PREPARATION OF PECTIN/5-AZACYTIDINE/CHLOROGENIC ACID NANOCOMPOSITE AND ITS USE AS CARDIAC TISSUE REPAIR SUPPLEMENTS

Abstract

The invention is focused on the synthesis of cardioprotective nanocomposite supplements that contain pectin, 5-azacytidine, and chlorogenic acid. The pectin was boiled and then crosslinked with calcium chloride in which 0.5-1.5 mg of each component was added separately. The solution was centrifuged for 20 min, while the pellet was further diluted in water. The nanocomposite prepared was found to protects the cardiac tissue against injury and aid in repair through tissue regeneration. This is characterized as a homogeneous preparation that indicates excellent stability, good entrapment efficiency, and hemobiocompatibility. The nanocomposite increased the antioxidant levels and reduced the oxidative stress, which therefore benefits in repairing of cardiac tissue.

Specification

Description:SUMMARY OF THE INVENTION
Out of various amounts of pectin (5 - 50 mg) and calcium chloride (10 mM) crosslinked , pectin at 25 mg crosslinked with calcium chloride was found to be optimal and was used for entrapping 5 azacytidine, chlorogenic acid or both.
For instance, pectin crosslinked with calcium chloride was labelled as 1, pectin crosslinked with calcium chloride and entrapped with 5 azacytidine was labelled as 2, pectin crosslinked with calcium chloride and entrapped with chlorogenic acid was labelled as 3, pectin crosslinked with calcium chloride and entrapped with 5 azacytidine and chlorogenic acid was labelled as 4.
To understand the release of drug when there is an increased intracellular concentration of calcium and the formation of gel nanocomposites of pectin crosslinked with calcium chloride and entrapped with 5 azacytidine and chlorogenic acid, here 100 mM concentration of calcium chloride was used and these nanocomposite gels were labelled as 5.
Composite labelled 1 had the hydrodynamic diameter of 461.4 d.nm and the zeta potential values were -13.9 mV.
Composite labelled 2, 3, 4 and 5 had the hydrodynamic diameter of 141.8 d.nm, 278.2 d.nm, 864.8 d.nm, 14.82 d.nm respectively and zeta potential of -16.1 mV, -18.8 mV, -18.8 mV, -3.11 mV respectively.
Lesser hydrodynamic diameter (d.nm) and zeta potential (mV) for composite labelled 5 indicates formation of composite gel due to increased uptake of calcium in the composite.
Composites labelled 1 to 5, also chlorogenic acid (labelled 6) and 5 azacytidine (labelled 7), 5 azacytidine and chlorogenic acid (labelled 8) were characterized by UV-Vis spectroscopy by shift in the spectra, increase or decrease in spectra and FTIR by changes in the molecular bonding vibrations.
Loading efficiency of 5 azacytidine in pectin nanocomposite (label 2) was found to be 85.35%%, loading efficiency of chlorogenic acid in pectin nanocomposite (label 3) was found to be 68.9%, loading efficiency of 5 azacytidine and chlorogenic acid in pectin nanocomposite (label 4) was found to be 84.82 % and 53.2% respectively, and loading efficiency of 5 azacytidine and chlorogenic acid in pectin nanocomposite gel (label 5) was found to be 84.73 % and 61.75% as determined by UV-Vis spectroscopy.
Label 2 to 6 was found to be hemobiocompatible as against other comonents.
Drug release studies as determined by chlorogenic acid release indicated that when there is an increase in the calcium concentration in nanocomposite gel, there was sustained release of chlorogenic acid.
DPPH scavenging activity of pectin, label 1 to 8 was found to be 58-88 %.
Ex vivo studies indicated the protective effect of nanocomposites against cardiac injury.
STATEMENT OF THE INVENTION
As a result, the current invention is primarily concerned with the use of 5 azacytidine and chlorogenic acid entrapped in pectin as supplements for cardiac injury, three crucial characteristics of this innovation are: a) Enhanced Biocompatibility: The use of pectin and chlorogenic acid in the nanocomposite formulation ensures that the nanocomposites are compatible with biological tissues and there is no significant toxicity to the tissue, b) Improved DPPH scavenging activity: the incorporation of pectin and chlorogenic acid provides significant free radical scavenging activity, Also, when there is an increase in the calcium concentration due to cardiac injury, the calcium will be entrapped in the nanocomposite and form gel and provide sustained and dual release of drugs from the composite gel, c) Protection against cardiac tissue injury, the ability of the nanocomposites to counter oxidative stress by nanocomposites can be an advantage to use these nanocomposites labelled as 4 as cardiac protective supplements.
Prepared pectin crosslinked with calcium chloride and entrapped with 5 azacytidine and chlorogenic acid nanocomposite labelled as 2 remained as a nanocomposite colloidal formation suitable for injecting and prepared pectin crosslinked with calcium chloride and entrapped with 5 azacytidine and chlorogenic acid labelled as 5 remained as a nanocomposite gel indicating that at the site of injury the nanocomposite s can form nanocomposite gels
In yet another embodiment of the present invention, pectin crosslinked with calcium chloride and entrapped with 5 azacytidine and chlorogenic acid nanocomposite labelled as 2 was characterized by UV-Vis spectroscopy and FTIR
In yet another embodiment of the present invention, pectin crosslinked with calcium chloride and entrapped with 5 azacytidine and chlorogenic acid nanocomposite labelled as 4 entrapment efficiency was 84.82 % for 5 azacytidine and 53.2% for chlorogenic acid.
In yet another embodiment of the present invention, DPPH scavenging activity of label 4 when compared to same concentration of 5 azacytidine or chlorogenic acid or both.
In yet another embodiment of the present invention, composite labeled 4 did not indicate any toxicity to erythrocytes indicating hemobiocompatibility.
In yet another embodiment of the present invention, composite labeled 4 protected against cardiac tissue injury, ex vivo.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The following examples are presented merely for the purpose of illustrative purposes and should not be interpreted as restricting the scope or applicability of the current invention. The invention is illustrated through these examples.
Example-1 5- 50 mg of pectin was dissolved in water of 6 mL heated at 50 oC and stirred continuously till it dissolved. The pH of the solution was maintained at 4.8 and to this 10 - 100 mM of calcium chloride was added and stirred. To this under sonication 0.75 - 1.5 mg of chlorogenic acid or 0.75 - 1.5 mg 5 azacytidine or both chlorogenic acid and 5 azacytidine was added. Total volume was 10 mL. all the samples were centrifuged at 3000 rpm at for 20 mins. The pellet and the supernatant were separated and stored. The pellet was diluted and made up to 10 mL using water and stored at 2-8 °C. To understand the shift in the peak of the formulated nanocomposite, UV-Vis spectrophotometric analysis was carried out and the spectra were recorded. The hydrodynamic diameter was done to understand the size distribution and zeta potential measurements were carried out to understand the degree of stability and the charge of the molecules that are bound on the surface. FT-IR studies on nanocomposites and drugs alone was done
Example-2 Hemolysis assay was carried out for all the composites and the components labelled 1 - 8 and the percentage of hemolysis was calculated by comparing with the erythrocytes incubated with SDS and only with medium. The DPPH free radical scavenging method was used to determine the antioxidant activity of nanocomposites and components labelled 1 to 8. A DPPH solution was added to a sample containing EGCG or nanogels. The absorbance at 517 nm was measured after which the percentage of DPPH scavenging activity was determined by a UV-Visible spectrophotometer. Drug release studies using dialysis tubing method was done by monitoring chlorogenic acid release and recording the absorbance using UV-Vis spectroscopy. The drug release studies was done for composites and composites gel label 4 and label 5.
Example-3 Ex vivo studies- In this, chicken heart was used for the evaluation of the protective effects of nanogels and the antioxidant potential of nanogels. The heart tissue was obtained and cut into smaller pieces, before being treated with a solution containing copper ascorbate. This solution, in its production, involved the use of PBS-sucrose buffer, which was then incubated for three hours. To ascertain the degree at which nanogels had an influence on the cardiac tissue, heart tissue was segmented into 8 groups and incubated with nanocomposites (Label 1-4) or components (label 6, 7 and 8) and compared with control (labelled as c) and induced alone (labelled as I) groups. The process included harvesting and cleaning the tissue of the heart, homogenizing it, and quantifying how much protein it contained. Measurings of the superoxide dismutase (U/mg), catalase (U/mg), glutathione peroxidase (U/mg), and LPO (nmol MDA/g) were performed with the aid of the tissue homogenate. Various procedures were used in conducting experiments for determining the catalase, glutathione peroxidase, and lipid peroxidation activities.
ADVANTAGES OF THE INVENTION
A simple method to entrap 5 azacytidine and chlorogenic acid in pectin nanocomposite and the ability to form gel is controlled by the presence of calcium concentration.
The main advantage of such nanocomposite gel is that it can release chlorogenic acid in a sustained manner
The presence of pectin in nanocomposite can aid in the formation of gel
The presence of 5 azacytidine in gel can aid in the differentiation of stem cells to cardiomyocytes and aiding tissue regeneration
The presence of 5 chlorogenic acid in the nanocomposite can aid in prevention of oxidative stress and protect the heart (cardioprotective) against cardiac injury and aid in cardiac tissue repair.
The pectin nanocomposite with 5 azacytidine and chlorogenic acid are biocompatible in nature.
The pectin nanocomposite with 5 azacytidine and chlorogenic acid size is in the nanometer region.
EXPLANATION OF FIGURES
Figure 1 - Fig1A represents the formation of nanocomposites and the encapsulation process of drug and the labelled counterpart of each group that has formed pectin nanocomposites with or without drug is represented in the vials and the gelability of the pectin nanocomposites at higher concentration is also represented in the figure 1A. Figure 1B indicates the UV-spectra of all the prepared nanocomposites and the shift indicates the presence of 5 -AZA or CA or both. Figure 1C represents the molecular changes of these nanocomposites when compared to the single component alone, thus indicating the structural changes with or without drugs. Figure 1D indicates the hydrodynamic diameter of 1 - 461.4 nm, 2- 141.8 nm, 3- 278.9 nm, 4 - 864.8 nm, 5- 141.82 nm and figure 1E indicates the zeta potential of 1 - -13.5 mV, 2- -16.1 mV, 3- -18.8 mV, 4 - -18.8 mV, 5- -3.11 mV (Example 1).
Figure 2. Antioxidant activity as determined by DPPH assay is represented in Figure 2B and is compared with the control as shown for all components for pectin and for all components labelled 1 to 8 and the percentage of antioxidant activity of pectin and 1 to 8 is found to be 58%, 62%, 71%, 79%, 83%, 82%, 84%, 85%, and 88% respectively and figure 2C indicates the drug release pattern of CA which is in a sustained manner when there is an increase in the intracellular concentration of calcium chloride. Figure 2D indicates hemolysis of pectin and for the groups 1 to 8, it can be seen that the group 2 to 6 did not have any hemolytic activity and the representative images are represented in the 2E (Example 2)
Figure 3. To further evaluate the cardio protective effect of the prepared nanocomposite and the counter component ex vivo studies on chicken cardiac heart tissue was conducted as represented in the figure 3A and the level of SOD, CAT, GPx and LPO are represented in figure 3B, figure 3C, figure 3D and figure 3E, respectively. From the figure it can be seen that the label 4 has got an very good antioxidant as well as free radical scavenging activity by an increase in SOD, CAT, GPx even after injury and decrease in LPO even after injury when treated with 4 (example 3).
, Claims:I/We Claim:
1. The pectin with 5 azacytidine and chlorogenic acid nanocomposite (label 4) for cardioprotective supplements comprises following characteristics:-
i) nanocomposite made of pectin with 5 azacytidine and chlorogenic acid was found to be in an homogeneous material with good stability as determined by the zetapotential measurement (-18.8 mV).
ii) nanocomposite made of pectin with 5 azacytidine and chlorogenic acid was found to be in nanometer range and the hydrodynamic diameter as determined by the DLS is 864.8 nm.
iii) nanocomposite made of pectin with 5 azacytidine and chlorogenic acid had entrapment efficiency of 84.82 % for 5 azacytidine and 53.2%for chlorogenic acid.
iv) nanocomposite made of pectin with 5 azacytidine and chlorogenic acid released chlorogenic acid in a sustained manner and was hemobiocompatible with no hemolysis monitored.
v) nanocomposite made of pectin with 5 azacytidine and chlorogenic acid improved antioxidant status DPPH scavenging activity (83%), reduced oxidative stress thus aiding in cardiac tissue repair.
2. Pectin with 5 azacytidine and chlorogenic acid nanocomposite (label 4) as claimed in claim 1, method of preparation comprising of steps:-
i) A process involving dissolving 5 - 50 mg of pectin that is heated at 50 oC and stirred till pectin dissolves.
ii) To prepare pectin nanocomposite containing 5 azacytidine and chlorogenic acid, first pectin is crosslinked with calcium chloride (10- 100 mM) and the solution is stirred.
iii) Later the solution is sonicated and under sonication 0.75 - 1.5 mg of 5 azacytidine and chlorogenic acid is added. The solution to separate pellet that is nanocomposite was centrifuged at 3000 rpm for 20 minutes, and supernatant separated.
iv) Following, the pellet was diluted and made up to 10 mL using water.

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