Patent Application
20140342009
The current disclosure provides non-acidic Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) and Boswellia oil fraction (BOIL) comprising BLPRE and BVOIL individually and their composition(s) obtained by combining with at least one biological agent or Nootropic agent for the prevention, control and treatment of brain related diseases comprising Attention-deficit Hyperactivity Disorder (ADHD) and memory deficits or to enhance brain functions such as cognition, memory, learning and communication. Importantly, the above fractions and compositions of the present disclosure help in improving brain health, brain function, and memory.
The current disclosure also provides a non-acidic Boswellia low polar gum resin extract fraction (BLPRE), a Boswellia volatile oil fraction (BVOIL), or a Boswellia oil fraction (BOIL) comprising BLPRE and BVOIL for increasing the bioavailability of biological agents. According to the current disclosure, BLPRE and BVOIL may be prepared by fractionation of BOIL.
The gum resin of Boswellia serrata(Burseraceae) plant has long been in use for the treatment of several diseases by the practitioners of Ayurvedic medicines in the Indian system of medicine. The extract of Boswellia was found to be a potent anti-inflammatory and anti-arthritic agent. The origin of anti-inflammatory actions of Boswellia gum resin and its extracts has been attributed to a group of triterpene acids called boswellic acids that were isolated from the gum resin of Boswellia serrata. Boswellic acids exert anti-inflammatory actions by inhibiting 5-lipoxygenase (5-LOX). 5-LOX is a key enzyme for the biosynthesis of leukotrienes from arachidonic acid. 3-O-Acetyl-11-keto-β-boswellic acid (AKBA) is biologically the most active component among its congeners, it being able to inhibit 5-LOX with an IC50 of 1.5 μM.
Boswellia gum resin and its extracts also demonstrated significant therapeutic improvements in human clinical trials confirming the anti-inflammatory effects shown in vitro and in vivo.
Worldwide aging of the population has increased the incidents of cognitive deficits, such as age-associated memory impairment and senile dementias, and this causes great disruptive impact on the life of the affected individuals. The “cholinergic hypothesis of learning” played a pivotal role in the development of drugs for degenerative diseases.
A disturbance of the cortical cholinergic system accompanied by a reduction of choline acetylase (reduced acetylcholine synthesis) is inter alia detectable biochemically in case of neurological diseases. Hence, there is a demand for a medicament whose active substance can ameliorate this disturbance and highly available at the target organ (brain) and which is well tolerated, particularly in long-term therapy.
Acetylcholinesterase (AChE) is an important enzyme to hydrolyze acetylcholine, a neurotransmitter mediating the activity of parasympathetic nerve, into choline and acetate. AChE is formed in the endoplasmic reticulum, and moves and functions in the cell membrane. AChE is distributed around cholinergic nerve, particularly much at the myoneural junction, and is found in the serum, liver and other tissues.
A wide range of evidence shows that acetylcholinesterase (AChE) inhibition can improve cognitive and mental functions through enhancing cortical cholinergic neurotransmission. The acetylcholinesterase (AChE) inhibitors increase the concentration of acetylcholine and help nerve cells to communicate better. The longer acetylcholine remains in the brain, the longer those cells can call up memories. The earliest known AChE inhibitors are physostigmine and tacrine.
However, clinical studies show that physostigmine has poor oral activity, brain penetration and pharmacokinetic parameters while tacrine has hepatotoxic side effects. Studies were thus focused on finding new types of acetylcholinesterase inhibitors that would overcome the disadvantages of these two compounds.
Donepezil and Rivastigmin inaugurate a new class of AChE inhibitors with longer and more selective action with manageable adverse effects but still small improvement of cognitive impairment. Galanthamine (Reminyl), an alkaloid isolated from Galanthus nivalis, is another recently approved AChE inhibitor for the treatment of Alzheimer's. It is selective, long acting, and reversible. Galanthamine produces beneficial effects in patients. Similarly, huperzine A, a novel Lycopodium alkaloid discovered from the Chinese medicinal plant Huperzia serratais a potent, reversible and selective inhibitor of AChE with a rapid absorption and penetration into the brain in animal tests. It exhibits memory-enhancing activities in animal and also in clinical trials.
Dementia with Lewy bodies (DLB) is a common cause of dementia. Changes in the acetylcholine system have been reported in brains of patients with DLB, which provides a rational basis for trials of acetylcholinesterase inhibitors in DLB.
Current treatment of dementia in Parkinson's disease (PD) is based on the compensation of profound cholinergic deficiency, as in recent studies with the cholinesterase inhibitors galantamine, donepezil and rivastigmine. It has also been shown that cholinesterase inhibitors can improve motor function in PD. The beneficial effect of cholinesterase inhibitors has been studied on patients suffering from Parkinson's disease and dementia.
Studies show that Wernicke-Korsakoff syndrome is associated with a persisting severe anterograde amnesia in which memory is not transferred from short-to long-term storage. It is believed to be a consequence of a thiamine-deficient state often found in alcohol abusers. The memory deficit has been attributed to a number of brain lesions (corpus mamillare and dorsomedial nucleus of the thalamus), loss of cholinergic forebrain neurons and serotonin-containing neurons. Many studies and case-reports suggest efficacy of acetylcholinesterase inhibitors in the Wemicke-Korsakoff-associated memory deficit. Studies also suggest that neurons in the nucleus basalis are at risk in thiamine deficient alcoholic.
The U.S. patent U.S. Pat. No. 5,720,975 relates to the use of incense (olibanum), incense extracts, substances contained in incense, their physiologically acceptable salts, their derivatives and their physiological salts, pure boswellic acid, of physiologically acceptable salts, of a derivative, of a salt of the derivative, for production of a medicament for the prevention or treatment of Alzheimer's disease.
US publication US20060177467A1 relates to the use of the hydrogenation products of frankincense (olibanum), its hydrogenated ingredients as well as physiologically acceptable salts and derivatives thereof and hydrogenated frankincense extracts for the production of a medicament for the prophylactic and/or therapeutic treatment of cerebral ischemia, cranial/brain trauma and/or Alzheimer's disease.
There is currently no prior art, to the best of the Applicants' knowledge, relating to the use of Boswellia non-acidic oil fractions and their compositions for the prevention, control and treatment of Memory and Cognition related diseases and enhancing brain functions.
Additionally, there are numerous pharmaceutical ingredients, herbal ingredients and biologically active molecules that are effective in vitro against a disease condition or disorder. However, several of these ingredients are not effective or not bioavailable in vivo, i.e., after administration to warm blooded animals. It is thus important to explore and identify safe and effective agents that help to increase the bioavailability of such ingredients. As set forth in the present disclosure, Boswellia non-acidic oil fractions have been found to increase bioavailability of a number of extracts, fractions, phytochemicals and compounds originating from plant, animal or microorganism sources.
There is currently no prior art, to the best of the Applicants' knowledge, relating to the use of Boswellia non-acidic oil fractions and their compositions for increasing the bioavailability of biological agents in warm blooded animals.
Various embodiments of the present disclosure provide use of compositions comprising Boswellia non-acidic fraction(s) selected from Boswellia low polar gum resin extract fraction (BLPRE) having a novel phytochemical composition, Boswellia volatile oil fraction (BVOIL), and a Boswellia oil fraction (BOIL) comprising BLPRE and BVOIL fractions, either individually or in combination. These compositions are useful for improving brain health and brain functions, which include but are not limited to cognition, memory, intelligence, motivation, attention, concentration, learning power and better communication. These compositions are also useful to alleviate disease conditions related to cognition and memory deficits and the like.
Various embodiments disclosed herein provide use of Boswellia non-acidic fraction(s) selected from Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL), and a Boswellia oil fraction (BOIL) comprising BLPRE and BVOIL, either individually or as compositions, to prevent, control and treat brain related diseases/disorders which include but not limited to senile dementia, multi-infarct dementia, dyslexia, aphasia, organic brain syndrome, myasthenia gravis, vascular dementia, mild cognitive impairment (MCI), Lewy body dementia, Wemicke-Korsakoff-syndrome, Alzheimer's, Parkinson's disease, Attention-deficit Hyperactivity Disorder (ADHD), hypoxia, anoxia, cerebrovascular insufficiency, epilepsy, myoclonus and hypocholinergic dysfunctions, to slowdown memory deterioration, functional loss and to treat memory impairment disorders, neurodegenerative disorders, and for controlling blood pressure and blood circulation in the brain.
Various embodiments disclosed herein relate to compositions comprising at least one component selected from Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) and non-acidic Boswellia oil fraction consisting of BLPRE and BVOIL in combination with at least one component selected from biological agent(s), Nootropic agent(s).
In another aspect, the disclosure provides compositions comprising at least one component selected from Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) and non-acidic Boswellia oil fraction (BOIL) consisting of BLPRE and BVOIL in combination with at least one component selected from Boswellia extract(s), fraction(s), extracts/fractions enriched in one or more boswellic acids, their salts or derivatives thereof.
In another aspect, the disclosure provides compositions comprising at least one component selected from Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) and non-acidic Boswellia oil fraction (BOIL) in combination with one or more agents selected from natural antioxidants, anti-inflammatory agents and immune modulators.
In another embodiment, the disclosure provides Boswellia low polar gum resin extract fraction (BLPRE) for increasing the bioavailability of biological agents.
In further embodiments, the disclosure provides compositions comprising at least one component selected from Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) obtained from Boswellia gum resin in combination with a biological agent, for increasing the bioavailability of biological agents in warm blooded animals in need thereof.
In various embodiments, the disclosure provides Boswellia derived bioenhancing agents for increasing the bioavailability of one or more biological ingredients or functional ingredients.
In certain embodiments, the disclosure provides Boswellia derived bioenhancing agents for increasing the bioavailability of one or more pharmaceutical drugs/synthetic drugs.
In various embodiments, the disclosure provides Boswellia derived bioenhancing agents for increasing the bioavailability of one or more Boswellia derived components.
In various embodiments, the disclosure provides non-acidic Boswellia derived bioenhancing agents for increasing the bioavailability of one or more acidic Boswellia derived components.
In certain embodiments, the disclosure provides Boswellia derived bioenhancing agents for increasing the bioavailability of one or more Curcuma derived components.
In various embodiments disclosed herein, a non-acidic Boswellia oil fraction selected from the group consisting of an intact Boswellia oil (BOIL) and a Boswellia volatile oil (BVOIL) may be produced by:
The gum resin of Boswellia has been very widely used since ancient times. The gum resin of various species of Boswellia such as Boswellia serrata, Boswellia carterii or Boswellia papyrifera is a complex mixture comprising Boswellia oil fraction (BOIL) containing essential oil/Boswellia volatile oil fraction (BVOIL) and non-acidic Boswellia low polar gum resin extract fraction (BLPRE); boswellic acids, sugars and polysaccharide fraction. The Boswellia serrata/Boswellia carterii/Boswellia papyrifera extracts widely available in the international markets are acidic fractions separated from the gum resin which are standardized to contain 65% or 85% total Boswellic acids by titrimetric method of analysis. During the execution of commercial process for regular Boswellia extracts derived from Boswellia serrata/Boswellia carterii/Boswellia papyrifera (85% total Boswellic acids), the acidic fraction, which contains predominantly triterpene acids including Boswellic acids is separated from the rest of gum resin components. The sugars and other polymeric materials get separated out into the aqueous phase during the enrichment process for total Boswellic acids. The remaining water immiscible low polar compounds are separated as Boswellia oil fraction/extract. These low polar compounds are either absent or present at very low concentration in both, commercial Boswellia extracts standardized to boswellic acids and Boswellia extracts selectively enriched in 3-O-acetyl-11-keto-β-Boswellic acid (AKBA).
The Boswellia non-acidic oil fractions BOIL, BVOIL, and BLPRE may be obtained in a number of ways. One method of obtaining the Boswellia non-acidic oil fractions BOIL, BVOIL, and BLPRE is outlined in
An alternative method of obtaining the Boswellia non-acidic oil fractions BOIL, BVOIL, and BLPRE is outlined in
A representative process for obtaining Boswellia oil comprises:
The process for obtaining Boswellia volatile oil (BVOIL) is through steam distillation or using high vacuum from Boswellia gum resin.
A representative process for obtaining Boswellia volatile oil comprises:
In an alternative process,
A representative procedure for obtaining Boswellia low polar gum resin extract (BLPRE) comprises:
Another representative procedure for obtaining Boswellia low polar gum resin extract (BLPRE) comprises:
Yet another representative procedure for obtaining Boswellia low polar gum resin extract (BLPRE) comprises:
A representative procedure for obtaining Boswellia serrata volatile oil (BsVOIL) comprises:
Yet another representative procedure for obtaining Boswellia carterii volatile oil (BcVOIL) comprises:
The representative processes for obtaining Boswellia volatile oil (BVOIL) from Boswellia serrata, Boswellia carterii are described above. However, a similar process or processes can be applied to any of the gum resin obtained from Boswellia species for producing Boswellia volatile oil (BVOIL).
A representative procedure for obtaining Boswellia serrata low polar gum resin extract (BsLPRE) comprises:
Alternatively, the BsLPRE can also be prepared by a process comprising:
A representative procedure for obtaining Boswellia carterii low polar gum resin extract (BcLPRE) comprises:
Alternatively, the BcLPRE can also be prepared by process comprising:
The representative processes for obtaining Boswellia low polar gum resin extract (BLPRE) from Boswellia serrata and Boswellia carterii are described above. However, a similar process or processes can be applied to any of the gum resin obtained from Boswellia species for producing the low polar gum resin extract.
In the above processes for obtaining BLPRE, BOIL, and/or BVOIL, the water immiscible organic solvent used for extraction of a Boswellia gum resin or for partitioning an alcohol extract may be, but is not limited to, 1,2-dichloroethane, hexane, dichloromethane, chloroform, ethyl acetate, n-butanol, methyl iso-butyl ketone (MIBK) or a suitable combination thereof. The alkali solution used for washing the organic solvent extract, or partitioning the alcohol extract, can be selected from Group-I or Group-II metal hydroxides, which include, but are not limited to, Sodium hydroxide, Potassium hydroxide, Calcium hydroxide, Magnesium hydroxide and mixtures thereof.
The said intact Boswellia oil (BOIL) or Boswellia volatile oil (BVOIL) or Boswellia low polar gum resin extract (BLPRE) constitute significant components in Boswellia gum resin. However, it has very limited commercial utility and it is mostly discarded as a waste material. Potential utilization of these fractions have been long overdue. It was found unexpectedly that Boswellia serrata low polar gum resin extract (BcLPRE), a fraction obtained after removing the volatile compounds from the Boswellia serrata oil, has several beneficial properties.
In our earlier Indian patent application 2229/CHE/2008 filed 15 Sep., 2008 and PCT application # PCT/IN2009/000505 filed 14 Sep., 2009 we disclosed synergistic compositions comprising AKBA enriched fraction and Boswellia serrata non-acidic extract (BNRE). BNRE composition and method of identification are also disclosed.
In our recent Indian patent application 394/CHE/2010 filed 15 Feb., 2010 we disclosed non Boswellic acid fraction and its synergistic compositions.
As a part of developing new agents for improving brain/mental function and alleviating disease conditions related to cognition and memory deficits, a large number of plant extracts have been screened for their inhibitory property on Acetylcholinesterase enzyme activity. The assay was performed in vitro by the method of Ellman et al., with minor modifications, using acetylthiocholine iodide as a substrate (Lee J. H., et. al. Arch Pharm Res 2004, 27(1): 53-56). It was found very unexpectedly that the non-acidic extract, Boswellia serrata oil (BsOIL), Boswellia serrata low polar gum resin extract (BsLPRE) fraction and Boswellia serrata volatile oil (BsVOIL) fractions, were potent inhibitors of acetylcholinesterase in vitro. BsLPRE for example potently inhibited acetylcholinesterase enzyme activity in vitro as shown in Table 2. BLPRE's in vitro efficacy against acetylcholinesterase enzyme is comparable to commercial drug Neostigmin. BsLPRE exhibited an IC50 value of 37.01 ng/mL compared to 43.29 ng/mL shown by neostigmin. Its acetylcholinesterase inhibitory activity was also evaluated by a cell based in vitro assay in Rat pheochromocytoma PC 12 cells. The inhibitory property of BsLPRE on the enzyme activity was assessed in β-amyloid peptide induced-rat pheochromocytoma PC 12 cells. Rat pheochromocytoma PC 12 cells were equally distributed with phenol red free Dulbecco's modified Eagle's red medium (DMEM) (Sigma Life Science, USA) containing 10% fetal bovine serum (FBS) in 24-well plate. Cells were pretreated separately with BLPRE and positive control Neostigmin for 1 h. Thereafter, cells were induced with 1 μg/mL of β-amyloid peptide (Calbiochem, USA) for 24 h at 37° C. After 24 h, cells were collected and washed twice with 1×PBS by centrifugation at 1200 rpm for 5 min at 4° C. The cell extracts were prepared in solubilization buffer and the cell lysates were analyzed for acetylcholine esterase (AChE) activity. The BsLPRE showed 25.3% inhibition at 100 ng/mL concentration, where as Neostigmin showed 49.1% inhibition at 20 ng/mL as summarized in Table 4.
In order to understand the chemical composition of BsLPRE, separation of BsLPRE was carried out using column chromatography and high performance liquid chromatography (HPLC), and several diterpenoid and triterpenoid compounds were isolated. The structures of the compounds were rigorously characterized using 1H NMR, 13C NMR, DEPT, HSQC and HMBC, Mass spectral data. The compounds so obtained and identified are guiol (1), nephthenol (2), serratol (3), diterpene X (4), lupeol (5), olean-12-ene-3β-ol (6), olean-12-ene-3α-ol (7), lanosta-8,24-diene-3α-ol (8) and urs-12-ene-3α-ol (9) as depicted in
The foregoing results manifest that BsLPRE is a novel composition comprising unique combination of sesquiterpenoids, diterpenoids and triterpenoids and other phytochemical(s). A compound tentatively identified as diterpene X (4) and compounds guiol (1), nepthenol (2) and Lanosta-8, 24-diene-3α-ol (8) are not known to be metabolites of Boswellia serrata gum resin.
The low polar gum resin extract of these as well as other Boswellia species comprise a composition having some similarity to that of Boswellia serrata. However, the low polar gum resin extract of Boswellia carterii (BcLPRE) has shown biological activity and synergistic effect very similar to that exhibited by BsLPRE as summarized in the following in vitro and in vivo studies. The experimental studies are discussed in the examples.
The acetylcholinesterase inhibitory of different boswellic acids was also evaluated in both enzyme based assay and cell based assay and the inhibitory activities are summarized in Tables 3 and 5.
Oxidative stress induced increased ROS is critical for neuronal damage, which is a serious complication with regard to brain health. Interestingly, the low polar gum resin extract of Boswellia serrata(BsLPRE) showed potent inhibition of reactive oxygen species (ROS) generation in RAW 264.7 mouse macrophages (Table 6). In addition, BsLPRE also showed protection from oxidative stress induced cytotoxic damage of human neuroblastoma cells. In a cell based assay, oxidative stress induced by H2O, showed potent cytotoxic effect on the proliferation of IMR32 human neuroblastoma cells. However, the treatment with BsLPRE significantly attenuates the proliferation index of IMR32 human neuroblastoma cells back to the normal level (Table 7). Hence the observations confirm that the low polar gum resin extract (BLPRE) offers protection from neuronal damage and support improving brain health.
The in vivo efficacy of BsLPRE on learning and memory improvement was proven in rats using elevated radial arm maze (RAM) method. Oral administration of BsLPRE (250 mg/kg) significantly (P<0.01) decreased the number of days required to make the rats learned as per set criteria and significantly (P<0.05) decreased the time taken to find the food by the learned rats in the elevated RAM model. The positive control Piracetin (150 mg/kg) also showed significant improvement in spatial learning like reduction in latency and Number of wrong entries, when compared with the control group and the results are as stated below (
Cell based and enzyme based in vitro anti-acetylcholinesterase studies were conducted on a broad array of Boswellia extracts standardized to boswellic acids and Boswellia serrata low polar gum resin extract (BsLPRE), in addition to other herbal extracts. The individual extracts and different combination of these extracts were tested for their efficacy to inhibit acetylcholinesterase enzyme. It was found surprisingly that a composition (composition-1) comprising a combination of 1) a Boswellia serrata extract containing 85% total boswellic acids (BSE85%) and 2) a Boswellia serrata low polar gum resin extract (BsLPRE) showed potent inhibition of acetylcholinesterase (AChE).
Hence, the foregoing shows that BOIL, BVOIL and BLPRE alone as well as in combination with Boswellic acid(s)/Boswellia extract(s) or fractions(s) containing boswellic acid(s)/extracts standardized to boswellic acids/one or more Nootropic agents are potent inhibitors of acetylcholinesterase and as such can be used for the prevention, control and treatment of cognitive disorders and improving memory and alleviating disease conditions related to cognition and memory deficits.
Pure boswellic acids and commercially available Boswellia serrata extract standardized to 85% boswellic acids have been used to demonstrate the subject matter disclosed herein. However, any Boswellia serrata standardized to 40%-100% total boswellic acids by titrimetric method of analysis or standardized to 30%-100% total boswellic acids by HPLC method of analysis can also be used.
Similarly, a composition (composition-34) containing low polar gum resin extract (BLPRE) in combination with α-mangostin offers better protection from neuronal damage (Table 6) and hence can improve brain health. In addition, composition-34 also showed better protection from oxidative stress induced cytotoxic damage of human neuroblastoma cells in a cell based assay (Table 7). This result further confirms the potential role of the composition containing BsLPRE in the improvement of brain health.
In the primary aspect, the disclosure provides non-acidic Boswellia low polar gum resin extract (BLPRE) fraction, Boswellia volatile oil (BVOIL) fraction and Boswellia oil fraction (BOIL) comprising BLPRE and BVOIL for improving mental condition/brain health, treating impaired memory and alleviating memory and cognition related disorders and other associated diseases in warm blooded animals.
In the other primary aspect the disclosure provides compositions comprising at least one fraction selected from Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) in combination with one or more biological agents or Nootropic agents for improving mental condition/brain health, treating impaired memory and alleviating memory and cognition related disorders and other associated diseases in warm blooded animal.
In another embodiment the disclosure provides, composition comprising at least one Boswellia derived non-acidic extract/fraction selected from Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) and non-acidic Boswellia oil fraction (BOIL) in combination with at least one component selected from biological agents, phytochemicals, vitamins, amino acids, minerals; pharmaceutically or dietetically acceptable excipients, vehicles, carriers and diluents or mixtures thereof for improving mental condition/brain health; enhancing brain functions such as cognition, memory, learning, communication; for treating impaired memory, and for preventing, control or treating memory and cognition related disorders/diseases.
In another embodiment the disclosure provides methods for improving brain health and brain functions such as cognition, memory, learning, communication or treating impaired memory in a subject or warm blooded animal in need thereof, wherein the method comprises supplementing the said subject or warm blooded animal with an effective dose of Boswellia derived non-acidic extract/fraction or their composition(s).
In another embodiment the disclosure provides methods for preventing, control or treating memory and cognition related disorders/diseases in a subject or warm blooded animal in need thereof, wherein the method comprises supplementing the said subject or warm blooded animal with an effective dose of Boswellia derived non-acidic extract/fraction or their composition(s).
In another embodiment the disclosure provides methods of preventing, control or treating memory and cognition related disorders/diseases, wherein memory and cognition related disorders/diseases include but not limited to senile dementia, multi-infarct dementia, dyslexia, aphasia, organic brain syndrome, myasthenia gravis, vascular dementia, mild cognitive impairment (MCI), Attention-deficient Hyperactivity Disorder (ADHD), Lewy body dementia, Wemicke-Korsakoff-syndrome, Alzheimer's, Parkinson's disease, hypoxia, anoxia, cerebrovascular insufficiency, epilepsy, myoclonus and hypocholinergic dysfunctions, memory impairment disorders and neurodegenerative disorders.
In another aspect, the disclosure provides Boswellia low polar gum resin extract (BLPRE) fraction, Boswellia volatile oil (BVOIL) fraction and Boswellia oil (BOIL) fraction comprising BLPRE and BVOIL individually or their composition(s) comprising useful for the prevention, control and treatment of brain related diseases comprising Attention-deficit Hyperactivity Disorder (ADHD) and memory deficits or to enhance brain functions such as cognition, memory, learning and communication. Importantly, the said fractions and compositions of the present disclosure help in making the brain healthy.
In another aspect, the disclosure provides compositions comprising at least one component selected from Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) and non-acidic Boswellia oil fraction (BOIL) in combination with at least one pharmaceutically/dietetically acceptable excipients/diluents, further optionally comprising one or more agents selected from natural antioxidants, anti-inflammatory agents and immune modulators.
In another embodiment the disclosure provides the composition comprising at least one Boswellia derived non-acidic extract/fraction in combination with at least one pharmaceutically/dietetically acceptable excipients/diluents, wherein said pharmaceutically or dietetically acceptable excipients, carriers, vehicles and diluents include but not limited to glucose, fructose, sucrose, maltose, lactose, yellow dextrin, white dextrin, silicon dioxide, microcrystalline cellulose powder, calcium stearate, magnesium stearate, sorbitol, stevioside, corn syrup, 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, nicotinamide, calcium pantothenate, 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 aspect, the disclosure provides Boswellia non acidic extracts/fractions selected from Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) and non-acidic Boswellia oil fraction (BOIL) and their compositions to prevent, control and treat brain related diseases/disorders which include but not limited to senile dementia, multi-infarct dementia, dyslexia, aphasia, organic brain syndrome, myasthenia gravis, vascular dementia, mild cognitive impairment (MCI), Lewy body dementia, Wernicke-Korsakoff-syndrome, Alzheimer's, Parkinson's disease, Attention-deficit Hyperactivity Disorder (ADHD), hypoxia, anoxia, cerebrovascular insufficiency, epilepsy, myoclonus and hypocholinergic dysfunctions, memory impairment disorders and neurodegenerative disorders
In another aspect, the disclosure provides Boswellia derived non-acidic extract/fraction or their composition(s) for improving mental condition/brain health by slowing down memory deterioration, functional loss, by inhibiting beta-amyloid plaque deposition, by controlling blood pressure and blood circulation in the brain.
In another aspect, the Nootropic agent(s) used for making the composition comprise one or more agent(s) selected from smart drugs, memory enhancers and cognitive enhancers; dietary supplements, herbal ingredients, nutraceuticals, functional ingredients and functional foods that improve mental functions such as cognition, memory, intelligence, motivation, attention and concentration.
In another aspect, the Nootropic agents can be selected from one or more components selected from the extract(s)/fraction(s)/phytochemicals derived from herbs including but not limited to Bacopa species, Curcuma species or Rosmarinus species.
In another aspect, the herbal ingredients that can be used for preparing compositions are selected from including but not limited to Boswellia serrata, Boswellia carterii, Bacopa monniera, Curcuma longa, Withania somnifera, Rosmarinus officinalis, Garcinia mangostana, α-mangostin, Annona squamosa and Sphaeranthus indices.
In another aspect of the disclosure, the Nootropic agents can be selected from extract(s)/fraction(s)/phytochemicals, extracts/fractions enriched in one or more phytochemicals selected from including but not limited to Bacopa monnieri, Withania somnifera, Emblica officinalis, Centella asiatica; extract or fraction enriched in one or more phytochemicals selected from including but not limited to Bacoside A3, Bacopaside II, Jujubogenin isomer of bacopasaponin C, Bacopasaponin C, Bacopaside I, Bacosine, Apigenin, Luteolin and Sitosterol-D-glucoside, curcumin, demethoxycurcumin, bisdemethoxycurcumin, monodemethylcurcumin, bisdemethylcurcumin, tetrahydrocurcumin, tetrahydrodemethoxycurcumin, tetrahydrobisdemethoxycurcumin and ar-turmerone, carnosic acid, rosmarinic acid, camphor, caffeic acid, ursolic acid, betulinic acid, rosmaridiphenol, rosmanol and their salts thereof.
In yet another aspect, the disclosure provides compositions comprising therapeutically effective combination of Boswellia oil (BOIL/Boswellia volatile oil BVOIL)/Boswellia low polar gum resin extract (BLPRE) in combination with at least one Boswellia derived component selected from the extract(s), fraction(s) enriched with one or more boswellic acids/pure boswellic acid compounds or Nootropic agents for improving memory, impaired memory and alleviating memory and cognition related disorders and other associated diseases.
In another aspect, the disclosure provides compositions for the cognition enhancement achieved through one or more biological actions comprising inhibition of Acetylcholinesterase, increase in Butyrylcholinesterase and inhibition of β-amyloid aggregation.
In yet another aspect, the disclosure further provides compositions comprising Boswellia oil (BOIL)/Boswellia volatile oil (BVOIL)/Boswellia low polar gum resin extract (BLPRE) and at least one component, derived from gum resin of Boswellia species, which include but not limited to α-boswellic acid, β-boswellic acid, 3-O-acetyl-α-boswellic acid, 3-O-acetyl-β-boswellic acid, 3-O-acetyl-11-keto-α-boswellic acid and 3-O-acetyl-11-keto-β-boswellic acid or mixtures thereof for improving memory, impaired memory and alleviating memory and cognition related disorders and other associated diseases.
In yet another important aspect, the disclosure further provides compositions comprising Boswellia serrata low polar gum resin extract (BsLPRE) or BsVOIL or BsOIL and at least one Boswellia derived component selected from the extracts or fractions enriched in or standardized to one or more compounds derived from the gum resin of Boswellia which include but not limited to α-boswellic acid, β-boswellic acid, 3-acetyl-α-boswellic acid, 3-acetyl-β-boswellic acid, 3-acetyl-11-keto-α-boswellic acid and 3-acetyl-11-keto-β-boswellic acid or mixtures thereof for improving memory, improving impaired memory and alleviating memory and cognition related disorders and other associated diseases.
In another aspect, the disclosure further provides compositions comprising Boswellia serrata low polar gum resin extract (BsLPRE) or BsVOIL or BsOIL and a Boswellia serrata extract standardized to 30-100% total boswellic acids by titrimetric method of analysis or 20-100% total boswellic acids by HPLC method of analysis.
In preferred aspect, the disclosure further provides compositions comprising Boswellia serrata low polar gum resin extract (BsLPRE) or BsVOIL or BsOIL and Boswellia serrata extract standardized to 85% total boswellic acids by titrimetric method of analysis or 65% total boswellic acids by titrimetric method of analysis.
In another preferred aspect, the disclosure provides compositions comprising Boswellia serrata low polar gum resin extract (BsLPRE) or BsVOIL or BsOIL and Boswellia serrata extract selectively enriched in AKBA concentration varying from 3-99% by HPLC method of analysis.
In other preferred embodiment, the disclosure further provides a process for producing the Boswellia low polar gum resin extract (BLPRE), which include extraction of the gum resin of Boswellia species with a water immiscible organic solvent followed by washing the organic solvent extract with an aqueous alkali solution such as aqueous potassium hydroxide, followed by water and brine, and then finally evaporating the organic layer under vacuum to obtain an oil, followed by removing the volatile compounds under high vacuum and temperature to obtain BLPRE. The water immiscible organic solvent can be selected from hexane, chloroform, dichloromethane, ethyl acetate, methyl isobutyl ketone or any other water immiscible solvent or mixtures thereof.
The process for producing the Boswellia serrata low polar gum resin extract (BsLPRE) is variable and the alternative process for example comprise, extracting the gum resin with alcohol or hydroalcohol, and then evaporating the organic solvent to optimum concentration of total solids and then adjusting the solution to pH to 9-11, followed by repeatedly extracting the solution with a low polar organic solvent and then evaporating the organic solvent followed by removing the volatiles under vacuum at high temperature to obtain BsLPRE.
In a further embodiment, the Boswellia serrata intact oil can also be used in place of BsLPRE for improving memory, impaired memory and alleviating memory and cognition related disorders and for making the compositions of the present disclosure.
The water immiscible organic solvent in the above process can be selected from the solvents but not limited hexane, chloroform, dichloromethane, ethyl acetate, methylisobutylketone, tert-butanol or any other water immiscible solvent.
The Boswellia serrate extract standardized to 30-100% total boswellic acids by a titrimetric method of analysis or 20-100% total boswellic acids by HPLC method of analysis can be prepared from the gum resin using a known procedure or obtained from a group of commercially available Boswellia serrata extracts standardized to boswellic acids.
In another aspect of the disclosure, the non acidic extracts Boswellia oil (BOIL), Boswellia volatile oil (BVOIL), Boswellia low polar gum resin extract (BLPRE) used for the demonstration of the disclosure can be obtained from the Boswellia species selected from Boswellia serrata, Boswellia carterii, Boswellia papyrifera, Boswellia ameero, Boswellia bullata, Boswellia dakielii, Boswellia dioscorides, Boswellia elongata, Boswellia frereana, Boswellia nana, Boswellia neglecta, Boswellia ogadensis, Boswellia pirottae, Boswellia poporiana, Boswellia rime, Boswellia sacra and Boswellia socotrana.
In another aspect of the disclosure one or more of the Curcuma species that can be used for making the compositions of the present disclosure can be selected from Curcuma longa, Curcuma aromatica, Curcuma domestica, Curcuma aeruginosa, Curcuma albicoma, Curcuma albiflora, Curcuma alismatifolia, Curcuma angustifolia, Curcuma elata, Curcuma ferruginea, Curcuma flariflora, Curcuma yunnanensis and Curcuma edoaria.
In yet another aspect of the present disclosure, BOIL, BVOIL or BLPRE alone or in combination with one or more Boswellia derived extracts selectively enriched in boswellic acids/commercially available boswellic extract(s) standardized to 50-100% total boswellic acids/Boswellia serrata extracts wherein AKBA concentration varies from 3-99% HPLC method of analysis and optionally contains one or more of pharmaceutically/nutraceutically/dietically acceptable excipient(s), diluents, salt(s), additive(s), natural antioxidants or natural anti-inflammatory agents.
In another aspect, the disclosure provides the usage of BOIL, BVOIL or BLPRE alone or their compositions as it is or in comminuted form and/or in unmodified form as granules or powder or paste or the active ingredients are formulated into a solid, semi-solid or liquid dosage form by adding a conventional biologically or pharmaceutically acceptable salt(s) or additive(s) or excipient(s).
In yet another aspect, the disclosure provides use of therapeutically effective amount of BOIL, BVOIL or BLPRE alone or their compositions with one or more biological agents or Nootropic agents for administration in a specific dosage form such as orally, topically, transdermally, parenterally or in the form of a kit to a subject or patient in need thereof. Specific dosage form for formulation of the compositions of the present disclosure include but not limited to 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; parenteral agents such as injections, intravenous drip and the like; suppositories; transdermal agents such as patches, topical creams and gel; ophthalmic agents and nasal agents.
In another aspect, the present disclosure provides compositions containing at least one extract/fraction selected from BOIL, BVOIL or BLPRE in combination with one or more functional ingredient(s) comprising herbal ingredients, dietary supplements, antioxidants, vitamins, minerals, amino acids, fatty acids, essential oils, fish oils, enzymes, glucosamine, Chondroitin and probiotics or their salts or mixtures thereof derived from plants or animals or microorganisms or chemical synthesis or semi-synthesis.
In a further aspect, the present disclosure provides BOIL, BVOIL or BLPRE alone or their compositions further optionally combined with effective amounts of one or more pharmaceutical/nutraceutical/dietically acceptable agents including but not limited to antioxidant(s), adaptogen(s), anti-acetylcholinesterase agent(s), anti-inflammatory agent(s), anti-diabetic agent(s), antiobese agent(s), antiatherosclerotic agent(s), bio-protectants and/or bio-availability enhancer(s) and trace metals.
The examples of the biologically/pharmaceutically acceptable carriers employed in the present disclosure include, but are not limited to, surfactants, excipients, binders, diluents, disintegrators, lubricants, preservatives, stabilizers, buffers and suspensions.
In alternative aspect of the disclosure, the BOIL, BVOIL or BLPRE alone or their compositions can be optionally delivered in the form of controlled release dosage forms; and by using techniques including nanotechnology, microencapsulation, colloidal carrier systems and other drug delivery systems. The said formulation can be designed for once a daily administration or multiple administrations per day.
In accordance to the present disclosure, the BOIL, BVOIL or BLPRE alone or their compositions 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.
In accordance to the present disclosure, the BOIL, BVOIL or BLPRE alone or their compositions 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 and treatment of brain related diseases/disorders.
In yet another embodiment, the composition can comprise 10%-99% by the weight of Boswellia serrata derived component selected from the extract(s) and fraction(s) enriched with one or more boswellic acids, pure boswellic acid compounds and mixtures thereof and 90%-10% by weight of Boswellia serrata low polar gum resin extract BsLPRE or BsVOIL or BsOIL.
During the search for bioenhancing agents, it was found that non-acidic Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) or Boswellia oil fraction (BOIL) comprising BLPRE and BVOIL enhance the bioavailability of bioactive agents. The compositions LI13108F containing Boswellia serrata low polar gum resin extract (BsLPRE; LI13115) and Boswellia serrata extract selectively enriched to 30% 3-O_acetyl-11-keto-β-boswellic acid (AKBA) and LI13119F containing Boswellia serrata volatile oil fraction (BsVOIL) and Boswellia serrata extract selectively enriched to 30% 3-O_acetyl-11-keto-β-boswellic acid (AKBA) were supplemented to Albino Wistar rats. The control group of animals was supplemented with Boswellia serrata extract selectively enriched to 30% AKBA. Blood samples were collected from all animals prior to oral administration of test products and at 0.5, 1, 2, 4, 8 and 12 hrs after oral administration. The comparative oral bioavailability of AKBA from these Boswellia products was evaluated by measuring the serum AKBA concentration for each test animal using LC-MS.
Surprisingly, both the compositions LI 13108 Fand LI 13119F showed better oral bioavailability with AUCs 14.08 and 11.23 respectively compared to AUC 9.825 shown by individual ingredient Boswellia serrata extract containing 30% AKBA (LI 13115). The bioavailability (in terms of AUC) of LI 13108F is 43.33% more than LI 13115. The bioavailability of LI 13119F is 14.33% more than that of LI 13115. The study details are summarized in example-5 and depicted in
To exert optimal therapeutic efficacy, an active substance should reach systemic circulation and site of its action in an effective concentration during the desired period. Improving bioavailability and reducing dosage frequency without losing therapeutic benefit is crucial in achieving therapeutic efficacy and patient compliance in chronic treatment regimes. The compositions disclosed herein achieve this objective by enhancing the oral bioavailability of AKBA in compositions containing BsLPRE.
The bioavailability enhancing effect of BsLPRE was further confirmed by evaluating the composition LI13124F1 containing BsLPRE and a novel curcumin compound called bisdemethylcurcumin (LI01008) in comparison with LI01008 alone in Alibino Wistar rats. Bisdemethylcurcumin is a potent curcuminoid, far superior to other naturally occurring curcuminoids with respect to antioxidant and other biological activities commonly exhibited curcumins. The composition LI13124F1 showed better bioavailability of LI01008 in serum samples compared to the animals supplemented with LI01008 alone. The serum samples of animals supplemented with LI13124F1 showed 75% better bioavailability compared to the serum samples of the animals supplemented with LI01008 alone. The experimental studies are discussed in example-6 and depicted in
The foregoing thus suggest that the non-acidic Boswellia low polar gum resin extract fraction (BLPRE), Boswellia volatile oil fraction (BVOIL) or Boswellia oil fraction (BOIL) comprising BLPRE and BVOIL enhance the bioavailability of bioactive agents. These bio-enhancing agents thus can be useful to improve the efficacy and reduce the dose of bioactive agents.
In an important aspect, the current disclosure provides bioenhancing agents selected from intact Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) obtained from Boswellia gum resin of Boswellia species for increasing the bioavailability of biological agents.
In an important aspect, the current disclosure provides compositions comprising one or more ingredients selected from intact Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) obtained from Boswellia gum resin of Boswellia species in combination with a biological agent for increasing the bioavailability of biological agent.
In another aspect, the current disclosure provides Boswellia derived bioenhancing agents for improving the bioavailability and/or bio-efficacy of nutraceuticals or dietary supplements is also relevant to animal health besides being important for humans.
In another aspect the current disclosure provides Boswellia derived bioenhancing agents for increasing the bioavailability of one or more biological ingredient(s) or functional ingredient(s).
In another aspect the current disclosure provides Boswellia derived bioenhancing agents for increasing the bioavailability of one or more Boswellia derived components.
In another aspect the current disclosure provides Boswellia derived bioenhancing agents for increasing the bioavailability of one or more Curcuma longa derived components.
In another aspect the current disclosure provides the method of using Boswellia derived bioenhancing agents for enhancing the bioavailability of biological agents.
In another aspect, the current disclosure provides bioenhancing agents, which function through one or more of the mechanisms comprising increasing the bioavailability, enhancing the serum concentration, improving gastrointestinal absorption, improving systemic utilization and improving cross over through certain biological barriers like respiratory lining, urinary lining, blood brain barrier and skin.
In another aspect, the current disclosure provides bio-enhancing agents Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) derived from the gum resin of Boswellia where in the gum resin can be obtained from one or more of the Boswellia species selected from Boswellia serrata, Boswellia carterii and Boswellia papyrifera.
In another aspect the current disclosure provides compositions for bioenhancing the activity of biological agents in warm blooded animals in need thereof.
In another aspect the current disclosure provides compositions comprising Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) for enhancing the bioavailability of nutraceutical or dietary ingredients in warm blooded animals in need thereof.
The nutraceutically/dietetically acceptable agents comprise one or more ingredients selected from phytochemicals, Nootropic agents, anti obese agents, anti-inflammatory agents, anti cholesterol agents, anti arthritic agents, anti diabetic agents, antimicrobial agents, anti fungal agents, anti cancer agents, anti hypertensive agents, analgesic agents, anti platelet aggregation agents, anti atherosclerotic agents, antioxidants, anti thrombotic agents, antibiotic agents, anti malarial agents, anti osteoporotic agents, probiotics agents, anti fungal agents, immune potentiating agents, anti viral agents, anti histamines, muscle relaxants, anti depressants, hypnotic agents and their salts thereof.
In another aspect the current disclosure provides composition(s) for increasing the bioavailability of one or more biological ingredient(s) selected from biologically active ingredient(s), functional ingredient(s), herbal ingredient(s), dietary supplement(s), nutrient(s), anti-oxidant(s), vitamin(s), mineral(s), amino acid(s), and oil(s) their mixtures obtained from plant(s)/animal(s)/microorganism(s)/synthesis/semi-synthesis.
The functional ingredient(s) comprise one or more ingredients selected from nutrients, dietary supplements, nutritional ingredients, herbal ingredients, phytochemicals, animal proteins, glucosamine, chondroitin, plant proteins, fruit extracts, animal extracts, algae extracts, probiotics and their salts thereof.
The herbal ingredient(s) comprise one or more ingredients selected from extracts/fractions/phytochemicals and their salts derived from Withania somnifera, Bacopa monniera, Boswellia species, Curcuma species, Centella asiatica, Sphaeranthus indicus, Annona squamosa, Holoptelia integrifolia, Piper betel, Dolichos biflorus, Moringa oleifera and Murraya koenigii.
The anti-oxidant(s) comprise one or more ingredients selected from vitamin A, vitamin C, vitamin E, alpha-carotene, trans-beta-carotene, betacryptoxanthin, lycopene, lutein/zeaxanthin, pine bark bioflavonals complex, germanium, selenium and zinc.
The vitamin(s) comprise one or more water soluble vitamins selected from vitamin B1, vitamin B2, niacinamide, vitamin B6, vitamin B12, folic acid and vitamin C; fat-soluble vitamins selected from vitamin A, vitamin D, vitamin E and vitamin K.
The mineral(s) comprise one or more minerals selected from calcium, iron, zinc, vanadium, selenium, chromium, iodine, potassium, manganese, copper and magnesium.
The amino acid(s) comprise one or more amino acids selected from lysine, isoleucine, leucine, threonine, valine, tryptophan, phenylalanine, methionine, L-selenomethionine and their mixtures thereof.
The oil(s) comprise one or more oils selected from omega-3 fatty acid, flaxseed oil, fish oils, krill oil, essential oils and volatile oils.
The biological activity of Boswellia derived compounds/phytochemicals that can be enhanced by bioenhancing agents include extracts of fractions standardized to one or more boswellic acids selected from α-Boswellic acid, β-Boswellic acid, 3-O-acetyl-α-Boswellic acid, 3-O-acetyl-β-Boswellic acid, 3-O-acetyl-11-keto-α-Boswellic acid, 11-keto-β-Boswellic acid and 3-O-acetyl-11-keto-β-Boswellic acid.
In another aspect, the current disclosure provides bio-enhancing agents selected from Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) derived from the gum resin of Boswellia for enhancing the bioavailability of extracts/fractions particularly standardized to 3-O-acetyl-11-keto-β-Boswellic acid (AKBA).
In another aspect, the current disclosure provides Boswellia derived agents and compositions for enhancing the bioavailability of the phytochemicals derived from Boswellia species including but not limited to boswellic acids selected from α-boswellic acid, β-boswellic acid, 3-acetyl-α-boswellic acid, 3-acetyl-β-boswellic acid, 3-acetyl-11-keto-α-boswellic acid and 3-acetyl-11-keto-β-boswellic acid or mixtures thereof.
The Boswellia species that can be used for producing the oil (BOIL) or volatile oil (BVOIL) or low polar gum resin extract (BLPRE) from the gum resin comprise one or more species selected from Boswellia serrata, Boswellia carterii, Boswellia payrijera. Boswellia ameero, Boswellia bullata, Boswellia dakielii, Boswellia dioscorides, Boswellia elongata, Boswellia frereana, Boswellia nana, Boswellia neglecta, Boswellia ogadensis, Boswellia pirottae, Boswellia popoviana, Boswellia rime, Boswellia sacra and Boswellia socotrana.
In another aspect, the current disclosure provides Boswellia oil or Boswellia volatile oil or Boswellia low polar gum resin extract for enhancing the bioavailability of one or more Curcuma derived extracts/fractions/components/phytochemicals that can be enhanced by bioenhancing agents include extracts of fractions standardized to selected from curcumin, demethoxycurcumin, bisdemethoxycurcumin, monodemethylcurcumin, bisdemethylcurcumin, tetrahydrocurcumin, tetrahydrodemethoxycurcumin, tetrahydro bisdemethoxycurcumin and ar-turmerone or mixtures thereof.
In another aspect, the current disclosure provides bio-enhancing agents Boswellia oil (BOIL), Boswellia volatile oil (BVOIL) and Boswellia low polar gum resin extract (BLPRE) derived from the gum resin of Boswellia for enhancing the bioavailability of extracts/fractions particularly standardized to curcumin or demethoxycurcumin or bisdemethoxycurcumin or mixtures thereof.
In another aspect, the current disclosure provides Boswellia derived bioenhancing agents and for enhancing the bioavailability of the one or more phytochemicals derived from Curcuma species selected from curcumin, demethoxycurcumin, bisdemethoxycurcumin, monodemethylcurcumin, bisdemethylcurcumin, tetrahydrocurcumin, tetrahydrodemethoxycurcumin, tetrahydro bisdemethoxycurcumin and ar-turmerone or mixtures thereof.
The Curcumin derived components that can be bioenhanced are derived from Curcuma longa, Curcuma aromatica, Curcuma domestica, Curcuma aeruginosa, Curcuma albicoma, Curcuma albiflora, Curcuma alismatifolia, Curcuma angustifolia, Curcumaelata, Curcuma ferruginea, Curcuma flaviflora, Curcuma yunnanensis and Curcuma zedoaria.
The following examples, which include various embodiments, will serve to illustrate the practice of the disclosed subject matter, and it should be understood that the particulars shown are by way of example and for purpose of illustrative discussion of certain embodiments of the invention; the following examples do not limit the scope of the invention.
The Boswellia serrata gum resin (100 g) was dispersed in 600 mL of methyl isobutyl ketone (MIBK) solvent and stirred at room temperature for 60 min. The insoluble gum materials were separated by filtration. The MIBK solution was extracted repeatedly with 2% KOH solution (3×200 mL) to remove the acidic compounds. The MIBK layer was then washed successively with water (400 mL) and brine (200 mL). The MIBK layer was evaporated under reduced pressure at 60-70° C. and the volatile components are removed from the oily residue under high vacuum at 75-110° C. to obtain BsLPRE as a viscous oil (12 g).
Alternatively, the gum resin (250 g) collected from Boswellia serrata was extracted with methanol (300 mL×3) and the combined methanol extract was concentrated. The residue (50 g) was dissolved in ethyl acetate (400 mL) and extracted thrice with 1N KOH (3×100 mL). The organic layer was washed with water (2×200 mL) and brine (200 mL) and evaporated to obtain Boswellia oil. The volatile compounds were evaporated from the oil under vacuum at high temperature (75-110° C.) to obtain 22 g of BsLPRE.
The BsLPRE was subjected to column chromatography over normal silica gel using solvents of increasing polarity starting from hexane to hexane/ethyl acetate mixtures to ethyl acetate. The identical fractions were combined based on TLC and combined fractions were subjected individually to column chromatography over silica gel using mixtures of hexane/ethyl acetate or hexane/acetone as eluents to obtain pure compounds. Some of the impure fractions were further subjected to preparative HPLC using a reversed phase C18 silica column to obtain pure compounds. The structures were established by analyzing the 1H NMR, 13C NMR, DEPT, HSQC and HMBC and mass spectral data and then comparing the data with that of known compounds. Nine of the prominent compounds are identified as guiol (1), nephthenol (2), serratol (3), diterpene X (4), lupeol (5), olean-12-ene-3β-ol (6), olean-12-ene-3α-ol (7), lanosta-8,24-diene-3α-ol (8) and urs-12-ene-3α-ol (9) as depicted in
Boswellia serrata extracts standardized to 85% or 65% total boswellic acids are commercially available. These extracts are standardized using titrimetric method of analysis. These extracts can be prepared using a known procedure. For example, by extracting the gum resin of Boswellia serrata using a water immiscible solvent and then selectively extracting the acidic compounds from the organic solvent extract using aqueous alkali solution through phase separation. Finally acidification of the alkali solution to precipitate the boswellic acids followed by vacuum drying to yield Boswellia serrata extract enriched to 85% boswellic acids (BE85%). Boswellia serrata extracts standardized to a selected concentration of total boswellic acids in the range of 40-100% by titrimetric method of analysis or 30-100% by HPLC method of analysis can be obtained by purification of the gum resin or the extracts or by dilution of higher grade material.
Acetylcholinesterase activity is measured using the substrate acetylthiocholine iodide, which is converted to thiocholine. The reaction of thiocholine with the chromogenic substrate Dithionitrobenzoic acid (DTNB) leads to the formation of a yellow anion, Nitrobenzoic acid, which absorbs strongly at 412 nm. Incubation was done for 10 min.
The AChE assay was performed by the method of Ellman et al, with minor modifications, using acetylthiocholine iodide as a substrate (Lee J. H., et. al. Arch Pharm Res 2004, 27(1): 53-56). Ellmans reaction mixture contains 0.5 mM acetylthiocholine iodide and 1 mM 5,5′-dithio-bis-(2-nitrobenzoic acid) in a 50 mM sodium phosphate buffer (pH 8.0). The assay mixture contained 50 μl of 50 mM phosphate buffer at pH—8.0, 30 μl of test substance (BsOIL or BsLPRE or BcLPRE or BsVOIL, different boswellic acids and positive control Neostigmin) at various concentrations and 20 μl of (100 mU/mL) enzyme. For blanks, enzyme was replaced with phosphate buffer. The reaction mixture was mixed thoroughly, 100 μl of Ellman's reagent was added and incubated at room temperature for 10 min. The absorbance was measured at 412 nm using microplate reader. The percentage inhibition of enzyme activity was calculated by comparing OD's of tests wells with that of control wells using the following formula. Calculations: % inhibition=[(control-sample)/control]×100. The results of Acetylcholinesterase inhibitory activity of BsOIL, BsLPRE, BcLPRE, BsVOIL and boswellic acids are summarized in Table 2 and 3.
The inhibitory property on the enzyme activity was assessed in β-amyloid peptide induced-rat pheochromocytoma PC 12 cells. Rat pheochromocytoma PC 12 cells were equally distributed with phenol red free Dulbecco's modified Eagle's red medium (DMEM) (Sigma Life Science, USA) containing 10% fetal bovine serum (FBS) in 24-well plate. Cells were pretreated with test agents (BsOIL, BsLPRE, BcLPRE, BsVOIL and different boswellic acids and positive control Neostigmin) for 1 h. Thereafter, cells were induced with 1 μg/mL of β-amyloid peptide (Calbiochem, USA) for 24 h at 37° C. After 24 h, cells were collected and washed twice with 1×PBS by centrifugation at 1200 rpm for 5 min at 4° C. Cell extracts were prepared in solubilization buffer (10 mM Tris, pH 7.2; 100 mM NaCl, 50 mM MgCl2, 1% Triton X-100). The cell extracts were used as samples for measuring the acetylcholine esterase (AChE) activity.
Cell extract samples (100 μl) were dispensed into each well of 96-well microtitre plate. Fifty micro litres of DTNB (Dithiobisnitro benzoate) solution was added to each well and incubated for 5 min at room temperature. After incubation, 50 μl of acetyl choline iodide solution was added to each well and absorbance was read immediately at 405 nm for 12 min at 2 min intervals. A standard curve was constructed by using serial concentrations of acetyl cholinesterase (0-100 mU). Total protein present in 100 μl aliquot of cell extract was calculated by Bradford method and the enzyme activity was normalized and expressed as unit activity per milligram of protein. Efficacy of test samples was expressed in terms of percent inhibition of AChE activity and compared with a standard drug, Neostigmin as the positive control.
Results:
Table 4 and Table 5 are summary of the acetyl cholinesterase inhibitory activities exhibited by various non acidic extracts from Boswellia serrata and Boswellia carterii (BsOIL, BsLPRE, BcLPRE and BsVOIL) and different boswellic acids. A standard drug, Neostigmin was used as the positive control for comparing the AChE inhibitory efficacies of the boswellia products.
Preparation of composition-1: Composition-1 was prepared by mixing unit doses of the following components; four parts of Boswellia serrata low polar gum resin extract (BsLPRE) (4 g) and one part of Boswellia serrata extract standardized to 85% total Boswellic acids (BSE 85%) (1 g).
Composition-2: Composition-2 was prepared by mixing unit doses of the following components; one part of Boswellia serrata volatile oil (BsVOIL) (1 g) and four parts of Boswellia serrata extract standardized to 85% total Boswellic acids (BSE 85%) (4 g).
Composition-3: Composition-3 was prepared by mixing unit doses of the following components; one part of Boswellia serrata non acidic oil (BsOIL) (1 g) and four parts of Boswellia serrata extract standardized to 85% total Boswellic acids (BSE 85%) (4 g).
Composition-4: Composition-4 was prepared by mixing unit doses of the following components; one part of Boswellia carterii low polar gum resin extract (BcLPRE) (1 g) and four parts of Boswellia serrata extract standardized to 85% total Boswellic acids (BSE 85%) (4 g).
Composition-5: Composition-5 was prepared by mixing unit doses of the following components; one part of Boswellia carterii volatile oil (BcVOIL) (1 g) and three parts of Boswellia carterii extract standardized to 85% total Boswellic acids (BCE 85%) (3 g).
Composition-6: Composition-6 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BcLPRE) (1 g), four parts of Boswellia serrata extract enriched with 20% of 3-O-acetyl-11-keto-β-Boswellic acid (AKBA) (4 g).
Composition-7: Composition-7 was prepared by mixing unit doses of the following components; one part of Boswellia carterii low polar gum resin extract (BcLPRE) (1 g), four parts of Boswellia carterii extract enriched with 20% of 3-O-acetyl-11-keto-β-Boswellic acid (AKBA) (4 g).
Composition-8: Composition-8 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BcLPRE) (1 g), four parts of Boswellia serrata extract enriched with 40% of 3-O-acetyl-11-keto-β-Boswellic acid (AKBA) (4 g).
Composition-9: Composition-9 was prepared by mixing unit doses of the following components; one part of Boswellia carterii low polar gum resin extract (BcLPRE) (1 g), three parts of Boswellia carterii extract enriched with 40% of 3-O-acetyl-11-keto-β-Boswellic acid (AKBA) (3 g).
Composition-10: Composition-10 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BcLPRE) (1 g) and three parts of Bacopa monniera standardized extract (3 g).
Composition-11: Composition-10 was prepared by mixing unit doses of the following components; one part of Boswellia serrata volatile oil fraction (BsVOIL) (1 g) and three parts of Bacopa monniera standardized extract (3 g).
Composition-12: Composition-12 was prepared by mixing unit doses of the following components; one part of non-acidic Boswellia serrata oil fraction (BsOIL) (1 g) and three parts of Bacopa monniera standardized extract (3 g).
Composition-13: Composition-13 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and four parts of Bacopa monniera water extract (4 g).
Composition-14: Composition-14 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and four parts of Bacopa monniera 90% methanol extract (4 g).
Composition-15: Composition-15 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and three parts of Bacopa monniera standardized extract (3 g).
Composition-16: Composition-16 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and four parts of Bacopa monniera extract standardized to 25% bacopasaponins (4 g).
Composition-17: Composition-17 was prepared by mixing unit doses of the following components; one part of Boswellia carterii low polar gum resin extract (BcLPRE) (1 g) and four parts of Bacopa monniera extract standardized to 25% bacopasaponins (4 g).
Composition-18: Composition-18 was prepared by mixing unit doses of the following components; one part of Boswellia papyrifera low polar gum resin extract (BpLPRE) (1 g) and four parts of Bacopa monniera extract standardized to 25% bacopasaponins (4 g).
Composition-19: Composition-19 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and four parts of Curcuma longa extract standardized to 95% total Curcuminoids (CLE 95%) (1 g).
Composition-20: Composition-20 was prepared by mixing unit doses of the following components; one part of Boswellia serrata volatile oil fraction (BsVOIL) (1 g) and four parts of Curcuma longa extract standardized to 95% total Curcuminoids (CLE 95%) (4 g).
Composition-21: Composition-21 was prepared by mixing unit doses of the following components; one part of non-acidic Boswellia serrata oil fraction (BsOIL) (1 g) and four parts of Curcuma longa extract standardized to 95% total Curcuminoids (CLE 95%) (4 g).
Composition-22: Composition-22 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and three parts of curcumin (3 g).
Composition-23: Composition-23 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and three parts of bisdemethylcurcumin (BDMC) (3 g).
Composition-24: Composition-24 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and four parts of Withania somnifera methanol extract (4 g).
Composition-25: Composition-25 was prepared by mixing unit doses of the following components; one part of Boswellia carterii low polar gum resin extract (BcLPRE) (1 g) and four parts of standardized Withania somnifera extract (4 g).
Composition-26: Composition-26 was prepared by mixing unit doses of the following components; one part of Boswellia low polar gum resin extract (BLPRE) (1 g) and four parts of standardized Rosmarinus officinalis extract (4 g).
Composition-27: Composition-27 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and four parts of standardized Rosmarinus officinalis extract (4 g).
Composition-28: Composition-28 was prepared by mixing unit doses of the following components; one part of Boswellia carterii low polar gum resin extract (BcLPRE) (1 g) and four parts of standardized Rosmarinus officinalis extract (4 g).
Composition-29: Composition-29 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BcLPRE) (1 g) and four parts of Rosmarinus officinalis extract standardized to 30% Rosmarinic acid (RA 30%) (4 g).
Composition-30: Composition-30 was prepared by mixing unit doses of the following components; one part of Boswellia carterii low polar gum resin extract (BcLPRE) (1 g) and three parts of Rosmarinus officinalis extract standardized to 30% Rosmarinic acid (RA 30%) (3 g).
Composition-31: Composition-31 was prepared by mixing unit doses of the following components; one part of Boswellia non acidic oil (BOIL) (1 g) and three parts of Garcinia mangostana methanol extract (3 g).
Composition-32: Composition-32 was prepared by mixing unit doses of the following components; one part of non-acidic Boswellia serrata oil (BsOIL) (1 g) and three parts of Garcinia mangostana methanol extract (3 g).
Composition-33: Composition-33 was prepared by mixing unit doses of the following components; one part of Boswellia serrata low polar gum resin extract (BsLPRE) (1 g) and three parts of Garcinia mangostana methanol extract (3 g).
Composition-34: Composition-34 was prepared by mixing unit doses of the following components; two parts of Boswellia serrata low polar gum resin extract (BsLPRE) (2 g) and one part of α-mangostin (1 g).
Composition-35: Composition-35 was prepared by mixing unit doses of the following components; four parts of Boswellia serrata low polar gum resin extract (BsLPRE) (4 g) and one part of α-mangostin (1 g).
Composition-36: Composition-40 was prepared by mixing unit doses of the following components; one part of Boswellia low polar gum resin extract fraction (BLPRE) (1 g) and three parts of Sphaeranthus indicus ethyl acetate extract (3 g).
Composition-37: Composition-40 was prepared by mixing unit doses of the following components; one part of non acidic Boswellia oil fraction (BOIL) (1 g) and four parts of Sphaeranthus indicus ethyl acetate extract (4 g).
Composition-38: Composition-40 was prepared by mixing unit doses of the following components; one part of Boswellia volatile oil fraction (BVOIL) (1 g) and four parts of Sphaeranthus indicus ethyl acetate extract (4 g).
Formation of ROS was measured using of the fluorescent probe DCFH-DA. The method is based on the incubation of the RAW 264.7 mouse macrophages with DCFH-DA, which diffuses passively through the cellular membrane. Intracellular esterase activity results in the formation of DCFH, which emits fluorescence when oxidized to 20, 70-dichlorofluorescein (DCF). Briefly, the cells (final concentration 2×106/ml suspension) were incubated with DCFH-DA (5 mM) in HEPES-buffered (20 mM) HBSS (CaCl2 1.26 mM, KCL 5.37 mM, KH2PO4 0.44 mM; MgCl2
0.49 mM, MgSO4 0.41 mM, NaCl 140 mM, NaHCO3 4.17 mM, Na2HPO4 0.34 mM) with glucose (5 mM) at 37° C. for 15 min. Following centrifugation, the extracellular buffer with DCFH-DA was exchanged with fresh buffer and the suspension was mixed gently. The cells (2×106/ml, 125 ml) were transferred to 250 ml wells (microtiter plate reader, 96 wells) containing 125 ml buffer with the different concentrations of test samples (α-mangostin, BsLPRE, Composition-34 and Composition-35) in presence or absence of 100 mM H2O2. Fluorescence was recorded using excitation wavelength 485 nm, emission wavelength 530 nm in a Modulus luminescence spectrometer (Turner Biosystems, USA) for 120 min. Results are calculated as area under the curve (AUC) and the percentage of inhibition of intracellular ROS generation was calculated from the cultures treated with H2O2. The results are summarized in Table 6.
Effect of BsLPRE or α-mangostin or their combination (Composition-34) on cell growth was tested in oxidative stress induced IMR32 human neuroblastoma cells SW 982 human synovial cells by using MTT based cell proliferation assay. Briefly, IMR32 human neuroblastoma cells were cultivated in Dulbecco's modified Eagle's red medium (DMEM) (Sigma Life Science, USA) containing 10% fetal bovine serum (FBS). Equal number of IMR32 cells was seeded in to each well of 96 well microplate and incubated at 37° C. with 5% CO2. The cells were treated with 250 M H2O2 in presence or absence of different concentrations of BsLPRE or α-mangostin or their combination (Composition-34) for 72 h. Control wells were supplemented with 0.05% DMSO. After 72 h of treatment, equal volume of MTT reagent (R&D Systems, USA) was added to each well and incubated for 4 h. Thereafter, 50 μl of solublization buffer (R&D Systems, USA) was added to each well to solubilize the colored formazan crystals produced by the reduction of MTT. After 24 h, the optical density was measured at 550 nm using microplate reader (Bio-Rad, USA). In each assay, the vehicle control and the treatments were done in quadruplicates. The average OD obtained in the vehicle control wells is considered as the cell proliferation index of 100. The results are summarized in Table-7 below.
Inhibition of acetylcholinesterase activity by herbal products and their combinations in Beta Amyloid protein induced PC12 cells: The acetylcholinesterase inhibitory activity of α-mangostin, BsLPRE and Composition-34, recorded in Table 8, was measured using the procedure described in example 7.
The animal study protocol was approved by institutional animal ethics committee. Sprague Dawley rats were acclimatized for one week and healthy rats were selected for the study. The selected rats were pre-trained in elevated radial arm maze (RAM) adapted rats were allocated to various treatment groups each containing eight rats. After completion of pre-training, the oral treatment was initiated to the animals and continued daily up to two weeks. During this treatment phase the rats were placed on the RAM for 10 min each to recognize the food pellets present in the three different colored arms. During training the spatial learning was estimated by measuring various parameters like number of days required to learn the task, latency in finding food and number of wrong entries/attempts. After this treatment and training the animals were given rest without treatment or training for one week (3rd week). On 4th week, the animals were treated with allocated doses of test products and memory retention test was assessed using the same animals by measuring latency and number of wrong entries. The data was analyzed using ANOVA followed by a suitable post-hoc test.
Oral administration of BsLPRE (250 mg/kg) significantly (P<0.01) decreased the number of days required to make the rats learned as per set criteria and significantly (P<0.05) decreased the time taken to find the food by the learned rats in the elevated RAM model. Piracetam showed significant improvement in spatial learning represented by reduction in latency and Number of wrong entries, when compared with the control group and the results are as stated below (
The Boswellia carterii gum resin (100 g) was dispersed in 600 mL of methyl iso butyl ketone (MIBK) solvent and stirred at room temperature for 60 min. The insoluble gum materials were separated by filtration. The MIBK solution was extracted repeatedly with 2% KOH solution (3×200 mL) to remove the acidic compounds. The MIBK layer was then washed successively with water (400 mL) and brine (200 mL). The MIBK layer was evaporated under reduced pressure at 60-70° C. and the volatile components are then removed from the oily residue under vacuum at 75-85° C. to obtain Boswellia carterii low polar gum resin extract or BcLPRE as a viscous oil (9.5 g).
Alternatively, the gum resin (250 g) collected from Boswellia carterii was extracted with methanol (300 mLx3) and the combined methanol extract was concentrated. The residue (50 g) was dissolved in ethyl acetate (400 mL) and extracted thrice with 1N KOH (3×100 mL). The organic layer was washed with water (2×200 mL) and brine (200 mL) and evaporated to obtain Boswellia oil. The volatile compounds were evaporated from the oil under vacuum at 75-85° C. to obtain 17.75 g of BcLPRE.
Albino Wistar rats were quarantined and healthy rats were selected for the study. The selected animals were acclimatized for 7 days prior to the study initiation in the allocated room. Animals employed for the study were randomized into various treatment groups, fasted overnight at free access to water, body weights were noted and individual doses were calculated based on the body weights. Blood samples were collected from all animals prior to oral administration of test products and at 0.5, 1, 2, 4, 8 and 12 hrs after oral administration. Collected blood samples were allowed to clot for 10 min and centrifuged at 4° C. at 1800 g for 10 min. The serum samples were deproteinized with 100 μL TCA (20%) and 1.8 mL of HPLC grade methanol, centrifuged at 4° C. at 1800 g for 10 min and supernatants were subjected to LCMS analysis for total AKBA. The Composition LI 13108F comprising Boswellia serrata extract selectively enriched to 30% 3-O-acetyl-11-keto-β-boswellic acid (AKBA) (LI 13115) and Boswellia serrata non-volatile oil (BcLPRE) in the ratio 2:1; and composition LI13119F comprising Boswellia serrata extract standardized to 30% AKBA and Boswellia serrata steam distilled oil (BVOIL) in the ratio 2:1 showed better oral bioavailability with Area under the curve (AUC) 14.08 and 11.23 respectively compared to individual Boswellia serrata extract standardized to 30% AKBA (LI 13115) (AUC: 9.825). The bioavailability [in terms of [AUC] of LI 13108F is 43.33% more than LI 13115. The bioavailability of LI 13119F is 14.33% more than that of LI 13115. The serum concentration of AKBA in animals of various treatment groups at various time points was summarized in Table 9. The serum concentration against time was plotted and the details are summarized in
LI13124F1Animals (Wistar Rats) were acclimatized for 7 days prior to study initiation. Six animals were divided randomly into 2 groups, each comprised of 3 animals. The body weights were noted and doses were calculated based on initial body weights. Animals were treated orally with 450 mg dose of a composition (LI13124F1) containing bisdemethylcurcumin (LI01008) and BsLPRE (LI13115) in 2:1 ratio or 300 mg/kg LI01008 as suspension in 0.5% CMC. Blood samples were collected before treatment and several time intervals after treatment at 0.5, 1, 2, 4, 6, 8 and 12 hours, as plotted in
As per the data, the bioavailability of LI01008 in the composition LI13124F1 is 75% better compared to that when LI01008 is administered alone.
It will be appreciated to those of ordinary skill in the art 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 687/CHE/2010 | Mar 2010 | IN | national |
| 688/CHE/2010 | May 2010 | IN | national |
This application is a divisional application claiming priority to U.S. patent application Ser. No. 13/616,283, filed on Sep. 14, 2012. U.S. patent application Ser. No. 13/616,283 is a continuation-in-part of international application PCT/IN2011/000170, filed on Mar. 14, 2011, which claims priority to Indian patent application 687/CHE/2010, filed Mar. 15, 2010; and also a continuation-in-part of international application PCT/IN2011/000364, filed on May 26, 2011, which claims priority to Indian patent application 688/CHE/2010, filed May 30, 2010. The entire disclosures of all prior applications are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13616283 | Sep 2012 | US |
| Child | 14451565 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IN2011/000170 | Mar 2011 | US |
| Child | 13616283 | US | |
| Parent | PCT/IN2011/000364 | May 2011 | US |
| Child | PCT/IN2011/000170 | US |
