Anti-mutagenic Activity of Salvia merjamie Extract Against Gemcitabine

Gemcitabine, is a nucleoside analogue antineoplastic used in chemotherapy. It is classified as an anti-metabolite in the subclass of pyrimidine analogues and metabolizes intracellularly to activate the diphosphate and triphosphate nucleosides (Yao et al., 2010; Suprasert et al., 2012). Gemcitabine inhibits thymidylate synthetase, leading to inhibition of DNA synthesis and cell death (Fowler et al., 2008). This anti-cancer drug is used clinically to treat various cancers, including breast cancer (Morandi, 2006), ovarian cancer (Lorusso et al., 2006), non-small cell lung cancer (Crino et al., 1999), pancreatic cancer (Burris et al., 1997), T-cell malignancies (Sallah, 2001), germ cell tumour (Einhorn et al., 1999), hepatocellular carcinomas (Kubicka et al., 2001), advanced squamous cell carcinoma of the head and neck (Catimel et al., 1994), cervical cancer (Mutch and Bloss, 2003), refractory transitional cell carcinoma of the bladder (Dalbagni et al., 2006) and peritoneal mesothelioma (Fracasso et. al., 1999). Gemcitabine has also been reported to have adverse effects including, haematological toxicity (Crombag et al., 2014) and pulmonary toxicity (Barlesi et al., 2004; Chi et al, 2012). Furthermore, gemcitabine has been reported to have mutagenic activity in male albino mice in vivo (Mohammed et al., 2009) and in human lymphocytes in vitro (Aydemir et al., 2005). Mutation is an essential factor


Khalid Mashay Alanazi
in carcinogenesis and the occurrence of cancers may be reduced by decreasing the rate of the mutations.The best approach to decrease the rate of mutation in humans is to avoid exposure to mutagens and carcinogens (Kim et al., 2000).Plant derived natural products have received considerable attention since ancient times due to their potent antioxidant activity and diverse pharmacological and anticancer properties (Ong et al., 1986;Owen et al., 2004;Omar, 2010;Patel et al., 2010;Karmakar et al., 2010;Al-Oqail et al., 2013;Al-Sheddi et al., 2014;Farshori et al., 2013 and2014) and there is, therefore, a pressing need to identify and investigate plant derived compounds with potential anti-carcinogenic and antimutagenic properties.Various studies have demonstrated that plant extracts have anti-mutagenic activity (Taneja et al., 2003;Meena et al., 2006;Agrawal and Pandey, 2009;Kumar et al., 2010) and the present study was designed to investigate the mutagenic effects of Salvia merjamie (Family: Lamiaceae)plant extracts against the mutagenic effects of gemcitabine.

Experimental animals
Inbred SWR/J male and female mice (10-12 weeks old and weight range 29.2-31.8gm) were used in the present study.Animals were obtained from the Experimental Animal Care Center, King Saud University, Riyadh and were maintained in an environmentally controlled room at a temperature of 22±1ºC, a relative humidity of 45±5 on a 10/14h light/dark cycle with standard food pellets and drinking water ad libitum.All experiments on animals were carried out according to the Guidelines of the Animal Care and Use Committee, King Saud University, Kingdom of Saudi Arabia.

Gemcitabine preparation
One gram of gemcitabine powder (BDH chemical) was dissolved in 100 ml of sterile normal saline, and then 30mg/kg body weight was applied.

Plant material
The flowering twig of Salvia merjamie growing wildly in nature was collected along with voucher specimens from Medina regions of Saudi Arabia.The plants were identified through consultation of the flora of Saudi Arabia (Chaudhary, 2001), and a specimen was submitted to the Herbarium of King Saud University in Riyadh, Saudi Arabia.The collected plant materials were rinsed thoroughly with tap water to remove extraneous contaminants and were then cut into small pieces, ovendried at 50°C until the dry weight stabilized, and ground into a powder with an electric grinder.A crude extract was prepared by macerating the powdered plant materials (1000 g) in 95% ethanol at room temperature for 1 week.Extracts were filtered and concentrated using a rotary evaporator at low temperature and pressure.The crude extracts were prepared in normal saline and were applied orally.
For each treatment group, mice were sacrificed after 24, 48 and 72h for analysis while anesthetized.

Chromosomal aberration test
Chromosome Preparations: Chromosomal preparations were performed following the methods of Preston et al. (1987) and Al-Hawary and Al-Saleh, 1989.
Slide preparations: A minimum of ten slides were prepared and distinctly identifiable metaphases were selected from each mouse.Each selected metaphase was examined using the 100xoil immersion objective of a Zeiss microscope in order to detect possible chromosomal aberrations.Prior to scoring the drug's effect on the chromosomes, the slides were covered and coded.The chromosomal aberrations scanned were: chromatid gaps (G), chromatid breaks (B), fragments (F), ring chromosomes (R), deletions (D), centromeric attenuation (CA), centric fusion (CF), pulverized chromosomes (PC), and End to End association (EE).According to the criterion of Matsuoka et al. (1979), a complete discontinuity narrower than the width of a chromatid was considered to be a gap.Photomicrographs of selected metaphases were taken under bright illumination usingthe 100xoil immersion objective and a 10xeyepiece.

Mitotic index
The mitotic index (MI) was determined using the protocol of Shubber and Juma (1999), scoring at least 1000 cells from each animal, and the MI was then calculated through the ratio of mitotic cells to interphase in 1000 cells.
Mitotic index (MI %)=Number of dividing cells / total No. of cells scored X 100

Statistical analysis
The results expressed as mean±SE were statistically analysed using a SAS computer program and a student-t test (Sokal and Rohlf, 1981).

Effects of Salvia merjamie extracts on chromosomal aberrations in mice bone marrow cells induced by gemcitabine
The results of the frequencies of chromosomal aberrations induced by gemcitabine and the preventive effects of Salvia merjamie extract are summarized in Tables 1-3 and Figure 1.A statistically significant doseand time-dependent effect of Salvia merjamie extract on chromosomal aberration was observed.As shown in Table 1, while gemcitabine increased the number of chromosomal aberrations, in comparison, the mice treated with Salvia merjamie at 100 and 150 mg/kg body weight for 24h exhibited a significantly decreased number of abnormal cells.The effect of Salvia merjamie extract was found to become more marked as the length of exposure increased.As shown in Table-2 and 3, however, a significant effect was observed even at the lowest dose, i.e.50 mg/kg body weight of Salvia merjamie extract.

Effects of Salvia merjamie extracts on changes in the mitotic index in mice bone marrow cells induced by gemcitabine
The effect of different concentrations of Salvia merjamie extract and gemcitabine on mice bone marrow mitotic index frequencies are shown in Tables 4-6.Compared to mice treated with gemcitabine alone, those treated with a combination of Salvia merjamie extract and gemcitabine showed a statistically significant increase in bone marrow mitotic indices.There was a significant (p>0.01)difference in the mitotic indices between all the studied groups.The mice treated with gemcitabine alone showed a mitotic index of 2.2% at 24h, whereas the mitotic index of mice treated with Salvia merjamie extract at100 and 150 mg/kg increased to 3.6% and 4.1%, respectively.Similarly, mice treated with100 and 150 mg/kg of Salvia merjamie extract had mitotic indices of3.5% and 3.7%, respectively, at 48h and 3.6% and 3.8%, respectively, at 72h, whereas those treated with gemcitabine alone had mitotic indices of 2.2% and 3.2% at 48h and 72h.There was no significant effect on the mitotic index of those mice treated 50 mg/kg of Salvia extract at any of the time intervals, however.

Discussion
Salvia herbs belong to the Labiatae family of plants, which includes nearly 900 species spread throughout the world (Mozafarian, 1996).Plants that belong to this family are well known for their pharmacological and other bioactivities, and have often been used in traditional medicine (Xu, 1990).Hohmann et al. (1999) and Zupko et al. (2001) reported the antioxidant activities of many species of Salvia and their active constituents in enzyme-dependent and enzyme-independent systems.Phytochemical analyses of Salvia species show the presence of many compounds, mainly belonging to the phenolic acids, phenolic glycosides, flavonoids, anthocyanins, coumarins, polysaccharides, sterols, terpenoids and essential oils (Ghannadi et al., 1999;Lu and Foo, 2002).Several species of Salvia have been used to treat microbial infections, cancer, malaria, inflammation, loss of memory, as well as to disinfect homes after sickness (Kamatou et al., 2008).The present investigation aimed to assess the anti-mutagenic activity of Salvia merjamie extracts against the mutagenic effects of gemcitabine in bone marrow cells.Chromosomal aberrations and a decline in the mitotic index are the most sensitive indicators of bone marrow damage (Giri et al., 1988;Natarajan et al., 1993;Smalinskiene et al., 2005) and, therefore, the experiment was designed to observe whether the toxic effects induced by gemcitabine, as revealed by chromosomal aberrations and the mitotic index, were neutralized by the administration of Salvia merjamie extracts.Such anti-mutagenic and immunomodulatory activities of Salvia merjamie in respect to the mutagenicity induced by gemcitabine have not yet been evaluated.
As this study has shown, gemcitabine causes disturbed homeostasis and the induction of biological stress, which is manifested by a sharp decline in the mitotic index and an elevation of chromosomal aberrations.Our results are similar to those of other studies into gemcitabine.Salem et al. (2012), for example, have shown the cytotoxic effects of gemcitabine, while Aydemir and Bilaloglu (2003) and Aydemir et al. (2005) have used the structural chromosomal aberration assay and micronucleus test system and Fowler et al. (2009) have used DNA polymerization to show its genotoxic potential.In this study, however, experimental animals treated with a single dose of gemcitabine (30 mg/ kg/day) but subsequently treated with Salvia merjamie for 24-72h showed a significant reduction in the mitotic index, indicating that Salvia merjamie is effective in reducing the mitotic index.The mice treated with gemcitabine and Salvia merjamie also showed a significant decrease in the number of chromosomal aberrations.The mitotic index of mice treated with Salvia merjamie extract in fact recovered to the point that it was equivalent to the mitotic index of the control group, and chromosomal damage was also significantly repaired by Salvia merjamie extract.It can be concluded, therefore, that Salvia merjamie extract protects mice bone marrow cells from gemcitabine induced mutation.

Table 4 . Effects of Salvia merjamie Extract on Mitotic Index of Bone Marrow Cells of SWR/J Mice After 24h of Injection
*Differences are statistically significant from the control group at p<0.01 *

Table 5 . Effects of Salvia merjamie Extract on Mitotic Index of Bone Marrow Cells of SWR/J Mice After 48h of Injection
*Differences are statistically significant from the control group at p<0.01 *

Table 6 . Effects of Salvia merjamie Extract on Mitotic Index of Bone Marrow Cells of SWR/J Mice After 72h of Injection
*Differences are statistically significant from the control group at p<0.01 *