1. A method of extracting essential oil from dead Aethoscytus foveolus (Dallas) bug, comprising the steps of: a. collection of 20 g dead bugs, washing with water, followed by 5 washing with 70% alcohol and again a wash with water; b. sieving, wrapping with tissue paper and drying the sample, followed by making a fine powder with liquid nitrogen; c. boiling the sample in the Soxhelt apparatus with defatting solvent at boiling point 50-60°C for 6 hours following the AOCS 10 method; d. centrifuging the sample at 12000-18000 rpm for 10-20 minutes and e. collecting the oil from the supernatant and storing at 4°C in a dark plastic container 15 2. The method as claimed in claim 1, wherein the defatting solvent is selected from the group n-hexane, petroleum ether and acetone.

3. The method as claimed in claim 1, wherein the amount of solvent in step c is 1:5, 100ml for 20g bugs. 20 4. The method as claimed in claim 1, wherein the AOCS method involves centrifuging twice for the final extraction of oil in supernatant.

5. The method as claimed in claim 1, wherein the 1:5 ratio of bugs and solvent leads to 44.50% oil yield. 25 6. A composition of essential oils extracted from the Aethoscytus foveolus (Dallas) bug, comprising of compounds 2- Bromotetradecanoic acid, 2-Hexadecanol, Tetratetracontane and Heptacosane.

7. The composition as claimed in claim 1 or 5, wherein the 30 composition has secondary metabolites- alkaloids, saponins, 24 hexose sugar, tannins, phenols, fat, glycosides, tyrosine and carbohydrates.

8. The composition as claimed in claim 1 or 5, wherein the compounds are identified by GC-MSs. 5 9. The composition as claimed in claim 1 or 5, wherein the composition shows (62.40±5.07) % inhibition of AChE in in vitro assay.

10. The composition as claimed in claim 1 or 5, wherein the composition has therapeutic potential against neurodegenerative 10 disorders.

The present invention relates to the therapeutic oil derived from dead
insects. More particularly, the present invention relates to the novel medicinal
oil extract from unexplored strain of Aethoscytus foveolus (Dallas) for the
5 management of neurodegenerative diseases.
BACKGROUND AND PRIOR ART
[0002] Entomophagy the practice of eating insects by humans is common to
cultures in most part of the world. Nearly two billion people regularly eat
10 insects in their basic meals and over 1,900 species are edible, such as beetles,
caterpillars, bees, wasps and ants are the most common and in them some
notable compounds are also identified. Besides their environmental,
economical and nutritional benefits; Insects have long been used in medicine,
both traditional and modern, sometimes with little evidence of their effectivene
15 [0003] Insect oils have been documented for nutritional, industrial and medicinal
applications viz. insecticidal, anti-parasitical, bactericidal, antiviral and
fungicidal. Across the globe, scientist are indulging in screening of these oils
for their possible usage in the management of varied types of disease arthritis
treatment, infections, pain, cancer, skin diseases etc. Chinese Black Mountain
20 Ant, Polyrhachis vicinais supposed to prolong life, to have anti-aging
properties, and to increase virility and fertility in traditional Chinese
therapy.The vast history of the efficacy of the Chinese ant as a tonic is well
documented in China. In alternative medicine parlance, that 'bug drugs' are on
the rise in modern medicine. In Ayurveda, Termite is said to cure a variety of
25 diseases such as ulcers, rheumatic diseases, and anemia.
[0004] Although the traditional eating of insects is usually associated with
continents such as Africa, Asia and Australia, Dr Wilkinson says ant drinks
have been used more recently in California as a hallucinogen and were actually
used in Britain in the middle Ages as a tonic for general ailments.
3
[0005] References have been made to the following patents:
[0006] CN101812378B relates to extraction of insect oil and a preparation
method thereof. The preparation method of the biodiesel comprises: extracting
insect oil from holometabolous insect larvae or pupae serving as raw materials
5 by using a solvent method; and subjecting the insect oil with a low acid value
(less than or equal to 2) or high acid value (more than 2) to ester exchange or
pre-esterification-ester exchange, separating phases to obtain coarse biodiesel,
purifying the coarse biodiesel by using an adsorption clarification technique or
a molecular distillation technique to obtain refined biodiesel. The method has
10 the advantages that: the raw materials are widely available, easy to breed and
low in cost; a new technical route for producing the biodiesel is provided; and a
novel approach for developing and utilizing insects is provided.
[0007] EP1123368B1 relates to a method for extracting lipid fractions from
marine and aquatic animal material by acetone extraction. The resulting non15 soluble and particulate fraction is preferably subjected to an additional solvent
extraction with an alcohol, preferably ethanol, isopropanol or t-butanol or an
ester of acetic acid, preferably ethyl acetate to achieve extraction of the
remaining soluble lipid fraction from the marine and aquatic animal material.
The remaining non-soluble particulate contents is also recovered since it is
20 enriched in proteins and contains a useful amount of active enzymes. Also
provided herein is a krill extract.
[0008] US5707673A relates to a solvent extraction process for the extraction of
an extractive from extractive-containing material employing in an extraction
zone operating under extraction conditions a process solvent, whereby a
25 miscella comprising a portion of the process solvent and a portion of the
extractive, and an extractive-depleted substrate is formed, the improvement to
which comprises: (a) removing the miscella from the extraction zone under
extraction conditions, (b) filtering the miscella by use of a microfiltration, an
ultrafiltration, a nanofiltration, or a reverse osmosis membrane, under
30 conditions which achieve a differential pressure across said membrane, to
4
separate the solvent in the miscella from the extractive in the miscella, and (c)
recycling under extraction conditions at least a portion of the separated solvent
to the extraction zone.
[0009] Thus in the view of above prior art, many drugs have been designed to
5 slow the disease progressions but, till today, no permanent solution has been
achieved. Heavy reliance on antibiotics, coupled with discomfort with insects
in Western culture limited the field of insect pharmacology until the rise of
antibiotic resistant infections sparked pharmaceutical research to explore new
resources Arthropods represent a rich and largely unexplored source of new
10 medicinal compounds.
[0010] In line to this, present invention relates to overcome the obstacles of the
prior art and emphasizes on the extraction of novel essential oil from the dead
bug for its possible usage in management of neurological diseases viz.
depression and Alzheimer’s disease. This invention has given us new insights
15 in drug development where insects’ derived oil may be used in the
management of neurological diseases.
[0011] The information disclosed in this background of the disclosure section is
only for enhancement of understanding of the general background of the
invention and should not be taken as an acknowledgement or any form of
20 suggestion that this information forms the prior art already known to a person
skilled in the art.
OBJECTS OF THE INVENTION
[0012] The principal object of the present invention is to extract medicinal oil
25 from Aethoscytus foveolus (Dallas) bug and a method of preparing thereof.
[0013] Another aspect of the present invention is to provide the novel therapeutic
oil from dead insects.
5
[0014] Still another object of the present invention is to provide the the novel oil
extract from the dead insect Aethoscytus foveolus (Dallas), free from any side
effect with anti- neurodegenerative properties.
[0015] Yet another object of the present invention is to provide the method for
5 preparing the said extract from the bug.
[0016] Another object of the present invention is to provide novel extract with oil
as an active ingredient with probable therapeutic efficacy in neurological
disorders.
[0017] Yet another aspect of the present invention is to extract active compounds
10 or proteins present in the oil for probable biological activities.
[0018] Another preferreable object of the present invention is to provide novel
medicinal oil that can be formulated in various dosage forms viz. liquid, solid
and powder form.
[0019] These and other objects and advantages of the present subject matter will
15 be apparent to a person skilled in the art after consideration of the following
detailed description taken into consideration with accompanying drawings in
which preferred embodiments of the present subject matter are illustrated.
SUMMARY OF THE INVENTION
20 [0020] In an important embodiment the present invention is to provide a novel
composition of medicinal oil from Aethoscytus foveolus (Dallas) bug and a
method of preparing thereof.
[0021] In yet another embodiment the present invention provides a method of
extracting essential oil from dead Aethoscytus foveolus (Dallas) bug,
25 comprising the steps of: a. collecting 20 g dead bugs, washing with water,
followed by washing with 70% alcohol and again a wash with water; b.
sieving, wrapping with sample with tissue paper and drying, followed by
6
making a fine powder with liquid nitrogen; c. boiling the sample in the Soxhelt
apparatus with defatting solvent at boiling point 50-60°C for 6 hours following
the AOCS (maerican Oil Chemist Society) method; d. centrifuging the sample
at 12000-18000 rpm for 20 minutes (twice); and e. collecting the oil from the
5 supernatant and storing at 4°C in a dark plastic container
[0022] In another embodiment the defatting solvent for oil extraction is selected
from the group n-hexane, petroleum ether and acetone.
[0023] In another embodiment the the amount of solvent for the extraction of oil
in 1:5 ration, meaning 100 ml of hexne for 20 g of bugs and oil is extracted by
10 AOCS method.
[0024] In a preferred embodiment the present invention provides a composition
of essential oil extracted from the Aethoscytus foveolus (Dallas) bug,
comprising of compounds 2-Bromotetradecanoic acid, 2-Hexadecanol,
Tetratetracontane and Heptacosane
15 [0025] In another embodiment the composition has secondary metabolitesalkaloids, saponins, hexose sugar, tannins, phenols, fat, glycosides, tyrosine
and carbohydrates and the compounds are identified by GC-MS.
[0026] In yet another embodiment the composition shows (62.40±5.07) %
inhibition of AChE assay with therapeutic potential against neurodegenerative
20 disorders.
[0027] The foregoing summary is illustrative only and is not intended to be in any
way limiting. In addition to the illustrative aspects, embodiments, and features
described above, further aspects, embodiments, and features will become
apparent by reference to the drawings and the following detailed description.
25 BRIEF DESCRIPTION OF THE DRAWINGS
[0028] It is to be noted, however, that the appended drawings illustrate only
typical embodiments of the present subject matter and are therefore not to be
considered for limiting of its scope, for the invention may admit to other
7
equally effective embodiments. The detailed description is described with
reference to the accompanying figures. Some embodiments of system or
methods in accordance with embodiments of the present subject matter are now
described, by way of example, and with reference to the accompanying figures,
5 in which:
[0029] Fig. 1 illustrates the Process of extraction of oil from insect, in accordance
with an embodiment of the present invention;
[0030] Fig. 2 illustrates the Effect of oil sample on AChE activity in zebra fish
hippocampus tissue, in accordance with an embodiment of the present
10 invention;
[0031] The figure depicts embodiments of the present subject matter for the
purposes of illustration only. A person skilled in the art will easily recognize
from the following description that alternative embodiments of the structures
and methods illustrated herein may be employed without departing from the
15 principles of the disclosure described herein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] While the embodiments of the disclosure are subject to various
modifications and alternative forms, specific embodiment thereof have been
20 shown by way of example in the figures and will be described below. It should
be understood, however, that it is not intended to limit the disclosure to the
particular forms disclosed, but on the contrary, the disclosure is to cover all
modifications, equivalents, and alternative falling within the scope of the
disclosure.
25 [0033] The terms “comprises”, “comprising”, or any other variations thereof used
in the disclosure, are intended to cover a non-exclusive inclusion, such that a
device, system, assembly that comprises a list of components does not include
only those components but may include other components not expressly listed
8
or inherent to such system, or assembly, or device. In other words, one or more
elements in a system or device proceeded by “comprises… a” does not,
without more constraints, preclude the existence of other elements or additional
elements in the system or device.
5 [0034] The present subject matter relates to the isolation and extraction of oil
derived from dead Aethoscytus foveolus (Dallas) bug and the method thereof.
[0035] Reference may be made to Figure 1 illustrating the Process of extraction of
oil from insect, in accordance with an embodiment of the present invention;
[0036] Reference may be made to Figure 2 illustrating the Effect of oil sample on
10 AChE activity in zebra fish hippocampus tissue, in accordance with an
embodiment of the present invention;
[0037] Methodology: Collection and authentication of bugs: A.foveolus bugs
were collected from different locations of Banasthali Vidyapith, Rajasthan
India. It is only found in Rajasthan, India. The species was identified by PUSA,
15 New Delhi. Since lamp light attract these bugs ( specially during rainy season
i.e.:- july-september). Dead bugs were collected from these areas early in the
morning (5-6AM) with the help of broom and pan and collected in a poly bag.
In the lab dust was separated with the help of sieve and samples were washed
properly with alcohol. Dead bugs were collected for the maintenance of
20 ecology, thus not affecting the environment.
[0038] Preparation of oil extract: A total of 20g of bugs were collected from
different places of Banasthali Vidyapith, Banasthali, Rajasthan, for oil
extraction. Oil was extracted with 100 ml n-hexane boiling point50-60°C from
the ground material in a Soxhlet apparatus for 6 hours following the AOCS
25 (American Oil Chemist Society) method (Adel et al., 2015 ) as shown in Figure
1. The obtained oil sample was stored at 4°C in a dark plastic container and
percentage yield of oil was estimated using a formula:
[0039]
9
[0040] Example 1: Phytochemical analysis of oil: Phytochemical analysis of oil
sample was performed to identify the presence of primary and secondary
biomolecules such as saponins, alkaloids, phenols, Hexose sugars, flavonoids,
5 amino acids and carbohydrates (Treaseet al.,1989). Tests were performed
using standard methods.
[0041] Test for alkaloid: Mayer’s test: 1mL of the oilsample was treated with
1mL of Mayer’s reagent and formation of cream precipitate was observed.
[0042] Wagner’s test:1mL of the oil sample was added to 1mL of Wagner’s
10 reagent and formation of reddish-brown precipitate was observed.
[0043] Dragendroff test:1mL of oil sample was treated with 1mL of
Dragendorff`s reagent, formation of orange brown precipitate was observed.
[0044] Hager’s test:1mL of the oil sample was treated with 1mL of Hager’s
reagent and formation of yellow precipitate was observed.
15 [0045] Test for tannins and phenolic compounds : Lead Acetate test:To 2 mL
of oil sample, few drops of lead acetate solution were added. Formation of
white colored precipitate indicated the presence of tannins and phenolic
compounds.
[0046] 5% Ferric Chloride test: To 2 mL of oil sample, few drops of 5% FeCl3
20 solution was added. Appearance of deep blue-black color indicated the
presence of tannins and phenolic compounds.
[0047] Test for carbohydrates: Molisch’s test: Few drops of alcoholic αnaphthol solution were mixed in 2-3 mL oil sample. The solution was agitated
properly and then few drops of conc. H2SO4 was added from side of the test
25 tubes. The appearance of violet ring at the junction of two liquids, showed the
presence of carbohydrates.
10
[0048] Fehling’s test: 1 mL of Fehling’s A and 1 mL Fehling’s B solutions were
mixed and boiled for a minute with oilsample and heated in boiling water bath
for 5-10 min. First yellow, then brick-red colored precipitate indicates
occurrence of reducing sugars.
5 [0049] Benedict’s test: Equal volume of oil sample and Benedict’s reagent
(alkaline solution of cupric ions) drop wise was mixed. Solution was heated in
boiling water bath for 4-5 min. The appearance of reddish-brown colored
shows the occurrence of carbohydrate.
[0050] Test for glycosides: Legal’s test: Oil sample was mixed with 1 mL
10 pyridine and 1 mL of sodium nitroprusside. Pink turns into red in color
indicated the occurrence of cardiac glycosides.
[0051] Keller-Killiani test:2 mL glacial acetic acid was mixed with few drops of
FeCl3, then dropwise conc. H2SO4 along the side of the test tubes was added.
Appearance of reddish-brown color at the junction of two liquid films and
15 upper layer appears in bluish-green color that indicates the occurrence of
cardiac glycosides.
[0052] Test for saponins : Foam test: 0.5 g of crude drug and extract was
dissolved in 10 mL of distilled water and shake vigorously for 10-15 min.
Formation of foam indicated the presence of saponins.
20 [0053] Test for lipids: Sudan test: A few drops of Sudan IV was added to 0.5
mL of oil sample. It is a dye that stains lipids. If there is no lipids present in the
test sample, then the dye will settle to the bottom.
[0054] Translucent spot test: 0.5 mL of oil sample was placed on a sheet of
paper. If the oil sample contains fats and oils, then due to diffraction of light a
25 translucent spot was produced on another side of paper.
[0055] Test for tyrosine: Millon’s test: A few drops of the Millon’s reagent
were added to the 0.5 mL of oil sample and heated gently. Appearance of
reddish brown coloration or precipitate indicates the presence of tyrosine.
11
[0056] Test for hexose sugar: Seliwanoff’s Test: This test is used for
identification of keto-hexoses or to distinguish between ketoses and aldoses.
To 1 mL of oil sample, 5 mL of Seliwanoff’s reagent (resorcinol in 6M HCl)
was added and boiled, formation of cherry red colour in presence of ketose
5 (fructose) due to formation of hydroxyl methyl furfural, which condensed with
resorcinol to produce cherry red colour.
[0057] Cobalt chloride Test: To 1.5 mL of oil sample 1 mL of cobalt chloride
solution was added, boiled for 2 min and cooled, to this 2-3 drops of sodium
hydroxide solution was added and observed for the change in colour. The
10 appearance of colourwas upper layer greenish blue and lower purplish layer
which indicated the presence of glucose, fructose or mixture glucose and
fructose respectively.
[0058] Example 2: GC-MS analysis of crude oil sample extracted from bug:
Oil sample was analyzed using GC–MSThermofisher scientific system, USA
15 Model- GC- Trace 1300 GC-MS-TXQ8000 with triple Quadripole
bearingautosampler- AI/AS 1310. The analysis was done oncolumn- TG-5 MS
(silica column) at temperature 330-350oC with split ratio100:1. The following
program was used: helium was used as the carrier gas at an injection
temperature 250 °C (with a split ratio of 10.0), ion-source temperature 230 °C
20 . The oven temperature was programmed from 80 °C (isothermal for 3 min),
with an increase of 10 °C /min, to 250 °C (isothermal for 5 min), then 15 °C
/min to 280 °C , ending with a 18 min isothermal at 350 °C . MS was
programmed to have a scan interval of 0.5 sec. All the mass spectra of the
identified peaks were compared with the spectra from the spectral library.
25 [0059] Example 3: In vitro assay: Estimation of AChE inhibitory assay: The
assay was carried out in isolated zebra fish hippocampus tissue. Inhibition of
acetylcholinesterase (AChE) enzyme activity was measured through UV/Visspectrophotometer employing the method described by Ellman et al.,1961.
AChE hydrolyse the acetyl choline into thiocholine which reacts with the
30 Ellman reagent also known as 5,5-dithiobis-2-nitrobenzoicacid (DTNB) to
12
form 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2nitrobenzoate which
can be easily identified in UV/Vis spectrophotometer at 412nm. To perform the
assay, firstly hippocampus tissue was homogenized and centrifuged. After
centrifugation, in a test tube 10 µL of supernatant aliquot was taken then 1710
5 µL of 50 mM sodium phosphate buffer, 20 µL of 10 mM of DTNB, 10 µL of
200 mM acetyl thiocholine iodide and 50 µL of oil sample was added in to it.
Same procedure was followed for the standard donepezil group at the place oil
sample. Afterwards the absorbance of all the samples was measured at 412 nm
(Owokotomoet al., 2015, Bahadoriet al., 2016).
10 [0060] The percentage enzyme inhibition was determined from the given
formula-
% inhibition = 100-change in sample absorbance/blank sample
absorbanceX100
The experiment was carried out in triplicates and data were analysed using
15 GraphPad Prism 5 statistical software.
[0061] Results: Oil sample was examined for presence of phytochemical
components using a battery of chemical tests. In addition, various components
available in oil was identified using GC-MS analysis. Possible therapeutic
application of oil sample was determined using in-vitro anti-acetylcholine
20 esterase assay to establish its role in the management of Alzheimer diseases
and related disorders.
Quantification of oil extract :The percentage of oil obtained from the collected
bug sample (20 g) is calculated below:
25
13
[0062] Phytochemical analysis of oil: The qualitative analysis of bioactive
phytochemical constituents of oil sample was performed to detect the presence
of secondary metabolites. The results of preliminary phytochemicals screening
of oil sample showed presence of alkaloids, saponins, hexose sugar, tannins,
5 phenols, fat, glycosides, tyrosine and carbohydrates(Table 1).
[0063] Table1. Phytochemical constituents present in oil sample extracted from
bug
S.no. Metabolites Present/Absent
1. Alkaloids
Mayer’s test
Wagner’s test
Hager’s test
Dragendorff’s test
Present
Absent
Present
Present
2. Test for tannins and phenolic
compounds:
Fecl3
Pott. Permanganate
Iodine solution
Lead acetate
HNO3
Absent
Present
Present
Absent
Present
3. Test for carbohydrates
Molish test
Fehling’s test
Benedict
Present
Absent
Absent
4. Test for glycosides
Legal test
Keller-Killiani test
Present
Absent
5. Test for saponins
Foam test Present
6. Lipid test
Fats and oils (on sheet)
Sudan red test
Present
Present
7. Test for tyrosine
Millon’s test Absent
8. Hexose sugar
Seliwanoff’s test
Cobalt chloride
Absent
Present
14
[0064] GC-MS analysis of crude oil sample extracted from bug: A total of
thirty-five compounds were identified by GC-MS from oil extracted from
bug.Amongst which few of the compounds have already known for various
biological activities (Table2).
5 [0065] Table 2.Some identified compounds with their biological activities
S.n
o
Name of compound Structure Mol. wt.
(g/mol)
Mol.
Formula
Biological
activities
1. Chromone,5-hydroxy6,7,8-trimethoxy-2,3-
dimethyl
280.27 C14H16O6 Not found
2. Methyl glycocholate,
3TMS derivative
O
HN
O
O
Si
H
H
O
Si
O
Si
HH
O
H
695 C36H69N06
Si3
Not found
3. Androstane-11,17-
dione,3-
[(trimethylsilyl)oxy]-
,17-[O-
(phenylmethyl)oxime],(
3à,5à)-
481.7 C29H43NO
3Si
Not found
15
4. Spirost-8-en-11-one, 3-
hydroxy-
,(3á,5à,14á,20á,22á,25R
)-
428 C27H40O4 Not found
5. 8H-Pyrano[3,4-
b]pyrimido[5,4-
d]furane,5,6-dihydro-4-
hydrazino-6,6-
dimethyl-2-methylthioO
N
N
O
S
H2N
NH
280 C12H16NO
2S
Not found
6. 1,4-Benzenediol, 2,6-
bis(1,1-dimethylethyl)-
222 C14H22O2 Not found
7. 4-Dehydroxy-N-(4,5-
methylenedioxy-2-
nitrobenzylidene)tyrami
ne
298.29 C16H14N2
O4
Not found
16
8. Betulin
HO
H
OH
H
H
H
Betulin
442.7 C30H50O2 Antiasthamatic,
Treatment in
urinary system
related disorders
9. 1,2-Benzenediol, 3,5-
bis(1,1-dimethylethyl)-
222.32 C14H22O2 Antibacterial
agents,
Antimalarials
10. 2-Hexadecanol 242.44 C16H34O In treatment of
dermatological
disorders ,
nervous
system and as
abronchodilators
11. Tetrapentacontane,
1,54-dibromo914 C54H108Br2 Not found
12. 2-Nonadecanone 2,4-
dinitrophenylhydrazine
462 C25H42N4
O4
Not found
13. Octatriacontyl
pentafluoropropionate
696 C41H77F5O
2
Not found
14. Oleic acid, 3-
(octadecyloxy)propyl
ester
593 C39H76O3 Not found
17
15. Z-5-Methyl-6-
heneicosen-11-one
322 C22H42O Not found
16. 9-Octadecenoic acid,
(2-phenyl-1,3-dioxolan4-yl)methyl ester, cis444 C28H44O4 Not found
17. 2-Bromotetradecanoic
acid
307.27 C14H27Br
O2
AntiParkinson drugs,
Antidepressant,
Antiviral,Antine
oplastic agents
18. Octadecane, 1,1'-[1,3-
propanediylbis(oxy)]bis
-
581.1
C39H80O2
For specific
purposes, not
provided for
groups
19. d-Mannitol, 1-
decylsulfonyl
335.32 C16H31Br
O2
For
osteoporosis,
antiasthamatic ,
for disorders
related to
urinary system
20. Hexadecanoic acid, 2-
bromo334 C16H31Br
O2
Not found
21. Stearic acid, 3-
(octadecyloxy)propyl
ester
594 C39H78O3 Not found
22. Octadecane, 3-ethyl-5-
(2-ethylbutyl)-
366 C26H54 Not found
18
23. Tetratetracontane
Tetratetracontane
619.2 C44H90 For treating
wounds, ulcers,
burns, scars,
keloids,
disorders of the
urinary system,
neurodegenerati
ve disorders of
the central
nervous
system, for
treating
Alzheimer's
disease or other
forms of
dementia
24. Heptadecane, 9-hexyl- 324 C23H48 Not found
25. Heptacosane, 1-chloro- 414 C27H55Cl Not found
26. 1,2-Propanediol, 3-
(octadecyloxy)-,
diacetate
428 C25H48O5 Not found
27. Heptacosane
Heptacosane 380.7 C27H56
Antiasthama
tic,
Bronchodilators
19
,for disorders of
the urinary
system
28. Tetrapentacontane,
1,54-dibromo914 C54H108Br2 Not found
29. 1,1,3,6-tetramethyl-2-
(3,6,10,13,14-
pentamethyl3-ethylpentadecyl)cyclohexane
448 C32H64 Not found
30. Tetrapentacontane,
1,54-dibromo914 C54H108Br2 Not found
31. 5HCyclopropa(3,4)benz(1,
2-e)azulen-5-one,
1,1a-à,1b-á,4,4a,7aà,7b,8,9,9a-decahydro7b-à,9
-á,9a-à-trihydroxy-3-
hydroxymethyl-1,1,6,8-
à-tet
ramethyl-4a-methoxy-,
9,9a-didecanoate
686 C41H66O8 Not found
32. Hexadecanoic acid,
1a,2,5,5a,6,9,10,10aoctahydro-5,5adihydroxy-4
-(hydroxymethyl)-
1,1,7,9-tetramethyl-11-
586 C36H58O6 Not found
20
oxo-1H
-2,8amethanocyclopenta[a]c
yclopropa[e]cyclod
ecen-6-yl ester,
[1aR-
(1aà,2à,5á,5aá,6á,8aà,9
à,10aà)]-
33. Octadecane, 1,1'-[1,3-
propanediylbis(oxy)]bis
-
580 C39H80O2 Not found
34. Tibolone, bis(tert.-
butyldimethylsilyl)
540 C33H56O2S
i2
Not found
35. Hexatriacontane
Hexatriacontane
507 C36H74 In treatment of
wounds, ulcers,
burns, scars,
keloids, for joint
disorders, e.g.
arthritis,
arthrosis ,Antiinfective, i.e.
antibiotics,
antiseptics,
chemotherapeuti
c
[0066] Estimation of AChE inhibitory assay: It is well known fact that in
Alzheimer’s disease (AD) level of acetylcholine gets decreases markedly
21
because of its rapid hydrolysis by acetylcholine esterase enzyme (AChE). The
increased AChE activity around the amyloid plaques causes the aggregation of
amyloid beta peptides into the form of fibrils and thus increases the plaques
cytotoxicity. The result obtained from in vitro AChE inhibitory assay clearly
5 indicates that oil sample has impressive anticholinesterase activity. The result
attained from the assay indicates that the oil sample exhibited better %
inhibition of AChE (62.40±5.07) when compared to standard drug donepezil
(53.08±3.65). Results of in vitro anticholinesterase activity are presented in
Table 3 and shown in Figure 2. Oil sample showed significant inhibition of
10 AChE which may be attributed to its lipophilicity which helped it in crossing
the blood brain barrier effectively and act at brain to exhibit its therapeutic
activity in the management of Alzheimers disease and related disorders.
[0067] Table 3: Effect of oil sample on AChE activity in fish hippocampus
tissue
S. No Groups % Inhibition
1. Donepezil 53.08± 3.65
2. Oil sample 62.40±5.07
15
[0068] Conclusion
This study is first report on extraction of oil from A.foveolus bug and
identification of various compounds from GC/MS analysis. Since lamp light
attract these bugs ( specially during rainy season i.e.:- july-september), dead bugs
20 were collected from the mentioned areas early in the morning (5-6AM) with the
help of broom and pan and collected in a poly bag. In the lab dust was separated
with the help of sieve and samples were washed properly with alcohol. Dead bugs
were collected for the maintenance of ecology, thus not affecting the environment.
Further, this oil was examined for the in vitro anticholinesterase activity which in
25 turn indicated its efficacy in the management of Alzheimer’s disease and related
neurodegenerative disorders. The oil sample indicated the presence of secondary
22
metabolites like alkaloids, saponins, hexose sugar, tannins, phenols, fat,
glycosides, tyrosine and carbohydrates. Further, GC-MS was performed to
identify the different compounds present in oil sample extracted from A.
foveolus.GC-MS indicated that oil sample possesses different compounds with
5 various biological activities. In major, most of the compounds identified from
GC-MS has already been reported as anti-Alzheimer agent.In view of availability
of these key compounds in oil sample, we evaluated the efficacy of oil in AD
using in-vitro assay i.e. AChE inhibitory assay.The result showed that oil
extractedfrom A.foveolus showed impressive inhibition (62.40±5.07) against
10 AChE when compared with donepezil (53.08±3.65), indicating it can be used as
therapeutic agent in the treatment of AD. Thus, future studies on the current topic
are therefore required to elucidate its more efficacious role in the management of
AD.
Although embodiments for the present subject matter have been described in
15 language specific to structural features, it is to be understood that the present
subject matter is not necessarily limited to the specific features described. Rather,
the specific features and methods are disclosed as embodiments for the present
subject matter. Numerous modifications and adaptations of the system/component
of the present invention will be apparent to those skilled in the art, and thus it is
20 intended by the appended claims to cover all such modifications and adaptations
which fall within the scope of the present subject matter.

We Claim:
1. A method of extracting essential oil from dead Aethoscytus
foveolus (Dallas) bug, comprising the steps of:
a. collection of 20 g dead bugs, washing with water, followed by
5 washing with 70% alcohol and again a wash with water;
b. sieving, wrapping with tissue paper and drying the sample,
followed by making a fine powder with liquid nitrogen;
c. boiling the sample in the Soxhelt apparatus with defatting
solvent at boiling point 50-60°C for 6 hours following the AOCS
10 method;
d. centrifuging the sample at 12000-18000 rpm for 10-20 minutes
and
e. collecting the oil from the supernatant and storing at 4°C in a
dark plastic container
15 2. The method as claimed in claim 1, wherein the defatting solvent
is selected from the group n-hexane, petroleum ether and
acetone.
3. The method as claimed in claim 1, wherein the amount of solvent
in step c is 1:5, 100ml for 20g bugs.
20 4. The method as claimed in claim 1, wherein the AOCS method
involves centrifuging twice for the final extraction of oil in
supernatant.
5. The method as claimed in claim 1, wherein the 1:5 ratio of bugs
and solvent leads to 44.50% oil yield.
25 6. A composition of essential oils extracted from the Aethoscytus
foveolus (Dallas) bug, comprising of compounds 2-
Bromotetradecanoic acid, 2-Hexadecanol, Tetratetracontane and
Heptacosane.
7. The composition as claimed in claim 1 or 5, wherein the
30 composition has secondary metabolites- alkaloids, saponins,
24
hexose sugar, tannins, phenols, fat, glycosides, tyrosine and
carbohydrates.
8. The composition as claimed in claim 1 or 5, wherein the
compounds are identified by GC-MSs.
5 9. The composition as claimed in claim 1 or 5, wherein the
composition shows (62.40±5.07) % inhibition of AChE in in
vitro assay.
10. The composition as claimed in claim 1 or 5, wherein the
composition has therapeutic potential against neurodegenerative
10 disorders.