Abstract: Fruit aqueous extract of Artemisia herba alba was studied for their effect on germination and seedling growth and for their antimutagenecity potentials. Germination percentage and total seedling length of A. herba alba reduced as the concentration of the aqueous fruit extract increased in both mature and immature. The toxicity of immature fruit aqueous extract against germination and seedling growth was stronger in comparison with that of mature. Moreover the inhibition of root length was more obvious than shoot length in both mature and immature fruit aqueous extract. In mutagenicity test, fruit aqueous extract of A. herba alba exhibited an anti-mutagenic activity. The percentage of anti-mutagenic increased with increasing aqueous extract concentration in both mature and immature fruit in the presence and absence of S9 mix. Anti-mutagenicity percentage was more obvious in immature fruit aqueous extract and in the present of S9 mix. The percentages of mutagenicty varied with fruit maturity and there was strong correlation between them. This relationship was coupled with the results of germination and seedling growth A. herba alba.
INTRODUCTION
Artemisia herba alba ASSO is a perennial shrub in desert and semi-deserts of the Middle East (Zohary, 1973) and its one of the medicinal plants that is used by bedouins (Al-Shamaony et al., 1994). Medicinal plants may contain bioactive compounds that posses anti-mutagenic activity (Marnewick et al., 2000; Tepsuwan et al., 1999) thereby may have a protective effect that reduce the onset of certain cancer.
Artemisia herba alba seed is a single fruit, that has mucilaginous cells which contain phytotoxic chemicals (Al-Charchafchi et al., 1987). These chemicals were toxic to the germination and seedling growth of other plants (Modallal and Al-Charchafchi, 2006) or even autotoxic (Al-Charchafchi et al., 1987). Many phytotoxic compounds like phenolics were isolated from the aqueous plant extracts (An et al., 2000).
Anticancer properties of some medicinal plants components are documented (Kusamran et al., 1998; Tepsuwan et al., 1999). Salmonella mutagenicity assay has been used to investigate the mutagenicity potential of many compounds by various means (Choi et al., 1996; Ikken et al., 1999). Plant phenolics have the ability to protect against mutagenicity and reduce genotoxicity of Salmonella typhimurium (Stagos et al., 2004; Belicova et al., 2001). Fruit phytochemicals have been isolated and identified as agents that block different stages in the carcinogensis process (Huang et al., 1994). These compounds may be interesting candidate class of chemopreventive compounds including antimutagenic and anticarcinogenic (Cardador-Martinez et al., 2002; Chlopcikova et al., 2004). In the present study, the biological activities of water soluble phytotoxins from mature and immature A. herba alba fruit on germination, growth and mutagenicity were investigated. At the same time, AMES test was utilized to study the antimutagenic activity of A. herba alba fruit aqueous extracts against Salmonella.
MATERIALS AND METHODS
Plant material: Fruits of A. herba alba were harvested from the medicinal and aromatic plant garden at Hashemite University in Jordan. Plant material was shade dried then ground and kept in glass jar at 5°C until use. Seeds of A. heba alba were harvested at maturity stage from the same garden and husked seeds were used in germination and seedling growth tests.
Extracts preparation: Hundred grams of dried powder from each mature and immature fruits of A. herba alba were extracted with one liter of boiled distilled water in a glass jar for 24 h at 20°C. The mixture has been shaken every 1 h. The mixture was placed in boiled water bath for 1 h, centrifuged at 6000 rpm for 15 min and filtered through micro-filter Millipore (0.2 μm). The filtrate designated as full strength (100%) and stored in a freezer (-50°C) prior to use for bioassay.
Germination and seedling growth test: Fifty husked seed of A. herba alba were germinated in petri dishes on Whatman filter paper with 5 mL of the aqueous extracts or distilled water as control. Three replicates were incubated in a randomized complete block design at 20°C in an incubator with fluorescent light. Germination criteria were the emergence of the radical through the pericarp. Germinated seeds were recorded and total seedling length was measured after 7 days of incubation. Total seedling length was measured using five seedlings taken randomly from each dish after seven days of incubation.
Antimutagenic activity test: Ames Salmonella Plates pre incubation assay was used to evaluate mutagenic activity (Gichner et al., 1994). All samples were tested in S. typhimurium strain TA100 with and without S9 mix (Putnam et al., 1999). The S9 mix was prepared from rats as previously described by Maron and Ames (1983). Mutagenicity for A. herba alba water extracts from mature and immature fruits was analyzed using H2O2 as a standard mutagen. Plates incubated for 48-72 h at 37°C. Plates were read using Artek colony counter. Positive and negative controls were carried out in addition to the test samples. Triplicate plates were made for each test.
The number of his + revertants (after substracting the spontaneous reversions) induced by H2O2 tested without extract were given 100% mutagenicity. The presence of remaining revertant in the presence of mature and immature extracts was calculated accordingly.
Statistical analysis: Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS 9.0.0; SPSS Inc, Chicago, Illinois, United States of America, 1998). Paired t-test was used to test the significance between means. Statistical significance was set at p< 0.05.
RESULTS
Germination and seedling growth: Germination percentage of Artemisia herba alba seeds were significantly decreased (p<0.05) as the concentration of the aqueous fruits extract (mature and immature) increased. The inhibition of germination was more pronounced at concentration above 12.5% (Table 1), while complete inhibition of seed germination was occurred at 75 and 100% of the full strength extract. Immature fruit extract showed higher inhibition in germination (p<0.05) as compared to mature fruit extract.
The total seedling length of A. herba alba also decreased with increasing concentration of aqueous fruit extract as indicated in Table 2. But no significant differences were shown between mature and immature fruit extract on the inhibition of total seedling length except in 25%. Therefore it is worthwhile to investigate which plant part (root or shoot) was mainly inhibited by crude aqueous fruit extracts; an experiment was administrated that determined the effect of mature and immature aqueous fruit extract on shoot and root length (Table 3 and 4, respectively). Both extracts had significant toxic inhibitors on root and shoot length, but the toxicity on the root length was higher than shoot.
| Table 1: | Effect of different concentration of A. herba alba
fruit (mature and immature) aqueous extract on its germination percentage |
| Table 2: | Effect of different concentration of A. herba alba
fruit (mature and immature) aqueous extract on its total seedling length |
| Table 3: | Effect of different concentrations of A. herba alba
mature fruit aqueous extract on its shoot and root length |
| Table 4: | Effect of different concentrations of A. herba alba
immature fruit aqueous extract on its shoot and root length |
| Table 5: | Effect of mature and immature extracts of A. herba alba
on the mutagenicity of H2O2 towards Salmonella
typhimurium TA100 in the presence of S9 mix. Results are mean±SE
from five repeats in each group |
| Table 6: | Effect of mature and immature extracts of A. herba
alba on the mutagenicity of H2O2 towards Salmonella
typhimurium TA100 in the absence of S9 mix. Results are mean±SE
from five repeats in each group |
Antimutagenic activity test: Ames assay was conducted with and without S9 mix on mature and immature fruit aqueous extract of A. herba alba to evaluate their antimutagenicity potentials against H2O2 using S. typhiumurium strain TA100 are shown in Tables 5 and 6, respectively. Both mature and immature aqueous extracts exhibit anti-mutagenic activity against H2O2 at doses ≤0.5 mg plate-1. Antimutagenicity percentages increase with increasing aqueous extract concentration of A. herba alba mature and immature fruit, but immature extract exhibit stronger anti-mutagenicity in presence and absence of S9 mix.
DISCUSSION
Artemisia herba alba fruit aqueous extracts exerted toxic effect on its germination and seedling growth. Thereby A. herba alba fruit extract may contain some toxic substances that reduced germination and seedling growth of its own (autotoxic) and other plant species such as Anabasis setifera (Modallal and Al-Charchafchi, 2006). Previous investigation reported that aqueous extract of some plant species contain phenolics or other toxic substances (Belicova et al., 2001). These phenolics inhibit the germination process (Blum et al., 1984; Al-Charchafchi et al., 1987), by their interference with indol acetic acid metabolism, or synthesis of protein and ion uptake by the plants (Castro et al., 1984). Therefore, A. herba alba might release some soluble phenolics to the environment which has a growth inhibitory effect on new seeding of A. herba alba or other plant species. This interpretation was in consistence with that reported by Modallal and Al-Charchafchi (2006) and Xu et al. (2003).
Moreover the fruit aqueous extracts of A. herba alba capable of inhibiting its root length more than shoot. This may be due to the direct contact between the root and phytotoxic compounds of the aqueous extracts which in turn may inhibit cell division (Rietjens and Alink, 2003) which is highly active in meristimatic tissue in the growing root.
The toxicity of the immature fruit aqueous extract on germination and seedling length was higher than mature which might be related to the difference in the amount and kinds of water soluble phytotoxins that released from fruit (Bewley and Black, 1998).
Both mature and immature extracts exhibit antimutagenic activity against H2O2 at doses ≤0.5 mg plate-1. Antimutagenicity percentage increases with increasing aqueous extract concentration of A. herba alba in both mature and immature. It has been mentioned earlier that phenolics reduce genotoxicity (Belicova et al., 2001), prevent early atherosclerotic lesions (Auger et al., 2004), chemoprotective against toxicity (Chlopcikova et al., 2004), inhibit cancer cell growth (Saleem et al., 2002) and exhibit antimutagenic activity (Malaveille et al., 1998; Stich et al., 1982). A. herba alba antimutagenicity may be due to presence of phenolic compounds that may have a role in modulation of mitogenic signaling regulation and induction of G1 arrest and apoptosis (Agarwal, 2000; Kampa, 2000), or an important role against DNA damage an mutations induced by reactive oxygen species (Stagos et al., 2004). The antimutagenicity may be polar in nature because both mature and immature extracts were extractable in water and exhibit antimutagenic activity. Higher antimutagenicity effect was shown in the presence of S9 mix, therefore suggested that these antimutagens may interact with some specific enzymes in the liver homogenates, which is necessary for activation of chemical mutagen (Tepsuwan et al., 1999).
This study showed that antimutagenicity was higher in immature fruit aqueous extract compared to mature. This might be due to higher phenolic concentration in immature fruit aqueous extract compared to mature (Modallal and Al-Charchafchi, 2006) or due to differences in kind of phenolics that were isolated from the fruit (Connor et al., 2002).
The results from this study suggest that toxicity of A. herba alba on germination, seedling growth was correlated with the total phenolic contents in the aqueous fruit extract. Phenolic compounds are well known potential phytotoxins (Al- Charchafchi et al., 1987) and exist as free forms, esters, or as glycosides when combined with sugars, that may be indirectly related to chemicals that is finally responsible for the observed toxic effect on germination and seedling growth (Seal et al., 2004). At the same time these phenolics exhibit some potential antimutagenic activity in presence or absence of S9 mix.
ACKNOWLEDGMENTS
The authors wish to thank the Hashemite University for the Financial Support of this project and Ms. Shereen Isa for her effort through the project.