Experiment Name: Analysis (ash content, moisture content, bulk density,

Polyphenol content, total extractive) of Tea.

i. Determination of Moisture:
The volatile material lost by a substance on heating the boiling temperature is called the moisture.
Actually it is the free water present in the material.
Method: About 5 gms of sample is taken in a Petridis. Then the sample is kept in the hot air oven at 104 0C
temperature. After a certain period the sample is taken out from the oven and the weight of the sample is
taken.
Calculation:
Moisture (percentage by mass) = 100*(M1-M2)/(M1-M)
Here,
M = mass of the empty dish (in gms)
M1 = Mass of the dish with the material before drying (in gms)
M2 = Mass of the dish with the material after drying (in gms)
i. Determination of Ash:
Principle: Ash is the residue remaining after a foodstuff is ignited until it is carbon free at a temperature not
exceeding red heat.
Procedure:
Ash content of sample is determined by using muffle furnace. Take 5-10 gm of sample in a silica crucible.
Churned it by using hot plate. Then keep it in Muffle furnace at 550 0C temperature for at least 3 hours.
After completion of ashing procedure the weight of the sample is taken
Calculation:
Weight of Crucible = a
Weight of Crucible + sample = b
Weight of Crucible + ash = c
Weight of sample = b - a
Weight of ash= c - a
% of Ash = (c-a)/(b-a)*100
i. Determination of Bulk Density:
 
Procedure:
Fill the measuring culinder with tea leaves and note the volume. Take out the tea leaves
and weigh it. Bulk density is cylinder is calculated as:-
 
Bulk Density (g/c.c) = (weight of Tea/Volume of Tea)
i. Determination of Estimation of Antioxidant(s)/ Polyphenol(s) in Tea

Principle: Antioxidants are essential for human health. Dietary antioxidants play an important role in controlling oxidative
stress. More than 5000 phytochemicals have been identified in plant foods and many more remain to be discovered. Phenolic
compounds have been proposed to be the potent and important contributors in reducing oxidative stress due to their antioxidant
activity. Excess free radical production underlies the pathogenesis of diseases like atherosclerosis, carcinogenesis, diabetes,
cataract and accelerated ageing. Within biological system there are at least four general sources of antioxidants: enzymes for
example superoxide dismutase, glutathione peroxidase and catalase; large molecules (albumin, ceruloplasmin,ferritin, other
protein); small molecules [ascorbic acid , gluthathione, uric acid, tocopherol, carotenoid, polyphenols]; and some hormones
(estrogen, angiotensin, melatonin. etc.)
Folin Ciocalteu Assay
Soluble phenolic compound (PC) were determined in sample extracts using the Folin-Ciocalteu reagent. Its principle is based
On the oxidation of phenolic compounds in alkaline medium with molybdenum and tungsten phosphate to form a blue colored
complex. The intensity of blue colored tungsten molybdenum complexes with polyphenols is measured spectrophotometrically
at the wavelength of 765nm. The value were expressed as equivalents of Gallic acid which is one of the most commonly, used
standards in phenolic estimations. Gallic acid has indeed been shown to be more stable and a pharmacologically active
antioxidant, quantitatively equivalent to many other phenolics and gives consistent and reproducible result.
NA2WO4/NA2MoO4 (phenol-MoW11O40)-4

Mo(VI)(yellow) + e Mo(V) (blue)

 Reagents required:

i. Methanol-water mixture(50:50)
ii. Folin-Ciocalteu reagent (1:10 diluted with distilled water)
iii. 1M Na2CO3 solution

iv. Methanol
v. Gallic acid
Procedure:
vi. Preparation of Standard curve of Gallic Acid: The standard curve using 0, 50, 100,150,200,250 mg/l concentrations of gallic
acid Methanol : Water (50:50, v/v)
vii. Preparation of Sample : Take 5g of sample and add it to 25ml Methanol. Keep the solution at 4 for 12 hr. Then centrifuge it
and use the clear supernatant estimation purpose
viii.Estimation of Total Phenolics : Take 0.5ml of methanolic extrat of sample gallic acid solution and mix with 5ml 1:10 dilute
Folin-Ciocalteu reagent and 4ml 1M aqueous sodium carbonate. This is allowed to stand for 15 mins and the total phenolics
is estimated by spectrophotometer at 765nm.
Calculation and Conclusion:
Total phenolics content of the sample is calculated from the standard curve of Gallic acid compare the result obtained with
standard values for the particular sample and conclude.
Calculation:

Y=0.0028X-0.2026
i. Determination of Estimation of Total Extractive:

 
Procedure:
1. Take 5 gm of Sample in 250 ml conical flask add 200 ml distilled water.
2. Reflux for 1 hr and allow cooling.
3. Transfer the mass into 250 ml volumetric flask and adjust the volume up to the mark with distilled water
4. Filter through filter paper. Filtrate contains the extractives.
5. Take 50 ml of aliquot into a porcelain dish and dry.