* Departments of Biology and
Chemistry, The Hong Kong University of Science and Technology, Hong Kong
Received March 6, 2000; accepted May 8, 2000
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ABSTRACT |
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Key Words: cytotoxicity; Ephedra; ephedrine; ephedrine-to-toxins ratio; herb preparations; ma-huang; MTT colorimetry..
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INTRODUCTION |
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Ephedrine-type alkaloids (ETA) are the active principles of ma-huang. Within these alkaloids, (-)-ephedrine (E) is the most abundant, constituting between 30 aand 90% of the total alkaloid content (Tyler, 1993). Other ETA include (+)-pseudoephedrine (PE), (-)-N-methylephedrine (NME), (+)-N-methylpseudoephedrine (MPE), (-)-norephedrine (NE), and (+)-norpseudoephedrine (NPE). The acute thermogenic and lipolytic effects of E on basal metabolism and/or on diet-induced thermogenesis have now been confirmed in lean, obese and post-obese humans. The chronic administration of E in clinical trials has been reported to induce weight loss in diet-restricted obese patients (Astrup et al., 1992
; Dulloo and Stock, 1993
). Large amounts of ma-huang have been recently used as a source of E in numerous dietary supplements formulated for weight reduction. However, the U.S. Food and Drug Administration has received many reports of poisoning and other serious side effects caused by the indiscriminate consumption of these ma-huangcontaining products (FDA, 1997
).
As ETA are the active principles, the quality of ma-huang is determined by the contents of total ETA, with higher contents indicating better quality. However, this practice of grading using only the total ETA contents as sole parameter is inadequate. The profile of alkaloids is also important, because although individual ETA has similar pharmacological activity, they vary significantly in potency (Cetaruk and Aaron, 1994; Dollery, 1991
). Both the contents and the profile of ETA in ma-huang vary with plant species and varieties (Cui et al., 1991
; Liu et al., 1993
; Zhang et al., 1989
), plant parts, (Chen and Schmidt, 1930
; Liu et al., 1993
), sex, seasons of harvest (Chen and Schmidt, 1930
; Kasahara et al., 1986
) and geographical origins (Zhang et al., 1989
). In order to accurately evaluate the quality of the crude drug, the quantitative analysis of individual ETA in ma-huang is urgently needed.
In addition to ETA, ma-huang also contains other phytoconstituents, which may modify its pharmacological and toxicological activities. Therefore, the toxicity of ma-huang, and hence the more than 800 reports of adverse effects received by the U.S. FDA associated with the use of ma-huangcontaining dietary supplements, cannot be totally accounted for by its ETA contents alone (FDA, 1997). A bioassay is needed to determine the total toxicity of ma-huang due to the combined effect of the alkaloids and other constituents.
In all of the published studies reviewed, the ma-huang samples were ground before extraction. This preparation method is similar to that used in preparing the marketed forms of ma-huang as they are exported from China to foreign countries, either as powder or as concentrated extract. However, these practices are different from the traditional use of ma-huang as documented in the Chinese literature (e.g., Chung-hua Jen Min Kung Ho Kuo Wei Sheng Pu Yao Tien Wei Yuan Hui, 1995). Traditionally, ma-huang was known to be used in whole-herb form and to be extracted for a longer period of time than other herbs in a prescription (Jan, 1993; Yang, 1993
). Also, with reference to Shang Han Lun, a classical Chinese pharmacopoeia, all herbs were only to be extracted once and then the extract was taken, without extracting the herbs any further (Wang, 1988
). However, the current practice is that all herbs are extracted twice and pooled together before taking. There has been no information found regarding the effects of these different ways of preparation on the biological activities of ma-huang.
In the present study, ma-huang was extracted with water under different conditions to examine the effect of several parameters, including grinding, boiling for 0.5 or 2 h, and extracting once or twice, on toxicity and drug extraction efficiency. For each extract, cytotoxicity to a battery of cell lines was measured using MTT colorimetry, and the contents of E, PE, NME, NE, and NPE were analyzed by HPLC. Having both the E (drug) contents and the cytotoxicity data, an optimal extraction process was identified in which a high drug-to-toxin ratio (DTR) was achieved.
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MATERIALS AND METHODS |
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Sample preparation.
The whole herb of ma-huang was ground into powdered form using a laboratory mill. Each 20.0 g aliquot of ma-huang, powdered or whole herb, was refluxed in 200 mL of double-distilled water for 0.5 h or 2 h. The extracts were collected and centrifuged twice at 4000 rpm at 20°C for 20 min to obtain the supernatant. These supernatants were named G1, G3, NG1, and NG3, respectively. Subsequently, the residue of each extraction was refluxed in another 200 mL of double-distilled water for another 0.5 h or 2 h. The extracts were collected and centrifuged twice at 4000 rpm at 20°C for 20 min to obtain the supernatant. These supernatants were named G2, G4, NG2 and NG4, respectively. All eight extracts were evaporated to dryness by rotary evaporation at 58°C and 0.125 atm, and reconstituted in Dulbecco's PBS to 90% of the final volume. These reconstituted extracts were neutralized to pH 7.4 and centrifuged twice at 4000 rpm at 20°C for 20 min to obtain the supernatant. Finally, all extracts were adjusted to a final volume of 20 mL (to attain a 1-g herb equivalent/mL) and stored at 20°C. At use, the frozen extracts were thawed until they reached room temperature. Table 1 shows the nomenclature of ma-huang extracts prepared under different conditions.
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Cell cultures.
Human hepatoblastoma cell line (HepG2) (ATCC# HB8065) and mouse neuroblastoma cell line (Neuro-2a) (ATCC# CCL131) were cultured in RPMI Medium 1640. Mouse fibroblastoma cell line LM(TK-) expressing human 1b and human
2A adrenergic receptors (L-alpha-1b and L-alpha-2A) (ATCC# CRL11139 and CRL11180) were cultured in Dulbecco's Modified Eagle Medium (DMEM). Chinese hamster ovary cell line CHO-K1 expressing rat ß3 adrenergic receptors (CHO-beta3) (ATCC# CRL11628) was cultured in F12 Nutrient Mixture (F12). All culture media were supplemented with 10% fetal bovine serum, 1% antibiotic and antimycotic solution (50,000 units/L of penicillin and 50 mg/L of streptomycin), and 2mM glutamine. Cells were incubated at 37°C in 5% carbon dioxide at 95% humidity.
MTT assay.
Cytotoxicity was determined by the MTT dye-reduction assay. The methodology described is a modification of the original MTT colorimetric assay developed by Mosmann (1983). Cells were harvested from maintenance cultures in the exponential phase and counted by a hemocytometer using trypan blue solution. The cell suspensions were dispensed (75 µL) in triplicate into 96-well culture plates at optimized concentrations of 1.5 x 105 cells/mL for HepG2, 2 x 104 cells/mL for L-alpha-1b and L-alpha-2A, 5 x 103 cells/mL for CHO-beta3, and 8 x 104 cells/mL for Neuro-2a, respectively. After a 48-h recovery period, the ETA standards or ma-huang extracts diluted with medium (25 µL) were added. For median inhibition concentration (IC50) determination, dose-response curves were conducted with a series of different concentrations of alkaloids or ma-huang extracts that were approximately equal to the IC50. To control wells, only culture medium (15 µL) with vehicle (10 µL PBS) was added. After an additional 72-h incubation period, the medium in each well was aspirated and replaced with 110 µL of MTT working solution (MTT stock solution mixed with medium to attain a final concentration of 0.5 mg/mL). The cells were incubated at 37°C for 4 h, and then the medium was aspirated and replaced with 100 µL DMSO to dissolve the formazan crystals formed. The culture plates were shaken for 5 min and the absorbance of each well was read at 570 nm with 750 nm as the reference wavelength.
The relative viability of the treated cells as compared to the control cells was expressed as the % cytoviability, using the following formula:
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IC50 was then determined by nonlinear regression analysis of the corresponding dose response curve.
Statistical analysis.
Data is presented as either means ± standard deviation (SD) or means ± standard error of the mean (SEM). Statistical analysis was performed using either paired t-test or analysis of variance (ANOVA). For ANOVA, pairwise comparisons between treatments were made using Tukey's Multiple Test Comparison.
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RESULTS AND DISCUSSION |
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In terms of E alone, the total amount obtained in 2 extractions ranged from 69.1 (NG3+4) to 69.9% (NG1+2) of the total ETA. This amount was within the range reported in the literature (30 to 90%) (Tyler, 1993). In a study by Tyler (1994), a tea prepared by steeping 2 g of ma-huang in 240 mL of boiling water for 10 min yielded a dose of 15 to 30 mg of E. In our study, 20.0 g of ma-huang was boiled in 200 mL of water, equivalent to steeping 2.0 g of ma-huang in 20 mL of water. The total amount of E obtained in two extractions ranged from 5.37 (NG1+2) to 6.23 (NG3+4) mg E/g herb. Hence, in our case, 2 g of ma-huang yielded 10.7 to 12.5 mg of E, which is less than those found by Tyler (1994), probably due to the smaller volume of water used in extraction in our study.
Cytotoxicity of E and Ma-huang Extracts Prepared under Different Conditions
Cytotoxicity to a battery of cells was evaluated by determining the IC50 values of different preparations to the cells. Using nonlinear regression analysis of the corresponding dose-response curves, the IC50 values of E and ma-huang extracts prepared under different conditions for a battery of cell lines were obtained and are shown in Figure 4. Tukey's Multiple Test Comparison showed that the IC50 values of E and ground ma-huang extracts (G1 through G4) were significantly lower than those of whole herb extracts (NG1 through NG4), with p < 0.001 for L-alpha-2A, p < 0.01 for HepG2, and p < 0.05 for L-alpha-1b, CHO-beta3, and Neuro-2a. The results indicate that E and ground ma-huang extracts were significantly more cytotoxic than the unground preparations. Grinding was found to be a crucial condition for increased toxicity.
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Among the adverse events associated with ma-huang-containing dietary supplements, 4 percent of the reports mentioned overt hepatitis (FDA, 1997; Nadir et al., 1996
). This observation prompted us to include HepG2 in the cytotoxicity assay, a substance widely used in cytotoxicity testing. Comparison with freshly isolated human adult hepatocytes has revealed that activities of cytochrome P-450-dependent, mixed-function oxidase (MFO) of HepG2 are 510-fold lower than in primary hepatocytes (Grant et al., 1988
). However, the activities of their conjugating enzymes, UDP-glucuronyltransferase (GT) and glutathione-S-transferase (GST), were similar (Duthie et al., 1988
). It was found that HepG2 retains many of the specialized functions normally lost in established hepatocytes in culture (Knowles et al., 1980
).
The cell lines L-alpha-1b, L-alpha-2A and CHO-beta3 were included in the MTT assay, as each of them expresses alpha-1, alpha-2, and beta-3 adrenergic receptors respectively, on which E basically acts.
While there were no significant differences in the IC50 values of E for different cell lines, the IC50 values of ma-huang extracts prepared under different conditions for Neuro-2a were significantly lower than those for L-alpha-1b and L-alpha-2A (both with p < 0.05), indicating that Neuro-2a cells were more sensitive to ma-huang extracts when compared with mouse fibroblastoma cells. This suggests that the toxins in ma-huang extracts may be more specific to neuronal cells. This result was consistent with the adverse events associated with dietary supplements containing ma-huang, as mentioned in the foregoing paragraphs. This difference in response to the same herbal extract suggests that using a battery of cell lines from different origins is of significance in the cellular approach to toxicity assessment of herbal products.
In our MTT assay, the IC50 value of bromobenzene on HepG2 was 0.5542 ± 0.0006 mM, which was 20-fold lower than that determined by previous investigators (10 mM) using a different assay protocol (Thabrew et al., 1997), indicating that our assay was considerably more sensitive than theirs. Because of their status as controlled substances (class IV), NME, MPE, NE, and NPE were not available for cytotoxicity determination. Cytotoxicity was therefore determined for only E and PE in HepG2. This is appropriate, as E and PE are the major alkaloids in ma-huang. The % cytoviability of equimolar concentrations (3.0 mM) of E and PE for HepG2 was 30.83% and 89.43% respectively (data not shown). A paired t-test showed that the cytotoxicity of E to HepG2 was significantly higher than that of PE (p = 0.0013).
In view of the abundance and the high potency of E, we used it as the reference compound in comparing cytotoxicity among ma-huang extracts prepared under different conditions. Indeed, when E is used chronically, it can cause cardiomyopathy, which is related to catecholamine-mediated cytotoxicity (Gualtieri and Harris, 1996; To et al., 1980
; Van Mieghem et al., 1978
).
The MTT AssayPros and Cons
The results of cytotoxicity testing can be determined by several endpoints, such as counting cells that include/exclude a dye, measuring the release of 51Cr-labeled protein after cell lysis, measuring the incorporation of radioisotopes ([3H]thymidine, [3H]uridine, [125I]iododeoxyuridine, and [3H]amino acid) during cell proliferation, and measuring the colorimetric changes of tetrazolium salts by metabolically active cells (McAteer and Davis, 1994; Wilson, 1986
). Among these assays, the radioisotope incorporation and colorimetric assays are most suitable for handling a large number of samples. However, radioisotope incorporation assays suffer from the hazards of handling and disposal of radioactive materials. For colorimetric assays using tetrazolium salts, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), apart from not involving radioactive materials, this salt is only cleaved by metabolically active cells, and therefore only living cells are assayed. The amount of formazan generated is directly proportional to the cell number over a wide range. These features permit micro-scale testing which reduces the amount of sample required and other resources needed. The results of the MTT assays can be optically visualized, and thus it is very useful if a rapid qualitative check is desired. Finally, the results obtained from MTT assays agree closely with those of [3H]thymidine incorporation assays (Mosmann, 1983
). A drawback of the colorimetric assay is that a few parameters, such as duration of MTT treatment, concentration of MTT used, and the number of test cells used have to be optimized. This drawback makes the direct inter-laboratory comparison of results difficult. It should be noted that the MTT assay measures the effects on the changes of enzyme activities that are affected by culture conditions. Therefore, standardized culture conditions must be maintained throughout the tests (Doostdar et al., 1988
).
Drug-to-Toxin Ratio (DTR)
The IC50 values of ma-huang extracts, normalized by their E contents, were calculated by multiplying the IC50 values of ma-huang extracts determined by MTT assay (in mg herb/mL) by their E contents obtained from HPLC analyses (in mg E/mg herb). They are shown in Figure 5. The IC50 values of all ma-huang extracts normalized by their E contents were lower than those of the pure E, indicating that E did not account for the entire toxicity and that there were other toxins present in the extracts. Tukey's Multiple Test Comparison showed that the IC50 values of ground ma-huang extracts normalized by their E (G1 through G4) were significantly lower than those of whole herb extracts (NG1 through NG4), with p < 0.001 for L-alpha-2A, CHO-beta3 and Neuro-2a, and p < 0.01 for HepG2 and L-alpha-1b. This showed that grinding significantly increased the extraction of toxins.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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NOTES |
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