U.S. patent application number 11/927923 was filed with the patent office on 2008-07-31 for agent and compositions comprising the same for inhibiting lipases and phospholipases in body fluids, cells and tissues.
This patent application is currently assigned to Reliance Life Sciences Pvt. Ltd.. Invention is credited to Arif Ansari, S. Harinarayana Rao, Shakti N. Upadhyay, Raman P. Yadav.
Application Number | 20080182787 11/927923 |
Document ID | / |
Family ID | 36640663 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080182787 |
Kind Code |
A1 |
Upadhyay; Shakti N. ; et
al. |
July 31, 2008 |
AGENT AND COMPOSITIONS COMPRISING THE SAME FOR INHIBITING LIPASES
AND PHOSPHOLIPASES IN BODY FLUIDS, CELLS AND TISSUES
Abstract
The present invention deals with a protein and compositions
comprising the same for inhibition of lipases and phospholipases in
the body fluids, cells, and tissues for the prevention and
treatment of metabolic syndrome, cardiovascular disorders, and
inflammatory diseases. The protein is either isolated from plant
species or synthesized or produced by recombinant DNA
technology.
Inventors: |
Upadhyay; Shakti N.; (Navi
Mumbai, IN) ; Yadav; Raman P.; (Navi Mumbai, IN)
; Ansari; Arif; (Navi Mumbai, IN) ; Rao; S.
Harinarayana; (Navi Mumbai, IN) |
Correspondence
Address: |
LACKENBACH SIEGEL, LLP
LACKENBACH SIEGEL BUILDING, 1 CHASE ROAD
SCARSDALE
NY
10583
US
|
Assignee: |
Reliance Life Sciences Pvt.
Ltd.
Mumbai
IN
|
Family ID: |
36640663 |
Appl. No.: |
11/927923 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11093637 |
Mar 30, 2005 |
7368529 |
|
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11927923 |
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60557624 |
Mar 30, 2004 |
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Current U.S.
Class: |
514/1.4 ;
514/1.9; 514/12.2; 514/16.6; 514/18.2; 514/6.9; 514/7.4;
530/324 |
Current CPC
Class: |
A61P 9/10 20180101; C07K
14/415 20130101; C12N 15/8247 20130101; A61P 3/10 20180101; A61P
19/02 20180101; A61P 29/00 20180101; A61K 38/00 20130101; A61P
25/16 20180101; A61P 9/00 20180101; A61P 3/04 20180101 |
Class at
Publication: |
514/12 ;
530/324 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; A61P 3/04 20060101
A61P003/04; A61P 3/10 20060101 A61P003/10; A61P 9/10 20060101
A61P009/10; A61P 19/02 20060101 A61P019/02; A61P 29/00 20060101
A61P029/00; A61P 9/00 20060101 A61P009/00; A61P 25/16 20060101
A61P025/16 |
Claims
1. A protein comprising 5-100 amino acid residues and having a
molecular weight ranging from 0.5-10 kD, with or without
glycosylation, the protein having an inhibitory or reducing effect
on lipase and phospholipase enzyme activities wherein the protein
has partial sequence ID as following SEQ. ID. NO. 1:
CGQQLRNISPPQRCPSLRQAVQLAHQQQGQGPQQVRQMYR, and is produced using any
suitable method of protein isolation, or synthesized or produced
through recombinant DNA technology.
2-3. (canceled)
4. The protein of claim 1, wherein the protein is isolated from
plant material.
5. The protein of claim 1, wherein the protein is isolated from the
Moringa species.
6. A composition for inhibition of lipases and phospholipases in
the body fluids, cells, and tissues comprising: a protein as
claimed in claim 1 in a therapeutically effective amount.
7. The composition of claim 6, wherein the protein is produced
using any suitable method of protein isolation, or synthesized or
produced through recombinant DNA technology.
8. The composition of claim 6, wherein the protein is isolated from
plant material.
9. The composition of claim 6, wherein the protein is isolated from
the Moringa species.
10. A composition for preventing or treating obesity, diabetes,
atherosclerosis, or another metabolic syndrome, the compositions
comprising a protein as claimed in claim 1.
11. The composition of claim 10, wherein the protein is produced
using any suitable method of protein purification, or synthesized
or produced through recombinant DNA technology.
12. The composition of claim 9, wherein the protein is isolated
from plant material.
13. The composition of claim 9, wherein the protein is isolated
from the Moringa species.
14. A composition for preventing or treating cardiovascular
disorders comprising a protein as claimed in claim 1.
15. The composition of claim 13, wherein the protein is produced
using any suitable method of protein purification, or synthesized
or produced through recombinant DNA technology.
16. The composition of claim 13, wherein the protein is isolated
from plant material.
17. The composition of claim 13, wherein the protein is isolated
from the Moringa species.
18. A composition for preventing or treating arthritis,
atherosclerosis, septic shock, and other inflammatory diseases
caused by the activation and/or the action of phospholipases, the
composition comprising a protein as claimed in claim 1.
19. The composition of claim 17, wherein the protein is produced
using any suitable method of protein purification, or synthesized
or produced through recombinant DNA technology.
20. The composition of claim 17, wherein the protein is isolated
from plant material.
21. The composition of claim 17, wherein the protein is isolated
from the Moringa species.
22. A composition for inhibiting or reducing accumulation of lipids
in monocytic cells, vascular cells, hepatocytes, and adipose
tissues, the composition comprising a protein as claimed in claim
1.
23. The composition of claim 21, wherein the protein is produced
using any suitable method of protein purification, or synthesized
or produced through recombinant DNA technology.
24. The composition of claim 21, wherein the protein is isolated
from plant material.
25. The composition of claim 21, wherein the protein is isolated
from the Moringa species.
26. A composition for preventing or treating cellular and tissue
damage caused by microbial pathogens secreting lipases and
phospholipases, the composition comprising a protein as claimed in
claim 1.
27. The composition of claim 25, wherein the protein is produced
using any suitable method of protein purification, or synthesized
or produced through recombinant DNA technology.
28. The composition of claim 25, wherein the protein is isolated
Previously Presented from plant material.
29. The composition of claim 25, wherein the protein is isolated
from the Moringa species.
30. A composition for skin and hair care and cosmetic Previously
Presented preparations comprising a protein as claimed in claim
1.
31. The composition of claim 29, wherein the protein is produced
using any suitable method of protein purification, or synthesized
or produced through recombinant DNA technology.
32. The composition of claim 29, wherein the protein is isolated
from plant material.
33. The composition of claim 29, wherein the protein is isolated
from the Moringa species.
34. A composition for use in veterinary medicine for the treatment
and prophylaxis of diseases caused or aggravated by lipase and
phospholipase activity in the body fluids, cells, and tissues, the
composition comprising a protein as claimed in claim 1.
35. The composition of claim 33, wherein the protein is produced
using any suitable method of protein purification, or synthesized
or produced through recombinant DNA technology.
36. The composition of claim 33, wherein the protein is isolated
from plant material.
37. The composition of claim 33, wherein the protein is isolated
from the Moringa species.
38. A formulation comprising of protein as claimed in claim 1
either alone or in combination with known pharmaceutically
acceptable and inert adjuvant, diluent or carrier.
39. The formulation comprising of protein as claimed in claim 1
either alone or in combination with at least one additional active
ingredient.
40. The formulation comprising of protein as claimed in claim 1 may
be administered once or a few times a day in an amount of about 10
to 2000 mg/day in terms of dry weight.
41. The formulation as claimed in claim 37, wherein the formulation
is adapted to be administered via a route selected from the group
consisting of: oral, intra-venous, intra-nasal, and
transdermal.
42. The formulation as claimed in claim 37, wherein the suitable
forms for oral administration include tablets, capsules, granules,
fine granules, spherules, syrups, and drinks, more preferably it is
in the form of spherules.
43. The formulation as claimed in claim 37, wherein the formulation
can be administered by following conventional dosage regimen.
44. The formulation as claimed in claim 37, wherein the formulation
can be administered by any modified release, controlled release or
timed release formulations.
45. A method for inhibition of lipases and phospholipases in the
body fluids, cells, and tissues comprising the step of
administering to a patient in need thereof a composition comprising
an effective amount of a protein as claimed in claim 4.
46. A method for inhibiting or reducing accumulation of lipids in
monocytic cells, vascular cells, hepatocytes, and adipose tissues,
the method of comprising the step of administering to a patient in
need thereof a composition comprising an effective amount of a
protein as claimed in claim 4.
47. A method for preventing or treating obesity, diabetes,
atherosclerosis, or another metabolic syndrome, the method
comprising the step of administering to a patient in need thereof a
composition comprising an effective amount of a protein as claimed
in claim 4.
48. A method for preventing or treating arthritis, septic shock,
and other inflammatory diseases caused by the activation and/or the
action of phospholipases, the method comprising the step of
administering to a patient in need thereof a composition comprising
an effective amount of a protein as claimed in claim 4.
49. (canceled)
50. A method for preventing and treating cellular and tissue damage
comprising administering about 10 to 2000 mg/day dry weight amount
of a protein at least once a day to a patient for the treatment of
cellular damage caused by microbial pathogens secreting lipases and
tissue damage caused by microbial pathogens secreting lipases and
phospholipases, wherein the protein is SEQ. ID. NO. 1:
TABLE-US-00009 GQQLRNISPPQRCPSLRQAVQLAHQQQGQGPQQVRQMYR
and including from about 40 to 100 amino acid residues.
51. A method as claimed in claim 50, for inhibition or reducing an
accumulation of lipids in the body comprising administering an
effective amount of the protein to a patient in need of a treatment
for lipid reduction.
52. The method as claimed in claim 50, for prevention or treatment
of increased lipase activity in the body comprising administering
an effective amount of the protein to a patient for treating
diseases or disorders with increased lipase activity.
53. The method as claimed in claim 50, for the prevention or
treatment of inflammation in the body comprising administering an
effective amount of the protein to a patient for treating
inflammatory diseases caused by the activation and/or the action of
phospholipases.
54. A method of treating disorders selected from the group
consisting of obesity, ischemic heart diseases, arteriosclerosis,
cerebrovascular dementia, diabetes, angiopathic Parkinson's
diseases, and inflammatory diseases requiring the use of a protein
for the inhibition of lipases, comprising: providing a person in
need thereof with an amount of about 10 to 2000 mg/day, dry weight
at least once a day of a protein for the inhibition of lipases
having a partial sequence ID as follows: TABLE-US-00010 SEQ. ID.
NO. 1: GQQLRNISPPQRCPSLRQAVQLAHQQQGQGPQQVRQMYR
and the protein consists essentially of 40 to 100 amino acid
residues; and wherein the protein is produced by using any suitable
method of protein isolation from plant material or method of
synthesis through recombinant DNA technology.
Description
[0001] (This invention claims priority from Provisional U.S. patent
Application with Ser. No. 60/557,624 filed on 30th Mar. 2004)
1. FIELD OF THE INVENTION
[0002] The present invention relates to a protein and composition
comprising the protein for inhibiting or reducing lipases and
phospholipase enzymes in body fluids, cells, and tissues. The
protein as described in the present invention and the composition
comprising of the same are useful for the prevention or treatment
of clinical manifestations and diseases caused as a consequence of
lipase and phospholipase enzyme activities in the body fluids,
cells, and tissues.
2. DESCRIPTION OF THE PRIOR ART
[0003] Lipases and phospholipases are key control elements in
mammalian metabolism. They share many common features that set them
apart from other metabolic enzyme classes, most importantly their
association with "two-dimensional" substrates, i.e., lipid
droplets, lipoproteins, phospholipid layers, biomembranes, and the
resulting implications for their cleavage mechanism and
regulation.
[0004] The pancreatic lipase (PL) is believed to be effective in
causing a partial hydrolysis of triglycerides to obtain fatty acids
and monoglycerides that, together with the bile acids, form
complexes, which are then absorbed through the intestinal mucosa.
Hepatic lipase (HL) and lipoprotein lipase (LPL) are the two major
lipolytic enzymes responsible for the hydrolysis of triglycerides
and phospholipids present in circulating plasma lipoproteins. Both
lipases are attached to the vascular endothelium via cell surface
proteoglycans. HL is primarily involved in the metabolism of
chylomicron remnants, intermediate density lipoproteins, and
high-density lipoproteins, whereas LPL catalyzes the hydrolysis of
triglycerides from chylomicrons and very low-density lipoproteins.
In addition to their traditional function as lipolytic enzymes, HL
and LPL appear to serve as ligands that mediate the interaction of
lipoproteins to cell surface receptors and/or proteoglycans.
[0005] Accumulation and distribution of triglyceride-rich
lipoprotein-associated fatty acids at extra-hepatic sites is
facilitated by LPL. The enzyme is also involved in several
non-lipolysis associated functions, including the cellular uptake
of whole lipoprotein particles and lipophilic vitamins. The
tissue-specific variations of LPL expression have been implicated
in the pathogenesis of various lipid disorders, obesity, and
atherosclerosis. LPL expressed by cells of the vascular wall,
particularly macrophages, have identified additional actions of the
enzyme that contribute to the promotion of foam cell formation and
atherosclerosis. Development of drugs specifically acting on the
cholesteryl ester transfer protein and lipoprotein lipase systems,
are being explored.
[0006] Over the past several years significant advances have been
made in our understanding of new, alternative mechanisms by which
HL and LPL modulate lipoprotein metabolism and the development of
atherosclerosis in vivo. Advances have also been made in our
understanding of the intravascular metabolism of triglyceride-rich
lipoproteins. It is now known that the complex extracellular
interactions of triglyceride-rich lipoprotein-associated
apolipoprotein E, lipoprotein lipase, and hepatic lipase with
heparan sulfate proteoglycans and lipoprotein receptors facilitate
the hepatocellular uptake of triglyceride-rich lipoproteins. Recent
studies have also revealed that the intracellular fate of
internalized triglyceride-rich lipoproteins is highly complex. The
dissociation of triglyceride-rich lipoprotein components within
intracellular endosomal compartments involves the recycling of
apolipoprotein E, whereas the remaining lipid core associated with
apolipoprotein B is susceptible to lysosomal degradation.
[0007] The high incidence of atherosclerosis in diabetic patients
has been correlated with LPL activity in macrophages. Accumulating
evidence indicates that LPL produced by macrophages in the vascular
wall may favor the development of atherosclerosis by promoting
lipid accumulation within the lesion.
[0008] The potential of lipases as drug targets for the treatment
of metabolic syndrome and cardiovascular disorders is increasingly
recognized. It is now believed that the front line therapy for
diseases related to lipid absorption and metabolism should be to
inhibit or reduce lipase activity in the body fluids, cells, and
tissues.
[0009] Lipase inhibitors have been reported from various natural
products, especially from microbial sources. The example of such
inhibitors include lipstatin and Panclicins A-E from Streptomyces
species or their synthetic derivatives that inhibit the hydrolysis
of triglycerides and cholesterol esters (Hochuli et al., Lipstatin,
and Inhibitor of Pancreatic Lipase, Produced by Streptornyces
Toxytricini, II. Chemistry and Structure Elucidation, J. Antibiot.
(Tokyo), 1987 August, 40(8):1086-91; Fernandez et al., Effects of
Tetrahydrolipstatin, a Lipase Inhibitor, on Absorption of Fat from
the Intestine of the Rat, Biochim. Biophys. Acta., 1989 February,
20, 1001(3):249-55; Yoshinari et al, Panclicins, Novel Pancreatic
Lipase Inhibitors, II. Structural elucidation, J. Antibiot.
(Tokyo), 1994 December, 47(12):1376-84) and is being used for
treatment of obesity (U.S. Pat. No. 5,540,917). Besides, a number
of molecules have been identified from plant sources including
tannins isolated from Cassia nomame (U.S. Pat. No. 5,629,338). LPL
has been shown to be involved in the pathogenesis of
atherosclerosis (Mead et al, Lipoprotein Lipase, a Key Role in
Atherosclerosis?, FEBS Lett., 1999 November, 26, 462(1-2):1-6).
Inhibition of LPL is believed to prevent the atherosclerotic
process (Zimmerman et al., Lipoprotein Lipase Mediates the Uptake
of Glycated LDL in Fibroblasts, Endothelial Cells, and Macrophages,
Diabetes, 50, 1643-1653, 2001).
[0010] Phospholipases specifically act on and hydrolyse membrane
phospholipids and generate mediators implicated in signal
transduction and inflammatory processes. The role of phospholipase
A2 (PLA2) is well known in the generation of arachidonic acid,
which is responsible for leukotriene and prostaglandin synthesis;
PLA2 inhibitors have been proposed as drugs for variety of
inflammatory and degenerative diseases. Lipoprotein-associated
phospholipase A2 has been shown to be involved in atherosclerosis
and its inhibition is being proposed for its treatment (Leach et
al., Lipoprotein-Associated PLA2 Inhibition--A Novel, Non-Lipid
Lowering Strategy for Atherosclerosis Therapy, Farmaco, 2001
January-February, 56(1-2):45-50).
3. SUMMARY OF THE INVENTION
[0011] The present invention relates to a protein and composition
comprising the same for inhibiting or reducing lipases and
phospholipase enzymes in body fluids, cells, and tissues. The
protein as described in the present invention and the composition
comprising of the same are useful for the prevention or treatment
of clinical manifestations and diseases caused as a consequence of
lipase and phospholipase enzyme activities in the body fluids,
cells, and tissues.
[0012] In the preferred embodiments the protein having lipase
inhibitory activity can be synthesized, produced by recombinant
technology or isolated from natural sources.
[0013] In the most preferred embodiments the protein is isolated
from the seeds of plant species belonging to Moringa genus.
[0014] In the preferred embodiments the compositions for inhibition
of lipases or phospholipases comprises of protein as described in
the present invention in a therapeutically effective amount and
pharmaceutically inert adjuvants, diluents or carriers.
[0015] In the preferred embodiments the compositions for inhibition
of lipases or phospholipases comprising of protein as described in
the present invention may also be combined with other active
ingredients.
[0016] The protein as described in the present invention or
composition comprising the same is believed to have the ability to
inhibit lipases and phospholipases under physiological conditions,
and thereby would have corresponding effectiveness for prevention
or treatment of metabolic syndrome, cardiovascular disorders, and
inflammatory diseases.
[0017] In the preferred embodiments the protein as described in the
present invention or the compositions comprising proteins are
useful as for inhibition of lipases and phospholipases in the body
fluids, cells, and tissues for the prevention and treatment of
metabolic syndrome, cardiovascular disorders, and inflammatory
diseases.
[0018] In another embodiments, the protein as described in the
present invention or the composition comprising a protein can be
used for prevention or treatment of metabolic disorders like
obesity, diabetes, and atherosclerosis.
[0019] In still another embodiments, the protein as described in
the present invention or the composition comprising a protein can
be used in inhibiting or reducing accumulation of lipids in
monocytic cells, vascular cells, hepatocytes, and adipose
tissues.
[0020] In yet another embodiments, the protein as described in the
present invention or the composition comprising a protein can be
used for prevention or treatment of inflammatory diseases, such as
arthritis, atherosclerosis, and septic shock, that are caused by
the activation and/or the action of phospholipases.
[0021] In yet another embodiments, the protein as described in the
present invention or the composition comprising a protein can be
used for skin and hair care and cosmetic preparations.
[0022] In yet another embodiments, the protein as described in the
present invention or the composition comprising a protein can be
used to prevent or treat cellular and tissue damage caused by
microbial pathogens secreting lipases and phopholipases.
[0023] In yet another embodiments on basis of lipase inhibitory
properties, the composition comprising a protein can be used in
veterinary medicine for the treatment and prophylaxis of diseases
caused or aggravated by lipase and phospholipase activity in the
body fluids, cells and tissues.
[0024] In preferred embodiments the present invention also provides
the pharmaceutical formulations comprising protein either alone or
a suitable pharmceautically acceptable adjuvant useful in
inhibition of lipases and phospholipases in the body fluids, cells,
and tissues for the prevention and treatment of metabolic syndrome,
cardiovascular disorders, and inflammatory diseases.
[0025] The present invention also provides the manner of
manufacture of medicaments comprising of protein as described in
the present invention in a therapeutically effective amount either
alone or in combination with pharmaceutically acceptable adjuvant.
The protein as described in the present invention may also be
combined with other active ingredients.
4. DESCPRITION OF THE INVENTION
[0026] The present invention relates to a protein containing 5-100
amino acid residues and having a molecular weight ranging from
0.5-10 kD, with or without glycosylation. The protein has
inhibitory or reducing effect on lipase and phospholipase enzyme
activities. The protein may be synthesized or produced through
recombinant DNA technology or it may isolated from plant
material.
[0027] The protein as disclosed in the present invention is
isolated from species belonging to genus Moringa, more preferably
it is isolated from seeds of plant Moringa. The protein can be
isolated by the method as disclosed herein later under the
examples.
[0028] The protein has partial sequence ID as following SEQ. ID.
NO. 1
TABLE-US-00001 CGQQLRNISPPQRCPSLRQAVQLAHQQQGQGPQQVRQMYR
[0029] The present invention also relates to the compositions for
inhibition of lipases or phospholipases comprising of protein as
described in the present invention in a therapeutically effective
amount and pharmaceutically inert adjuvants, diluents or carriers.
The compositions for inhibition of lipases or phospholipases
comprising of protein as described in the present invention may
also be combined with other active ingredient or ingredients.
[0030] The protein as described in the present invention or
composition comprising the same is believed to have the ability to
inhibit lipases and phospholipases under physiological conditions,
and thereby would have corresponding effectiveness for prevention
or treatment of metabolic syndrome, cardiovascular disorders, and
inflammatory diseases.
[0031] The protein as described in the present invention or the
compositions comprising protein are useful for inhibition of
lipases and phospholipases in the body fluids, cells, and tissues
for the prevention and treatment of metabolic syndrome,
cardiovascular disorders, and inflammatory diseases.
[0032] In the preferred embodiments the protein as described in the
present invention or the composition comprising a protein can be
used for prevention or treatment of metabolic disorders like
obesity, diabetes, and atherosclerosis.
[0033] In further aspects the protein as described in the present
invention or the composition comprising a protein can be used in
inhibiting or reducing accumulation of lipids in monocytic cells,
vascular cells, hepatocytes, and adipose tissues.
[0034] In still other aspects, the protein as described in the
present invention or the composition comprising a protein can be
used for prevention or treatment of inflammatory diseases, such as
arthritis, atherosclerosis, and septic shock, that are caused by
the activation and/or the action of phospholipases.
[0035] In still another aspects, the protein as described in the
present invention or the composition comprising a protein can be
used to prevent or treat cellular and tissue damage caused by
microbial pathogens secreting lipases and phopholipases.
[0036] In yet another aspects, the protein as described in the
present invention or the composition comprising a protein can be
used for skin and hair care and cosmetic preparations.
[0037] The protein as described in the present invention or
composition comprising the same can be administered in any
conventional oral, buccal, nasal, by inhalation spray in unit
dosage form, parenteral, (for example, intravenous, intramuscular,
subcutaneous intrastemal or by infusion techniques), topical (for
example, powder, ointment or drop), transdermal, intracisternal,
intravaginal, intraperitoneal, intravesical, or rectal. In another
aspect of the invention, the compound of the present invention and
at least one other pharmaceutically active agent may be
administered either separately or in the pharmaceutical composition
comprising both. It is generally preferred that such administration
be oral. However, if the subject being treated is unable to
swallow, or oral administration is otherwise impaired or
undesirable, parenteral or transdermal administration may be
appropriate.
[0038] The protein as described in the present invention or
composition comprising the same can be administered in the form of
any modified release, controlled release or timed release
formulations.
[0039] Accordingly, formulations according to the present invention
for reducing lipase and phospholipase activity in body fluids,
cells, and tissues will comprise, as the essential active
ingredient, the protein of the present invention.
[0040] In preferred embodiments the present invention provides
formulations for reducing lipase and phospholipase activity in body
fluids, cells, and tissues comprising the protein of the present
invention, it can be formulated either alone or in combination with
a known pharmaceutically acceptable and inert adjuvant, diluent or
carrier.
[0041] A formulation comprising the protein according to the
present invention can be formulated together with one or more
routine additives, carriers, assistants, and the like. It can be
formulated for oral administration and can be used in the field of
pharmaceuticals. Examples of suitable forms for oral administration
include tablets, capsules, granules, fine granules, spherules,
syrups, and drinks. In the preferred embodiments it is formulated
in the form of spherules. In most preferred embodiments the
spherules are enteric coated. Examples of suitable carrier
materials are water, gelatin, gum arabic, lactose, starch,
magnesium stearate, talc, vegetable oils, polyalkyleneglycols,
petroleum jelly, etc. The pharmaceutical preparations can be made
up in a solid form (e.g., as tablets, degrees, suppositories or
capsules) or in a liquid form (e.g., as solutions, suspensions or
emulsions). The pharmaceutical preparations may be sterilized
and/or may contain adjuvants such as preservatives, stabilizers,
wetting agents, emulsifiers, salts for varying the osmotic
pressure, or buffers. They can also contain other therapeutically
valuable substances.
[0042] For the preparation of a formulation according to the
present invention, the essential ingredients are mixed with one or
more pharmaceutically-acceptable vehicles, carriers, excipients,
binders, antiseptics, anti-oxidants, stabilizers, taste corrigents,
buffers, and the like, followed by formation into a desired unit
dosage form.
[0043] Examples of adjuvants that can be incorporated in tablets,
capsules, or the like, upon formulation according to the present
invention include: binders such as gum arabic, corn starch, and
gelatin; lubricants such as magnesium stearate; excipients, such as
crystalline cellulose; swelling agents, such as gelatinized starch
and arginic acid; sweeteners, such as sucrose, lactose, and
saccharin; and taste corrigents, such as peppermint and cherry.
Upon formulation into capsules, a liquid carrier, such as oil, can
also be incorporated together with the above adjuvants.
[0044] Furthermore, other materials can be added as a coating agent
or to change the physical form of the preparation. For example,
tablets can be coated with shellac, sugar, or any acidic pH
resistant polymer. Syrups and elixirs can be added with sucrose as
a sweetener, methylparaben or propylparaben as an antiseptic,
and/or peppermint or orange flavor as a taste corrigent.
[0045] According to the present invention, the formulation can be
used as a medicament for lowering total serum lipid cholesterol,
and for the treatment of obesity, ischemic heart diseases,
arteriosclerosis, cerebrovascular dementia, diabetes, angiopathic
Parkinson's diseases, inflammatory diseases, and the like.
[0046] The formulation described in the present invention may be
administered once or a few times a day in an amount of about 10 to
2000 mg/day in terms of dry weight.
[0047] The active ingredients of the formulation according to the
present invention can be added to various foods for the reduction
of the serum lipid level or the total blood cholesterol level or
accumulation of fat in tissues. Examples of foods to which the
active ingredients according to the present invention can be added
include tea beverages, juice, coffee, drinks, carbonated beverages,
chewing gum, candies, caramels, chocolates and ice creams.
[0048] In additional aspects the protein or the composition
comprising of protein as described in the present invention may be
useful as veterinary medicine for the treatment and prophylaxis of
diseases caused or aggravated by lipase and phospholipase activity
in the body fluids, cells, and tissues in animals.
[0049] This invention further relates to a method for inhibiting or
reducing lipase and phospholipase activity in body fluids, cells,
and tissues by administration of a formulations comprising of the
protein. The present invention is based on our discovery that the
protein is a potent inhibitor of lipases and phospholipases using
specific enzyme assays as disclosed herein later. Further, the
invention is also based on our observation that the protein remains
stable during formulation and thereby it would retain its
activity.
[0050] The further aspects and features of the present invention
are illustrated in the following non-limiting examples:
5. EXAMPLES
Example 1
[0051] Isolation of Lipase Inhibitory Protein from Moringa
Seeds:
[0052] 100 gm-powdered seeds were soaked in 1 litre MQ water for 48
hrs at room temperature. The extract was filtered through Whatman
filter paper. The filtrate was concentrated using lyophlizer
(-50.degree. C.) for two days to get the protein isolate. Protein
was estimated by using Bradford method.
[0053] Result: 10 mg powdered contained 494 .mu.g protein.
Example 2
[0054] SDS-PAGE (17%) was performed with the protein isolated from
Moringa seeds and stained with Coomassi blue. 5.+-.1 kd band was
cut and transferred to a siliconized tube and washed and destained
in 200 .mu.L 50% methanol overnight. The gel pieces were dehydrated
in acetonitrile, rehydrated in 30 .mu.L of 10 mM dithiolthreitol in
0.1 M ammonium bicarbonate and reduced at room temperature for 0.5
h. The DTT solution was removed and the sample alkylated in 30
.mu.L 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room
temperature for 0.5 h. The reagent was removed and the gel pieces
dehydrated in 100 .mu.L acetonitrile. The acetonitrile was removed
and the gel pieces rehydrated in 100 .mu.L 0.1 M ammonium
bicarbonate. The pieces were dehydrated in 100 .mu.L acetonitrile,
the acetonitrile removed and the pieces completely dried by vacuum
centrifugation. The gel pieces were rehydrated in 20 ng/.mu.L
trypsin in 50 mM ammonium bicarbonate on ice for 10 min. Any excess
trypsin solution was removed and 20 .mu.L 50 mM ammonium
bicarbonate added. The sample was digested overnight at 37.degree.
C. and the peptides formed extracted from the polyacrylamide in two
30 .mu.L aliquots of 50% acetonitrile/5% formic acid. These
extracts were combined and evaporated to 25 .mu.L for MS
analysis.
[0055] The LC-MS system consisted of a Finnigan LCQ ion trap mass
spectrometer system with a Protana nanospray ion source interfaced
to a self-packed 8 cm.times.75 um id Phenomenex Jupiter 10 um C18
reversed-phase capillary column. 0.5-5 .mu.L volumes of the extract
were injected and the peptides eluted from the column by an
acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.25
.mu.L/rnin. The nanospray ion source was operated at 2.8 kV. The
digest was analyzed using the double play capability of the
instrument acquiring full scan mass spectra to determine peptide
molecular weights and product ion spectra to determine amino acid
sequence in sequential scans.
[0056] The Partial Sequence ID of the Protein was Identified as
Shown Below:
TABLE-US-00002 CGQQLRNISPPQRCPSLRQAVQLAHQQQGQGPQQVRQMYR
Example 3
[0057] Lipase Inhibitory Activity of Moringa Seed Protein in the
Presence of Synthetic Substrate:
[0058] Lipase Assay:
[0059] Enzyme assay was performed by method described by Winkler
and Stuckmann, 1979, with modification where there was use of
pancreatic lipase. Assay was designed, using a 96-well format. The
substrate used in this assay was p-nitrophenol palmitate (Sigma,
Cat No-N-2752). 4.5 mg of p-nitrophenol palmitate was dissolved in
200 .mu.l of N, N-dimethylformamide (Sigma, Cat No, D-4551) and
volume made up to 10 ml with 0.1 M Ph 8.0-phosphate buffer. Lipase
(Sigma, Cat No, L-3126) sample was prepared by dissolving the
enzyme in 0.1M-phosphate buffer at a concentration of 5mg/ml. The
reaction mixture consisted of substrate solution-150 .mu.L;
phosphate buffer (pH 8.0, 0.1 M) -40 .mu.l and lipase solution-10
.mu.l. The reaction mixture was incubated at 37.degree. C. and
optical density was measured at 405 nm after incubation. Enzyme
activity was presented in the form of international unit (KU). One
enzyme unit of lipase is defined as that quantity releasing 1 nm of
free phenol from the substrate (p-nitro phenol palmitate) ml/min
under the standard assay condition (Winkler K. W. and Stuckman M,
1979 Glycogen hyaluronate and some other polysaccharides greatly
enhances the formation of exolipase by Serratia marcescens, J. of
Bacteriology 138: 663-670). It is derived from standard graph of
p-nitro phenol.
[0060] Lipase Inhibition by Moringa Seed Protein:
[0061] Inhibition assay was performed in a dose dependent manner.
The concentration of the protein checked 40 .mu.g-0.156 .mu.g/ml
reaction mixture. The assay was similar to assay described above
except 40 .mu.l of inhibitor solution was used instead of phosphate
buffer in control. Released p-nitro phenol was recorded at 405 nm.
Enzyme inhibition was presented in the term of percentage
inhibition simply on the basis of change in international unit
(IU), which was calculated from standard graph.
[0062] Result:
TABLE-US-00003 TABLE 1 Sample type % Inhibition Control (only
enzyme) 0.00 Protein, 40 .mu.g/ml 100.00 Protein, 20 .mu.g/ml
100.00 Protein 10 .mu.g/ml 100.00 Protein 5.0 .mu.g/ml 99.51
Protein 2.5 .mu.g/ml 91.25 Protein 1.25 .mu.g/ml 77.70 Protein
0.625 .mu.g/ml 62.362 Protein 0.312 .mu.g/ml 41.918 Protein 0.156
.mu.g/ml 0.00
[0063] Conclusion: Moringa protein was able to inhibit the
pancreatic lipase even at very low concentration.
Example 4
[0064] Inhibitory Activity of Moringa Seed Protein in the Presence
of Natural Substrate:
[0065] Lipase activity was also measured by titrating free fatty
acids liberated in the reaction mixture by following a modified
method of "Ishiia C et al., 1988, Inhibition of lipase by proteins
and their inhibitory mechanism, Nippon Shokuhin Kogyo Gakkaishi, 35
(6), 430-439. The reaction was performed in tubes by shaking at
37.degree. C. for 30 min. the reaction mixture was prepared with
1.5 ml of Mcllvaine buffer, pH-7.0, 0.24 ml of olive oil, and 0.5
ml of inhibitor solution and water all in final volume of 5.5 ml .
After 5 min preincubation, the reaction was then started by adding
0.5 ml of enzyme solution (5.0 mg) . The reaction was then stopped
by the addition of 10 ml mixed solution of n-propyl alcohol:
petroleum ether (1:4), and mixture was then shaken vigorously for 2
min 1 ml of upper layer was pippetted and titrated with 0.02 M
alcoholic KOH using phenolphthalein as an indicator. The standard
reaction mixture was prepared as described except that buffer
substituted the inhibitor solution. Fatty acid released .mu.M/min
under standard assay condition was considered as one international
unit (W) of enzyme.
[0066] Result
TABLE-US-00004 TABLE 2 Sample type % Inhibition Control 1, Heat
killed enzyme 0.00 Control 2, Active enzyme 00.00 Enzyme + Protein
(300 .mu.g/ml) 90.10 Enzyme + Protein (150 .mu.g/ml) 90.10 Enzyme +
Protein (75.0 .mu.g/ml) 70.00 Enzyme + Protein (37.5 .mu.g/ml)
50.00 Enzyme + Protein (18.75 .mu.g/ml) 0.00
[0067] Conclusion: Moringa seed protein was able to inhibit the
pancreatic lipase during the hydrolysis of natural substrate olive
oil.
Example 5
[0068] Effect of Trypsin on Lipase Inhibitory Activity of Moringa
Seed Protein:
[0069] The lipase inhibitory activity was performed after tryptic
cleavage of Moringa seed protein. The isolated protein (500
.mu.g/ml estimated by Bradford method) was incubated with trypsin
(0.5%) in 50 mM, Tris buffer, pH-9.0 & sample buffer for 24 hrs
at 37.degree. C. in 1:1:2 ratio. After incubation all the samples
were allowed for thermal inactivation at 70.degree. C. for 10 min.
The lipase inhibitory assay was performed as described earlier
except 40 .mu.l of test solution (trypsin treated/untreated) was
used instead of phosphate buffer in control. Released p-nitro
phenol was recorded at 405 nm. Lipase activity was presented in the
term of percentage inhibition simply on the basis of change in
international unit (IU), which was calculated from standard
graph.
[0070] Result
TABLE-US-00005 TABLE 3 Sample type % Inhibition Control, (only
lipase) 0.00 Lipase + Protein 100.00 Lipase + Protein treated with
18.17 trypsin
[0071] Conclusion: Moringa seed protein lost the lipase inhibitory
activity after tryptic cleavge which suggests the lipase inhibitory
activity present of protein.
Example 6
[0072] Protection of Moringa Seed Protein from Trypsin with Soya
Protein.
[0073] Isolation of Soya Protein from Soybean Seed
[0074] In a 2 litre glass beaker charge 500 gm soybean seed with
1.5 litre Milli Q water and heated the beaker in water bath at
65.degree. C. (external temperature) for 90 min. after 90 min soya
bean seed extract was filtered and cooled to room temperature. The
filtrate was concentrated using lyophlizer (-50.degree. C.) for two
days. Crude protein stuck to the wall of round bottle flask and was
scratched using spatula to fine powder. Total solid powder protein
obtained was 12.5 gm with hygroscopic property. Protein was
estimated by using Bradford method.
[0075] Result: 10 mg solid contained 18.52 .mu.g protein
[0076] Protein Protection Assay
[0077] Moringa seed protein was protected against trypsin performed
using various concentration of soya protein. The Moringa seed
protein(49.1 .mu.g/100ul estimated by Bradford method) was
incubated with 100 .mu.l trypsin (0.5%) solution and various
concentration 100 .mu.l of soya protein in 50 mM, buffer, pH-9.0
& sample buffer for 24 hrs at 370.degree. C. in 1:1:1:1 ratio.
After incubation all the samples were allowed for thermal
inactivation at 70.degree. C. for 10 min. The assay was performed
as described earlier except 40 .mu.l of test solution
(treated/untreated) was used instead of phosphate buffer in
control. Released p-nitro phenol was recorded as OD at 405 nm.
Lipase activity was presented in the term of percentage inhibition
simply on the basis of change in international unit (IU), which was
calculated from standard graph.
TABLE-US-00006 TABLE 4 Sample type % Inhibition Control, (only
lipase) 0.00 Lipase + Moringa seed protein 100.00 Lipase + Moringa
seed protein treated with trypsin 18.17 Lipase + Moringa seed
protein treated with trypsin + soya 100.00 protein (1.852 ug)
Lipase + Moriga seed protein treated with trypsin + soya 89.91
protein (0.926 ug) Lipase + Moringa seed protein treated with
trypsin + soya 32.08 protein (0.463)
Example 7
[0078] Moringa seed protein ( 50.0 gms ), Microcrystalline
Cellulose (336.0 gms ) & Lactose ( 84.0 gms ) were passed
through 40# separately, & geometrical mixed in a suitable mixer
for 15 minutes. 10% starch paste was prepared using Maize starch
(30.0 gms ) & required amount of water. Starch Paste was added
in the mixer & mixing was continued until dough was formed with
a consistency suitable for extrusion. The dough mixture was charged
into a Fuji Paudal EXDS--60 extruder fitted with a 0.8 mm radial
screen. The extrude were dried using drying oven at 40.degree. C.
for 8 hrs. Spheronise using Fuji Paudal Q230, dry these at
40.degree. C. over night. The dried spherules were sifted through
sieves, the spherules passed through 24 no. & retained over
mesh n0. 40 for further processing.
[0079] Coating was done in two steps viz 1) Seal coating 2) Enteric
coating.
[0080] Seal coating was done by using HPMC as a film forming
polymer & enteric coating was done with the help of Eudragit L
30 D 55 as an enteric polymer & Yellow iron oxide as coloring
agent.
[0081] The spherules were filled in size 0 hard gelatin transparent
capsules.
[0082] The in-vitro protein analysis was done on Coated Spherules
as well as on Filled capsules by using Brad ford Method.
[0083] Analytical Data:
TABLE-US-00007 TABLE 5 Sr. Blank OD Absorbance at Absorbance
Conc.ug/ No Std/Sample (A) 570 nm (B) (B - A) ml 1. 2 ul 0.2525
0.369 0.1165 0.055 2. 3.5 ul 0.2525 0.488 0.2355 .0096 3. 7.0 ul
0.2525 0.603 0.3505 .0192 4. 14.5 ul 0.2525 0.746 0.4935 0.397 5.
18.0 ul 0.2525 0.876 0.6145 0.493 6. Sample A* 0.2525 0.774 0.5215
A 7. Sample B** 0.2525 0.7375 0.4850 B Sample A* = Spherules sample
Sample B** = Filled Capsules sample Graph was plotted Conc. v/s
Absorbance R = 0.950458 Equation obtained: Y = 1.13616 * X +
0.0682681, Where Y is absorbance & X is concentration in ug/ul
From calculation Sample A* = 0.3990 ug/ul Sample B** = 0.3668
ug/ul
[0084] From the above data it can be inferred that the protein does
not get destroyed during the formulation process.
Example 8
[0085] Animal Study.
[0086] Short term animal experiment was performed with Moringa seed
protein along with or without Soya protein (isolated earlier) in
Wistar male rat in four groups. Male Wistar administered orally to
the rat was deprived of food overnight, 1.2 ml of lipid emulsion
(10 ml sunflower oil in water) or lipid emulsion 1.2 ml with
various formulation in define groups (Group: control, Group-1: 50
mg soya seed extract solid (2.5 mg protein), Group-2: Soya 50 mg
solid extract contained 92.6 .mu.g protein & Group-3: 50 mg
Moringa solid extract (2.5 mg protein) & 50 mg soya solid
extract ( 92.6 .mu.g protein). Blood sample was taken and the
plasma triacylglycerol concentration was determined.
[0087] Triglyceride Concentration (mg/dl)
[0088] Mean value:
TABLE-US-00008 0 hr 1 hr 2 hr 3 hr Control 69.16667 70.16667
76.16667 105.5 Group 1 72.33333 70.16667 99.83333 115.5 Group 2 102
57.83333 82.83333 99 Group 3 77 59.33333 79.66667 84.83333
Sequence CWU 1
1
1140PRTMoringa 1Cys Gly Gln Gln Leu Arg Asn Ile Ser Pro Pro Gln Arg
Cys Pro Ser1 5 10 15Leu Arg Gln Ala Val Gln Leu Ala His Gln Gln Gln
Gly Gln Gly Pro 20 25 30Gln Gln Val Arg Gln Met Tyr Arg 35 40
* * * * *