KR0000086
KAERI/RR-1912/98
Development of Food Science and Technology by Radiation
Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation
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31/ 30 KAERI/RR-1912/98
Development of Food Science and Technology by Radiation
Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation
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- ix - SUMMARY
I. Project Title
Development and Hygiene of Functional Foods with Immunomodulation Activity Using Radiation
II. Objective and Importance of the Project
The health and long-life have been an important issue with elevation of standard of living. Despite the impressive accomplishments of the modern pharmacological industry, a large number of diseases remain for which treatment is of limited benefit. As a result, there is an increasing need for the development of so-called "alternative treatment by natural products" and health foods, for which predictable pharmacologic activity and therapeutic efficacy can be documented. Concerning maintenance of health, prevention of diseases and inhibition of aging have been important theme. Therefor, the topics of "food and health" have been an important issue. Many kinds of substances are concerned with regulation of physiological function and maintenance of homeostasis, and they might be supplied by various foods, and food stuffs from plant might be important their resources. Natural products such as herbal medicines have only recently begun to receive some attention as possible regulators of host defense mechanism for preventing disease and recovering damage. Especially, the regulation of immunity and hemopoiesis have might be a important theme in prevention of disease, inhibition of aging and overcoming radiation damage. And also, toxicological safety in sanitization of food resources for sanitary production and circulation of functional foods should be confirmed. The present study was performed to develop functional foods with immunomodulation activity overcoming declined immunity and hemopoiesis, and to develop techniques for sanitization of function food resources with gamma- irradiation.
- x - . Scope and Contents of the Project
1. Selection of test materials We selected 9 herbs and 6 decoctions of the Oriental medicinal prescriptions, concerning usefulness of herbs as foods, quantities of the medicinal plants cultured in Korea, and reports on the effects.
2. Screening of the immunomodulation components and preparing the provisional products - Screening of the immunomodulation components in herbs - Investigating synergistic effects of immune cell activation by combined treatments - Investigating the effects of the prescriptions on immune cell activation - Making combination of the herbs for preparing the provisional products and investigating the effects of the fractions on immune cell activation - Investigating the immunomodulation activity in vivo - Verifying stability of immunomodulation activity of gamma-irradiated samples
3. Investigation on the hematopoietic effects of functional foods using radiation and preparation of the provisional products - Selection of 6 energy tonic or blood-building decoction of Oriental medicinal prescriptions as candidate foods - Evaluating the effects of the 6 prescriptions and its ingredients in irradiated mice • Jejunal crypt survival assay • Endogenous spleen colony formation assay • Apoptosis inhibition assay - Verifying stability of the biological activity of the irradiated prescriptions • Jejunal crypt survival assay
- xi - • Endogenous spleen colony formation assay • • ' • • Apoptosis inhibition assay - Making combination of the herbs for preparing the provisional products on the basis of data on the effects of the prescriptions and its ingredients
4. Search for the active components and studies on the mechanism of the hematopoiesis improvement foods - Isolation and culture of bone marrow stromal cells - Search for active components of the hematopoiesis improvement foods - Investigating growth augmentation of nonadherent precursor cells - Analysis of cytokine expression in stromal cells - Analysis of nitric oxide production in macrophages - Analysis of cytokine expression in macrophages
5. Development of techniques for the sanitation of functional food resources (herbs) by gamma-irradiation - Sanitization test of herbs by gamma-irradiation - Verifying stability of active components in gamma-irradiated herbs • HPLC patterns of paeoniflorin in gamma-irradiated Paeonia Radix • HPLC patterns of decursin in gamma-irradiated Angelica gigantis Radix - Verifying safety of gamma-irradiated herbs in respect of genotoxicity • No mutagenicity of total extracts in Salmonella reversion assay • No induction of micronuclei in CHO cells by total extracts • No mutagenicity of paeoniflorin in Salmonella reversion assay • No induction of micronuclei in CHO cells by paeoniflorin - Verifying stability of the biological activity of the gamma-irradiated herbs • No change in antimutagenic activity of the extracts in Salmonella reversion assay and in micronucleus assay • No change in electron donating activity to DPPH
- xii - IV. Results of the Project
1. Screening of the immunomodulation components and preparing the provisional products
- Screening of the immunomodulation components in herbs In order to screen immunomodulator, we selected 9 herbs and 6 decoctions of the Oriental medicinal prescriptions. In the test of immune cell proliferation by herbal extracts in vitro, 1, 3 and 2 herbs showed high, moderate and slight effect, respectively.
- Investigating synergistic effects of immune cell proliferation by combined treatments In the test of immune cell proliferation by 10 combinations of herbal extracts in vitro, 6 combinations showed higher effects than sum of that by single extract.
- Investigating the effects of the prescriptions on immune cell activation In the test of immune cell proliferation by 6 decoctions of energy tonic or blood-building prescriptions in vitro, Si-Jun-Zi-Tang, Bu-Zhong-Yi-Qi -Tang, San-Ling-Bai-Shu-San showed slight effects. But the effects were lower than that of previous combinations made in this study.
- Making combination of the herbs for preparing the provisional products and investigating the effects of the fractions on immune cell activation On the basis of the previous results, 2 combinations of herbs were made, and extracted into fractions. In the test of immune cell proliferation by the fractions in vitro, 2 fractions showed high effects.
- xiii - - Investigating the immunomodulation activity in vivo It was tested in a hemolytic plaque assay and in graft versus host reaction whether in vivo treatment of the extracts could influence the secretion of antibodies against sheep red blood cells(SRBC) and cellular immune response against transplanted allogenic immune cells, respectively. It turned out that intraperitoneal injection of the extract of 1 single herb or 2 herbal combinations increased the number of antibody-secreting cells remarkably. Splenomegaly, due to graft versus host reaction, was also increased by intraperitoneal treatment of the extract of 1 single herb or 1 herbal combinations.
- Verifying stability of immunomodulation activity of gamma-irradiated samples The immunomodulation ativity of irradiated herbs was shown to the same level of that of non-irradiated samples, in vitro and in vivo.
2. Investigation on the hematopoietic effect of functional foods using radiation and preparing the provisional product.
- The effect of Oriental prescriptions and its ingredients • Augmentation of jejunal crypt survival Pretreatment with Si-Wu-Tang, Bu-Zhong-Yi-Qi-Tang, Kuei-Pi-Tang, Rensen, Shengma, Danggui, Baishaoyao, Gancao, Chaihu, Baifuling, Baibiandou, Sharen, Jiegeng, Suanzaoren or Muxiang extract resulted in a significant increase in the number of surviving crypts compared with those in the irradiation control. • Augmentation of endogenous spleen colony formation The numbers of spleen colonies was higher 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 than in mice esposed to radiation only.
- xiv - • Apoptosis inhibition 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.
- The stability of biological activity of irradiated Oriental medical 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.
3. Search for the active components and studies on the mechanism of the hematopoiesis improvement foods
- Search for active components of the hematopoiesis improvement foods in the culture of bone marrow stromal cells At the first experiment, we established 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 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 exhabited cytotoxicity at
- xv - 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 mixture of 2 strain extracts than by addition of single strain extract.
- Investigating growth augmentation of nonadherent precursor cells Nonadherent supernatant-derived mononuclear cells harvested from long termed stromal cell culture in the presence or absence of complex extracs. In the previous and cooprative experiments, we selected 3 or 4 strains complex extracts which increased the number of nonadherent mononuclear cells.
- Induction of cytokine expression in stromal cells Different cytokine expression patterns were observed stromal cellls cultured in the presence or absence of 3 to 4 strains mixture extracts which support differentiation of nonadherent cells.
- Induction of nitric oxide production in macrophages 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- y 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 A^-monomethyl-L-arginine. In addition, these extracts induced the same effects on the peritoneal macrophages.
- Induction of cytokine expression in macrophages Altered patterns of cytokine mRNA expression - IL-1/?, IL-6, LT and iNOS - were observed in the stromals cells cultured with extracts of herbal plant.
- xvi - 4. Development of techniques for the sanitation of functional food resources (herbs) by gamma-irradiation
- Sanitization of herbs by gamma-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 10 kGy.
- Verifying stability of active components in gamma-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/ml 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% (C.V=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. • 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 chromatographyCtolune^ether, hexane: EtOAc). And then structures were confirmed using the *H and 13ONMR
- XVII - data. The quantitative analysis of decursin was performed in the high performance liquid chromatographic (HPLC) methods using reverse phase columns(Nova Pak Cis) 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.
Verifying safety of gamma-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 10 kGy. • 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, 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.
- xviii - • 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 10 kGy. - 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 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.
- Verifying stability of the biological activity of the gamma-irradiated herbs • 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 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 ojficinale 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.
- xix - • 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.
V. Application Plan of the Project Results
On the basis of the results from this study on screening modulator of immunity and hemopoiesis from herbs and obtaining the synergistic effects by combination treatments, some combinations of herbs were selected in expectation of higher effects. In the further studies, we would to evaluate the effects of the combinations, to confirm toxicological safety, and to prepare the provisional products for foods. And then, the functional foods with immunomodulation activity would be developed, and would be applied to overcoming the declined immunity and hemopoiesis caused by various factors. - Applied in developments of functional foods for modulation of the declined immunity and hemopoiesis for treatment of aging, cancer, adult diseases, radiation damages 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 elevation of quality of public living.
- xx - CONTENTS
Chapter 1. Introduction 1
Chapter 2. Status of home and foreign technology development ? 4 Section 1. Foreign status of functional food development 4 Section 2. Home status of functional food development • • • • 5 Section 3. Assessment of the States 6
Chapter 3. Contents, Methods and Results 8 Section 1. Screening of the immunomodulation components and preparing the provisional products • • 8 1. Contents and Methods • 8 a. Animals • 8 b. Preparation of samples • • • • 8 c. Irradiation of samples 8 d. MTT assay 9 e. Isolation and culture of splenic lymphocytes • 9 f. Lymphocyte proliferation 9 g. Hemolytic plaque forming cell assay • • • , 9 h. Graft vs Host reaction 10 2. Results and Discussion • • • 10 a. Screening of the immunomodulation components, in herbs • • • • 11 b. Investigating synergistic effects of immune cell activation by combined treatments 11 c. Investigating the effects of the prescriptions on immune cell activation 11 d. Making combination of the herbs for preparing the provisional products and investigating the effects of the fractions on immune cell activation 11
- xxi - e. Investigating the immunomodulation activity in vivo 11 f. Verifying stability of immunomodulation activity of gamma- irradiated samples 12
Section 2. Investigation on the hematopoietic effects of functional foods using radiation and preparation of the provisional products • • 28 1. Contents and Methods 28 a. Animal 28 b. Preparation of samples • • • 28 c. Irradiation 28 d. Jejunal crypt survival assay • 29 e. Endogenous spleen colony formation assay 29 f. Apoptosis inhibition assay 30 2. Results and Discussion 30 a. Evaluating the effects of the 6 prescriptions and its ingredients in irradiated mice 30 b. Verifying stability of the biological activity of the irradiated prescriptions 31 c. Making combination of the herbs for preparing the provisional products on the basis of data on the effects of the prescriptions and its ingredients 31
Section 3. Search for the active components and studies on the mechanism of the hematopoiesis improvement foods 56 1. Contents and Methods 56 a. Animal 56 b. Isolation and culture of bone marrow stromal cells 56 c. Search for active components of the hematopoiesis improvement foods • 56 d. Isolation and culture of nonadherent precursors of blood cells • • 56
- XXII - e. Culture of cell lines and peritoneal macrophage 56 f. Measurement of nitric oxide production in macrophages • • • • • 57 g. Measurement of LPS in samples 57 h. RT-PCR method • • • • 57 2. Results and Discussion • 60 a. Culture of bone marrow stromal cells 60 b. Search for active components of the hematopoiesis improvement foods 60 c. Investigating growth augmentation of nonadherent precursor cells • 60 d. Measurement of cytokine expression in stromal cells • • • • • • -61 e. Measurement of nitric oxide production in macrophages 61
Section 4. Development of techniques for the sanitation of functional food resources (herbs) by gamma-irradiation 79 1. Contents and Methods 79 a. Sanitization test of herbs by gamma-irradiation • • • 79 b. Stability test of active components in gamma-irradiated herbs • • 80 c. Safety test of gamma-irradiated herbs in respect of genotoxicity 81 d. Stability test of the biological activity of the gamma-irradiated herbs 81 2. Results and Discussion 82 a. Sanitization of herbs by gamma-irradiation 82 b. Verifying stability of active components in gamma-irradiated herbs • • 83 c. Verifying safety of gamma-irradiated herbs in respect of genotoxicity 84 d. Verifying stability of the biological activity of the gamma- irradiated herbs 85
- XXIII - Chapter 4. Goal achievement and External Contribution • • 117
Chapter 5. Application plan • 120
Chapter 6. References
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- 8 - . MTT Assay
* 1X105 cells/well^ ^fJES. . microplate^ CCVincubateHH 2Q& aU^fS^. z\ X\g_Wr ^£lS ^7>«>^ 1< ^^ 4-§- MTT dye(3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide)!- 2.5 mg/ml^ ^£S ^7}^H 4^>1^> «fl6j=^: ^ acidic isopropanol(0.07 N HCl in absolute isopropanol)-£ ^7]-sH ^^€ dye ^^S^l- 2X105 cells/well^] MTT Assay* ^ s|^^ 4(99,100). C57BL/6 ^>-f^ HBSS<^1 HBSSS. . Trypan blue 4- ^^ (lymphocyte proliferation test) S^l- 2xiO5/well5] ^5.^ microplate^l 3H-thymidine# 7>^r ^ 4^1^ c] yfl^ cell harvester* ^7^}^4. Scintillation cocktail^: 2.5 /3-scintillation counters 3H-Thymidine incorporation ^£# cpm ^S #^S ^^(Hemolytic plaque cell forming assay) 0.25 - 9 - ml/mouse(25grH 47<^> *>$4. =L t]-,g-v£- SRBC 2xi087lj/mouset- 2. $\$ 20% SRBC O.lml4 0.5% agarose 1.6 mil- ^I^^ri^ 3<>H dishi pourit ^f > ^^> CO2-incubatoH^ ufl^^SH^-. ^ 4^- ji^CGPC, 1/70)1- %7}*}^ 2 PFC/spleen^-S SH*S>$i4(101-104). ^^ (Graft us. Host Reaction) BDFKC57BL/6XDBA/2) 4^1 recipients *}•%••$•%±*\, grafts C57BL/6 i- 1 x 1077fl/0.2ml/mouse^ 7°i# 4^^4(0.2 ml/mouse)l *5^>^4. 7^ ^ wl^-^- T) Tfl-i- ^Jl ^1^3}-^ «!•§:•§• Ji^^V^ spleen index! ?ll#Sr&4(105-107). allogenic (spleen weight/body weight) Spleen index = syngeneic (spleen weight/body weight) 2. fe 1000 ~ 2000 ^: 1000 ~ 2000 ^g/ml, ^-7>3]^ 300 ^g/ml, <^2:fe 2000 ~ 4000 fig/ml, ^^^ 300 jug/ml, ^>4^ 500 ~ 1000 /ig/ml ^1^4(Table 1.1). - 10 - 1.2, Table 1.3). 1.4). 27H ^^4(Table 1.5, Table 1.6). ^ SRBC^l (1) ^*11 ^S^^^ #7^-j:3j- ^^ ^ l&iH^flO^)^- 2^4 >«^7fl2:^-51 ^## 1.7, Fig. 1.1, Table 1.8, Fig. 1.2, Table 1.9, Fig. 1.3). (2) 1.10, Table 1.11). - 11 - (1) 10 1.12). (2) 10 1.7, Fig. .1.1, Table 1.8, Fig. 1.2, Table 1.9). - 12 - Table 1.1. Lymphocyte proliferation by water soluble fraction of Korean medicinal herbs Concentration cpm Sample (pt g/ml) Day 2 Day 3 Day 4 Negative Control 4176 ± 310 3927 ± 463 1934 + 227 4000 16443 ± 263 80194 ± 2718 98724 + 14946 Angelica gigas Nakai 2000 35531 ± 3886 133752 ± 7188 89084 ± 9474 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 A/Tallinn 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 sessiliflorus 300 36732 ± 1714 88694 ± 5865 84553 ± 8608 150 32120 ± 2488 61646 ± 3519 40795 ± 5481 4000 21019 ± 2937 65616 ± 7256 83247 ± 6235 2000 24130 ± 1648 47957 ± 3553 50679 ± 6083 Houttuynia cordata 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 - 13 - Table 1.2. Lymphocyte proliferation by water extracts of KJ-1, KJ-2, KJ-3, KJ-4, KJ-5 and KJ-6 in H-thymidine uptake assays Concentration cpm Sample (mg/ml) 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 KJ-4 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 - 14 - Table 1.3. Lymphocyte proliferation by water extracts of Kl, K2, K3 and K4 in H-thymidine uptake assays Concentration cpm Sample (mg/ml) Day 2 Day 3 Kl 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 K2 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 K3 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 K4 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 - 15 - Table 1.4. Lymphocyte proliferation by water extracts of several prescriptions of Oriental medicine in 3H-thymidine uptake assays a Concentration H-thymidine uptake (cpm) Sample (mg/ml) 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. - 16 - Table 1.5. Lymphocyte proliferation by water extracts of KJ-6 + El or E9 or El + E9 in 3H-thymidine uptake assays cpm Sample f^ rvn f* pn tr*£i 1"i on wv/LiLsCiiL-l CtllUil 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 + 0.00075 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 - 17 - Table 1.6. Lymphocyte proliferation by water extracts of HIM-I-O, HIM-I-Wi, HIM-II-0 and HIM-II-Wi in 3H-thymidine uptake assays Concentration cpm Sample (mg/ml) Day 2 Day 3 HIM-I-0 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-Wi 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-II-0 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-Wi 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 5136 ± 57 3437 ± 681 - 18 - Table 1.7. Effect of Angelica gigas Nakai 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.) 2.0xi077fl 60, 102, 65 ( 75.7) 2,163 7 3.7xiO 7fl 9, 18, 20 (15.7) 830 Saline 7 4.9xiO 7fl 68, 69, 44 (60.3) 4,221 6.2xiO67fl 3, 96, 89 (91) 806 4.4xlO77fl 93, 99, 113 (101.7) 6,393 8.2X1O77H Angelica gigas 151, 128, 135 (139.5) 16,166 20 (non-irradiated) 7 5.7xiO 7fl 97, 101, 81 (93) 7,573 7 4.8xiO 7fl 53, 60, 53 (55.3) 3,792 7 6.3 x 10 7fl 78, 96, 91 (88.3) 7,947 Angelica gigas 7 4.5xiO 7fl 123, 103, 115 (113.7) 7,309 (irradiated 20 6.9 x 1077fl 90, 90, 93 (91.0) 8,970 10 kGy) 6.8xiO77l] 60, 36, 31 (42.3) 4,109 - 19 - 10000 - 8000 - T •..•..'....- . • • 6000 - • • 4000 - ..':•;••? onnn T 1 .••• ... 0 — Control Angelica gigas Angelica gigas non-irradiation irradiation (OkGy) (lOkGy) Fig 1.1. Effect of non-irradiated(o kGy) and irradiateddO kGy) Angelica gigas on the secretion of antibody tested by hemolytic plaque forming cell assay (Mean ± S.E.) - 20 - Table 1.8. Effect of KJ-1 on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage No. of plaque/7 X105 Sample Splenocyte/spleen PFC/spleen (mg/kgB.W.) splenocyte 1.3 X 107 cell/ml 14, 17, 19 (16.7) 311 3.2 X 107 cell/ml 69, 52, 64 (61.7) 2,820 4.6 X 107 cell/ml 26, 45, 42 (37.7) 2,477 Saline 8.5 X 107 cell/ml 32, 34, 36 (34) 4,128 5 X 107 cell/ml 73, 71, 63 (69) 4,927 5.7 X 107 cell/ml 44, 52, 38 (44.67) 3,638 1.4 X 108 cell/ml 54, 50, 50 (51.3) 10,260 7.5 X 107 cell/ml 49, 58, 46 (51) 5,457 1.1 X 108 cell/ml 69, 48, 58 (58.3) 9,159 KJ-1 120 + 120 (non-irradiated) 9.7 X 107 cell/ml 36, 34, 44 (38) 5,266 9.3 X 107 cell/ml 56, 56, 56 (56) 7,776 9.2 X 107 cell/ml 54, 44, 60 (56.67) 7,448 6.4 X 107 cell/ml 56, 58, 52 (55.3) 5,056 7.4 X 107 cell/ml 90, 90, 76 (85.3) 9,018 KJ-1 9.8 X 107 cell/ml 44, 38, 38 (38.67) 5,414 (irradiated, 120 + 120 7 10 kGy) 8.4 X 10 cell/ml 38, 70, 90 (66) 7,920 9.2 X 107 cell/ml 44, 42, 62 (49.3) 6,480 8 1.1 X 10 cell/ml 42, 48, 78 (56) 8,800 - 21 - 10000 8000 - $ 6000 -I in 4000 - 2000 - Control KJ-1 KJ-1 non-irradiation irradiation (OkGy) (lOkGy) Fig 1.2. Effect of non-irradiated(0 kGy) and irradiateddO kGy) KJ-1 on the secretion of antibody tested by hemolytic plaque forming cell assay (Mean ± S.E.) - 22 - Table 1.9. Effect of KJ-2 on the secretion of antibody tested by hemolytic plaque forming cell assay Dosage No. of plaque/7 X105 Sample Splenocyte/spleen PFC/spleen (mg/kgB.W.) splenocyte 4.6 X 107 cells/ml 75, 51, 62 (62.7) 4,118 8.6 X 107 cells/ml 32, 29, 30 (30.3) 3,726 7.5 X 107 cells/ml 11, 25, 23 (19.7) 2,110 7.6 X 107 cells/ml 23, 18, 24 (21.7) 2,357 Saline 8.1 X 107 cells/ml 35, 34, 28 (32.3) 3,737 9.8 X 107 cells/ml 21, 23, 26 (23.3) 3,262 7.0 X 107 cells/ml 27, 39, 17 (27.7) 2,770 5.7 X 107 cells/ml 27, 31, 28 (28.7) 2,336 1.2 X 108 cells/ml 65, 45, 56 (55.3) 9,485 1.1 X 108 cells/ml 133, 102, 100 (111.7) 17,548 7.5 X 107 cells/ml 44, 48, 47 (46.3) 4,959 1.1 X 108 cells/ml 45, 56, 44 (48.3) 7,313 KJ-2 120 + 26 7.3 X 107 cells/ml 70, 69, 64 (67.7) 7,061 6.4 X 107 cells/ml 47, 45, 52 (48) 4,387 9.4 X 107 cells/ml 59, 36, 30 (41.7) 5,600 1.3 X 108 cells/ml 33, 43, 44 (40) 7,428 - 23 - 10000 8000- 6000- CO 4000- 2000 - Control Fig 1.3. Effect of KJ-2 on the secretion of antibody tested by hemolytic plaque forming cell assay (Mean ± S.E.) - 24 - Table 1.10. Effect of Angelica gigas on Graft vs. Host reaction Group Donor Recipient Sex spleen w.t/body w.t.( x 10~3) Spleen index Saline BDF1 BDF1 M 2.333 ± 0.233 1.00 F 3.413 ± 0.434 1.00 Saline C57BL/6 BDF1 M 4.595 ± 0.714 1.970 F 4.227 ± 0.180 1.238 Angelica gigas C57BL/6 BDF1 M 7.633 ± 1.362 3.271" (non-irradiated) lOOmg/Kg B.wt. F 8.673 ± 1.577 2.541* Angelica gigas C57BL/6 BDF1 M 5.445 ± 0.686 2.334* (irradiated, 10 kGy) lOOmg/Kg B.wt. F 6.303 ± 1.383 1.847 * p<0.05 as compared with control. ** p<0.01 as compared with control. - 25 - Table 1.11. Effect of KJ-1 on Graft us. Host reaction Group Donor Recipient Sex spleen w.t./body w.tXxiO'3) Spleen index BDF1 BDF1 M 3.613 ± 0.076 1.00 Saline F 4.200 ± 0.849 1.00 C57BL/6 BDF1 M 4.243 ± 0.517 1.1743 Saline F 4.890 ± 0.451 1.1543 C57BL/6 BDF1 M 7.403 ± 0.536 2.0489** KJ-1 (60mg/kg B.wt.) F 7.707 ± 0.327 1.8349** ** p<0.01 as compared with control. - 26 - Table 1.12. Lymphocyte proliferation by water soluble fraction of korean medicinal herbs irradiate with gamma ray (lOkGy) Concentration cpm Sample ( M g/ml) Day 2 Day 3 Day 4 Negative Control 4399 ± 161 3163 ± 736 2063 ± 1274 2000 25437 ± 5754 69841 ± 5054 105111 ± 13405 Angelica gigas Nakai 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 Makino 500 39710 ± 5578 63767 ± 1750 78767 ± 7647 600 3732 ± 589 1968 ± 159 7340 ± 594 Paeonia japonica 300 11720 ± 65 10563 ± 1380 7214 ± 284 Miyabe 150 13304 ± 313 12761 ± 1636 5828 ± 210 600 18694 ± 4486 59417 ± 8281 111496 ± 9058 Acanthopanax 300 42967 ± 2694 141888 ± 1117 141057 ± 1113 sessiliflorus Seemann 150 44938 ± 2243 115309 ± 2585 109994 ± 3198 1000 7476 ± 441 5058 ± 473 3765 ± 2 Crataegus pinnatifida 500 7724 ± 330 5891 ± 835 4345 ± 414 Bunge 250 7328 ± 73 4716 ± 105 3057 ± 907 Positive Control PHA 2 96651 ± 10836 112050 ± 18939 64295 ± 20552 LPS 20 239137 ± 640 76815 ± 3325 16604 ± 3626 - 27 - i. Jg. Ol-g-£] (2Gy) , apoptosis 4 apoptosis 4. ^, 80°C 3-g-sH - 28 - 60 : CO, am 2 kGy^ #^3. 10 free radical dosimeter^- eerie cerous dosimeter-i- yj-A}Aj 2A]-71 (Gamma-cell Elan 3000, Nordion International, Canada) ^ r ^('?4^:-i::l0.9Gy/min)-§ -g- ^^^l^^^fe 12Gy, Ml^ ^ 6.5Gy apoptosis #^l . ICR *Fr-^t- A z]- 36 , A 8-1071] 5] 4 4. i 25] ^^ A 4-fit 5) Bouin - 29 - . Apoptosis # Carnoy's Jl^^i Jl^^l?] ^ zj- ^-f^1^ 8-107fl^ ± § 2. ±3. -^-sl^Slfe ^.JLiij-l- M-^-^4 (Fig. 2.1, Table 2.1-2.6). (2) - 30 - %•*} (Fig. 2.2, Table 2.7-2.12). (3) Apoptosis lfe apoptosis (Fig. 2.3, Table 2.13 2.18). Z]- A]^^ (1) 3.46)1 4^ (2) (Table 2.19). (3) ^tf(Table 2.20). (4) Apoptosisl!^ apoptosis 4(Table 2.21). A] § - 31 - B Fig. 2.1. 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( x 40). (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. - 32 - Table 2.1. Effect of Si-Wu-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.252 ± 6.051 Irradiation control (12Gy) 38.484 ± 4.335 Si-Wu-Tang(lmg/head, twice IP, at 36 and 12hrs. 77.980± 16.101* before irradiation) + irradiation Untreated control 157 ±14.819 Irradiation control 21.82 ±12.314 Shoudehuang(lmg/head, twice I.P. at 36 and 12hrs. 31.34 ± 1.759 before irradiation) •'-irradiation Chuanxiongdmg/head, twice IP. at 36 and 12hrs. 2.667 ±22.007 before irradiation)+irradiation Dangguidmg/head, twice IP. at 36 and 12hrs. 50.538 ± 6.089** before irradiation)+irradiation Baishaoyaodmg/head, twice IP. at 36 and 12hrs. 49.05 ±12.642*** 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. - 33 - Table 2.2. Effect of Si-Jun-Zi-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.252 ±6.051 Irradiation control(12Gy) 38.484±4.335 Si-Jun-Zi-Tang(lmg/head, twice IP. at 36 and 12hrs. 27.402±6.118 before irradiation) + irradiation Untreated control 163.1 ±12.341 Irradiation control 19.67±6.085 Rensan(lmg/head, twice IP. at 36 and 12hrs. 37.8 ±10.134** before irradiation) + irradiation Gancaodmg/head, twice IP. at 36 and 12hrs. 59.775 ±26.494** before irradiation) + irradiation Baifuling(lmg/head, twice IP. at 36 and 12hrs. 50.375 ± 9.746* before irradiation) + irradiation Baizhu(lmg/head, twice IP. at 36 and 12hrs. 26.625 ± 8.643 before irradiation) + irradiation *p<0.0001 as compared with irradiation control group. **p<0.005 as compared with irradiation control group. - 34 - Table 2.3. 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 157.252 ± 6.051 Irradiation control 38.484 ± 4.335 Shi-Quan-Dai-Bu-Tang (lmg/head, twice I.P. at 37.623 ± 8.100 36 and 12hrs. before irradiation) + irradiation Untreated control 157 ±14.819 Irradiation control 21.82 ± 12.314 Shoudehuang (lmg/head, twice I.P. at 36 and 12hrs. 31.34 ±1.759 before irradiation) + Irradiation Chuanxiong (lmg/head, twice I.P. at 36 and 12hrs. 42.667 ±22.007 before irradiation) + Irradiation Danggui (lmg/head, twice I.P. at 36 and 12hrs. 50.538 ±6.089** before irradiation) + Irradiation Baishaoyao (lmg/head, twice I.P. at 36 and 12hrs. 49.05 ±12.642* before irradiation) + Irradiation Rensan (lmg/head, twice I.P. at 36 and 12hrs. 41.947 ± 5.134* before irradiation) + Irradiation Gancao (lmg/head, twice I.P. at 36 and 12hrs. 61.922±24.094* before irradiation) + Irradiation Baifuling (lmg/head, twice I.P. at 36 and 12hrs. 54.622 ± 3.746*** before irradiation) + Irradiation Baizhu (lmg/head, twice I.P. at 36 and 12hrs. 29.772 ± 4.643 before irradiation) + Irradiation Huagqui (lmg/head, twice I.P. at 36 and 12hrs. 28.883 ±15.558 before irradiation) + Irradiation Rougui (lmg/head, twice I.P. at 36 and 12hrs. 40.067 ±21.043 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. - 35 - Table 2.4. 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 157.252 ± 6.051 Irradiation control (12Gy) 38.484 ± 4.335 Bu-Zhong-Yi-Qi-Tang (lmg/head, twice IP. at 64.723 ± 4.228* 36 and 12hrs. before irradiation) + irradiation Untreated control 157.00 ±14.81 Irradiation control (12Gy) 19.673+ 6.085 Rensan( lmg/head, twice I.P. at 36 and 12hrs. before 41.937 ± 4.789** irradiation) + irradiation Gancao(lmg/head, twice I.P. at 36 and 12hrs. before 61.958±24.257** irradiation) + irradiation DangguK lmg/head, twice I.P. at 36 and 12hrs. before 54.896± 7.873*** irradiation) + irradiation Baizhudmg/head, twice I.P. at 36 and 12hrs. before 31.521 ± 7.974 irradiation) + irradiation HuangqK lmg/head, twice IP. at 36 and 12hrs. before 26.7 ±15.558 irradiation) + irradiation Chenpi(lmg/head, twice IP. at 36 and 12hrs. before 30.45 ±13.391 irradiation) + irradiation Shengma(lmg/head, twice I.P. at 36 and 12hrs. before 40.96 ± 4.957**** Irradiation) + irradiation Chaihudmg/head, twice I.P. at 36 and 12hrs. before 35.3 ± 6.538**** 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. - 36 - Table 2.5. 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 157.252± 6.051 Irradiation control (12Gy) 38.484 ± 4.335 San-Ling-Bai-Shu-San 47.804 ±12.730 (lmg/head, twice I.P. at 36 and 12hrs. before irradiation) + irradiation Untreated control 164.237± 8.314 Irradiated control 19.637 ± 6.085 Rensen (lmg/head, twice I.P. at 36 and 12hrs. 37.8 ±10.134** before irradiation)+irradiation Baizhudmg/head, twice IP. at 36 and 12hrs. 26.625 ± 8.643 before irradiation)+irradiation Fuling(lmg/head, twice I.P. at 36 and 12hrs. 45.186 ±12.579** before irradiation)+irradiation Gancao( lmg/head, twice I.P. at 36 and 12hrs. 59.775 ±26.494** before irradiation)+irradiation Yiyiren(lmg/head, twice I.P. at 36 and 12hrs. 30.542 ±15.248 before irradiation)+irradiation baibiandou(lmg/head, twice I.P. at 36 and 12hrs. 31.107 ± 8.951* before irradiation)+irradiation Shanyao(lmg/head, twice I.P. at 36 and 12hrs. 32.729 ±13.795 before irradiation)+irradiation Lianroudmg/head, twice I.P. at 36 and 12hrs. 42.05 ±26.355 before irradiation)+irradiation Sharen( lmg/head, twice I.P. at 36 and 12hrs. 49.375 ±17.562** before irradiation)+irradiation Jiegeng( lmg/head, twice I.P. at 36 and 12hrs. 47.07 ±19.659** before irradiation)+irradiation *p<0.05 as compared with irradiated control group. **p<0.005 as compared with irradiated control group. - 37 - Table 2.6. Effect of Kuei-Pi-Tang and its ingredients on intestinal crypt survival in irradiated mice (M±SD) Groups Crypts per circumference Untreated control 157.252 ± 6.051 Irradiation control (12Gy) 38.484 ± 4.335 Kuei-Pi-Tang 62.131 ±14.659* (lmg/head, twice IP. at 36 and 12hrs. Before irradiation) + irradiation Untreated control 164.241 ± 8.314 Eradiation control(12Gy) 19.637± 6.085 DangguK lmg/head, twice I.P. at 36 and 12hrs. 51.2 ±11.952** before irradiation)+iiradiation Longyanrou(lmg/head, twice IP. at 36 and 12hrs. 27.279 ± 6.980 before irradiation) +iiradiation Suanzaoren(lmg/head, twice IP. at 36 and 12hrs. 30.696± 8.217*** before irradiation)+iiradiation Yuanzhi(lmg/head, twice I.P. at 36 and 12hrs. 25.786 ± 8.439 before irradiation)+iiradiation RensanC lmg/head, twice I.P. at 36 and 12hrs. 37.8 ±10.134* before irradiation)+iiradiation Huangqi(lmg/head, twice I.P. at 36 and 12hrs. 26.7 ±15.558 before irradiation)+iiradiation Baizhudmg/head, twice IP. at 36 and 12hrs. 26.625 ± 8.643 before irradiation)+iiradiation Fuling(lmg/head, twice I.P. at 36 and 12hrs. 50.375 ± 9.746* before irradiation)+iiradiation Muxiang(lmg/head, twice I.P. at 36 and 12hrs. 28.951 ± 7.766*** before irradiation)+iiradiation Gancaodmg/head, twice I.P. at 36 and 12hrs. 59.775 ±26.494* before irradiation)+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. - 38 - Fig. 2.2. The macroscopic finding of endogenous spleen colonies formed after irradiation. Mice were exposed to whole body irradiation with single doses of 6.5 Gy. Nine days after irradiation, the spleens were removed and fixed in Bouin's solution. - 39 Table 2.7. Effect of Si-Wu-Tang and its ingredients on endogenous spleen colony formation in irradiated mice (M±SD) Groups Number of colony Irradiation control(6.5Gy) 3.5 ±4 Si-Wu-Tang (2mg/ml of drinking water, for 7days 10.0 ±7.730* before irradiation) + irradiation Si-Wu-Tang(lmg/head, twice IP. at 36 and 12hrs. 8.667±5.244* before irradiation) + irradiation Irradiation + 4.668 ±6.144 Si-Wu-Tang (2mg/ml of drinking water, for 9days) Irradiation+ 8.625 ±5.999* Si-Wu-Tang (lmg/head, single IP. 30min. after irradiation) Irradiation control 1.125 ± 1.246 ShoudehuangUmg/head, twice IP. at 36 and 12 hrs. 3.375+ 2.875* before irradiation) + irradiation Chuanxiong(lmg/head, twice IP. at 36 and 12 hrs. 9.375± 15.729 before irradiation) + irradiation Dangguidmg/head, twice IP. at 36 and 12 hrs. 6.5 ± 6.302* before irradiation) + irradiation Baishaoyao( lmg/head, twice IP. at 36 and 12 hrs. 15 ±17.330* before irradiation) + irradiation *p<0.05 as compared with irradiation control group. - 40 - Table 2.8. 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(6.5Gy) 4.268 ±3.976 Si-Jun-Zi-Tang (2mg/ml of drinking water, for 7days) 4.814 ±2.818 + irradiation Si-Jun-Zi-Tang (lmg/head, twice I.P. at 36 and 12hrs 6.152±3.606 before irradiation) + irradiation Irradiation + 5.282 ±4.229 Si-Jun-Zi-Tang(2mg/rnl of drinking water, for 9days) Irradiation + 3.694 ±2.482 Si-Jun-Zi-Tang(lmg/head,single I.P. 30min. after irradiation) Irradiation control 1.125+1.246 Rensan( lmg/head, twice I.P. at 36 and 12 hrs. 9.5 ± 6.777* before irradiation) + irradiation Gancao( lmg/head, twice IP. at 36 and 12 hrs. 1 ± 1.773 before irradiation) + irradiation Baifuling( lmg/head, twice I.P. at 36 and 12 hrs. 6.75 ±10.553 before irradiation) + irradiation Baizhu( lmg/head, twice IP. at 36 and 12 hrs. 3 ± 5.398 before irradiation)+ irradiation *p<0.005 as compared with irradiation control. - 41 - Table 2.9. 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 (6.5Gy) 4.5 ±2.510 Shi-Quan-Dai-Bu-Tang 2.857 ± 1.773 (2mg/ml of drinking water, for 7days) + irradiation Shi-Quan-Dai-Bu-Tang 3.833 ± 3.251 (lmg/head, twice IP. at 36 and 12hrs. before irradiation) + irradiation Irradiation + Shi-Quan-Dai-Bu-Tang 2.333 ± 1.633 (2mg/ml of drinking water, for 9days) Irradiation + 4.571 ± 3.259 Shi-Quan-Dai-Bu-Tang (lmg/head, single IP. 30min. after irradiation) Irradiation control 1.125± 1.246 Shoudehuang (lmg/head, twice I.P. at 36 and 12 hrs. 3.375 ± 2.875* before irradiation) + irradiation Chuanxiong(lmg/head, twice I.P. at 36 and 12 hrs. 9.375 ±15.729 before irradiation) + irradiation Dangguidmg/head, twice IP. at 36 and 12 hrs. 6.5 ± 6.302* before irradiation) + irradiation Baishaoyao(lmg/head, twice I.P. at 36 and 12 hrs. 15 ±17.330* before irradiation) + irradiation Rensan(lmg/head, twice I.P. at 36 and 12 hrs. 9.5 ± 6.777** before irradiation) + irradiation Gancao(lmg/head, twice IP. at 36 and 12 hrs. 1 ± 1.773 before irradiation) + irradiation Baifuling(lmg/head, twice I.P. at 36 and 12 hrs. 6.75 ± 10.553 before irradiation) + irradiation Baizhu( lmg/head, twice I.P. at 36 and 12 hrs. 3 ± 5.398 before irradiation) + irradiation Huangquidmg/head, twice I.P. at 36 and 12 hrs. 13.375 ±14.745* before irradiation) + irradiation Rouguidmg/head, twice I.P. at 36 and 12 hrs. 8.432 ±12.738 before irradiation) + irradiation *p<0.05 as compared with irradiation control group. **p<0.005 as compared with irradiation control group. - 42 - Table 2.10. 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 (6.5Gy) 5 ± 3.024 Bu-Zhong-Yi-Qi-Tang (2mg/ml of drinking water, 5.667± 4.093 for 7days) + irradiation Bu-Zhong-Yi-Qi-Tangdmg/head, twice IP. at 36 and 12hrs. 15.375 ±11.096* before irradiation) + irradiation Irradiation + Bu-Zhong-Yi-Qi-Tang (2mg/ml 6 ± 6.782 of drinking water, for 9days) Irradiation + Bu-Zhong-Yi-Qi-Tang (lmg/head, 5.778± 3.154 single I.P. 30min. after irradiation) Irradiation control (12Gy) 2.111 ± 1.692 Rensan(lmg/head, twice IP. at 36 and 12hrs. before 10.1 ± 6.777** irradiation) + irradiation Gancao(lmg/head, twice IP. at 36 and 12hrs. before 1.935± 2.547 irradiation) + irradiation Danggui(lmg/head, twice I.P. at 36 and 12hrs. before 7.2 ± 6.237* irradiation) + irradiation BaizhuC lmg/head, twice IP. at 36 and 12hrs. before 4.375 ± 6.938 irradiation) + irradiation Huangqi(lmg/head, twice IP. at 36 and 12hrs. before 15.107± 16.759* irradiation) + irradiation Chenpi(lmg/head, twice IP. at 36 and 12hrs. before 2.778± 3.032 Irradiation) + irradiation Shengma( lmg/head, twice IP. at 36 and 12hrs. before 4.778 ± 5.239 Irradiation) + irradiation Chaihu(lmg/head, twice IP. at 36 and 12hrs. before 7.444 ± 6.784* irradiation) + irradiation *p<0.05 as compared with irradiation control group. **p<0.005 as compared with irradiation control group. - 43 - Table 2.11. 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 (6.5Gy) 2.625 ± 0.916 San-Ling-Bai-Shu-San 3.5 ± 2.330 (2mg/ml of drinking water, for 7days) + irradiation San-Ling-Bai-Shu-San(lmg/head, two IP. 5.375± 3.253* at 36 and 12hrs. before irradiation) + irradiation Irradiation + San-Ling-Bai-Shu-San 2.75 ± 3.105 (2mg/ml of drinking water, for 9days) Irradiation + San-Ling-Bai-Shu-San 6.625 ± 8.847 (lmg/head, single IP. 30min. after irradiation) Irradiation control 2.111 ± 1.682 Rensen (lmg/head, twice IP. at 36 and 12hrs. 10.364 ± 6.972** before irradiation)+irradiation Baizhu(lmg/head, twice IP. at 36 and 12hrs. 3.986 ± 5.247 before irradiation)+irradiation Fuling( lmg/head, twice IP. at 36 and 12hrs. 7.736 ±10.486 before irradiation)+irradiation Gancao( lmg/head, twice IP. at 36 and 12hrs. 1.989 ± 1.294 before irradiation)+irradiation Yiyiren( lmg/head, twice IP. at 36 and 12hrs. 12.625 ±13.405* before irradiation)+irradiation baibiandouC lmg/head, twice IP. at 36 and 12hrs. 5.445 ± 8.233 before irradiation)+irradiation ShanyaodmgAead, twice IP. at 36 and 12hrs. 1.778± 2.729 before irradiation)+irradiation Lianrou(lmg/head, twice IP. at 36 and 12hrs. 4 ± 3.354 before irradiation)+irradiation Sharen(lmg/head, twice IP. at 36 and 12hrs. 13.486 ±17.694 before irradiation)+irradiation Jiegeng(lmg/head, twice IP. at 36 and 12hrs. 2.778 ± 2.167 before irradiation)+irradiation *p<0.05 as compared with irradiation control group. **p<0.005 as compared with irraidation control group. - 44 - Table 2.12. 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 (6.5Gy) 3.556 ± 2.789 Kuei-Pi-Tang 5.3 ± 2.983 (2mg/ml of drinking water, for 7days) + irradiation Kuei-Pi-Tang 5.667± 4.093 (lmg/head, twice I.P. at 36 and 12hrs. before irradiation) + irradiation Irradiation + 4.2 ± 3.360 Kuei-Pi-Tang (2mg/ml of drinking water, for 9days) Irradiation + 3.667 ± 2.915 Kuei-Pi-Tang (lmg/head, single IP. 30min. after irradiation) Irradiation control (12Gy) 1.125± 1.246 DangguKlmg/head, twice I.P. at 36 and 12hrs. 6.5 ± 6.302* before irradiation)+iiradiation Longyanroudmg/head, twice I.P. at 36 and 12hrs. 3.125± 7.356 before irradiation)+iiradiation Suanzaorendmg/head, twice I.P. at 36 and 12hrs. 10.459 ±12.088* before irradiation)+iiradiation Yuanzhi(lmg/head, twice IP. at 36 and 12hrs. 5.57 ± 8.22 before irradiation)+iiradiation Rensan(lmg/head, twice I.P. at 36 and 12hrs. 9.5 ± 6.678** before irradiation)+iiradiation Huangqi(lmg/head, twice I.P. at 36 and 12hrs. 13.375± 14.745* before irradiation)+iiradiation Baizhu(lmg/head, twice I.P. at 36 and 12hrs. 3 ±5.398 before irradiation)+iiradiation Fuling( lmg/head, twice I.P. at 36 and 12hrs. 6.75 ±10.553 before irradiation)+iiradiation Muxiang(lmg/head, twice I.P. at 36 and 12hrs. 3.792 ± 4.032 before irradiation)+iiradiation Gancao(lmg/head, twice I.P. at 36 and 12hrs. 1 ± 1.773 before irradiation)+iiradiation * p<0.05 as cpmpared with irradiation control group. ** p<0.005 as compared with irradiation control group. - 45 - P 0 ••*>*'. '* IT B Fig. 2.3. Intestinal crypts of mice 6 hours after exposure to gamma radiation. (A) Exposure to 2Gy gamma radiation. Cells exhibiting pyknosis of nuclei (arrow) are seen. H-E staining, X 330. (B) In situ end labelling(ISEL) demonstrating numerous apoptotic nuclei and bodies in the crypts. ISEL, chromogen diaminobenzidine, hematoxylin counterstaining, X 330. Table 2.13. 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(2Gy) 4.540 ±0.646 5.111 ±0.529 Si-Wu-TangClmg/head, twice IP. at 36 and 12hrs. 3.6 ±0.184** 3.919±0.214* before irradiation) + irradiation Untreated control 0.068 ±0.032 0.084 ±0.024 Irradiation control 4.688± 1.138 4.938 ±1.194 Shoudehuang(lmg/head, twice IP. at 36 and 12 hrs. 3.1 ±0.975 3.369± 1.080 before irradiation) + irradiation Chuanxiong(lmg/head, twice IP. at 36 and 12 hrs. 2.519±0.335** 2.856±0.478** before irradiation) + irradiation Danggui(lmg/head, twice IP. at 36 and 12 hrs. 3.861 ±0.469 4.081 ±0.387 before irradiation) + irradiation Baishaoyaodmg/head, twice IP. 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. - 47 - Table 2.14. 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 (2Gy) 4.540±0.646 5.111 ±0.529 Si-Jun-Zi-Tang(lmg/head, twice I.P. at 36 and 3.756± 1.131 4.077 ± 1.119 12hrs. before irradiation)* irradiation Untreated control 0.068 ±0.032 0.084+0.024 Irradiation control 4.688± 1.138 4.938± 1.194 Rensan(lmg/head, twice I.P. at 36 and 12 hrs. 2.769 ±0.208 3.126 ±0.382* before irradiation)* irradiation Gancao(lmg/head, twice IP. at 36 and 12 hrs. 5.128± 1.296 5.488± 1.956 before irradiation) + irradiation Baifuling(lmg/head, twice I.P. at 36 and 12 hrs. 2.981 ± 0.391 * 3.331 ± 0.335* before irradiation) + irradiation Baizhu(lmg/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. - 48 - Table 2.15. 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 cells per crypt Groups Base Total Untreated control 0.071 ±0.035 0.091 ±0.031 Irradiation control (2Gy) 4.540 ±0.646 5.111 ±0.529 Shi-Quan-Dai-Bu-Tang (lmg/head, 3.888 ±0.593 4.275 ±0.628 twice IP. at 36 and 12hrs. before irradiation) + irradiation Untreated control 0.068 ±0.032 0.084 ±0.024 Irradiation control 4.688 ±1.138 4.938 ±1.194 Shoudehuang(lmg/head, twice IP. at 36 and 12 hrs. 3.1 ±0.975 3.369 ±1.080 before irradiation) + irradiation Chuanxiong (lmg/head, twice I.P. at 36 and 12 hrs. 2.519 ±0.335* 2.856 ±0.478* before irradiation) + irradiation Danggui(lmg/head, twice IP. at 36 and 12 hrs. 3.861 ±0.469 4.081 ±0.387 before irradiation) + irradiation Baishaoyao(lmg/head, twice IP. at 36 and 12 hrs. 2.594 ±0.464* - 2.806 ±0.429* before irradiation) + irradiation Rensan(lmg/head, twice IP. at 36 and 12 hrs. 2.769 ±0.208 3.126±0.382* before irradiation) + irradiation Gancao( lmg/head, twice I.P. at 36 and 12 hrs. 5.128 ±1.296 5.488 ±1.956 before irradiation) + irradiation Baifuling(lmg/head, twice I.P. at 36 and 12 hrs. 2.981 ±0.391* 3.331 ±0.335* before irradiation) + irradiation Baizhu(lmg/head, twice IP. at 36 and 12 hrs. 4.224 ±1.71 4.568 ±1.379 before irradiation) + irradiation Huangqui(img/head, twice I.P. at 36 and 12 hrs. 3.244 ±0.490 3.581 ±0.453 before irradiation) + irradiation RouguKlmg/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. - 49 - Table 2.16. 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 (2Gy) 4.540 ±0.646 5.111 ±0.529 Bu-Zhong-Yi-Qi-Tang (lmg/head, 3.438 ±0.161* 3.8 ±0.176** twice I.P. at 36 and 12hrs. before irradiation) + irradiation Untreated control 0.071 ±0.036 0.091 ±0.032 Irradiation control (12Gy) 4.688±1.138 4.938±1.194 Rensan(lmg/head, twice IP. at 36 and 12hrs. 2.769±0.208* 3.126±0.382* before irradiation) + irradiation Gancaodmg/head, twice IP. at 36 and 12hrs. 5.246±1.835 5.743±3.136 before irradiation) + irradiation Dangguidmg/head, twice IP. at 36 and 12hrs. 3.812±0.625 4.194±0.124 before irradiation) + irradiation Baizhu(lmg/head, twice IP. at 36 and 12hrs. 4.189± 1.905 4.224±3.285 before irradiation) + irradiation Huangqidmg/head, twice IP. at 36 and 12hrs. 3.244±0.490 3.581 ±0.453 befor irradiation) + irradiation Chenpidmg/head, twice IP. at 36 and 12hrs. 3.288±0.165 3.6 ±0.177 before irradiation) + irradiation Shengma( lmg/head, twice IP. at 36 and 12hrs. 2.388±0.449** 2.619±0.452* before irradiation) + irradiation Chaihudmg/head, twice IP. at 36 and 12hrs. 2.944±0.405* 3.275±0.448* before 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. - 50 - Table 2.17. Effect of S an -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 (2Gy) 4.540 ±0.646 5.111 ±0.529 San-Ling-Bai-Shu-San (lmg/head, 2.2 ±0.704* 2.475 ±0.820* twice I.P. at 36 and 12hrs. before irradiation) + irradiation Untreated control 0.071 ±0.036 0.091 ±0.032 Irradiated control (2Gy) 4.688± 1.138 4.938± 1.194 Rensen (lmg/head, twice I.P. at 36 and 12hrs. 2.769 ±0.208* 3.712 ±0.692* before irradiation)+irradiation Baizhudmg/head, twice I.P. at 36 and 12hrs. 3.988 ±2.724 4.113 ±3.719 before irradiation)+irradiation Fuling(lmg/head, twice I.P. at 36 and 12hrs. 2.981 ±0.391* 3.331 ±0.335* before irradiation)+irradiation Gancao( lmg/head, twice I.P. at 36 and 12hrs. 5.456 ±2.679 5.875 ±3.524 before irradiation)+irradiation Yiyiren( lmg/head, twice I.P. at 36 and 12hrs. 4.825 ±2.986 5.25 ±4.986 before irradiation)+irradiation baibiandou(lmg/head, twice I.P. at 36 and 12hrs. 4.95 ±1.830 5.282 ±2.372 before irradiation)+irradiation Shanyao( lmg/head, twice I.P. at 36 and 12hrs. 3.294± 1.852 3.763 ±1.090 before irradiation)+irradiation Lianrou( lmg/head, twice I.P. at 36 and 12hrs. 5.244 ±1. 5.632 ±2.368 before irradiation)+irradiation Shren(lmg/head, twice I.P. at 36 and 12hrs. 3.325± 1.917 3.125± 2.317 before irradiation)+irradiation Jiegeng( lmg/head, twice I.P. at 36 and 12hrs. 6.488 ±2.453 7.269 ±1.194 before irradiation)+irradiation *p<0.05 as compared with irradiation control group. - 51 - Table 2.18. 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 (2Gy) 4.540 ±0.646 5.111 ±0.529 Kuei-Pi-Tang 3.763 ±0.475 4.081 ±0.447* (lmg/head, twice I.P. at 36 and 12hrs. before irradiation) + irradiation Untreated control 0.063 ±0.025 0.081 ±0.036 Iiradiation control(12Gy) 4.088± 0.423 4.481 ±0.262 DangguKlmg/head, twice I.P. at 36 and 12hrs. 2.974 ±1.625 3.407 ±1.489 before irradiation)+iiradiation Longyanrou(lmg/head, twice I.P. at 36 and 12hrs. 1.788±0.334**** 2.038± 0.383**** before irradiation )+iiradiation Suanzaoren(lmg/head, twice I.P. at 36 and 12hrs. 2.844 ±0.487** 3.250 ±0.594** before irradiation) +iiradiation YuanzhK lmg/head, twice I.P. at 36 and 12hrs. 2.944 ±0.754* 3.163 ±0.697* before irradiation)+iiradiation Rensan(lmg/head, twice I.P. at 36 and 12hrs. 2.739 ±0.682* 3.281 ±0.743* before irradiation)+iiradiation Huangqi(lmg/head, twice I.P. at 36 and 12hrs. 3.144 ±0.647 3.624 ±0.727 before irradiation)+iiradiation Baizhu(lmg/head, twice I.P. at 36 and 12hrs. 3.388 ±2.280 3.656 ±3.285 before irradiation)+iiradiation Fuling( lmg/head, twice I.P. at 36 and 12hrs. 2.743 ±0.843* 3.274 ±0.945* before irradiation)+iiradiation Muxiang( lmg/head, twice I.P. at 36 and 12hrs. 2.106 ±0.624*** 2.238±0.717*** before irradiation )+iiradiation Gancaodmg/head, twice I.P. at 36 and 12hrs. 4.858 ±2.194 5.481 ±3.136 before irradiation)+iiradiation * p<0.05 as compared with irradiation control group. ** p<0.0I as compared with irradiation control group. *** p<0.005as compared with irradiation control group. **** p<0.0005 as compared with irradiation control group. - 52 - Table 2.19. Results on 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(lOkGy) 161.435 ± 7.361 Unirradiated Bu-Zhong-Yi-Qi-Tang 150 ± 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 (12Gy) 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. - 53 - Table 2.20. Results on the endogenous spleen colonies in irradiated mice treated with irradiated or unirradiated Oriental medical prescriptions (M±SD) Groups Number of colony Irradiation(6.5Gy) without prescription 0.375 ± 0.518 Unirradiated Si-Wu-Tang + irradiation 3.554 ± 3.615* Irradiated Si-Wu-TangQOkGy) + 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 ± 0.707* Irradiated San-Ling-Bai-Shu-San + irradiation 1.333 ± 1* *p<0.05 as compared with irradiation without prescription group. - 54 - Table 2.21. Results on 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(lOkGy) 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(2Gy) 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. **p<0.01 as compared with irradiation without prescription group. - 55 - 1. 7\. 20% FCS - RPMI1640 «1| 2 - 2-3^°^ el-. RAW264.7^ - 56 - proteose peptone^ JL 4 lipopolysaccharide(LPS)£^ ^ NO2) NO3, NO ^ Hfl<#oJHJ ^^E]7] nfl^-ofl ^A^A]^ ELISA reader^ #^ 96 well plate well^ 5xiO47|)^ ^ ELISA Titer Tek plated Griess Reagent(l:l, v/v, N-naphthylethylendiamine 0.1% in H2O, sulfanilarnide 1% in 5% H3PO4)* ^7}^JL 10^-^t ^^-^1 ^l^ ^ ELISA reader (Molecular Devicer)^ 550nmi^ NaNO2# £^r S)^ >^>. LPS # ^ ^^7fl tfl^ Sf-^H ^-i-^l S.^±= lipopolysdccharide^ AA 7£%°] 7}^$: Amoeboyte lysate assay(E-TOXATE, sigma)* -g-^4 amoebocyte lysatel- 1 : IS. sl^^l^ ^"f^r^l ^Ji, 3 olnfl -g-oflo] ]^ ^^ LPSJSL fe cytokine^r RNAzol B(Biotecx lab)l- °]^-§>^ total RNA-& €«]*H, ^^ 7}z] cytokine £| primerl- dl^-^ RT-PCR ^AS mRNA - 57 - 7} ^ random hexamer^l oligo(dN)6^- primer^ ^1-Jl dNTP's, RNase inhibitor, MMLV reverse transcriptase ^-t ^7)-^ <&%• ^-§-^4 ^o] 6 V-g-Al^ first strand cDNA# •t^sf^^-. 1 #-§-^ 75°C, reverse transcriptaset- 1:#^^- *]?A ^ PCR^: W template DNAS °l-§-^>^4. °1 template DNA^f Taq polymerase ^-i- 94°C 1^:, 55°C 30^, 72°C l^ri-H 35cycleS. ^*J ^i^># ethidium bromide(0.05jMg/ml)7r ^7>Jg 1.2% agarose - 58 - Sequences of the oligonucleotide primers used for PCR amplification of cytokines and iNOS mRNA mRNA Primer sequence51 Product(bp) Sense 5'-GCAACTGTTCCTGAACTCA-3' IL-l/S 165 Antisense 5'-CTCGGAGCCTGTAGTGCAG-3' Sense 5'-AACAGCGCACCCACTTCAA-3' IL-2 382 Antisense 5'-TTGAGATGATGCTTTGACA-3' Sense 5'-GCAGCTCTATTGTCAAGGAG-3' IL-3 216 Antisense 5'-GCAGAGTCATTCGCAGATGTAG-3' Sense 5'-TAGTTGTCATCCTGCTCTT-3' IL-4 404 Antisense 5'-CTACGAGTAATCCATTTGC-3' Sense 5'-TTCCTCTCTGCAAGAGACT-3' IL-6 532 Antisense 5'-TGTATCTCTCTGAAGGACT-3' Sense 5'-ATCTTCCATGTTTCTTTTAGA-3' IL-7 450 Antisense 5'-TATACTGCCCTTCAAATTTTTATT-3' Sense 5' -TCCTT AATGC AGGACTTTAAGGGTTACTTG-3' IL-10 256 Antisense 5'-GACACCTTGGTCTTGGAGCTTATTAAAATC-3' Sense 5'-CAGAAGCTAACCATCTCCTGGTTTG-3' IL-12 394 Antisense 5'-TCCGGAGTAATTTGGTGCTtCACAC-3' Sense 5'-GCTTGACTACTCTTCTGGAC-3' SCF 354 Antisense 5'-CTGCTGTCATTCCTAAGGG-3' Sense 5' - AACFCTACACACTFCATCT-3' IFN-y 342 Antisense 5'-TGCTCATTGTAATGCTTGG-3' Sense 5'-GGCAGGTCTACTTTGGAGTCATTGC-3' TNF-a 308 Antisense 5' -ACATTCGAGGCTCCAGTGAATTCGG-3' Sense 5'-TCAGAAGCACTTGACCCAT-3' LT 322 Antisense 5'-AAGTCCCGGATACACAGACT-3' Sense 5'-ACTGTACAACCGCAGTAATACGG-3' IGIF 434 Antisense 5'-AGTGAACATTACAGATTTATCCC-3' Sense 5' -CATGGCTTGCCCCTGGAAGTTTCTCTTCAAAG-3' iNOS 754 Antisense 5' -GCAGCATCCCCTCTGATGGTGCCATCG-3' Sense 5'-GTTGGATACAGGCCAAGACTTTGTTG-3' HPRT 165 Antisense 5'-GATTCAACTTGCGCTCATCTTAGGC-3' Sense 5'-GTGGGCCGCTCTAGGCACCA-3' 0 -actin 240 Antisense 5'-TGGCCTTAGGGTTCAGGGGG-3' - 59 - 2. <£^€ 7\. # #^#^]£(bone marrow stem cells)4 i(stromal cells)44 ^ 7}*|JL ig. 3.1). 14. , 87H 44 ^£iS ^7HH 2-3 ig. 3.2A, 3.2B). 44 f-?)i ^7}^>^ tiflo^ ^, ^^^ils^ ^H 44^ i41- #^§H 4 4(Fig. 3.3A, 3.3B). ZL ^4, 444 27M ^^-4 ^^-i- ^r° *1, ^^^ 44 4 4 50 4. it, ^^^L ^^S ^r^> 1^t!: 14 11^ f4# ^.% 14 - 60 - ig. 3.4). ^ 31-I- P>. tfl^AHioflA^ ^A};^- ^(Nitric Oxide)^ ^ NO ^^-i: -ff-SSV^l ^-§1-^1 ^>, IFN N0 ^-i- -n-S^I-^31, iNOS IL-1,3, IL-6, LT, iNOS^l mRNA (1) RAW264.7Afl3L°1! 10, LPS(115,116) LPS1- ^^-^.S. ^el§}^-i: ^^ NO2"^£7l- 10/zM ^H ^. xtfl ig. 3.6B). # - 61 - IFN-yi /ml °a *fl <$# #£fJHr 7J0.5. q-E]-tftKFig. 3.6C). ofeH IFN-r 4 LPS^I (2) | ^ 9^ ^ ^^fl ll f }&\i % g})) IFN fe IFN-r# ^^-^S. ^el^l-^-i- nfl -S.4 NO2"^£7> sf 10 ig. 7A). £•& IFN-7^ ^L ^oz{- ^-^ IFN-7 , 10, 1- ^r 9X9X3., SL7}s\£\ ^-^7} , 10, lOOU/mlS fe^r^r^- NO2"^£7> ^71-^-i- ^ ^ $a&4(Fig. 3.7B). ig. 3.8). ig. 3.9) (3) c -7^- SL7W£\ NO^^°H tfl^ ^^^47]-, i7f3^ #^# #i LPS7> LPS 3.10A). LPS gr^k ^^-1- ^61-^-71 ^slj Amebocyte r 62 - lysate assay(E-TOXATE, sigma)* °l-§-«H LPS^H-t #^$4. n ^4 .2. \91 lOpg/ml ol«}^ LPS7]- ^^ ^ilSL 44 LPSi ^tb 3H c>\i ^^-S ^4^4. 4w^S. NO l 7] ig. 3.10B). (4) tn^Afli^ iNOS mRNA tfl^^i^ IFN-7 5]- SL7}3f\, 7} ^^'s)^7l t41^i, oliSH^r ^^Sf^^l- >S^t>fe JLi?l iNOSQnduceable nitric oxide synthease)^l mRNA^:^^- RT-PCR^^-S. ^- ^fe IFN-r1?} ig. 3.11). ^, ^^^fl ^ (5) 4^ A>o]S7l-^l^ mRNA mRNA t^ir 2:44^4(120,121). n ^14 tflSnH H1*H IL-ljff, IL-6, LT T?£| A}O]£7}.O] mRNA Tg-^o] ^7|-§}.^ ^^. ^- ^ ^^4(Fig. 3.12). (6) ^44 , IFN-/4 ig. 3.13). - 63 - Fig. 3.1. Morphology of stromal cells derived mouse bone marrow cells. The bone marrow cells (lxlO6 cells/2ml) from BALB/c mice were cultured for 14 days in 10% FCS-RPMI1640 medium. - 64 - Effect of various plants extracts on stromal cells growth 100 90 80 o 70 X 60 50 jmbe r —> c 40 Cel l 30 20 10 0 1 10 100 1000 Concentrations, ug/ml Fig. 3.2A. Effect of various plant extracts in stromal cells growths. The bone marrow cells(lxl06cells/ml) from BALB/c mice were cultured for 14 days in 10% FCS-RPMI1640 medium contained with various plant extracts at 37 °C, 5% CO2 and their viable cells were counted by Trypan blue staining. - 65 - 0^2= 100 80 60 40 ( 20 -/ X w 0 0 1 10 100 1000 Fig. 3.2B. Effect of various plant extracts on stromal cells growth. - 66 - 300 r 250 <*><$. <\ <$•$. Q <<). <^ <& 300 250 0W« 1 o K88S o 1 o — 200 1 1 1 of 1150 1 1 1 I E 1 1 1 c100 I o 1 § 50 1 1 1 II I$ 1 1 1 S3t 1t I Plant Extracts Fig. 3.3A. Synergistic effect of plant extracts on stromal cells growth. The bone marrow cellsd x 106/ml) from BALB/c mice were cultured for 14days 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. - 67 - 300 250 o o o 200 Plant Detracts 300 250 o o o 200 Plant Extracts Fig. 3.3B. Synergistic effect of plant extracts on stromal cells growth. The bone marrow cells(lxlO6/ml) 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. - 68 - _ 10 Q O in O 6 • 4 • to 2 ^— • • • • • • 0 1,1 • • • • , • s Num l °o 5 "£ ^ g. Ce l b ?. ^ X b ^. i t i Plant extracts, 1 00^g/ml Fig. 3.4. Effect of herbal plants mixture extract on proliferation of nonadherent cells. Nonadherent supernatant-derived mononuclear cells harvested from long-termed stromal cell culture in the presence or absence of mixture extracts. - 69 Marker IL-3 IL-4 IL-7 SCF TNF-ot p-actin Control Kl Fig. 3.5. RT-PGR analysis of IL-3, IL-4, IL-7, SCF and TNF- a mRNA expression by 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 bromide. - 70 - A. Control B. 19 14 • c o o 2 2 o c •t—« • —. CD 9 -1 & iO KD Water-extract V\feiter-extract water-extract concentration, ^.g/ml concentration, concentration, n,g/ml Fig. 3.6. Effect of Acanthopanicis Cortex extracts on NO synthesis in RAW 264.7 cell lines. The cells (5xlO4/well) were cultured with LPS(l/*g/ml) and/or IFN- r (lOU/ml). After 48hr of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 71 - , lOUrrt 2D i -o-syp-t) » 1 1= C o ion , i '•a 4—' If c o o o c o o concen t I /// o Z 5 •- o: J/J 0 1 10 1C0 V\feter-extract concentration, IRshf, U/ni jjg/rrl Fig. 3.7. Dose-dependent effect of Acanthopanicis Cortex extracts on NO synthesis in IFN- y -treated RAW 264.7 cell lines. The cells (5 x 104/well) were cultured with various concentrations of IFN- y. After 48hr of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 72 - +IFN-7, 10U/ml on -»-gl9l i -|-t7| 2 15 //• A X 1 ion , re //• y // o concent r S 5 1 n 0 1 10 100 Water-extract concentration, ng/ml Fig. 3.8. Effect of varrious herbal plant extract on NO synthesis in IFN-7 treated RAW 264.7 cell lines. The cells (5x lOVwell) were cultured with various herbal plant extracts in the presence of IFN- y (lOU/ml). After 48hr of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 73 - NO2 concentration, I SI Fig. 3.10. Effect of Acanthopanicis Cortex extracts and NOS inhibitors on nitrite accumulation in the cultured medium of RAW 264.7 cell lines. The cells (5><104/well) were cultured with various concentrations of Acanthopanicis Cortex extracts or NGMMA. After 48hr of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 75 - Marker IL-10 IL-2 IL-4 IL-6 IL-10 IL-12 Maker LT IFN- y TNF- a IGIF iNOS HPRT Control Fig. 3.11. RT-PCR analysis of IL-1/?, IL-2, IL-4, IL-6, IL-10, IL-12, LT, IFN-7, TNF-ff, IGIF and iNOS mRNA expression by 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 bromide. - 76 - t : Marker Control IFN+^fl IFN+J2.7rs} IFN+§"?1 IFN- y iNOS HPRT Fig. 3.12. RT-PCR analysis of iNOS mRNA expression by 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 bromide. - 77 - primery rracrophage 12 10 c 8 o '•»-> CCj •*-> Co o • c O -- O X • s - k.- z: :••:•• m "1 1 "":" 1 1 J1L :' ••'.' V Fig. 3.13. Effect of various herbal plant extracts on NO synthesis in IFN- r -treated mouse peritoneal macrophages. The cells (5 x 104/well) were cultured with various herbal plant extracts (100/ig/ml) for 48hr in the presence or absence of IFN- 7. After 48hr of culture, NO release was measured by the Griess method. Nitrite released into the medium is presented as the mean ± S.D. - 78 - 1. (1) (2) "1^# ^ ^1^1- 3*Rr 4 ^S.°ll ^^=51 ^5^ 0.1% peptone H 3S] ti}^- ^Al^>^4. 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Al^^ofl ^^r^^-g-^ 0.5 ml (tflA>^Aj Si-A]?}±r 7^-foil^ S9 mix(124,125) 0.5 ml), Als.^-^ o.l ml 4 Oxoid nutrient <5fl o.l ml# ^) histidine/biotin°l ^7}^ top agar(45°C)t- 2 ml 7>s)-ji minimal glucose agar plate -SH ^ - 80 - (2) n Aj^ofl Af-g-^ jc^.^.^.^i^. Chinese hamster ovary (CHO) *fl*l*r 10% fetal bovine serum ^-§- %7}*]f(\ RPMI 1640 «1)*l3. 5% CO2* ^ 4 ^^ 37ts] CO2 IncubatoHl^ HflWS^. ^^^ CHO 43. 8xlO 7flt- Flaskette(19.8x51.8 mm, NuncHl 4^«H 2°^ cytochalasin B(Cyt-B; 3 Ug/ml, Aldrich)^ f-^1] ^7|-§}j ^r ^l-Al^KFenech and Morley(127)^l cytokinesis-block method). 75 mM KC1 -§-^-i- ^e]«H 43.9 *§%*}?1 4^ JI^S^(methanol : acetic acid, 3:1)1S JL^A]^ :f ^yl^S^^-S. ^1-^4. >H|aa-&-i- 3% Giemsa <3 S9 l^ 20% «1 •§:)!• ^ Cyt-B* ^7J-eV^ \%X\ (1) Salmonella typhimurium benzo( a (2) CHO CHO benzo( (3) ml-§- 50 250 - 81 - (SL7}v\, o^a : 3*1 #*! 2S] «]:^-)^#*>Jl <*| 4 (Whatman No. 44)^ ^70 °C -§-^HH rotary evaporators. #«?HF^SH 80 ml 5. ^-$4. °1 methanol/acetone(l:l)£«*!•§: *H*1 ^A*}^] 2*1 # ^<& ^^-i 1-1: 200 ml£] ^^-^ri -§-*T|A]^4. n^jl <^£ ^#^H 30 : 30 ml ^^r + 6 ml DMSO)^1 -8-«l|«rSil^.^, 3 3-8-(Electron Donating Ability ; ^-picryl hydrazyl)<>ll tfl$ ^i^}^ DPPH 16 mgt 100 ml absolute ethanoli -§-*fl^: ^ ^^-^r 100 ml# 7]-*>jl filter paper(No. 1, Whatman)^. . °1 ^^ 5 mil ^J^H ^#1- 1 mil- 7]-^: $• vortex mixerS 30 528 ^ A A EDA(%) = ( 1 - ) x 100 B B 2. (D (2) 4- 2.5, 5, 10 KGy 5KGy -O.M-, 10 KGy 4.1) - 82 - 4. (1) paeoniflorin ^ ^r MeOHS ^#*V ^ H2O, Et2O5. £^fJl 4^1 *rf"i- n-BuOHS. ) ^-^1-ir silicagel column chromatographyS CHCla : MeOH 9:13]- 5 SiO2 column chromatography# crude paeoniflorin-i: paeoniflorin# Et2O:MeOH HPLC(Table 4.2)5. 13C-NMR # 5t ^^^>^4(Table 4.3). paeoniflorin^ *}<&4(129). (2) ^>^ SA> 3-^ils.^-^ ^^^r paeoniflorin^ HPLC^l ^ ^^ o-ethoxybenzamidel- ^4S^#^S HPLC 4.4). Paeoniflorin^: retention time°l 7.915, ^^r 10.914i^i 44 ^#3 ^4. lOkGy 2:4 4^^- «12>M- paeoniflorin ^^^-^4 €4 2.59%4 2.42%7> 44 S^-s]^ chromatogram^ patterni 4 S. ^4€- ^^-7]- ^#£]4 &&4(Table 4.4). (3) ^?1^ decursin 4 decursin angelate^ ^-B] 2 5 ^T ]^! -n-^.^-^-^1 decursin ^ decursinol angelatefe SiO2 column chromatography (tolune: ether, Hexane: EtOAc)S. ^-2]*|-^-5.H(Scheme 4.1), HPLCS. ££•» $^%5L, % 13C- NMR ^ l-^^-^ signal^ 4.5, Table 4.6). (4) Decursin^]- decursinol angelate^ ^^f- ^1^:^ 3.84 4.(H&JL ^#7]^ 320 - 44>R^-^ reverse phase column°fl>Hfe acetonitrile: H2O 7} column (Shim-pack CLC-ODS(M))^^ - 83 - normal phase column^ fe hexane: EtOAc 84. Decursin^r 200ug - lOOOug/ml 4(Table 4.7). (5) #*}# 2*1- ^^^1 -n-M^-S! decursin^ decursinol angelate HPLC ^4 y]^Al- ^^ -fi-3L^.^-^-^^4fe decursin^ 0.27%^ S. decursinol angelate^ # 4.8). (1) Salmonella typhimurium TA98, TA100,TA102 fe Ames tesHH ^>-id(10-KGy) (Table 4.9-416); (2) CHO KGy) 4.17-424).." (3) Paeoniflorin°l #^^lol^^€- iL^l^l ^^-^: Ames test°lH paeoniflorin^: 3^ 57fl pattern^ 4.25). - 84 - (4) Paeoniflorin°l CHO AfSull^^ , binucleated cells paeoniflorin 7} ^#£]^1 ^^Jl ^ 4.26). (1) *1^#4| tfl« benzo(a)pyreneAS Salmonella typhimurium TA98, TA100 4.27, Table 4.28). (2) CHO CH0 A\]g.6\}*} benzo(a)pyreneAS (3) DPPH<^1 tfl^- 4.30). - 85 - Table 4.1. Effects of gamma irradiation on the growth of total aerobic bacteria in herbs Total aerobic bacteria(CFU/g sample) Korean name 0 KGy 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 50 0 0 0 - 86 - Table 4.2. Analytical condition of paeoniflorin 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-> 100 gradient Detection 230nm Flow rate(ml/min) 1 Column Temp.CC) room Retention time(min) 7.982 - 87 - Table 4.3. 13C-NMR data for paeoniflorin c Paeoniflorin Standard 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 r 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 - 88 - Table 4.4. The content of paeoniflorin of Paeonia japanica Miyabe Sample Content Paeoniflorin(%) Radiation , S.D C.V(%) (mg )x (mgx) (n=3x) OkGy 500 12.95 2.59 ; 0.01 0.22 paeoniflorin lOkGy 500 12.1 2.42 0.02 0.45 - 89 - The root of Angelica gigas Nakai(500g) 70% EtOH 2L CHC13: H2O (1:1) 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 4.1. Isolation of decursinol angelate and decursin from the root of Angelica gigas Nakai - 90 - Table 4.5. spectral data of deciirsinol angelate and decursin (in CDCls) Proton decursinol angelate decursin 3 6.244(9.5) 6.234(9.5) 4 7.594(9.5) 7.594(9.5) 5 6.80,s 6.80,s 8 7.16,s 7.16,s 3' 5.13t(4.8) 5.09,t(4.8) 4' 2.904d(17.6,4.8) 2.874d(17.6,48) 3.234d(17.6,4.8) 3.20,dd(17.6,4.8) 1.38,s 1.36,s 1.40,s 1.39,s 2" - 5.76,m 3" 6.11,qq(7.2,1.4) - 2"-CH3 1.844(1-4) - 3"-CH3 - 2.154(1.2) 4" 1.894(7.2) 1.884(1.2) - 91 - 1 *3 Table 4.6. C-NMR spectral data of decursinol angelate and decursin (in CDCb) Carbon decursinol angelate decursin 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 - 92 - Table 4.7. Analytical condition of decursinol angelate and decursin A B Instrument Waters HPLC Waters HPLC System 2690 separation module 2690 separation module Pump 2690 pump 2690 pump 996 photodiode array 996 photodiode array Detector detector detector Column Nova Pak C18 Nova Pak Silica Mobile Phase Acetonitrile: H2O 6: 4 Hexane:EtOAc 9:1 Detection 320nm 320nm Flow rate(ml/min) 1 2 Comumn Temp. CO room room Retention time(min) decursin 3.869 8.057 decursinol angelate 4.046 7.422 C Instrument Shimadzu HPLC System CBM-lOA Pump LC-10AD Detector SPD-lOA Shim-Pack Column CLC-ODS(M)25 Mobile Phase Acetonitrile: H2O 6: 4 Detection 254nm Flow rate(ml/min) 1 Comumn Temp. (°C) 30 °C Retention time(min) decursin 13.717 decursinol angelate 14.333 - 93 - Table 4.8. The content of decursin and decursinol angelate of Angelica gigas Nakai Sample Content Decursin(%) Radiation , , S.D C.V(%) (mg v) (mg s) (n=2x) OkGy 3002 129.71 4.32 0.008 0.09 decursin lOkGy 3012 138.12 4.59 0.025 0.27 decursinol OkGy 3018 80.1 2.65 0.0007 0.01 angelate lOkGy 3019 80.25 2.66 0.005 0.09 - 94 - Table 4.9. Revertant colonies in the S. typhimurium reversion assay with methanol-soluble fraction of r -irradiated Paeonia japonica Miyabe Number of revertant Test aIrradi Dose S9 Mix colonies(His+) per plate Material ation (jug/plate) TA98 TA100 TA102 DMSO 24 ± 10 246 ± 18 299 ± 24 Test GI(50%f 25 ± 3 227 ± 1 297 ± 44 material GI(50%)/3 20 + 1 209 ± 17 308 ± 16 GK50%)/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 ± 1 GI(50%)/27 20 ± 1 226 ± 1 382 ± 21 GI(50%)/81 27 ± 8 206 ± 12 349 ± 11 GK50%)b 27 + 1 259 ± 28 334 ± 23 GI(50%)/3 24 + 2 213 ± 8 368 ± 9 GK50%)/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 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). - 95 - Table 4.10. Revertant colonies in the S. typhimurium reversion assay with water-soluble fraction of r -irradiated Paeonia japonica Miyabe Number of revertant Test aIrradi Dose S9 Mix colonies(His+) per plate Material ation (jug/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 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). - 96 - Table 4.11. Revertant colonies in the S. typhimurium revertant assay with water soluble of r -irradiated Angelica gigas Nakai Number of revertant Dose Test "Irradiation S9 Mix colonies(His*) per plate Material (^g/plate) 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) 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) and 2-AF (2-aminofluorene) were used as positive controls for the corresponding strains. - 97 - Table 4.12. Revertant colonies in the S. typhimurium revertant assay with water-soluble of y -irradiated Cnidium officinale Makino Number of revertant Test Dose + Irradiation S9 Mix colonies(His ) per plate Material (/ig/plate) TA100 TA98 H20 - - 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) H20 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) 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) and 2-AF (2-aminofluorene) were used as positive controls for the corresponding strains. - 98 - Table 4.13. Revertaht colonies in the S. typhimurium reversion assay with methanol-soluble fraction of y -irradiated Curcuma longa Linne Number of revertant Test aIrradi Dose S9 Mix colonies (His*) per plate Material ation (jag/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 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). - 99 - Table 4.14. Revertant colonies in the S. typhimurium reversion assay with water-soluble fraction of 7 -irradiated Curcuma longa Linne Number of revertant Test Irradi Dose S9 Mix colonies(His+) per plate Matenal ation (fig/plate) TA98 TA100 TA102 H2O 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). - 100 - Table 4.15. Revertant colonies in the S. typhimurium reversion assay with methanol-soluble fraction of y -irradiated Scutellaria baikalensis George Number of revertant Test aIrradi Dose S9 Mix colonies (His*) per plate Material ation (.fig/plate) TA98 TA100 TA102 DMSO 22 ± 2 61 ± 3 161 ± 3 Test GI(50%)b 20 ± 6 154 ± 4 268 ± 39 material GK50%)/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 ± 4 169 ± 1 277 10 GK50%)/3 16 ± 3 154+7 275 22 GI(50%)/9 17 ± 1 165 ± 1 289 16 GI(50%)/27 18 ± 6 151 ± 19 305 1 GI(50%)/81 19 ± 5 142 ± 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 ± 5 221 ± 13 357 ± material GK50%)/3 32 ± 6 219 ± 3 367 ± GK50%)/9 31 ± 4 224 ± 8 336 ± 18 GI(50%)/27 32 ± 6 215 ± 21 353 ± 38 GI(50%)/81 25 ± 1 200 ± 1 305 ± 3 b GI(50%) 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 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). - 101 - Table 4.16. Revertant colonies in the S. typhimurium reversion assay with water-soluble fraction of y -irradiated Scutellaria baikalensis George Number of revertant Tool1 Co" I TTTO/'I111 dUIl Dose colonies(His*) per plate Matenal ation (jug/plate) TA98 TA100 TA102 H20 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 H20 24 ± 5 252 ± 9 323 ± 28 H20 - + 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 ± 2 287 ± 28 353 ± 43 + + 500 38 ± 2 258 ± 23 307 ± 12 + + 167 32 ± 5 223 ± 9 313 ± 11 + + 56 29 ± 1 224 ± 8 327 ± 11 + + 19 28 ± 1 215 ± 13 293 ± 4 2-AF -. + 10 1648 ±350 874 ± 47 606 ± 50 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). - 102 - Table 4.17. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with methanol-soluble fraction of 7 -irradiated Paeonia japonica Miyabe Total No. of CB cells Total MN/1000 Test S9 Dose b 0 Irradiation with n MN NO. of cells material mix (jug/ml) 0 1 2 3 4 MN (Mean±S.D) H2O 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 IrradiationUO 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. - 103 - Table 4.18. Frequency of micronuclei(MN) in cytokinesis-blocked GHO cells following treatment with water-soluble fraction of r -irradiated Paeonia japonica Miyabe b Cone. Cells Cells with MN No. of MN/1000 cells' Material IRa S9 (fi g/xni) w/o MN MN (Mean ± S.D.) 1 2 3 4 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 72 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 MMC 0.1 - 2712 247 33 8 0 337 112.3 ± 16.1 H2Q + 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 MMC 0.1 + 2656 298 38 7 1 399 133.0 ± 20.5 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. - 104 - Table 4.19. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of 7 -irradiated Angelica gigas Nakai Total No. of CB cells Total MN/1000 Test " . S9 Dose b TIrradiatio na with n MN NO. of cells0 material mix (#g/ml) 0 1 2 3 4 MN (Mean±S.D) H20 - - 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 H20 - + 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 B(a)P - + 20 2648 304 37 6 0 400 133.3 ±21.4 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. 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. - 105 - Table 4.20. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of 7 -irradiated Cnidium officinale Makino Total No. of CB cells Total MN/1000 Test S9 Dose 1> Irradiation with n MN NO. of cellsc material mix (^g/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 H2O - + 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(a)P - + 20 2653 304 37 6 0 396 132.0 ±19.4 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. 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. - 106 - Table 4.21. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with methanol-soluble fraction of y -irradiated Curcuma longa Linne Total No. of CB cells Total MN/1000 Test . S9 Dose Irradiation with n MNb NO. of cells0 material mix (#g/ml) 0 12 3 4 MN (Mean±S.D) H2O 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 H2O 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. 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. - 107 - Table 4.22. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water-soluble fraction of y -irradiated Curcuma longa Linne Total No. of CB cells Total MN/1000 Test " . . S9 Dose Irradiatio nB with n MN' NO. of cells' mix (jug/ml) material 0 1 2 3 4 MN (Mean±S.D) 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 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. MMC (Mitomycin C) and B(a)P (benzo(a)pyrene) were used as positive controls. - 108 - Table 4.23. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with methanol-soluble fraction of r -irradiated Scutellaria baikalensis George Total No. of CB cells Total MN/1000 Test S9 Dose 1 Irradiation8 with n MN NO. of cells0 mix (fig/ml) material 0 1 2 3 4 MN (Mean±S.D) 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 BU)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. MMC (Mitomycin C) and B(a)P (benzo(a)pyrene) were used as positive controls. - 109 - Table 4.24. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water-soluble fraction of y -irradiated Scutellaria baikalensis George Total No. of CB cells Total MN/1000 Test . S9 Dose b Irradiation with n MN1 NO. of cells0 material mix (jug/ml) 0 1 2 3 4 MN (Mean±S.D) 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 H20 - +• 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 Irradiation(10 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. - 110 - Table 4.25. Revertant colonies in the S. typhimurium reversion assay with paeoniflorin isolated from Paeonia japonica Miyabe. Number of revertant colonies (His')per plate Test material S9 Mix Dose/plate) TA98 TA100 H2O lOOul 43 54 (48) 170 184 (177) Test material 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 20ug 1088 1014 (1051) Na-Azide 1.5ug 954 864 (909) + lOOul 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 + lOug 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 - 111 - Table 4.26. Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with paeoniflorin isolated from Paeonia japonica Miyabe Cells without Frequency of the cells with MN MN/1000 a Material S9mix Dose/plate MN(0) 1 2 3 4 (Mean ± S.D.) H2O - 2938 57 4 1 0 22.7 ±6.1 - 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 - O.lug 2716 202 30 6 1 94.7 ±12.8 H2O - 2964 52 1 0 0 19.0±3.9 + 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 0 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(or)p + 20ug 2743 233 27 5 1 102.0 ±14.9 MMCCMitomycin C) and B( a )P (benzo( a )pyrene) were as positive controls a Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored. - 112 - Table 4.27. Antimutagenic effect of water-soluble fractions of 7 -irradiated Curcuma longa Linne, Paeonia japonica Miyabe, Scutellaria baikalensis George in the S. typhimurium reversion assay Cone. of colonies/ plate Inhibition ratio Material IR1 S9 mix Bp. No. (ji g/plate) TA98 {%) H20 - 41 43 37 (40) H2O + 41 41 45 (42) C. longd1 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. japo4 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* 750 + + 122 87 110 (106) 74.1 + 750 + + 110 115 96 (107) 73.7 - 750 + 48 47 42 (46) + 750 + 45 46 43 (45) B(a)P 20 + .+ 287 297 283 (289) 1 7 -irradiation(10 KGy); 2 benzo( a )pyrenei 3 Curcuma longa linne; 4 Paeonia japonica Miyabe; D Scutellaria baikalensis George - 113 - Table 4.28. Antimutagenic effect of water-soluble fractions of y -irradiated Curcuma longa Linne, Paeonia japonica Miyabe, Scutellaria baikalensis George in the S. typhimunum reversion assay Cone. „ No. of colonies/ plate Inhibition Material IR1 S9 mix RP2 (/jt g/plate) ratio {%) TA100 H20 183, 197, 201 (194) H2O + 246, 223, 214 (228) C. longa3 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.4 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. Baik5 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(or)P 20 370, 457, 468 (432) 1 r -irradiationdO KGy); 2 benzo( a )pyrene; 3 Curcuma longa linne; 4 Paeonia japonica Miyabe; 0 Scutellaria baikalensis George - 114 - Table 4.29. Antirriutagenic effects of water-soluble fractions of r -irradiated Paeonia japonica Miyabe, Curcuma longa Linne and Scutellaria baikalensis George in the micronucleus(MN) assay using cytokinesis-blocked CHO cells 3 , Cone. , Cells Cells with MN No. of MN/1000 cells Material IR1 S9 BP2 (ft g/ml) w/o MN 1 2 3 4 MN (Mean ± S.D.) H2O 2946 51 22 1 0 58 29.3 ± 4.3 C. longa4 - 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. baik6 - 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 1 7 -irradiation(10 KGy); 2 benzo( a )pyrenei 3Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1,000 binucleated cells were scored Curcuma longa linne; 5 Paeonia japonica Miyabe; 6 Scutellaria baikalensis George - 115 - Table 4.30. Effects of gamma irradiation in electron donating ability of herbs. 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Department of Food Irradiation, KAERI) Kim, Sung-Ho(Chonnam Univ.), Yee, Sung-Tae(Sunchon Univ.) Publication Publication Taejon Publisher KAERI 1999 Place Date Page 165 p. 111. & Tab. Yes(#), No ( ) Size A4 Note Classified Open(#), Restricted( Report Type Research Report Class Document Sponsoring Org. Contract No. Abstract (15-20 Lines) In searching modulators of immunity and hemopoiesis among natural products, being used as foods, 6 herbs exhibited lymphocyte proliferation in vitro, and 6 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 4 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 herb combinations was confirmed in mice. In culture of bone marrow cells, the changes of cytokine expression patterns by herb mixture extracts were observed. In the further studies, we would to evaluate the effects of the herb combinations, to identify the active component, to confirm toxicological safety, and to prepare the provisional products for foods. And then, the functional foods with immunomodulation activity would be developed, and would be applied to overcoming the declined immunity and hemopoiesis caused by various factors. Subject Keywords Immunomodulation, Hematopoietic cells, Natural products, Herbs, (About 10 words) Intestinal crypt, Bone marrow cells, 2| -^•L O I "T" 1 ^3-4 (~\A —I 1 1. o| an #¥0l|Ai All O [ ~T~-\ /\ r ^•* *-'"l —r^- ;H^-A|-So| "^T 2. o| y^ HI — ^i=iO]|Ai A|1S|- Ol -^ »»iot age 3. s,^l# ^l^ ^X|O1| ^ AI^ omiUP.