Abstract: Iron chelating activity of 16 extracts from 11 medicinal plants has been determined to find alternative sources with lower side effects in thalassemic patients. Thalassemia is characterized by iron overload and chelation therapy reduces iron-related complications and thereby improves quality of life and overall survival. Because of poor oral bioavailability, short plasma half-life and severe side effects of available chelators, this screening may be useful in this area. Extracts were prepared by soaking dry material of the selected plant in appropriate solvent. Phenol and flavonoid content of the extract were measured by Folin Ciocalteu and AlCl3 colorimetric assays. Phenol content of the extracts varied between 8.4-194.9 mg g-1 of extract. The highest chelating activity was found in aerial parts of Leonurus cardiana and Grammosciadium platycarpum which had high amount of phenol and flavonoid contents. All extracts contained various amount of flavonoids from 5.9 to 90.9 mg g-1 of extract. Weak correlations were found between phenolic and flavonoids contents and iron chelatory activity with R2 = 0.40. Extracts with high phytochemicals and chelating activity can be candidate as a good source of new agents for thalassemic patients.
INTRODUCTION
Patients with chronic anemias such as thalassemia, require regular blood transfusions in order to improve both quality of life and survival. Humans are unable to eliminate the iron released from the breakdown of transfused red blood cells and the excess iron is deposited as hemosiderin and ferritin in the liver, spleen, endocrine organs and myocardium. The accumulation of toxic quantities of iron causes tissue damage and leads to complications such as heart failure, endocrine abnormalities like diabetes, hypothyroidism, liver failure and ultimately early death (Taher et al., 2006; Rund and Rachmilewitz, 2005; Loukopoulos, 2005). Thalassemia major is characterized by anemia, iron overload, further potentiation of ROS and damage to major organs, especially the cardiovascular system. Oxidative stress is ultimately involved in endothelial dysfunction, a condition which is evident in adults suffering from various cardiovascular diseases including thalassemia (Shinar and Rachmilewitz, 1990; Hebbel et al., 1990; Grinberg et al., 1995). Antioxidant and other supportive therapies protect RBC against oxidant damage (Kukongviriyapan et al., 2008; Filburn et al., 2007). Also, a higher rate of LDL oxidation in thalassemia patients is due to a lower concentration of vitamin E and C in the LDL particles. Enrichment with vitamins E and C was effective in preventing LDL oxidation in patients with thalassemia (Rachmilewitz et al., 1979; Livrea et al., 1996). Iron chelators mobilize tissue iron by forming soluble, stable complexes that are then excreted in the feces and/or urine. Chelation therapy reduces iron-related complications and thereby improves quality of life and overall survival (Shinar and Rachmilewitz, 1990; Hebbel et al., 1990). In addition, brain iron dysregulation and its association with amyloid precursor protein plaque formation are implicated in Alzheimer's Disease (AD) pathology and so iron chelation could be considered a rational therapeutic strategy for AD (Reznichenko et al., 2006). The poor oral bioavailability, short plasma half-life and severe side effects of available chelators are still not optimal (Hebbel et al., 1990; Grinberg et al., 1995; Kukongviriyapan et al., 2008; Filburn et al., 2007; Rachmilewitz et al., 1979; Livrea et al., 1996). Within this context and taking in consideration the relative paucity of iron chelating agents it is not surprising that clinical scientists put a great effort towards finding any potentially useful sources in order to obtain the maximum possible benefit with the least possible harm (Loukopoulos, 2005; Ebrahimzadeh et al., 2006, 2007; Mahmoudi et al., 2007; Pourmorad et al., 2007). For thousands of years, mankind has known about the benefit of drugs from nature. Plant extracts, for the treatment of various ailments, were highly regarded by the ancient civilizations. Even today, plant materials remain an important resource for combating illnesses. In this study, some medicinal plants traditionally used for management of diseases were selected and their phenol and flavonoid content and iron chelating activities were evaluated.
MATERIALS AND METHODS
This study was performed from the spring and the end of autumn 2008 in Pharmaceutical Sciences Research Center, School of pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.
Chemicals: Gallic acid, quercetin, EDTA and other necessary agents were purchased from Merck and Fluka companies. All other chemicals and reagents used were of the highest commercially available purity.
Preparation of extracts: A brief description of the plants can be found in Table 1. Plant parts were collected from Sari forest, Iran, in 2008 and identified by Dr. Bahman Eslami. vouchers have been deposited in the Sari School of Pharmacy herbarium. Hundred grams each of the dried specific part of plant was soaked in desired solvent for 3 days in room temperature. The solvent was evaporated under reduced pressure and then lyophilized. The resulting solid masses were preserved in 4°C.
Determination of total phenolic compounds and flavonoid contents: Total phenolic compound contents were determined by the Folin-Ciocalteau method according to our recently published paper (Ebrahimzadeh et al., 2008a, b). The standard curve was prepared using 50 to 250 mg mL-1 solutions of gallic acid in methanol-water (1:1, v/v). Total phenol values are expressed in terms of gallic acid equivalent (mg g-1 of dry mass), which is a common reference phenolic compound.
Flavonoid content of each extract was determined by following colorimetric method (Ebrahimzadeh et al., 2008c; Nabavi et al., 2008). The calibration curve was prepared by preparing quercetin solutions at concentrations 12.5 to 100 μg mL-1 in methanol.
Metal chelating activity: The chelation of ferrous ions by extracts was estimated by method of (Ebrahimzadeh et al., 2008b; Nabavi et al., 2009). The percentage inhibition of ferrozine-Fe2+ complex formation was calculated as:
[(A0- As)/ As] x
100 |
| Table 1: | The studied plants and their medicinal uses |
where A0 was the absorbance of the control and As was the absorbance of the extract/standard. Na2 EDTA was used as positive control.
Statistical analysis: Results are presented as Mean±SD. Statistical analysis were performed by Student's t-test. The values of p<0.05 were considered significant.
RESULTS AND DISCUSSION
Total phenols measured by Folin Ciocalteu reagent in terms of gallic acid equivalent. Phenol content of the extracts varied between 8.4-194.9 mg g-1 of extract. The highest amounts were found in aerial parts of Artemisia absinthium (194.9±14.51) and fruits extract of Hyoscyamus squarrosus (178.90±9.02). They showed good iron chelatory activity (Table 2). The flavonoid content of Ferula assa-foetida aerial parts extract in terms of quercetin equivalent was 90.9±6.3. Leaves extract (at Flowering stage) of Eryngium caucasicum Trautv also contained high amount of flavonoids (60.12±5.31). Both of them showed good iron chelatory activity (Table 2). The chelating of Fe2+ by extracts was estimated by the method of Dinis et al. (Ebrahimzadeh et al., 2008b). The highest chelating activities were found in aerial parts of Leonurus cardiana and Grammosciadium platycarpum with IC50 = 0.02±0.00 and 0.079±0.00, respectively. Weak correlations were found between phenolic and flavonoids contents and iron chelatory activity with R2 = 0.40 (Fig. 1, 2).
| Table 2: | Total phenol and flavonoid content and iron chelating IC50 of the herbs studied. |
| *mg gallic acid equivalent g-1 extract, **mg quercetin equivalent g-1 extract, Data presented as Mean±SD | |
It has been recognized that flavonoids show antioxidant activity and their effects on human nutrition and health are considerable. The mechanisms of action of flavonoids are through scavenging or chelating process (Kessler et al., 2003; Cook and Samman, 1996). Phenolic compounds are a class of antioxidant compounds which act as free radical terminators (Shahidi and Wanasundara, 1992). The compounds such as flavonoids, which contain hydroxyl functional groups, are responsible for antioxidant effect in the plants (Das and Pereira, 1990; Younes, 1981). According to our study, the content of these phytochemical compounds in studied extracts have direct relation with iron chelating activity.
The chelating of Fe2+ by extracts was estimated by the method of (Ebrahimzadeh et al., 2008b). Ferrozine can quantitatively form complexes with Fe2+. However, in the presence of chelating agents, the complex formation is disrupted with the result that the red color of the complex is decreased.
| Fig. 1: | Correlation between phenol contents and iron chelating activity in 16 tested extracts |
| Fig. 2: | Correlation between flavonoids contents and iron chelating activity in 16 tested extracts |
Measurement of colour reduction, therefore, allows the estimation of the chelating activity of the coexisting chelator. The transition metal ion, Fe2+ possess the ability to move single electrons by virtue of which it can allow the formation and propagation of many radical reactions, even starting with relatively non-reactive radicals (Aboul-Enein et al., 2003). The main strategy to avoid ROS generation that is associated with redox active metal catalysis involves chelating of the metal ions. Leonurus cardiana and Grammosciadium platycarpum, the most active extracts interfered with the formation of ferrous and ferrozine complex, suggesting that it has chelating activity and captures ferrous ion before ferrozine. The highest chelating activities was found in aerial parts of Leonurus cardiana with IC50 = 0.02±0.00 which was comparable to the reference compound EDTA (IC50 = 0.017 mg mL-1). A weak direct relation between this activity and the content of active compounds, phenols and flavonoids were found in persent study (Fig. 1, 2).
The interaction between flavonoids and iron ion has been published previously (Mira et al., 2002). It is suggested that this chelating activity depends on the flavonoid structures and may be a proposal explanation for antioxidant activity of flavonoids (Mira et al., 2002). In our recently published paper, in spite of some relation between chelating activity and phenol and flavonoids, no direct correlation was found between these contents and chelating activity in tested plants (p>0.001). Some plants with high phenol and flavonoid contents showed very weak chelating activity but some with high phenol and flavonoid contents showed good chelating activity. This lack of relationship is in agreement with other literature (Ghasemi et al., 2009; Heinonen et al., 1998; Assimopoulou et al., 2006; Nickavar et al., 2007). It is known that o nly flavonoids with a certain structure and particularly hydroxyl position in the molecule can act as proton donating and show radical scavenging activity (Mensor et al., 2001; Hou et al., 2003). Furthermore, the extracts are very complex mixtures of many different compounds with distinct activities (Mensor et al., 2001; Hou et al., 2003). In this study, a weak direct relation was found (R2 = 0.40). So the chelating activity of these extracts may be a function of these compounds.
CONCLUSION
All extracts showed a variety of activity and phytochemical compounds in this study, but Leonurus cardiana can be observed as a potent iron chelating source for further investigation.
ACKNOWLEDGMENT
This research was Financial supported by a grant from the research council of Mazandaran University of Medical Sciences, Iran.