Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant

profile to suggest further study

Section A-Research paper

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex

vivo and antioxidant profile to suggest further study
Adarsh Kumar Pathak*1, Bhuwanendra Singh1
1
Shri Venkateshwara University

Gajraula, Uttar Pradesh, India

*Corresponding Author

154, Newada, Kajgaon, Jaunpur

Uttar Pradesh, India - 222002

drpathak125@gmail.com

9838046617

Abstract

Background: In this article, we constructed a very novel carrier in the form of nanoparticle

which is core shell structure and applied for the delivery of the curcumin to examine the

toxicity and dose profile of the drug. Side by side its controlled release behavior of curcumin

due to use of this carrier i.e. Silica nanoparticle. The nanocomposite structure was prepared

by the simple process the mesoporous silica nanoparticle seen to be higher surface area. It

also provided a large accessible volume for the good absorption of the drug.

Keywords:

Curcumin Loaded Mesoporous Silica Nanoparticle, Nanoparticle, Anti-oxidant

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

1. Background

According to the WHO estimates, India had 32 million diabetic subjects in the year 2000 and

this number would increase to 80 million by the year 2030 [1]. According to International

diabetes federation, India had 50.8 million diabetic adults in the year 2010 and this would

increase to 87 million by the year 2030. The prevalence of diabetes is five times higher in

urban population than in rural area, due to urbanization and high in southern region as

compared to northern and eastern region of the country [2]. Health care economics of

diabetes is less explored discipline in India. The lack of access to health care services,

national welfare schemes and health insurance coverage for diabetes makes the treatment

unaffordable resulting in late diagnosis and increased cost in treatment of diabetes and early

onset of complications. As per the current diabetic estimate of 50.8 million diabetics in India,

the recent study states that the expenditure towards direct and indirect cost incurred would be

$31.9 billion while the allotted national health budget for the fiscal year 2009-2010 was a

meagre of $4.5 billion.

2. Material and Methods

2.1 Material

Cetyltrimethylammonium bromide (CTAB) (ottochemica purity ≥ 99%),

Tetraethylorthosilicate (TEOS) (Alfaaesar chemicals limited purity≥ 98%), Ethanol

(Mercpurity ≥99%), Dionised water, Sodiumhydroxide, Concentrated Hydrochloric acid,

Polaxamer, Sodiumdihydrogen Orthophosphate dehydrate (Fisher scientific Purity ≥98),

Acetonitrile HPLC grade, Methanol (CDH purity 99.5%), Curcuminoid (Gift sample

Karnataka antibiotic)

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

2.2 Method

2.2.1 Preparation method of Silica nanoparticles

The different amount of the silica nanoparticle was prepared by slight change in the process

as reported (Hom et al., 2010) by taking different concentration of CTAB (2.8 gm), 2.0 M

Sodium hydroxide (3.9 mL), and water (100mL) putted it at 80 0C for 30 min. when fully

clear solution is observed then TEOS 3.3 g is rapidly added with help of injection and rapid

stirring nearly 600 rpm after continuous stirring for four minutes there is observance of white

precipitate the temperature maintained at 80oC for 2.5 hours. Then the product was diluted

three times with distilled water nearly (300mL) and filtered simultaneously. Then it is washed

with methanol and water solution in ratio (2:5) two times further its acid extraction was done

with the methanol (100mL) conc. Hydrochloric acid (1mL) mixture and previously prepared

sample nearly 3.3 g at 60oC for 6.5 h using hot plate. The resulted sample was then washed

with water and methanol several time until all the surfactant (CTAB) were removed and then

the solid product was collected by the centrifugation at 2000 rpm (CPR-30 Plus, REMI,

India). The process was done with different concentration of the above used chemicals to

obtain different types of the mesoporous silica nanoparticles MCM-NPs and the given table

shows the concentration of the chemicals and name of sample obtained.

2.2.2 In vitro release study through High performance liquid Chromatography analysis

Liquid chromatograph Shimadzu LC-2010 CHT (M/s Schimadzu Co. Ltd., Chiyoda ku,

Tokyo, Japan) equipped with 4.6 mm × 250 mm Merck HPLC column RP-18, ODS with

particle size 5 micron and PDA detector with 284 nm wavelengths was used for the

determination of in vitro release study. About the 5 mg of the sample MCM-NPs-C-CAR and

5 mg pure CAR was suspended in the 2mL of the 1% sodium lauryl sulphate (SLS) with

phosphate buffer saline having pH 7.4 with cellulose membrane of fixed cut off (MW cut-off

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

5000, Hi-Media) separately. The dialysis bags were placed in 50 mL of the phosphate buffer

saline(7.4pH) solution (sink condition) with magnetic stirring at 75rpm and then 1.5 mL

aliquots was extracted at different time intervals and replaced with fresh buffer solution of

same amount 1.5mL and after 24hrs the sample was analyzed with reverse phase HPLC C18

column [3-4]. The used solvent was carbinol of HPLC grade with water of HPLC grade and

acetonitrile of HPLC grade (50:50:80 v/v) and before using it was filtered with membrane

filter of 0.22µm and flow rate was maintained 1mL/min the experiment was revised double

time for the analysis of variability obtained with each time. The obtained chromatogram was

analyzed further.

2.2.3 Ex vivo study

2.2.3.1 Percentage live red blood cells (RBC) count study

As per suggested protocol percentage live RBC was calculated by subtracting the percent

haemolysis from the total haemolysed sample i.e., haemolysis by distilled water the

percentage of live blood cells was calculated of samples naïve drug CAR, blank MCM-NPs-

C and final loaded drug MCM-NPs-C-CAR. Human blood sample was calculated from the

healthy human 8 mL within the EDTA storage vial and then the blood sample was

centrifuged at 2500 rpm (R-4C DX, REMI, India) and then the RBC was collected and

instantly suspended into normal saline solution (0.9%w/v). Then sample which should be

analyzed prepared of (20ppm) and placed 4mL each sample and then equal amount of RBC

are placed to each samples and let it for the incubation period of the 30 minutes and after

incubation it was centrifuged and supernatant was analyzed as it is by ultraviolet visible

spectrophotometer (Cary series-100, UV-visible spectrophotometer, AgilentTech.) [5,6,7].

RBCs in distilled water considered as 100 percent haemolysis or no live RBCs left and the

absorbance of distilled water is taken as a reference for other samples

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

% RBC Live = % haemolysis DW - %haemolysis of Samples

For % Haemolysis = [as ÷a100]×100

Where,

as=absorbance of sample

a100= absorbance of distilled water

In the above report time dependent percent RBCs live are counted i.e., 30 minutes,

12hoursand 24hrs respectively.

2.2.3.2 Protein binding study

A Solution of BSA was prepared (2% w/w) of PBS saline of pH 7.4 for the study. CAR

(1mg) and MCM-NPs-C-CAR (equivalent to 1 mg drug) were added to 1 mL of BSA

solution and packed into dialysis bags, separately. These dialysis bags were dipped into 20

mL of PBS of pH 7.4 under stirring at 37±1 °C. Samples were analyzed

spectrophotometrically which were taken at the intervals of 1 h, 2 h, 3 h, 4 h, 5 h and 6 h. The

calculations of percent protein drug binding were performed, as per the equation [8-9].

2.2.4 In-Vitro Antioxidant Study by DPPH Method

DPPH free radical scavenging assay were used for determining antioxidant activity of

HAF/HLO as mentioned by Nithianitham et al and Zuraini et al with some modifications.

10mg/mL stock solution of HAF/HLO was prepared. Different dilution of HAF /HLO (20

μLto 100 μL) was taken and was diluted up to 1 mL with methanol. Then 1mL of each

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

dilution was added with 2 mL of 0.004% (w/v) DPPH solution. This mixture was vortexed,

kept inside the incubator for 30 minutes in dark, and spectrophotometric absorbance was

measured at 425 nm. 80% (v/v) methanol was used as blank solution. Ascorbic acid was used

as the standard compound for comparative study. All measurements were done in triplicate.

Following formula was used to calculate DPPH free radical scavenging activity:

Scavenging activity (%) =

Here, control =0.004 % (w/v) DPPH solution; sample = HAF/HLO

The result was reported as IC50 value and ascorbic acid equivalents (AAE, mg/g) of

HAF/HLO

2.2.5 In-vitro-release study by HPLC analysis

It is observed that the almost 95 percent of the drug release in 6hrs and the MCM-NPs-C-

CAR favours sustained release profile and it is seen that almost 75 percent of the drug

released in the media take almost 12hrs and after 20hrs the release pattern was seen constant.

The in vitro drug release is explained (Fig-1) by the first order kinetics and (r 2=0.9132)

showing the good class of release of the drug.

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

Release Profile of CAR and MCM-NPs-C-CAR

120

100
Percent Release

80

60

40

20

0
0 5 10 15 20 25

Time(hrs)

Figure 1 Release Profile of CAR and MCM-NPs-C-CAR

2.2.6 Ex-vivo study

2.2.6.1 Percentage live RBCs count

The live RBCs are counted in percent for the confirmation of the formulation that what is the

haemolytic percentage and result of distilled water, normal saline, CAR, MCM-NPs-C and

the final formulation MCM-NPs-C-CAR is giving the result 0%,99.14%,98.63%,99.66%and

98.8% respectively (Fig-2). Distilled water is taken as the reference sample with no live

RBCs and all the formulations are dissolved in the normal saline and volume was also make

up with the same saline. Therefore, the above stated result support the good behaviour in

reference to the haemolytic toxicity. The result also suggest that the silica Nano-formulations

are very fortunate means for the targeted drug delivery.

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

Figure 2 Percentage of Live RBCs Count

2.2.6.2 Protein binding study

The biding efficiency of the pure drug CAR and the drug loaded silica nanoparticle MCM-

NP-C-CAR at time interval of 0 to 6 hrs and it is observed to be (74.54%) in case of the pure

drug and (62.31%) observed in the drug loaded nanoparticle (Fig-3). It is confirming with this

data that the protein binding is lesser in drug loaded silica nanoparticles which reveals that

the prepared formulation has very good penetration power to the cell. Therefore, the

significant difference is observed between the CAR and MCM-NP-C-CAR.

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

Figure 3 Percentage of Protein Binding

Antioxidants have the ability to scavenge free radicals in the human body and have been

suggested to contribute to the protective effect of plant-based foods on diseases such as

cardiovascular disease, cancer, and type 2 diabetes. However, evidence from supplementation

studies using various antioxidants, including vitamin C, vitamin E, carotenoids, zinc, or

selenium, does not support the hypothesis that antioxidants decrease risk of these diseases.

Intervention studies highlight a lack of information on the safety of sustained intakes of

moderate to high doses of micronutrient supplements and suggest that long-term harm cannot

be ruled out, particularly in smokers. The observed values of HAF’s scavenging activity at

different concentrations were depicted as the plotted graph. IC50value of HAF and ascorbic

acid were calculated as 114.24 μg/mL and 26.86 μg/mL respectively.

Values represent the mean ± SEM (n = 3);

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Eur. Chem. Bull. 2023,12(10), 6457-6467

Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

3. Conclusions

The proposed plan concludes that the prepared formulation with nano drug delivery with

curcumin shows high efficiency to combat with the diseases and it is also intervein with all

the activities the invitro release of the nanoparticles shows the sustained release pattern and

percent hemolysis is very low so it is very good to administer the formulation and also patient

compliance. There is also the lower protein binding platform and their antioxidant profile

suggestive to the formulation for recommendation and they are highly suggestive to the

patient and further recommended to animal study model.

Abbreviations

MCM-NPS-C Mesoporous Silica Nanoparticles

MCM-NPs-C-CUR Curcumin loaded Mesoporous Silica Nanoparticles

UV Ultra violet

FTIR Fourier transform infrared analysis

SEM scanning electron microscopy

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Preparation of curcumin-loaded mesoporous silica and its evaluation of ex vivo and antioxidant
profile to suggest further study

Section A-Research paper

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