Evaluation of DNA damage of hydro-alcoholic and aqueous extract of Echium amoenum and Nardostachys jatamansi.

BACKGROUND: Today most of herbal medicines are marketing without any standard safety profiles. Although common assumption is that these products are nontoxic but this assumption may be incorrect and dangerous, so toxicological studies should be done for herbal drugs. According to the frequent use of Echium amoenum as immunostimulant and useful in conditions including pain, cough, sore throat and arthritis, and Nardostachys jatamansi as tranquilizer and sleep inducer and evidences of some toxicities, we assessed the probable effect of their extracts on DNA of hepG2 cells using the comet assay.
MATERIALS AND METHODS: Different concentrations of above extracts of the plants are incubated with hepG2 cells for 24 h. A mixture of cell suspension and agarose gel were put on slides, then slides were embedded in a lysing solution and were put in electrophoresis buffer (pH = 13). Then the electrophoresis procedure took place in an alkaline solution and after neutralization stage, colorization was done by ethidium bromide and comets were observed using a fluorescence microscope. At least 100 cells of each sample were evaluated and three parameters including comet length, percent of DNA in tail, and tail moment were assessed.
RESULTS: Both Aqueous and hydro-alcoholic extract of E. amoenum were genotoxic in the concentrations of 25 mg/ml and aqueous and hydro-alcoholic extract of N. jatamansi were genotoxic in the concentrations 5 and 10 mg/ml, respectively.
CONCLUSIONS: Although E. amoenum and N. jatamansi are highly used in medicine, these herbs have genotoxic effects in determined concentrations and they should be used cautiously.

INTRODUCTION

Today most of herbal medicines are marketing without any standard safety and toxicological trials. Although common assumption is that these products are nontoxic but the true is that, this assumption is incorrect and dangerous, so toxicological studies should be done for herbal drugs. According to the frequent use of Echium amoenum and Nardostachys jatamansi and evidences of their active toxic compounds, we made an effort to assess probable effect of aqueous and hydro-alcoholic extract of E. amoenum and N. jatamansi on DNA of hepG2 cells using the comet assay.[1]

In many countries N. jatamansi (belongs to the family Valerianaceae) is traditionally used in mental disorders, insomnia, and disorders of blood and circulatory system.[2]

N. Jatamansi contains numerous chemical compounds such as, β-eudesmol, elemol, β-sitosterol, angelicin, jatamansinol, valrenone, valtrate, valerenic acid, and didrovaltrate. From them the most important is valeranon which is responsible for the sedative effect of N. Jatamansi.[34]

E. amoenum (belongs to the family Boraginaceae) is one of the most important medicinal plants in Iranian traditional medicine.[5] Petals of E. amoenum have rosmarinic acid, alkaloids, flavonoid aglycons, volatile oils, and anthocyanidine.[6]

This herb is used in Iranian traditional medicine as tranquilizer, anti-inflammatory, analgesic, heart tonic, anti-tussive diuretic, anxiolytic, and sedative.[578]

In the recent two decades, cytotoxicity and genotoxicity of these herbs drew much attention. E. amoenum has been thought to have potential toxicity because of the presence of pyrrolizidine alkaloids.[910] There are some evidence suggesting that N. jatamansi is cytotoxic and probably genotoxic due to its valtrate and didrovaltrate components.[4]

Two types of variations in the genetic material of somatic cells which might lead to aging are DNA damage and mutations. DNA damages consist of a variety of chemical disorders in polynucleotide structure of the double helix, such as pyrimidine, apurinic sites, crosslinks, and both large and small chemical additions, named adducts.[11]

DNA damage has a basic role in most of human diseases including cancer.[12] An accurate, fast and sensitive method is required to evaluate DNA damage in clinical studies, this method should be able to monitor DNA repair properly.[13]

Several different tests such as MN (Micronucleus test), UDS assay (unscheduled DNA synthesis), intracellular ROS assay, 8-OHdG assay, Ames assay, and comet assay have been used to assess the rate of DNA damage or genotoxicity. Comet assay or single cell gel (SCGE) electrophoresis was used in the current study to determine DNA damage.[1415161718]

SCGE has been established as a simple, rapid, cheap, flexible and most importantly sensitive method to detect DNA damage in individual cells.[19]

This assay is particularly notable since it requires little equipments. The SCGE can estimate single and double-strand breaks in DNA of any eukaryotic cells caused by a variety of physical, chemical, and biological agents. The alkaline comet assay was described in 1998 by Singh et al. and since 1998, some modifications have increased its sensitivity.[13]

The comet assay is built on the foundation that DNA damage decreases the size of DNA fragments. This effect could be evaluated by utilization of electrophoretic field to lysed cells. The spotted DNA fragments create a typical comet-shaped migration pattern.[19]

Many studies about the efficacy of E. amoenum and N. jatamansi, their chemical compounds and effective substance have been published that introduce these two herbs much beneficial and harmless, without any notification to their adverse effects and toxicity. A few assessments have been done about their toxicity but, the important question is that, is this point of view correct or are these two herb really without harmfulness and nontoxic in any concentration therefore the necessity of study about their harmfulness or harmlessness are feeling.

In this study, we sought to determine the effects of hydro-alcoholic and aqueous extract of N. jatamansi and E. amoenum on DNA of hepG2 using the comet assay method. Also the relationship between amount of damage in DNA and concentration of the extract of these two herbs was studied.

MATERIALS AND METHODS

Material

Desiccated petals of E. amoenum and rhizomes of N. jatamansi were purchased from a local medicine market in Isfahan, Iran and were identified by the experts. Ethidium bromide, Tris, Triton X-100, H2O2, NaCl, EDTA, NaOH, and NaH2PO4 were purchased from Merck Chemical Co. LMA (low melting agarose), NMA (normal melting agarose), and Na2HPO4 and KCl were purchased from Sigma Chemical Co.

Preparation of the extracts

Extraction by maceration method

200 g of dried powder of each plant was used for extraction by ethanol (70%). After filtration, the residue was extracted for three times. Then all extract were concentrated by rotary evaporator and were freeze dried.[20]

Extraction by decoction method

E. amoenum and N. jatamansi were poured into hot water (90 centigrade) and were put in water bath and mixed for 30 minutes. Then these were filtered and freeze dried.[21]

Cell culture

In the present study, we used human hematoma (hepG2) cells to investigate the genotoxicity of E. amoenum and N. jatamansi. The hepG2 cell line was obtained from Pasture Institute of Iran and was cultured in RPMI medium (contain 10% fetal bovine serum) under 5% CO2 at 37΀C in micro filter plates.

Cells were incubated with different concentrations of the extraction of E. amoenum and N. Jatamansi or media for 24 hours. Then the upper medium of each well was thrown away and after trypsinization, they were neutralized by the medium and were poured in five different falcons and centrifuged. Then 1 ml of medium was added to each falcon to use for next stages of the comet assay.

Comet assay

Only cell suspensions with viabilities of more than 90% were used for determination of DNA damage. 0.4 ml cell suspension was added to 1.2 ml of one percent low melting point agarose. After solidification, the slides were submerged in a cold fresh lysing solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, and 200 mM NaOH with pH 10) for 40 minutes. Then, the slides were put in an alkaline solution (NaOH 10 N, EDTA 200 mM with pH 13) for 40 min to permit unwinding of DNA. Electrophoresis was done in the same buffer for 40 min at 25 V and 300 mA. After this stage, the slide was neutralized by 0.4 M Tris (pH 7.5). Afterwards the slides were stained using 100 μl of ethidium bromide (EtBr, 20 μg/ml). Finally, the images were taken by fluorescence microscope and at least 100 randomly selected cells (33 cells from each of the three replicate slides) were analyzed per sample. Parameters of Tail moment (% DNA in tail × Tail length), Tail length, and percent of DNA in tail, the most frequently used parameters in the comet assay, were used in this study.[22]

Statistical analysis

Results are presented as mean ± SD from at least three separate experiments. Statistical analysis of data was carried out by one-way ANOVA followed by Tukey post hoc test. All statistical analyses were performed using the statistical software package Sigma Stat 3.1. A difference was considered significant at P < 0.05.

RESULTS

Genotoxic effects of hydro-alcoholic and aqueous extracts of E. amoenum and N. jatamansi were studied. Aqueous extract of N. jatamansi was incubated with hepG2 in cell culture. This test was done at two concentrations of 1 and 5 mg/ml and was compared with control group. Three parameters of Tail length, percent of DNA in tail, and Tail moment were checked. Our findings showed that the aqueous extract of N. jatamansi at a concentration of 5 mg/ml increased Tail length, %DNA in tail, and Tail moment with a significant difference compared with the control group. For concentration of 1 mg/ml only Tail length and Tail moment were significantly increased, so this extract would be genotoxic at a concentration of 5 mg/ml. (P < 0.05) [Figure 1]. For hydro-alcoholic extract of N. jatamansi, different concentrations were in contact with hepG2. The results are shown in Figure 2. This diagram demonstrates that at concentrations of 1, 5, and 10 mg/ml, all parameters are significantly increased, concentration dependently, and the concentrations of 5 mg/ml and 10 mg/ml have significant difference when compared with the control group. According to these results this extract would be genotoxic at concentration of greater than 5 mg/ml (P < 0.05) [Figure 2].

Figure 1

The effect of aqueous extract of N. jatamansi on Tail length, %DNA in tail, and Tail moment of hepG2 cells. Results are presented as mean ± SD from at least three separate experiments, *= P < 0.05

Figure 2

The effect of hydro-alcoholic extract of N. jatamansi on Tail length, %DNA in tail, and Tail moment of HepG2 cells. Results are presented as mean ± SD from at least three separate experiments, *= P < 0.05

Aqueous extract of E. amoenum significantly and concentration dependently increase all the three parameters. The diagrams of Tail length, %DNA, Tail moment against concentration of aqueous extract of E. amoenum were reviewed. A significant difference is observed between concentration of 10 μg/ml, 100 μg/ml, 500 μg/ml, 10 mg/ ml, 25 mg/ml for tail moment (Tail moment = %DNA in tail × Tail length) when compared with the control, similar results can be seen for %DNA and the most significant one is seen at concentration of 25 mg/ml. Consequently, the genotoxic effect was seen at concentrations greater than 25 mg/ml (P < 0.05) [Figure 3].

Figure 3

The effect of aqueous extract of E. amoenum on Tail length, %DNA in tail, and Tail moment of hepG2 cells. Results are presented as mean ± SD from at least three separate experiments, *= P < 0.05

Also exposure of hepG2 cells to hydro-alcoholic extract of E. amoenum significantly and concentration dependently increased all the three parameters tested. The diagram of tail length demonstrates the significant difference between the concentration of 25 mg/ml and control group. Therefore, the hydro-alcoholic extract of E. amoenum is genotoxic at concentrations above 25 mg/ml (P < 0.05) [Figure 4].

Figure 4

The effect of hydro-alcoholic extract of E. amoenum on Tail length, %DNA in tail, and Tail moment of hepG2 cells. Results are presented as mean ± SD from at least three separate experiments, *= P < 0.05

DISCUSSION

DNA damage could be detected by the single cell gel electrophoresis (SCGE) or comet assay, a fast, flexible, simple, and sensitive technique. During the last 20 years, the comet assay has been used to discover primary DNA damages such as double-strand breaks, single-strand breaks, alkali-labile sites in complete repair sites, and crosslinks.[23]

In this study, we used comet assay to find if E. amoenum and N. jatamansi have any genotoxicity. The comet assay has expanded an internationally well-known method to assay DNA damage in various cell types during the last two decades. It is believed that the comet assay is still growing in use and has high potential to be use in clinical and cancer research.[13] According to the frequent use of E. amoenum and N. jatamansi and evidence of their active toxic compounds, we made an effort to assess probable effects of aqueous and hydro-alcoholic extracts of E. amoenum and N. jatamansi on DNA of hepG2 cells using the comet assay. Regarding the fact that in extraction by water and alcohol, different compounds are separated, obtained result for each type of extract could be different from each other. Our findings showed that aqueous and hydro-alcoholic extracts of N. jatamansi increased Tail length and Tail moment significantly and concentration dependently indicating the genotoxicity of this plant and its capability of DNA damage. Similarly, aqueous and hydro-alcoholic extracts of E. amoenum increased Tail length, %DNA and Tail moment of hepG2 cells significantly and concentration dependently. This in vitro study was done on hepG2 cell culture and there is a long way to be generalized to in vivo and determine the amount of plant that would affect human hepatoma. It is important to perform an in vivo study to recognize the amount of the plant should be consume orally to create concentrations of the herb extract that is in genotoxic for hepG2 cells and to conclude if the amount of herb which is normally consumed is equal to amount of herb that is needed to make genotoxic effect and on the basis of this case decide about that normal use are genotoxic or not.

This study showed that mentioned concentrations of these herbs are toxic but more studies are required to determine the toxic effects of these herbs when they are used according to their traditionally common amounts. So our finding should serve as scientific guidance for clinical therapy of these two herbs preparation and more studies should be done about their harmlessness or their harmfulness in common dose but the obvious matter is that it is better to use them cautiously.

CONCLUSION

In conclusion, our findings revealed that hydro-alcoholic and aqueous extracts of E. amoenum and N. Jatamansi can cause DNA damage which make it necessary to reconsider the safety of using these plants and need of more toxicological studies in this regard.