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about 50-80% of bile acid content consisted of cholic acid in the substitutes. These
values are in agreement with Yan et al. (
2007
), who previously reported that substi-
tutes contained much higher levels of cholic acid than the natural preparations.
Based on principal components analysis (PCA), Kong et al. (
2010
) concluded that
natural
C
.
Bovis
and its substitutes underwent different manufacturing procedures,
leading to significant differences in cholic acid content. We suggest that cholic acid
is likely added intentionally during the manufacturing process of the substitutes;
therefore, cholic acid serves as a predictable marker of the substitutes. Moreover,
the high content of cholic acid likely contributes to the toxicity of the substitutes
towards cardiac myocytes and fibroblasts (Takahashi et al.
2010
) .
Although most natural
C
.
Bovis
preparations (Aus-1, Aus-2, Brazil, Mexico, and
Kenya) contain less than 5 mmol/g taurine, Cul-2 and Art-1 contain more than
200 mmol/g of the amino acid. Taurine has been identified as a characteristic amino
acid component of
C
.
Bovis
. It has been previously shown that the ratio of taurine to
free amino acids and total amino acids of
C
.
Bovis
is significantly higher than in
other crude animal drugs. According to Hashimoto et al. (
1994
) , the taurine content
of
C
.
Bovis
is about 3.6-11.6 mmol/g, a range consistent with the value reported in
this study. On the other hand, some substitutes contain more than 200 mmol/g of the
cardioprotective amino acid (Wójcik et al.
2012
; Yamori et al.
2009,
2010
) . It also
appears to be added to the preparation of the substitutes and could serve as a marker
to distinguish natural samples from their substitutes.
15.3.3
The Inorganic Composition of Natural C . Bovis
and Its Substitutes
We also focused on the levels of certain inorganic ingredients using ICP-MS. The
relative concentration of 9 elements that were present in the sample extracts at a
concentration exceeding 1 mg/l are shown in Fig.
15.2
. Natural
C
.
Bovis
contained
more copper, manganese, and zinc than the substitutes. On the other hand, the
substitutes were characterized by high levels of iron, magnesium, and calcium.
According to Okasaka (
2008
), in vitro cultured
C
.
Bovis
is made from bilirubin,
some bile acids, ferrous sulfate, magnesium sulfate, cattle bile, water, and other
additives. Thus, the elements added during the manufacturing process could also
serve as markers to distinguish the substitutes from natural
C
.
Bovis
.
15.3.4
The Spectroscopic Analysis of Natural C . Bovis
and Its Substitutes
We have previously reported differences between natural
C
.
Bovis
and its substitutes
using Mössbauer analysis (Azuma et al. submitted). In the present study, we ana-
lyzed the spectroscopic characteristics of the extract samples using KURRI-LINAC
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