Title of Invention	A BIOENHANCER AND BIOAVAILABILITY FACILITATOR COMPOSITION
Abstract	The invention relates to a non-alkaloid compound that is plant derived glycoside "glycyrrhizin" as a highly potent bio-enhancer of activity and availability of antibiotics and other drugs including anti-infective and anticancer agents. The molecule of invention facilitates the absorption/uptake of antibiotics and other molecules across the cell membrane in plant and animal cells as well as Gram-positive and Gram-negative bacteria and therefore can be used as a drug facilitator or bioenhancer molecule to increase the affectivity of drugs and nutraceutical agents. The compound having no antimicrobial or cytotoxic activity of its own, is a safe candidate to reduce the drug dosage towards circumventing the problem of drug resistance and the other side effects in anti-infective and anti-cancer therapies. The claimed composition comprises [i] a bioenhancer or bioavailability facilitator selected from the group consisting of isolated and purified Glycyrrhizic acid, isolated and purified Glycyrrhizin or mixtures thereof and [ii] an agent selected from one or more nutraceuticals, anti-bacterial, anti-fungal, anti-tuberculosis, and anti-cancer agents; wherein the concentration of [i] is in the range of 0.1 to 10.0% of the weight of the nutraceutical agents, 0.05 to 50.0% of the weight of the anti-bacterial and anti-tuberculosis agents, 0.25 to 20.0% of the weight of the anti-fungal agents and 90.9 to 99.9% of the weight of the anti-cancer compounds.

Title of Invention

A BIOENHANCER AND BIOAVAILABILITY FACILITATOR COMPOSITION

Abstract

The invention relates to a non-alkaloid compound that is plant derived glycoside "glycyrrhizin" as a highly potent bio-enhancer of activity and availability of antibiotics and other drugs including anti-infective and anticancer agents. The molecule of invention facilitates the absorption/uptake of antibiotics and other molecules across the cell membrane in plant and animal cells as well as Gram-positive and Gram-negative bacteria and therefore can be used as a drug facilitator or bioenhancer molecule to increase the affectivity of drugs and nutraceutical agents. The compound having no antimicrobial or cytotoxic activity of its own, is a safe candidate to reduce the drug dosage towards circumventing the problem of drug resistance and the other side effects in anti-infective and anti-cancer therapies. The claimed composition comprises [i] a bioenhancer or bioavailability facilitator selected from the group consisting of isolated and purified Glycyrrhizic acid, isolated and purified Glycyrrhizin or mixtures thereof and [ii] an agent selected from one or more nutraceuticals, anti-bacterial, anti-fungal, anti-tuberculosis, and anti-cancer agents; wherein the concentration of [i] is in the range of 0.1 to 10.0% of the weight of the nutraceutical agents, 0.05 to 50.0% of the weight of the anti-bacterial and anti-tuberculosis agents, 0.25 to 20.0% of the weight of the anti-fungal agents and 90.9 to 99.9% of the weight of the anti-cancer compounds.

Full Text	Field of the Invention The invention provides novel compositions containing extracts and compounds obtained from the plant Glycyrrhiza glabra, useful as bio-enhancers and bioavailability facilitators for a variety of molecules including anti-infective, anticancer agents and nutritional compositions. This invention also relates to a method for enhancing the biological availability, proficiency and effectivity of drugs, Pharmaceuticals, nutraceuticals and other related compounds including amino acids, vitamins and other nutritional elements and ions. The present invention has direct implication in drastically reducing the dosage of antibiotics, drugs and anti-cancer agents while increasing the efficiency of absorption of nutritional elements. Background of the invention Since the invention of penicillin in 1928 by Alexander Flemming and their wider availability in the 1940s, further variants of existing and new antibiotics have been on the trial. Compared to the situation in 1954 when two million pounds of antibiotics were produced in the US, the present figure exceeds 50 million pounds. Antibiotics work either by killing bacteria (bacteriocidal) or by inhibiting growth (bacteriostatic) and their bioactivities have transformed human ability to treat many infectious diseases that previously were considered certain killers. It is estimated that globally humans consume above 250 million doses of antibiotics annually and 20%-50% of that use is unnecessary depending on the class of antibiotic. Further, widespread use of antibiotics promotes spread of antibiotic resistance many a times leading to multiple drug resistance. The total amount of antibiotic in the given dosage used for treatment of an infection generally is much higher than what is actually required to control a given population of parasite in the infected individual. This is so because all antibiotic given to the patient in a therapy does not reach the target site. This may be due to (i) lower absoiption in the gut membrane when taken orally (ii) restrictive uptake by the target microbe or (iii) operation of efflux pump leading to indiscriminate extrusion of the antibiotics or therapeutic molecules. Thus, large portions of the drugs we apply are wasted and only a miniscule percentage is being targeted to the infective microbes. But even worse part is that the unutilized drug / antibiotic amount remains as a load in the body and environment. This then acts as a selection pressure, facilitating emergence of drug resistance in parasites leading to their predominance in the niche and ultimately resulting into failure of antibiotics against resistant infections. Additionally, such a situation leads to side effects, illness and reduction in life expectancy being more acute in the older population. One of the ways that has been feasible to reduce drug dosage is the occurrence of synergism between different therapeutic agents. However, even in such a situation if both the molecules have the antibiotic property, the problem of continued selection pressure on microbes is still likely to continue. Therefore, the need is for molecules, which by themselves are not microbicidal but when present with a drug or active molecule, enhance its activity and availability (bioenhancers). These molecules by their presence will not exert any selection pressure for the mutants to emerge resistant against them and on the other hand could reduce the dosage of antibiotics or drugs so that their ill effects are minimized. This way resistance development process will be substantially delayed ultimately leading to enhanced life-span of the novel and existing antibiotics. Such drug / molecule facilitators should have novel properties like non-toxic to humans, animals or plants, should be effective at a very low concentration in a combination, should be easy to formulate and most importantly enhance uptake/absorption and activity of the drug molecules. This can lead to development of judicious and strategic concentrations of antibiotics with specific bioenhancers to improve availability of the drug right up to the target for effectively controlling the infectious organisms. The present invention was the result of planned experiments to provide a novel method for improving activity and bioavailability of antibiotics, drugs and other molecules using a plant glycoside "Glycyrrhizin" in different formulations. The bioavailability of nutrients and enhancement of antibiotics/drugs effectivity is relevant to human, plant as well as animal health and thus the compositions and methods of the invention are also intended to be used in agriculture and veterinary practice. Description of related Art Use of ayurvedic preparation "trikatu" dates back to the period between the seventh century B.C. and the sixth century A.D., which is a Sanskrit word meaning three acrids. It refers to a combination of three elements namely, black pepper (Piper nigrum Linn.), long pepper (Piper longum Linn.) and ginger (Zingiber officinale Rose.). It is believed that the use of "trikatu", and its constituents individually as well as collectively, enhances the bioavailability of a number of drugs. In specific studies carried out on animals as well as human volunteers, it was noted that the active component responsible for the increase in bioavailability of various drugs was piperine (United States Patent 5,616,593 and 5,972,382). Till today, thus, the known documented bio-availability enhancer is piperine and a series of inventions related to this compound have been described by several prior art investigators. Though the compound piperine has been reported to be enhancing the bioavailability of drugs, nutrients and vitamins, still a proper formulation for the combination is yet to come to the market. Beside this, the compound piperine being an alkaloid would find restricted use for the reasons of its typical action. The Applicants sought to provide a non-alkaloid molecule capable of bio-enhancing the availability of anti-infective, nutritional and anti-cancer compounds like 'taxoP produced by the yew tree in microscopic amounts. The Applicants screened a large number of the available extracts and known compounds in the laboratory at the Central Institute of Medicinal & Aromatic Plants, Lucknow, U.P., India (CIMAP), particularly those, which by themselves did not exhibit any antibacterial property. After extensive experimentation, it was found that out of 70 pure compounds tested, only one plant compound 'glycyrrhizin', a glycoside enhanced the killing activities of different antibiotics on three diverse kinds of bacteria including Escherichia coli, Bacillus subtilis and Mycobacterium smegmatis. The compound is generally isolated from the roots and stolon of the plant Glycyrrhiza glabra and is a known sweetener and hepatoprotective drug. Licorice (the extract from the root of the plant) is used to flavor foods, tobacco, drinks, medicines and sweets. It has expectorant action to treat bronchitis and can reduce inflammation, allergies, asthma, gastritis, peptic ulcers, rheumatism, and sore throat. It helps the liver to detoxify drugs and is used for treatment of liver disease. Licorice strengthens the immune system, stimulates the adrenal gland, and is diuretic and laxative. Licorice from roots of Glycyrrhiza glabra, contains glycyrrhizin, which is 50 times sweeter than sugar. Primary uses for licorice include treatment for peptic ulcers and stomach ailments, respiratory and intestinal passages. Licorice makes a wonderful expectorant expelling mucus from the chest area. The compound when studied by the Applicants also showed enhancement in the activity of known azole class antifungal agents like clotrimazole against infectious fungal strain of Candida albicans. More important was the obvious enhancement in the cell division inhibitory activity against the breast cancer cell line MCF-7. Accordingly, the Applicants have sought to increase the absorption of anti-infective, nutritional and anti-cancer compounds by the body and increase their utilization by the body by providing novel compositions containing extracts from the plant Glycyrrhiza glabra. Objects of the invention The main object of the invention is to provide novel compositions comprising extracts and compounds from the plant Glycyrrhiza glabra useful as a bioenhancer and capable of improving or enhancing the bio-availability of drugs such as antibiotics, anti-infective agents and of nutritional compounds. Another object of the invention is to provide compositions comprising extracts and compounds from the plant Glycyrrhiza glabra useful as bio-enhancers and bioavailability facilitators, thus enabling effective transport of drugs and molecules across biological membranes to reach target sites. Still another object is to provide methods for enhancing the bio-availability of drugs molecules and nutritional compounds to the target site in the body. Summary of the Invention Accordingly the present invention provides a bioenhancer and bioavailability facilitator composition comprising: (i) a bioenhancer or bioavailability facilitator selected from the group consisting of isolated and purified Glycyrrhizic acid, isolated and purified Glycyrrhizin or mixtures thereof; (ii) an agent selected from one or more nutraceuticals, anti-bacterial, anti-fungal, antituberculosis, and anti-cancer agents; wherein the concentration of [i] is in the range of 0.1 to 10.0% of the weight of the nutraceutical agents, 0.05 to 50.0% of the weight of the anti-bacterial and anti-tuberculosis agents, 0.25 to 20.0% of the weight of the anti-fungal agents and 90.9 to 99.9% of the weight of the anti-cancer compounds. Detailed description Accordingly, the invention provides a composition comprising an effective amount of an extract or compound obtained from the plant Glycyrrhiza glabra and useful as a bio-enhancer and bioavailability facilitator together with a therapeutically effective amount of one or more nutraceuticals, antibiotics, anti-infective agents and anti-cancer agents. In an embodiment, extract obtained from the leaves, stem, roots or stolon of Glycyrrhiza glabra, glycyrrhizic acid or glycyrrhizin, or a combination thereof. In still another embodiment, glycyrrhizin and glycyrrhizic acid are obtained from the plant Glycyrrhiza glabra. In yet another embodiment, the anti-infective agents are selected from anti-bacterial, anti-fungal and anti-tuberculosis agents. In yet another embodiment, the extract from the roots of Glycyrrhiza glabra is licorice. In an embodiment, the compound as bioavailability enhancer facilitates the transport of the compound(s) across the biological membranes to reach the target site for better effectivity/activity. In an embodiment, the concentration of glycyrrhizin ranges from 0.05 to 50% of the weight of the anti-bacterial compounds. In still another embodiment, the concentration of glycyrrhizin ranges from 0.10 to 10% of the weight of the nutraceutical compounds. In yet another embodiment, the concentration of glycyrrhizin ranges from 0.25 to 20% of the weight of the anti-fungal agents. In another embodiment, the anti-bacterial agent is selected from the group consisting of p-Iactams, macrolides, quinolones, fluoro-quinolones, aminoglycosides, glycopeptides, rifamycins, folate inhibitors, tetracyclines, anti-tuberculosis agents and other similar microbicidal compounds. In still another embodiment, the anti-fungal agent used is selected from the group comprising of polyene, triazole, imidazole, clotrimazole and other fungicidal compounds. In still another embodiment of the present invention bioenhancer improves the uptake of anti-infectives, anticancer agents and other molecules when glycyrrhizin is provided prior to the treatment with these molecules. In yet another embodiment of the present invention the anticancer agents include paclitxel (taxol) or other cell division inhibitors controlling the growth and multiplication of tumor/cancer cells. In yet another embodiment of the present invention living system used is selected from bacteria, fungi or any living cells. In yet another embodiment of the present invention the bioenhancer element is used in pure form as glycyrrhizin or as precursor/derivatives like glycyrrhizic acid. In yet another embodiment of the present invention the bioenhancer element is administered as licorice in form of aqueous/alcoholic extracts, decoction or direct root flakes/chips/sticks through wetting by any means. In yet another embodiment, the invention provides a pharmaceutical composition comprising of the plant compound glycyrrhizin and a bio-active chemical compound selected from the group consisting of anti-bacterial, anti-fungal, anti-cancerous and other similar compounds including nutraceuticals wherein, the glycyrrhizin concentration ranges from 0.05 to 50% of the weight of the nutraceuticals compound used. In yet another embodiment, the anti-bacterial agent is selected from the group consisting of p-lactams, macrolides, quinolones, fluoro-quinolones, aminoglycosides, glycopeptides, rifamycins, folate inhibitors, tetracyclines, anti-tuberculosis agents and other similar microbicidal compounds wherein, the glycyrrhizin concentration ranges from 0.05 to 50% of the weight of the anti-bacterial compound used. In yet another embodiment, the macrolides comprise erythromycin, clarithromycin, azithromycin and other similar compounds. In yet another embodiment, the quinolones and fluoroquinolones used are selected from the group consisting of nalidixic acid, norfloxacin, ciprofloxacin, sparfloxacin and similar other compounds. In yet another embodiment of the present invention the aminoglycosides used are selected from the group consisting of streptomycin, gentamicin, amikacin and other similar compounds. In yet another embodiment of the present invention the glycopeptides used are vancomycin, teicoplanin and other similar compounds. In yet another embodiment of the present invention the rifamycins used are rifampicin and other similar compounds. In yet another embodiment of the present invention the anti-tuberculosis agents used are isoniazid, pyrazinamide, ethambutol and other similar compounds. In yet another embodiment of the present invention the folate inhibitors are tnmethoprim, sulphonamides and other similar compounds. In yet another embodiment, the p-Lactams comprise penicillin, flucloxacillin, cloxacukkub, methicillin, cephalosporins, carbapenenms and similar compounds. In yet another embodiment, other similar microbicidal compounds are streptogramins and oxazolidinones. In another embodiment the anti-fungal agent is selected from the group consisting of polyene, tnazole, imidazole, clotrimazole and other fungicidal compounds wherein, the glycyrrhizin concentration ranges from 0.25 to 20% of the weight of the antifungal compound used. In yet another embodiment the polyenes used are amphoterecin B, nystatin and other similar compounds. In yet another embodiment the triazoles used are fluconazole, itraconazole and other similar compounds. In yet another embodiment the imidazoles used are ketoconazole and other similar compounds. In yet another embodiment other fungicidal compounds are griseofulvin and terbinafine. In yet another embodiment, the anti-cancer agent is selected from the group consisting of Paclitaxel (Taxol), Docetaxel (Taxotere), Vinblastine (Velban), Vincristine (Oncovin), Vinorelbine (Navelbine) and other similar compounds used in cancer therapy. In another embodiment, the levels of glycyrrhizin ranges from 10 to 10,000 folds of the weight of the anti-cancer compound used. In another embodiment, the nutraceuticals used to increase the bio-activity and bioavailability are selected from the group consisting of vitamins, amino acids, hormones and other nutritional supplements. In yet another embodiment, the pharmaceutical compositions comprising of the plant compound glycyrrhizin in form of liquorice / dried root with the bioactive chemical compound(s) to increase their bioactivity and bioavailability wherein, the glycyrrhizin in form of liquorice preparation or dried root is given as one gram equivalent to 40 mg of glycyrrhizin. The invention also provides a method for enhancing the bio-availability of herbal extracts, anti-infective agents, anti-cancer and nutraceutical compounds/compositions across biological membranes in living systems; said method comprising the steps of administering to a subject in need thereof, a composition comprising an effective amount of the bioenhancer obtained from the plant Glycyrrhiza glabra, and any one of anti-infective agents, anti-cancer agents and nutraceutical compounds. In an embodiment, the bio-enhancer is selected from the extract of Glycyrrhiza glabra, glycyrrhizin, glycyrrhizic acid obtained from Glycyrrhiza glabra or combinations thereof. In yet another embodiment, administration of the composition circumvents the side effects of chemotherapy by substantially reducing the dosages of therapeutic agents comprising of anti-bacterial, anti-fungal and anti-cancerous agents wherein the side effects of those agents can be bone marrow suppression, liver and kidney toxicity, pulmonary scarring, high fevers, skin reactions, Nausea and vomiting, hair loss, skin rash, mouth sores, diarrhoea, loss of appetite and confusion, lethargy and ambulation 10 problems (ataxia), anaphylactic reactions (consisting of fast heart rate, wheezing, lowered blood pressure and facial edema, cerebellar dysfunction (consists of slurring of speech, walking problems, as well as eye motion problems), loss of periods (menses), ovarian failure can occur, painful soft tissue ulcers, cardiotoxicity, hypersensitivity reactions, abnormal blood pressure, sweating, bleeding, shortness of breath and many related problems. In still another embodiment, the bioenhancer improves the uptake of anti-infectives, anticancer agents and other molecules when glycyrrhizin is provided prior to the treatment with these molecules. The invention also provides use of the extract or the compound obtained from the plant Glycyrrhiza glabra, as a bioenhancer and bioavailability and facilitator of nutritional compounds and drugs and molecules selected from anti-infective and anticancer agents. The invention is described in detail herein below with reference to the following drawings wherein : Fig. 1 : represents the structural formula of Glycyrrhizic acid; and Fig.2 : represents the structural formula of Glycyrrhizin Some of the embodiments of the invention are illustrated herein below by the following bioassays and examples which are provided merely to illustrate the invention and should not be construed as limitations on the inventive concept embodied herein. The methodology followed for screening of this compound (Glycyrrhizic acid and Glycyrrhizin) included specifically designed bioassays as follows: 1. Assay for bio-enhancement of anti-infective agents a) The minimum inhibitory concentration (MIC) of any antibiotic or drug molecule is determined against Escherichia coli (ATCC 10536), Bacillus subtilis (ATCC 6015 11 ) and Mycobacterium smegmatis (ATCC 14468) and Candida albicans (ATCC 10231 ) in broth and disc diffusion assays. b) The antibiotics or antifungal agents at concentrations 1/10, 1/8, 1/6, 1/4, 1/3, 1/2 and equal to MIC are added alone and in combination with the test compound at varying concentrations in broth assays to evaluate the comparative level of inhibition and similarly at varying concentrations in disc diffusion assay. c) The combinations showing enhancement in terms of higher activity than antibiotic alone for the inhibition of bacterial growth (large inhibition zone in disc diffusion and higher killing at lower concentration in broth assay) are picked up for future testing. d) In broth assays the activity is quantified by counting number of viable cells in a given treatment and converted in fold enhancement by the combination treatment compared to antibiotic/drug alone in the killing percentage of cells. e) The pretreatment assay is followed to determine whether the bioenhancer which may be either an extract obtained from the plant Glycyrrhiza glabra, glycyrrhizin or glycyrrhizic acid, is required along with antibiotic to enhance its activity or even its withdrawal after treatment or prior to antibiotic treatment would benefit. For this, the cells are treated with the bioenhancer/compound for 4 to 8 hours and then washed free of it by centrifugation and washing in sterile water. This is followed by treatment with antibiotic/drug as in steps (b) to (d) above. f) The results of the bio-assays carried out are represented in the tables provided herein below wherein tables 1-6 represent the activity of glycyrrhizin. Tables 7-9 depict the activity of glycyrrhizic acid and tables 10-11 depict the activity of licorice. 2. Assay for bio-enhancement of anti-cancer agents a) MCF-7 (Breast cancer cell line) is inoculated at a density of about 0.1 X 106 cells in MEM medium in the wells of 24 well plate. b) The used up medium in these cultures is replaced with fresh medium after 18 hours in each well. 12 c) The test compound (s) is added at desired concentrations in different wells just after the medium replacement. d) Observations are recorded on the cell count after 36 hours for which the following steps are required. i. The medium is removed from the wells. ii. The wells are rinsed with 1ml PBS (Phosphate buffer saline). iii. To each well 500 jul of freshly prepared trypsin solution (0.1% in PBS) is added, iv. Trypsin solution is removed after 30 seconds and the plate is gently tapped till the cells are released from the plate surface, v. Fresh 1 ml of MEM growth medium is added and agitated with a pipette to obtain a cell suspension, vi. 10 \|il of cell suspension is taken on the haemocytometer and a cover glass is placed over the counting chamber, vii. The number of viable cells is counted in 5 big squares and the reading is taken from 5 microscopic fields to determine the average, viii. The cell count (titre per ml) in the original sample is then calculated as average count X 103. Composition of Minimum Essential Medium (MEM): 100 ml MEM powder (Sigma-Aldrich, USA) = 0.96 g HEPES Buffer (Sigma -Aldrich, USA) = 0.26 g Sodium Bicarbonate = 0.22 g Penicillin G = lOmg Streptomycin = 20 mg Gentamycin = 5 mg Foetal Calf Serum = 15 ml Distilled water = 85 ml 3. Unavailability tests through biological membrane 13 a) A specially designed U-tubes of glass consisting of two component (opposite-L type) were used in which one open end of a L-shaped was tapered to fit within the untapered end of the other L-tube. b) The membrane of goat gut (initial part) was stretched and fixed to act as the barrier between the two ends such that by joining the two L-tubes, a U-tube was made. c) Sterile distilled water was then filled in both the sides to equal height/level. d) The antibiotic/compound was added to the donor tube (tapered) and through spectro-photometer the transfer of molecule was observed using UV and visible absorption maxima of the respective molecules by taking the OD at defined wavelengths. Examples In the next step of elucidation of the enhancer action the applicants experimented with the killing activities of different antibiotics against the bacteria singly and in combination with the test compound glycyrrhizin following the method described above. These experiments are to be considered with assay for bioenhancement of the compounds glycyrrhizin/glycyrrhizic acid and licorice, as described earlier. When the bacteria were grown in presence of the compound as such no significant killing was observed. In all the experiments the glycyrrhizin concentration was kept at 1 jag/ml, unless it is specifically mentioned. Example 1. Glycyrrhizin mediated enhancement in the killing action of antibiotics against Gram-negative bacterium Escherichia coli. Table 1 Antibiotics Rifampicin Concentration Hg/ml 10 Survival fraction of viable cells upon treatment with antibiotic alone (a) 0.42 Survival fraction of viable cells upon treatment with antibioticglycyrrhizin combination (b) 0.11 Folds enhancement in antibiotic activity (a/b) 3.8 14 Rifampicin Nalidixic acid Tetracycline Tetracycline Ampicillin Ampicillin 20 8 2 4 6 8 0.28 0.19 0.42 0.27 0.38 0.013 0.02 0.02 0.22 0.03 0.03 0.006 14.0 9.5 1.9 9.0 12.6 2.1 Example 2. Glycyrrhizin mediated enhancement in the killing action of antibiotics against Gram-positive bacterium Bacillus subtilis. Table 2 Antibiotics Nalidixic acid Nalidixic acid Tetracycline Concentration Hg/ml 6 8 1 Survival fraction of viable cells upon treatment with antibiotic alone (a) 0.50 0.27 0.10 Survival fraction of viable cells upon treatment with antibiotic- glycyrrhizin combination (b) 0.11 0.04 0.02 'Folds enhancemen t in antibiotic activity (a/b) 4.50 6.75 5.00 Example 3. Glycyrrhizin mediated enhancement in the killing action of antibiotics against bacterium Mycobacterium smegmatis Table 3 Antibiotics Rifampicin Nalidixic Acid Tetracycline Concentratio n ug/ml 0.5 4 0.5 Survival fraction of viable cells upon treatment with antibiotic alone (a) 0. 56 X 10'4 0.70 0.3 Survival fraction of viable cells upon treatment with antibiotic- glycyrrhizin combination (b) 0.086 X 10'4 0.12 0.2 Folds enhancement in antibiotic activity (a/b) 6.5 7.7 1.5 From the above experiments it was deduced that the potency of the antibiotic is increased when applied along with the compound glycyrrhizin. It was interesting to note that the same results were observed when the bacterial cells were pretreated with glycyrrhizin for 4-8 hours and then the cells were washed free of the glycyrrhizin by centrifugation and resuspending the cells in fresh medium followed by treatment with respective antibiotics. This implicated some changes induced by glycyrrhizin on the bacterial surface/membrane making it more receptive to the antibiotics and hence enhancing bioavailability and ultimately activity/bactericidal effect of the given antibiotic. 15 Example 4: Glycyrrhizin mediated enhancement observed in disc diffusion assays for the killing action of antifungal agent against Candida albicans. Table 4 Clotrimazole jj4Lg/ disc) 0 0 4 4 Glycyrrhizin (Hg/disc) 0 1 0 1 Zone of inhibition (mm) 0 0 7 11 Example 5 Glycyrrhizin mediated enhancement in the activity of anticancerous agent 'Taxol" Table 5 Taxol Concentration (jag/ml) 0.01 0.02 0.05 Without taxol Control Control (glycyrrhizin) Initial titre of viable cells O.IXIO'6 0.1X 10'6 0.1X10'6 0.1 X 10'6 0.1X10'6 Final titre of viable cells upon treatment with taxol alone 0.05 X 106 0.04 X 106 0.03 X 106 Final titre of viable cells upon treatment with taxol-glycyrrhizin combination 0.025 X 106 0.024 X 106 0.021 X 106 Final titre 0.16 X 10'6 0.12 X10'6 Example 6 Glycyrrhizin mediated enhancement in the bioavailability across the biological membrane Table 6 Compound(s) in the donor tube Rifampicin Rifampicin + Glycyrrhizin Wave length (nm) A340 A475 A340 A475 A36o OD measured as Absorbance (specific to the compound maxima) across the membrane in receiving tube after I h r 0.0039 0.0015 0.0070 0.0100 0.0250 2hr 0.0275 0.0148 0.0638 0.0395 0.0556 3hr 0.0371 0.0210 0.1465 0.0575 0.0714 4hr 0.0440 0.0297 0.2090 0.1362 0.1283 5hr 0.0906 0.0334 0.3424 0.2365 0.1370 6hr 0.1165 0.0394 0.3930 0.2853 0.1595 Vitamin B- 12 VitaminB-12 Aj60 ASBO A530 0.0250 0.0582 0.0556 0.0627 0.0714 0.1058 0.1283 0.2051 0.1370 0.2616 0.1595 0.2984 Here, the applicants observed glycyrrhizin-mediated enhancement in the cell division inhibitory action of anti-cancer agent taxol in the animal cell culture experiments using cancerous cell line MCF-7. As evident in Table 5 above, the anticancerous activity of Taxol' in terms of inhibiting the growth and multiplication of MCF-7 cancer cells was markedly enhanced by at least 5 folds. In these experiments, the cancerous cells growth inhibition by Taxol (@ 0.01 u.g/ml) in presence of glycyrrhizin (@ 1 )J,g/ml) was higher than even the treatment with taxol (@ 0.05 (j.g/ml) alone. Example 7:: Glycyrrhizic acid mediated enhancement in the killing action of antibiotics against Escherichia coli in broth assay. Table 7 Antibiotics Rifampicin Concentratio n^g/ml 20 Survival fraction of viable cells upon treatment with antibiotic alone (a) 0.57 Survival fraction of viable cells upon treatment with antibiotic glycyrrhizic acid combination (b) 0.281 Folds enhancement in antibiotic activity (a/b) 2.03 Table 8 Glycyrrhizic acid mediated enhancement in the killing action of antibiotics against Escherichia coli in disc diffusion assay. Rifampicin (^g/disc) Glycyrrhizic acid (jig/disc) Net zone of inhibition (mm) 17 0 0 500 500 0 4 0 4 0 0 2 3 Table 9 Glycyrrhizic acid mediated enhancement in the killing action of antibiotics against Mycobacterium smegmatis in disc diffusion assay. Rifampicin (ng/disc) 0 0 0.4 0.4 Glycyrrhizic acid (jj.g/disc) 0 4 0 4 Net zone of inhibition (mm) 0 0 3 4 In the above experiments, the Applicants used Glycyrrhizic acid to test the potency of this acid in enhancing the activity of antibiotics. The Applicants observed that the killing action the antibiotics (such as Rifampicin) is enhanced when used with Glycyrrhizic acid. Table 7 shows that when the cells were treated with Rifampicin alone, the survival fraction was about 0.57. However, the survival fraction of cells when treated with a combination of rifampicin and glycyrrhizic acid was much less i.e. about 0.281. This shows that the combination of glycyrrhizic acid with rifampicin enhances the availability of rifampicin to the cells. Similarly, the results of the disc diffusion assay also show that the combination of glycyrrhizic acid with rifampicin is more effective against Mycobacterium smegmatis than glycyrrhizic acid or rifampicin used singly. Example 8: Licorice (25 (ug/ml) mediated enhancement in the killing action of Rifampicin against Gram-negative bacterium Escherichia coli. Table 10 18 Antibiotics Rifampicin Concentration Hg/ml 10 Survival fraction of viable cells upon treatment with antibiotic alone (a) 0.420 Survival fraction of viable cells upon treatment with antibiotic glycyrrhizic acid combination (b) 0.135 Folds enhancement in antibiotic activity (a/b) 3.10 Table 11 Licorice (25 \|4.g/ml) mediated enhancement in the killing action of antibiotics against Gram-positive bacterium Bacillus subtilus. Antibiotics Nalidixic acid Concentration Hg/ml 6 Survival traction of viable cells upon treatment with antibiotic alone (a) 0.500 Survival fraction of viable cells upon treatment with antibiotic glycyrrhizic acid combination (b) 0.121 *Folds enhancement in antibiotic activity (a/b) 4.10 Tables 10 and 11 show the bio-enhancing effects observed in the use of licorice, the extract from the roots of Glycyrrhiza glabra. As can be seen from Tables 10, the survival rate of bacteria treated with the antibiotic Rifampicin is more than when treated with a combination of Rifampicin and licorice. Similarly, Table 11 shows that the survival rate of the bacteria treated with nalidixic acid is less than when treated with a combination of nalidixic acid and licorice. The results in Tables 10 and 11 demonstrate the use of licorice as an effective bio-enhancer and bioavailability facilitator. In the experiments related to transport across the mammalian gut membrane, it was observed that the compound glycyrrhizin enhances the transport of antibiotics e.g. Rifampicin, Tetracycline, Nalidixic acid, Ampicillin and vitamins like B-l and B-12 across the gut as well as artificial membrane. The enhancement in transport is approximately 2 to 6 folds. Several medicinal plants are cited for use in Indian system of medicine for various activities such as immunostimulant, adaptogenic, anticancer, antimalarial, antimicrobial, angiogenetic, hepatoprotective, hypolinidemic, hypoglycemic, memory enhancing etc. The genus Glycyrrhiza (Mulethi) has attracted sizable importance for compound(s) with hepatoprotective ability. In Glycyrrhiza glabra (family, 19 Leguminosae) 12 different varieties (Biovars) are known. Among these only three namely typica, glandilifera and violacea are commonly used for the pharmaceutical and confectionary requirements. Glycyrrhiza glabra var. typica is the source of Spanish Liquorice and is sweeter than other types. Glycyrrhiza glabra var. glandilifera is the source of Russian liquorice, which is moderately sweet and some what bitter, also more or less acrid. Glycyrrhiza glabra var. violacea is known as the Persian or Iranian liquorice of commerce and has thick roots. The name Glycyrrhiza (popularly known as mulethi in India) is of Greek origin meaning sweet root. Cultivation of this species is mostly limited to US, Eastern Europe and several CIS of former USSR and scattered in western and northern India. The roots of the plant Glycyrrhiza glabra are highly useful as these contain the important glycoside Glycyrrhizin for its hepatoprotective ability. The root extracts also contain flavonoids, saponins, sterols, amino acids, gums essential oils, sugars and starches. The plant takes at least 4-5 years to achieve substantial production of glycyrrhizin in the roots. Generating sufficient planting material for commercial cultivation is thus desirable for harvesting required quantity of Glycyrrhizin on sustainable basis. This molecule (Glycyrrhizin) is a known sweetener and hepatoprotective drug. It helps the liver to detoxify drugs and is given for liver disease. The molecule is known to be strengthening the immune system, stimulates the adrenal gland, and is diuretic and laxative. This is also used to treat peptic ulcers and stomach ailments, respiratory and intestinal passages. Considering the wide use of the molecule in traditional system of medicine and the level of toxicity well tested, this can be used along with the antibiotics and other molecule of importance to enhance the efficacy on the target. This molecule (glycyrrhizin) in the Applicant's study facilitates the transport/uptake of various compounds including antibiotics, antifungal and anticancer agents, vitamins and amino acids etc. across the membrane thereby increasing the absorption or bioavailability of various biological systems including bacteria, animal and plant cells. This thus has immense importance for absorption of the drugs, Pharmaceuticals, nutraceutical and other related compounds and ions by the cells and hence huge potential of being used as drug/molecule facilitators. 20 Great emphasis now is being laid towards quality assurance of crude drugs from plants sources widely used in the Indian system of medicine. The scientific study of traditional medicines, derivation of drugs through bioprospection and systematic conservation, domestication and cultivation of the concerned medicinal plants has assumed great importance in the present day context when more and more people prefer safe and effective medicines at affordable price for curing their ailments. The present invention enlarges the scope and use of the natural glycoside glycyrrhizin in therapeutical and nutraceutical application. These plants synthesizing multi-use molecules are Clycyrrhiza sps and Abarus sps. The preferred plant is Glycyrrhiza glabra which is propagated through underground rhizome. This demands large amount of planting material which may be possible by micro propagation in vitro for its commercial cultivation. Agrotechnology and suitable cultivars for its cultivation in India are therefore required through screening of germplasm and genetic selection. At CIMAP a genetically superior variety of Glycyrrhiza glabra var. typica named as 'Misree' is available for cultivation with better yield of glycyrrhizin (Kukreja A K, Tyagi B R, Muni Ram and Sushil Kumar (1997) Licorice cultivar - Mishree. Journal oj Medicinal and Aromatic Plants Sciences. 19 : 732-733). Procedure for isolation of quality glycyrrhizin the main compound from the root extract. Of the plant Glycyrrhiza glabra var. typica was also standardized. For this purpose we choose the plant Glycyrrhiza glabra var. typica 'Misree' (the released variety of CIMAP) and commercially available plant material. Fresh roots were harvested from the plant and shade dried. Commercial dried roots were purchased from the market. The protocol followed for the isolation was as follows: Protocol: 1. Plant material (250 gram) was dipped in 900 ml of Butanol: glacial acetic acid : ethyl alcohol (1:1:1) for 48 hours. 2. This was filtered through filter paper and concentrated in vacuums rotavapour. 3. The solid material thus obtained was dissolved in acetic acid (180 ml) and treated with diethyl ether 2 litre (10 times, each with 200 ml) to remove impurity as precipitate which was filtered out. 21 4. Then the liquor in diethyl ether was evaporated to 250 ml and fractionated through liquid - liquid fractionation twice in water, followed by discarding the water fraction. 5. This diethyl ether fraction was further concentrated in vacuum rotavapour to obtain the compound. 6. The compound was hydrolyzed with 80 ml of NaOH (NaOH 5% in Methaol) at 80°-90°C for Ihour. 7. The hydrolyzed reaction mixture was acidified (100 ml of glacial acetic acid and 5 ml of concentrated HC1) to pH 3.6 to form a ppt which was filtered and washed with ethanol. After isolation, the compound was checked for the purity compared to the standard glycyrrhizin obtained from Sigma chemical Inc. Preparation of the test solution: One gram of the isolated compound was extracted in 20 ml chloroform for 15 min. After filtration the filtrate was discarded. The marc was refluxed for 1 hour with 30 ml of 0.5 M F^SO,}. The unfiltered mixture was then cooled and extracted with chloroform (2X20ml) and the combined CHCb extract was concentrated. The residue was dissolved in 1.0 ml CHCh : Methanol (1:1) mixture. Reference solution: Five milligram of glycyrrhizin was refluxed with 20 ml 0.5 M HbSO,}. This was cooled and extracted with chloroform (2X10ml). The combined CHCb extract was evaporated and the residue was dissolved in 1.0 ml CHCIs: Methanol (1:1) mixture. Solvent system : Toluene : ethyl acetate: glacial acetic acid (12.5 : 7.5:0.5) Procedure: The test and reference solutions were applied in two different tracks on a precoated silica gel GF254 plate (5X15 cm) of uniform thickness (0.2 mm). The plate was developed in the solvent system to a distance of 12 cm. Scanning: The plate was scanned densitometrically at 254 nm in both the tracks and the fingerprint profiles were recorded. Visualization of spots: (i) Under UV 254 nm. (ii) The plate was sprayed with anisaldehyde-sulfuric acid reagent and heated at 110°C for 5-10 min. 22 Evaluation: (i) Under UV 254 nm light (before spraying):one spot (0.41) was visible in the sample solution track, corresponding to (Rf 0.41) Glycyrrhetic acid of reference track. (ii) In daylight (after spraying): Gylcyrrhetic acid was visible as a dark violet spot in both reference and test solution tracks. The above experiments indicate a sound procedure of isolating fairly pure glycyrrhizin from the root of the plant variety 'Misree' of CIMAP as well as market available dry root samples. By this procedure about 0.1 to 0.25% of glycyrrhizin could be isolated. The protocol can be scaled up or scaled down depending upon the initial starting material. Advantages 1. The plant extract of Glycyrrhiza glabra and glycyrrhizin in the Applicant's study facilitates the transport/uptake of various compounds including antibiotics, antifungal and anticancer agents, vitamins and amino acids etc. across the membrane thereby increasing the absorption or bioavailability of various biological systems including bacteria, animal and plant cells. This thus has immense importance for absorption of the drugs, Pharmaceuticals, nutraceutical and other related compounds and ions by the cells and hence huge potential of being used as drug/molecule facilitators. 2. The present invention enlarges the scope and use of the natural glycoside Glycyrrhizin in therapeutical and nutraceutical application. 3. Glycyrrhizin is nontoxic to humans, animals or plants, is effective at very low concentrations in a combination, easy to formulate and enhances the uptake/absorption and activity of drug molecules. Thus the novel use of this compound as described in the present invention is safe and effective for bioenhancement of activity of drugs. 23 4. Glycyrrhizin by its presence will not exert any selection pressure for the mutants to emerge resistant against them and reduce the dosage of antibiotics or drugs so that their ill effects are reduced. Claims 1. A composition comprising an effective; amount of an. extract or compound obtained; from the plant Glycyrrliiza glabra and. usefui.as a bio-enhancer anicf'bioavaHability facilitator together with a therapeutically effective amount of one or more 'antibiotics, anti-infective agents or anti-cancer agents. 2. A composition as claimed in claim 1 containing an extra'ci of leaves, siem, roots of stolon of Glycyrrhiza glabra, glycyrchizic acid or glycyrrhizin, or a combination thereof. ; 3. A composition as claimed in'claim 1. wherein glycyrrhrzin and glycyrrhteic acid are obtained from the plant Glycyrrhiza glabra. 4. A composition as claimed in claim I, wherein the' extract of roots Of Glycyrrhiza glakra. is licorice. • . ; 5. A composition as claimed in claim 1, wherein the anti-infective agents are selected from ami -bacterial, anti-fungal and anti-tuberculosis agents. 6. A composition as claimed in claim 1, wherein the concentration of glycyrrliizih ranges from 0.05 to SOwcight % of the anti-bacterial compounds. 7. A composition as claimed in claim 1, wherein the concentration of glycyrrliiz'.in' ranges from 0.25 to 20weight % of the anti-fungal' agents. 8. A composition as claimed in claim 5, wherein die-anti-bacterial, agents ure selected from the group consisting of p-lactams, macrolides, quitiolones, fluoro-quinblones, aminoglycosides, glycopeptides, rifamycins, rotate inhibitors, tetracyclines. anti- tuberculosis agents or microbicidal compounds. • . 9. A composition as claimed in claim 9, wherein" the quinolones and fluoro-quinolones are selected from the group consisting of naUdixic acid,inorfloxacin, ciproflaxacii>,. sparfloxacin or similar otiicr compounds. 10. A composition as claimed in claim 9, wherein the ß-Lactams are selected from the group comprising penicillin, flucloxacillin, cloxacukkub, methicillin, ceptialosporlns or carbapenenms. 1J.A composition as claimed in. claim 9, wherein; the macrolides are splected from erythromycih, clarithromycin;, azithromycin:or other similar compounds. 12. A composition as claimed in claim 9, wherein the aminoglycosides are selected from the group consisting of streptomycin, gentamicin, amikactn or similar compounds. • 13. A composition as claimed in claim 9, wherein the glycopeptides are selected from varicomycin, teicoplahin or other similar compounds. 14. A composition us claimed in claim '9, wherein the rit'amycins arc. rif'ampicin or similar compounds. 15. A composition as claimed in claim 9, wherein the folate inhibitors are- selected from trimethoprim, sulphonamides or similar compounds. 16. A composition as claimed in claim 9, wherein the mi'crbbiocidal compounds tut selected from slreptogramins and oxazolidinones. 17. A composition as claimed in claim 5, wherein the anti-tuberculosis• agents are selected from the group comprising isoniazid,' pyrazmamide, ethambutol or similar compounds.' 18. A composition as claimed in claim 5,'wherein the anti-fungal agents are selected from the group consisting of polyene, triazole, imidazole, clolrimaziolc or fungicidal compounds. 19. A composition as claimed in claim 19, wherein'the'polyene is selected from nystatin or similar compounds. 20. A composition as claimed. in claim 19, wherein the iriazole is selected from fluconazole, itraconazole and or similar compounds. 21. A composition as claimed in claim 19, wherein the imidazole is selected from ketoconazole or similar compounds. 22. A composition as claimed in claim 19, wherein the fungicidal'compound: is selected from griseofulvjn or terbinafine. 23'. A composition as claimed in claim I, wherein; the anti-cancer, agent is selected from ihe group consisting of Paclitaxel (Taxol), DocetaxeJ- (Takotere), . Vinblasiine (Velban), Vincristine (Oncovin) or Vinorelbine (Navelbirie). . . 24. A composition as claimed in claim I, wherein the concentration-of glycyrrhizin ranges from 10 to 10,000 folds of the. weight of the anti-cancercompound. ;• 25. A composition as .claimed in claim 1, wherein licorice preparation from1 .one gram of dried root is equivalent to 40 mg of glycyrrhizin. 26. A method for enhancing the bio-availability of anti-infective agents or anti-cancer using herbal extracts or compounds derived from the exrtract across biological membranes in living systems; said method comprising the-steps-of administering to a subject in need thereof, a composition comprising an effective amount of ihe. bioenhancer obtained from the plant Glycyrrhiza glabra, and any one of anti-infective agents or, anti-cancer agents.. . ... 27. A method as claimed in claim 28, wherein, the bio-enhancer is selected .from the extract of Glycyrrhiza glabra, glycyrrhizin, glycyrrhizic acid or-combinations thereof. 28. A method as claimed in claim 28, wherein the bioenhancer is obtained from the roots, stem, stolon or leaves of the p[&nt-Glycyrrhiza glabra. 29. A method as claimed in claim -28, wherein the anti-infective agents are selected from . anti-bacterial, anti-fungal, or anti-tuberculosis agents. 30. A method as claimed in claim. 31, wherein the anti-bacterial agents are selected .from the group comprising P-lactams, macrolides, .quiriolones, fluoro-quinolones, aminbglycbsides. glycopeptides, rifamycins, folate inhibitors, litracyclines:. aati-tuberculosis agents or' microbicidal compounds. 31. A method as claimed in claim 31, wherein-concentration of glycyrrhizin ranges from 0.05 to 50 weight % of the anti-bacterial agents. • : 32. A method as claimed in cliim 32, wherein'the quinqlones and fluoroquinplones are selected from the group consisting of nalidixic acid, riorfloxacin. ciprofloxacin, sparfloxacin or similar other compounds. , 33. A method as' claimed in claim 32, wherein thelp-Lactams .are selected fi-om the group consisting of penicillin, flucloxacillin; cloxacukkub, rnethicUIiri, cephulosporins or carbapenenms. • : 34. A method as claimed in claim 32;, wherein the macrplides are selected from erythromycin, clarithromycin, azithromycin or other similar compounds. 35. A method as claimed in claim 32, wherein1 the' aminoglycosides-are selected from the group consisting of streptomycin, gentamicin, a-mjkaein or:sirnttar compounds. 36. A method as claimed in claim 32, wherein the glycopeptides are selected from vancomycin, teicoplanin or other similar compounds. . . • 37. A method as claimed in claim ,32, wherein the rifamycins .are rifumpicin or similar compounds. . 38. A method as claimed in claim 32, wherein the anti-tubierQulosik agents selected from the group consisting of isoniazid, pyrazinamide, etharhbuiol .or other similar compounds. . . •'• 39. A method as claimed in claim 31, wherein concentration ofgiycyrrhizin ranges- from 0,25 to 20 weight % of the anti-fungal agents.. . 40. A method as claimed in claim 31, wherein the anri-fungaJ agenris selected from the group consisting of polyene, triazole; imidazole, clotriihazob or other fungiciclal compounds. • ' .. ' : • 41. A method as claimed in claim 28, wherein, the anti-cancer agent is selected from the group consisting of PacJitaxel (Tiixol), Dbcetaxe'J'(TaxoteTe); .Vinblastine (Velban), Vincristine (Oncovin) or Vinorelbirie (Navelbine):. 42. A method as claimed in cfaim 31, wherein the concentration of gtycyrrhiiin ranges ;• from 10 to 10,000 folds of the weight of the anti-cancer agentl ; 43. A method as claimed'in!-claim 27, wherein- administration !of the composition- circumvents the side effects of chemotherapy by substantially reducing the dosages .of therapeutic agents comprising of anti-bacterial, anti-fungal and anti-cancerous agents wherein, the side effects of those agents can be bone marrow suppression, liwer and kidney toxicity, pulmonary scarring, high.- fevers, .skin reactions, Nausea and vomiting, hair loss, skin rash, mouth sores, diarrhoea, loss of appetite and confusion, lethargy and ambulation problems (ataxia)i anaphylactic reactions (consisting «F fast heart rate, wheezing, lowered blood pressure and facial edema, cerebellar dysfunction (consists of slurring of speech, -walking problems, as we'll as eye rtiotio'n problems), loss of periods .(menses), ovarian failure can occur, painful soft-1 tissue ulcers, cardiotoxicity, hypersensitivity reactions, abnormal blood pressure, sweating,, bleeding, shortness of breath and many related problems 44. A method as claimed in claim 27, wherein,-the bioenhancer improves the uptake of anti-infectives, or anticancer agents when glycyrrhizin is provided prior .to the treatment with these molecules. 45. Use of the-extract or the compound obtained from the plant.Gfycjvr/ij^a-g/aArfl, -as a bioenhancer and bioavailability and facilitator drugs molecules selected from anti- infective and anti-cancer agents.

Full Text

Field of the Invention
The invention provides novel compositions containing extracts and compounds
obtained from the plant Glycyrrhiza glabra, useful as bio-enhancers and
bioavailability facilitators for a variety of molecules including anti-infective, anticancer
agents and nutritional compositions. This invention also relates to a method for
enhancing the biological availability, proficiency and effectivity of drugs,
Pharmaceuticals, nutraceuticals and other related compounds including amino acids,
vitamins and other nutritional elements and ions. The present invention has direct
implication in drastically reducing the dosage of antibiotics, drugs and anti-cancer
agents while increasing the efficiency of absorption of nutritional elements.
Background of the invention
Since the invention of penicillin in 1928 by Alexander Flemming and their wider
availability in the 1940s, further variants of existing and new antibiotics have been on
the trial. Compared to the situation in 1954 when two million pounds of antibiotics
were produced in the US, the present figure exceeds 50 million pounds. Antibiotics
work either by killing bacteria (bacteriocidal) or by inhibiting growth (bacteriostatic)
and their bioactivities have transformed human ability to treat many infectious
diseases that previously were considered certain killers. It is estimated that globally
humans consume above 250 million doses of antibiotics annually and 20%-50% of
that use is unnecessary depending on the class of antibiotic. Further, widespread use
of antibiotics promotes spread of antibiotic resistance many a times leading to
multiple drug resistance. The total amount of antibiotic in the given dosage used for
treatment of an infection generally is much higher than what is actually required to
control a given population of parasite in the infected individual. This is so because all
antibiotic given to the patient in a therapy does not reach the target site. This may be
due to (i) lower absoiption in the gut membrane when taken orally (ii) restrictive
uptake by the target microbe or (iii) operation of efflux pump leading to
indiscriminate extrusion of the antibiotics or therapeutic molecules. Thus, large
portions of the drugs we apply are wasted and only a miniscule percentage is being
targeted to the infective microbes. But even worse part is that the unutilized drug /
antibiotic amount remains as a load in the body and environment. This then acts as a
selection pressure, facilitating emergence of drug resistance in parasites leading to
their predominance in the niche and ultimately resulting into failure of antibiotics
against resistant infections. Additionally, such a situation leads to side effects, illness
and reduction in life expectancy being more acute in the older population. One of the
ways that has been feasible to reduce drug dosage is the occurrence of synergism
between different therapeutic agents. However, even in such a situation if both the
molecules have the antibiotic property, the problem of continued selection pressure on
microbes is still likely to continue. Therefore, the need is for molecules, which by
themselves are not microbicidal but when present with a drug or active molecule,
enhance its activity and availability (bioenhancers). These molecules by their presence
will not exert any selection pressure for the mutants to emerge resistant against them
and on the other hand could reduce the dosage of antibiotics or drugs so that their ill
effects are minimized. This way resistance development process will be substantially
delayed ultimately leading to enhanced life-span of the novel and existing antibiotics.
Such drug / molecule facilitators should have novel properties like non-toxic to
humans, animals or plants, should be effective at a very low concentration in a
combination, should be easy to formulate and most importantly enhance
uptake/absorption and activity of the drug molecules. This can lead to development
of judicious and strategic concentrations of antibiotics with specific bioenhancers to
improve availability of the drug right up to the target for effectively controlling the
infectious organisms.
The present invention was the result of planned experiments to provide a novel
method for improving activity and bioavailability of antibiotics, drugs and other
molecules using a plant glycoside "Glycyrrhizin" in different formulations.
The bioavailability of nutrients and enhancement of antibiotics/drugs effectivity is
relevant to human, plant as well as animal health and thus the compositions and
methods of the invention are also intended to be used in agriculture and veterinary
practice.
Description of related Art
Use of ayurvedic preparation "trikatu" dates back to the period between the seventh
century B.C. and the sixth century A.D., which is a Sanskrit word meaning three
acrids. It refers to a combination of three elements namely, black pepper (Piper
nigrum Linn.), long pepper (Piper longum Linn.) and ginger (Zingiber officinale
Rose.). It is believed that the use of "trikatu", and its constituents individually as well
as collectively, enhances the bioavailability of a number of drugs. In specific studies
carried out on animals as well as human volunteers, it was noted that the active
component responsible for the increase in bioavailability of various drugs was
piperine (United States Patent 5,616,593 and 5,972,382). Till today, thus, the known
documented bio-availability enhancer is piperine and a series of inventions related to
this compound have been described by several prior art investigators. Though the
compound piperine has been reported to be enhancing the bioavailability of drugs,
nutrients and vitamins, still a proper formulation for the combination is yet to come to
the market. Beside this, the compound piperine being an alkaloid would find restricted
use for the reasons of its typical action.
The Applicants sought to provide a non-alkaloid molecule capable of bio-enhancing
the availability of anti-infective, nutritional and anti-cancer compounds like 'taxoP
produced by the yew tree in microscopic amounts.
The Applicants screened a large number of the available extracts and known
compounds in the laboratory at the Central Institute of Medicinal & Aromatic Plants,
Lucknow, U.P., India (CIMAP), particularly those, which by themselves did not
exhibit any antibacterial property. After extensive experimentation, it was found that
out of 70 pure compounds tested, only one plant compound 'glycyrrhizin', a glycoside
enhanced the killing activities of different antibiotics on three diverse kinds of
bacteria including Escherichia coli, Bacillus subtilis and Mycobacterium smegmatis.
The compound is generally isolated from the roots and stolon of the plant Glycyrrhiza
glabra and is a known sweetener and hepatoprotective drug.
Licorice (the extract from the root of the plant) is used to flavor foods, tobacco, drinks,
medicines and sweets. It has expectorant action to treat bronchitis and can reduce
inflammation, allergies, asthma, gastritis, peptic ulcers, rheumatism, and sore throat.
It helps the liver to detoxify drugs and is used for treatment of liver disease. Licorice
strengthens the immune system, stimulates the adrenal gland, and is diuretic and
laxative. Licorice from roots of Glycyrrhiza glabra, contains glycyrrhizin, which is 50
times sweeter than sugar. Primary uses for licorice include treatment for peptic ulcers
and stomach ailments, respiratory and intestinal passages. Licorice makes a wonderful
expectorant expelling mucus from the chest area.
The compound when studied by the Applicants also showed enhancement in the
activity of known azole class antifungal agents like clotrimazole against infectious
fungal strain of Candida albicans. More important was the obvious enhancement in
the cell division inhibitory activity against the breast cancer cell line MCF-7.
Accordingly, the Applicants have sought to increase the absorption of anti-infective,
nutritional and anti-cancer compounds by the body and increase their utilization by the
body by providing novel compositions containing extracts from the plant Glycyrrhiza
glabra.
Objects of the invention
The main object of the invention is to provide novel compositions comprising extracts
and compounds from the plant Glycyrrhiza glabra useful as a bioenhancer and
capable of improving or enhancing the bio-availability of drugs such as antibiotics,
anti-infective agents and of nutritional compounds.
Another object of the invention is to provide compositions comprising extracts and
compounds from the plant Glycyrrhiza glabra useful as bio-enhancers and
bioavailability facilitators, thus enabling effective transport of drugs and molecules
across biological membranes to reach target sites.
Still another object is to provide methods for enhancing the bio-availability of drugs
molecules and nutritional compounds to the target site in the body.
Summary of the Invention
Accordingly the present invention provides a bioenhancer and bioavailability facilitator composition
comprising:
(i) a bioenhancer or bioavailability facilitator selected from the group consisting of isolated
and purified Glycyrrhizic acid, isolated and purified Glycyrrhizin or mixtures thereof;
(ii) an agent selected from one or more nutraceuticals, anti-bacterial, anti-fungal, antituberculosis,
and anti-cancer agents;
wherein the concentration of [i] is in the range of 0.1 to 10.0% of the weight of the nutraceutical
agents, 0.05 to 50.0% of the weight of the anti-bacterial and anti-tuberculosis agents, 0.25 to 20.0% of
the weight of the anti-fungal agents and 90.9 to 99.9% of the weight of the anti-cancer compounds.
Detailed description
Accordingly, the invention provides a composition comprising an effective amount of
an extract or compound obtained from the plant Glycyrrhiza glabra and useful as a
bio-enhancer and bioavailability facilitator together with a therapeutically effective
amount of one or more nutraceuticals, antibiotics, anti-infective agents and anti-cancer
agents.
In an embodiment, extract obtained from the leaves, stem, roots or stolon of
Glycyrrhiza glabra, glycyrrhizic acid or glycyrrhizin, or a combination thereof.
In still another embodiment, glycyrrhizin and glycyrrhizic acid are obtained from the
plant Glycyrrhiza glabra.
In yet another embodiment, the anti-infective agents are selected from anti-bacterial,
anti-fungal and anti-tuberculosis agents.
In yet another embodiment, the extract from the roots of Glycyrrhiza glabra is
licorice.
In an embodiment, the compound as bioavailability enhancer facilitates the transport
of the compound(s) across the biological membranes to reach the target site for better
effectivity/activity.
In an embodiment, the concentration of glycyrrhizin ranges from 0.05 to 50% of the
weight of the anti-bacterial compounds.
In still another embodiment, the concentration of glycyrrhizin ranges from 0.10 to
10% of the weight of the nutraceutical compounds.
In yet another embodiment, the concentration of glycyrrhizin ranges from 0.25 to 20%
of the weight of the anti-fungal agents.
In another embodiment, the anti-bacterial agent is selected from the group consisting
of p-Iactams, macrolides, quinolones, fluoro-quinolones, aminoglycosides,
glycopeptides, rifamycins, folate inhibitors, tetracyclines, anti-tuberculosis agents and
other similar microbicidal compounds.
In still another embodiment, the anti-fungal agent used is selected from the group
comprising of polyene, triazole, imidazole, clotrimazole and other fungicidal
compounds.
In still another embodiment of the present invention bioenhancer improves the uptake
of anti-infectives, anticancer agents and other molecules when glycyrrhizin is
provided prior to the treatment with these molecules.
In yet another embodiment of the present invention the anticancer agents include
paclitxel (taxol) or other cell division inhibitors controlling the growth and
multiplication of tumor/cancer cells.
In yet another embodiment of the present invention living system used is selected
from bacteria, fungi or any living cells.
In yet another embodiment of the present invention the bioenhancer element is used in
pure form as glycyrrhizin or as precursor/derivatives like glycyrrhizic acid.
In yet another embodiment of the present invention the bioenhancer element is
administered as licorice in form of aqueous/alcoholic extracts, decoction or direct root
flakes/chips/sticks through wetting by any means.
In yet another embodiment, the invention provides a pharmaceutical composition
comprising of the plant compound glycyrrhizin and a bio-active chemical compound
selected from the group consisting of anti-bacterial, anti-fungal, anti-cancerous and
other similar compounds including nutraceuticals wherein, the glycyrrhizin
concentration ranges from 0.05 to 50% of the weight of the nutraceuticals compound
used.
In yet another embodiment, the anti-bacterial agent is selected from the group
consisting of p-lactams, macrolides, quinolones, fluoro-quinolones, aminoglycosides,
glycopeptides, rifamycins, folate inhibitors, tetracyclines, anti-tuberculosis agents and
other similar microbicidal compounds wherein, the glycyrrhizin concentration ranges
from 0.05 to 50% of the weight of the anti-bacterial compound used.
In yet another embodiment, the macrolides comprise erythromycin, clarithromycin,
azithromycin and other similar compounds.
In yet another embodiment, the quinolones and fluoroquinolones used are selected
from the group consisting of nalidixic acid, norfloxacin, ciprofloxacin, sparfloxacin
and similar other compounds.
In yet another embodiment of the present invention the aminoglycosides used are
selected from the group consisting of streptomycin, gentamicin, amikacin and other
similar compounds.
In yet another embodiment of the present invention the glycopeptides used are
vancomycin, teicoplanin and other similar compounds.
In yet another embodiment of the present invention the rifamycins used are rifampicin
and other similar compounds.
In yet another embodiment of the present invention the anti-tuberculosis agents used
are isoniazid, pyrazinamide, ethambutol and other similar compounds.
In yet another embodiment of the present invention the folate inhibitors are
tnmethoprim, sulphonamides and other similar compounds.
In yet another embodiment, the p-Lactams comprise penicillin, flucloxacillin,
cloxacukkub, methicillin, cephalosporins, carbapenenms and similar compounds.
In yet another embodiment, other similar microbicidal compounds are streptogramins
and oxazolidinones.
In another embodiment the anti-fungal agent is selected from the group consisting of
polyene, tnazole, imidazole, clotrimazole and other fungicidal compounds wherein,
the glycyrrhizin concentration ranges from 0.25 to 20% of the weight of the antifungal
compound used.
In yet another embodiment the polyenes used are amphoterecin B, nystatin and other
similar compounds.
In yet another embodiment the triazoles used are fluconazole, itraconazole and other
similar compounds.
In yet another embodiment the imidazoles used are ketoconazole and other similar
compounds.
In yet another embodiment other fungicidal compounds are griseofulvin and
terbinafine.
In yet another embodiment, the anti-cancer agent is selected from the group consisting
of Paclitaxel (Taxol), Docetaxel (Taxotere), Vinblastine (Velban), Vincristine
(Oncovin), Vinorelbine (Navelbine) and other similar compounds used in cancer
therapy.
In another embodiment, the levels of glycyrrhizin ranges from 10 to 10,000 folds of
the weight of the anti-cancer compound used.
In another embodiment, the nutraceuticals used to increase the bio-activity and bioavailability
are selected from the group consisting of vitamins, amino acids, hormones
and other nutritional supplements.
In yet another embodiment, the pharmaceutical compositions comprising of the plant
compound glycyrrhizin in form of liquorice / dried root with the bioactive chemical
compound(s) to increase their bioactivity and bioavailability wherein, the glycyrrhizin
in form of liquorice preparation or dried root is given as one gram equivalent to 40 mg
of glycyrrhizin.
The invention also provides a method for enhancing the bio-availability of herbal
extracts, anti-infective agents, anti-cancer and nutraceutical compounds/compositions
across biological membranes in living systems; said method comprising the steps of
administering to a subject in need thereof, a composition comprising an effective
amount of the bioenhancer obtained from the plant Glycyrrhiza glabra, and any one of
anti-infective agents, anti-cancer agents and nutraceutical compounds.
In an embodiment, the bio-enhancer is selected from the extract of Glycyrrhiza
glabra, glycyrrhizin, glycyrrhizic acid obtained from Glycyrrhiza glabra or
combinations thereof.
In yet another embodiment, administration of the composition circumvents the side
effects of chemotherapy by substantially reducing the dosages of therapeutic agents
comprising of anti-bacterial, anti-fungal and anti-cancerous agents wherein the side
effects of those agents can be bone marrow suppression, liver and kidney toxicity,
pulmonary scarring, high fevers, skin reactions, Nausea and vomiting, hair loss, skin
rash, mouth sores, diarrhoea, loss of appetite and confusion, lethargy and ambulation
10
problems (ataxia), anaphylactic reactions (consisting of fast heart rate, wheezing,
lowered blood pressure and facial edema, cerebellar dysfunction (consists of slurring
of speech, walking problems, as well as eye motion problems), loss of periods
(menses), ovarian failure can occur, painful soft tissue ulcers, cardiotoxicity,
hypersensitivity reactions, abnormal blood pressure, sweating, bleeding, shortness of
breath and many related problems.
In still another embodiment, the bioenhancer improves the uptake of anti-infectives,
anticancer agents and other molecules when glycyrrhizin is provided prior to the
treatment with these molecules.
The invention also provides use of the extract or the compound obtained from the
plant Glycyrrhiza glabra, as a bioenhancer and bioavailability and facilitator of
nutritional compounds and drugs and molecules selected from anti-infective and anticancer
agents.
The invention is described in detail herein below with reference to the following
drawings wherein :
Fig. 1 : represents the structural formula of Glycyrrhizic acid; and
Fig.2 : represents the structural formula of Glycyrrhizin
Some of the embodiments of the invention are illustrated herein below by the
following bioassays and examples which are provided merely to illustrate the
invention and should not be construed as limitations on the inventive concept
embodied herein.
The methodology followed for screening of this compound (Glycyrrhizic acid and
Glycyrrhizin) included specifically designed bioassays as follows:
1. Assay for bio-enhancement of anti-infective agents
a) The minimum inhibitory concentration (MIC) of any antibiotic or drug molecule is
determined against Escherichia coli (ATCC 10536), Bacillus subtilis (ATCC 6015
11
) and Mycobacterium smegmatis (ATCC 14468) and Candida albicans (ATCC
10231 ) in broth and disc diffusion assays.
b) The antibiotics or antifungal agents at concentrations 1/10, 1/8, 1/6, 1/4, 1/3, 1/2
and equal to MIC are added alone and in combination with the test compound at
varying concentrations in broth assays to evaluate the comparative level of
inhibition and similarly at varying concentrations in disc diffusion assay.
c) The combinations showing enhancement in terms of higher activity than antibiotic
alone for the inhibition of bacterial growth (large inhibition zone in disc diffusion
and higher killing at lower concentration in broth assay) are picked up for future
testing.
d) In broth assays the activity is quantified by counting number of viable cells in a
given treatment and converted in fold enhancement by the combination treatment
compared to antibiotic/drug alone in the killing percentage of cells.
e) The pretreatment assay is followed to determine whether the bioenhancer which
may be either an extract obtained from the plant Glycyrrhiza glabra, glycyrrhizin
or glycyrrhizic acid, is required along with antibiotic to enhance its activity or
even its withdrawal after treatment or prior to antibiotic treatment would benefit.
For this, the cells are treated with the bioenhancer/compound for 4 to 8 hours and
then washed free of it by centrifugation and washing in sterile water. This is
followed by treatment with antibiotic/drug as in steps (b) to (d) above.
f) The results of the bio-assays carried out are represented in the tables provided
herein below wherein tables 1-6 represent the activity of glycyrrhizin. Tables 7-9
depict the activity of glycyrrhizic acid and tables 10-11 depict the activity of
licorice.
2. Assay for bio-enhancement of anti-cancer agents
a) MCF-7 (Breast cancer cell line) is inoculated at a density of about 0.1 X 106 cells
in MEM medium in the wells of 24 well plate.
b) The used up medium in these cultures is replaced with fresh medium after 18
hours in each well.
12
c) The test compound (s) is added at desired concentrations in different wells just
after the medium replacement.
d) Observations are recorded on the cell count after 36 hours for which the following
steps are required.
i. The medium is removed from the wells.
ii. The wells are rinsed with 1ml PBS (Phosphate buffer saline).
iii. To each well 500 jul of freshly prepared trypsin solution (0.1% in PBS) is
added,
iv. Trypsin solution is removed after 30 seconds and the plate is gently tapped
till the cells are released from the plate surface,
v. Fresh 1 ml of MEM growth medium is added and agitated with a pipette to
obtain a cell suspension,
vi. 10 |il of cell suspension is taken on the haemocytometer and a cover glass
is placed over the counting chamber,
vii. The number of viable cells is counted in 5 big squares and the reading is
taken from 5 microscopic fields to determine the average,
viii. The cell count (titre per ml) in the original sample is then calculated as
average count X 103.
Composition of Minimum Essential Medium (MEM): 100 ml
MEM powder (Sigma-Aldrich, USA) = 0.96 g
HEPES Buffer (Sigma -Aldrich, USA) = 0.26 g
Sodium Bicarbonate = 0.22 g
Penicillin G = lOmg
Streptomycin = 20 mg
Gentamycin = 5 mg
Foetal Calf Serum = 15 ml
Distilled water = 85 ml
3. Unavailability tests through biological membrane
13
a) A specially designed U-tubes of glass consisting of two component (opposite-L
type) were used in which one open end of a L-shaped was tapered to fit within the
untapered end of the other L-tube.
b) The membrane of goat gut (initial part) was stretched and fixed to act as the
barrier between the two ends such that by joining the two L-tubes, a U-tube was
made.
c) Sterile distilled water was then filled in both the sides to equal height/level.
d) The antibiotic/compound was added to the donor tube (tapered) and through
spectro-photometer the transfer of molecule was observed using UV and visible
absorption maxima of the respective molecules by taking the OD at defined
wavelengths.
Examples
In the next step of elucidation of the enhancer action the applicants experimented with
the killing activities of different antibiotics against the bacteria singly and in
combination with the test compound glycyrrhizin following the method described
above. These experiments are to be considered with assay for bioenhancement of the
compounds glycyrrhizin/glycyrrhizic acid and licorice, as described earlier. When the
bacteria were grown in presence of the compound as such no significant killing was
observed. In all the experiments the glycyrrhizin concentration was kept at 1 jag/ml,
unless it is specifically mentioned.
Example 1. Glycyrrhizin mediated enhancement in the killing action of antibiotics
against Gram-negative bacterium Escherichia coli.
Table 1
Antibiotics
Rifampicin
Concentration
Hg/ml
10
Survival fraction
of viable cells
upon treatment
with antibiotic
alone (a)
0.42
Survival fraction of
viable cells upon
treatment with
antibioticglycyrrhizin
combination (b)
0.11
*Folds
enhancement
in antibiotic
activity
(a/b)
3.8
14
Rifampicin
Nalidixic acid
Tetracycline
Tetracycline
Ampicillin
Ampicillin
20
8
2
4
6
8
0.28
0.19
0.42
0.27
0.38
0.013
0.02
0.02
0.22
0.03
0.03
0.006
14.0
9.5
1.9
9.0
12.6
2.1
Example 2. Glycyrrhizin mediated enhancement in the killing action of antibiotics
against Gram-positive bacterium Bacillus subtilis.
Table 2
Antibiotics
Nalidixic
acid
Nalidixic
acid
Tetracycline
Concentration
Hg/ml
6
8
1
Survival fraction of
viable cells upon
treatment with
antibiotic alone
(a)
0.50
0.27
0.10
Survival fraction of
viable cells upon
treatment with
antibiotic- glycyrrhizin
combination (b)
0.11
0.04
0.02
'Folds
enhancemen
t in
antibiotic
activity
(a/b)
4.50
6.75
5.00
Example 3. Glycyrrhizin mediated enhancement in the killing action of antibiotics
against bacterium Mycobacterium smegmatis
Table 3
Antibiotics
Rifampicin
Nalidixic Acid
Tetracycline
Concentratio
n
ug/ml
0.5
4
0.5
Survival fraction of
viable cells upon
treatment with
antibiotic alone
(a)
0. 56 X 10'4
0.70
0.3
Survival fraction of
viable cells upon
treatment with
antibiotic- glycyrrhizin
combination (b)
0.086 X 10'4
0.12
0.2
*Folds
enhancement
in antibiotic
activity
(a/b)
6.5
7.7
1.5
From the above experiments it was deduced that the potency of the antibiotic is
increased when applied along with the compound glycyrrhizin. It was interesting to
note that the same results were observed when the bacterial cells were pretreated with
glycyrrhizin for 4-8 hours and then the cells were washed free of the glycyrrhizin by
centrifugation and resuspending the cells in fresh medium followed by treatment with
respective antibiotics. This implicated some changes induced by glycyrrhizin on the
bacterial surface/membrane making it more receptive to the antibiotics and hence
enhancing bioavailability and ultimately activity/bactericidal effect of the given
antibiotic.
15
Example 4: Glycyrrhizin mediated enhancement observed in disc diffusion assays for
the killing action of antifungal agent against Candida albicans.
Table 4
Clotrimazole
jj4Lg/ disc)
0
0
4
4
Glycyrrhizin
(Hg/disc)
0
1
0
1
Zone of inhibition
(mm)
0
0
7
11
Example 5 Glycyrrhizin mediated enhancement in the activity of anticancerous agent
'Taxol"
Table 5
Taxol Concentration
(jag/ml)
0.01
0.02
0.05
Without taxol
Control
Control (glycyrrhizin)
Initial titre
of viable
cells
O.IXIO'6
0.1X 10'6
0.1X10'6
0.1 X 10'6
0.1X10'6
Final titre of viable
cells upon treatment
with taxol alone
0.05 X 106
0.04 X 106
0.03 X 106
Final titre of viable cells
upon treatment with
taxol-glycyrrhizin
combination
0.025 X 106
0.024 X 106
0.021 X 106
Final titre
0.16 X 10'6
0.12 X10'6
Example 6 Glycyrrhizin mediated enhancement in the bioavailability across the
biological membrane
Table 6
Compound(s) in
the donor tube
Rifampicin
Rifampicin
+ Glycyrrhizin
Wave
length
(nm)
A340
A475
A340
A475
A36o
OD measured as Absorbance (specific to the compound maxima)
across the membrane in receiving tube after
I h r
0.0039
0.0015
0.0070
0.0100
0.0250
2hr
0.0275
0.0148
0.0638
0.0395
0.0556
3hr
0.0371
0.0210
0.1465
0.0575
0.0714
4hr
0.0440
0.0297
0.2090
0.1362
0.1283
5hr
0.0906
0.0334
0.3424
0.2365
0.1370
6hr
0.1165
0.0394
0.3930
0.2853
0.1595
Vitamin B- 12
VitaminB-12
Aj60
ASBO
A530
0.0250
0.0582
0.0556
0.0627
0.0714
0.1058
0.1283
0.2051
0.1370
0.2616
0.1595
0.2984
Here, the applicants observed glycyrrhizin-mediated enhancement in the cell division
inhibitory action of anti-cancer agent taxol in the animal cell culture experiments
using cancerous cell line MCF-7. As evident in Table 5 above, the anticancerous
activity of Taxol' in terms of inhibiting the growth and multiplication of MCF-7
cancer cells was markedly enhanced by at least 5 folds. In these experiments, the
cancerous cells growth inhibition by Taxol (@ 0.01 u.g/ml) in presence of glycyrrhizin
(@ 1 )J,g/ml) was higher than even the treatment with taxol (@ 0.05 (j.g/ml) alone.
Example 7:: Glycyrrhizic acid mediated enhancement in the killing action of
antibiotics against Escherichia coli in broth assay.
Table 7
Antibiotics
Rifampicin
Concentratio
n^g/ml
20
Survival fraction
of viable cells
upon treatment
with antibiotic
alone (a)
0.57
Survival
fraction of
viable cells
upon treatment
with antibiotic
glycyrrhizic
acid
combination
(b)
0.281
*Folds
enhancement in
antibiotic
activity (a/b)
2.03
Table 8
Glycyrrhizic acid mediated enhancement in the killing action of antibiotics against
Escherichia coli in disc diffusion assay.
Rifampicin (^g/disc) Glycyrrhizic acid (jig/disc) Net zone of inhibition
(mm)
17
0
0
500
500
0
4
0
4
0
0
2
3
Table 9
Glycyrrhizic acid mediated enhancement in the killing action of antibiotics against
Mycobacterium smegmatis in disc diffusion assay.
Rifampicin (ng/disc)
0
0
0.4
0.4
Glycyrrhizic acid (jj.g/disc)
0
4
0
4
Net zone of inhibition
(mm)
0
0
3
4
In the above experiments, the Applicants used Glycyrrhizic acid to test the potency of
this acid in enhancing the activity of antibiotics. The Applicants observed that the
killing action the antibiotics (such as Rifampicin) is enhanced when used with
Glycyrrhizic acid. Table 7 shows that when the cells were treated with Rifampicin
alone, the survival fraction was about 0.57. However, the survival fraction of cells
when treated with a combination of rifampicin and glycyrrhizic acid was much less
i.e. about 0.281. This shows that the combination of glycyrrhizic acid with rifampicin
enhances the availability of rifampicin to the cells. Similarly, the results of the disc
diffusion assay also show that the combination of glycyrrhizic acid with rifampicin is
more effective against Mycobacterium smegmatis than glycyrrhizic acid or rifampicin
used singly.
Example 8: Licorice (25 (ug/ml) mediated enhancement in the killing action of
Rifampicin against Gram-negative bacterium Escherichia coli.
Table 10
18
Antibiotics
Rifampicin
Concentration
Hg/ml
10
Survival fraction
of viable cells
upon treatment
with antibiotic
alone (a)
0.420
Survival fraction of
viable cells upon
treatment with antibiotic
glycyrrhizic acid
combination (b)
0.135
*Folds
enhancement
in
antibiotic
activity
(a/b)
3.10
Table 11
Licorice (25 |4.g/ml) mediated enhancement in the killing action of antibiotics against
Gram-positive bacterium Bacillus subtilus.
Antibiotics
Nalidixic acid
Concentration
Hg/ml
6
Survival traction of
viable cells upon
treatment with
antibiotic alone (a)
0.500
Survival fraction of
viable cells upon
treatment with
antibiotic
glycyrrhizic acid
combination (b)
0.121
*Folds enhancement
in antibiotic activity
(a/b)
4.10
Tables 10 and 11 show the bio-enhancing effects observed in the use of licorice, the
extract from the roots of Glycyrrhiza glabra. As can be seen from Tables 10, the
survival rate of bacteria treated with the antibiotic Rifampicin is more than when
treated with a combination of Rifampicin and licorice. Similarly, Table 11 shows that
the survival rate of the bacteria treated with nalidixic acid is less than when treated
with a combination of nalidixic acid and licorice. The results in Tables 10 and 11
demonstrate the use of licorice as an effective bio-enhancer and bioavailability
facilitator.
In the experiments related to transport across the mammalian gut membrane, it was
observed that the compound glycyrrhizin enhances the transport of antibiotics e.g.
Rifampicin, Tetracycline, Nalidixic acid, Ampicillin and vitamins like B-l and B-12
across the gut as well as artificial membrane. The enhancement in transport is
approximately 2 to 6 folds.
Several medicinal plants are cited for use in Indian system of medicine for various
activities such as immunostimulant, adaptogenic, anticancer, antimalarial,
antimicrobial, angiogenetic, hepatoprotective, hypolinidemic, hypoglycemic, memory
enhancing etc. The genus Glycyrrhiza (Mulethi) has attracted sizable importance for
compound(s) with hepatoprotective ability. In Glycyrrhiza glabra (family,
19
Leguminosae) 12 different varieties (Biovars) are known. Among these only three
namely typica, glandilifera and violacea are commonly used for the pharmaceutical
and confectionary requirements. Glycyrrhiza glabra var. typica is the source of
Spanish Liquorice and is sweeter than other types. Glycyrrhiza glabra var.
glandilifera is the source of Russian liquorice, which is moderately sweet and some
what bitter, also more or less acrid. Glycyrrhiza glabra var. violacea is known as the
Persian or Iranian liquorice of commerce and has thick roots. The name Glycyrrhiza
(popularly known as mulethi in India) is of Greek origin meaning sweet root.
Cultivation of this species is mostly limited to US, Eastern Europe and several CIS of
former USSR and scattered in western and northern India. The roots of the plant
Glycyrrhiza glabra are
highly useful as these contain the important glycoside Glycyrrhizin for its
hepatoprotective ability. The root extracts also contain flavonoids, saponins, sterols,
amino acids, gums essential oils, sugars and starches. The plant takes at least 4-5 years
to achieve substantial production of glycyrrhizin in the roots. Generating sufficient
planting material for commercial cultivation is thus desirable for harvesting required
quantity of Glycyrrhizin on sustainable basis.
This molecule (Glycyrrhizin) is a known sweetener and hepatoprotective drug. It helps
the liver to detoxify drugs and is given for liver disease. The molecule is known to be
strengthening the immune system, stimulates the adrenal gland, and is diuretic and
laxative. This is also used to treat peptic ulcers and stomach ailments, respiratory and
intestinal passages. Considering the wide use of the molecule in traditional system of
medicine and the level of toxicity well tested, this can be used along with the
antibiotics and other molecule of importance to enhance the efficacy on the target.
This molecule (glycyrrhizin) in the Applicant's study facilitates the transport/uptake of
various compounds including antibiotics, antifungal and anticancer agents, vitamins
and amino acids etc. across the membrane thereby increasing the absorption or
bioavailability of various biological systems including bacteria, animal and plant cells.
This thus has immense importance for absorption of the drugs, Pharmaceuticals,
nutraceutical and other related compounds and ions by the cells and hence huge
potential of being used as drug/molecule facilitators.
20
Great emphasis now is being laid towards quality assurance of crude drugs from
plants sources widely used in the Indian system of medicine. The scientific study of
traditional medicines, derivation of drugs through bioprospection and systematic
conservation, domestication and cultivation of the concerned medicinal plants has
assumed great importance in the present day context when more and more people
prefer safe and effective medicines at affordable price for curing their ailments. The
present invention enlarges the scope and use of the natural glycoside glycyrrhizin in
therapeutical and nutraceutical application. These plants synthesizing multi-use
molecules are Clycyrrhiza sps and Abarus sps. The preferred plant is Glycyrrhiza
glabra which is propagated through underground rhizome. This demands large
amount of planting material which may be possible by micro propagation in vitro for
its commercial cultivation. Agrotechnology and suitable cultivars for its cultivation in
India are therefore required through screening of germplasm and genetic selection. At
CIMAP a genetically superior variety of Glycyrrhiza glabra var. typica named as
'Misree' is available for cultivation with better yield of glycyrrhizin (Kukreja A K,
Tyagi B R, Muni Ram and Sushil Kumar (1997) Licorice cultivar - Mishree. Journal
oj Medicinal and Aromatic Plants Sciences. 19 : 732-733).
Procedure for isolation of quality glycyrrhizin the main compound from the root
extract. Of the plant Glycyrrhiza glabra var. typica was also standardized. For this
purpose we choose the plant Glycyrrhiza glabra var. typica 'Misree' (the released
variety of CIMAP) and commercially available plant material. Fresh roots were
harvested from the plant and shade dried. Commercial dried roots were purchased
from the market. The protocol followed for the isolation was as follows:
Protocol:
1. Plant material (250 gram) was dipped in 900 ml of Butanol: glacial acetic acid
: ethyl alcohol (1:1:1) for 48 hours.
2. This was filtered through filter paper and concentrated in vacuums rotavapour.
3. The solid material thus obtained was dissolved in acetic acid (180 ml) and
treated with diethyl ether 2 litre (10 times, each with 200 ml) to remove
impurity as precipitate which was filtered out.
21
4. Then the liquor in diethyl ether was evaporated to 250 ml and fractionated
through liquid - liquid fractionation twice in water, followed by discarding the
water fraction.
5. This diethyl ether fraction was further concentrated in vacuum rotavapour to
obtain the compound.
6. The compound was hydrolyzed with 80 ml of NaOH (NaOH 5% in Methaol)
at 80°-90°C for Ihour.
7. The hydrolyzed reaction mixture was acidified (100 ml of glacial acetic acid
and 5 ml of concentrated HC1) to pH 3.6 to form a ppt which was filtered and
washed with ethanol.
After isolation, the compound was checked for the purity compared to the standard
glycyrrhizin obtained from Sigma chemical Inc.
Preparation of the test solution: One gram of the isolated compound was extracted
in 20 ml chloroform for 15 min. After filtration the filtrate was discarded. The marc
was refluxed for 1 hour with 30 ml of 0.5 M F^SO,}. The unfiltered mixture was then
cooled and extracted with chloroform (2X20ml) and the combined CHCb extract was
concentrated. The residue was dissolved in 1.0 ml CHCh : Methanol (1:1) mixture.
Reference solution: Five milligram of glycyrrhizin was refluxed with 20 ml 0.5 M
HbSO,}. This was cooled and extracted with chloroform (2X10ml). The combined
CHCb extract was evaporated and the residue was dissolved in 1.0 ml CHCIs:
Methanol (1:1) mixture.
Solvent system : Toluene : ethyl acetate: glacial acetic acid (12.5 : 7.5:0.5)
Procedure: The test and reference solutions were applied in two different tracks on a
precoated silica gel GF254 plate (5X15 cm) of uniform thickness (0.2 mm). The plate
was developed in the solvent system to a distance of 12 cm.
Scanning: The plate was scanned densitometrically at 254 nm in both the tracks and
the fingerprint profiles were recorded.
Visualization of spots:
(i) Under UV 254 nm.
(ii) The plate was sprayed with anisaldehyde-sulfuric acid reagent and heated
at 110°C for 5-10 min.
22
Evaluation:
(i) Under UV 254 nm light (before spraying):one spot (0.41) was visible in
the sample solution track, corresponding to (Rf 0.41) Glycyrrhetic acid of
reference track.
(ii) In daylight (after spraying): Gylcyrrhetic acid was visible as a dark violet
spot in both reference and test solution tracks.
The above experiments indicate a sound procedure of isolating fairly pure glycyrrhizin
from the root of the plant variety 'Misree' of CIMAP as well as market available dry
root samples. By this procedure about 0.1 to 0.25% of glycyrrhizin could be isolated.
The protocol can be scaled up or scaled down depending upon the initial starting
material.
Advantages
1. The plant extract of Glycyrrhiza glabra and glycyrrhizin in the Applicant's
study facilitates the transport/uptake of various compounds including
antibiotics, antifungal and anticancer agents, vitamins and amino acids etc.
across the membrane thereby increasing the absorption or bioavailability of
various biological systems including bacteria, animal and plant cells. This thus
has immense importance for absorption of the drugs, Pharmaceuticals,
nutraceutical and other related compounds and ions by the cells and hence
huge potential of being used as drug/molecule facilitators.
2. The present invention enlarges the scope and use of the natural glycoside
Glycyrrhizin in therapeutical and nutraceutical application.
3. Glycyrrhizin is nontoxic to humans, animals or plants, is effective at very low
concentrations in a combination, easy to formulate and enhances the
uptake/absorption and activity of drug molecules. Thus the novel use of this
compound as described in the present invention is safe and effective for
bioenhancement of activity of drugs.
23
4. Glycyrrhizin by its presence will not exert any selection pressure for the
mutants to emerge resistant against them and reduce the dosage of antibiotics
or drugs so that their ill effects are reduced.

Claims
1. A composition comprising an effective; amount of an. extract or compound obtained;
from the plant Glycyrrliiza glabra and. usefui.as a bio-enhancer anicf'bioavaHability
facilitator together with a therapeutically effective amount of one or more 'antibiotics,
anti-infective agents or anti-cancer agents.
2. A composition as claimed in claim 1 containing an extra'ci of leaves, siem, roots of
stolon of Glycyrrhiza glabra, glycyrchizic acid or glycyrrhizin, or a combination
thereof. ;
3. A composition as claimed in'claim 1. wherein glycyrrhrzin and glycyrrhteic acid are
obtained from the plant Glycyrrhiza glabra.
4. A composition as claimed in claim I, wherein the' extract of roots Of Glycyrrhiza
glakra. is licorice. • . ;
5. A composition as claimed in claim 1, wherein the anti-infective agents are selected
from ami -bacterial, anti-fungal and anti-tuberculosis agents.
6. A composition as claimed in claim 1, wherein the concentration of glycyrrliizih
ranges from 0.05 to SOwcight % of the anti-bacterial compounds.
7. A composition as claimed in claim 1, wherein the concentration of glycyrrliiz'.in'
ranges from 0.25 to 20weight % of the anti-fungal' agents.
8. A composition as claimed in claim 5, wherein die-anti-bacterial, agents ure selected
from the group consisting of p-lactams, macrolides, quitiolones, fluoro-quinblones,
aminoglycosides, glycopeptides, rifamycins, rotate inhibitors, tetracyclines. anti-
tuberculosis agents or microbicidal compounds. • .
9. A composition as claimed in claim 9, wherein" the quinolones and fluoro-quinolones
are selected from the group consisting of naUdixic acid,inorfloxacin, ciproflaxacii>,.
sparfloxacin or similar otiicr compounds.

10. A composition as claimed in claim 9, wherein the ß-Lactams are selected from the group comprising penicillin, flucloxacillin, cloxacukkub, methicillin, ceptialosporlns or carbapenenms.
1J.A composition as claimed in. claim 9, wherein; the macrolides are splected from erythromycih, clarithromycin;, azithromycin:or other similar compounds.
12. A composition as claimed in claim 9, wherein the aminoglycosides are selected from
the group consisting of streptomycin, gentamicin, amikactn or similar compounds. •
13. A composition as claimed in claim 9, wherein the glycopeptides are selected from
varicomycin, teicoplahin or other similar compounds.
14. A composition us claimed in claim '9, wherein the rit'amycins arc. rif'ampicin or similar
compounds.
15. A composition as claimed in claim 9, wherein the folate inhibitors are- selected from
trimethoprim, sulphonamides or similar compounds.
16. A composition as claimed in claim 9, wherein the mi'crbbiocidal compounds tut
selected from slreptogramins and oxazolidinones.
17. A composition as claimed in claim 5, wherein the anti-tuberculosis• agents are
selected from the group comprising isoniazid,' pyrazmamide, ethambutol or similar
compounds.'
18. A composition as claimed in claim 5,'wherein the anti-fungal agents are selected from
the group consisting of polyene, triazole, imidazole, clolrimaziolc or fungicidal compounds.
19. A composition as claimed in claim 19, wherein'the'polyene is selected from nystatin
or similar compounds.
20. A composition as claimed. in claim 19, wherein the iriazole is selected from
fluconazole, itraconazole and or similar compounds.
21. A composition as claimed in claim 19, wherein the imidazole is selected from
ketoconazole or similar compounds.

22. A composition as claimed in claim 19, wherein the fungicidal'compound: is selected from griseofulvjn or terbinafine.
23'. A composition as claimed in claim I, wherein; the anti-cancer, agent is selected from
ihe group consisting of Paclitaxel (Taxol), DocetaxeJ- (Takotere), . Vinblasiine
(Velban), Vincristine (Oncovin) or Vinorelbine (Navelbirie). . .
24. A composition as claimed in claim I, wherein the concentration-of glycyrrhizin
ranges from 10 to 10,000 folds of the. weight of the anti-cancercompound. ;•
25. A composition as .claimed in claim 1, wherein licorice preparation from1 .one gram of
dried root is equivalent to 40 mg of glycyrrhizin.
26. A method for enhancing the bio-availability of anti-infective agents or anti-cancer
using herbal extracts or compounds derived from the exrtract across biological
membranes in living systems; said method comprising the-steps-of administering to a
subject in need thereof, a composition comprising an effective amount of ihe.
bioenhancer obtained from the plant Glycyrrhiza glabra, and any one of anti-infective
agents or, anti-cancer agents.. . ...
27. A method as claimed in claim 28, wherein, the bio-enhancer is selected .from the
extract of Glycyrrhiza glabra, glycyrrhizin, glycyrrhizic acid or-combinations thereof.
28. A method as claimed in claim 28, wherein the bioenhancer is obtained from the roots,
stem, stolon or leaves of the p[&nt-Glycyrrhiza glabra.
29. A method as claimed in claim -28, wherein the anti-infective agents are selected from .
anti-bacterial, anti-fungal, or anti-tuberculosis agents.
30. A method as claimed in claim. 31, wherein the anti-bacterial agents are selected .from the
group comprising P-lactams, macrolides, .quiriolones, fluoro-quinolones, aminbglycbsides.
glycopeptides, rifamycins, folate inhibitors, litracyclines:. aati-tuberculosis agents or'
microbicidal compounds.
31. A method as claimed in claim 31, wherein-concentration of glycyrrhizin ranges from
0.05 to 50 weight % of the anti-bacterial agents. • :

32. A method as claimed in cliim 32, wherein'the quinqlones and fluoroquinplones are
selected from the group consisting of nalidixic acid, riorfloxacin. ciprofloxacin,
sparfloxacin or similar other compounds. ,
33. A method as' claimed in claim 32, wherein thelp-Lactams .are selected fi-om the group
consisting of penicillin, flucloxacillin; cloxacukkub, rnethicUIiri, cephulosporins or
carbapenenms. • :
34. A method as claimed in claim 32;, wherein the macrplides are selected from
erythromycin, clarithromycin, azithromycin or other similar compounds.
35. A method as claimed in claim 32, wherein1 the' aminoglycosides-are selected from the
group consisting of streptomycin, gentamicin, a-mjkaein or:sirnttar compounds.
36. A method as claimed in claim 32, wherein the glycopeptides are selected from
vancomycin, teicoplanin or other similar compounds. . . •
37. A method as claimed in claim ,32, wherein the rifamycins .are rifumpicin or similar
compounds. .
38. A method as claimed in claim 32, wherein the anti-tubierQulosik agents selected from
the group consisting of isoniazid, pyrazinamide, etharhbuiol .or other similar
compounds. . . •'•
39. A method as claimed in claim 31, wherein concentration ofgiycyrrhizin ranges- from
0,25 to 20 weight % of the anti-fungal agents.. .
40. A method as claimed in claim 31, wherein the anri-fungaJ agenris selected from the
group consisting of polyene, triazole; imidazole, clotriihazob or other fungiciclal
compounds. • ' .. ' : •
41. A method as claimed in claim 28, wherein, the anti-cancer agent is selected from the
group consisting of PacJitaxel (Tiixol), Dbcetaxe'J'(TaxoteTe); .Vinblastine (Velban),
Vincristine (Oncovin) or Vinorelbirie (Navelbine):.

42. A method as claimed in cfaim 31, wherein the concentration of gtycyrrhiiin ranges ;•
from 10 to 10,000 folds of the weight of the anti-cancer agentl ;
43. A method as claimed'in!-claim 27, wherein- administration !of the composition-
circumvents the side effects of chemotherapy by substantially reducing the dosages .of
therapeutic agents comprising of anti-bacterial, anti-fungal and anti-cancerous agents
wherein, the side effects of those agents can be bone marrow suppression, liwer and
kidney toxicity, pulmonary scarring, high.- fevers, .skin reactions, Nausea and
vomiting, hair loss, skin rash, mouth sores, diarrhoea, loss of appetite and confusion,
lethargy and ambulation problems (ataxia)i anaphylactic reactions (consisting «F fast
heart rate, wheezing, lowered blood pressure and facial edema, cerebellar
dysfunction (consists of slurring of speech, -walking problems, as we'll as eye rtiotio'n
problems), loss of periods .(menses), ovarian failure can occur, painful soft-1 tissue
ulcers, cardiotoxicity, hypersensitivity reactions, abnormal blood pressure, sweating,,
bleeding, shortness of breath and many related problems
44. A method as claimed in claim 27, wherein,-the bioenhancer improves the uptake of
anti-infectives, or anticancer agents when glycyrrhizin is provided prior .to the
treatment with these molecules.
45. Use of the-extract or the compound obtained from the plant.Gfycjvr/ij^a-g/aArfl, -as a
bioenhancer and bioavailability and facilitator drugs molecules selected from anti-
infective and anti-cancer agents.

Documents:

00226-delnp-2003-abstract.pdf

00226-delnp-2003-claims.pdf

00226-delnp-2003-correspondence-others.pdf

00226-delnp-2003-description (complete)-19-05-2008.pdf

00226-delnp-2003-description (complete)-22-05-2008.pdf

00226-delnp-2003-description (complete).pdf

00226-delnp-2003-form-1.pdf

00226-delnp-2003-form-18.pdf

00226-delnp-2003-form-2.pdf

00226-delnp-2003-form-3.pdf

226-DELNP-2003-Abstract-(05-03-2008).pdf

226-DELNP-2003-Abstract-19-05-2008.pdf

226-DELNP-2003-Abstract-22-05-2008.pdf

226-DELNP-2003-Abstract-27-05-2008.pdf

226-DELNP-2003-Claims-(05-03-2008).pdf

226-DELNP-2003-Claims-19-05-2008.pdf

226-DELNP-2003-Claims-22-05-2008.pdf

226-DELNP-2003-Correspondence-Others-(05-03-2008).pdf

226-DELNP-2003-Correspondence-Others-(11-03-2008).pdf

226-DELNP-2003-Correspondence-Others-19-05-2008.pdf

226-DELNP-2003-Correspondence-Others-22-05-2008.pdf

226-DELNP-2003-Correspondence-Others-27-05-2008.pdf

226-DELNP-2003-Form-1-(05-03-2008).pdf

226-DELNP-2003-Form-1-22-05-2008.pdf

226-DELNP-2003-Form-2-19-05-2008.pdf

226-DELNP-2003-Form-3-(05-03-2008).pdf

226-DELNP-2003-PCT-210-(05-03-2008).pdf

226-DELNP-2003-PCT-409-22-05-2008.pdf

226-DELNP-2003-Petition-137-(11-03-2008).pdf

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Patent Number

220700

Indian Patent Application Number

00226/DELNP/2003

PG Journal Number

30/2008

Publication Date

25-Jul-2008

Grant Date

02-Jun-2008

Date of Filing

25-Feb-2003

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

Inventors:

#	Inventor's Name	Inventor's Address
1	SHASANY AJIT KUMAR	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
2	SINGH MONIKA	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
3	AWASTHI SOUMYA	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
4	KUMAR SUSHIL	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
5	ARYA JAI SHANKAR	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
6	SHASANY AJIT KUMAR	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
7	SINGH MONIKA	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
8	AWASTHI SOUMYA	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
9	KUMAR SUSHIL	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS
10	ARYA JAI SHANKAR	CENTRAL INSTITUTE OF MEDICANAL AND AROMATIC PLANTS

PCT International Classification Number

A61K

PCT International Application Number

PCT/IN00/0083

PCT International Filing date

2000-08-31

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	PCT/IN00/00083	2000-08-31	India