Determination of aristolochic acids I and II in Brazilian sugar cane spirit infusions “milhomem” commonly used in northeast Brazil as popular drinks

Aristolochic acids (AA) are phytochemicals found in plants of the genus Aristolochia belonging to the family Aristolochiaceae. These compounds bear a nitrophenanthrene carboxylic acid skeleton and are reported to be carcinogenic, mutagenic, and nephrotoxic. Sugar cane spirit infusions containing Aristolochia species are commonly used in Brazil as popular drinks, in total absence of scientific information. The presence aristolochic acids was confirmed in samples collected in popular markets of the city of Aracaju, Sergipe, Brazil. The aristolochic acids quantitative estimation was made in five samples of sugar cane spirit infusions obtained from different places of that city and were performed by high-performance liquid chromatography. The samples analyzed contained aristolochic acids I and II in concentrations ranging between 1.96 and 6.10 μg/ml for AA I and 2.22 and 11.55 μg/ml for AA II. The immediate banning of such popular drinks is recommended in view of the danger to ingest aristolochic acids, botanical products containing aristolochic acids or herbal products containing plants belonging to Aristolochiaceae family.


Introduction
Many plants have been used for centuries in Brazilian folk medicine to treat several human ailments.
Different types of preparations, such as water infusions (referred to as "teas"), hydroalcoholic extracts and juices among others are usual on these therapies. "Garrafadas" are mixed medicinal plants extracts that use sugar cane spirit, a typical Brazilian alcoholic beverage called "aguardente" or "cachaça" or wine as vehicle [1] .
A very popular herbal preparation referred to as "milhomem" (obtained by infusion of several Aristolochia plant species, such as A. cymbifera, A gigantea, A. birostris with sugar cane spirit) is used to treat liver and Revista Fitos. Rio de Janeiro. 2020; 14(1): 38-44 | e-ISSN: 2446-4775 | www.revistafitos.far.fiocruz.br | CC-BY 4.0 Determination of aristolochic acids I and II in Brazilian sugar cane spirit infusions ("milhomem") commonly used in northeast Brazil as popular drinks Silva, Kaplan, Alviano et al stomach disorders, snake bites, fever and ulcer for example [2][3][4] . With about 490 species spreading around the tropical areas [5] plants of genus Aristolochia (Aristolochiaceae) are known to contain nitrophenanthrene carboxylic acid compounds known as aristolochic acids (AA).
Aristolochic acids are strongly recorded as responsible for the nephrotoxic and genotoxic effects associated with the use of some herbal preparations [6,7] . The first evidence about the nephrotoxicity of aristolochic acids I and II was published in 1963 [8] . In addition, some types of cancer specially occurring in the urinary tract have also been linked to the ingestion of AA's present in these products [9,10] . Several analogs of AA are described, but aristolochic acids I and II (FIGURE 1) have particular significance for their common occurrence [11,12] .    3 , Aristolochic Acid III methyl ester R 1 = OCH 3 , R 2 , R 4 = H, R 3 = OCH 3 Aristolochic Acid IV R 1 = OCH 3 , R 2 = H, R 3 =OCH 3 , R 4 = CH 3 , Aristolochic Acid IV methyl ester R 1 = OH, R 2 , R 4 = H, R 3 = OCH 3 Aristolochic Acid IVa R 1 , R 2 = OCH 3 , R 3 , R 4 = H, Aristolochic Acid V Notwithstanding the fact that FDA suggested the removal of herbal products suspected to contain aristolochic acids from the market since 2001 [13] , some of these products are still available for consumption [14] . Taking into consideration the risk of using products containing AA's, this work aims to

General
Reagents and solvents were PA grade (Tedia Brazil) except those used for HPLC, analysis that were UV/HPLC grade (Merck). The ultrapurified water used was obtained with a Milli-Q apparatus (Millipore).
Aristolochic acids I and II standards were obtained from Sigma-Aldrich. Analytical data was processed using

HPLC analysis conditions
An HPLC Agilent 1200 series equipped with photodiode array detector was used for sample analysis. The method was as follows: column: RP18 Waters XTerra, 4.6 mm x 250 mm, 5 μm particle size; mobile phase, aqueous 0.5 % acetic acid (A) and methanol 0.5% acetic acid (B); gradient elution: 60% to 100% of B in 21 minutes; flow rate, 1.0 mL/min.; injection volume: 20 µL. UV detection, 254, 300 and 320 nm; retention times AAI: 22.6 min and AAII: 19.9 min. The retention times and UV spectra of samples and standards were compared in order to confirm identification [15] .   The quantitation results are summarized in TABLE 1, where it is seen the simultaneous occurrence of both aristolochic acids in all of them. The concentrations found ranged between 1.96 and 6.10 µg/mL for AAI and 2.22 and 11.55 µg/mL for AAII. In the present work we have set the UV detector to wavelengths 300 and 320 nm, respectively for AAII and AAI, instead of 224 and 254 nm as described by Schaneberg & Khan [14] .

Results and Discussion
The choice was made to allow absorbance detection in peak maxima (see FIGURE 2). The quantitative analysis of the "milhomem" infusion samples collected in Aracaju, Brazil showed an expressive content of both AAI and AAII. These results suggest that a systematic assessment of the content of aristolochic acids has also to be done in the following species, all of them used as medicinal plants in birostris is not popularly referred to as "milhomem". All the others are known as "milhomem" [16] .
Although regulatory agents, such as FDA in 2001, advised about the risk of using products suspected to contain AA's, several herb products are marketed without control. In Brazil, it is easy to access these plants in popular markets either as fresh plants or as in preparations such as the ones analyzed here. Many Aristolochia species are popularly recommended as medicines to treat snakebites, malaria, intestinal worms and so on [2,[18][19][20][21] .
Governmental agencies of different parts of world are banning products suspect to containing AA's. Several countries, such as UK, USA, Canada, Australia and New Zealand have suggested to reduce or prohibit the sale of herbal products containing aristolochic acids [13,22,23] .

Conclusion
Aristolochic acids I and II were detected and quantified in samples of herbal preparations ("milhomem" sugar cane spirit infusions) collected in several popular markets in Aracaju, Sergipe, Brazil. In spite of the risks of consuming such preparations, the "milhomem" sugar cane spirit infusions has been used as a very popular drink, maybe due to the lack of scientific information on its dangers. The results found in the present work highlights the need that Brazilian public health authorities issue a warning on health risks involved in such practice. Thus, an urgent communication of these results to the community is strongly recommended and necessary to ensure a safe use of Brazilian medicinal plants.