Protective effects of Rosmarinus tomentosus ethanol extract on thioacetamide-induced liver cirrhosis in rats.
The capability of an ethanol extract of Rosmarinus tomentosus to protect rat liver in an experimental model of cirrhosis induced by thioacetamide (TAA) has been evaluated. Four groups of rats were used: Two of them received 300 mg TAA/l in the drinking water for 3 months while the other two, which served as controls, were given water ad libitum. During the same period and for each one of the treatments, one group received a semi-purified (SP) diet and the other one was fed the same diet supplemented with 1% of the dry residue obtained from R. tomentosus ethanol extract (SP + E). There was a significant reduction of TAA toxicity in rats fed the SP + E diet, as assessed by plasma and liver biochemical markers, and by liver histopathology. Plasma total protein concentration was restored, urea concentration and plasma alkaline phosphatase and [gamma]-glutamyl-transferase activities were reduced. A significant correction of plasma fatty acids concentrations was also evident. Hepatic alkaline phosphatase and [gamma]-glutamyl-transferase activities were significantly reduced in animals fed SP + E diet and glucose-6-phosphatase activity was significantly enhanced. The results suggest that R. tomentosus ethanol extract administered in the diet affords protection against TAA-induced cirrhosis, preventing most of the histological changes and functionality alterations own to this experimental pathology.
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Keywords: Rosmarinus tomentosus; Hepatotoxicity; Cirrhosis; Thioacetamide
Among liver diseases, cirrhosis is an important and common cause of human mortality in many western countries. It is the end-stage of most liver pathologies of different aetiologies and leads to chronic liver dysfunction, accompanied by important metabolic alterations. To date, no treatment has revealed efficient to prevent or to avoid the progress of this pathology and, in most instances, patients receive treatment for complications derived from the disease (Mavier and Mallat, 1995).
Different vegetal species have been described for providing active principles that protect against liver damage in experimental animal models (Liu et al., 1993, 1995; Scott, 1999). Ethanol extract of the endemic species Rosmarinus tomentosus has been previously reported by our group for its antihepatotoxic effects in experimental animal models of acute liver damage induced by CC[l.sub.4] (Navarro et al., 1993) and thioacetamide (TAA) (Galisteo et al., 2000).
The experimental liver damage induced by long exposure and/or high doses of TAA results in histological and biochemical changes that present most similarities with the human disease (Zimmermann et al., 1987). Fatty acids profile is also modified in rats with cirrhosis induced by TAA (Moreira et al., 1995; Fontana et al., 1998).
Based on our previous results on acute models of liver toxicity, in the present study we have looked for the effects of the oral dietary administration of R. tomentosus ethanol extract in an experimental model of cirrhosis induced by the intake of TAA in the drinking water for 3 months. We have analyzed liver histology, functional biochemical parameters and plasma fatty acids profile, to see whether our extract prevents this experimental pathology and how.
Materials and methods
R. tomentosus specimens were collected during its flowering period in Azud de Velez (Velez de Benaudalla, Granada, Spain), an area where this endemic species did not overlap with others of the same genus. They were authenticated and a voucher specimen was deposited at the Herbarium of the Faculty of Pharmacy, University of Granada (GDA) under catalogue number 19741. The aerial parts of the plant were separated from the rest and dried at room temperature over 48 h. Five hundred grams of this plant material was extracted with ethanol in a Soxhlet device. The extract was concentrated at reduced pressure to obtain a dry residue, which had a yield of 29.2% w/w. The phytochemical groups present in this dry residue were qualitatively determined by methods previously described (Farnsworth, 1966). The main groups found, namely triterpenes, phenolic acids [e.g. rosmarinic acid and picrosalvin (= carnosol)] and flavonoids, were also quantified. Triterpenes were assayed by colorimetric analysis, using ursolic acid as standard, as previously described (Hiai et al., 1975). Phenolic acids content was determined according to the method of Nichiforesco and Coucou (1965). The flavonoids content was measured by the method of Fernandez Costa (1982). The relative contents of these groups in the dry residue were 33.6%, 4.2% and 1.2%, respectively. The essential oil content of the extract was 1.7% v/w, and camphor was its main component (80%). HPLC fingerprint of the ethanol extract is shown in Fig. 1 (HPLC DAD; Column RP18, 5 [micro]m, 125 x 4 mm, Merck; Solvent A: water + 10 ml 0.1 N [H.sub.3]P[O.sub.4]/l, Solvent B: acetonitrile; Flow rate 1.0 ml/min; Detection 270 nm; Injection 10 [micro]l ethanol extract; Gradient: 10-30% B in 10 min, 30% B for 5 min, 30-60% B in 7 min, 60% B for 3 min, 60%-90% B in 5 min). Adult female Wistar rats (Interfauna Iberica, Barcelona, Spain) were kept in wire-bottomed cages at a controlled temperature (21 [+ or -] 2 [degrees]C) and a 12 h light-dark cycle for at least a week before the treatment. The animals were randomly divided into four groups of ten individuals. Two of these groups received a semi-purified (SP) diet (Table 1) (ILAR, 1979), while the other two were fed the same diet supplemented with 1% of the dry residue obtained from R. tomentosus ethanol extract (SP + E). For each type of diet, one of the groups received 300 mg TAA/l in the drinking water for 3 months, and the other was given water during the same period. No differences in the consumption of drinking water containing TAA was observed between the groups fed the SP and the SP + E diets (animals of both groups received approximately 25 mg TAA/day/kg body wt). We will refer the groups fed SP diet as C (control) and T (TAA), while those receiving SP + E will be represented as CE (control) and TE (TAA). All over the treatment period, pair-fed control rats were given the same amount of diet as that consumed by the TAA-treated rats on the previous day, to equalise the nutritional intake of the groups. After the 3-month treatment and 3 additional days in which animals of the intoxicated groups received water to eliminate TAA acute effects, rats were fasted over 18 h before sacrifice. Animals were anaesthetised intraperitoneally with 1.25 g/kg body wt. of urethane. Blood was collected by abdominal aorta artery punction with sterile heparinized syringes, and centrifuged at 3000g for 15 min at 4 [degrees]C to obtain plasma. Livers were immediately removed, washed, weighed, and divided, as well as plasma, in aliquots, which were immediately frozen and stored at -80 [degrees]C until their analysis. Liver samples from the left lobe were obtained for light microscopy observation. They were fixed in 4% paraformaldehyde in phosphate-buffered solution for 3 days, dehydrated in ethanol, and embedded in paraffin by conventional methods. Sections of 5 [micro]m thick were stained with periodic acid-Schiff reagent and examined under a light microscope. Plasma protein concentration was determined using a Spinreact kit. Urea levels in plasma were measured by means of a Dipal kit. Plasma ASAT and ALAT activities were determined using Boehringer kits. ALP and [gamma]-GT activities were assayed in plasma and soluble fraction of liver homogenates, using Boehringer kits. Hepatic glucose-6-phosphatase (G-6-Pase) was determined in soluble fraction of liver homogenates (Harper, 1965). Liver ASAT and ALAT activities were assayed by the methods of Bergmeyer and Bernt (1974). Glutamate dehydrogenase (GDH) was measured in liver homogenates as described by Schmidt (1974). Threonine deaminase (TD) was assayed by the method of Burns (1971). Hepatic protein concentration was measured according to Bradford (1976). Plasma fatty acids were extracted and methylated in a one-step reaction (Lepage and Roy, 1986), and fatty acids methyl esters were quantified by gas-liquid chromatography in a Hewlett Packard chromatograph as previously described (Moreira et al., 1995).
[FIGURE 1 OMITTED]
Results are expressed as means [+ or -]SEM. Comparison between means values were made by a two-way analysis of variance to evaluate the combined effects of two variables (diet and TAA) and a posteriori Bonferroni tests. p < 0.05 was considered statistically significant. All data were evaluated for statistical significance using the BMDP software (Los Angeles, CA, USA) (Dixon et al., 1990).
At the beginning of the experiment the rats weighed 160-175 g, and after 3 months animals of groups C and CE reached body weights of 235.4 and 239.2 g, respectively. Chronic intake of TAA significantly decreased the body weight gain of animals from groups T and TE compared to those of groups C and CE. There was no difference of body weight between TAA-intoxicated rats (T and TE groups) (Table 2). Livers of animals of T and TE groups were significantly heavier compared to their respective controls, C and CE groups. No difference was seen between C and CE groups. Liver weight of animals from TE group was significantly decreased compared to the T group.
TAA administration for 3 months led to micronodular cirrhosis. Rat livers of T group showed regenerative parenchyma nodules surrounded by septa of fibrous tissue with a significant increase in fat storing cells, Kupffer cells and bile ductules (Fig. 2B). Livers of group TE animals showed an appreciably histological regeneration (Fig. 2C) compared to those of the T group. They showed a reduced extent and development of fibrous septa and an increase in the extension of normal hepatic parenchyma, and in no case led to the nodular parenchyma transformation own to the cirrhotic process. No histological differences were observed between healthy animals (groups C and CE) (Fig. 2A).
Intake of TAA produced a significant decrease of plasma total protein and increase of urea concentration compared to the C group. A reversal of both parameters was observed in plasma of the TE group rats (Fig. 3). Concerning polyunsaturated fatty acids, the extract treatment is accompanied of a decrease down to normal values of 18:2n-6 and a simultaneous rise of 20:4n-6, together with a certain recovery of 18:3n-6 and an increase of values of 22:6n-3 with respect to the controls. The extract decreases the total of monounsaturated fatty acids in the TE group, and also increases the total of polyunsaturated fatty acids on the series n-6 (n-6 > 18C) (Table 3). Plasma enzyme activities are shown in Table 4. No differences between the intoxicated groups and their respective controls were shown concerning plasma aminotransferases activities. However, the intake of supplemented extract diet (CE and TE groups) produced a diminution in ASAT and ALAT plasma activities compared to the groups fed the SP diet (C and T groups). Plasma ALP and [gamma]-GT activities rose significantly in TAA-intoxicated rats (T and TE groups) respect to their controls (C and CE). Animals of group CE showed significantly lower levels of ALP activity compared to the C group, while no differences were observed between both groups concerning [gamma]-GT activity. Both parameters were significantly decreased in TE rats compared to the intoxicated animals fed SP diet (group T).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Hepatic ALP and [gamma]-GT activities were significantly increased in T and TE animals, whereas G-6-Pase was markedly reduced compared to C and CE groups (Table 5). Impact of TAA on ALP, [gamma]-GT and G-6-Pase activities, was significantly reduced in TE group animals compared to the T group. Hepatic ALAT, GDH and TD activities were significantly diminished in the TAA-treated groups (T and TE) compared to their respective controls (C and CE). Animals of TE group showed significant increases of ALAT and GDH activities compared to the T group.
The model of liver cirrhosis induced in rats by oral administration of TAA for 3 months produces many of the alterations own to the human cirrhosis (Zimmermann et al., 1987; Fontana et al., 1996). In the present work, the livers of rats that received TAA showed signs of cirrhosis, and there was a general alteration of different biochemical plasma and liver markers in the cirrhotic rats. Body weight of cirrhotic animals was significantly lower compared to animals of control group, although they were paired fed, showing a decrease in the body weight gain caused by chronic administration of TAA. This effect could be associated to alterations in nutrient absorption and metabolic utilization, efficiency that has been described in the same experimental model of TAA-induced cirrhosis (Ortega et al., 1997). Liver weight was significantly increased in cirrhotic rats, and that was also the case of mean relative liver weight. Although rats of group TE did not recover healthy rats body weight, liver weight and also mean relative liver weight were significantly reduced compared to the cirrhotic animals, suggesting a protective effect of the dietary R. tomentosus extract.
The main histological finding of this study was that dietary R. tomentosus ethanol extract influenced the recovery of liver structure in rats with liver cirrhosis induced by TAA. A marked reduction in fibrosis extent and a decrease of stellate and myofibroblast-like cells was evident in rats that received SP + E diet compared to those fed SP diet.
In our study, rats of group T registered significantly lower plasma total protein levels compared to those of the healthy group (C group), as described in other TAA-intoxication models (Trennery and Waring, 1983). This alteration could be related to the induction of ubiquitin-associated protein degradation by TAA toxic stress (Andersen et al., 1981). A significant increase of plasma urea concentration accompanied hypoproteinemia, probably related to the induction of the amino-acid oxidative metabolism. Both parameters were completely reversed in the rats that received the R. tomentosus ethanol extract in their diet during the period of TAA-intoxication. Rat liver [[DELTA].sup.6]-desaturase, a key enzyme in the synthesis of long chain polyunsaturated fatty acids, is inhibited by high concentrations of linoleic acid (Brenner, 1971). This fatty acid was significantly increased in plasma of TAA-cirrhotic rats, running in parallel with a reduced level of 20:4n-6 and 22:6n-3 fatty acids, in whose synthesis is involved [[DELTA].sup.6]-desaturase, suggesting that this metabolic pathway could be inhibited by chronic administration of TAA. Animals of group TE showed plasma levels of 20:4n-6 and 22:6n-3 fatty acids significantly increased compared to those of TAA-cirrhotic rats, and their 18:2n-6 levels were restored until control values, suggesting that ethanol extract components might act in this pathway, by reversing inhibition of [[DELTA].sup.6]-desaturase. Plasma ALP activity, was increased more than two-fold in the T group. Although it remained elevated in TE group, it was in a significantly lower extent. [gamma]-GT activity was also increased in plasma by the chronic effect of TAA. This marker of hepatobiliary diseases decreased significantly in rats of the TE group. All of these results point to a protective effect of dietary extract in the liver, and support the histological data found.
TAA chronic administration induced important changes in liver enzyme activities: ALAT, GDH and TD activities were significantly decreased in cirrhotic animals compared to those of control group. Liver ALAT and GDH were significantly increased in animals of TE group, although normal values were not completely restored, while low TD activity persisted. G-6-Pase is the liver enzyme responsible of glucose release. Its decreased activity in human cirrhosis contributes to the carbohydrate intolerance in alcoholic patients (Sotaniemi et al., 1985). This enzyme is also significantly decreased in our study. Animals of the TE group registered significantly higher G-6-Pase activity compared to the T group, suggesting an improvement of the carbohydrate metabolism. Hepatic ALP and [gamma]-GT were significantly increased in cirrhotic rats, as previously described (Xiangnong et al., 2002), but dietary intake of R. tomentosus ethanol extract over the TAA-administration period to induce experimental cirrhosis resulted also in a marked recovery of both enzymatic activities.
TAA is metabolized by the liver cytochrome P4502E1 (Wang et al., 2000) enzymes, rendering sulfone and sulfoxide derivatives which are apparently responsible of structural proteins and enzyme inactivation (Chieli and Malvaldi, 1984). Besides, TAA toxicity mechanism was related to alterations in NADPH-cytochrome P450 reductase activity and to nitric oxide synthesis (Cascales et al., 1991; Diez-Fernandez et al., 1997). The hepatocellular metabolism of potent inflammation mediators, such as the leukotrienes, is also impaired in TAA-induced cirrhosis (Dargel, 1995). The molecular mechanism by which R. tomentosus ethanol extract protects against liver damage is still unknown, but it should be directly related to its chemical composition. Its main components are triterpenes, flavonoids and phenolic acids. It is well established that many triterpenoid molecules afford protection against chemical-induced hepatotoxicity, by inhibition of cytochrome P450 (CYP) levels (Liu et al., 1995), particularly CYP2E1 (Jeong, 1999). Furthermore, phenolic acids, as rosmarinic and caffeic acid, picrosalvin (= carnosol) and most flavonoids, are powerful antioxidants and possess also interesting anti-inflammatory activities, as they have been described, as inhibitors of leukotriene B4 (LTB4) formation, enhancers of prostaglandin [E.sub.2] (Kimura et al., 1987) and inhibitors of nitric oxide release (Wadsworth and Koop, 1999). The protective effect of R. tomentosus ethanol extract could be attributed to a complex mechanism involving the inhibition of CYP by its triterpenic derivatives (Liu et al., 1995; Jeong, 1999), and the antioxidant and anti-inflammatory properties of its phenolics, phenolic acids and flavonoids.
In conclusion, our results demonstrate that R. tomentosus ethanol extract administered in the diet simultaneously to TAA-intoxication appears to protect rat liver against cirrhosis. This dietary extract contributes to prevent the important histological changes produced by TAA-induced cirrhosis as well as liver functionality alterations by reducing, in an important manner, many of the altered plasma and liver biochemical markers of this experimental pathology.
We thank ABBOT Laboratories (Granada, Spain) for providing the SP diet. This work was supported by the Fondo de Investigaciones Sanitarias del Ministerio de Sanidad y Consumo of Spain (FIS) project 92/0762. Milagros Galisteo was supported by Ministerio de Educacion y Ciencia, Spain.
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M. Galisteo (a,*), A. Suarez (b), M.P. Montilla (a), M.I. Fernandez (c), A. Gil (b), M.C. Navarro (a)
(a) Facultad de Farmacia, Departamento de Farmacologia, Universidad de Granada, Granada, Spain
(b) Facultad de Farmacia, Departamento de Bioquimica y Biologia Molecular, Universidad de Granada, Granada, Spain
(c) Facultad de Ciencias, Departamento de Biologia Celular, Universidad de Granada, Granada, Spain
Received 13 February 2004; accepted 2 June 2004
*Corresponding author. Tel.: +34958243889; fax: +34958248964.
E-mail address: email@example.com (M. Galisteo).
Table 1. Semipurified diet composition Composition g/kg of Chemical % (w/w) diet composition Calcium 220.5 Proteins 21 caseinate Cellulose 80 Carbohydrates 64.8 Sugar 150 Fat composition 3.9 Corn starch 455.8 Water 7.6 Oil mixture (a) 37.5 Minerals (b) 2.7 Minerals 50 Vitamins (b) 0.02 Vitamins 0.2 L-Methionine 4 Choline chloride 0.2 (a) Mixture of olive oil (66%), soya oil (23%) and refined coconut oil (11%). (b) The mineral and vitamin mixture contained the following (g/kg of mix): calcium diphosphate, 2.00; magnesium chloride, 2.00; potassium phosphate, 7.00; magnesium sulfate, 1.1; manganous sulfate, 0.166; ferric lactate, 152.2; zinc sulfate, 0.06; cupric sulfate 5[H.sub.2]O, 0.021; potassium iodate, 0.0026; sodium selenite. 5[H.sub.2]O, 0.000331; chromic potassium sulfate 12[H.sub.2]O, 0.0192; thiamine HCl, 0.006; riboflavin, 0.006; pyridoxine HCl, 0.007; nicotinic acid, 0.03; calcium pantothenate, 0.016; folic acid, 0.002; biotin, 0.02; cyanocobalamin, 0.0001; retinol acetate, 0.0012; cholecalciferol, 0.00025; all-rac-[alpha]-tocopherol, 0.1; phyloquinone, 0.001. Table 2. Effects of thioacetamide and R. tomentosus ethanolic extract intake on the body and liver weight of rats Treatment Body weight (g) Liver weight (g) C 235.40 [+ or -] 4.97 (a) 5.36 [+ or -] 0.20 (a,b) T 186.50 [+ or -] 8.37 (b) 6.47 [+ or -] 0.30 (c) CE 239.25 [+ or -] 7.46 (a) 4.82 [+ or -] 0.29 (a) TE 198.50 [+ or -] 4.47 (b) 5.59 [+ or -] 0.20 (b) Treatment Liver wt. x 100/body wt. C 2.27 [+ or -] 0.05 (a) T 3.52 [+ or -] 0.17 (c) CE 2.12 [+ or -] 0.07 (a) TE 2.82 [+ or -] 0.10 (b) Data are expressed as mean [+ or -] SEM, Means among treatment groups sharing unlike superscripts are significantly different, p < 0.05. Table 3. Plasma fatty acid composition (g/100 g total fatty acids) from control and TAA-treated rats, fed SP and SP + E diets Fatty acid C T 16:0 17.16 [+ or -] 0.36 (a) 19.19 [+ or -] 0.34 (c) 24:0 0.92 [+ or -] 0.05 (a) 0.69 [+ or -] 0.04 (b) 16:1n-9 0.36 [+ or -] 0.01 (a) 0.44 [+ or -] 0.03 (b) 18:2n-6 7.77 [+ or -] 0.36 (a) 8.98 [+ or -] 0.29 (b) 18:3n-6 0.46 [+ or -] 0.02 (a) 0.76 [+ or -] 0.09 (b) 20:4n-6 30.35 [+ or -] 0.21 (a) 26.80 [+ or -] 0.63 (b) 22:6n-3 2.80 [+ or -] 0.06 (a) 2.89 [+ or -] 0.10 (a) Fatty acid CE TE 16:0 17.34 [+ or -] 0.20 (a) 17.80 [+ or -] 0.29 (a) 24:0 0.87 [+ or -] 0.05 (a) 0.82 [+ or -] 0.04 (a) 16:1n-9 0.34 [+ or -] 0.03 (a) 0.36 [+ or -] 0.02 (a) 18:2n-6 7.28 [+ or -] 0.26 (a) 7.77 [+ or -] 0.25 (a) 18:3n-6 0.52 [+ or -] 0.02 (a) 0.63 [+ or -] 0.04 (b) 20:4n-6 28.97 [+ or -] 0.63 (a) 30.55 [+ or -] 0.70 (a) 22:6n-3 2.96 [+ or -] 0.09 (a) 3.57 [+ or -] 0.19 (b) Data are expressed as mean [+ or -] SEM. Means among treatment groups sharing unlike superscripts are significantly different, p < 0.05. Table 4. Effect of thioacetamide and R. tomentosus ethanolic extract intake for 3 months on plasma enzymatic activities in rats Groups ASAT (U/l) ALAT (U/l) C 60.6 [+ or -] 4.3 (b) 27.2 [+ or -] 2.3 (b) T 65.7 [+ or -] 3.7 (b) 23.9 [+ or -] 1.7 (b) CE 46.0 [+ or -] 2.4 (a) 15.5 [+ or -] 1.2 (a) TE 46.9 [+ or -] 2.4 (a) 16.4 [+ or -] 1.0 (a) Groups ALP (U/l) [gamma]-GT (U/l) C 98.7 [+ or -] 2.0 (b) 2.96 [+ or -] 0.15 (a) T 268.7 [+ or -] 16.7 (d) 7.26 [+ or -] 0.62 (c) CE 81.1 [+ or -] 2.8 (a) 2.37 [+ or -] 0.23 (a) TE 170.8 [+ or -] 13.2 (c) 4.10 [+ or -] 0.43 (b) Data are expressed as mean [+ or -] SEM. Means among treatment groups sharing unlike superscripts are significantly different, p < 0.05. ASAT, aspartate aminotrasnferase; ALAT, alanine aminotransferase; ALP, alkaline phosphatase; [gamma]-GT, [gamma]-glutamyl-transferase. Table 5. Effect of thioacetamide and R. tomentosus ethanolic extract intake for 3 months on liver enzymatic activities in rats Groups ASAT ALAT GDH C 1402.6[+ or -]53.6 (a) 399.3[+ or -]19.0 (a) 263.3[+ or -] 12.5 (a) T 1310.2[+ or -]81.1 (a) 173.6[+ or -]14.7 (d) 171.4[+ or -] 8.4 (c) CE 1416.2[+ or -]88.4 (a) 316.3[+ or -]20.3 (b) 273.9[+ or -] 12.4 (a) TE 1360.4[+ or -]71.7 (a) 218.6[+ or -]15.1 (c) 213.6[+ or -] 12.2 (b) Groups TD ALP C 17.4[+ or -]1.3 (a) 2.2[+ or -]0.2 (a) T 6.7[+ or -]0.3 (b) 15.3[+ or -]0.5 (c) CE 16.1[+ or -]1.9 (a) 2.4[+ or -]0.3 (a) TE 7.6[+ or -]0.7 (b) 11.2[+ or -]0.5 (b) Groups [gamma]-GT G-6-Pase C 1.4[+ or -]0.1 (a) 20.8[+ or -]0.8 (a) T 22.7[+ or -]2.2 (c) 10.7[+ or -]0.6 (c) CE 1.0[+ or -]0.1 (a) 21.1[+ or -]1.0 (a) TE 13.0[+ or -]1.7 (b) 15.0[+ or -]1.1 (b) Data are expressed as mean [+ or -] SEM. Means among treatment groups sharing unlike superscripts are significantly different, p < 0.05. ASAT, aspartate aminotrasnferase; ALAT, alanine aminotransferase; GDH, glutamate dehydrogenase; TD, threonine deaminase; ALP, alkaline phosphatase; [gamma]-GT, [gamma]-glutamyl-transferase; G-6-Pase, glucose-6-phosphatase. All these liver enzyme activities are expressed in pmol/min/mg prot except for ALP, [gamma]-GT and G-6-Pase activities, which are expressed in nmol/min/mg prot.
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|Author:||Galisteo, M.; Suarez, A.; Montilla, M.P.; Fernandez, M.I.; Gil, A.; Navarro, M.C.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jan 1, 2006|
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