Jurkst:iene v., simoniene G., Kondrot:as A., Jankauskiene K. Kaunas Unrversrty of mcdicrne,
M;ckev!CiRJS 9, Kaunas, 3000-LT, Lithuania
T,B LYMPHOCYTE SUBSETS IN INFLUENCE OF PHYTOIMMUNOSTIMULATORS (ECHINACEA PURPUREA (L.) MOENCH AND DESMODIUM CANADENSE DC.) One of the urgent problems in immunology is the establishment of effective immunostimulants. Natural plant preparations are being searched for they are introduced into medical practice. The number of diseases related to the lesion of the immune system increases. The main problem of immunopathology is the increase of lesions of the immune system that is primary and secondary immunodeficiencies, infectious, allergic and autoimmune diseases, tumors, rejection of transplants and other ( 5, I 0). Secondary immunodeficiencies dominate and manifest in postnatal period or adults. They are usually treated by immunotherapy, which is based on substitutive preparations that compensate the defective chain of the immune system. That is why secondary immunodeficiencies are divided into several groups. Our patients often suffered from chills. This can be understood as secondary immunodeficiencies. which was due to hypovitaminosis, emotional stress, inadequate nutrition, disorders of hormonal balance. Secondary immu(?0 221
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nodeficiencies often can be cau'>ed by bacterial and viral infections and manifests in lesion of respiratory organs, skin and mucous membranes. Immunodeficiencies can be corrected only when it is clear which chain of factors of svstemic or local immunity is dysfunctional (4, 7, 12). Synthetical or natural immunostimulants are expensive and cause side effects. Long - term use of synthetical drugs, may result in leucopenia, skin allergy, dizziness, agranulocytosis. For this reason while using this medicine it is necessary to control leucocytes and lymphocytes. Jmmunostimubnts produced from natural products. such as immunoglobulin, arc expensive and cause side effects -- allergic reactions (I). To avoid this immunostimulants - preparations of Echinacea purpurea (L.) Moench and Desmodium canadense DC. may be used. Natural materials found in these plants stimulate the immune system, enhance phagocytosis, Le. increase the activity of phagocyies, which destroy pathogene agents of the disease: viruses, bacteria, fungi. These preparations decrease the possibility of recurrence of the disease and the number of complications (3, 9). Desmodium canadense D.C. preparation is mostly known for its antiviral effect, but it also stimulates the immune system. Desmodium canadense DC. is little known herb, originated from the North America, but it can be cullivatcd in our climate zone. The experimental research of the properties of HD preparation suggested that it effectively stimulates cellular and humor immune system (8). The aim of the our study was to reveal immunomodulative properties of preparations of Echinacea purpurca (L.) Moench and Desmodium canadense DC. The people participating in the preclinical study, were treated with preparation of Echinacea which was made by pharmacy. The preparation was being instilled (20 - 30 ml) into a negligible quantity of room temperature water and was being used thee times per day before a meal. The calculations for subpopulations of lymphocytes were carried both-before treatment and after four weeks of treatment. As control subjects, we chose 11 - 15 age children and 18 · · 20 agematched healthy 21 subjects, not to be associated with immunologic abnormalities. Investigated groups using preparation Echinacea purpurea (L) Moench for 4 weeks and preparations of Desmodium canadense DC. -- Helepin D, 1 tabl./day for three days. Combinations of two or three monoclonal antibodies conjugated with fluorochromes (Becton Dickinson) were used for lymphocyte subset analysis. Staining of the whole anticoagulated peripheral blood was performed following manufacturer's recommendations. 100 ~tl of peripheral blood was placed in each tube with 20 !11 of each monoclonal antibody and incubated for 15 m in at room temperature in the dark. Then erythrocytes were lysed using FACS R
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Lysing Solution (Becton Dickinson). The cells were washed twice Cell WASH solution and fixed with Cell FIXr\1 (Becton Dickinson). The cells were kept at 4° C until flow cytomctric analysis was performed.
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fill Before using the preparations illl After using preparations of Echinacea purpurea
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o~ Desmodium canaden:.:_ ___ j
Fig. 1. Count (%)of lymphocyte subsets in patients before and after using phytoimmunostimulators Lymphocyte subset analysis was performed with a gate on lymphocytes. The white blood cell count required for determination of absolute numbers of cells was obtained by counting the cells in haemocytometer (2). Differences between mean values were determined using two-tailed Student's test. Differences between variances were determined using Fisher's test. A p-value of 0.05 or less was considered statistically significant. After treatment a statistical significant increase in leukocytes, Tlymphocytes, B-lymphocytes, T-helpers, index CD4+/CD8+, NK cells, T+B+NK was being measured. The increase in T-helpers and remote changes in T-·suppressors indicate a positive imunomodulative effect (Fig. I, 2.).
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Fig. 2. Index CD4+/CD8+ (0,94-before using the preparations; 1,27-after using preparations of Echinacea purpurea; I ,09-after using preparations of Dcsmodium canadense) The surface of T -lymphocytes has cell function regulating antigenic structures and receptors. The preparations of Echinacea stimulate non-specitic response factors, T-lymphocytes, T-helpers; increase the secretion of IL-2, the growth and differentiation factor of T-cells, the mediator of cell immunity protecting the immune system. T helpers (Th) receive information about alien antigens from macrophages and with lL-2 stimulate other macrophages and Blymphocytes together activate other helping cells. B lymphocytes are stimulated by activating the recognition of the antigen and its delivery to Thl and Th2. The result is the intensified secretion of antibodies or the production of body strengthening interleukines Il-2, TNF, gama INF. Lipopolysacharides activate B cells, but there is evidence proving that they are also activated by lectins found in Echinacea. In their action, the differentiation of B cells from lymphoblasts accelerates and IgM antibodies are formed. They positively affect microorganisms. lgM joins a target cell, dissolves it and agglutinates with an attached complement !gM is the antibody of the immune system protecting against bacteria. The primary response starts with an antigen activating B lymphocytes via the receptors of IgM ( 6, 10 -- 11 ). The preparations of Echinacea purpurea (L.) Moench and Desmodium canadense DC. are to be considered effective non-specific immunostimulators, i.e. stimulate T and B immune system, increase lymphocytes, T helpers, CD4+/CD8+ index, NK and other cells (13). Conclusions. 1. The number of cells had a tendency to decrease dming a chills. 2. The general number ofT and B cells, T helpers, CD4+/CD8+index, NK cells, T +B+NK marked a statistically significant increase after four weeks of treatment using preparation of Echinacea purpurea. 3. The percentage CDJ+, CD4+, CD 19+, and CD4 i·/CD8+ index increased after using preparation ofDesmodium canadense DC.
Bibliography 1. KoHJl,pOTac A., lOpKwTeHe B., 5l.HyJli1C B. l13MeHeHue aKTHBHOCTI1 HCKOTOpblX IIOKa3aTeiiei1 HeC!ICilHqHI'ICCKOH peaKTHBHOCTH opraHJ13M3 f10Jl BmH!HHeM npenapaTOB (Echinacea Moench) // YlMMyHonorml, annepronorH» 11 ImcpeKTOJlOf'l151. -1999. - N~ l.- c. 84 - 85. 2. HoBHKOB )].. K., HPBHKOBa B. M. OueHKa HMMyaoro cTaTyca. -- MHHCK B!ne6cK, 1997. 3. CaMopo.noB B. H., f1ociieJlOB c. B., MouceeBa r. <D. 11 /:(p. <l>I1MOXI1M11'1eCKI1i1 cocTaB npe.n:cTaBureneif po,aa exuHaue» (Echinacea Moench) 11 ero r!JapMaKOJJOriPJCCKHe csoi1cTBa (o61op) // XHM.-r!JapMau. )f(ypH.- 1996.- T. 30.N~ 4. --C. 32-- 37. 4. Abdullah T. A strategic call to utilize Echinacea-garlic in flu-cold seasons 1/J. Natl. Med. Assoc.- 2000.- Vol. 92. N 1.- P. 48-51 5. Astin J. A. Why patients use alternative medicine. Results of a national study// JAMA.- 1998.- Vol. 279. -N~ 19. P. 1548-1553. 6. Bauer R. Echinacea - Drogen -- Wirkungen und Wirkssubstanzcn // Z. Arztl. Forbild. Jena. - 1996. -Vol. 90.- N~ 2. - P. Ill - 115. 7. Gunning K., Sttclc P. Echinacea fo the prevention of upper respiratory trakct infections 1/ J. Fam. Pract.- 1999.- Vol. 48. N~ 2.-- P. 93 8. Kondrotas A., Janulis V.,Simoniene G., Jurkstiene V. The Neutrophil activation in vivo and in vitro by preparations, obtained from Desmodium Canadense D. C. and Echinacea Purpura// Immunol. Lett. (IMLED). - 1997. Vol. 56. - N~ l - 3.- P. 431. 9. Kondrotas A., Stankaitis V., Janulis V., Simoniene G. A new preparation, got from Desmodium Canadense as stimulatos of T lymphocytes // Baltic Immunological Society Meeting: Abstracts. 11 - 12. IX. 1995. Riga- Jurmala. ·· Riga, 1995.- P. 29. I 0. Mcl chart D., Walther E., Linde K. et al. Echinacea root extracts for the prevention of upper respiratory tract infections: a double-blid, placebo --controlled randomized trial 1/ Arch. Fam. Med.- 1998.- Vol. 7. - N~ 6.- P. 541 - 545. ll.Pepping J. Echinacea //Am. J. Health Syst. Phann. -- 1999.- Vol. 16.- N~ 2.- P. 121- 122. 12. Perry N. B., van Klink J. W., Burges E. J. et al. Alkamide levels in Echinacea purpurea: effects of processing, drying and storage I/ Planta Med. 2000.- Vol. 66.- N~ I.- P. 54-56. 13. Simoniene G., Janulis V., Kondrotas A., Maciulyte L. lmmunostimulative properties of a new plant source preparation // Inter. J. Immunorehabil. -- 1999. N~ 12.--- Suppl. -- P. 65.
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