4.6 ሇનೱ่խࣝ֎ߕݤ
4.7.1. ࡑෳኬࠔೀ
! ! ڗᕴᗐໆऊ 1 mg ޑࡑෳኬࠔྋనܭኬࠔύǴ٩ׇуΕ 95% Όᎇǵ99%Ό ᎇϷคНҘᎇ෧ᓸᐚᕭǴ٬ኬࠔଳᔿǴӆаϿໆคНҘᎇஒኬࠔΕНှᆅύǴ٠ аޜܜѐନคНҘᎇǶ
4.7.2. Οࢧᎉለ (trifluoroacetic acid, TFA) Нှ
! ! уΕ 0.5 mL 4 M TFA Нྋన (ەܭჴᡍВଛᇙ) ԿНှᆅύǴஒᕉნܜԋ
ޜǴа 100ɗଳϸᔈ 3 λਔǴ෧ᓸᐚᕭѐନ TFA НྋనԿᕉნࣁύ܄ (аቶҔ ၂રෳۓ pH ॶ=7)Ǵа 1 mL ຬપНൺྋНှౢނǴ 0.45 μm nylon ᘠጢ (Nylon, 0.45 μm, Aglela Technologies) ၸᘠǴа HPAEC-PAD سϩǴϩύ܄ᗐచҹࣁ 19 mM NaOH (֖ 1 mM barium acetate) НྋనǴࢬೲࣁ 0.5 mL/minǴКჹ L-arabinoseǵ L-fucoseǵD-galactoseǵD-glucoseǵD-mannoseǵL-rhamnoseǵD-xylose ྗࠔޑ ᅉ੮ਔ໔ǵճҔӚݢঢ়ޑᑈϩय़ᑈीᆉрൂᗐವԸಔԋ (mol%)Ƕ
4.7.3.Ҙ
Ҙ✊ϯϸᔈ (Methanolysis)! ! ਥᏵ Deruiter (1992)ǵBertaud (2002) Ϸ Talaga (2002)ΓϐୖԵБݤǴڗᕴᗐ ໆऊ 1 mg ޑࡑෳኬࠔྋనܭޜНှᆅύǴ෧ᓸᐚᕭԿଳǴуΕ 1 mL 2 M HCl/MeOHǴଛᇙБݤࣁǴܭӇύஒ 1.41 mL acetyl chloride ᄌуΕคНҘᎇ ύǴ٠аคНҘᎇۓԿ 10 mLǶஒᕉნܜԋޜࡕǴܭ 80ɗଳϸᔈ 12 λਔǴ ෧ᓸᐚᕭѐନคНҘᎇǴуΕ 1 mL 2 M TFAǴஒᕉნܜԋޜǴܭ 100ɗଳϸ ᔈ 1 λਔǴ෧ᓸᐚᕭѐନ TFA НྋనԿᕉნࣁύ܄ (аቶҔ၂રෳۓ pH ॶ=7)Ǵ а 1mL ຬપНൺྋНှౢނǴ 0.45 μm nylon ᘠጢၸᘠǴа HPAEC-PAD سϩ
Ǵϩύ܄ᗐచҹӕ 4.5.2ǹለ܄ᗐϩచҹࣁ 75 mM NaOH (֖ 1 mM barium acetate ک 150 mM sodium acetate) НྋనǴࢬೲࣁ 1 mL/minǴКჹ D-galacturonic acid Ϸ D-glucuronic acid ྗࠔޑᅉ੮ਔ໔ǵճҔӚݢঢ়ޑᑈϩय़ᑈीᆉрൂᗐ ವԸಔԋ (mol%)(Deruiter et al., 1992; Bertaud et al., 2002; Talaga et al., 2002)Ƕ
4.7.4. ϩس
! ! ଯਏૈᚆηҬඤቫس (high performance anion exchange chromatography with PAD,HPAEC-PAD ᖼԾ Jasco, Ino., Japan.)ǵଯਏૈᚆηҬඤቫߥៈᆅࢊǴ CarboPac PA1 guard column (2% polystyrene cross-linked with divinylbenzene, 4 mm i.d.Ø5 cm, 10 μm) ϷϩᆅࢊǴCarboPac PA1 (2% polystyrene cross-linked with
divinylbenzene, 4 mm i.d.Ø5 cm, 10 μm) ֡ᖼԾ Dionex, Sunnyvale, CAǶ
4.8 ϩ
ϩηໆෳۓ4.8.1. ࡑෳኬࠔೀ
! ! ڗᕴᗐໆऊ 1 mg ϐжෳኬࠔྋనǴ 1.2 μm PVDF ᘠጢ (PVDF, Dia.25 mm, Critical)ၸᘠഢҔǶ
4.8.2. ྗࠔ
! ! ଛᇙ 1 mg/mL dextran (2Ø106Da)ǵglucose ᆶ pullulans ྗࠔྋన (P5, 5.9Ø103 DaǹP10, 0.96Ø104 DaǹP20, 2.11Ø104 DaǹP50, 4.71Ø104 DaǹP100, 1.07Ø105 Daǹ P200, 2.00Ø105DaǹP400, 3.75Ø105DaǹP800, 7.08Ø105Da)Ǵ 0.45 μm nylon ᘠጢ ၸᘠഢҔǶ
4.8.3. ϩس
! ! ଯ ਏ ૈ ϩ η ᑔ ቫ س (high performance size exclusion chromatography, HPSEC)Ǵᔅၮଌسࣁ PU-980 Plus quatermary-gradient pump (Jasco, Inc., Japan)ǹ ݙΕᡏᑈ 200 μLǴݙΕᏔࣁ 7215 Cotati (California, U.S.A.)ǹୀෳᏔࣁשෳۓ Ꮤ, Shodex, RI-71 refractive index detector (Showa Denko, Tokyo, Japan)ǹߥៈᆅࢊ
TSK guard column PWH (7.5 mm i.d.Ø7.5 cm,12 μm)ϷϩᆅࢊǴTSK 4000 PWXL column (7.5 mm i.d.Ø30 cm,17 μm) Սᖄ TSK 3000 PWXL column (7.5 mm i.d.Ø30 cm,17 μm) (Tosoh, Tokyo, Japan.)Ǵᆅࢊྕࡋࣁ 80ɗǴၗϩسࣁ SISC32 (ૻ
ިҽԖज़ϦљǴѠчѱǴѠ)Ƕࢬࢱచҹࣁ 0.3 M NaNO3(֖ 0.02% NaN3)Ǵࢬ ೲ 0.5 mL/minǶ
4.9 HPSEC Ս
Սᖄ ELISA assay! ! ڗᕴᗐໆऊ 1 mg ϐжෳኬࠔྋనǴ 1.2 μm PVDF ᘠጢ (PVDF, Dia.25 mm, Critical) ၸᘠࡕ HPSEC ϩࡕаϩࢤԏᏔ (RediFrac ᖼԾ GE health, Uppsala, Sweden.) аᆅ 10 ᅀ (0.29 mL-0.31 mL) ՉԏǴаচన܈ีញ 100 ७ࡕݙܭ 96 ϾዬǴՉ ELISA ϩǶࣁΑஒ ELISA ϩკᆶ HPSEC ϐቫკՉӝٳǴ
ዴᇡ ELISA ϩკϐ distribition coefficient (DC)ǶዴᇡϐБݤࣁݙΕ 10 mg/mL ϐ blue dextranǴаϩࢤԏᏔࡪྣॊБݤԏǴෳۓ OD 630 nm ϐ֎ӀॶǴջ ёளޕ blue dextran ໒ۈࢬрϐᆅኧǴ४ᆅ܌ԏϐᡏᑈǴջࣁ VoǶVt ߾аᑗࡋ
ीෳໆځשǴа HPSEC ࢬࢱనᘜ႟ǴёаෳளࢬࢱనᆶНঢ়ϐ໔࣬౦ޑש
ॶǴڗНঢ়ᆅǴ४ᆅ܌ԏϐᡏᑈǴջࣁ VtǴҗԜ Vo ᆶ Vt ॶջёෳளϩࢤ ԏᏔϐ DC ॶǴ٠ёаᆶ HPSEC ϐ RI კӝٳǶ
ಃϖǵġीϩ
! ! ჴᡍኧᏵ่݀֡аѳ֡ॶ²ྗৡ߄ҢǴኧᏵϩа SAS 9.3 း೬ᡏ аᎅМཥӭᡂୱෳᡍݤ (Duncan’s new multiple range test) ՉᔠۓǴP ॶ
<0.05 ຎࣁԖᡉৡ౦Ƕ
Ҵ
Ҵǵġ่݀ᆶፕ
ಃകǵġНྋ܄όёϯӭᗐ
ಃġǵНྋ܄όёϯӭᗐϩϐಔԋϩ
! ! ՋࢩୖϐНనࣁԜᅿ१ޑЬाឪ१БԄǴНނКٯࣁচϐ 58.2%Ǵಉӭᗐࣁ 25.04%ǴԜಉӭᗐ֖Ԗೈқ፦ 1.18%ǴஒಉӭᗐՉ࿚నࢉՅǴ ևᙔ๋ՅёፕՋࢩୖಉӭᗐύ֖ԖεໆޑᐘણǶࣁΑᕕှΓୖ१܌ឪΕޑ ӭᗐ܄፦Ǵҁჴᡍஒ ಉӭ ᗐа protease բҔѐନೈқ፦ࡕǴ ᆶ α-amylaseǵ amyloglucosidase ՉբҔǴёஒಉӭᗐϩࣁёϯӭᗐ (digestible polysaccharide) ᆶНྋ܄όёϯӭᗐ (water-soluble nondigestible polysaccharide)Ƕёϯӭᑗ
җဟᑗ਼ϯ䁙-ၸ਼ϯނ䁙 (glucose oxidase-peroxidase, GOD-POD) ෳۓǴ֖
glucoseǴෳࢂҗ α-amylase ᆶ amyloglucosidase ܌Нှޑᐘણ่ᄬǴ(1,4;1,6)-α-D-glucanǶሇનНှࡕ܌ளϐНྋ܄όёϯӭᗐǴᆶಉӭᗐ࣬КǴځൂᗐಔԋ ύ glucose ֖ໆεࣁ෧ϿǴҗ 93.86%फ़Կ 5.19%ǶёϯӭᗐǴЬाࣁᐘણ܌ᄬ ԋǴࣁ (1,4;1,6)-α-D-glucanǴэಉӭᗐϐ 95.7%ǴНྋ܄όёϯӭᗐ߾э 4.3%
(݅, 2012)ǶஒНྋ܄όёϯӭᗐаᚆηҬඤቫᆅࢊϩǴܭ 20 mM Tris ֖ NaCl ᐚࡋ 0ǵ0.1ǵ0.18ǵ0.3 M ϐࢬࢱనؑගǴёளѤঁϩ F1ǵF2ǵF3 ᆶ F4 (კ ΜѤ)ǶᚆηҬඤᐋિϩᚆБݤࣁ٩Ᏽځႝ܄ǴF1 ϩҗᡶᐚࡋࣁ 0 M ܌ؑග рǴ⾺ᗐለ֖ໆե (߄)Ǵаύ܄ᑗ arabinose (18.92%) ᆶ galactose (68.53%)ࣁ Ь (߄Β)ǶF2 ࣁᡶᐚࡋ 0.1 M ܌рǴಔԋϝа arabinose (47.35%) ᆶ galactose (40.71%) ࣁЬǴෳԜΒϩࣁ݀ጤ RG-I ่ᄬϐϩЍ AGIǶᒿࢬࢱనᡶᐚࡋຫ ଯǴࢬࢱрޑӭᗐԖႝ܄ຫଯǴᡶᐚࡋ 0.18 M ࢬࢱрϐ F3 ϩǴځ⾺ᑗለ֖
ໆэᕴᗐໆϐ 35.30 %ǶF4 ࣁᡶᐚࡋ 0.3 M ܌ࢬࢱрٰޑϩǴа galacturonic acid ࣁЬाಔԋǴ⾺ᗐለэᕴᗐໆ 197.13%ǴচӢࣁ F4 ࢂҗଯໆޑъ٢ᑗ⾺ለ܌ಔԋǴ ՠࢂаᕴᗐໆޑෳۓݤǴለ܄ᑗคݤНှևՅǴե F4 ϐᕴᗐ֖ໆǶF3 ᆶ F4
ࣣ֖Ԗ glucuronic acidǴࡺࣁ RG-I ϐ AGII ϩЍǶ
߄ǵНྋ܄όёϯӭᗐҗ DEAE ቫ܌ϩᚆޑѤᅿϩቫӭᗐϐ୷ҁಔԋǶ Table 1. Chemical compositions of four fractions from water-soluble nondigestible polysaccharides separated by DEAE chromatography.
Fraction %
aProtein /Carbohydrate ratio Uronic acid in carbohydrate
dProtein
b:Carbohydrate
c(%)
F1 18.51±6.68 1:9.11 1.36±0.01
F2 19.90±3.03 1:9.14 1.04±0.02
F3 28.28±2.67 1:6.83 35.30±0.07
F4 33.31±6.61 1:3.76 197.13±1.55
aOn basis of carbohydrate contents of four fractions.
bValues from Coomassie blue method (Bio-red protein assay reagent ) using BSA as standard.
cValues from phenol-sulfuric acid method using glucose as standard.
dOn carbohydrate basis and the uronic acid values from m-hydroxydiphenyl method using galacturonic acid as standard.
eMeans²standard deviation, n=3.
߄ΒǵНྋ܄όёϯӭᗐҗ DEAE ቫ܌ϩᚆϐѤঁϩӭᗐځӚձൂᗐಔ ԋ (݅, 2012)Ƕ
Table 2. Monosaccharide molar compositions of four fractions from water-soluble nondigestible polysaccharides separated by DEAE chromatography.
Fraction Molar ratio of sugar composition (%)
aAra Gal Glc Man Rha GalA GlcA
F1 18.92 68.53 5.41 7.14 ND ND ND
F2 47.35 40.71 4.55 7.39 ND ND ND
F3 55.18 30.72 ND ND 4.23 6.33 3.55
F4 21.31 17.71 7.96 ND 9.16 43.54 0.3
კΜѤǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩკǶ
Figure 14. Elution profile of water-soluble nondigestible polysaccharides of P.
quinquefolius on DEAE chromatography, eluted with 20mM Tris and a stepwise
gradient of NaCl.ಃ
ಃΒġǵНྋ܄όёϯӭᗐύӚӭᗐϩϐϩηໆ
! ! ࣁᕕှӭᗐϐϩѲǴ٬Ҕ HPSEC ՉϩηໆϩǶӭᗐڀԖፄᚇޑ่ᄬǴค
ൂϩηໆǴϩηঁኧόֹӄ࣬ӕǴѸаኧໆѳ֡ϩηໆ (number average molecular weight, Mn) Ϸख़ໆѳ֡ϩηໆ (weight average molecular wight, Mw) ٰ ߄ҢǴځύΞаख़ໆѳ֡ϩηໆၨૈж߄ӭᗐޑ܄ǴٿޣޑКॶࣁ polydispersity index, PI (Mw/Mn) ߾߄ҢϩηໆޑϩѲǶ
! ! ஒՋࢩୖНྋ܄όёϯӭᗐаᚆηҬඤᐋિϩᚆϐ F1ǵF2ǵF3 ᆶ F4 ϩೀ RAW 264.7 ႵѮᏘಒझǴว F3 ᆶ F4 ϩӧᐚࡋ 100 μg/mL аᡉڈ ᐟ TNF-α ᆶ NO ғԋ(݅,2012)Ƕϩ F1-F4 ϩϐϩηໆ (კΜϖ-კΜΖ)Ǵ F1ǵF2 ϩځ Mw ࣁ 71ǵ115 kDaǴPI ࣁ 3.62 ᆶ 2.01ǹF3 ᆶ F4 ࣁ 273ǵ172 kDaǴ PI ࣁ 3.87 ᆶ 2.94 (߄Ο)ǶڀԖڈᐟಒझᐟનғԋϐ F3 ᆶ F4 ϩځϩηໆϷ PI ࣣ
εܭ F1 ᆶ F2ǴԜ่݀ёჹᔈܭ 2002 ԃ Shin ΓஒڗϐΓୖӭᗐ (ϩηໆ 150 kDa) ڈᐟѮᏘಒझǴӧᐚࡋ 100 μg/mL аܴᡉڈᐟ TNF-α Ϸ NOǴܭ 10 ug/mL аڈᐟ IL-1βǵIL-6 ᆶ IFN-γ ϐғԋ(Shin et al., 2002)ǶKabat ᆶ Bezer ࡰрǴጋ ᆒޑϩηໆεܭ 90 kDa ჹΓᜪڀԖխࣝלচ܄ǴԶϩηໆλܭ 50 kDa ਔ߾όڀ խࣝڈᐟ܄(Kabat and Bezer, 1958)Ƕ1999 ԃ Yamada ᆶ Kiyohara(Yamada and Kiyohara, 1999) ගрύᛰύڀԖࢲϯံᡏࢲ܄ϐӭᗐǴъኧаځϩηໆεܭ 128 kDaǴ߄ҢӭᗐϐϩηໆελϷ PI ॶࣁ،ۓځڈᐟಒझᐟનᆶࢂցڀԖխࣝࢲ
܄ϐᜢᗖӢનǶ
კΜϖǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F1 ϩηໆკǶ Figure 15. HPSEC elution profiles of fraction one of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.
კΜϤǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F2 ϩηໆკǶ Figure 16. HPSEC elution profiles of fraction two of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.
კΜΎǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F3 ϩηໆკǶ Figure 17. HPSEC elution profiles of fraction three of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.
კΜΖǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F4 ϩηໆკǶ Figure 18. HPSEC elution profiles of fraction four of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.
߄ΟǵНྋ܄όёϯӭᗐҗ DEAE ቫ܌ϩᚆϐѤᅿϩӭᗐځӚձϩη ໆǶ
Table 3. The molecule weight of four fractions water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatographya
Fraction Molecular weight of fraction (ൈ
)
bMn Mw PI
F1 0.20±0.00 0.71±0.01 3.62±0.08
F2 0.57±0.00 1.15±0.01 2.01±0.01
F3 0.70±0.03 2.73±0.20 3.87±0.11
F4 0.58±0.00 1.72±0.00 2.94±0.02
aThe value was determined by HPSEC with pullulans as standards.
bMn: number average weight (kDa); Mw: weight average weight; PI: polydispersity index (Mw/Mn).
cMeans²standard deviation, n=3.
ಃ
ಃΒകǵġሇનೱ่խࣝ֎ߕϩݤനϯచҹϩ
! ! ሇનೱ่խࣝ֎ߕϩݤ (enzyme linked immunosorbent assay, ELISA)Ǵࣁ ᙖҗלᡏ-לচ໔ڀ܄ޑᒃکΚǴՉϩ᠘ۓǶҔܭڰۓלচ܈לᡏǴ٬Ҕ ڀԖமᗖ่Κޑ൳ᅿ༟፦ӵᆫशΌ (polystyrene)(Leininger et al., 1966; Catt and Tregear, 1967)ǴҞჹܭځᗖ่БԄۘ҂ܴᕕǴෳࢂҗ౧НϷᒃНΚᆶϐ࣬
ϕբҔ٬ځၲ֎ߕૈΚ(Dillman and Miller, 1972; Shirahama and Suzawa, 1985)Ƕ
ಃġǵCoating buffer ϐ pH ॶᆶᚆηமࡋჹלচ֎ߕϸᔈϐቹៜ
! ! לচᙖҗ౧НբҔǵᓉႝЇΚϷΥቺґᅟΚڰۓܭ 96 ϾዬǴࣁளޕྋᏊৡ ౦ࢂցቹៜלচϐڰۓૈΚǴஒלচϩձྋܭόӕྋᏊύǴ่݀ᡉҢǴלচྋܭ ᕗለፂన (phosphate buffer) ܈ HPSEC ࢬࢱన (0.3 M NaNO3 ֖ 0.02% NaN3)Ǵ ځ֎Ӏॶᆶᐚࡋևጕ܄ᜢ߯ǴऩஒלচྋܭΒԛᇃᚖНǴ߾όڀԖጕ܄ᜢ߯Ǵෳ
ࢂྋᏊύޑᡶᜪᚆηԖշܭלচڰۓܭ 96 Ͼዬ (კΜΐ-კΒΜ)Ƕ
! ! לচڰۓܭ༟߄य़ϐ֎ߕϸᔈࢂᙖҗஒלচྋܭեᚆηமࡋޑᡵ܄ፂྋ న(Voller et al., 1979)ǴלচᙖҗᒃНϷ౧Н࣬ϕբҔǴڗ،ܭ coating buffer ޑ pH ॶᆶᚆηமࡋ(Shirahama and Suzawa, 1985)Ƕջ٬ӭੰࢥלচёаҥջ֎ߕԿ༟
߄य़(Voller et al., 1979)Ǵޑ coating buffer ىаߦלচ֎ߕԿ༾ϾዬǴԶל চ֎ߕܭ༟߄य़ޑำࡋ߾ޔௗቹៜלচ،ۓՏ (epitope) ޑཞѨᆶց(Bruck et
al., 1982; McCullough et al., 1985)Ƕࣁࡌҥ coating buffer ύ pH ॶᆶᚆηமࡋჹܭ
לচ֎ߕܭ༾ϾዬϐቹៜǴჴᡍаᕗለፂనࣁЬǴׯᡂ pH ॶᆶ NaCl ᐚࡋՉࣴزǶ
! ! ٬ҔѤᅿྗࠔϷჹᔈϐלᡏǶGum arabic Ьाࣁ arabinogalactanǴڀԖ proteinǴ ࣁፄᚇ่ᄬǴᒣᇡޑלᡏࣁ LM2ǶCitrus pectinǴڀԖ 85% GalAǴ֖Ԗεໆለ܄
ᑗǴჹᔈϐלᡏࣁ LM5Ƕ4-O-methyl-glucuronoxylanǴа LM10 לᡏᆶϐᒣᇡǶӧ gum arabic ᆶ LM2 ᒃکΚϸᔈύǴpH ॶᆶלচ֎ߕόڀ҅࣬ᜢ܄ǶCitrus pectin ࣁ
ለ܄ᑗǴᕗለፂనܭ pH ॶܭ 6.4 ਔԖനଯޑ֎ӀॶǴЪܴᡉӧ pH ॶࣁ 3.4 Կ 6.4 ϐለ܄ᕉნΠځ֎ӀॶၨமᡵᕉნࣁଯǴෳҗܭለ܄ᑗޑ pKa=3-5Ǵܭለ܄
ᕉნΠځႝጨǴܰ֎ߕܭ 96 ϾዬǶឦܭύ܄ᑗޑ(1ʈ5)-α-L-arabinan ᆶ xylose: GlcA=10:1 ޑ 4-O-methyl-glucuronoxylanǴҗܭᎉለਥᚆηޑ pKa=3-5Ǵӧ pH 6.4-9.4 ޑᕉნΠᎉለਥှᚆࣁ COOˇǴԖճ֎ߕܭ༾ϾዬǶᕉნύϐ pH ॶ ౦ਔǴׯᡂלচ่ᄬϐႝ܄Ϸᆶ༾ϾዬϐѾΚǴԶቹៜלচᆶ༾Ͼዬޑ֎
ߕ (კΒΜΒ)Ƕ
! ! ֎ߕϸᔈёᙖҗ NaCl ᐚࡋϲԶගϲǴቚуᚆηமࡋჹלচ֎ߕܭӭኧ౧Н ᆫӝނӵ polystyrene Ԗ҅࣬ᜢ܄ǴԶ polystyrene Ψࢂ 96 ϾዬޑЬाǶࣁ
ᡶࡋჹלচ֎ߕޑቹៜǴ٬Ҕόӕ NaCl ᐚࡋޑᕗለፂనǶӧ gum arabic ᆶ LM2 ᒃکΚϸᔈύǴNaCl ᐚࡋᆶלচ֎ߕόڀ҅࣬ᜢǶCitrus pectin ޑ֎ߕΚڙ҂బу NaCl ޑಔձቹៜၨځдಔձଯǴځ֎ӀॶܴᡉၨեǴԶځᎩᡶࡋᡂϯჹܭځ֎ߕ բҔคᡉৡ౦Ƕ0-0.8M ϐᡶࡋճܭ (1ʈ5)-α-L-arabinanǴࠅ෧Ͽ citrus pectin ᆶ 4-O-methyl-glucuronoxylan Ԗለ܄ᑗϐ֎ߕǶ3.2M ᆶ 4.0M NaCl ϐଯᡶࡋё
ܴᡉቚу 4-O-methyl-glucuronoxylan ֎ߕǶቚу֎ߕϸᔈޑЬӢෳࣁלচϷ༾Ͼ ዬ໔ޑᓉႝΚ (electrostatic) ᆶѾΚ (repulsive force) ӢᚆηமࡋቚуԶύکǴቚ уלচ֎ߕ (კΒΜΟ)Ƕ
კΜΐǵόӕᐚࡋ gum arabic ྋܭΒԛᇃᚖНᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ
(R2=0.1586)Ƕ
Figure 19. Immunoaffinity of LM2 on gum arabic in ddH2O with different concentrations (R2=0.1586).
კΒΜǵόӕᐚࡋ gum arabic ྋܭᕗለፂనᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ
(R2=0.9924)Ƕ
Figure 20. Immunoaffinity of LM2 on gum arabic in phosphate buffer with different concentrations (R2=0.9924).
კΒΜǵόӕᐚࡋ gum arabic ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%
NaN3) ᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ (R2=0.998)Ƕ
Figure 21. Immunoaffinity of LM2 on gum arabic in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R2=0.998).
1Values followed by different letters in the same group are significantly different (Duncan’s test p<0.05).
კΒΜΒǵόӕ pH ॶϐᕗለፂనჹܭ gum arabicǵcitrus pecitnǵ(1ʈ5)-α-L-arabinan ᆶ 4-O-methyl-glucuronoxylan Ϸൂਲ਼לᡏ LM2ǵLM5ǵLM6 ᆶ LM10 ϐ
֎ӀॶቹៜǶ
Figure 22. Immunoaffinity of LM2, LM5, LM6 and LM10 on gum arabic, citrus pectin, (1ʈ5)-α-L-arabinan and 4-O-methyl-glucuronoxylan in phosphate buffer with different pH values.
1Values followed by different letters in the same group are significantly different (Duncan’s test p<0.05).
კΒΜΟǵόӕෛϯ໊ᐚࡋϐᕗለፂనჹܭ gum arabicǵcitrus pecitnǵ(1ʈ5)-α-L-arabinan ᆶ 4-O-methyl-glucuronoxylan Ϸჹᔈϐൂਲ਼לᡏ LM2ǵLM5ǵLM6 ᆶ LM10 ϐ֎ӀॶቹៜǶ
Figure 23. Immunoaffinity of LM2, LM5, LM6 and LM10 on gum arabic, citrus pectin, (1ʈ5)-α-L-arabinan and 4-O-methyl-glucuronoxylan in phosphate buffer with different concentrations of NaCl.
ಃ
ಃΒġǵଯᏊໆ㸃ރਏᔈ (High dose hook effect)
! ! ! ! ࣁளޕൂਲ਼לᡏᆶόӕלচᐚࡋՉխࣝᒃکΚϸᔈϐ֎Ӏॶޑጕ܄ጄ ൎǴஒϖᅿൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM10 ᆶ LM20 ᆶჹᔈϐלচՉೱុ
ีញǴ٠ஒלচϩձྋှܭᕗለፂనϷ HPSEC ࢬࢱన (კΒΜѤ-კΒΜϖ)Ƕ
่݀ᡉҢǴᐚࡋଯܭ 20 μg/mLǴ֎Ӏॶόևጕ܄ᜢ߯Ƕҗᘜϩύޑ،ۓ߯
ኧ R2ॶፕځጕ܄ᜢ߯Ƕᐚࡋϟܭ 0.1~100 μg/mL ጄൎϣǴR2ॶပܭ 0.08-0.60Ǵ֎Ӏॶᆶלচᐚࡋόڀጕ܄ᜢ߯Ǵלᡏᐚࡋϟܭ 0.1~20 μg/mL ໔ǴR2ॶ
ࣣܭ 0.93 аǴԖၨӳޑጕ܄࣬ᜢ (კΒΜϤ-კΟΜϖ)Ƕଯᐚࡋኬࠔޑ֎Ӏॶ
ၨեᐚࡋኬࠔٰளեǴԜຝࣁଯᏊໆ㸃ރਏᔈ (high dose hook effect )ǴЬाว ғܭሇનೱ่խࣝ֎ߕϸᔈǴ२ԛวܭ 1974 ԃǴMiles ΓаΓᜪՈమ៓ೈқ
(human serum ferritin) ᆶีញ 500ǵ1000ǵ10000 Ϸ 25000 ७ϐלচ ferritin Չ two site immunoradiometric assayǴ่݀ᡉҢଯᐚࡋלচǴջีញ 500 ७ϐಔձǴ ځ᠐ॶၨځдಔձեǶԜਏᔈҭܭ۳ࡕวܭᖏխࣝᔠෳᅟᆾᆶဍዦǴ ӵ macroprolactinoma ύϐ prolactin (St-jean et al., 1996; Frieze et al., 2002), prostate specific antigen(Charrie et al., 1995; Furuya et al., 2001), hepatoblastoma ύ alpha-fetoprotein(Jassam et al., 2006), Ϸ metastatic medullary thyroid carcinoma ύޑ calcitonin (Leboeuf et al., 2006)Ǵѿלচᐚࡋຬၸלচ،ۓՏ֖ໆǴջ㸃ރ⸣
ॶ (hook threshold)Ǵෞᚆלচ (free antigen) ջکᆶלᡏೱ่ޑלচᝡݾǴࡺ
לচᐚࡋቚуਔǴр՟ࢂԶߚ (paradoxical) ޑեϸᔈ่݀(Jassam et al., 2006)Ƕவόӕᐚࡋჹᔈ֎Ӏॶϐ่݀ᡉҢǴଯᏊໆ㸃ރਏᔈჹܭ֎Ӏॶϐቹៜཱུ
εǴऩ٬ҔᒃکΚϸᔈՉۓໆਔǴஒלচᐚࡋีញܭጕ܄ጄൎϣǶ
კΒΜѤǵόӕᐚࡋྗࠔྋܭᕗለፂనᆶൂਲ਼לᡏᒃکΚ่݀Ƕ
Figure 24. Immunoaffinity of monoclonal antibodies on antigens in phosphate buffer with different concentrations. LM2 and gum arabic (Ʌ), LM5 and citrus pectin (Ʉ), LM6 and (1→5)-α-L-arabinan (ɐ*- LM10 and 4-O-methyl-glucuronoxylan (ɏ*-LM20 and citrus pectin (Ɏ*/!
კΒΜϖǵόӕᐚࡋྗࠔྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02% NaN3) ᆶ
ൂਲ਼לᡏᒃکΚ่݀Ƕ
Figure 25. Immunoaffinity of monoclonal antibodies on antigens in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations. LM2 and gum arabic (Ʌ), LM5 and citrus pectin (Ʉ), LM6 and (1→5)-α-L-arabinan (ɐ*- LM10 and 4-O-methyl-glucuronoxylan (ɏ*- LM20 and citrus pectin (Ɏ*/!
კΒΜϤǵόӕᐚࡋ gum arabic ྋܭᕗለፂనᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ
(R2=0.9993)Ƕ
Figure 26. Immunoaffinity of LM2 on gum arabic in phosphate buffer with different
კΒΜΎǵόӕᐚࡋ gum arabic ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%
NaN3) ᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ (R2=0.9502)Ƕ
Figure 27. Immunoaffinity of LM2 on gum arabic in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R2=0.9502).
კΒΜΖǵόӕᐚࡋ citrus pectin ྋܭᕗለፂనᆶൂਲ਼לᡏ LM5 ϐᒃکΚϩ
(R2=0.9993)Ƕ
Figure 28. Immunoaffinity of LM5 and on citrus pectin in phosphate buffer with different concentrations (R2=0.9993).
კΒΜΐǵόӕᐚࡋ citrus pectin ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%
NaN3) ᆶൂਲ਼לᡏ LM5 ϐᒃکΚϩ (R2=0.9905)Ƕ
Figure 29. Immunoaffinity of LM5 on citrus pectin in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R2=0.9905).
კΟΜǵόӕᐚࡋ(1→5)-α-L-arabinan ྋܭᕗለፂనᆶൂਲ਼לᡏ LM6 ϐᒃکΚ ϩ (R2=0.9565)Ƕ
Figure 30. Immunoaffinity of LM6 on (1→5)-α-L-arabinan in phosphate buffer with different concentrations (R2=0.9565).
კΟΜǵόӕᐚࡋ(1→5)-α-L-arabinan ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02% NaN3) ᆶൂਲ਼לᡏ LM6 ϐᒃکΚϩ (R2=0.9388)Ƕ
Figure 31. Immunoaffinity of LM6 on (1→5)-α-L-arabinan in HPSEC ࢬࢱన (0.3M NaNO3and 0.02% NaN3) with different concentrations (R2=0.9388).
კΟΜΒǵόӕᐚࡋ 4-O-methyl-glucuronoxylan ྋܭᕗለፂనᆶൂਲ਼לᡏ LM10 ϐᒃکΚϩ (R2=0.9587)Ƕ
Figure 32. Immunoaffinity of LM10 on 4-O-methyl-glucuronoxylan in phosphate buffer with different concentrations (R2=0.9587).
კΟΜΟǵόӕᐚࡋ 4-O-methyl-glucuronoxylan ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02% NaN3) ᆶൂਲ਼לᡏ LM10 ϐᒃکΚϩ (R2=0.9957)Ƕ
Figure 33. Immunoaffinity of LM10 on 4-O-methyl-glucuronoxylan in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R2=0.9957).
კΟΜѤǵόӕᐚࡋ citrus pectin ྋܭᕗለፂనᆶൂਲ਼לᡏ LM20 ϐᒃکΚϩ
(R2=0.996)Ƕ
Figure 34. Immunoaffinity of LM20 on citrus pectin in phosphate buffer with different
კΟΜϖǵόӕᐚࡋ citrus pectin ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%
NaN3) ᆶൂਲ਼לᡏ LM20 ϐᒃکΚϩ (R2=0.9995)Ƕ
Figure 35. Immunoaffinity of LM20 on citrus pectin in HPSEC eluent (0.3M NaNO3
and 0.02% NaN3) with different concentrations (R2=0.9995).
ಃ
ಃΟകǵġНྋ܄όёϯӭᗐύӚӭᗐϩϐൂਲ਼לᡏᒃکΚ่݀
! ! ҞςԖӭኧޑൂਲ਼לᡏҔаᒣᇡނಒझᏛόӕޑ݀ጤ่ᄬǴҭว߄݀
ጤӭᅿלচ،ۓՏ(Willats et al., 2001)ǶҁፕЎ٬Ҕᒣᇡ݀ጤӭᗐϐൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ՉխࣝᒃکϸᔈǶ
! ! F1Ǵᆶ LM5 ڀԖᒃکϸᔈǴൂᗐಔԋЬाࣁ arabinose (18.92%) Ϸ galactose (68.53%)Ǵෳࣁ arabinogalactan type I ่ᄬ (კΟΜϤ)ǶF2 ϩǴarabinose ᆶ galactose К ٯ ࣁ 1.16:1 Ǵ Ъ ჹ ܭ LM5 ᆶ LM6 ڀ Ԗ ᒃ ک ϸ ᔈ Ǵ ෳ ࣁ rhamnogalacuronan I (RGI) ϐ arabinogalactan type I (AGI) Ϸ linear arabinan ϩЍ (კΟΜΎ)ǴԜ่݀ёаჹᔈډൂᗐಔԋǶՋࢩୖਥӧՉ௦ԏࡕаӀᚼᠴ
ՉߥӸǴෳނύ rhamnogalacturonase ᆶ galacturonase Нှ RG-I ϐЬ༸ᆶϩЍ
ϩǴ٬ RGI ϷϩЍϩᘐࣁλТࢤǴಔԋ F1 ᆶ F2Ǵόᆶ༾ႝϐϩǶ
! ! F3 ϩǴҗᒃکϸᔈ่݀ෳǴϩηໆεޑୱࣁ AGI Ϸ AGII ่ᄬǴԶ linear
arabinan ߾ቶݱϩѲǴԜෳёҗൂᗐಔԋКٯளډᡍǶF3 ֖Ԗ 3.55% GlcAǴ ᡉҢࣁ AGII ่ᄬǶarabinose : galactose=1.80:1ǴऩԖ arabinogalactan type I Ϸ type II ่ᄬǴarabinose ֖ໆᔈ၀όଯܭ galactose ߈ 2 ७ǴځᎩޑ arabinose ёૈ
ࢂҗ linear arabinan ϩЍ܌ගٮ (კΟΜΖ)ǶF4 ϩЬाࣁለ܄ᑗǴჹܭ LM2ǵ LM5ǵLM6 ӕኬڀԖᒃکϸᔈǴΨёவൂᗐКٯளޕځڀԖ AGIǵAGII Ϸ linear arabinan ϐϩЍ่ᄬǴՠࢂ֖ໆܴᡉК F3 եǶF4 Ьाჹܭ LM19 Ԗமਗ਼ϐᒃکϸ ᔈǴF3 ჹځϐૻဦ߾ၨ১ (კΟΜΐ)ǶLM19 ᆶ LM7ǵLM20ǵJIM7 ࣣࣁ anti-homogalacuronan (HG) לᡏǴHG Ҙ୷✊ϯϐำࡋϷՏ߾،ۓלᡏ໔ᒣᇡלচ
،ۓՏϐৡ౦ǶClausen ӧ 2003 ԃஒ hexagalacturonan ܭόӕՏௗҘ୷ (Clausen et al., 2003)ǴLM7 ᒣᇡޑ่ᄬࣁٿঁҘ୷✊ϯ୷ϐ໔ǴѤঁೱុόڀҘ୷
✊ϯޑ galacturonanǶJIM5 (לচ،ۓՏӕ LM19)ǴᒣᇡΟঁೱុόڀҘ୷✊ϯ ޑ galacturonanǴԶ JIM7 (לচ،ۓՏӕ LM20) ߾ሡԖೱុ܈໔႖௨ӈϐΟԿ Ѥঁ methyl esterified galacturonan ωૈᆶϐᒣᇡǶᕴکٰᇥǴѤᅿ homogalacturonan ޑלᡏᒣᇡޑ partially methyl esterified homogalacturonan Ӛό࣬ӕǴ٩ྣ܌ሡϐ HG Ҙ୷✊ϯำࡋᆶஏࡋ௨ӈǴҗեԿଯࣁ LM7ǵLM19ǵJIM7ǵLM20Ƕ
ġ ġ F3 ᆶ F4 ϩᆶѤᅿ anti-homogalacturonan לᡏϐᒃک่݀Ǵჹܭ LM19 ڀ ԖᒃکϸᔈǴ߄ҢٿঁϩࣣԖ homogalacturonan ่ᄬǴ٠ЪΨࢂൂᗐಔԋύ galacturonic acid ϐЬाٰྍǴҗ LM19 ჹܭלচޑᒣᇡёаளޕǴF3 ᆶ F4 ܌֖
Ԗޑ HG ่ᄬࣁϿໆҘ୷✊ϯϐ homogalacturonanǶHG ଆ߃ࢂаଯҘ୷✊ϯޑރ ᄊӝԋǴՠࢂނϣޑ pectin methylesterase ჹܭ HG ՉѐҘ୷✊ϯ (de-esterified)ǴόӕҘ୷✊ϯำࡋቹៜځғނཀကǴញрޑԾҗ♐୷ (free carboxyl groups) ᆶᚆηᗖ่܈ᆶ݀ጤᗐՉҬᖄǴቹៜԋጤ܄፦ᆶ݀ጤӭᗐޑ่ᄬǶ ќБय़Ǵа KDO ෳۓ F3ǵF4ǴڀԖ II ่ᄬǴԜ่ᄬࢂҗለ✊ᆶځд RG-II ่ᄬᖄ่Ǵԋ RG-RG-II ΒᆫᡏǴࣁಒझᏛமࡋٰྍǴቹៜ݀ጤᖄᆛϐϾࢰЁκϷ ቸ܄Ǵ F1-F4 Ѥঁϩჹܭ LM10 ࣣόڀԖᒃکϸᔈǴЪൂᗐಔԋύ҂Ԗ xyloseǴ
ࡺԜѤϩόڀԖ xylan ܈ xylogalacturonan ่ᄬǴԶ xylose ӧނύЬाӸӧъ ᠼᆢનǴҗԜፕǴՋࢩୖਥό֖ъᠼᆢનǴځӭᗐЬाࣁ݀ጤӭᗐǶ
! ! а่݀ᡉҢǴՋࢩୖНྋ܄όёϯӭᗐޑЬाԋϩࣁ݀ጤӭᗐǴऊԖ 39%
ޑό܈༾ႝϐӭᗐёૈࣁ݀ጤӭᗐύڀԖଯࡋϩЍޑ RGI ༧ϐफ़ှТࢤǴ ٠ෳՋࢩୖӭᗐύ 61%ޑ F3 ᆶ F4 ϩǴࣁނಒझᏛ݀ጤ่ᄬޑЬाಔԋǴ ٠Ъҗ HGǵRGI ᆶ RGII ่ᄬǴಔԋ݀ጤᖄᆛǴԶ،ۓ߃ભಒझᏛޑமࡋǵቸ
܄ᆶфૈ܄Ƕ
კΟΜϤǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F1 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩǶ Figure 36. HPSEC elution profile of fraction one of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6, LM7, LM10, LM19, LM20 and JIM7 affinity.
კΟΜΎǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F2 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩǶ Figure 37. HPSEC elution profile of fraction two of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6,LM7, LM10, LM19, LM20 and JIM7 affinity.
კΟΜΖǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F3 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩǶ Figure 38. HPSEC elution profile of fraction three of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6,LM7, LM10, LM19,
კΟΜΐǵՋࢩୖНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐ F4 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩǶ Figure 39. HPSEC elution profile of fraction four of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6,LM7, LM10, LM19, LM20 and JIM7 affinity.
߄ѤǵНྋ܄όёϯӭᗐа DEAE ቫϩᚆϐѤঁϩڀԖϐ݀ጤ่ᄬំ
߄Ƕ
Table 4. The pectin structure of the four fractions of water-soluble nondigestible polysaccharide of P. quinquefolius L. on DEAE column
Fraction HGa XGAb RGI
arabinan AGI AGII RGII
F1 ˇˇ ˇˇ ˇˇ ˁˁ ˇˇ ˇˇ
F2 ˇˇ ˇˇ ˁˁ ˁˁ ˇˇ ˇˇ
F3 ˁˁ ˇˇ ˁˁ ˁˁ ˁˁ ˁˁ
F4 ˁˁ ˇˇ ˁˁ ˁˁ ˁˁ ˁˁ
Defined by the data of monoclonal polysaccharide composition and the immunoaffinity with mAbs and KDO determination.
aHomogalacturonan.
bXylogalacturonan.
ഌ
ഌǵġ่ፕ
! ! ҁࣴز่ӝሇનНှǵᚆηᐋિቫǵଯਏૈϩηᑔቫݤᆶൂਲ਼לᡏխࣝ
ᒃکϸᔈѤᅿמೌǴаՋࢩୖࣁෳ၂চǴࡌҥёӕϩӭᅿНྋ܄ӭᗐޑמೌ
ѳѠǶ
! ! Ջࢩୖಉӭᗐа 95.7%ϐ 1,4;1,6-α-D-glucan ёϯӭᗐǴᆶ 4.3%Нྋ܄ό ёϯӭᗐ܌ಔԋǶНྋ܄όёϯӭᗐᚆηҬඤᐋિቫϩᚆёϩࣁ F1ǵ F2ǵF3 ᆶ F4 ѤঁϩǶҁࣴز٬Ҕଯਏૈϩηᑔቫسᆶൂਲ਼לᡏᒃکϸᔈམ ଛൂᗐಔԋෳНྋ܄όёϯӭᗐѤঁϩϐ่ᄬǶ
! ! לচޑ֎ߕϸᔈቹៜ ELISA ่݀ǶGum arabic ྋܭΒԛᇃᚖНύคݤ֎ߕ ܭ 96 ϾዬǴྋܭᕗለፂన܈ HPSEC eluent ߾όቹៜځ֎ߕǶלচϐ܄ (ႝ
܄) Ӣࣁ coating buffer ޑᚆηமࡋᆶ pH ॶׯᡂځ่ᄬԶቹៜځ֎ߕǴԶϸᔈ Կ֎Ӏॶϐৡ౦ǶคፕҺՖ pH ॶ܈ᡶࡋࣣόׯᡂ gum arabic ϐ֎ߕǶύ܄ᑗϐ (1→5)-α-L-arabinan ߾ӧ pH=6.4-9.4 ᆶ҂బу NaCl ϐ phosphate buffer Ԗന٫ϐ֎
ӀॶǴෳҗܭᎉለਥᚆηޑ pKa=3-5Ǵӧ pH 6.4-9.4 ޑᕉნΠᎉለਥှᚆࣁ COO―Ǵ Ԗճ֎ߕܭ༾ϾዬǶለ܄ᑗ citrus pectin ᆶԖ GlcA ϐ 4-O-methyl-glucuronoxylan ӧ pH=6.4-9.4 ޑ phosphate buffer ύǴӢࣁለ܄ᑗޑ pKa=3-5Ǵܭለ܄ᕉნΠځႝ
ጨǴቚу֎ߕΚǶ҂బу NaCl ϐᕗለፂనफ़ե citrus pectin ֎ߕǴԶ 4-O-methyl-glucuronoxylan ϐന٫ፂనᡶࡋࣁ 4.0 MǶ
! ! ଯᏊໆ㸃ރਏᔈҭࣁቹៜ ELISA ჴᡍౢғଵ܄ϐӢનǴלᡏᐚࡋϟܭ 0.1~100 μg/mL ጄൎϣǴ֎Ӏॶᆶלচᐚࡋόڀጕ܄ᜢ߯ǴԶᐚࡋϟܭ 0.1~20 μg/mL ໔Ǵځ R2ॶࣣܭ 0.93 аǴԖၨӳޑጕ܄ᜢ߯ǴࡺӧՉխࣝᒃکΚϸᔈ ਔǴஒלচᐚࡋีញܭጕ܄ጄൎϣǶ
! ! F1 ϩЬाჹܭ LM5 ڀԖᒃکϸᔈǴа arabinose (18.92%) ᆶ galactose (68.53%) ܌ಔԋǴෳࣁ rhamnogalacturonan I ϐ arabinogalactan type I ่ᄬǶ F2 ϩёаҗ LM5 Ϸ LM6 ܌ᒣᇡǴځ arabinose ᆶ galactose ϐКٯࣁ 1.16:1Ǵ
ෳନΑڀԖ AGI ่ᄬѦǴԖޔ arabinan ϩЍǶF3 ёҗ LM5ǵLM6 ᒣᇡǴෳ
ࣁ AGI Ϸޔ arabinan ่ᄬϐѦǴLM2 ᆶ LM19 ޑᒃکϸᔈҭёෳځڀԖ AGII ᆶ partially methyl esterified HGǶF4 Ьाҗለ܄ᑗᄬԋǴLM2 Ϸ LM19 ᒣᇡዴᇡڀ Ԗ AGII ᆶϩҘ୷✊ϯϐ HG ่ᄬǴԜѦǴLM5 ᆶ LM6 ϐᒃکϸᔈ่݀ҭගٮ ڀԖ AGI ᆶ arabinan ่ᄬǶѤঁϩჹܭ LM10 ࣣࣁॄϸᔈǴ٠ӧൂਲ਼לᡏύค xylose ᔠрǴ߄ҢόڀԖ xylose ϩЍ܈ xylogalacturonanǶՋࢩୖНྋ܄όёϯ ӭᗐޑЬाԋϩࣁ݀ጤӭᗐǴऊԖ 39%ޑό܈༾ႝϐӭᗐёૈࣁ݀ጤӭᗐ ύڀԖଯࡋϩЍޑ RGI ༧ϐफ़ှТࢤǴ٠ෳՋࢩୖӭᗐύ 61%ޑ F3 ᆶ F4 ϩǴҗ HGǵRGI ᆶ RGII ่ᄬǴಔԋ݀ጤᖄᆛǴࣁނಒझᏛ݀ጤ่ᄬޑЬाಔ ԋǶ
! ! ҁࣴزࡌҥаѤᅿמೌಔԋϐӕϩسǴගٮѳѠаϩ݀ጤӭᗐޑ
ቻǴࣁᘉቚԜسϐᔈҔ܄ǴѤᅿמೌࣣё٩ྣόӕ܄ޑ݀ጤӭᗐՉፓᆶ অׯǶԜϩسᗨςගٮזೲᔠෳ݀ጤӭᗐϐѳѠǴ࣪εໆޑϩਔ໔Ǵՠ
ࢂ൩аԖޑൂਲ਼לᡏǴࠅόૈԖਏᒣᇡ܌Ԗ݀ጤӭᗐޑ่ᄬǴෳচӢࣁ݀ጤӭ ᗐ่ᄬፄᚇǴלচ،ۓՏϐᒣᇡᆄӢҥᡏ่ᄬԶጨ܈֖ໆϼϿคݤᒣᇡǶ ऩाஒ݀ጤӭᗐϐ่ᄬֹӄှǴѸаϩηໆϐελஒ݀ጤӭᗐՉ׳
ޑϩǴ܈ёаගଯᆶൂਲ਼לᡏᒃکϐૈΚǶќѦǴёаஒόӕൂਲ਼לᡏ ᒣᇡϐୱǴϩձԏࡕڈᐟѮᏘಒझǴёаᕕှӭᗐ่ᄬᆶڈᐟಒझᐟન ғԋϐ࣬ᜢ܄Ƕ҂ٰ׆ఈૈஒӭᗐϩηໆǵᆶൂਲ਼לᡏᒃک่݀аϷڈᐟಒझᐟન ϐૈΚᆕӝϩǴࡌᄬֹӭᗐ่ᄬᆶխࣝࢲ܄ϐϩКၨၗǶ
ࢠ
immunomodulator ginsan induces resistance to experimental sepsis by inhibiting Toll-like receptor-mediated inflammatory signals. Eur. J. Immunol. 2006, 36, 37-45.Anderson, M. A.; Sandrin, M. S.; Clarke, A. E., A high proportion of hybridomas raised to a plant extract secrete antibody to arabinose or galactose. Plant Physiol.
1984, 75, 1013-1016.
Aspinall, G. O., Chemistry of cell wall polysaccharides. In The biochemistry of plants, Preiss, J., Ed. Academic Press: New York, 1980; Vol. 3, pp 473-500.
Assinewe, V. A.; Amason, J. T.; Aubry, A.; Mullin, J.; Lemaire, I., Extractable polysaccharide of Panax quinquefolius L. (North American ginseng) root stimulate TNFDproduction by alveolar macrophage. Phytomedicine 2002, 9, 398-404.
Attele, A. S.; Wu, J. A.; Yuan, C. S., Ginseng Pharmacology. Biochem. Pharmacol.
1999, 58, 1958-1963.
Aubertin, A. M.; Tondre, L.; Lopez, C.; Obert, G.; Kirn, A., Sodium dodecyl sulfate-mediated transfer of electrophoretically separated DNA-binding proteins. Anal.
Biochem. 1983, 127-134, 127.
Baker, S.; Barr, D. B.; Driskell, W. J.; Beeson, M. D.; Needham, L. L., Quantification of selected pesticide metabolites in human urine using isotope dilution
high-performance liquid chromatography/tandem mass spectrometry. J. Expo. Anal.
Environ. Epidemiol. 2000, 10, 789-798.
Barr, D. B.; Barr, J. R.; Maggio, V. L.; Whitehead Jr. , R. D.; Sadowski , M. A.; Whyatt , R. M.; Needham, L. L., A multi-analyte method for the quantification of
contemporary pesticides in human serum and plasma using high-resolution mass spectrometry. J. Chromatogr. B 2002, 778, 99-111.
Battaiger, B.; Newhall, W. J.; Jones, R. B., The use of Tween 20 as a blocking agent in
the immunological detection of proteins transferred to nitrocellulose membranes.
J. Immunol. Methods 1982, 55, 297-307.
Benishin, C. G.; Lee, R.; Wang, L. C. H.; Liu, H. J., Effects of ginsenoside Rb1 on central cholinergic metabolism. . Pharmacology 1991, 42, 223-229.
Benishin, C. G., Actions of ginsenoside Rb 1 on choline uptake in central cholinergic nerve endings. Neurochem. Int. 1992, 21, 1-5.
Bertaud, F.; Sunberg, A.; Holmbom, B., Evaluation of acid methanolysis for analysis of wood hemicelluloses and pectins. Carbohydr. Polym. 2002, 48, 319-324.
Biagini, R. E.; Bernstern, I. L.; Gallagher, J. S.; Moorman, W. J.; Brooks, S.; Gann, P.
H., The diversity of reaginic immune responses to platinum and palladium metallic salts. J. Allergy Clin. Immunol. 1985, 76, 794-802.
Biagini, R. E.; Henningsen, G. M.; MacKenzie, B.; Sanderson, W. T.; Robertson, S.;
Baumgardner, E. S., Evaluation of acute immunotoxicity of alachlor in male F344/N rats. Bull. Environ. Contam. Toxicol. 1993, 50, 266-273.
Biagini, R. E.; Tolos, W.; Sanderson, W. T.; Henningsen, G. M.; MacKenzie, B., Urinary biomonitoring for alachlor exposure in commercial pesticide applicators by immunoassay. Bull. Environ. Contam. Toxicol. 1995, 54, 245-250.
Biagini, R. E.; Sammons, D. L.; Smith, J. P.; MacKenzie, B. A.; Striley, C. A. F.;
Semenova, V.; Steward-Clark, E.; Stamey, K.; Freeman, A. E.; Quinn, C. P.;
Snawder, J. E., Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins. Clin. Vaccine
Immunol. 2004, 11, 50-55.
Biagini, R. E.; Striley, C. A. F.; Snawder, J. E., Chapter11ǺImmunochemical
techniques in biological monitoring. In Immunoassay and other bioanalytical
techniques Emon, J. M. V., Ed. CRC press: United States, 2006; pp 265-286.
Blumenkrantz, N.; Asboe-Hansen, G., New method for quantitative-determination of uronic acids. Anal. Biochem. 1973, 54, 484-489.
Bradford, M. M., Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
Bruck, C.; Mathot, S.; Protetelle, D.; Berte, C.; J-D., F.; Heridn, P.; Burny, A.,
Monoclonal antibodies define eight independent antigenic regions on the bovine leukaemia virus (BLV) envelope glycoprotein gp 51. Virology 1982, 122, 342-352.
Butler, J. E.; Ni, L.; Nessler, R.; Joshi, K. S.; Suter, M.; Rosenberg, B.; Chang, J.;
Brown, W. R.; Cantarero, L. A., The physical and functional behavior of capture
Carabias-Martınez, R.; Garcıa-Hermida, C.; Rodrıguez-Gonzalo, E.; Soriano-Bravo, F.
E.; Hernandez-Mendez, J., Determination of herbicides, including thermally labile phenylureas, by solid-phase microextraction and gas chromatography–
mass spectrometry. J. Chromatogr. A 2003, 1002, 1-12.
Carpita, N. C.; Gibeaut, D. M., Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 1993, 3, 1-30.
Cassab, G. I.; Varner, J. E., Immunocytolocalization of extensin in developing soybean seed coats by immunogold-silver staining and by tissue printing on nitrocellulose paper. J Cell Biol 1987, 105, 2581-2588.
Catt, K.; Tregear, G. W., Solid-phase radioimmunoassay in antibody-coated tubes.
Science 1967, 158, 1570-1572.
Charrie, A.; Charriere, G.; Guerrier, A., Hook effect in immunometric assays for prostate-specific antigen. Clin. Chem. 1995, 41, 480-481.
Chen, F. D.; Wu, M. C.; Wang, H. E.; Hwang, J. J.; Hong, C. Y.; Huang, Y. T.; Yen, S.
H.; Ou, Y. H., Sensitization of a tumor, but not normal tissue, to the cytotoxic effect of ionizing radiation using Panax Notoginseng extract. Am. J. Chin. Med.
2001, 29, 517-524.
Chin, J. J. C., Monoclonal antibodies that immunoreact with a cation-stimulated plant membrane ATPase. Biochem. J 1982, 203, 51-54.
Clarke, A. E.; Anderson, R. L.; Stone, B. A., Form and function of arabinogalactans and arabinogalactan-proteins. Phytochemistry 1979, 18, 521-540.
Clausen, M. H.; Willats, W. G. T.; Knox, J. P., Synthetic methyl hexagalacturonate hapten inhibitors of anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. Carbohydrate research 2003, 338, 1797-1800.
Condit, C. M., Developmental expression and localization of petunia glycine-rich protein 1. Plant Cell 1993, 5, 277-288.
Cramer, S. M.; Brooks, C. A., Ion-exchange displacement chromatography of proteins.
In Chromatography in Biotechnology, ACS Symposium Series 529, Horvith, C.;
Ettre, L. S., Eds. American Chemical Society: Washington, DC, 1993; pp 27-42.
Czop, J. K.; Gurish, M. F.; Kadish, J. L., Production and isolation of rabbit anti-idiotypic antibodies directed against the human monocyte receptor for yeast E-glucans. . J. Immunol. 1990, 145, 995-1001.
Dankwardt, A., Immunochemical assays in pesticide analysis. In Encyclopedia of
Analytical Chemistry, Chichester, M. R., Ed. Wiley: New York, 2000; pp 1-27.
David, H.; Bade, P.; David, A.; Savy, C.; Demazy, C.; Van Cutsem, P., Pectins in walls of protoplast-derived cells imbedded in agarose and alginate beads. Protoplasma
1995, 186, 122-130.
De Blas, A. L.; Cherwinski, H. M., Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies. Anal. Biochem. 1983, 133, 214-219.
Deruiter, G. A.; Schols, H. A.; Voragen, A. G. J.; Rombouts, F. M., Carbohydrates analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with tfa hydrolysis is superior to 4 other methods. Anal. Biochem. 1992, 207, 176-185.
Dey, P. M.; Brinson, K., Plant cell-walls. Adv. Carbohydr. Chem. Biochem. 1987, 42, 265-382.
Dillman, W. J.; Miller, I. F., On the adsorption of serum proteins on polymer membrane surfaces. J. Colloid Interface Sci. 1972, 44, 221-241.
Doco, T.; Williams, P.; Vidal, S.; Pellerin, P., Rhamnogalacturonan II, a dominant polysaccharide in juices produced by enzymic liquefaction of fruits and vegetables. Carbohydr. Res. 1997, 297, 181-186.
Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F., Colorimetric method for determination of sugars and realted substances. [Articles]. Analytical
Chemistry 1956, 28, 350-356.
Engvall, E.; Perlmann, P., Enzyme-linked immunosorbent assay, ELISA. J. Immunol.
1972, 109, 129-135.
Evans, P. T.; Holaway, B. L.; Malmberg, R. L., Biochemical differentiation in the tobacco flower probed with monoclonal antibodies. Planta 1988, 175, 259-269.
Frieze, T. W.; Mong, D. P.; Koops, M. K., "Hook effect" in prolactinomas: case report and review of literature. Endocr Pract 2002, 8.
Furuya, Y.; Cho, S.; Ohta, S.; Sato, N.; Kotake, T.; Masai, M., High dose hook effect in serum total and free prostate specific antigen in a patient with metastatic prostate cancer. J. Urol. 2001, 166, 213.
Gao, Q. P.; Kiyohara, H.; Cyong, J. C.; Yamada, H., Characterization of anti-complementary acidic heteroglycans from the leaves of Panax
ginseng.C.A.Meyer. Carbohydr. Res. 1988, 181, 175-187.
Gao, Q. P.; Kiyohara, H.; Yamada, H., Further structural studies of anti-complementary acidic heteroglycans from the leaves of Panax ginseng C.A.Meyer. Carbohydr
Research 1990, 196, 111-125.
Gao, Q. P.; Kiyohara, H.; Cyong, J. C.; Yamada, H., Chemical properties and anti-complementary activity of heteroglycans from the leaves of Panax ginseng C. A.
Meyer. Planta Med. 1991, 57, 132-136.
Gershoni, J. M.; Palade, G. E., Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal.
Gillis, C. N., Panax ginseng pharmacology: a nitric oxide link? Biochem. Pharmacol.
1997, 54, 1-8.
Gooding, K. M.; Regnier, F. E., Size exclusion chromatography. In HPLC of Biological
Macromolecules: Methods and Applications, Gooding, K. M.; Regnier, F. E.,
Eds. Marcel Dekker: New York, 1990; pp 47-75.Hines, C. J.; Deddens, J. A.; Striley, C. A. F.; Biagini, R. E.; Shoemaker, D. A.; Brown, K. K.; Mackenzie, B. A.; Hull, R. D., Biological Monitoring for Selected
Herbicide Biomarkers in the Urine of Exposed Custom Applicators: Application of Mixed-effect Models. Ann. Occup. Hyg. 2003, 47, 503-517.
Holst, G. J.; Clarke, A. E., Quantification of arabinogalactan-protein plant extracts by single radial gel diffusion. Anal. Biochem. 1985, 148, 446-450.
Honda, S.; Sugino, H.; Asano, T.; Kakinuma, A., Activation of the alternative pathway of complement by an anti-tumor (1ʈ3)-E-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140 and its lower molecular weight and
carboxymethylated derivatives. Immunopharmacology 1986, 11, 29-37.
Hoson, T.; Masuda, K.; Sone, Y.; Misaki, A., Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles. Plant Physiol. 1991, 95, 551-557.
Hoson, T.; Masuda, Y.; Nevins, D. J., Comparison of the outer and inner
epidermis.Inhibition of auxin-induced elongation of maize coleoptiles by glucan antibodies. Plant Physiol. 1992, 98, 1298-1303.
Huisman, M. H.; Brýll, L. P.; Thomas-Oatesc, J. E.; Haverkamp, J.; Schols, H. A.;
Voragena, A. J., The occurrence of internal (1ʈ5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances. Carbohydr. Res. 2001, 330, 103-114.
Inngjerdingen, K.; Patel, T. R.; Chen, X.; Kenne, L.; Allen, S.; Morris, G. A.; Harding, S. E.; Martsumoto, T.; Diallo, D.; Yamada, H.; Michaelsen, T. E., Immunological and structural properties of a pectic polymer from Glinus oppositifolius.
Glycobiology 2007, 17, 1299-1310.
Inouhe, M.; Nevins, D. J., Inhibition of auxin-induced cell elongation of maize
coleoptiles by antibodies specific for cell wall glucanases. Plant Physiol. 1991,
96, 426-341.
Ishii, T., Pectic polysaccharides from bamboo shoot cell walls. Mokuzai Gakkaishi
1995, 41, 669-676.
Ishii, T., O-acetylated oligosaccharides from pectins of potato tuber cell walls. Plant
Physiol. 1997, 113, 1265-1272.
Jackson, C. L.; Dreaden, T. M.; Theobald, L. K.; Tran, N. M.; Beal, T. L.; Eid, M.; Gao, M. Y.; Shirley, R. B.; Stoffel, M. T.; Kumar, M. V.; Mohnen, D., Pectin induces
apoptosis in human prostate cancer cells: correlation of apoptotic function with pectin structure. Glycobiology 2007, 17, 805-819.
Jacobs, M.; Gilbert, S. F., Basal localization of the presumptive auxin transport carrier in pea stems. Science 1983, 220, 1297-1300.
Jarvis, M. C.; Apperley, D. C., Chain conformation in concentrated pectic gels: evidence from 13C NMR. Carbohydr. Res. 1995, 15, 131-145.
Jassam, N.; Jones, C. M.; Briscoe, T.; Horner, J. H., The hook effect: a need for constant vigilance. Ann. Clin. Biochem. 2006, 43, 314-317.
Jensen, J. K.; Sørensen, S. O.; Harholt, J.; Geshi, N.; Sakuragi, Y.; Møller, I.;
Zandleven, J.; Bernal, A. J.; Jensen, N. B.; Sørensen, C., Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis. Plant Cell 2008, in press.
Jermyn, M. A.; Yeow, Y. M., A class of lectins present in the tissues of seed plants. Aust.
J. Plant Physiol. 1975, 2, 501-531.
Johnson, D. A.; Gautch, J. W.; Sportsman, J. R.; Elder, J. H., Improved technique utilizing non-fat dried milk for analysis of proteins and nucleic acids transferred to nitocellulose. Gene Anal. Tech. 1984, 1, 3-8.
Jones, L.; Seymour, C. B.; Knox, J. P., Localization of pectic galactan in tomato cell walls using a monoclonal antibody specific to (1ʈ4)-ß-D-galactan Plant
Physiol. 1997, 113, 1405-1412.
Köhler, G.; Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256, 495-497.
Kabat, E. A.; Bezer, A. E., The effect of variation in molecular weight on the antigenicity of dextran in man. Arch. Biochem. Biophys. 1958, 78, 306-318.
Kaku, H.; Shibata, S.; Satsuma, Y.; Sone, Y.; Misaki, A., Interactions of D-L-arabinofuranose-specific antibody with plant polysaccharides and its histochemical application. Phytochemistry 1986, 25, 2041-2047.
Karkalas, J., An improved enzymic method for the determination of native and modified starch. J. Sci. Food.Agric. 1985, 36, 1019-1027.
Kenarova, B.; Neychev, H.; Hadjiivanova, C.; Petkov, V. D., Immunomodulating activiry of ginsenoside Rg1from Panax ginseng. Jpn. J. Pharmacol. 1990, 54, 447-454.
Khan, M.; Bajpai, V. K.; Anasari, S. A.; Kumar, A.; Goel, R., Characterization and localization of fluorescent Pseudomonas cold shock protein(s) by monospecific polyclonal antibodies. Microbiol. Immunol. 2003, 47, 895-901.
Kikuchi, A.; Edashige, Y.; Ishii, T.; Satoh, S., A xylogalacturonan whose level is
dependent on the size of cell clusters is present in the pectin from cultured carrot
King, M. L.; Murphy, L. L., Role of cyclin inhibitor protein p21 in the inhibition of HCT116 human colon cancer cell proliferation by American ginseng (Panax quinquefolius) and its constituents. Phytomedicine 2010, 17, 261-268.
Kiyohara, H.; Yamada, H.; Otsuka, H., Unit structure of anti-complementary
Kiyohara, H.; Yamada, H.; Otsuka, H., Unit structure of anti-complementary