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Title:
SULPHATED CHELATING AGENTS
Document Type and Number:
WIPO Patent Application WO/2014/097284
Kind Code:
A1
Abstract:
This invention refers to a series of substances according to formula (I): and its synthesis process. The subtances according to formula I above, known as sulphated chelating agents have the ability to enlarge/loosen simple cell membranes or common organic membranes. Furthermore, these subtances have the potential as biocides raw material, with very low toxicity to mammals. These features make the substances of this invention become very useful on many applications.

Inventors:
WARDOYO HARYANTO (ID)
Application Number:
PCT/ID2013/000009
Publication Date:
June 26, 2014
Filing Date:
November 22, 2013
Export Citation:
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Assignee:
WARDOYO HARYANTO (ID)
International Classes:
C07C305/04; C07C305/10; A61P31/04; A61P39/04; C07C303/24
Domestic Patent References:
WO2002081487A12002-10-17
Foreign References:
US20080227987A12008-09-18
US20030055007A12003-03-20
Other References:
PETER CLAESSON: "Ueber die Aetherschwefelsäuren der mehrsäurigen Alkohole und der Kohlehydrate nebst einigen Bemerkungen über die Constitution der letzteren", JOURNAL FUER PRAKTISCHE CHEMIE, vol. 19, no. 2, 1879, LEIPZIG, pages 1 - 34, XP002720747, ISSN: 0021-8383
KREMS I J ET AL: "The Reaction of Lauric Acid Esters with Sulfuric Acid", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, ACS PUBLICATIONS, US, vol. 81, 1 January 1959 (1959-01-01), pages 1620 - 1627, XP002222376, ISSN: 0002-7863, DOI: 10.1021/JA01516A028
H. HAQUE; A. D. RUSSELL: "Effect of Chelating Agents on the Susceptibility of Some Strains of Gram-Negative Bacteria to Some Antibacterial Agents", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, August 1974 (1974-08-01), pages 200 - 206, XP055014630, DOI: doi:10.1128/AAC.6.2.200
KAUR P; VADEHRA DV: "Effect of certain chelating agents on the antibacterial action of silver nitrate", J HYG EPIDEMIOL MICROBIOL IMMUNOL., vol. 32, no. 3, 1988, pages 299 - 306, XP055496431
A. HINTON JR.; K.D. INGRAM: "Comparison of the Antibacterial Activity of Chelating Agents Using the Agar Diffusion Method", INTERNATIONAL JOURNAL OF POULTRY SCIENCE, vol. 9, no. 11, 2010, pages 1023 - 1026
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Claims:
Claim

Formula I

wherein:

Xi / X are selected from: Hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives, where Xi , X2 , X3 / could be similar or different atoms, moleculs or groups.

Rx, Rz are selected from: Hydrogen, hydroxy, halide, alkyl Ci- 20 alkylene C1-20 / alkyl alcohol Ci-2cw aliphatic or branched subtituted or unsubstituted, aryl, cycloalkyl subtituted or unsubstituted, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives.

M: are Hydrogen, or group I, group II, transition group cation that are pharmaceutically acceptable,

n: intreger from 0-3

a : intreger

and/or its isomer, enantiomer, stereotiomer, salt, solvate., hydrate that are pharmaceutically acceptable.

2. A Compound according to claim 1, whereas the subtances are selected out of:

- Tetra Hydroxy Ethyl Di Sulphate

- Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate

- Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate

- Tetra Hydroxy Isoamyl 1 , 2 , 3 , 4-Tetra Sulphate

- Ethyl hexa sulphate acid

Subtances according to claim 1 or 2, whereas M is natrium

4. A Compound according to claim 1-3, whereas the said subtances are selected out of:

- Tetra Hydroxy Ethyl Di Sulphate Di Natrium

- Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate Tetra Natrium - Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate Tri Natrium

- Tetra Hydroxy Isoamyl 1 , 2 , 3, 4-TetraSulphate Tetra Natrium.

- Ethyl Hexa Sulphate-Hexa Natrium ( EHS- 6Na ) .

5. A Compound according to one of the above claims used as chelating agent.

6. A Compound according to one of the above claims whereas the said subtances have the ability to open/loosen cell wall membranes and/or another organic membranes.

7. A Compound according to one of the above claims whereas the said subtances can be used as biocide.

8. Synthesis of a compound according to formula I, that is consist of :

- reacting keton/aldehyde/carboxylate compound in formula II formula II

where :

XI, X2, X3, Rx, Rz, and a as per claim 1 definition above; with sulphuric acid (H2S04) or sulphonic acid (H2SO3) and/or sulfur trioxide (SO3) or sulfur dioxide (SO?) ,

9. Synthesis of a compound according to formula I, from formula II subtances as per claim 8, of which the preferred formula II subtances are formaldehide, acetaldehide, formic acid, asetic acid and oxalic acid.

10. A method to break or loosen the hydroxy-hydroxy bound on peptidoglycan or hydroxyl-amide bound on protein that compose cell wall membrane or another organic membrane by mean of utilizing the sulphate chelating agent of this invention, whereas the said substance enlarge the membrane porosity that turns the membrane into totally permeable on both of its sides . 11. A method according to claim 10, whereas the said sulphate chelating agent can cause the lysis of membrane.

12. Use of the compound according to claim 1 as the membrane loosening or opening agent.

13. Use of the compound according to claim 1 as biocide.

14. Use of the compound according to claim 1 as chelating agent .

Description:
Description SULPHATED CHELATING AGENTS

Field of the invention

This invention is related to the Sulphate chelating agents that have the capability to enlarge the membrane porosity of simple microorganism cell and its synthesis proces .

Background of the invention

Many types of chelating agents are commonly known and have been applied in various industry. Chelating agent are chemicals contain donor atoms that have the capability to bind metallic atoms in coordinative complex bound that turn into cyclic structural subtances known as chelating complex or in its simple term : a chelat. The chelation technology was developed from some naturally occurred chemicals that contain naturally or purposely added metallic ions. The use of chelating agent might give a way to control or manipulate the metallic ions in the system to perform the expected effect. The chelating complex subtances formed, from the interaction of some metallic ion with some chelating agent use to have significantly different characteristics/properties either from its original ions or the chelating agent itself. Therefore its characteristics or properties could be modified.

Therefore, the chelating agents are the very effective subtances in the formation of the complex subtances with the metallic cations and also with the organic salts in order to prevent them act as simple hidrated cations. Common example of chelating agent is. Ethylene Di Amine Tetra Acetic acid (EDTA) (1) and its derivatives that will form the complex subtances with most of M 2+ and M 3+ types of cations . HO - C \ C -OH

N CH 2 _ CH 2 N

HO - C ^ \ C -OH

O O

EDTA

It has been known also that gluconic acid and some others hydroxide acid perform similar properties.

There are many features of the chelating reaction that become the basic of chelating agents various aplications.

The first, chelation provide a mechanism to control the concentration of the free metallic ions via dissociation equilibrium between chelating agent and the said metallic ions. The related application as the sequestration process that reduces several properties of some metal without eliminate it from the existing system or phase, solubilization is a process that elaborate undissolved component phase become soluble in the said medium, and daparing, a condition where the addition or removal of several metallic ions result insignificance change of the said ionic concentration inside the solution which totally depending on the accurate control of the chelating agent concentration.

All of the above features and properties have been chemically applied in industry.

For example, the sequestration of the metallic ions could be used to control the water hardness. Then solubilization is used to dissolve the boiler scale, heat exchange equipment scale and the hard scale in the pipe.

In the minning industry, solubilization utilizes chelating agents to extract the metal from the metal ores, also to be used to clean the contaminated area.

Daparing with the chelating agents find its use to supply the metallic ions micro nutrient for the biological growth at very low stabilized concentration.

Secondly, on some applications as the chelating ligand catalyst which really has the catalytic activity dan has been applied as catalyst on the unsymmetrical pharmaceutical subtances synthesis.

Finally, chelating agents have also been used for human medication. For example, the removal/cleaning 'with the chelating agent with its chelation termonation, including the curing on the lead (Pb) poisoning and other metals with EDTA (Ethylene Di Amine Tetra Acetic acid) here lead almostly chelated to EDTA, therefore could be removed from the system.

In the publication by H. HAQUE AND A. D. RUSSELL "Effect of Chelating Agents on the Susceptibility of Some Strains of Gram-Negative Bacteria to Some Antibacterial Agents", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1974, page. 200- 206, explain that some chelating agents improve the activity of beta-lactam in the bacterial inhibition of P. aeruginosa strain through the metallic removal from the nutrient medium and some chelating agent could increase the bacterial sensitivity against some antibiotics.

In the other journal published by Kaur P, Vadehra DV. , "Effect of certain chelating agents on the antibacterial action of silver nitrate." J Hyg Epidemiol Microbiol Immunol. 1988; 32 (3) : 299-306, explain that EDTA and EGTA when apllied together with AgN0 3 significantly increase the antibacterial action against Staphylococcus aureus , where EDTA and EGTA also increase the sensitivity of Staphylococcus aureus bacteria that is resistant against AgNC>3.

In the publication by A. Hinton Jr. and K.D. Ingram, "Comparison of the Antibacterial Activity of Chelating Agents Using the Agar Diffusion Method" International Journal of Poultry Science 9 (11) : 1023-1026, 2010 explain that the addition of EDTA chelating agent for the cleaning agent formulation in the poultry treatment could improve the cleaning capability that has the antimicrobial activity helping to reduce poultry corpses contamination.

But until now, there is no publication describing that EDTA itself alone can perform as an antibacterial agent, due to the fact that chelating agents in some cases have the ability to influence simple cell membranes or other organic membranes .

In the US 2003/0055007 patent publication, Al was disclosing about some lignin sulphonate substance that has the antiviral activity against HIV and also act as an antibacterial agent, but there was no further explanation about the inhibition mechanism of the said subtances as the antiviral and antibacterial agent.

Therefore, it needs the development of new chelating agents and its derivatives that are non toxic or less toxic and induce no negative or less negative effect into environment, these features will significantly increase the application of the said chelating agents .

Unexpectedly, the inventor has discover such a new chelating agent that act alone as antibacterial agent. The inventor has invent a series of sulphate chelating agents that have the antibacterial activity by enlarging the cell membranes porosity of simple cells and the other organic membranes. The membrane porosity enlargement mechanism for simple cell or other organic membranes occurred due to the reaction of the sulphate groups of some sulphate chelating agent with the hydroxyl group (OH " ) or ammine group (N¾ ~ ) in the peptide bound of modular bound structural of a protein or modular bound structural of a peptidoglycan and their similarities as the main ingredient of the simple cell wall membranes or other organic membranes.

Brief description of the invention

Moreover, these invented subtances have the capability as common chelating agent or subtances that capable to open/enlarge the membrane porosity of either simple cell membrane wall or another organic membranes, also have the potential biocidal property, and it has been discovered that the said subtances have a very low toxicity against mamal and also easy to be degraded in the environment. All these features put the said subtances become very usefull in many aplications .

In one manifestation the present invention provides all the subtances have the structure as of formula I

Formula I

wherein :

Xi, X 2 , X 3 , are selected from : Hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives, where Xi, X 2 , X3, could be similar or different atoms, moleculs or groups.

Rx, Rz are selected from: Hydrogen, hydroxy, halide, alkyl Ci- 20 / alkylen C 1 - 20 alkyl alcohol Ci- 2 o f aliphatic or branched subtituted or non substituted, aryl, cycloalkyl subtituted or non substituted, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives. M: are Hydrogen, or group I, group II, transition group cation that are pharmaceutically acceptable,

n : intreger from 0-3

a : intreger

and/or its isomer, enantiomer, stereotiomer, salt, solvate, hydrate

In other preferred embodiment, this invention provide some subtances selected from:

- Tetra Hydroxy Ethyl Di Sulphate

- Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate

- Tri Hydroxy Isobuthyl 1,2,3-Tri Sulphate

- Tetra Hydroxy Isoamyl 1, 2, 3, -Tetra Sulphate

In the further embodiment, the preferred subtances is where M is Natrium.

In the further embodiment, including its isomer, enantiomer, stereotiomer, salt, solvate, hydrate of Formula I.

In the further embodiment, the preferred subtances are:

- Tetra Hydroxy Ethyl Di Sulphate Di Natrium

- Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate Tetra Natrium

- Tri Hydroxy Isobuthyl 1,2,3-Tri Sulphate Tri Natrium

- Tetra Hydroxy Isoamyl 1, 2, 3, 4-Tetra Sulphate Tetra Natrium

In such invention embodiment provides some sulphate chelating agent represent by the above Formula I that has antibacterial activity by enlarging the membrane porosity of simple cells or another organic membranes. The membrane porosity enlargement mechanism for simple cell or other organic membranes occurred due to the reaction of the sulphate groups of some sulphate chelating agent with the hydroxyl group (OH " ) or ammine group (NH2 " ) in the peptide bound of modular bound structural of a protein or modular bound structural of a peptidoglycan and their similarities as the main ingredient of the simple cell wall membranes or other organic membranes. In another embodiment, this invention provide some series sulphate chelating agents represent by the above Formula I that can be used as biocide.

Further embodiment of this invention is providing the synthesis process of the said sulphate chelating agent represent by Formula I, that consist of :

- reacting keton/aldehyde/carboxylate in Formula II:

Formula II

Where :

XI, X2, X3, Rx, Rz, and a as have already defined in the above Formula I;

With sulphuric acid (H 2 S0 4 ) or sulphonic acid (H2SO3) and/or sulfur triokside (S0 3 ) or sulfur diokside (S0 2 )as it is and/or might be following by polymerization to get the desired length of polymer chain.

In the preferred embodiment of this invention, the said new subtances I can form a complex substance with metal.

In the further aspect of this invention describing the method of membrane porosity enlargement of simple cells or another organic membranes with some effective ammount of this invention. In the preferred embodiment of this invention, the new subtances of this invention to be discovered have the capability to modify membrane of simple cells or organic membranes by mean enlarging significantly the membrane porosity. By not theoretically fully supported, it is postulated due to sulphate group electronegativity of this sulphate chelating agents will tense the hydroxy-hydroxy bound of peptidoglycan and/or hydroxy-ammine bound of protein that compose cell membranes or organic membranes. The capability of these said subtances are proven by mean of opening/enlarging membrane porosity of some microorganisms simple cell, such as : bacteria cell, algae, fungi, and similar with some concentration level worked on the membrane modification mechanism from semi permeable into permeable membrane therefore it is become possible for another ligand insertion into target microorganism and/or simple cell; also possible that the microrganism cell wall membranes become totally permeable result on the microorganism death due to cell internal lysis or dorment/latent condition of the target microorganism. It is postulated that this said mechanism works for almost all of microorganism having cell membranes.

Brief description of the figures

Figure 1 : Describe the absorbance curve Menggambarkan kurva absorbansi Tetra Hydroxy Ethyl Di Sulphate Di Natrium (THES) against NH 3 FeS0 4 volume at pH 7.

Fig 2a and 2b : are the titration curves show the comparation of chelating capability between THES and EDTA, where it needs less THES concentration in comparation to EDTA.

Fig 3 : SEM pictures result of pure CaC0 3 crystals with its rhombus structure and CaCC>3 crystals after chelated with Tetra Hydroksy Ethyl Di Sulphate Di Natrium (THES) at pH 7.

Figure 4 : Transversal section of Staphylococcus aerus single cell (x 35, 000), from left to right : Normal cell, in contact with 0.05 % THES, in contact with 1% THES (tetra hydroxy ethyl di sulphate) at pH 7.

Fig 5 : Transversal section of E. Colli single cell (x 25, 000), from left to right : Normal cell, in contact with 0.05 % THES, in contact with 1% THES (tetra hydroxy ethyl di sulphate) at pH 7.

Fig 6 : Outer view of Staphylococcus aerus cell wall/membranes, from left to right : Normal cell, in contact with 1% THES (tetra hydroxy ethyl di sulphate) at pH 7.

Fig 7 : Outer view of E. Colli, cell wall /membranes , from left to right : Normal cell, in contact with 1% THES (tetra hydroxy ethyl di sulphate) at pH 7.

Detail description of the invention

In such embodiment of subtances have the formula

Formula I

wherein :

Xi / ¾ / are selected from : Hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives, where Xi, X2, X3, could be similar or different atoms, moleculs or groups.

Rx, Rz are selected from: Hydrogen, hydroxy, halide, alkyl Ci- 20 / alkylene C1-20 alkyl alcohol Ci-20, aliphatic or branched subtituted or unsubstituted, aryl, cycloalkyl subtituted or unsubstituted, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives.

M: are Hydrogen, or group I, group II, transition group cation that are pharmaceutically acceptable,

n: intreger from 0-3 a : intreger

and/or its isomer, enantiomer, stereotiomer, salt, solvate, hydrate that are pharmaceutically acceptable.

In the other preferred embodiment of subtances derived from Formula I where M is hydrogen.

In the most preferred embodiment of substance is

- Tetra Hydroxy Ethyl Di Sulphate

- Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate

Tri Hydroxy Isobuthyl 1,2,3-Tri Sulphate

- Tetra Hydroxy Isoamyl 1 , 2 , 3 , 4-Tetra Sulphate

- Ethyl Hexa Sulphate acid (EHS)

and/or its isomer, enantiomer, stereotiomer, salt, solvate, hydrate that are pharmaceutically accepted.

In the other preferred embodiment of such subtances from Formula I, where M is Natrium.

In the preferred embodiment of the above subtances are : - Tetra Hydroxy Ethyl Di Sulphate Di Natrium

- Tetra Hydroxy Buthyl 1 , 2 , 3 , 4 Tetra Sulphate Tetra Natrium

Tri Hydroxy Isobuthyl 1,2,3-Tri Sulphate Tri Natrium

a +

Tetra Hidroxy Isoamyl 1, 2 , 3, 4-Tetra Sulphate Tetra Natrium.

Ethyl Hexa Sulphate-Hexa Natrium (EHS-6Na) and/or its isomer, enantiomer, stereotiomer, salt, solvate, hydrate that are pharmaceutically accepted.

Furthermore, the formed alkyl sulphate subtances derived from formula I are still enabled to be polymerized to form longer chain.

In the preferred embodiment of this invention covers breaking or loosening the hydroxyl-hydroxyl bound of peptidoglycan or hydroxyl-ammine bound of protein that compose cell wall membranes or organic membranes by mean utilizing some sulphate chelating agent that result on the enlargement of the membrane porosity, therefore the membrane become permeable on both of its sides. The capability of the said subtances was proven by opening/enlargement the microorganism simple cell's membrane, such as : bacteria, algae, fungi, virus, etc with some concentration level that works on the cell wall membrane modification from semi permeable membrane into more and more permeable, therefore it becomes possible to insert another ligands inside the microorganism or the cell wall membrane become totally permeable that result on the microorganism death and/or microorganism dorment condition. It is postulated the said mechanism works for all types of microorganism that has cell membrane.

Definition

The following terms are used throughout the specification and have the following meanings unless otherwise specified:

"Alkyl" means carbon atom chains having the designated number of carbon atoms which can be either straight chain or branched. Examples of alkyl include but are not limited to, methyl, ethyl, propyl, butyl, isobutyl, and the like.

"Alkenyl" means carbon atom chains having the designated number of carbon atoms which can be either straight chain or branched and which contain at least one double bond. The alkenyl compounds may have more than one such double bond and the orientation about each double bond is independently either cis or trans. Examples of alkenyl include, but are not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl and the like.

Alkynyl" means carbon atom chains having the designated number of carbon atoms which can be either straight chain or branched and which contain at least one triple bond.

As used herein the term "aryl" means single, polynucleic conjugated and fused residues of aromatic hydrocarbon or aromatic heterocyclic ring systems, exampleas of aryl include, but are not limited to phenyl, naphthyl, fluorenyl, pyrinyl, pyridyl, pyrollyl, and the like.

Cycloalkyl termination refers to non-aromatic aliphatic ring moiety of 3-20 mono-cyclic, bicyclic, or polycyclic carbon atoms. Cycloalkyl can be bicycloalkyl , polycycloalkyl, branched, or spiroalkyl. One or more of the rings might have one or more double bond but no such ring has fully conjugated pi-electron system. Example, without limitation, of cycloalkyl group are: cyclopropane, cyclobutane, cyclopentane, cyclopenthenyl , cyclohexane, cyclohexadiene , adamanthane, cycloheptane , cycloheptatriene, and the like.

Sulphite structure could be described with three equal resonance structures. In every resonance structure, the sulphur atom has a double bond with one oxygen atom with zero/neutral charge, and the single sulphur atom is bound into two other oxygen atoms that each has formal charge of -1. It is also found free electrons pair on Sulphur atom, therefore the predicted structure by VSEPR theory is a pyramid trigonal similar to amonia (NH3) . Sulphate termination refers to sulphur as the centered atom surrounded by four equal oxygen atoms in tetrahedral structure. Sulphur atom in its oxidation state +6 while the four oxygen atoms, each in its oxidation state -2. Sulphate ion carry negative charge and two of them are the alkaline conjugate of bisulphate (or hydrogen sulphate) , HS0 4 ~ , as the alkaline conjugate of H 2 SO , sulphuric acid.

O 0 0 "

II II I

< *s 0 - ^ ^ → ^s ¾0

cr cr 0-

Sulphonate termination refers to an ester of sulphonic acid. An ester with the common formula R0S0 2 R' is a sulphonic ester. The common structures of sulphonic ester shown by the structur s below:

(covalen form)

[ionic form)

Nitrogen derivatives termination refers to carbon subtances that contain nitrogen atom, cyclical or aliphatic, straight or branched chain, if it has cyclical structure, then it could be in fussion or non fussion form, could be aromatic or non aromatic.

Phosphorous derivatives termination refers to carbon subtances that contain phosphorous atom, cyclical or aliphatic, straight or branched chain, if it has cyclical structure, then it could be in fussion or non fussion form, could be aromatic or non aromatic.

Pharmaceutically accepted salts refer to alkaline addition types of salts synthesized by adding some alkaline subtances to the said subtances in this invention. The common pharmaceutically accepted salts are: natrium, kalium, calcium or zink.

In the specification, the termination of substitued means that a group may be further substituted with one or more groups selected from alkyl, alkenyl, alkunyl, aryl, fluoro, chloro, bromo, hydroxyl, alkoxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl or two or more subtituens may, together with the carbon atoms to which they are attached from a 5 or 6 membered aromatic or non aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen and sulphur.

Main selection of the preferred subtances of this invention are:

a. Tetra Hydroxy Ethyl Di Sulphate

b. Tetra Hydroxy Ethyl Di Sulphate Di Natrium

c. Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate

d. Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate Tetra

Natrium.

e. Tri Hydroxy Isobuthyl 1,2,3-Tri Sulphate

f. Tri Hydroxy Isobuthyl 1,2,3-Tri Sulphate Tri Natrium g. Tetra Hydroxy Isoamyl 1, 2, 3, 4-Tetra Sulphate

h. Tetra Hydroxy Isoamyl 1, 2, 3, 4-Tetra Sulphate Tetra

Natrium . Synthesis of the subtances of formula (1) or (2) are of below :

- reacting keton/aldehyde/carboxylate compound in formula III below :

Formula III wherein :

Xi, X 2 , X3, are selected from : Hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives, where Xi, X 2 , X3, could be similar or different atoms, moleculs or groups.

Rx, Rz are selected from: Hydrogen, hydroxy, halide, alkyl Ci_ 20 , alkylen C1-20 alkyl alcohol C1-20 / aliphatic or branched subtituted or unsubstituted, aryl, cycloalkyl subtituted or unsubstituted, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives;

a: intreger;

with sulphuric acid (H 2 S0 4 ) and/or sulphonic acid (H 2 S0 3 ) and/or sulphur trioxide (SO3) and/or sulphur dioxide (S0 2 ) ,

Examples of the synthesis of some subtances within this invention : Example 1: Synthesis of Tetra Hydroxy Ethyl Pi Sulphate (THES)

225 g of oxalic acid crystal was diluted in 150 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 250 ml of 98% H 2 S0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100°C at atmospheric pressure. Upon completion of H 2 S0 4 acid addition, the solution is still heated with agitation until the next 180 minutes with the installation of vertical pipe condensor for reflux purposses to maintain the volume of the liquid until a very clear solution is achieved.

Then the liquid temperature was raised to 90 - 100°C. Evaporation of the solvent followed by crystallization result in formation of the desired subtance as an off white powder with the following properties : melting point >. 200°C, boiling point > 400°C, specific gravity = 1.86, water solubility 48 %. Example 2 : Synthesis of Tetra Hydroxy Ethyl Pi Sulphate-Pi Natrium (THES-2Na)

90 g of oxalic acid crystal was diluted in 150 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 100 ml of 98% H 2 S0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry heated with agitation until the next 180 minutes with the installation of vertical pipe condensor for reflux was in the range of 90-100°C at atmospheric pressure. Upon completion of H2SO acid addition, the solution is still heated with agitation until the next 180 minutes with the installation of vertical pipe condensor for reflux purposses to maintain the volume of the liquid until a very clear solution is achieved.

To this clear solution was added dropwise a solution of 73 g NaOH flakes in 75 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 50 + 20°C. Upon completion of addition, the temperature was adjusted slowly to ambient and the solution was held agitated in the ambient temperature for the next 60 minutes .

Evaporation of the solvent followed by crystallization result in formation of the desired subtance as an off white powder with the following properties: melting point > 200°C, boiling point > 400°C, specific gravity = 2.2, water solubility 31 %. Example 3: Synthesis of Tetra Hydroxy Butyl 1 , 2 , 3 , 4-Tetra Sulphate

184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 230 ml of 98% H 2 S0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100°C at atmospheric pressure. Upon completion of H2SO 4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Then the temperature was adjusted slowly to ambient and the solution was held agitated in the ambient temperature for the next 60 minutes.

Upon 60 minutes agitation completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35 - 40 % of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into + 10 °C by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate acid crystals, the separated crystals then oven dried at + 70-80°C until the constant weight reached.

Example 4: Synthesis of Tetra Hidroxy Butyl 1 , 2 , 3 , 4-Tetra Sulphate-Tetra Natrium

184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 230 ml of 98% H 2 S0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100°C at atmospheric pressure. Upon completion of H2SO 4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. To this clear solution was added dropwise a solution of 170 g NaOH flakes in 200 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 50 + 20°C. Upon completion of addition, the temperature was adjusted slowly to ambient and the solution was held agitated in the ambient temperature for the next 60 minutes.

Upon 60 minutes agitation completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35 - 40 % of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into + 10 °C by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate Tetra Natrium crystals, the separated crystals then oven dried at + 70-80°C until the constant weight reached.

Example 5: Synthesis of Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate :

138 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 173 ml of 98% H 2 S0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100°C at atmospheric pressure. Upon completion of H2SO4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Next, prepare methanol solution of 34 g methanol 96 % technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly.

Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate .

Upon the filtration completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35 - 40 % of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into + 10 °C by ice water bath. The resulting suspension then is filtrated to separate the Tri Hydroxy Butyl 1,2,3 Tri Sulphate acid; the separated crystals then oven dried at + 70-80°C until the constant weight reached.

Example 6: Synthesis of Tri Hidroxy Isobutyl 1,2,3-Tri Sulphate-3Natrium

138 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 173 ml of 98% H 2 SO 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100°C at atmospheric pressure. Upon completion of H 2 SO acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Next, prepare methanol solution of 34 g methanol 96 % technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly. Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate .

To this clear solution was added dropwise a solution of

130 g NaOH flakes in 200 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 50 + 20°C.

Upon completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35 - 40 % of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into + 10 °C by ice water bath. The resulting suspension then is filtrated to separate the Tri Hydroxy Butyl 1,2,3 Tri Sulphate-3Natrium crystals; the separated crystals then oven dried at + 70-80°C until the constant weight reached.

Example 7: Synthesis of Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulphate

184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 230 ml of 98% H 2 SO4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100°C at atmospheric pressure. Upon completion of H2SO4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Next, prepare methanol solution of 34 g methanol 96 % technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly. Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate .

Upon the filtration completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35 - 40 % of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into + 10 °C by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulphate acid; the separated crystals then oven dried at + 70-80°C until the constant weight reached. Example 8: Synthesis of Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulfat-4Natrium

184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 230 ml of 98% H 2 S 0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100°C at atmospheric pressure. Upon completion of H 2 S0 4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved.

Next, prepare methanol solution of 34 g methanol 96 % technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly.

Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate .

To this clear solution was added dropwise a solution of 170 g NaOH flakes in 200 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 50 + 20°C.

Upon completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35 - 40 % of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into + 10 °C by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulphate- 4Natrium; the separated crystals then oven dried at + 70-80°C until the constant weight reached.

Example 9: Synthesis of Ethyl Hexa Sulphate (EHS)

75 g of oxalic acid crystal was diluted in 75 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 250 ml of 98% H 2 S0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry heated with agitation until the next 180 minutes with the installation of vertical pipe condensor for reflux was in the range of 90-100°C at atmospheric pressure. Upon completion of H 2 S0 4 acid addition, the solution is still heated with agitation until the next 120 minutes with the installation of vertical pipe condensor for reflux purposses to maintain the volume of the liquid until a very clear solution is achieved; then the vertical pipe condensor was released, the resulting solution is still heated with another 30 minutes agitation to ensure complete and homogenous sulphatation reaction.

Then the liquid temperature was raised to 100 - 105°C. Evaporate half of the solvent volume followed by crystallization result in formation of the desired subtance as an off white powder with the following properties : melting point > 21CTC, boiling point > 400 U C, specific gravity = 1.87, water solubility 48 % and very higroscopic .

Example 10: Synthesis of Ethyl Hexa Sulphate-Hexa Natrium (EHS-6Na)

75 g of oxalic acid crystal was diluted in 75 ml of water and the resulted slurry heated to 40-50°C. To this slurry was added dropwise 250 ml of 98% H 2 S0 4 . The rate of acid addition was controlled to ensure that the temperature of the slurry heated with agitation until the next 180 minutes with the installation of vertical pipe condensor for reflux was in the range of 90-100°C at atmospheric pressure. Upon completion of H 2 S0 4 acid addition, the solution is still heated with agitation until the next 120 minutes with the installation of vertical pipe condensor for reflux purposses to maintain the volume of the liquid until a very clear solution is achieved; then to this clear solution was added dropwise a solution of 185 g NaOH flakes in 190 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 50 + 20°C within one hours time. Then the vertical pipe condensor was released, the resulting solution is still heated with another 30 minutes agitation to ensure complete and homogenous neutralization reaction.

Then the liquid temperature was raised to 100 - 105°C. Evaporate half of the solvent volume followed by crystallization result in formation of the desired subtance as an off white powder with the following properties : melting point > 360°C, boiling point > 400°C, specific gravity = 2.21, water solubility 29 % and higroscopic.

Analytical method :

Analytical method to determine Tetra Hydroxy Ethyl di Sulphate-di Natrium (THES-2Na) , utilizing Iron's chelating agent Test Principle :

Active matter of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-2Na) was determined by chelating reaction of residual metallic ion with several anionic surfactans that prevent this anionic surfactants dissolved in the solution result in the turbid solution measured by its turbidity and/or absorbance spectrophotometer . Materials and Equipments:

Materials :

Reagent grade NH 3 FeS0 4 .2H 2 0, reagent grade Natrium Laurier Ethoxylate sulphate (Na-SLES), Caustic Soda flakes, aquadess.

Equipments :

Visible Spectrofotometer , cuvet, erlen meyer, beaker glass, pipete, scale weight, magnetic stirrer. Method:

I. Absorbance:

1) Add 2 gr Na-SLES into 100 cc aquades, stir with magnetic stirrer for 15 minutes until clear solution (1) reached.

2) Add on 1,0 cc of tested chelating agent (THES-2Na) , stir with magnetic stirrer for 15 minutes until homogenous solution(2) .

3) Adjust the pH of the solution above into 7 with Caustic

Soda flakes.

4) Prepare 50 cc 0,1 M NH3FeS04 solution in the 100 cc beaker glass .

5) the cuvet with the solution (2), record the absorbance at 450 nm. 6) Titration starts with the addition of 1,0 cc 0,1 NH3FeS04 solution into solution(2), stir with magnetic stirrer for 2 minutes until homogenous solution (3).

7) Fill the cuvet with the solution (3), record the absorbance at 450 nm.

8) Repeat step 5 and 6 until end point of 0,1 M NH3FeS04 reached .

End point and calculation

I. Absorbance method:

1) Absorbance reading will show significant increases, until some points, then the reading will decrease.

2) When this absorbance reading suddenly jump from the increasing trend into decreasing trend, the end point of the titration has reached.

3) Continue the NH3FeS04 solution addition by another 2 or 3 cc volume to get the smooth curve and/or representative data for accurate interpolation of the end point volume.

4) Draw the absorbance curve versus titrant volume addition then determine the end point volume by this curve or using all absorbance and NH3FeS04 volume addition through Lagrance interpolation formula to get the end point volume.

5) Calculate the concentration or active matter of Tetra Hydroxy Ethyl Di Sulphate Di Natrium (THES- 2Na) using the formula below :

THES 2Na content = (V ep x M NH3FeS04 x MW THES 2Na x 3,9)/(V sample x sample SG x 1,000)

where

V ep end point volume in cc

NH3FeS04 : molarity NH3FeS04 : 0,1 M BM THES 2Na : molecule weight THES 2Na: 330

V sampel : sample volume of tested chelant in cc

Example :

Take 1,0 cc of 35% Tetra Hidroksi Etil Di Sulfat Di Natrium (THES-2Na) solution as sample, density of 1,1 g/cc. Then add on Na-SLES, titrate with 0,1 M NH3FeS04; the absorbance record shown in the Table 1 below: Table 1

From Table 1 above, the absorbance curve versus volume NH3FeS04 trend is shown in Figure 1. From Figure 1, the end point volume is 3,5 ml, Molarity

NH3FeS04 is 0,1 M, M THES-2Na is 330 and sample volume of 1,0 cc with SG 1.1 : THES-2Na concentration = 3,2 cc x 0,1 M x 330 x 3,9/(1,0 x 1,15 x 1,000) = 0,358 = 35,8 % of chelating agent.

Charactheristics of this new invention subtances

These subtances of this new invention are preferred to have the charactheristics as below:

a. Tetra Hydroxy Ethyl Di Sulphate (THES)

b. Tetra Hydroxy Ethyl Di Sulphate Di Natrium

c. Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate Molecular weight 506

Crystal density (g/cuCm) 2.28

Solubility @ 25 °C (%) 39.6

pH of 30 % solution <0.0

Melting point (°C) 242

Boiling point (°C) >400

Colour White brownies

Crystal charactheristic Very hygroscopic d. Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate Tetra Natrium

e. Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate

f. Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate Tri Natrium

Molecular weight 459 Crystal density (g/cuCm) 2.35

Solubility @ 25 °C (%) 36.2

pH of 30 % solution 0.0-1.0

Melting point (°C) 242

Boiling point (°C) >400

Colour Off white

Crystal charactheristic hygroscopic g. Tetra Hydroxy Isoamyl 1 , 2 , 3 , 4 -Tetra Sulphate

Tetra Hydroxy Isoamyl 1 , 2 , 3 , 4-Tetra Sulphate Tetra Natrium.

i. Asam Etil Heksa Sulfat (EHS)

Molecular weight 606

Crystal density (g/cuCm) 1,87

Solubility @ 25 °C (%) 48% pH of 30 % solution 0.0

Melting point (°C) >210 U C

Boiling point (°C) >400°C

Colour Off white

Crystal charactheristic hygroscopic j . Etil Heksa Sulfat-Heksa Natrium ( EHS- 6Na )

Molecular weight 738

Crystal density (g/cuCm) 1,87

Solubility @ 25 °C (%) 48%

pH of 30 % solution 0.0-0.5

Melting point (°C) >210°C

Boiling point (°C) >400°C

Colour Off white

Crystal charactheristic hygroscopic

Ability to enlarge the simple cell membrane

The subtances of this invention were found to have the ability to modify the cell membrane or organic membrane by means enlarging/loosening membrane porosity significantly. By not totally theoretically supported, it was postulated due to the sulphate group electronegativity in the said subtances as the chelating agent will loosen the hydroxy-hydroxy bound of a peptidoglycan or hydroxy-ammine bound of a protein that compose the membrane cell nor organic membrane. The ability of these subtances to enlarge/loosen the membrane porosity were proven by opening/enlarging the microorganism cell membrane, ie : bacteri, algae, fungi, virus, etc with such concentration level that works on the cell wall membrane modification from the semi permeable into more permeable until it is possible to insert another ligands inside the microorganism or the cell wall membrane becomes totally permeable that result on the microorganism death or its latent position. It has been tought that the said mechanism works for all microorganism that has the cell membrane. The said ability to modify the simple cell membrane permeability will be shown by the mechanism and testing data below:

EDTA THES

Method :

The ability of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES) as a Sulphate chelating agent to open/loosen the cell membrane was tested againts two types of bacterias, ie: Staphylococcus aerus (gram positive) that has two cell membrane (the outer membrane as the outer cell wall and the inner membrane) and E. Colli (gram negative) that has three cell membranes. First, the Minimum Inhibitory Concentration (MIC) of each bacterias was determined, then the bacteria membrane alteration after contacting with THES were observed by Electron Microscope (TEM and SEM) with the THES concentration range below and above MIC. Antibacterial activity was tested using disc-diffussion method by checking the clear zone from the paper disc inhibition that contains THES . The growth curve were made by means examining the bacterial culture on the broth media.

Materials and equipments:

Materials :

Difco brand nutrient broth, aquadistilata, THES (tetra hydroxy ethyl di sulphate di natrium) as the tested subtance, glutaraldehyde, paraformaldehyde, albumin serum bovin (BSA) , buffer phosphate pH 7, NaCl 0.9 %, white LR resin, blue toluidine, Natrium citrate trihydrate, NaOH 0,1N, uranil acetate, lead nitrate (Pb(NC>3)2) , parafin, transparan capsule, glass cutter, grid, collodion, gold powder.

Equipments :

Incubator, autoclave, petri dish, ose, spectrophotometer

(Bausch&Lomb) , vibrating incubator, glassware, ultra mikrotom, knife maker, visible microscope, grid pad, transmission electron microscope (Philips), scanning electron microscope (Hitachi) .

Table 2 : Result of Biocide effication plate test of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-Di Natrium)

Total Plate Count Killing

No Biocide dosing (Colony/ml) efficiency

Total Plate Count

Microorganism

1 Blanco/Control 2400 0

2 0.05 % THES-di Natrium 132 94.5

3 0.1 % THES-di Natrium 115 95.2

4 0.5 % THES-di Natrium 101 95.8

5 1.0 % THES-di Natrium 80 96.7

E.Coli 1 Blanco 3900 0

2 0.025 % THES-di Natrium 2010 48.5

3 0.05 % THES-di Natrium 1150 70.5

4 0.1 % THES-di Natrium 1050 73.1

5 0.5 % THES-di Natrium 970 75.1

6 1.0 % THES-di Natrium 720 81.5

7 5.0 % THES-di Natrium 630 83.8

Staphylococcus Aerus

1 Blanco 980 0

2 0.025 % THES-di Natrium 296 69.8

Test on Microorganism:

Staphylococcus aerus ATCC 6538, Escherichia coli ATCC 9637 were supplied from Chemotherapy Laboratory of Pharmaceutical Department, Bandung Institute of Technology (ITB) .

Bacteria preparation

The testing bacterias were growth on the aquaeus medium and incubated for 24 hours at 37°C.

Tested Biocide preparation

The tested Biocide was diluted in distillate water at various gradually decreased concentration.. This gradual concentration variation is necessary to determine MIC (Minimum Inhibitory Concentration) and the bacteriostatic/bactericidal properties of the tested Biocide.

Determination of Killing method (bacteriostatic and bactericidal properties)

The testing was done on the aquaeus medium contains the tested subtance with the concentration 5, 10 and 20%. The turbidity of the cultured medium then will be measured every 30 minutes until 270 minutes with spectrophotometer .

4. Preparation of microbe cells for electron microscope examination

Staphylococcus aereus and Escherichia coli were growth on the aquaeus medium within 24 hours, then mixed with tetra hydroxy ethyl di sulphate di sodium (THES) with the final concentration 0,6; 5 and 10 %. This system mixtures then was placed under vibrating incubator for several hours. Microbe cells then were separated and washed, first with NaCl 0,9 % then with distilled water. Cells then were utilized for the preparation of electron microscope testing.

5. Preparation for Scanning Electron Microscope (SEM) examination

Bacteria cells were suspended into 4% collodion. The said suspensions then were fixed on some metal disc and were coated with the gold powder. Then were examined with electron microscope and pictured.

6. Preparation for Transmitance Electron Microscope (TEM) examination

a) The prepared microbes in (5) were fixed into a mixture of formaldehyde 2% and glutaraldehyde 0.5% for 2 hours at 4°C.

b) The above mixtures then was three times washed with pH 7 phosphate buffer, then were diluted into bovin serum albumin (BSA) 1/5 for embedded preparation, while leave glutaraldehyde hardening at room temperature .

c) After leaving dehydrated in alcohol, the white LR resin then will be infiltrated.

d) Embeded process were done at white LR resin following by polymerization process at 60°C for 48 hours time. The polymerization product as hardened capsules then are ready to be sliced,

e) Making of semi thin and ultra thin slices.

Slices of microbe sample were made by utilizing automatic ultra microtom. To get the precised and acurate slices of sample, semi thin slice were made and coloured with blue toluidine then were examined under the visible microscope. When the right slices were obtained, ultra . thin slices were prepared and were made contrast with uranil acetate and lead citrate. The dried ultra thin slices then can be examined with Transmitance Electron Microscope.

Testing results

Minimum inhibitory Concentration (MIC)

MIC of tetra hydroxy ethyl di sulphate di natrium againts E.coli and S. aureus are shown in Table 3.

Table 3 : Antibacterial activity of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-Di Natrium)

Minimum

Inhibitory

Concentration

No Microorganisme (MIC) (%)

Gram Positive Bacteria

1 Bacillus Subtilis ATCC 0, 234

6633

2 Staphylococcus Aerus ATCC 0,234

6538

3 Staphylococcus Epidermidis 0, 234

ATCC 12228 Gram Negative Bacteria

1 Acinetobacter Anitratus 0, 115

2 Escherichia Coli ATCC 0, 234

25922

3 Pseudomonas Aeruginosa 0,115

ATCC 27853

4 Klebsiella Pneumoniae 0, 234

5 Salmonella Typhii 0, 115

Fungi

1 Aspergillus Niger 0, 937

2 Candida Albicans 0,469

The test done on the pH range of 7-7,5 with the sample of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-Di Natrium) 30% in aquaeus solution.

From the transversal section (TEM) of Staphylococcus aerus cell, can be observed between normal cell that still contains cytoplasm and other organic subtances (dark sight) and the partly empty cells that has already lost its cytoplasm liquid due to lysis out of cell wall membrane (transparent sight), in fact the E. Colli cell that has more layers cell wall membranes also experienced the said internal cell liquid lysis out of its cell wall membrane layers, although the MIC of E. Colli cell was slightly higher than Staphylococcus aerus cell.

From the outer surface of bacteria's cell wall observation through Scanning Electron Microscope (SEM) , it was shown that the outer cell wall membrane of bacteria looked wrinkled/corrugated due to the wider cell wall surface compared to normal cell wall for both Staphylococcus aerus nor E. Colli.