INTRODUCTION:

Conventional formulation is changing gradually into smart formulation. Metabolic disorders are brought on by longterm highcalorie food intake¹. Metabolic syndrome, is encouraged by the onset of chronic inflammation and oxidative stress². The World Health Organization suggests cutting back on calories. Foods that are high in calories but poor in nutrients should be avoided since they increase the body's calorie intake without providing any beneficial effects³.

Figure 1. Significance of lyophilization

OBJECTIVES:

The antioxidant activity and low calorie profile of Stevia rebaudiana Bertoni. leaves assist the people to move towards a state of finest health. Total phenolic contents and total flavonoid contents of stevia leaves extracts get impacted by the method of drying. Lyophilization removes water without heat to prevent product degradation during drying⁴. Thus product’s quality and stability are enhanced. This prompted us for the development of “A lyophilization method which may yield the stevia leaves extract having profound antioxidant activity⁵.

MATERIALS AND METHODS:

Stevia rebaudiana Bertoni. leaves (family: Asteraceae) were cultivated and authenticated (IU/PHAR/HRB/ 22/03) at Faculty of Pharmacy, Integral University, Lucknow, India.

Extraction and Drying:

Air dried stevia leaves were grinded into a coarse powder. 20g powder in 100mL distilled water was subjected to aqueous extraction by decoction to yield to dark brown extract.

The extract was divided into two parts and dried via lyophilization and evaporation respectively.

The dried extracts were analyzed for chemical constituents⁶ and in-vitro antioxidant activity⁷ initially and after a storage period of 6 months (25±3°C) and relative humidity of 30±5%.

Total Phenolics Content by Folin Ciocalteu Assay:

500 microliter of aqueous extract solution (100μg/mL), 2.0 milliliters of water, 500 microliters of FolinCiocalteu reagent were assorted and incubated at room temperature for 5 minutes. 1000 microliters of sodium carbonate (10%) were mixed and placed in the dark at 22±2°C for 1 hour. Golden yellow colour changed to greenish blue and absorbance noted at 765nm. Standard curve of an aqueous gallic acid (yellowish white, 20 - 100μg/mL) was used todetermine the total phenolics content as equivalents of gallic acid⁸.

Total Flavonoids Content:

500μL aqueous extract solution (100μg/mL), 1500μL methanol, 500μL aluminum chloride (10%), 500μL sodium acetate (1M) mixed with 2 mL water. Mixture was incubated in dark at 22±2°C for 60 minutes and absorbance measured at 415nm. Quercetin (yellow powder in methanol, 20 - 100μg/mL) standard graph was utilized to derive the total flavonoid content of sample as equivalents of quercetin⁹.

Quercetin content in Extracts:

Standard quercetin stock methanolic solution (100 μg/mL) was diluted to obtain solutions (50, 20, 10, 5, 2 ng/mL). 10 ng/mL solution was scanned for the determination of the absorption maxima. Standard curve was obtained using absorbance recorded at 386 nm. The concentration of test solutions was determined from the standard graph¹⁰.

In vitro antioxidant assay of extracts:

Working solutions were prepared at the time of the experiment by dilution of DPPH stock solution (50 μg/mL) with methanol to obtain the absorbance at 517 nm below one. Extract stock methanolic solution (100 μg/mL) was prepared in methanol and diluted to obtain test solution (50, 40, 30, 20, 10μg/mL). Three mL of DPPH solution was taken in a quartz cuvette and absorbance recorded at 517 nm. 1 mL of extract solution was added to DPPH solution, kept in dark for incubation at 22±2°C for 60 minutes and absorbance recorded at 517nm by UV-Visible spectrophotometer. The percentage radical scavenging activity was taken equal to [(Control absorbance – Extract absorbance)/Control absorbance] x 100. IC50 value (Extract solution providing fifty percentage radical scavenging activity) was obtained from the graph¹¹.

Statistical Analysis:

Statistical variation amid groups was measured using GraphPad Prism 8.4.3.686 software. Readings are expressed as mean±SEM for three observations per set. Statistical difference between groups was measured by non parametric paired t-test, where discrepancies are non significant for p ˃ 0.05, significant (*) for p ≤ 0.05, very significant (**) for p ≤ 0.01, highly significant (***) for p ≤ 0.001 on comparing test group to reference /control group.

RESULTS:

Table 1. Drying Parameters

Extract	Duration of Drying	Temperature Utilized	% Loss on Drying
Conventionally dried Extract	10 hours	110 – 115°C	6.48
Lyophilized Extract	10 hours	- (40 – 45)°C	8.83

Table 2. Properties of Dried Extracts

Extract	Colour	Odour	Appearance	% Yield
Conventionally dried Extract	Dark Brown	Characteristic	Sticky Viscous Mass	3.72
Lyophilized Extract	Greenish Brown	Characteristic	Amorphous powder	4.38

Table 3. Total Phenolics Content (mg GAE/g dry weight)

Standard

Test

Alteration

p value (Level of significance)

Conventionally dried Extract

(6.267 ± 0.1943)

Lyophilized Extract

(10.093 ± 0.2473)

+ 3.826

0.000817

(Highly significant)

Table 4. Total Flavonoids Content (mg Quercetin equivalent/g dry weight)

Standard

Test

Alteration

p value

(Level of significance)

Conventionally dried Extract

(08.2643 ± 0.3726)

Lyophilized Extract

(11.1912 ± 0.2491)

+3.1269

0.000927

(Highly significant)

Table 5. 2,2-diphenyl-1-picrylhydrazyl inhibition assays (IC50, µg/mL)

Test

Standard

Alteration

p value

(Level of significance)

Conventionally dried Extract

(36.2105 ± 0.0428)

Ascorbic acid

(15.4834 ± 0.1728)

- 20.7271

0.002241

(Very significant)

Lyophilized Extract

(29.3784 ± 0.3042)

- 13.895

0.000942

(Highly significant)

…Results

8

DRIED EXTRACTS ANALYZED AFTER A STORAGE FOR 6 MONTHS:

Table 6. Properties of Dried Extracts after a storage period of 6 months

Extract	Colour	Odour	Appearance
Conventionally dried Extract	Dark Blackish Brown	Rotten	Sticky Viscous Mass
Lyophilized Extract	Greenish Brown	Characteristic	Amorphous

Table 7. Total Phenolics Content (mg GAE/g dry weight) after a storage period of 6 months

Standard

Test

Alteration

p value

(Level of significance)

Conventionally dried Extract

(4.873 ± 0.2486)

Lyophilized Extract

(9.458 ± 0.1572)

+ 4.585

0.002459

(Very significant)

…Results

9

Table 8. Total Flavonoids Content (mg Quercetin equivalent/g dry weight) after a storage period of 6 months

Standard

Test

Alteration

p value (Level of significance)

Conventionally dried Extract

(06.943 ± 0.4731)

Lyophilized Extract (10.471 ± 0.3491)

+ 3.528

0.000846

(Highly significant)

Table 9. 2,2-diphenyl-1-picrylhydrazyl inhibition assays (IC₅₀, µg/mL) after a storage period of 6 months

Test

Standard

Alteration

p value

(Level of significance)

Conventionally dried Extract

(49.4718 ± 0.1573)

Ascorbic acid

(15.2486 ± 0.12483)

- 34.2232

0.0007128

(Highly significant)

Lyophilized Extract

(35.7182 ± 0.0642)

- 20.4696

0.0008726

(Highly significant)

Table 10. Alteration in quercetin content (mg/g dried extract)

Month

Conventionally dried Extract

(Standard)

Lyophilized Extract

(Test)

Alteration

p value

(Level of significance)

8.173 ± 0.0012

9.341 ± 0.0073

+ 1.168

0.0008103

(Highly significant)

7.053 ± 0.0008

9.128 ± 0.0016

+ 2.075

0.0005406

(Highly significant)

Figure 2. Quercetin Content (ng/mL) in the extracts

Figure 3. Quercetin Content (ng/mL) after a storage of 6 months

Figure 4. Thermal degradation of quercetin

DISCUSSION:

Search of novel molecules^12,13 has been complemented by surveys¹⁴ and investigation¹⁵ of natural products^16,17. Aqueous extract of Stevia leaves was obtained and dried using lyophilization/heat evaporation. Dried extracts were analyzed for chemical constituents and in-vitro antioxidant activity initially and after a storage period of 6 months¹⁸. The lyophilized extract exhibited enhanced chemical constituents and free radical scavenging as compared to heat dried extracts¹⁹. After the storage period of 6 months²⁰Heat treated extract displayed a deviation from the initial values. This indicated decomposition of the active constituents due to the traces of water present in the extract. Quercetin is highly sensitive to temperature^21,22. The thermal degradation begins at low temperature. Products I (2-(3’,4’-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one), II ((3’,4’,5’,6’-tetrahydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one), III ((3’,4’,5’-trihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one) are obtained at 35⁰C. During the thermal treatment (130⁰C, 2 hr), quercetin, I and II are totally degraded and lead to the formation of IV (2-(3,4-dihydroxyphenyl)-2-oxoacetic acid), V (2,4,6-trihydroxybenzoic acid)²³, Non significant changes in the colour, odour, chemical constituents and in-vitro antioxidant are detected in the lyophilized extract and proved its stable nature²⁴. Altering antioxidant activity ofextracts may be a result of variation in hydrolysable constituents and antioxidant network mediated synergistic effects due to the hydrolysable constituents²⁵. Thus lyophilization reduces dose of administration and stability of the formulations^26,27,28 i.e. beneficial in development of user friendly dosage forms^29,30.

Figure 5. Alteration in total phenolics content on storage

Figure 6. Alteration in 2,2-diphenyl-1-picrylhydrazyl inhibition (IC₅₀, µg/mL) on storage

Figure 7. Alteration in total flavonoids content on storage

CONCLUSION:

Lyophilization of the aqueous extract of Stevia rebaudiana leaves results in higher total phenolic contents, total flavonoid contents and superior DPPH inhibition activity. Consequently, utilization of lyophilized extract may be beneficial in the development of stevia based nutraceuticals utilized in free radical mediated disorders.

ACKNOWLEDGMENTS:

Investigators are thankful to Respected Prof. S.W. Akhtar, Chancellor, Integral University, Lucknow; Dr. Syed Nadeem Akhtar, Pro Chancellor, Integral University, Lucknow; Prof. Javed Musarrat, Vice Chancellor, Integral University, Lucknow and Prof. Mohammad Haris Siddiqui, Registrar, Integral University, Lucknow for providing excellent support for carrying out the work (Manuscript communication Number: IU/R&D/2023-MCN0001794). Dr. Shom Prakash Kushwaha is grateful to Shri Parameshwar Foundation, Basti, India for the Shri Parameshwar Foundation - Post Doctoral Fellowship (SPF/2022/APR/0A1).

DECLARATION OF CONFLICT OF INTEREST:

None.

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Received on 22.03.2023 Modified on 10.01.2024

Research J. Pharm. and Tech 2024; 17(10):4717-4721.

DOI: 10.52711/0974-360X.2024.00727