Antimutagenic Effect of Mulberry Leaf Extract

1. Mulberry leaves (MLs) source and reagents

Mulberry (Morus alba L.) leaf powder (certified by the Republic of Korea, Ministry of Food and Drug Safety) was purchased from Yakcho Wellbeing Teuku manufactured by Jaechen Hakdeul, in Ha-Myun, Gapyung-Gun, Kyunggi -Do, Korea. Reagents such as 2-aminoanthracene (2-AA) used as an indirect mutagen control, 4-nitroquinoline-noxide (4-NQ) used as a direct mutagen control, agar, Lhistidine, and D-biotin used in bacterial cell culture were from Sigma-Aldrich (St. Louis, MO, USA). All reagents and solvents used in this study were analytical grade. The S-9 mixture, a preparation containing xenobiotic metabolizing enzyme complexes of the rat liver, was purchased from the Molecular Toxicology (Boone, NC, USA) and stored at –70℃ until use.

2. Bacterial strain

The bacterial strain used in this test was Salmonella typhimurium TA98, a frameshift mutant, acquired from the Gene Bank of the Korean Research Institute of Bioscience and Biotechnology. The genotype was verified before performing each experiment. The genotype strain check was performed using the following criteria: Histidine dependence, biotin dependence, biotin and histidine dependence, presence of the rfa marker, uvr B deletion, ampicillin resistance, and spontaneous mutant frequency, according to the method described by Maron and Ames (1983).

The TA98 strain used in this test has HisD3052 allele mutation among histidine-requiring strains. This strain has enhanced sensitivity by the cell wall lipopolysaccharide (LPS), and the passage of the mutant substance is easily carried out, deep rough (rfa) mutation, uvr B deletion mutation and insertion of plasmid pKM101. Therefore, TA98 strain has more reactivity and inhibition rate than other histidine-requiring strains. After the genotype verification, the Salmonella strain was cultured overnight in Oxoid nutrient broth No. 2, before freezing at -80℃ with the addition of 0.09㎖ of the dimethylsulfoxide (DMSO) for each 1㎖ culture medium.

3. Preparation of the crude extracts and solvent extracted fractions

MLs powder, 100 g, was added to 70% ethanol solution, brought to 1ℓ, and extracted for 3 days and repeated three times. The crude extract was filtrated, concentrated by evaporation, and freeze-dried. The MLs crude extract was successively extracted with increasing polarity of solvent. Crude extracts were dissolved and sonicated in 200 ㎖ of distilled water, which was then added to the 200 ㎖ of diethyl ether for 12 - 24 h with stirring. After complete removal of the aqueous layer, the diethyl ether layer was filtered, concentrated, and freeze-dried. Chloroform was added to the aqueous layer and treated in a similar manner as the diethyl ether fractionation. In succession, a dichloromethane and butanol fractions were produced using the same procedure. Once four solvent fractions were generated from the crude MLs extract, the remaining aqueous layer was also filtered, concentrated, and freezedried. The residue from each of the four freeze-dried solvent fractions were then dissolved in dimethyl sulfoxide and the material from the aqueous layer was dissolved in phosphate buffer solution (pH 7.4) at the concentration of 40㎎/㎖. Finally, each fraction was passed through a 0.45㎛ membrane filter and stored at −80℃ until use.

4. Collection of CSCs

Forty 3R4F standard cigarettes were smoked under ISO smoking conditions using a RM-20 automatic smoking machine (Heinr Borgwaldt, Hamburk, Germany) and the following parameters: Puff volume, 35㎖; puff time, 2 min; puff duration, 1 min; butt length from the paper tip was + 3㎜. CSCs was collected via isopropanol solvent extraction by using a Cambridge filter.

5. Mutagenicity test

Mutagenicity was tested according to the method described by Maron and Ames (1983) and OECD guide line (Test no. 471) using the crude extract and each of the solvent fractions from the MLs powder.

4-NQO, a direct mutagen, a positive control, and 2-AA, an indirect mutagen, a negative control, were both used at 0.5㎍/plate in this test. The test sample treatment concentrations were 125, 250, 500, 1,000, and 2,000㎍/ plate. To each plate we added either 500㎕ of 0.2M sodium phosphate buffer (pH 7.4) in the case of direct mutagenicity test or S-9 liver mixture for the indirect mutagenicity test, which is capable of mutagenic activation of substances. For plating, Salmonella typhimurium TA 98 strain (1 - 2 × 10⁹ cells/㎖) were suspended in 100 ㎕ culture medium and 100㎕ of test sample or control were was added in a sterilized tube, voltex-mixed for 3 - 5 s, placed on a shaking incubator for 30 min at 37℃, and then, added to 10㎖ of top agar with 1㎖ of 0.5 mM histidine plus biotin solution, voltex-mixed, spread evenly on minimal glucose agar, and cultured for 48 h in a 37℃ incubator.

The number of revertant colonies in each plate was determined using an automatic colony counter. Concurrent positive and negative controls were performed in all experiments. All testing was conducted using triplicate plates for each of the experimental concentrations.

6. Antimutagenic test

An antimutagenic test was also performed on the crude extract and each solvent fraction of MLs according to the method described by Maron and Ames (1983).

Salmonella typhimurium TA98 strain (1 - 2 × 10⁹ cells/㎖) was suspended in 100㎕ culture medium. Separately, 200㎍ of CSCs and 500㎕ of S-9 fraction were added into a sterilized cap tube with 50㎕ of test sample, voltex-mixed for 3 - 5 s, and pre-incubated for 30 min in a 37℃ shaking incubator, after which time, the incubation mixture was added to 10 ㎖ of top agar plus 1㎖ of 0.5 mM histidine and biotin solution, voltex-mixed, spread evenly over minimal glucose agar, and cultured for 48 h in a 37℃ incubator. The number of revertant colonies in each plate was determined using an automatic colony counter. Concurrent positive and negative controls were utilized in all experiments.

All testing was conducted using triplicate plates at each of the experimental concentrations. Inhibition was calculated as follows:

$$ \text{Inhibition (\%) = (M- B) / (M - A)\hspace{0.17em}}\times 100$$

where, M= number of revertant colonies by mutagen, A = number of spontaneous revertant colonies, B = number of revertant colonies when the sample and mutagen were both added to the test strains.

7. Statistical analysis

All data are presented as means ± standard deviation. Data analysis was performed using StatView version (Statview Software, Cary, NC, USA). The statistical analysis between two groups was performed using the Fisher's protected least significant difference test or Scheffe's F test. Statistical significance was defined as when the rvalue difference between two groups was less than 0.01.

1. Recovery data

For the crude extract from mulberry (Morus alba L.) powder, 35.1 g of solids was recovered from 100 g of powder dissolved with 1ℓ of 70% ethanol solution (35.1%). The recovery from each solvent extraction was calculated based on the weight of crude extract fractionated by each of the four organic solvents, and after concentration.

The total yield (g/g of crude extract) for the diethyl ether, chloroform, dichloromethane, butanol, and remaining water fraction were 12.8%, 6.4%, 2.8%, 19.9% and 58.1%, respectively.

2. Selection of CSCs concentration

An appropriate CSCs (cigarette smoke condensates) concentration in culture medium for investigation of the antimutagenic effect of the crude extract and solvent extracted fractions was determined. The total amount of CSCs produced from smoking 40 3R4F standard cigarettes was 0.42 g.

Fig. 1 shows the change of revertant colony number with CSCs concentration in the culture medium inoculated with Salmonella typhimurium TA98 strain, from 25㎍/plate to 1,600㎍/plate. The revertant colony number was 31.0 ± 3.0 in the control not treated with CSCs, but was 347.3 ± 56.6 in the control treated with 2-AA (positive control). The number of revertant colonies was 41.0±3.5, 47.7±2.5, 115.3±8.2, 359.0±23.5, 555.3±44.9, 482.7±78.0 and 109.3±12.5 when CSCs were present in the culture medium at the concentrations of 25, 50, 100, 200, 400, 800 and 1,600㎍ per plate, respectively, increasing at concentrations up to 400㎍ of CSCs: Thereafter, the number of revertants decreased when CSCs were present at 800㎍ or more. One explanation is that more than 400㎍ CSCs in the culture medium may be toxic to Salmonella typhimurium TA98.

Fig. 1

Dose response curve of CSCs mutagenicity (+S-9 mix).

Therefore, we chose the treatment concentration of CSCs to 200㎍/plate for evaluating the inhibitory effect of the MLs crude extract and each solvent fractions against mutagenicity caused by CSCs.

3. Mutagenic activity of MLs crude extracts

In order to investigate whether MLs crude 70% ethanol extract has inherent mutagenic activity, we added the extract to the culture medium inoculated with Salmonella typhimurium TA98 at 125㎍/plate to 2㎎/plate. The results are shown in Table 1. The number of spontaneous revertant colonies in the negative control was 27.3 ± 3.5 with S-9 mix and 17.7 ± 1.5 without S-9 mix, respectively. The number of revertant colonies was 448.0±8.5 when treated the 0.5㎍ of 4-NQO, a direct mutagen, and was 524.7±10.7 when treated the 0.5㎍ of 2-AA, an indirect mutagen.

Table 1

Mutagenicity on 70% ethanol extract extracted from MLs.

These results are consistent with those of Maron and Ames (1983), indicating our system is operating in a manner similar to published results. The number of revertant colonies did not show any significant difference compared to negative control and was not more than two times different from the negative control when MLs crude extract was tested with S-9 mix and without S-9 mix. Therefore, we conclude that MLs crude extract itself is not mutagenic to Salmonella typhimurium TA98.

4. Mutagenic activity of MLs solvent extracted fractions

We also investigated whether the MLs solvent extracted fractions have mutagenic activity. Table 2 shows the change of revertant colony number when each MLs solvent fraction was tested at 1,000㎍/plate in culture medium inoculated with Salmonella typhimurium TA98, with and without S-9 mix.

Table 2

Mutagenicity on solvent fractions separated from MLs 70% ethanol extract with polarity.

The number of revertant colonies did not show a significant difference in comparison to negative control when treated each of the diethyl ether, chloroform, dichloromethane, butanol and water extracted fractions of MLs up to 1,000㎍/plate either with or without S-9 mix. Therefore, we further concluded that none of the solvent extracted fractions of MLs crude extract has direct or indirect mutagenic activity.

5. Antimutagenic effect of MLs crude extracts

In order to investigate whether mulberry crude extract has an antimutagenic effect on CSCs, the change of revertant colony number was measured with MLs crude extract in culture medium at a concentration of 500㎍ with S-9 mix and 200㎍/plate CSCs (Table 3).

Table 3

Inhibitory effects of MLs 70% ethanol extract against mutagenicity of CSCs.

The spontaneous revertant colony number of Salmonella typhimurium TA98 was 34.3±2.5, and the number of revertant colonies was 362.4±52.5 in the presence of CSCs at 200㎍/plate in same culture medium. Moreover, the number of revertant colony was 37.3±4.8 when the MLs crude extract and CSCs were both present, giving a rate of inhibition of mutagenicity of 99.1%.

6. Antimutagenic effect of MLs solvent extracted fractions

We also investigated whether 5 each solvent fractions from MLs crude 70% ethanol extract has an antimutagenic effect on CSCs (Table 4). When treated with 1㎎/plate of material from each solvent fraction and 200㎍ of CSCs in the culture medium at the same time, the inhibition of mutagenicity against CSCs was 99.0%, 0.0%, 0.0%, 0.0% and 0.1% for the diethyl ether, chloroform, dichloromethane, butanol, and water fractions of MLs crude 70% ethanol extract, respectively.

Table 4

Inhibitory effects of solvent fractions separated from MLs 70% ethanol extract against the mutagenicity of CSCs.

Therefore, the chloroform, dichloromethane, butanol, and water fractions did not show an antimutagenic effect in Salmonella typhimurium TA98 strain. Only the diethyl ether fraction of MLs extracts had an antimutagenic effect against CSCs.

7. Concentration dependence for the antimutagenic effect of the diethyl ether fraction

We further investigated the effective minimal concentration of the material in the diethyl ether fraction capable of producing an antimutagenic effect.

Table 5 shows the results of the antimutagenic assay when the diethyl ether fraction was tested at concentrations from 31.3㎍/plate to 1,000㎍/plate in culture medium inoculated with Salmonella typhimurium TA98 together with S-9 mix and CSCs at 200㎍/plate. These data confirm the antimutagenic effect of diethyl ether fraction against CSCs and showed that the effect increased as treatment concentration of diethyl ether layer increased in the culture medium. The inhibition of the mutagenicity was 97.1% in the presence of 1㎎/plate of diethyl ethyl fraction, which is approximately the same as the previous result for 500㎍/plate. The antimutagenicity data, therefore, suggest that more advanced studies such as the separation, analysis, and determination of chemical components responsible for the antimutagenic activity in the diethyl ether fraction of MLs crude extract may be needed.

Table 5

Inhibitory effects on the diethyl ether fraction separated from MLs 70% ethanol extract against the mutagenicity of CSCs with concentration.

In the case of Morus alba L., Chang et al. (2016) recently reported that the fruit does not produce subchronic oral toxicity or genotoxicity at concentrations greater than 1,000㎎/㎏ in a 90 day toxicity study of using Sprague-Dawley rats. On the contrary, there are numerous reports of antimutagenic activity by various plant extracts. In one study, 800㎍ of the hexane fraction from cinnamon bark ethanol extract produced over 98% inhibition when the antimutagenic effect was evaluated against B[a]P using Salmonella typhimurium TA98 strain (Jeong et al., 1998). Other reports where Salmonella typhimurium TA98 was used, include where: 200 ㎕ of a water extract of comfrey (Symphytum officinale) showed a 50% inhibition of mutagenicity induced by B[a]P (Ham et al., 1992), 100㎍ of a butanol extracted fraction from Ixeris dentata methanol extract showed over 90% inhibition of the mutagenicity induced by B[a]P (Kim et al., 2002a), and 100㎍ of a butanol extracted fraction from Kalopanax pictus Nakai crude methanol extract showed 74.5% inhibition (Kim et al., 2002b). The inhibition of mutagenicity by 1-aminopyrene was over 90% at 15㎎ of methanol extract of Curcuma sessilis and Punica granatum flowers grown in Thailand (Wongwattanasathien et al., 2010). An extract of tea grown in Brazil inhibited 2- aminofluorene mutagenicity in the Salmonella typhimurium TA98 (Horn and Vargas, 2008). Although many of these reports used the same bacterial mutant as the present study, an accurate comparison of efficacy based on test concentrations between various plants extracts is impossible because of the differences in methods of handling and preparing the fractions of each of the medicinal herbs, and, in many cases, the percent inhibition was given without providing the data on revertant colony number.

It is known that substances that inhibit mutagenicity can be cancer suppressing due to the ability to reduce formation of DNA adducts between the mutagen and DNA (Park et al., 2016). Plant-derived compounds may also interfere with the absorption of carcinogens into the body. We propose that the constituents of the diethyl ether fraction of MLs crude 70% ethanol extract may have anticancer activity based on one of two mechanisms: first, they may be metabolically activated by xenobiotic metabolizing enzymes, such as those present in liver fractions, that react with CSCs mutagens directly: or, second, constituents in the fraction may play an important role in promoting the DNA repair system involved in removing DNA damage caused by CSCs toxins or their metabolites.