KR0000208
KAERI/RR-2028/99
Development of Radiation Food and Biotechnology
Development of Functional Foods for Radiation Workers
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(4) ^^5] decursin ^ decursin angelate^l ^-2] ^^ 44^1 -n-Sl^-& xvii - (5) ^3 HPLC ^ ^# Decursin^ decursin angelate^ H^f- Al^ol 3.84 4.0°lSiJi ^iM^ 320 nmi^i 2ltfl^^-£l- 4M-M reverse phase column^] *\ fe acetonitrile: °J:3i^^ O^AJ-co iumn (Shim-pack CLOODS(M)HH $}A^, normal phase columni^^- Hexane: EtOAc -§- . Decursin^ 200ug - lOOOug/ml ^H (6) #*VS ^A> ^^^ -n-Jl^^:^! decursin^l- decursinol angelate HPLC ^^ ^^I^f U1^4 ^^^ -B-^Aou@- ^^14^ decursin^ 0.27%^ decursinol angelatefe ^^ V. 7fl - xvin - SUMMARY I. Project Title Development of Functional Foods for Radiation Workers II. Objective and Importance of the Project The radiation damage has been demonstrated since discovery of X-rays. As the diversity of radiation used in medicine, agriculture, industry, biochemical research and military operation increases, the risk from exposure including total body or local organs damage will be elevated. Thus, the protection of individuals against radiation damage is an important issue. Synthetic compounds have been studied for their ability to protect against the adverse effects of ionizing radiation since the original observation of radioprotection. Subsequently, many compounds have been tested for their ability to modify the effect of radiation. Despite some toxic effects, newer compounds are currently under investigation as possible adjuvants in the radiation treatment of cancer. Natural products such as herbal medicines have only recently begun to receive some attention as possible modifiers of the radiation response. Especially, the regulation of immunity and hemopoiesis have might be a important theme in prevention of disease and overcoming radiation damage. The present study was performed to develope non-toxic functional foods with activity of imrnunomodulation, hematopoiesis augmentation and stem cell protection from radiation. The effects of several eatable herbs and Oriental prescriptions were evaluated, and the mixtures of herbs were made to prepare provisional products with higher activity in all respects. On the other hand, it is required by industrial world to apply the irradiation technology for hygienic purpose that is usually performed by chemical preservatives. So the safety of irradiated functional resources was evaluated in respects of biological activity, genetic toxicity and preservation of active components. - xix - III. Scope and Contents of the Project 1. Development of functional foods for modulation of host defence A. Scope and strategy 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) Strategy for development of provisional products To develope non-toxic functional foods with activity of immunomodulation, hematopoiesis augmentation and stem cell protection from radiation. Several eatable herbs and Oriental prescriptions were screened, and the mixtures of herbs were made to prepare provisional products with higher activity in all respects. 1.1 Screening of food materials (1) Screening of food materials with immunomodulation activity - Screening of the immunomodulation components from herbs - Investigating synergistic effects of immune cell activation by combined treatments - Investigating the effects of the prescriptions on immune cell activation - Investigating the immunomodulation activity in vivo (2) Screenig of food materials with hematopoiesis improvement activity - Isolation and culture of bone marrow stromal cell - Search for food materials and investigating synergistic effects by combined treatments - Analysis of nitric oxide production in macrophages - xx - (3) Screening of food materials for protection of stem cells from radiation - Selection of 6 energy-tonic or blood-building decoction of Oriental medicinal prescriptions as candidate foods - Evaluating their effects on jejunal crypt survival in irradiated mice - Evaluating their effects on endogenous spleen colony formation in irradiated mice - Evaluating their effects on apoptosis of jejunal crypt cells of irradiated mice 1.2 Development of provisional products for modulation of host defence (1) Preparation of herb mixture and investigation of their effects - Preparation of 4 mixtures of herbs which expected to have the higher activity - Evaluation of the immunomodulation activity of the mixtures - Analysis of effects of the mixtures on the hematopoietic system - Evaluation of the effects of the mixtures on the stem cell protection in irradiated mice (2) Selection of effective mixtures as prescriptions and analysis of their effects - Evaluation of immunomodulation activity of the two prescriptions - Analysis of effects of the prescriptions on the hematopoietic system - Determination of effects of the prescriptions on the stem cell protection in irradiated mice (3) Development of the provisional products and acute toxicity test - Preparation of two provisional products with higher activity of immunomodulation, hematopoiesis augmentation and radioprotection - Acute toxicity test of two provisional products xxi - 2. Security for application of gamma-irradiation techniques to sanitization of functional food resources (1) Sanitization of functional food resources (herbs) by irradiation - Selection of test materials - Determination of the irradiation dosage for sanitization (2) Verifying stability in the biological activity of the irradiated herbs - Verifying stability in immunomodulation activity of gamma-irradiated herbs - The stability in radioprotection activity of irradiated Oriental medicinal prescriptions - No changes in antimutagenic activity and electron donating activity of the irradiated samples (3) Verifying safety of irradiated herbs in respect of genotoxicity - No mutagenicity of the extracts of irradiated herbs in Salmonella reversion assay - No induction of micronuclei in CHO cells by the extracts of irradiated herbs (4) Verifying stability of active components in irradiated herbs - HPLC patterns of paeoniflorin in gamma-irradiated Paeonia Radix - No mutagenicity of paeoniflorin in Salmonella reversion assay - No induction of micronuclei in CHO cells by paeoniflorin - HPLC patterns of decursin in gamma-irradiated Angelica gigantis Radix - XXll - IV. Results of the Project 1. Development of functional foods for modulation of host defence 1.1 Screening of food materials (1) Screening of food materials with immunomodulation activity In order to screen immunomodulator, we selected nine herbs and six decoctions of the Oriental medicinal prescriptions. In the test of immune cell proliferation by herbal extracts in vitro, six herbs showed some effect. The synergistic effects of combined treatments was investigated in immune cell proliferation test in vitro. Among ten combinations of herbal extracts, six combinations showed higher effects than sum of those by single extract. In the test of immune cell proliferation by six 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 of herbal extracts made in this study. 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. (2) Screening of food materials with hematopoiesis improvement activity At the first experiment, we estabilished long-termed culture methods of bone marrow stromal cells for proliferation and differentiation of stem cells. We conformed stromal cells alive with long spikes and adherence to culture - xxiii - dish. The cultures were maintained for a period of 3 or 4 weeks for study of continuous growth by weekly removal of half of the medium from the flask and replenishment with fresh growth medium supplemented with 8 strains of herbal plant extracts. And selected some extracts which support maximal proliferation of stromal cells. All extracts exhibited cytotoxicity at high concentrations. In the next experiments, we cultured stromal cells with mixture of 2 strain extracts for the synergic effects. The proliferation of stromal cells was increased rather by the addition of combination of 2 strain extracts than by addition of single strain extract. We tested the production of nitric oxide by mouse macrophages cell line to verify the effect of herbal plant extracts. Stimulation of macrophages cell line with 4 strains extracts with the treatments of recombinant interferon- / resulted in increased nitric oxide synthesis in a dose-dependent manners. Four strains of herbal plant had no effect on nitric oxide synthesis by itself. Nitric oxide production was inhibited by NG-monomethyl-L-arginine. In addition, these extracts induced the same effects on the peritoneal macrophages. Altered patterns of cytokine mRNA expression - IL-1/5, IL-6, LT and iNOS - were observed in the stromals cells cultured with extracts of herbal plant. (3) Screening of food materials for protection of stem cells from radiation The effects of Oriental prescriptions and its ingredients on the stem cell survival were investigated in mice irradiated with gamma-rays. In the jejunal crypt survival test, 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. In the endogenous spleen colony formation test, the numbers of spleen colonies was higher in the mice that received Si-Wu-Tang, Bu-Zhong-Yi-Qi-Tang, San-Ling-Bai-Shu-San, Rensen, Huangqi, Danggui, Baishaoyao, Chaihu, Suanzaoren, Yiyiren or Shoudehuang extract and radiation - xxiv - than in mice esposed to radiation only. In the apoptosis assay, the number of cells with nuclei positively stained for apoptosis was decreased in the groups of mice pretreated with Si-Wu-Tang, Bu-Zhong-Yi-Qi-Tang, San-Ling-Bai-Shu-San, Kuei-Pi-Tang, Rensen, Shengma, Baishaoyao, Chaihu, Baifuling, Longyanrou, Suanzaoren, Muxiang, Chuanxiong or Yuanzhi compared with those of irradiation control group. 1.2 Development of provisional products for modulation of host defence (1) Preparation of herb mixtures and investigation of their effects On the basis of the previous results, we prepared 4 mixtures of herbs (herb mixtures; HM-I, HM-II, HM-III, HM-IV) which expected to have the activity oj immunomodulation , hematopoiesis augmentation and radioprotection, and investigated the effects of the extracts. Firstly, we tested the immunomodulation activity of the prescriptions. In the test of immune cell proliferation in vitro, 4 mixtures showed significant effects, in order of HM-III, HM-I, HM-IV and HM-II. Secondary, we tested the effects of the prescriptions on the hematopoietic system. To investigate the effects on growth of nonadherent precursor cells, nonadherent supernatant-derived mononuclear cells harvested from long termed bone marrow cell culture in the presence or absence of the extracts. Two mixtures(HM-I, HM-II) increased the number of nonadherent mononuclear cells. When the stromal cells cultured in the presence or absence of HM-I extract, different cytokine expression patterns were observed, which support differentiation of nonadherent cells. Thirdly, we tested the effects of the prescriptions on the stem cell protection in irradiated mice. In the gamma-irradiated mice, the effects of the 4 mixtures on the stem cell survival were investigated. In the Jejunal crypt assay, - xxv - pretreatment with HM-II and HM-IV resulted in a significant increase in the number of surviving crypts compared with those in the irradiation control.In endogenous spleen colony formation assay,the numbers of spleen colonies was higher in the mice that received extracts of the 4 mixtures and radiation than in mice exposed to radiation only. In apoptosis assay,the number of cells with nuclei positively stained for apoptosis was decreased in the groups of mice pretreated with HM-I, HM-II and HM-IV compared with those of irradiation control group. In micronucleus assay with mouse bone marrow cell,the formation of radiation-induced micronuclei in reticulocyte was inhibited by treatment of HM-I, HM-II and HM-IV. (2) Selection of effective mixtures as prescriptions and analysis of their effects We selected the HM-I and HM-II as effective prescriptions and performed the experiments for evaluating the radioprotective effect of water extract, ethanol fraction, methanol fraction and polysaccharide fraction of each prescription. Firstly, we tested the immunomodulation activity of the prescriptions. In the lymphocyte proliferation test, the two fractions of HIM-I-Pi and HIM-II-Pi exhibited high effects. Especially, HIM-I-Pi showed ver high effects. It was tested in vivo whether the extracts could influence the secretion of antibodies against sheep red blood cells(SRBC) and cellular immune response against allogenic immune cells, respectively. In the hemolytic plaque assay, intraperitoneal injection of the water extract of HIM-1 increased the number of antibody-secreting cells by 1.8 times. In the graft versus host reaction, splenomegaly was also increased in mice intraperitoneally injected with the polysaccharide fraction of HIM-I and HIM-II by 2 and 1.5 times, respectively. In the test of mixed lymphocyte reaction, the spleen cells from C57BL/6 mice treated with fraction HHVI-I-E showed slightly increased reaction against allogenic lymphocytes of DBA/2. And all fraction of HIM-II increased - xxvi - significantly the reaction, especially, the reaction was increased by 3 times in mice treated with polysaccharide, methanol, ethanol fractions. Since the polysaccharide fractions of HIM-I and HIM-II showed high activity of immunomodulation, we mixed the water extract with its polysaccharide fraction to develope more effective prescriptions with higher activity of immunomodulation, hematopoiesis augmention and radioprotection. The mixture of extract and polysaccharide fraction increased the lymphocyte proliferation remarkably. And the optimum concentration was declined. Secondary, we tested the effects of the prescriptions on the hematopoietic system. The effects of HIM-I and HIM-II fractions on the adherent stromal cell growth were investigated. The proliferation of stromal cells were increased at 20 - 40 times with fraction of HIM-I-W+Pi, HIM-II-W, Po, Pi and W+Pi. The mRNA expressions of IL-10 and LT were induced and IGIF increased by HIM-I fractions in the bone marrow cells. The effects of HIM-I and HIM-II fractions on the differentiation of progenitor to macrophages and erythrocytes were invetigated. In the macrophage differentiation, any of HIM-I fractions had no effected even in the presence of GM-CSF. But all of HIM-II fractions induced the differentiation of progenitors into primary macrophages. The diferentitaion of erythrocytes was increased rather by HIM-II than by HIM-I fractions in the presence of EPO(erythropoietin). The proliferation of irradiated bone marrow cells were measured in the presence or absence of HIM-I and HIM-II fractions. Some fractions of HIM-I and HIM-II increased the proliferation of bone marrow cells at the low(4Gray) y -ray irradiation. But these effects were not seen at the high(8Gray) y -ray irradiation. Thirdly, we tested the effects of the prescriptions on the stem cell protection in irradiated mice. In jejunal crypt assay, pretreatment with water extract of HIM-I and water extract and methanol fraction of HIM-II resulted in a significant increase in the number of surviving crypts compared with those in the irradiation control. In endogenous spleen colony formation assay, the - xxvii - numbers of spleen colonies was higher in the mice that received polysaccharide fraction of HIM-I or HIM-II and radiation than in mice exposed to radiation only. In apoptosis assay, the number of cells with nuclei positively stained for apoptosis was decreased in the groups of mice pretreated with water extract, ethanol fraction arid polysaccharide fraction of HIM-I, and water extract, methanol fraction and polysaccharide inner fraction of HIM-II compared with those of irradiation control group. In the analysis of peripheral blood, the numbers of erythrocytes, leukocytes and thrombocytes were increased slightly in all of the treated group. In micronucleus assay with human peripheral blood lymphocyte in vitro, the formation of radiation-induced micronuclei was inhibited by treatment of HIM-II. In survival test, the survival rate and mean survival time of the groups treated with water extract and polysaccharide fractions of HIM-I and HIM-II within 30 days after the treatment were far better than the irradiation control group. (3) Development of the provisional products and acute toxicity test On the basis previous results, we prepared the mixture of each prescription and its polysaccharide fraction, and served as the provisional product-1 and -2, which have the higher activity of immunomodulation, hematopoiesis augmentation and radioprotection, and investigated the effects of the extracts. The LD50/14 was higher than 2 g per kg of body weight in the group of treated with provisional product 1, the LD50/7 was 0.4 g per kg of body weight in the group injected intraperitoneally with provisional product 2 and this result indicated that the provisional products are nontoxic prescriptions. In further research, the active components will be identified, and the mechanism of the protection will be studied. And studies on practical application of the provisional products will be performed. - xxvm - 2. Security for application of gamma-irradiation techniques to sanitization of functional food resources (1) Sanitization of functional food resources (herbs) by irradiation We selected 15 herbs, concerning usefulness of herbs as foods, quantities of the medecinal plants cultured in Korea. In order to determine the irradiation dosage for sanitization, the microorganisms remained in the irradiated samples were investigated by growth test. Any viable microorganisms were not found in samples irradiated with 10k Gy. (2) Verifying stability in the biological activity of the irradiated herbs - Verifying stability in immunomodulation activity of gamma-irradiated herbs The immunomodulation activity of irradiated herbs was shown to the same level of that of non-irradiated samples, in vitro and in vivo. - The stability in radioprotection activity of irradiated Oriental medicinal prescriptions No adverse effects on the parameters examined were found in the groups treated with irradiated prescriptions. All of the prescriptions protected the jejunal crypts, increased the formation of endogenous spleen colony and reduced the frequency of radiation-induced apoptosis. In the majority of cases, the findings obtained in experimental animals treated with irradiated prescriptions did not differ from those in the animals treated with unirradiated prescriptions. - No changes in antimutagenic activity of the extracts in Salmonella reversion assay There was no changes in antimutagenic activity of the extracts of irradiated Paeonica japonica Miyabe, Angelica gigas Nakai, Cnidium officinale Makino, Curcuma longa Linne, Scutellaria baikalensis George in - xxix - the reverse mutation of Salmonella by benzo(a)pyrene comparing with that of non-irradiated samples. - No change in antimutagenic activity of the extracts in CHO cells There was no changes in antimutagenic activity of the extracts of irradiated Paeonica japonica Miyabe, Angelica gigas Nakai, Cnidium officinale Makino, Curcuma longa Linne, Scutellaria baikalensis George in the micronucleus induction by benzo(a)pyrene during cell division comparing with that of non-irradiated samples. - No change in electron donating activity to DPPH There was no changes in electron donating activity of the extracts of irradiated herbs comparing with that of non-irradiated samples. (3) Verifying safety of irradiated herbs in respect of genotoxicity - No mutagenicity of the extracts of irradiated herbs in Salmonella reversion assay The mutagenicity of extracts of irradiated Paeonica japonica Miyabe, Angelica gigas Nakai, Cnidium officinale Makino, Curcuma longa Linne, Scutellaria baikalensis George was examined in Salmonella reversion assay (Ames test). The increase of the revertant colony formation by the extracts did not appreciated in both direct non-activated and indirect activated test. From these results, any direct or indirect mutagen might be not formed in the herbs by gamma-irradiation of lOKGy. - No induction of micronuclei in CHO cells by the extracts of irradiated herbs The micronucleus induction in cell division by the extracts of irradiated Paeonica japonica Miyabe, Angelica gigas Nakai, Cnidium officinale Makino, Curcuma longa Linne, Scutellaria baikalensis George was examined by cytokinesis-block method. The frequency of micronuclei in the test group was not significantly different from negative control group, - xxx - and similar to that of non-irradiated sample in both direct non-activated and indirect activated test. From these results, aberration in nucleus division might be not induced by the extracts of irradiated herbs. (4) Verifying stability of active components in irradiated herbs - HPLC patterns of paeoniflorin in gamma-irradiated Paeonia Radix The paeoniflorin isolated from the radix of Paeonia japonica Miyabe was mesuared using NMR and the structure was confirmed by comparing the data obtained with that of a previous report. The peak area ratio of paeoniflorin was proportional to its concentration from 100 to 500 ug/m£ under conditions described with a regression line of Y=12452.39X+96934.29 and a correlation coefficient of 0.998. The results of the determination of paeoniflorin in irradiated Paeonia radix showed that paeoniflorin contents (average of 3 replications) were of 2.42% (CM-0.22%) by the irradiated sample and 2.59%(C.V=0.45%) by the non-irradiated sample. Chromatograms of paeoniflorin was similar with non-irradiated samples. Hence both samples showed good agreement, so the stability of paeoniflorin in irradiated p. radix was admitted with no doubt. It was found that paeoniflorin did not discompose. - No mutagenicity of paeoniflorin in Salmonella reversion assay The mutagenicity of paeoniflorin isolated from irradiated Paeonica japonica Miyabe was examined in Salmonella reversion assay. The number of revertant colony of each strain did not increased by paeoniflorin. From these results, any mutagen might be not formed in the herbs by gamma-irradiation of lOKCy. - No induction of micronuclei in CHO cells by paeoniflorin The induction of micronuclei by paeoniflorin isolated from irradiated Paeonica japonica Miyabe was examined in cytokinesis-blocked binucleated - xxxi - cells. The frequency of micronuclei in the test group was not signigicantly different from negative control group, and similar to that of non-irradiated sample in both direct non-activated and indirect activated test. From these results, aberration in nucleus division might be not induced by extract of irradiated Paeonica japonica Miyabe. - HPLC patterns of decursin in gamma-irradiated Angelica gigantis Radix From the root of Angelica gigas Nakai, decursin and decursinol angelate were isolated by silica gel column chromatography(tolune:ether, hexane: EtOAc). And then structures were confirmed using the 1H and 13ONMR data. The quantitative analysis of decursin was performed in the high performance liquid chromatographic (HPLC) methods using reverse phase columns(Nova Pak C18) and normal phase columns(Shim-pack CLC-ODS(M)). The decursin contents in gamma-irradiated Angelica Radix were parallel with the yields of non-irradiated sample and the HPLC patterns of gamma-irradiated Angelica Radix also similar with non-irradiated samples. V. Application Plan of the Project Results - Applied as function foods for overcome of radiation damage and reduction of side effects in radiotherapy. - Applied as functional foods for modulation of the declined immunity and hemopoiesis for treatment of cancer, adult diseases and stress. - Applied as techniques in developments of various functional foods from natural products including herbs. - Applied in augmentation of quality and diversity of food industry. - Contributed to activation of related industry conforming toxicological safety and efficacy stability of irradiated foods. - Contributed to improvement of quality of public living. - XXXll - CONTENTS Chaptet 1. Introduction 1 Chapter 2. Status of home and foreign technology development 3 Section 1. Foreign status of functional food development 3 Section 2. Home status of functional food development 5 Section 3. Assessment of the status 6 Chapter 2. Contents, Methods and Results 7 Section 1. Contents and Methods 7 1. Development of functional foods for modulation of host defence 7 A. Scope and strategy of the project 7 (1) Selection of test materials 7 (2) Strategy for development of provisional products 7 (3) Sample preparation 7 B. Screening of food materials with immunomodulation activity 8 (1) Animals 8 (2) Isolation and culture of splenic lymphocytes 8 (3) MTT Assay 8 (4) Lymphocyte proliferation test 9 (5) Hemolytic plaque forming cell assay 9 (6) Graft vs. Host reaction 9 C. Screening of food materials with hematopoiesis improvement activity • • • 10 (1) Animal ••- • 10 (2) Reagents • 10 (3) Detection of LPS in samples 11 (4) Isolation and culture of bone marrow stromal cells 11 (5) Screening of food materials with growth improvement of stromal cells ••••11 (6) Determination of growth improvement of non-adherent cells 11 (7) Culture of cell lines and peritoneal macrophages 11 (8) Measurement of nitric oxide production in macrophages 12 (9) RT-PCR method 12 (10) Preparation of GM-CSF and IL-3 13 xxxui - (11) Proliferation of bone marrow cells into macrophages and erythrocytes •••13 (12) Culture of bone marrow cells from irradiated mice 13 C. Screening of food materials for protection of stem cells from radiation •••14 (1) Animal 14 (2) Irradiation of mice 14 (3) Jejunal crypt survival assay 15 (4) Endogenous spleen colony formation assay 15 (5) Apoptosis of jejunal crypt cells 15 (6) Micronucleus test in bone marrow cells of mice 16 (7) Hematological analysis 16 (8) Micronucleus test in human lymphocytes 17 (9) Mice survival assay 18 (10) Acute toxicity test of provisional products 18 2. Security for application of gamma-irradiation techniques to sanitization of functional food resources 18 A. Sanitization of functional food resources(herbs) by irradiation 18 (1) Selection of test materials 18 (2) Irradiation of test materials 18 (3) Test of microorganism death 19 B. Verifying stability in the biological activity of irradiated herbs 19 (1) Verifying stability in immunomodulation activity of irradiated herbs 19 (2) The stability in radioprotection activity of irradiated Oriental medicinal prescriptions 19 (3) The stability in antimutagenic activity and electron donating activity • • 19 C. Verifying safety of irradiated herbs in respect of genotoxicity 20 (1) No mutagenicity of the irradiated herbs in Salmonella reversion assay --20 (2) No induction of micronuclei in CHO cells by irradiated herbs 21 D. Verifying stability of active components in irradiated herbs 21 (1) Sample preparation 21 (2) Isolation of active components 22 (3) Analysis of NMR 22 (4) Determination of active components in HPLC 22 Section 2 Results and Discussion 23 - xxxiv - 1. Development of functional foods for modulation of host defence 23 1-1. Screening of food materials 23 A. Screening of food materials with immunomodulation activity 23 (1) Screening of the immunomodulation components from herbs 23 (2) Investigating synergistic effects of immune cell activation by combined treatments 23 (3) Investigating the effects of the decoctions on immune cell activation 23 (4) Investigating the immunomodulation activity in vivo 24 B. Screening of food materials with hematopoiesis improvement activity 37 (1) Establishment of culture of bone marrow stromal cells 37 (2) Search for food materials and investigating synergistic effects by combined treatments 37 (3) Analysis of nitric oxide production in macrophages 37 C. Screening of food materials for protection of stem cells from radiation •••54 (1) Evaluating effects on jejunal crypt survival in irradiated mice 54 (2) Evaluating effects on endogenous spleen colony formation in irradiated mice • 54 (3) Evaluating effects on apoptosis of jejunal crypt cells of irradiated mice ••••54 1-2. Development of provisional products for modulation of host defence 76 A. Preparation of herb mixtures and investigation of their effects 76 (1) Preparing mixtures of herbs for the higher activity 76 (2) Evaluation of the immunomodulation activity of the mixtures 76 (3) Analysis of effects of the mixtures on the hematopoietic system 76 (4) Evaluation of the effects of the mixtures on the stem cell protection in irradiated mice • 77 B. Selection of effective mixtures as prescriptions and analysis of their activity 86 (1) Evaluation of immunomodulation activity of the two prescriptions 86 (2) Analysis of effects of the prescriptions on the hematopoietic system •••87 (3) Determination of effects of the prescriptions on the stem cell protection in irradiated mice 90 C. Development of the provisional products and acute toxicity test 126 (1) Preparation of two provisional products with higher activity of immunomodulation, hematopoiesis augmentation and radioprotection 126 - xxxv - (2) Acute toxicity test of two provisional products 126 (3) Plan in further research 127 2. Security for application of gamma-irradiation techniques to sanitation of functional food resources 129 A. Sanitization of functional food resources (herbs) by irradiation 129 (1) Selection of test materials 129 (2) Determination of the irradiation dosage for sanitization 129 B. Verifying stability in the biological activity of the irradiated herbs 131 (1) Verifying stability in immunomodulation activity of irradiated herbs 131 (2) The stability in radioprotection activity of irradiated Oriental medicinal prescriptions 131 (3) No changes in antimutagenic activity and electron donating activity of the irradiated samples 132 C. Verifying safety of irradiated herbs in respect of genotoxicity 156 (1) No mutagenicity of the extracts of irradiated herbs in Salmonella reversion assay 156 (2) No induction of micronuclei in CHO cells by the extracts of irradiated herbs 156 D. Verifying stability of active components in irradiated herbs 167 (1) Isolation and identication of paeoniflorin in irradiated Paeonia Radix •• 167 (2) HPLC patterns of paeoniflorin in irradiated Paeonia Radix 167 (3) No mutagenicity of paeoniflorin in Salmonella reversion assay 167 (4) No induction of micronuclei in CHO cells by paeoniflorin 167 (5) Isolation of decursin and decursin angelate in irradiated Angelica gigantis Radix 168 (6) HPLC conditions for Angelica gigantis Radix 168 (7) HPLC patterns of decursin in irradiated Angelica gigantis Radix 168 Chapter 4. Goal Achievement and External Contribution 179 Chapter 5. Application Plan 182 Chapter 6. References 183 - xxxvi - 2 ^ -^-Lfl • ^ 7]^7fl«J- «i% 3 *l] 1 ^ ^ 7l#7fl^ W 3 *)] 2 3 ^ °A^7m^r^ ^fl-§- ^ ^4 7 *ll 1 ^ ^^^fl^- ^ ^^ 7 ^^ ^ 7Mif- ?m 7 ^i^= 7 (1) Al^jg. Al^ 7 (2) 7}x|f £# ^5* 7 (3) AlS-^sCt^r ^## ^ ^^ ^lS) 7 M-. ^^^^1 A1^^^ ^AJ] ^ Jl^^^ 8 (1) ^^^-# 8 (2) ti]^x^ ^e|> yflo^ 8 (3) MTT Assay 8 (4) ^^Ilil-^s:} #^ (lymphocyte proliferation test) 9 (5) ^*H^£} ^7££-3\ $AA (Hemolytic plaque cell forming assay) 9 (6) ^*H 4^^ ^^^-8-^1 f7oi-i4 %q 9 ^ 7}^^^ 10 (1) ^^^-# 10 (2) 4-g-Al^ 10 (3) LPS ^£ #^^ 11 (4) ^ ^^^1S^ £5] ^ -B-^l 11 (5) ^ ^r^^5. ^^#^1 ^f-i^ ^ 11 (6) til^-^-^ 2:1 ^^-^5. ^^£4 7^ 11 (8) cfl^^lS^l^ °^^S)-^4i ^^ #^ 12 (9) RT-PCR1^ 12 - xxxvii - (10) GM-CSF ^ IL-3 §«1 13 (12) yov4^i 24 4-T-^ tt^li afl0^ 13 (1) ^^^# 14 (2) uj-^^6]] c|] "6]- ^"A]->y 2: A]- 14 n^ ^s^i-rS- ABSAI^ is ( A\ til ^1"iJl ^-?- ^ All A^ --?] S^-"S^ A~| A| "5H 1 f-\ (5) i-o^-Q- ^115.-2] Apoptosis ^-^ 15 (6) D}--f-— s"ir-Aili# ol-§-?): i^]A]^ 16 \ (/ I i —- r* —i o —I T^ -*H" I T3 XO \U/ I I • O T— S. I T3 1U ^lvjy ^r 1|~5~"—I t) o —i o xi r J-O (2) •£!.§-£j t9"4/^ 2^1- 18 (3) ^]^^ Aj-^i AI^ 19 (1) (2) : (3) 7T--plA| 2:A|- -T^JI.^1 -^SljiL^ Oj-^-iA-l 7-1 ^ ig (1) Salmonella typhimurium 5"^# °1 -§•?}• "1" 91 ^ °] -cl Ac! Al ^ 20 (2) -TT£ <3^r OJ -&H 2Z (Q) tLjd-§1-x-1 7] 7I Jf ^ 22 (A) 7VplA:| ^-AV ^Jj^t^fl^l -2-t^^r- HPTP? 7*1 efc 99 I Tt / rj r -j I 1 I O —I n ~"^f I I it ^ ^r ^3 JL XJ. Jl ,^V ,y .^r 'w ^*j' • f if i - xxxviii - 4 2 ^ ^14 ^ 5Lzt 23 1. $n yoH7l^ ^ 7^Aj *\W$\ 7M& 7^ 23 1-1. « yoH7l^ ^ ^^fl 1H 23 7>. ^^ ^#iM ^A^ ^ 3^.2.3. ^-^£.4 ^ 23 (1) ^^^£€^2}- #^ ^ 23 (2) *)g.2\ 3:^^-§-AS. ^^^]i*^S)-^ AJ-TI4 ^-^1 23 (3) 7l^ ^^^-^l^l ^-MISL^SJ)- 14 ^-^1 23 (4) ^^jifl ^^ ^^1 S.4 7^ 24 M ^#fl ^ ^ ^ 37 (1) #^ #^li(stromal cells)S| afl«g= JE.^ ^-^ 37 (2) 2:171^ ^^1 ^f-:£^ ^^ ^ A] ^.3:^-0.5. AJ^I4 ^ 37 (3) tfl^^lss] O_]A>3}. ^^(Nitric oxide) ^^ ^tfl ^^ 71 AB 37 54 (1) ^Aj-Aj SA> 4-f^^A-l i^-^- ^^^7]. Ai^dbAfl %*S\ 54 (2) ^4^1 2:4 4-T-^iAi til^-^ 3:1^13. ^^"^^ ^7} +\^4i4 ^m ...54 (3) ^-g- ^]S^1 Apoptosis -B-^^^ll ^^i^fl ^An 54 1-2. ^^1 ycH7]^f ^^1 a^-^]^^ 7}^1§ 7fl^r 76 7}. ^ ^1/2:17}^ ^ ^ ^#7l^ i tij-X ^SL 2:^^]# S.#/JL^^ ^ 76 (1) ^M. S^-^^ ^^ 76 (2) ^iL a^-^#S] ig^Hli %^s± S-^Q^ 76 (3) ^il ^i^^-i-s] 2:17]^ ^^lS3]- ^ 76 (4) ^Ji 2:^-^-f-^ €#7i/ili yo^s4 ^^ 77 (1) -B-3L 2:-t^^5] ^^^-^^ ^^ *^S1- 14^^ 86 (2) -^-JL 2:^f-£] 3:171^ ^^ ^ S] 4 5-^^/7}^^^ 87 (3) •%-§. 2:tM^ ^#^^1 ^#7l^lS ifJ-sJLSl- ^^ 90 126 (1) 7>^fl# 2^ ^^ 126 (2) 7MH-3 ^"AjS-Aj ^A> 126 (3) ^>7l cf^] ^^7]]^ 127 - xxxix - 2. 7l^ ^€3L£l ^^Sfl- %% #*># 247] # 3-§- 7>^^ ^-iL 129 7K #*]-# oj-g- 71^ ^#€^(^M)^ -$-^^71^1 ^7fl 129 (1) *}5-^ 129 (2) *H# 41! ## ^ 129 ^^ 131 (1) #4^1 2:4 $.3-$ #^42. #^^ S4^1 °^^^ ^^ 131 (2) 7»A] £./.> ^g.o^ 2:l7]l/7fl^2:^ €#7l^5L yo^i^ °J^^ ^^-131 (3) ^"^^ 2:AJ- ^^.Sl ^ElJL^ ^^^ 7j^ 132 24 ^.s.^ -ft-#^^3 °Y#s$ 7j^ 156 (1) ^1^^#^°1 1-^€°1€^^: iL°)^l ^^-# ^ 156 (2) ^1S-^##^1 ^-#^15. ^^ f1^ ^^4 -ft-^l^l^l fev^# 7^^ 156 tf (1) xj-ojo) paeoniflorin ^^ f-^ 167 (2) ^-P}-^ 24 *]-<>}: O.^J=L3 ^e]*> paeoniflorin^ HPLCi ^*> °J^^ ^^ -167 (3) Paeoniflorin<>1 1-^^°1€^# Ji^l^l ^^-^ Ames tesH^i ^^ 167 (4) Paeoniflorin<>1 i^-ft-^:^^: iLo]^| ^^^- 7^^ 167 (5) ^^1^1 decursin 4 decursin angelate^ ^2] ^^ 168 (6) ^s] HPLC ^^ 2?1 ^^ 168 (7) #*Ri 24 ^^ -frJl^^-'y decursin4 decursinol angelate^l HPLC ^^ 168 4 ^ ^^-7fl^ s-5. ^^5. ^ c}]^ 7M5E. 179 ^1 1 ^ ^^71]^ ^-a ^^i£ 179 7-11 2 ^ cfl^ 7HE 181 5 # ^^711^:^4^ #-§-7fl^ 182 6 ^> ^-n§-«i 183 - xl - M 1947} t Roentgen °1 300 Gy) s]^ 31 ^^(10~30 Gy) ^7} A1-POT-§}711 si31, ^^^^(4-8 Gy) ^^7]^ Z\Z}% S,4t}7\] 5)O] 30°^ ^1^1 ^7} *}^}7]} 5)31, Gy) Patt -.(1)o] thiol t ^43^-^ thiol7l-& dipyridamole, adenosine monophosphate f- l ^ f ^^-t ^*>^ glucan interleukin-l# *]%$: ^^ 4^^1, ^ (granulocyte colony stimulating factor, GM-CSF)4 ^-^ 2:1 ^4(2-13). — 1 — $14. $14(14,15). $14(16-40). $14. $14(41-85). ^^ ^ si7] 7M ^^c] o^ ^oj^^. 444 A]ofl -2-^^. ^1^1-^ 4^4 ^^^ 71 ^S] - 2 - ^}] ^ ^ofl tfl*v cs[, ^^: -§"°flAi dipyridamole, adenosine monophosphate, tetracychlorodecaoxide, adeturon, deoxyspergualin f- glucan ^-^1 3|-«!-l:^4 interleukin-l# wl^-^ ^^ ^r^"^l, necrosis factor, TNF), z\-^^^^"^^#^1 ^1^1- (granulocyte colony stimulating factor, GM-CSF)£f ^^ 2l^]S ^^ -fKE.*ll, J:^^ f-^ $14(2-13). 1988\1 iti colony forming unit-spleen, endogenous A spleen colony assay, white blood cell count ^ 'HHT?-!-^ ^3:^ $^ik i§-i: ^] $14(11, 41-50). -^ 1986^44 3 - ^4534. 4^44 ^ (functional 1990^44 -l- 7 (86, 87). designer 44 ^c 4 E ^^1 ^4(86, 88). ^ A.D. Little4^ i# 1992\£ 100^1: 2000^i:e 1,000^ 1-, 20054 616^^, 365^ §14(89-91). 4 - 4 - *&*}#*] 31 6) designer foods^l 7>^ - 5 - 5U4. o) 4. "^^ 71 4. >]4. °H 44, 71 ^ - 6 - MI 1. (l) (2) 7]^ 4. ZL (3) - 7 - methanoKMeOH)-!: 7>sfl 7,500rpm EtOH ^^1S *>Sl-5-^ ^^i#^ D.W°fl (D ^ C57BL/6, BDFKC57BL/6XDBA/2) mouse 22±2°C, ^£^r 55~60%S -fMsM light and dark cycled 12^1?} (2) HI^-^S^- &?\, C57BL/6 4-fi« ^ ^^r^BJ HBSSofl 2s] ^^^^4. Trypan blue ^^^^.S. ^li^l- #^^r ^ afl^^ CO2-incubateri *\ afl ^§}^4(99). (3) MTT Assay l- 1X105 cells/well^ ^SS. 96-well microplatei 0 ^ CO2-incubateri^ 2^^- Bfl ^^^^. A *\%-% ^£^5L ^7}e>^ i«a^ A A°^ A% MTT dye(3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide)* 2.5mgM4 ^5-S ^7f^H 4^1^: ^°^ ^ acidic isopropanol(0.07 N HC1 in absolute isopropanol)^: ^7>e}^ ^-^^ dye* ^^^^ ELISA - 8 - reader^ ^#JE(OD570-OD630)f- #^H A 2xl05cells/well^ ^:ES 4#^^ 4 A1S# ^£^5- ^7}-SH MTT Assay *Wf 534(100, 101). (4) ^^AflSH-3^- #^ (lymphocyte proliferation test) S^-t 2xlO5/well^l ^£S 96 well microplatei ^-^§}JI A o -§- CO2-incubateH^ 2^, 3°^, 4°d ^Q «fl ^>55i4. wfl^ ^ well1^ 1.5/iCi^ 3H-thymidine^r 7>*> ^ 4^]# ^ yfl^^r^ji cell harvester# ^7^> §|cf. Scintillation cocktail^: 2.5 -scintillation counters. 3H-Thymidine incorporation ^5.# cpm^-S. -^^^ (101). (5) ^-^11^^^ ^^S.^ ^^(Hemolytic plaque cell forming assay) 0.25m£ 8 /mouse(25g)^] ^-70I-^A>§|-O|^I ^ 4^.^ SRBC 2x 10 7l]/mouse# ^^^4^>i 0.1 m^)4 20% SRBC 0.1 m£4 0.5% agarose 1.6m£«- ^l^^ti^ 41 ^^1 dishi pourft ^ 2^1^} CCVincubatoHH tifl^§}^4. 3. 4-§- 3i^l(GPC, 1/70)1- ^l^H 2^1 ^r plaque# > ^ fl §>$|4(101 ~ 104). (6) (7» ^^^l7i] ^^ cfl^- Afls^ ig*}«>-§-(Graft vs. Host Reaction) BDFl(C57BL/6XDBA/2) 4-fi» recipients. Al-g-s}^^^, graftfe C57BL/6 lxl077l]/0.2 me/moused 25]^^ .2 me/mouse)# - 9 - •>!]* 43- spleen index 4(105-107). all enic( spleen Spleen index °g ^ weight/body weight) syngeneic(spleen weight/body weight) Lymphocyte Reaction) C57BL/6 0.25 responder cellsS 4-§-*f^-^-^J stimulator cells^ DBA/2 5] til ^]5.-1- <£^ A|-g-^|-^c}. 4 responder cells^- stimulator cellsl- 96-well 5 flat bottom microplate^ 2X1O 7H^) ^JL 37 °C, 5% CO2 incubatori^ 4^^} «H oo^l-^4. ^r^>7l^[ 4^1 ^> ^^ 3H-thymidine(1.5 ^Ci/welD^- ^7}§]-^ afloj:^ ^4. ^^ $• cell-harvesterS *\]5.% ^r^*>^ scintillation cocktail^ "43. y3 -scintillation counter^ °]-o-'5'rci H-thymidine incorporation £-8- cpm-5-5L :^ A. (l) (2) RPMI 1640«l]^, FCS(fetal calf serum)4 antibiotic-Antimycotic^ GibcoBRL(Grand Island, NY, USA)sl4 ^^--i; A|-g-§}« o^, proteose peptone 4 LPSfe Difco Laboratories (Detroit, Michihan, USA)^4 ^^ 4-§-^-^4. NGMMA, 2ME(2-mercaptoethanol), sodium bicarbonate (NaHCO3), - 10 - N-1-naphthyl-ethylendiamine^- Sulfanilamide^ SigmaS)4 lift "S^-S-i *]--§-$ X\<$°- promegaSlAl- ^flf-^- (3) LPS &#*1 JZS^r lipopolysaccharide^ ^#^ 10 Amoeboyte lysate assay(E-TOXATE, sigma)# ° 4 amoebocyte lysate# 1 : IS. $\q^*\ ^]^^°fl ^JL, 3 LPS7]- (4) #^ ^ 20% FCS - RPMI1640 (5) (6) (7) - 11 - proteose peptone!- 2 m^ ^4*1-Jl PBS 10 (8) lipopolysaccharide(LPS)££^r 4^ *]^li-^ ^-^^ 4^ ^6> L-argininei ^ NO2, NO3, NO ^- ELISA reader^ #^ 96 well plate well^ 5xlO47fl^! ^<>] $4. IFN-7 4 LPS, ^7HU 48*] # ¥ B ELISA Titer Tek plated Griess Reagent(l:l, v/v, N-naphthylethylendiamine 0.1% in H2O, sulfanilamide 1% in 5% H3PO4)* ^7j-*fji 10^-^b 41^1 Ho^l^ ^ ELISA reader (Molecular Devicer)S NaNO2l- (9) RT-PCR'g ^$t^l] ^7>S -fKE£]fe- cytokine^: RT-PCR^# 4-§-«V^ #^§}$i4. RNAzol BCBiotecx lab)!- °1 -g-^>°^ total RNAt- ^^§>^, ^ z] 7}x] cytokinel-^ primer RT-PCR ^£ mRNA °^ #^ RNA1- 65°Ci^ 10^-?> ^ ^ 1A1^> aV-g-A]^ first strand cDNAt- ^-^t}^4. o] a>-g-«^^: 75°C, 10^ 71-^^H reverse transcriptase* lr%AjSl- ^ ^ PCR-I: #& template DNAS °1 -§-«!• ^4. °l template DNA4 Taq polymerase f-^] 5Ef-^. i£-g- ^f-^-i- 9 12 - 4°C 1C 55°C 302:, 72°C 1£^14 35cycle5L ^3 ethidium bromide(0.05 m/nl)7\ ^%7}Q 1.2% agarose geH (10) GM-CSF ^ IL-3 ^«1 3}-^ <£%<$ 4^-t GM-CSF , GM-CSF^r murine GM-CSF 10% FCS - RPMI1640 Hfl4i4 3°^ afl GM-CSFS Af^-§}^rf. tfl^^i ^ % 4§453# ) # , £v}t}±r M]3.^<& WEHI-3* 1.5% FCS - RPMI1640 wfl 5 (11) #^ >-f^ #^i^i ^^)£^. ^^*> tq-^-, 100 mm 6X10 "4JL, IL-3# 10%s}7]] ^7>tb 4-S-, GM-CSF^r S^ii 44 0, 5, 20% 44 1.8X105 7fl ^ji EPO(sigma)# 0, 300, 1000 (12) ^14^^ ^4^1 ^4^ 8 Gy4 #35.^1 4 Gy 4 €^^ - 13 - 4. ^, 2.3xiO67l] 2:47] (Gamma cell Elan 3000, Nordion International, Canada)!- Co r^(^l^^: 10.9 Gy/min)^: ^1^1 Gy), ^5.^(6.5 Gy) ^ ^ Gy) ^^, apoptosis (1) apoptosis (2) 2:A}7] (Gamma-cell Elan 3000, Nordion International, Canada) 60Co /^(AJ^^-: 10.9 Gy/min)^: ±^ -§- ^^A]^6flAife 12 Gy, j ^ #^A!^O]|A^ 6.5 Gy Ji apoptosis #^A!*HA]^ 2 GyS. 13j 1- #%*}-7l ^*H 8 Gy# 54^^JI, 5 Gyl- ^A] 3 Gy^i - 14 - (3) ^^-§1-^4. ICR 4-f^l" 4 ^ 64^ 36 ^ ^ 3.5^^1 (4) 4 ^ 36 ^ 12*1# ^di ^J-ifls. 2S| Bouin (5) i^^g- 43.$ Apoptosis # 36 ^ 12*R> ^i 47o^^ 231 Camoy's i^0J]i ul^*]?l ^ A ^ 4^ 44^ Sis}l ^.%% ^>1- - 15 - -M4 4314. 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Hi^ : #^^.^r, mean platelet volume, plateletcrit, platelet distribution width (8) Ficoll-Hypaque gradient^^^-S- ^i^l HBSSi ^r4 $• 15% heat inactivated fetal calf serum, L-glutamine, 2-mercaptoethanol3il- %>«7|- ^7}Q RPMI1640 ufl^l^ ^-^1^4. ^^^fe multi-well tissue culture plate(Corning, No. 25820, NY)# ^1"S-«H WH^] m^^ 5 5 xlO 7l)£] ^£S 37°C, 5% CO2 tffl^HH wfl^^}^^-^ phytohaemagglutinin (PHA, Sigma, MO, 25F-0640)^r polystylene tube(Corning, No. 25310-15, ^ PHA^7} ^^o]] 3 GyS] 60Co -^# 1090 cGy/min^ -id^^-S. IS) (Gammacell 3000 Elan, Nordion International Co., Canada)*B!4. 2:^-^1 10 ^ Cytokinesis-blocked method Cytochalasin B(Cyt-B, Aldrich Chemical Co.)-fe- dimethylsulphoxide°]l mi^ 2 >O]| afl^l m£^- 4 /zg ^ cytocentrifugel Diff-Quik -g-^^1 ^^*>^tf. 50% - 17 - Graph PAD In Plot program^ (9) n>-fi (10) 6*1-33 4-f^l- Aj-^-^^o,^ ^^^o^^^ ^cfl^^- n^ kg^- 10 . 50% 2. 71 (1) A] 2.1). (2) : 60Co, 10*1 CD-I: *l-§-«}^ ^^i^ A1^>^ 2 kGy^ ^^^S. 10 ^ ^ free radical dosimeter^ eerie cerous dosimeter# l§f4 - 18 - (3) *H# ^1^1- 34fe A A1S.o)l IH^ ^5^ 0.1% peptone ^# 7p<& ^ °-^ 4 -g-^H 3*1 aV^ ^=H4534. S.7]^ 3H5£ APHAS^IH 44 plate countagar(Difco laboratories, Detroit, MI, USA)* 4-§-«H 30°C°lH 1-2^3: afloj: ^ ^ Aj^si ^^-^. 7j|^§j.^o_^; ^£ ^ ^^-^1^ potato dextrose agar(Difco)!- 4-§-*H #5€ 10% tartaric acid^ pH# 3.5S S^t!: ^ ^^^ 6.5. 25OC^1^1 5-7°^^: *V&% $• ^]^4$I4. 3.2]5L ^^^^ desoxycholate agar(Difco)* ^l-§-«> pour plate method^ 37°C°114 1-2°^^ *V 4. (1) (2) 24 4-f^ ^r^ X (3) ^ (7» Salmonella typhimurium 3-^» benzo( a)pyrene# ^7r* 244^4(109-113) - 19 CHO CHO benzo( a ^ 2:44534(114-115) (4) 500 ^Jl 250 mi$] ^^ ^^^^ ^^^^^ DPPH 16mg^- 100 m^ absolute ethanoHl -§-*fl«t :f ^^-^ 100 m£# 44^- filter paper(No. 1, Whatman)^ ^44^4. °] ^^ 5ml1 ^4^fl ^##1 mm 44 ^ vortex mixerS 30 44 B 4. (1) Salmonella typhimurium 5^# ^1^^ ^14 Hfl^l, ^14 ^ S9 mix^l 2:^4 ^^ Marton & Ames^l - 20 - ^ (109, 110W 4^4. A11H 4-§-?F S^fe Salmonella typhimurium TA98, TA100, TA102 °1&A^ A ^$\ histidine $L={->& ^ -fj-^*^ ^91^ %• ^liH 4-§-*>^4. tflA]-%^^.A]?l^ &fe 3-fo] A]^|°- standard plate incorporation tests., ^A^^SM^lfe ^-fofl-fe- pouring^[7] *H 30£-# ^wjafl ^•sl-fe preincubation tests *l*3s>&4. ^lH^H Sl^f-g-^ 0.5 mi (tflA>%^ ^1^1 fe ^-T-^fe S9 mixdll, 112) 0.5 mi, ^l^-§-^ o.l ml 4 Oxoid nutrient brotholH 12A1^> tifl^Al^l ^tifl^ofl 01 mij^ ,go] y}^7\} vortex*}S^. tflA}^ ^^ ^^ ^^-ife «>S (£fl4#^SM7l^ ^-f^l^r 30^:?> 37°CiA-] ^ 4^-) histidine/biotin^»1 ^7}sl top agar(45°C)# 2 m^ 7}%}5L 33,1} vortex*}^ minimal glucose agar plate ^"i ^- ^ revertant colony# ^}^}^4(113). (2) W A]^oi] A}^-^ ^^.^.^-AflSfe- Chinese hamster ovary (CHO) AHi<>l^^.^. «]] ^l^r 10% fetal bovine serum ¥# ^7>A]?1 RPMI 1640 n*\S. 5% CO2# ^^ 4 *>^r 37M CO2 Flaskette(19.8x51.8 mm, NuncHl cytochalasin B(Cyt-B; 3 -i: ^^^^-(Fenech and Morley(114)^l cytokinesis-block method). 75 mM KC1 -g-^# ^sl*}^ ^l£# ^^1?1 4-§- ^^ ^ (methanol : acetic acid, 3:1)AS JI^A]^ ^ ^71^2:^ 0.5. ^§^4. ^IIM 3% Giemsa ^ 6.5)AS °J^*H ^-^-^^1^AS ^:#*}il4. ^A>#^ ^^*}^1 A]^ ^, A|^^^3f S9 mix(tifl^si 20% ^l-ir)* ^ -^itr till^lS 51^:*>Jl Cyt-Bl- ^7>*>^ 18A] (1) - 21 - (2) ^A]§}JI column chromatography^ (3) 7l7l ^-^['H-NMR ^ 13C-NMR^- Bruker DRX 300 NMR spectrometer(300MHz, 7 ^£1- ^«H HPLC [Waters^ Shimazu]^-^ ^7A^ A (4) - 22 - 1. 1-1. 7\. ^f (l) rg-^ ^ 1000~2000 1000-2000 ^g/m£, ^.71-31]^r 300 2000-4000 300 tr 500-1000 fig/mi °1^4(Table 1.1). (2) 6 7M^ 1.2, Table 1.3). (3) 1.4). - 23 - (4) ^l^^H ^<^Ml£ *^2f M# iL°l^r i^i SRBCi (7V) 1.5, Fig. 1.1, Table 1.6, Fig. 1.2, Table 1.7, Fig. 1.3). 1.8, Table 1.9). - 24 - Table 1.1. Lymphocyte proliferation by water extracts of Korean medicinal herbs Concentration cpm Sample Day 2 Day 3 Day 4 Negative Control 4176 ± 310 3927 ± 463 1934 ± 227 4000 16443 ± 263 80194 ± 2718 98724 ± 14946 2000 35531 ± 3886 133752 ± 7188 89084 ± 9474 Angelica gigas Nakai 1000 53894 ± 3502 137322 ± 1485 83335 ± 8563 500 44003 ± 1447 106311 ± 7222 72747 ± 999 250 34173 ± 717 53977 ± 9019 56731 ± 7385 4000 21809 ± 1276 82618 ± 7957 61353 ± 4797 2000 31057 ± 2503 84790 ± 2280 78523 ± 11090 Cnidium officinale 1000 38036 ± 1769 82421 ± 4828 71807 ± 8175 500 32956 ± 1601 63556 ± 3233 62795 ± 5214 250 24907 ± 665 37266 ± 4040 29605 ± 2107 600 8165 ± 302 9415 ± 1490 6888 ± 1681 Paeonia japonica Miyabe 300 10231 ± 303 9069 ± 1460 6927 ± 700 150 8320 ± 385 6792 ± 975 4967 ± 398 600 20586 ± 755 38589 ± 2688 33612 ± 4005 Acanthopanax sessiliflorum 300 36732 ± 1714 88694 ± 5865 84553 ± 8608 150 32120 ± 2488 61646 ± 3519 40795 ± 5481 4000 21019 ± 2937 65616 ± 7256 83247 ± 6235 Houttuynia cordata 2000 24130 ± 1648 47957 ± 3553 50679 ± 6083 Thunberg 1000 22409 ± 146 31851 ± 1983 30640 ± 1975 500 15594 ± 347 18778 ± 1587 18600 ± 4106 1000 8976 ± 469 10525 ± 1429 8360 ± 2 Crataegus pinnatifida 500 9176 ± 519 9658 ± 861 5916 ± 98 Bunge 250 8086 ± 1184 5557 ± 90 5452 ± 1508 125 6085 ± 282 5173 ± 807 3080 ± 390 Positive Control PHA 2 81707 ± 4528 109975 ± 15272 57713 ± 16464 LPS 20 204040 ± 7976 126492 ± 3687 26687 ± 3729 - 25 - Table 1.2. Lymphocyte proliferation by combination of water extracts from herbs, KJ-1, KJ-2, KJ-3, KJ-4, KJ-5 and KJ-6, in 3H-thymidine uptake assays Concentration cpm Sample (mg/m-O Day 2 Day 3 KJ-1 1 + 1 89010 ± 7810 243923 ± 6075 0.6 + 0.6 253372 ± 2918 167923 ± 474 0.3 + 0.3 196753 ± 7394 107176 ± 1544 0.15 + 0.15 144051 ± 16408 150219 ± 7107 KJ-2 1 + 1 52569 ± 5183 166432 ± 11645 0.6 + 0.6 199619 ± 4451 247817 ± 1544 0.3 + 0.3 127662 ± 6863 148224 ± 17828 0.15 + 0.15 70358 ± 3091 51712 ± 6385 KJ-3 1 + 0.5 83285 ± 7613 181601 ± 13078 0.6 + 0.24 208157 ± 5331 176298 ± 3604 0.3 + 0.12 138389 ± 7288 140542 ± 2185 0.15 + 0.06 97134 ± 3091 95442 ± 62 2 + 0.3 16137 ± 9392 21697 ± 4296 1 + 0.15 102450 ± 1117 141661 ± 4719 0.5 + 0.075 130237 ± 1883 148716 ± 14986 0.25 + 0.0375 110320 ± 2844 134710 ± 4456 0.125 + 0.01875 76321 ± 4712 91176 ± 10561 KJ-5 1 + 0.5 60690 ± 5288 94157 ± 6813 0.6 + 0.24 145674 ± 435 180215 ± 6182 0.3 + 0.12 102889 ± 8912 112671 ± 6288 0.15 + 0.06 79981 ± 2629 79316 ± 1148 KJ-6 1 + 1 + 0.5 74377 ± 2171 194373 ± 13043 0.6 + 0.6 + 0.24 228840 ± 20243 156681 ± 10082 0.3 + 0.3 + 0.12 190142 ± 9919 174287 ± 504 0.15 + 0.15 + 0.06 148258 ± 7701 124252 ± 12660 Cell 4836 ± 425 2846 ± 256 - 26 - Table 1.3. Lymphocyte proliferation by combination of water extracts from herbs, KJ-6 + El or E9 or El + E9, in 3H-thymidine uptake assays cpm Sample Concentration Day 2 Day 3 KJ-6 + El + 0.096 1567 ± 124 1309 ± 810 + 0.048 37803 ± 431 74921 ± 12348 + 0.024 89126 ± 6946 295745 ± 8734 + 0.012 147320 ± 4443 384944 ± 11683 + 0.006 261187 ± 2696 411186 ± 34308 + 0.003 241834 ± 23906 415115 ± 29333 + 0.0015 239822 ± 2121 422010 ± 975 + 0.0007£ 245313 ± 1610 413695 ± 6582 + 0.000375 246516 ± 6206 398840 ± 20934 KJ-6 + E9 + 1 8528 ± 2432 9032 ± 1913 + 0.5 53809 ± 2541 158201 ± 6744 + 0.4 130603 ± 2485 339654 ± 19723 + 0.2 200361 ± 1160 389537 ± 14412 + 0.1 222446 ± 21361 372476 ± 30099 + 0.05 241700 ± 7944 380232 ± 3446 + 0.025 247257 ± 18514 382613 ± 30327 KJ-6 + El + E9 + 0.006 +• 0.4 131238 ± 8792 344643 ± 18915 + 0.2 213304 ± 12323 382203 ± 4045 + 0.1 235477 ± 5228 363332 ± 30103 + 0.05 246947 ± 8321 359717 ± 5756 KJ-6 + El + E9 + 0.003 + 0.4 118761 ± 3973 316041 ± 15113 + 0.2 203306 ± 7223 366163 ± 8854 + 0.1 232771 ± 10005 372993 ± 15306 + 0.05 250588 ± 1515 350545 ± 45947 KJ-6 + El + E9 + 0.0015 + 0.4 136669 ± 9617 341612 ± 16998 + 0.2 195224 ± 9749 376687 ± 18975 + 0.1 226251 ± 5375 349780 ± 44592 + 0.05 241524 ± 316 391264 ± 25191 KJ-6 + El + E9 + 0.00075 + 0.4 128743 ± 5312 328386 ± 622 + 0.2 189851 ± 16756 352408 ± 44690 + 0.1 230391 ± 3004 380919 ± 11073 + 0.05 246435 ± 5433 377206 ± 22438 Cell 3896 ± 468 2890 ± 119 KJ-6 : 0.6 mg + 0.6 mg + 0.24 mg - 27 - Table 1.4. Lymphocyte proliferation by water extracts of several prescriptions of Oriental medicine in H-thymidine uptake assays Concentration ^H-thymidine uptake(cpm) Sample (mg/ni-d) Day 2 Day 3 Medium Control 4338 ± 829 2444 ± 293 Si-Wu-Tang 2 1404 ± 214 451 ± 91 0.4 2843 ± 314 4185 ± 191 0.08 6016 ± 151** 5644 ± 659** 0.016 2828 ± 1828 2894 ± 272 0.003 2277 ± 56 1870 ± 144 Si-Jun-Zi-Tang 2 489 ± 48 181 ± 71 0.4 9519 ± 159** 10942 ± 682** 0.08 7890 ± 170 8734 ± 526 0.016 4484 ± 53 3327 ± 373 0.003 2064 ± 235 2011 ± 652 Bu-Zhong- Yi~ Qi~ Tang 2 397 ± 150 190 ± 50 0.4 5743 ± 200 11475 ± 330 0.08 9927 ± 2149** 15016 ± 109** 0.016 8389 ± 1410 8810 ± 634 0.003 3911 ± 219 3188 ± 416 San-Ling-Bai-Shu-San 2 736 ± 303 364 ± 115 0.4 12514 ± 1377** 15530 ± 508** 0.08 9638 ± 299 8439 ± 86 0.016 4150 ± 11 3923 ± 673 0.003 2910 ± 567 2238 ± 139 Shi- Quan-Dai-Bu-Tang 2 468 ± 6 163 ± 39 0.4 4557 ± 883 4955 ± 155 0.08 6610 ± 305** 3509 ± 2747 0.016 4030 ± 327 2909 ± 279 0.003 2286 ± 237 2305 ± 530 ** P<0.01, as compared with medium control. - 28 - Table 1.5. Effect of Angelica gigas on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage Sample Splenocyte/spleen No. of plaque/7 x 10° splenocyte PFC/spleen (rag/kgB.W.) 2.0xi077H 60, 102, 65 ( 75.7) 2,163 3.7X10'7fl 9, 18, :>0 (15.7) 830 Saline 4.9 x lO77fl 68, 69, 44 (60.3) 4,221 6.2xiO67fl 88 96, 89 (91) 806 4.4xiO77fl 93, 99, 113 (101.7) 6,393 8.2 x lO77fl 151, 128, 135 (139.5) 16,166 Angelica gigas 20 5.7 x lO77fl 97, 101, 81 (93) 7,573 4.8xiO77l] 53, 60, 53 (55.3) 3,792 12000 10000 - - -C/splee n 4000 - 2000 u Control Angelica gigas Fig 1.1. Effect of Angelica gigas on the secretion of antibody tested by hemolytic plaque forming cell assay (M±S.E.) - 30 - Table 1.6. Effect of KJ-1 on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage Sample Splenocyte/spleen No. of plaque/7 x 10° splenocyte PFC/spleen (mg/kgB.W.) 1.3 x 107 cell/m£ 14, 17, 19 (16.7) 311 3.2 x 107 cell/roe 69, 52, 64 (61.7) 2,820 4.6 x 107 cell/me 26, 45, 42 (37.7) 2,477 Saline 8.5 x 107 cell/me 32, 34, 36 (34) 4,128 5 x 107 cell/me 73, 71, 63 (69) 4,927 5.7 x 107 cell/m£ 44, 52, 38 (44.67) 3,638 1.4 x 108 cell/m£ 54, 50, 50 (51.3) 10,260 7.5 x 107 cell/m« 49, 58, 46 (51) 5,457 1.1 x 108 cell/m« 69, 48, 58 (58.3) 9,159 KJ-1 120 + 120 9.7 x 107 cell/m£ 36, 34, 44 (38) 5,266 9.3 x 107 cell/ntf 56, 56, 56 (56) 7,776 9.2 x 107 cell/me 54, 44, 60 (56.67) 7,448 - 31 - 9000 8000 T 7000 c 6000 CO •| 5000 O 4000 T °~ 3000 I 2000 1000 0 Control KJ-1 Fig 1.2. Effect of KJ-1 on the secretion of antibody tested by hemolytic plaque forming cell assay (M±S.E.) - 32 - Table 1.7. Effect of KJ-2 on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage Sample Splenocyte/spleen No. of plaque/7 x 10 splenocyte PFC/spleen (mg/kgB.W.) 4.6 X 107 cells/m-0 75, 51, 62 (62.7) 4,118 8.6 X 107 cells/mi 32, 29, 30 (30.3) 3,726 7.5 X 107 cells/m£ 11, 25, 23 (19.7) 2,110 7.6 X 107 cells/m£ 23, 18, 24 (21.7) 2,357 Saline 8.1 X 107 cells/M 35, 34, 28 (32.3) 3,737 9.8 X 107 cells/m£ 21, 23, 26 (23.3) 3,262 7.0 X 107 cells/m£ 27, 39, 17 (27.7) 2,770 5.7 X 107 cells/mi 27, 31, 28 (28.7) 2,336 1.2 X 108 cells/m£ 65, 45, 56 (55.3) 9,485 1.1 X 108 cells/m-2 133, 102, 100 (111.7) 17,548 7.5 X 107 cells/m-K 44, 48, 47 (46.3) 4,959 1.1 X 108 cells/m-K 45, 56, 44 (48.3) 7,313 KJ-2 120 + 26 7.3 X 107 cells/m£ 70, 69, 64 (67.7) 7,061 6.4 X 107 cells/m£ 47, 45, 52 (48) 4,387 7 9.4 X 10 cells/m^ 59, 36, 30 (41.7) 5,600 1.3 X 108 cells/ing 33, 43, 44 (40) 7,428 - 33 - 10000 8000 - g 6000 - U 4000 - 2000 - Control KJ-2 Fig 1.3. Effect of KJ-2 on the secretion of antibody tested by hemolytic plaque forming cell assay (M±S.E.) - 34 - Table 1.8. Effect of Angelica gigas on Graft vs. Host reaction Group Donor Recipient Sex spleen w.t./body w.t.(xlOs) Spleen index M 2.333 ± 0.233 1.00 Saline BDF1 BDF1 F 3.413 ± 0.434 1.00 M 4.595 ± 0.714 1.970 Saline C57BL/6 BDF1 F 4.227 ± 0.180 1.238 M 7.633 ± 1.362 3.271" 10mo0, mg/k/ID*g B.wt.. C57BL/6 BDF1 F 8.673 ± 1.577 2.541* * p<0.05 as compared with control. ** p<0.01 as compared with control. - 35 - Table 1.9. Effect of KJ-1 on Graft vs. Host reaction spleen w.t./body Group Donor Recipient Sex Spleen index w.t.(xKT3) M 3.613 ± 0.076 l.C Saline BDF1 BDF1 4.200 ± 0.849 1.00 M 4.243 ± 0.517 1.1743 Saline C57BL/6 BDF1 4.890 ± 0.451 1.1543 M 7.403 ± 0.536 2.0489" KJ-1 C57BL/6 BDF1 (60 mg/kg B.wt.) 7.707 ± 0.327 1.8349* ** p<0.01 as compared with control. - 36 - 4. (1) #^r #^1S(stromal cells)^ «!)<$ #^MlS(bone marrow stem cells) 3 f^ (stromal cells)^ ^#4-§-°l ^fl-f T!^4^ ^4^31 &4U16). £5}-4 ^Hl lS«> ^V^^lSl- ^-7]^} Bfl^-t ^ SJfeSJ - ig. 1.4). (2) 7H ig. 1.5A, > l, 87M 1.6A, 1. 50% (3) iflAMlS^ "i^^- €i(Nitric oxide) -2.5-fe NO'^-i: -H-S^H ^4534 ^, IFN-r \JL, iNOS & - 37 - 4. att o]^ ,$££• 42.^7} 44 47 (7\) 10, 4. °m^5L ^7>5] ^4(Fig. 1.7A). ^H] cfl^^lS^ NCHJ-^-i: $SL*}^ ^A^-S. <£$& LPS(121, 122)5]- 0_7>S]l- *^j ^5l^}5|l- ufl NOS^o] ^71-^fe^l- ^oj-ioj-^^, ZL £.7l- 10 IFN-7* IFN 2.7} 100 fig/mi °i «fl ^^1 ^-4i«l-fe 7A±S- M-B})ot4(Fig. 1.7C). 44^i IFN-/ 4 LPSi ^1^1 ^^r*l - (4) 4-§--5.S. NO^^i 4^]^r IFN- r IFN-rl- #^-°-3. ^el«]-5i^ nfl ^4 i7>3i|i " =L I4fe C] 3:71] 444^, SL 100 ^g/ltl£^]^rr IFN-rt" #^°-S- ^B]§}^^; nfl il4 NO2"^£7> 4 10 S«V IFN-7 4 2-7}*\7} 14 -f^ IFN-7 7} 100 , 10, 100 }^ - 38 - &, ]]^ ^} 100 //g/M(££: ^^ *fl ) H ^ IFN" , 10, 100 nfl 1.9). ig. 1.10) n-efl LPS^l -Sltt ^4 °^ ^£ il7l nfl^4f xg-^ #^##i LPS7J- 4444 ^$J:^-^, ^71-4^ ^5L7> ^OJ-S. LPS ^ NO2" ^£iL4 -2-^1^ 4^4^ ^^-1: jl 3l^a]o_S ^.71-Sl #^##o]j tfl^V LPS Amebocyte lysate assay(E-TOXATE, sigma)# ^1^ #^ *M1*]?1 10 pg/m£ °14^ LPS7]- IFN-7 ^]«v ^6] 4^ ^^s ^4^4. 4^s NO NGMMAl- ^e]s}-8-i- 14 NGMMA^] ^£7} ^^r^s. IFN-7 4 ?A°-£- 444 IFN-7 4 (ef) tfl^^S.51 iNOS mRNA tfl^^S^fl IFN-74 $L7\V\, ^, , , A /8$$%7) n&*\), °|«Hfe oa^}Sl-Ui* ^^4^r Ii6J iNOSQnduceable nitric oxide synthease)^] mRNA^-^^r RT-PCR^AS. ^4^4(125). o. 1 - 39 - , IFN-/4 ^*fl* ig. 1.12). ^, (4) mRNA ^^# 2:4^^4(126, 127). a ^4 ^2^1 o)^f^ IL-1/?, BL-6, LT ig. 1.13). , IFN-7 ig. 1.14). - 40 - 1.1. Morphology of stromal cells derived from mouse bone marrow cells. The bone marrow cells (lxl()" cel]s/2:nP) from BALB/c mice were cultured for 14 days in 10% PCS RPMIIOK) medium. Effect of various plant extracts on stromal cell growth 100 90 80 70 o o X 60 CO _a 50 ZE3 C el l 40 o 30 20 10 0 0 1 10 100 1000 Concentrations, |ug/ml Fig. 1.5A. Effect of various plant extracts on stromal cell growth. The bone marrow cells (1 x 106 cells/mO from BALB/c mice were cultured for 14 days in 10% FCS-RPMI1640 medium contained with various plant extracts at 37 °C, 5% CO2 and their viable cells were counted by try pan blue staining. - 42 - 100 60 40 20 1 10 100 1000 100 80 60 40 20 ^r 0 _x 0 1 10 100 1000 Fig. 1.5B. Effect of various plant extracts on stromal cell growth. (Cell numbers, X1000) - 43 - Fig. 1.6A. Synegistic effect of combination of plant extracts on stromal cell growth. The bone marrow cellsQxlO6 cells/m£) from BALB/c mice were cultured for 14 days in 10% FCS-RPMI1640 medium contained with each optimal growth concentration of various plant extracts and their viable cells were counted by trypan blue staining. - 44 - 300 250 o |200 150 E i 100 CD o 50 V \? \P \P \P \? NiP V ^ip V Plant Extracts Fig. 1.6B. Synergistic effect of combination of plant extracts on stromal cell growth. The bone marrow cellsQxlO6 cells/inO from BALB/c mice were cultured for 14 days in 10% FCS-RPMI1640 medium contained with each optimal growth concentration of various plant extracts and their viable cells were counted by trypan blue staining. - 45 - 3 NO2 concentration, /*M trq o o I- O CD o «Q O "; 3 91 n> •-« "i NO2 concentration, O o •^e si It (/) NO2 concentration, o o CD § o ^ CD |O 3X CO B.-HR A MRhy, lOLKrrl 2D 1 -•-27H1 /; k -•-27H100 / / J I a. 15 SS. 15 - / // i ion , ion , f / co ra t / / 10 10 1/ \ concen t concen t / i i CM CM o 5 o 5 7 | : i 0 o 7 (D 1 10 100 0 1 10 100 i/Vater-extract concentration, IFN-7, LJ/ml \&j/rml Fig. 1.8. Dose-dependent effect of Acanthopanax sessiliflorum extract on NO synthesis in IFN- 7 -treated RAW 264.7 cells. The cells (5X104 cells/well) were cultured with various concentrations of IFN- y. After 48 hrs of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 47 - 1 10U/ml 15 c o V-» +-> 10 O o o oCM z •—• 1 10 100 Water-extract concentration, (ig/ml Fig. 1.9. Effect of various herbal plant extracts on NO synthesis in IFN- / treated RAW 264.7 cells. The cells (5 x 104 cells/well) were cultured with various herbal plant extracts in the presence of IFN- 7 (10 U/ M). After 48 hrs of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 48 - A. §9i 20 3. / e n ratio n T icen t i' / 8 10 / o'CM z: / 5 ; If 0 ( () 1 1C) 100 IFN-y, U/rri Fig. 1.10. Dose-dependent effect of various herbal plant extracts on NO synthesis in IFN- r treated RAW 264.7 cells. The cells(5xlO4 cells/well) were cultured with various concentrations of herbal plant extracts. After 48 hrs of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 49 - Fig. 1.11. Effect of Acanthopanax sessiliflorum extract and NOS inhibitors on nitrite accumulation in the cultured medium of RAW 264.7 cells. The cells(5xlO4 cells/well) were cultured with various concen- trations of Acanthopanax sessiliflorum extract or NGMMA. After 48 hrs of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 50 Marker Control IFN+^l IFN+£.7]-3) IFN+%?1 IFN- iNOS r-* • HPRT Fig. 1.12. RT-PCR analysis of iNOS mRNA expression in macrophage cell line. Total RNA was reverse transcribed to cDNA. Equivalent amounts of cDNA were amplified by PCR. PCR products were electrophoresed through 2% agarose gel containing ethidium bromid - 51 - Marker IL-1/3 IL-2 IL-4 [L-6 IL-10 11,-12 Maker LT IFN-r 1M-a IGIF iNOS HPRT Control Fig. 1.13. RT-PCR analysis of IL-10, IL-2, IL-4, IL-6, IL-10, IL-12, LT, IFN-7, TNF-a, IGIF and iNOS mRNA expression in stromal cells. Total RNA was reverse transcribed to cDNA. Equivalent amounts of cDNA were amplified by PCR. PCR products were electrophoresed through 2% agarose gel containing ethidium bromid. - n'P. - primary macrophage Fig. 1.14. Effect of various herbal plant extracts on NO synthesis in IFN- y treated mouse peritoneal macrophages. The cells(5xlO4 cells/well) were cultured with various herbal plant extracts(lOOjug/m-0 for 48 hrs in the presence or absence of IFN- 7. After 48 hrs of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 53 - tfl--gg- Gy), ^^£^1^(6.5 Gy) ^ ^i %& 4S\ apoptosis (1) (Fig. 1.15, Table 1.10-1.15) (2) ^"A>^ 24 4-0.^0114 Hl^-lfl 21 Alii ^^^^ ^7} ^j ; o). Hfe °J^, 3§-7l, ^^, Xlfe S41- M-Bf^4 (Fig. 1.16, Table 1.16-1.21) (3) db^-g- >H1S^ Apoptosis ^1^^-S -n-^^$l^r apoptosis 1k*& ^^lJl41- M-^-^14 (Fig. 1.17, Table 1.2 2-1.27). 54 - • •,'•*:• -', %• Ki"'-^i"' A B Fig. 1.15. Photomicrograph of transverse sections of mouse jejunum. (A) Irradiation control. At 3.5 days after exposure to gamma-rays of 12 Gy, mouse jejunum was collected, and stained with H-E solution(x40). (B) Treatment group. Jejunum collected from mouse treated with effective prescription or ingredient before or after irradiation(12 Gy). The number of surviving crypts was significantly increased comparing with those in the irradiation control. - 55 - Table 1.10. Effect of Si-Wu-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.25 ± 6.05 Irradiation control (12 Gy) 38.48 ± 4.34 Si-Wu-Tang(l mg/head, twice I.P. at 36 and 77.98 i 16.10 12 hrs. before irradiation) + irradiation Untreated control 157.00 ± 14.82 Irradiation control 21.82 ± 12.31 ShoudehuangQ mg/head, twice I.P. at 36 and 31.34 ± l./b 12 hrs. before irradiation) +irradiation Chuanxiongd mg/head, twice I.P. at 36 and Z.bl ± zz.01 12 hrs. before irradiation)+irradiation Dangguid mg/head, twice I.P. at 36 and 12 hrs. before irradiation)+irradiation Baishaoyaod mg/head, twice I.P. at 36 and 49.05 ± 12.64 12 hrs. before irradiation)+irradiation *p<0.0005 as compared with irradiation control group. **p<0.001 as compared with irradiation control group. ***p<0.005 as compared with irradiation control group. - 56 - Table 1.11. Effect of Si-Jun-Zi-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.25 ± 6.05 Irradiation control(12 Gy) 38.48 ± 4.34 Si-Jun-Zi-Tang(l mg/head, twice I.P. at 36 and 27.40 ± 6.12 12 hrs. before irradiation) + irradiation Untreated control 163.10 ± 12.34 Irradiation control 19.67 ± 6.09 RensanQ mg/head, twice I.P. at 36 and „ 37.80 ± 10.13 12 hrs. before irradiation) + irradiation Gancaod mg/head, twice I.P. at 36 and 59.78 ± 26.49 12 hrs. before irradiation) + irradiation BaifulingQ mg/head, twice I.P. at 36 and 50.38 ± 9.75 12 hrs. before irradiation) + irradiation BaizhuQ mg/head, twice I.P. at 36 and „„ „ , 26.63 ± 8.64 12 hrs. before irradiation) + irradiation *p<0.0001 as compared with irradiation control group. **p<0.005 as compared with irradiation control group - 57 - Table 1.12. Effect of Shi-Quan-Dai-Bu-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.25 ± 6.05 Irradiation control 38.48 ± 4.34 Shi-Quan-Dai-Bu-Tang (lmg/head, twice I.P. at 37.62 i 8.10 36 and 12 hrs. before irradiation) + irradiation Untreated control 157.00 ± 14.82 Irradiation control 21.82 ± 12.31 Shoudehuang (1 mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + Irradiation Chuanxiong (1 mg/head, twice I.P. at 36 and 12 hrs. * 42.67 ± 22.01 before irradiation) + Irradiation Danggui (1 mg/head, twice I.P. at 36 and 12 hrs. . . , T . 50.54 ± 6.09 before irradiation) + Irradiation Baishaoyao (1 mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + Irradiation Rensan (1 mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + Irradiation Gancao (1 mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + Irradiation Baifuling (1 mg/head, twice I.P. at 36 and 12 hrs. 04.62 ± 3.75 before irradiation) + Irradiation Baizhu (1 mg/head, twice I.P. at 36 and 12 hrs. _^ before irradiation) + Irradiation Huagqui (1 mg/head, twice I.P. at 36 and 12 hrs. „.„ , ,ncr ^o.oo — ib.ob before irradiation) + Irradiation Rougui (1 mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + Irradiation p<0.005 as compared with irradiation control group. p<0.001 as compared with irradiation control group. p<0.0001 as compared with irradiation control group. Table 1.13. Effect of Bu-Zhong-Yi-Qi-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.25 ± 6.05 Irradiation control (12 Gy) 38.48 ± 4.34 Bu-Zhong-Yi-Qi-Tang (ling/head, twice I.P. at 36 and 12 hrs. before irradiation) + irradiation 64.72 ± 4.23* Untreated control 157.00 ± 14.81 Irradiation control (12 Gy) 19.67 ± 6.09 Rensand mg/head, twice IP. at 36 and 12 hrs. before 41.94 ± 4.79** irradiation) + irradiation Gancaod mg/head, twice I.P. at 36 and 12 hrs. before 61.96 ± 24.26" irradiation) + irradiation DangguiQ mg/head, twice I.P. at 36 and 12 hrs. before 54.90 ± 7.87*** irradiation) + irradiation BaizhuQmg/head, twice I.P. at 36 and 12 hrs. before 31.52 ± 7.97 irradiation) + irradiation HuangqiQ mg/head, twice I.P. at 36 and 12 hrs. before 26.70 ± 15.56 irradiation) + irradiation Chenpid mg/head, twice I.P. at 36 and 12 hrs. before 30.45 ± 13.39 irradiation) + irradiation Shengmad mg/head, twice I.P. at 36 and 12 hrs. before 40.96 ± 4.96**** Irradiation) + irradiation Chaihud mg/head, twice I.P. at 36 and 12 hrs. before 35.30 ± 6.54**** irradiation) + irradiation *p<0.0001 as compared with irradiation control group. **p<0.005 as compared with irradiation control group. ***p<0.0005 as compared with irradiation control group **"p<0.05 as compared with irradiation control group. - 59 - Table 1.14. Effect of San-Ling-Bai-Shu-San and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.25 ± 6.05 Irradiation control (12 Gy) 38.48 ± 4.34 San-Ling-Bai-Shu-Sand mg/head, twice IP. at 36 47.80 ± 11.16 and 12 hrs. before irradiation) + irradiation Untreated control 164.24 ± 8.31 Irradiated control 19.64 ± 6.09 Rensen (1 mg/head, twice I.P. at 36 and 12 hrs. OT on -*- mio** , . . ,...... 37.80 — 10.lo before ltradiationj+irradiation Baizhud mg/head, twice I.P. at 36 and 12 hrs. „„ _» , „„„ 26.63 ± 8.64 before irradiationj+irradiation Fulingd mg/head, twice I.P. at 36 and 12 hrs. »* before irradiation)+irradiation Gancaod mg/head, twice I.P. at 36 and 12 hrs. ». . . . •,. ... \ • ,. ... 59.78 ± 26.49 before irradiationj+irradiation Yiyirend mg/head, twice I.P. at 36 and 12 hrs. oncr. , ,_„._ 3O.b4 3: lb.2b before irradiation)+irradiation Baibiandoud mg/head, twice I.P. at 36 and 12 hrs. , before irradiation)+irradiation Shanyaod mg/head, twice I.P. at 36 and 12 hrs. „„„ , ,. . ... 61.15 3: lc5.o1OOUA before irradiation j+irradiation Lianroud mg/head, twice I.P. at 36 and 12 hrs. .„ „ , n_ „„ 42. U5 — 2b.o6 before irradiation)+irradiation Sharend mg/head, twice I.P. at 36 and 12 hrs. „ before irradiation)+irradiation Jiegengd nig/head, twice I.P. at 36 and 12 hrs. 4707 + iQfifi** before irradiation)+irradiation *p<0.05 as compared with irradiated control group. **p<0-005 as compared with irradiated control group. 60 - Table 1.15. Effect of Kuei-Pi-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.25 ± 6.05 Irradiation control (12 Gy) 38.48 ± 4.34 Kuei-Pi-Tang(l mg/head, twice I.P.at 36 and 12 hrs. 62.13 ± 14.66 Before irradiation) + irradiation Untreated control 164.24 ± 8.31 Iiradiation control(12 Gy) 19.64 ± 6.09 Dangguid mg/head, twice I.P. at 36 and 12 hrs. „, before irradiation)+iiradiation LongyanrouQ mg/head, twice IP. at 36 and 12 hrs. Z t.Zo — 6.98 before irradiationj+iiradiation Suanzaorend mg/head, twice I.P. at 36 and 12 hrs. „„ before irradiationj+iiradiation Yuanzhid mg/head, twice I.P. at 36 and 12 hrs. before irradiationj+iiradiation Rensand mg/head, twice I.P. at 36 and 12 hrs. u x .,..'.,.. 37.80 ± 10.13 before irradiationj+iiradiation Huangqid nig/head, twice I.P. at 36 and 12 hrs. n^ ™ . 26.70 ^ 15.56 before irradiation)+iiradiation Baizhud mg/head, twice I.P. at 36 and 12 hrs. Zb.bS ± 8.64 before irradiationj+iiradiation Fulingd mg/head, twice I.P. at 36 and 12 hrs. r ' 50.38 ± 9.75 before irradiation)+nradiation Muxiangd mg/head, twice I.P. at 36 and 12 hrs. , r ..',.. 28.95 ± 7.77 before irradiationj+nradiation Gancaod mg/head, twice I.P. at 36 and 12 hrs. , c . • • • 59.78 ± 26.49 before irradiationj+iiradiation p<0.005 as compared with irradiation control group. p<0.0005 as compared with irradiation control group. ***p<0.05 as compared with irradiation control group. - 61 - Fig. 1.16. The macroscopic finding of endogenous spleen colonies formed after irradiation. Mice were exposed to whole body irradiation with single dose of 6.5 Cy. Nine days after irradiation, the spleens were removed and fixed in Bouin's solution. - 62 - Table 1.16. Effect of Si-Wu-Tang and its ingredients on endogenous spleen colony formation in irradiated mice (M±SD) Groups Number of colony Irradiation control(6.5 Gy) 3.50 ± 4.00 Si-Wu-Tang(2 mg/m£ of drinking water, for 7 days before irradiation) + irradiation Si-Wu-Tang (1 mg/head, twice IP. at 36 and 12 hrs. ,..,.,.. 8.67 ± 5.24 before irradiation) + irradiation Irradiation + Si-Wu-Tang (2 mg/m£ of drinking water, for 9 days) ' ~ Irradiation + Si-Wu-Tang(l mg/head, single IP. „...... 8.63 ± 6.00* 30min. after irradiation) Irradiation control 1.13 ± 1.25 Shoudehuangd mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + irradiation Chuanxiongd mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + irradiation Dangguid mg/head, twice I.P. at 36 and 12 hrs. 6.50 ± 6.30 before irradiation) + irradiation Baishaoyaod mg/head, twice I.P. at 36 and 12 hrs. . ' 15.00 ± 17.33 before irradiation) + irradiation *p<0.05 as compared with irradiation control group. 63 - Table 1.17. Effect of Si-Jun-Zi-Tang and its ingredients on endogenous spleen colony formation in irradiated mice (M±SD) Groups Number of colony Irradiation control (6.5 Gy) 4.27 ± 3.98 Si-Jun-Zi-Tang (2 tag/mi of drinking water, for 7 , s ,. .. 4.81 ± 2.82 days) + irradiation Si-Jun-Zi-Tang (1 mg/head, twice IP. at 36 and 12 , . , . T. .;.'.. 6.15 ± 3.616 hrs before irradiation) + irradiation Irradiation + Si-Jun-Zi-Tang(2 mg/m£ of drinking 5.28 ± 4.23 water, for 9 days) Irradiation + Si-Jun-Zi-Tang(l mg/head, single I.P. 3.69 — 2.48 30 min. after irradiation) Irradiation control 1.13 ± 1.25 Rensand mg/head, twice I.P. at 36 and 12 hrs. before irradiation) + irradiation Gancaod mg/head, twice I.P. at 36 and 12 hrs. + before irradiation) + irradiation BaifulingQ mg/head, twice I.P. at 36 and 12 hrs. 6.75 ±10.55 before irradiation)+ irradiation BaizhuQ mg/head, twice I.P. at 36 and 12 hrs. 3.00 ± 5.40 before irradiation)+ irradiation *p<0.005 as compared with irradiation control. - 64 - Table 1.18. Effect of Shi-Quan-Dai-Bu-Tang and its ingredients on endogenous spleen colony formation of irradiated mice at ninth day after irradiation (M±SD) Groups Number of colony Irradiation control (6.5 Gy) 4.50 ± 2.51 Shi-Quan-Dai-Bu-Tang + (2 mg/m-C of drinking water, for 7 days) + irradiation Shi-Quan-Dai-Bu-Tang (1 mg/head, twice I.P. at 36 and 12 hrs. 3.83 ± 3.25 before irradiation) + irradiation Irradiation + Shi-Quan-Dai-Bu-Tang (2 mg/m-f! of drinking water, for 9 days) ' ~~ Irradiation + Shi-Quan-Dai-Bu-Tang (1 mg/head, + „ 9fi single IP. 30 min. after irradiation) ' ~~ Irradiation control 1.13 ± 1.25 ShoudehuangQ mg/head, twice IP. at 36 and 12 hrs. + , before irradiation) + irradiation ' ~~ Chuanxiongd mg/head, twice IP. at 36 and 12 hrs. before irradiation) + irradiation . — . Dangguid mg/head, twice IP. at 36 and 12 hrs. + _ » before irradiation) + irradiation . — . Baishaoyaod mg/head, twice IP. at 36 and 12 hrs. + , before irradiation) + irradiation ' ~~ Rensand mg/head, twice IP. at 36 and 12 hrs. + fi7o** before irradiation) + irradiation ' ~~ Gancaod mg/head, twice IP. at 36 and 12 hrs. + before irradiation) + irradiation ' ~ Baifulingd mg/head, twice IP. at 36 and 12 hrs. „ __ , .. __ beforu t e irradiationJ- 4.- )\ + irradiatio.r 4.- n 6.75 ± 10.55 Baizhud mg/head, twice IP. at 36 and 12 hrs. before irradiation) + irradiation ' "~ Huangquid mg/head, twice IP. at 36 and 12 hrs. + M7,-* before irradiation) + irradiation ' ~~ Rouguid mg/head, twice IP. at 36 and 12 hrs. + before irradiation) + irradiation . _ . *p<0.05 as compared with irradiation control group. **p<0.005 as compared with irradiation control group. - 65 - Table 1.19. Effect of Bu-Zhong-Yi-Qi-Tang and its ingredients on endogenous spleen colony formation of colonies of irradiated mice at ninth day after irradiation (M±SD) Groups Number of colony Irradiation control (6.5 Gy) 5.00 ± 3.02 Bu-Zhong-Yi-Qi-Tang(2 mg/m£ of drinking water, for 5.67 ± 4.09 7 days) + irradiation Bu-Zhong-Yi-Qi-Tang(l mg/head, twice IP. at 36 and 12 hrs. before irradiation) + irradiation Irradiation + Bu-Zhong-Yi-Qi-Tang (2 mg/m£ of 6.00 ± 6.78 drinking water, for 9 days) Irradiation + Bu-Zhong-Yi-Qi-Tang (1 mg/head, single 5.78 ± 3.15 IP. 30 min. after irradiation) Irradiation control (12 Cy) 2.11 ± 1.69 Rensand mg/head, twice IP. at 36 and 12 hrs. before „ 10.10 i 6.78 irradiation) + irradiation GancaoQ mg/head, twice IP. at 36 and 12 hrs. before irradiation) + irradiation Dangguid mg/head, twice IP. at 36 and 12 hrs. before 7.20 ± 6.24 irradiation) + irradiation Baizhud mg/head, twice IP. at 36 and 12 hrs. before 4.38 i 6.94 irradiation) + irradiation Huangqid tug/head, twice IP. at 36 and 12 hrs. before irradiation) + irradiation Chenpid mg/head, twice IP. at 36 and 12 hrs. before 2.78 — 3.03 Irradiation) + irradiation Shengmad mg/head, twice IP. at 36 and 12 hrs. before 4.78 i 5.24 Irradiation) + irradiation Chaihud mg/head, twice IP. at 36 and 12 hrs. before , irradiation) + irradiation *p<0.05 as compared with irradiation control group. **p<0.005 as compared with irradiation control group. - 66 - Table 1.20. Effect of San-Ling-Bai-Shu-San(Sam-Ryung-Baek-Chul-San) on endogenous spleen colony formation of irradiated mice at ninth day after irradiation (M±SD) Groups Number of colony Irradiation control (6.5 Gy) 2.63 ± 0.92 San-Ling-Bai-Shu-San(2 mg/m£ of drinking water, o CQ + n 33 for 7 days) + irradiation San-Ling-Bai-Shu-San(l mg/head, two IP. at 36 + » and 12 hrs. before irradiation) + irradiation ' ~ Irradiation + San-Ling-Bai-Shu-San(2 mg/m£ of 2 75 + 311 drinking water, for 9 days) Irradiation + San-Ling-Bai-Shu-San(l mg/head, r ~o , o O£r b.uo — 0.0D single IP. 30 mm. after irradiation) Irradiation control 2.11 ± 1.68 Rensen (1 mg/head, twice IP. at 36 and 12 hrs. + fiQ7«» before irradiation)+irradiation ' ~ Baizhud mg/head, twice IP. at 36 and 12 hrs. o nq + cor before irradiation)+irradiation ' ~~ Fulingd mg/head, twice IP. at 36 and 12 hrs. 774 + IQ4Q before irradiation)+irradiation ' ~ Gancaod rag/head, twice IP. at 36 and 12 hrs. 1 QQ + 1 9Q before irradiation)+irradiation ' ~ Yiyirend mg/head, twice IP. at 36 and 12 hrs. IOCQ + IQ/II* before irradiationHirradiation l -bd> ~ L6A[ baibiandoud mg/head, twice IP. at 36 and 12 hrs. 545 + 023 before irradiation)+irradiation ' ~ ShanyaoCl mg/head, twice IP. at 36 and 12 hrs. , ?o + o 7o before irradiation)+irradiation " ~ ' Lianroud mg/head, twice IP. at 36 and 12 hrs. . ^ + before irradiation)+irradiation " ~ Sharend mg/head, twice IP. at 36 and 12 hrs. i^AQ + \i m before irradiation)+irradiation . _ . Jiegengd mg/head, twice IP. at 36 and 12 hrs. 2 70 + 017 before irradiation)+irradiation ' ~ ' *p<0.05 as compared with irradiation control group. **p<0.005 as compared with irraidation control group. - 67 - Table 1.21. Effect of Kuei-Pi-Tang and its ingredients on endogenous spleen colony formation of irradiated mice at ninth day after Irradiation (M±SD) Groups Number of colony Irradiation control(6.5 Cy) 3.56 ± 2.79 Kuei-Pi-Tang(2 mg/mi. of drinking water, for 7 RQn + 9QS days) + irradiation °-JU ~ Z"y8 Kuei-Pi-Tang(l mg/head, twice IP. at 36 and 12 + hrs. before irradiation) + irradiation ' ~ Irradiation + Kuei-Pi-Tang (2 mg/ml! of drinking A™ + o oc water, for 9 days) " ~ Irradiation + Kuei-Pi-Tang (1 nig/head, single I.P. ^Q-] + 2 92 30 min. after irradiation) ' ~ Irradiation control(12 Cy) 1.13 ± 1.25 DangguHl mg/head, twice I.P. at 36 and 12 hrs. . _. , „ -., , . . ,. ,. > .. ,. ,. 6.50 ± 6.30 before irradiation)+nradiation Longyanroud mg/head, twice I.P. at 36 and 12 hrs. ^IO + 7ofi before irradiation)+iiradiation Suanzaorend mg/head, twice I.P. at 36 and 12 hrs. + 19m« before irradiation)+iiradiation ' ~ Yuanzhid mg/head, twice I.P. at 36 and 12 hrs. ,_ „ + „ „„ before irradiation)+iiradiation Rensand mg/head, twice I.P. at 36 and 12 hrs. + before irradiation)+iiradiation ' ~ Huangqid mg/head, twice I.P. at 36 and 12 hrs. + , before irradiation)+iiradiation ' ~ Baizhud mg/head, twice I.P. at 36 and 12 hrs. „„„ + n .„ before irradiation)+iiradiation Fulingd mg/head, twice I.P. at 36 and 12 hrs. fi —- + ir)c-c before irradiation)+iiradiation Muxiangd mg/head, twice I.P. at 36 and 12 hrs. „ _ + before irradiation)+iiradiation ' ~ Gancaod mg/head, twice I.P. at 36 and 12 hrs. , , ...... l.UU 3: 1.it before irradiatio n x)+nradiation p<0.05 as compared with irradiation control group. p<0.005 as compared with irradiation control group. V '• ' 1 _ V * T". i, Fig. 1.17. Intestinal crypts of mice 6 hours after exposure to gamma radiation. (A) Exposure to 2 Gy gamma radiation. Cells exhibiting pyknosis of nuclei(arrow) are seen. H-E staining, X330. (B) In situ end labelling (ISEL) demonstrating numerous apoptotic nuclei and bodies in the crypts. ISEL, chromogen diaminobenzidine, hematoxylin counterstaining, X330. - 69 - Table 1.22. Effect of Si-Wu-Tang and its ingredients on incidence of cell death by apoptosis in crypt of intestine following irradiation (M ± SD) Apoptotic cell per crypt Groups Base Total Untreated control 0.071 ± 0.035 0.091 ± 0.031 Irradiation control(2 Gy) 4.540 ± 0.646 5.111 ± 0.529 Si-Wu-Tang(l mg/head, twice I.P. at 36 and 12 hrs. 3.6 ± 0.184** 3.919 ± 0.214* before irradiation) + irradiation Untreated control 0.068 ± 0.032 0.084 ± 0.024 Irradiation control ± 1.138 4.938 ± 1.194 Shoudehuangd mg/head, twice IP. at 36 and 12 hrs. 3.1 ± 0.975 3.369 ± 1. before irradiation) + irradiation ChuanxiongQ mg/head, twice IP. at 36 and 12 hrs. 2.519 ± 0.335" 2.856 ± 0.478** before irradiation) + irradiation Dangguid mg/head, twice I.P. at 36 and 12 hrs. 3.861 ± 0.469 4.081 ± 0.387 before irradiation) + irradiation Baishaoyaod mg/head, twice I.P. at 36 and 12 hrs. 2.594 ± 0.464** 2.806 ± 0.429* before irradiation) + irradiation *p<0.01 as compared with irradiation control group. **p<0.05 as compared with irradiation control group. - 70 - Table 1.23. Effect of Si-Jun-Zi-Tang and its ingredients on incidence of cell death by apoptosis in crypt of intestine following irradiation (M±SD) Apoptotic cell per crypt Groups Base Total Untreated control 0.071 ±0.035 0.091 ±0.031 Irradiation control(2 Gy) 4.540 ±0.646 5.111 ±0.529 Si-Jun-Zi-Tang(l mg/head, twice IP. at 36 and 12 3.756+1.131 4.077 ±1.119 hrs. before irradiation) + irradiation Untreated control 0.068 ±0.032 0.084 ±0.024 Irradiation control 4.688 ±1.138 4.938± 1.194 RensanQ mg/head, twice I.P. at 36 and 12 hrs. 2.769 ±0.208 3.126 ±0.382* before irradiation)+ irradiation Gancaod mg/head, twice I.P. at 36 and 12 hrs. 5.128 ±1.296 5.488± 1.956 before irradiation) + irradiation BaifulingQ mg/head, twice I.P. at 36 and 12 hrs. 2.981 ±0.391* 3.331 ±0.335* before irradiation) + irradiation Baizhud tug/head, twice I.P. at 36 and 12 hrs. 4.224 ±1.71 4.568 ±1.379 before irradiation)* irradiation *p<0.05 as compared with irradiation control. - 71 - Table 1.24. Effect of Shi-Quan-Dai-Bu-Tang and its ingredients on incidence of cell death by apoptosis in crypt of intestine following irradiation (M±SD) Apoptotic cell per crypt Groups Base Total Untreated control 0.071 ± 0.035 0.091 ± 0.031 Irradiation control(2 Gy) 4.540 ± 0.646 5.111 ± 0.529 Shi-Quan-Dai-Bu-Tang(l mg/head, twice I.P. at 36 3.888 ± 0.593 4.275 ± 0.628 and 12 hrs. before irradiation) + irradiation Untreated control 0.068 ± 0.032 0.084 ± 0.024 Irradiation control 4.688 ± 1.138 4.938 ± 1.194 Shoudehuangd mg/head, twice I.P. at 36 and 12 hrs. 3.1 ± 0.975 3.369 ± 1.080 before irradiation) + irradiation ChuanxiongQ mg/head, twice I.P. at 36 and 12 hrs. 2.519 ± 0.335* 2.856 ± 0.478* before irradiation) + irradiation Dangguid mg/head, twice I.P. at 36 and 12 hrs. 3.861 ± 0.469 4.081 ± 0.387 before irradiation) + irradiation Baishaoyaod mg/head, twice I.P. at 36 and 12 hrs. 2.594 ± 0.464* 2.806 ± 0.429* before irradiation) + irradiation Rensand mg/head, twice I.P. at 36 and 12 hrs. 2.769 ± 0.208 3.126 ± 0.382* before irradiation)* irradiation Gancaod mg/head, twice I.P. at 36 and 12 hrs. 5.128 ± 1.296 5.488 ± 1.956 before irradiation) + irradiation Baifulingd mg/head, twice I.P. at 36 and 12 hrs. 2.981 ± 0.391* 3.331 ± 0.335* before irradiation) + irradiation Baizhud mg/head, twice I.P. at 36 and 12 hrs. 4.224 ± 1.710 4.568 ± 1.379 before irradiation) + irradiation Huangquid mg/head, twice I.P. at 36 and 12 hrs. 3.244 ± 0.490 3.581 ± 0.453 before irradiation) + irradiation Rouguid mg/head, twice I.P. at 36 and 12 hrs. 5.092 ± 1.472 5.243 ± 1.856 before irradiation) + irradiation *p<0.05 as compared with irradiation control group. - 72 - Table 1.25. Effect of Bu-Zhong-Yi-Qi-Tang and its ingredients on incidence of cell death by apoptosis in crypt of intestine following irradiation (M±SD) Apoptotic cell per crypt Groups Base Total Untreated control 0.071 ± 0.035 0.091 ± 0.031 Irradiation control(2 Cy) 4.540 ±0.646 5.111 ± 0.529 Bu-Zhong-Yi-Qi-Tang(l mg/head, twice IP. at 36 and 3.438 ± 0.161* 3.80 ± 0.176" 12 hrs. before irradiation) + irradiation Untreated control 0.071 ± 0.036 0.091 ± 0.032 Irradiation control(12 Cy) 4.688 ± 1.138 4.938 ± 1.194 Rensand mg/head, twice IP. at 36 and 12 hrs. before 2.769 ± 0.208* 3.126 ± 0.382* irradiation) + irradiation Gancaod mg/head, twice IP. at 36 and 12 hrs. before 5.246 ± 1.835 5.743 ± 3.136 irradiation) + irradiation Dangguid mg/head, twice IP. at 36 and 12 hrs. before 3.812 ± 0.625 4.194 ± 0.124 irradiation) + irradiation Baizhud mg/head, twice IP. at 36 and 12 hrs. before 4.189 ± 1.905 4.224 ± 3.285 irradiation) + irradiation Huangqid rug/head, twice IP. at 36 and 12 hrs. before 3.244 ± 0.490 3.581 ± 0.453 irradiation) + irradiation Chenpid mg/head, twice IP. at 36 and 12 hrs. before 3.288 ± 0.165 3.60 ± 0.177 irradiation) + irradiation Shengmad mg/head, twice IP. at 36 and 12 hrs. before 2.388 ± 0.449** 2.619 ± 0.452* irradiation) + irradiation Chaihud mg/head, twice IP. at 36 and 12 hrs. before 2.944 ± 0.405* 3.275 ± 0.448* irradiation) + irradiation *p<0.05 as compared with irradiation control group. **p<0.005 as compared with irradiation control group. ***p<0.01 as compared with irradiation control group. - 73 - Table 1.26. Effect of San-Ling-Bai-Shu-San(Sam-Ryung-Baek-Chul-San) on incidence of cell death by apoptosis in crypt of intestine following irradiation (M±SD) Apoptotic cell per crypt Groups Base Total Untreated control 0.071 ± 0.035 0.091 ± 0.031 Irradiation control (2 Gy) 4.540 ± 0.646 5.111 ± 0.529 San-Ling-Bai-Shu-San (1 mg/head, twice I.P. at 36 2.20 ± 0.704* 2.475 ± 0.820* and 12 hrs. before irradiation) + irradiation Untreated control 0.071 ± 0.036 0.091 ± 0.032 Irradiated control (2 Gy) 4.688 ± 1.138 4.938 ± 1.194 Rensen (1 mg/head, twice I.P. at 36 and 12 hrs. 2.769 ± 0.208* 3.712 ± 0.692* before irradiation)+irradiation Baizhud mg/head, twice I.P. at 36 and 12 hrs. 3.988 ± 2.724 4.113 ± 3.719 before irradiation)+irradiation Fuling(1 mg/head, twice I.P. at 36 and 12 hrs. 2.981 ± 0.391* 3.331 ± 0.335* before irradiation )+irradiation Gancaod mg/head, twice I.P. at 36 and 12 hrs. 5.456 ± 2.679 5.875 ± 3.524 before irradiation)+irradiation YiyirenCl mg/head, twice I.P. at 36 and 12 hrs. 4.825 ± 2: 5.25 ± 4.986 before irradiation)+irradiation Baibiandoud mg/head, twice I.P. at 36 and 12 hrs. 4.95 ± 1.830 5.282 ± 2.372 before irradiation)+irradiation Shanyaod mg/head, twice I.P. at 36 and 12 hrs. 3.294 ± 1.852 3.763 ± 1.090 before irradiation)+irradiation Lianroud mg/head, twice I.P. at 36 and 12 hrs. 5.244 ± 1. 5.632 ± 2.368 before irradiation)+irradiation Shren(l mg/head, twice I.P. at 36 and 12 hrs. 3.325 ± 1.917 3.125 ± 2.317 before irradiation)+irradiation Jiegengd mg/head, twice I.P. at 36 and 12 hrs. 6.488 ± 2.453 7.269 ± 1.194 before irradiation)+irradiation *p<0.05 as compared with irradiation control group. - 74 - Table 1.27. Effect of Kuei-Pi-Tang and its ingredients on incidence of cell death by apoptosis in crypt of intestine following irradiation (M±SD) Apoptotic cell per crypt Groups Base Total Untreated control 0.071 ± 0.035 0.091 ± 0.031 Irradiation control (2 Gy) 4.540 ± 0.646 5.111 ± 0.529 Kuei-Pi-Tang(l mg/head, twice IP. at 36 and 12 3.763 ± 0.475 4.081 ± 0.447* hrs. before irradiation) + irradiation Untreated control 0.063 ± 0.025 0.081 ± 0.036 Iiradiation control(12 Gy) 4.088 ± 0.423 4.481 ± 0.262 DangguiQ mg/head, twice IP. at 36 and 12 hrs. 2.974 ± 1.625 3.407 ± 1.489 before irradiation)+iiradiation LongyanrouQ mg/head, twice IP. at 36 and 12 hrs. 1.788 ± 0.334*** 2.038 ± 0.383**** before irradiation)+iiradiation SuanzaorenU mg/head, twice IP. at 36 and 12 hrs. 2.844 ± 0.487** 3.250 ± 0.594** before irradiation)+iiradiation Yuanzhid mg/head, twice IP. at 36 and 12 hrs. 2.944 ± 0.754* 3.163 ± 0.697* before irradiation)+iiradiation Rensand mg/head, twice IP. at 36 and 12 hrs. 2.739 ± 0.682* 3.281 ± 0.743* before irradiation)+iiradiation Huangqid mg/head, twice IP. at 36 and 12 hrs. 3.144 ± 0.647 3.624 ± 0.727 before irradiation)+iiradiation Baizhud mg/head, twice IP. at 36 and 12 hrs. 3.388 ± 2.280 3.656 ± 3.285 before irradiation)+iiradiation Fulingd mg/head, twice IP. at 36 and 12 hrs. 2.743 ± 0.843* before irradiation )+iiradiation 3.274 ± 0.945* Muxiangd mg/head, twice IP. at 36 and 12 hrs. 2.106 ± 0.624*** 2.238 ± 0.717*** before irradiation)+iiradiation Gancaod mg/head, twice IP. at 36 and 12 hrs. 4.858 ± 2.194 5.481 ± 3.136 before irradiation)+iiradiation p<0.05 as compared with irradiation control group. p<0.01 as compared with irradiation control group. p<0.005as compared with irradiation control group. p<0.0005 as compared with irradiation control group. - 75 - 1-2. 7\. (1) (herb mixture, HM)^- (2) 47H H) 1.28). (3) HM-I 3. # 1.18). 7}^ - 76 - (4) EM-N7} 1.29). 47H 1.30). (4) d Apoptosis HM-I, HM-n HM-IV7]- ^- ^^$lfe apoptosis -frit HM-I4 apoptosis -R-^: (Table 1.31). HM-I, HM-E ^ HM-IV7]- ^. 14 ig. 1.20, Table 1.32). - 77 - Table 1.28. Lymphocyte proliferation by water extracts of herb mixtures, HM-I, HM-H, HM-m and HM-PV in 3H-thymidine uptake assays Concentration cpm Sample (mg/m-d) Day 2 Day 3 HM-I 0.33 20678 ± 2343 15093 ± 2370 0.11 21135 ± 400 15316 ± 4569 0.03 12695 ± 2602 11571 ± 773 0.01 7183 ± 589 5766 ± 288 0.003 5446 ± 600 3996 ± 165 HM-n 0.33 213 ± 33 84 ± 30 0.11 4595 ± 312 3281 ± 472 0.03 10340 ± 801 7651 ± 59 0.01 7235 ± 1177 5074 ± 1619 0.003 5063 ± 704 3284 ± 159 HM-ffl 0.33 27292 ± 136 24448 ± 4448 0.11 30099 ± 1586 24093 ± 3916 0.03 17928 ± 305 14279 ± 592 0.01 8592 ± 192 6749 ± 119 0.003 5504 ± 136 4353 ± 368 HM-IV 0.33 9649 ± 731 6097 ± 1149 0.11 20835 ± 825 15278 ± 704 0.03 11529 ± 793 8743 ± 63 0.01 6426 ± 772 5367 ± 470 0.003 5107 ± 621 3811 ± 132 Cell 4338 ± 829 2444 ± 293 - 78 - 20 -g 15 in O S 10 = o I JLl o V <5> \ \\ w Plant extracts, Fig. 1.18. Effect of herbal mixture extracts on proliferation of nonadherent cells. Nonadherent supernatant-derived mononuclear cells harvested from long-termed bone marrow cell culture in the presence or absence of mixture extracts - 79 - Marker IL-3 IL-4 IL-7 SCF TNF-a p-catin Control !•! HM-I >••'•• lifill-e . Fig. 1.19. RT-PCR analysis of IL-3, IL-4, IL-7, SCF and TNF- ff mRNA expression in stromal cells. Total RNA was reverse transcribed to cDNA. Equivalent amounts of cDNA were amplified by PCR. PCR products were electrophoresed through 2% agarose gel containing ethidium bromid. - 80 - Table 1.29. Effects of the Herb mixtures on intestinal crypt survival in irradiated mice (M±SD) Group Crypt per circumference Unirradiated control 156.452 ± 8.351 Irradiation control (12 Gy) 26.833 ± 4.082 HM- I + irradiation 35.794 ± 19.038 HM-II + irradiation 40.075 ± 10.709* HM-III + irradiation 36.606 ± 10.565 HM-PV + irradiation 35.874 ± 8.665* *p<0.05 as compared to the irradiation control group. - 81 - Table 1.30. Effects of the prescriptions on endogenous spleen colony formation in irradiated mice (M±SD) Group Number of colonies Irradiation control (6.5 Gy) 1.75 ± 2.252 HM- I + irradiation 9.556 ± 8.904* HM-II + irradiation 12 ± 6.124*** HM-m + irradiation 6.222 ± 3.701** HM-IV + irradiation 6.889 ± 5.11* p<0.05, ** p<0.01, *** p<0.001 as compared to the irradiation control group. Table 1.31. Effects of the Herb mixtures on incidence of cell death by apoptosis in crypt of intestine following irradiation (M + SD) Apoptotic cells per crypt Group Base Total Normal control 0.062 ± 0.032 0.084 ± 0.024 Irradiation control (2 Gy) 3.688 ± 0.444 3.906 ± 0.536 HM- I + irradiation 2.212 ± 0.415" 2.444 ± 0.368" HM-II + irradiation 2.300 ± 0.712* 2.463 ± 0.807* HM-m + irradiation 2.481 ± 0.668* 2.731 ± 0.875 HM-IV + irradiation 1.556 ± 0.077*** 1.675 ± 0.050** p<0.05, ** p<0.005, *** p<0.0005 as compared to the irradiation control group. Fig. 1.20. Photomicrograph of micronucleated polychromatic erythrocyte (arrow). Diff-Quik stain, x 1000. Table 1.32. Effects of the herb mixtures on radiation-induced micronuclei in mouse bone marrow cells Group Percentage of micronucleKM ±SD) Unirradiated control 0.167 ± 0.408 Irradiation control (5 Gy) 8.933 ± 3.043 HM- I + irradiation 4.733 ± 2.093* HM-H + irradiation 3.733 ± 1.282" HM-m + irradiation 6.133 ± 2.481 HM-IV + irradiation 5.567 ± 1.546* p<0.05, ** p<0.05, *** p<0.001 as compared to the irradiation control group. n)t- z]-^ -B-S. ^f-^1 ^(herbal immunomodulator mixture) HIM- I , HIM-II3. ^i^^^^f. ^i^^l -n-5- 2:^-^1-i- HIM-I34 HIM-n^l water extract(W), methanol fraction(M), ethanol fraction(E) ^ polysaccharide fraction(P)-i: 412:*} (1) (7]-) (HIM-I-Pi, HIM-n-Pi)^^ ^£ i4# 1.33). 4 ^^^ITJI ^^^i(allogenic lymphocyte)^ 4. HIM-I-W ^^ A]6fl^ ot . St!:, HIM-I-Pi ^^ Ajoflfe $} 2.2«fl, HIM-n-Pi ^^ AH^. ^ 1.34-1.36). ^ ^^«V-§-(Graft versus host reaction, GVH): HIM-II-Pit- ^^ - 86 - 1.37-1.39). 4S.^ ^^«>-g-(Mixed lymphocyte reaction, MLR): HIM-I4 HM-n^l A ^^•^ ^^ ^-f^S^-B] £3$ A^A=t$\ T^0]?}] ^^^]i£(allogenic lymphocyte)0!] $]& MLR (mixed lymphocyte reaction)^ H 1.40). (4) ^4^ %AJ^ SL4^tfl v ^ES, ^4^a^ HIM-14 ^- iL$|Ji ^^ ^£5. ^^^4(Table 1.41, 1.42). (2) (7V) HIM-I4 - i^lfe ^-^^ HIM- HIM-n-Pi °]^4(Fig. 1.21). ^^)6fl ^l^lfe HIM-I4 ^1-^ i4t #^§>$i4. 3. 14, HIM-14 HiM-n^l ^ ^-^i- ?ofl wi^fl ^^^ ^v^^s^» ^7M?ife i47f oife- ^A^ 4 HIM-I-H4 HIM-E-W, Po, Pi ^ W+Pi ^-^l-^- ^ - 87 - 40*1144 ig. 1.22). HIM- , IL-64 A & ^1, HIM-I-Pi, W+Pi £3H ^ IGIFdnterferon-r inducing factor)^ ^ HIM-I-W+Pi . LT(lymphotoxin)^ ^2:^^ , HIM-I-W+Pi (Fig. 1.23). -, HIM-H4 A 14^ HIM-M] til ^-O] reverse transcriptase DNA polymerase^l 4. Slfe 7}^} GM-CSF4 IL-37}- ^ ig. 1.24). % 7} GM-CSF4 IL-31- nfl, HIM-I4 HM-0 5] A &%•£ ^A #, GM-CSF1- ^7>«H ^^ ^^H^ HM-I4 HIM-II5} 5} ^-Sf^^i ^ ^51 fl^l^l fe^^S ^^€4. 4^-^-S GM-CSF^l 5% ^fe 20%# ^7>*>JIJ HIM-I4 HIM-E ^1 4 ^-^^ 0.01, 0.1, 1 GM-CSF^l °oH ^7f%>^^ tfl^ . ^-^1, HIM-H-Po, Pi, W+Pi ^r ig. 1.25, 1.26). fe- ^^H1?]:, GM-CSF A ^^°1 ^^ ^^^: *l-*lfe-*l ^ §: -^-^^1-fe #^^ ^^^ EPO(erythropoietin)* , HM-I4 HIM-II^1 A gr'^Sr ^7}*}<^ ^-Sf- f ig. 1.27). a 14, HIM-I^l ^-f^ EPO1- 300, 1000 U/ral -n-M, E, Po)7> ^:#5l^ZL, ^-§1, HIM-E -Pi ^"^^^ tfl (Fig. 1.28, 1.29). ^, HIM-n51 ^^^ #=- 5£^ EPO ^^fl §foflA-] AT-^JT^ ^-51 ^##^1 HIM-I4, 47H HIM- n 51 ^-^oi ^^o| w. 147} 4 Gy ^ HIM-I . ^-^1, HIM-I-M, Pi ^^^ ^-f ^^*1 ^l^^l- ^7|-A]^4. ^ HIM-E^I ^^-i^r ^^1^^-S Gyi ^#€ 4-f^^ ^i?^ tio^>^ii t#s]^ &^ 4-f^^l tfl^i «1^ HIM-I4 HIM-IIS] 7-1 <>] a^ ^-^#^1 8 7}^] ^^#(HIM-I-W, HIM-H-M, E, W+Pi)^- 1.30, 1.31). ^1^^ 14S., HIM-I4 HIM-n ^^r^^l A . Z1S14, (3) if*L 3:^-^1 f-^ J:4 ^^o] ^4^. £cfl^; HIM-I4 HM-n ^ •f-^.5. -{d^^l-Ji °11-^1 water extract(W), methanol fraction (M), ethanol fraction(E) ^ polysaccharide fraction(P)^: (7]-) HIM-I-W, HIM-H-W ^ HIM-n-M * 44^4(Table 1.43, 1.44). - 90 - HiM-i-E-i- «1 HIM-I-Pi^ HIM-H-M ^ HIM-E-Pi ^^^i^ ^ JL4 (Table 1.45, 1.46). Apoptosis HIM-I-W, E, Pi^ HIM-n-W, M, Pi 1.47, 1.48). 1.49, 1.50). } ^Af ^ HIM-E1- ig. 1.32, Table 1.51, 1.52). HIM-I-W7> 1.67a]], HIM-I-Pi 7\ 1.5*1], HIM-E-W7J- l.24afl, HIM-n-Pi7> 1.33*113 AJ^^- (Table 1.53, 1.54). - 91 - Table 1.33. Lymphocyte proliferation by fractions of HIM-I and HIM-II in 3H-thymidine uptake assays Concentration cpm Sample (mg/miO Day 2 Day 3 HIM-I-W 0.4 48891 ± 5581 66274 ± 8892 0.2 60227 ± 2033 64996 ± 4977 0.1 47218 ± 3195 38798 ± 182 0.05 36691 ± 1875 25874 ± 1664 HIM-I-Pi 0.6 68284 ± 1391 92029 ± 712 0.3 268113 ± 8272 281177 ± 20977 0.15 245759 ± 4487 258466 ± 20718 0.075 184754 ± 10507 179601 ± 16667 HIM- H -W 0.08 14880 ± 1733 7951 ± 1082 0.04 22796 ± 398 13558 ± 1913 0.02 17894 ± 1389 10662 ± 202 0.01 14986 ± 462 8003 ± 227 HIM- n -Pi 0.4 15959 ± 180 4706 ± 801 0.2 45874 ± 2933 23648 ± 633 0.1 42271 ± 4670 104493 ± 8275 0.05 71883 ± 3043 79477 ± 2585 0.025 49774 ± 1364 45702 ± 1468 Cell control 5136 ± 57 3437 ± 681 HIM- I -W: water extract of HIM- I HIM- I -Pi: inner part of plysaccharide fraction of HIM- I - 92 - Table 1.34. Effect of water extract of HIM-I-W on the secretion of antibody tested by hemolytic plaque forming cell assay _ . Dosage No. of plaque/7 x 10 , Sample , . . Splenocyte/spleen , PFC/spleen (mg/kg B.W.) splenocyte 1.48 x 10* cells/m£ 77, 87, 93 (86) 18,117 1.38 x 1OK cells/mi 104, 100, 108 (104) 20,498 Saline 1.44 x 10s cellsM 92, 120, 82 (98) 20,159 1.54 x 10a cells/M 70, 113, 76 (86) 18,986 1.52 x 10B cells/m« 128, 157, 175 (153.3) 33,281 1.86 x l0b cells/ntf 120, 124, 106 (116.7) 31,007 HIM-I-W 400 1.98 x l0b cells/me 180, 172, 119 (157) 44,415 1.94 x 108 cells/me 96, 157, 123 (125.3) 34,721 - 93 - Table 1.35. Effect of fraction HIM-I-Pi on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage No. of plaque/7 x 10° Sample Splenocyte/spleen PFC/spleen (mg/kg B.W.) splenocyte 6.47 x 10' cells/m£ 30, 34, 40, (34.7) 3,206 8.25 x 10' cells/m£ 30, 30, 26 (28.7) 3,384 5.5 x 10' cells/nrf 32, 50, 54 (45.3) 3,561 Saline 7.2 x 10' cells/ni£ 54, 30, 52 (45.3) 4,661 6.47 x 10' cells/me 34, 34, 30 (32.7) 3,021 5.75 x 10' cells/me 34, 24, 20 (26) 2,135 1.08 x 1OM ce]\s/mt 52, 54, 58 (54.7) 8,440 8.75 x 10' cells/me 92, 90, 80 (87.3) 10,913 7.38 x 10' cells/mi 58, 56, 42 (52) 5,481 HIM-I-Pi 60 3.03 x 10' cells/mi! 3, 88, 76 (77.3) 8.81 x 10' cells/me 30, 28, 40 (32.7) 4,117 7.47 x 10' cells/me 62, 60, 60 (60.7) 6,477 - 94 - Table 1.36. Effect of fraction HIM-H-Pi on the secretion of antibody tested by hemolytic plaque forming cell assay _ , Dosage , No. of plaque/7 X10 ™-w , Sample , ,, _. ,, . Splenocyte/spleen , ^ Pbw spleen (mg/kg B.W.T ) splenocyte 4.2 x ioY cells/me 57, 64, 47 (56.0) 3,360 2.4 x 10' cells/mH 55, 48, 43 (48.7) 1,670 Saline 1.7 x 10' cells/me 70, 92, 98 (86.7) 2,107 3.8 x ioY cells/me 63, 70, 46 (59.7) 3,242 3.6 x 10' cells/m£ 83, 95, 85 (87.7) 4,508 HIM- n -Pi 5 4.6 x 10' cells/m£ 64, 44, 50 (52.7) 3,462 6.4 x 10' cells/me 54, 67, 75 (65.3) 5,968 4.4 x 10' cellsM 64, 60, 94 (72.7) 4,572 - 95 - Table 1.37. Effect of fraction HIM-I-Pi on Graft vs. Host reaction spleen w.t/body Group Donor Recipient Sex -, Spleen index w.t.( x 10"3) BDF1 BDF1 M 2.565 ± 0.163 1.00 Saline 3.447 ± 0.317 1.00 C57BL/6 BDF1 M 3.135 ± 0.361 1.222 Saline F 4.9225 ± 0.729 1.428 C57BL/6 BDF1 M 6.243 ± 0.6124 2.434 HIM-I-Pi 60 mg/kg B.wt. 8.638 ± 0.671 2.506 - 96 - Table 1.38. Effect of fraction HIM- II -Pi on Graft vs. Host reaction spleen w.t./body Group Donor Recipient Sex , Spleen index w.t.(xio3) BDF1 BDF1 M 2.32 ± 0.208 1.00 Saline 3.05 ± 0.117 1.00 C57BL/6 BDF1 M 3.21 ± 0.309 1.384 Saline 4.324 ± 0.05 1.418 C57BL/6 BDF1 M 5.01 ± 0.624 2.159 HIM- H -Pi 60 mg/kg B.wt. 6.58 ±1.17 2.157 - 97 - Table 1.39. Effect of water extracts HIM-I-W and HIM-II-W on Graft vs. Host reaction Spleen w.tTbody „ , Group Donor Recipient Sex 3 w.t.(xiO" ) Spleen index M 2.15 ± 0.49 1.00 Saline BDF1 BDF1 F 3.55 ± 0.07 1.00 M 3.70 ± 0.28 1.72 Saline C57BL/6 BDF1 F 4.35 ± 0.92 1.22 M 4.05 ± 0.21 1.88 HIM-I-W C57BL/6 BDF1 (lOOmg/kg B.W.) F 5.80 ± 0.78 1.63 M 4.07 ± 1.80 1.89 HIM- E -W C57BL/6 BDF1 (lOmg/kg B.W.) F 5.50 ± 0.26 1.55 Table 1.40. Mixed lymphocyte reaction of splenocytes obtained from the C57BL/6 mice injected with fractions of HIM-I and HIM- II Sample Dose cpm Saline 6,169 ± 1,035 HIM-I-W 20mg/kg B.W. 5,295 ± 557 HIM-I-Pi 15mg/kg B.W. 4,133 ± 704 HIM-I-M 4mg/kg B.W. 5106 ± 417 HIM-I-E 4mg/kg B.W. 8685 ±1113 HIM-n-W 0.6mg/kg B.W. 9417 ± 338 HIM- n -Pi 6mg/kg B.W. 13556 ± 2544 HIM-H-M 2mg/kg B.W. 24829 ± 3520 HIM-n-E 2mg/kg B.W. 18057 ± 745 As responder cells, the splenocytes from C57BL/6 mice injected with HIM-I and II fractions are used. ; as stimulator cells, splenocytes of the DBA/2 mice y -irradiated with 3,000 rad were used. - 99 - Table 1.41. Comparsion of the lymphocyte proliferation by fraction HIM-I-Pi q and fraction mixture HIM-I-W+Pi in H-thymidine uptake assays Concertration cpm Sample (mg/iM) Day 2 Day 3 HIM-I-Pi 0.6 184520 ± 1306 116842 ± 1945 0.3 252962 ± 1331 189210 ± 1526 0.15 286814 ± 3578 280838 ± 1430 0.075 329647 ± 1764 333709 ± 3560 0.038 286396 ± 1373 299438 ± 1645 HIM-I-W+Pi 0.075 60221 ± 2001 53222 ± 2382 0.038 166888 ± 999 167843 ± 220 0.019 192604 ± 5199 237489 ± 3765 0.008 163111 ± 1689 185948 ± 1792 Cell control 8669 ± 718 6954 ± 909 HIM- I -W+Pi: mixture of water extract and polysaccharide fraction from HIM- I . - 100 - Table 1.42. Comparsion of the lymphocyte proliferation by fraction HIM-IT -Pi and fraction mixture HIM-II-W+Pi in H-thymidine uptake assays Concertration cpm Sample (mg/mt) Day 2 Day 3 HIM- II -Pi 0.05 88706 ± 33 62921 ± 3487 0.025 107009 ± 194 126879 ± 497 0.013 71433 ± 2620 66499 ± 4448 0.0063 59240 ± 5587 57690 ± 6606 0.0031 44184 ± 3536 41554 ± 9463 HIM- D -W+Pi 0.01 15269 ± 670 5265 ± 2 0.005 38286 ± 211 25763 ± 1995 0.0025 42848 ± 3550 37173 ± 5066 0.0013 35285 ± 3484 34084 ± 2099 Cell 9213 ± 585 7765 ± 374 HIM- II -W+Pi: mixture of water extract and polysaccharide fraction from HIM- II. - 101 - Fig. 1.21. Effect of herbal plant mixture extracts on proliferation of nonadherent cells. Nonadherent supernatant-derived mononuclear cells harvested from long-termed bone marrow cell culture in the presence or absence of mixture extracts. - 102 - Hm-I i— x CD z; E 2 CD o 1 _ .. 1 ... Ill II 1• 1 § § f 8 § if if if Concentration, jj.g/rti Him-ll |3 = 2 o> O 1 0 I § 5 f ~ s « ? .e ^e Concentration, Fig. 1.22. Effect of herbal plant mixture-extracts on proliferation of stromal cells. Bone marrow cells(1.7xiO6 cells/well) were cultured for 10 days in the presence or absence of herbal plant mixture-extracts. Nonadherent cells were depleted from stromal cells culture and adherent viable cells were counted by try pan blue staining. - 103 - marker Control Him- I -W IIim~ I -M Him IE Him 1 Pi Him-I -W-I'i IL-6 IGIF LT 'ik1!1 i-iiii-M i !••«.: •-' »••'': :> -actin marker Control Him II W Him II M Him II K Him II Po Him IT I'i Him II W'I'i -actin mil Fig. 1.23. RT-PCR analysis of IL-6, IL-10, IGIF, TNF-a, LT and /5-actin mRNA expression in bone marrow cells. Bone marrow cells were incubated for 4 hours in the presence or absence of herbal plant mixture~extracts(100 iig/mi). Total RNA was reverse transcribed to cDNA. Equivalent amounts of cDNA were amplified by PCR. PCR products were electrophoresed through 2% agarose gel containing ethidium bromid. - 104 - I1" i IS I*«lii3i"i l it ij i O'il'it fl I ill * l"' I'll » J IIII ' ^1 ' * r^.* pl r •I!1!' 'f'*i'i" I'*! '- ' '*" ** ' *" *''i***J*"^ '' "W ' Fig. 1.24. Morphology of macrophage colony derived from bone marrow cells. Nonadherent bone marrow cells were cultured for 0 day(A), 3 days(B), 5 days(C) and 7 days(D) in the presence of IL-3(10% : WEHI-3 supernatant) and GM-CSF(20% : GM-CSF gene transfected cell culture supernatant). - 105 - G M -CS F (0%) V Concentration, ng/ml G M -CS F 5%) 5 3 2 1 n . . 1 1 1 • 1 . • • 1 • I • 1 • 1 X* Concentration, ng/ml G M -CS F (2 0%) 1 III lll.ll.ll III V- Vs Concentration, ng/ml Fig. 1.25. Effects of HIM-I on differentiation of bone marrow cells into primary macrophages. Nonadherent bone marrow cells(6xlO4 cells/well) were cultured for 1 week in the presence or absence of IL-3(10% : WEHI-3 cell culture supernatant) and GM- CSF (5%, 20% : GM-CSF gene transfected cell line culture supernatant). Total cells were counted by trypan blue staining. - 106 - GM-CSF(O%) 5 x 4 _. 2 O • • 1 f ° £ 5 Concentration, GM-CSF (5%) x 4 if & 3 £ tm Ill III III I.I Concentration, GM-CSF (20%) 5 x 4 « I 3 |- 1 • . 2 . Ill h o ll 1 1 t: tz IllI IIII ilIIlI 1I1I 1 1 *" £ ° Concentration, ng/n Fig. 1.26. Effects of HIM-II on differentiation of bone marrow cells into primary macrophages. Nonadherent bone marrow cells(6xlO4 cells/well) were cultured for 1 week in the presence or absence of IL-3(10% : WEHI-3 cell culture supernatant) and GM-CSF (5%, 20% : GM-CSF gene transfected cell line culture supernatant). Total cells were counted by trypan blue staining. - 107 - .1 f V <• ••"''* --'.;:• 1. 2i. Morphology ()f crythrocyle colony derived from bone marrow cells. Nonadherenl bone marrow cells were cultured for 0 day(A), 3 days(B), o days(C) and 7 days(D) in the presence of IL 3 (10% : WEIII-3 cell culture supernatant) and Kl'O (erythro])oietin, 1000 l7m(0. 108 Cell Numbers, X1(f Cell Numbers, X10 Cell Numbers, X10 CO O5 03 oo co •• 3 8 03 \ O CO H rt O i=j CO o _, CD 8." S o s 3 03 H 3 o CD co c? X °CD 3 03 3 £> CD ^ ^ 3 § O O^ N/5. O CD ^ g M R 8 g CL CD CJ" -"-• ^ ""3 CO t-1 3 3 ° ^ o CO CO p- CO O CD 3 CD O o\ w S" Co ntro I EPO, 300U/ml EPO , 1 OOOU/ml Fig. 1.29. Effects of HIM-II on differentiation of bone marrow cells into erythrocytes. Nonadherent bone marrow cells(1.8x 105 cells/well) were cultured for 1 week in the presence or absence of IL-3(10% : WEHI-3 cell culture supernatant) and EPO (ery thropoietin : 300 U/m£, 1000 U/m-£). Total cells were counted by trypan blue staining. - no - Co ntro I 20 ~ 15 10 t II Concentration, Radiatio n 4 G ray " 1.5 Concentration, ng/ml Radatio n 8 G ray 1.5 o tt iJL I Concentration, Fig. 1.30. Effects of HIM-I on proliferation of 7 -ray irradiated bone marrow cells. Irradiated BALB/c mice were sacrificed and isolated bone marrow cells(2.3 x 106 cells/well) were cultured for 9 days in the presence or absence of herbal plant mixture-extracts. Total cells were counted by try pan blue staining. - ill - Contro 1 20 o 15 X || • 1 || I. ,1 11 Cel l Numb e D e n c 1 II II II II II III > o Concentration , ng/ml Radiation 4 G ray 2 o X 15 • to o II _ a 1 1 | | z • 1 1 0.5 1 1 • l o II II II • I II 1 Concentratio i, up/ml Radiation 8 G ray O x 1-5 1 1 - 11 . 1 •I 1 l.l. ll ll .1 ill Concentration, ng/ml Fig. 1.31. Effects of HIM-II on proliferation of 7 -ray irradiated bone marrow cells. Irradiated BALB/c mice were sacrificed and isolated bone marrow cells(2.3xlO6 cells/well) were cultured for 9 days in the presence or absence of herbal plant mixture-extracts. Total cells were counted by trypan blue staining. - 112 - Table 1.43. Effects of the fractions of HIM- I on intestinal crypt survival in irradiated mice (M±SD) Group Crypt per circumference Unirradiated control 157.252 ± 6.051 Irradiation control (12 Cy) 21.285 ± 9.710 HIM- I -W(l nig/animal) + irradiation 35.121 ± 10.995* HIM- I -M(0.66 mg/animal) + irradiation 41.162 ± 10.011 HIM- I -E(0.16 mg/animal) + irradiation 32.701 ± 9.176 HIM- I -PK0.17 mg/animal) + irradiation 26.037 ± 18.! HIM- I -W: water extract of HIM- I . HIM- I -M: methanol fraction of HIM- I . HIM- I -E: ethanol fraction of HIM- I . HIM- I -Pi: inner part of polysaccharide fraction of HIM- I . The fractions were given i.p. at 36 and 12 hours before irradiation. p<0.05 as compared to the irradiation control group. - 113 Table 1.44. Effects of the fractions of HIM- II on intestinal crypt survival in irradiated mice (M±SD) Group Crypt per circumference Unirradiated control 157.252 ± 6.051 Irradiation control (6.5 Gy) 21.285 ± 9.710 HIM-n-W(l mg/animal) + irradiation 34.400 ± 9.810* HIM-II-M(0.63 mg/animal) + irradiation 34.808 ± 9.810* HIM-n-E(0.083 mg/animal) + irradiation 29.800 ± 8.438 HIM-H-Po(0.084 mg/animal) + irradiation 28.350 ± 14.140 HIM-H-Pi(0.20 mg/animal) + irradiation 20.839 ± 13.398 HIM-n-W: water extract of HIM-II. HIM-n-M: methanol fraction of HIM-II. HIM- H -E: ethanol fraction of HIM- E. HIM-H-Po: outer polysaccharide fraction of HIM-II. HIM-II-Pi: inner polysaccharide fraction of HIM- II. The fractions were given i.p. at 36 and 12 hours before irradiation. p<0.05 as compared to the irradiation control group. - 114 - Table 1.45. Effects of the fractions of HIM- I on endogenous spleen colony formation in irradiated mice(M±SD) Group Number of colonies Irradiation control (6.5 Gy) 2.667 ± 3.240 HIM- I -W(l mg/animal) + irradiation 8.222 ± 6.741* HIM- I -MC0.66 mg/animal) + irradiation 12.222 ± 10.733* HIM- I -E(0.16 mg/animal) + irradiation 6.556 ± 7.732 HIM- I -PK0.17 mg/animal) + irradiation 14.222 ± 9.744" HIM- I -W: water extract of HIM- I . HIM- I -M: methanol fraction of HIM- I . HIM- I -E: ethanol fraction of HIM- I . HIM- I -Pi: inner part of polysaccharide fraction of HIM- I . The fractions were given i.p. at 36 and 12 hours before irradiation. p<0.05, ** p<0.005 as compared to the irradiation control group. - 115 - Table 1.46. Effects of the fractions of HIM-II on endogenous spleen colony formation in irradiated mice(M±SD) Group Number of colonies Irradiation control (6.5 Gy) 2.667 ± 3.240 HIM-n-W(l mg/animal) + irradiation 10.125 ± 9.833* HIM-n-M(0.63 mg/animal) + irradiation 8.788 ± 3.420" HIM- H -E(0.083 mg/animal) + irradiation 10.889 ± 8.328* HIM- II -Po(0.084 mg/animal) + irradiation 14.556 ± 10.175" HIM- II -Pi(0.20 mg/animal) + irradiation 15.222 ± 9.230" HIM-n-W: water extract of HIM-II. HIM-n-M: methanol fraction of HIM-EL HIM- H -E: ethanol fraction of HIM- E. HIM- II -Po: outer polysaccharide fraction of HIM- II. HIM- II -Pi: inner part of polysaccharide fraction of HIM- II. The fractions were given i.p. at 36 and 12 hours before irradiation. p<0.05, " p<0.005 as compared to the irradiation control group. - 116 - Table 1.47. Effects of the fractions of HIM- I on incidence of cell death by apoptosis in crypt of intestine following irradiation(M±SD) Apoptotic cells per crypt Group Base Total Normal control 0.071 ± 0.035 0.091 ± 0.031 Radiation control (2 Gy) 2.875 ± 0.267 3.138 ± 0.332 HIM- I -W(l mg/animal) + irradiation 2.250 ± 0.154* 2.438 ± 0.148* HIM- I -M(0.66 mg/animal) + irradiation 2.419 ± 0.382 2.869 ± 0.406 HIM- I -E(0.16 mg/animal) + irradiation 1.975 ± 0.106** 2.394 ± 0.165* HIM- I -Pi(0.17 mg/animal) + irradiation 1.681 ± 0.105* 2.013 ± 0.003* HIM- I -W: water extract of HIM- I . HIM- I -M: methanol fraction of HIM- I . HIM- I -E: ethanol fraction of HIM- I . HIM- I -Pi: inner part of polysaccharide fraction of HIM- I . The fractions were given i.p. at 36 and 12 hours before irradiation. *p<0.01, **p<0.001, ***p<0.0005 as compared to the irradiation control group. - 117 - Table 1.48. Effects of the fractions of HIM-II on incidence of cell death by apoptosis in crypt of intestine following irradiation (M±SD) Apoptotic cells per crypt Group Base Total Normal control 0.071 ± 0.035 0.091 ± 0.031 Radiation control (2 Gy) 2.875 ± 0.267 3.138 ± 0.332 HIM-n-W(l mg/animal) + irradiation 2.056 ± 0.133*** 2.456 ± 0.138" HIM-n-M(0.63 mg/animal) + irradiation 1.873 ± 0.364*** 2.144 ± 0.367" HIM-n-E(0.083 mg/animal) + irradiation 2.331 ± 0.348* 2.906 ± 0.433 HIM-II-Po(0.084 mg/animal) + irradiation 2.206 ± 0.323* 2.763 ± 0.304 HIM-n-Pi(0.20 mg/animal) + irradiation 1.769 ± 0.151* 2.206 ± 0.247* HIM- II -W: water extract of HIM- II. HIM-H-M: methanol fraction of HIM-II. HIM-n-E: ethanol fraction of HIM-II. HIM-EI-Po: outer polysaccharide fraction of HIM-II. HIM- II -Pi: inner part of polysaccharide fraction of HIM- II. The fractions were given i.p. at 36 and 12 hours before irradiation. *p<0.05, "p<0.01, *"p<0.005, ****p<0.0005 as compared to the irradiation control group. - 118 - Table 1.49. Hematological values in irradiated mice administered with the fractions of HIM- I on day 25 after irradiation (M±SD) Group T T * i. Test Unit Unirradiated Irradiation HIM- I -W HIM- I -M HIM- I -Pi control control + irradiation + irradiation + irradiation Erythrocyte M/fd 10.11 ±0.24 6.70 ±1.01 7.74 ±2.46 8.15±0.45* 7.63±1.18 Hemoglobin g/de 16.8 ±0.85 9.63 ±2.07 10.75 ±2.67 11.53 + 0.28 10.78 ±1.23 MCV fi 58.25 ±3.89 58.58 ±5.42 61.85 ±9.73 60.58 ±4.88 61.78 ±5.71 MCH pg 16.6 ±0.42 14.28 ±1.58 14.25 ±1.58 14.2 ±0.77 14.2 ±1.25 MCHC g/d£ 28.55 ±1.06 24.4 ±1.44 23.18±1.18 23.45 ±0.88 23.08 ±2.07 Hematocrit % 58.95 ±5.30 39.6 ±9.19 46.1 ±10.01 49.18 ±1.47 46.63 ±2.49 Thrombocyte K/fd 637 ±228 530 ±329 954 ±348 872 ±161 873 ±260 Leukocyte K/fd 10.45±1.51 1.70 ±0.29 2.97±1.19 1.96 ±0.69 2.71 ±0.52* Neutrophil K/fd 2.49 ±0.56 0.32 ±0.05 0.60 ±0.22* 0.63 ±0.27 1.03 ±0.26** Lymphocyte K/fd 7.43 ±1.80 1.13 ±0.26 2.05 ±1.47 1.09 ±0.37 1.34 ±0.34 Monocyte K/fd 0.30 ±0.18 0.22 ±0.05 0.31 ±0.16 0.23 ±0.09 0.30 ±0.11 Eosinophil K/ni 0.19 ±0.05 0.02 ±0.02 0.01 ±0.01 0.01 ±0.01 0.03 ±0.02 Basophil K/fd 0.06 ±0.02 0.01 ±0.01 0.00 ±0.00 0.00 ±0.00 0.01 ±0.01 HIM- I -W: water extract of HIM- I . HIM- I -M: methanol fraction of HIM- I . HIM- I -Pi: inner part of pplysaccharide fraction of HIM- I . The fractions were given i.p. at 36 and 12 hours before irradiation. *p<0.05, **p<0.01 as compared to the irradiation control group. - 119 - Table 1.50. Hematoiogical values in irradiated mice administered with the fractions of HIM-II on day 25 after irradiation (M±SD) Group Test Unit Unirradiated Irradiation HIM-n-W HIM-n-M HIM- n -Pi control control + irradiation + irradiation + irradiation Erythrocyte M/fd 10.11 ±0.24 6.70 ±1.01 7.43 ±0.97 7.88 ±0.15 7.46 ±0.72 Hemoglobin g/d£ 16.80 ±0.85 9.63 ±2.07 10.25 ±1.64 11.6 ±0.3 11.68 ±0.85 MCV it 58.25 ±3.89 58.58 ±5.42 57.65 ±2.27 58.9 ±2.08 64.95 ±4.11 MCH pg 16.60 ±0.42 14.28 ±1.58 13.75 ±0.54 14.73 ±0.21 15.7 ±0.76 MCHC g/M 28.55 ±1.06 24.4 ±1.44 23.93 ±1.72 25.0 ±1.22 24.23 ±1.19 Hematocrit % 58.95 ±5.30 39.6 ±9.19 42.63 ±4.09 46.4 ±1.4 48.25 ±2.94 Thrombocyte K/id 637 ±228 530 ±329 930± 73 780 ±202 976 ±171 Leukocyte K/fd 10.45 ±1.51 1.70 ±0.29 2.14±0.31 2.61 ±0.69 2.22 ±1.28 Neutrophil K/fd 2.49 ±0.56 0.32 ±0.05 0.61 ±0.17 0.7 ±0.19** 0.63 ±0.48 Lymphocyte K/fd 7.43 ±1.80 1.13±0.26 1.23 ±0.09 1.50 ±0.53 1.30 ±0.69 Monocyte K/fd 0.30±0.18 0.22 ±0.05 0.29 ±0.09 0.39 ±0.13* 0.29 + 0.18 Eosinophil K/fd 0.19 ±0.05 0.02 ±0.02 0.01 + 0.01 0.02±0.02 0.01 + 0.01 Basophil K/fd 0.06 ±0.02 0.01 ±0.01 0.00 ±0.00 0.01 ±0.01 0.00 ±0.00 HIM- II -W: water extract of HIM- II. HIM-n-M: methanol fraction of HIM-II. HIM-II-Pi: inner part of polysaccharide fraction of HIM-II. The fractions were given i.p. at 36 and 12 hours before irradiation. *p<0.05, **p<0.01 as compared to the irradiation control group. - 120 - ip^ Fig. 1.32. Photomicrograph of cytokinesis-blocked human lymphocyte containing a micronucleus (arrow). Diff-Quik stain, X1000. - 121 - Table 1.51. Micronuclei (MN) per 500 cytokinesis-blocked human lymphocytes treated with radiation and water extract of HIM- II in vitro No. of cells Number of MN per cell Total number of Group without MN 1 2 3 4 MN Sample 1 Unirradiated control 497 3 3 HIM-II (30 fig/vA) 497 2 1 4 HIM-n (10 ftg/rd) 497 3 3 Irradiation control (3 Gy) 271 155 63 11 314 HIM- n (30 pg/mt) + irradiation 303 164 33 230 HIM- H (10 ng/ml) + irradiation 351 119 26 4 183 Irradiation + HIM-H (30 U%/XRIL) 288 149 54 8 1 285 Irradiation + HIM-D (10 ug/mi] 299 141 51 9 270 Sample 2 Unirradiated control 496 4 4 HIM-n (30 ug/rd) 497 3 3 HIM-II (10 ^g/m£) 496 3 1 5 Irradiation control (3 Gy) 303 138 43 13 3 275 HIM- II (30 ixg/mi) + irradiation 342 131 24 3 188 HIM- n (10 fig/ml) + irradiation 332 130 37 1 207 Irradiation + HIM-H (30 ^gM) 318 128 49 5 241 Irradiation + HIM- n (10 fig/mi) 308 133 54 5 256 Sample 3 Unirradiated control 497 3 3 HIM-n (30 (jg/id) 496 4 4 HIM-H (10 jig/ml) 496 4 4 Irradiation control (3 Cy) 280 159 46 14 1 297 HIM- H (30 ug/ml) + irradiation 332 143 23 2 195 HIM-II (10 mM) + irradiation 348 117 34 1 188 Irradiation + HIM- U (30 ue/mi) 329 128 41 2 216 Irradiation + HIM-H (10 /xg/mH,) 330 125 43 2 217 - 122 - Table 1.52. Frequency of micronuclei in binucleated human lymphocytes treated with radiation and water extract of HIM- E in vitro Micronuclei per 500 cytokinesis-blocked cells Group (M±SD) Unirradiated control 3.333 ± 0.577 HIM- n (30 fig/mi) 3.667 ± 0.577 HIM-n (10 vgMH) 4.00 ± 1.000 Irradiation control (3 Gy) 295.333 ± 19.553 HIM- II (30 mMH) + irradiation 204.333 ± 22.502* HIM- E (10 fig/ml) + irradiation 192.667 ± 12.662* Irradiation + P2 (30 jig/mi) 244 ± 29.614 Irradiation + P2 (10 iig/ml) 247.667 ± 27.465 *p<0.01 as compared to the irradiation control group. - 123 - Table 1.53. Effect of the fractions of HIM- I on 30-day lethality in irradiated mice (M±SD) Group Survival rate (%) Mean survival time (days) Unirradiated control 30.000 ± 0.000 Irradiation control (8 Gy) 33.33 18.055 ± 8.795 HIM- I -W (1 mg/animal) + irradiation 55.56 21.944 ± 9.396 HIM- I -M (0.66 mg/animal) + irradiation 28.57 17.429 ± 8.465 HIM- I -Pi (0.17 mg/animal) + irradiation 50.00 21.611 ± 8.793 HIM- I -W: water extract of HIM- I . HIM- I -M: methanol fraction of HIM- I . HIM- I -Pi: inner part of polysaccharide fraction of HIM- I . The fractions were given i.p. at 36 and 12 hours before irradiation. - 124 - Table 1.54. Effect of the fractions of HIM-II on 30-day lethality in irradiated mice (M±SD) Group Survival rate (%) Mean survival time (days) Unirradiated control 30.000 ± 0.000 Irradiation control (8 Cy) 33.33 18.055 ± 8.795 HIM-II-W (1 mg/animal) + irradiation 41.18 19.649 ± 8.077 HIM-n-M (0.63 mg/animal) + irradiation 26.67 17.467 ± 8.043 HIM-II-Pi (0.20 mg/animal) + irradiation 44.44 20.556 ± 8.880 HIM- II -W: water extract of HIM- n. HIM-n-M: methanol fraction of HIM-IL HIM-II-Pi: inner polysaccharide fraction of HIM-II. The fractions were given i.p. at 36 and 12 hours before irradiation. - 125 - 4- (1) 4f-3 S^ 3H«H 2^-1: -fi-S HIM-I^l M, E, P HIM-I , Apoptosis ^-(HIM-I, f-o.^ ^^^$14. ^r, HIM-I-Wi f-1, HIM-H-Wi HIM-E-Pi (2) 2 g olAo>o. , 7Hlf- 10 g 1.55). ; U g/kg 0.5 g/kg BW - 126 - (3) , 50 mM tris-HCl buffer(pH 7.0)5. *%%^ DEAE-Cellulose(CD column(5xll cm)0!] •%: £*H?i ^ ^v^ buffer^ Ji 2N £*HM li Jl Sepharose CL-4B^-^-S gel permeation chromatography# 45} ^el§}ji %^ assay* ' GC, GC-MS, NMR^ H1fe HIM-I ^ HIM-E^ #^##(W)4 methanol - 127 - Table 1.55. Acute toxicity of provisional product 1 and 2 in mice LD50 (mg/kg body weight) Treatment Route of administration Male Female Oral intubation > 10,000 M0.O0O Provisional product 1 Intraperitoneal injection > 2,000 > 2,000 Oral intubation > 10,000 > 10,000 Provisional product 2 Intraperitoneal injection 920 408 - 128 2. 7]^ ^€5-^ 3^3-* 3$ #*]-# 2:47] 7} ^H ^^H ethylene oxide(EO) £f^ f-£ #53:4^ l:^, f-^SJJ-, 2^> ±°S 7} A>-g-o] ^]§> WI-JL ^4. ojofl rcj-ej- Afl^L^ ^^S)- 7] i $14(1-4). 10 7K (1) (2) ^H# 4^ ^ -44-l: 2.5, 5, 10 k'Gy 5 kGy 2:4 O_4, 10 kGy 2:Af ^6fl^ S^ A) 2.1). - 129 - Table 2.1. Effects of gamma irradiation on the growth of total aerobic bacteria in herbs Total aerobic bacteria(CFU/g sample) Korean name 0 kCy 2.5 kGy 5 kGy 10 kGy 2,016,000 2,100 0 0 300,000 233 0 0 0 0 0 0 9,250 600 133 0 3,675,000 8,000 133 0 1,100 133 0 0 *> 2,472,000 120,833 49,200 0 1,590,000,000 533,367 14,866 0 9,720,000 151,633 50,933 0 5,265,000 144,833 0 850 0 0 0 100 0 0 0 120 0 0 0 0 0 0 0 7\A 50 0 0 0 - 130 - (1) #4# 24 A (7» 10 kGy^l #*}#-k 2:4?!: ^1^(5^^ ^-g-7}^ • >S^fl)oflA-] 2.2). 10 2.3, Fig. 2.1, Table 2.4, Fig. 2.2, Table 2.5). (2) # (7]-) 2,A} (Table 2.6). 2.7). - 131 - (4) Apoptosis^^ Al^ 2:4 ^SL^f- ^f-^^H apoptosis 4(Table 2.8). (3) benzo( a )pyreneAS. Salmonella typhimurium TA98, TA100 2.9, Table 2.10). CHO ^Ii^l4 benzo( 2.11). (4) DPPHN cfl^: 2.12). - 132 Table 2.2. Lymphocyte proliferation by water extracts of korean medicinal herbs irradiated with gamma ray (10 kGy) Concentration cpm Sample (jUg/lM) Day 2 Day 3 Day 4 Negative Control 4399 ± 161 3163 ± 736 2063 ± 1274 2000 25437 ± 5754 69841 ± 5054 105111 ± 13405 Angelica gigas 1000 39039 ± 451 106608 ± 160 90345 ± 11632 500 43874 ± 1716 90492 ± 8461 77852 ± 7338 2000 23963 ± 183 85752 ± 1232 89289 ± 1038 Cnidium officinale 1000 30421 ± 1283 79626 ± 7569 84836 ± 7384 500 39710 ± 5578 63767 ± 1750 78767 ± 7647 600 3732 ± 589 1968 ± 159 7340 ± 594 Paeonia japonica 300 11720 ± 65 10563 ± 1380 7214 ± 284 150 13304 ± 313 12761 ± 1636 5828 ± 210 600 18694 ± 4486 59417 ± 8281 111496 ± 9058 Acanthopanax sessiliflorum 300 42967 ± 2694 141888 ±1117 141057 ±1113 150 44938 ± 2243 115309 ± 2585 109994 ± 3198 1000 7476 ± 441 5058 ± 473 3765 ± 2 Crataegus pinnatifida 500 7724 ± 330 5891 ± 835 4345 ± 414 250 7328 ± 73 4716 ± 105 3057 ± 907 Positive Control PHA 96651 ± 10836 112050 ± 18939 64295 ± 20552 LPS 20 239137 ± 640 76815 ± 3325 16604 ± 3626 - 133 - Table 2.3. Effect of gamma-irradiated Angelica gigas on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage Sample Splenocyte/spleen No. of plaque/7 X 10D splenocyte PFC/spleen (mg/kg B.W.) 2.0xl077fl 60, 102, 65 ( 75.7) 2,163 3.7xiO77fl 9, 18, 20 (15.7) 830 Saline 4.9xiO/7fl 68, 69, 44 (60.3) 4,221 88, 96, 89 (91) 806 4.4xl077fl 93, 99, 113 (101.7) 6,393 8.2 x 1O77U 151, 128, 135 (139.5) 16,166 Angelica gigas 20 (non-irradiated) 5.7X1O77H 97, 101, 81 (93) 7,573 4.8 x 1077fl 53, 60, 53 (55.3) 3,792 6.3 x 1077H 78, 96, 91 (88.3) 7,947 Angelica gigas 4.5 x 1077|l 123, 103, 115 (113.7) 7,309 (irradiated 20 7 10 kCy) 6.9 x 1O 7H 90, 90, 93 (91.0) 8,970 6.8xiO7?U 60, 36, 31 (42.3) 4,109 - 134 - 12000 r 10000 8000 CD 6000 O u_ 4000 2000 0 Control Angelica gigas Angelica gigas Non-irradiation Irradiation (0 kGy) (10 kGv) Fig 2.1. Effect of non-irradiation(0 kGy) and irradiatedQO kGy) Angelica gigas on the secretion of antibody tested by hemolytic plaque forming cell assay (M±S.E.) - 135 - Table 2.4. Effect of irradiated KJ-1 on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage No. of plaque/7 x 105 Sample Splenocyte/spleen PFC/spleen (mg/kg B.W.) splenocyte 1.3 x 107 cell/me 14, 17, 19 (16.7) 311 3.2 x 107 cell/me 69, 52, 64 (61.7) 2,820 4.6 x 107 cell/me 26, 45, 42 (37.7) 2,477 Saline 8,5 x 107 cell/roe 32, 34, 36 (34) 4,128 5 x 107 cell/me 73, 71, 63 (69) 4,927 5,7 x 107 ceWmX, 44, 52, 38 (44.67) 3,638 1.4 x 108 cell/m£ 54, 50, 50 (51.3) 10,260 7.5 x 107 cellM 49, 58, 46 (51) 5,457 8 KJ-1 1.1 x 10 cell/m« 69, 48, 58 (58.3) 9,159 120 + 120 (non-irradiated) 9.7 x 107 cell/ml 36, 34, 44 (38) 5,266 9,3 x 107 cell/mH 56, 56, 56 (56) 7,776 9.2 x 107 ceWmi 54, 44, 60 (56.67) 7,448 6.4 x 107 cell/mH 56, 58, 52 (55.3) 5,056 7.4 x 107 cell/nrf 90, 90, 76 (85.3) 9,018 KJ-1 9.8 x 107 cell/me 44, 38, 38 (38.67) 5,414 (irradiated, 120 + 120 8.4 x 107 cell/me 38, 70, 90 (66) 7,920 10 kGy) 9.2 x 107 cell/mi 44, 42, 62 (49.3) 6,480 1.1 x 108 cell/me 42, 48, 78 (56) 8,800 - 136 - 9000 8000 - r—I—, T 7000 - c 6000 f 5000 tn O 4000 - X 3000 2000 1000 0 Contra KJ-1 KJ-1 Non-irradiation Irradiation (0 kCv) (10 kCv) Fig 2. 2. Effect of non-irradiation(0 k.Gy) and irradiateddO kGy) KJ-1 on the secretion of antibody tested by hemolytic plaque forming cell assay (M±S.E.) - 137 - Table 2.5. Effect of irradiated Angelica gigas on Graft vs. Host reaction spleen w.t./body Group Donor Recipient Sex Spleen index w.t.(xiO3) Saline BDF1 BDF1 M 2.333 ± 0.233 1.00 3.413 ± 0.434 1.00 Saline C57BL/6 BDF1 M 4.595 ± 0.714 1.970 4.227 ± 0.180 1.238 C57BL/6 BDF1 M 7.633 ± 1.362 Angelica gigas 3.271" (non-irradiated) 100 mg/kg B.wt. 8.673 ± 1.577 2.541* Angelica gigas C57BL/6 BDF1 M 5.445 ± 0.686 2.334* (irradiated, 10 kGy) 1.847 100 mg/kg B.wt. 6.303 ± 1.383 * p<0.05 as compared with control. ** p<0.01 as compared with control. - 138 Table 2.6. The intestinal crypt survival in irradiated mice treated with irradiated or unirradiated Oriental medical prescriptions (M± SD) Groups Crypts per circumference Normal control 156.25 ± 6.051 Unirradiated Si-Wu-Tang 153.341 ± 7.312 Irradiated Si-Wu-Tang(10 kGy) 161.435 ± 7.361 Unirradiated Bu-Zhong-Yi-Qi-Tang 150.00 ± 4.768 Irradiated Bu-Zhong-Yi-Qi-Tang 166.917 ± 5.428 Unirradiated San-Ling-Bai-Shu-San 149.546 ± 6.456 Irradiated San-Ling-Bai-Shu-San 163.748 ± 7.384 Irradiation(12 Gy) without prescription 19.860 ± 8.010 Unirradiated Si-Wu-Tang + irradiation 35.749 ± 11.620* Irradiated Si-Wu-Tang + irradiation 34.84 ± 13.917* Unirradiated Bu-Zhong-Yi-Qi-Tang + irradiation 35.641 ± 14.593* Irradiated Bu-Zhong-Yi-Qi-Tang + irradiation 30.356 ± 7.546* Unirradiated San-Ling-Bai-Shu-San + irradiation 38.204 ± 15.418* Irradiated San-Ling-Bai-Shu-San + irradiation 31.789 ± 11.433 *p<0.05 as compared with irradiation without prescription group. - 139 - Table 2.7. The endogenous spleen colony formation in irradiated mice treated with irradiated or unirradiated Oriental medical prescriptions (M± SD) Groups Number of colony Irradiation(6.5 Gy) without prescription 0.375 ± 0.518 Unirradiated Si-Wu-Tang + irradiation 3.554 ± 3.615* Irradiated Si-Wu-Tang(10 kGy) + irradiation 2.844 ± 2.877* Unirradiated Bu-Zhong-Yi-Qi-Tang + irradiation 4.667 ± 5.268* Irradiated Bu-Zhong-Yi-Qi-Tang + irradiation 4.551 ± 4.502* Unirradiated San-Ling-Bai-Shu-San + irradiation 1.00 ± 0.707* Irradiated San-Ling-Bai-Shu-San + irradiation 1.333 ± 1.00* *p<0.05 as compared with irradiation without prescription group. - 140 - Table 2.8. The incidence of cell death by apoptosis in intestinal crypts of irradiated mice treated with irradiated or unirradiated Oriental medical prescriptions (M±SD) Apoptotic cells per crypts Groups Base Total Normal control 0.062 ± 0.025 0.084 ± 0.031 Unirradiated Si-Wu-Tang 0.053 ± 0.035 0.061 ± 0.041 Irradiated Si-Wu-Tang(10 kGy) 0.061 ± 0.025 0.072 ± 0.041 Unirradiated Bu-Zhong-Yi-Qi-Tang 0.054 ± 0.028 0.061 ± 0.051 Irradiated Bu-Zhong-Yi-Qi-Tang 0.072 ± 0.012 0.082 ± 0.041 Unirradiated San-Ling-Bai-Shu-San 0.042 ± 0.024 0.052 ± 0.034 Irradiated San-Ling-Bai-Shu-San 0.052 ± 0.025 0.064 ± 0.034 Irradiation(2 Gy) without prescription 5.663 ± 1.734 6.244 ± 2.085 Unirradiated Si-Wu-Tang + irradiation 2.563 ± 0.266 2.931 ± 0.243* Irradiated Si-Wu-Tang + irradiation 2.663 ± 0.575 3.013 ± 0.700* Unirradiated Bu-Zhong-Yi-Qi-Tang + irradiation 1.944 ± 0.811 2.194 ± 0.887* Irradiated Bu-Zhong-Yi-Qi-Tang + irradiation 2.738 ± 0.904 3.025 ± 1.021* Unirradiated San-Ling-Bai-Shu-San + irradiation 2.581 ± 0.746 2.956 ± 0.694* Irradiated San-Ling-Bai-Shu-San + irradiation 2.838 ± 0.964 3.275 ± .1.115* *p<0.05 as compared with irradiation without prescription group. K*p<0.01 as compared with irradiation without prescription group - 141 - Table 2.9. Antimutagenic effect of water-soluble fractions of r -irradiated Curcuma longa Linne, Paeonia japonica Nakai, Scutellaria baicalensis Georgi in the S. typhimurium reversion assay Cone, No. of colonies/ plate Inhibition ratio Material IR1 S9 mix BP (//g/plate) TA98 H2O 41 43 37 (40) H2O 41 41 45 (42) C. longa3 3500 103 121 116 (113) 71.3 3500 112 96 109 (106) 74.1 3500 44 48 42 (45) 3500 46 43 47 (45) P. japo 7500 54 66 71 (63) 91.5 7500 59 66 78 (68) 89.5 7500 43 49 48 (47) 7500 56 45 51 (51) S. Baik.D 750 122 87 110 (106) 74.1 750 110 115 96 (107) 73.7 750 48 47 42 (46) 750 45 46 43 (45) 20 287 297 283 (289) 1 7 -irradiationdO kCy); 2 benzo( a )pyrene; 3 Curcuma longa Linne; 4 Paeonia japonica Nakai Scutellaria baicalensis Georgi - 142 - Table 2.10. Antimutagenic effect of water-soluble fractions of 7 -irradiated Curcuma longa, Paeonia japonica, Scutellaria baicalensis in the S. typhimurium reversion assay Cone, No. of colonies/ plate Inhibition Material IR1 S9 mix BP2 ratio (%) (//g/plate) TA100 H2O 183, 197, 201 (194) H2O 246, 223, 214 (228) C. longa 2,000 274, 265, 259 (266) 81.4 2,000 290, 255, 280 (275) 77.0 2,000 219, 228, 206 (218) 2,000 197, 207, 229 (211) P. japo. 7,500 243, 285, 242 (257) 85.4 7,500 264, 231, 265 (253) 87.8 7,500 214, 240, 249 (234) 7,500 231, 257, 235 (241) S. Baik: 200 311, 278, 303 (297) 66.2 200 284, 293, 300 (292) 68.6 200 275, 252, 259 (262) 200 264, 280, 258 (267) B(ff)P 20 370, 457, 468 (432) 7 -irradiationQO kGy); benzo( a )pyrene; Curcuma longa, 4 Paeonia japonica', ° Scutellaria baicalensis - 143 - Table 2.11. Antimutagenic effects of water-soluble fractions of 7 -irradiated Paeonia japonica, Curcuma longa and Scutellaria baicalensis in the micronucleus(MN) assay using cytokinesis-blocked CHO cells r~ ~ r- 11 Cells with MN , ,,, „ 3 Cone. XT I/imn 1 Cone. 2 Cells No. of MN/1000 cells Material IR S9 BP2 w/oMN j 2 3 4 MN (Mean ± S.D.) H2O - - 2946 51 22 1 0 58 29.3 ± 4.3 C. longa - 330 + + 2717 256 20 3 0 305 101.7 ± 11.1 C. longa + 330 + + 2746 228 24 2 0 282 94.0 ± 14.7 P. japo5 - 500 + + 2822 158 13 1 0 187 62.3 ± 13.7 P. japo. + 500 + + 2803 183 10 3 0 212 70.7 ± 11.5 S. baik.6 - 7 + + 2779 201 17 3 0 244 81.3 ± 10.1 S. baik. + 7 + + 2777 193 28 3 0 258 86.0 ± 14.7 B(a)P + 5 - + 2618 342 29 9 2 435 145.0 ± 9.2 7 -irradiation(10 kGy); benzo( a )pyrene; 3 Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored 4 Curcuma longa ', n Paeonia japonica ', b Scutellaria baicalensis - 144 - Table 2.12. Effects of gamma irradiation in electron donating ability of herbs. Electron donating abilities(O.D) Herbs Irradiation Water extracts ME/AC extracts 0 kCy 57.24%/lm£ 58.26%/lm£ (1003*0 Angelica gigas 10 kGy 59.82%/lm£ 66.20%/lmH (1003*0 0 kGy . 24.93%/lm£ 57.68%/0.2m£ (10s)*}) Cnidium officianle 10 kGy 16.59%/lm£ 82.28%/0.2m£ (103*1) 0 kGy 56.38%/lm£ 42.73%/0.2m£ (1003*1) Acanthopanax sessiliflorum 10 kGy 66.44%/lm£ 38.96%/0.2m£ (1003*1) 0 kGy 43.39%/0.2m£ 89.16%/1 m« (10003*0 Paeonia japonica 10 kGy 53.62%/0.2m£ 82.27%/lm£ (10003*1) 0 kGy 59.91%/1 m« 82.28%/0.2m£ (103*0 Crataegus pinnatifida 10 kGy 69.32%/! m? 83.10%/0.2me (103*0 - 145 - Table 2.13. Revertant colonies in the S. typhimurium reversion assay with water-soluble fraction of r -irradiated Paeonia japonica Number of revertant Test Irradiat Dose S9 mix colonies(His') per plate Material ion (yUg/plate) TA98 TA100 TA102 H2O 23 ± 9 170 ± 6 297 ± 58 Test 15,000 33 ± 4 166 ± 12 302 ± 20 material 5,000 23 ± 1 156 ± 11 335 ± 16 - 1,667 27 ± 4 159 ± 1 339 ± 8 - 556 19 ± 7 154 ± 11 310 ± 13 - 185 24 ± 2 167 ± 11 307 ± 2 + - 15,000 31 ± 11 158 ± 9 308 ± 7 + 5,000 30 ± 2 154 ± 11 322 ± 30 + 1,667 18 ± 1 141 ± 1 304 ± 9 + - 556 18 ± 6 143 ± 20 299 ± 4 + - 185 20 ± 2 154 ± 13 313 ± 13 NPD 20 2340 ± 92 Na-Azid 1.5 1229 ±103 MMC 0.5 5189 ± 306 H2O 24 ± 4 227 ± 7 309 ± 14 H2O - + 31 ± 3 257 ± 6 342 ± 16 Test - + 15,000 31 ± 3 315 ± 25 394 ± 3 material - + 5,000 25 ± 8 254 ± 11 382 ± 18 - + 1,667 25 ± 3 258 ± 6 367 ± 12 + 556 32 ± 12 246 ± 10 351 ± 25 - + 185 20 ± 1 235 ± 14 321 ± 16 + + 15,000 29 ± 3 301 ± 6 411 ± 6 + + 5,000 24 ± 1 226 ± 6 378 ± 8 + + 1,667 26 ± 10 229 ± 1 350 ± 35 + + 556 25 ± 11 247 ± 11 329 ± 13 + + 185 25 ± 1 223 ± 25 319 ± 11 2-AF - + 10 1641 ±158 909 ± 78 531 ± 57 a IrradiationdO kGy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMC(mitomycin C) and 2-AF (2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean ± S.D. of revertant colonies per plate in duplicate experiments except for negative control groups(triplicate). - 146 - Table 2.14. Revertant colonies in the 5. typhimurium reversion assay with methanol-soluble fraction of 7 -irradiated Paeonia japonica Number of revertant Test Irradia Dose S9 mix colonies(His*) per plate Material tion (/zg/plate) TA98 TA100 TA102 DMSO 24 ± 10 246 ± 18 299 ± 24 Test GI(50%)b 25 ± 3 227 ± 1 297 ± 44 material GI(50%)/3 20 ± 1 209 ± 17 308 ± 16 GI(50%)/9 22 ± 1 214 ± 22 298 ± 2 - GI(50%)/27 27 ± 8 244 ± 4 326 ± 43 - GI(50%)/81 23 ± 2 232 ± 30 307 ± 11 + - GI(50%)b 27 ± 1 213 ± 8 332 ± 9 + - GI(50%)/3 24 ± 2 222 ± 10 297 ± 23 + GI(50%)/9 29 ± 2 254 ± 7 341 ± 7 + GI(50%)/27 28 ± 5 238 ± 4 330 ± 34 + - GI(50%)/81 18 ± 5 218 ± 9 310 ± 33 NPD 20 1897 ± 40 Na-Azid 1.5 994 ±138 MMC 0.5 5840 ±161 DMSO 25 ± 4 224 ± 19 337 ± 9 DMSO - + 31 ± 4 245 ± 10 351 ± 18 Test - + GI(50%)b 28 ± 1 236 ± 17 337 ± 37 material - + GI(50%)/3 31 ± 6 214 ± 1 350 ± 13 - + GI(50%)/9 29 ± 5 224 ± 4 338 ± i - + GI(50%)/27 20 ± 1 226 ± i 382 ± 21 + GI(50%)/81 27 ± 8 206 ± 12 349 ± 11 + + GI(50%)b 27 ± 1 259 ± 28 334 ± 23 + + GI(50%)/3 24 ± 2 213 ± 8 368 ± 9 + + GI(50%)/9 29 ± 2 223 ± 1 383 ± 28 + + GI(50%)/27 28 ± 5 231 ± 9 328 ± 6 + + GI(50%)/81 18 ± 5 214 ± 21 342 ± 8 2-AF - + 10 1090 ± 95 772 ± 74 553 ± 63 a Irradiation (10 kCy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMCCmitomycin C) and 2-AF(2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean ± S.D. of revertant colonies per plate in duplicate experiments except for negative control groups(triplicate). - 147 - Table 2.15. Revertant colonies in the S. typhimurium revertant assay with water soluble of 7 -irradiated Angelica gigas Number of revertant Test Dose "Irradiation S9 mix colonies (His*) per plate Material TA100 TA98 H2O 140 157 146 (148) 43 43 33 (40) Test 5,000 219 221 (220) 56 49 (53) material 1,650 170 139 (155) 39 45 (42) 550 134 159 (147) 39 43 (41) 180 152 145 (149) 41 36 (39) 61 128 154 (141) 41 44 (43) 5,000 200 210 (205) 40 44 (42) 1,650 141 159 (150) 31 33 (32) 550 132 142 (137) 31 35 (33) 180 151 147 (149) 37 31 (34) 61 141 121 (131) 30 32 (31) Na-Azid 1.5 1121 1269(1195) NPD 20 2021 1968(1995) H2O 240 236 260 (245) 32 31 29 (31) Test 5,000 325 327 (326) 32 34 (33) material 1,650 246 258 (252) 34 32 (33) 550 261 250 (256) 27 34 (31) 180 236 243 (240) 30 34 (32) 61 228 237 (233) 25 35 (30) 5,000 312 292 (302) 33 38 (36) 1,650 237 249 (243) 26 31 (29) 550 246 235 (241) 26 27 (27) 180 240 229 (235) 32 32 (32) 61 236 238 (237) 41 27 (34) 2-AF 10 792 732 (762) 2444 2203 (2324) a IrradiationdO kGy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide) and 2-AF(2-aminofluorene) were used as positive controls for the corresponding strains. - 148 - Table 2.16. Revertant colonies in the S. typhimurium revertant assay with water-soluble of y -irradiated Cnidium officinale Number of revertant Test Dose aIrradiation S9 mix colonies(His') per plate Material (^g/plate) TA100 TA98 H2O 172 173 173 (173) 40 40 37 (39) Test 5,000 185 182 (183) 36 41 (38) material 1,650 148 179 (164) 41 33 (37) 550 151 142 (147) 34 37 (35) 180 180 160 (170) 38 40 (39) 61 168 148 (154) 42 41 (41) 5,000 214 195 (205) 31 39 (35) 1,650 181 172 (177) 44 34 (39) 550 174 166 (170) 29 36 (33) 180 181 149 (165) 34 42 (38) 61 155 146 (151) 29 34 (32) Na-Azid 1.5 1121 1269(1195) NPD 20 2021 1968(1995) H2O 141 146 (143) 59 48 51 (53) Test 5,000 159 179 (169) 62 64 (63) material 1,650 141 140 (141) 50 58 (54) 550 133 135 (134) 55 49 (52) 180 144 140 (142) 49 55 (52) 61 136 152 (144) 54 50 (52) 5,000 156 150 (153) 48 48 (48) 1,650 150 147 (149) 60 61 (61) 550 142 141 (142) 47 56 (52)- 180 142 140 (141) 46 60 (53) 61 132 135 (134) 55 48 (52) 2-AF 10 792 732 (762) 2444 2203 (2324) IrradiationdO kCy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide) and 2-AF(2-aminofluorene) were used as positive controls for the corresponding strains. - 149 - Table 2.17. Revertant colonies in the 5. typhimurium reversion assay with water-soluble fraction of 7 -irradiated Curcuma longa Number of revertant Test Irradia Dose S9 mix colonies (His+) per plate Material tion (jug/plate) TA98 TA100 TA102 H20 26 + 5 241 ± 19 310 ± 33 Test 7,500 32 ± 4 275 ± 3 391 ± 8 material 2,500 27 ± 1 252 ± 23 371 ± 23 833 27 ± 1 237 ± 12 359 ± 10 278 24 ± 3 238 ± 4 306 ± 39 93 22 ± 4 240 ± 13 297 ± 25 7,500 22 ± 1 256 ± 17 384 ± 14 2,500 28 ± 1 219 ± 4 351 ± 1 833 25 ± 4 225 ± 4 312 ± 8 278 25 ± 5 232 ± 22 276 ± 10 93 25 ± 3 241 ± 18 280 ± 20 NPD 20 2164 ± 55 Na-Azid 1.5 1136 ± 54 MMC 0.5 5205 ± 138 H2O 23+3 214 ± 9 294 ± 6 H2O 36 ± 8 255 ± 19 324 ± 15 Test 7,500 33 ± 8 228 ± 13 344 ± 12 material 2,500 35 + 10 256 ± 3 336 ± 16 833 34 ± 4 239 ± 7 315 ± 74 278 35 ± 8 250 ± 18 342 + 33 93 37 ± 1 227 ± 6 308 ± 52 7,500 34 + 4 243 ± 20 354 11 2,500 34 ± 6 261 ± 27 346 11 833 33 + 4 245 ± 38 320 41 278 30 ± 6 222 ± 3 312 13 93 38 ± 2 254 ± 35 324 34 2-AF 10 1186 ± 25 762 ± 59 589 81 a Irradiation(10 kGy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMC(mitomycin C) and 2-AF(2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean ± S.D. of revertant colonies per plate in duplicate experiments except for negative control groups (triplicate). - 150 - Table 2.18. Revertant colonies in the S. typhimurium reversion assay with methanol-soluble fraction of y -irradiated Curcuma longa Number of revertant Test aIrradia Dose S9 mix colonies(His*) per plate Material tion (^g/plate) TA98 TA100 TA102 DMSO 24 ± 4 170 6 297 ± 58 Test 40.0 34 ± 2 166 12 302 ± 20 material 13.3 28 ± 7 156 11 335 ± 16 4,4 32 ± 4 159 1 339 ± 8 1.3 28 ± 1 154 11 310 ± 13 0.4 24 ± 2 167 11 307 ± 2 40.0 26 ± 6 158 ± 9 308 ± 7 13.3 25 ± 1 154 ± 11 322 ± 30 4,4 24 ± 1 141 ± 1 304 ± 9 1.3 23 ± 1 143 ± 20 299 ± 4 0.4 24 ± 3 154 ± 13 313 ± 13 NPD 20 2049 ± 40 Na-Azid 1.5 1243 ± 38 MMC 0.5 5110 ± 139 DMSO 31 ± 6 202 ± 23 295 ± 11 DMSO 34 ± 5 225 ± 16 329 ± 15 Test 40.0 25 ± 4 217 ± 7 353 ± 24 material 13.3 30 ± 8 217 ± 2 328 ± 25 4,4 33 ± 7 206 ± 4 313 ± 17 1.3 35 ± 2 202 ± 11 328 ± 18 0.4 35 ± 7 223 ± 2 325 ± 18 40.0 30 ± 10 215 ± 13 304 ± 10 13.3 31 ± 3 212 ± 2 320 ± 8 4,4 25 ± 8 208 ± 7 313 ± 14 1.3 34 ± 3 212 ± 6 281 ± 28 0.4 29 ± 4 218 ± 13 278 ± 2 2-AF 10 1247 ± 57 711 ± 30 488 ± 47 IrradiationdO kCy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMC(mitomycin C) and 2-AF (2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean ± S.D. of revertant colonies per plate in duplicate experiments except for negative control groups (triplicate). - 151 - Table 2.19. Revertant colonies in the S. typhimurium reversion assay with water-soluble fraction of 7 -irradiated Scutellaria baicalensis a Number of revertant Test Irradia Dose S9 mix colonies (His) per plate Material tion (^g/plate) TA98 TA100 TA102 H2O 27 ± 6 203 ± 18 317 ± 21 Test 1,500 34 ± 7 202 ± 11 346 ± 8 material 500 28 ± 4 210 ± 9 320 ± 13 167 30 ± 8 188 ± 6 313 ± 12 56 20 ± 2 203 ± 15 323 ± 16 19 29 ± 11 208 ± 4 308 ± 1 1,500 25 ± 4 189 ± 6 360 ± 1 500 28 ± 1 196 ± 6 317 ± 10 167 32 ± 2 177 ± 6 330 ± 5 56 32 ± 9 185 ± 11 313 ± 4 19 31 ± 4 192 ± 4 327 ± 20 NPD 20 2226 ±173 Na-Azid 1.5 1362 ±126 MMC 0.5 5265 ±228 24 ± 5 252 ± 9 H2O 323 ± 28 H2O 38 ± 4 270 ± 14 333 ± 74 Test 1,500 38 ± 4 273 ± 5 372 ± 31 material 500 43 ± 4 256 ± 1 355 ± 4 167 30 ± 1 266 ± 6 342 ± 69 56 38 ± 4 258 ± 6 318 ± 29 19 42 ± 13 224 ± 16 297 ± 13 1,500 27 ± 287 ± 28 353 ± 43 500 38 ± 258 ± 23 307 ± 12 167 32 ± 223 ± 9 313 ± 11 56 29 ± 224 ± 8 327 ± 11 19 28 ± 215 ± 13 293 ± 4 2-AF 10 1648 ±350 874 ± 47 606 ± 50 IrradiationdO kGy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMC(mitomycin C) and 2-AF(2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean ± S.D. of revertant colonies per plate in duplicate experiments except for negative control groups(triplicate). - 152 - Table 2.20. Revertant colonies in the S. typhimurium reversion assay with methanol-soluble fraction of y -irradiated Scutellaria baicalensis Number of revertant Test Irradia Dose S9 mix coloniesdnuV) per plate Material tion (//g/plate) TA98 TA100 TA102 DMSO 22 ± 2 61 ± 3 161 ± 3 Test GI(50%)b 20 ± 6 154 ± 4 268 ± 39 material GI(50%)/3 18 ± 2 162 ± 7 305 ± 12 GI(50%)/9 21 ± 6 168 ± 8 287 ± 21 GI(50%)/27 19+8 152 ± 17 294 ± 18 GI(50%)/81 13 ± 1 153 ± 4 292 ± 8 GI(50%)b 16 ± 169 ± 1 277 ± 10 GI(50%)/3 16 ± 154 ± 7 275 ± 22 GI(50%)/9 17 ± 165 ± 1 289 ± 16 GI(50%)/27 18 ± 151 ± 19 305 ± 1 GI(50%)/81 19 ± 142 ± 33 281 ± 1 NPD 20 2178 ±197 Na-Azid 1.5 1227 ±173 MMC 0.5 5520 ±356 DMSO 24 ± 2 221 ± 23 297 ± 15 DMSO 35 ± 3 241 ± 26 335 ± 8 Test GI(50%)b 26 ± 221 ± 13 357 ± 4 material GI(50%)/3 32 ± 219 ± 3 367 ± 1 GI(50%)/9 31 ± 224 ± 8 336 ± 18 GI(50%)/27 32 ± 215 ± 21 353 ± 38 GI(50%)/81 25 ± 200 ± 1 305 ± 3 GI(50%)b 26 ± 6 217 ± 4 363 11 GI(50%)/3 34 ± 2 216 ± 4 344 23 GI(50%)/9 29 ± 4 190 ± 8 340 59 GI(50%)/27 31 ± 6 171 ± 8 336 4 GI(50%)/81 32 ± 1 194 ± 2 312 6 2-AF 10 1687 ±121 871 ± 19 523 81 Irradiation(10 kGy of Co-60 gamma-ray) was treated to the medicinal herb before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMC(mitomycin C) and 2-AF(2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean ± S.D. of revertant colonies per plate in duplicate experiments except for negative control groups (triplicate). - 153 - Table 2.21. Revertant colonies in the S. typhimurium reversion assay with water extract of 7 -irradiated Houttuynia cordata colonies (/z/s')per plate Test a S9 Dose Number of revertant Irradiation Material mix (/ig/plate) TA 98 TA 100 H2O - 29 44 38 (37) 178 144 175 (166) Houttuynia 5000 43 42 (43) 185 179 (182) cordata 1650 32 26 (29) 156 174 (165) - 550 37 38 (38) 143 155 (149) - 180 27 36 (32) 162 138 (150) - 61 40 41 (41) 164 166 (165) + 5000 34 47 (39) 190 173 (182) + 1650 37 37 (37) 165 157 (161) 550 30 37 (34) 152 149 (151) + - 180 29 44 (37) 162 141 (152) + 61 28 31 (30) 152 170 (161) NPD 20 2021 1968 (1995) Na-Azide 1.5 1121 1269 (1195) H20 - + - 66 52 46 (55) 232 222 235 (230) Houttuynia - + 5000 53 53 (53) 236 237 (237) cordata - + 1650 54 65 (60) 240 223 (232) - + 550 50 50 (50) 232 226 (229) - + 180 44 46 (45) 242 238 (240) - + 61 55 59 (57) 237 208 (223) + + 5000 67 54 (61) 242 238 (240) + + 1650 47 62 (55) 217 242 (230) + + 550 44 42 (43) 215 221 (218) + + 180 54 49 (52) 226 246 (236) + + 61 53 49 (51) 222 216 (219) 2-AF - + 10 792 732 (762) 2442 2203 (2324) d Irradiation(10 kCy of Co-60 gamma-ray) was treated to the herbs before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide) and 2AF(2-aminofluorene) were used as positive controls for the corresponding strains. - 154 - Table 2.22. Revertant colonies in the S. typhimurium reversion assay with water extract of 7 -irradiated Lycium chinense Miller + Test Dose Number of revertant colonies(/zzs )per plate Irradiation8 S9 mix Material (/zg/plate) TA 98 TA 100 H2O 22 25 23 (23) 150 178 155 (161) Lycium 5000 24 27 (26) 157 177 (167) chinense 1650 21 20 (21) 168 157 (163) 550 20 26 (23) 147 152 (150) 180 14 20 (17) 149 140 (145) 61 18 16 (17) 128 135 (132) 5000 29 27 (28) 193 168 (181) 1650 21 18 (20) 160 163 (162) 550 24 19 (22) 124 160 (142) 180 22 22 (22) 141 160 (151) 61 18 17 (18) 130 143 (137) NPD 20 2021 1968 (1995) Na-Azide 1.5 1121 1269 (1195) H2O 38 35 47 (40) 162 151 147 (153) Lycium 5000 47 42 (45) 181 173 (177) chinense 1650 44 38 (41) 158 151 (155) 550 43 42 (43) 155 145 (150) 180 37 34 (36) 151 146 (149) 61 41 41 (41) 143 142 (143) 5000 46 45 (46) 180 169 (175) 1650 43 34 (39) 162 166 (164) 550 37 43 (40) 160 155 (158) 180 43 40 (42) 134 143 (139) 61 49 43 (46) 135 140 (138) 2-AF 10 792 732 (762) 2442 2203 (2324) IrradiationQO kGy of Co-60 gamma-ray) was treated to the herbs before extraction. NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide) and 2AF(2-aminofluorene) were used as positive controls for the corresponding strains. - 155 - (1) Salmonella typhimurium TA98, TA100, TA102 S"^£| 4^1" ^Ml- *]•%•*} Ames test(129~132)i^ ^^^i(10 kGy) 2.13-2.22). S-n-^1- 4S.91 CHO 4^iAi i^l-n-^1^(133-138)^: <>}•%•* kGy) 2.23-2.32). - 156 - Table 2.23. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water-soluble fraction of 7 -irradiated Paeonia japonica Cone. Cells Cells with MNb No. of MN/1000 cells0 Material IRa gg w/o MN 1 4 MN (Mean ± S.D.) H20 2,940 56 4 0 0 64 21.3 ± 5.0 Test 1500 2925 68 7 0 0 82 27.3 ± 7.8 material 500 2944 47 5 0 0 57 19.0 ± 3.6 150 2943 50 7 0 0 64 21.3 ± 2.5 1500 2934 60 6 0 0 24.0 ± 12.0 500 2951 44 5 0 0 54 18.0 ± 2.6 150 2934 57 2 2 0 67 22.3 ± 4.7 24 MMC 0.1 2712 33 8 0 337 112.3 ± 16.1 7 H2O 2944 53 2 1 0 60 20.0 ± 4.9 "Test 1500 2938 54 8 0 0 70 23.3 ± 2.5 material 500 2934 59 .7 0 0 73 24.3 ± 4.5 150 + 2927 66 3 1 0 75 25.0 ± 4.6 + 1500 + 2951 48 1 0 0 50 16.7 ± 2.1 + 500 + 2954 45 1 0 0 47 15.7 ± 2.5 + 150 + 2957 43 0 0 0 43 14.3 ± 2.1 29 MMC - 0.1 + 2656 38 7 1 399 133.0 ± 20.5 8 IrradiationdO kGy of Co-60 gamma ray) was treated to the medicinal herb before extraction. Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. c Number of MN/1,000 binucleated cells in the triplicated experiments. MMC(Mitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 157 - Table 2.24. Frequency of micronuclei (MN) in cytokinesis-blocked CHO cells following treatment with methanol-soluble fraction of 7 -irradiated Paeonia japonica Total No. of CB cells with n T Dose b MN/1000 cells0 Test a S9 MN Irradiation No. of material mix (ptg/ml) (Mean±S.D) 0 1 2 3 4 MN H20 2,938 58 3 1 0 67 22.3 ± 5.8 Test 500 2,941 50 7 2 0 70 23.3 ±6.8 material 150 2,942 53 5 0 0 63 21.0 ± 3.4 - 50 2,939 54 6 1 0 69 23.0 ± 2.9 500 2,932 60 7 1 0 77 25.6 ± 5.4 150 2,936 59 4 1 0 70 23.3 ± 2.5 50 2,937 55 6 2 0 73 24.3 ± 4.1 MMC 0.1 2,720 237 38 5 0 328 109.3 ±17.1 H2O - + 2,943 54 2 1 0 61 20.3 ± 4.7 Test - + 500 2,925 67 6 2 0 85 28.3 ± 7.8 material - + 150 2,960 36 4 0 0 44 14.7 ± 3.8 - 50 2,956 42 2 0 0 46 15.3 ± 4.5 500 2,946 47 7 0 0 62 20.7 ± 5.1 150 2,966 30 4 0 0 38 12.7 ± 5.7 50 2,957 43 5 0 0 53 17.7 ± 1.5 B(a )P - + 20 2,650 309 36 4 1 397 132.3 ±15.4 a IrradiationdO kGy of Co-60 gamma ray) was treated to the medicinal herb before extraction. b Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. 0 Number of MN/1,000 binucleated cells in the triplicated experiments. MMC(Mitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 158 - Table 2.25. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of / -irradiated Angelica gigas Total No. of CB cells Total MN/1000 Dose b Test S9 with n MN 0 Irradiation8 No. of cells material mix 0 1 2 3 4 MN (Mean±S.D) H2O 2937 42 3 0 0 66 22.0 ±5.7 Test 100 2942 40 2 0 0 64 21.3 ±3.8 material 300 2949 52 5 0 0 54 18.0 ±3.6 - 1000 2919 74 6 1 0 89 29.7 ±3.1 100 2946 65 5 2 0 58 19.3 ±3.1 + 300 2917 72 9 0 0 89 29.7 ±2.3 1000 2930 78 4 1 0 81 27.0 ±11.5 MMC 0.1 2704 237 38 5 0 346 115.3 ±19.6 H2O - + 2944 58 3 1 0 60 20.0 ±4.6 Test - + 100 2945 52 1 1 0 58 19.3 ±5.5 material - + 300 2938 47 1 0 0 68 22.7 ±2.5 - 1000 2929 56 7 1 0 87 29.0 ±9.2 100 2949 58 3 0 0 55 18.3 ±3.1 + 300 2921 58 11 2 0 89 29.6 ±2.1 + 1000 2919 69 10 0 0 90 30.0 ±5.0 BU)P 20 2648 304 37 6 0 400 133.3 ±21.4 a IrradiationdO kCy of Co-60 gamma ray) was treated to the medicinal herb before extraction. Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. c Number of MN/1,000 binucleated cells in the triplicated experiments. MMC(Mitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 159 - Table 2.26. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of 7 -irradiated Cnidium officinale Total No. of CB cells with Total MN/1000 Test S9 Dose b 8 n MN 0 Irradiation No. of cells material mix ing/ml) 0 1 2 3 4 MN (Mean±S.D) H2O - 2954 42 3 0 0 49 16.3 ±2.1 Test 100 2954 40 2 0 0 48 16.0 ±5.6 material 300 2943 52 5 0 0 62 20.7 ±4.0 - 1000 2919 74 6 1 0 89 29.7 ±3.1 + 100 2930 65 5 2 0 81 27.0 ±11.5 + 300 2919 72 9 0 0 90 30.0 ±5.0 + 1000 2917 78 4 1 0 89 29.7 ±2.3 MMC 0.1 2720 237 38 5 0 328 109.3 ±17.1 H20 + 2938 58 3 1 0 67 22.3 ±5.8 Test + 100 2964 52 1 1 0 57 19.3 ±3.9 material + 300 2952 47 1 0 0 49 16.3 ±6.7 - + 1000 2937 56 7 1 0 70 23.3 ±2.5 + + 100 2939 58 3 0 0 64 21.3 ±2.5 + + 300 2929 58 11 2 0 87 29.0 ±9.2 + + 1000 2921 69 10 0 0 89 29.6 ±2.1 B(ff)P + 20 2653 304 37 6 0 396 132.0 ±19.4 a IrradiationdO kCy of Co-60 gamma ray) was treated to the medicinal herb before extraction, b Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. c Number of MN/1,000 binucleated cells in the triplicated experiments. MMC(Mitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 160 - Table 2.27. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water-soluble fraction of y -irradiated Curcuma longa Total No. of CB cells with n Total Test S9 Dose MNb MN/1000 cells' 8a No. of material Irradiation (Mean±S.D) 0 1 MN H2O 2,938 57 4 1 0 68 22.7 ± 6.1 Test 1,000 2,953 43 4 0 0 51 17.0 ± 3.6 material 300 2,949 46 5 0 0 56 18.7 ± 3.1 100 2,948 50 2 0 0 54 18.0 ± 2.6 1,000 2,943 52 4 0 0 60 20.0 ± 4.0 300 2,962 36 2 0 0 40 13.3 ± 2.5 100 2,955 38 7 0 0 52 17.3 ± 5.7 MMC 0.1 2,709 249 34 7 1 342 114.3 ±15.8 H2O 2,964 52 1 1 0 57 19.0 ± 3.9 Test 1,000 2,952 47 1 0 0 49 16.3 ± 6.7 material 300 2,937 56 7 1 0 70 23.3 ± 2.5 100 2,939 58 3 0 0 64 21.3 ± 2.5 1,000 2,954 42 3 0 0 49 16.3 ± 2.1 300 2,954 40 2 0 0 48 16.0 ± 5.6 100 2,943 52 5 0 0 62 20.7 ± 4.0 B(a)P 20 2,653 304 37 6 0 396 132.0 ±19.4 IrradiationdO kGy of Co-60 gamma ray) was treated to the medicinal herb before extraction. Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. Number of MN/1,000 binucleated cells in the triplicated experiments. MMCXMitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 161 - Table 2.28. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with methanol-soluble fraction of y - irradiated Curcuma longa Total No. of CB cells with Total 0 Test S9 Dose b MN/1000 cells Irradiationa n MN No. of material mix (jUg/mC) (Mean±S.D) 0 1 2 3 4 MN H20 2,938 59 2 1 0 66 22.0 ±5.7 Test 150 2,920 64 16 0 0 87 29.0 ±9.2 material 50 2,945 46 5 2 0 89 29.6 ±2.1 - 15 2,919 74 6 1 0 89 29.7 ±3.1 + 150 2,919 72 9 0 0 90 30.0 ± 5.0 50 2,917 78 4 1 0 89 29.7 ± 2.3 15 2,930 65 5 2 0 81 27.0 ±11.5 MMC 0.1 2,704 256 32 6 2 346 115.3 ±19.6 H20 - + 2,944 53 2 1 0 60 20.0 ±4.6 Test - + 150 2,945 52 3 0 0 58 19.3 ±5.5 material - + 50 2,938 58 5 0 0 68 22.7 ±2.5 - 15 2,949 48 3 0 0 54 18.0 ±3.6 + 150 2,949 47 4 0 0 55 18.3 ±3.1 50 2,946 50 4 0 0 58 19.3 ±3.1 + 15 2,942 54 5 0 0 64 21.3 ±3.8 B(a)P - + 20 2,648 311 35 5 1 400 133.3 ±21.4 a IrradiationdO kGy of Co-60 gamma ray) was treated to the medicinal herb before extraction. Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. c Number of MN/1,000 binucleated cells in the triplicated experiments. MMC(Mitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 162 - Table 2.29. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water-soluble fraction of y -irradiated Scutellaria baicalensis Total No. of CB cells with n JL ULcli 0 Dose b MN/10 00 cells Test a S9 MN Irradiation NO. of material mix (jag/roe) (Meari±S.D) 0 1 2 3 4 MN H2O 2,938 59 2 1 0 66 22.0 ± 3.9 Test 500 2,920 64 16 0 0 106 32.0 ± 9.5 material 150 2,945 46 5 2 0 62 20.7 ± 3.1 - 50 2,924 66 4 2 0 80 26.7 ± 3.1 + 500 2,913 78 6 3 0 110 32.3 ± 11.5 + — 150 2,944 51 5 0 0 61 20.1 ± 4.2 50 2,934 62 2 2 0 71 23.7 ± 3.8 MMC 0.1 2,722 234 35 8 1 332 110.7 ±12.4 H2O - + 2,946 51 2 1 0 58 19.3 ± 4.3 Test - + 500 2,943 53 3 0 0 59 19.7 ± 3.8 material - + 150 2,962 37 1 0 0 39 13.0 ± 4.6 + 50 2,967 29 4 0 0 37 12.3 ± 3.2 + + 500 2,939 56 5 0 0 66 22.0 ± 2.6 + + 150 2,960 33 6 1 0 48 16.0 ± 2.6 + + 50 2,957 39 4 0 0 47 15.7 ± 5.7 B( a )P - + 20 2,719 239 36 9 0 338 112.7 ±17.5 a IrradiationQO kGy of Co-60 gamma ray) was treated to the medicinal herb before extraction. b Total number of cytokinesis-blocked (CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. c Number of MN/1,000 binucleated cells in the triplicated experiments. MMC(Mitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. 163 - Table 2.30. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with methanol-soluble fraction of y - irradiated Scutellaria baicalensis Total No. of CB cells with n X ULCU b 0 Test a S9 Dose MN MN/1000 cells Irradiation NO. of material mix (tig/xd) (Mean±S.D) 0 1 2 3 4 MN H2O 2,941 54 4 1 0 65 21.7 ± 7.2 Test 150 2,936 56 8 2 0 78 26.0 ± 5.9 material 50 2,936 69 4 1 0 70 23.3 ± 6.1 - 15 2,932 60 6 2 0 78 26.0 ± 4.1 + 150 2,929 62 9 0 0 70 23.3 ± 3.3 50 2,927 68 4 1 0 79 26.3 ± 3.0 + - 15 2,932 61 5 2 0 77 25.7 ± 2.8 MMC 0.1 2,720 239 34 7 0 328 109.3 ± 19.7 H2O - + 2,944 53 2 1 0 60 20.0 ± 4.4 Test - + 150 2,934 60 6 0 0 72 24.0 ± 3.6 material - + 50 2,944 51 5 0 0 61 20.3 ± 2.5 - + 15 2,945 50 4 1 0 61 20.3 ± 4.7 + + 150 2,949 49 2 0 0 53 17.7 ± 3.5 + + 50 2,955 38 5 2 0 48 16.0 ± 3.0 + + 15 2,955 40 4 1 0 51 17.0 ± 4.2 B( a )P - + 20 2,706 244 40 9 1 355 118.3 ±14.6 a IrradiationdO kGy of Co-60 gamma ray) was treated to the medicinal herb before extraction. b Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. c Number of MN/1,000 binucleated cells in the triplicated experiments. MMCXMitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 164 - Table 2.31. Frequency of Micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of 7 -irradiated Acanthopanax sessiliflorum Cells Frequency of the Cone. No. of MN/1000 cells0 Material IRa S9 without cells with MN (tig/id) MN (Mean ±S.D.) MN 1 2 3 4 H20 - - 2916 74 9 1 0 95 31.7 ± 10.1 Acanthopanax 1000 2883 106 11 1 0 128 42.7 ± 11.2 sessiliflorum 300 2926 68 5 1 0 91 27 ± 3.6 100 2913 78 8 0 0 97 32.3 ± 10.0 - 30 2922 73 5 0 0 83 27.7 ± 1.5 - 10 2905 90 5 0 0 110 33.3 ± 7.6 + - 1000 2894 93 13 0 0 119 39.7 ± 2.5 + - 300 2917 77 5 1 0 90 30 ± 3 + - 100 2924 71 5 0 0 81 27 ± 2.6 + - 30 2923 73 4 0 0 81 27 ± 1 + - 10 2934 60 5 1 0 63 24.3 ± 6.4 MMC - - 0.1 2720 237 38 5 0 328 109.3 ± 17.1 S9 - + 2939 60 1 0 0 62 20.7 ± 3.1 Acanthopanax - + 1000 2908 82 8 2 0 104 34.7 ± 5.5 sessiliflorum - + 300 2926 68 4 2 0 82 27.3 ± 6.7 - + 100 2924 66 4 2 0 80 26.7 ± 3.1 - + 30 2930 65 5 2 0 81 •• 27.0 ± 11.5 - + 10 2925 68 7 0 0 82 27.3 ± 7.8 + + 1000 2926 61 11 2 0 89 29.7 ± 10.0 + + 300 2943 51 6 0 0 63 21 = 2.6 + + 100 2928 66 5 1 0 79 26.3 - 2.70 + + 30 2928 66 4 1 1 81 27.9 = 5.70 + + 10 2931 60 7 1 1 81 28.1 ± 5.30 B(ff)P - + . 20 2648 311 35 5 1 400 133.3 ± 21.4 IrradiationdOkGy of Co-60 gamma ray) was treated to the medicinal herb before extraction. Number of MN/1000 binucleated cells in the triplicate experiments in which 1,000 cells were scored. MMCCMitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 165 - Table 2.32. Frequency of Micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of 7 -irradiated Lycium chinense Frequency of the cells with Cells No. of MN/1000 cells0 Material IRa without MN S9 ^ MN (Mean ± S.D.) MN 1 2 3 4 H20 - 2925 68 5 2 0 84 28.0 ± 4.4 Lycium - 3000 2933 64 3 0 0 70 23.3 ± 2.1 chinense - 1000 2911 84 5 0 0 94 31.3 ± 5.9 - - 300 2939 51 9 1 0 71 24.0 ± 3.6 + - 3000 2919 73 8 0 0 89 29.7 ± 1.5 - 1000 2932 66 1 1 0 71 23.7 ± 2.9 - 300 2932 58 9 0 0 76 25.3 ± 3.2 MMC 0.1 2720 237 38 5 0 328 115.3 ± 19.6 S9 2939 60 1 0 0 62 20.7 ± 3.2 Lycium + 3000 2924 72 4 0 0 80 26.7 ± 3.1 Chinense - + 1000 2950 41 8 1 0 60 20.0 ± 5.3 - + 300 2934 60 6 0 0 72 23.5 ± 5.8 + 3000 2930 67 2 1 0 74 24.7 ± 1.5 + 1000 2944 46 8 2 0 68 22.7 ± 5.5 + 300 2934 60 4 2 0 74 24.6 ± 2.3 BU)P + 20 2653 304 37 6 0 396 132.0 ± 19.4 a IrradiationdO kGy of Co-60 gamma-ray) was treated to the herbs before extraction. b Number of MN/1000 binucleated cells in the triplicate experiments in which 1,000 cells were scored. MMC(Mitomycin C) and B( a )P(benzo( a )pyrene) were used as positive controls. - 166 - ef. (1) 4^^ paeoniflorin &?\ ^ •§-£?!: 44 4 200g# MeOHS ^#*> ^ H2O, n-BuOHS. £3«H °1 ^-^1-i: silicagel column chromatography^ CHC13 : MeOH 9:14 5:15. AA -§-#*>^l4. 5:1 -§-#°J]# 4^1 SiO2 column chromatography-1- r^l ^Al^j-°i crude paeoniflorin-i- ^$i4. 2. paeoniflorin-i- Et2O:MeOH (10:1)5. ^M^^H HPLC(Table 2.33)^ 13C-NMR ^-sM ^ T1^* -i-^§f^4(Table 2.34). ^^ft paeoniflorin^ (2) ^M^ 2^f 3-^.^5^-EJ ^-s)^- paeoniflorin^l HPLC<>11 ^ o-ethoxybenzamidel- ^^a^l-^S. §><^ HPLCS 2.35). Paeoniflorin^ retention time°l 7.915, ^ 10.914^^ AA ^#s] 0J4. 10 kGy 24 3-^=4 paeoniflorin $ ^^^ €4 2.59%4 2.42%7> AA chromatogram^l pattern^^S. ^4^ ^Sj-4 ^t"£]x] ^^4(Table 2.35). (3) PaeoniflorirH #^^^1€^# &°]x] ^#^r Ames paeoniflorin# 3«1]^ 5/fl ^£^ §f^ ^^ ^-^tfls^ofl «!§]) ^7>£)^ ^$^JI, til2:A} 4 -B-^tb pattern^ -f 2.36). (4) Paeoniflorin< CHO ^]Stil]°oM]Ai , binucleated cells paeoniflorin ^7> ^H°l °J - 167 - ^$9X A (Table 2.37). (5) ^n^ decursin ^f decursin angelate^] ^^ ^^•^ -n-S^-§r(?] decursin2}- decursinol angelatefe- SiO2 column chromato- graphy(tolune: ether, Hexane: EtOAc)S £3*]-8-^(Scheme 2.1), HPLC3- £ 1 13 , H> C-NMR ^ £#3H signal^: 2.38, Table 2.39). (6) ^s] HPLC ^ Decursin^ decursinol angelate^ ^^f- ^]^:ol 3.84 4.0°}9XJL ^#7l^ 320 ^i ^tfl^^-s.-!- M-Bf^^.^ reverse phase column0!]^fe- acetonitrile: H2O 7\ ^31^ -g-i^l^Jl, *1^M ^^ ^l^^l^ ^^^-^i 1-^^ ?A°-S. ^3]^ ^ ^AJ- column (Shim-pack CLC-ODS(M)HH # normal phase column^*!fe hexane: EtOAc -§-nfl7%ofl 5i4. Decursin^ 200 //g~1000 cf (Table 2.40). (7) #*KI 2^]- ^71^1 -B-JL^^-^1 decursin3f decursinol angelate^ HPLC kGy) ^-^^ «12:A> ^-^^1 -fi-JL^^ ^f!4fe decursin^l \ 0.27%^ £. ^ decursinol angelatefe ^-^j-^ 2:4 2.41). - 168 - Table 2.33. HPLC analytical condition of paeoniflorin HPLC Condition Instrument Shimadzu HPLC System CBM-10A Pump LC-10AD Detector SPD-10A Column Shim-Pack CLC-ODS(M)25 (4.6X250) Mobile Phase Acetonitrile: H2O 20-MOO gradient Detection 230nm Flow rate(m£/min) Column Temp. CO room Retention time(min) 7.982 - 169 - Table 2.34. 13ONMR data for paeoniflorin C number Paeoniflorin( S ) Standard( S ) 1 85.0 86.0 2 43.6 44.6 3 104.7 105.8 4 42.3 43.7 5 22.0 23.4 6 87.5 88.8 7 70.2 71.5 8 61.2 61.3 9 100.0 101.5 10 19.1 19.7 1' 129.7 130.4 2'6' 129.0 129.8 3'5' 128.7 128.7 4' 133.4 133.2 7' 165.7 166.4 1" 98.6 100.2 2" 73.4 74.6 3" 76.8 78.2 4" 70.2 71.5 5" 76.8 78.2 6" 60.4 60.7 - 170 - Table 2.35. The content of paeoniflorin of Paeonia japonica Sample Content Paeoniflorin(%) Radiation S.D C.V(%) (nig) (mg) (n=3) 0 kGy 500 12.95 2.59 0.01 0.22 paeoniflorin 10 kGy 500 12.1 2.42 0.02 0.45 - 171 - Table 2.36. Revertant colonies in the S. typhimurium reversion assay with paeoniflorin isolated from Paeonia japonica Number of revertant colonies (His')per plate Test material S9 mix Dose TA98 TA100 H2O 100 id 43 54 (48) 170 184 (177) paeoniflorin lOmM 40 50 (45) 176 151 (163) 3.3mM 56 35 (45) 182 160 (171) l.lmM 65 45 (55) 162 157 (159) 360uM 48 38 (43) 166 136 (151) 120uM 49 43 (46) 131 149 (140) NPD 20/zg 1088 1014 (1051) Na-Azide 954 864 (909) paeoniflorin 37 38 (38) 178 145 (166) lOmM 47 46 (46) 197 199 (198) 3.3mM 49 46 (47) 170 167 (168) l.lmM 35 43 (39) 203 170 (186) 360uM 51 37 (44) 162 172 (167) 120uM 48 45 (46) 174 182 (178) 2-AF 10/ig 2912 2916 (2914) 1614 1724 (1669) NPD(4-nitro-o-phenylenediamine), Na-Azide(sodium azide), 2-AF(2-amino fluorene) were used as positive controls for the corresponding strains - 172 - Table 2.37. Frequency of micronuclei (MN) in cytokinesis-blocked CHO cells following treatment with paeoniflorin isolated from Paeonia japonica Cells without Frequency of the cells with MN MN/1000 a Material S9mix Dose MN(0) 1 2 3 4 (Mean ± S.D.) H2O 2938 57 4 1 0 22.7 ±6.1 paeoniflorin 15mM 2953 43 4 0 0 17.0±3.6 - 5mM 2949 46 5 0 0 18.7 + 3.1 - 1.6mM 2943 52 4 0 0 20.0 ±4.0 - 0.58mM 2962 36 2 0 0 13.3 ±2.5 - 0.19mM 2955 38 7 0 0 17.3 ±5.7 MMC 0.1vg 2716 202 30 6 1 94.7 ±12.8 H2O 2964 52 1 0 0 19.0 ±3.9 paeoniflorin + 15mM 2952 47 1 0 0 16.3 ±6.7 + 5mM 2939 58 3 0 0 21.3 ±2.5 + 1.6mM 2954 42 3 0 a. 16.3±2.1 + 0.58mM 2954 40 2 0 0 16.0 ±5.6 + 0.19mM 2943 52 5 0 0 20.7 ±4.0 B( a )p + 20^g 2743 233 27 5 1 102.0±14.9 MMC(Mitomycin C) and B( a )P (benzo( a )pyrene) were as positive controls Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. - 173, - The root of Angelica gigas(500g) 70% EtOH 2L CHC13: H2O (11) H2O CHCI3 SiO2 column chromatography tolune: diethylether (1:1-9:1) decursinol angelate and decursin mixture SiO2 column chromatography hexane: EtOAc (15:1- 9:1) decursinol angelate decursin Scheme 2.1. Isolation of decursinol angelate and decursin from the root of Angelica gigas - 174 - Table 2.38. ^-NMR spectral data of decursinol angelate and decursin(in CDCls) Proton number Decursinol angelate ( S ) Decursin( 8) 3 6.24, d(/=9.5Hz) 6.23, d(/=9.5Hz) 4 7.59, d(/=9.5Hz) 7.59, d(/=9.5Hz) 5 6.80, s 6.80, s 7.16, s 7.16, s 3' 5.13, t(/=4.8Hz) 5.09, t(/=4.8Hz) 4' 2.90, dd(/=17.6Hz, 4.8Hz) 2.87, dd(/=17.6Hz, 4.8Hz) 3.23, dd(/=17.6Hz, 4.8Hz) 3.20, dd(/=17.6Hz, 4.8Hz) gem(CH;j)2 1.38, s 1.36, s 1.40, s 1.39, s 2" 5.76, m 3" 6.11, dd(7=7.2H; 2"-CH3 1.84, d(/=1.4Hz) 3"-CH3 - 2.15, d(/=1.2Hz) 4" 1.89, d(/=7.2Hz) 1.88, d(/=1.2Hz) - 175 - Table 2.39. C-NMR spectral data of decursinol angelate and decursin (in CDCb) Carbon Decursinol angelate( 8 ) Decursin( 8 ) 2 161.2 161.3 3 113.3 113.2 4 143.1 143.2 5 128.6 128.7 6 115.4 115.9 7 156.4 156.4 8 104.6 104.6 9 154.2 164.1 10 112.8 112.8 2' 27.9 27.6 3' 77.1 76.6 4' 70.0 69.0 gem(CH3)2 23.2 23.2 25.1 25.0 1" 167.1 165.7 2" 127.3 115.6 3" 139.5 158.5 4" 15.8 27.5 2"-CH3 20.5 3"-CH3 20.3 - 176 - Table 2.40. Analytical condition of decursinol angelate and decursin HPLC Condition A Condition B Instrument Waters HPLC Waters HPLC System 2690 separation module 2690 separation module Pump 2690 pump 2690 pump 996 photodiode array 996 photodiode array Tfc O"t"O C \ C\ T" detector detector Column Nova Pak C18 Nova Pak Silica Mobile Phase Acetonitrile: H2O 6: 4 Hexane:EtOAc 9:1 Detection 320nm 320nm Flow rate(m£/min) 1 2 Comumn Temp. CO room room Retention time(min) decursin 3.869 8.057 decursinol angelate 4.046 7.422 HPLC Condition C Instrument Shimadzu HPLC System CBM-10A Pump LC-10AD Detector SPD-lOA Column Shim-Pack CLC-ODS(M)25 Mobile Phase Acetonitrile: H2O 6: 4 Detection 254nm Flow rateW/min) 1 Comumn Temp. CO 30 °C Retention time(min) decursin 13.717 decursinol angelate 14.333 177 - Table 2.41. The content of decursin and decursinol angelate of Angelica gigas . Sample Content Decursin(%) Radiation S.D C.V(%) (mg) (mg) (n=2) 0 kGv 3002 129.71 4.32 0.008 0.09 decursin 10 kGy 3012 138.12 4.59 0.025 0.27 0 kCy 3018 3.1 2.65 0.0007 0.01 decursinol angelate 10 kCy 3019 1.25 2.6 0.005 0.09 - 178 - (stem cell) 7H - 179 - (%) (%) -giS4 £X| Al§ AJqtX(| SA-n» (6#) a^ (6#) -fc^l^: 15 S -fe^^S: 20 E ^ ®°W Sf^ i# (61) -^5|#S: 1 Zd - gj-A|-A|iA|- n^^o||Ai 80 100 (5,^# 1 Zi) 4 SS7|A1|S COO ^^| 7|#0|£j &s ^tWI5E */2^a#: 2 #) % ^ -a^n a^ ^5!) 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Stamdard Report No. INIS Subject Code Report No. Report No. KAERI/RR-2028/99 Title / Subtitle Development of Functional Foods for Radiation Workers Project Manager Jo, Sung-Kee (Dept. of Radiation Food & Biotechnology, KAERI) and Department Researcher and Yu, Young-Beob, Park, Hae-Ran, Byun, Myung-Woo, Yang, Jae-Seung (Dept. Department of Radiation Food & Biotechnology, KAERI) Kim, Sung-Ho(Chonnam Univ.), Yee, Sung-Tae(Sunchon Univ.) Publication Publication Taejon Publisher KAERI 1999 Place Date Page 238p. 111. & Tab. Yes(#), No ( ) Size 21cm x 29.7cm Note Classified Open(#), Restricted( Report Type Research Report Class Document Sponsoring Org. Contract No. Abstract (15-20 Lines) In searching modulators of immunity and hematopoiesis among natural products, being used as foods, six herbs exhibited lymphocyte proliferation in vitro, and six exhibited augmentation of hematopoietic cell growth. The combined treatments showed synergistic effects of lymphocyte proliferation and of hematopoietic cell growth. On the other hand, we found four effective Oriental medicinal prescriptions, used as energy tonic or blood-building decoctions, for survival and regeneration of hematopoietic cells and for protection of stem cells of intestinal crypt in irradiated mice. On the basis of these results, extracts from combinations of herbs were made in expectation of higher effects in the three respects. The immunomodulation activity by the two combinations of herbs was confirmed in mice. In culture of bone marrow cells, growth improvement of non-adherent precursor and induction of cytokine expression by herb mixture extracts were observed. In evaluation of fractions, polysaccharide fraction showed modulation of immunity and hematopoiesis, and methanol fraction showed stem cell protection from radiation. On the basis of the results, we made two provisional products by addition of polysaccharide fraction to the water extract. In further research, the active components would be identified and the functional foods would be developed for overcoming of declined immunity and radiation damage. For security of sanitation by irradiation, the stability in activity of irradiated resources was confirmed. Subject Keywords Immunomodulation, Hematopoietic cells, Radioprotection, (About 10 words) Natural products, Herbs, Intestinal crypt, Bone marrow cells, nr 21 —1 —i 1J '— 7IL?^I- ~J 1 ^Ci td All (J y 1. 0| ili _I_L A^ LT -^f ^1\ ' 1 ^~I~ "11 ^1 a, s °J M q-. 2. Ol i Ai A a a >° »>8 Of o §[• L[|2° bl-lT 3. F3 i i (01A1te omgqcK