FIELD OF THE INVENTION;
This invention relates to a process for the extraction of polyphenols from
green tea leaves.
This invention relates to a process for the membrane based extraction of
polyphenols from green tea leaves using only water as solvent.
BACKGROUND OF THE INVENTION:
Tea is a beverage widely consumed throughout the world and it is
characterized by its high content of polyphenols. The anti-carcinogenic
effects of tea and its polyphenols have been demonstrated in numerous
animal studies involving tumors of the lung, digestive tract, prostate,
mammary glands, and skin. Several chemical constituents of green tea
have been identified. The variation in chemical composition is further
complicated with fermentation and other treatments. The most abundant
components of green tea are polyphenols, such as gallic acid and
catechin, and their derivatives. Dried green tea leaves generally contain
8-12% total polyphenols. Green tea also contains caffeine, theanine,
lignin, organic acids, protein, chlorophyll etc. The most abundant
components of green tea are polyphenols, such as gallic acid and
catechin, and their derivatives e.g., theogallin, gallocatechin, epiatechin,

and epigallo catechin. With fermentation, catechins partially change into
oligomeric quinones, including theaflavine, theaflavine acid and
thearubigene, or to non-water-soluble flavonoids such as quercetin,
kaempferol and myrecetin. Green tea polyphenols are potent antioxidant
compounds and may increase the activity of antioxidant enzymes. Green
tea also blocks the formation of cancer-causing compounds such as
nitrosamines, suppresses the activation of carcinogens, and detoxifies
and traps cancer-causing agents. Human studies also point to the
concept that green tea can prevent some forms of cancer, most
specifically GI cancers (stomach, small instine, pancreas, colon), lung
cancer, and estrogen-related cancers including most breast cancers. In
vitro studies of breast cancer have shown that green tea inhibits the
interaction of tumor promoters, hormones, and growth factors with their
receptors, producing growth arrest of the tumor. Epigallocatechin gallate
(EGCG) is the most powerful of these catechins. The antioxidant activity
of green tea EGCG is about 25-100 times more potent than vitamins C
and E. One cup of green tea may provide 10-40 mg polyphenols and has
antioxidant activity greater than a serving of broccoli, spinach, carrots or
strawberries.
Thus there is a growing importance in the investigation on the extraction
of the major polyphenols, from fresh green tea leaves. There have been
various methods used in the production of green tea polyphenols. One of

the most conventional methods of extraction of green tea polyphenols is
by extraction using methanol and ethanol and then separation using
membrane technology or by adsorption. Extraction of polyphenols from
green tea leaves is also done using microwave assisted extraction where
tea leaves are infused with solvents and then the suspensions are
irradiated with microwaves. Another method involves a supercritical fluid
extraction technique coupled with an absorption system to extract
polyphenol from green tea leaves. The extraction is done by super critical
carbon dioxide. US patent no. 5532012 discloses a method for
preparation of purified tea components involving pre-concentration by
cream separation and solubilization followed by medium pressure
chromatography and/or preparative HPLC. Another process for
producing a natural antioxidant from tea leaves is disclosed in US patent
no. 4673530 which comprises of treating tea leaves with a solvent
selected from the group consisting of hot water (preferably 80°C -100 °C),
a 40-75% aqueous solution of methanol, a 40-75% aqueous solution of
ethanol and a 30-80% aqueous solution of acetone to obtain an extract-
containing solution; followed by further washing the extract-containing
solution with chloroform to obtain a washed extract. The washed extract
is then combined with an organic solvent to transfer the washed extract
into the organic solvent. Next step is to remove the organic solvent and
drying the resulting extract.

However, all the methods mentioned hereinbefore use organic solvents
(e.g. methanol) with varying degrees of toxicity. So far, complete removal
of organic solvents has not been achieved, and even slight traces of these
solvents render the polyphenol powder toxic and unfit for human
consumption. Thus, polyphenols produced by these methods may not be
suitable for use with edible products. Therefore, a method using only
water as solvent will have greater acceptance, especially in
pharmaceutical and health food applications. In addition, the separation
process involved to achieve solvent-free polyphenol powder is extremely
costly and the time requirement for removal of trace amount of solvents
is very high. Another process with uses HPLC for production of
polyphenols makes it inherently costly. The cost of the HPLC equipment
is very high, the overall production process is time consuming, and thus
bulk production using HPLC is not a very viable proposition keeping
industry scale operations in mind.
Therefore, the need exists in the industry, to propose a method which is
less expensive, fast and which can be adapted easily for mass production
of polyphenols from green tea leaves.

OBJECT OF THE INVENTION:
This invention proposes a process for the extraction of polyphenols from
green tea leaves, which is cost effective and can produce the polyphenols
on a larger scale.
Moreover, this invention proposes a process for the extraction of
polyphenols from green tea leaves, which is fast and eco-friendly.
Another objective of this invention is to propose a process for the
extraction of polyphenols from green tea leaves which does not involve
toxic chemicals and costly processing equipment.
These and other objectives of the invention will be apparent from the
ensuing description, when read in conjunction with the accompanying
drawings.
SUMMARY OF THE INVENTION:
This invention provides a process for the extraction of green tea
polyphenols using water as solvent. The process involves the steps of
infusion, centrifugation, two membrane separation i.e. ultrafiltration and
nanofiltration and freeze drying of the retenate to obtain the green tea

polyphenols. The membrane separation techniques used herein is cost-
effective processes and do not involve any harmful chemicals, and leaves
no harmful waste products.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS;
Fig 1 shows a schematic diagram of the process.
Fig. 2 shows a schematic diagram of the membrane separation set-up.
Fig 3 is a graph showing the variation of permeate flux during
ultrafiltration at 8 atm applied pressure.
Fig 4 is a graph showing the variation of permeate flux during
nanofiltration at 10 atm applied pressure.
Fig. 5 shows the variation of polyphenol concentration in the retenate of
nanofiltration.

DESCRIPTION OF THE INVENTION;
In accordance with this invention, the process for the extraction of
polyphenols from green tea leaves involves the steps of (i) infusion (ii)
centrifugation (iii) two membrane separation steps, namely ultrafiltration
and nanofiltration and (iv) freeze drying of the retentate of the
nanofiltration step to produce Green Tea Polyphenol Powder. The
schematic diagram of the process is provided in Fig. 1 of the
accompanying drawings.
Infusion is carried out to extract polyphenols using water as solvent. The
typical infusion temperature is in the range of 65 to 75°C. Centrifugation
is used to remove all large suspended particles left in the water extract
after infusion. Centrifugations carried out for 30 to 40 minutes at 7000
to 8000 rpm. The water extract after centrifugation is subjected to
ultrafiltration. During ultrafiltration, the smaller components
preferentially permeate through membrane while the large species,
mainly proteins and its derivatives are retained. The permeate contains
majority of the polyphenols and is subjected to nanofiltration. In this
step, the polyphenols are concentrated. The schematic diagram of the
batch membrane separation set -up is shown in Fig 2.

Water extract from the centrifugation step is the feed for ultrafiltration
step. A batch cell (Fig. 2 of the accompanying drawings) is used for
utrafiltration. The batch cell (1) is made up of stainless steel. It has a
stirrer assembly with the stirrer (2) rotated using an external motor (3).
The set up can be pressurized using a N2 gas cylinder (4). The feed
solution (F) is pressurized at 6-10 atm with stirring speed varying from
800 to 1600 RPM. A polyethersulfone membrane (5) of molecular weight
cut-off (MWCO) 50000, obtained from M/s Permionics, (India), is placed
on a porous stainless step support (6) and is used for filtration. The
permeate (7) is collected from the bottom of the cell. The permeability of
the membrane is found to be 1.7 x1010 m/Pas using distilled water and
the effective filtration area is 34.2 cm2.
The permeate from the ultrafiltration is collected and subjected to
nanofiltration. The solution is pressurized to 10-14 atms with stirring
speed varying from 800-1600 RPM. A membrane of molecular weight cut-
off (MWCO) 200, obtained from M/s Genesis Membrane Sepratech Pvt.
Ltd., Mumbai 400 083, India, is placed on the porous stainless steel
support. The permeability is found to be 1.9x 1012 m/Pas using distilled
water. The effective filtration area is 24.2 cm2. Permeate is collected from
the bottom of the cell. Finally the retentate of the nanofiltration step is
freeze dried to produce green tea polyphenol powders.

Figures 3 and 4 describe the variation of permeate flux with time for UF
and NF experiment, respectively, at different stirring speeds. It is clear
from both the figures that the permeate flux declines sharply with time
and takes about 20 minutes for UF and 10 minutes for NF to attain a
steady state valve. This is due to the phenomena of concentration
polarization, i.e., the accumulation of solution particles over the
membrane surface which grows in time. The growth of concentration
boundary layer over the membrane surface is arrested by the external
turbulence and ultimately a steady state value is reached. The thickness
of the deposited layer over the membrane surface is lower at higher
stirrer speed due to enhanced turbulence. This trend is clear from Figs 3
and 4.
The variation of polyphenol concentration in the retentate of
nanofiltration at various operating pressure is presented in Fig. 5 at
different stirrer speeds. It may be observed that the polyphenol
concentration varies in a range of 0.8 to 1.5 g/1 for various combinations
of operating conditions.
The powder thus obtained is tested for its polyphenols and protein
content and the results are provided below.

Spectrophotometric analysis of polyphenols
The total tea polyphenols is estimated by Acid Butanol Assay for
Proanthocyanidins method. The procedure involves measurement of
absorbance caused by a specific reaction of the phenolic groups. ECGC is
a phenolic compound containing hydroxyl groups that react with a
chemical reagent to produce a colour, the intensity of which is measured
by a spectrophotometer. Using ECGC as a standard, it is possible to
measure the relative polyphenol content of tea and fruit extracted. The
samples are diluted and the reagents are added as specified in the
procedure. Full reaction and color development requires a minimum of
one and half hours for completion. The absorbance is then measured at
550 nm. A regression analysis is performed on the known standard
solution (ECGC) to develop the standard calibration curve and the final
polyphenol content for unknown sample is determined from the standard
calibration curve.
Spectrophotometric analysis of proteins
The protein present in tea is estimated by Bradford Analysis. It is fairly
accurate and samples that are out of range can be retested within
minutes. This assay is based on the binding specificity of the dye
Coomassie Brilliant Blue-G 250 for protein molecules but not for other

cellular constituents. This organic dye binds specifically to tyrosine side
chains. The binding of the dye to protein shifts the peak absorbance of
the dye. Unbound Coomassie Blue absorbs light maximally at a
wavelength of 465 nm, while the absorption maximum is at 595 nm
when the dye is bound to protein. The absorbance of light by the dye-
protein complex at 595 nm is proportional to the amount of protein
bound (over a limited range); i.e. there is a linear relationship between
absorbance and the total protein concentration of the sample over a
narrow range. The Bradford assay is more sensitive to bovine serum
albumin which is found in tea and fruit extracts.
Membrane separation technique offers a cost effective process that can
be used to produce Green Tea Polyphenol Powder of high purity. The
benefits of this process over the other conventional processes lie on the
fact that it can be used in mass production of the desired material
involving low equipment cost. These membrane processes are pressure
driven membrane modules and mounted in different array designs to
optimize the process. Energy costs involve only pressurizing the
membrane modules. For separation of water soluble substances such as
polyphenols in this case, membrane separation is effective as it does not
involve any harmful chemicals and the side products can be recycled
without leaving any waste products.

The purity of the final product in terms of the EGCG content is found to
be in range of 50-70% which is of high commercial value. The apparatus
used is very cheap compared to the HPLC set ups. The process is
absolutely green and hence the product of this process can serve the food
and edible products industry in a much better way then any other
existing polyphenol production process.

WE CLAIM;
1. A process for the extraction of polyphenols from green tea leaves
comprising the steps of water infusion of the green tea leaves,
centrifugation of the infusion followed by membrane separation
steps of ultrafiltration and nanofiltration to obtain a retenate
rich in polyphenols and freeze drying the retentate to obtain
polyphenols in powdered form.
2. The process as claimed in claim 1, wherein infusion is effected
using hot water at a temperature of 65 to 75°C.
3. The process as claimed in claim 1, wherein the infusion is
centrifuged for a period of 30 to 40 minutes.
4. The process as claimed in claim 1, wherein the infusion is
centrifuged at a speed of 7000 to 8000 rpm.
5. The process as claimed in claim 1, wherein the infusion is
optionally subjected to a step of cloth-filtration before being
centrifuged.
6. The process as claimed in claim 1, wherein for the step of
ultrafiltration, the feed solution is pressurized to 6 to 10 atm.

7. The process as claimed in claim 1, wherein ultrafiltration is
effected at a stirring speed varying from 800 to 1600 RPM.
8. The process as claimed in claim 1, wherein ultrafiltration is
effected by a polyethersulfone membrane of molecular weight
cut-off (MWCO) 50,000.
9. The process as claimed in claim 1, wherein for the step of
nanofiltration the solution is pressurized to 10 to 14 atm.
10. The process as claimed in claim 1, wherein the step of
nanofiltration, is effected at a stirring speed of 800 to 1600
RPM.
11. The process as claimed in claim 1, wherein nanofiltration is
effected by a membrane of molecular weight cut-off (MWCO)
200.
12. In this process, Green tea polyphenol powder having a purity of
50 to 70% is produced.

This invention provides a process for the extraction of green tea polyphenols using water as solvent. The process involves the steps of
infusion, centrifugation, two membrane separation i.e. ultrafiltration and
nanofiltration and freeze drying of the retenate to obtain the green tea polyphenols. The membrane separation techniques used herein is cost-effective processes and do not involve any harmful chemicals, and leaves no harmful waste products.