WO2019021143A1

WO2019021143A1 - L- histidine molecule based hydrogel

Info

Publication number: WO2019021143A1
Application number: PCT/IB2018/055448
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

An anti-microbial hydrogel comprising n-number of metal organic coordinated compound with one silver ion sandwiched between two L-histidine molecules, arranged in a particular manner, with π-π stacking in the upper and the lower side with silver ion in a sandwiched in between, in the form of multiple intertwined thread like nanofibers of varying thickness between 180nm to 195nm, and with more than 95% entrapped water molecules and a little dissolved alkali, as a polymer like structure, having antimicrobial activity and having physical and chemical properties as described and claimed herein.

Description

L- HISTIDINE MOLECULE BASED HYDROGEL

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. L- histidine is one such amino acid. But 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 L- histidine had a prominent role in the regeneration of skin and improves regenerating skin- breaking 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 bio- compatability. However, to prepare these polymers high temperature and pressure are required thus increasing production cost. Whereas 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(Poly(methyl methacrylate)). In this process of preparation, organic solvents 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.

PCT/JP2009/000337 is for a liquid composition comprising a silver L-histidine complex with germicidal activity, in which L-histidine co-ordinates 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 non-suited. 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. . FIELD OF THE INVENTION

The present invention pertains to L-histidine molecule based hydrogel, which has a high antimicrobial activity for domestic and industrial use, including in the health care industry.

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 2^nd step consists of preparation of silver nitrate stock solution. These two steps are performed simultaneously but separately. The 3 step pertains to the mixing of the two solutions prepared in step 1 and 2. The 4^th 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 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 L-histidine 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.

Ste -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 at 300 rpm during the said time. Here mixing by stirring is done and not sonications as there are any undissolved 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 L- histidine 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 sodium hydroxide within the quantity specified in step-2 plays a critical role in the formation of a hydrogel. If the quantity of sodium hydroxide used is less than that specified in step-2 then no gel is formed and the resultant product is only a liquid form. Similarly if sodium hydroxide 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 sodium hydroxide is mixed in step 1 and not in step 3. Further, in view of the complete dissolution property of silver nitrate in water, sodium hydroxide is not mixed in step 2. Therefore, the hydroxyl ion such as in sodium hydroxide 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 as 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 L- histidine molecule in the said compound undergoes π-π stacking in the upper side as well as lower side and 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-fibres of thickness varying from 185nm to 195nm. Therefore the self- assembling of the said compound 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 hydrogel in gel form.

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, shaken well and kept aside without any disturbance, for 10 to 15 hrs when hydrogel in gel form is formed. 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, shaken well and kept aside without any disturbance, for 10 to 15 hrs when hydrogel in gel form is formed.

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, shaken well and kept aside without any disturbance, for 10 to 15 hrs when hydrogel in gel form is formed.

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 turbid colloidal precipitate and not a gel. Further, if the quantity of L-histidine used is between 0.1 parts to 0.24 parts then the black color gel is obtained. Whereas if the quantity of L-histidine used in 0.25 to 2 parts then a transparent gel is obtained. The following Table- 1 illustrates the above. Varying the quantity of L-histidine:

Table 1

Similarly, the use of silver nitrate in the said specified quantity also affects 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 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 a transparent form. Whereas the use of silver nitrate in 1 part leads to the formation of a black gel. The following Table-2 illustrates the above. Varying the quantity of Silver nitrate:

Table 2

Similarly, the use of a specified quantity of L-histidine and silver nitrate which affects the hydrogel formation. 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 hydrogel (referred to as, 'the product') thus obtained by the above-said the process has thermal, physical, chemical and biological properties, in particular antimicrobial properties.

1. Thermal reversibility analysis:

About 2.5 ml of the product is taken in a 3 ml cuvette and placed into a sample holder, and then the fluorescence spectrum is measured for all the samples at room temperature. Thereafter, the product is heated to 55°C and the temperature constantly maintained for about 5 minutes. By heating the product from 25 °C to 55 C it is seen that the fluorescence intensity decreases. The said behaviour is observed for all these product. When heating is cut off the sample undergoes atmospheric air cooling slowly to regain its fluorescence intensity, nearing to its original intensity. This property of the product is called as fluorescent thermal reversibility characteristic. The said product has the fluorescence thermal reversibility of 20 to 34 minutes for L-histidine within the specified quantity of 0.25 to 2 parts respectively.

Fluorescence thermal reversibility:

Table 3

The said product has the fluorescence thermal reversibility of 30 to 9 minutes for use of silver nitrate in the specified quantity of 0.25 to 1 parts respectively.

2. Thermo gravimetric Analysis:

This analysis is performed using thermo gravimetric analyzer. This analysis was done to determine the weight loss of the product and to thereby confirm that one of the ingredients is water. The product is heated in the analyzer at, 100 C. When the temperature is gradually increased at a constant rate to incur a thermal reaction, the thermo gravimetric data collected from the thermal reaction is compiled into a graph. The X-axis shows the temperature and Y- axis shows the initial mass of the product. This percentage is graphically represented by a TGA curve as in fig- 1. The data obtained reveals that when the product is heated, it losses about 95% of its weight, during temperature between 40 to 100°C. This is the evaporation range of water, thereby confirming that 95% of the content of the product is water.

3. Thermal Degradation Test:

This test was done in a thermal oven maintained at a temperature of 50°C. Where initially

2g of the product was kept heated at 50°C and weight loss checked at an interval of 1 hour for 20 hours. The weight loss of the product obtained is plotted graphically as in fig-2 whereby it can be seen that the loss of water by evaporation from the product when the temperature is maintained constantly at 50°C is an exponential decay as in fig-2. This test confirms that with constant heating during the said period the product gradually losses weight due to the evaporation of liquid present in it which is in the range of water thus indicating and confirming the presence of water. Further 40-100°C is the evaporation temperature of water. Therefore this test once again confirms the presence of water in the product.

4. Surface analysis for obtaining the surface morphology of the product:

This analysis is done by using atomic force microscopy (AFM) to ascertain the surface morphology of the product. The product with the specific composition of L-histidine 0.5g and silver nitrate 0.25g is used in the AFM imaging studies. Initially a small amount of the product was pickled on the Highly Ordered Pyrolytic Graphite (HOPG) substrate by using a needle. Then this substrate is dried at room temperature for about 48 hours without any interference of dust particles. After drying the substrate is mounted on the AFM sample holder. Then the distance between the substrate and AFM scanning tip is maintained constant during the imaging process. This analysis reveals the surface morphology of the product. The test revealed the 2D and 3D surface morphology of the product as in fig-3 & 4 showing multiple intertwined thread like nanofibers of thickness varying from 180nm to 195nm, on the surface.

5. Field emission scanning electron microscope analysis: This analysis is done to ascertain the nano structure morphology of the product. Surface morphology of the product was analyzed by CARL ZEISS-SIGMA Instrument with an operating voltage of 100 kV. The product were pasted on the sample holder (Aluminum) sputtered with gold nanoparticles. After the sputtering, the product was focused by the filament in a low- pressure environment. Secondary electrons were detected by Bruker-EDS detector to identify the percentage of elements present in the product. The analysis shows multiple intertwined threads like nano fiber of thickness varying between 185nm to 195nm as in fig-5. These nano structural threads were found responsible for the formation of the said product.

6. Rheology test:

This test was done to ascertain the visco elasticity of the product. The changes in shear stress at different shear rate with different temperatures were ascertained and the same is shown graphically in fig-6. A small quantity (~lg) of the product was placed into the sample holder and kept at rest for 5 min before the start of shearing. After that, the shear stress of the product was measured according to the applied shear rate. The change of shear stress was measured by the increase of shear rate. The shear rate was measured from 0-1500/s and the shear stress measured from 0-115 Pa for all the samples at different temperatures. These experiments were done using different quantity of silver nitrate and L-histidine at different temperatures. The results showed that by varying silver nitrate quantity with a constant amount of L-histidine (0.5g) at different temperature as in (Fig.6) the shear stress and shear rate varied. It is found that higher the quantity of silver nitrate used, higher is the shear stress and vice versa. Further, When the test was performed by varying L-histidine quantity with a constant amount of silver nitrate (0.25g) at different temperatures (Fig.7),it was found that higher L- histidine quantity gives lower shear stress with lower shear rate and vice versa. This behaviour indicated the thermal instability of the product with respect to temperature when L-histidine quantity is varied.

Therefore based on the above analysis, it is showed that the shear stress and viscosity of the product with respect to shear rate is graphically shown below;

From figure 6 (a) it is evident that, at 25° C (room temperature), the product with the composition of silver nitrate 0.25 g and L-histidine 0.5g (black color in the graph) showed a shear stress of 24 Pa at the shear rate of 965 per second. The said product showed the viscosity of 273 Pa.S. The product with the composition of silver nitrate 0.5 g and L-histidine 0.5g (red color in the graph) showed a shear stress of 73 Pa at the shear rate of 965 per second. The said product showed the viscosity of 1600 Pa.S. The product with the composition of silver nitrate 0.75 g and L-histidine 0.5g (blue color in the graph) showed the shear stress of 92 Pa at the shear rate of 965 per second. The said product showed the viscosity of 5770 Pa.S. The product with the composition of silver nitrate 1.0 g and L-histidine 0.5g (pink color in the graph) showed the shear stress of 61 Pa at the shear rate of 965 per second. The said product showed the viscosity of 1810 Pa.S.

From figure 6 (b) it is evident that at 37° C ( human physiological body temperature), the product with the composition of silver nitrate 0.25 g and L-histidine 0.5g (black color in the graph) showed the shear stress of 6.43 Pa at the shear rate of 965 per second. The said product showed the viscosity of 479 Pa.S. The product with the composition of silver nitrate 0.5 g and L- histidine 0.5g (red color in the graph) showed the shear stress of 55.1 Pa at the shear rate of 965 per second. The said product showed the viscosity of 944 Pa.S. The product with the composition of silver nitrate 0.75 g and L-histidine 0.5g (blue color in the graph) showed the shear stress of 88.3 Pa at the shear rate of 965 per second. The said product showed the viscosity of 6450 Pa.S. The product with the composition of silver nitrate 1.0 g and L-histidine 0.5g (pink color in the graph) showed the shear stress of 69.8 Pa at the shear rate of 965 per second. The said product showed the viscosity of 1330 Pa.S.

Further, from figure 6 (c) it is evident that at 50° C which corresponds to the wound temperature in humans, animals Eg. diabetic wound), the product with the composition of silver nitrate 0.25 g and L-histidine 0.5g (black color in the graph) showed the shear stress of 2.45 Pa at the shear rate of 965 per second. The said product showed the viscosity of 1610 Pa.S. The product with the composition of silver nitrate 0.5 g and L-histidine 0.5g (red color in the graph) showed the shear stress of 43.8 Pa at the shear rate of 965 per second. The said product showed the viscosity of 373 Pa.S. The product with the composition of silver nitrate 0.75 g and L- histidine 0.5g (blue color in the graph) showed the shear stress of 96.5 Pa at the shear rate of 965 per second. The said product showed the viscosity of 27300 Pa.S. The product with the composition of silver nitrate 1.0 g and L-histidine 0.5g (pink color in the graph) showed the shear stress of 55.6 Pa at the shear rate of 965 per second. The said product showed the viscosity of 19500 Pa.S.

The same rheology analysis was carried out by varying the amount of L-histidine with the constant amount of silver nitrate. The results have been graphically shown in Fig 7.

From figure 7 (a) it is evident that at 25° C, the product with the composition of L- histidine 0.25g and silver nitrate 0.25 g (black color in the graph) showed the shear stress of 36.4 Pa at the shear rate of 965 per second. The said product exhibited the viscosity of 594 Pa.S. The product with the composition of L-histidine 0.5g and silver nitrate 0.25 g (red color in the graph) showed the shear stress of 24 Pa at the shear rate of 965 per second. The said product showed the viscosity of 273 Pa.S. The product with the composition of L-histidine

0.75g and silver nitrate 0.25 g (blue color in the graph) showed the shear stress of 11.5 Pa at the shear rate of 965 per second. The said product showed the viscosity of 500 Pa.S. The product with the composition of L-histidine 1.0 g and silver nitrate 0.25 g (pink color in the graph) showed the shear stress of 14.6 Pa at the shear rate of 965 per second. The said product showed the viscosity of 171 Pa.S.

From figure 7 (b) it is evident that at 37° C, the product with the composition of L- histidine 0.25g and silver nitrate 0.25 g (black color in the graph) showed the shear stress of 26.8 Pa at the shear rate of 965 per second . The said product showed the viscosity of 366 Pa.S. The product with the composition of L-histidine 0.5g and silver nitrate 0.25 g (red color in the graph) showed the shear stress of 6.43 Pa at the shear rate of 965 per second. The said product showed the viscosity of 479 Pa.S. The product with the composition of L-histidine 0.75g and silver nitrate 0.25 g (blue color in the graph) showed the shear stress of 10 Pa at the shear rate of 965 per second. The said product showed the viscosity of 649 Pa.S. The product with the composition of L-histidine 1.0 g and silver nitrate 0.25 g (pink color in the graph) showed the shear stress of 8.33 Pa at the shear rate of 965 per second. The said product showed the viscosity of 287 Pa.S.

From figure 7 (c)it is evident that at 50° C, the product with the composition of L- histidine 0.25g and silver nitrate 0.25 g (black color in the graph) showed the shear stress of 14 Pa at the shear rate of 965 per second . The said product showed the viscosity of 146 Pa.S. The product with the composition of L-histidine 0.5g and silver nitrate 0.25 g (red color in the graph) showed the shear stress of 2.45 Pa at the shear rate of 965 per second. The said product showed the viscosity of 1610 Pa.S The product with the composition of L-histidine 0.75g and silver nitrate 0.25 g (blue color in the graph) showed the shear stress of 5.04 Pa at the shear rate of 965 per second. The said product showed the viscosity of 6000 Pa.S. The product with the composition of L-histidine 1.0 g and silver nitrate 0.25 g (pink color in the graph) showed the shear stress of 4.17 Pa at the shear rate of 965 per second. The said product showed the viscosity of 4220 Pa.S.

7. Cytotoxicity and cell viability test:

This test was done to ascertain the cytotoxicity and cell viability of the product. An in vitro cytotoxicity test was performed for the product as per ISO 10993:5 standard. The culture medium from the L929 cell was replaced with fresh medium. The product at different concentration in triplicate was added on the cells. After incubation at 37± 1° C for 18 h, MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)(lmg/ml) were added in all the wells and incubated for 4h. After incubation, DMSO (Dimethyl sulfoxide) was added in the wells and read at 570 nm using photometer. Cytotoxicity and cell viability were calculated by the following formula. cmird ···· tie&feiti

The fixed grade for cytotoxicity and cell viability studies as per the said ISO standard is as shown in table 4 below:

Table 4:

The cytotoxicity and cell viability experiments for the product for varying silver nitrate quantities and fixed L-histidine quantity with sample code is as shown in Table-5 below;. Amount of the components in the product:

Table 5

S.No Sample Amount of Ag Amount of L- code (g) histidine (g)

1. Al 0.25 0.5

2. A2 0.5 0.5 3. A3 0.75 0.5

4. A4 1.0 0.5

5. HI 0.25 0.25

6. H2 0.25 0.75

7. H3 0.25 1.0

The product sample used for the said test is from 5 to 100 μg. Each serial number corresponding to one sample code which inturn pertains to specific quantity of silver nitrate and L-histidine used as shown in Table-5. Serial number 1 to 4 of Table-5 pertains to analysis using a fixed amount of L-histidine for varying the silver nitrate quantity. Similarly, serial number 5, 6, 7 pertains to analysis using fixed amount of silver nitrate but by varying quantity of L-histidine.

Based on the enumeration in Table-5 sample code for each cytotoxicity, cell viability and cytotoxic reactivity is calculated using the above said formula, where the results as in Table-6 were obtained. The cytotoxic reaction, to the said results based on the ISO standard grades is as furnished in Table-6 below.

Cytotoxicity of Hydrogel

Table 6

Sample particulars Cytotoxicity Cell viability Cytotoxic

Description Cone ^g) (%) (%) Reactivity

code

5 10 90 Slight

25 14 86 Slight

Al

50 15 85 Slight

75 21 29 Mild

100 23 77 Mild

5 2 98 Slight

25 8 92 Slight

50 11 89 Slight

A2

75 22 88 Mild

100 29 81 Mild

A3 5 0 >99 None 25 0 >99 None

50 0 >99 None

75 0 >99 None

100 0 >99 None

5 1 99 None

25 4 96 Slight

A4 50 6 94 Slight

75 6 94 Slight

100 6 94 Mild

5 9 94 Slight

1 25 16 84 Mild

rli

50 32 68 Mild

75 47 53 Mild

100 49 51 Mild

5 10 90 Slight

25 39 61 Mild

H2 50 63 37 Moderate

75 71 29 Moderate

100 74 26 Moderate

5 0 >99 None

25 0 >99 None

H3 50 0 >99 None

75 7 93 Slight

100 38 62 Mild

From the results furnished in Table-6, it is evident that for a product quantity from 5 to 50μg, for sample code A 1 showed the slight cytotoxic reactivity with less than 20% of cytotoxicity and more than 80% of cell viability is seen. For the said product quantity from 75 to 100μg showed the mild cytotoxic reactivity with less than 25 % of cytotoxicity and more than 28% of cell viability is seen.

Similarly for a product quantity from 5 to 50μg, for sample code A2 showed the slight cytotoxic reactivity with less than 15% of cytotoxicity and more than 85% of cell viability is seen. For the said product quantity from 75 to 100μg showed mild cytotoxic reactivity with less than 30 % of cytotoxicity and more than 80% of cell viability is seen. For a product quantity from 5 to 10(^g, for sample code A3, there was no cytotoxic reactivity with 0% of cytotoxicity and above 99% of cell viability.

For a product quantity from 5 to 10(^g, for sample code A4 slight/none cytotoxic reactivity with less than 10% of cytotoxicity and more than 90% of cell viability was observed . For a product quantity from 5 to 50μg, for sample code HI mild cytotoxic reactivity with less than 35% of cytotoxicity and more than 65% of cell viability is observed. The said product quantity from 75 to 100μg showed mild cytotoxic reactivity with less than 45 % of cytotoxicity and more than 50% of cell viability.

For a product quantity from 5 to 50μg, for sample code H2 slight/mild/moderate cytotoxic reactivity with less than 65% of cytotoxicity and more than 70% of cell viability is observed. For the said product from 75 to 100μg showed moderate cytotoxic reactivity with less than 75 % of cytotoxicity and more than 25% of cell viability is seen.

For a product quantity from 5 to 50μg, for sample code H3, no cytotoxic reactivity that is 0% of cytotoxicity and above 99% of cell viability is observed. The said product from 75 to 100μg showed slight/mild cytotoxic reactivity with less than 40 % of cytotoxicity and more than 60% of cell viability.

8. Anti microbial analysis

The antimicrobial analysis reveals that the inventive product has 97% reactivity to Escherichia coli (E.Coli) and Staphylococcus aureus (S. aureus) and 98% for other microbes. The following Table-7 illustrates the same: Antimicrobial activity of the product:

Table 7

A bacterial inoculum of 10⁵ (Colony-forming unit) CFU / ml concentration of 24 h culture was kept as positive control. To the other set of said inoculum 1 ml of product sample was added. The sterile nutrient broth was kept as negative control/ blank. All the above-mentioned sets were incubated at 37°C for 24 hr. Post incubation Optical Density (OD) values were measured at 625 nm. Antibacterial activity was determined using the formula.

% non-viable bacteria= (l-(OD625 Sample/OD625 Control))* 100 When the result as in table 7 was achieved, showing that the poly-microbial activity of the product is between 58 to 98% depending on the quantity of silver nitrate present in the product. Similarly, the antibacterial activity of the product varied from 57 to 97% for the said two bacteria.

Advantages of the product:

1. Easy applicability due to the gel form.

2. The product being in gel form can directly be used in healthcare as an anti-microbial as a disinfectant, wound cleaning and as a curative in view of its anti-microbial properties. 3. The product gel has antimicrobial properties making it useful for varied activities where anti-microbial product that too in gel form is required.

Claims

CLAIMS We claim,

1. A hydrogel which comprises n-number of metal organic coordinated compound in a polymer like structure;

a. with one silver ion sandwiched between two L-histidine molecules,

b. arranged in a particular manner, with π-π stacking in upper side and lower side and liner attachment on the other side with silver ion sandwiched inbetween, c. as multiple inter-twined thread like nanofibers of thickness varying between 180nm to 195nm,

d. with 95% of water molecules containing dissolved alkali, entrapped in between the nanofiber threads,

e. having fluorescence thermal reversibility, shear stress, shear rate and viscocity as described herein, and

f. having more than 99% cell viability and antimicrobial activity as described herein.

2. An invention as in claim 1 where the fluorescence thermal reversibility of the product is 20 to 34 minutes when 0.25 to 2 parts respectively of L-histidine is used as raw material.

3. An invention as in claim 1 where the fluorescence thermal reversibility of the product is 30 to 9 minutes when 0.25 to 1 part respectively of silver nitrate is used as raw material.

4. An invention as in claim 1 where 95 % of its content is water.

An invention as in claim 1, where at 25 °C, the product's shear rate is 965 per second, and a. shear stress 24 Pa, viscosity 273 Pa.S, when 0.5 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

b. shear stress 73 Pa, viscosity 1600 Pa.S, when 0.5 parts of L-Histidine and 0.50 parts of silver nitrate are used as raw materials in preparing the product,

c. shear stress 92 Pa, viscosity 5770 Pa.S, when 0.5 parts of L-Histidine and 0.75 parts of silver nitrate are used as raw materials in preparing the product,

d. shear stress 61 Pa, viscosity 1810 Pa.S, when 0.5 parts of L-Histidine and 1.0 part of silver nitrate are used as raw materials in preparing the product.

An invention as in claim 1, where at 37 °C, the product's shear rate is 965 per second, and a. shear stress 6.43 Pa, viscosity 479 Pa.S, when 0.5 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

b. shear stress 55.1 Pa, viscosity 944 Pa.S, when 0.5 parts of L-Histidine and 0.50 parts of silver nitrate are used as raw materials in preparing the product,

c. shear stress 88.3 Pa, viscosity 6450 Pa.S, when 0.5 parts of L-Histidine and 0.75 parts of silver nitrate are used as raw materials in preparing the product,

d. shear stress 69.8 Pa, viscosity 1330 Pa.S, when 0.5 parts of L-Histidine and 1.0 part of silver nitrate are used as raw materials in preparing the product.

An invention as in claim 1, where at 50 °C, the product's shear rate is 965 per second, and a. shear stress 2.45 Pa, viscosity 1610 Pa.S, when 0.5 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product, b. shear stress 43.8 Pa, viscosity 373 Pa.S, when 0.5 parts of L-Histidine and 0.50 parts of silver nitrate are used as raw materials in preparing the product,

c. shear stress 96.5 Pa, viscosity 27300 Pa.S, when 0.5 parts of L-Histidine and 0.75 parts of silver nitrate are used as raw materials in preparing the product,

d. shear stress 55.6 Pa, viscosity 19500 Pa.S, when 0.5 parts of L-Histidine and 1.0 part of silver nitrate are used as raw materials in preparing the product.

An invention as in claim 1, where at 25 °C, the product's shear rate is 965 per second, and a. shear stress 36.4 Pa, viscosity 594 Pa.S, when 0.25 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

b. shear stress 24 Pa, viscosity 273 Pa.S, when 0.5 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

c. shear stress 11.5 Pa, viscosity 500 Pa.S, when 0.75 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

d. shear stress 14.6 Pa, viscosity 171 Pa.S, when 1.0 part of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product.

An invention as in claim 1, where at 37°C, the product's shear rate is 965 per second, and a. shear stress 26.8 Pa, viscosity 366 Pa.S, when 0.25 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

b. shear stress 6.43 Pa, viscosity 479 Pa.S, when 0.5 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product, c. shear stress 10 Pa, viscosity 649 Pa.S, when 0.75 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

d. shear stress 8.33 Pa, viscosity 287 Pa.S, when 1.0 part of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product.

10. An invention as in claim 1, where at 50°C, the product's shear rate is 965 per second, and a. shear stress 14 Pa, viscosity 146 Pa.S, when 0.25 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

b. shear stress 2.45 Pa, viscosity 1610 Pa.S, when 0.5 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

c. shear stress 5.04 Pa, viscosity 6000 Pa.S, when 0.75 parts of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product,

d. shear stress 4.17 Pa, viscosity 4220 Pa.S, when 1.0 part of L-Histidine and 0.25 parts of silver nitrate are used as raw materials in preparing the product.

11. An invention as in claim 1 where the cytotoxicity of the product is 0% and cell viability is above 99%m when 0.75 parts of silver nitrate and 0.5 L - histidine are used as raw materials in preparing the said product.

12. An invention as in claim 1 where the cytotoxicity of the product is 0% and cell viability is above 99%m when 0.75 parts of silver nitrate and 0.5 L - histidine are used as raw materials in preparing the said product.

13. An invention as in claim 1 having polymicrobial properties is in the range of 58 to 98 %.

14. An invention as in claim 1 having antibacterial activity for Escherichia coli (E.Coli) bacteria is 57 to 97%.

15. An invention as in claim 1 having antibacterial activity for Staphylococcus aureus (S. aureus) is 56 to 97%.