Nobuaki Takahashi 1) and Akari Takahashi-Takaya 1) 1) Shingen Medical Co., Ltd.

Transcription

1 A determinative experiment of molecular structure concerning heated glucose possessing cancer-preventing function Nobuaki Takahashi 1) and Akari Takahashi-Takaya 1) 1) Shingen Medical Co., Ltd.

2 Summary of the preceding meeting 1) We have showed here about the study of D-(+)-glucose that some new cancer-protecting substance(s ) is produced by highly heat treatment. 2) In HPLC pattern, its substance was illustrated as a mountain spot. As the eluted solution of this spot showed a dark brown color, we named this spot a dark brown mountain or glucose. 3) The growth inhibition of solid cancer which was derived from the cancer stem cell-like cell W14 was brought by highly heat treatment for glucose. As the heated glucose did not suppress directly the cell proliferation of W14, this inhibition appeared to be brought through innate immunological system, for the used nude mouse did not posses the thymus.

3 Effect of commercially available caramel and D-(+)- glucose one on the proliferation of W14 cells, using MTT assay Commercial caramel 30 Glucose caramel Concentration (micro/ml) Concentration (micro/ml)

4 Gel Filtration of Glucose Caramel and Determination of Inhibitory Active Fraction 1.2 茶色が濃くなったグルコースカラメル A230(nm) Absorbance, A230 (nm) (%) Inh ibit ion rat e Gel filtration by Sephadex G Fraction Number 0

5 Growth Inhibitory Effect of Fraction No. 6 & 7 of Caramel- Gel Filtration on W14-Solid Cancers Control Fr. No. 6&7 Fr. No. 13&14 V=839 (mm 3 ), 163, 864 Town water Fr. No. 6&7 Fr. No. 13&14

6 Determination of Active Part in The Active Fraction, No. 6&7 by HPLC 100( %) Control Inh ibit ion rat e part part part Nongrowth W14- solid cancers

7 Subjective In the preceding meeting, we reported that, when glucose has been heated, it become to acquire a cancer-preventing function. We introduce here a determinative experiment of molecular structure on the active minimum size of heated glucose, because changed glucose by heating develops polymerization and builds several molecular-size materials.

8 Materials & Methods WFB Introduction to W14 cells Volume = (minor axis) 2 x (major axis) x 0.5 Volume: 1,000 mm 3 + H-ras onco. = W14 + CD44 Glucose caramel One week Three weeks Start of water drinking assay (conc. of test materials=200 μ /ml) Injection of 10 5 cells of W14 Water drinking assay Finish (One mouth)

9 Energy stream to live Solar energy Ecological system Thermal energy Photosynthesis Respiration Once, we cached fire. Glucose ( ぶどう糖 ) Heating Cooking Unknown research area Several new substances 1 levoglucosan 2 DB glucose (Fucan-like sub.) 3? 4?

10 Inhibitory effect of fractionated heated-glucose by Sephadex G-50 Inhibitory Effect of Renual DBG by Sephadex G A 254 (nm) A254 (nm) Fraction Number Fraction Number

11 30,000 Gel Type Fractionation Range of Globular Proteins (Da) Molecular Weight 20,000 10,000 Sephadex G-50 G-50 1,500-30,000 G-25 1,000-5,000 No. 12 (14,000) 5,000? Sephadex G-25 No. 14 (8,000) Fraction Number

12 Polymerization of D- (+)-glucose (m.w. 180) by Heating Dehydration in polymer synthesis Hydrolysis in polymer degradation

13 Elution pattern of HPLC by 20 μl injection of each glucose (200μg/ml) A254nm (100%) Normal glucose (10%) min A254nm (methanol gradient) Heated glucose

14 The molecular structure of one peak (4min) was solved. Town waterlevoglucosan Heated levoglucosan The molecule was levoglucosan (C 6 H 10 O 5, m.w. 160). Although levoglucosan didn t depress the growth of solid cancer by W14, its growth somewhat inhibited by heating.

15 A difference in the growth of W14-solid cancer A Change of Volume Average by HPLC Peak Fractions between two peaks of HPLC fractions Volume (mm 3 ) Volume (mm3) min peak control 8 min peak Days after plantation of W14 cells Days from Transplantation of W14 cells

16 It is known that sugar is changed to furan polymer by heating (cited from Wikipedia). Furan polymer Sugar=Glucose+Fructose Accordingly, heated glucose (DB-glucose) also appears to be a kind of furan compounds.

17 Inhibitory effect of heated glucose on the growth of solid cancer in human lung cancer cell (LU65A) by oral administration Volume (mm 3 ) Volume (mm 3 ) Days Heated glucose effects a initial phase of cancer growth. P<0.05* P<0.1 Control, n=5 Exp. group, n=4 P< Days

18 Heated glucose produces an effect to the initiation phase of cancer growth.

19 Conclusions: 1) Glucose was changed at least to four substances by heating. 2) Heated glucose (Dark Brown Glucose) appears to be one kind of furan polymer. 3) Heated glucose produces an inhibitory effect to the initiation phase of cancer growth.

20 About Lavender Water A. Takahashi-Takaya 1) A subsequent study in heated glucose possessing a function of caner protection 2) A report of Dr. A. Takahashi-Takaya, in special to improvement effect of lavender water on atopic-like dermatitis 3) An additional report about MMP10, which gene activity is significantly depressed by fucoidan from brown algae

21 Occurrence of a glycoside glucose to some poly-con Nobuaki Takahashi and Akari Takaya

22 molecule in the process from ensed compound by heating (Sapporo Inst., Shingenmedical Co., Ltd)

23 Summary of the preceding meeting 1) We have showed here about the study of D-(+)-glucose that some new cancer-protecting substance(s ) is produced by highly heat treatment. 2) In HPLC pattern, its substance was illustrated as a mountain spot. As the eluted solution of this spot showed a dark brown color, we named this spot a dark brown glucose. 3) The growth inhibition of solid cancer which was derived from the cancer stem cell-like cell W14 was brought by highly heat treatment for glucose. As the heated glucose did not suppress directly the cell proliferation of W14, this inhibition appeared to be brought through innate immunological system, for the used nude mouse did not posses the thymus.

24 Subjective In the preceding meeting, we reported that, when glucose has been heated, it become to acquire a cancer-preventing function. We introduced in the last one that an active mlecular size in the heated glucose reached around ten thousand by a mean of polymerization. We searched next a precursor molecule to its large size compund.

25 As One Conclusion The glycoside composing of glucose and hydroxymethylfurufural of aglycone was detected as some possible precursor by MS and NMR analysis. Hydroxymethylfurufural

26 1st Point of Presentation: Elution pattern of HPLC by 20 μl injection of each glucose (200μg/ml) A254nm (100%) Normal glucose (10%) min A254nm (methanol gradient) Heated glucose

27 2nd Point Effect of commercially available caramel and D-(+)- glucose one on the proliferation of W14 cells, using MTT assay Commercial caramel 60 Glucose caramel Concentration (micro/ml) Concentration (micro/ml)

28 3rd Point: Inhibitory effect of heated glucose on the growth of solid cancer in human lung cancer cell (LU65A) by oral administration Volume (mm 3 ) Volume (mm 3 ) Days Heated glucose effects a initial phase of cancer growth. P<0.05* P<0.1 Control, n=5 Exp. group, n=4 P< Days

29 Materials & Methods Introduction to W14 cells (Cancer stem cell-like cell) WFB Volume = (minor axis) 2 x (major axis) x 0.5 Volume: 1,000 mm 3 Glucose caramel ( 0.1g glucose in 1ml water treated with 180, 2hrs) + H-ras onco. = W14 + CD44 One week Three weeks Start of water drinking assay (conc. of test materials=200 μ /ml) Injection of 10 5 cells of W14 Water drinking assay Finish (One mouth)

30 Results: Study of Active Molecular Size 1.2 Gel Filtration of Glucose Caramel and 茶色が濃くなったグルコースカラメル Determination of Inhibitory Active Fraction A230(nm) Absorbance, A230 (nm) (%) Inh ibit ion rat e 0 Gel filtration by Sephadex G-25 0 Fraction Number Fraction Number

31 Growth Inhibitory Effect of Fraction No. 6 & 7 of Caramel- Gel Filtration on W14-Solid Cancers Control Fr. No. 6&7 Fr. No. 13&14 V=839 (mm 3 ), 163, 864 Town water Fr. No. 6&7 Fr. No. 13&14

32 Determination of Active Part in The Active Fraction, No. 6&7 by HPLC 100( %) Control Inh ibit ion rat e part part part Nongrowth W14- solid cancers

33 Inhibitory effect of fractionated heated-glucose by Sephadex G-50 Inhibitory Effect of Renual DBG by Sephadex G A 254 (nm) A254 (nm) Fraction Number Fraction Number

34 30,000 Gel Type Fractionation Range of Globular Proteins (Da) Molecular Weight 20,000 10,000 Sephadex G-50 G-50 1,500-30,000 G-25 1,000-5,000 No. 12 (14,000) 5,000? Sephadex G-25 No. 14 (8,000) Fraction Number

35 2hrs Active High Molecule- HPLC Pattern of Heated Glucose A Search of Precursor 30min A254nm NMR 解析 Short Heating-HPLC Pattern of Heated Glucose C 12 H 16 O 8 C 6 H 12 O 6 C 6 H 10 O 5 C 18 H 25 O 13 C 6 H 10 O 5 C 18 H 23 O 12 C 6 H 10 O 5 C 12 H 15 O 8 (C 12 H 16 O 8 ) MS 分析

36 It is known that sugar is changed to furan polymer by heating (cited from Wikipedia). Furan polymer Sugar=Glucose+Fructose Accordingly, heated glucose (trade name, furan D glucose) also appears to be a kind of furan compounds.

37 Heated glucose produces an inhibitory effect to the initiation phase of cancer growth.

38 Conclusions: 1) Glucose was changed at least to four substances by heating. 2) Heated glucose (Furan D Glucose) appears to be one kind of furan polymer. 3) Heated glucose produces an inhibitory effect to the initiation phase of cancer growth.

39 The Significance of This Study Energy stream to live Solar energy Ecological system Once, we cached fire. Heating Cooking Photosynthesis Respiration Glucose ( ぶどう糖 ) Several new substances 1 levoglucosan Thermal energy Unknown research area 2 some heated glucose (fucanlike sub.) 3? 4?

40 Future Plan: An Experiment on The Artificial Synthesis of Cancer-Preventing Furan Compound from 5- (Hydroxymethyl)furfural and/or Its Derivatives

41 COI Disclosure Information Lead Presenter/Responsible Researcher Nobuaki Takahashi I have no financial relationships to disclose.