The present invention relates to A novel compound, difurocurcumenonol and a process for the preparation/isolation thereof from the mangoginger (Curcuma amada Roxb.) rhizome" The present invention particularly relates to the isolation and characterization of difurocumenonol, a novel antibacterial and antioxidant compound hitherto unreported from the chloroform extract of mangoginger rhizome by chromatographic technique.
Curcuma amada Roxb., commonly called mango ginger, belongs to the family Zingiberaceae. It is a perennial herb with rhizomes having characteristic odour of raw mangoes. The main use of mango ginger is in the manufacture of pickles. The rhizomes find extensive use in the indigenous system of medicine. According to Ayurveda, the ancient herbal medicine of India, the rhizome is appetizer, alexteric, antipyretic, aphrodisiac and laxative. It is useful in biliousness, itching, skin diseases, bronchitis, asthma, hiccough and inflammation due to injuries. According to Unani systems of medicine, the root has a bitter sharp taste. It is diuretic, maturant, emollient, expectorant, antipyretic and appetizer. It is useful in inflammation troubles in the mouth, ear, gleet, and ulcers on the male sex organs, scabies, lumbago and stomatitis (N.B.Shankaracharya, 1982).
Why natural antimicrobials and antioxidants? WHO reported 80 % of the world population chiefly rely on traditional medicine and a major part of the traditional therapies involve the use of plant extracts and their active constituents. Due to the indiscriminate use of antimicrobial drugs, the microbes have developed resistance to antibiotics. This has created immense clinical problems in treatment of infectious diseases. In addition to this problem, antibiotics are sometimes associated with adverse effects on host, which include hypersensitivity, depletion of beneficial gut & mucosal microbes, immunosuppressions and allergic reactions. There is an increased interest to identify antibacterial molecules from natural source, which has preventive or curative properties with less or no adverse effect on health. In this regard fruit and vegetables are identified as a rich source of phytochemicals or nutraceuticals. This is one such successful attempt to identify and characterize a natural and novel antibacterial from mango ginger, an underutilized endemic tuber crop of India.
Reference may be made to Gurdeep Singh, Om Prakash Singh, M.P. de Lampanosa and C. Catalan, Indian perfumer (2003): 47 (2), 143-146, wherein they have studied the chemical composition of mangoginger rhizome oil. The draw back is that, there are no reports on the
antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Gurdeep Singh, Om Prakash Singh and Sumitra Maurya, Prog. Crystal Growth and Charect.(2QQ2): 45, 75-81, wherein they have investigated the chemical composition and biocidal activity of essential oils from different Curcuma species. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Mujumdar, A. M, Naik, D. G., Dandge, C. N., Puntambekar, H. M. Ind. J. Pharmacology (2000): 32, 375-377, wherein they have reported the anti inflammatory activity of ethanol extract of Curcuma amada Roxb. in albino rats. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Srinivasan, M. R. and Chandrasekhara, N., Nutr.Res. (1993): 13, 1183-1190, wherein they have reported the effect of mangoginger (Curcuma amada Roxb.) rhizome on Triton WR-1338 induced hyperlipidemia and plasma lipases activity in the rats. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Srinivasan, M. R. and Chandrasekhara, N., J. Food Sci. Tech. (1992): 29, 130-132, wherein they have reported the effect of mangoginger (Curcuma amada Roxb.) rhizome on lipid status in normal and hypertriglyceridemic rats. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Alapati Srinivasa Rao, Bandaru Rajanikanth and Ramachandran Seshadri, J. Agri. Food. Chem. (1989): 37, 740-743, wherein they have reported volatile aroma components of Curcuma amada Roxb. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of
difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Achyut, S. Gholap and Chriranjib Bondyopadhyay. J. Agri. Food Chem. (1984): 32, 57-59, wherein they have characterized mango like aroma in Curcuma amada Roxb. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to S. Sumathi and T.N. Pattabiraman, Indan J. Biochem. Biophys. (1975): 12, 383-385, wherein they have studied the protease inhibitors from tubers and bubs. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Mahendra Kumar Jain and Rajendra Kumar Mishra... Ind. J. Chem, (1964): 2, 39, wherein they have reported the chemical composition of acetone extract. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
Reference may be made to Dutt, S. and Tayal, J. N., Ind. Soap J. (1941): 7, 200-205, wherein they have reported chemical examination of essential oils derived from the rhizomes of Curcuma amada Roxb. The draw back is that, there are no reports on the antibacterial activity of chloroform extract and isolation of difurocumenonol, a novel antibacterial and antioxidant compound from chloroform extract of mango ginger rhizome.
The main objective of the present invention is to provide a process for the isolation of difurocumenonol, a novel antibacterial and antioxidant compound from the mango ginger (Curcuma amada Roxb.) rhizome powder, which obviates the drawbacks as detailed above.
Another objective of the present invention is to use the chloroform extract of the mango ginger rhizome powder as a source of antibacterial and antioxidant principle.
Still another objective of the present invention is to isolate a bioactive principle from the chloroform extract by repeated silica gel column chromatography using different solvent gradients from non-polar to polar in succession.
Yet another objective of the present invention is to characterize the isolated bioactive principle by UV, IR, LC-MS and 2D-NMR analysis.
Still another objective of the present invention is to determine the antibacterial activity of the isolated difurocumenonol against Bacillus cereus, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus, Streptococcus faecalis and Yersinia enterocolitica by agar-well diffusion method.
Yet another objective of the present invention is to determine the antioxidant activity of the isolated difurocumenonol by DPPH radical scavenging activity, superoxide radical scavenging activity and lipid peroxidation inhibitory activity.
Still another objective of the present invention is to determine the solubility of the isolated difurocumenonol in solvents like chloroform, isopropyl alcohol, n-butyl alcohol, ethyl acetate, acetone, methanol, ethanol, water and DMSO. Accordingly, the present invention provides novel compound, difurocurcumenonol [13, 15, 23, 25-tetrahydroxy-l, 5, 10, 14, 17, 21-hexamethyl-7, 19-dioxahexacyclo (13.9.1.0 2' 14.0 °.0 I6'24.0 18'22) pentacosa-4 (8), 5, 11, 16 (24), 18 (22), 20-hexaen-3-one], the said compound having the following structure, structure # 01:
Structure # 01
In an embodiment the novel compound, difurocurcumenonol is isolated from mangoginger (Curcuma amada Roxb.) rhizome.
In an embodiment of the present invention the novel compound, difurocurcumenonol has the following spectral characteristic data, given hereunder:
a) UV: (chloroform): 242nm
b) IR: (DMSO): 3442 cm"1 (OH stretching), 2995 cm"1 (Alkyl CH stretching),
1659 cm"1 (C = O stretching), 1437 cm"1 and 1055 cm"1
c) LC-MS:498M'2'M/e279and219
d) 2D-HMQCT NMR a:
13C NMR (125 MHz, CDC13): 5 19.0(1-CH3), 14.5 (5-CH3), 21.8(10-CH3), 14.4(14-CH3), 20.2(17-CH3), 14.4(21-CH3), 38.3(9-CH2), 55.5(2-CH), 144.0(6-CH), 29.5(10-CH), 136.1(1 1-CH), 1 15.5(12-CH), 56.5(13-CH), 38.0(17-CH), 144.1(20-CH), 50.8(23-CH), 65.5(25-CH), 197.8(3C=0), 43.1(C-1), 42.0(C-14), 97.2(C-15), 142.1(C-18), 106.2(C-22)and 116.2(C-24).
1 H NMR (500 MHz, CDC13): 8 1.48(1-H), 0.65(5-H), 0.83(10-H), 0.75(14-H), 2.10(17-H), 0.72(21-H), 2.40(9-H), 1.15(2-H), 7.05(6-H), 1.25(10-H), 4.45(1 1-H), 6.75(12-H), 3.95(13-
H), 2.42(17-H), 7.10(20-H), 3.50(23-H) and 4.55(25-H), wherein 'a' stand for indicating that some of the assignments are interchangeable
In an embodiment of the present invention the novel compound, difurocurcumenonol, has radical scavenging(antioxidant) activity and antibacterial (antibiotic) activity.
In an embodiment of the present invention the novel compound, difurocurcumenonol, has radical scavenging (antioxidant) activity by DPPH radical scavenging activity at an ICso of 180u£/ml
In an embodiment of the present invention the novel compound, difurocurcumenonol, has superoxide radical scavenging(antioxidant) activity at an ICso value of about 114)j.g/ml.
In an embodiment of the present invention the novel compound, difurocurcumenonol, has lipid peroxidation inhibitory(antioxidant) activity with an IC.so alue of about 61u.g/ml.
In an embodiment of the present invention the novel compound, difurocurcumenonol, has antibacterial (antibiotic) activity, against bacteria selected from the group consisting of Bacillus cereus, Enterobacter aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella lyphi, Streptococcus faecalis and Yersinia enterocolilica, with an inhibition zone of 18mm, 13mm, llmm, llmm, 17mm, 16mm, 12mm, 15mm and 12mm respectively.
In an embodiment of the present invention the novel compound, difurocurcumenonol, has following solubility characteristics:
(Table Removed)
An embodiment of the present invention provides a pharmaceutical composition, useful effecting radical scavenging(antioxidant) activity in subjects mammals, comprising a novel compound, difurocurcumenonol, optionally along with pharmaceutically accepted salts, diluents and other excipients selected from the group consisting of carriers, colorants, flow modifiers and stabilizers.
Another embodiment of the present invention provides a pharmaceutical composition, useful effecting antibacterial (antibiotic) activity in subjects mammals, comprising a novel compound , difurocurcumenonol ,optionally along with pharmaceutically accepted salts, diluents and other excipients selected from the group consisting of carriers, colorants, flow modifiers and stabilizers.
Another embodiment of the present invention provides a pharmaceutical composition of novel compound, difurocurcumenonol, wherein said composition is useful for effecting antibacterial (antibiotic) activity particularly against bacteria selected from the group consisting of Bacillus cereus, Enterobacter aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus faecalis and Yersinia enterocolitica
Another embodiment of the present invention provides a pharmaceutical composition of novel compound, difurocurcumenonol, wherein said composition is used in the form of oral, parental, buccal and ocular administration.
Another embodiment of the present invention provides a process for the preparation/isolation of a novel compound, difurocurcumenonol, having the following structure, structure # 01:
structure # 0 1
from the mangoginger (Curcuma amada Roxb.) rhizome, the said process comprises the steps of:
a) washing and slicing of mango ginger rhizomes by a known method;
b) drying of sliced mango ginger rhizomes at a temperature in the range of 40
deg. C to 55 deg. C for a period raging from 36 to 48 hours in order to
obtain a dried mango ginger slices;
c) powdering dried mango ginger slices obtained in step (b) in order to obtain
mango ginger powder having mesh size in the range of 60 to 80;
d) extracting the mango ginger powder obtained in step (c) using organic
solvents hexane followed by chloroform for a period ranging from 24 to
48 hours in order to obtain chloroform extract; AND
e) conducting repeatedly silica gel (60- 200 mesh) column chromatography followed by elution using following solvents:

Solvents

Ratio (v/v)

hexane and different 95: 5 to 5: 95
gradients of hexane:
chloroform
chloroform: ethyl acetate 95: 5 to 5: 95
ethyl acetate: acetone 95: 5 to 5: 95
acetone: methanol 95: 5 to 5: 95
in order to isolate the novel compound, difurocurcumenonol.
Another embodiment of the present invention provides a process for the preparation/isolation of a novel compound, difurocurcumenonol, wherein the novel compound obtained has radical scavenging(antioxidant) activity and antibacterial (antibiotic) activity.
Another embodiment of the present invention provides a process for the preparation/isolation of a novel compound, difurocurcumenonol, wherein the novel compound obtained has radical scavenging (antioxidant) activity by DPPH radical scavenging activity at an ICsoof 180jig/ml
Another embodiment of the present invention provides a process for the preparation/isolation of a novel compound, difurocurcumenonol, wherein the novel compound obtained has superoxide radical scavenging(antioxidant) activity at an ICjo value of about 114ug/ml.
Another embodiment of the present invention provides a process for the preparation/isolation of a novel compound, difurocurcumenonol, wherein the novel compound obtained has lipid peroxidation inhibitory(antioxidant) activity with an ICso alue of about 6 lug/ml.
Another embodiment of the present invention provides a process for the preparation/isolation of a novel compound, difurocurcumenonol, wherein the novel compound obtained has antibacterial (antibiotic) activity, against bacteria selected from the group consisting of Bacillus cereus, Enterobacter aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus faecalis and Yersinia enterocolitica, with an inhibition zone of 18mm, 13mm, 11mm, 11mm, 17mm, 16mm, 12mm, 15mm and 12mm respectively.
In yet another embodiment of the present invention, the isolated difurocumenonol is soluble in chloroform, isopropyl alcohol, n-butyl alcohol, ethyl acetate, acetone, methanol, ethanol and DMSO and insoluble in water.
A typical procedure consists of collection of mangoginger rhizome, slicing and drying in hot air oven at 40 °C to 55 °C for a period of 36 hours to 48 hours, powdering in apex grinder to 60 mesh to 80 mesh after which 100 g of mangoginger rhizome powder was extracted with solvents such as hexane followed by chloroform and solvent was removed by distillation to obtain 11.6 and 3.8 g of crude extracts respectively. The chloroform extract was tested for the antibacterial activity by agar-well diffusion method against Bacillus cereus, Enterobacter aerogenes, Escherichia coll, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus, Streptococcus faecalis and Yersinia enterocolitica and showed potential antibacterial activity against Bacillus cereus, Micrococcus luteus and Streptococcus faecalis with an inhibition zone of 14 mm, 13mm and 13 mm respectively.
About 14g of chloroform extract was mixed with equal quantity of silica gel (60-120 mesh) and subjected to silica gel column chromatography and eluted with hexane, and linear gradient of hexane: chloroform (75: 25 to 0: 100 v/v), chloroform: ethyl acetate (75: 25 to 0: 100 v/v), ethyl acetate: acetone (75: 25 to 0: 100 v/v) and acetone: methanol (75: 25 to 0: 100 v/v). About 82 fractions measuring 100 ml were collected and concentrated by using rotavapour. All the fractions were analyzed by TLC and pooled into 5 groups (Fraction No. 1-5) and tested for the antibacterial activity by agar-well diffusion assay. The fraction No. 3 showed potential antibacterial activity (maximum inhibition zone) hence, it was selected for
further purification. About 3.5g of bioactive fraction No. 3 was subjected to column (450 x 20) chromatography using silica gel (60-120 mesh). The column was eluted stepwise with hexane, and linear gradient of hexane: chloroform (90: 10 to 0: 100 v/v), chloroform: ethyl acetate (90: 10 to 0: 100 v/v), ethyl acetate: acetone (90: 10 to 0: 100 v/v) and acetone: methanol (90: 10 to 0: 100 v/v). About 48 fractions of 50 ml each were collected, analyzed by TLC and pooled into four fractions (Fraction No. T- 4'). These fractions were tested for the antibacterial activity by agar-well diffusion assay. The Fraction No. 2' showed potential antibacterial activity (maximum inhibition zone) and was selected for further purification. About 800 mg of bioactive fraction No. 2' was subjected to column (600 x 15) chromatography using silica gel (100-200 mesh). The column was eluted stepwise with linear gradient of hexane: chloroform (90: 10 to 0: 100 v/v), chloroform: ethyl acetate (95: 05 to 0: 100 v/v), ethyl acetate: acetone (90: 05 to 0: 100 v/v). About 28 fractions of 25 ml each were collected and pooled into three fractions (1" - 3") by analyzing TLC profile. The 2" fraction showed a single spot in TLC profile, and reverse phase HPLC on C-18 column with water and methanol (40:60 v/v) as mobile phase showed single peak.
The purified fraction No. 2" was tested for antibacterial activity and showed inhibition against Bacillus cereus, Enterobacter aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luleus, Pscudomonas aeruginosa, Salmonella typhi, Streptococcus faecalis and Yersinia enlerocolitica with an inhibition zone of 18mm, 13mm, llmm, llmm, 17mm, 16mm, 12mm, 15mm and 12mm respectively.
The purified fraction No.2" was tested for antioxidant activity and showed DPPH radical scavenging activity, superoxide radical scavenging activity and lipid peroxidation inhibitory activity with an ICso of 180(ag/ml, 114|ag/ml and 61u,g/ml respectively.
A flow chart for the isolation of difurocumenonol
Selection of mature healthy mangoginger rhizomes
I
Washing, drying in hot air oven at 40 °C to 55 °C for a period of 36 hours to 48 hours
Powdering to 60 mesh to 80 mesh
I
Solvent extraction using hexane followed by chloroform
I
Chloroform extract
I
Silica gel (60-120 mesh) column chromatography I (Linear gradients of Hexane, chloroform, ethyl acetate, acetone and methanol)
I
Bioactive enriched fraction
I
Silica gel (60-120 mesh) column chromatography II (Linear gradients of Hexane, chloroform, ethyl acetate, acetone and methanol)
I
Bioactive enriched fraction
I
Silica gel (100-200 mesh) column chromatography III (Linear gradients of chloroform, ethyl acetate, acetone and methanol)
I
Difurocumenonol, a novel compound
The following examples are given by way of illustration of the present invention and therefore should not be constructed to limit the scope of the present invention.
EXAMPLE 1
The mature healthy and fresh mangoginger rhizomes were procured, washed, sliced and dried at 48 °C in hot air oven for 36 hours. Dried slices were powdered to 60 mesh in an apex grinder. About 100 g of dry mangoginger powder was extracted with hexane followed by chloroform and the solvent was removed by distillation to obtain 11.6 g and 3.8 g of crude extracts respectively. About 14 g of chloroform extract was subjected to column
chromatography using silica gel (60-120 mesh) and eluted with hexane, and linear gradient of hexane: chloroform (75: 25 to 0: 100 v/v), chloroform: ethyl acetate (75: 25 to 0: 100 v/v), ethyl acetate: acetone (75: 25 to 0: 100 v/v) and acetone: methanol (75: 25 to 0: 100 v/v). The fractions were analyzed by TLC and pooled into 5 groups (Fraction No. 1-5) and tested for the antibacterial activity by agar-well diffusion assay. The active fraction 3 was subjected to further purification using silica gel (60-120 mesh) column chromatography and eluted stepwise with hexane, and linear gradient of hexane: chloroform (90: 10 to 0: 100 v/v), chloroform: ethyl acetate (90: 10 to 0: 100 v/v), ethyl acetate: acetone (90: 10 to 0: 100 v/v) and acetone: methanol (90: 10 to 0: 100 v/v). Fractions were collected, analyzed by TLC and pooled into four fractions. The active fraction No.2"was subjected to silica gel (100-200 mesh) column chromatography and eluted stepwise with linear gradient of hexane: chloroform (90: 10 to 0: 100 v/v), chloroform: ethyl acetate (95: 05 to 0: 100 v/v), ethyl acetate: acetone (90: 05 to 0: 100 v/v). The fractions were collected and pooled into three fractions (l'\ 2" and 3") by TLC profile. The 2" fraction showed a single spot in TLC profile and showed single peak in reverse phase HPLC on C-18 column with water and methanol (40:60 v/v) as a mobile phase.
EXAMPLE 2
UV visible spectrum was recorded on Shimadzu UV-160A instrument at room temperature. Region from 200-800 nm was employed for scanning. About 2mg the isolated bioactive compound dissolved in 20ml was used for recording the spectrum. IR spectrum was recorded on Perkin-Elmer Spectrophotometer (spectrum 2000) at room temperature. Region from 4000-400cm"' was employed for scanning. About 3mg of the isolated bioactive compound dissolved in 1ml of DMSO was used for recording the spectrum. A 2DHMQC1 NMR spectrum was recorded on Bruker DRX 500 NMR instrument operating at 500 MHz for 'H and 125 MHz for I3C at room temperature. Region from 0-12 ppm for 'H and 0-220 ppm for 13C was employed for scanning; and signals were referred to TMS. About 30mg the isolated bioactive compound dissolved in 2ml CDCb was used for recording spectra. The isolated bioactive compound was subjected to spectral characterization and the data are given as follows:
UV: (chloroform): 242nm
IR: (DMSO): 3442 cm"1 (OH stretching), 2995 cm'1 (Alkyl CH stretching),
1659 cm"1 (C = O stretching), 1437 cm'1 and 1055 cm"1 LC-MS: 498 M42' M/e 279 and 219
2D-HMQCT NMRa:
I3C NMR (125 MHz, CDC13): 5 19.0(1-CH3), 14.5 (5-CH3), 21.8(10-CH3), 14.4(14-CH3), 20.2(17-CH3), 14.4(21-CH3), 38.3(9-CH2), 55.5(2-CH), 144.0(6-CH), 29.5(10-CH), 136.1(11-CH), 115.5(12-CH), 56.5(13-CH), 38.0(17-CH), 144.1(20-CH), 50.8(23-CH), 65.5(25-CH), 197.8(3C=0), 43.1(C-1), 42.0(C-14), 97.2(C-15), 142.1(C-18), 106.2(C-22)and 116.2(C-24).
1 H NMR (500 MHz, CDC13): 5 1.48(1-H), 0.65(5-H), 0.83(10-H), 0.75(14-11), 2.10(17-H), 0.72(21-H), 2.40(9-H), 1.15(2-H), 7.05(6-H), 1.25(10-H), 4.45(11-H), 6.75(12-H), 3.95(13-H), 2.42(17-H), 7.10(20-H), 3.50(23-H) and 4.55(25-H). a- some of the assignments are interchangeable
The compound exhibited UV maxima at 242 nm indicating the presence of double bonds. IR spectral data showed O-H stretching at 3442 cm"1, alkyl stretching at 2995 cm"1 and carbonyl stretching at 1067 cm"1 indicating the presence of OH and carbonyl groups. A detailed Two Dimensional Heteronuclear Quantum Coherence Transfer NMR Spectra was recorded along with straight one-dimensional *H and 13C NMR spectra, which gave clear indication of the structure of the compound. The compound showed as many as three CH3 groups as singlets. Other three CH3 signals were observed as doublets indicated that they were attached to CH carbons. The corresponding 13C signals for the six CH3 groups were also observed. The region between l.lppm and 2.4ppm indicated quite a lot of CH and CH2 signals. It was difficult to resolve the multiplicities. The region between 3.5ppm and 4.5 ppm showed (in 'H spectra) CH signals attached to OH groups. Corresponding I3C signals for these CH groups were also observed. A carbonyl signals at 197.8 ppm was observed. Some quaternary carbons and aromatic carbons in the region 97.2 ppm to 116.2 ppm were observed indicating the presence of aromatic carbons. Olefmic carbon signals were observed at 136.1 ppm. Around 144.1 ppm some aromatic signals were also observed, which indicated that they might come from a furan ring. Corresponding 'H signals were also recorded. LC-MS data showed a parent molecular ion peak at 498 and M/e peaks at 219 and 279. All these spectral characters indicated that the molecule resembled difurocumenone with slight variation with
respect to substituted groups. Hence, based on the spectral data we deduced the structure to be a probable precursor of difurocumenone and has designated it as a difurocumenonol {13, 15, 23, 25-tetrahydroxy-l, 5, 10, 14, 17, 21-hexamethyl-7, 19-dioxahexacyclo (13.9.1.0 2' 14.0 4,8 0 16,24 0 18,22) pentacosa_4 (^ 5? j^ 16 (24), 18 (22), 20-hexaen-3-one}.
Structure of difurocumenonol
EXAMPLE 3
The isolated bioactive compound was tested for the antioxidant activity by DPPH radical scavenging assay. The sample was dissolved in ethanol and different concentrations were mixed with 0.8 ml of Tris-HCl buffer (pH 7.4), to which 1 ml of DPPH (SOO^iM in ethanol) was added. The mixture was shaken and left to stand for 30 minutes. Absorbance of the resulting solution was measured at 517 nm in a UV-visible spectrophotometer. ICjo represents the 50 % the radicals scavenged by the test sample. The isolated bioactive compound showed DPPH radical scavenging activity with an ICso of 180|j.g/ml.
EXAMPLE 4
The isolated bioactive compound was tested for super oxide radical scavenging activity. The superoxide radicals were generated in 1 ml of Tris-HCl buffer (0.02 M, pH 8.3) containing 0.1 mM NADH, 0.1 mM NBT, lOuM PMS and purified compound. The colour
reaction of superoxide radicals and NBT was detected at 560nm in UV-visible spectrophotometer. The isolated bioactive compound showed superoxide radical scavenging activity with an ICjo 114(j,g/ml.
EXAMPLE 5
The isolated bioactive compound was tested for lipid peroxidation inhibitory activity. Egg lecithin (3mg/ml Phosphate buffer, pH 7.4) was sonicated and different concentration of the isolated bioactive compound was added to 1 ml of liposome mixture, lipid peroxidation was induced by adding lOul of FeC^ (400mM), and lOul of L-ascorbic acid (200mM). After incubation for 1 hour at 37 ° C, the reaction was stopped by adding 2 ml of 0.25 N HC1 containing 15% TCA and 0.375% TBA and the reaction mixture was boiled for 15 minutes, cooled and centrifuged. The absorbance of the supernatant was recorded at 532 nm. The isolated bioactive compound showed lipid peroxidation inhibitory activity with an ICso of 61u,g/ml.
EXAMPLE 6
The antibacterial activity of isolated bioactive compound was tested by agar-well diffusion method. The antibacterial activity was tested by seeding 100(4,1 of microbial suspension (107 CFU/ml) to the Petri plates containing nutrient agar. Wells (8 mm diameter) were made in these plates using a sterile cork borer. About 75u.l of isolated bioactive compound (2mg/ml) was added into the wells using sterilized dropping pipettes and diffusion was allowed at room temperature for 2 h. The plates were incubated at 37 °C for 24 h. Respective proper controls of solvent extracts were maintained. Diameter of the inhibition zones was recorded. The isolated bioactive compound showed antibacterial activity against Bacillus cereus, Enterobacter aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus faecalis and Yersinia enterocolitica with an inhibition zone of 18mm, 13mm, llmm, 11mm, 17mm, 16mm, 12mm, 15mm and 12mm respectively.
EXAMPLE 7
The solubility of difurocumenonol was tested by dissolving about 1 mg of pure compound in different solvents like chloroform, isopropyl alcohol, n-butyl alcohol, ethyl acetate, acetone, methanol, ethanol, water and DMSO. Difurocumenonol is soluble in all the solvents tested except water, in which it is not soluble. Difurocumenonol was greenish yellow in colour when dissolved in DMSO.
Novelties of the present invention are:
1. Crude chloroform extract of mango ginger rhizome can be used as an antibacterial
principle.
2. Difurocumenonol isolated from mango ginger rhizome is a new bioactive molecule,
which has not been reported so far.
3. Difurocumenonol can be used as an antibacterial principle.
4. Difurocumenonol can be used as antioxidant principle.
5. Difurocumenonol is insoluble in water and soluble in chloroform, isopropyl alcohol, n-
butyl alcohol, ethyl acetate, acetone, methanol, ethanol and DMSO.
The main advantages of the present invention are:
1. It employs a simple and inexpensive process for the isolation of difurocumenonol.
2. Difurocumenonol isolated is a new compound, which has not been reported so far.
3. Difurocumenonol was found to have antibacterial activity against Bacillus cereus,
Enterobacter aerogenes, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus
luteus, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus faecalis and Yersinia
enter ocolitica.
4. Difurocumenonol was found to have good antioxidant activity and showed DPPH radical
scavenging activity, superoxide radical scavenging activity and lipid peroxidation
inhibitory activity
5. Difurocumenonol isolated was found to be insoluble in water and soluble in chloroform,
isopropyl alcohol, n-butyl alcohol, ethyl acetate, acetone, methanol, ethanol and DMSO.

WE CLAIM:
1) A novel compound, difurocurcumenonol [13, 15, 23, 25-tetrahydroxy-l, 5, 10, 14, 17, 21-hexamethyl-7, 19-dioxahexacyclo (13.9.1.0 2' I4.0 4'8.0 I6'24.0 18'22) pentacosa-4 (8), 5, 11, 16 (24), 18 (22), 20-hexaen-3-one], having the following structure, structure # 01:
(Structure Removed)
2. The novel compound, difurocurcumenonol, as claimed in claim 1, wherein the said compound is isolated from mangoginger (Curcuma amada Roxb.) rhizome.
3. A process for the preparation of a novel compound, difurocurcumenonol, having the following structure, structure #01 and as claimed in claim 1 from the
mangoginger (Curcuma amada Roxb.) rhizome, the said process comprises the steps of:
a) washing and slicing of mango ginger rhizomes by a known method;
b) drying of sliced mango ginger rhizomes at a temperature in the range of 40 deg. C to 55 deg. C for a period raging from 36 to 48 hours in order to obtain a ried mango ginger slices;
c) powdering dried mango ginger slices obtained in step (b) in order to obtain mango ginger powder having mesh size in the range of 60 to 80;
d) extracting the mango ginger powder obtained in step (c) using organic solvents hexane followed by chloroform for a period ranging from 24 to 48 hours in order to obtain chloroform extract; and
e) conducting repeatedly silica gel (60- 200 mesh) column chromatography followed by elution using following solvents in the ratio of (v/v)
hexane and different gradients of hexane: chloroform: 95: 5 to 5: 95;
chloroform: ethyl acetate: 95: 5 to 5: 95;
ethyl acetate: acetone : 95: 5 to 5: 95;
acetone: methanol: 95: 5 to 5: 95;
in order to isolate the novel compound, difurocurcumenonol.