.

ARTICLE INFO

Keywords: Crataegus laevigata (Rosaceae)

Neutrophil function

Elastase

Coronary heart disease

Randomized, double blind placebo controlled trial

ABSTRACT

Crataegus laevigata is a medicinal plant most commonly used for the treatment of heart failure and psychosomatic disorders. Based on previous experimental findings, this double-blind placebo-controlled study was aimed at finding beneficial effects of C. laevigata on biomarkers of coronary heart disease (CHD). The study included 49 diabetic subjects with chronic CHD who were randomly assigned to the treatment for 6 months with either a micronized flower and leaf preparation of C. laevigata (400 mg three times a day) or a matching placebo. Blood cell count, lipid profile, C-reactive protein, neutrophil elastase (NE) and malondialdehyde were analyzed in plasma at baseline, at one month and six months. The main results were that NE decreased in the C laevigata group compared to the placebo group. In the C laevigata group, baseline figures (median and interquartile range) were 35.8 (4.5) and in the placebo group 31 (5.9). At the end of the study, values were 33.2 (4.7) ng/ml and 36.7 (2.2) ng/ml, respectively; p<0.0001. C. laevigata, added to statins, decreased LDL cholesterol (LDL-C) (mean [+ or -] SD) from 105 [+ or -] 28.5mg/dl at baseline to 92.7 [+ or -] 25.1 mg/dl at 6 months (p = 0.03), and non-HDL cholesterol from 131 [+ or -] 37.5mg/dl to 119.6 [+ or -] 33 mg/dl (p < 0.001). Differences between groups did not reach statistical significance at 6 months. No significant changes were observed in the rest of parameters. In conclusion, C. laevigata decreased NE and showed a trend to lower LDL-C compared to placebo as add-on-treatment for diabetic subjects with chronic CHD.

[c] 2010 Elsevier GmbH. All rights reserved.

Introduction

Reviews of herbal remedies recently published, including references to Crataegus laevigata (Poir.) DC, have claimed to increase the information about their efficacy, safety and potential interactions with conventional therapies before establishing specific therapeutic recommendations (De Smet 2002; Miller et al. 2004; Expert Consensus Document 2005). The beneficial effects of C laevigata observed in basic and animal research on cardiovascular disorders justifies more human studies.

Flowers, leaves and fruit of Crataegus spp. contain polyphenols, mainly procyanidins and flavonoids. Standardizing preparations depending on their procyanidin concentration has been recommended, since these molecules are related to many of its pharmacological effects (Chatterjee et al. 1997). Crataegus has a positive inotropic effect and, unlike other synthetic inotropic drugs, prolongs the refractory period (Joseph et al. 1995). Its antiarrhythmic effect has been observed in vitro and in ischemia-reperfusion animal models (Popping et al. 1995). Myocardial protection has also been demonstrated during ischemic conditions (Al Makdessi et al. 1996; Jayalakshmi et al. 2006; Veveris et al. 2004). One potential therapeutic use could be the atherothrombotic disease. In fact, in vitro inhibition of neutrophil elastase (NE) (Chatterjee et al. 1997), and secretory phospholipase A2 (sPLA2) (Ahumada et al. 1997), as well as anti complementary activity (Shahat et al. 1996), have been shown. A tincture of Crataegus was shown to have hypocholesterolemic action in atherogenic diet fed rats by increasing bile acid excretion and depressing hepatic cholesterol synthesis (Rajendran etal. 1996). C laevigata also decreased oxidative stress and lipid peroxidation, not only with the entire plant but also with their isolated compounds (Quettier-Deleu Voiselle et al. 2003). We have previously reported that a C laevigata dry extract modified in vitro neutrophil functions, with a dose-dependent reduction of fMLP-induced NE release, superoxide anion generation, leukotriene B4 production and lipopolysaccharide-induced generation of tumour necrosis factor-alpha and interleukin-8. C laevigata also produced a dose-dependent inhibition of in vitro neutrophil migratory capacity (Dalli et al. 2008). In humans, the Crataegus extract WS 1442 improved symptoms and exercise parameters in heart failure (Weikl et al. 1996; Tauchert 2002; Zapfe 2001). A meta-analysis of randomized, placebo-controlled trials suggested a benefit favouring the treatment with Crataegus over placebo (Pittler et al. 2003). Recently, the SPICE trial randomized 2681 patients with NYHA class II or III congestive heart failure to treatment with the Crataegus extract WS 1442 or placebo, added on top of conventional medication. No differences were observed in the primary end point (a composite of sudden cardiac death, death due to progressive heart failure, fatal myocardial infarct (Ml), nonfatal MI, or hospitalization due to heart failure progression), after one year of treatment. Nevertheless, in the subgroup of patients with ejection fraction (EF) between 25 and 35%, a 39.7% reduction of sudden cardiac death was observed (Holubarsch et al. 2008). Neutrophils infiltrate the postischemic myocardium and cause much of the myocardial dysfunction associated with this condition (Kyne et al. 2000). In the coronary arteries, they are involved from the initial steps of fatty streak formation until the rupture of the advanced atherosclerotic plaques. NE has the potential to preferentially disrupt the elastic network but also collagen, fibronectin, proteoglycan and laminin fibres (Owen and Campbell 1999). NE is elevated in patients with coronary heart disease (CHD) and is correlated with the complexity and severity of the coronary stenosis in patients with unstable angina (Amaro et al. 1995). The present study was aimed at finding new therapeutic options for C laevigata in secondary prevention, by measuring C laevigata treatment-induced changes compared to placebo in lipid parameters, oxidative stress, and plasmatic NE levels, in diabetic patients with chronic CHD, in which the atherothrombotic process is more aggressive.

Subjects and methods

Patients

Subjects were eligible for participation if they were between 45 and 75 years old and had been previously diagnosed of type 2 diabetes mellitus and chronic CHD. Patients were recruited from the outpatient clinic of our hospital. Diagnosis of diabetes mellitus was established with a fasting blood sugar test [greater than or equal to] 126mg/dl, HbAlc > 6.5% or hypoglycaemic treatment. Chronic CHD was considered with prior diagnosis of unstable angina or myocardial infarction, and at least one coronary lesion [greater than or equal to] 70%, revascularized or not. Patients had been asymptomatic within the previous 6 months (NYHA class I). Exclusion criteria were: HbAlc > 10%, malignancy, cerebrovascular, kidney, pulmonary or liver disease, treatment with non steroidal anti-inflammatory drugs, and having taken vitamins, dietary or herbal supplements three months before entry. All types of alcohol beverages, fruit juices or tea infusions were prohibited during the study period. The habit of smoking was defined as a consumption of at least one cigarette a day in the last three months, hypertension with blood pressure > 140/90 mm Hg or pharmacological treatment, dyslipemia was considered if cholesterol > 200mg/dl, triglycerides > 150mg/dl or pharmacological treatment. The last dosage change of hypolipemic or hypotensive treatment was made at least 3 months before the start of the study. The study was conducted in accord with the Declaration of Helsinki and following guidelines for Good Clinical Practice. All subjects gave written informed consent to participate. The study was approved by the Local Ethics Committee and Sanitary Authorities.

Protocol

Visits and blood tests took place the morning after an overnight fasting at baseline, and one month and six months after treatment. A medical history check and a physical exam were carried out in the first visit. The analytical variables included were blood ceil count, and levels of glucose, urea, creatinine, electrolytes, high sensitive C-reactive protein (CRP), lipids, neutrophil elastase (NE) and mal-ondialdehyde (MDA). Eligible patients were assigned to C laevigata or placebo in a double-blind fashion by computer-generated numbers. C laevigata was administered by means of micronized flowers and leaves, standardized for a content of 5% of procyanidins and 2% of flavonoids (Crataesor[R], Soria Natural SL, Spain). According to the information provided by the manufacturer, procyanidin quantification was made as follows: 1 g of the C laevigata preparation was extracted four times with 25 ml of acetone/water (70/30) by stirring at room temperature for 15min. Extraction was followed by centrifugation at 200 g for 5 min and the supernatant was collected. The precipitate was reextracted. All extracted material was mixed and filtered on paper. 10 ml of extraction was evaporated to dryness, not exceeding 30[degrees]C. Dried residue was dissolved in a mixture of n-BuOH-HCl (95:5). After heating in a water bath under reflux and stirring for 110 min, the solution was cooled to room temperature and made up to 100ml with n-BuOH-HCl 37% (95:5). The absorbance was measured in the spectrophotometer at [lambda] = 540 nm, considering the n-BuOH-HCl as a white reference. Procyanidin content (expressed as cyanidin chloride) was derived from the formula:

% procyanidins = E x 4.115 / b

where E = absorbance and b = weight of dried powdered tissue (Kartnigetal. 1993).

The HPLC analysis of the flavonoids in C laevigata was performed with an apparatus Agilent 1100 with programmable gradient. The column used was an Agilent XOB-C18. The flow rate was 1 ml/min. The concentration of the main flavonoids was: vitexin-2-O-rhamnoside 14.2 mg/g, chlorogenic acid 3.1 mg/g, hyperoside 2 mg/g, hesperidin 1.4 mg/g, quercitrin 1.1 mg/g, caffeic acid 0.7 mg/g, myricitrin 0.2 mg/g, isovitexin 0.1 mg/g. The HPLC profile analysis is shown in Fig. 1. Placebo consisted of microcrys-talline cellulose with appropriate masking conditions. Cellulose powder was coloured green, similar to Crataegus, and both treatments were identically packaged and supplied to study subjects by the Hospital Pharmacy Department. Each patient took 400 mg of either C. laevigata or placebo, three times a day, for 6 months added to their conventional treatment. No modification of concomitant medications was allowed during the study period. All participants underwent counselling to ensure their adherence to the study medication and maintenance of their usual life style (diet and physical activity) throughout the study period. Compliance was carried out by pill counting.

Laboratory data

Blood cell count and biochemical parameters were analyzed in the core laboratory of the hospital. Levels of cholesterol and triglycerides in plasma were measured by automatic procedures using Vitros diagnostic products (Ortho-Clinical Diagnostics, Rochester, NY). The methodologies for triglycerides (Vitros TRIG Slides) and total cholesterol (Vitros CHOLSlides) are based on enzymatic methods as previously described (Spayd et al. 1978; Allain et al. 1974). HDL cholesterol (HDL-C) was measured by means of the VITROS dHDL slide assay based on methodology previously proposed (Allain et al. 1974; Burstein et al. 1970). LDL cholesterol (LDL-C) was calculated by the Friedewald formula (Friedewald et al. 1972). CRP was measurer with a high-sensitive immunoassay and determinations were performed with the Behring Nephelometer BN II (Dade Behring, Germany), according to the method described elsewhere (Rifal et al. 1999). Plasma aliquots were fib ozen and stored at -80 [degrees] until further analysis. MDA was analyzed by HPLC following Wong et al.'s (1987) method, as described in Oltra et al. (2001). NE was measured by the Human Elastase ELISA kit (HyCult Biotechnology b.v., Uden, The Netherlands) according to the manufacturer's instructions. Each sample was analyzed in at least two independent experiments with duplicate samples.

[FIGURE 1 OMITTED]

Statistical analysis

The Kolmogorov-Smirnov test was used to determine whether or not the data were normally distributed. Normally distributed variables are expressed as mean [+ or -] SD, and not normally distributed variables are expressed as median and interquartile range. Due to the small sample size non-parametric test were used. Differences between continuous and discontinuous parameters at baseline were tested using the Mann-Whitney U test or Chi-squared test, respectively. Intra-group comparisons among baseline values and values at 1 and 6 months were performed by the Friedman's test. The statistical significance of the differences between variables in the placebo and C laevigata groups at baseline and after treatment was analyzed using the Kruskal-Wallis test. Patients who withdrew prior to the first month follow-up visit were not included in the efficacy analysis. Missing data were not replaced. Statistical significance was set at p < 0.05. The analyses were performed using SPSS version 15.0.

Results

Subjects

A total of 49 type 2 diabetic patients with chronic CHD, were included. They had a mean age of 62 [+ or -] 8 years in the C laevigata group and 60 [+ or -] 8 years in the placebo group. Males were predominant in both groups, 20 (83%) in the C laevigata group and 18 (80%) in the placebo group. Concomitant medication was as follows: aspirin (100%), statins (84%), angiotensin-converting enzyme inhibitors (ACE1) (70%), betablockers (55%), calcium channel blockers (14%) and nitrates (20%), with no differences between either group. Patients' clinical and analytical data are shown in Table 1. At the end of the treatment period, 45 patients were available for the efficacy evaluation. Four patients were prematurely withdrawn from the study within the first 3 weeks (three in the placebo group and one in the C laevigata group), therefore no analytical data were recorded. In the placebo group, one patient presented unstable angina three weeks after randomization, another one referred abdominal discomfort and dizziness, and the last one suffered from upper respiratory tract infection. In the C laevigata group, one patient had digestive intolerance that disappeared after treatment withdrawal. All other participants completed the study with no side effects and good tolerance to C laevigata or placebo. As mentioned before, there were no changes in usual diet, regular medical treatment, and the body mass index was unchanged throughout the study period. Blood pressure did not change significantly in either group. In the Crataegus group subjects took 93% of capsules, and in the placebo group 91%. One patient in each group had a treatment compliance of Less than 80%.
Table 1

Demographic and analytical data of patients by treatment groups.

 C. laevigata (n Placebo (n = p value
 = 24) 21)

Age (year) 61.3 [+ or -] 60.4 [+ or -] 0.58
 8.3 8.2

Male (n%) 20 (83%) 18 (85%) 0.91

Coronary risk factors (n) 2.8 [+ or -] 3.1 [+ or -] 0.46
 0.7 0.9

Coronary lesions (n) 2A [+ or -] 0.8 1.7 [+ or -] 0.15
 0.7

Leukocyte count (x 6.9 [+ or -] 7.1 [+ or -] 0.85
[10.sup.3] /[mu] l) 1.4 2.3

Glucose (mg/dl) 151.6 [+ or -] 169.8 [+ or -] 0.35
 50.5 70.0

Statins (%) 85 80 0.68

ACEI (%) 55 80 0.09

Calcium-channel blockers 29 33 0.87
(%)

Values presented are mean [+ or -] SD or percentage frequency, as
appropriate. ACEI: angiotensin-converting enzyme inhibitors.


Blood cell count and lipid profile

There were neither changes in haemoglobin, erythrocytes, platelets or leukocytes, including neutrophils, nor in plasma concentrations of urea, creatinine or electrolytes. A significant reduction in the concentration of total cholesterol (p<0.05), LDL-C (p = 0.03) and non-HDL cholesterol (non-HDL-C) (p = 0.001) was observed with C. laevigata supplementation compared to baseline values. At the end of the study, LDL-C was reduced a mean of 13mg/dl (11%) and non-HDL-C a mean of 13 mg/dl (10%) compared to baseline. According to the statistical analysis of differences between both groups, active therapy compared to placebo resulted in a trend to lower total cholesterol (p = 0.11), LDL-C (p = 0.054) and non-HDL-C (p = 0.13). No differences were observed in HDL-C or triglycerides. One patient in the placebo group and two patients in the Crataegus group had hypertriglyceridemia, explaining the higher standard deviation and the non-estimation of LDL-C in these cases. The rest of subjects had triglyceride levels below 210mg/dl (Table 2).
Table 2

Change of analytical parameters from baseline in the C laevigata and
placebo groups.

 Baseline 1 month 6 months
 (n = 45) (n-45) (n-45)

Leukocyte count
(x [10.sup.3] /
[mu] l)

 C laevigata 6.9 [+ or -] 6.9 [+ or -] 7.1 [+ or -]
 1.4 1.3 1.0

 Placebo 7.1 [+ or -] 7.1 [+ or -] 73 [+ or -] 2.4
 2.3 2.3
Neutrophil count
(x [10.sup.3] /
[mu] l)

 C.laevigata 4.3 [+ or -] 4.1 [+ or -] 4,1 [+ or -]
 1.0 0.9 0.7

 Placebo 4.5 [+ or -] 4.1 [+ or -] 4.1 [+ or -]
 1.9 0.9 1.5

Glucose
(mg/cll)

 C laevigata 151.6[+ or 155.5 [+ or 1543 [+ or -]
 -]50.5 -]52.0 44.6

 Placebo 169.8 [+ or -] 172.9 [+ or -] 161.2 [+ or -]
 70.0 77.0 57.2

TC(mg/dl)

 C laevigata 177.1 [+ or -] 171.4 [+ or -] 166.8 [+ or -]
 32.1 33.3 29.5 (a)

 Placebo 174.3 [+ or -] 173.6 [+ or -] 175.2 [+ or -]
 20.6 21.5 25.0

LDL-C (mg/dl)

 C. laevigata (n 102.2 [+ or -] 94.9 [+ or -] 893 [+ or -]
 = 22) 25.8 22.3 20.2 (b)

 Placebo (n = 97.9 [+ or -] 98.9 [+ or -] 1023 [+ or -]
 20) 15.9 15.9 21.1

HDL-C (mg/dl)

 C. laevigata (n= 47.0 [+ or -] 47.2 [+ or -] 47.2 [+ or -]
 22) 12.0 11.5 10.8

 Placebo (n = 45.2 [+ or -] 43.7 [+ or -] 45.7 [+ or -]
 20) 9.0 9.2 8.7

Non-HDL-C
(mg/dl)

 C laevigafa 131.0 [+ or -] 124.8 [+ or -] 119.6 [+ or -]
 (n=22) 37.5 37.4 33.0 (c)

 Placebo (n = 130.1 [+ or -] 129.9 [+ or -] 128.3 [+ or -]
 20) 23.9 22.4 26.0

Triglycerides
(mg/dl)

 C laevigata 142.7 [+ or -] 142.6 [+ or -] 140.8 [+ or -]
 105.5 109.3 104.0

 Placebo 197.1 [+ or -] 185.0 [+ or -] 174.9 [+ or -]
 213.3 210.8 231.1

Neutrophil
elastase
(ng/ml)

 C taevigafa 35.8 (4.5) 33.2 (5.5) 33.2 (4.7) (d)

 Placebo 31.0 (5.9) 32.6 (5.4) 36.7 (2.2) (e)

MDA (ng/ml)

 C laevigata 0.9 [+ or -] 0.7 [+ or -] 0.8 [+ or -]
 0.4 0.4 0.4

 Placebo 1.0 [+ or -] 0.9 [+ or -] 0.9 [+ or -]
 0.5 0.3 0.3

Hs-CRP(mg/l)

 C. laevigata 2.5 [+ or -] 3.5 [+ or -] 2.6 [+ or -]
 2.5 4.6 3.4

 Placebo 43-7.5 2.6 [+ or -] 3.2 [+ or -]
 2.5 4.3

Values are the mean [+ or -] standard deviation except for elastase,
expressed as median (interquartile range).
(a) p<0.05 vs. baseline.
(b) p = 0.03 vs. baseline.
(c) p<0.001 vs. baseline.
(d) p<0.0001 vs. placebo group.
(e) p<0.0001 vs. baseline.


Lipid peroxidation and C-reactive protein

MDA was assessed as expression of lipid peroxidation. No significant change was observed after the treatment period with either C laevigata or placebo compared to baseline values. No significant differences were found in CRP between C laevigata and placebo groups at the end of the study (Table 2).

Neutrophil elastase

Plasmatic NE levels were generally comprised within a homogeneous and relatively narrow range, with only three patients in the placebo group and two patients in the Crataegus group presenting higher levels at baseline. These high levels returned to normal range in the C laevigata group but not in the placebo group, in which the mean value increased significantly along the study period. Individ-ual data for both groups are displayed in Fig. 2. At the end of the study, comparison between groups showed a significant decrease of NE with C laevigata treatment compared to placebo (Fig. 3).

Discussion

The main findings of this pilot trial are that C. laevigata decreases NE in plasma and shows a trend to decrease LDL-C values compared to placebo, in type 2 diabetic patients with chronic CHD under standard treatment, including statins. Most human studies with Crataegus extracts have been addressed to the treatment of heart failure with results favouring C laevigata extracts over placebo in terms of increment of exercise capacity and improvement of the double product, but these studies were carried out before the ACEI or betablockers era (Weikl et al. 1996; Tauchert 2002; Zapfe 2001; Pittler et al. 2003). In the SPICE trial, a C laevigata extract, on top of usual treatment for heart failure, reduced sudden cardiac death in the subgroup of patients with EF higher than 25% (Holubarsch et al. 2008). In animal models, C laevigata yielded myocardial protection in ischemic conditions (Al Makdessi et al. 1996; Veveris et al. 2004; Jayalakshmi et al. 2006) and showed antiarrhythmic effects in an animal model of ischemia-reperfusion (Al Makdessi et al. 1999). Since there is no information from human studies, the present work was aimed at identifying beneficial effects derived from C laevigata on top of conventional treatment, on some clinical and analytical parameters, related to secondary prevention of CHD. With regard to blood pressure, no significant changes were observed compared to placebo, probably because patients were normotensive under treatment with many different drugs, mainly ACEI, followed by betablockers and calcium channel blockers. A weak hypotensive effect with Crataegus curvisepala has been described as a monotherapy (Asgary et al. 2004), In a placebo-controlled study with another Crataegus formulation added to prescribed hypotensive drugs, a significant reduction in diastolic blood pressure could be observed (Walker et al. 2006). One of the benefits of C laevigata observed in the present study was its trend to reduce total cholesterol, LDL-C and non-HDL-C, which deserves further investigation because the small sample limits the clarification of this point and a possible interaction with statins cannot be ruled out. Interestingly, this was observed in patients on statins and with low cholesterol baseline levels. No significant changes in either HDL-C or triglycerides were seen. Rajendran et al. (1996) had previously observed a hypolipemic effect in mice fed with a tincture of C laevigata, which produced an increase in the hepato-cyte LDL receptor activity, an increase in cholesterol degradation into biliary acids, and a reduction of cholesterol biosynthesis. In traditional Chinese medicine, a C laevigata berry extract has been used as a hypolipemic agent (La Cour et al. 1995). Chen et al. tested a special drink made of C. laevigata extract, vitamins C, Bl, B2, iron and zinc on 30 subjects, resulting in a reduction of total cholesterol and LDL-C, without modification of HDL-C and triglycerides. They also observed a decrease in plasmatic MDA, perhaps not attributable only to Crataegus due to the associated vitamin content (Chen et al. 1995). Another natural product rich in procyanidins, red grape juice, has demonstrated recently to have hypolipemic effects both on hemodialysis patients and healthy subjects (Castilla et al. 2006). Decreased [Cu.sup.2+]-induced LDL oxidation has been demonstrated with C. laevigata in vitro (Quettier-Deleu Voiselle et al. 2003). In the present study in humans, no changes in MDA were observed in either of the two treatment groups. It can be hypothesized that statin-associated treatment in 85% of the subjects and baseline low LDL-C could have attenuated a possible effect of the experimental drug. The same comments could be made concerning CRP, also considering the impact of statins on this parameter. Our results confirm, for the first time, that C laevigata can reduce plasmatic NE in humans but only under experimental conditions, as we have already demonstrated (Dalli et al. 2008). The anti-elastolytic activity of C. laevigata was initially described in vitro by Chatterjee et al., through direct enzyme inhibition. This effect was only reproduced by the procyanidin fraction, not by the flavonols or flavones of the plant extract (Chatterjee et al. 1997). The anti-elastolytic action of C laevigata is shared by other plants, but only under experimental conditions (Tring et al, 2009), not yet in humans. Ahumada et al. (1997) demonstrated that the isolated triterpenic fraction of C monogyna inhibited leukocyte migration in a mouse model of peritonitis induced by carrageenan, and a weak in vitro inhibition of the phospholipase A2. Although not entirely related to our results, it can be mentioned that previous reports associate elastase with phospholipase A2 in the inflammatory process, both in vascular endothelium (Ginsburg et al. 1993), and traqueal epithelium (Kosuke et al. 2007).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

In the present study, only a few subjects in each group presented high elastase levels at baseline. Similar figures were reported by Amaro et al. (1995), who observed high elastase levels only in a small number of subjects with complex coronary lesions compared to subjects with simple lesions. After treatment with C laevigata, high elastase levels returned to normal values, however, after placebo treatment, they remained elevated and even increased over time. Not only the two outliers drove the findings for the treatment group but also a majority of subjects showed a decrease. The observed results could be relevant because elastase has been demonstrated to be involved at the beginning, progression and thrombotic complications of atherotrombosis (Martin-Ventura et al. 2007). Culprit coronary lesions contain neutrophils that can be identified by means of antibodies against elastase and myeloperoxidase (Naruko et al. 2002). In the Edinburgh Artery Study, patients with angina pectoris with leukocyte elastase levels in the top fertile were at increased risk of thrombotic cardiovascular events compared to the lowest tertile, after adjustment by cardiovascular risk factors such as age, sex, insulin resistant syndrome, and previous myocardial infarction (Tzoulaki et al. 2007). In diabetic patients, high elastase levels in plasma have been shown to be related to neoangiogenesis (Piwowar et al. 2000). Elastase, not only degrades elastin and some collagen fibres but also can modulate the activity of cytokines, thus increasing metalloprotease synthesis and regulating protease-activated receptors (Wiedow and Meyer-Hoffert 2005; Pham 2006; Shamamian et al. 2001; Leclercq et al. 2007). It has been suggested that protease activity retention in the arterial wall could be a new therapeutic target in atherosclerosis (Houard et al. 2006). Rudolph et al. (2007) recently reported high plasma levels of myeloperoxidase and elastase in both ischemic and nonischemic cardiomyopathy. Regarding the results of the present study a possible neutrophil modulation of C laevigata in subjects with heart failure could be hypothesised. The patients in our study received C laevigata or placebo on top of the best available therapy, including aspirin, statins, ACEI, betablockers or nitrates. These drugs, however, may not prevent the intraplaque progressive expansion that can be prevented by, for example, lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibition, as has been recently published (Serruys et al. 2008). Certain molecules may be persistently elevated after an acute myocardial infarction and may not be effectively counteracted by conventional treatment, i.e., neutrophil elastase (Caimi et al. 2005). This could be related to our observation of an increase of NE in the placebo group. Modulation of the inflammatory cells within the arterial wall is a new therapeutic target that merits further investigation. Until new synthetic molecules are available, an amount of naturally occurring substances, mainly phytochemicals, whose pharmacological effects are well documented in basic and animal research, could be tested in humans. Herbal supplements are increasingly used in patients with cardiovascular diseases in western countries but there is scarce scientific information about their benefits, risks or pharmacological interactions (Wood et al. 2003). These conclusions can be applied to C. laevigata, one of the most used herbal remedies at cardiovascular level. The present study has shown that C laevigata is safe and well tolerated when administered to diabetic subjects with chronic CHD under usual treatment for ischemia and secondary prevention. Further investigation would be required to confirm the results herein obtained and the possible therapeutic implications on secondary prevention of CHD. It can be concluded that a micronized flower and leaf preparation of C. laevigata has been demonstrated, for the first time, to decrease plasmatic NE levels, along with a trend to lower LDL-C in diabetic patients with chronic CHD, compared to placebo. These benefits were observed on top of conventional treatment. No significant side effects were observed.

Limitations

The hypolipemic effect of C laevigata was observed in a reduced group of subjects when comparing figures at baseline and after treatment. Compared to placebo, only a trend to decrease cholesterol, LDL-C and non-HDL-C was observed. The results are certainly not enough to establish definite conclusions, nevertheless, it is in accordance with previously published studies and, for the first time, it was observed in subjects under treatment with statins and low LDL-C levels at baseline. Although the estimate of the LDL-C was not direct but calculated by the Friedewald formula, the exclusion of those subjects with triglycerides upper to 210mg/dl in any of the determinations guarantees the reliability of the results.

No pharmacokinetic studies were made in the present investigation, and no references of this type of studies with Crataegus species have been conducted in humans. The only available information comes from cocoa and grape procyanidins detected in the plasma of human subjects after acute consumption, which has been related to the observed effects (Holt et al. 2002; Neukam et al. 2007).

Funding

The study was supported by grants from Soria Natural Laboratories and the Spanish Instituto de Salud Carlos III (RETICS to GSTt RD06/0045/0006).

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Abbreviations: NE, nentrophil elastase; sPLA2, secretory phospholipase A2, Lp-PLA2, lipoprotein-associated phospholipase A2; fMLP, N-formyl-rnethionyllencyl-phenylalanine; NYHA, New York Heart Association; EF, ejection fraction; MI, myocardial infarction; CHD, coronary heart disease; HbAl c, glycollated haemoglobin AIC; CRP, C-reactive protein; MDA, malondialdehyde; ACEI, angiotensin-converting enzyme inhibitors.

Tel.: +34 963868466; fax: +34 963868466.

E-mail address: edalli@hotmail.es (E. Dalli).

0944-7113/$ - see front matter[C] 2010 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2010.11.011

E.Dalli (a) *, E.Colomer (a), M.C. Tormos (b), J. Cosin-Sales (a), J.Milaea (c), E. Esteban (a), G. Saez (b)

* Corresponding author at: Servicio de Cardiologia, Hospital Asociado Universetario Arnan de Vilanova, C/San Clemente 12, 46015 Valencia, Spain.

(a) Department ofCardiofogy,, Associated University HospitalArenau de Vilanova, Valencia, Spain

(b) Department of Biochemistry and Molecular Biology, School of Medicine, University of Valencia,Valencia, Spain

(c) Department of Pharmacology, School of Medicine. University of Valencia,,Valencia, Spain
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Author:Dalli, E.; Colomer, E.; Tormos, M.C.; Cosin-Sales, J.; Milara, J.; Esteban, E.; Saez, G.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUSP
Date:Jun 15, 2011
Words:5662
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