**4.3. Antimutagenic activity of bamboo vinegars**

**4.2. Mutagenicity of bamboo vinegars**

and TA100 without or with S9 mix.

**Specimens Diluting percent content (%)**

56 Bamboo - Current and Future Prospects

**Specimens Diluting percent content (%)**

Bamboo vinegars

Bamboo vinegars

Unit: %. 1

Ames *et al*. report that the number of spontaneous revertants induced by the specimen is less than that for the control group by more than two times; the specimen has no mutagenicity [32]. **Table 3** shows the mutagenicity results for the bamboo vinegar collected at temperatures of 90–92°C with a diluting percent content of 25.00, 20.00, 13.33 and 10.00% for *S. typhimurium* TA98 and TA100 without S9 mix and with S9 for a diluting percent content of 20.00, 13.33 and 10.00%. The spontaneous revertants of Blank are 65 for TA98 and 132 for TA100 and of bamboo vinegars without S9 mix are from 41 to 53 for TA98 and from 109 to 129 for TA100. With S9 mix, the spontaneous revertants of Blank and bamboo vinegars are 69–76 for TA98

**Table 2.** Survival of diverse bamboo vinegars collected at different temperatures toward *Salmonella typhimurium* TA98

Survival (%) = (the bacterial count of test group/the bacterial count of control group) × 100.

**Collection temperature (°C)**

−S9 33.33 95.16<sup>1</sup> 85.42 89.86 85.58 91.69 90.18

S9 25.00 88.81 92.83 90.53 85.46 76.36 70.81

−S9 33.33 109.12 90.76 95.92 112.16 109.79 104.98

S9 25.00 89.79 96.39 103.87 122.07 86.94 89.12

**Over 80 90–92 99–102**

25.00 115.54 88.91 99.13 80.04 99.13 105.82 20.00 86.43 109.11 94.43 84.27 84.64 86.20 13.33 95.30 88.11 99.53 90.20 99.53 90.56 10.00 106.58 94.69 99.56 109.42 101.16 84.53

20.00 95.40 84.84 89.53 86.75 86.53 106.65 13.33 92.12 120.27 94.53 111.60 98.66 104.95 10.00 84.19 99.79 92.39 87.00 94.73 83.29

**120–123 145–150 80–150**

25.00 104.13 115.42 90.86 99.82 112.82 102.42 20.00 111.19 115.69 107.04 123.40 124.23 108.62 13.33 114.93 118.91 109.61 102.42 95.88 128.20 10.00 113.75 111.47 122.63 96.44 127.98 119.71

20.00 113.32 90.36 89.11 89.27 85.19 89.74 13.33 126.73 115.78 143.39 140.28 84.85 97.83 10.00 117.83 99.02 120.54 98.09 102.71 131.10

**TA98 TA100 TA98 TA100 TA98 TA100**

**TA98 TA100 TA98 TA100 TA98 TA100**

If mutagenicity occurred in a treated material, the results of the antimutagenic assay would be confused due to increased or decreased numbers of revertants of TA98 and TA100 [42]. Hence, without S9 mix a diluting percent content of 25.00–10.00% and with S9 mix at 20.00–10.00% a diluting percent content are selected for the antimutagenic activity. NQNO and AFB<sup>1</sup> are used as direct mutagens requiring metabolic activation and indirect acting mutagen, respectively


1 MR (mutagenicity ratio) = induced revertants per plate/spontaneous revertants per plate (control).

**Table 4.** Mutagenicity ratio of diverse bamboo vinegars collected at different temperatures toward *Salmonella typhimurium* TA98 and TA100 without or with S9 mix.

for TA98 and 843–1202 (68.97–53.14%) for TA100 from bamboo vinegar at 90–92°C with different diluting percents. The results also show that the higher diluting percent content, the greater the inhibition, as well as, no matter what the vinegar is, the inhibition for TA100 is greater than that of TA98. Moreover, the inhibition of the bamboo vinegars collected at all different temperatures against the NQNO for diluting percent contents of 25.00, 20.00, 13.33 and

**Table 5.** Antimutagenic activity of diverse bamboo vinegar collected at the temperature of 90–92°C toward *Salmonella* 

Inhibition (%) = [1–(number of His+revertants in the presence of the test vinegar—number of spontaneous revertants)/

(number of His+revertants in the absence of the test vinegar—number of spontaneous revertants)] × 100.

**Bamboo vinegar (90–92°C)**

25.00 807 30.18 425 77.65 20.00 841 27.04 446 76.05 13.33 891 22.33 594 64.80 10.00 985 13.49 616 63.15

20.00 1114 40.63 843 68.97 13.33 1292 30.44 1012 61.53 10.00 1319 28.88 1202 53.14

NQNO (1 μg/plate) Blank2 11283 0.00 1445 0.00

(5 μg/plate) Blank 1824 0.00 2406 0.00

Spontaneous revertants 65 132

Spontaneous revertants 76 140

Blank (the control group) is added without either bamboo or wood vinegars.

**TA98 Inhibition1) (%) TA100 Inhibition (%)**

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59

at 20.00, 13.33 and 10.00% is shown in **Table 6**.

(37.43–75.24%), they are better

toward TA100 is greater than that

toward TA98

The inhibition of the bamboo vinegars to TA98 is 0.19–30.18% for NQNO and 0.92–40.63%

than those for TA98. The antimutagenic activity to NQNO is effective for bamboo vinegars collected at diverse temperatures with a diluting percent content of 25.00% or less, and for

The identified compounds of bamboo vinegars collected from different temperatures in the category of 80–150°C, over 80, 90–92, 99–102, 120–123 and 145–150°C are analyzed by gas chromatography-mass spectroscopy [15, 38, 44]. The acid, phenol, ketone and other

, it is also effective at a 20.00% or less diluting percent content. Furthermore, the bamboo

. For TA100 against NQNO (18.18–81.77%) and AFB<sup>1</sup>

toward TA98. It is also indicated that the inhibition of the vinegars against AFB<sup>1</sup>

**4.4. Effect of bamboo vinegars' compounds on preliminary safety evaluation**

vinegars show that the inhibitory effect on NQNO or AFB<sup>1</sup>

and TA100 is better than that against NQNO.

10.00% or against the AFB1

*typhimurium* TA98 and TA100 with S9 mix.

**Mutagens Diluting percent** 

**content (%)**

for AFB1

AFB1

1

2

3 Mean.

AFB1

[37]. Doses of mutagens, 1 μg for NQNO and 5 μg for AFB<sup>1</sup> , are selected from a dose-response curve of a preliminary experiment [43]. The His+revertants of strain are less than those of the control group (Blank), indicating that with antimutagenic activities. Meanwhile, the inhibitory effect of the specimen is expressed by inhibition (%), and the higher the inhibition, the more effective the antimutagenic activities [40]. The inhibitory effects for one of the antimutagenic activity results for the bamboo vinegar collected at temperatures of 90–92°C with a diluting percent content of 25.00, 20.00, 13.33 and 10.00% for NQNO and at 20.00, 13.33 and 10.00% for AFB1 are summarized in **Table 5**.

The His+ revertants of strain against the NQNO in Blank (without bamboo vinegars) are 1128 for TA98, and 1445 for TA100, for AFB1 : they are 1824 for TA98 and 2406 for TA100. The spontaneous revertants without NQNO are 65 for TA98 and 132 for TA100 and without AFB<sup>1</sup> are 76 for TA98 and 140 for TA100. The His+ revertants of strain (inhibition,%) against the NQNO for bamboo vinegar at 90–92°C with different diluting percent contents are 807–985 (30.18–13.49%) for TA98 and 425–616 (77.65–63.15%) for TA100. For AFB<sup>1</sup> , they are 1114–1319 (40.63–28.88%)


1 Inhibition (%) = [1–(number of His+revertants in the presence of the test vinegar—number of spontaneous revertants)/ (number of His+revertants in the absence of the test vinegar—number of spontaneous revertants)] × 100. 2 Blank (the control group) is added without either bamboo or wood vinegars.

3 Mean.

[37]. Doses of mutagens, 1 μg for NQNO and 5 μg for AFB<sup>1</sup>

are summarized in **Table 5**.

for TA98 and 425–616 (77.65–63.15%) for TA100. For AFB<sup>1</sup>

for TA98, and 1445 for TA100, for AFB1

TA98 and TA100 without or with S9 mix.

for TA98 and 140 for TA100. The His+

10.00% for AFB1

**Specimens Diluting percent content (%)**

58 Bamboo - Current and Future Prospects

**Specimens Diluting percent content (%)**

Bamboo vinegars

Bamboo vinegars

Unit: %. 1

The His+

curve of a preliminary experiment [43]. The His+revertants of strain are less than those of the control group (Blank), indicating that with antimutagenic activities. Meanwhile, the inhibitory effect of the specimen is expressed by inhibition (%), and the higher the inhibition, the more effective the antimutagenic activities [40]. The inhibitory effects for one of the antimutagenic activity results for the bamboo vinegar collected at temperatures of 90–92°C with a diluting percent content of 25.00, 20.00, 13.33 and 10.00% for NQNO and at 20.00, 13.33 and

**Table 4.** Mutagenicity ratio of diverse bamboo vinegars collected at different temperatures toward *Salmonella typhimurium*

MR (mutagenicity ratio) = induced revertants per plate/spontaneous revertants per plate (control).

**Collection temperature (°C)**

−S9 25.00 0.721 1.08 0.81 0.98 − −

S9 20.00 1.10 1.47 0.90 1.39 0.88 1.57

−S9 25.00 0.74 0.63 0.68 0.50 0.62 0.80

S9 20.00 1.18 1.19 1.32 1.32 1.16 0.83

**Over 80 90–92 99–102**

20.00 0.68 1.02 0.64 0.91 0.65 0.90 13.33 0.70 0.86 0.81 0.83 0.72 0.78 10.00 0.61 0.93 0.63 0.87 0.75 1.00

13.33 0.91 1.51 0.96 1.45 0.89 1.62 10.00 1.04 1.42 0.97 1.47 0.96 1.66

**120–123 145–150 80–150**

20.00 0.81 0.62 0.59 0.54 0.67 0.76 13.33 0.52 0.57 0.57 0.50 0.68 0.72 10.00 0.69 0.77 0.69 0.56 0.73 0.60

13.33 1.14 1.29 1.22 1.26 1.21 1.04 10.00 1.00 1.28 1.20 1.40 1.35 1.05

**TA98 TA100 TA98 TA100 TA98 TA100**

**TA98 TA100 TA98 TA100 TA98 TA100**

revertants of strain against the NQNO in Blank (without bamboo vinegars) are 1128

taneous revertants without NQNO are 65 for TA98 and 132 for TA100 and without AFB<sup>1</sup>

bamboo vinegar at 90–92°C with different diluting percent contents are 807–985 (30.18–13.49%)

: they are 1824 for TA98 and 2406 for TA100. The spon-

revertants of strain (inhibition,%) against the NQNO for

, are selected from a dose-response

, they are 1114–1319 (40.63–28.88%)

are 76

**Table 5.** Antimutagenic activity of diverse bamboo vinegar collected at the temperature of 90–92°C toward *Salmonella typhimurium* TA98 and TA100 with S9 mix.

for TA98 and 843–1202 (68.97–53.14%) for TA100 from bamboo vinegar at 90–92°C with different diluting percents. The results also show that the higher diluting percent content, the greater the inhibition, as well as, no matter what the vinegar is, the inhibition for TA100 is greater than that of TA98. Moreover, the inhibition of the bamboo vinegars collected at all different temperatures against the NQNO for diluting percent contents of 25.00, 20.00, 13.33 and 10.00% or against the AFB1 at 20.00, 13.33 and 10.00% is shown in **Table 6**.

The inhibition of the bamboo vinegars to TA98 is 0.19–30.18% for NQNO and 0.92–40.63% for AFB1 . For TA100 against NQNO (18.18–81.77%) and AFB<sup>1</sup> (37.43–75.24%), they are better than those for TA98. The antimutagenic activity to NQNO is effective for bamboo vinegars collected at diverse temperatures with a diluting percent content of 25.00% or less, and for AFB1 , it is also effective at a 20.00% or less diluting percent content. Furthermore, the bamboo vinegars show that the inhibitory effect on NQNO or AFB<sup>1</sup> toward TA100 is greater than that toward TA98. It is also indicated that the inhibition of the vinegars against AFB<sup>1</sup> toward TA98 and TA100 is better than that against NQNO.

#### **4.4. Effect of bamboo vinegars' compounds on preliminary safety evaluation**

The identified compounds of bamboo vinegars collected from different temperatures in the category of 80–150°C, over 80, 90–92, 99–102, 120–123 and 145–150°C are analyzed by gas chromatography-mass spectroscopy [15, 38, 44]. The acid, phenol, ketone and other


and AFB1

**5. Conclusion**

1 μg/plate and aflatoxin B1 (AFB<sup>1</sup>

in the portion of drinks or/and food.

Address all correspondence to: alexhlin@mail.ncyu.edu.tw

NQNO and AFB<sup>1</sup>

**Author details**

University, Chiayi, Taiwan

19780646836

Han Chien Lin

**References**

with 5 μg/plate in *S. typhimurium* TA98 and TA100. This is because the antimicro-

Preliminary Safety Evaluation of Bamboo Pyrolysis Products: Charcoal and Vinegar

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61

bial activity is affected by some phenol compounds [45]. In this report, the main fractions of bamboo vinegars are phenol compounds, 57.87–65.98% [38]. It is inferred that the phenol

The biological action of bamboo charcoal and vinegars is evaluated by *Salmonella* mutagenesis assay, as a safety evaluation and antimutagenic activity. The charcoal can be expected to be similar to the materials of edible natural colorants and/or natural drying agents, because the water activity of charcoal is below 6.0, the Pb base of charcoal is below 40 ppm, and the bamboo charcoals have no toxicity and mutagenicity toward *Salmonella typhimurium* TA98 and TA100. For the bamboo vinegars, the main percent content of the phenol compounds for bamboo vinegars is phenol (5.93–16.60%) and 2-methoxy-phenol (8.27–16.39%). The diluting percent content of vinegars is lower than 20.00% or less with S9 mix and 33.33% or less without S9 mix in cytotoxicity and mutagenicity toward *Salmonella typhimurium* TA98 and TA100 because the rest of the bacterium (Survival) these percent contents is higher than 80% of the control group (Blank), and the mutagenicity ratio (MR) is less than for the control group by more than two times. The diluting percent content of vinegars of 20.00% or less expressed the amount-dependent inhibitory effect against both the mutagenicity of 4-nitroquinoline-N-oxide (NQNO) with

It is suggested that bamboo vinegars with a diluting percent content to the least extent of 20.00% or less have no cytotoxicity and mutagenicity, and their antimutagenic activity with

reference of functional additives, due to their prospective applications in human care, such as

Department of Wood Based Materials and Design, College of Agriculture, National Chiayi

[1] Austin R, Ueda K. Bamboo. New York: John Weatherhill Inc; p. 1970. 215 Record Number:

) with 5 μg/plate in *Salmonella typhimurium* TA98 and TA100.

is effective. The bamboo vinegars in the extent of this report can be as the

compounds may partially account for the biological action of bamboo vinegars.

1 Inhibition (%) = [1–(number of His+revertants in the presence of the test vinegar—number of spontaneous revertants)/ (number of His+revertants in the absence of the test vinegar—number of spontaneous revertants)] × 100.

**Table 6.** Inhibition of diverse bamboo vinegars collected at different temperatures toward *Salmonella typhimurium* TA98 and TA100 with S9 mix.

compounds of bamboo vinegars are about 10.65–20.09%, 57.87–65.98%, 10.13–18.76% and 9.66–15.30, respectively. The acid compounds included butanoic acid, 2-methoxyethyl acetate, 4-hydroxy-butanoic acid and 4-hydroxy-3-methoxy-butanoic acid. The maximum fraction of acid compounds is the bamboo vinegar collected from 80 to 150°C. The phenol (5.93–16.60%), 2-methoxy-phenol (8.27–16.39%) and 4-ethyl-phenol (3.68–9.48%) are the main fractions of phenol compounds for bamboo vinegars. For ketone compounds, the 2-hydroxy-3-methyl-2-cyclopentenone-1-one, 2, 3-dimethyl-2-cyclopentenone-1-one and maltol can be measured for bamboo vinegars collected at all temperatures. The maximum fraction of ketone compounds is the bamboo vinegar collected from 120 to 123°C.

According to the former, results of safety evaluation (**Tables 1** and **2**; **Tables 3** and **4**) and antimutagenic activity (**Tables 5** and **6**) are present in diverse bamboo vinegars, the mutagenicity is occurred in the diluting percent content of vinegars that are higher than 20.00%, and without S9 mix is at 33.33%, but the diluting percent content of vinegars of 20.00% or less, expressed the amount-dependent inhibitory effect against both NQNO with 1 μg/plate and AFB1 with 5 μg/plate in *S. typhimurium* TA98 and TA100. This is because the antimicrobial activity is affected by some phenol compounds [45]. In this report, the main fractions of bamboo vinegars are phenol compounds, 57.87–65.98% [38]. It is inferred that the phenol compounds may partially account for the biological action of bamboo vinegars.
