Title of Invention

A PROCESS FOR THE PREPARATION OF PHTHALIDES FROM CELERY SEED OIL

Abstract

The present invention relates to a process for the preparation of phthalides from celery seed oil. Celery (Apium graveolens L. var. dulce [Mill.] Pers.), a herb, grown as a biennial or as an annual, is cultivated as a popular vegetable, for the green and blanched leaf stalks and to a limited extent for the edible thickened roots and crowns. The essential oil of celery seed includes d-limonene (> 60%), selinene (10-20) and pthalides (1-4%). The pthalides are reported as 3-n-butyl phthalide, sedanenolide and sedanolide. The phthalide enriched nutraceutical fraction in highly concentrated form (> 95 %) from celery seed oil is obtained using a simple, yet effective and selective solvent -solvent partition followed by further purification by fractional distillation under reduced pressure.

Full Text

The present invention relates to a process for the preparation of phthalides from celery seed oil. Celery (Apium graveolens L. var. dulce [Mill.] Pers.), a herb, grown as a biennial or as an annual, is cultivated as a popular vegetable, for the green and blanched leaf stalks and to a limited extent for the edible thickened roots and crowns (Simon, J.E., A.F. Chadwick and L.E. Craker. 1984. Herbs: An Indexed Bibliography. 1971-1980. The Scientific Literature on Selected Herbs, and Aromatic and Medicinal Plants of the Temperate Zone. Archon Books, 770 pp., Hamden, CT). In India, celery is mainly grown in Rajasthan and Punjab. Celery seed is ovate and brown. It has a characteristic odour and a warm aromatic taste. The pungent seed is used in salads, soups, stews, vegetable dishes, meat dishes, and celery salt (a mixture of table salt and ground celery seed). Celery seeds contain about 3% volatile oil and 16% fixed oil. They are usually crushed by a plate mill/hammer mill and then subjected to hydro distillation/steam distillation for extraction of essential oil. The essential oil of celery seed includes d-limonene (> 60%), selinene (10-20) and pthalides (1-4%). The pthalides are reported as 3-n-butyl phthalide, sedanenolide and sedanolide. The fixed oil includes the fatty acids petroselinic, oleic, linoleic, myristic, palmitic, palmitoleic, stearic, and myristoleic. Oil of celery seed is sometimes adulterated with celery chaff oil or di-limonene and other terpenes from less expensive essential oils. The phthalides, 3-n-butyl phthalide, sedanenolide and sedanolide are separable by GC analysis. The compounds are identified by their characteristic molecular ions (M+) at m/z 190, 192 and 194 respectively [Uhlig J W, Chang A and Jen J J, J. Food Science, 52 (3), 658-660, 1987]. Further, the fragmentation pattern for n-butyl phthalide is 133(100 %), 105 (38 %), 77 (20 %), 134 (10 %) and 190 (4 %). Sedanenolide is characterized by its mass spectrum consisting of peaks at 107 (100 %), 108 (22 %), 79 (19 %), 192 (17 %), 77 (16 %), 133 (9 %), and 135 (5 %). The mass spectrum of sedanolide consists of peaks at 108(100 %), 79 (15 %), 80 (15 %), 109 (12 %), 137 ( 4 %), and 194 (1.7 %). As a medicinal plant, celery has been used as an aphrodisiac, anthelmintic, antispasmodic, carminative, diuretic, emmenagogue, laxative, sedative, stimulant, and tonic. The plant is used against asthma, bronchitis, and rheumatism. Large amounts of the volatile oil can produce sedation and irritation that may be responsible for attributed antispasmodic properties. Celery is known to cause photodermatitis and contact dermatitis. Celery has also shown hypoglycemic activity. Wild celery usually refers to Vallisneria spiralis L, an aquatic perennial plant of the Hydrocharitaceae family. It is reported in Ayurvedic pharmacopia for treatment of headaches and as a diuretic and spasmolytic. Preparations of celery are also used for blood purification, for regulating elimination of the bowels, for glandular stimulation, and as cure in case of gall and kidney stones. It is also used for weight loss during malnutrition, for loss of appetite, exhaustion and as prophylactic for nervous unrest. The celery seed extracts have been shown to possess anti-inflammatory properties. The pthalides from celery are the most significant bio-active compounds exhibiting many health benefits like protection against cancer, high blood pressure and cholesterol. Sedanolide has been reported to be the most active of the pthalides in the reduction of tumours in laboratory animals. The sedanolide being the major flavour-impact compound of celery volatile oil, possessing the above mentioned health benefits, an enriched sedanolide fraction will be desirable and useful for the treatment of ailments like hypertension and heart ailments. Antioxidant, cyclooxygenase and topoisomerase inhibitory activities have been shown to be associated with compounds including sedanolide isolated from Apium graveolens Linn, seeds (R A Momin / M G Nair, Phytomedicine, 2002). The methanolic extract of Apium graveolens seeds was investigated for bioactive compounds and resulted in the isolation and characterization of mosquitocidai. nematicidal, and antifungal compounds sedanolide (1), senkyunolide-N (2), and senkyunolide-J (3) [Momin. Rafikali A.; Nair, Muraleedharan G. J.Agric.Food Chem. 2001, 49: 1 142 - 145]. Compounds 1-3 gave 100 % mortality at 25, 100, and 100 ug mL"1, respectively on the nematode, PanagreHus redivivus. Compound 1, showed 100 % mortality at 50 jjg mL"1 on nematode, Caenorhabditis elegans, and fourth-instar mosquito larvae, Aedes aegyptii. Also, they inhibited the growth of Candida aibicans and Candida parapsilasis at 100 ug mL"1. 3-n-Butyl phthalide, and sedanolide, isolated from celery seed oil exhibited high activities to induce the detoxifying enzyme glutathione S-transferase (GST) in the target tissues of female A/J mice (Zheng GQ, Kenney PM, Zhang J, Lam LK, Nutr Cancer, 1993, 19:77-86). These compounds (20 mg/dose every two days for a total of 3 doses) increased GST activity 4.5-5.9 and 3.2-5.2 times over the controls in the mouse liver and small intestinal mucosa, respectively. They were further tested for their ability to inhibit benzo[a]pyrene- (BP) induced tumorigenesis in mice. After treatment with 3-n-butyl phthalide and sedanolide, the tumor incidence was reduced from 68% to 30% and 11%, respectively. About 67% and 83% reduction in tumor multiplicity was also observed with 3-n-butyl phthalide and sedanolide, indicating that 3-n-butyl phthalide and sedanolide were both active in tumor inhibition and GST assays, suggesting a correlation between the inhibitory activity and the GST-inducing ability. The phthalides are known to determine the characteristic odor of celery. The results suggest that phthalides, as a class of bioactive natural products occurring in edible umbelliferous plants, may be effective chemopreventive agents. Reference may be made to the US patent No. 5916565, 1999 wherein a preparation from specific herbal phytochemicals from ginger, cayene, turmeric, yucca, devil's claw, nettle leaf, black cohosh, alfalfa and celery seeds were used to treat prophylaxis and the therapy of joint and connective tissue disorders in vertebrates. Reference may be made to a US patent NO. 6352728, 2002 "extracts of celery seed for the treatment of pain; inflammation and gastrointestinal irritation" wherein biologically active extracts of celery seed are produced by controlled ethanolic extraction, distillation, drying, further processing by supercritical fluid extractions followed by column fractionation and desolventisation. The extracts have been evaluated for treatment and prevention of inflammation and gastrointestinal irritation. The drawback of the above process is that it employs a capital-intensive technology consisting of multiple steps of isolation and purification. Reference may be made to US patent No. 6576274, 2003, "extracts of celery seed for the prevention and treatment of pain, inflammation and gastrointestinal irritation" wherein a biologically active celery seed extract produced by a supercritical fluid extraction of an ethanolic extract of fresh celery. Supercritical fluid extraction carried out at temperatures ranging from 30-40° C between 100-300 mm atm. Using carbon dioxide admixed with methanol. The extract obtained after super critical fluid extraction was subjected to column chromatography using 1-20% ether in petroleum ether as eluant and further fractionation with reducing agent LiAIH4. The drawback of the above process is that it employs several steps of isolation / purification and utilizes expensive technology. The main objective of the present invention is to provide a process for the preparation of phthalides from celery seed oil, which obviates the drawbacks of the processes detailed above. Accordingly the present invention provides a process for the preparation of phthalides from celery seed oil comprising; a) subjecting celery (Apium graveolens L.) seed volatile oil, produced by known techniques such as steam distillation, to a process of solvent partition, with an aqueous polar solvent selected from aqueous ethanol having 55-72% to obtain two layers, b) extracting the lower layer with a solvent selected from ethylene dichloride or ethyl acetate followed by desolventing under reduced pressure of upto 10mm to obtain a phthalide rich fraction, c) subjecting the above phthalide rich fraction to fractional distillation under reduced pressure in the range of 1-0.1mm Hg to obtain a highly enriched phthalides, In an embodiment of the process the phthahde rich fraction has a purity as high as 90-98% phthahdes In an another embodiment of the process the phthahdes is useful as nutraceutical and a flavourant The process consists of following steps: Celery seed oil having limonene (70-80%), (3-selinene 15% and pthalides (17%) is taken and subjected to solvent-solvent partition The celery seed oil is partitioned with aqueous ethanol or ethyl acetate The upper phase contains limonene and selinene as major components and the lower phase contains pthalides and small amount of selinene, which on further fractional distillation yields phthahdes in high purity Novelty Obtaining the phthahde enriched nutraceutical fraction in highly concentrated form (> 95 %) from celery seed oil using a simple, yet effective and selective solvent -solvent partition followed by further purification by fractional distillation under reduced pressure The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention Example 1 Celery seed oil (50 g) was partitioned with ethanol (65%, 100 ml) and the two layers were separated The upper layer was collected and analysed by GC By GC analysis, it was found to contain limonene (81%), selinene (14%) and pthalides (0 8%) The lower layer was again partitioned with ethylene dichlonde The extraction of lower layer with ethylene dichlonde (50 ml) was repeated twice The ethylene dichlonde layer was dried over anhydrous sodium sulphate and concentrated in a flash evaporator until all the solvent was removed The residue (1.15 g) was analysed by GC and was found to contain n-butyl phthalide (66%), sedanenolide (22%) and sedanolide (1%) respectively with minor amount of selinene (4.2%). n-butyl phthalide MS (m/z) : 190(4%), 133 (100%), 105 (30%), 77 (16%), 134 (12%). sedanenolide MS (m/z) : 192(18%), 107 (100%), 79 (27%), 77 (28%), 108 (8%), 133(8%), 135(6%). sedanolide MS (m/z) : 194 (2%), 108 (100%), 79 (35%), 80 (22%), 109 (13%), 107 (10%), 81 (10%), 77 (10%), 137 (4%). Example 2 Celery seed oil (50 g) was partitioned with ethanol (65%, 100 ml) and the two layers were separated. The upper layer was collected and analysed by GC. By GC analysis, it was found to contain limonene (81%), selinene (14%) and pthalides (0.8%). The lower layer was again partitioned with ethylene dichloride. The extraction of lower layer with ethylene dichloride (50 ml) was repeated twice. The ethylene dichloride layer was dried over anhydrous sodium sulphate and concentrated in a flash evaporator until all the solvent was removed. The residue (1.15 g) was analysed by GC and was found to contain butyl phthalide (66%), sedanenolide (22%) and sedanolide (1%) respectively with minor amount of selinene (4.2%). Further, the phthalide fraction (1.15 g) was subjected to fractional distillation at a vacuum of 0.1 mm. Hg to remove the selinene. The residue after the removal of selinene, had the composition: n-butyl phthalide (73%), sedanenolide (26%) and sedanolide (1.5%). Example 3 Celery seed oil (50 g) was partitioned with 65% ethanol and two layers collected separately and the upper layer was collected and analysed by GC. By GC analysis it was found to contain limonene 81%, selinene 14% and pthalides 0.8%. The lower layer was again partitioned with ethyl acetate. The extraction of lower layer with ethyl acetate (50 ml) was repeated twice. The ethyl acetate layer was dried over anhydrous sodium sulphate and concentrated in a flash evaporator until all the solvent was removed. The residue (1.05 g) was analysed by GC and was found to contain butyl phthalide (65%), sedanenolide (20%) and sedanolide (1 %) respectively with 4.0% of selinene. Main advantages of the process: ? The process proposed employs seed oil as the starting material which is not dependent on availability and procurement of fresh celery seeds. ? The process employs inexpensive and simple technology compared to capital- intensive supercritical fluid methodology as mentioned in earlier reported methods (patents). ? The method gives enrichment of phthalides to the extent of 95 %. We claim, 1. A process for the preparation of phthalides from celery seed oil comprising a) characterized in partitioning celery {Apium graveolens L.) seed volatile oil with an aqueous polar solvent selected ethanol having 55-72% ethanol to obtain two layers, b) partitioning the lower layer with a solvent selected from ethylene dichloride or ethyl acetate followed by desolventing under reduced pressure of upto 10mm to obtain a phthalide rich fraction, c) subjecting the above phthalide rich fraction to fractional distillation under reduced pressure in the range of 1-0.1mm Hg to obtain a highly enriched phthalides, 2. A process as claimed in claim 1, wherein the phthalide rich fraction has a purity of 90-98% phthalides. 3. A process as claimed in claims 1-2, wherein the phthalides is useful as nutraceutical and a flavourant. 4. A process for the preparation of phthalides from celery seed oil, substantially as herein described with reference to the examples accompanying the specification.

Documents:

593-DEL-2004-Abstract-(13-09-2010).pdf

593-del-2004-abstract.pdf

593-DEL-2004-Claims-(13-09-2010).pdf

593-del-2004-claims.pdf

593-DEL-2004-Correspondence-Others-(13-09-2010).pdf

593-del-2004-correspondence.pdf

593-del-2004-description.pdf

593-DEL-2004-Form-3-(13-09-2010).pdf

593-del-2004-form1.pdf

593-del-2004-form2.pdf

593-del-2004-form3.pdf

593-del-2004-form5.pdf