A determinative experiment of molecular structure concerning heated glucose possessing cancer-preventing function

 

Nobuaki Takahashi1) and Akari Takahashi-Takaya1)

1) Shingen Medical Co., Ltd.

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.

0

30

60

90

120

1 2 3 4

0

30

60

90

120

1 2 3 4

Effect of commercially available caramel and D-(+)-glucose one on the proliferation of W14 cells, using MTT assay

800. ���400����200����100

���Concentration (micro/ml)���

800. ���400����200����100

���Concentration (micro/ml)���

Commercial caramel Glucose caramel

茶色が濃くなったグルコースカラメル

A2

30

(nm

)

0

0.3

0.6

0.9

1.2

Fraction Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

100

50

0

（％）Inhibition rate

Fraction Number

Abs

orba

nce,

A23

0 (n

m)

Gel filtration by Sephadex G-25

Gel Filtration of Glucose Caramel and Determination of Inhibitory Active Fraction

Growth Inhibitory Effect of Fraction No. 6 & 7 of Caramel- Gel Filtration on W14-Solid Cancers

V=839 (mm3), 163, 864

Town water Fr. No. 6&7 Fr. No. 13&14

Control Fr. No. 6&7 Fr. No. 13&14

① ② ③

① ② ③

Inhibition rate

100(%) Control

① part

② part

③ part

Non-growth

Determination of Active Part in The Active Fraction, No. 6&7 by HPLC

W14-solid cancers

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.

Subjective

Materials & Methods Introduction to W14 cells

+ CD44

�One week�

�Three weeks�

Volume: 1,000 mm 3

Volume = (minor axis) 2 x (major axis) x 0.5

Start of water drinking assay (conc. of test materials=200�μ /ml)

Finish

(One mouth)

WFB

+ H-ras onco.= W14

Injection of 105 cells of W14

Glucose caramel

Water drinking assay

Glucose

（ぶどう糖）

Heating

Cooking

Several new substances1 levoglucosan

2 DB glucose (Fucan-like sub.)

3 ?

4 ?���

Solar energy

Thermal energy

Photosynthesis

Respiration

Energy stream to live Once, we cached fire.

Unknown research area

Ecological system

Inhibitory Effect of Renual DBG by Sephadex G-50

A2

54

(nm

)

0

0.23

0.45

0.68

0.9

Fraction Number

⑦⑨

⑫⑭

Inhibitory effect of fractionated heated-glucose by Sephadex G-50

Fraction Number

A25

4 (nm

)

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

��1���3���5���7���9���11���13���15���17

10,000

20,000

30,000

No. 12 (14,000)

No. 14 (8,000)

Mol

ecul

ar W

eigh

t

Fraction Number

5,000

Sephadex G-50

Sephadex G-25

G-50 1,500-30,000

G-25 1,000-5,000

Gel Type Fractionation Range of Globular Proteins (Da)

？

Dehydration in polymer synthesis

Hydrolysis in polymer degradation

Polymerization of D-(+)-glucose (m.w. 180) by Heating

���

����

A254nm

min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Elution pattern of HPLC by 20 μl injection of each glucose (200μg/ml)

Normal glucose

Heated glucose

(10%)

(100%)

(methanol gradient)A254nm

Town waterLevogluco-san

Heated levogluco-san

The molecular structure of one peak (4min) was solved.

The molecule was levoglucosan (C6H10O5, m.w. 160). Although levoglucosan didn’t depress the growth of solid cancer by W14, its growth somewhat inhibited by heating.

A Change of Volume Average by HPLC Peak FractionsV

olum

e (m

m3

)

0

200

400

600

800

Days from Transplantation of W14 cells

contro

l9 min

peak

8 min peak

A difference in the growth of W14-solid cancer between two peaks of HPLC fractions

Days after plantation of W14 cells

Volu

me

(mm

3 )

It is known that sugar is changed to furan polymer by heating (cited from Wikipedia).

Sugar=Glucose+Fructose

Furan polymer

Accordingly, heated glucose (DB-glucose) also appears to be a kind of furan compounds.

0

25

50

75

100

0

150

300

450

600

Inhibitory effect of heated glucose on the growth of solid cancer in human lung cancer cell (LU65A) by oral administration

Days

Days

Volu

me

(mm

3 )

Volu

me

(mm

3 )

P<0.05*

P<0.1

P<0.2

3 7 10 14 17

21 24 27

Heated glucose effects a initial phase of cancer growth.

Cont

rol,

n=5

Exp.

grou

p, n

=4

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

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.

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

Occurrence of a glycoside glucose to some poly-con

Nobuaki Takahashi and Akari Takaya

molecule in the process fromdensed compound by heating

(Sapporo Inst., Shingenmedical Co., Ltd)

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.

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.

Subjective

Hydroxymethylfurufural ヒドロキシメチルフルフラール

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

���

����

A254nm

min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

1st Point of Presentation: Elution pattern of HPLC by 20 μl injection of each glucose (200μg/ml)

Normal glucose

Heated glucose

(10%)

(100%)

(methanol gradient)A254nm

0

30

60

90

120

1 2 3 4

0

30

60

90

120

1 2 3 4

Effect of commercially available caramel and D-(+)-glucose one on the proliferation of W14 cells, using MTT assay

800. ���400����200����100

���Concentration (micro/ml)���

800. ���400����200����100

���Concentration (micro/ml)���

Commercial caramel Glucose caramel

2nd Point

0

25

50

75

100

0

150

300

450

600

3rd Point: Inhibitory effect of heated glucose on the growth of solid cancer in human lung cancer cell (LU65A) by oral administration

Days

Days

Volu

me

(mm

3 )

Volu

me

(mm

3 )

P<0.05*

P<0.1

P<0.2

3 7 10 14 17

21 24 27

Heated glucose effects a initial phase of cancer growth.

Cont

rol,

n=5

Exp.

grou

p, n

=4

Materials & Methods

+ CD44

�One week�

�Three weeks�

Volume: 1,000 mm 3

Volume = (minor axis) 2 x (major axis) x 0.5

Start of water drinking assay (conc. of test materials=200�μ /ml)

Finish

(One mouth)

WFB

+ H-ras onco.= W14

Injection of 105 cells of W14

Glucose caramel ( 0.1g glucose in 1ml water treated with 180℃, 2hrs)

Water drinking assay

Introduction to W14 cells (Cancer stem cell-like cell)

茶色が濃くなったグルコースカラメルA

23

0(n

m)

0

0.3

0.6

0.9

1.2

Fraction Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

100

50

0

（％）Inhibition rate

Fraction Number

Abs

orba

nce,

A23

0 (n

m)

Gel filtration by Sephadex G-25

Gel Filtration of Glucose Caramel and Determination of Inhibitory Active Fraction

Results: Study of Active Molecular Size

Growth Inhibitory Effect of Fraction No. 6 & 7 of Caramel- Gel Filtration on W14-Solid Cancers

V=839 (mm3), 163, 864

Town water Fr. No. 6&7 Fr. No. 13&14

Control Fr. No. 6&7 Fr. No. 13&14

① ② ③

① ② ③

Inhibition rate

100(%) Control

① part

② part

③ part

Non-growth

Determination of Active Part in The Active Fraction, No. 6&7 by HPLC

W14-solid cancers

Inhibitory Effect of Renual DBG by Sephadex G-50

A2

54

(nm

)

0

0.23

0.45

0.68

0.9

Fraction Number

⑦⑨

⑫⑭

Inhibitory effect of fractionated heated-glucose by Sephadex G-50

Fraction Number

A25

4 (nm

)

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

��1���3���5���7���9���11���13���15���17

10,000

20,000

30,000

No. 12 (14,000)

No. 14 (8,000)

Mol

ecul

ar W

eigh

t

Fraction Number

5,000

Sephadex G-50

Sephadex G-25

G-50 1,500-30,000

G-25 1,000-5,000

Gel Type Fractionation Range of Globular Proteins (Da)

？

Short Heating-HPLC Pattern of Heated Glucose

A254nm

Active High Molecule- HPLC Pattern of Heated Glucose

C6H10O5 C18H25O13

C6H10O5

C12H15O8

(C12H16O8)

C18H23O12

C6H10O5

C6H12O6

C12H16O8

2hrs

30min

ヒドロキシメチルフルフラール

NMR解析

MS分析

A Search of Precursor

It is known that sugar is changed to furan polymer by heating (cited from Wikipedia).

Sugar=Glucose+Fructose

Furan polymer

Accordingly, heated glucose (trade name, furan D glucose) also appears to be a kind of furan compounds.

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

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.

Conclusions:

Glucose

（ぶどう糖）

Heating

Cooking

Several new substances1 levoglucosan

2 some heated glucose (fucan-like sub.)

3 ?

4 ?���

Solar energy

Thermal energy

Photosynthesis

Respiration

Energy stream to liveOnce, we cached fire.

Unknown research area

Ecological system

The Significance of This Study

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

Future Plan:

COI Disclosure Information

Nobuaki Takahashi

�I have no financial relationships to disclose.

Lead Presenter/Responsible Researcher