U.S. patent application number 15/621437 was filed with the patent office on 2017-10-05 for anti dengue activity of cissampelos pareira extracts.
The applicant listed for this patent is DEPARTMENT OF BIOTECHNOLOGY, MINISTRY O SCIENCE AND TECHNOLOGY, INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY, SUN PHARMACEUTICAL INDUSTRIES LIMITED. Invention is credited to Pradip Kumar BHATNAGAR, Rajeev DUGGAR, Anil KANAUJIA, Chandra Kant KATIYAR, Seema KHAN, Navin KHANNA, Pawan Kumar PAREEK, Rajendra RAUT, Navin Kumar SHARMA, Gyanesh SHUKLA, Yogendra SINGH, Smita SINGHAL, Ruchi SOOD, Kona SRINIVAS, Sathyamangalam SWAMINATHAN, Dilip Jatashankar UPADHYAY.
Application Number | 20170281704 15/621437 |
Document ID | / |
Family ID | 41831617 |
Filed Date | 2017-10-05 |
United States Patent
Application |
20170281704 |
Kind Code |
A1 |
BHATNAGAR; Pradip Kumar ; et
al. |
October 5, 2017 |
ANTI DENGUE ACTIVITY OF CISSAMPELOS PAREIRA EXTRACTS
Abstract
The present invention relates to the anti dengue activity of the
cissampelos pareira extracts. Pharmaceutical compositions
comprising extracts of cissampelos pareira and processes for the
preparation of extracts of cissampelos pareira are also
provided.
Inventors: |
BHATNAGAR; Pradip Kumar;
(Exton, PA) ; KATIYAR; Chandra Kant; (Gurgaon,
IN) ; KHANNA; Navin; (New Delhi, IN) ;
UPADHYAY; Dilip Jatashankar; (Kalyan, IN) ;
SWAMINATHAN; Sathyamangalam; (Hyderabad, IN) ;
SRINIVAS; Kona; (East Godavari, IN) ; SHARMA; Navin
Kumar; (Northwest Delhi, IN) ; KANAUJIA; Anil;
(Kanpur, IN) ; SOOD; Ruchi; (New Delhi, IN)
; SINGHAL; Smita; (Gautam Buddh Nagar, IN) ;
SHUKLA; Gyanesh; (Jalaun, IN) ; DUGGAR; Rajeev;
(Jodhpur, IN) ; PAREEK; Pawan Kumar; (Bikaner,
IN) ; SINGH; Yogendra; (Gurgaon, IN) ; KHAN;
Seema; (New Delhi, IN) ; RAUT; Rajendra; (New
Delhi, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SUN PHARMACEUTICAL INDUSTRIES LIMITED
INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY
DEPARTMENT OF BIOTECHNOLOGY, MINISTRY O SCIENCE AND
TECHNOLOGY |
Mumbai
New Delhi
New Delhi |
|
IN
IN
IN |
|
|
Family ID: |
41831617 |
Appl. No.: |
15/621437 |
Filed: |
June 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13145732 |
Jan 10, 2012 |
9707261 |
|
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PCT/IB2010/050299 |
Jan 23, 2010 |
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15621437 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/00 20180101;
Y02A 50/30 20180101; A61K 36/59 20130101; Y02A 50/475 20180101;
A61P 31/14 20180101; Y02A 50/385 20180101; A61P 31/12 20180101 |
International
Class: |
A61K 36/59 20060101
A61K036/59 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 2009 |
IN |
141/DEL/2009 |
Claims
1. An extract of cissampelos pareira for treating dengue virus
infection.
2. An extract of claim 1, which is selected from methanol,
methanol:water (50:50) or aqueous extract.
3. An extract of claim 1, which is selected from a fraction of
methanol extract.
4. The fraction of claim 3, which is selected from hexane,
chloroform, dichloromethane, butanol, aqueous, ethyl acetate or
acetone fraction.
5. A pharmaceutical composition comprising an extract of
cissampelos pareira along with one or more of pharmaceutically
acceptable carriers, excipients or diluents for treating dengue
virus infection.
6. A method of treating dengue virus infection comprising
administering a therapeutically effective amount of an extract of
cissampelos pareira.
7. A method of treating dengue virus infection comprising
administering a pharmaceutical composition comprising an extract of
cissampelos pareira along with one or more of pharmaceutically
acceptable carriers, excipients or diluents.
8. Use of an extract of cissampelos pareira in the manufacture of a
medicament for treating dengue virus infection.
9. An extract of cissampelos pareira for treating dengue virus
infection, which is prepared by a process comprising: (a)
extracting the plant mass of cissampelos pareira with one or more
solvents selected from water, alcohols, ketones, esters,
halogenated hydrocarbons, petroleum fractions, or mixture(s)
thereof, (b) concentrating the extract, and (c) drying the
extract.
10. An extract of cissampelos pareira for treating dengue virus
infection, which is prepared by a process comprising: (a)
extracting the plant mass of cissampelos pareira with one or more
solvents selected from water, alcohols, ketones, esters,
halogenated hydrocarbons, petroleum fractions or mixture(s)
thereof, (b) concentrating the extract, (c) adding water, (d)
partitioning the extract with one or more solvents selected from
water, petroleum fractions, halogenated hydrocarbons, esters,
ketones, alcohols, ethers or mixture(s) thereof, and (e) drying the
extract.
11. An extract of cissampelos pareira for treating dengue virus
infection, which is prepared by a process comprising: (a)
extracting the plant mass of cissampelos pareira with one or more
solvents selected from water, alcohols, ketones, esters,
halogenated hydrocarbons, petroleum fractions or mixture(s)
thereof, (b) concentrating the extract, (c) extracting the extract
with one or more solvents selected from one or more solvents
selected from water, alcohols, ketones, esters, halogenated
hydrocarbons, petroleum fractions or mixture(s) thereof, and (d)
drying the extract.
12. A process for the preparation of extracts of cissampelos
pareira, the process comprising: (a) extracting the plant mass of
cissampelos pareira with one or more solvents selected from water,
alcohols, ketones, esters, halogenated hydrocarbons, petroleum
fractions, or mixture(s) thereof, (b) concentrating the extract,
and (c) drying the extract, wherein the extract is used for
treating dengue virus infection.
13. A process for the preparation of extracts of cissampelos
pareira, the process comprising: (a) extracting the plant mass of
cissampelos pareira with one or more solvents selected from water,
alcohols, ketones, esters, halogenated hydrocarbons, petroleum
fractions, or mixture(s) thereof, (b) concentrating the extract,
(c) adding water, (d) partitioning the extract with one or more
solvents selected from water, petroleum fractions, halogenated
hydrocarbons, esters, ketones, alcohols, ethers, or mixture(s)
thereof, and (e) drying the extract, wherein the extract is used
for treating dengue virus infection.
14. A process for the preparation of extracts of cissampelos
pareira, the process comprising: (a) extracting the plant mass of
cissampelos pareira with one or more solvents selected from water,
alcohols, ketones, esters, halogenated hydrocarbons, petroleum
fractions, or mixture(s) thereof, (b) concentrating the extract,
(c) extracting the extract with one or more solvents selected from
one or more solvents selected from water, alcohols, ketones,
esters, halogenated hydrocarbons, petroleum fractions, or
mixture(s) thereof, and (d) drying the extract, wherein the extract
is used for treating dengue virus infection.
15. The process of claim 9, 10, 11, 12, 13 or 14 wherein alcohol is
selected from methanol, ethanol, propanol, isopropanol or butanol,
ketone is selected from acetone or methyl isobutyl ketone, ester is
selected from methyl acetate or ethyl acetate, halogenated
hydrocarbon is selected from chloroform, dichloromethane or
ethylene dichloride, ether is diethyl ether and petroleum fraction
is selected from hexane, petroleum ether or heptane.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the anti dengue activity of
cissampelos pareira extracts. Pharmaceutical compositions
comprising extracts of cissampelos pareira and processes for the
preparation of extracts of cissampelos pareira are also
provided.
BACKGROUND OF THE INVENTION
[0002] Dengue viruses (DEN1-4), mosquito-borne members of the
family Flaviviridae, are human pathogens of global significance. Of
the 1 million annual cases of dengue haemorrhagic fever/dengue
shock syndrome, about 2-5% are fatal. Currently, there is no
vaccine or antiviral drug to treat DEN infections (Ann, N.Y. Acad.
Sci., 951, p. 262-271 (2001), and Clinical Microbiology Reviews,
July, p. 480-496 (1998)).
[0003] Herbal medicines have emerged as a unique approach for
meeting the need for safe, effective and relatively inexpensive new
remedies for a variety of disorders. These represent the fastest
growing segment among all of alternative medicine. The herbal
medicines are produced in different forms, which range from crude,
decocted herbs to refined, concentrated and standardized extracts.
The health benefit from taking those herbals also varies with the
quality of the products and the knowledge of consumers on the
products. Some of the products have to be used under a physician's
supervision, particularly those indicated for serious diseases
although the majority of herbal medicines are generally safe.
[0004] Many plants have been scientifically evaluated on
experimental models as anti-viral agents viz. Acacia nilotica for
inhibitory effects on Hepatitis C virus protease (Hussein et. al.,
Phytotherapy, Res., 14(7) p. 510-16 (2000)), Andrographis
paniculata for HIV-1 inhibitory activity (Reddy et. al., Nat. Prod.
Res., 19(3): p. 223-30, (2005)), Areca catechu for inhibitory
activity on the plaque formation of Herpes simplex virus type-1
(Hattori et. al., Phytotherapy Res., 9, p. 270-276 (1995)), and
also for inhibition of Hepatitis B virus DNA polymerase (Chung et.
al., Phytotherapy Res, 9, p. 429-434 (1995)), Azadirachta indica
for inhibition of replication of Dengue virus type 2 (Parida et.
al., J. Ethnopharmacol., 79, p. 273-78 (2002)), Glycyrrhiza glabra
for inhibition of growth and cytopathology of several unrelated DNA
and RNA viruses and also for inactivating herpes simplex virus
irreversibly (Pompei et. al., Nature, 281(5733): p. 689-90 (1979)),
Ocimum basilicum for inhibition of DNA viruses viz. herpes virus
(HSV); adenovirus (ADV); Hepatitis B and also inhibition of RNA
viruses viz. coxsackie virus B1 (CVB1), enterovirus 71 (EV 71)
(Chiang et. al., Clin. Exp. Pharm. Physiol., 32, p. 811-16 (2005)),
Phyllanthus amarus for suppression of Hepatitis B surface antigen
(HBsAg) gene expression in human hepatoma cells (Yeh et. al.,
Antiviral Res., 20, p. 185-92 (1993)), and for HBsAg clearance
(Thyagarajan et. al., Lancet, p. 764-66 (1988)), and Terminalia
chebula against herpes simplex virus type 1 infection (Kurokawa et.
al., Antiviral Res., 27, p. 19-37 (1995)). Further, antiviral
agents of plant origin, being non-toxic and inexpensive, can have
easy acceptability (Parida et al, J. Ethnopharmacol., 79, p.
273-278 (2002)).
[0005] Though the association of several herbal extracts with
antiviral activity is well documented, however, a systematic search
for anti-DEN virus activity in plant extracts has not been
undertaken so far. It was earlier elucidated that extracts from
different parts of plants may yield a source for antiviral
compounds (Herrmann et. al., Proc. Soc. Exp. Biol. Med., 124, p.
865-74 (1967)). This prompted several workers to undertake a
concerted search for antiviral compounds of plant origin that in
turn culminated in a report which showed that a number of plants
exhibited efficacy to suppress the growth of several viruses (Aswal
et. al., Ind. J. Exp. Biol., 34, p. 444-67 (1996)).
[0006] Cissampelos pareira Linn (Family: Menispermaceae, English
Name: Velvet Leaf, Hindi Name: Patha, Sanskrit Name: Ambasthaki) is
a climbing shrub distributed throughout the warm parts of Asia,
East Africa and America and common in India and Ceylon. It is
common in warm and dry regions of tropical and sub-tropical parts
of India up to an altitude of about 1500 m. It is found in Himachal
Pradesh, Chota Nagpur, Bihar, West Bengal, Punjab, Rajasthan
particularly in the east of Aravalli, hilly forests of Marathwada,
Konkan, Deccan, Bababuden hills of Mysore, Tamil Nadu (Ayurvedic
Pharmacopoeia of India, First Edition, Part 1, Vol 1, p. 92-93;
Govt of India, Ministry of Health and Family Welfare, Dept of
Indian System of Medicine and Homoeopathy, New Delhi; The Wealth of
India, A Dictionary of Indian Raw Materials and Industrial
Products, Raw Materials, Vol II, Council of Scientific and
Industrial Research, Delhi; Database on Medicinal Plants Used In
Ayurveda, Vol 2, Central Council for Research in Ayurveda and
Siddha, Dept of Indian System of Medicine and Homoeopathy, New
Delhi).
[0007] Cissampelos pareira Linn is an Ayurvedic medicinal plant
used traditionally for the treatment of a number of diseases. It is
said to be bitter, astringent, anthelmintic, carminative,
stomachic, digestive, anti-inflammatory, diuretic, febrifuge,
expectorant, galactogogue and bitter tonic. It is useful in
dyspepsia, abdominal pain, diarrhoea, dysentery, fever, cough,
coryza, asthma and lactation disorders (Database on Medicinal
Plants Used In Ayurveda, Vol 2, Central Council for Research in
Ayurveda and Siddha, Dept of Indian System of Medicine and
Homoeopathy, New Delhi).
[0008] Dengue fever is not mentioned as such in the Ayurvedic
classical textbooks; however, many types of "Jwara" have been
enumerated in these textbooks along with their signs and symptoms.
It is often difficult to correlate one type of "Jwara" as a
clinical counter-part of a particular type of "fever" mentioned in
the contemporary science. In classical Ayurvedic textbooks, some of
the signs and symptoms of Dengue fever do correlate with a type of
fever referred to as viz. "Vata-Pittaja Jwara". The plants which
have been mentioned to be used in a condition known as
"Vata-Pittaja Jwara", "Vataj Jwara" and "Pittaj Jwara" in the
classical textbooks of Ayurveda were selected and their Ayurvedic
attributes were studied which basically falls under the headings
viz. "Rasa"; "Guna"; "Veerya"; "Vipaaka"; and "Dosha-Karma". All
these attributes were thoroughly studied for each and every plant
and a hypothesis was developed wherein it was postulated that any
herb which at least possess viz. Tikta and/or Kashaya Rasa, Laghu
and/or Tikshna Guna, Ushna or Sheeta Veerya, having Katu Vipaaka
and Vata and/or Pitta Shaamaka would be useful in alleviating signs
and symptoms correlating with Dengue fever. Some of the attributes
viz. Katu Rasa and Ruksha Guna were in addition to the attributes
which were proposed in the hypothesis. Therefore, on the basis of
the proposed hypotheses and the presence of proposed, as well as,
additional Ayurvedic attributes thereupon, cissampelos pareira Linn
(Ambasthaki/Patha) was selected to be undertaken for studying its
therapeutic effect in Dengue Fever.
SUMMARY OF THE INVENTION
[0009] In one aspect of the invention, there are provided extracts
of cissampelos pareira.
[0010] In another aspect of the invention, a pharmaceutical
composition comprising an extract of cissampelos pareira along with
one or more of pharmaceutically acceptable carriers, excipients or
diluents is provided.
[0011] In another aspect of the invention, a process for the
preparation of extracts of cissampelos pareira is provided.
[0012] In another aspect of the invention, there is provided a
method of treating dengue virus infection.
[0013] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects and
advantages of the invention will be apparent from the
description.
DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows flow diagram for the bioassay guided
fractionation process.
[0015] FIG. 2 shows anti-dengue activity of methanolic extract of
c. pareira against Dengue serotype 3 (Den-3) by Virus titer
reduction Assay.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention provides extracts of cissampelos
pareira, wherein the extracts have anti dengue activity.
[0017] A bioassay guided fractionation approach for the plant
material leading to the identification of active extracts and
fractions is provided. The process includes preparing different
extracts of cissampelos pareira, subjecting the extracts for the
bioactivity (primary screening--conventional Plaque Reduction
Neutralization Test (PRNT) Assay, secondary screening--modified
Plaque Reduction Neutralization Test (PRNT) Assay and tertiary
screening--Virus Titer Reduction Assay). Active extracts were
further subjected to fractionation by one or more of solvents and
each fraction was evaluated for the bioactivity.
[0018] The one or more solvents for extraction may be, for example,
water; alcohols, for example, methanol, ethanol, propanol,
isopropanol or butanol; ketones, for example, acetone or methyl
isobutyl ketone; esters, for example, methyl acetate or ethyl
acetate; halogenated hydrocarbons, for example, chloroform,
dichloromethane or ethylene dichloride; petroleum fractions, for
example, hexane, petroleum ether or heptane; or mixture(s)
thereof.
[0019] The one or more solvents for fractionation may be, for
example, water; petroleum fractions, for example, hexane, petroleum
ether or heptane; halogenated hydrocarbons, for example,
chloroform, dichloromethane or ethylene dichloride; esters, for
example, ethyl acetate or methyl acetate; ketones, for example,
acetone or methyl isobutyl ketone; alcohols, for example, butanol;
ethers, for example, diethyl ether; or mixture(s) thereof.
[0020] In another aspect of the invention, processes for the
preparation of extracts from cissampelos pareira are provided. The
processes include extracting the plant mass of cissampelos pareira
with one or more solvents from non polar to polar range,
concentrating the extract, and drying the extract, or extracting
the plant mass of cissampelos pareira with one or more solvents
from non polar to polar range, concentrating the extract, adding
water and partitioning the extract with one or more solvents from
non polar to polar range, and drying the extract, or extracting the
plant mass of cissampelos pareira with one or more solvents from
non polar to polar range, concentrating the extract, extracting the
extract with one or more solvents from non polar to polar range,
and drying the extract.
[0021] The solvents for extraction may be, for example, water;
alcohols, for example, methanol, ethanol, propanol, isopropanol or
butanol; ketones, for example, acetone or methyl isobutyl ketone;
esters, for example, methyl acetate or ethyl acetate; halogenated
hydrocarbons, for example, chloroform, dichloromethane or ethylene
dichloride; petroleum fractions, for example, hexane, petroleum
ether or heptane; or mixture(s) thereof.
[0022] The solvents for partitioning may be, for example, water;
petroleum fractions, for example, hexane, petroleum ether or
heptane; halogenated hydrocarbons, for example, chloroform,
dichloromethane or ethylene dichloride; esters, for example, ethyl
acetate or methyl acetate; ketones, for example, acetone or methyl
isobutyl ketone; alcohols, for example, butanol; ethers, for
example, diethyl ether; or mixture(s) thereof.
[0023] In another aspect of the invention, there is provided a
method of treating dengue virus infection.
[0024] Pharmaceutical compositions comprising extracts of
cissampelos pareira, along with one or more of pharmaceutically
acceptable carriers, excipients or diluents are provided, which may
be administered to a mammal for treatment of dengue virus infection
by any route, which effectively transports the active compound to
the appropriate or desired site of action such as oral, nasal,
pulmonary, transdermal or parenteral (rectal, subcutaneous,
intravenous, intraurethral, intramuscular or intranasal). The
choice of pharmaceutical carrier, excipient or diluent can be made
with regard to the intended route of administration and standard
pharmaceutical practice.
[0025] The extracts of cissampelos pareira include (a) the extracts
obtained by extraction of plant mass of cissampelos pareira with
one or more solvents, and (b) the fractions obtained by
partitioning of the extracts of step (a) with one or more
solvents.
[0026] "Plant mass of cissampelos pareira" refers to whole plant,
which includes, aerial parts, for example, fruits, flowers, seeds,
leaves, branches, stem bark, stem or heartwood, and root.
[0027] While the following examples are provided to certain
embodiments of the invention, these are not intended to be limiting
to the scope of the invention.
EXAMPLE 1
Preparation of Methanol Extract
[0028] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at a temperature ranging from
room temperature to the boiling point of the solvent for about 16
hours. The extract was filtered and stored in a container. Again,
300 liters of methanol was added into the extractor and extraction
was done at temperature ranging from room temperature to the
boiling point of the solvent for about 16 hours. The extract was
filtered and stored in a container. Again, 300 liter of methanol
was added into the extractor and extraction was done at temperature
ranging from room temperature to the boiling point of the solvent
for about 16 hours. The methanolic extracts were combined and
concentrated to maximum under reduced pressure at low temperature.
The extract was down loaded into stainless steel trays (ss) and
dried in high vacuum oven at room temperature for about 16 hours to
18 hours. [0029] Yield=6%-15%
EXAMPLE 2
Preparation of (Methanol:Water: 50:50) Extract
[0030] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. A mixture of methanol:water (250
liter:250 liter) was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, a mixture of methanol:water (150
liter:150 liter) was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, a mixture of methanol:water (150
liter:150 liter) was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The hydro alcoholic
extracts were combined and concentrated to maximum under reduced
pressure at low temperature, down loaded the extract into ss trays
and dried in high vacuum oven at room temperature for about 16
hours to 18 hours. [0031] Yield=10%-25%
EXAMPLE 3
Preparation of Aqueous Extract
[0032] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Water (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, water
(300 liter) was added into the extractor and extraction was done at
temperature ranging from room temperature to the boiling point of
the solvent for about 16 hours. The extract was filtered and stored
in a container. Again, water (300 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The aqueous extracts were combined and concentrated to maximum
under reduced pressure at low temperature, down loaded the extract
into ss trays and dried in high vacuum oven at room temperature for
about 16 hours to 18 hours. [0033] Yield=15%-30%
EXAMPLE 4
Preparation of Hexane Fraction
[0034] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 300
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 300 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
suspended in water (50 liter) and partitioned with hexane (40
liter). Hexane layer was separated and collected in a container.
The process was repeated for three more times and combined hexane
layer was dried over a drying agent. It was concentrated under
reduced pressure and down loaded into ss trays and dried in high
vacuum oven at room temperature for about 16 hours to 18 hours.
[0035] Yield=1.0%-3.0%
EXAMPLE 5
Preparation of Chloroform Fraction
[0036] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 300
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 300 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
suspended in water (50 liter) and partitioned with hexane (40
liter). The hexane layer was separated and collected in a
container. The process was repeated for three more times and
combined hexane layer was dried over a drying agent and
concentrated. The remaining aqueous layer was partitioned with
chloroform (40 liter) and chloroform layer was separated and
collected in a container. The process was repeated for three more
times and the combined chloroform layer was passed through a drying
agent. The chloroform layer was concentrated under reduced pressure
and down loaded into ss trays and dried in high vacuum oven at room
temperature for about 16 to 18 hours. [0037] Yield=0.20%-1.0%
EXAMPLE 6
Preparation of Dichloromethane Fraction
[0038] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 300
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 300 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
suspended in water (50 liter) and partitioned with hexane (40
liter) and hexane layer was separated and collected in a container.
The process was repeated for three more times and combined hexane
layer was dried over a drying agent and concentrated. The aqueous
layer was partitioned with dichloromethane (40 liter) and
dichloromethane layer was separated and collected in a container.
The process was repeated for three more times and the combined
dichloromethane layer was passed through a drying agent. The
dichloromethane layer was concentrated under reduced pressure and
down loaded into ss trays and dried in high vacuum oven at room
temperature for about 16 hours to 18 hours. [0039]
Yield=0.22%-1.2%
EXAMPLE 7
Preparation of Dichloromethane Fraction
[0040] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 300
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 300 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
suspended in water (50 liter) and partitioned with dichloromethane
(40 liter) and dichloromethane layer was separated and collected in
a container. The process was repeated for three more times and the
combined dichloromethane layer was passed through a drying agent.
The dichloromethane layer was concentrated under reduced pressure
and down loaded into ss trays and dried in high vacuum oven at room
temperature for about 16 hours to 18 hours. [0041]
Yield=1.0%-1.75%
EXAMPLE 8
Preparation of Butanol Fraction
[0042] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 300
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 300 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
suspended in water (50 liter) and partitioned with hexane (40
liter) and hexane layer was separated and collected in a container.
The process was repeated for three more times and the combined
hexane layer was dried over a drying agent and concentrated.
Aqueous layer was partitioned with chloroform (40 liter) and
chloroform layer was separated and collected in a container. The
process was repeated for three more times and the combined
chloroform layer was passed through a drying agent and
concentrated. Aqueous layer was partitioned with butanol (40 liter)
and the butanol layer was separated and collected in a container.
The process was repeated for three more times and the combined
butanol layer was dried over a drying agent. The butanol layer was
concentrated under reduced pressure and down loaded into ss trays
and dried in high vacuum oven at room temperature for about 16
hours to 18 hours. [0043] Yield=2.0%-4.5%
EXAMPLE 9
Preparation of Aqueous Fraction
[0044] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 200
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 200 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
suspended in water (50 liter) and partitioned with hexane (40
liter) and hexane layer was separated and collected in a container.
The process was repeated for three more times and combined hexane
layer was dried over a drying agent and concentrated. The aqueous
layer was partitioned with chloroform (40 liter) and the chloroform
layer was separated and collected in a container. The process was
repeated for three more times and the combined chloroform layer was
passed through a drying agent and concentrated. The aqueous layer
was partitioned with butanol (40 liter) and the butanol layer was
separated and collected in a container. The process was repeated
for three more times and the combined butanol layer was dried over
a drying agent and concentrated. The exhausted aqueous layer was
concentrated under reduced pressure and down loaded into ss trays
and dried in high vacuum oven at room temperature for about 16
hours to 18 hours. [0045] Yield=2.5%-6.0%
EXAMPLE 10
Preparation of Ethyl Acetate Fraction
[0046] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 300
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 300 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
suspended in water (50 liter) and partitioned with ethyl acetate
(40 liter) and the ethyl acetate layer was separated and collected
in a container. The process was repeated for three more times and
the combined ethyl acetate layer was dried over a drying agent.
Ethyl acetate layer was concentrated under reduced pressure and
down loaded into ss trays and dried in high vacuum oven at room
temperature for about 16 hours to 18 hours. [0047]
Yield=2.0%-4.0%
EXAMPLE 11
Preparation of Acetone Fraction
[0048] Pulverized cissampelos pareira aerial parts (100 kg) were
charged into the extractor. Methanol (500 liter) was added into the
extractor and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours.
The extract was filtered and stored in a container. Again, 300
liter of methanol was added into the extractor and extraction was
done at temperature ranging from room temperature to the boiling
point of the solvent for about 16 hours. The extract was filtered
and stored in a container. Again, 300 liter of methanol was added
into the extractor and extraction was done at temperature ranging
from room temperature to the boiling point of the solvent for about
16 hours. The methanolic extracts were combined and concentrated to
maximum under reduced pressure at low temperature. The extract was
extracted with acetone (50 liter) at temperature ranging from room
temperature to the boiling point of the solvent for about 2 hours
to 3 hours. The extract was filtered and stored in a container. The
process was repeated for three more times. The extract was down
loaded into ss trays and dried in high vacuum oven at room
temperature for about 16 hours to 18 hours. [0049]
Yield=2.0%-3.5%
EXAMPLE 12
Biological Activity
[0049] [0050] (i) Primary Screening--Conventional Plaque Reduction
Neutralization Test (PRNT) Assay
[0051] The antiviral activity of extracts was assayed using the
PRNT assay (Khanam et. al., Am. J. Trop. Med. Hyg., 74(2) p.
266-277 (2006), LLCMK2 cells (monkey kidney cell line) in 24 well
format were seeded at a concentration of 1.times.10.sup.5/well.
Infection was done with approximately 50 plaque forming units (PFU)
of dengue virus (DENV) (all four serotypes DENV-1, DENV-2, DENV-3,
and DENV-4) at a multiplicity of infection (MOI) of 1. Viruses were
separately pre-incubated with an equal volume of extracts in the
range of 100-0.33 mcg/ml. After overnight preincubation at
4.degree. C., the viruses/extracts were diluted to a final volume
of 200 .mu.L with Dulbecco's Modified Eagles Medium (DMEM) plus 2%
heat inactivated Fetal calf serum (.DELTA.FCS) and used to infect a
single well of 24-well plate. Each dilution was assayed in
duplicate wells and the virus/extract preincubation mixture was
prepared as a master mixture sufficient for two wells. After
adsorption for two hours, the inoculum was aspirated off and the
cells were overlaid with 1.25% methylcellulose in DMEM plus 6%
.DELTA.FCS (1 mL/well). Appropriate controls were set up in
parallel with negative control (mock--not infected) wells receiving
200 .mu.L of DMEM plus 2% .DELTA.FCS and positive control wells
receiving (DEN-1, 2, 3 and 4 viruses separately), pre-incubated
with DMEM plus 2% .DELTA.FCS, instead of extracts. Plates were
incubated at 37.degree. C. in a humidified 5% CO.sub.2 incubator.
On day 6 post-infection, the cells were fixed with 1 mL of 4%
formaldehyde solution at room temperature for two hours. Wells were
washed with tap water and then stained with 1:40 diluted stock of
2% crystal violet solution in 20% ethanol for 30 minutes. Plaques
revealed after staining were counted and the extract dilution
resulting in 50% reduction in plaque count (with reference to the
number of plaques generated by the virus in the absence of
extract), was expressed as PRNT.sub.50 titer.
[0052] Methanolic extract showed activity against all the four
serotypes of Dengue in conventional assay with PRNT.sub.50 values
in the range of 1.2-11.1 mcg/ml. PRNT.sub.50 values of
hydroalcoholic and aqueous extracts in conventional assay against
all the serotypes were >100 mcg/ml. [0053] (ii) Secondary
Screening--Modified Plaque Reduction Neutralization Test (PRNT)
Assay
[0054] LLCMK2 cells in 24 well format were seeded at a
concentration of 1.times.10.sup.5/well. Seeded cells were infected
with approximately 50 .mu.l of virus with 50 (1.times.) plaque
forming units (PFU) of all 4 serotypes at a MOI of 1. After
adsorption for two hours at 37.degree. C. and 5% CO.sub.2, the
inoculum was aspirated. Wells were washed with phosphate buffered
saline (PBS) and infected cells were exposed to different
concentrations of extracts (200-0.82 mcg/ml) for a period of 24
hours. After 24 hours of incubation at 37.degree. C. and 5%
CO.sub.2, extract was aspirated out and the cells were overlaid
with 1% methylcellulose in DMEM plus 6% .DELTA.FCS (1 ml/well).
Each dilution was assayed in duplicate wells. Plates were incubated
at 37.degree. C. in a humidified 5% CO.sub.2 incubator for 6 days.
On day 6 post-infection, the overlay was gently decanted and the
cells were fixed with 1 mL of 4% formaldehyde solution at room
temperature for one hour. Wells were washed with tap water and then
stained with 1:40 diluted stock of 2% crystal violet solution in
20% ethanol for 30 minutes. Plaques revealed after staining were
counted and the extract dilution resulting in 50% reduction in
plaque count (with reference to the number of plaques generated by
the virus in the absence of extract), was expressed as the
PRNT.sub.50 titer.
[0055] Methanolic extract showed PRNT.sub.50 values in the range of
78-125 mcg/ml in modified assay against all the four serotypes.
However, hydroalcoholic and aqueous extracts were inactive up to
200 mcg/ml. [0056] (iii) Tertiary Screening--Virus Titer Reduction
Assay
[0057] Virus Titer Reduction Assay was performed by following
Puig-Basagoiti et al., Antimic. Agents and Chemotherapy, 50(4) p.
1320-1329 (2006). Vero cells in 24 well format were seeded at a
concentration of 1.times.10.sup.5/well. Seeded cells were infected
with approximately 50 .mu.l of virus with 50 (1.times.) plaque
forming units (PFU) of all 4 serotypes at a MOI of 1. After
adsorption for two hours at 37.degree. C. and 5% CO.sub.2, the
inoculum was aspirated. Wells were washed with PBS and infected
cells were exposed to growth medium (DMEM plus 2% .DELTA.FCS) with
different concentrations of extracts (200-0.82 mcg/ml) along with
negative control (mock--not infected) wells receiving DMEM plus 2%
.DELTA.FCS and positive control wells receiving DEN-3 viruses, for
a period of 9 days. 20 .mu.l of supernatant sample was withdrawn at
different days intervals, i.e., 0, 3, 6, 9 days and frozen down
till further processing.
[0058] The aliquoted samples were serially diluted in DMEM plus 2%
.DELTA.FCS medium prior to addition to the wells containing
confluent cells (in duplicate or triplicate). The viruses were
allowed to adsorb for two hours, followed by washing with
phosphate-buffered saline. After adsorption for two hours, the
inoculum was aspirated off and the cells were overlaid with 1.25%
methylcellulose in DMEM plus 6% .DELTA.FCS (1 mL/well). The plates
were incubated at 37.degree. C. in a humidified 5% CO.sub.2
incubator for 6 days. On day 6 post-infection, the overlay was
gently decanted and the cells were fixed with 1 mL of 4%
formaldehyde solution at room temperature for one hour. Wells were
washed with tap water and then stained with 1:40 diluted stock of
2% crystal violet solution in 20% ethanol for 30 minutes. Plaques
revealed after staining were counted and log reduction in plaque
titer at different time points vs different tested concentrations
were calculated as compared to virus control.
[0059] Kill kinetic profile of methanolic extract indicated a 2 log
reduction on day 3 at 66.66 mcg/ml against Den-3 serotype. On day
6, at 2.46 and 7.4 mcg/ml concentration of extract, viral load was
reduced by 1-1.5 log. [0060] (iv) Cytotoxicity Assays
[0061] Cytotoxicity of methanolic extract was assessed by
incubating HEpG-2 cells (Human liver hepatoma cell line) with a six
point dilution of extract (concentration range of 200 mcg/ml-0.82
mcg/ml) in culture medium RPMI (Roswell Park Memorial Institute) 2%
.DELTA.FCS in 96-well plates for 3 days, corresponding to the
incubation period of cells with extracts in the primary and
secondary screens. After 72 hours, cell viability was measured by
cellular metabolism of MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
(Markland W. et al., Antimic. Agents and Chemotherapy, 44(4) p.
859-866 (2000)). Growth index (GI) 50 was found to be 90
mcg/ml.
* * * * *