Effect of Oral Application of an Immunomodulating Plant Extract on Influenza Virus Type A Infection in Mice

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Extracts
• TLC
• HPLC
• Mice
• Virus
• Experimental design
• Statistics
• Results
• Discussion
• References

Abstract

The influence of the oral administration of an aqueous-ethanolic extract of a mixture of Thujae occidentalis herba, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix, on the course of Influenza A virus infection in Balb/c mice was investigated. The extract was administered to mice via the drinking water for 14 days starting 6 days before intranasal infection with Influenza A virus. The progress of infection was recorded during a time range of 21 days. Parameters for the evaluation of antiviral activity were survival rate and mean day to death. In a further set of experiments infected mice were sacrificed on defined days. Determination of consolidation score and virus titer were performed for each lung. The data show that the oral treatment with the extract induced a statistically significant increase in the survival rate, prolonged the mean survival time and reduced lung consolidation and virus titer. The experiments demonstrate that the plant immunomodulator given 6 days before exposure is a potent inhibitor of Influenza A virus pathology in vivo.

Introduction

The immunomodulating action of extracts of several plants, such as Coneflower species (Echinacea spec.), Yellow Indigo (Baptisia tinctoria (L.) Vent.) and American Arborvitae (Thuja occidentalis L.) is well known. If used for prophylaxis these extracts can decrease the incidence rate of respiratory tract infections in man and, if therapeutically used, alleviate the symptoms of these diseases, shorten their duration and reduce the frequency of recurrences [1], [2], [3].

The intention of this study was to investigate whether these effects, observed in clinical studies, can be objectivized in animal experiments. Therefore we examined the effects of the oral application of an aqueous-ethanolic extract from Echinaceae purpureae radix, Echinaceae pallidae radix, Baptisiae tinctoriae radix and Thujae occidentalis herba (Thujae summitates) on the course of Influenza A virus infection in mice, recording pathological parameters and survival rates. We used a mixture of plants for extraction because it has been shown in animal experiments, that the immunostimulating effect was potentiated by combining different immunomodulating plants [4]. In order to obtain data that are relevant for human therapy the extract was applied orally in dosages that correspond to the human therapeutic range under consideration of the dissimilar murine metabolic acitivity.

Materials and Methods

Plant material

Dried parts of following plant drugs were used (suppliers in brackets): roots of Echinacea purpurea (L.) Moench (Dieter Müller, Germany), roots of Echinacea pallida (Nutt.) Nutt. (Berghof-Kräuter GmbH, Germany), roots of Baptisia tinctoria (L.) Vent. (Paul Müggenburg GmbH, Germany) and shoots of Thuja occidentalis L. (Alfred Galke GmbH, Germany). The plant drugs were checked for identity and purity.

Extracts

The drugs were extracted by exhaustive percolation at room temperature using 66.5 kg of roots of Echinacea purpurea and Echinacea pallida, respectively, 177 kg roots of Baptisia tinctoria and 36 kg of shoots of Thuja occidentalis with 3800 l of 30 % (V/V) EtOH. This procedure was identical with the manufacturing specification for Esberitox® N (DER 1 : 11) The characterization of the extract by TLC and HPLC has already been published [5].

TLC

Figure [1] shows two thin layer chromatograms (A and B) which were performed to verify the identity by marker compounds in the crude extract. Maackiain (Rf 0.69) and umbelliferone (Rf 0.58) served as reference substances for Thujae occidentalis herba resp. Baptisiae tinctoriae radix. TLC A (silica gel), EtOAc, AcOH, CH₃COOH, H₂O = 100 : 11 : 11 : 27. Detection after spraying with 1 % (m/V) 2-aminoethyl diphenyl borate in MeOH and 5 % (V/V) PEG 400 in MeOH under UV (366 nm).

Cichoric acid (Rf 0.95) and echinacoside (Rf 0.37) were used as marker compounds for Echinaceae purpureae radix and Echinaceae pallidae radix. TLC B (silica gel), HCOOH, HCOOEt, toluene = 9 : 30 : 30. Detection as above.

HPLC

HPLC was carried out with Spherisorb ODS I 250 mm × 4 mm, particle size 5 μm; gradient system: mobile phase A: C₂H₃N with 1 % 0.1 N H₃PO₄ and mobile phase B: H₂O with 1 % 0.1 N H₃PO₄, gradient: 0 - 20.0 min: 5 → 25 % A with total flow 1.0 ml/min, 20.0 - 20.1 min: 25 → 100 % A with total flow 1.0 ml/min, 20.1 - 35.0 min: isocratic 100 % A with total flow 1.5 ml/min, 35.0 - 35.1 min: 100 → 5 % A with total flow 1.5 ml/min, 35.1 - 50.0 min: isocratic 5 % A with total flow 1.5 ml/min; injection volume: 30 μl; UV detection at 254 nm and 330 nm. The corresponding HPLC profiles are shown in Figs. [2] and 3.

Mice

Specific pathogen-free female Balb/c mice (18 - 21 g) were obtained from the Institute of Pathology, Department for Experimental Animals in Karlsburg, Germany. The animals were housed under standard conditions with food and water ad libitum. All animals were maintained in conditions at 23 ± 1 °C in alternating light/dark cycle (Uni-protect Luftstromschrank-Ehret 6MBH). The keeping and handling of the animals corresponded to the principles of laboratory animals care under consideration of EU-guideline (Amtsblatt EU L358 (86/609/EW6) [22].

Animal experiments were approved by the local German animal care and oversight committee. The permit for the experiment was given with approval by the ethical committee of the ”Ministerium für Landwirtschaft und Naturschutz Mecklenburg-Vorpommern” under the registration number VI 522a-7221-31-1-003/98.

Virus

Influenza virus type A/WSN33/London was propagated in chorio-allantoic cavities of 10-days old embryonated hen eggs for 48 h. The yielded virus suspension was titrated in MDCK cells and stored at -70 °C. The titer of the viral suspension was 10^-6.5.

Experimental design

Mice (68 in all) were anesthized by intraperitoneal injection of 100 μl of an anesthetic mixture (Xylazin hydrochloride and Ketamin hydrochloride) and then exposed to 50 μl of virus suspension by intranasal instillation. Virus challenge was 10^3.5 TCID₅₀ (tissue culture infectious dose 50 %), resulting in a 90 % death rate.

The extract was applied to mice (34 mice per group) via the drinking water in a daily dose of 150 μl/kg body weight in filtered, autoclaved tap water. It was administered for 14 days beginning 6 days pre-virus infection to 7 days after infection. The control group (34 mice per group) received autoclaved tap water containing a comparable amount of ethanol (0.03 %).

The progress of infection was recorded during a time range of 21 days. The evaluation of the protective effects was based on survival rate, mean survival time in days, the lung consolidation score, histopathological analysis and the estimation of virus titer. Lung pathology was examined as described previously [6].

To evaluate survival time and survival rate 14 animals of each group were used. Additional 20 mice per group were used for the monitoring of lung consolidation and virus titer. Data result from 4 animals per day per group.

In brief, each 4 mice per group were sacrificed at various times post infection (day 2, 3, 5, 7, 16) by cervical dislocation. The score of lung consolidations was determined on a scale ranging from 0 (normal) to 4 (maximal consolidation). The histopathological sections of each lung were analysed by two observers without knowledge of the treatment group or the time of sacrifice. The extent of the inflammatory responses was also scored from 0 to 4.

For estimation of virus titer the pulmonary tissue (half of a lung each, n = 4) was homogenized by sonification in MEM and incubated on MDCK cells for 48 h at 37 °C. The supernatants from the wells were tested for the presence of infectious viruses by hemagglutination assay using 0.25 % guinea pig erythrocyte suspension. The cells were fixed and tested with FITC-labelled influenza A virus specific antibodies in a modified focus assay [7].

Statistics

The statistical evaluation was performed using the log-rank-test (approximated χ² -test), p < 0.01 vs. control.

Results

It was shown that the oral treatment with the plant extract induced a statistically significant increase in the survival rate of the infected mice. 6 days after infection 85.7 % of the treated mice had survived, compared to only 42.8 % of the mice in the control group. On day 21 the survival rate of the treated mice was 50 %, and 7.1 % in the group of control mice only (Fig. [4]). The mean day to death (MDD) was calculated as 6.98 for the mice of the control group and 16.38 for the mice treated with the plant extract (Table [1]).

Five days after infection the macroscopic evaluation of lung consolidation, scored from 0 to 4, was 1.12 for the treated animals, 3.12 for the control mice, however. The scores for the histologically observed perivascular and peribronchiolar infiltrations, typical for influenza A infections, were determined as 0.68 on day 2 p. i. (post infectionem) and 1.62 on day 5 p. i. in the verum group. The scores in the control group were 1.8 on day 2 p. i. and increased to 2.88 on day 5 p. i. (Table [1]).

The virus titer in the lung tissue determined by the focus assay on day 5 p. i. was 4.75 (log₁₀) for the verum group and 5.5 for the control group. In addition the hemagglutination titers of the lung extracts of the infected mice were significantly lowered by the therapy with the herbal extract. They ranged from day 2 to day 5 after infection between 1 : 15 and 1 : 26 in the treated group and between 1 : 100 and 1 : 63 in the control group (Fig. [5]).

Discussion

The results show clearly that oral administration of an aqueous-ethanolic extract of a mixture of Thujae occidentalis herba, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix in human therapeutically relevant dosages exerts a protective effect on Influenza A virus-infected mice. The treatment with the extract induced a statistically significant increase in the survival rate, prolonged the mean survival time and reduced pathological lung consolidation and virus titers. These effects could possibly be attributed to the antiviral components of the extract and/or to its immunostimulating activity.

Several studies have shown that extracts of herbs and roots of Echinacea purpurea and other Echinacea species, macromolecular components [8], [9], [10] or caffeoylconjugates isolated from these extracts [11] administered prior to a virus infection make cultivated cells resistant to an infection with influenza and many other viruses. Similar observations were made with extracts from shoots of Thuja occidentalis, fractions thereof [12] and with components of the herbal drug such as podophyllotoxin lignans [13] and copper chelates of thujaplicines [14]. Almost all of these experiments were performed in vitro using concentrations that are probably not relevant for human therapy, where low dosages of extracts are commonly applied orally. In vivo, the antiviral activity of these herbal extracts and of their components has not been shown before and it is not clear yet whether the substances mentioned above can be absorbed in quantities that are sufficient to directly inhibit the replication of viruses.

The in vitro immunostimulating effect of the extracts of the mentioned plants is well known. It was shown that the mixture extract used in our experiments or its high molecular mass fraction, containing polysaccharides and glycoproteins, increases the proliferation rate of mouse spleen cells, the secretion of IL-1, IL-6 and TNF_α by mice macrophages and human monocytes, the production of IL-2, IFN_{α/ β} and IgM by mouse spleen cells, the synthesis of nitrogen oxide (NO) by alveolar macrophages of mice, the phagocytotic activity of several types of macrophages and microphages and the number of antibody secreting spleen cells of mice, evaluated by hemolytic plaque assay [15], [16], [17], [18], [19].

The immunostimulating activity of the mixture extract could also be proven in vivo. After oral administration it enhances ex vivo the phagocytosis activity of granulocytes and macrophages [20], the capability of mouse Peyer`s plaques cells (cells of aggregated intestinal lymphatic follicles) and spleen cells to change to antibody secreting cells, and the production of IL-2, GM-CSF and IFN_γ by mouse spleen cells. Furthermore when administered orally it increases the antibody response of immunosuppressed mice against sheep red blood cells [5], [18].

Therefore one may assume, that the positive effect of this extract exerted on the infected mice is mediated first of all by its immunostimulating activity and that the direct antiviral activity is rather of secondary importance. That has to be confirmed in further studies.

The results of Mori et al. [21], who tested the effect of a TJ-41, a Japanese herbal medicine consisting of spray-dried hot water extracts of a mixture of ten medicinal plants, on the survival of mice infected with Influenza virus, support this theory. TJ-41 was found to increase the survival rate and to inhibit lung consolidation. The effects were correlated to an augmentation of IFN_α in bronchoalveolar lavage fluid. The authors postulated that the plant extract exerts its inhibitory effects on an Influenza virus infection via enhancement of the host immune responses.

These results explain the mode of action of medicinal preparations based on herbal extracts in respiratory tract infections of humans with an immunomodulating activity. Because under normal circumstances the infections of man are not as massive as under experimental conditions, one may suppose that the prophylactic and therapeutic effects will be even more impressive in medical praxis.

References

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• 16 Beuscher N, Scheit K H, Bodinet C, Egert D. Modulation der körpereigenen Abwehr durch polymere Substanzen von Baptisia tinctoria und Echinacea purpurea . In: Masihi KN, Lange W, editors Immunotherapeutic Prospects of Infectious Diseases. Berlin; Heidelberg; Springer 1990: 56-63
  Search in Google Scholar
• 17 Bodinet C, Freudenstein J. Effects of perorally applicated Esberitox® on cytokine induction and antibody response against sheep red blood cells in mice. Planta Med: 46th Ann Congress of the Soc for Med Plant Res: 1998 Vienna; Abstracts, J20
  Search in Google Scholar
• 18 Bodinet C. Immunpharmakologische Untersuchungen an einem pflanzlichen Immunmodulator. Dissertation Greifswald; 1999
  Search in Google Scholar
• 19 Vömel T. Der Einfluss eines pflanzlichen Immunstimulans auf die Phagozytose von Erythrozyten durch das retikulohistiozytäre System der isolierte perfundierten Rattenleber.  Arzneimittelforschung. 1985;  35 1437-9
  PubMedSearch in Google Scholar
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• 21 Mori K, Kido T, Daikuhura H, Sakakibara I, Sakata T, Shimizu K, Amagaya S, Sasaki H, Komatsu Y. Effect of Hochu-ekki-to (TJ-41), a Japanese herbal medicine, on the survival of mice infected with influenza virus.  Antiviral Res. 1999;  44 103-11
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  Search in Google Scholar

Dr. Cornelia Bodinet

Schaper & Brümmer GmbH & Co. KG

Bahnhofstr. 35

38259 Salzgitter

Germany

Email: cornelia.bodinet@schaper-bruemmer.de

Fax: +49-5341-307-713

References

• 1 Melchart D, Linde K, Worku F, Bauer R, Wagner H. Immunomodulation with Echinacea - a systematic review of controlled clinical trials.  Phytomedicine. 1994;  1 245-54
  PubMedSearch in Google Scholar
• 2 Wagner H. Pflanzliche Immunstimulanzien. Zur Prophylaxe von Erkältungskrankheiten.  Z Phytother. 1996;  17 79-95
  PubMedSearch in Google Scholar
• 3 Wüstenberg P, Köhler G, Stammwitz U, Henneicke-von Zepelin H H. Phytopharmakon zur Immunmodulation.  Dtsch Apoth Ztg. 2000;  140 2189-97
  PubMedSearch in Google Scholar
• 4 Wagner H, Jurcic K. Immunologische Untersuchungen von pflanzlichen Kombinationspräparaten.  Arzneimittelforschung. 1991;  35 1069-75
  PubMedSearch in Google Scholar
• 5 Bodinet C, Freudenstein J. Effects of an orally applied aqueous-ethanolic extract of a mixture of Thujae occidentalis herba, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix on antibody response against sheep red blood cells in mice.  Planta Med. 1999;  65 695-9
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Mentel R, Wegner U. Evaluation of the efficacy of 2′,3′-dideoxycytidine against adenovirus infection in a mouse pneumonia model.  Antiviral Res. 2000;  47 79-87
  CrossrefPubMedSearch in Google Scholar
• 7 Mentel R, Matthes E, Janta-Lipinski M, Wegner U J. Fluorescent focus reduction assay for the screening of antiadenoviral agents.  J Virol Methods. 1996;  59 99-104
  CrossrefPubMedSearch in Google Scholar
• 8 Beuscher N, Bodinet C, Willigmann I, Egert D. Immunmodulierende Eigenschaften von Wurzelextrakten verschiedener Echinacea-Arten.  Z Phytother. 1995;  16 157-66
  PubMedSearch in Google Scholar
• 9 Bodinet C, Willigmann I, Beuscher N. Host-resistance increasing activity of root extracts from Echinacea species.  Planta Med. 1993;  59 A 672-3
  PubMedSearch in Google Scholar
• 10 Wacker A, Hilbig W. Virushemmung mit Echinacea purpurea .  Planta Med. 1978;  33 89-102
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Cheminat A, Zawatzky R, Becker H, Brouillard R. Caffeoylconjugates from Echinacea species: Structure and biological activity.  Phytochemistry. 1988;  27 2787-94
  CrossrefPubMedSearch in Google Scholar
• 12 Beuscher N, Kopanski L. Purification and biological characterization of antiviral substances from Thuja occidentalis .  Planta Med. 1986;  52 555-6
  PubMedSearch in Google Scholar
• 13 Gerhäuser C, Leonhardt K, Tan G T, Pezzuto I M, Wagner H. What is the active antiviral principle of Thuja occidentalis? .  Pharm Pharmacol Lett. 1992;  2 127-30
  PubMedSearch in Google Scholar
• 14 Miyamoto D, Kusagaya Y, Endo N, Sometani A, Takeo S, Suzuki T, Arima Y, Nakajima K, Suzuki Y. Thujaplicine-copper chelates inhibit replication of human influenza viruses.  Antiviral Res. 1998;  39 89-100
  CrossrefPubMedSearch in Google Scholar
• 15 Bauer R, Wagner (ed) H. Echinacea. Ein Handbuch für Ärzte, Apotheker und andere Naturwissenschaftler. Stuttgart; Wiss. Verlagsges. mbH 1990
  Search in Google Scholar
• 16 Beuscher N, Scheit K H, Bodinet C, Egert D. Modulation der körpereigenen Abwehr durch polymere Substanzen von Baptisia tinctoria und Echinacea purpurea . In: Masihi KN, Lange W, editors Immunotherapeutic Prospects of Infectious Diseases. Berlin; Heidelberg; Springer 1990: 56-63
  Search in Google Scholar
• 17 Bodinet C, Freudenstein J. Effects of perorally applicated Esberitox® on cytokine induction and antibody response against sheep red blood cells in mice. Planta Med: 46th Ann Congress of the Soc for Med Plant Res: 1998 Vienna; Abstracts, J20
  Search in Google Scholar
• 18 Bodinet C. Immunpharmakologische Untersuchungen an einem pflanzlichen Immunmodulator. Dissertation Greifswald; 1999
  Search in Google Scholar
• 19 Vömel T. Der Einfluss eines pflanzlichen Immunstimulans auf die Phagozytose von Erythrozyten durch das retikulohistiozytäre System der isolierte perfundierten Rattenleber.  Arzneimittelforschung. 1985;  35 1437-9
  PubMedSearch in Google Scholar
• 20 Beuscher N. Über die medikamentöse Beeinflussung zellulärer Resistenzmechanismen im Tierversuch. Aktivierung von Peritonealmakrophagen der Maus durch pflanzliche Reizkörper.  Arzneimittelforschung. 1982;  32 134-8
  PubMedSearch in Google Scholar
• 21 Mori K, Kido T, Daikuhura H, Sakakibara I, Sakata T, Shimizu K, Amagaya S, Sasaki H, Komatsu Y. Effect of Hochu-ekki-to (TJ-41), a Japanese herbal medicine, on the survival of mice infected with influenza virus.  Antiviral Res. 1999;  44 103-11
  CrossrefPubMedSearch in Google Scholar
• 22 Zutphen L FM, Baumans V, Beynen A C. Grundlagen der Versuchstierkunde. Gustav Fischer Verlag Stuttgart, Jena, New York,; 1995
  Search in Google Scholar

Dr. Cornelia Bodinet

Schaper & Brümmer GmbH & Co. KG

Bahnhofstr. 35

38259 Salzgitter

Germany

Email: cornelia.bodinet@schaper-bruemmer.de

Fax: +49-5341-307-713