WO2019021144A1

WO2019021144A1 - Process of preparation of l-histidine molecule based hydrogel by gelation of water

Info

Publication number: WO2019021144A1
Application number: PCT/IB2018/055449
Authority: WO
Inventors: S Syed JAFFER
Original assignee: Rangasamy Naidu Educational Trust
Priority date: 2017-07-22
Filing date: 2018-07-22
Publication date: 2019-01-31

Abstract

A process of hydrogel preparation by gelation of water with only one amino acid, which is Lhistidine, where during gelation for 15 hours, multiple thread-like nano fibers of n- number of metal-organic co-ordinated polymers come together by self-assembling during the stable period and rearrange themselves to entrap water molecules during such rearrangement, within their spaces to form hydrogel. L-histidine stock solution and silver nitrate stock solution in the ration of 1:1 is mixed by constant stirring at 300rpm for 8 to 15 minutes and then gelated as mentioned above.

Description

PROCESS OF PREPARATION OF L-HISTIDINE MOLECULE BASED HYDROGEL BY GELATION OF WATER

BACKGROUND OF THE INVENTION

Proteins are high polymers classified as polyamides. The monomers from which they are derived are called alpha amino carboxylic acids. There are about 23 amino acids found in proteins, a-amino acids are required by human and animal body for growth and metabolism. Lhistidine is one such amino acid. But L-histidine is not synthesized in the human or animal body and hence has to be injected or consumed through L-histidine rich food. L-histidine is required for formation of protein and for various metabolic reactions in the body. L-histidine is especially required for growing young children. It influences several growth-related actions in the body.

Studies of L-histidine in wound healing were also conducted and it revealed that Lhistidine had a prominent role in the regeneration of skin and improve regenerating skinbreaking strength in rats. Recent researchers in bio-technology and related fields focus on tissue engineering and regeneration by creating hybrid materials for tissue engineering and regeneration. The process for producing these types of hybrid materials was carried out by using different types of polymers which having cross-linking properties, as this is essential for gel formation. Example poly (vinyl alcohol) and poly [furmarate-co-(ethylene glycol)], poly (ethylene oxide). These polymers are water soluble and pose some toxicity which is due to its artificial synthesis and biocompatability. However, to prepare these polymers high temperature and pressure are required thus increasing production cost. Whereas is in the present invention hydrogel is prepared from this polymer at room temperature and atmospheric pressure and using only one amino acid namely L-histidine .

Further, the exciting literature teaches that hydrogel is prepared using amino acid with the tag of 9-fluorenylmethyl (Fmoc), Bolaamphiphilic, lipids, peptide form and with polymers like PMMA. In this process of preparation organic solvent are required for preparing hydrogel. Whereas under the present invention process hydrogel is prepared using only distilled water and L-histidine without any organic solvent polymers.

JP2000256365A teaches a water soluble silver complex having anti-microbial and antifungal property obtained by the reaction of silver oxide with 2-pyrrolidone-5- carboxylic acid or L-histidine. The resultant product is a milky white powder 3

PCT/JP2009/000337 is for a liquid composition comprising a silver- L-histidine complex with germicidal activity, in which L-histidine co-ordinate with silver ion along with a hydrocarboxylic acid and polycarboxylic acid and their salts. JP2010198479A pertains to an anti-microbial composition containing a polyalcohol based anti-bacterial agent and silver based anti microbial agent, where a number of polyalcohol based anti-microbial agents have been claimed.

JP2011195582A pertains to a composition having a silver amino acid complex. It also contains hydro-carboxylic acid and at least one member selected from polycarboxylic acid groups. It also contains glucuronic acid including citric acid groups. The said prior arts have certain limitations and cannot be used where gel form of the product is required. They do not teach the process of making hydrogel i.e. as a gel. The ingredients used in the present invention are also different from those mentioned in the prior art. The process of production is also totally different. Further, none of these prior art documents teach the process of making a hydrogel with antimicrobial activity, as a gel, by gelation, using L- histidine and Silver Nitrate and involving a mono valent non toxic alkali metal hydroxide in the specific proportion and manner as described and claimed herein. The products obtained using these prior art documents are either a powder, liquid or solid and not in gel form, and are hence not suitable for domestic and industrial use where the gel form of the product is required and suited and the other forms like powder, liquid or solid are nonsuited. So the present invention addresses these short comings in the prior art and produces a hydrogel in gel form which is the only suitable product for certain applications in domestic and industrial areas such as in treatment with gels in pharma and bio-technology industries.

FIELD OF THE INVENTION

The present invention pertains to the process of production of L-histidine molecule based hydrogel with high antimicrobial activity for domestic, and industrial use, including healthcare industry.

SUMMARY OF THE INVENTION

The present invention pertains to a process for the preparation of hydrogel by gelation of water. Only one amino acid namely L-histidine is used. In this process, L-histidine stock solution and silver nitrate stock solution prepared separately are mixed in equal proportions and then allowed to gelate for 15 hours, when multiple thread like nano fibers are formed. These multiple thread like nano fibers come together by self-assembling during the stable period, rearrange themselves, and entrap water molecules contained some amount of dissolved alkali, during such rearrangements in their spaces to form hydrogel. This hydrogel is prepared from above said method, which has very high antimicrobial property.

DESCRIPTION OF THE INVENTION

L-histidine molecule based hydrogel is prepared by this inventive process, which consists of four steps. The first step pertains to the preparation of L-histidine stock solution. The 2nd step consists of preparation of silver nitrate stock solution. These two steps are performed simultaneously but separately. The 3rd step pertains to the mixing of the two solutions prepared in step 1 and 2. The 4th step is the final step which pertains to gelation from the above process, to obtain a metal organic coordinated compound in gel form. The detailed process is as described below.

Proteins are natural polymers. They are derived from a-amino acids which are monomers that makeup the entire protein structure. Proteins are found in all living cells. They are the principal constituents of skin, muscle, tendons, ligaments, nerve cells, blood cells, enzymes, antibodies and many hormones. There are about 23 aminoacids found in proteins of which some of them are essential for the proper growth of young animals and children. These aminoacids cannot be synthesised by the animal/human body but have to be supported externally as a supplement. L-histidine is one such essential aminoacid. Step-1: The first step of this inventive process involves the dissolution of L-histidine in distilled water in the presence of a monovalent non-toxic alkali metal hydroxide such as sodium hydroxide (NaOH) which provides the alkaline pH medium. An alkaline pH medium is required to increase the formation of carboxylate ion which is required to enhance the dissociation of Lhistidine in distilled water. The solution is then sonicated for 5 minutes. About 0.10 to 2 parts of L-histidine, which are a white color powder is dissolved in 9 to 15 parts of distilled water in the presence of 0.1 to 1 part of sodium hydroxide. Sonication is then done to ensure complete dissolution of L-histidine in distilled water to get a homogeneous solution. Sonication is done for 5 minutes within which time complete dissolution takes place.

Step-2:

In this step silver nitrate stock solution is prepared by dissolving 0.25 to 1 parts of silver nitrate (AgN03) which is a white color crystal, in 6 to 10 parts of water and then sonicating the solution for 5 minutes. Sonication is done to ensure complete dissolution of silver nitrate in distilled water. Within this time of 5 minutes, complete dissolution takes place. Step-3:

The L-histidine solution prepared in step-1 is mixed with the silver nitrate solution prepared in step-2 in the ratio of 1 :1. Constant stirring of the solution for 8 to 15 minutes does the mixing. The rate of stirring is maintained between 200 to 400 rpm during the said time. Here mixing by stirring is done and not sonication as there are no un-dissolved solids. Sodium hydroxide is used to increase the solubility of L-histidine in water. By the addition of sodium hydroxide, the pH of the solution goes up to 7.5 to 8. The solubility of Lhistidine increases at high pH value. Any other monovalent non-toxic alkali metal hydroxides such as potassium hydroxide can also be used instead of sodium hydroxide. Sodium hydroxide is preferred to other said hydroxide because it is cost effective. The use of said alkali within the quantity specified in step-2 plays a critical role in the formation of a hydrogel. If the quantity of said alkali used is less than that specified in step-2 then no gel is formed and the resultant product is only a liquid from. Similarly, if said alkali is used above quantity specified in step-2 then the resultant product will be in solid form and not in gel form.

The presence of hydroxyl ion in the said range in the L-histidine solution increases the solubility of L- histidine in distilled water. Because of which an effective reaction takes place between the L-histidine homogeneous solution and silver nitrate homogeneous solutions when mixed in the ratio of 1 :1 and stirred as mentioned above to obtain a complete reaction between the two solutions. It is for this reason that the said hydroxyl ion such as in said alkali is mixed in step 1 and not in step 3. Further, in view of the complete dissolution property of silver nitrate in water, said alkali is not mixed in step 2. Therefore the hydroxyl ion such as in said alkali plays a critical role in the gel formation only when used in step 1 and not in step 3 or 2. In other words, the addition of the hydroxyl ion in step 1 is to ensure complete solubility of L-histidine before it reacts with silver nitrate in step-3. Therefore, this aspect is a prime factor in the gel formation. The carboxylate ion in L-histidine has a hydrophobic aromatic region and a hydrophilic carboxylate end. Addition of hydroxyl ion such as NaOH leads to the formation of more carboxylate ion, which ensures the complete dissociation of L-histidine in distilled water during sonication In this step, the mixing of the said two solutions leads to the formation of a metal organic coordinated compound, which is a polymer like structure as represented below. This metal organic coordinated compound consists of one silver ion sandwiched between two L- histidine molecules. This acts like a monomer to form a polymer like structure. In the resultant reaction n-number of metal organic coordinated compounds, propagate to form a polymer like structure, where a silver ion is sandwiched between two L-histidine molecules.

Step-4

During this step gelation is done, whereby the polymer like structure obtained in step-3 above is left undistributed for 15 hours at room temperature and normal atmospheric pressure. During this time the polymer like structure undergoes rearrangement of the comprising metal organic coordinated compounds. During this rearrangement, the aromatic region of the Lhistidine molecule in the said compound undergoes π-π stacking in the upper side as well as lower side and also linear attachment on the other side with a silver ion in a sandwiched manner. This is due to the self-assembling of these said compounds leading to the formation of multiple thread-like nano-fibers of thickness varying between 185nm to 195nm. Therefore the selfassembling of the said compounds and the resultant propagation of the thread like nano-fibers entrapping water molecules in between the nanofibre threads during the self assembling of the nano-fiber threads leads to the formation of a hydrogel.

The color of the hydrogel obtained varies from black to transparent depending upon the quantity of L-histidine used in the range of 0.10 to 2 parts. If the quantity of L-histidine used is less than 0.10 parts then no gel is obtained and the resultant product is a turbid colloidal liquid. Similarly, if the quantity of L-histidine used is above 2 parts than the resultant product is a turbid colloidal precipitate and not a gel. Further, if the quantity of L- histidine used is between 0.1 part to 0.24 parts then a black color gel is obtained. Whereas if the quantity of L-histidine used in 0.25 to 2 parts then a transparent hydrogel is obtained. The following Table- 1 illustrates the above;

Varying the quantity of L-histidine:

Table -1

Quantity Observation

S.No Sample Lhistidine Silver NaOH Water After 15 After 15

Code (g) nitrate (g) (ml) min hr

(g)

1. HI 0.1 0.25 0.4 20 Black Black colloidal gel formed

2. H2 0.2 0.25 0.4 20 Colloidal Gel formed

3. H3 0.5 0.25 0.4 20 Colloidal Gel formed

4. H4 0.7 0.25 0.4 20 Colloidal Gel formed

5. H5 1 0.25 0.4 20 Colloidal Gel formed

6. H6 1.5 0.25 0.4 20 Liquid & No gel solid formed

7. H7 2.0 0.25 0.4 20 Liquid & No gel solid formed

8. H8 2.1 0.25 0.4 20 Liquid & No gel solid formed

9. H9 2.5 0.25 0.4 20 Liquid & No gel solid formed

10. H10 3.0 0.25 0.4 20 Liquid & No gel solid formed

Similarly, the use of silver nitrate in the said specifies quantity also affect the hydrogel formation. If no silver nitrate is used then no gel is formed and no antimicrobial activity is obtained. If the quantity of silver nitrate used is less than 0.25 parts then the resultant product is colloidal and not a gel. Similarly, if the quantity of silver nitrate used is more than 1 part then the resultant product is a black colored turbid precipitate and not a gel. The use of silver nitrate between 0.25 to 0.99 parts results in the formation of hydrogel in transparent form. Whereas the use of silver nitrate in 1 part leads to the formation of a black gel. The use of silver nitrate above 1 upto 1.25parts/grams does not give a proper gel. It is a transitional between gel and colloidal 10 and is black in color. The use of silver nitrate above 1.25 parts does not result in gel formation but only a black colloidal phase is obtained. The following table-2 illustrates the above.

Varying the quantity of Silver nitrate:

Table -2

Quantity Observation

S.No Code Silver Lhistidine NaOH Water After 15 After 15 nitrate (g) (g) (ml) min hr

(g)

1. Agl 0.1 0.5 0.4 20 Liquid Colloid phase

2. Ag2 0.25 0.5 0.4 20 Colloidal Gel formed

3. Ag3 0.5 0.5 0.4 20 Colloidal Gel formed 4. Ag4 0.7 0.5 0.4 20 Colloidal Gel formed

5. Ag5 1 0.5 0.4 20 Black Black gel colloidal formed

6. Ag5 1.25 0.5 0.4 20 Black Black solution colloidal phase

From the above it is seen that the use of a specified quantity of L-histidine and silver nitrate directly affects the hydrogel formation. Further, the quantity of sodium hydroxide used in step-1 (0.1 to 1 part) also directly affects the hydrogel formation as already detailed above. The best method of performing the inventive process is as detailed below:

Example 1:

0.5 g of L-histidine with 0.4g of NaOH is added and mixed with 10 ml of distilled water after that this solution is sonicated for 5 min. 0.5 g of silver nitrate is dissolved in 10 ml of distilled water and this solution is sonicated for 5 min. Then these two solutions are mixed together, by constant stirring at 300 rpm for 8 to 15 minutes as described herein shaken well and kept aside without any disturbance, which gives the hydrogel.

Example 2:

0.5 g of L-histidine with 0.4g of NaOH is added and mixed with 10 ml of distilled water after that this solution is sonicated for 5 min. 0.6 g of silver nitrate is dissolved in 10 ml of distilled water and this solution is sonicated for 5 min. Then these two solutions are mixed together, by constant stirring at 300 rpm for 8 to 15 minutes as described herein shaken well and kept aside without any disturbance, which gives the hydrogel.

Example 3:

0.5 g of L-histidine with 0.4g of NaOH is added and mixed with 10 ml of distilled water after that this solution is sonicated for 5 min. 0.7 g of silver nitrate is dissolved in 10 ml of distilled water and this solution is sonicated for 5 min. Then these two solutions are mixed together, by constant stirring at 300 rpm for 8 to 15 minutes as described herein shaken well and kept aside without any disturbance, which gives the hydrogel.

Advantages of the inventive process:

1. No toxic polymers are formed during the entire production process thus rendering it safe for domestic and industrial use including in health care industry.

2. Raw materials used are non-toxic, and

3. The cost of the process is very less thus being economically viable.

Claims

CLAIMS We claim,

1. A process of gelation of water for production of hydrogel comprising of n number of metal- organic coordinated polymers where L-histidine stock solution and silver nitrate stock solution are mixed in the ratio of 1 : 1, by constant stirring at 200 to 400 rpm for 8 to 15 minutes followed by gelation for 15 hours at room temperature and normal atmospheric pressure to obtain multiple thread like nanofibers of entrapped water molecules with dissolved alkali, which during this process come together by self assembling during the stable period and rearrange themselves and entrap water molecules during such rearrangement within their spaces to form hydrogel.

2. A process as in claim 1 where the L-histidine stock solution is obtained by dissolving 0.10 to 2 parts of L-histidine in 9 to 15 parts of distilled water in the presence of 0.1 to 1 part of a monovalent non-toxic alkali metal hydroxide by sonication for 5 minutes.

3. An invention as in claim 1 where 0.1 to 1 part of silver nitrate is dissolved in 6 to 10 parts of distilled water and sonication for 5 minutes.

4. An invention as in claim 1 where the solution obtained in claim 2 and the solution obtained in claim 3 are mixed in the ratio of 1 : 1 by constant stirring at 300rpm for 8 to 15 minutes.

5. An invention as in claim 1 where the n number of metal organic coordinated compound formed by the reaction caused due to the mixing of the solutions mentioned in claim 2 and claim 3 as in claim 4, is kept stable for 15 hours at room temperature and normal atmospheric pressure to obtain multiple thread like nanofibers entrapping water with dissolved alkali, in their spaces.

6. An invention as in claim 1 and 5 where the multiple thread like nano-fibers come together by self assembling during the stable period and rearrange themselves and entrap water molecules during such rearrangement, undergoing gelation within their spaces to form the said compound in gel form as hydrogel.