WO2004056444A1

WO2004056444A1 - A process for extraction of antioxidant principles from pomegranate fruit waste

Info

Publication number: WO2004056444A1
Application number: PCT/IB2002/005489
Authority: WO
Inventors: Somaradhya Mallikarjunaradhya; Anand Pandurang Kulkarni; Soundar Divakar
Original assignee: Council Of Scientific And Industrial Research
Priority date: 2002-12-19
Filing date: 2002-12-19
Publication date: 2004-07-08
Also published as: AU2002348732A1

Abstract

The present invention relates to a process for the extraction of antioxidant principles from pomegranate fruit waste viz. path ana carpellary membrane; particularly relates to the extraction of an antioxidnt principle from pomegranate fruit waste viz. pith and carpellary membrane by chromatography.

Description

A PROCESS FOR EXTRACTION OF ANTIOXIDANT PRINCIPLES FROM POMEGRANATE FRUIT WASTE.

Technical Filed The present invention relates to a process for the extraction of an antioxidant principles from pomegranate fruit waste viz. pith and carpellary membrane.

The process particularly relates to the extraction of antioxidant principle from pomegranate fruit waste viz. pith and carpellary membrane by chromatography. Background Art Bioactive compounds from plant material form the major source of designer foods or nutraceuticals or functional foods. Antioxidants form the current area of interest under functional foods or nutraceuticals. Literature survey makes an overwhelming case for the existence of a relationship between high blood level of antioxidant nutrients and lowered incidence of disease. In addition antioxidant act as food preservative by preventing rancidity of food. Synthetic antioxidants such as butylated - hydroxyanisole (BHA), butylated - hydroxytoluene (BHT) have restricted use in foods as these synthetic antioxidants at effective or at higher dosage are suspected to be carcinogenic. Therefore the importance of the search for and exploitation of natural antioxidants, especially of plant origin has greatly increased in recent years.

Reference may be made to Noda Y.; Kaneyuki T.; Mori A. and Packer L., J. Agric.

Food Chem., 2002, 50, 166-171, wherein they have reported antioxidant activities of pomegranate fruit extracts and its anthocyanidins. The drawback is that pith and carpellary membrane of pomegranate fruit are not included in the study.

Reference may be made to applicants unpublished Indian patent application No 392/DEL/2001, March 29, 2001 by Singh R.P.; Jayaprakasha G. K. and Sakariah, K.K., wherein they have described a process for the extraction of antioxidants from pomegranate peel. The drawback in this work is that pith and carpellary membrane have not been included in the process and bioactive compounds have not been identified.

Reference may be made to Gil M. I.; Tomas-Barberan F. A.; Hess-Pierce B.; Holcroft D. M. and Kader A. A., J. Agric. Food Chem. 2000, 48, 4581-4589 wherein they have reported punicalagin and punicalin from pomegranate fruit juice. The drawback is that pith and carpellary membrane of pomegranate fruit are not included in the study.

CONFIRMATION G0PY Reference may be made to Aviram M. and Dornfeld L.; United States Patent, number 6,361,807; March 26, 2002, wherein they have provided pomegranate extracts and methods of use thereof, particularly an antioxidative composition from pomegranate. The drawback in this work is that the bioactive compounds have not been identified. Reference may be made to Aviram M. and Dornfeld L.; United States Patent, number 6,375,993; April 23, 2002, wherein they have provided pomegranate extracts and methods of use thereof, particularly an antioxidative composition from pomegranate. The drawback in this work is that the bioactive compounds have not been identified.

Reference may be made to Aviram M. and Dornfeld L.; United States Patent, number 6,387,418; May 14, 2002, wherein they have provided pomegranate extracts and methods of use thereof, particularly an antioxidative composition from pomegranate. The drawback in this work is that the bioactive compounds have not been identified.

Objects of the invention

The main object of the present invention is to provide a process for the extraction of antioxidant principles from the pomegranate fruit waste viz. pith and carpellary membrane which obviates the drawbacks as detailed above. Summary of the invention

Accordingly, the present invention provides a process for the extraction of an antioxidant principle from pomegranate fruit waste viz. pith and carpellary membrane which comprises collection of pomegranate fruit waste viz. pith and carpellary membrane from healthy mature fruits, drying in hot air oven at 48°C for a period of 36 hours, powdering in apex grinder to 60 mesh after which 500-g of pomegranate pith and carpellary membrane powder was extracted with suitable solvents such as methanol and solvent was removed by distillation to obtain 310 g of crude extract which was mixed with equal quantity of silica gel (60-120 mesh) and subjected to silica gel column chromatography and eluted with chloroform : ethyl acetate : methanol (100:0:0, 30:20:50) as solvent system, fractions collected and solvent was removed from the fractions by distillation and pooled in to different groups based on thin layer chromatographic(TLC) analysis and assayed for DPPH free radical scavenging activity, the fraction which showed maximum free radical scavenging activity was labeled as active fraction I which showed DPPH free radical scavenging activity with IC₅0 of 15.74 μg/ml reaction mixture and was subjected to silica gel column chromatography for further purification and eluted with chloroform : ethyl acetate (100:0, 0:100 ) followed by ethyl acetate : methanol (100:0, 50:50) as solvent system, fractions collected and solvent was removed from the fractions by distillation and pooled in to different groups based on TLC analysis and major fraction was further purified by fractional crystallization using solvents like isopropyl alcohol, acetone, chloroform and ethyl acetate after which it was lyophilized and assayed for DPPH free radical scavenging activity, the purified fraction showed DPPH free radical scavenging activity with IC₅o of 10.86 μg/ml reaction mixture and was labeled as active fraction II and further purified by reverse phase HPLC on C- 18 column with water : methanol : acetic acid (70:25:05) as mobile phase, fractions collected and lyophilized to obtain 11 g of the said antioxidant principle which showed DPPH free radical scavenging activity with IC₅₀ of 16.66 μg/ml reaction mixture, the purity of which as checked by TLC showed a single major spot and reverse phase HPLC on C-18 column with water, methanol and acetic acid as mobile phase showed single peak.

Detailed description of the invention

Accordingly, the present invention provides a process for the extraction of antioxidant principles from matured pomegranate fruit wastes viz. pith and carpellary membrane, the said process comprising steps of: a. collecting pit and carpellary membrane of healthy matured pomegranate fruits, drying in hot air oven, grinding to obtain a powder, b. extracting the poser of step (a) with an organic solvent to obtain an extract, c. removing the solvent from the extract of step (b) to obtain under extract, d. mixing the extract of step (c) with an equal amount of silica gel (60-120 mesh), e. purifying the step (d) mixture by subjecting to silica gel chromatography, eluting with a gradient mixture of chloroform, ethyl acetate: methanol (100:0:0 to 30:20: 50) collecting pooling the identical fractions on the toxic of TLC, f. pooling fraction for antioxidant activity to obtain bioactive fraction I, g. subjecting of the active fraction 1 to silica gel column chromatograph and eluted with chloroform with a gradient mixture of chloroform (100:0, to 0:100) followed by elution with ethyl acetate: methanol (100:0 to 50:60), h. pooling the elected fraction of step (g) on the basis of TLC to obtain a major fraction, i. purifying major fraction of step (h) by fractional crystallization using an organic solvent following by lyophilization to obtain bioactive fraction II, and j. further purifying bioactive fraction II of step (i ) to obtain bioactive principle III.

In an embodiment, the drying in step (a) is carried out in hot air oven at a temperature ranging from 45°-50° C for time of 30h to 40 h.

Another embodiment, the dried material in step (a) is ground to about 60 to 80 mesh size using apex grinder. Still another embodiment, the organic solvent used in step (b) is selected from a group consisting of methanol, ethanol, isopropanol or their mixture thereof.

Yet another embodiment, the extract obtained in step (c), may be used as a source of antioxidant principle.

Yet another embodiment, the bioactive fraction I obtained in step (f), may be used as source of antioxidant-principle.

In yet another embodiment, the bioactive fraction II obtained step (i), may be used as source of antioxidant principle.

Yet another embodiment, the organic solvent used in step (i) is selected from a group consisting of isopropyl alcohol, acetone, chloroform and ethyl acetate or mixture thereof.

One more embodiment of the invention relates to free radial scavenging activity of bioactive fraction I, which has IC₅₀ value in the range of 14.79 to 16.18μg/ml.

In yet another embodiment, the free radial scavenging activity of bioactive fraction I have IC₅₀ value of 15.74 μg/ml.

Still another embodiment, the free radial scavenging activity of bioactive fraction II has IC₅₀ value in the range of 10.4 to 11.96 μg/ml

Still another embodiment, the free radial scavenging activity of bioactive fraction II has IC₅₀ value of 10.864 μg/ml

In another embodiment of the invention, the bioactive principle III has IC₅₀ value in the range of 16.47 to 18.28 which is higher than bioactive fraction I and II. Another embodiment, the bioactive principle III has IC5₀ value of 16.66 which is higher than bioactive fraction I and II.

Still another embodiment, the antioxidant principle obtained is soluble methanol DMSO and H₂O.

Still another embodiment, the said antioxidant principle has UN absorption maxima at 217, 260 and 362nm.

In another embodiment of the present invention, the dry powder of pith and carpellary membrane was extracted with suitable solvent like methanol and solvent was removed by distillation. This crude extract showed DPPH free radical scavenging activity with IC₅o of 8.33 μg/ml reaction mixture and crude extract may be used as a source of the said antioxidant principle.

In yet another embodiment of the present invention, the said antioxidant principle may be separated and purified from crude extract by silica gel (60-120 mesh) column chromatography using various combinations of solvents like chloroform: ethyl acetate: methanol (100:0:0, 30:20:50) to obtain active fraction I which showed DPPH free radical scavenging activity with IC₅₀ of 15.74 μg/ml reaction mixture and may be used as a source of the said antioxidant principle.

In still another embodiment of the present invention, the said antioxidant principle may be further purified from active fraction I by silica gel (60-120mesh) column chromatography using chloroform and ethyl acetate combination (100:0, 0:100) followed by ethyl acetate: methanol (100:0, 50:50) as solvent system and may be further purified by fractional crystallization using isopropyl alcohol, chloroform, ethyl acetate and acetone as solvents to obtain active fraction II which showed DPPH free radical scavenging activity with IC₅o of 10.86 μg/ml reaction mixture and may be used as a source of the said antioxidant principle.

In still yet another embodiment of the present invention, the said antioxidant principle may be further separated and purified from active fraction II by reverse phase

HPLC on C-18 column using water, methanol and acetic acid (70:25:05) as mobile phase and lyophilized. Purified antioxidant principle showed DPPH free radical scavenging activity with IC₅o of 16.66-μg/ml reaction mixture. A general flowchart for the process of extraction of an antioxidant principle is given below.

Selection of mature healthy pomegranate fruits

Collection of pith a Ind carpellary membrane

Drying in hot air oven at 48°C for 36 hours

Powder Iing (60mesh)

, Sol,ve +nt extracti ^■on

Concentrat Iion by distillation Crude extract

Silica gel column chromatography-I (chloroform, ethyl acetate and methanol)

Active fraction-I

Silica gel column chromatography-II (chloroform, ethyl acetate and methanol)

Fractiona Il crystallization

Activ Ie fraction -II

Prepar Iative HPLC (water, methanol and acetic acid)

Antioxidant principle

The structure of antioxidant principle was determined by UN, IR, 2D-ΝMR [Two-

Dimensional Heteronuclear multiple quantum coherence transfer spectroscopy

(2DHMQCT)] and mass spectrometry (GC-MS, MALDI-MS).

UN visible spectrum was recorded on Shimadzu UN-160A instrument at room temperature. Region from 200-800 nm was employed for scanning. About 2mg sample 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^_1 was employed for scanning. About 7mg sample dissolved in 1ml of DMSO was used for recording the spectrum.

2DHMQCT NMR spectra recorded on Broker DRX500 NMR instrument operating at 500 MHz for 1H and 125 MHz ¹³C at room temperature. Region from 0-12 ppm for ^!H and 0-200 ppm for carbon was employed for scanning, signals were referred to tetramethylsilane to within ± 0.01 ppm. About 30mg sample dissolved in 0.5ml DMSO-d₆ was used for recording spectra.

The following examples are given by way of illustration of the present invention only and should not be construed to limit the scope of invention.

EXAMPLE -1

The mature healthy pomegranate fruits were harvested from orchard, peeled off manually, pith and carpellary membrane collected separately, washed and dried at 48 °C in hot air oven for 36 hours. Dried pith and carpellary membrane was powdered to 60 mesh in an apex grinder. About 500 g dry powder of pith and carpellary membrane of pomegranate fruit was extracted with methanol using soxhlet apparatus, solvent was removed by distillation to obtain 310g of the concentrated extract. This was used as a source of the antioxidant principle. Its antioxidant capacity was determined by DPPH method. DPPH

(1,1 - diphenyl - 2 - picrylhydrazyl) a stable free radical used to monitor the free radical scavenging abilities of various antioxidants. To analyze free radical scavenging capacity, increasing concentrations of extract was mixed with lml of DPPH (500μM) and 0.8 ml of tris buffer (pH-7.4), change in optical density followed kinetically in spectrophotometer at

517nm. The crude extract showed antioxidant activity with IC₅o of 8.33 μg/ml reaction mixture. EXAMPLE 2

The crude extract was further subjected to purification on silica gel (60- 120mesh)column chromato graph and eluted with various combinations of solvents such as chloroform : ethyl acetate : methanol (100:0:0, 30:20:50). Solvent was removed from fractions by distillation and pooled in to different groups based on TLC analysis, lyophilized, dissolved in ethyl alcohol and assayed for antioxidant activity by DPPH method. Out of 6 fractions, the fraction labeled 6 showed maximum free radical scavenging activity with IC₅o of 15.74 μg/ml reaction mixture and was labeled as active fraction I and may be used as a source of the said antioxidant principle. EXAMPLE- 3

The active fraction I was further subjected to purification on silica gel (60-120 mesh) column chromatograph and eluted with chloroform: ethyl acetate (100:0, 0:100) followed by ethyl acetate: methanol (100:0, 50:50). Solvent was removed from fractions by distillation and pooled based on TLC analysis, then lyophilized. The major fraction (100% ethyl acetate) was further purified by fractional crystallization using suitable solvents like isopropyl alcohol, acetone, chloroform and ethyl acetate and lyophilized. The purified fraction showed DPPH free radical scavenging activity with IC₅o of 10.86 μg/ml reaction mixture and it was labeled as active fraction II and may be used as a source of the said antioxidant principle.

EXAMPLE-4

The active fraction II was further purified by reverse phase HPLC on C-18 column with water : methanol : acetic acid (70:25:05) as mobile phase. The fractions were collected, lyophilized and assayed for DPPH free radical scavenging activity. The fraction which showed maximum DPPH free radical scavenging activity with IC₅o of 16.66μg/ml reaction mixture was labeled as antioxidant principle. The purity of which as checked by TLC showed a major single spot. The purity was also ascertained by analytical reverse phase HPLC with water : methanol : acetic acid (70:25:05) as mobile phase wherein a single major peak was obtained.

The characteristic spectroscopic data obtained for antioxidant principle is as follows.

UN data (MeOH): - 217nm, 260nm, and 362nm. IR data (DMSO): - 3430cm^"1 (OH stretching), 2988cm^"1, 1660cm^"1 (carbonyl stretching), 1027cm^"1.

ΝMR data:-

The ΝMR data were obtained from two Dimensional Heteronuclear Multiple Quantum

Coherence Transfer spectra (2D-HMQCT).

Solvent DMSO-d₆. Frequency 500 MHz for 1 H and 125 MHz for ¹³C.

The atoms marked prime are from punicalin The unprimed atoms are from punicalagin

MASS SPECTROMETRIC DATA

GC-MS data (Peracetylated compound): -

Retention times:- 19.64min, 19.75min, 20.26min

19.64min:- 43(CH₃-CO-), 98(Glucopyranoside ring), 115, 127, 157, 169 (All from galloyl group).

19.75 min:- 43(CH₃-CO-), 98(Glucopyranoside ring), 115, 127, 157, 169 (All from galloyl group).

20.26min:- 43(CH₃-CO-), 98(Glucopyranoside ring), 115, 127, 157, 169 (All from galloyl group). MALDI-MS (Matrix Assisted Laser Desorption lonization Mass Spectrometry) DATA

(Matrix used:- -cyano - 4 - hydroxycinnamic acid) MS Fragments :- 1109 (hydrated parent ion, punicalagin), 605 (gallagic acid). SPECIFIC ROTATION (1% MeOH)

[ ]^D ₂₅ = +6.0

Structures of the compounds The novelty of the present invention is to isolate an antioxidant principle from the pomegranate fruit waste viz. pith and carpellary membrane.

The main advantages of the present invention are

1. It provides a natural and inexpensive source that is pomegranate fruit waste, (pith and carpellary membrane) for the production of an antioxidant.

2. It employs a simple extraction and cliromatographic procedure to purify the antioxidant principle from the crude mixture.

3. It provides a potent natural antioxidant principle.

Claims

1. A process for the extraction of antioxidant principles from matured pomegranate fruit wastes viz. pith and carpellary membrane, the said process comprising steps of: a. collecting pit and carpellary membrane of healthy matured pomegranate fruits, drying in hot air oven, grinding to obtain a powder, b. extracting the poser of step (a) with an organic solvent to obtain an extract, c. removing the solvent from the extract of step (b) to obtain under extract, d. mixing the extract of step (c) with an equal amount of silica gel (60-120 mesh), e. purifying the step (d) mixture by subjecting to silica gel chromatography, eluting with a gradient mixture of chloroform, ethyl acetate: methanol (100:0:0 to 30:20: 50) collecting pooling the identical fractions on the toxic of TLC, f. pooling fraction for antioxidant activity to obtain bioactive fraction I, g. subjecting of the active fraction 1 to silica gel column chromatograph and eluted with chloroform with a gradient mixture of chloroform (100:0, to 0:100) followed by elution with ethyl acetate: methanol (100:0 to 50:60), h. pooling the elected fraction of step (g) on the basis of TLC to obtain a major fraction, i. purifying major fraction of step (h) by fractional crystallization using an organic solvent followed by lyophilizing to obtain bioactive fraction II, and j. further purifying bioactive fraction II of step (i ) by reverse phase HPLC with water: methonol: acetic acid (70:25:05) as mobile phase to obtain bioactive principle III.

2. A process of claim 1 wherein in step (a) the drying is carried out in hot air oven at a temperature ranging from 45 -50 C for period of 30 to 40 hours.

3. A process of claim 1, wherein in step (a) the dried material is ground to about 60 to 80 mesh size using apex grinder.

4. A process of claim 1, wherein in step (b) the organic solvent used, is selected from a group consisting of methanol, ethanol, isopropanol and/or mixture thereof.

5. A process of claim 1, wherein in step (c) the extract obtained, may be used as a source of antioxidant-principle.

6. A process of claim 1, wherein in step (f) bioactive fraction I obtained, may be used as source of antioxidant-principle.

7. A process of claim 1, wherein in step (i) bioactive fraction LI obtained, may be used as source of antioxidant principle.

8. A process of claim 1, wherein the step (i) the organic solvent used in selected from a group containing of isopropyl alcohol, acetone, chloroform and ethyl acetate or mixture thereof.

9. A process of claim 1, wherein the crude extract exhibit free radical scavenging activity with IC₅₀ value 8.33μg/ml

10. A process of claim 1 wherein, free radial scavenging activity of bioactive fraction I has IC50 value in the range of 14.79 to 16.18μg/ml.

11. A process of claim 1 wherein, the free radial scavenging activity of bioactive fraction I has IC₅₀ value of 15.74 μg/ml.

12. A process as claim 1, wherein, the free radial scavenging activity of bioactive fraction II has IC₅₀ value in the range of 10.4 to 11.96 μg/ml

13. A process as claim 1, wherein the free radial scavenging activity of bioactive fraction II has IC₅₀ value of 10.864 μg/ml

14. A process of claim 1, wherein the free radial scavenging activity of the bioactive principle III has IC₅₀ value in the range of 16.47 to 18.28 which is higher than bioactive fraction I and II.

15. A process of claim 1, wherein free radial scavenging activity of the bioactive principle III has IC₅o value of 16.66 which is higher than bioactive fraction I and II.

16. A process of claim 1 wherein the antioxidant principle obtained is soluble methanol DMSO and H₂O.

17. A process of claim 1 wherein the said antioxidant principle has UN absorption maxima at 217, 260 and 362nm.