7-HYDROXYFRULLANOLIDE AND ITS ANALOGS FOR PREVENTION,
CONTROL AND TREATMENT OF METABOLIC DISORDERS
Field of Invention;
The present invention discloses biologically active ingredient(s) comprising atleast one
component selected from 7-hydroxyfrullanolide, its analog(s) or the herbal extract(s) and
fraction(s) standardized to 7-hydroxyfrullanolide or its analog(s) or mixtures thereof as a
biologically active ingredient or their compositions, optionally containing one or more of
pharmaceutically or dietetically acceptable diluents, vehicles, carriers and actives or
mixtures thereof for the prevention, control and/or treatment of one or more of the
metabolic disorders selected from metabolic syndrome, obesity, diabetes, endothelial
dysfunction and other disease indications related thereto.
The invention also relates to the amelioration of one or more of the biological marker
proteins or metabolic processes related to metabolic syndrome, obesity and other
metabolic disorders by 7-hydroxyfrullanolide or its analog(s) or the extract(s)/ fraction(s)
standardized to 7-hydroxyfrullanolide or its analog(s) or mixtures thereof or their
compositions.
Background of the invention;
Obesity is a medical condition in which excess body fat has accumulated to the extent
that it may have an adverse effect on health, leading to reduced life expectancy and/or
increased health problems. Body mass index (BMI), a measurement which compares
weight and height, defines people as overweight (pre-obese) when their BMI is between
25 kg/m2 and 30 kg m2, and obese when it is greater than 30 kg/m2. Obesity increases the
likelihood of various diseases, particularly heart disease, type 2 diabetes, breathing
difficulties during sleep, certain types of cancer, and osteoarthritis. It is a leading
preventable cause of death worldwide, with increasing prevalence in adults and children,
and authorities view it as one of the most serious public health problems of the
21st century.
Metabolic Syndrome also known as Syndrome X, insulin resistance syndrome and
DysMetabolic Syndrome is a condition, wherein a group of diseased states, which
increase atherosclerosis, stroke and diabetes. It was first described by Reaven in 1988 as a
cluster of interrelated common clinical disorders, including obesity, insulin resistance,
glucose intolerance, hypertension and dyslipidemia.
A criteria for diagnosing Metabolic Syndrome was established by The Adult Treatment
Panel-Ill (ATP-III) of the National Cholesterol Education Program in 2001. Five Criteria
were selected by this Panel to identify individuals with Metabolic Syndrome including
abdominal obesity, impaired fasting glucose, high triglyceride (TG), low HDL cholesterol
(HDL-C) concentrations and increased blood pressure. Metabolic Syndrome is diagnosed,
if any three of the components are present in an individual.
A lot of research is being carried out over a decade to develop agents to control Metabolic
Syndrome. The application of metabolic markers for the control of this syndrome has also
been attempted.
Based on the information cited above and several other documents, the inventors of the
present invention have felt the need for the development of an effective compound or
composition which can efficiently be used for the prevention, control and treatment of
obesity, metabolic syndrome and several other associated and related diseases.
3a -hydroxy-5a,9 -dimethyl-3-methylene-3a, 4, 5, 5a, 6, 7, 8, 9 -octahydro-3H-naphthol
[1,2-b] furan-2-one (7-a-Hydroxy-4,l l (13)-eudesmadien-12,6-olide or 7-
hydroxyfrullanolide) is a natural compound isolated from the flower heads of
Sphaeranthus indicus. 7-hydroxyfrullanolide (7-HF) is a sesquiterpene compound.
None of the published literature describes anti-obese or anti-metabolic syndrome potential
of 7-HF. The amelioration of biomarkers or biological processes related to metabolic
disorders such as obesity, metabolic syndrome and other disease conditions associated
with metabolic syndrome by 7-hydroxyfrullanolide also not known.
Some of the Patent literature of Sphaeranthus is quoted below:
US patent US7635494 relates to a novel herbal composition comprising an extract of
flowering and fruiting heads of the plant, Sphaeranthus indicus. The said extract of
Sphaeranthus indicus contains a compound, 3a-hydroxy-5a,9-dimethyl-3-methylene-
3a,4,5,5a,6,7,8,9b-octahydro-3H-naphtho[ 1,2-b]furan-2-one (7-Hydroxy-4, 11(13)-
eudesmadien-12,6-olide/7-hydroxyfrullanolide) (compound 1), as a bioactive marker. The
application also relates to a composition comprising 3a-hydroxy-5a,9-dimethyl-3-
methylene-3a,4,5,5a,6,7,8,9b-octahydro-3H-naphtho[l,2-b]furan-2-one (compound 1) as
an active ingredient, methods of manufacture of the said compositions, methods of
administration of the said compositions to a subject in need of treatment for an
inflammatory disorder. The publication also disclosed tumor necrosis factor-a (TNF-a)
and interleukin (IL-1, IL-6, IL-8) inhibitory activity of the said compositions.
PCT Publication WO07036900A2 relates to a novel herbal composition comprising an
extract of flowering and fruiting heads of the plant, Sphaeranthus indicus containing 3ahydroxy-
5a,9-dimethyl-3-methylene-3a,4,5,5a,6,7,8,9b-octahydro-3H-naphtho[l,2-
b]furan-2-one (7-Hydroxy-4,l l(13)-eudesmadien-12,6-olide), as a bioactive marker and
relates to methods of manufacture of the said compositions.
PCT Publication WO06016228A2 relates to a compound or group of compounds present
in an active principle derived from plants of the species Sphaeranthus, for the preparation
of pharmaceutical formulations or food supplements for the prophylaxis and/or treatment
of tumor diseases. The said invention furthermore relates to a novel method for the
isolation of an active principle from Sphaeranthus plant parts which are effective in
prophylaxis and/or treatment of cancers.
According to our knowledge, there is no prior art relating to the usage of 7-
hydroxyfrullanolide or its analogs and their compositions for the amelioration of
metabolic markers or for the control, prevention and treatment of diseased conditions
associated with or related to obesity, metabolic Syndrome and other metabolic disorders.
Summary of the invention:
In the primary embodiment, the invention provides biologically active ingredient(s)
comprising atleast one component selected from 7-hydroxyfrullanolide, its analog(s);
extract(s) and fraction(s) containing 7-hydroxyfrullanolide or its analog(s) or both; or
mixtures thereof for the prevention, control and/or treatment of one or more metabolic
disorders.
In another embodiment the invention provides biologically active composition
comprising at least one component selected from the list comprising 7-
hydroxyfrullanolide, its analog(s); the extract(s) or fraction(s) containing 7-
hydroxyfrullanolide/its analog(s) or both; or mixture(s) thereof as an active in
combination with one or more ingredients selected from other biologically active
components derived from plants, animals and microorganisms; pharmaceutically or
dietetically acceptable active ingredients, vitamins, amino acids, minerals, vehicles,
carriers and diluents or mixtures thereof for the prevention, control and/or treatment of
one or more metabolic disorders.
In yet another embodiment, the invention provides biologically active ingredient(s) or
their composition(s) for the amelioration of the expression/production of one or more
biological marker proteins related to metabolic disorders.
In yet another embodiment, the invention provides compositions comprising atleast one
component selected from 7-hydroxyfrullanolide, its analogs, the extract(s) and fraction(s)
standardized to 7-hydroxyfrullanolide or its analogs or mixtures thereof as an active
ingredient and atleast one component selected from pharmaceutically or dietetically
acceptable phytochemical actives, plant extracts, diluents, vehicles, carriers and actives or
mixtures thereof for the control, prevention and treatment of metabolic disorders, which
include but not limited to metabolic syndrome or obesity, and/or one or more disease
indications related to or associated with metabolic syndrome.
In still another embodiment, the invention provides pharmaceutical or dietary supplement
or food ingredient selected from 7-hydroxyfrullanolide, its analog(s) and the extract(s)
and fraction(s) standardized to 7-hydroxyfrullanolide alone or its analogs or mixtures
thereof or their composition(s) for the amelioration of the expression or production of one
or more biological marker proteins related to or associated with metabolic syndrome,
obesity and other disease conditions associated with metabolic syndrome including but
not limited to Peroxisome proliferator-activated receptor gamma (PPARy), Adipose
Differentiation Related Protein (ADRP), adipocyte CD36, Macrophage CD36, Monocyte
Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding
protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (P3AR), Perilipin,
Adiponectin, Protein tyrosine phosphatase- IB (PTP-1B), Metalloproteinase-1 (MMP-1),
Matrix Metalloproteinase-3 (MMP-3) and Matrix Metalloproteinase-1 3 (MMP-1 3).
In the other embodiment, the invention provides method(s) for the prevention, control
and/or treatment of metabolic disorders, which include but not limited to obesity, over
weight, diabetes, arteriosclerosis, cardiovascular diseases, hypertension,
hypercholesteremia, hyperlipidemia, triglyceridemia, metabolic syndrome, endothelial
dysfunction and other metabolic disorders in warm blooded animals, wherein the method
comprises of administering to a warm blooded animal in need thereof an effective amount
of a component selected from 7-hydroxyfrullanolide, its analog(s), the extract(s) and
fraction(s) containing 7-hydroxyfrullanolide alone or its analogs or mixtures thereof or
their composition(s).
Brief Description of Figures:
Figure I : Illustration of anti-adipogenic activity of 7-hydroxyfrullanolide (7-HF). 7-HF
decreases the lipid content in mouse adipocytes by inhibiting adipogenesis process.
Photomicrographs show lipid accumulation in Oil Red O stained 3T3-L1 adipocytes
treated with either 0.1% DMSO as the vehicle control, or 1 g/ml 7-HF. Arrows indicate
the lipid vesicles in the cytoplasmic compartment of adipocytes.
Figure II: Illustration of prolipolytic activity of 7-hydroxyfrullanolide (7-HF). Bar
diagram represents 7-HF increases glycerol release into the cell culture supernatant in a
dose dependent manner. Equal number of 3T3-L1 mouse pre-adipocytes was
differentiated and maintained in post differentiation medium to obtain mature adipocytes.
The mature adipocytes were treated with 2.5 and 5.0 g ml of 7-HF for 2 h. Lypolytic
potential of 7-HF was evaluated by measuring the released glycerol in the culture
supernatant by Glycerol assay reagent. Each bar represents the mean ± SD of released
glycerol (n= 6). P value of <0.05 was considered as statistically significant (Students t
test).
Figure III: Amelioration of marker proteins of Adipogenesis and lipolysis by 7-
hydroxyfrullanolide (7-HF). Figure illustrates modulation of marker proteins of
adipogenesis and lipolysis processes by 7-HF in 3T3-L1 adipocytes. Representative
immuno blots indicate down-regulation of various marker proteins such as PPARy,
ADRP, CD36, aP2, and perilipin. The 3T3-L1 mouse pre-adipocytes were allowed to
differentiate in absence or presence of 1 ug/ml of 7-HF. Vehicle control cultures received
only similar concentrations of DMSO. Expression of actin protein was evaluated in each
blot as the internal control. Expression of each protein was measured densitometrically
and normalized with actin expression. The comparative levels protein expressions are
represented as bar diagrams (side panels).
Figure IV: Illustrates Representative photomicrographs show 7-HF inhibits lipid
accumulation in high glucose induced macrophage cells of an in vitro model of
atherosclerosis. The J774 mouse macrophage cells were exposed to high glucose (600
mg/dL) for 5 days in presence or absence of 1 of 7-HF. Control cultures (A)
received low glucose (100 mg/dL). B and C represent the macrophage cells supplemented
with 600mg/dL of glucose alone or in combination with 1 g l of 7-HF, respectively.
Figure V: Illustration of the down-regulation of high glucose induced CD36 expression
in macrophage cells 7-HF. The J774 mouse macrophage cells were exposed to high
glucose (600 mg/dL) for 5 days in presence or absence of 1 g/ml of 7-HF. Control
culture received low glucose (100 mg/dL). Representative immuno-blot assay
demonstrates down regulation of CD36 protein and expression of actin protein is
considered as the internal control. Bar diagram shows the CD36 expression normalized
with actin protein (lower panel).
Figure VI: Bar diagram • represents increased nitric oxide production in human
endothelial cells induced by 7-HF in a dose dependent manner. Equal number of human
endothelial cells was treated with various concentrations of 7-HF (0.1, 0.25, 0.50, 1.00
ng/ml respectively) as indicated for 24h. The control cultures received 0.01% (v/v)
DMSO as the vehicle. Culture supernatants were collected and the nitrite concentrations
were estimated quantitatively by Griess reagent. Each bar represents mean ± SD of nitrite
concentration () (n=5). * indicates significance, p < 0.01 (vs. control).
Figure VII: Bar diagram represents inhibition of PTP-1B activity in 3T3-L1 cells. 7-HF
inhibits PTP-1B activity in 3T3-L1 cells in a dose dependent manner. Equal number of
3T3-L1 mouse pre-adipocytes was treated with either 1.0 or 2.5 Dg/ml of 7-HF for 48 h.
Cells were treated with 50 Na V0 3 as a positive control. PTP-1B activity in cell
lysates was analyzed based on the hydrolysis of p-nitrophenyl phosphate (pNPP)
substrate. Each bar represents the mean ± SD of enzyme activity in pmol/min/Dg of cell
lysate protein (n=5). * indicates significance at P < 0.05 (vs. control), and # indicates P <
0.001 (vs. control).
Figure VIII: Representative immunoblot showing over expression of adiponectin protein
in 3T3-L1 adipocytes treated with 1 g/ml of 7-hydroxyfrullanolide (7-HF). Protein
expressions were densitometrically analyzed and normalized with the actin expression.
Bar diagram in each panel shows normalized protein expressions in arbitrary units. In bar
diagrams, the bars represent protein expressions in cells treated with vehicle control (a)
and 7-HF (b).
Figure IX: Figure represents the natural analogs of 7-hydroxyfrullanolide isolated from
Sphaeranthus indicus.
Figure X: Figure represents the semi-synthetic analogs of 7-hydroxyfrullanolide.
Figure XIA: Bar diagrammatic representation of body mean weight gain in HFD induced
metabolic syndrome model of SD rats supplemented without (1) or with (2) ethyl acetate
extract of Sphaeranthus indicus (SIE) from week-1 to week-8 of treatment. Each bar
represents mean ± SD, *p < 0.05.
Figure XIB: Line diagrammatic representations of body weight gain in diet induced
metabolic syndrome model of SD rats supplemented with (2) or without (1) 7-
hydroxyfrullanolide. Each line indicates change in mean body weight gain during eightweek
treatment period.
Figure XII: Bar diagrammatic representation of increase in serum adiponectin
concentration in diet induced metabolic syndrome model of Sprague Dawley rats. Each
bar indicates mean ± SD of serum adiponectin concentration at 0 day and after 56 days of
treatment with either vehicle (1) or ethyl acetate extract of Sphaeranthus indicus (SIE) (2)
as indicated in the diagram. N=6, * indicates statistical significance (t-test, 8 weeks vs. 0
week).
Figure : Bar diagrammatic representation of reduction of HOMA Index in ethyl
acetate extract of Sphaeranthus indicus (SIE) supplemented metabolic syndrome model of
Sprague Dawley rats. Each bar indicates mean ± SD of HOMA Index (arbitrary units) at 0
week and at 8 weeks of supplement with either vehicle (1) or 250 mg/kg of SIE (2). N=6;
* indicates statistical significance (t-test, SIE group vs. control at 8 weeks).
Detailed description of the invention:
Obesity is excess body weight for a particular age, sex and height as a consequence of
imbalance between energy intake and energy expenditure. The primary causes of obesity
are either due to overeating, inadequate exercise or eating disorder, some genetic
disorders, underlying illness (e.g. hypothyroidism), certain medications or sedentary
lifestyle. Obesity increases the risk of many diseases and health conditions such as
hypertension, dyslipidemia (for example, high total cholesterol or high levels of
triglycerides), type 2 diabetes, coronary heart disease, stroke, gallbladder disease,
osteoarthritis, sleep disorders, respiratory problems, tumors (endometrial, breast, and
colon), arteriosclerosis and heart failure.
Metabolic syndrome is a condition involving a set of disorders that enhances the risk of
heart disease. The major components of metabolic syndrome are excess weight, the
cardiovascular parameters (high blood pressure, dyslipidemia, high levels of triglycerides
and low levels of HDL in the blood), atherosclerosis, diabetes and insulin resistance. A
subject suffering with several of these components, i. e. metabolic syndrome is highly
prone to heart disease, though each component is a risk factor. Even though several
classes of drugs are available in the market for the treatment of different components of
Metabolic Syndrome and many of them are associated with a number of side effects, very
few medicines are available to treat Metabolic Syndrome and none of them are
comprehensive in addressing all the associated diseases. Hence there exists a great
medicinal need for developing agents for the prevention, control and treatment against
metabolic syndrome, obesity, diabetes, hypertension and atherosclerosis especially using
safe and beneficial natural compounds.
Keeping this in mind the inventors have conducted extensive research investigation
involving several in vitro and in vivo experiments on several plant extracts, fractions and
pure compounds and found surprisingly that administration of 7-hydroxyfrullanolide (7-
HF) in a therapeutically effective amount in cell based studies potently ameliorated
metabolic processes which include inhibition of adipogenesis and promotion of adipolysis
(lipolysis). In addition, 7-HF was also found to potently modulate the expression and
production of many bioactive protein molecules related to different components of
metabolic disorders such as obesity and metabolic syndrome. These unexpected aspects
of the present invention are
Modulation of metabolic processes by 7-Hydroxyfrullanolide (7-HF)
Adipogenesis : Adipogenesis is the differentiation and proliferation of pre-adipocytes into
major adipocytes or fat cells and it has been one of the most intensely studied models of
cellular differentiation. In the adipogenesis process, proliferation of preadipocytes or
precursor fat cells is followed by the differentiation of these cells into mature adipocyte
phenotype. The nuclear receptor PPAR-is expressed predominantly in adipose tissue,
where it is known to play a critical role in adipocyte differentiation and fat deposition.
Adipocytes secrete proteins exhibiting either beneficial (leptin, adiponectin) or
deleterious effects (angiotensinogen). A disturbance in the balance between these various
secreted factors, in association with the effect of secretory products from macrophages
(cytokines), leads to the development of metabolic syndrome.
Lipolysis : Lipolysis is the breakdown of stored lipid in adipocytes. p3-Adrenoreceptor
agonists can stimulate lipolysis in the white adipose tissue and thermogenesis in the
brown adipose tissue. Adipose tissue lipolysis is the catabolic process leading to the
breakdown of triglycerides stored in fat cells and release of fatty acids and glycerol. The
proteins involved in the lipolytic process constitute drug targets for the treatment of
obesity and the metabolic syndrome.
Thus the phytochemical agents having the adipogenesis and lipolysis activities could be
useful in the treatment of obesity, metabolic syndrome and other metabolic disorders.
We have disclosed the compositions and synergistic compositions of Sphaeranthus
indicus in our earlier Indian provisional application no. 224/CHE/2009 filed on February
2nd, 2009 and PCT application no. PCT/TN2010/000053 filed on February 1st, 2010. The
ameliorative effect of biomarkers expression such as PPAR-, ADRP, CD 36, aP2, A
and Perilipin by Sphaeranthus indicus ethanol extract (SIE) along with 7-
hydroxyfrullanolide (LI054A01) was also disclosed.
In a cell based assay, 7-HF potently inhibited lipid accumulation in 3T3-L1 human
adipocyte cells as depicted in Figure I. 7-Hydroxyfrullanolide (1) exhibited 52.5%
inhibition of lipid accumulation at 1 g/ l concentration in 3T3-L1 Human pre-adipocyte
cells in a cell based in vitro assay. In addition, 7-HF also inhibited lipid accumulation in
high glucose induced J774 mouse macrophage cells of an in vitro model of
atherosclerosis as depicted in Figure IV. Further, 7-Hydroxyfrullanolide potently
enhanced lipolysis in 3T3-L1 Human pre-adipocyte cells. 7-HF showed 47.8% increase in
lipolysis at 5 ΐ concentration in an in vitro cell based assay.
Amelioration of biologically active metabolic biomarker protein by 7-HF:
Adipocytes and macrophages play important role in the pathogenesis of metabolic
syndrome and disease components associated with it. They both share a number of
common features, including the ability to phagocytize and kill microorganisms and to
secrete cytokines such as tumor necrosis factor (TNF) and interleukin-l(IL-l). Critical
transcription factors in adipocytes involved in regulating the expression of cytokines,
inflammatory molecules, and fatty acid transporters are also expressed and have similar
biologic roles in macrophages. The adipocytes, in addition to accumulating fat during the
obesity development, produce and circulate several low molecular weight bioactive
protein molecules having powerful effects throughout the body. These protein markers
are related to different components of metabolic disorders such as obesity and metabolic
syndrome. The expression and production of several of these metabolic markers, which
include but not limited to PPAR-, Adipose Differentiation Related Protein (ADRP),
CD36, Adipocyte Fatty-Acid-Binding Protein (aP2/FABP4/A-FABP), Beta-3 adrenergic
receptor (P3-AR), adiponectin and Perilipin, become abnormal during obesity and
metabolic syndrome and other disease conditions associated with metabolic syndrome.
A brief description of some of the metabolic biomarker molecules that are involved in the
pathogenesis and control of metabolic syndrome and the disease conditions associated is
outlined below:
Peroxisome proliferator-activated receptor-y (PPAR-): Peroxisome proliferator-activated
receptor-(PPAR-) is a nuclear receptor that plays a pivotal role in obesity and diabetes.
An increase in adipose tissue mass can be the result of the production of new fat cells
through the process of adipogenesis and the deposition of increased amounts of
cytoplasmic triglyceride or lipid droplets per cell. In the adipogenesis process,
proliferation of preadipocytes or precursor fat cells is followed by the differentiation of
these cells to the mature adipocyte phenotype. PPAR-is expressed predominantly in
adipose tissue, wherein it is known to play a critical role in adipocyte differentiation and
fat deposition.
Adipose differentiation related protein (ADRP): ADRP is a 50 kD protein and it's mRNA
(ADRP mRNA), which is 1.7 Kb in size, is expressed at high level in adipose tissue. The
expression of ADRP is very low in undifferentiated adipocytes, but ADRP mRNA
reaches 50 to 100-fold in few hours after the onset of adipose differentiation process. The
above thus indicate the possible role of ADRP in the formation or stabilization of lipid
droplets in adipocytes and other cells. ADRP specifically enhances uptake of long chain
fatty acids by adipose tissue. Hence ADRP is an important target to identify the
compounds that can potentially control obesity and diabetes through regulation of the
expression of ADRP.
Adipocyte CD36: CD36 is a common protein marker expressed by both adipocytes and
macrophages. The CD36 expressed in adipocytes is known to function as a fatty acid
transporter (FAT). It is a scavenger receptor that binds and internalizes oxidized LDL (Ox
LDL) in macrophages. CD36 also functions as a long-chain fatty acid (LCFA) transporter
to facilitate the uptake of LCFAs in adipocytes. The CD36 expression is up-regulated by
PPAR during the differentiation of both types of cells. It is also shown that the adipocytes
can endocytose and lysosomally degrade Ox LDL, a process mainly mediated by CD36.
The CD36 null animals thus found to show significant decrease in binding and uptake of
oxidized low density lipoprotein and showed significant increase in fasting levels of
cholesterol, nonesterified free fatty acids, and triacylglycerol.
Fatty-Acid-Binding Protein (aP2/FABP4): FABPs are molecular chaperones linked to
metabolic and inflammatory pathways. Different members of the FABP family exhibit
unique patterns of tissue expression/distribution and are expressed most abundantly in
tissues involved in active lipid metabolism. FABPs play numerous functions. As lipid
chaperones, for example, FABPs may actively facilitate the transport of lipids to specific
compartments in the cell, such as to the lipid droplet for storage; to the endoplasmic
reticulum for signaling, trafficking and membrane synthesis; to the mitochondria or
peroxisome for oxidation. A-FABP is abundantly present in human serum and it may play
a central role in the development of major components of the metabolic syndrome such as
obesity, type 2 diabetes and cardiovascular diseases, through its distinct actions in
adipocytes and macrophages. Hence aP2 is an important marker for metabolic disorders.
Perilipin: Perilipin is a protein that forms a coating around the lipid droplets in
adipocytes. It is a protective coating against body's natural lipases, such as hormonesensitive
lipase, that breaks triglycerides into glycerol and free fatty acids by a process
called lipolysis. Perilipin [PLIN] may play key role in obesity. Following -adrenergic
receptor activation, protein kinase A (PKA) hyperphosphorylates perilipin localized at the
surface of the lipid droplet. Phosphorylated perilipin changes conformation and
translocate away from the lipid droplet, exposing the stored lipids to hormone-sensitive
lipase-mediated hydrolysis of triglycerides (lipolysis) to release non esterified fatty acids
(NEFA). Perilipin is thus an important regulator of lipid storage, lipolysis and energy
balance and is an important target for developing anti-obesity drugs.
The inventors have thus evaluated the efficacy of 7-hydroxyfrullanolide in the modulation
of the above metabolic biomarkers that are primarily responsible for the adipogenesis
processes, insulin resistance in type 2 diabetes, obesity, metabolic syndrome and other
metabolic disorders using an immunoblot assay in 3T3-L1 adipocytes. It was found
surprisingly that 7-hydroxyfrullanolide potently ameliorated the levels of several
adipocyte differentiation markers such as Peroxisome proliferator-activated receptor
gamma (PPARy), ADRP, CD36, Fatty Acid Binding Protein 4 (aP2/FABP4) and
intracellular lipid droplet surface associated protein (perilipin) (Figure ) in a dose
dependent manner. This unexpected result confirms the potential of 7-HF to control,
prevent and treat metabolic disorders through modulating multiple disease targets.
Efficacy of 7-hvdroxyfrullanolide in the improvement of cardiovascular health
Macrophage CD36: CD36 is a prototypic member of the class B scavenger receptor
family. It is widely expressed on the surface of monocytes and macrophages, and
mediates uptake of oxidized low-density lipoprotein (Ox-LDL) as well as play a role in
diverse cellular processes including foam cell formation, fatty acid transport, engulfment
and removal of senescent cells, suppression of angiogenesis, and cell-matrix interactions.
As such it can be an important risk factor of cardiovascular disease and a potential
molecular maker of atherosclerosis. Hyperglycemia-induced synthesis of CD36 in
macrophages has been associated with increased uptake of Ox-LDL by macrophages and
foam cell formation in atherosclerotic lesions in people with diabetes.
Inhibition of CD36 protein expression in high glucose induced J774 macrophage cells in
presence or absence of 7-HF was evaluated using immunoblot assay and the results are
summarized in Figure V. The CD36 levels were significantly enhanced in the cells
treated with glucose. However, these levels were reduced back towards their base values
in the cells treated with 7-HF when compared to the untreated control cells. This
unexpected observation provides support in favor of the potential use of 7-HF for the
prevention, control and treatment of cardiovascular diseases.
Nitric oxide (NO): Nitric oxide (NO) is a key biological molecule that, either directly or
through intracellular signaling, stimulates host defenses in the immune system, maintains
blood pressure in the cardiovascular system and modulates neural transmission in the
brain. NO is an activator of soluble guanylyl cyclase, which converts guanosine
triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) and leads to
vasodilatation and inhibition of leukocyte and platelet activation. As the biologically
active component of endothelium-derived relaxing factor, NO plays critical roles in the
maintenance of vascular homeostasis. Hence, compounds that enhance the NO levels can
have potential health benefits in humans. However, the volatile nature of NO makes it
unsuitable for most detection methods. In the cell, NO undergoes a series of reactions
with several molecules present in biological fluids and is eventually metabolized to nitrite
(NO 2 ) and nitrate (NO 3
") . Hence measuring the nitrite level gives an indication of nitric
oxide. The ability of 7-HF to modulate the NO level was evaluated in a human
endothelial (HE) cellular model and found surprisingly significant and dose dependent
increase in NO concentration in cell culture supernant following administration of the HE
cells with 7-HF. The data is summarized in Figure VI.
Inhibition of Protein tyrosine phosphatase IB (PTP-IB) by 7-HF:
Resistance to the hormone insulin is the hallmark of type 2 diabetes and obesity. Protein
tyrosine phosphatase IB (PTP-IB) is regarded as a physiological negative regulator of
insulin signal transduction in insulin sensitive cells such as adipocytes, muscle cells and
hepatocytes. In insulin resistant diabetes and obesity, the PTB-1B is over expressed and
its enzyme activity is increased. Over expression of PTP-IB decreases insulin receptor
and IPvS-1 Phosphorylation and hence produces insulin resistance. Silencing of PTP-IB
gene in an animal study astonishingly provided resistance from developing type 2
diabetes. Therefore, inhibition of PTP-IB has recently been emerged as a potential target
to treat type 2 diabetes. The ability of 7-HF in modulating PTP-IB was evaluated in 3T3-
Ll mouse preadipocytes. Surprisingly, the adipocytes treated with 7-HF in the present
invention exhibited significant inhibition of PTP-IB activity as summarized in Figure
Vn. This observation thus indicates that 7-HF can also be used as a potential therapeutic
intervention to prevent, control and treat type 2 diabetes and insulin resistance.
Modulation of adiponectin by 7-HF:
Adiponectin is an important adipokine hormone exclusively secreted from the adipose
tissue and it modulates a number of metabolic processes including glucose homeostasis
and lipid metabolism. It is known that low levels of adiponectin are associated with
disease states such as obesity, diabetes and cardiovascular disease. Administration of
adiponectin was proven to be beneficial in animal models of diabetes, obesity and
atherosclerosis. High plasma concentrations of adiponectin were also found to associate
with lower risk of Myocardial Infarction in men. Therefore, adiponectin has been
established as a promising marker of obesity, metabolic syndrome and other metabolic
disorders. The modulation of adiponectin protein by 7-HF in 3T3-L1 adipocytes was
evaluated in Western immunoblot assay. The cell culture, treatment protocol and
immunoblot assay methodology were as per the standard protocol. 7-HF showed potent
upregulation of adiponectin protein expression in 3T3-L1 mature adipocytes as depicted
in Figure VIII, manifesting its potential use in the prevention, treatment and control of
metabolic disorders, such as obesity, insulin resistance or Type 2 diabetes and endothelial
dysfunction.
It was quite unexpected and surprising to see that a single ingredient, 7-
hydroxyfrullanolide could be able to modulate the marker proteins related to many
disease conditions associated with metabolic disorders. This suggests that 7-
hydroxyfrullanolide could be a potential therapeutic agent to prevent, treat and control
metabolic syndrome, obesity, diabetes, atherosclerosis, endothelial dysfunction, chronic
kidney disease (CKD) and other metabolic disorders in animals and humans.
Several natural analogs, namely compound-1 to compound-9 and their structures are
depicted as those with numbers 3 to 11 respectively in Figure IX, have been isolated as
congeners of 7-hydroxyfrullanolide from Sphaeranthus indicus alcohol extract and their
structures characterized using IR, 1H NMR, 1 C NMR and Mass Spectral data. Several of
these natural analogs have shown potent anti-adipogenesis activity in the cell based assay.
Out of these compound-2(4), compound-3(5), compound-5(7), compound-7(9) and
compound-8(10) are found to be new to the best of our knowledge. The anti-adipogenic
potential of some of these analogs are summarized in Table-1.
Similarly, several synthetic analogs of 7-hydroxyfrullanolide, named sequentially as
compound- 10 to compound-23 in the present invention, have been prepared using a semi
synthetic process and their structures are depicted as those with numbers 12 to 25
respectively as in Figure X. Their biological activity was evaluated in a cell based assay.
These compounds also exhibited antiadipogenic potential and inhibited lipid
accumulation in human 3T3-L1 adipocytes as summarized in Table-l.
The extracts containing 7-hydroxyfrullanolide and/or one or more of the analogs of 7-
hydroxyfrullanolide also showed potent anti-adipogenic activity in 3T3-L1 Human preadipocyte
cells. The ethyl acetate extract of Sphaeranthus indicus (SIE) containing 1%
of 7-hydroxyfrullanolide, for example, showed 65.9% inhibition of lipid accumulation at
10 g/mL concentration in human 3T3-L1 pre-adipocyte cells. Further, SIE accelerated
the lipolysis by 26.7% at 25 g/mL as indicated by the percentage increase in glycerol
release in the lipolysis assay.
The efficacy shown by SIE in vitro models was further evaluated in an in vivo rat model
of metabolic syndrome (MS). Metabolic syndrome condition was experimentally induced
in male Sprague Dawley rats by feeding the rats with high fat, high cholesterol, high salt
and high sucrose diet for eight weeks. After eight weeks of induction period, the rats were
randomly divided into two groups with six animals in each group and the treatment group
animals were supplemented orally with 250 mg/kg body weight of SIE in 10 mL of 0.5%
CMC in water for further 8 weeks. The control group of animals received only the vehicle
(10 mL kg of 0.5% CMC in water) during this period. Body weight of individual animal
was recorded weekly for the entire duration of the study. Mean body weights for the
treatment group and control group were determined. The body weight gain was calculated
at the end of 1st week, 4th week and 8th week after initiation of treatment in comparison
to initial body weight. SIE at a dose of 250 mg kg exhibited highly potent and statistically
significant (p<0.01) reduction in body weight gain (66.04%) in comparison to control
group as summarized in figures XIA & XIB.
Assessment of serum adiponectin in MS rats: The serum adiponectin concentration in the
treatment and control groups of animals was assessed using double antibody based
sandwich rat adiponectin ELISA kit. The data revealed that daily supplementation of SIE
at 250 mg/kg body weight for 8 weeks resulted in significant (p=0.00618) improvement
in serum adiponectin concentration, when compared to the baseline as summarized in
Figure XII. The control group, however, did not show such improvement in serum
adiponectin concentration.
Supplementation of SIE at 250 mg/kg also resulted in improvement in fat profile with
15.3, 12.7 and 22.9 percentage reductions respectively in serum cholesterol, LDL and
triglycerides. This is well corroborated with its efficacy observed in improvement of
adiponectin levels.
Homeostasis Model Assessment (HOMA):
-The HOMA index was calculated based on serum insulin and glucose levels using the
formula: Fasting insulin concentration (/mL) Fasting glucose concentration
(mmol/L)/22.5. The data presented in Figure manifested that compared to the
control group, supplementation of a daily dose of 250 mg kg of ethyl acetate extract of
Sphaeranthus indicus (SIE) for 8-weeks resulted in significant reduction of HOMA index.
The Homeostatic Model Assessment (HOMA) is widely considered as a measure of
insulin resistance and beta cell function in clinical research. The data indicates that (SIE)
can be a therapeutic agent to improve insulin sensitivity and -cell function.
Based on the above animal study, it is obvious that ethyl acetate extract of Sphaeranthus
indicus (SIE) not only reduces obesity but also ameliorates various symptoms of
metabolic syndrome including body weight gain, visceral and organ fat deposition and
improves lipid profile, glucose homeostasis, insulin resistant type-II diabetes, -cell
function etc.
The foregoing thus suggest that 7-hydroxyfrullanolide, its analogs, the extracts/fractions
containing 7-hydroxyfrullanolide or its analogs or both or mixtures thereof or their
compositions can be potential pharmaceutical/ dietary supplement/food ingredient for the
control, prevention and treatment of one or more metabolic disorder(s) and/or for the
amelioration of the expression/production of one or more of the biological markers
related to metabolic disorders.
The present invention comprises different aspects cited below:
For the purpose of this invention, the word "component" or "biologically active
ingredient" widely used in the specification and claims of the present invention refer to 7-
hydroxyfrullanolide alone, or one or more of its analog(s); or the extracts or fraction
standardized to 7-hydroxyfrallanolide or analog(s) or both; or mixtures thereof. The word
"component" or "biologically active ingredient" are used interchangeably through out the
specification and the same may be appreciated as such by the person skilled in the art.
The word "composition" used in the specification and claims of the present invention
refers to combination of one or more of 7-hydroxyfrullanolide or one or more of its
analog(s); or the extracts or fraction standardized to 7-hydroxyfrallanolide or analog(s) or
both; or mixtures thereof with one or more of other biologically active components,
vehicles, carriers and diluents etc.
The phrase "other biologically active components" refers to extract(s) or fraction(s) or
compound(s) derived from plants, animals and microorganisms.
In an important embodiment, the invention provides biologically active ingredient(s)
selected from one or more of 7-hydroxyfrullanolide alone, its analog(s) and the extract(s)
or fraction(s) containing 7-hydroxyfrullanolide alone/its analog(s) or both or mixture(s)
thereof as an active for the control, prevention and treatment of one or more metabolic
disorder(s) and/or for the amelioration of the expression/production of one or more of the
biological markers related to metabolic disorders.
In another important embodiment, the invention provides biologically active ingredient(s)
compositions comprising at least one component selected from 7-hydroxyfrullanolide, its
analog(s), the extract(s) or fraction(s) containing 7-hydroxyfrullanolide/its analog(s) or
both or mixture(s) thereof as an active in combination with one or more selected from
biologically actives derived from plants, animals and microorganisms, pharmaceutically
or dietetically acceptable active ingredients, vitamins, aminoacids, minerals, vehicles,
carriers and diluents or mixtures thereof for the prevention, control and treatment of one
or more metabolic disorder(s) and/or for the amelioration of the expression/production of
one or more of the biological markers related to metabolic disorders.
In the other important embodiment, the metabolic disorders to be controlled/prevented/
treated by the biologically active ingredient(s) or compositions described comprise
obesity, over weight, diabetes, arteriosclerosis, cardiovascular diseases, hypertension,
hypercholesteremia (LDL, HDL, VLDL), hyperlipidemia, triglyceridemia, metabolic
syndrome, endothelial dysfunction and other metabolic disorders.
In the other embodiment, the invention provides biologically active ingredient(s) selected
from one or more of the components 7-hydroxyfrullanolide, its analog(s) and the
extract(s) or fraction(s) containing 7-hydroxyfrullanolide alone or its analog(s) or
mixture(s) thereof or their composition(s) for the amelioration of the expression or
production of one or more biological marker proteins related to or associated with
metabolic syndrome, obesity and other disease conditions associated with metabolic
syndrome including but not limited to Peroxisome proliferator-activated receptor gamma
(PPARy), Adipose Differentiation Related Protein (ADRP), adipocyte CD36,
Macrophage CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL),
adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor
(P3AR), Perilipin, Adiponectin Protein tyrosine phosphatase- IB (PTP-1B),
Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3) and Matrix
Metalloproteinase- 13 (MMP- 13).
In another embodiment of the invention the 7-hydroxyfrullanolide and its analog(s)
mentioned in the previous embodiments are of synthetic or semi-synthetic origin or
natural origin, wherein the natural origin can be any plant species that produces 7-
hydroxyfrullanolide or its analog(s) or mixtures thereof, more selectively Sphaeranthus
indicus.
In another embodiment the invention provides the extract(s) and fraction(s) comprising 7-
hydroxyfrullanolide or its analogs or mixtures thereof, wherein these extracts or fraction
can be derived from any plant species that produces 7-hydroxyfrullanolide or its analog or
mixtures thereof, more selectively Sphaeranthus indicus.
In another embodiment the invention provides biologically active ingredient(s) comprises
of the extracts and fractions containing 7-hydroxyfrullanolide or its analogs or mixtures
thereof wherein the said extracts and fractions contain 7-hydroxyfrullanolide or its
analog(s) or mixtures thereof in the range of 0.001% to 100%, preferably 0.01 to 99%.
In another embodiment, the invention provides biologically active ingredient(s)
compositions wherein the percentage of the extract or fraction standardized to 7-
hydroxyfrullanolide or its analog(s) or both varies in the range from 0.01% to 99%,
preferably 1% to 90% by weight in the composition.
In the other embodiment, the invention provides extracts, fractions and compositions
comprising 7-hydroxyfrullanolide or its analog(s) for the control, prevention and
treatment of one or more metabolic disorder(s), wherein the concentration of 7-
hydroxyfrullanolide or its analog(s) or mixtures thereof varies in the range from 0.01% to
99.9%.
In the other embodiment, the invention provides biologically active ingredient(s)
compositions comprising at least one component selected from 7-hydroxyfrullanolide, its
analog(s), the extract(s) or fraction(s) containing 7-hydroxyfrullanolide or its analog(s) or
mixture(s) thereof as an active for the control, prevention and treatment of one or more
metabolic disorder(s), wherein the concentration of active in the composition varies in the
range from 0.001% to 99.9%, preferably 0.01 to 95% by weight.
In a further embodiment the invention provides analogs of 7-hydroxyfrullanolide as
described above for the control, prevention and treatment of metabolic disorder(s)
and/or for the amelioration of the expression/production of one or more of the
biological marker(s) related to metabolic disorder(s), where in the analogs comprises
of the compounds represented by the general formula I given below:
Formula I
Wherein Ri, R2, R3, R4 and R are each independently selected from H, hydrogen,
hydroxy, halogen, -OORn, alkoxy, -OC(0)Ri 2 and C(0)Ri ; optionally Ri and R2
are taken together to form a ketone (=0).
The tricyclic ring system consisting of one or two or three double bonds.
Optionally R2 and R3 together form a double bond;
Optionally R3 and R4 together form a double bond;
Optionally R3 and R5 together form a double bond;
Optionally R and R together form a double bond;
Optionally R and R9 together form a double bond;
Further optionally R3 and R5together form an epoxide ring
R7 is selected from hydrogen, hydroxy, halogen, alkoxy and -OC(0 )R 2; R8 is
selected from hydrogen, hydroxy, halogen, alkoxy, -OC(0)Ri 2, - C(0 )R 2 and
NRi3Ri4; R9 is selected from hydroxy, alkyl, cycloalkyl, alkoxy, aryl,
heterocyclyl, halogen, -OC(0)Ri 2, -C(0)Ri 2, azido and -NR13Ri4, -S(0) mRi5, -
OS(O) mRi5j wherein m is 0, 1 or 2;
Rio and R are each independently selected from hydrogen, alkyl, halogen, ORi ,
-NH R 2 and SRi2; wherein R is selected from hydrogen, alkyl and -C(0)R ,2 or
Rio and together form one of ketone (=0), thioketone (=S), imine (=NH) and
selenoketone (=Se);
R1 is selected from hydrogen and alkyl;
Ri3 and i4 are each independently selected from hydrogen, alkyl, cycloalkyl,
aralkyl, aryl, heterocyclyl, -C(0)Ri 2 and -C(S)NHRi 2; or Ri3 and Ri4 together
with the N atom to which they are bonded, form a 5-, 6-, or 7-membered
heterocyclic ring, optionally having one or more additional heteroatoms selected
from O, N, S and Se;
R15 is selected from hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl
X is selected from O, NH, S and Se.
The 7-hydroxyfrullanolide or its analogs used for the prevention, control or treatment of
obesity, metabolic syndrome and other metabolic disorders or for making the composition
of the present invention can be naturally derived from plant species or can be produced
through synthesis or semisynthesis.
In other embodiment, the natural analogs of 7-hydroxyfrullanolide described above
comprises of frullanolide/eudesmanoid sesquiterpene compounds selected from but not
limited to frullanolides, 7-hydroxyfrullanolide (1); 1la,13-dihydro-3a,7a-dihydroxy-4,5-
epoxy-6p,7-eudesmanolide ; 1la,13-dihydro-7a-acetoxy -3 -hydroxy-6p,7-eudesm-4-
enolide;3-keto- -eudesmol; 11a,13-dihydro-3a,7a-dihydroxyeudesm-4-en-6a, 12-olide;
lla,13-dihydro-3a,7a-dihydroxyfrullanolide; 1la,13-dihydro-7a,13-
dihydroxyfrullanolide; lla,13-dihydro-7a-hydroxy-13-methaoxyfrullanolide (8); 2a,7adihydroxy-
4-en-l l,13-dihydroeudesm-6,l 2-olide; 2a-hydroxycostic acid; 3-keto-7ahydroxyeudesm-
4-en-6,l 2-olide (cryptomeridiol); 4-epicryptomeridiol; sphaeranthanolide
(11); 2a-hydroxysphaerantholide; 2a-acetoxysphaerantholide; 2a,7adihydroxysphaerantholide;
2a-acetoxy-7a-hydroxysphaerantholide; 2a-acetoxy-5ahydroxyisosphaerantholide,
eudesmanolide dimer [compound-2, (4)],
(3aR,5aR,9aR,9bR)-decahydro-9a-hydroperoxy-3a-hydroxy-5a-methyl-3,9-
dimethylenenaphtho[l,2-b]furan-2(9bH)-one [compound-3, (5)], (3aS,5aR,9bR)-
3,3a,4,5,5a,6,7,8-octahydro-3,5a,9-trimethylnaphtho[1,2-b]furan-2(9bH)-one [compound-
4, (6)], (R)-5,5a,6,7-tetrahydro-3,5a,9-trimethylnaphtho[1,2-b]furan-2(4H)-one
[compound-5, (7)], (3R,3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-3-
(methoxymethyl)-5a,9-dimethylnaphtho[l,2-b]furan-2(9bH)-one [compound-6, (8)], 2-
((3R,8aR)-l ,2,3,7,8,8a-hexahydro-5,8a-dimethylnaphthalen-3-yl)acrylic acid [compound-
7, (9)], (3aR,5aR,9bS)-3-((6-amino-9H-purin-9-yl)methyl)-3,3a,4,5,5a,6,7,8-octahydro-
3a-hydroxy-5a,9-dimethylnaphtho[1,2-b]furan-2(9bH)-one [compound-8, (10)],
(3R,3aR,5aR,8R,9bS)-8-((2R,3S,4R,5R)-tetrahydro-3,4,5-trihydroxy-6-(hydroxymethyl)-
2H-pyran-2-yloxy)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-3,5a,9-trimethylnaphtho[l,2-
b]furan-2(9bH)-one [compound-9, (11)].
In another embodiment, the invention provides biologically active ingredient(s)
selected from 7-hydroxyfrullanolide or their analog(s) or their compositions as
described above, wherein the synthetic and semi-synthetic analogs of 7-
hydroxyfrullanolide comprises (3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a,8-
dihydroxy-5a,9-dimethyl-3-methylenenaphtho[l,2-b]furan-2(9bH)-one [compound10,
(12)], (3aR,5aS,9bS)-3a,4,5,5a,6 ,7-hexahydro-3a-hydroxy-5a,9-dimethyl-3-
methylenenaphtho[l,2-b]furan-2,8(3H,9bH)-dione [compound-1 1, (13)], (R)-
2,4,5,5a,6 ,7-hexahydro-5a,9-dimethyl-2-oxonaphtho[l,2-b]furan-3-carbaldehyde
[compound-12, (14)], 4,5-epoxy -7-hydroxyfrullanolide [compound-1 3, (15)], (R)-3-
(bromomethyl)-5,5a,6 ,7-tetrahydro-5a,9-dimethylnaphtho[l,2-b]furan-2(4H)-one
[compound- 14, (16)], (3aR,5aR,9bS)-3,3a,4,5,5a,6 ,7,8-octahydro-3a-hydroxy-
3,5a,9-trimethylnaphtho[l,2-b]furan-2(9bH)-one [compound- 15, (17)], (2E)-
(3aR,5aR,9bS)-2,3,3a,4,5,5a,6 ,7,8,9b-decahydro-3a-hydroxy-3,5a,9-trimethyl-2-
oxonaphtho[l,2-b]furan-8-yl 3-(2,5-dimethoxyphenyl)acrylate [compound-16, (18)],
(3aR,5aR,9bS)-3,3a,4,5,5a,6 ,7,8-octahydro-3a-hydroxy-5a,9-dimethyl-3-
(morpholinomethyl)naphtho[ 1,2-b]furan-2(9bH)-one [compound- 17, (19)],
(3aR,5aR,9bS)-3,3a,4,5,5a,6 ,7,8-octahydro-3a,8-dihydroxy-5a,9-dimethyl-3-
(morpholinomethyl)naphtho[ 1,2-b]furan-2(9bH)-one [compound- 18, (20)],
(3aR,5aR,9bS)-3,3a,4,5,5a,6 ,7,8-octahydro-3a-hydroxy-5a,9-dimethyl-3-((piperidinl-
yl)methyl)naphtho[l,2-b]furan-2(9bH)-one [compound-19, (21)], (5aR)-
5,5a,6 ,7,8,9-hexahydro-9-hydroxy-5a,9-dimethyl-3-((piperidin-lyl)
methyl)naphtho[l,2-b]furan-2(4H)-one [compound-20, (22)], (3aR,5aR,9bS)-3-
((4H-l,2,4-triazol-4-yl)methyl)-3,3a,4,5,5a,6 ,7,8-octahydro-3a-hydroxy-5a,9-
dimethylnaphtho[l,2-b]furan-2(9bH)-one [compound-21, (23)], (3aR,5aR,9bS)-
3,3a,4,5,5a,6 ,7,8-octahydro-3a-hydroxy-5a,9-dimethyl-3-((piperazin- -
yl)methyl)naphtho[l,2-b]furan-2(9bH)-one [compound-22, (24)] and piperazine bis-
7-hydroxyfrullanolide [compound-23, (25) represented by the structure numbers 12
through 25 respectively as depicted in Figure X.
In another embodiment, the invention provides the extracts and fractions derived from
Sphaeranthus indicus containing 7-hydroxyfrullanolide/other frullanolide(s)/eudesmanoid
sesquiterpene(s)/other phytochemicals for the prevention, control and treatment of
obesity, metabolic syndrome and other metabolic disorders or for making the
compositions described above comprises, 7-hydroxyfrullanolide/other
frullanolide(s)/eudesmanoid sesquiterpene(s)/other phytochemicals or mixture thereof
varies in concentration range of 0.001% to 100%, preferably 0.01 to 99%.
In another embodiment, the concentration of the active compound 7-
hydroxyfrullanolide/other frullanolide(s)/eudesmanoid sesquiterpene(s)/ other
phytochemicals in the compositions comprising Sphaeranthus indicus derived component
as described in the previous embodiments varies in the range from 0.001% to 99%,
preferably 0.01 to 95% by weight.
In another embodiment of the invention, the other biologically active components used
for making the compositions comprise components having any health benefit selected
from but not limited to anti-diabetic activity, anti-hyperlipidemic activity, anti-obesity
activity, anti-hypertensive activity, anti-platelet aggregation activity, anti-infective
activity, anti-atherosclerotic activity and anti-inflammatory activity, anti-oxidant activity
and bio-enhancing activity.
I another embodiment, the invention provides biologically active ingredient(s) selected
from 7-hydroxyfrullanolide, its analogs, the extracts and fractions containing 7-
hydroxyfrullanolide or its analogs or mixtures thereof, derived from Sphaeranthus
indicus, or their composition, wherein said extract(s) or active fraction(s) or active
compound(s) or phytochemicals or mixtures thereof are derived from atleast one of the
plant parts selected from but not limited to leaves, flower heads, fruits, stem, bark, root,
whole plant or mixtures thereof, preferably flower heads.
In another embodiment of the invention, biologically active ingredient(s) and their
compositions as described in previous embodiments, wherein said 7-hydroxyfrullanolide,
it natural analogs, the extract(s) or active fraction(s) containing 7-hydroxyfrullanolide or
it natural analog(s) or mixtures thereof or phytochemicals or mixtures thereof derived
from Sphaeranthus indicus are obtained through extraction using solvents selected from
one or more of organic solvents, alcohols, hydroalcohols, water or mixtures thereof or
those followed by partitions and/or chromatography.
The examples of the biologically or pharmaceutically acceptable excipients, vehicles and
carriers employed in the present invention include, but are not limited to, surfactants,
binders, diluents, disintegrators, lubricants, preservatives, stabilizers, buffers, suspensions
and drug delivery systems.
The examples of the biologically or pharmaceutically acceptable excipients, carriers and
diluents comprise glucose, fructose, sucrose, maltose, yellow dextrin, white dextrin,
aerosol, microcrystalline cellulose, calcium stearate, magnesium stearate, sorbitol,
stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic
acid, L-ascorbic acid, dl-alpha-tocopherol, glycerin, propylene glycol, glycerin fatty ester,
poly glycerin fatty ester, sucrose fatty ester, sorbitan fatty ester, propylene glycol fatty
ester, acacia, carrageenan, casein, gelatin, pectin, agar, vitamin B group, nicotinamide,
calcium pantothenate, amino acids, calcium salts, pigments, flavors, preservatives,
distilled water, saline, aqueous glucose solution, alcohol, propylene glycol and
polyethylene glycol, various animal and vegetable oils, white soft paraffin, paraffin and
wax.
In another embodiment, the invention provides biologically active ingredient(s) or their
composition(s) as claimed in preceding embodiments, wherein said component or
composition is administered orally, topically or parenterally or by inhalation to a subject
or mammal or warm blooded animal in need thereof.
In another embodiment, the invention provides biologically active ingredient(s) or their
composition(s) as claimed in preceding embodiments, wherein said components or
compositions can be formulated into any suitable formulation like oral agents such as
tablets, soft capsule, hard capsule, soft gel capsules, pills, granules, powders, emulsions,
suspensions, syrups, pellets, food, beverages, concentrated shots, drops and the like; and
parenteral agents such as injections, intravenous drip and the like; suppositories; and
transdermal agents such as patches, topical creams and gel; ophthalmic agents; nasal
agents; and food or beverages.
In another embodiment, the invention provides a method for the
control/prevention/treating of a metabolic disorder selected from but not limited to
obesity, over weight, diabetes, arteriosclerosis, cardiovascular diseases, hypertension,
hypercholesteremia, hyperlipidemia, triglyceridemia, metabolic syndrome, endothelial
dysfunction and other metabolic disorders in a mammal or warm blooded animal in need
thereof, wherein the method comprises administering a therapeutically effective amount
of atleast one biologically active ingredient(s) selected from 7-hydroxyfrullanolide, its
analog(s), the extract(s) or fraction(s) comprising 7-hydroxyfrullanolide/its analogs or
both as an active or mixtures thereof or their compositions as described in preceding
embodiments.
In another embodiment , the invention provides a method of promoting lipolysis and/or
inhibiting adipogenesis comprising administering to a subject or mammal or warm
blooded animal in need thereof a therapeutically effective quantity of atleast one
biologically active ingredient(s) selected from 7-hydroxyfrullanolide, its analog(s), the
extract(s) or fraction(s) comprising 7-hydroxyfrullanolide/its analog(s) or both or
mixtures thereof as an active or their compositions as described in the preceding
embodiments.
In another embodiment, the invention provides a method of using biologically active
ingredient(s) selected from 7-hydroxyfrullanolide or its analogs; the extract(s) or
fraction(s) comprising 7-hydroxyfrullanolide or its analog(s) or both or their compositions
for the amelioration of the expression or production of biological markers selected from
but not limited to PPAR-, C-reactive protein (CRP), Adipose Differentiation Related
Protein (ADRP), adipocyte CD36, macrophage CD36, Monocyte Chemotactic protein
(MCP-1), Oxidized LDL, Adipocyte Fatty-acid-Binding Protein (aP2/FABP4/A-FABP),
Beta-3 adrenergic receptor (P3-AR), adiponectin, Perilipin, Protein tyrosine phosphatase
IB (PTP IB), Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3) and
Matrix Metalloproteinase-1 3 (MMP-13).
In a further embodiment of the present invention, the components selected from 7-
hydroxyfrullanolide, its analogs; the extract(s) or fraction(s) or mixtures thereof derived
from Sphaeranthus indicus comprising 7-hydroxyfrullanolide as an active ingredient or
their compositions as described above can be optionally combined with bio-availability
enhancing agents selected from but not limited to extract(s), fraction(s), pure
compound(s) derived from Piper nigrum or Piper longum, and Stevia rebaudiana..
In alternative embodiment of the invention, the components selected from 7-
hydroxyfrullanolide or its analogs or compositions comprising the extract(s), fraction(s),
active compound(s) or phytochemical(s) or mixtures thereof derived from Sphaeranthus
indicus comprising 7-hydroxyfrullanolide as an active ingredient or their compositions
claimed in the present invention are delivered in the form of controlled release tablets,
using controlled release polymer-based coatings by the techniques including
nanotechnology, microencapsulation, colloidal carrier systems and other drug delivery
systems known in the art. The said formulation can be designed for once a daily
administration or multiple administrations per day.
In other embodiment of the invention, the components selected from 7-
hydroxyfrullanolide or its analogs or the extracts or fractions containing 7-
hydroxyfrullanolide or their compositions described/claimed in the present invention can
also be formulated into or added to existing or new food and beverage form(s) and animal
feeds as a healthy food or beverage or feed for prevention, control or treatment of one or
more of the diseases including but not limited to obesity, diabetes, hypertension,
cardiovascular diseases, neurological disorders, Alzheimer's, cognitive disorders,
oxidative stress, skin disorders, aging of the skin, UV irradiated damage,
hypercholesterolemia, variations of LDL, HDL & VLDL, hyperlipidemia,
triglyceridemia, immune deficiency, cancer, metabolic syndrome, for bringing about
weight loss effectively, for producing lean body mass, for using during weight loss
program as well as for other metabolic disorders.
In other embodiment, the invention provides the use of ingredient(s) or composition(s) for
prevention, control and treatment of one or more diseases several diseases or disease
conditions including but not limited to obesity, diabetes, hypertension, atherosclerosis,
cardiovascular diseases, neurological disorders, Alzheimer's, cognitive disorders,
oxidative stress, skin disorders, aging of the skin, UV irradiated damage,
hypercholesterolemia, variations of LDL, HDL & VLDL, hyperlipidemia,
triglyceridemia, immune deficiency, cancer, metabolic syndrome, for bringing about
weight loss effectively, for producing lean body mass, for using during weight loss
program as well as for other metabolic disorders.
In other embodiment, the invention provides a method of prevention/ control /treatment
of one or more metabolic disorders selected from obesity, over weight, diabetes,
atherosclerosis, arteriosclerosis, cardiovascular diseases, hypertension,
hypercholesterolemia, variations of LDL, HDL, VLDL, hyperlipidemia, triglyceridemia,
metabolic syndrome, endothelial dysfunction and other metabolic disorders in a mammal
or warm blooded animal in need thereof, wherein the method comprises administering to
mammal or warm blooded animal a therapeutically effective amount of atleast one
biologically active ingredient(s) from 7-hydroxyfrullanolide, its analog(s), the extract(s)
or fraction(s) comprising 7-hydroxyfrullanolide or its analog(s) or both as an active or
mixtures thereof or their compositions.
In other embodiment, the invention provides a method of promoting lipolysis and/or
inhibiting adipogenesis in a subject or mammal or warm blooded animal in need thereof
comprising administering to said subject or mammal or warm blooded animal a
therapeutically effective quantity of atleast one biologically active ingredient(s) selected
from 7-hydroxyfrullanolide, its analog(s), the extract(s) or fraction(s) comprising 7-
hydroxyfrullanolide or its analog(s) or both or mixtures thereof as an active or their
compositions.
In other embodiment the invention provides a method of amelioration of the expression or
production of atleast one biological marker selected from PPAR-, C-reactive protein
(CRP), Adipose Differentiation Related Protein (ADRP), adipocyte CD36, macrophage
CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL, Adipocyte Fatty-acid-
Binding Protein (aP2/FABP4/A-FABP), Beta-3 adrenergic receptor (P3-AR), adiponectin,
Perilipin, Protein tyrosine phosphatase IB (PTP IB), Metalloproteinase-1 (MMP-1),
Matrix Metalloproteinase-3 (MMP-3) and Matrix Metalloproteinase-1 3 (MMP-13) in a
subject or mammal or warm blooded animal in need thereof, wherein the method
comprises administering to the subject or mammal or warm blooded animal a biologically
active ingredient(s) selected from 7-hydroxyfrullanolide or its analogs; the extract(s) or
fraction(s) containing 7-hydroxyfrullanolide or its analog(s) or both or mixtures thereof in
their composition(s).
The unexpected and superior ameliorating effects of 7-hydroxyfrullanolide or its analogs
or the extracts/fraction containing 7-hydroxyfrullanolide or mixtures thereof their
*
compositions claimed in the present invention are illustrated by the following nonlimiting
examples:
Example 1
Sphaeranthus indicus ethyl acetate extract (SIE): Sphaeranthus indicus flower heads (2.2
kg) were charged into a pilot extractor and extracted with ethyl acetate (22 L) at reflux
temperature for 2 h. The extract was filtered and the spent raw material was re-extracted
twice with ethyl acetate (2 x 13 L) under similar conditions. The combined extract was
fine filtered and concentrated over a climbing film evaporator to obtain residue (174 g).
The ethyl acetate extract showed 11% of 7-hydroxy-4, 11 (13)-eudesmadien-12, 6-olide
(7-hydroxyfrullanolide) by HPLC method of analysis.
Example 2
Preparation of methanol extract of Sphaeranthus indicus and isolation of 7-
hydroxyfrullanolide and its natural analogs: Sphaeranthus indicus flower heads (7 kg)
were taken into a pilot extractor and extracted with methanol (56 L) at 80°C temperature
for 2 h. The extract was filtered and the spent raw material was re-extracted twice with
methanol (2 x 40L) under similar conditions. The combined extract was fine filtered and
concentrated under vacuum to obtain a residue ( 1 kg). The methanol extract was
suspended in water ( 1 L) and extracted with ethyl acetate (3 x 1.5 L). The combined
organic layer was evaporated under vacuum and the residue (300 g) was subjected to
column chromatography over silica column using eluants of increasing polarity from
hexane to acetone. The fractions eluted with 20% acetone/hexane were combined and
evaporated under vacuum to give a residue (fraction-I; 49 g). The fractions eluted with
40% acetone/hexane were combined and evaporated under vacuum to give a residue
(fraction-II; 35 g). The fraction eluted with 60% acetone/hexane was evaporated under
vacuum to give a residue (fraction-Ill).
The fraction-I was subjected to re-chromatography over silica column using solvents of
increasing polarity from chloroform to ethyl acetate. The fraction (3 g) eluted with
chloroform was evaporated and the residue subjected to repeated chromatography over
silica gel using ethyl acetate/hexane mixture to obtain turmerone (2); 60 mg. The
fractions eluted of the fraction-I column with 2-5% ethyl acetate/chloroform were
combined and evaporated, and the residue was subjected to repeated chromatography
over silica gel using acetone/hexane and chloroform/hexane mixtures to yield compound-
1 (3); 40 mg and compound-2 (4); 50 mg. The fractions eluted with 5-10% ethyl
acetate/chloroform mixtures were evaporated under vacuum to obtain 7-
hydroxyfrullanolide (1); 13 g). The fractions obtained on elution with 15% ethyl
acetate/chloroform mixture were evaporated and the residue was subjected to further
chromatography over silica gel using same solvent system to obtain compound-3 (5); 20
mg. The fraction eluted with 20% ethyl acetate/chloroform was subjected to repeated
chromatography on silica column using ethyl acetate/chloroform mixtures, followed by
final purification on HPLC to obtain compound-4 (6); 24 mg.
The fraction-II obtained of the main column was subjected to re-chromatography over
silica column using chloroform and ethyl acetate/chloroform mixtures as eluants. The
fractions eluted with chloroform and 5% ethyl acetate/chloroform mixture were combined
and evaporated. The residue (5 g) was re-purified on silica column again using ethyl
acetate/chloroform mixtures and the fraction eluted with 2% ethyl acetate/chloroform was
evaporated under vacuum to provide compound-5 (7); 15 mg. The fraction eluted with
10% ethyl acetate/chloroform mixture was evaporated to yield a further quantity (3 g) of
7-hydroxyfrullanolide. The fraction (12 g) eluted with 20% ethyl acetate/chloroform
mixture was subjected to further purification on silica column using acetone/hexane
mixtures and the fraction so obtained using 10% acetone/hexane was re-purified on silica
column using ethyl acetate/chloroform mixtures to obtain compound-6 (8); 100 mg. The
other fraction obtained on elution with 20% acetone/hexane mixture furnished compound-
7 (9); 20 mg upon evaporation of the solvent. The fraction (5g) eluted with 60% ethyl
acetate/chloroform mixture was subjected to further purification on silica column using
ethyl acetate to obtain compound-8 (10); 30 mg.
The fraction-Ill obtained of the main column was purified on silica column using
methanol/ethyl acetate mixtures and the fraction eluted with 5% methanol/ethyl acetate
upon evaporation of the solvent yielded compound-9 (11); (1500 mg).
7-hydroxyfrullanolide (1): NMR (400 MHz, CDC13): 6.24 (s,lH), 5.80 (s 1H), 5.02
(s,lH), 2.12 (m,2H), 1.68 (m,lH), 1.95 (td J=l 2.4,3.2Hz,lH), 1.80 (m,lH), 1.68 (m,lH)
1.65 (m,lH) , 1.52 (td; J=14.,3.1Hz,lH), 1.42 (m,2H), 1.35 (m,lH), 1.77 (s,3H ), 1.07
(s,3H); NMR C (100 MHz, de-DMSO): 168.99, 144.84, 140.41, 126.97, 120.91, 81.42,
76.02, 38.85, 34.97, , 33.15, 32.77, 31.57, 26.15, 19.88, 18.21; LCMS (ESI, positive
scan): m/z 249 (M+H) +.
ar-Turmerone (2): NMR (CDC13, 400 MHz): 7.10 (4H, s), 6.02 (IH, t, J = 1.2 Hz),
3.28 (lH,sextet, J = 7.6 Hz), 2.70 (IH, dd, J = 6.0, 15.6 Hz), 2.60 (IH, J = 8.4, 15.6 Hz),
2.31 (3H, s), 2.10 (3H, d, J = 0.8 Hz), 1.85 (3H, d, J = 0.8 Hz), 1.24 (3H, d, J = 7.2 Hz);
1 C NMR (CDC13, 400 MHz): 199.77, 154.84, 143.71, 135.51, 129.08, 126.65, 124.15,
52.70, 35.32, 29.67, 27.53, 21.95, 20.91, 20.64.
3,3a,4,5,5a,6,7,8-octahydro-5a,9-dimethyl-3-methylenenaphtho[l,2-b]furan-2(9bH)-one
[Compound-1 (3)]: NMR (CDC13, 400 MHz): 6.16 (IH, s), 5.57 (IH, s), 5.26 (IH, d,
J = 5.6Hz), 2.95 (IH, m), 2.09 (2H, ), 1.83 (IH, ), 1.76 (3H, s), 1.61 - 1.71 (4H, m),
1.51 - 1.23 (9H, m), 1.08 (3H, s); LCMS: 232 (M+H) + +ve ion mode.
Compound-1 (structure: 3)
Eudesmanolide dimer [Compound-2, (4)]: NMR (CDC13, 400 MHz): 6.20 (IH, s),
3.28 (IH, dd, J = 6.0, 12.8Hz), 2.74 (IH, ), 2.52 (2H, m), 2.23 (2H, m), 2.14 (IH, m),
2.10 (IH, ), 2.04 (IH, m), 2.02 (IH, ), 1.99 (IH, m), 1.92 (IH, ), 1.88 (3H, s), 1.84
(IH, m), 1.78 (IH, m), 1.74 (IH, ), 1.68 (IH, s), 1.68 (3H, s), 1.61 (IH, m), 1.60 (IH,
m), 1.54 (IH, m), 1.48 (IH, m), 1.41 (IH, m), 1.38 (IH, m), 1.19 (2H, m), 1.06 (3H, s),
0.98 (3H, s); 13C NMR (CDC13, 100MHz): 206.32, 177.13, 175.07, 140.21, 139.85,
138.13, 127.88, 88.69, 82.78, 52.61, 52.46, 51.26, 39.99, 38.39, 38.14, 37.74, 33.82,
33.05, 32.45, 32.40, 27.74, 27.13, 26.37, 25.38, 24.13, 20.84, 20.63, 19.73, 19.61, 18.51,
18.36; LCMS: 478 (M+Na) +, +ve ion mode.
Compound-2 (structure: 4)
(3aR,5aR,9aR,9bR)-decahydro-9a-hydroperoxy-3a-hydroxy-5a-methyl-3,9-
dimethylenenaphtho[l,2-b]furan-2(9bH)-one [Compound-3, (5)]: NMR (CDC13, 400
MHz): 7.82 (IH, br s), 6.19 (IH, s), 5.80 (IH, s), 5.39 (IH, s), 5.29 (IH, s), 4.75 (IH,
s), 3.32 (IH, br s), 2.42-2.26 (4H, m), 1.88 -1.98 (4H, m), 1.63 - 1.78 (12H, m), 1.37 -
1.42 (2H, m), 1.16-1.09 - 1.16 (4H, ), 1.04 (3H, s), 0.84 - 0.94 (4H, ); , C NMR
(CDC13, 400MHz): 167.87, 143.34, 142.57, 119.70, 115.98, 86.99, 78.38, 75.69, 38.13,
35.97, 32.36, 30.35, 29.67, 21.92, 20.29; LCMS: 279(M-H) negative ion mode.
Compound-3 (structure: 5)
(3aS,5aR,9bR)-3,3a,4,5,5a,6,7,8-octahydro-3,5a,9-trimethylnaphtho[l,2-b]furan-2(9bH)-
one [Compound-4, (6)]: NMR (CDC1 , 400 MHz): 5.30 (IH, s), 4.90 (lH,s), 4.80
(IH, d, J=5.2Hz), 3.77 (IH, t, J=5.2Hz), 2.79 (IH, q, J=7.2), 1.87 (IH td, J=4.4Hz), 1.815
(3H, s), 1.61 (IH, m), 1.59 (IH, ), 1.52 (2H, ), 1.48 (IH, m), 1.45 (IH, ), 1.27 (IH,
m), 1.18 (IH, ), 1.04 (3H, d, J=7.2Hz), 0.92 (3H, s); 1 C NMR (CDC13, 400 MHz): 
176.66, 139.38, 130.01, 79.39, 75.92, 67.30, 47.30, 34.46, 33.87, 33.14, 27.38, 24.40,
23.60, 17.04, 6.798.
Compound-4 (structure: 6)
(R)-5,5a,6,7-tetrahydro-3,5a,9-trimethylnaphtho[ 1,2-b]furan-2(4H)-one [Compound-5,
(7)]: NMR (CDC13, 400 MHz): 5.83 (1H, m), 2.65 (2H, m), 2.30 - 2.35 (1H, m), 2.16
- 2.23(2H, m), 2.13 (3H,s), 1.89 (3H, s), 1.841 - 1.60 - 1.84 (7H, m), 1.48 - 1.54 (2H, m),
1.28 - 1.46 (3H, m), 1.08 (3H, s); LCMS: m/z 233 (M+H) +, +ve ion mode.
Compound-5 (structure: 7)
(3R,3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-3-(methoxymethyl)-5a,9-
dimethylnaphtho[l,2-b]furan-2(9bH)-one [Compound-6, (8)]: NMR (CDC13, 400
MHz): 5.01 (1H, s), 3.88 (1H, dd, J = 5.2, 9.6 Hz), 3.74 (1H, t, J = 11.2 Hz), 3.40 (3H,
s), 3.18 (1H, dd, J = 4.8, 10.8Hz), 1.92 - 2.15 (4H, m), 1.79 (3H, s), 1.60 - 1.68 (3H, m),
1.40 - 1.50 (4H, m), 1.05 (3H, s); LCMS: m/z 303 (M+Na) + +ve ion mode.
Compound-6 (structure: 8)
2-((3R,8aR)- 1,2,3,7,8,8a-hexahydro-5,8a-dimethylnaphthalen-3-yl)acrylic acid
[Compound-7, (9)]: NMR (CDC13, 400 MHz): 6.32 (1H, s), 5.71 (1H, s), 5.55 (1H,
bs), 5.38 (1H, bs), 3.43 (1H. m), 2.25 (3H, t J = 7.8, 11.2Hz), 2.03 - 2.09 (3H, m), 1.78
(3H, s), 1.63 (5H, ), 1.47 (2H, d, J = 5.2Hz), 1.00 (3H, s); LCMS: m/z 231 (M-H) -ve
ion mode.
Compound-7 (structure: 9)
(3aR,5aR,9bS)-3-((6-amino-9H-purin-9-yl)methyl)-3,3a,4,5,5a,6,7,8-octahydro-3ahydroxy-
5a,9-dimethyInaphtho[l,2-b]furan-2(9bH)-one [Compound-8, (10)]: NMR
(DMSO, 400MHz): 8.22 (2H, d J = 6Hz), 7.22 (2H, bs), 5.47 (IH, s), 5.08 (IH, s), 4.51
(2H, d, J = 7.2Hz), 3.58 (IH, t, J = 7.2, 14Hz), 2.07 - 2.14 (3H, m), 1.78 - 1.82 (3H, ),
1.75 (3H, s), 1.34 - 1.66 (10-H,
Compound-8 (structure: 10)
(3R,3aR,5aR,8R,9bS)-8-((2R,3S,4R,5R)-tetrahydro-3,4,5-trihydroxy-6-(hydroxymethyl)-
2H-pyran-2-yloxy)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-3,5a,9-trimethylnaphtho[l,2-
b]furan-2(9bH)-one [Compound-9, (Sphaeranthanolide; 11): NMR (400MHz,
d4MeOH): 5.01 (d; J=l.lHz, IH), 4.38 (d; J=7.2Hz, IH) 4.142 (d; J=1.6Hz,lH), 3.91 (dd;
J=12.7, 5.2Hz, IH), 3.71 (dd; J=12.7, 1.9Hz, IH), 3.39 (ddd; J=9, 7.81 Hz, IH), 3.33
(m,lH), 3.20 (m,lH), 3.19 (dd; J=7.8, 9Hz, IH), 2.82 (q; J=7.2Hz, IH), 1.97 (s, 3H), 1.91
(m, IH), 1.85 (m,lH), 1.70 (m,lH), 1.69 (m,lH) , 1.65 (m,lH), 1.59 (m, 2H), 1.43 (m,
H), 1.17 (d; J=7.2Hz, 3H); NMR C (100 MHz, t-MeOH): 178.22, 139.21, 133.94,
101.88, 81.54, 78.27, 78.01, 77.88, 75.99, 75. 07, 72.00, 63.02, 49.21, 35.25, 34.66,
25.42, 24.27, 24.25, 17.24, 7.24; LCMS (ESI, positive scan): m z 429.21 (M+H) +
Example 3
Oxidation of 7-hvdroxyfrullanolide to produce (3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-
octahydro-3a,8-dihydroxy-5a,9-dimethyl-3-methylenenaphtho[l,2-b]furan-2(9bH)-one
[compound- 10, (12)] and (3aR.5aS.9bSV3a.4.5.5a.6.7-hexahvdro-3a-hvdroxv-5a,9-
dimethyl-3-methylenenaphtho[l,2-b]furan-2,8(3H,9bH)-dione [compound- 11, (13) : A
mixture of 7-hydroxyfrullanolide (lg, 0.00403mol) and lOmL of ethanol was taken in a
RB flask and treated slowly with Se0 2 (1.4g, 0.0126mol) at room temperature, The
reaction mixture (RM) was then stirred at 70°C for 12h and then poured into ice-cold
water. The mixture was extracted with EtOAc and the organic layer washed with brine,
dried over Na S0 4 and concentrated. The residue (1.2g) was subjected to chromatography
eluting with chloroform followed by 2% and 5% methanol/chloroform mixtures as
eluants. The fractions eluted with 5% methanol/ chloroform were monitored, the
fractions containing the compounds were combined and evaporated to obtain 700 mg of
compound-10 [(12), 70%] and 50mg of compound-11 [(13), 4.7%].
Compound-10: NMR(CDC13, 400MHz); 6.29 (lH,s), 5.84 (IH, s), 4.99(1H, s), 4.08
(IH, d, J = 3.2 Hz), 3.10 (IH, brs), 2.22 (IH, brs), 2.06 (IH, tt, J = 3.2, 14.4Hz), 1.94
(3H, s), 1.90 (IH, m), 1.66 - 1.79 (3H, m), 1.55 (IH, m), 1.28 - 1.44 (2H, m), 1.08
(3H,s). C NMR (CDC13, 100MHz); 168.0, 142.9, 138.2, 130.9, 120.7, 80.3, 68.4, 33.6,
32.5, 32.1, 31.1, 26.6, 24.2, 16.8; LC/MS: 287 (M+Na)+, 303(M+K) + positive ion mode.
Compound-1 1: NMR(CDC13, 400MHz); 6.398(lH,s), 5.952(1H, s), 5.138(1H, s),
2.711(2H, m), 2.583-2.482(2H, m), 2.09-1.94(2H, m), 1.913(3H, s), 1.773-1.6 17(2H, m),
1.538-1.431(1H, m), 1.284(3H, s). LC MS: 261(M-HV negative ion mode.
Example 4
Oxidation of 7-a-hvdroxyfrullanolide to produce (R)-2.4.5.5a,6.7-hexahydro-5a,9-
dimethyl-2-oxonaphtho[l,2-b1furan-3-carbaldehvde [compound-12, (14)1: A mixture of
2.0 g of 7-hydroxyfrullanolide (0.00806 mol) and 40ml of dichloromethane was taken in
a Round Bottom (RB) flask and treated with 39g of silica gel adsorbed Jones reagent
(13ml of Jones reagent was adsorbed on 26gm of silica gel). The reaction mixture was
stirred at room temperature. After 3h, the mixture was filtered and the solid was washed
with chloroform. The combined filtrate was concentrated and the residue (2.0g) was
subjected to silica column chromatography using EtOAc/hexane mixtures as eluant. The
fractions eluted with 10% EtOAc/hexane were monitored and the fractions containing the
oxidized product were evaporated to obtain 50mg of Compound-12 [(14), 2.3%] as a
colorless solid.
Compound-12: NMR(CDC13, 400MHz): 9.98 ( IH, s), 6.15 (IH, m), 3.45 (IH, dt, J
= 4.0, 20.0 Hz), 2.96 (IH, p, J = 8.8 Hz), 2.19 (3H, t, J = 1.2Hz), 1.73 (2H, dd, J = 3.2,
9.6 Hz), 1.61 (2H, dd, J = 3.6, 8.4 Hz), 1.14 (3H, s); LC/MS: 245 (M+H) +, 267 (M+Na)+
positive ion mode.
Example 5
Epoxidation of 7-HF to obtain 4,5-epoxv-7-hvdroxvfrullanolide [compound- 13, (15)1:
A mixture of 7-HF (300 mg, 0.0012 mol) and 5mL of methylene chloride in a round
bottomed flask was treated slowly with m-chloroperbenzoic acid (417 mg, 0.0024 mol)
and the contents stirred at rt. After 1.5h, the reaction mixture was poured into ice-water
and the mixture was extracted with EtOAc. The EtOAc layer was washed with NaHC0 3
solution followed by brine, dry over Na2S0 4 and concentrated under vacuum. The residue
(300mg) was purified on a silica column using acetone/hexane mixtures. The fractions
eluted with 30% acetone/hexane were monitored on TLC and the fractions containing
Compound- 13 (15) were combined and evaporated under vacuum to form a solid (71.5
mg).
Compound-13: H NM (C ,400MHZ): 6.25(1H, s), 5.84 (1H, s), 3.91 (1H, s), 2.56
(1H, s), 2.009-1.944 - 2.01 (3H, m), 1.91-1.74 (5H, m), 1.59 - 1.69 (4H, m), 1.43 (1H,
m), 1.38 (3H, s), 1.32-1.27 (3H, ), 1.12 (3H, s), 0.97 (1H, m); , C NMR (CDC13,
100MHz): 167.9, 143.6, 121.1, 83.5, 75.8, 63.7, 62.7, 33.7, 32.0, 31.9, 31.0, 27.7, 19.9,
19.7, 15.2; LC MS: 263(M-H) negative ion mode.
Example 6
Treatment of 7-HF with N-bromosuccinimde ( BS to obtain (R)-3-(bromomethvD-
5,5a,6,7-tetrahydro-5a,9-dimethylnaphtho[ 1,2-b1furan-2(4H)-one [compound-14, (16) :
To a mixture 7-Hydroxyfrullanolide (500 mg, 0.002 mol) in carbon tetrachloride (5 mL)
was slowly added 720 mg (0.004 mol) of NBS at Room Temperature (RT). After lh, the
reaction mixture was poured into ice water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over Na S0 4 and evaporated under vacuum. The
residue (500 mg) was subjected to column chromatography over silica gel using ethyl
acetate/hexane mixtures. The fraction eluted with ethyl acetate/hexane was evaporated to
obtain compound- 4 [(16); 200 mg, 32.2%]
Compound-14: NMR (CDC13,400MHz): 5.95 (1H, m), 4.21 (2H, s), 2.83 - 2.88 (1H,
m), 2.67 - 2.76 (1H, m), 2.32 - 2.40 (1H, m), 2.22 - 2.27 (1H, m), 2.14 (3H, m), 1.71
(2H, m), 1.57 (2H, m), 1.1 (3H, m).
Example 7
Reduction of 7-a-HF with Pd/CaCOVethanol to obtain (3aR.5aR.9bS)-3.3a.4.5.5a.6.7.8-
octahydro-3a-hydroxy-3,5a,9-trimethylnaphtho[l,2-b]furan-2(9bH)-one [compound- 15.
am
To a mixture of 7- a-HF (500mg) in 10 ml of ethanol in a RB flask under stirring was
added lOmg of Pd on CaC0 3. The RB flask is flushed with H2 gas and the stirring
continued under H2 atmosphere. After lh, the reaction mixture was filtered through super
cell. The filtrate was concentrated under vacuum and the residue (500 mg) was subjected
to column chromatography on silica gel using ethyl acetate/chloroform mixtures to yield
200 mg (40%) of Compound-15 (17).
Compound-15: NMR(CDC13, 400MHz): 4.96 (1H, d, J = 0.8 Hz), 2.78 (1H, q, J =
7.2Hz), 2.08 - 2.14 (3H, m), 1.81 (2H, m), 1.79 (3H, s), 1.64 (1H, m), 1.37 - 1.56 (5H,
m), 1.20 (3H, d, J = 7.2 Hz), 1.05 (3H, s); LC MS: 273(M+Na) + positive ion mode.
Example 8
Synthesis of (2E)-(3aR.5aR.9bSV2.3.3a.4.5.5a.6 7.8.9b-decahvdro-3a-hvdroxv-3.5a,9-
trimethyl-2-oxonaphtho[ 1,2-b]furan-8-yl 3-(2, -dimethoxyphenyDacrylate [compound-
16. (18)1: A solution of (3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a,8-dihydroxy-5a,9-
dimethyl-3-methylenenaphtho[l,2-b]furan-2(9bH)-one (compound-10; 300 mg, 0.001 13
mol), 2, 5-dimethoxy cinnamic acid (280 mg, 0.013 mol), 6mL of methylene dichloride
(MDC), 50 mg of dimethylaminopyridine (DMAP) was taken in a RB flask and treated
slowly with a solution of 3 mg of dicyclohexylcarbodiimide (DCC) (0.0017 mol)
dissolved in 4mL of MDC under stirring at 0°C. The RM was then allowed to RT and
continued the stirring for 2h. The RM was then poured into ice-cold water and the
mixture extracted with EtOAc. The organic layer was washed with brine, dried over
Na2S0 4 and concentrated under vacuum. The residue (600 mg) was subjected to column
chromatography on silica column using acetone/hexane mixtures to obtain 120 mg of
compound- 16 (18, 24%) in the fraction eluted with 25% of acetone/hexane.
Compound-16: NMR (CDC13 400MHz): 7.91 (1H, d, J = 16.0Hz), 7.44 (1H, d, J =
8.4Hz), 6.50 (1H, dd, J = 8.4, 2.4 Hz), 6.46 (1H, d, J = 16.0 Hz), 6.48 (1H, d, J = 2.0Hz),
5.34 (1H, d, J = 4.0Hz), 3.88 (3H, s), 3.84 (3H, s), 2.52 (1H, q, J = 7.6Hz), 2.05 (2H, ),
1.848(3H, s), 1.84 (2H, ), 1.68 (2H, m), 1.47 (1H, m), 1.138(1H, m), 1.34 (3H, d, J =
7.6Hz), 1.23 (1H, m), 1.10 (3H, s); LC/MS: 479.3 (M+Na) + positive ion mode, 455 (MH)
491 (M+2H 20-H) " negative ion mode.
Example 9
Reaction of 7-HF with morpholine to obtain (3aR.5aR.9bS)-3.3a.4.5.5a.6.7.8-octahydro-
3a-hydroxy-5a,9-dimethyl-3-(mo holinomethyl)naphtho[l,2-b]furan-2(9bH)-one
[compound- 17, (19Y|:
To a solution of 7-hydroxyfrullanolide (300 mg, 0.0012mol) in 5mL of THF in a RB flask
was added slowly (87 L, 0.0014mol) under stirring at RT. After 1.5 h, the
RM was poured into ice-cold water and the mixture extracted with EtOAc. The' organic
layer was washed with brine, dried over Na2S0 4 and concentrated under vacuum. The
residue (300 mg) was purified on silica column using acetone/chloroform mixtures to
obtain 150 mg (yield 38%) of compound- 17 (19) in the fractions eluted with 15%
acetone/chloroform.
Compound-17: NMR (CDC13, 400MHz): 5.02 (1H, s), 3.74 (2H, m), 3.67 (2H, ),
3.08 (1H, dd, J = 7.6, 8.8 Hz), 2.84 (1H, d, J = 7.6 Hz), 2.84 (1H, d, J = 8.8 Hz), 2.73
(2H, m), 2.44 (2H, m), 2.09 (2H, m), 1.91 (1H, dt, J = 4.0, 14.0 Hz), 1.79 (3H, m), 1.705-
1.58 - 1.70 (4H, m), 1.40 - 1.47 (3H, m), 1.06 (3H, s); LC/MS: 336.4(M+H) +,
358.4(M+Na) + positive ion mode.
Example 10
Reaction of (R 2A5.5a.6 J-hexahydro-5a.9-dimethyl-2-oxonaphtho[ ,2-b]furan-3-
carbaldehyde_[compound- 10, (12)1 with to produce (3aR,5aR,9bS)-
3,3a,4,5,5a,6,7,8-octahydro-3a,8-dihydroxy-5a,9-dimethyl-3-
A mixture of
300 mg (0.001 mol) of compound-10 (12)] and (0.169 niL, 0.00135 mol) in
5 L of THF in a RB flask was stirred at RT for one and half hours and poured in ice
cold water. The mixture was extracted with EtOAc and the organic layer washed with
brine, dried over sodium sulfate and concentrated under vacuum. The residue was
subjected to chromatography on silica column using acetone/chloroform mixtures. The
fraction eluted with 40% acetone/chloroform yielded 100 mg (yield: 25%) of compound-
18 (20).
Compund-18: NMR(CDC1 , 400MHz): 4.95 (1H, s), 3.95 (1H, d, J = 3.2 Hz) 3.74
(2H, m), 3.67 (2H, ), 3.08 (1H, dd, J = 6.0, 10.0 Hz), 2.84 (2H, m), 2.72 (2H, ), 2.44
(2H, m), 2.04 (1H, dt, J = 3.2, 13.6 Hz), 1.98 (1H, ), 1.96 (3H, s), 1.60 - 1.89 (6H, m),
1.35 - 1.48 (2H, m), 1.04 (3H, s).
Example
Reaction of 7-HF with piperidine to produce (3aR.5aR.9bS)-3.3a.4.5.5a.6.7.8-octahvdro-
3a-hydroxy-5a,9-dimethyl-3-((piperidin- 1-yl)methyl)naphtho[ 1,2-b]furan-2(9bH)-one
[compound- 19, (21)1:
To a mixture of 7-HF (300 mg, 0.0012mol) in 5mL of THF in a RB flask was slowly
added piperidine (0.143mL, 0.0014mol) at rt and the mixture stirred for 1.5 h. The
reaction mixture was then poured into ice cold water and the mixture extracted with
EtOAc. The organic layer was washed with brine, dried over Na2S0 4 and evaporated
under vacuum. The residue (300 mg) was subjected chromatography over silica column
using methanol/chloroform mixtures. The fraction eluted with 40% of acetone/hexane
yielded compound- 19 [(21), yield 44%].
Compound-19: NMR(CDC1 3, 400MHz): 5.02 (1H, s), 3.10 (1H, dd, J = 5.6, 10.8
Hz), 2.85 (1H, d, J = 11.2 Hz), 2.79 (2H, dd, J = 5.6, 11.2Hz), 2.70 (2H, brs), 2.41 (2H,
brs), 2.17 (3H, s), 2.151-2.06 (3H, m), 1.81 - 1.85 (2H, m), 1.79 (3H, s), 1.56 - 1.71 (4H,
m), 1.505- 1.38 - 1.50 (3H, m), 1.05 (3H, s).
Example 12
Reaction of 4.5-epoxy-7-hydroxy frul lanolide [compound- 13, (15)] with piperidine to
produce (5aR)-5,5a,6,7,8,9-hexahydro-9-hydroxy-5a,9-dimethyl-3-((piperidin-lvl)
methvl)naphthori,2-blfuran-2(4H)-one fcompound-20, (22)]: To a mixture of epoxide
[compound-13, (15); 300mg, 0.001 13mol] in 5mL of THF in a RB flask was slowly
added piperidine (1441_,, 0.0013mol) at Room Temperature (RT) and the mixture stirred
for 24h. Then the Reaction Mixture (RM) was poured in to ice-cold water and the mixture
was extracted with EtOAc. The organic layer was washed with brine, dried over Na S0 4
and concentrated under vacuum. The residue (400mg) was purified on a silica column
using acetone/chloroform mixtures. The fraction eluted with 30% acetone/chloroform
yielded compound-20 [(22); 150mg) with a percentage yield 40%.
Compound-20: NMR (CDC13 > 400MHz): 3.27 (2H, s), 2.93 (1H, m), 2.27 (1H, m),
2.41 (4H, m), 1.90 (1H, m), 1.63 - 1.85 (6H, m), 1.55 - 1.59 (6H, m), 1.53 (3H, s), 1.46
(4H, m), 1.2 1 (3H, s). LC/MS: 332 (M+H)+ positive ion mode
Example 13
Reaction of 7-HF with 1. 2. 4- triazole to produce (3aR.5aR.9bS)-3-((4H-1.2.4-triazol-4-
yl)methyl)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydro
2(9bH)-one rcompound-21 (23) : To a mixture of 7-HF (300 mg, 0.0012mol) in 5 mL of
THF in a RB flask was slowly added 1, 2, 4 - triazole (0.1 g, 0.0014 mol) at RT and the
mixture stirred for 1.5 h. The reaction mixture was then poured into ice-cold water and
the mixture extracted with EtOAc. The organic layer was washed with brine, dried over
a2 S0 and evaporated under vacuum. The residue (300 mg) was subjected
chromatography over silica column using methanol/chloroform mixtures. The fraction
eluted with 40% of acetone/hexane yielded compound-21 [(23), yield 79%].
Compound-21: H NMR(CDCl , 400MHz): 8.21 (1H, s), 8.02 (1H, s), 5.10 (1H, s), 5.06
(1H, s), 4.82 (1H, dd, J = 2.4, 14.4 Hz), 4.36 (1H, dd, J = 11.2, 14.8 Hz), 3.19 (1H, dd, J
= 2.8, 11.2 Hz), 2.16 (1H, m), 2.08 (1H, dd, J = 6.0, 17.6 Hz), 1.90 (2H, ), 1.80 (3H, s),
1.77 (2H, m), 1.47 (3H, m), 1.07 (3H, s). LC/MS: 318(M+H) +, 340(M+Na) +, 356(M+K) +
positive ion mode.
Example 14
Reaction of 7-HF with piperazine to produce (3aR.5aR,9bS)-3,3a.4,5,5a,6.7,8-octahydro-
3a-hydroxy-5a,9-dimethyl-3-((piperazin-l-yl)methyl)naphtho[l,2-b]furan-2(9bH)-one
rcompound-22. (24)1:
To a mixture of 7-HF (300 mg, 0.0012mol) in 5mL of THF in a RB flask was slowly
added piperazine (624mg, 0.0072mol) at RT and the mixture stirred for 1.5 h. The
reaction mixture was poured into ice cold water and the mixture extracted with EtOAc.
The organic layer was washed with brine, dried over Na2S0 4 and evaporated under
vacuum. The residue (300 mg) was subjected chromatography over silica column using
acetone/hexane mixtures. The fraction eluted with 30% acetone/hexane yielded 170 mg of
compound-22 [(24), yield 5 %].
Compound-22: NMR (CDC13, 400MHz): 5.02 (1H, s), 3.06 (1H, m), 2.94 (5H, m,),
2.71 (2H, brs), 2.73 (2H, m), 2.40 (2H, brs), 2.154-2.03 (2H, m), 1.87 (1H, td, J = 3.6,
14.0 Hz), 1.79 (3H, m), 1.56 - 1.71 (4H, m), 1.38 - 1.49 (4H, m), 1.06 (3H, s); LC/MS:
335(M+H) + positive ion mode.
Example 1
Reaction of 7-HF with piperazine to produce piperazine bis-7-hydroxyfrullanolide
fcompound-23. (25)]:
To a mixture of 7-HF (300 mg, 0.001 2mol) in 5mL of THF in a RB flask was slowly
added piperazine (124 mg, 0.0014 mol) at RT and the mixture stirred for 1.5 h. The
reaction mixture was then poured into ice cold water and the mixture extracted with
EtOAc. The organic layer was washed with brine, dry over Na2 S04 and evaporated under
vacuum. The residue (300 mg) was subjected chromatography over silica column using
acetone/hexane mixtures. The fraction eluted with 30% of acetone/hexane yielded 30 mg
of compound-23 (25).
Compound-23: NMR (CDC13, 400MHz): 5.01 (2H, s), 3.06 (2H, dd, J = 5.2, 1.2
Hz), 2.87 (2H, d, J = 13.2 Hz), 2.80 (4H, dd, J = 5.6, 13.2 Hz), 2.1 1 (4H, m), 1.91 (2H,
td, J = 2.8, 13.2 Hz), 1.80 (2H, m), 1.79 (6H, s), 1.39 - 1.70 (18H, ), 1.05 (6H, s); C
NMR (CDC13, 100MHz): 174.2, 140.3, 126.7, 79.7, 77.5, 77.2, 53.2, 49.3, 39.5, 35.0,
33.5, 33.2, 1 .7, 25.9, 25.4, 19.4, 18.2; LC/MS: 583(M+H) +, 605(M+Na) + positive ion
mode.
Example 16
Assessment of inhibition of lipid accumulation in differentiated adipocytes by 7-
hydroxyfrullanolide (7-HF): One hundred thousand 3T3-L1 Human pre-adipocyte cells in
Dulbecco's Modified Eagles Medium (DMEM) containing 10% Fetal Bovine Serum
(FBS) were taken into each well of a 24-well plate and incubated for 48h at 37°C and 5%
C0 2. The differentiation of pre-adipocyte cells was initiated in a differentiation medium
containing 0 g ml insulin, 1.0 dexamethasone, and 0.5 mM isobutylmethylxanthine
(IBMX) for 48h. After this the medium was replaced by DMEM containing lOug/ml
insulin and incubated for 3 days. Then the differentiating cells were treated with 1.0 or 2
or 2.5 g/ml of 7-hydroxyfruIlanolide (1) or different natural analogs (structure numbers
3 to 11) or semi-synthetic analogs (structure numbers 12 to 25) of 7-HF. The cells were
maintained in the medium for another 3-5 days. The cells incubated with 0.1% DMSO
were considered as the vehicle control. After the incubation period, cells were washed
with phosphate buffered saline (PBS) and fixed with 10% buffered formalin for lh at
room temperature. One small aliquot of cell suspension was separated for cell counting in
hemocytometer chamber. Fixed cells were stained with Oil Red O solution to measure the
cellular neutral lipid accumulation. Briefly, cells were washed with PBS, fixed with 10%
buffered formalin and stained with Oil Red O solution (0.5 g in 100 ml isopropanol) for
10 min. After removing the staining solution, the dye retained in the cells will be eluted
into isopropanol and OD measured at 550 nm. The inhibition of fat accumulation in the
treated cells was compared with the mock treated differentiated adipocytes. The treated
and control cells were also analyzed and compared for inhibition of lipid accumulation
visually under microscope and recorded digitally in suitable image capture system. The
anti-adipogenic activity by 7-HF is depicted in Figure I and the percentage inhibition of
lipid accumulation shown by 7-HF and its natural and semi-synthetic analogs is
summarized in Table-1.
Table-1
Concentration
S. No Compound % inhibition
g L)
1 7-HF (1) 1.0 52.5
2 2.5 63.5
Compound- 1 2.5 19.4
4 Compound-3 2.5 40.5
5 Compound-4 2.5 10.0
6 Compound- 10 2.0 45.0
7 Compound- 11 2.0 8.0
8 Compound- 2 2.0 55.0
Compound- 3 2.0 79.7
10 Compound- 14 2.0 14.0
11 Compound- 7 2.0 36.2
12 Compound- 18 2.5 133
13 Compound- 9 2.0 23.3
14 Compound-20 2.0 7
15 Compound-22 1.0 15
16 Compound-23 1.0 15.2
Example 17
Assessment of pro-lipolvtic activity of 7-hvdroxyfrullanolide (1 and its analogs in
differentiated adipocytes: The lipolytic activity was assessed in mature adipocytes as per
the procedure of Chemicon International, USA, by measuring free glycerol secreted into
the culture medium. One hundred thousand 3T3-L1 Human pre-adipocyte cells in
Dulbecco's Modified Eagles Medium (DMEM) containing 10% Fetal Bovine Serum
(FBS) were taken into each well of a 24-well plate and incubated for 48h at 37°C and 5%
C0 2. The differentiation of pre-adipocyte cells was initiated in a differentiation medium
containing 10 g ml insulin, 1.0 dexamethasone, and 0.5 mM isobutylmethylxanthine
(IBMX). The cells were differentiated for 5 days and then the culture medium was
removed. The monolayer was washed twice with wash solution (Hank's balanced salt
solution), and then 250 of incubation solution (Hank's balanced salt solution plus 2%
bovine serum albumin) was added to the wells in triplicate in presence or absence of 7-
hydroxyfrullanolide or its analogs or the extracts containing 7-HF, and the cells were
further incubated for 16 h. To measure lipolysis, 200 of free glycerol assay reagent
was added to 25 of culture supernatants and controls containing glycerol standard.
The samples and the controls were incubated for 15 min, and the absorbance was read at
540 nm. A standard curve constructed from the glycerol was used to calculate the
concentration of free glycerol in the culture supernatants. The percentage increase in
glycerol concentration in the sample solutions compared to the control containing the
known concentrations of glycerol corresponds to the percentage acceleration of lipolysis
by test compound. The percentage increase in lipolysis accelerated by 7-HF was found to
be 47.8% at 5 concentration. The data is summarized in Figure II. The data for
other compounds is summarized in Table-2.
Table-2
Concentration % acceleration
S. No Compound
g mL) of lipolysis
1 7-HF (1) 5 47.8
2 Compound- 0 44.6
3 Compound-3 5 39.6
7 Compound- 12 5 45.2
8 Compound- 13 5 52.8
Example 18
Inhibition of Peroxisome proliferator-activated receptor gamma PPAR ) , Adipose
Differentiation Related Protein ADRP . CD36. adipocyte fatty acid binding protein (aP2)
and Perilipin in 3T3-L1 adipocytes by 7-hydroxyfruUanoUde (7-HFV.
Experimental protocol: Mouse pre-adipocyte 3T3-L1 cells are maintained in Dulbecco's
Modified Eagles Medium (DMEM) supplemented with 2 mM glutamine, 4.5g/L glucose
and 10% fetal bovine serum. Equal number of cells was plated in each well of 24-well
culture plates. Cells were pre-treated separately with 1 g mL 7-hydroxyfrullanolide for
2h and followed by addition of differentiation medium containing 500 nM insulin, 1.0
dexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX) for 48h. Thereafter,
cells were further incubated with post differentiation medium (DMEM containing 100
nM insulin) in presence or absence of 7-HF. Finally, the cells were harvested, washed
with chilled phosphate buffered saline and lysed with the lysis buffer. The protein extracts
were clarified at 14,000g for 20 min. Protein content was measured in Bradford method
by using Coomassie blue dye and cell lysates were stored in aliquots at -80°C until further
use. The modulation of adipocyte differentiation markers such as Peroxisome
proliferator-activated receptor gamma (PPARy), CD36, adipocyte fatty acid binding
protein (aP2); and intracellular lipid droplet surface associated protein, perilipin
expression were evaluated by immunoblot assay.
Inhibition of protein expression of biomarker molecules adipocytes in presence or
absence of 7-hydroxyfrullanolide (7-HF) was evaluated in immunoblot assay. Briefly,
equal amount of cell lysates proteins were resolved in 7.5% SDS-PAGE; thereafter, the
proteins were transferred to nitrocellulose membrane. After blocking the non-specific
sites, the membrane was incubated with either anti-PPARy, or anti-CD36, or anti-aP2, or
anti-ADRP, or anti-perilipin antibody. Finally, the specific immuno-reactive bands were
developed with West-pico chemiluminescent substrate (Pierce Biotechnology, IL, USA),
and the immunoblot images were recorded in a Kodak Image Station (Kodak, USA).
Band intensities were calculated densitometrically and normalized with expression of
actin in respective samples. The data is summarized in Figure III.
Example 1
Inhibition of CD36 production by 7-hvdroxyfrullanolide (7-HF) in macrophage cells :
Experimental protocol: This was evaluated in glucose induced J774, mouse macrophage
cells. Briefly, the cells were cultured in DMEM with 2 mM Glutamine, 100 U/mL
penicillin, 100 mg/mL streptomycin and 10% fetal bovine serum (Hyclone, Logan, UT).
Equal number of cells was seeded into 35 mm petri dishes (Corning, USA) one day
before the experiment. The culture media was replaced with fresh, glucose free DMEM
supplemented with 10% fetal bovine serum. 7-HF was diluted at 1 g and all cultures
were pre-incubated for 2 hours at 5% C0 2 at 37°C, and then incubated with 600 mg/dL of
glucose for 5 days. Representative photomicrographs showing inhibition of lipid
accumulation by 7-HF in high glucose induced macrophage cells of an in vitro model of
atherosclerosis are shown in Figure IV. The control culture was supplemented with 100
mg/dL glucose. The cells were harvested and lysed with lysis buffer. Cell lysates were
clarified at 14,000g. Protein concentration was measured by Bradford method.
Inhibition of CD36 protein expression in high glucose induced J774 macrophage cells in
presence or absence of 7-HF was evaluated in immunoblot assay. Briefly, equal amount
of cell lysates proteins were resolved in 7.5% SDS-PAGE; thereafter, the proteins were
transferred to nitrocellulose membrane. After blocking the non-specific sites, the
membrane was incubated with CD36 antibody (R&D Systems, Minneapolis, MN).
Finally, the specific immuno-reactive bands were developed with West-pico
chemiluminescent substrate (Pierce Biotechnology, IL, USA), and the immunoblot
images were recorded in a Kodak Image Station (Kodak, USA). Band intensities were
calculated densitometrically and normalized with expression of actin in respective
samples. The results are summarized in Figure V.
Example 20
Nitrite assay protocol
Equal number (5000 cells) of human endothelial cells was plated in each well of a 96-
well cell culture plate. The cells were treated with various concentrations (0.1, 0.25, 0.5
and 1.0 ng/ml) of 7-HF for 24h. The control cultures received 0.01% (v/v) DMSO as the
vehicle. After 24h, the culture supernatants were collected and mixed with equal volume
of Griess reagent [1:1 mixture of NED solution (0.1% N-l-napthylethulenediamine
dihydrochloride in water) and Sulfanylamide solution (1% sulfanilamide in 5%
phosphoric acid)]. The reaction was allowed for 10 min at room temperature. Finally, the
color reaction was read at 550 nm in a micro-plate reader (BioRad, USA). Known
concentrations of sodium nitrite were reacted to obtain a standard curve. Modulation of
nitrite production in 7-HF treated cultures was quantitatively assessed by extrapolating
the absorbance readings obtained from the test samples into the standard plot. The data is
summarized in Figure VI.
Example 2 1
Inhibition of PTP-1B activity by 7-HF: Equal number of 3T3-L1 mouse preadipocytes
was seeded into cell culture dishes. After 24h, the cells were treated either with different
concentrations of 7-HF or 50 DM Sodium vanadate (Na2V0 3 for further 48h. Thereafter,
the washed cells were lysed with cell lysis buffer and the clarified at 14,000 g for 10 min
at 4°C. The protein concentrations were calculated by Bradford method and the cell
lysates were reacted with equal volume of substrate reagent containing 10 mM pnitrophenyl
phosphate (pNPP). After lh incubation at 37°C, the reaction was stopped with
N NaOH and the developed color was read at 405 nm. The specific enzyme activity was
calculated by using an extinction coefficient of 1.78 x 04 M 'cm _ for pNPP at A405. The
inhibition shown by 7-HF is depicted in Figure VII.
Example 22
Modulation of adiponectin by 7-hvdroxyfrullanolide (7-HF): Modulation of adiponectin
protein by 7-hydroxyfrullanolide (7-HF) in 3T3-L1 adipocytes was evaluated in Western
immunoblot assay. The cell culture, treatment protocol and immunoblot assay
methodology were the same as described above in Example 18. Figure VIII summarizes
the enhancement of adiponectin protein expression in 3T3-L1 mature adipocytes by 7-HF.
Example 23
In vivo efficacy of Sphaeranthus indicus ethyl acetate extract (SIE) against metabolic
disorders:
Efficacy of the Sphaeranthus indicus ethyl acetate extract (SIE) was tested in high fat,
high cholesterol, high salt and high sucrose diet induced model of metabolic syndrome.
Induction: A batch of 12 Sprague Dawley Rats was randomly divided into 2 groups, each
comprised of 6 animals. Animals were acclimatized for 7 days prior to study initiation.
Metabolic syndrome was induced by feeding the rats with the metabolic syndrome diet
containing 32 g of roasted bengal gram, 27 g of sucrose, 17 g of milk powder, 5 g of
mineral salt mixture, 1 g of yeast, 2 g of butter, 11 g of groundnut oil and 5 g of
cholesterol per 100 g of the diet for 8 weeks.
Treatment: Following 8 weeks induction phase, the animals were treated orally (using
oral feeding gavage) with allocated test substance or vehicle daily for 8 weeks. The
treatment group animals were supplemented orally with 250 mg/kg body weight of SIE in
10 mL of 0.5% CMC in water for further 8 weeks. The control group of animals received
only the vehicle (10 mL of 0.5% CMC in water) during this period. During the treatment
phase, all animals were provided with the standard rodent diet till the end of the study.
Body weights: Body weight of individual animal was recorded weekly for the entire
duration of the study. Mean body weights for the treatment group and control group were
determined. The body weight gain was calculated at the end of 1st week, 4th week and 8th
week after initiation of treatment in comparison to initial body weight. In comparison to
the control group, SIE at 250 mg/kg dose exhibited highly potent and statistically
significant (p<0.01) reduction in body weight gain (66.04%) in comparison to control
group. The results of body weight gain for the treatment groups and control group are
summarized in figures XIA & XIB.
Fat tissue weight: Abdominal and epididymal fat were isolated and weighed at the
termination of the study and the results were represented in Table-3. Abdominal and
epididymal fat weights in the treatment group are lower, when compared to those in the
control group. The total fat was significantly reduced (p<0.05) in the treatment group
supplemented with ethyl acetate extract of Sphaeranthus indicus (SIE).
Weight of fat tissues isolated from abdomen and epididyma area of rats.
TabIe-3
Values expressed as mean weight ± SD
Serum Biochemistry: Blood sampling was done via sinus orbital plexus under mild
anesthesia, before induction, before initiation of treatment and after completion of
treatment. Various biochemical parameters including lipid profile were evaluated using
biochemistry reagents supplied by Human, Germany, in an automated clinical chemistry
analyzer HumaStar300, Make: Human, Germany. Mean values of the biochemical
parameters especially serum cholesterol levels and triglycerides levels were estimated
before induction, after induction/before treatment and after treatment. Supplementation of
ethyl acetate extract of Sphaeranthus indicus (SIE) at 250 mg kg resulted in improvement
in fat profile with 15.3, 12.7 and 22.9 percentage reductions respectively in serum
cholesterol, LDL and triglycerides.
Estimation of Biomarker Adiponectin: The serum adiponectin concentration for the
control and treatment groups of animals were assessed using double antibody based
sandwich rat adiponectin ELISA kit. The assay was performed following the instructions
provided by the manufacturer (Linco Research, USA). The sensitivity of the assay is
0.155 ng/ml. Adiponectin assay revealed that supplementation of SIE at a dose of 250
mg day kg body weight for 8-weeks resulted in significant (p=0.00618) improvement in
serum adiponectin concentration, in comparison with the baseline. The control group,
however, did not show improvement in serum adiponectin concentration. The results are
summarized in Figure XII.
The Homeostasis Model Assessment (HOMA): The HOMA index was calculated based
on serum insulin and glucose levels, using the following formula:
Fasting insulin concentration (ϋ /mL) * Fasting glucose concentration (mmoI/L)/22.5.
The supplementation of treatment group of rats with a daily dose of 250 mg/kg body
weight for 8-week treatment period resulted in significant reduction of HOMA index
compared to control group. The data is presented in Figure XIII.
Those of ordinary skill in the art will appreciate that changes could be made to the
embodiments described above without departing from the broad inventive concept
thereof. It is understood, therefore, that this invention is not limited to the particular
embodiments or examples disclosed, but is intended to cover modifications within the
objectives and scope of the present invention as defined in the specification.
We claim:
1. Biologically active ingredient(s) comprising atleast one component selected from 7-
hydroxyfrullanolide, its analog(s); extract(s) and fraction(s) containing 7-
hydroxyfrullanolide or its analog(s) or both; or mixtures thereof for the prevention,
control and/or treatment of one or more metabolic disorders.
2. A biologically active composition comprising at least one component selected from
the list comprising 7-hydroxyfrullanolide, its analog(s); the extract(s) or fraction(s)
containing 7-hydroxyfrullanolide/its analog(s) or both; or mixture(s) thereof as an active
in combination with one or more ingredients selected from other biologically active
components derived from plants, animals and microorganisms; pharmaceutically or
dietetically acceptable active ingredients, vitamins, aminoacids, minerals, vehicles,
carriers and diluents or mixtures thereof for the prevention, control and/or treatment of
one or more metabolic disorders.
3. The biologically active ingredient or their composition according to claims 1 and 2,
wherein said metabolic disorders comprise obesity, over weight, diabetes, arteriosclerosis,
cardiovascular diseases, hypertension, hypercholesteremia, hyperlipidemia,
triglyceridemia, metabolic syndrome, endothelial dysfunction and other metabolic
disorders.
4. The biologically active ingredient(s) or their composition according to claims 1 and 2,
for the amelioration of the expression/production of one or more biological marker
proteins related to metabolic disorders.
5. The biologically active ingredient(s) or their composition according to claim 4,
wherein said biological marker proteins comprise Peroxisome proliferator-activated
receptor gamma (PPARy), Adipose Differentiation Related Protein (ADRP), adipocyte
CD36, Macrophage CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-
LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic
Receptor (P3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase- B (PTP-1B),
Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3), and Matrix
Metalloproteinase- 13 (MMP-13).
6. The biologically active ingredient(s) or their composition according to claims 1,2 and
4, wherein the analog(s) of 7-hydroxyfrullanolide comprises of the compound(s)
represented by the general formula I given below;
Formula I
Wherein Ri, R2, R3, » and R5 are each independently selected from hydrogen,
hydroxy, halogen, -OOR]2, alkoxy, -OC(0)R 12 and C(0)R 2; optionally and R2
are taken together to form a ketone (=0).
The tricyclic ring system consists of one or two or three double bonds.
Optionally R2 and R3 together form a double bond;
Optionally R3 and together form a double bond;
Optionally R3 and R5 together form a double bond;
Optionally R5 and R 6 together form a double bond;
Optionally R8 and R9 together form a double bond;
Further optionally R3 and R5together form an epoxide ring
R7 is selected from hydrogen, hydroxy, halogen, alkoxy and -OC(0)Ri 2; R8 is
selected from hydrogen, hydroxy, halogen, alkoxy, -OC(0)Ri 2, -C(0)Ri and
NRi3Ri4; R9 s selected from hydroxy, alkyl, cycloalkyl, alkoxy, aryl,
heterocyclyl, halogen, -OC(0)Ri 2, -C(0)R 12, azido and -NRi R 4, -S(0) mR , -
OS(O) mRi5; wherein m is 0, 1 or 2;
Rio and R are each independently selected from hydrogen, alkyl, halogen,
OR(6, -NH Ri2 and SRi 2 ; wherein R i is selected from hydrogen, alkyl and -
C(0)Ri2 or Rio and R together form one of ketone (=0), thioketone (S), imine
(NH) and selenoketone (Se);
R12 is selected from hydrogen and alkyl;
Ri3 and R i4 are each independently selected from hydrogen, alkyl, cycloalkyl,
aralkyl, aryl, heterocyclyl, -C(0)R 12 and - C(S)NHRi 2 ; or R and R i4 together
with the N atom to which they are bonded, to form a 5-, 6-, or 7-membered
heterocyclic ring, optionally having one or more additional heteroatoms selected
from O, N , S and Se;
i 5 is selected from hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl
X is selected from O, NH, S and Se
7. The biologically active ingredient(s) or their composition according to claim 6
wherein said 7-hydroxyfrullanolide or its analog(s) or mixtures thereof can be of natural,
synthetic or semi-synthetic origin.
8. The biologically active ingredient(s) or their composition according to claim 7,
wherein the natural origin is of any plant species that produces 7-hydroxyfrullanolide or
its arialog(s) or mixtures thereof.
9. The biologically active ingredient(s) or their composition according to claim 7,
wherein the natural analogs of 7-hydroxyfrullanolide comprises of frullanolides; 1la, 13-
dihydro-3a,7a-dihydroxy-4,5-epoxy-6p,7-eudesmanolide; 11a,13-dihydro-7a-acetoxy-
3 -hydroxy-6p,7-eudesm-4-enolide; 3-keto- -eudesmol; 1la,13-dihydro-3a,7ctdihydroxyeudesm-
4-en-6a,12-olide; lla,13-dihydro-3a,7a-dihydroxyfrullanolide;
1la,13-dihydro-7a,13-dihydroxyfrullanolide; 1la,13-dihydro-7a-hydroxy-13-
methaoxyfrullanolide; 2a,7a-dihydroxy-4-en-l l,13-dihydroeudesm-6,12-olide; 2ahydroxycostic
acid; 3-keto-7a-hydroxyeudesm-4-en-6,12-olide (cryptomeridiol); 4-
epicryptomeridiol; sphaeranthanolide; 2a-hydroxysphaerantholide; 2aacetoxysphaerantholide;
2a,7a-dihydroxysphaerantholide; 2a-acetoxy-7ahydroxysphaerantholide;
2a-acetoxy-5a-hydroxyisosphaerantholide; eudesmanolide
dimer (compound-2); (3aR,5aR,9aR,9bR)-decahydro-9a-hydroperoxy-3a-hydroxy-5amethyl-
3,9-dimethylenenaphtho[ 1,2-b]furan-2(9bH)-one (compound-3); (3aS,5aR,9bR)-
3,3a,4,5,5a,6,7,8-octahydro-3,5a,9-trimethylnaphtho[ 1,2-b]furan-2(9bH)-one (compound-
4); (R)-5,5a,6,7-tetrahydro-3,5a,9-trimethylnaphtho[l,2-b]fiiran-2(4H)-one (compound-
5), (3R,3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-3-(methoxymethyl)-5a,9-
dimethylnaphtho[l,2-b]furan-2(9bH)¾>ne (compound-6); 2-((3R,8aR)-l ,2,3,7,8,8ahexahydro-
5,8a-dimethylnaphthalen-3-yl)acrylic acid (compound-7); (3aR,5aR,9bS)-3-
((6-amino-9H-purin-9-yl)methyl)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-5a,9-
dimethylnaphtho[ 1,2-b]furan-2(9bH)-one (compound-8); (3R,3aR,5aR,8R,9bS)-8-
((2R,3S,4R,5R)-tetrahydro-3,4,5-trihydroxy-6-(hydroxymethyl)-2H-pyran-2-yloxy)-
3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-3,5a,9-trimethylnaphtho[l,2-b]furan-2(9bH)-one
(compound-9) or mixtures thereof, preferably 7-hydroxyfrullanolide.
10. The biologically active ingredient(s) or their composition according to claim 7,
wherein the synthetic/semi-synthetic analogs of 7-hydroxyfrullanolide comprises
(3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a,8-dihydroxy-5a,9-dimethyI-3-
methylenenaphtho[l,2-b]furan-2(9bH)-one (compound- 10); (3aR,5aS,9bS)-3a,4,5,5a,6,7-
hexahydro-3a-hydroxy-5a,9-dimethyl-3-methylenenaphtho[l,2-b]furan-2,8(3H,9bH)-
dione (compound- 11); (R)-2,4,5,5a,6,7-hexahydro-5a,9-dimethyl-2-oxonaphtho[l,2-
b]furan-3-carbaldehyde (compound-12); 4,5-epoxy-7-hydroxyfrullanolide (compound-
13); (R)-3-(bromomethyl)-5,5a,6,7-tetrahydro-5a,9-dimethylnaphtho[l,2-b]furan-2(4H)-
one (compound- 14; (3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-3,5a,9-
trimethylnaphtho[ 1,2-b]furan-2(9bH)-one (compound- 15); (2E)-(3aR,5aR,9bS)-
2,3,3a,4,5,5a,6,7,8,9b-decahydro-3a-hydroxy-3,5a,9-trimethyl-2-oxonaphtho[l,2-b]furan-
8-yl 3-(2,5-dimethoxyphenyl)acrylate (compound- 16); (3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-
octahydro-3a-hydroxy-5a,9-dimethyl-3-(mo holinomethyl)naphtho[l,2-b]furan-2(9bH)-
one (compound- 17); (3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a,8-dihydroxy-5a,9-
dimethyl-3-(morpholinomethyl)naphtho[l,2-b]furan-2(9bH)-one (compound- 18);
(3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-5a,9-dimethyl-3-((piperidin-lyl)
methyl)naphtho[l,2-b]furan-2(9bH)-one (compound- 19); (5aR)-5,5a,6,7,8,9-
hexahydro-9-hydroxy-5a,9-dimethyl-3-((piperidin-l-yl)methyl)naphtho[l,2-b]furan-
2(4H)-one (compound-20); (3aR,5aR,9bS)-3-((4H- 1,2,4-triazol-4-yl)methyl)-
3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-5a,9-dimethylnaphtho[l,2-b]furan-2(9bH)-one
(compound-21), (3aR,5aR,9bS)-3,3a,4,5,5a,6,7,8-octahydro-3a-hydroxy-5a,9-dimethyl-3-
((piperazin-l-yl)methyl)naphtho[l,2-b]furan-2(9bH)-one (compound-22) and piperazine
bis-7-hydroxyfrullanolide (compound-23) as substantially depicted in figure X by
structures 12 through 25.
11. The biologically active ingredient (s) or their composition according to claims 1 and
2, wherein said ,7-hydroxyfrullanolide or its analog(s) or fractions or mixtures thereof are
derived from any plant species that produces 7-hydroxyfrullanolide or its analog(s) or
mixtures thereof, preferably Sphaeranthus indicus.
12. The biologically active ingredient(s) or their composition according to claims 1 and 2,
wherein said extract(s) and fraction(s) standardized to 7-hydroxyfrullanolide or its
analog(s) or mixtures thereof are derived from any plant species that produces 7-
hydroxyfrullanolide or its analog(s) or mixtures thereof, preferably Sphaeranthus indicus.
13. The biologically active ingredient(s) comprising atleast one component selected from
7-hydroxyfrullanolide, its analog(s); extract(s) and fraction(s) containing 7-
hydroxyfrullanolide or its analog(s) or both; or mixtures thereof according to claims 1 and
4, wherein the said extracts and fractions contain 7-hydroxyfrullanolide or its analog(s) or
mixtures thereof in the range of 0.001% to 100%, preferably 0.01 to 99%.
14. The biologically active composition according to claims 2 and 4, wherein the
concentration of 7-hydroxyfrullanolide or its analog(s) or mixtures thereof in the
compositions varies in the range from 0.001% to 99%, preferably 0.01 to 95% by weight.
15. The biologically active composition according to claims 2 and 4, wherein the
percentage of the extract or fraction standardized to 7-hydroxyfrullanolide or its analog(s)
or both varies in the range from 0.01% to 99%, preferably 1% to 90% by weight in the
composition.
16. The biologically active composition according to claim 2, wherein the other
biologically active components used for making the compositions comprise extr'act(s),
fraction(s), active compound(s), phytochemical(s) or powder(s) derived from plant(s),
animal(s) or microorganisms having one or more health benefits selected from but not
limited to anti-diabetic activity, anti-hyperlipidemic activity, anti-obesity activity, ant i
hypertensive activity, anti-platelet aggregation activity, anti-infective activity, antiatherosclerotic
activity, anti-inflammatory activity, anti-oxidant activity and bioenhancing
activity.
17. The biologically active ingredient(s) or their compositions according to claims 1 and
2, can be optionally combined with bio-availability enhancing agents selected from but
not limited to extract(s), fraction(s), pure compound(s) derived from Piper nigrum or
Piper longum or Stevia rebaudiana.
18. The biologically active ingredient(s) or their compositions according to claims 1 and
2, wherein the 7-hydroxyfrullanolide, its analogs; extracts and fractions containing 7-
hydroxyfrullanolide or its analogs or mixtures thereof are derived from Sphaeranthus
indicus, wherein said extract(s) or active fraction(s) or active compound(s) or
phytochemicals or mixtures thereof are derived from atleast one of the plant parts selected
from leaves, flower heads, fruits, stem, bark, root, whole plant or mixtures thereof,
preferably flower heads.
19. The biologically active ingredient(s) or their compositions according to claim 2,
wherein, the pharmaceutically or dietetically acceptable excipients, vehicles and carriers
comprise surfactants, binders, diluents, disintegrators, lubricants, preservatives,
stabilizers, buffers, suspensions and drug delivery systems.
20. The biologically active ingredient(s) or their compositions according to claim 19,
wherein the pharmaceutically or dietetically acceptable excipients, carriers and diluents
comprise glucose, fructose, sucrose, maltose, yellow dextrin, white dextrin, aerosil,
microcrystalline cellulose, calcium stearate, magnesium stearate, sorbitol, stevioside, corn
syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic
acid, dl-alpha-tocopherol, glycerin, propylene glycol, glycerin fatty ester, poly glycerin
fatty ester, sucrose fatty ester, sorbitan fatty ester, propylene glycol fatty ester, acacia,
carrageenan, casein, gelatin, pectin, agar, vitamin B group, nicotinamide, calcium
pantothenate, amino acids, calcium salts, pigments, flavors, preservatives, distilled water,
saline, aqueous glucose solution, alcohol, propylene glycol and polyethylene glycol,
various animal and vegetable oils, white soft paraffin, paraffin and wax.
21. The biologically active ingredient(s) or its composition(s) according to claims 1, 2
and 4, wherein said ingredient(s) or composition(s) is administered orally, topically,
parenterally or by inhalation to a subject or mammal or warm blooded animal in need
thereof.
22. The biologically active ingredient(s) or its composition(s) according to claim 21,
wherein said ingredient(s) or composition(s) is administered once daily or multiple
administrations per day.
23. The biologically active ingredient(s) or its composition(s) according to claims 1, 2
and 4, wherein said ingredient(s) or composition(s) can be formulated as oral agents such
as tablets, soft capsule, hard capsule, soft gel capsules, pills, granules, powders,
emulsions, suspensions, syrups, pellets, food, beverages, concentrated shots, drops and
the like; and parenteral agents such as injections, intravenous drip and the like;
suppositories; and transdermal agents such as patches, topical creams and gel; ophthalmic
agents; nasal agents; and food or beverages.
24. The biologically active ingredient(s) or its composition(s) according to claims 1, 2
and 4, wherein the said ingredient(s) or their composition(s) are delivered in the form of
controlled release tablets, using controlled release polymer-based coatings by the
techniques including nanotechnology, microencapsulation, colloidal carrier systems and
other drug delivery systems.
25. The biologically active ingredient(s) or its composition(s) according to claims 1 and
2, wherein the said ingredient or their composition(s) can be formulated into or added to
existing or new food and beverage form(s) as a healthy food for warm blooded animals.
26. Use of biologically active ingredient or its composition(s) according to claims 1 and
2 for prevention, control and/or treatment of one or more diseases or conditions including
but not limited to obesity, diabetes, hypertension, arteriosclerosis, cardiovascular
diseases, neurological disorders, Alzheimer's, cognitive disorders, oxidative stress, skin
disorders, aging of the skin, UV irradiated damage, hypercholesterolemia,
hyperlipidemia, triglyceridemia, immune deficiency, metabolic syndrome, for bringing
about weight loss effectively, for producing lean body mass, for using during weight loss
program as well as for other metabolic disorders.
27. A method of preventing/ controlling /treating one or more metabolic disorders
selected from obesity, over weight, diabetes, arteriosclerosis, cardiovascular diseases,
hypertension, hypercholesterolemia, hyperlipidemia, triglyceridemia, metabolic
syndrome, endothelial dysfunction and other metabolic disorders in a mammal or warm
blooded animal in need thereof, wherein the method comprises administering to mammal
or warm blooded animal a therapeutically effective amount of atleast one biologically
active ingredient or its composition(s) according to claims 1 and 2.
28. A method of promoting lipolysis and/or inhibiting adipogenesis in a subject or
mammal or warm blooded animal in need thereof comprising administering to said
subject or mammal or warm blooded animal a therapeutically effective quantity of atleast
one biologically active ingredient or its composition(s) according to claims 1 and 2.
29. A method of amelioration of the expression or production of atleast one biological
marker selected from PPAR-, C-reactive protein (CRP), Adipose Differentiation Related
Protein (ADRP), adipocyte CD36, macrophage CD36, Monocyte Chemotactic protein
(MCP-1), Oxidized LDL, Adipocyte Fatty-acid-Binding Protein (aP2/FABP4/A-FABP),
Beta-3 adrenergic receptor (P3-AR), adiponectin, Perilipin, Protein tyrosine phosphatase
B (PTP IB), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-
3) and Matrix Metalloproteinase-1 3 (MMP-1 3) in a subject or mammal or warm blooded
animal in need thereof, wherein the method comprises administering to the subject or
mammal or warm blooded animal atleast one biologically active ingredient or its
composition(s) according to claims 1 and 2.