INTERACTION OF SEVERAL TRADITIONAL MEDICINE PREPARATIONS WITH AMINOPYRINE IN RAT HEPATOCYTES
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1 INTERACTION OF SEVERAL TRADITIONAL MEDICINE PREPARATIONS WITH AMINOPYRINE IN RAT HEPATOCYTES H.H. Abas, H.B. Saringat and A.A.Rozana The study looks at the in vitro interaction of three Malay (Air Gamat Asli Langkawi, Faizol Tonik and Air Jamu Pak Tani ) and three Chinese (Yomeishu, Ginseng & Polygona Root Extractum and King To Nin Jiom Pei Pa Kao ) traditional medicine preparations aminopyrine in rat liver hepatocytes. Aminopyrine N-demethylase activity was determined by measuring the quantity of formaldehyde formed. Results indicate that Faizol Tonik a and Air Jamu Pak Tamu increase the activity of aminopyrine N-demethylase significantly (P<0.05) whereas Air Gamat Asli Langkawi produces no significant change in the enzymatic activity when compared to control. With the Chinese traditional medicine preparations, all three preparations investigated increase the activity of aminopyrine N-demethylase significantly (P<0.05). Thus, several of the traditional medicine preparations tested were found to affect the metabolism of aminopyrine in-vitro and whether the biovailability of aminopyrine in-vivo can be similarly affected by these preparations remains to be investigated INTRODUCTION Systematic botanical, chemical and pharmacological studies of medicinal plants are being actively conducted in many universities and research institutions in Malaysia. Major portions of research are being conducted on crude plant extracts, soluble plant fractions and pure compounds. Literature search indicated that there are still limited research done on traditional medicine preparations that are available in the market. Most traditional medicine preparations consumed by the public, irrespective of racial origin, usually contain more than one plant extracts as their active ingredients. The therapeutic outcome or adverse effect/s of these preparations thus could have been derived from the summation of all the ingredients in the traditonal medicine preparations. The analysis of these preparations available in the market is of more immediate relevance with respect to the health benefit or risk to consumer. Furthermore, such information is much needed by the relevant government authorities responsible in overseing the registration of all traditional medicine preparations as required under the National Drug Control Legislation. This preliminary study aims to investigate possible in vitro interactions between three Malay and three Chinese traditional medicine preparations available in the market with aminopyrine utilising the rat liver hepatocytes. Article V, May 1998 Page 1
2 Interaction Of Traditional Medicine Preparations With Aminopyrine In Rat Hepatocytes ARBEC, May 1998 MATERIAL AND METHODS Chemicals Used All chemicals used were of analytical grade purity. Collagenase (type IV) and aminopyrine were obtained from the Sigma Chemical Co., St. Louis, MO, USA. The medicinal preparations were obtained from local medicine shops. Air Gamat Asli Langkawi was manufactured by Keluaran Gamat Asli Langkawi, Langkawi, Kedah; Faizol Tonik was manufactured by Kilang Faizol Tonik, Kuala Kangsar, Perak and Air Jamu Pak Tani was manufactured by Perusahaan Jamu Arjuna, Batu Pahat, Johor; Yomeishu was manufactured by Yomeishu Seizo Co. Ltd., Tokyo, Japan; Ginseng & Polygona Root Extractum was manufactured by Tianjin Medicine and Health Products, China and King To Nin Jiom Pei Pa Kao was manufactured by Nin Jiom Medicine Manufacturing (Hong Kong) Limited. Preparation of Hepatocytes and Liver Enzyme Assay Forty to sixty-weeks old normal male rats of the Sprague Dawley strain, bred in the School of Pharmaceutical Sciences, Universiti Sains Malaysia were used. All rats weighing between g were fed a normal laboratory chow (Gold Coin ) ad libitum and had free access to tap water. Isolated hepatocytes were prepared by using the modified collagenase perfusion technique (Hussin and Skett, 1988). The animals were anaesthetised with ether and the liver perfused in-situ through the hepatic portal vein with calcium-free Hank's Balanced Salt Solution (HBSS) for about 15 minutes followed by the collagenase buffer (HBSS supplemented with 4 mm calcium chloride and collagenase 0.5 mg/ml) until the liver appeared to have broken up. The liver was removed from the animal and the cells dispersed in the calcium-free HBSS using a steel forcep. The cell suspension was filtered through gauze and centrifuged at 200g for 5 minutes (in a Hettich Universal centrifuge) to clean the parenchymal cells of debris. The supernatant was removed and cells suspended in incubation medium (HBSS supplemented with lg/l glucose, 100mg/L MgS0 4, 100mg/L MgC1 2 and 185mg/L CaCl 2 ; ph 7.4). The cells were then counted using a hemocytometer and assayed for viability using trypan blue exclusion method. Average yield was about 5 x 10 7 cells/g liver and viability was routinely greater than 90 % for all preparations. Equal volume (0.5 ml) of serial dilutions of the traditional medicine preparations were added to petri dishes containing aminopyrine (25nM final concentration), 2 x 10 6 hepatocytes and incubation medium (final volume of 10 ml). Control petri dishes contain the same but had the traditional medicine preparations replaced with distilled water. The highest volume of the traditional medicine preparations was chosen so as not to interfere with spectrophotometric measurement. The petri dishes were then incubated in C0 2 incubator (Queue Systems Inc./Model 2710) for 18 minutes at 37 C. Aminopyrine N-demethylase activity was determined by measuring the quantity of formaldehyde formed according to the colorimetric method of Nash (1953). Statistical Analysis Results were presented as means ± S.D. Statistical test within groups were analysed using Student t-test from a computerised Statswork software. Article V, May 1998 Page 2
3 RESULTS AND DISCUSSION The pharmacological or adverse effect/s that resulted from the consumption of any traditional medicine preparations is derived from the combination of effects of the total ingredients in a preparation. This is well differentiated from the consumption of allopathic medicine whereupon the content and dose of an active ingredient in any particular dosage form is well quantitated due to more stringent quality control. The traditional medicine preparations available in the market usually contain more than one plant extracts or other biological sources. Figure 1 indicates that at dilutions higher than 1:8, Air Jamu Pak Tani significantly increase the activity of aminopyrine N-demethylase in the rat liver. Undiluted volume of the same preparation exerted a 90 % activity increase above control (P < 0.001) whereas at lower dilutions (< 1: 16) no significant effect (P > 0.05) was produced. It is not known however which of the I I natural plant extracts used in the concoction of Jamu Pak Tani is the active ingredient producing the effect observed. Air Gamat Asli Langkawi, a single component concoction, showed no significant effect on aminopyrine N-demethylase activity when compared to control (Figure 2). Compared to control, serial dilutions of this preparation exerted no significant influence on the liver enzyme activity. Faizol Tonik exerted significant increase in aminopyrine Ndemethylase activity (Figure 3). At one-eighth and one-fourth dilutions, the enzyme activity was increased by about 16 % above control (P < 0.05); at half dilution, 41% above control (P < 0.001) and undiluted preparation increased the enzyme activity by 96 % above control (P < 0.001). King To Nin Jiom Pei Pa Kao also increased aminopyrine Ndennethylase activity (Figure 4). Undiluted preparation exerted a 61% increase in enzyme activity when compared to control (P < 0.001) whereas one-eighth and higher dilutions had no effect on the enzyme activity. Undiluted Yomeishu significantly increased aminopyrine Ndemethylase activity by 92 % (P < 0.001). The enzyme activity significantly increased with decreasing dilutions of Yomeishu and dilutions of one-sixteenth and higher exerted no significant effect on the enzyme activity with-respect to control (Figure 5). Only half dilution and undiluted Ginseng & Polygona Root Extractum exerted significant increase in aminopyrine N-demethylase activity (34 % and 91% respectively) (Figure 6). One-fourth and higher dilutions showed no significant effect on the enzyme activity when compared to control. With the exception of Air Gamat Asli Langkawi all the other five preparations investigated in this study have more than one plant source. As documented on the bottle container, Air Jamu Pak Tani and Faizol Tonik have I I and 17 ingredients natural sources respectively; Yomeishu, King To Nin Jioin Pei Pa Kao and Ginseng & Polygona Root Extractum have 14, 16 and 2 herbal ingredients respectively. Article V, May 1998 Page 3
4 Interaction Of Traditional Medicine Preparations With Aminopyrine In Rat Hepatocytes ARBEC, May 1998 Figure 1 : In-vitro effect of Air Jamu Pak Tani an aminopyrine N-demethylase activity in normal male rat hepatocytes Figure 2 - In-vitro effect of Air Gamat Asli Langkawi on aminopyrine Article V, May 1998 Page 4
5 Figure 3 - In-vitro effect of Faizol Tonik on aminopyrine N-demethylase activity in normal male rat hepatocytes Figure 4 - In-vitro effect of King To Nin Jiam Pei Pa Kao on aminopyrine Article V, May 1998 Page 5
6 Interaction Of Traditional Medicine Preparations With Aminopyrine In Rat Hepatocytes ARBEC, May 1998 Figure 5 - In-vitro effect of Yomeishu on aminopyrine Figure 6 - In-vitro effect of Ginseng and Polygona root extractum on aminopyrine Article V, May 1998 Page 6
7 Aminopyrine undergoes phase I N-demethylation process in the liver in the presence of cytochrome P-450 N-demethylase. The increase in the aminopyrine N-dernethylase activity in-vitro as illustrated in this study may increase the metabolism of aminopyrine and tentatively reduces the latter bioavailability. Other clinical drugs such as tricyclic antidepressants such as imipramine, desipramine; neuroleptic drugs such as phenothiazine, chlorpromazine and thioridazine; procainamide, cocaine, morphine and salicylamide are also known to undergo N-- demethylation in-vivo. Thus there is also a possibility that the traditional medicine preparations investigated in this study may increase the metabolism of these clinical drugs. However, it should be stressed that the effect here is only seen in-vitro and a similar effect in-vivo needs to be investigated. In conclusion, the study indicated that all the traditional medicine preparations investigated (except Air Garnat Asli Langkawi ) significantly increase aminopyrine N-demethylase activity in rat hepa-tocytes in-vitro. A possibility exists that similar interactions may occur in-vitro with other drugs that undergo hepatic phase I N-demethylation. Whether this effect is similarly produced in-vivo still needs further investigation. ACKNOWLEDGEMENT The authors acknowledge the research grant No. 131/0210/0520 provided by the Universiti Sains Malaysia, Penang, for making this study possible. REFERENCES Hussin, A.H. and Skett, P Lack of effect of insulin in hepatocytes isolated from streptozotocin-diabetic male rats. Biochem. Pharmac. 37: Nash, T The colorimetric estimation of formaldehyde by means of the Hantzsch Reaction. Biochem. J, 55: Article V, May 1998 Page 7
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