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14
with 10 mg/kg AA, AA-I and AA-II every 3 leptic. In 2011, the National Drug Commit-
days for 19 days had pathological changes, tee removed KK from every herbal recipe on
i.e. liver and kidney cell enlargement and le- the List of Herbal Medicinal Products A.D.
5
sions. For an acute toxicity study, rats and 2011. However, this crude drug can still be
mice treated orally or intravenously with high- bought from local herbal dispensaries. So the
dose AA died from acute renal failure within detection of AA is needed for herbal medi-
6
15 days. Due to these toxicities, many coun- cine manufacturers in order to avoid using
tries have eliminated any drugs or herbal prod- AA-containing herbal raw materials. Thin
ucts that contain AA. Therefore, several ana- layer chromatography (TLC) is one of the
lytical methods including high performance chromatographic techniques for the separa-
7-10
liquid chromatography (HPLC), capillary tion and identification of mixture compounds
11 12
zone electrophoresis and UHPLC have based on the use of appropriate stationary
been developed to detect these toxic com- and mobile phases. This technique is simple,
pounds in products that are suspected of cost-effective and reliable. In this study we,
containing AA. However, such previous therefore, attempted to optimize a simple and
methods have several limitations such as the rapid TLC method to detect AA-I in KK
ionization of the carboxylic acid group when samples. Furthermore, we also tried to de-

using alkaline mobile phase in reverse-phase tect AA-I in Yahom Nawakod (or
HPLC analysis, the inconvenience to control Homnawakod, HNK) Ayurved Siriraj, a herbal
pH and power supply in capillary zone elec- formula that contains KK, which is a repre-
trophoresis, the use of expensive tools and sentative sample of complex multiple herbal
solvents, the requirements of many steps in formulas. Yahom Nawakod is a famous
sample preparation when analyzing with remedy for the treatment of nausea, vomit-
HPLC, etc. In this study, we modified the ing, and vertigo among elderly persons and
method of AA detection using the TLC tech- has been included on the List of Herbal Me-
nique of Ohno T. et al as it was a more con- dicinal Products since A.D. 2006.

venient, faster and cheaper procedure that
could be an alternative method for detecting Materials and Methods
13
such compounds.
1. Chemicals and plant materials
In Thailand, Krai-kruea (KK), a dried root
of either Aristolochiapierrei Lecomte or All reagents were of analytical grade,
Aristolochia tagala Cham. (Aristolochiaceae), including AA-I and AA-I sodium salt (AA-I
has been used as an ingredient of many tra- salt) purchased from Sigma; acetic acid, 25

ditional herbal medicines. It is used as an % ammonia solution and acetonitrile from
antipyretic, tonic, appetizer, anti-inflamma- Merck; chloroform, methanol and ethyl ac-
tory, muscle relaxant, appetizing, and ana- etate from BDH, Scharlauand Riedel-deHaen,