FORM 2
THE PA TENTS ACT. 1970
(39 OF 1970}
&.
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
[SECTION 10; RULE 13]
"A PROCESS FOR EXTRACTION OF NEEM LEAVES1

APPLICANT: GODREJ CONSUMERS PRODUCT LTD.
NATIONALITY: COMPANY INCORPORATED UNDER THE
COMPANIES ACT, 1956.
ADDRESS: PIROJSHANAGAR, EASTERN EXPRESS
HIGHWAY, VIKHROLL MUMBAI- 400 079,
MAHARASHTRA STATE. INDIA
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THIS
INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED: -



Field of Invention
The present invention relates to a process of extraction of neem leaves. More

particularly, the present invention relates to a process involving microwave-
assisted extraction of neem leaves using water, salt-water, glycerine-water,
surfactant-water and hydrotrope system. The extracts obtained by this method are
clear with characteristic odor and could be directly incorporated into cosmetic and
pharmaceutical formulations.
Background
The use of microwave energy for carrying out herbal extraction has become a well
established procedure. (Letellier M., Budzinski H., Microwave assisted extraction
of organic compounds, Analusis, 1999, 27, 259-271; Seifert P.,-Bertram Ch.,
Chollet D.. Microwave extraction of botanicals: A high tech green approach.
SOFW Journal, 2000, 126, 3-9: Gabriel C.: Gabriel S. Grant E. H.T Ben S. J.
Halstead, Mingos D. M. P.. Dielectric parameters to microwave dielectric heating,
Chemical Society Reviews. 1998, vol. 27. 213-223.) Microwave extraction follows
the same principle as the maceration and percolation using thermal energy, but the
speed of breaking up of a plant cell and tissues takes place in a very short time as
compared to conventional heating. This reduced processing time as compared to
conventional heating is not only an economically beneficial process but because of
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short microwave exposure time, there is less risk of degradation of plant
constituents or actives.
Microwave extraction (MW) requires proper optimization of the following
parameters: MW heating time, MW power level (wattage), nature and size of plant
material and plant parts to solvent ratio. By optimization and handling of these
parameters, the selectivity, quality and the yield of the resulting extract can be
controlled. Before the MW extraction of the plant material it is necessary that the
plant material have to be brought into small, uniform particles so as to increase the
contact surface and improve-the extraction results. Short mixing time with the
extracting solvent and swelling for a short time is useful and important to improve
the quality of final extraction results.
US patent 6,545,167 describes extraction of actives such as nimbin by an alcoholic
extraction of neem seeds. They also use chlorinated hydrocarbon solvents for
extraction after alcoholic extraction step. The extraction process time was about 2
to 24 hrs,
US patent 5,370,873 describes extraction of neem leave using soxhlet apparatus
and use organic of solvents such as alcohol, acetone, pyridine and mixtures these
solvents with water for extraction. The time for extraction was about 24 hrs. The
extracts are used for pharmaceutical preparations.
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US patents 5,900,493; 5,756,773; 5,663,374; 5,395,951 and 5,602,261 describes
extraction of actives from tieem seed .using combination of solvents such as
methanol, hexane. acetone and petroleum ether in sequential manner. The
extraction process time was about 2 to 4 hrs. The neem extracts are used for pest
control.

US patent 4,515, 785 describes extraction of neem bark for actives using organic
solvents such as methanoL ethanol and benzene.
All of these patents describe use of hazardous organic solvents like methanol,
ethanol, ethyl acetate, petroleum ether, hexane, benzene for extraction of neem
with long extraction process time.
To overcome the use of hazardous organic solvent in our inventive process we use
water, salt water, glycerine water, surfactant -water and hydrotrope system as a
vehicle for extraction. By using microwave heating instead of conventional
process, extraction time has been reduced dramatically in our process.
The aforesaid processes employ no hazardous organic solvents for extraction of
herbal or plant materials.
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Object of the present invention
The present invention obviates the aforesaid drawbacks by providing a process
developed to control environmental pollution. Thus the primary goal of the present
invention is to achieve neem leaves extraction using the most eco-friendly,
abundantly available and economical solvent - water.
Abstract
The present invention relates to a process for extraction of bioactives from neem
leaves comprising the steps of:
a) drying said neem leaves;
b) using water, salt-water, glycerine-water, surfactant-water or
hydrotrope water system or a combination thereof as extracting
vehicle
c) applying m icrowa ve radiation
d) filtering the extract
e) adding 0.5 % to 1 % of formaldehyde solution.
Detailed Description of the Invention
The present invention discloses a process of microwave-assisted extraction of
neem leaves using water, salt-water, glycerine water, surfactant-water and
hydrotrope water system, comprising the steps of:
a) Drying neem leaves.
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b) Adding either water, salt-water, glycerine water, surfactant-water and
hydrotrope water system as extracting vehicle.
c) Applying microwave radiation.
d) Filtration.
e) Addition of about 0.5% of formaldehyde solution.
The optimization of microwave extraction time is done by UV spectroscopy. It is
observed that microwave extraction gives extract of superior quality in very short
time. The extract is most suitable for use in cosmetic and toiletries formulations.
The aforesaid process of microwave extraction avoids the use of conventional
heating and organic solvents like methanol, ethanol. ethyl acetate, petroleum ether,
hexane, chloroform that are otherwise commonly used as vehicles for herbal and
plant extraction. Furthermore, the process of extraction established herein is easy,
economical and a fast method of microwave extraction of neem leaves. The
extracts obtained by this method are clear with characteristic odor. The MW batch
process of extraction within just 270 sec has been developed by us it is free of
organic solvents and hence it is environmentally 'green' process. The extracts are
directly incorporated into cosmetic and toiletries formulations as they are free of
organic solvents.
In the instant invention described herein, microwave extraction of neem leaves is
optimized for following parameters:
1 -. Use of extraction vehicle like water, salt water, glycerine-water, surfactant -
water and hydrotrope system.
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2. MW heating time and MW power level (wattage).
3. Nature and size of plant material and plant parts to solvent
4. Cosmetic aesthetic of neem extract is obtained from formulators point.
The present invention is now explained with reference to the following examples.
However, the said examples do not limit the scope of the invention.
Illustrative examples
Reagents and Materials
1. Pre air-dried Neem leaves.
2. Water, O.1 to 5%, preferably 1% salt-water, O.I to 5%, preferably J %
glycerine-water, O.I to 5%, preferably 1 % surfactant-water (AOS water
solution (sodium salt of alpha olefin sulfonate)) or 1 to 55%, preferably
50% hydrotrope water system (Na-pTSA (sodium salt of para toluene
acid)) are used as solvent for extraction.
3. Merck pre-coated TLC plates, ethyl acetate and toluene.
4. Samsung microwave kitchen oven (M1630W), 2450MHz, MW output
600W.
5. UV-Visible Shimadzu spectrophotometer (UV-160A model).
6. Anchrom UV visualizing chamber.
Example 1:
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In a 125 mL conical flask 5 g of clean, shredded and dried neem leaves of about
0.2-0.5 cm length are taken. To the flask 100 mL of distilled water is added as
extracting solvent. The conical flask is covered with a glass funnel which works as
a condenser. This system is (hen irradiated in Samsung microwave kitchen oven
for predetermined time. The MW extraction of neem leaves is monitored by UV
spectroscopy. After every MW irradiation cycle time of initial 30 sec and later 60
sec interval, 0.1 ml of neem sample extract is withdrawn from the conical flask,
diluted to 'lOml in a volumetric flask by distilled water. The absorbance is noted at
207 nm wavelength to check for extraction of active after microwave exposure.
After the mw extraction the extract is filtered through the filter paper and stored
into airtight glass bottles for further studies. About 0.5 % of formaldehyde solution
is added to this extract as preservative.
Example 2:
In a 125 mL conical flask 5 g of clean, shredded and dried neem leaves of about
0.2-0.5 cm length are taken. To the flask 100 mL of 1 % NaCl distilled water
solution is added as extracting vehicle. The conical flask is covered with a glass
funnel which works as a condenser. This system is then irradiated in Samsung
microwave kitchen oven for predetermined time. The MW extraction of neem
leaves is monitored by "UV spectroscopy. After every MW irradiation cycle time of
initial 30 sec and later 60 sec interval, 0.1 ml of neem sample extract is withdrawn
from the conical flask, diluted to 10ml in a volumetric flask by distilled water and
absorbance was noted at 207nm wavelength. After the mw extraction the extract is
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filtered through the filter paper and stored into airtight glass bottles for further
studies. About 0.5 % of formaldehyde solution is added to this extract as
preservative.
Example 3:
In a 125 mL conical flask 5 g of clean, shredded and dried neem leaves of about
0.2-0.5 cm length are taken. To the flask 100 mL of 1 % glycerine distilled water
solution is added as extracting vehicle. The conical flask is covered with a glass
funnel which works as a condenser. This system is then irradiated in Samsung
microwave kitchen oven for predetermined time. The MW extraction of neem
leaves is monitored by UV spectroscopy. After every MW irradiation cycle time of
initial 30 sec and later 60 sec interval. O.1 ml of neem sample extract is withdrawn
from the conical flask, diluted to 10ml in a volumetric flask by distilled water and
absorbance is noted at 207nm wavelength. After the mw extraction the extract is
filtered through the filter paper and stored into airtight glass bottles for further
studies. About 0.5 % of formaldehyde solution is added to this extract as
preservative.
Example 4:
In a 125 mL conical flask 5 g of clean, shredded and dried neem leaves of about
0.2-0.5 cm length are taken. To the flask 100 mL of 50 % NapTSA distilled water
(hydrotrope) solution is added as extracting vehicle. The conical flask is covered
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10
with a glass funnel which works as a condenser. This system is then irradiated in
Samsung microwave kitchen oven for predetermined time. The MW extraction of
neem leaves is monitored by UV spectroscopy. After every MW irradiation cycle
time of initial 30 sec and later 60 sec interval, 0.1 ml of neem sample extract was
withdrawn from the conical flask, diluted to 10ml in a volumetric flask by distilled
water and absorbance is noted at 350nm wavelength. After the mw extraction the
extract are filtered through the filter paper and stored into airtight glass bottles for
further studies. About 0.5 % of formaldehyde solution is added lo this extract as
preservative.
As the 50% NapTSA hydrotrope solution it self strongly absorbs between 200 to
270 nm, the mw extractions are monitored at 350 nm where absorption due to
hydrotrope solution is marginal.
Example 5:
In a 125 mL conical flask 5 g of clean, shredded and dried neem leaves of about
0.2-0.5 cm length are taken. To the flask 100 mL of 1 % AOS distilled water
(surfactant) solution is added as extracting vehicle. The conical flask is covered
with a glass funnel which works as a condenser. This system is then irradiated in
Samsung microwave kitchen oven for predetermined time. The MW extraction of
neem leaves is monitored by UV spectroscopy- After every MW irradiation cycle
time of initial 30 sec and later 60 sec interval, 0.1 ml of neem sample extract was
withdrawn from the conical flask, diluted to lOml in a volumetric flask by distilled
water and absorbance is noted at 350nm wavelength. After the mw extraction the
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extract is filtered through the filter paper and stored into airtight glass bottles for
further studies. About 0.5 % of formaldehyde solution is added to this extract as
preservative.
As the 1% AOS surfactant solution it self strongly absorbs between 200 to 270 nm,
the mw extractions are monitored at 350 nm where absorption due to AOS solution
is marginal.
Example 6:
The experiment is carried out to compare microwave heating ability with that of
conventional heating. In a 125 mL conical flask 5 g of clean, shredded and dried
neem leaves of about 0.2-0,5 cm length are taken. To the flask 100 mL of distilled
water is added as extracting solvent. The conical flask is covered with a glass
funnel which works as a condenser. This system is then heated conventionally on
hot plate for 60 min i.e. the system is boiled for 60 min. The conventional
extraction of neem leaves is monitored by UV spectroscopy. After every 6 min
time interval, 0.1 ml of neem sample extract is withdrawn from the conical flask,
diluted to 10ml in a volumetric flask by distilled water and absorbance is noted at
207nm wavelength. After the extraction the extract is filtered through the filter
paper and stored into airtight glass bottles for further studies. About 0.5 % of
formaldehyde solution is added to this extract as preservative. The results are pH
of extract is 5.57, %solid is J .520, TLC Rf is 0.51, nature is turbid and odor is little
burnt.
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It is observed that 48 min time of conventional heating is required to get
comparable extraction result as those are achieved by microwave heating of
270sec.
The neem extracts from all the different vehicles were analyzed for pH, percentage
solids, thin layer chromatography (TLC), clarity/ color, and odor and shown in
Table 1.
Table 1: Analysis of neem leaves extracts

Vehicle Water !%NaCl- 1 % glycerine 50% 1% ACS
water water NapTSA- water
water
pH 5.53 5.52 5.51 5.67 8.57

%solid 1.467 2.522 1.530 6.330 2.532
TLC (Ethyl 0.51 0.51 0.52 0.50 0.51
acetate:
toluene 1:1)
Rf
Clarity Clear Clear Clear Clear Clear
Odor Characteristic Characteristic Characteristic Characteristic Characteristic
Very good quality of neem leaves extract is obtained under MW exposure with all
the vehicles. The procedure is very simple and reproducible results are obtained. It
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is found that 1%NaCl-water solution is better than the other vehicles for extraction
(figure 1). This may be because in the presence of NaCl superheating of water
takes place thereby accelerating the MW extraction due to ionic currents under
MW irradiation.

Figure 1: Comparison of different extraction system under MW exposure.
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Figure 2: Conventional extraction system using water as a solvent.
Advantages:
1. Environmentally benign solvent system such as for neem leaves
extraction under microwave irradiation.

2. Reduction in extraction time of neem leaves is achieved by using
microwave heating.
3. Cosmetic aesthetic of neem leaves extract is obtained from formulators
point.
4. The neem leaves extracts are directly incorporated into cosmetic and
toiletries formulations as they are free of organic solvents.
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We Claim:
1. A process for extraction of bioactives from neem leaves comprising the
steps of:
a) drying said neem leaves;
b) using water, salt-water, glycerine-water, surfactant-water or
hydrotrope water system or a combination thereof as extracting
vehicle:
c) applying microwave radiation:
d) filtering the extract; and
e) adding 0.5 % to 1 % of formafdehyde solution,

2. A process as claimed in Claim 1, wherein said extracting vehicle in step (b)
includes water, 0.1 to 5% salt-water, 0.1 to 5% glycerine-water, 0.1 to 5%
surfactant-water or 1 to 55% hydrotrope water system.
3. A process as claimed in Claim 2, wherein said extracting vehicle includes
1% salt-water, 1 % glycerine-water, 1 % surfactant-water or 50 % hydrotrope
water system.
4. A process for extraction of bioactives from neem leaves substantially as
herein described with reference to the examples accompanying the specification.
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Dated this 13th day of April 2006.

S-3/PATENT/GODREJ/NEW PATENT/NEEM EXTRACnON/CS FINAL
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