NL2005573C2

NL2005573C2 - Method for the production of glycosides from bulbs and use of the glycosides thus produced.

Info

Publication number: NL2005573C2
Application number: NL2005573A
Authority: NL
Inventors: Robert Verpoorte; Andrea Lubbe; Hendrikus Gude
Original assignee: Stichting Dienst Landbouwkundi; Academisch Ziekenhuis Leiden
Priority date: 2010-10-26
Filing date: 2010-10-26
Publication date: 2012-04-27
Also published as: WO2012057617A2; WO2012057617A3

Description

METHOD FOR THE PRODUCTION OF GLYCOSIDES FROM BULBS AND USE OF THE GLYCOSIDES THUS PRODUCED

The present invention relates to a method for producing sugars, in particular glycosides, from bulbs. The 5 invention further relates to the glycosides thus obtained and to the use thereof. The invention relates in particular to isolation of the glycoside tuliposide, in particular tuliposie B, from tulip bulbs.

Tulips produce compounds called tulipalins and 10 tuliposides. Tulipalins are five-carbon rings that can be described as a-methylene-y-butyrolactones. Tuliposides are glycosides, consisting of glucose with one or more tulipalins attached as side chains in an open configuration. The side chains can be released from the tuliposides either 15 by pH-dependant or enzyme-mediated lactonization to form tulipalin A or B.

Six tuliposides, and two tulipalins have been isolated from various tulip tissues, e.g. flowers or stems. The structures of these tuliposides are shown in figure 1.

20 Tuliposides A and B are widely distributed in the genus

Tulipa, with different ratios of the different kinds being reported. Tulips in section Leiostemones usually contain larger amount of tuliposide B than A, while those in section Eriostemones have large amounts of both A and B with not 25 consistent interrelationship between the -two.

Tuliposide D occurs in large amounts in T.patens, and only in trace amounts in other species. Tuliposide F was first isolated from T.turkestania and has also been found in all taxa investigated since. The name Tuliposide C was given 30 to a compound from tulip in an early study, but since this compound was never fully characterized, the next tuliposide that was completely elucidated was named tuliposide D.

Tulipalins can be produced from the tuliposide by 2 and enzymatic or chemical method. Tuliposide A and tulipalin A have allergenic properties, and are responsible for the skin irritation often suffered by tulip harvesters (called "tulip fingers"). Tulipalin B and the other tuliposides are 5 not allergenic to humans but these compounds all play a protective function to the plant. Tulipalin A concentration increases after fungal .infections, and slow fungal penetration into the plant tissue. Tuliposide A is not active against fungus F.oxysporium, but tulipalin A is. It 10 was thus first believed that tuliposides store tulipalins for release only after infection or mechanical damage, but it was later shown that free tulipalins also occur at low concentrations in healthy plants.

At present the production of tuliposides involves 15 extraction from tulip tissue e.g. flowers and stems. This involves several steps to separate the compound from the plant matrix and obtain a tuliposide in pure form.

From Shigetomi, K.et al. (Phytochemistry. 2010 Feb;71(2-3):312-24) it is known that 6-tuliposide B is a 20 secondary metabolite occurring specifically in tulip anthers. Christensen LP and Kristiansen K (Contact Dermatitis. 1999 40(6):300-9) describe the isolation of tuliposide B from tulip tissue by means of RP-HPLC.

US patent application US2003170653 discloses a 25 biological method for the production of tuliposide A and its intermediates. In this publication a biosynthetic pathway to tulipalin A is described, and genes encoding key enzymes in this pathway were cloned. The patent describes the use of these gene sequences in recombinant yeast or E.coli cells to 30 produce tuliposide A or tulipalin A.

Tulipalin A and B have in common the a-methylene-γ-butyrolcatone structure. a-Methylene-y-butyrolactones have been prepared synthetically using petroleum-based 3 ingredients. This is expensive and not environmentally friendly or sustainable, and a natural source of these compounds is now desired.

In a recent paper, Kato and co-workers purified an 5 enzyme from tulip bulbs that converts tuliposides to tulipalins by removing the sugar part (Kato, Shoji et al. 2009, Bioscience Biotechnology and Biochemistry 73(8): 1895-1897). In a paper published shortly after, the purified enzyme was used to convert tuliposide A and B to tulipalin A 10 and B, respectively (Kato, Yoshida et al. 2009, Tetrahedron Letters 50(33); 4751-4753).

Isolated tuliposides and tulipalins have been tested for various bioactivities. Most of these compounds were found to have antifungal, antibacterial and/or 15 insecticidal activities. Tulipalin A was found to be insecticidal against thrips (Thrips palmi, F.occidentalis, F. intonsa) and mite (Tetranchus urticae) and to also have an antifungal activity. Tulipalin B was described to be antibacterial against various human pathogens. Both 20 tuliposide A and tuliposide B are described to be antifungal. Tuliposide B even has an activity against some fungicide-tolerant strains and was also found to be antibacterial against gram positive and gram negative bacterial strains.

25 In order to benefit from the known activities of the tuliposides the need exists to produce these compounds in high amounts in a substantially pure form.

In the research that led to the present invention it was surprisingly found that when-tulip bulbs are induced 30 to produce a gum, for example with ethylene gas, or by applying lanolin paste containing ethepon or indole-3-acetic acid, this gum is a useful source for the production of tuliposides and in particular 6-tuliposide B. It was 4 furthermore found that a KH2PO4 buffer-methanol (1:1; pH=6) extract of the gum consisted of a compound present in almost pure form. The compound extracted from the tulip gum has been identified as 6-tuliposide B by one- and two-5 dimensional proton nuclear magnetic resonance (1H NMR).

Certain tulip cultivars are known to produce large amounts of gum in the bulbs when infected with the fungus Fusarium oxysporium. Gum production can also be induced by applying the gas ethylene, or lanolin paste containing 2-10 chloroethyl phosphonic acid (ethepon) or indole-3-acetic acid (IAA) to the bulbs after harvest. In the past, studies on the composition of tulip gum reported it to consist mainly of polysaccharides (Demunk and Saniewski 1989, Horticulture 40(2): 153-162; Saniewski, Miyamoto et al.

15 2007, Floriculture ad Ornamental Biotechnology 1(1): 34-40).

The compound 6-tuliposide B has not been reported in tulip gum before.

The tuliposides can thus be extracted from the gum in one or two easy steps using polar conditions, such as 20 water or methanol, optionally followed by a purification step, such as chromatography (size exclusion or adsorption column chromatography, centrifugal partition chromatography) or liquid-liquid partitioning. This process is a novel way of obtaining 6-tuliposide B from a natural source in pure 25 form to use as is, or to use for the production of tulipalins. The optional purification step is for obtaining a pure product, in particular a product that is >95% pure.

Based on the above finding the invention now provides a method for the production of sugars, in 30 particular glycosides, from bulbs, comprising the steps of: a) inducing gummosis in the bulbs; b) optionally separating the gum from the bulbs to obtain the gum; 5 ' ·* c) optionally extracting the sugar from the gum or the bulbs under polar or semipolar conditions.

The method of the invention can be used for the production of sugars in general and glycosides in 5 particular. When the bulb is a tulip the glycosides to be extracted are tuliposides, such as tuliposide A, tuliposide B, tuliposide D and tuliposide F, and tulipalins. The tuliposide is in particular tuliposide B.

-The word "bulb" is a term well-known to the person 10 skilled in the art and refers to an underground vertical shoot that has modified leaves (or thickened leaf bases) that are used as food storage organs by a dormant plant. The leaf bases may resemble scales, or they may overlap and surround the centre of the bulb as with the onion. According 15 to the invention the method can be performed with any bulb that is capable of gummosis. Examples of bulbs capable of gummosis are Allium bulbs, such as garlic, hyacinth bulbs, iris bulbs (Kamerbeek & De Munk 1976, Scientia Horticulturae 4:101-115), grape hyacinth (Muscari aremniacum) bulbs 20 (Myamoto et al. 2010, J Plant Res 123:363-370).

The induction of gummosis can be achieved in various ways. In one embodiment, the gum forms in response to Fusarium infection. In another embodiment gummosis is induced by ethylene treatment or application of jasmonic 25 acid or jasmonate. According to another embodiment, ethephon (2-chloroethyl-dioxido-oxophosphorane), which, upon metabolism by the plant, is converted into ethylene, is used to induce gummosis. The bulbs of Allium species produce gum as a consequence of mechanical injury. Hyacinths (and 30 related species) can produce gum after infestation by the bacteria Dickeya chrysanthemi (formerly known as Erwinia chrysanthemi).

Gummosis can also be induced by various stress 6 factors, in particular stress factors that lead to internal ethylene production, in particular wounding, pressure, humidity and extreme temperatures.

Before extracting the glycosides from the gum, the 5 gum can be separated from the bulb. Alternatively, the sugars, in particular glycosides, can be extracted from the intact bulb, i.e. the b(ulb that has the gum still attached to it.

-.In the case of tulip, the gum is sticky and clear 10 and comprises polysaccharides and tuliposides, in particular tuliposide B. It is possible to use the gum as such, for example as an antimicrobial or antifungicidal coating, for example to coat seeds or other material that needs to be protected against microorganisms. For this application it is 15 thus not necessary to perform the extraction.

The polar to medium polar solvents of the invention that are used for extracting the glycoside from the gum can be any known polar or semipolar solvent and are in particular selected from water, methanol, acetone, DMSO 20 or mixtures of two or more of these. Other suitable solvents are ionic liquids and deep eutectic solvents. The extraction can be performed under pressurized conditions or microwave or ultrasound assisted.

The extraction can be performed at any pH. In 25 order to keep the sugar attached to the tulipalin, the extraction is however suitably made at a neutral or acidic pH.

As was described already above, various uses for 6-tuliposide B have been described in the literature.

30 US patent application 2008/0262082 relates to the very effective antimicrobial properties of the compounds 3-(-)-tulipalin B or acetylated-S-(-)-tulipalin B).

Tulipalin A and B have in common the a-methylene- 7 γ-butyrolcatone structure. These are very useful structures and are used as synthetic intermediates in the production of several bioactive agents, antimutageriic agents, insect repellants, electrolyte solutions of lithium-ion batteries 5 and momomers of bioplastics as described in Kato, Yoshida et al. 2009 Tetrahedron Letters 50(33): 4751-4753). The production of plastics from these compounds is particularly interesting, as copolymers using a-methylene-γ-butyrolacfeones as building blocks have been found to be very 10 resistant to weathering and solvents, as well as having desirable optical properties.

The glycosides obtained by the method of the invention and in particular the tuliposides, more in particular 6-tuliposide B, can be used for the production of 15 a-methylene-y-butyrolactones as building block for e.g. bioplastics and for antimicrobial applications.

According to a further aspect thereof the invention relates to the new use of the glycosides., in particular the tuliposides, more in particular tuliposide B 20 as a herbicide and for germination inhibition. Tuliposide B can occur in two forms, 6-tuliposide B and 1-tuliposide B, depending on the position at which the side chain is attached to the glucose molecule (either carbon 1 or 6 of glucose). The invention relates to both forms.

25 The process of tuliposide production by inducing gummosis in tulips is a simple, environmentally friendly way to obtain a useful compound with many applications. In a broader sense, the production of useful compounds by inducing gummosis also applies to other plants, such as 30 garlic, hyacinth, grape hyacinth, iris, Allium.

The invention will be further illustrated in the Examples that follow and that are given for illustration purposes and are not intended to limit the invention in any 8 - way.

FIGURES

Figure 1: (A) The structure of tulipalins and tuliposides isolated from tulips (From Christensen & 5 Kristiansen 1999); (B) The structure of 6-tuliposide B with numbering of the carbon atoms.

Figure 2: 1H NMR spectrum of 6-tuliposide B.

Figure 3: J-resolved spectrum of 6-tuliposide B.

10 Figure 4: COSY spectrum of 6-tuliposide B.

Figure 5: Phytotoxicity assay. Shown is the weight of seedlings with and without treatment with 6-tuliposide B.

Figure 6: HPLC chromatogram of tuliposides extracted from tulip gum (cultivar Apeldoorn induced to 15 produce gum 1 week after harvesting) by methanol detected by UV detector at 208 nm. Mobile phase was 100% water for 30 minutes at 1 mL/min. Compounds in chromatogram: 6-tuliposide B (1.54 min), 6-tuliposide A with β-form of glucose (8.24 min), 6-tuliposide A with α-form of glucose (12.95 min).

20 Figure 7: HPLC chromatogram of 6-Tuliposide B

isolated from methanol extract of Yokohama tulip gum, using C18 SPE.

EXAMPLES EXAMPLE 1 25 Production of tuliposides

Induction of gummosis in tulip bulbs

Experiments were performed to induce gummosis in tulip bulbs of two different cultivars. Bulbs were placed in a sealed chamber containing a known amount of ethylene gas.

30 Ethylene gas was replenished every 24 hours. To enhance gum production, some bulbs were mechanically injured by piercing with a needle. Bulbs were treated for different lengths of time to see if there was an effect on gum yield.

9

The results are shown in Table 1 below.

Table 1

Cultivar Time Weight Ethylene I Days needle Weight Yield after bulbs (ppm) treated injured gum (g) gum (% __harvest (g)______w/w) c; Meidoorn 1 week 825.7__30__2__No__3.6 0.43 ^ Apeldoorn 1 week 852.7__30__2__Yes_ 4.8 o.57

Apeldoorn 6 weeks 805.0__80__5__No_ 4.8 0.59

Apeldoorn 6 weeks 805,0 * 80__5__Yes_ 9.6 ~Ï7Ï9

Yokohama 6 weeks 860.0 80_ 5 No 19.9 2.31 I Yokohama 6 weeks ~860.0 80 5 ~Yes . 22.2 2.58

From the results it can be seen that the time when 10 gummósis is induced plays a role in how much gum is produced. Different cultivars produce different amounts of gum in response to ethylene treatment. Mechanically injuring the bulbs increases gum production, but different cultivars seem to respond differently to this treatment.

15

Identification of tuliposides in tulip gum

The gums produced, by different cultivars of tulips were analyzed to confirm the presence of 6-tulipos'ide B.

This compound was found to be present in all three gum 20 samples analyzed (Apeldoorn 1 week after harvest, Apeldoorn induced 6 weeks after harvest, Yokohama induced 6 weeks after harvest). Another tuliposide, 6-tuliposide A was also detected in the Apeldoorn gum induced 1 week after harvest.

The identity of both tuliposides was confirmed by ΧΗ NMR and 25 HPLC-MS.

Extraction of tuliposide B from tulip bulbs

Tulip gum from Apeldoorn bulbs induced to produce gum 1 week after harvesting was extracted with a variety of polar to medium-polar solvents. The extracts were analyzed 30 by HPLC and UV detection (at 208 nm). Peak areas of tuliposide B and tuliposide A were compared between extracts to determine which extraction solvents extracted the most of the glycoside(s) from the gum. An example of an HPLC chromatogram of a tulip gum extract is shown below in j 10 -

Figure 6.

From these results it can be seen that while all the solvents tested were able to extract tuliposides, methanol and water extracted the most tuliposides from the 5 gum. Different solvents had extracted tuliposide A and B from the gum in different proportions.

Table 2 shows Peak areas of tuliposides extracted from tulip gum using different solvents.

10 Table 2

Extraction 6-tuliposide B le-tuliposide A

solvent_

Methanol 2051 : 3138

Methanol-water 721 905 (1:1)

Water 1186 676

Ethanol__852__1242 15 Peak area of compounds detected at 208 nm

Acetonitrile "Ï9 360

Acetone 59 170

Butanol__120__ 154 EXAMPLE 2

Isolation of 6-tuliposide B

20 To obtain 6-tuliposide B in pure form, gum produced by inducing gummosis in bulbs of tulip cultivar Yokohama was extracted with methanol. Three grams of gum was extracted three times with 50 mL of methanol. The methanol was filtered through filter paper and evaporated under 25 reduced pressure at 40°C. This yielded 63.6 mg of dry extract. The dry extract was dissolved in distilled water to a concentration of 4 mg/mL.

6-Tuliposide B was isolated using reverse phase (C18) solid phase extraction (SPE). A SPE cartridge 30 containing 500 mg of C18 solid phase was conditioned with 5 mL of methanol, followed by 5 mL of distilled water, at a flow rate of 1 mg/mL. One mL of gum extract was loaded onto lithe cartridge. The extract was eluted with 5 mL of distilled water. The water eluent was collected, and evaporated under reduced pressure at 40°C, which yielded 25 mg of eluent.

Analysis by HPLC showed the presence of 6-tuliposide B (with 5 alpha- and beta-glucose forms separated as two peaks using new Diol column) in pure form after cleanup with solid phase extraction.

Figure 7 shows the water eluent of SPE cleanup, containing 6-tuliposide B, beta- and alpha-glucose forms now 10 separated on diol column. The HPLC mobile phase was: 0-5 min 0% methanol, 5-35 min 0-100% methanol, 35-45 min 100% methanol, 45-50 min 100-0% methanol.

EXAMPLE 3

Tuliposide B identification 15 Extraction for 1H NMR determination

Approximately 50 mg of tulip gum was dissolved in 1.5 mL of a solution of methanol-d* (99.80%) and phosphate (KH2PO4) buffer (pH 6.0) in deuterium oxide (1:1, pH 6.0) containing 0.01% trimethylsilylproprionic acid sodium salt-d* 20 (TMSP, w/w) as an internal standard for calibration of chemical shift. The solution was ultrasonicated for 10 minutes, followed by centrifugation at 13000 rpm for 10 minutes. An aliquot of 1 mL of the supernatant was collected and 800 pL transferred to a 5 mm NMR tube for 1H NMR 25 measurement.

NMR measurement 1H NMR spectra were recorded with a Bruker AV 500 spectrometer (Bruker, Karlsruhe, Germany). For each sample 64 scans were recorded using the following parameters: 0.167 30 Hz/point, pulse width (PW) 4.0 ps and relaxation delay (RD) = 1.5 s. FIDs were fourier transformed with LB = 0.3 Hz.

Manual phase adjustment and baseline correction were applied i 12 as well as calibration with internal standard TMSP to 0.0

ppm. Two-dimensional J resolved NMR spectra and 1H-1H

correlated spectroscopy (COSY) spectra were also recorded.

Signals in the 1H NMR spectrum were found to 5 correspond to those reported for 6-tuliposide B in the literature (Christensen 1999, Phytochemistry 51 : 969-974), with small differences-in chemical shift values due to·the use of a different NMR solvent. Integrals from the anomeric proton (Er—1: δ 5.19 and 4.59 ppm) showed that the ♦ 10 equilibrium between the a- and β-forms of 6-tuliposide B was

approximately 4:6 in this solvent. Table 3 shows the assignment of NMR signals to protons of 6-tuliposide B

Table 3

Η δ (ppm) integral sP^t j (Hzj COSY

pattern 3' a 5.98 Ϊ s " 3'b 5.75 Ϊ s 1792, 2.75 ~T' 3.83 ‘ Ϊ dd .4.35, 8.05 2.92, 2.76 Ü'a 2792 Ï dd 14.8, 4.35 2.76, 3.83, 5.75 5'b 2.76 1 dd 14.8, 8.05 2.92, 3.83, 5.75 p-glucose: 20_________ 1 4.59 0.6 d 8.2 3.20 2 3.20 dd 8.8, 8.2 4.59, 3.44 3 3.44 t 8.86 . 3.20 *4 3752 d 11.86 3770 5 3.70 m 3.52, 3.88, 4.03 25------ 6a 3.88 dd * 12.3, 2.0 4.03, 3.70 6b 4.03 dd 12.3, 2.0 3.88, 3.70 a-glucose: ~ 13 5.19 0.4 Td 3.75 3.48 ~2 1748 I üd 3.75, 9.8 5Λ9, 1 3.63 t ÊTl 3.24 5 Ί 3.24 ' ~ '“t 971 3.63

1 ' " 3.48 " ÏÜ 3.73, 3.69 I

~6a 3.73 : dd 12.0, 5.0 3.69 ~6b ” 3.69 I dd 12.0, 2.0 3.73 10 EXAMPLE 4

Phytotoxicity assay

Arabidopsis thaliana seeds were germinated and grown on Murashige & Skoog medium. Week-old seedlings were treated with a 0.5 mg/mL {1.7 mMol) solution of 6-tuliposide 15 B (94% pure according to HPLC) in methanol (n=4). Control treatments were also carried out with methanol. Treated and control seedlings were weighed after 7 days. On average tuliposide treated seedlings weighed 70% less than control seedlings (T-test, P = 0.0058).

20 These results show that 6-tuliposide B has phytotoxic activity.

Claims

A method for producing sugars, in particular glycosides, from spheres, comprising the steps of: a) inducing gum dose in a sphere; b) optionally separating the gum and the bulb to obtain the gum; c) optionally extracting the sugar from the gum under polar or semi-polar conditions.

The method of claim 1, wherein the sphere is a sphere capable of gumosis.

Method according to claim 2, wherein the bulb is selected from tulips, Allium, in particular garlic, hyacinth, blue grape, iris.

The method according to any of claims 1-3, wherein gum dose is induced by infection, stress factors, chemical compounds, etc.

The method of claim 4, wherein the infection includes naturally occurring infection or active infection with

20 Fusarium.

The method of claim 4, wherein the stress factors are factors that induce internal ethylene production and in particular include injury, pressure, humidity, high or low temperatures, etc.

The method of claim 4, wherein the chemical compound is a plant hormone, particularly selected from jasmonic acid, jasmonate, IAA (indole-3-acetic acid), ethylene, or a compound capable of releasing a plant hormone, or ethephon (2- chloofethylphosphonic acid).

A method according to any of claims 1-7, wherein the glycoside to be isolated is tuliposide B.

9. Use of tuliposide B to inhibit seed germination.