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Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation

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Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation KR0000086 KAERI/RR-1912/98 Development of Food Science and Technology by Radiation Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation 7] 3Z|- 71 31/ 30 KAERI/RR-1912/98 Development of Food Science and Technology by Radiation Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation 7] 7l 1999. 4. •ftl.J3.Sl C3 TT =?• SI ^ "a 'o 2. ?i o| ^ € °J =? 0| ii. Aj-s] jfe n £ 71 ^ III - i - 2. 3. Apoptosis Apoptosis 4. cells)^ wfl^ 2-cI - ii - <£#%• ^(Nitric Oxide) 5.7]^^ ^#db7jK^^^)^j 7jv\# *]•%• 1-^ 2:A} ^j 2:A} 4^^ -a-Sl^^- ^^^4 HPLCS. Paeoniflorin<>l 1-^^°]€A^ £-°)z\ #•%••£ Ames test°lM ^^ . Paeoniflorin^l ^^- CHO DPPHi IV. -i°fl4 SRBCi : 10 : 10 2. - IV - Apoptosis ^. apoptosis €3*5= Apoptosis apoptosis - v - 3. marrow stem cells)^1 f^ £(stromal cells) ^ ^aj-g-o] nfl-f ^A^JL ^ZJ-SIJI 5^4. 87M 50% - vi - ^, 37H -2-S.fe NO ^A^ - , ^3^.S NO ^S^^: -^-S^l-^Jl, iNOS ^*MS1 NGMMi IL-liff, IL-6, LT, iNOS^) mRNA 4. 7l^>g ^ , SIS. ^ ^^-^1^-, 1^t^S M-^f^ £4^ ^4, 10 KGy 54 paeoniflorin^- £e|, ^^I*>^ HPLC^ 97%^ 13C-NMR AT*! paeoniflorin^ retention timeo] 7.915, - VI! ~ 1 44 ^#5] $4. lOkGy 24 4^4 paeoniflorin ^ ^^^ 14 2.59%4 2.42%7> chromatogram^ pattern0!]^H 1141- -<?l decursin4 decursin angelatefe SiO2 column chromatographyS. ^-^«>^l*.H, HPLC* ^f-Sfl ^ signal-i- E-$*1^ ylJI^H 3- ^2:1- ^^^-^i 1:51 tb ^^-S ^"^^^ ^^l- column (Shim-pack CLC-ODS(M))*fH #^r ^-el^-i- i-^^^A^, normal phase column^ 4fe Hexane: EtOAc -§-*1l7lHH ^^tb ^-^^^r ^.^ ^r&4- 4D}^i 2: 4(10kGy) ^^^ «12:4 ^^^ -frjL^^- ^^=^4fe decursin^l decursinol angelate^ Salmonella typhimurium TA98, TA100, TA102 ^^^ 4^1"^^oll- °l-§- Sj-fe Ames testi4 444(10 KGy) ^S^l CHO KGy) - Paeoniflorin^ #^^°l€^^r S-°14 &-§-# Ames tesH4 paeoniflorin-i- ^^fF ^4, 4 Paeoniflorino] CHO 24 4^H ^-s!€ paeoniflorin $7} 444, - viii - x\g,«\ benzo(a)pyrene-°-S. Salmonella typhimurium TA98, TAlOO -CHO CHO DPPHi V. § 71| 71 7] - ix - SUMMARY I. Project Title Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation II. Objective and Importance of the Project The health and long-life have been an important issue with elevation of standard of living. Despite the impressive accomplishments of the modern pharmacological industry, a large number of diseases remain for which treatment is of limited benefit. As a result, there is an increasing need for the development of so-called "alternative treatment by natural products" and health foods, for which predictable pharmacologic activity and therapeutic efficacy can be documented. Concerning maintenance of health, prevention of diseases and inhibition of aging have been important theme. Therefor, the topics of "food and health" have been an important issue. Many kinds of substances are concerned with regulation of physiological function and maintenance of homeostasis, and they might be supplied by various foods, and food stuffs from plant might be important their resources. Natural products such as herbal medicines have only recently begun to receive some attention as possible regulators of host defense mechanism for preventing disease and recovering damage. Especially, the regulation of immunity and hemopoiesis have might be a important theme in prevention of disease, inhibition of aging and overcoming radiation damage. And also, toxicological safety in sanitization of food resources for sanitary production and circulation of functional foods should be confirmed. The present study was performed to develop functional foods with immunomodulation activity overcoming declined immunity and hemopoiesis, and to develop techniques for sanitization of function food resources with gamma- irradiation. - x - . Scope and Contents of the Project 1. Selection of test materials We selected 9 herbs and 6 decoctions of the Oriental medicinal prescriptions, concerning usefulness of herbs as foods, quantities of the medicinal plants cultured in Korea, and reports on the effects. 2. Screening of the immunomodulation components and preparing the provisional products - Screening of the immunomodulation components in herbs - Investigating synergistic effects of immune cell activation by combined treatments - Investigating the effects of the prescriptions on immune cell activation - Making combination of the herbs for preparing the provisional products and investigating the effects of the fractions on immune cell activation - Investigating the immunomodulation activity in vivo - Verifying stability of immunomodulation activity of gamma-irradiated samples 3. Investigation on the hematopoietic effects of functional foods using radiation and preparation of the provisional products - Selection of 6 energy tonic or blood-building decoction of Oriental medicinal prescriptions as candidate foods - Evaluating the effects of the 6 prescriptions and its ingredients in irradiated mice • Jejunal crypt survival assay • Endogenous spleen colony formation assay • Apoptosis inhibition assay - Verifying stability of the biological activity of the irradiated prescriptions • Jejunal crypt survival assay - xi - • Endogenous spleen colony formation assay • • ' • • Apoptosis inhibition assay - Making combination of the herbs for preparing the provisional products on the basis of data on the effects of the prescriptions and its ingredients 4. Search for the active components and studies on the mechanism of the hematopoiesis improvement foods - Isolation and culture of bone marrow stromal cells - Search for active components of the hematopoiesis improvement foods - Investigating growth augmentation of nonadherent precursor cells - Analysis of cytokine expression in stromal cells - Analysis of nitric oxide production in macrophages - Analysis of cytokine expression in macrophages 5. Development of techniques for the sanitation of functional food resources (herbs) by gamma-irradiation - Sanitization test of herbs by gamma-irradiation - Verifying stability of active components in gamma-irradiated herbs • HPLC patterns of paeoniflorin in gamma-irradiated Paeonia Radix • HPLC patterns of decursin in gamma-irradiated Angelica gigantis Radix - Verifying safety of gamma-irradiated herbs in respect of genotoxicity • No mutagenicity of total extracts in Salmonella reversion assay • No induction of micronuclei in CHO cells by total extracts • No mutagenicity of paeoniflorin in Salmonella reversion assay • No induction of micronuclei in CHO cells by paeoniflorin - Verifying stability of the biological activity of the gamma-irradiated herbs • No change in antimutagenic activity of the extracts in Salmonella reversion assay and in micronucleus assay • No change in electron donating activity to DPPH - xii - IV. Results of the Project 1. Screening of the immunomodulation components and preparing the provisional products - Screening of the immunomodulation components in herbs In order to screen immunomodulator, we selected 9 herbs and 6 decoctions of the Oriental medicinal prescriptions. In the test of immune cell proliferation by herbal extracts in vitro, 1, 3 and 2 herbs showed high, moderate and slight effect, respectively. - Investigating synergistic effects of immune cell proliferation by combined treatments In the test of immune cell proliferation by 10 combinations of herbal extracts in vitro, 6 combinations showed higher effects than sum of that by single extract. - Investigating the effects of the prescriptions on immune cell activation In the test of immune cell proliferation by 6 decoctions of energy tonic or blood-building prescriptions in vitro, Si-Jun-Zi-Tang, Bu-Zhong-Yi-Qi -Tang, San-Ling-Bai-Shu-San showed slight effects. But the effects were lower than that of previous combinations made in this study. - Making combination of the herbs for preparing the provisional products and investigating the effects of the fractions on immune cell activation On the basis of the previous results, 2 combinations of herbs were made, and extracted into fractions. In the test of immune cell proliferation by the fractions in vitro, 2 fractions showed high effects. - xiii - - Investigating the immunomodulation activity in vivo It was tested in a hemolytic plaque assay and in graft versus host reaction whether in vivo treatment of the extracts could influence the secretion of antibodies against sheep red blood cells(SRBC) and cellular immune response against transplanted allogenic immune cells, respectively. It turned out that intraperitoneal injection of the extract of 1 single herb or 2 herbal combinations increased the number of antibody-secreting cells remarkably. Splenomegaly, due to graft versus host reaction, was also increased by intraperitoneal treatment of the extract of 1 single herb or 1 herbal combinations. - Verifying stability of immunomodulation activity of gamma-irradiated samples The immunomodulation ativity of irradiated herbs was shown to the same level of that of non-irradiated samples, in vitro and in vivo. 2. Investigation on the hematopoietic effect of functional foods using radiation and preparing the provisional product. - The effect of Oriental prescriptions and its ingredients • Augmentation of jejunal crypt survival Pretreatment with Si-Wu-Tang, Bu-Zhong-Yi-Qi-Tang, Kuei-Pi-Tang, Rensen, Shengma, Danggui, Baishaoyao, Gancao, Chaihu, Baifuling, Baibiandou, Sharen, Jiegeng, Suanzaoren or Muxiang extract resulted in a significant increase in the number of surviving crypts compared with those in the irradiation control. • Augmentation of endogenous spleen colony formation The numbers of spleen colonies was higher
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