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Chế phẩm tạo trầm “TDV-01”
Trầm Hương và Kỳ Nam
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Trên 100 kg kỳ nam giữa rừng.....
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Trang trại trầm hương.
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Hương trầm Việt Nam lại bay xa.
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Chuyện về một người tỷ phú và một người......
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United States Patent
Blanchette , et al.
6,848,211
February 1, 2005

 
Description
 

Agarwood is a resinous wood substance that is produced by the tree as a nonspecific host response to wounding, insects and/or microbial invasion. The resin contains tree extractives that have aromatic terpenes present. As discussed above, Aquilaria is unique in that it produces phloem bundles within the xylem. This network of phloem and parenchyma cells produce and distribute the resin around affected areas as a tree defense reaction. It was previously thought that only old trees could produce resin.

The present inventors have determined how agarwood forms in nature and have used this information to produce agarwood in young cultivated Aquilaria trees. The trees may be grown, for example, in a home or cooperative garden, on a plantation, or in a greenhouse. The present technique for producing agarwood has been used on young plantation-grown trees, about 3 to 8 years old. The technique can be performed on trees that are older than this, although for economic reasons, it is beneficial to use younger trees. The inventors have found that two factors are needed in order to induce the production of agarwood in Aquilaria trees: (1) an open wound in the tree, and (2) this open wound must be aerated.

Trees are wounded with a drill to make a hole into the xylem. Many types of wounds were tried and a hole that cuts across the xylem is needed. The size of the hole is not important. Of prime importance is that the hole must not be closed by callus tissue. Small drill wounds may close by new wood growth within one year in fast-growing tropical trees. To insure that the wound does not close, a region of cambium can be cut around the drill wound. This removes the cambium and delays callus formation and wound closure. This process needs to be done repeatedly if the wound appears to be closing. Another method is to insert a sturdy plastic, bamboo, wood or other organic material, or metal tube or pipe (with holes made all along the sides of the tube) into the drill wound leaving it protruding out of the tree. See FIGS. 2A and 2B. As the tree grows, the tube prevents the tree from closing the wound. These and any other methods to prevent wound closure can be used.

In order to maximize agarwood production, one can disturb the tree cells that line the hole after the hole is made. It was seen that the more disruption of live cells, the greater the internal area where agarwood formed. If a hole is drilled, agarwood forms only around the edge of the wound (and only while aeration occurs). If a drill wound is made and substances that kill some of the tree cells (resin-inducing agents) are introduced, the resin forms over a much larger area. See FIG. 3. Many different resin-inducing substances can be used, such as NaCl, ferric chloride, ferrous chloride, chitin, formic acid, etc. Also microbes can be inoculated into the tree to induce a more intense host response. Species of fungi, taxonomically from the Deuteromycota, Ascomycota and Basidiomycota groups that were isolated from agarwood columns in old growth trees growing in Viet Nam were inoculated into the test trees. The presence of some fungi may help keep the wound open and disrupt live cells of the tree, therefore acting as an inducing agent. The sesquiterpenes produced in agarwood from naturally occurring resin in old growth trees and in young plantation-grown trees were chemically analyzed, and the resin was the same.

In one embodiment of the invention, the method would include a series of drill wounds made in a spiral up the tree (30 to 100+ per tree). Each wound is separated by a short interval. Each wound receives a tube, such as a plastic tube, with many holes in its walls. The tube is inserted into the drill wound and left to extend out from the tree 2 to 15 cm. The tube would also contain an inducing agent.

Another method would be to drill holes that are spiraled up the tree and the inducing agent is added directly into the wounds. Each wound would be scribed to cut away a patch of cambium around the hole. The wounds would be inspected over time and new scribing and/or drilling used to keep the hole open. Trees may be grown in nature or in greenhouses for agarwood production.

The following examples are intended to illustrate but not limit the invention.

EXAMPLES

Trees, approximately 4-5 years old, growing in plantations in An Giang Province or Phu Quoc Island, Vietnam were used for experiments 1 to 8.

Example 1

Trees were wounded by making six ax wounds into the main trunk on each tree. Trees harvested after six months were cut and split through the wounded area. The area of discoloration was measured on the exposed longitudinal plane. Resin formation (agarwood) occurred (if it occurred) in a small zone between the wounded, discolored wood and the live unaltered wood. After 6 months, the mean area of discoloration of six ax wounds was 6.1 cm.sup.2 ranging from 1.6 to 9.1 cm.sup.2 and no distinct area of resin formation was seen. After 21 months, the mean area of six ax wounds was 0.3 cm.sup.2 ranging from 0.0 to 1.5 cm.sup.2 and no resin was apparent. The results from this study show that wounding trees with an ax and making shallow surface wounds does not produce agarwood.

Example 2

Trees were wounded with a 5/8 inch drill to a depth of approximated 5 cm. Six wounds were made in a spiral fashion on the trunk of the tree approximately 20 cm apart. One of the six wounds served as a control and the other 5 wounds were filled with agarwood (approximately 0.5 g per wound) from a tree in the forest with naturally produced agarwood. This tree had been cut by poachers but some parts of the tree remained and some fresh agarwood was obtained from it. Small sections of the agarwood were cut and put into the drill wounds. After 6 and 18 months trees were harvested and evaluated with the following results.
Wound Treatment Area of discoloration
After 6 months
1 Control 11.7
2 agarwood 27.6
3 agarwood 22.1
4 agarwood 15.6
5 agarwood 12.6
6 agarwood 16.0
After 18 months
1 Control 18.4
2 agarwood 16.9
3 agarwood 18.8
4 agarwood 19.1
5 agarwood 15.1
6 agarwood 17.6


The amount of discoloration was somewhat greater than when ax wounds were used and a very small region of what appeared to be resin was found at the interface between discolored wood and sound wood. However, trees rapidly closed and the agarwood formation process did not progress as seen in the relatively small areas of discoloration present after 18 months.

Example 3

Trees were wounded with a 5/8 inch drill to a depth of approximated 5 cm. Six wounds were made in a spiral fashion on the trunk of the tree approximately 20 cm apart. One of the six wounds served as a control and the other 5 wounds were inoculated with pure cultures of fungi isolated from fresh agarwood obtained from natural forests in Vietnam. Five different fungi, representing different species of Ascomyota and Deuteromycota, were used. The genus and species was not determined but culture morphology indicated each fungus represented different genera. Only cultures proving to be successful for stimulating agarwood were to be identified to species after field data was obtained. Fungi were grown on sterile oats supplemented with malt extract broth for added nutrients. Cultures were grown for 3 weeks on the oat/malt extract substrate. Drill wounds inoculated with the fungi were filled with the fungus/oat inoculum. After 6, 18 and 21 months the trees were harvested and the following results were obtained.
Wound Treatment Area of discoloration
After 6 months
1 Control untreated 24.8
2 Isolate F-4 20.2
3 Isolate F-5 17.7
4 Isolate F-9 14.6
5 Isolate F-24 15.6
6 Isolate F-32 11.7
After 18 months
1 Control untreated 8.8
2 Isolate F-4 7.7
3 Isolate F-5 12.5
4 Isolate F-9 8.6
5 Isolate F-24 1.6
6 Isolate F-32 13.3
After 21 months
1 Control untreated 10.2
2 Isolate F-4 8.6
3 Isolate F-5 11.4
4 Isolate F-9 10.8
5 Isolate F-24 9.9
6 Isolate F-32 11.7


The results indicate that the common fungi associated with fresh agarwood in Vietnam did not significantly stimulate agarwood to form. The area of discoloration and intermediate area of very slight amounts of resin production was similar between the non-inoculated drill wounds and wounds receiving pure cultures of fungi. No significant increases in agarwood production occurred over the three harvest dates.

Example 4

Trees were wounded with a 5/8 inch drill to a depth of approximated 5 cm. Six wounds were made in a spiral fashion on the trunk of each tree approximately 20 cm apart. Two of the six wounds served as controls and the other 4 wounds were inoculated with different types of nutrient growth media (used to culture microorganisms in the laboratory) or soil.

Treatments included:

Control no treatment

Difco Malt Extract (ME) approximately 0.1 g added per wound

Difco mycological agar (MYCO) approximately 0.1 g added per wound

Difco yeast extract (YE) approximately 0.1 g added per wound

Soil approximately 0.25 grams of soil from the plantation where the tree was growing was added per wound
Wound Treatment Area of discoloration
After 12 months
1 Control no treatment 16.7
2 ME 11.2
3 MYCO 12.3
4 YE 8.1
5 Soil 7.3
6 Control no treatment 6.4
After 21 months
1 Control no treatment 6.0
2 ME 7.2
3 MYCO 16.8
4 YE 10.8
5 Soil 10.5
6 Control no treatment 12.6


These results indicate that no inducement of agarwood was found when just nutrients used to grow fungi in culture were used. Soil placed into the drill wound also did not stimulate agarwood formation. All wounds had evidence of wound closure from surface cambium cells and the included phloem cells of the xylem produced new cells to close the wounds from within the drill hole.

Example 5

To test if different types of compounds could stimulate agarwood production, 5/8 inch drill wounds were made approximately 5 cm into the main trunk of young plantation trees and different materials used to treat the drill wounds. Chemicals used were from Sigma Chemicals Inc. St. Louis, Mo. or Mallinckrodt Inc. Paris, Ky. One drill wound per tree received no treatment and others received one of the following treatments:

Chitosan--purified chitin approximately 0.2 g of chitin was added per wound

Formic acid--approximately 0.1 g of formic acid was added per wound

Sodium chloride--approximately 0.2 g added per wound

Cellobiose--approximately 0.2 grams added per wound

Lime--approximately 0.1 g calcium carbonate added per wound

Trees were harvested after 12, 18 and 21 months.
Wound Treatment Area of discoloration
After 12 months
1 Control no treatment 11.2
2 Chitosan 9.0
3 Formic acid 28.5
4 NaCl 49.3
5 Lime 10.0
6 Cellobiose 9.9
After 18 months
1 Control no treatment 25.4
2 Chitosan 22.1
3 Formic acid 18.6
4 NaCl 44.7
5 Lime 18.7
6 Cellobiose 20.3
After 21 months
1 Control no treatment 11.1
2 Chitosan 3.8
3 Formic acid 31.8
4 NaCl 36.4
5 Lime 9.7
6 Cellobiose 7.4


These results indicate that the reaction area within a tree and area of discoloration can be increased with compounds that kill live parenchyma cells around the wounded region of the xylem. Deposits of agarwood resin were formed at the edges of the discolored regions. Substances like formic acid that have a low pH and NaCl that has a high pH both can disrupt live cells and induce greater amounts of agarwood than the control wounds. As the tree grows and wounds are closed the affected area decreases. Other substances like chitosan, cellobiose and lime do not increase the area of discoloration within the tree at the concentrations tested. However, if added at amounts that are detrimental to live cells adjacent to the wounded area it did have an effect. Microscopic observations indicate that the cells around wounds treated with NaCl or formic acid react extensively and phloem cells are filled with resin. These cells do not have the ability to produce cambial initial cells and wound closure is delayed. Substances that affect the live parenchyma cells and phloem cells in the xylem stimulate resin production and induce agarwood production. They also inhibit wound closure from the outer cambium as well as new cells formed by the included phloem.

Example 6

Analysis of the chemical composition of natural agarwood and experimentally produced agarwood was done by identification of the sesquiterpenes present. Samples were extracted in methylene dichloride at 37.5.degree. C. and nitrogen used to reduce the volume to no less than 0.1 ml. A methylating agent was added before injecting into a Hewlett-Packard 5890 gas chromatograph with a 15 m.times.0.25 mm DB-1 column. The injector temperature was 280.degree. C. After four minutes the initial column temperature of 50.degree. C. was raised at 10C/min to 340.degree. C. The eluent was detected with a Hewlett-Packard 5972 mass selective detector with the interface at 280.degree. C.

Samples of low, medium and high quality agarwood obtained commercially from Singapore merchants had levels of sesquiterpenes that ranged from 0.3 to 10% of the sample. Sesquiterpenes included, aromadendrene, .beta.-selinene, .gamma.-cadinene, .alpha.- and .beta.-guaiene. The levels of sesquiterpenes from samples of agarwood from the experimental trees were at 0% (control wounds) to 1.5% (NaCl treated drill wound treatment after 12 months). Sesquiterpenes included aromadendrene, .alpha.-selinene, .gamma.-cadinene, .alpha.- and .beta.-guaiene and .alpha.-humulene.

Example 7

Multiple, small drill wounds were made at three locations on each tree to observe the effect of wound size and multiple wounds on agarwood formation. Twenty holes approximately 5mm in diameter were drilled approximately 5 cm into the xylem of the tree. Four rows of five wounds were made approximately 2 cm apart. Groups of wounds were made at 30 cm intervals on different sides of the tree. A 10.times.10 cm area of the bark was cut out around each group of 20 wounds. Trees were harvested after 18 months. The area of discoloration caused by the small drill wounds coalesced together resulting in a large area of discoloration with some agarwood resin formed along the edges. The multiple wounds served to disrupt normal functioning of cells around the wounds and wound closure was delayed. Single small drill wounds made in a tree would not produce significant amounts of agarwood resin since they would close rapidly, but groups of multiple wounds made closely together on the tree disrupted the normal functioning of the xylem and stimulated agarwood production.

Example 8

Fourteen plantation grown trees on Phu Quoc Island were used to test different treatments. Each tree received 8 wounds that were spiraled up the main trunk of the tree separated by approximately 20 cm. A range of different types of wounds and treatments as well as controls were made on each tree. Each treatment was made at a different location on each tree to insure that location of wound did not have an effect. Trees were harvested after 15 months and taken to the laboratory for analyses.

Treatments included:

1. A surface wound approximately 5.times.5 cm was made by cutting the bark and removing the bark tissue to expose the xylem. No drill hole was made.

3. A 5/8 inch drill wound was made approximately 5 cm into the xylem and a 5.times.5 cm section of the bark removed around the drill hole and approximately 0.3 g sterile Aquilaria sawdust added to the drill wound.

4. Same as #3 but with approximately 0.3 g of a 1:1 ratio of ferrous chloride and sterile Aquilaria sawdust added to the drill wound.

5. Same as #3 with approximately 0.3 g of a 1:2 ratio of NaCl and sterile Aquilaria sawdust added to the drill wound.

6. Same as #3 but with approximately 0.3 g of a 1:4 ratio of Difco yeast extract and sterile Aquilaria sawdust.

7. Same as #3 but with approximately 0.3 g of a 1:1 ratio of sodium bisulfite and sterile Aquilaria sawdust.

8. Same as #3 but with approximately 0.3 g of a 1:2:4 ratio of Difco nutrient broth: Difco malt extract: sterile Aquilaria sawdust.

After 15 months, 8 trees were harvested and brought to the laboratory for analyses. The area of discoloration and resin formation was determined by splitting the tree through the wounded region and area affected measured using an image analyzer. Area (cm.sup.2) was determined for all wounds in the longitudinal plane. Each value is a mean of 8 wounds.
Wound Treatment Area of discoloration/resin
1 Surface wound 1.4
2 Drill wound 46.3
3 Surface wound and drill wound 27.3
4 wounds/ferrous chloride 112.5
5 wounds/NaCl 48.1
6 wounds/yeast extract 28.6
7 wounds/sodium bisulfite 182.0
8 wounds/nutrient media 34.3


This study showed the effectiveness of several treatments for the production of appreciable amounts of agarwood in young Aquilaria trees. Surface wounds do not produce agarwood. Deep penetrating wounds may produce some agarwood as long as the wound site remains open. Compounds that challenge the living cells around the drill wounds in the xylem (e.g. NaCl, sodium bisulfite, ferrous chloride, and any other chemicals that disrupt the normal functioning of living tree cells) increase the area of discoloration within the tree and the amount of agarwood resin formed.

Example 9

Young trees growing at two sites, in home gardens and on agricultural land (Kon Tum, Vietnam and Nui Cam, Vietnam), received 8 wounds per tree. Drill wounds 5/8 inch diameter were drilled approximately 5 cm into the main trunk of each tree in a spiral fashion separated by approximately 20 cm. All drill wounds were scribed to remove a 5.times.5 cm area of bark around the wound. Fungal treatments consisted of three different types of Basidiomycota obtained from Aquilaria trees in Vietnam. These isolates were not identified to genus but culture morphology indicated they were different genera. Cultures were grown on sterile rice supplemented with malt extract. Cultures were grown for three weeks before inoculation.

Treatments included:

1. Control wound receiving sterile rice

2. Fungal culture 97-14-5

3. Fungal culture 97-13-7

4. Fungal culture 97-11-25

5. Control wound receiving 5 g sterile Aquilaria sawdust

6. Approximately 0.5 g of a 1:4 ratio of sodium bisulfite and sterile Aquilaria sawdust

7. Approximately 0.5 g of a 1:2 ratio of salicylic acid in sterile Aquilaria sawdust

8. Approximately 0.5 g of a 1:4 ratio of ferrous chloride in sterile Aquilaria sawdust

After 15 months two trees were harvested from each location and brought to the laboratory for analyses. The mean area for each type of wound was calculated.
Wound Treatment Area of Discoloration cm.sup.2
Location Kon Tum
1 Control 22.7
2 97-14-5 14.5
3 97-13-7 18.0
4 97-11-25 17.5
5 Control 11.1
6 Sodium bisulfite 52.3
7 Salicylic acid 16.9
8 Ferrous chloride 18.4
Location Nui Cam
1 Control 15.5
2 97-14-5 18.2
3 97-13-7 31.2
4 97-11-25 20.9
5 Control 20.6
6 Sodium bisulfite 56.9
7 Salicylic acid 25.5
8 Ferrous chloride 23.3


This study indicated that the three different basidiomycetous fungi used did not result in extremely large areas of discoloration and the area was not very different from control wounds. It also indicated that the reduced concentration of sodium bisulfite used in comparison to example 8 caused a reduced amount of discoloration and agarwood resin. Although less area of discoloration was found, agarwood resin did form at the discolored/sound wound interface. When this wood was removed from around the wound and burned it produced a distinctive agarwood aroma. Some compounds such as salicylic acid and ferrous chloride did not produce large areas of discoloration and apparently must be applied in sufficient concentration to affect the living cells in the xylem adjacent to the drill wound. For example, in this experiment ferrous chloride was used in a 1:4 ration with sawdust and moderate amounts of discoloration were observed. In previous experiments it was used in a 1:1 ratio with greater amounts of reaction and agarwood production in the wounded xylem.

Example 10

Iron nails placed into the experimental trees showed a small but significant amount of agarwood when harvested after 6, 15, or 18 months. Reactions observed in the xylem indicate that iron stimulates resin production and iron nails or other sources of iron affect agarwood formation.

Example 11

Young trees 5-6 years old growing at two locations, Nui Cam and Kon Turn, Vietnam, were wounded by making 5/8 inch drill wounds into the xylem to a depth of approximately 5.0 cm. Wounds were placed in a spiral up the tree separated by approximately 10 cm. A 5.times.5 cm area of bark was removed from around the wound and a plastic tube inserted into the wound. The plastic tube had aeration holes drilled into the sides and shallow grooves made into its surface to facilitate air movement along the tube when inserted into the tree (see FIG. 3). The tube extended out from the tree approximately 10 cm so the hole will not close for many years. This tube will insure that the wound will stay open and air will be available to the inner wounded xylem. In addition to wounds receiving only the tubes, other wounds with tubes inserted had a combination of 1:1:3 sodium bisulfite, Difco yeast extract and iron powder added (the iron powder was a 99.6% Fe powder manufactured by J.T. Baker Inc. Phillipsburg, N.J.). Other compounds that cause a localized disruption of the normal functioning of xylem and phloem cells can also be used to prevent internal included phloem from producing secondary cells (that could close the wound from the inside of the drill wound) and to disrupt a greater area of cells in the xylem. As long as the wound remains open to the air, agarwood will progressively accumulate. The greater the disruption of live cells around the wound (without killing the tree) the greater the agarwood production. The tree must remain alive for agarwood to form, as dead trees do not form agarwood. As the tree grew new wood, additional holes were made to allow the zone of agarwood to move into the new xylem (FIG. 4).

Example 12

Trees located in two locations, Nui Cam and Kon Turn, Vietnam, were wounded using a 5/8 inch drill to a depth of approximately 5 cm. Wounds were placed in a spiral fashion on the tree from the ground line up into the crown. Wounds were placed 3 to 5 cm apart. Over time, the wounds were rewounded to keep the wounds open. This was done whenever the wounds appeared to have any wound closure. In these areas of Vietnam they were checked and rewounded every 2-3 months. Trees were wounded with 30 to 70 wounds depending on the size of the tree. These studies show that repeated mechanical wounds that are made deep into the xylem kept the wounds open by preventing external cambial wound closure and internal secondary cell growth by the included phloem. Localized areas of agarwood accumulated immediately adjacent to the wound as long as the wound remained open.

All publications, patents and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the scope of the invention.


 


 




 
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