Title of Invention

"A PROCESS FOR ARTIFICIALLY INDUCING AGAR FORMATION IN TREES BELONGING TO THE GENUS AQUILARIA"

Abstract

The present invention relates to a modified agar obtained from trees of Aquilaria Spp., especially A. agalocha by chemically stressing the trees followed by distillation of wood of the said trees resulting in the formation of essential oil. The present invention also relates to a process for artifically inducing chemical solution of dipyridinium salt in said trees by a delivery system, resulting into agar formation in the said trees within a year.

Full Text

The present invention relates to a process for artificially inducing agar formation in trees belonging to the genus Aquilaria of the family Thymelaceae. Aquilaria is a small genus of trees, fairly widely distributed in the Indo-Malaysian region, in Southern China, and in certain parts of Japan. Of it's species, Aquilaria agallocha Roxb. Syn. A. Malaccensis Lam., commonly known as agarwood, aloewood, eaglewood, and in many Indian languages as agru or agar, is specially important as a source of a highly valuable and precious essential oil from it's wood. However, in the normal course of it's growth, the tree does not produce this prized essential oil. Agarwood is white or pale yallow, and is practically devoid of any odour. On the other hand, when the tree comes under attack by fungi, which generally happens to older trees on injury, the wood develops irregular patches and streaks charged with an odoriferous oleoresin. Depending on the extent of resin induction the color of the wood patches and streaks varies from almost black to light brown. Usually trees around 50 years old contain well-developed black patches in the bole of the tree, especially at junctions where branches fork out from the main stem. The well-formed black portion, which usually has an irregular shape, is normally chiselled out, and is sold as such as incense and fetches good price. The essential oil is normally distilled from the remaining resin-bearing wood, which has little value as incense. Several attempts have been made in the past to induce agar formation in healthy trees. Fungal species associated with the development of resin in the infected trees have been identified as those belonging mostly to the genra Aspergillus, Fusarium, and Pencillium (The Wealth of India, Raw Materials. Vol 1, pp.328-330. Publication & Information Directorate, CSIR, New Delhi 1985). Experiments were carried out to artificially infect healthy trees with these fungi, but no significant results were obtained (B. C. Gulati, Agarwood Oil; Survey of Indian Essential Oils, CSIR leaflet No.l, 1959). More recently, attempts have been made to induce agar formation by application of certain chemicals, but without success (Indian perfumer. 1977,22,151). The present invention overcomes the problem faced by the prior art. It is the objective of the present invention to find a novel process by which agar production can be induced in trees belonging to the genus Aquilaria. STATEMENT OF INVENTION According to the present invention there is provided a process for artifically inducing agar formation in trees of Aquilaria Spp., especially A. agalocha, comprising the following steps of: (a) selecting a tree of Aquilaria Spp., especially A. agalocha having a heart wood formation; (b) making a chemical solution of dipyridinium salt such as diquat and paraquat wherein the concentration of the active component in the chemical solution varies from 0.2 to 5.0% on weight to weight basis; (c) applying the plant with the said solution by delivery system as herein described to render it capable of producing agar within a year. In accordance with the present invention there is also provided a process for the induction of agar formation in trees of the genus, Auilaria especially A. agalocha which comprises treating the plant with chemicals that produce stress in the plant as evidenced by appearance of signs of senescence. SUMMARY OF THE INVENTION Plants come under stress from a variety of reasons, ranging from mechanical injury, attack by predators, infection by microorganisms, to climatic and nutritional strain. Stress can also be generated by the application of certain chemicals. Under all these situations, plants try to offset the onslaught by adapting various strategies, including de novo synthesis of certain defence chamicals. The inventors have explored, with success, the propensity of certain plant species to synthesis, de novo, certain chemicals as defence agents, to induce agar formation in Aquilaria agallocha. DETAILED DESCRIPTION OF THE INVENTION Majority of the compounds constituting the essential oil of agar are sesquiterpene alcohols, aldehydes and ethers (see e.g. K. R. Varma, M.L. Maheshwari, and S.C. Bhattacharyya, Tetrahedron. 1965, 21, 115; M. Ishihara, T. Tsuneya, and K. Uneyama, Phtochemisty. 1993, 33, 1147; and references cited in these articles), which do not occur in the uninfected wood, nor are these normal metabolises or microorganisms identified on the infected trees. Thus, these compounds must be phytoelexins, that is stress metabolises, which are de novo synthesised by the / organism (the tree, in the present discussion) to fight the fungal attack. There is enough documentation on phytoelexins, in general (see e.g. V.D. Gross, Forytschr. chem. orcr. Naturst. . 1977, 34, 187; A. A. Bell, Ann. Rev. Plant Phvsiol. 1981, 32, 21; J.A. Bailey and J.W. Mansfield, Phytoelexins, Blackie, Glasgow, 1982) . Several chemicals are known to cause stress in plants, as evidenced by defoliation, stunted growth, or heightened susceptibility to fungal, insect attack to the treated plant (see e.g. F.C. Steward and A.D. Krikorian, Plants. Chemicals and Growth, pp.232, Academic Press, New York, 1971). Of these, dipyridyls such as Diquat (6-7-dihydrodipyridol[1,2-a:2,1'-C] pyrazinediium dibromide, Structure 1), and Paraquat (1,1'-dimethyl-4-4'- bipyridinium dichloride, Structure 2) appeared most promising. These compounds are non-selective contact herbicides, and have been shown to act by generating superoxide ion radicals and/or hydroxyl radicals; this, in turn, leads to cellular damage, and other cytochemical changes reminiscent of normal senescence (0. J. Schwarz in: Plant Growth Regulating Chemicals, vol. II. editd. by L.G. Nickell, CRC Press, Boca Raton, 1983). Another class of compounds, the so-called ethylene generators, such (Formula Removed) as ethephon (2-chlorethyl phosphonic acid), which are used to stimulate latex flow in the rubber tree, was also selected. It has been found that the dipyridyls are indeed capable of inducing agar formation, and further work has been carried out with this class of compounds. Since the main purpose of these investigations was to induce agar formation artificially, in contrast to natural resinification which occurs sporadically in 20-50 years old or older trees, healthy trees, six to eight years old, were selected for chemical treatment. However, trees of any age with at least some heart wood formation can be treated. Any of the useful methods of application of a chemical into the xylem wood can be employed. Preferably, these include: (i) debarking an area approximately 1 inch wide and 1/3 of the circumference of the tree, and applying the chemical solution to run-off, (ii) feeding through a lateral root, (iii) injection of a solution of the chemical through the bark by a suitable means, such as a hypodermic syringe, (iv) boring one or more holes of suitable diameters into the xylem, and applying a particular volume of the solution into the hole by any suitable means, (v) making a V-notch, sloping inwardly, through the bark into the xylem, and pouring a dipyridyl solution into it. Two dipyridinium salts, suitably formulated, are available commercially [Diquat, Paraquat, as depicted in Structures 1, 2, above, and sold under various Trade names, e.g. Reglone (diquat), Gramaxone (paraquat), of Imperial Chemical Industries, United Kingdom. Any suitable concentration of any one of these compounds, which does not kill the tree can be applied. Usually, a 0.2 to 5% of the reagent (wt/wt basis, prepared by suitably diluting the commercial product with water) is appropriate. Total amount of the solution to be fed to a single tree is, again determined by the condition that the tree does not die. It has been found that approximately 50 to 200 ml of 1% solution of paraquat can be tolerated by a tree. This, of course, is circumscribed by the state of health of the tree, as well as the season during which the treatment is given. In Assam, in India, best period for treatment is from October to March end. Treatment invariably leads to defoliation within a month or so, followed by reappearance of the foliage slowly. Agar formation can be detected within a year, as evidenced by the appearance of brown to dark brown streaks within the bole, initiating at the point where the application of the chemical was made and travelling upwards in the trunk. Harvesting of the tree can be done after a year, or preferably, after several years. In the latter eventuality, one or more additional treatments with the chemical can be given, provided the tree can withstand additional amounts. The formation of agar was confirmed by steam distillation of the wood from the treatment tree, and identification of it's constituents by gas chromatography coupled with mass spectrometry. The invention will now been described with reference to the following example which should not be construed to limit the invention in any manner whatsoever. EXAMPLE: A set of three trees, all approximately eight years old and having a girth of 50 to 60 cm at the base and usually branched at a height of 120 to 150 cm, were selected for chemical treatment. In this set of experiments, feeding was done with a paraquat solution. A commercial preparation of paraquat, Gramoxone (ICI, UK) , was diluted with distilled water to prepare a stock solution of 1% (wt/wt). One tree was fed through the root. For this, earth was dug up close to the base and then proceeding away from it, and thus exposing a stout lateral root. A 100 ml stock solution of paraquat was taken in a 200 ml tall, wide-mouthed bottle, and the tip of the exposed root was dipped in this solution, and secured in place by a split rubber stopper. The exposed root along with the bottle was then covered with earth. The other two trees were fed through holes drilled in the bole of the trees. For this, three holes of 1.5 cm diameter, and about 1.5 to 2.0 cm deep reaching the xylem, and equally spaced in a staggered manner along the height of the trunk, were drilled in the tree by a suitable device. The stock paraquat solution was then fed through these holes with the help of flexible plastic tubes, suitably secured in place. Each tree received a total of 50 ml of the solution spread over a period of three years. The Aquilaria agallocha forms in Hojai, stem from the vision of and foresightedness of Mr. Ajmal Ali Haji Abdul Mazid. He started systematic plantation of Aquilaria agollocha so as to serve as inexhaustible and renewable source for constant and undisturbed supply of agarwood oil for coming generations. The experiments were carried out in the Applicant Company's Aquilaria agallocha farms located at Hojai (Assam, India), during the months of December and January. Within a month of paraquat application, the trees started shedding leaves, and became completely denuded within a week or so. The foliage reappeared slowly, and the trees appeared to have recovered. The trees were harvested after a year to determine if agar formation has taken place. After felling the trees, the canopy was removed, and the trunk wook split length-wise. In each of these trees, there were numerous brown streaks running along the length of the bole. For comparison, an untreated tree was similarly cut down and the stalk split: the wood was perfectly pale white without any trace of brown veins. To confirm that brown streaks are indeed due to agar formation, the coloured wood was cut apart, and subjected to steam distillation in the traditional manner (see e.g.: B.C. Gulati, vide supra). A yield of 0.5% of essential oil, with the typical agar oil odour was obtained. For further confirmation, this product was subjected to GC-MS analysis, using a Hewlett Packard quadrupole instrument. The analysis was carried out at a temperature of 110° using a silica capillary column coated with PEG as the stationary phase. The result was compared with a similar analysis of an authentic sample of agar oil obtained from natural agarwood of Assam origin. The general appearance of the two chromatograms was essentially similar. Specifically, "main" components appearing at the following retention times (minutes) were common: 21.10, 26.59, 28.15, 43.50, 45.20, 46.02, 47.13, 48.30, 51.11, 54.67, 56.73, 96.61. However, there were several "minor" peaks in the two chromatographs, which do not correspond. An important difference in the two materials is the almost total absence of the compound (m/z: 222; identified by mass spectrometry as 10-epi- y -eudesmol) with retention time 39.12 (min) from the oil obtained from the paraquat treated tree. These differences are unimportant, as composition of essential oils can vary considerably depending on the source of the raw material. The above data illustrates that agar formation can be induced artificially. We claim:- 1. A process for artificially inducing agar formation in trees of Aquilaria Spp., especially A. agalocha, comprising the following steps of: (a) selecting a tree of Aquilaria Spp., especially A. agalocha having a heart wood formation; (b) making a chemical solution of dipyridinium salt such as diquat and paraquat wherein the concentration of the active component in the chemical solution varies from 0.2 to 5.0% on weight to weight basis; (c) applying the plant with the said solution by delivery system as herein described to render it capable of producing agar within a year. 2. A process as claimed in claim 1, wherein the delivery system is through lateral roots. 3. Agar obtained by the process as claimed in claim 1. 4. A process for artificially inducing agar formation in trees of aquilaria Spp., substantially as herein described with reference to the foregoing examples.

Documents:

1716-del-1996-abstract.pdf

1716-del-1996-claims.pdf

1716-del-1996-corrspondence-others.pdf

1716-del-1996-corrspondence-po.pdf

1716-del-1996-description (complete).pdf

1716-del-1996-form-1.pdf

1716-del-1996-form-13.pdf

1716-del-1996-form-19.pdf

1716-del-1996-form-2.pdf

1716-del-1996-form-3.pdf

1716-del-1996-form-4.pdf

1716-del-1996-gpa.pdf

1716-del-1996-petition-137.pdf