FOL-02

FOL-2 Method Title: METHOD FOR THE ESTIMATION OF TOTAL PHENOLIC CONTENT USING THE FOLIN- C ASSAY Submitted by: Jyotish Srivastava Submitted by Email: j.srivastava@omniactives.com Enclosures: 1 Submitter notes: Attach methodology for "METHOD FOR THE ESTIMATION OF TOTAL PHENOLIC CONTENT USING THE FOLIN- C ASSAY" along with the Validation data on excel sheet.

METHOD FOR THE ESTIMATION OF TOTAL PHENOLIC CONTENT USING THE FOLIN- C ASSAY

1.0 Title: Estimation of Total Phenolic content in Salacia Species using the Folin-C (Folin Ciocalteu) Assay.

2.0 Scope: This method is used for Estimation of Total Phenolic content in Salacia Species using the Folin-Ciocalteu Assay.

3.0 Principle: Phenolics include simple phenols, phenolic acids (benzoic and cinnamic acid derivatives), coumarins, flavonoids, stilbenes, hydrolyzable and condensed tannins, lignans, and lignins. These compounds are among the most widely occurring secondary metabolites in the plant kingdom, acting mainly as phytoalexins, attractants for pollinators, and contributors to plant pigmentation, antioxidants, and protective agents against UV light, among others. Although quantitative determination of polyphenols is hampered by their structural complexity and diversity, several methods have been used to determine polyphenols in plant extracts. Assuming that quantification of individual polyphenols does not adequately reveal the proportion of polymeric procyanidins, and then spectrophotometry in the ultraviolet region may be a useful tool to help resolve this problem. Colorimetric reactions are widely used in the UV/VIS spectrophotometric method, which is easy to perform, rapid, reproducible, reliable and applicable in routine laboratory use, and low-cost. However, it is important that colorimetric assay need to use a reference substance, and then this method measures the total concentration of phenolic hydroxyl groups in the plant extract against reference standard. Polyphenols in plant extracts react with specific redox reagents (Folin-Ciocalteu’s phenol reagent) to form a blue complex that can be quantified by visible-light spectrophotometry. The reaction forms a blue chromophore constituted by a phosphotungstic- phosphor- molybdenum complex, where the maximum absorption of the chromophores depends on the alkaline solution and the concentration of phenolic compounds.The reaction generally provides accurate and specific data for several groups of phenolic compounds.

4.0 Reagents:

Gallic acid >99% (Make: Sigma)

•

• Folin-Ciocalteu’s phenol reagent (Make: Merck)

Sodium Carbonate (Make: Merck)

•

• Methanol HPLC Grade (Make: Rankem)

HPLC grade Water (Make: Rankem)

•

5.0 Apparatus:

• UV-Visible Spectrophotometer (Make: Shimadzu UV-1800)

Ultrasonicator (Make: PCI analytics)

•

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METHOD FOR THE ESTIMATION OF TOTAL PHENOLIC CONTENT USING THE FOLIN- C ASSAY

Water Bath (Make: PCI analytics)

•

• Weighing Balance (Make: Sartorius BSA224S-CW)

• 100 mL Volumetric Flask (Make: Borosil Class-A)

• 50 mL Volumetric Flask (Make: Borosil Class-A)

• 25 mL Volumetric Flask (Make: Borosil Class-A)

• 15 mL Test tubes (Make: Borosil Class-A)

• 10mL,5 mL, 2mL, 1mL Pipettes (Make: Borosil Class-A)

6.0 Calibration: All Equipments and glassware’s used for performing the analysis should be calibrated.

7.0 Procedure:

7.1. Preparation of reagents:

• 10% Folin-Ciocalteu’s Phenol reagent: Pipette out 10mL of Folin-Ciocalteu’s phenol reagent in 100 mL Volumetric Flask and Make up to volume with water and Sonicate & stored under dark. This solution can be stored for 15 days.

• 7.5% Sodium carbonate Solution: Weigh accurately 7.5gm of sodium carbonate in 100mL volumetric flask , Add 50mL of Water , Shake and Sonicate till it dissolve , then make up the volume up to the mark with water.

7.2. Preparation of Blank:

• Take 1mL of Water in to 15mL test tube and add 5 ml of 10%Folin-Ciocalteu’s phenol reagent and add 4 mL of 7.5% Sodium Carbonate solution. Keep the test tubes in dark condition for 1hour.This blank solution should be used for auto zeroing the UV-Visible spectrophotometer at 765nm.

7.3. Preparation of Standard Solution:

• Weigh accurately 50mg of Gallic acid in to a 50mL volumetric Flask, add 25mL of Methanol and Sonicate for 5 min & make up the volume up to the mark with methanol . (Stock –A Concentartion1000ppm).

Pipette out solution from stock-A and prepare a Four different concentration ranges from 5-100ppm and plot the linearity graph. The R 2 value (Co-relation coefficient) should be greater than 0.99.

7.4. Preparation of Sample:

• Raw Material Solution : Weigh accurately 2.00gm of Powdered sample passed through 40mesh (425µm) in Round bottom flask and add about 50mL of Methanol and Sonicate for 10min, followed by Reflux in water bath at 80 0 C for 5min using Air condenser. Decent the upper layer in to 100mL Volumetric Flask. Again add 40mL of methanol in round bottom flask & Reflux in water

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METHOD FOR THE ESTIMATION OF TOTAL PHENOLIC CONTENT USING THE FOLIN- C ASSAY

bath at 80 0 C for 5min. Decent the supernant in to 100mL volumetric Flask containing previous solution. Allow to cool the solution and make up to the mark with methanol (Solution-A). Pipette out 2.0mL of Solution-A and transfer it into 25mL volumetric flask Make up to the mark with methanol (Solution-B) Further pipette out 1mL from Solution-B in to a 15mL test tubes and add 5 ml of Folin-Ciocalteu’s phenol reagent followed by 4 mL of 7.5% Sodium Carbonate solution. Close & Keep the test tubes in dark condition up to 1hour for colour development. Take the absorbance at 765nm against Blank using UV-Visible spectrophotometer. • Extract containing 15% Polyphenols: Weigh accurately 150-200mg of Sample extract in 50mL volumetric flask and add 25mL of methanol , Sonicate for 10min and make up to the mark with Methanol(Solution-A) Pipette out 2.0mL of Solution-A and transfer it into 25mL volumetric flask. Make up to the mark with methanol (Solution-B). Further pipette out 1mL from Solution-B in to a 15mL test tubes and add 5 ml of Folin-Ciocalteu’s phenol reagent followed by 4 mL of 7.5% Sodium Carbonate solution. Keep the test tubes in dark condition for 1hour for colour development. Take the absorbance at 765nm against Blank using UV- visible spectrophotometer.

8.0 Calculation: Total Phenolic content in Salacia Species is calculated by below mentioned formula,

A Sample

D standard

% Purity of Standard

%Total phenolic content = ---------- x ----------- x A Standard D sample

Where: A Sample A Standard D sample D standard

= Absorbance of sample at 765nm = Absorbance of standard at 765nm = Dilution of sample in mg per mL = Dilution of standard in mg per mL

9.0 Controls (Precaution):

• Prepared Folin-Ciocalteu’s phenol reagent can be used for 15 days if it stored under dark condition.

• Sodium carbonate solution should be prepared freshly and used.

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METHOD FOR THE ESTIMATION OF TOTAL PHENOLIC CONTENT USING THE FOLIN- C ASSAY

Results of validation: Following test were conducted to verify the performance of the method as per the requirement of AOAC SMPR 2015.009.

10.0

10.1. Analytical Range: Analytical range was demonstrated on reference standard. Different concentrations ranging from 5-100ppm were prepared and measure the absorbance.

Sr.no.

Concentration (ppm)

Absorbance

1

5

0.101

2

25

0.325

3

50

0.651

4

100

1.273

Calibration Curve of Gallic acid

1.4

1.2

1

y = 0.012x + 0.028 R² = 0.999

0.8

0.6

Abosrabance

0.4 Absorbance

0.2

0

0

50

100

150

Concentartion in ppm

Conclusion: The Co-relation coefficient (R 2 ) of 0.999 confirms that the concentrations ranges are linear and follows the Beers lambert’s laws.

10.2. Limit of Quantification (LOQ): Based on Linearity study a concentration of 5 ppm is found to be the LOQ. Six different preparation of 5ppm were analyzed and the relative standard deviation (RSD) is within 10%. Confirming LOQ to be 5ppm 10.3. Recovery: Recovery study was conducting by adding known amount (80 to 120% of working range) of standard in both the raw material & extract. The samples were analyzed using the methodology mentioned above (7.4). The recovery is within 80 to 110%.

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METHOD FOR THE ESTIMATION OF TOTAL PHENOLIC CONTENT USING THE FOLIN- C ASSAY

For Raw material:

Range

% Accuracy

102

80%

99

100% 120%

104

For extract:

Range

% Accuracy

98

80%

105 101

100% 120%

10.4. Repeatability: Repeatability study of Raw material and Extract were carried out by preparing and analyzing same sample at three different times. The limit of % RSD should be in the range of ≤9%.

For Raw material:

Sample

%Phenols

Average

%RSD

OA150152-Trial-1 OA150152- Trial-2 OA150152- Trial-3

2.46 2.47 2.39

2.44

1.85

For Extract:

Sample

%Phenols

Average

%RSD

SREE-00-080202002- Trial -1 SREE-00-080202002- Trial -2 SREE-00-080202002- Trial -3

22.77 22.16 22.26

22.40

1.47

Conclusion: The %RSD is in the range of 1.78 & 1.47 respectively for Raw material & extract.

10.5. Stability of the extract: Stability study of the Salacia extract was conducted both at real time (25° C±2° C and 60% RH±5% RH) and accelerated (40° C±2° C and 75% RH±5% RH). The extract is found to be stable confirming the ruggedness of the methodology (Different day & Different analysis).

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Estimetion of Total Phenolic content using the Folin-C assay

Validation Data Of Polyphenol content by Folin Ciocalteu's reagent in Salacia

Analytical Range:

1)

Gallic acid Concentartion in ppm Absorbance

Sr.No.

1 2 3 4

5

0.101 0.325 0.651 1.273

25 50

100

Calibration curve of Gallic acid

0 0.2 0.4 0.6 0.8 1 1.2 1.4

1.273

y = 0.0124x + 0.0285 R² = 0.9996

0.651

0.325

Absorabance

0.101

0

20

40

60

80

100

120

Concentartion in ppm

Limit of Quantification:

2)

Weight Of Standard (mg)

Absorbance of 5ppm concentration

50.6 50.2 51.2 49.5 50.4 50.2

0.101 0.109 0.118 0.101 0.102 0.098

Average

50.35

0.10 0.01 7.07

Standard Deviation

0.56 1.11

% RSD

Recovery For Raw material (B.No-OA150152) Range (%)

3)

Theoratical Value

Practical Value

% Accuracy

80

1.96 2.46 2.95

2.00 2.44 3.08

102.04

100 120

99.19

104.41

For Extract (B.No-SREE-00-080202002) Range (%)

Theoratical Value

Practical Value

% Accuracy

80

17.60 22.26 25.90

17.23 23.45 26.22

97.90

100 120

105.35 101.24

Repeatability

4)

Gallic acid

II Dilution

Weight Dilution

50.6

0.5

50 99

10

Purity

Absorbance

0.651

II-Dilution

Sample Details

Sample wt

Dilution

Absorbance

Result (%)

OA150152-T1 OA150152-T2 OA150152-T3

2076.70 2011.20 2005.80

50 50 50

2 2 2

25 25 25

1.063 1.033 0.996

2.46 2.47 2.39 2.44 0.05 1.85

Average

SD

%RSD

SREE-00-080202002-T1 SREE-00-080202002-T2 SREE-00-080202002-T3

175.30 173.00 169.40

50 50 50

2 2 2

25 25 25

0.830 0.797 0.784

22.77 22.16 22.26 22.40

Average

SD

0.33 1.47

%RSD

5) Stability Study of Salacia Extarct B.No.: OASR081214L23

Stability Study of Salacia Extarct B.No.: OASR081214L23

Real time stability (25 °C ± 2 °C /60%RH ± 5% RH)

Accelarated Stability (40 °C ± 2 °C /75%RH ± 5% RH)

Real time Withdrawal months

Accelarated Withdrawal months

Initial Month

Initial Month

20.03 20.47 19.99 19.98 20.07 19.49 19.34

20.03 20.24 20.12 20.21

1 month 2 month 3 month 4 month 5 month 6 month

3 month 9 month 12 month

18 18.5 19 19.5 20 20.5 21 21.5 22

Stability Data

Accelerated

Real Time

% Polyphenols

0

2

4

6

8

Months