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             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,
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