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Evidence for immunotoxic effects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse
International Journal of Immunopharmacology 22 (2000) 229±236 www.elsevier.com/locate/ijimmpharm
Evidence for immunotoxic eects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse E. Koch*, H. Jaggy, S.S. Chatterjee Department of Pharmacology and Department of Natural Product Chemistry, Dr Willmar Schwabe GmbH & Co, Arzneimittel, 76209, Karlsruhe, Germany Received 4 May 1999; accepted 21 September 1999
Abstract Allergic reactions due to contact with dierent parts of the ancient tree Ginkgo biloba L. have repeatedly been reported. Provocation tests in patients and animal experiments have identi®ed alkylphenols such as ginkgolic acids as causative constituents. Leaf extracts from Ginkgo are widely used to treat peripheral or cerebral circulatory disorders and Alzheimer's disease. Since alkylphenols are also present in leaves, potential allergic and other immunological hazards of such preparations have to be carefully controlled. Thus, we have evaluated if the popliteal lymph node assay (PLNA) in the mouse may represent a suitable model for the detection of constituents with immunotoxic properties in a complex mixture of biologically active agents such as plant extracts. Subplantar injection (2 mg) of a crude aqueous-ethanolic extract from Ginkgo leaves caused a signi®cant lymphoproliferative reaction (LPR) in the ipsilateral popliteal lymph node. PLNA-active compounds in this extract could be enriched in the lipophilic phase by liquid±liquid partition between heptane and water. Chemical analysis of the heptane extract revealed the presence of a high concentration of alkylphenols (approx. 30%) and further subfractionation indicated that the enlargement of the popliteal lymph node was mainly due to the content of ginkgolic acids. This presumption was corroborated by observing a similar LPR following injection of a puri®ed mixture of ginkgolic or hydroginkgolic acids. Thus, our experiments con®rm that Ginkgo leaf extracts may contain constituents with immunotoxic properties, underlining the need to apply adequate production procedures to guarantee the completest possible removal of these compounds. The PLNA appears to represent a simple test model for the detection, characterisation and control of ingredients with potential immunotoxic side eects in complex herbal drugs. # 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Ginkgo biloba extract EGb 761; Alkylphenols; Ginkgolic acids; Popliteal lymph node assay; Contact dermatitis
* Corresponding author. Tel.: +49-721-4005-356; fax: +49721-4005-150. E-mail address: [email protected] (E. Koch). 0192-0561/00/$20.00 # 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 9 9 ) 0 0 0 8 0 - 6
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E. Koch et al. / International Journal of Immunopharmacology 22 (2000) 229±236
Fig. 1. Chemical structure of alkylphenols isolated from Ginkgo biloba leaves.
1. Introduction Allergic skin reactions, stomatitis and proctitis following contact with or consumption of Ginkgo fruits (Ginkgo biloba L.) have repeatedly been reported in the literature [1±7]. On the basis of provocation tests and the close structural similarity with the highly allergenic urushiols from Anacardiaceae, alkylphenols present in G. biloba (anacardic or ginkgolic acids, cardols and cardanols) have been considered as the inducing cause for this hypersensitivity response [3,6,8,9]. Experimentally, the high sensitizing potential of ginkgolic acids has been con®rmed by delayed hypersensitivity testing in guinea pigs [9±11]. Extracts of Ginkgo leaves are widely prescribed drugs for the treatment of peripheral or cerebral circulatory disorders as well as vascular and Alzheimer type dementia. As ginkgolic acids and cardanols (Fig. 1) have also been detected in Ginkgo leaves [12±15], the presence of these substances has to be expected in crude extracts. Thus, it was the aim of the present study to examine whether the popliteal lymph node assay (PLNA) in the mouse is a suitable test system for the detection of allergenic constituents in such preparations and possibly can be used to control for the elimination of these compounds during the production of Ginkgo extracts. 2. Materials and methods 2.1. Preparation of extracts and extract fractions Starting material for the ®rst set of exper-
Fig. 2. Production and composition of a crude aqueous-ethanolic extract from dried green Ginkgo leaves. This total extract was further fractionated by liquid±liquid partition between heptane and water.
iments described later was a crude aqueous-alcoholic extract obtained by extracting dried green Ginkgo biloba leaves with 60% w/w ethanol. A lipophilic fraction of this extract was prepared by liquid±liquid partition between heptane and water. The production and composition of the crude total extract and the heptane soluble fraction are shown in Fig. 2. Further fractionation studies were performed with a heptane extract from the so called ``decanter sludge'' which is eliminated as water insoluble material from the primary 60% w/w aqueous acetone extract during production of the standardized G. biloba extract EGb 761 [16]. This extract was separated into seven fractions by Sephadex LH-20 column chromatography using dichloromethane as eluent. Yields and contents of ginkgolic acids and cardanols of these fractions are shown in Fig. 3. 2.2. Preparation of ginkgolic and hydroginkgolic acids A mixture of pure ginkgolic acids containing 13:0, 15:1 and 17:1 alkylated salicylic acids in a proportion of 13.5, 41.7 and 44.8%, respectively, was obtained by a multistep puri®cation scheme
E. Koch et al. / International Journal of Immunopharmacology 22 (2000) 229±236
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Fig. 3. Flow diagram for the chromatographic fractionation of a heptane extract from the decanter sludge, which is eliminated during the production of the standardized Ginkgo biloba extract EGb 761. n.a., not analyzable; substance was not detected by thin layer chromatography and quanti®cation by HPLC was compromised by interference with unidenti®ed compounds. b.d.l., below detection limit.
from the above mentioned decanter sludge. This sludge contains the most lipophilic components of G. biloba leaves. A series of column chromatographic procedures (twice with Sephadex LH-20 in 95% w/w ethanol, Lichroprep RP-18 (40± 63 mm) in 60% v/v ethanol and ®nally radially compressed silica cartridge (32±63 mm) in chloroform) led to a semipure mixture of ginkgolic acids which still contained some cardanols. The dierent separation steps were monitored by thin layer chromatography (TLC) on silica in heptane/ethyl acetate 60+40. Ginkgolic acids and cardanols were detected by UV absorbance at 245 nm and staining with vanillin phosphoric acid. Pure ginkgolic acids (melting point 338C) were isolated by removing the cardanols on silica (35±70 mm) impregnated with heptane/triethylamine 98+2 in ethyl acetate/heptane/triethylamine 25+75+0.5. Hydroginkgolic acids were prepared by hydrogenation of a portion of the described mixture of ginkgolic acids in 96% v/v ethanol at normal
pressure and room temperature for 48 h using 5% palladium on charcoal as catalyst. The completeness of the reaction was monitored by mass spectrometry. After ®ltration of the catalyst and distillation of the ethanol the hydroginkgolic acids were obtained as waxy solids with a melting point of 758C. 2.3. Popliteal lymph node assay (PLNA) Male C57BlI/6 mice, weighing approx. 18±24 g, were used in all tests (Charles River, Sulzfeld, Germany). The animals were housed under standard environmental conditions (temperature 20± 228C, relative humidity 55%, light on/o 06:00/ 18:00) with free access to food and water. For the induction of a lymphoproliferative reaction (LPR), the test substances in 10 ml DMSO were injected subplantar into the right hind paw. The animals in the control group received an injection of 10 ml DMSO only. The crude ethanolic-aqueous extract and the heptane soluble fraction as
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well as diphenylhydantoin, which was used as a positive reference substance, were administered at doses of 2 mg. The doses of the dierent heptane extract fractions corresponded in each case to their yield obtained from the original material. Seven days after injection, the animals were killed by cervical dislocation under ether anaesthesia. Both popliteal lymph nodes were removed and placed on damp ®lter-paper in petri dishes after complete removal of adhering tissue. The weight of the lymph nodes was immediately determined using an electronic balance (Sartorius A210P, GoÈttingen, Germany) with a precision of 0.1 mg. The extent of the LPR was assessed by means of the weight dierence between the ipsi- and contralateral popliteal lymph nodes. All results are given as means 2 SEM. The Student's t-test was used for statistical analysis and P < 0.05 was considered to indicate statistical signi®cance. 3. Results The local application of DMSO led only to a small enlargement of the lymph nodes. In contrast, the subplantar injection of 2 mg of the crude aqueous-ethanolic extract from Ginkgo leaves resulted in a clear and statistically signi®cant LPR in the ipsilateral popliteal lymph node when compared to the DMSO control (Table 1). A massive lymph node hyperplasia, almost com-
parable to the LPR observed after injection of diphenylhydantoin, was noticed after administration of the heptane soluble fraction obtained from the aqueous-ethanolic extract (Table 1). While the crude extract contained 5.5% ginkgolic acids, the liquid±liquid partition resulted in an approximately ®ve-fold increase in the concentration of these compounds in the heptane fraction (24.6%). These results indicated that ginkgolic acids may be responsible for the LPR observed. To further verify this presumption, we separated a heptane extract from the decanter sludge by gel chromatography into seven dierent fractions which yielded between 2.7 and 33% of the original material (Fig. 3). When injected at doses proportional to their share in the heptane extract only fractions 1 and 6 induced an enlargement of the ipsilateral popliteal lymph node. Interestingly, fraction 6 caused a LPR almost identical to that of the starting material (Fig. 4). On analysis, fraction 6 (yield: 33%, dose injected: 660 mg) contained approximately 50% ginkgolic acids but no cardanols were found. In fraction 1 neither of these alkylphenols could be detected by TLC. Subplantar injection of fraction 6 caused a dosedependent enlargement of the local popliteal lymph node over a range of 8±500 mg. A very similar response was observed when puri®ed mixtures of ginkgolic or hydroginkgolic acids were administered at comparable doses (Fig. 5).
Table 1 Eect of subplantar injection (2 mg/paw) of a crude aqueous-ethanolic extract from dried green Ginkgo leaves and a heptane soluble fraction produced by liquid±liquid partition on the weights of the ipsi- and contralateral popliteal lymph nodes of mice. Diphenylhydantoin (2 mg/paw) was used in some experiments as reference substance Group
N
Lymph node (LN) weight (mg; mean2SEM) Contralateral LN
Ipsilateral LN
LPR (mg)
Control (DMSO) Aqueous-ethanolic extract Heptane soluble fraction
10 10 10
0.7920.06 0.6920.03 0.8420.07
1.0220.05 2.3020.18 4.1820.58
0.2320.07 1.6120.17 3.3420.58
Control (DMSO) Diphenylhydantoin
10 10
0.7620.06 1.0020.09
1.3920.11 5.8520.43
0.6320.10 4.8520.43
P < 0.05 vs control.
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Fig. 4. Eect of subplantar injection of a heptane extract from the decanter sludge and fractions from it on the LPR in the ipsilateral popliteal lymph node of mice. In each case, the injected dose of the dierent fractions corresponded to their yield from the original extract. Each group consisted of nine animals. P < 0.05 vs control.
4. Discussion For regulatory purposes and experimental detection of contact allergens, usually test models utilizing guinea pigs are employed. All these procedures are based on a bi-phasic test protocol, in which the animals are initially sensitized and later re-exposed to the test substance to elicit a hypersensitivity reaction. In principle, the ®nal stage of the various techniques is the visual evaluation of the epidermal changes produced. In addition to the subjective assessment of the allergic reaction, these procedures have other serious disadvantages. For example, the interpretation of test results following local application of irritant or coloured chemicals is dicult. More objective and reproducible test results can be achieved using newer test systems in which the primary lymphocyte reaction in the aerent lymph nodes after local application of test substances is evalu-
ated in experimental animals. As a result of the immunological recognition of low-molecular allergens and the subsequent reaction of T-lymphocytes, there is a direct correlation between the extent and duration of lymphocyte proliferation and the sensitizing properties of test substances. As immunological changes are measured during the induction phase of the immune response, the assessment of the local lymph node hyperplasia has the additional advantage that not only substances with allergenic but also those with autoimmune properties can be detected [17±20]. Since its introduction, comprehensive practical experience with the PLNA has shown that this procedure is well suited to test chemicals for immunotoxic properties, such as allergens or substances with autoimmune potential [21]. With appropriate modi®cations, substances with immuno-stimulant or immuno-suppressive action can also be detected. Of over 60 drugs which
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Fig. 5. Eect of subplantar injection of fraction 6 from a heptane extract and puri®ed mixtures of ginkgolic or hydroginkgolic acids on the LPR in the ipsilateral popliteal lymph node of mice. The injected doses of fraction 6 were calculated on the basis of a ginkgolic acid content of 49.1%. After injection of the vehicle (DMSO) a LPR of 0.4820.09 and 0.6320.10 mg, respectively, was measured in both experiments. Each group consisted of 10 animals. Generally, a statistically signi®cant (P < 0.05) LPR was observed after application of ginkgolic acids at a dose of 100 mg/paw and above when compared to the vehicle control.
have been tested by means of the PLNA, the majority of the compounds in which immunological side-eects in man are known could be clearly identi®ed. Only a few cases of false positive results were found. Furthermore, detailed investigations of the false negative results revealed that the immunotoxic eects of these compounds are exclusively due to metabolites which do not appear to be formed by local injection of test substances. Based on the experimental ®ndings and the fact that the PLNA is quick, simple and inexpensive to carry out, its application for the testing of chemicals for immunotoxic eects was recommended by the International Workshop on Immunotoxicology [18,19]. In the present investigation, substances with potential immunotoxic side-eects in an aqueousethanolic extract of Ginkgo leaves could be demonstrated using the PLNA. Further separ-
ation of this total extract by liquid±liquid partition and Sephadex LH-20 column chromatography of a heptane extract indicated that ginkgolic acids are obviously the main constituents with allergenic properties in the crude extract. This presumption was corroborated by observing a similar LPR following injection of puri®ed mixtures of ginkgolic or hydroginkgolic acids. However, as fraction 6 of the heptane extract caused a somewhat stronger response as the puri®ed ginkgolic or hydroginkgolic acids, we can not exclude that crude Ginkgo extracts besides these substances may contain other constituents with potential immunotoxic eects. The observation that fraction 1, in which no ginkgolic acids or cardanols could be detected by TLC, induced a statistically signi®cant LPR is in agreement with this suggestion. As described for numerous reference sub-
E. Koch et al. / International Journal of Immunopharmacology 22 (2000) 229±236
stances [18], the LPR after injection of ginkgolic or hydroginkgolic acids was clearly dose-dependent. Although usually doses of 0.5±2 mg of known allergenic substances are required to induce a positive LPR [17±19], a signi®cant lymph node swelling was already observed after the injection of 100 mg of ginkgolic or hydroginkgolic acids. This observation emphasizes the high sensitizing potential of ginkgolic acids and demonstrates that the presence of a double bond in the alkyl side chain is not required for their immunotoxic action. To verify our test system, we have applied diphenylhydantoin as positive reference substance in some of our experiments. Diphenylhydantoin is used as an anticonvulsant and has been reported to induce various lymphoproliferative disorders in patients. In accordance with other investigators [21,22], we regularly observed a positive popliteal lymph node reaction after subplantar injection of this compound under our experimental settings. Using a modi®ed Freund's complete adjuvant technique Hausen [11] recently reported that he was unable to sensitize female guinea pigs by three intradermal injections (0.1±0.15 ml each at six injection sites) of a Ginkgo leaf extract containing 1000 ppm ginkgolic acids. From this result he concludes that Ginkgo extracts containing between 10 and 200 ppm ginkgolic acids with certainty bear no risk to induce a type IV allergic reaction and that such low amounts are all the more negligible for producing ¯ares in G. biloba-sensitive individuals. In our view this conclusion is not appropriate. Although it is reasonable to assume that the mechanism of sensitization is similar in humans and the usual animal models, there is no evidence that relative potency in the guinea pig is the same as or similar to that in humans. Furthermore, it is important to remember that a threshold determined in an animal model relates to the animal and is completely dependent on the test conditions used to de®ne it [23]. In addition, guinea pig contact sensitivity tests generally require that irritating concentrations of the test substance are applied during the induction phase [24]. As guinea pigs could be successfuly
235
sensitized by application of pure ginkgolic acids at a concentration of 0.19% there is strong evidence that the author did not use an optimal sensitization protocol when applying the Ginkgo extract at a concentration of only 0.375%. For patients involved, sensitization to low-molecular compounds can have serious consequences as this process is irreversible and already exceptionally low concentrations of the triggering substance may induce a recurrence of symptoms on re-exposure to the allergenic substance [25]. Under this aspect and with respect to the fact that no contribution of ginkgolic acids to the therapeutic eect of Ginkgo extracts has been demonstrated [26], it is not admissible to take dose-eect relations into consideration for the bene®t-risk assessment of the alkylphenol content of Ginkgo extracts. The relatively small number of allergic side-eects observed after oral intake of Ginkgo preparations [27] is probably due to the fact that mainly extracts are marketed which comply with the requirements of the monograph of the Commission E of the former German Health Authority (Bundesgesundheitsamt, BGA) according to which extracts are not allowed to contain more than 5 ppm of ginkgolic acids. As this limit value is technically easy to achieve higher concentrations of alkylphenols are not acceptable from the viewpoint of drug safety. As cross reactions between urushiols in Anacardiaceae and alkylphenols in G. biloba have repeatedly been observed in human and animal studies [3,6,9], the therapeutic use of Ginkgo extracts which contain ginkgolic acids above the established limit value may constitute a particular hazard to patients in North America, where between 50 and 85% of the population is sensitized to members of the Anacardiaceae or cashew family which includes plants like poison ivy, poison oak, poison sumac, cashew, mango, Japanese lacquer, and Indian marking nut [28]. The present investigation emphasizes the importance of maintaining satisfactory production conditions, in order to guarantee the completest possible removal of allergenic alkylphenols from Ginkgo extracts. The PLNA appears to represent a simple test procedure for the detection, charac-
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terization and elimination control of constituents with potential immunotoxic side eects in complex plant extracts.
[16] [17]
References [1] Kawamura J. UÈber die chemischen Bestandteile der Frucht von Ginkgo biloba (I. Mitteilung). Jap J Chem 1928;3:89±108. [2] Saito J. Pharmakologische Untersuchung des Ginkgogifts, des Dermatitiserregers in Ginkgo biloba L. Tohoku. J Exp Med 1930;16:413±23. [3] Sowers WF, Weary PE, Collins OD, Cawley EP. Ginkgo-tree dermatitis. Arch Derm 1985;91:452±6. [4] Becker LE, Skipworth GB. Ginkgo tree dermatisis, stomatitis and proctitis. J Am Med Assoc 1975;231:1162. [5] JiraÂsek L, Brozkova L. ProfesionaÂlni kontaktni precitlivelost na Ginkgo biloba L. (Occupational contact hypersensitivity to Ginkgo biloba L.). Cs Derm 1981;56:366±73. [6] Nakamura T. Ginkgo tree dermatitis. Contact Dermatitis 1985;12:281±2. [7] Tomb RR, Foussereau J, Sell Y. Mini-epidemic of contact dermatisis from ginkgo tree fruit (Ginkgo biloba L.). Contact Dermatitis 1988;19:281±3. [8] Adawadkar PD, El Sohly MA. Isolation, puri®cation and antimicrobial activity of anacardic acids from Ginkgo biloba fruits. Fitoterapia 1981;52:129±35. [9] Lepoittevin JP, Benezra C, Asakawa Y. Allergic contact dermatitis to Ginkgo biloba L.: relationship with urushiol. Arch Dermatol Res 1989;281:227±30. [10] Saito F. Contact dermatitis due to Ginkgo fruit pulp: clinical survey, patchtest, LTT and animal sensitization. Hifu (Suppl) 1987;4:155±68. [11] Hausen BM. The sensitizing capacity of ginkgolic acids in guinea pigs. Am J Contact Dermatitis 1998;9:146±8. [12] Gellerman JL, Schlenk H. Methods for isolation and determination of anacardic acids. Anal Chem 1968;40:739± 43. [13] Cartayrade A, Bourgeois G, Balz JP, Carde JP. The secretory apparatus of Ginkgo biloba: structure, dierentiation and analysis of the secretory product. Trees 1990;4:171±8. [14] Verotta L, Peterlongo F. Selective extraction of phenolic components from Ginkgo biloba extracts using supercritical carbon dioxide and o-line capillary gas chromatography/mass spectrometry. Phytochem Anal 1993;4:178± 82. [15] Irie J, Murata M, Homma S. Glycero-3-phosphate dehy-
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drogenase inhibitors, anacardic acids, from Ginkgo biloba. Biosci Biotech Biochem 1996;60:240±3. DeFeudis FV. Ginkgo biloba extract (EGb 761). From chemistry to the clinic. Wiesbaden: Ullstein Medical, 1998. KammuÈller ME, Thomas C, De Bakker JM, Bloksma N, Seinen W. The popliteal lymph node assay in mice to screen for the immune disregulating potential of chemicals Ð a preliminary study. Int J Immunopharmacol 1989;11:293±300. Descotes J. The popliteal lymph node assay: a tool for studying the mechanisms of drug-induced autoimmune disorders. Toxicol Lett 1992;64/65:101±7. Bloksma N, Kubicka-Muranyi M, Schuppe HC, Gleichmann E, Gleichmann H. Predictive immunotoxicological test systems: suitability of the popliteal lymph node assay in mice and rats. Crit Rev Toxicol 1995;25:369±96. Hilton J, Kimber I. The murine local lymph node assay. In: O'Hare S, Atterwill CK, editors. In vitro toxicity testing protocols. Totowa, USA: Humana Press, 1995. p. 227±35. Gleichmann E. Studies on the mechanism of drug sensitization: T-cell-dependent popliteal lymph node reaction to diphenylhydantoin. Clin Immunol Immunopathol 1981;18:203±11. Marintchev A, Mirtcheva J, Sidjimov A, Haimova M. Induction of lymphoproliferative popliteal lymph node reaction by hydantoin derivatives: structure±activity relationships. Int J Immunopharmacol 1995;17:981±4. Basketter DA, Cookman G, Gerberick GF, Hamaide N, Potokar M. Skin sensitization thresholds: determination in predictive models. Food Chem Toxicol 1997;35:417± 25. Basketter DA. Guinea pig predictive tests for contact hypersensitivity. In: Dean JH, Luster MI, Munson AE, Kimber I, editors. Immunotoxicology and immunopharmacology. New York: Raven Press, 1994. p. 693±702. Merk H Haut. In: Marquardt H, SchaÈfer SG, editors. Lehrbuch der Toxikologie. Mannheim: Wissenschaftsverlag, 1994. p. 271±86. Jaggy H, Koch E. Chemistry and biology of alkylphenols from Ginkgo biloba L. Pharmazie 1997;52:735±8. Woerdenbag HJ, Van Beek TA. Ginkgo biloba. In: De Smet PAGM, Keller K, HaÈnsel R, Chandler RF, editors. Adverse eects of herbal drugs, vol. 3. Berlin: Springer± Verlag, 1997. p. 51±66. Benezra C. Plants causing adverse skin reactions. In: Plant ¯avonoids in biology and medicine II: biochemical, cellular, and medical properties. New York: Alan R. Liss Inc, 1988. p. 395±400.
Evidence for immunotoxic eects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse E. Koch*, H. Jaggy, S.S. Chatterjee Department of Pharmacology and Department of Natural Product Chemistry, Dr Willmar Schwabe GmbH & Co, Arzneimittel, 76209, Karlsruhe, Germany Received 4 May 1999; accepted 21 September 1999
Abstract Allergic reactions due to contact with dierent parts of the ancient tree Ginkgo biloba L. have repeatedly been reported. Provocation tests in patients and animal experiments have identi®ed alkylphenols such as ginkgolic acids as causative constituents. Leaf extracts from Ginkgo are widely used to treat peripheral or cerebral circulatory disorders and Alzheimer's disease. Since alkylphenols are also present in leaves, potential allergic and other immunological hazards of such preparations have to be carefully controlled. Thus, we have evaluated if the popliteal lymph node assay (PLNA) in the mouse may represent a suitable model for the detection of constituents with immunotoxic properties in a complex mixture of biologically active agents such as plant extracts. Subplantar injection (2 mg) of a crude aqueous-ethanolic extract from Ginkgo leaves caused a signi®cant lymphoproliferative reaction (LPR) in the ipsilateral popliteal lymph node. PLNA-active compounds in this extract could be enriched in the lipophilic phase by liquid±liquid partition between heptane and water. Chemical analysis of the heptane extract revealed the presence of a high concentration of alkylphenols (approx. 30%) and further subfractionation indicated that the enlargement of the popliteal lymph node was mainly due to the content of ginkgolic acids. This presumption was corroborated by observing a similar LPR following injection of a puri®ed mixture of ginkgolic or hydroginkgolic acids. Thus, our experiments con®rm that Ginkgo leaf extracts may contain constituents with immunotoxic properties, underlining the need to apply adequate production procedures to guarantee the completest possible removal of these compounds. The PLNA appears to represent a simple test model for the detection, characterisation and control of ingredients with potential immunotoxic side eects in complex herbal drugs. # 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Ginkgo biloba extract EGb 761; Alkylphenols; Ginkgolic acids; Popliteal lymph node assay; Contact dermatitis
* Corresponding author. Tel.: +49-721-4005-356; fax: +49721-4005-150. E-mail address: [email protected] (E. Koch). 0192-0561/00/$20.00 # 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 9 9 ) 0 0 0 8 0 - 6
230
E. Koch et al. / International Journal of Immunopharmacology 22 (2000) 229±236
Fig. 1. Chemical structure of alkylphenols isolated from Ginkgo biloba leaves.
1. Introduction Allergic skin reactions, stomatitis and proctitis following contact with or consumption of Ginkgo fruits (Ginkgo biloba L.) have repeatedly been reported in the literature [1±7]. On the basis of provocation tests and the close structural similarity with the highly allergenic urushiols from Anacardiaceae, alkylphenols present in G. biloba (anacardic or ginkgolic acids, cardols and cardanols) have been considered as the inducing cause for this hypersensitivity response [3,6,8,9]. Experimentally, the high sensitizing potential of ginkgolic acids has been con®rmed by delayed hypersensitivity testing in guinea pigs [9±11]. Extracts of Ginkgo leaves are widely prescribed drugs for the treatment of peripheral or cerebral circulatory disorders as well as vascular and Alzheimer type dementia. As ginkgolic acids and cardanols (Fig. 1) have also been detected in Ginkgo leaves [12±15], the presence of these substances has to be expected in crude extracts. Thus, it was the aim of the present study to examine whether the popliteal lymph node assay (PLNA) in the mouse is a suitable test system for the detection of allergenic constituents in such preparations and possibly can be used to control for the elimination of these compounds during the production of Ginkgo extracts. 2. Materials and methods 2.1. Preparation of extracts and extract fractions Starting material for the ®rst set of exper-
Fig. 2. Production and composition of a crude aqueous-ethanolic extract from dried green Ginkgo leaves. This total extract was further fractionated by liquid±liquid partition between heptane and water.
iments described later was a crude aqueous-alcoholic extract obtained by extracting dried green Ginkgo biloba leaves with 60% w/w ethanol. A lipophilic fraction of this extract was prepared by liquid±liquid partition between heptane and water. The production and composition of the crude total extract and the heptane soluble fraction are shown in Fig. 2. Further fractionation studies were performed with a heptane extract from the so called ``decanter sludge'' which is eliminated as water insoluble material from the primary 60% w/w aqueous acetone extract during production of the standardized G. biloba extract EGb 761 [16]. This extract was separated into seven fractions by Sephadex LH-20 column chromatography using dichloromethane as eluent. Yields and contents of ginkgolic acids and cardanols of these fractions are shown in Fig. 3. 2.2. Preparation of ginkgolic and hydroginkgolic acids A mixture of pure ginkgolic acids containing 13:0, 15:1 and 17:1 alkylated salicylic acids in a proportion of 13.5, 41.7 and 44.8%, respectively, was obtained by a multistep puri®cation scheme
E. Koch et al. / International Journal of Immunopharmacology 22 (2000) 229±236
231
Fig. 3. Flow diagram for the chromatographic fractionation of a heptane extract from the decanter sludge, which is eliminated during the production of the standardized Ginkgo biloba extract EGb 761. n.a., not analyzable; substance was not detected by thin layer chromatography and quanti®cation by HPLC was compromised by interference with unidenti®ed compounds. b.d.l., below detection limit.
from the above mentioned decanter sludge. This sludge contains the most lipophilic components of G. biloba leaves. A series of column chromatographic procedures (twice with Sephadex LH-20 in 95% w/w ethanol, Lichroprep RP-18 (40± 63 mm) in 60% v/v ethanol and ®nally radially compressed silica cartridge (32±63 mm) in chloroform) led to a semipure mixture of ginkgolic acids which still contained some cardanols. The dierent separation steps were monitored by thin layer chromatography (TLC) on silica in heptane/ethyl acetate 60+40. Ginkgolic acids and cardanols were detected by UV absorbance at 245 nm and staining with vanillin phosphoric acid. Pure ginkgolic acids (melting point 338C) were isolated by removing the cardanols on silica (35±70 mm) impregnated with heptane/triethylamine 98+2 in ethyl acetate/heptane/triethylamine 25+75+0.5. Hydroginkgolic acids were prepared by hydrogenation of a portion of the described mixture of ginkgolic acids in 96% v/v ethanol at normal
pressure and room temperature for 48 h using 5% palladium on charcoal as catalyst. The completeness of the reaction was monitored by mass spectrometry. After ®ltration of the catalyst and distillation of the ethanol the hydroginkgolic acids were obtained as waxy solids with a melting point of 758C. 2.3. Popliteal lymph node assay (PLNA) Male C57BlI/6 mice, weighing approx. 18±24 g, were used in all tests (Charles River, Sulzfeld, Germany). The animals were housed under standard environmental conditions (temperature 20± 228C, relative humidity 55%, light on/o 06:00/ 18:00) with free access to food and water. For the induction of a lymphoproliferative reaction (LPR), the test substances in 10 ml DMSO were injected subplantar into the right hind paw. The animals in the control group received an injection of 10 ml DMSO only. The crude ethanolic-aqueous extract and the heptane soluble fraction as
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well as diphenylhydantoin, which was used as a positive reference substance, were administered at doses of 2 mg. The doses of the dierent heptane extract fractions corresponded in each case to their yield obtained from the original material. Seven days after injection, the animals were killed by cervical dislocation under ether anaesthesia. Both popliteal lymph nodes were removed and placed on damp ®lter-paper in petri dishes after complete removal of adhering tissue. The weight of the lymph nodes was immediately determined using an electronic balance (Sartorius A210P, GoÈttingen, Germany) with a precision of 0.1 mg. The extent of the LPR was assessed by means of the weight dierence between the ipsi- and contralateral popliteal lymph nodes. All results are given as means 2 SEM. The Student's t-test was used for statistical analysis and P < 0.05 was considered to indicate statistical signi®cance. 3. Results The local application of DMSO led only to a small enlargement of the lymph nodes. In contrast, the subplantar injection of 2 mg of the crude aqueous-ethanolic extract from Ginkgo leaves resulted in a clear and statistically signi®cant LPR in the ipsilateral popliteal lymph node when compared to the DMSO control (Table 1). A massive lymph node hyperplasia, almost com-
parable to the LPR observed after injection of diphenylhydantoin, was noticed after administration of the heptane soluble fraction obtained from the aqueous-ethanolic extract (Table 1). While the crude extract contained 5.5% ginkgolic acids, the liquid±liquid partition resulted in an approximately ®ve-fold increase in the concentration of these compounds in the heptane fraction (24.6%). These results indicated that ginkgolic acids may be responsible for the LPR observed. To further verify this presumption, we separated a heptane extract from the decanter sludge by gel chromatography into seven dierent fractions which yielded between 2.7 and 33% of the original material (Fig. 3). When injected at doses proportional to their share in the heptane extract only fractions 1 and 6 induced an enlargement of the ipsilateral popliteal lymph node. Interestingly, fraction 6 caused a LPR almost identical to that of the starting material (Fig. 4). On analysis, fraction 6 (yield: 33%, dose injected: 660 mg) contained approximately 50% ginkgolic acids but no cardanols were found. In fraction 1 neither of these alkylphenols could be detected by TLC. Subplantar injection of fraction 6 caused a dosedependent enlargement of the local popliteal lymph node over a range of 8±500 mg. A very similar response was observed when puri®ed mixtures of ginkgolic or hydroginkgolic acids were administered at comparable doses (Fig. 5).
Table 1 Eect of subplantar injection (2 mg/paw) of a crude aqueous-ethanolic extract from dried green Ginkgo leaves and a heptane soluble fraction produced by liquid±liquid partition on the weights of the ipsi- and contralateral popliteal lymph nodes of mice. Diphenylhydantoin (2 mg/paw) was used in some experiments as reference substance Group
N
Lymph node (LN) weight (mg; mean2SEM) Contralateral LN
Ipsilateral LN
LPR (mg)
Control (DMSO) Aqueous-ethanolic extract Heptane soluble fraction
10 10 10
0.7920.06 0.6920.03 0.8420.07
1.0220.05 2.3020.18 4.1820.58
0.2320.07 1.6120.17 3.3420.58
Control (DMSO) Diphenylhydantoin
10 10
0.7620.06 1.0020.09
1.3920.11 5.8520.43
0.6320.10 4.8520.43
P < 0.05 vs control.
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Fig. 4. Eect of subplantar injection of a heptane extract from the decanter sludge and fractions from it on the LPR in the ipsilateral popliteal lymph node of mice. In each case, the injected dose of the dierent fractions corresponded to their yield from the original extract. Each group consisted of nine animals. P < 0.05 vs control.
4. Discussion For regulatory purposes and experimental detection of contact allergens, usually test models utilizing guinea pigs are employed. All these procedures are based on a bi-phasic test protocol, in which the animals are initially sensitized and later re-exposed to the test substance to elicit a hypersensitivity reaction. In principle, the ®nal stage of the various techniques is the visual evaluation of the epidermal changes produced. In addition to the subjective assessment of the allergic reaction, these procedures have other serious disadvantages. For example, the interpretation of test results following local application of irritant or coloured chemicals is dicult. More objective and reproducible test results can be achieved using newer test systems in which the primary lymphocyte reaction in the aerent lymph nodes after local application of test substances is evalu-
ated in experimental animals. As a result of the immunological recognition of low-molecular allergens and the subsequent reaction of T-lymphocytes, there is a direct correlation between the extent and duration of lymphocyte proliferation and the sensitizing properties of test substances. As immunological changes are measured during the induction phase of the immune response, the assessment of the local lymph node hyperplasia has the additional advantage that not only substances with allergenic but also those with autoimmune properties can be detected [17±20]. Since its introduction, comprehensive practical experience with the PLNA has shown that this procedure is well suited to test chemicals for immunotoxic properties, such as allergens or substances with autoimmune potential [21]. With appropriate modi®cations, substances with immuno-stimulant or immuno-suppressive action can also be detected. Of over 60 drugs which
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Fig. 5. Eect of subplantar injection of fraction 6 from a heptane extract and puri®ed mixtures of ginkgolic or hydroginkgolic acids on the LPR in the ipsilateral popliteal lymph node of mice. The injected doses of fraction 6 were calculated on the basis of a ginkgolic acid content of 49.1%. After injection of the vehicle (DMSO) a LPR of 0.4820.09 and 0.6320.10 mg, respectively, was measured in both experiments. Each group consisted of 10 animals. Generally, a statistically signi®cant (P < 0.05) LPR was observed after application of ginkgolic acids at a dose of 100 mg/paw and above when compared to the vehicle control.
have been tested by means of the PLNA, the majority of the compounds in which immunological side-eects in man are known could be clearly identi®ed. Only a few cases of false positive results were found. Furthermore, detailed investigations of the false negative results revealed that the immunotoxic eects of these compounds are exclusively due to metabolites which do not appear to be formed by local injection of test substances. Based on the experimental ®ndings and the fact that the PLNA is quick, simple and inexpensive to carry out, its application for the testing of chemicals for immunotoxic eects was recommended by the International Workshop on Immunotoxicology [18,19]. In the present investigation, substances with potential immunotoxic side-eects in an aqueousethanolic extract of Ginkgo leaves could be demonstrated using the PLNA. Further separ-
ation of this total extract by liquid±liquid partition and Sephadex LH-20 column chromatography of a heptane extract indicated that ginkgolic acids are obviously the main constituents with allergenic properties in the crude extract. This presumption was corroborated by observing a similar LPR following injection of puri®ed mixtures of ginkgolic or hydroginkgolic acids. However, as fraction 6 of the heptane extract caused a somewhat stronger response as the puri®ed ginkgolic or hydroginkgolic acids, we can not exclude that crude Ginkgo extracts besides these substances may contain other constituents with potential immunotoxic eects. The observation that fraction 1, in which no ginkgolic acids or cardanols could be detected by TLC, induced a statistically signi®cant LPR is in agreement with this suggestion. As described for numerous reference sub-
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stances [18], the LPR after injection of ginkgolic or hydroginkgolic acids was clearly dose-dependent. Although usually doses of 0.5±2 mg of known allergenic substances are required to induce a positive LPR [17±19], a signi®cant lymph node swelling was already observed after the injection of 100 mg of ginkgolic or hydroginkgolic acids. This observation emphasizes the high sensitizing potential of ginkgolic acids and demonstrates that the presence of a double bond in the alkyl side chain is not required for their immunotoxic action. To verify our test system, we have applied diphenylhydantoin as positive reference substance in some of our experiments. Diphenylhydantoin is used as an anticonvulsant and has been reported to induce various lymphoproliferative disorders in patients. In accordance with other investigators [21,22], we regularly observed a positive popliteal lymph node reaction after subplantar injection of this compound under our experimental settings. Using a modi®ed Freund's complete adjuvant technique Hausen [11] recently reported that he was unable to sensitize female guinea pigs by three intradermal injections (0.1±0.15 ml each at six injection sites) of a Ginkgo leaf extract containing 1000 ppm ginkgolic acids. From this result he concludes that Ginkgo extracts containing between 10 and 200 ppm ginkgolic acids with certainty bear no risk to induce a type IV allergic reaction and that such low amounts are all the more negligible for producing ¯ares in G. biloba-sensitive individuals. In our view this conclusion is not appropriate. Although it is reasonable to assume that the mechanism of sensitization is similar in humans and the usual animal models, there is no evidence that relative potency in the guinea pig is the same as or similar to that in humans. Furthermore, it is important to remember that a threshold determined in an animal model relates to the animal and is completely dependent on the test conditions used to de®ne it [23]. In addition, guinea pig contact sensitivity tests generally require that irritating concentrations of the test substance are applied during the induction phase [24]. As guinea pigs could be successfuly
235
sensitized by application of pure ginkgolic acids at a concentration of 0.19% there is strong evidence that the author did not use an optimal sensitization protocol when applying the Ginkgo extract at a concentration of only 0.375%. For patients involved, sensitization to low-molecular compounds can have serious consequences as this process is irreversible and already exceptionally low concentrations of the triggering substance may induce a recurrence of symptoms on re-exposure to the allergenic substance [25]. Under this aspect and with respect to the fact that no contribution of ginkgolic acids to the therapeutic eect of Ginkgo extracts has been demonstrated [26], it is not admissible to take dose-eect relations into consideration for the bene®t-risk assessment of the alkylphenol content of Ginkgo extracts. The relatively small number of allergic side-eects observed after oral intake of Ginkgo preparations [27] is probably due to the fact that mainly extracts are marketed which comply with the requirements of the monograph of the Commission E of the former German Health Authority (Bundesgesundheitsamt, BGA) according to which extracts are not allowed to contain more than 5 ppm of ginkgolic acids. As this limit value is technically easy to achieve higher concentrations of alkylphenols are not acceptable from the viewpoint of drug safety. As cross reactions between urushiols in Anacardiaceae and alkylphenols in G. biloba have repeatedly been observed in human and animal studies [3,6,9], the therapeutic use of Ginkgo extracts which contain ginkgolic acids above the established limit value may constitute a particular hazard to patients in North America, where between 50 and 85% of the population is sensitized to members of the Anacardiaceae or cashew family which includes plants like poison ivy, poison oak, poison sumac, cashew, mango, Japanese lacquer, and Indian marking nut [28]. The present investigation emphasizes the importance of maintaining satisfactory production conditions, in order to guarantee the completest possible removal of allergenic alkylphenols from Ginkgo extracts. The PLNA appears to represent a simple test procedure for the detection, charac-
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terization and elimination control of constituents with potential immunotoxic side eects in complex plant extracts.
[16] [17]
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