|Title of Invention||
A COMPOSITION FOR ENHANCING BIOAVAILABILITY OF DRUGS/NUTRACEUTICALS.
|Abstract||The invention relates to the isolation and preparation of an active fraction from plant Cuminum cyminum, its further purification and standardization as chemically defined entity (ies) with their intended use as drug bioavailability enhancer for the drugs belonging to therapeutic categories such as antimicrobial, antifungal, anti-viral, antitubercular, antileprosy, anti-inflammatory, antiarthritic, cardiovascular, antihistaminics, respiratory distress relieving drugs, immunosuppressants, anti-ulcerogenic, anti-cancer, CNS drugs, corticosteroids, nutraceuticals in compositions to be administered orally/parenterally, topically, inhalations (including nebulizers), rectally, vaginally in human beings and/or veterinary conditions.|
|Full Text||The present invention is directed to isolation/ preparation of an active molecule and/ or a fraction from the plant Cuminum cyminum which includes their isolation, purification and characterisation and methods of using such products to enhance bioavailability of drugs, natural products and essential nutraceuticals. The present invention is intended to enhance the bioavailability/ bioefficacy of drugs which are poorly bioavailable or given for a long period of time and are expensive and toxic. The present invention also relates to the use of bioavailability enhancers - also termed as bioenhancers or BE and methods of their preparation which include their isolation from a natural source and obtaining the final product in the form of a pure isolate and /or a fraction with all the components in a chemically characterized or their fingerprint profiled form.
DISTINGUISHING FEATURES and ADVANTAGES
There is a great interest and medical need for the improvement of bioavailability of a large number of drugs which are (a) poorly bioavailable, (b) given for long periods, and are (c) toxic and expensive. Maximizing oral bioavailability is therapeutically important because the extent of bioavailability directly influences plasma concentrations as well as therapeutic and toxic effects resulting after oral drug administration. Poorly bioavailable drugs remain sub-therapeutic because a major portion of a dose never reaches the plasma or exerts its pharmacological effect unless and until very large doses are given which may lead to serious side effects. Any significant improvement in bioavailability will result in lowering the dose or the dose frequency of that particular drug. Besides, inter-subject variability is inversely correlated with the extent of bioavailability.
Many therapeutic treatments are also accompanied by loss of essential nutraceuticals in the course of therapy. The present invention improves nutritional status by increasing bioavailability/bioefficacy of various nutraceuticals also which include metals and vitamins.
Several approaches have been adopted in the past to maximize oral bioavailability, such as (a) micronization, (b) polymorphic or crystal size and form selection, (c) solubilization of lesser soluble drugs by way of chemical modifications, complexation and use of co-solvents/surfactants, (d) targeted delivery of drug at the site of action, (e) controlled drug delivery by film coating or use of polymeric matrices for sustained release of drugs, (f) prodrug approach, and (g) microencapsulation using liposomes.
This is a new approach of increasing the bioavailability of drugs including poorly bioavailable drugs. A single major alkaloidal constituent from peppers (piperine) was found to be responsible for bioavailability enhancing effect. Influence of piperine was extensively studied on anti-TB drugs. It was determined that in combination with piperine the dose of rifampicin can be reduced by about 50% while retaining the therapeutic efficacy of this anti-TB drug at par with the standard dose (450 mg). Based on these findings several other reputed plants were evaluated for bioavailability/bioefficacy enhancing activity. Polar and non-polar extracts of parts of a few plants viz., Zingiber officinalis, Carum carvi and Cuminum cyminum increased significantly (25-300%), the bioavailability of a number of classes of drugs, for example, but not limited to, antibiotics, antifungals, anti-virals, anticancer, cardiovascular, CNS, anti-inflammatory/anti-arthritic, anti-TB/antileprosy, anti-histaminic/, corticosteroids, immunosppressants. Such extracts either in presence or absence of piperine have been found to be highly selective in their bioavailability/bioefficacy enhancing action.
Description of the preferred embodiment
The present invention is directed to isolation/ preparation of an active molecule and a fraction from the plant Cuminum cyminum. which includes their isolation, purification and characterisation and methods of using such products to enhance bioavailability of drugs, natural products and essential nutraceuticals. The products of the present invention viz., an active molecule and a fraction enhances bioavailability/ bioefficacy of certain drugs, natural products and essential nutraceuticals. The chemical name and structure of the active molecule is shown in Fig 1. HPLC fingerprint of the active molecule and the fraction is shown in Fig 2 and 3 respectively. The compound (Fig 1) though known has been for the first time reported to be useful as an effective bioavailability enhancer
Physical and chemical data of as 3',5-Dihydroxy flavone-7-O-p-D-galacturonide-4'-O-p-D-glucopyranoside
Greenish yellow powder (H2O:EtOH), soluble in H2O , m.p. 270°C decompose.
UV Xmax. nm
MeOH 256.5, 267.5 sh, 350
NaOMe 265.5, 393, 5
A1C13 273, 327.5 sh, 429.5
A1C13 / HC1 266, 358
'HNMR (DM so-d6):
8 3.08-3.75 (m, 17H, sugar protons), 4.50 (d, 1H, J= 7.21 Hz, H-l/7/), 5.21 (d, 1H, J= 6.82 Hz, H-l77 ), 6.42 (bs, 1H, H-6), 6.65 (bs, 1H, H-8), 6.81 (d, 1H, J=8.42, H-57), 7.09 (s, 1H, H-3), 7.35 (q, 1H, J= 8.42 and 1.8 Hz, H-67), 7.80 (bs, 1H, H-27).
5 165.36 (C-2) , 104.01 (C-3), 183.19 (C-4), 160.87 (C-5), 99.41 (C-6), 163.09 (C-7), 96.31 (C-8), 157.65 (C-9), 106.50 (C-10), 122.36 (C-l7 ) ,114.10 (C-2 '), 145.37 (C-3 '), 148.74 (C-47) ,116.81 (C-57), 120.75 (C-6 7), 101.21 (C-l77), 73.25 (C-277), 77.23 (C-377), 70.72 (C-477), 76.89 (C-577), 62.02 (C-677), 103.39 (C-l777), 75.02 (C-2777 and C-4/7/), 77.71 (C-3 77/) 82.07 (C-5777) ,176.44 (C-6777).
On the basis of above data the compound has been identified as 3',5-Dihydroxy flavone-7-O-p-D-galacturonide-4'-0-p-D-glucopyranoside (Fig. 1).
The products of the invention act by any one or more than one of the following ways: (a) Promoting the absorption of drugs from GIT, (b) Inhibiting or reducing the rate of biotransformation of drugs in the liver or intestines, (c) Modifying the immune system in a way that the overall requirement of the drug is reduced substantially, (d) Increasing the penetration or the entry into the pathogens even where they become persistors within the macrophages such as for Mycobacterium tuberculosis and such others. This eventually ensures the enhanced killing of these organisms well secured within the places otherwise inaccessible to the active drug, (e) Inhibiting the capability of pathogens or abnormal tissue to reject the drug e.g., efflux mechanisms frequently encountered with anti-malarial, anti-cancer and anti-microbial drugs, (f) Modifying the signalling process between host and pathogen ensuring increased accessibility of the drugs to the pathogens, (g) Enhancing the binding of the drug with the receptors like proteins, DNA, RNA, etc., in the pathogen, thus potentiating and prolonging its effect leading to enhanced antibiotic activity against pathogens, (h) Besides above plausible modes of action, the bioenhancer agents may also be useful for promoting the transport of nutrients and the drugs across the blood brain barrier, which could be of immense help in the control of diseases like cerebral infections, epilepsy and other CNS problems.
Primarily, but not exclusively, the invention enhances the carrier mediated entry of drugs and also the passive diffusion and the active transport pathways in the tissue which are responsible for transporting physiological substances such as nutraceuticals to their target sites. As applicable to any mechanism of action the products of this invention contribute in a synergistic and /or additive manner so that most drugs and nutraceuticals in presence of the products described in the present art are more bioavailable as a result of one or more of these mechanisms. As a preferred embodiment, the active molecule and the fraction increase the plasma levels and bioefficacy of certain categories of drugs and nutraceuticals by 80 - 220 % over the effect that results from normal intake of therapeutic and nutraceutical products.
The ratio (w/w) of an effective bioenhancer (fraction / active molecule) in combination with a drug/ nutraceutical may vary in the range of 0.1 to 300 %.
The bioavailability of drugs and nutraceuticals is also relevant to animal health besides being important for humans. The invention therefore is also intended to be used in veterinary preparations.
EXAMPLES: The following examples are intended to demonstrate some of the preferred embodiments and in no way should be construed so as to limit the scope of the invention. Any person skilled in the art can design more formulations, which may be considered as part of the present invention. Example 1:
Cuminum cyminum seeds (0.5 kg) were ground to a coarse powder and then extracted with deionised water at 98± 1°C for 2 hrs. Extraction process was repeated four times using total of 3.1 litres water (1 x 1 litre + 3 x 0.7 Litre, four extractions). All the four extracts were pooled. The pooled extract was centrifuged, followed by vacuum filtration through a celite bed. The clear filtrate was lyophilized to get greenish yellow amorphous powder (yield 88 gm, 17. 6 %). The dry extract was dissolved in deionised water (500 mL) and partitioned between n-BuOH (6 x 500 mL) and H2O. The n-BuOH extract was concentrated on a rotavapour under reduced pressure at 65°C (residue 11.0 gm). n-BuOH free aqueous extract was freeze dried (residue 75.0 gm) and subjected to adsorption chromatography. Aqueous extract residue was dissolved in minimum quantity of H2O and adsorbed on SiO2 gel, 60-120 mesh (150 gm) . Solvent was completely removed to get free flowing material. A glass column of 1.5 inch dia was packed with 100 gm SiO2 gel, 60-120 mesh in EtOAc. The adsorbed material was charged in the column over the packed SiO2 gel.. The column was eluted with EtOAc and then with EtOH by gradually increasing the %age of H20 in EtOH. In all 420 fractions of 70 mL each were collected and pooled on the basis of TLC pattern using BuOH (B) : AcOH (A) : H20 (W) (4:1:5) as developing solvent. Spots were visualized by spraying with freshly prepared Borinate-PEG solution [ 1% solution of 2-aminoethyl diphenylborinate in MeOH and 5% solution of polyethylene glycol 4000 in EtOH ( mixed 1:1 v/v before spraying)]. Fraction no. 81-167 (eluted in EtOH and 10% H2O in EtOH) showed same TLC pattern. These fractions were pooled, dried and then dissolved in minimum quantity of water. Crystallisation was carried out by the addition of EtOH in small portions, supernatant was drained off and
residue was washed with aq. EtOH. Residue was repeatedly crystallized from H2O : EtOH. A yellow powder (70 mg) soluble in H2O was thus obtained. Compound Rf 0.28 solvent system B : A : W (4:1:5) was identified as 3',5 -dihydroxy flavone 7-O-(3-D-galacturonide-4'-O-p-D-glucopyranoside.
Cuminum cyminum seeds (0.5 kg) were ground to a coarse powder. The powder was soaked in 50% aqueous ethanol (1.0 L) for 16 hrs. The marc was extracted three times more under same conditions using 0.7 L of extraction solvent each time. The pooled extract was clarified by vacuum filtration through a celite bed. The extract thus obtained was concentrated at 60 ± 2°C on a rotavapour. The EtOH free extract was lyophilized to get a greenish yellow powder (88 gm, 17.60%). 80 gm of the extract was extracted by heating on a steam bath respectively with
1. CHC13 (2X200mL)
2. 10%EtOHinCHCl3 (1x200)
3. 20% EtOH in CHC13 (Ix 200 mL)
4. 30% EtOH in CHC13 ( Ix 200 mL)
5. 40% EtOH in CHC13 ( Ix 200 mL)
6. 50% EtOH in CHC13 (1x200 mL)
7. 60% EtOH in CHC13 ( Ix 200 mL)
8. 70% EtOH in CHC13 (Ix 200 mL)
9. EtOH (6 x 200 mL)
10. EtOH + 10% H2O (1 x 200 mL)
The insoluble residue left after extraction with 10% water in EtOH was then extracted at room temperature with EtOH + 20% H2O (3 x 500 mL). The left over fraction (25 gm) was subjected to adsorption chromatography. It was adsorbed on silica gel (60-120 mesh 70 gm). Solvent was completely removed to get free flowing material. A glass column of 1.5 inch dia was packed with 70 gm SiOa gel 60-120 mesh in EtOH. The adsorbed extract
was charged in the column. The column was eluted with EtOH by gradually increasing the %age of tbO. In all 94 fractions of 70 mL each were collected and pooled on the basis of TLC pattern using B:A:W (4:1:5) as developing solvent. Spots were visualized by spraying the TLC plate with Borinate PEG spray reagent. Fractions 56-80 homogeneous on TLC were pooled, dried and charged on a Sephadex LH-20 column. Column was eluted with water then with EtOH to produce two fractions of 200 and 500 mL respectively. The first fraction was purified repeatedly (three times) on Sephadex LH-20 column. TLC homogeneous fractions containing target compound were pooled and residue was repeatedly crystallized from HiO : EtOH. A yellow powder (50 mg) soluble in water was obtained. Compound Rf 0.28 , Solvent system : B : A : W (4 : 1 : 5) was identified 3',5 -dihydroxyflavone 7-O-p-D-galacturonide-4'-O-p-D-glucopyranoside.
Cuminum cyminum seeds (100 gm) were ground to a coarse powder. Coarse powder was extracted with deionised water at 98 ± 1°C for 2 hrs. Extraction process was repeated four times using total water (200 +3x 100 mL four extractions). All the four extracts were centrifuged, followed by vacuum filtration through celite bed. The clear filtrate was lyophilized to get a greenish yellow amorphous powder (yield 17.0 gm , 17%). Aqueous extract residue was dissolved in deionised water (100 mL) and partitioned between n-BuOH (6 x 100 mL) and HiO . The n-BuOH extract was concentrated on a rotavapour under reduced pressure at 65 C (residue 2.3 gm). n-BuOH free aqueous extract was freeze dried (residue 13.9 gm). The aqueous residue was dissolved in HPLC grade HaO (15 mg/mL) and subjected to further purification by preparative HPLC under following conditions:
Column : RP-18, length 10 cmx2 cartridge with guard
Column dia : 1.5 cm
Sample concentration : 15 mg/mL
Injection volume : 4 mL
Mobile phase : CH3CN: H20 (1:9)
Flow rate : 10 mL/min.
Xmax : 271 nm
Run Time : 50 minutes
Pooled target fraction was concentrated under reduced pressure and crystallized from
: EtOH to afford a yellow powder 110 mg , compound Rf 0.28 , solvent system B :
A : W (4 : 1 : 5) and was identified as 3',5-dihydroxy flavone 7-O-p-D-galacturonide-4'-0-p-D-glucopyranoside.
Example 4 :
Cuminum cyminum seeds (0.5 kg) were ground to a coarse powder. Coarse powder was defatted with pet. ether 60-80 (1.0 litre) by Soxhlet extraction for 8 hrs. Pet. ether extract was discarded . The marc was dried and then extracted with EtOH (1.0 litre ) by Soxhlet extraction for 16 hrs. The EtOH extract was also discarded. The marc was then extracted with 50% aqueous EtOH at room temperature for 16 hrs each time (Total solvent used 1 litre + 4 x 0.5 litre, five extractions). All the five extracts were pooled , concentrated on a rotavapour (residue 67 gm). This residue was dissolved in a minimum quantity of water and adsorbed on silica gel 60-120 mesh (125 gm). A glass column of 1.5 inch dia was packed with silica gel 60-120 mesh (100 gm) in EtOH. The adsorbed extract was charged in the column. Elution was carried out with solvents by gradually increasing the %age of H20. Each fraction of 50 mL was collected . The fractions (148-190) eluated in 10% H2O in EtOH were pooled and subjected to further purification by preparative HPLC using following conditions.
Column :RP-18, length 10 cmx2 cartridge with guard
Column dia :2.5 cm
Sample concentration : 15 mg/mL
Injection volume : 4 mL
Mobile phase :CH3CN: H2O (1:9)
Flow rate : 10 mL/min.
Xmax :271 nm
RunTime :50 minutes
Pooled target fraction was concentrated under reduced pressure and crystallized from H2O : EtOH to afford a yellow powder (60 mg) soluble in water, compound Rf 0.28 , solvent system B : A : W (4 : 1 : 5) and was identified as 3',5 -dihydroxyflavone 7-O-P-D-galacturonide-4'-O-p-D-glucopyranoside.
EXAMPLE 5. List of drugs cited as some of the examples for the purpose of the present invention.
Example 5 (i) Doses of drug used and % Enhancement of Bioavailability. C. Drugs Category: I. Antibiotics:
IV. CNS drugs:
VII. Anti-inflammatory/ antiarthritic:
VII. Anti-TB/ Antileprosy drugs:
VIII. Anti-histamines/respiratory disorders:
IX. IX. Corticosteroids:
1. A composition for enhancing bioavailability of a drug / nutraceutical, said composition
a) an active drug / nutraceutical selected from the group comprising of
antibiotics, anti-fungal drugs, antiviral drugs, anticancer drugs, cardiovascular disorder drugs, CNS disorders drugs, antiinflammatory / antiarthritic drugs, anti-TB / anti-leprosy drugs, anti-histamines / respiratory disorder drugs, corticosteroids, immuno-suppressants and anti ulcer drugs,
b) and, a bioenhancer selected from a fraction obtained from Cuminum
cyminum having formula 1, where in w/w ratio of the bioenhancer to the drug / nutraceutical is in the range of 0.1 to 300.
3',5-Dihydroxy flavone 7-0-P-D-galacturonide-4'-0-(3-D-glucopyranoside
2. A composition as claimed in claim 1, wherein said bioenhancer increases
bioavailability of the drug / nutraceutical by 80-220%.
3. A composition as claimed in claim 1, wherein said antibiotic is selected from the group
comprising of fluroquinolones, macrolides, cephalosporins, penicillins and
4. A composition as claimed in claim 3, wherein said fluroquinolone is selected from the
group comprising of ciprofloxacin, p-floxacin, o-floxacin and norfloxacin.
5. A composition as claimed in claim 3, wherein said macrolide is selected from the group comprising of erythromycin, roxythromycin and azithromycin.
6. A composition as claimed in claim 3, wherein said cephalosporin is selected from the group comprising of cefalexin, cefadroxil and cefatrioxone.
7. A composition as claimed in claim 3, wherein said penicillin is selected from the group comprising of amoxycillin and cloxacillin.
8. A composition as claimed in claim 3, wherein said aminoglycoside used is amikacin.
9. A composition as claimed in claim 3, wherein said antifungal drug is selected from the group comprising of fluconazole, amphotericin B and ketoconazole.
10. A composition as claimed in claim 3, wherein said antiviral drug is selected from the group comprising of acyclovir and zidovudine.
11. A composition as claimed in claim 3, wherein said CNS drug is selected from the group comprising of alprazolam and haloperidol.
12. A composition as claimed in claim 3, wherein said anti-cancer drug is selected from the group comprising of methotrexate, 5-fluorouracil, doxorubicin and cisplatin.
13. A composition as claimed in claim 3, wherein said cardiovascular disorder drug is selected from the group comprising of amlodipine, atenolol and propranolol.
14. A composition as claimed in claim 3, wherein said anti-inflammatory / antiarthritic drug is selected from the group comprising of diclofenac, piroxicam, nimesulide and rofecoxib.
15. A composition as claimed in claim 3, wherein said anti-TB / antileprosy drug is selected from the group comprising of rifampicin, dapsone, ethionamide and cycloserine.
16. A composition as claimed in claim 3, wherein said anti-histamines / respiratory disorder drug is selected from the group comprising of salbutamol, theophylline and loratidine.
17. A composition as claimed in claim 3, wherein said corticosteroid is selected from the group comprising of prednisolone, dexamethasone and betamethasone.
18. A composition as claimed in claim 3, wherein said immunosuppressant is selected from the group comprising of cyclosporin A and tacrolimus.
19. A composition as claimed in claim 3, wherein said anti-ulcer drug is selected from the group comprising of ranitidine, cimetidine and omeprazole.
|Indian Patent Application Number||2226/DEL/2004|
|PG Journal Number||31/2010|
|Date of Filing||08-Nov-2004|
|Name of Patentee||COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH|
|Applicant Address||RAFI MARG, NEW DELHI-110001, INDIA.|
|PCT International Classification Number||A61K 35/78|
|PCT International Application Number||N/A|
|PCT International Filing date|