PREPARATION OF DRIED PLANT MATERIAL HAVING AN INCREASED CONTENT OF PHYLLODULCIN

Abstract

The present invention relates to the cultivation (preferably indoor) of Hydrangea species for increased production of phyllodulcin and the provision of a plant material, preferably a dried plant material, having an increased content of phyllodulcin.

Claims

1-13. (canceled)

14. A method for preparing dried plant material comprising at least 2.5 wt. % of phyllodulcin equivalents, based on a total weight of the dried plant material, the method comprising: (a) providing a head or shoot cutting of Hydrangea macrophylla; (b) rooting the head or shoot cutting; (c) using a hydroponic, aeroponic, or fogponic system, cultivating the rooted head or shoot cutting under filtered or shaded natural light to obtain a cultivated plant, and fertilizing the cultivated plant with 5 to 250 grams of a nutrient solution per 100 L of water, the nutrient solution comprising: (i) 5 to 15 wt. % nitrogen source, (ii) 2 to 10 wt. % phosphorus source, (iii) 5 to 25 wt. % potassium source, and (iv) 1 to 20 wt. % magnesium source; (d) harvesting stem or leaves from the cultivated plant after a cultivation period of 20 to 200 days; (e) drying the harvested stem or leaves at a temperature of 20 to 110 C.; and (f) obtaining the dried plant material comprising at least 2.5 wt. % of phyllodulcin equivalents.

15. The method of claim 14, wherein the Hydrangea macrophylla is Hydrangea macrophylla ssp. serrata.

16. The method of claim 14, wherein the Hydrangea macrophylla ssp. serrata is a variety selected from Oamacha, Amacha, Amagi-Amacha, and hybrids or breeds thereof.

17. The method of claim 14, wherein the cultivated plants are exposed to artificial stress at least once during cultivation.

18. The method of claim 17, wherein the artificial stress is selected from targeted UV light exposure, chemical stress, or a combination thereof.

19. The method of claim 18, wherein the artificial stress is exposure to jasmonate.

20. The method of claim 19, wherein the cultivated plants are exposed to jasmonate by foliar application of the jasmonite, by adding jasmonate to water used to water the plant, or by application of the jasmonite in a gaseous form.

21. The method of claim 19, wherein the cultivated plants are exposed to 0.1 mM to 10 mM of the jasmonite.

22. The method of claim 14, wherein the rooting of the head or shoot cutting is carried out in a substrate that is free from soil and peat.

23. The method of claim 22, wherein the substrate is selected from vermiculite, rock wool, coconut fiber, perlite, pon, seramis, volcanic ash, volcanic ash granules, Miscanthus fiber, zeolith, lava, pumice, or mixtures thereof.

24. The method of claim 14, wherein the cultivated plant is cultivated at a temperature of 15 to 30 C.

25. The method of claim 14, wherein the cultivated plant is cultivated with artificial illumination for 12 to 19 hours per day.

26. The method of claim 14, wherein a plurality of cultivated plants is cultivated and wherein the plurality of cultivated plants is cultivated with a planting density of 25 plants/m.sup.2 or less.

27. The method of claim 14, wherein the cultivated plant is cultivated in an ebb and flow, nutrition film, drip irrigation, trickle irrigation, deep water, aeroponic, or aquaponics system.

28. The method of claim 14, wherein the harvesting of stem or leaves from the cultivated plant in (d) is repeated at least once, wherein each harvest is at least 20 days after an immediately prior harvest.

29. The method of claim 14, wherein the stem or leaves harvested (d) and dried in (e) are divided into leaf or stem material by air separation, air floating, or air sieving, to obtain a plant material comprising at least 75 wt. % of leaf material.

30. Dried plant material obtained by the method of claim 14.

31. The dried plant material of claim 30, wherein the dried plant material comprises at least 2.5 wt. % of phyllodulcin equivalents and less than 2.0 wt. % of hydrangenol equivalents, based on the total weight of the dried plant material.

32. The dried plant material of claim 31, wherein the dried plant material comprises less than 20 wt. % of residual moisture, based on the total weight of the dried plant material.

Description

DESCRIPTION OF FIGURES

[0065] FIG. 1 shows the phyllodulcin content (+/ standard deviation, aglycon, determined after fermentation of the dried and re-humidified young leaves with UPLC) after treatment with different concentrations of methyljasmonate (MeJ): directly after MeJ application; C=control: 0 mM; V1: 0.1 mM, V2: 0.5 mM, V3: 1 mM; V4: 10 mM MeJ (n=24 m p=0.01; different characters means statistical significance).

[0066] FIG. 2 shows the influence of targeted fertilization and cultivation in the hydroponic system with the genotype Amagi Amacha.

[0067] FIG. 3 shows the influence of up to five MeJ-applications (5 mM) in the course of up to 8 days. Plants were sampled one day after the last application, genotype Oamacha.

[0068] FIG. 4 shows the influence of up to nine MeJ-applications. At each sampling date, samples were taken from control and MeJ-treated plants (18 composite samples for each group at each sampling date, except for the last sampling date after nine applications, where 90 individual plants of each group were sampled).

[0069] FIG. 5 shows the influence of four MeJ-applications (5 mM solution). Plants were sampled one day after the last application, the genotype is Oamacha.

[0070] FIG. 6 shows influence of four MeJ-applications (5 mM solution). Plants were sampled one day after the last application, the genotype is Amagi Amacha.

[0071] FIG. 7 shows the influence of four MeJ-applications (5 mM solution). Plants were sampled one day after the last application, the genotype is Amagi Amacha.

[0072] FIG. 8 shows the influence of UV-A light and MeJ-applications (5 mM solution). Plants were sampled one day after the last application.

[0073] FIG. 9 shows the influence of UV-A light and MeJ-applications (5 mM solution). Plants were sampled three days after the last application.

[0074] FIG. 10 shows spectral intensity of the LED panels with UV-A peak (A) vs. the wavelength of the used LED panels without UV-A peak (B).

EXAMPLES

Example 1: Quantification of Phyllodulcin and Hydrangenol Equivalents

[0075] For the quantification of phyllodulcin and hydrangenol equivalents, the sampled leaves were dried at 40 C. for 72 h. Subsequently, samples were homogenized using a mortar, moistened and fermented before being analyzed. Fermentation was carried out by adding water (200 L) and finally stopped with methanol (1800 L), followed by ultrasonic extraction for 30 minutes and filtration (membrane filter Chromafil XtraPTFE-20/25). UPLC analyses of samples were performed on a Waters Acquity UPLC I-Class System equipped with an Acquity UPLC &A PDA detector and a commercially available reversed phase C18 column (Luna Omega 1.6 m Polar C18 50x 2,1 mm). A binary solvent system consisting of acidified water (0.1% formic acid) and acetonitrile was used. Detection wavelength was at 254 nm and chromatographic data were processed by Empore 3 Pro 2010.

Example 2: Cultivation of Hydrangea macrophylla ssp. serrata Oamacha Treated With Methyl Jasmonate

[0076] Rooted cuttings (propagated by the company Ktterheinrich, Lengerich) were placed in a greenhouse of the experimental farm of the Osnabrck University of Applied Sciences, Campus Haste (Department of Agricultural and Landscape Architecture). After one week of acclimatization, the plants were potted in 13 cm pots (capacity about 1 liter) filled with clay substrate from Klasmann-Deilmann GmbH. The substrate consisted of 80% peat as well as clay and a base fertilization of 210 mg nitrogen, 150 mg phosphate, 270 mg potassium, 100 mg magnesium and 150 mg sulfur per liter, as well as trace elements. The plants were placed in a quadrangle with 56 plants/m.sup.2 and the ventilation was set to 18 C. at night and 20 C. during the day.

[0077] Six weeks later, 25 experimental plants were selected and placed on a table with a fleece mat and MyPex film in the experimental design.

[0078] Due to the high outdoor temperatures and high irradiation, the greenhouse cell was shaded throughout the experiment. Before and during the experiment, the plants were watered manually. In addition, the liquid fertilizer Ferty 3 Mega from Planta Dngermittel GmbH was applied once a week at a concentration of 0.5 wt.-% as a foliar application during the entire cultivation period, with the exception of the trial period.

[0079] Afterwards, the plants were exposed to different concentrations of methyl jasmonate. Leaf samples were taken from all plants before the first methyl jasmonate application in order to exclude the possibility that the plants differed a priori with respect to their phyllodulcin content and to ensure that this previous (invasive) sampling of the plants (i.e. clipping off the leaves) did not bias the results.

[0080] It could be observed that the phyllodulcin content increases after application of different concentrations of methyl jasmonate (FIG. 1).

Example 3: Cultivation of Hydrangea macrophylla ssp. serrata Amagi Amacha in a Hydroponic System

[0081] The starting material for the hydroponic trials are plants of Hydrangea macrophylla ssp. serrata of the genotype Amagi Amacha. The selected genotype comes from the tea hydrangea collection of the company Ktterheinrich Hortensienkulturen in Lengerich. After the seedlings were rooted, 75 plants of the genotype Amagi Amacha were potted into the agrivermiculite substrate of the company Floraguard and transferred into a hydroponic system. This hydroponic system is a nutrient film technique system (NFT). This technique describes a hydroponic system that supplies water and nutrients to the plants through a thin nutrient film.

[0082] After the seedlings had acclimatized, these plants were supplied with the fertilizer solution Hakaphos blau (10 wt.-% nitrogen, 4 wt.-% phosphate, 7 wt.-% potassium and 2 wt.-% magnesium) from Compo Expert.

[0083] For the first five weeks, the plants were cultivated in the NFT system for 24 h with a 0. 1 wt.-% Hakaphos blau nutrient solution and a pH of 5.8. After another two weeks, the nutrient solution was increased to 0.2 wt.-% Hakaphos blau. Afterwards, YaraTera KRISTA MAG (magnesium nitrate flakes, low chloride, sulfate free, fully water soluble, 15 wt.-% MgO) was added to the 0.2 wt.-% Hakaphos blau. The pH was maintained at 5.8 for the entire time.

[0084] At an average temperature of 18-22 C. and humidity of about 60%, plants were illuminated for 16 hours with the P1-500-VIS LED lamp from Future LED at 238 mol/(m.sup.2*s) (sunlight equivalent PAR). A watering treatment with Neudomk (Neudorff company) against fungus gnat larvae was applied in the third week of culture. Likewise, the plants had to be treated against aphids and spider mites in the fourth week of culture, here the agents Vertimec pro from Syngenta and Spuzit from Progema, both acaricides and insecticides with contact and feeding effect, were used. Regular inspection of the plants for new infestations and diseased leaves was carried out weekly. The cleanliness of the NFT plant and the adjacent area was also controlled.

[0085] Plant sampling was conducted at three time points following the fertilizer amendment. The samples were transferred to a drying cabinet in the laboratory for analysis, here the plants were dried at 40 C. for 48 hrs. The plants developed good and formed more and larger roots than observable in the peat substrate of Example 1.

[0086] The samples prepared for UPLC measurement were mortared uniformly with a pestle. After mortaring, a representative sample was weighed into a cap (2 mL), on average between 10 mg and 17 mg. Then 0.2 mL of water was added and the sample was incubated for 2 h at 40 C. Afterwards, 1.8 mL of methanol was added to the sample and in a further step, the sample was extracted in an ultrasonic bath for 30 min. After extraction, the sample had to be passed through a 0.2 m membrane filter before loading into UPLC. Quantification of hydrangenol (HG) and phyllodulcin (PD) was determined by UPLC with external calibration in mg/mL.

[0087] The detection of the substances was carried out via the rentention time and by means of a UV detector. The results can be seen in FIG. 2.

Example 4: Phyllodulcin Accumulation After Repeated Methyl Jasmonate Application

[0088] 15 plants (or 90 plants for trials with nine times of methyl jasmonate application) of Oamacha hydrangeas cultivated as described in Example 3, were treated at different time points (one time, three times, five times, seven times and nine times) with 5 mM methyl jasmonate. It should be tested, how many methyl jasmonate applications it takes to reach a saturation point beyond which further applications have no significant effect on the phyllodulcin content in the dry matter.

[0089] Physiological effects such as leaf weight, growth rate, dry matter, photosynthetic performance, diseases and healthy growth of applications of <10 mM MeJA could not be detected in any of the trials.

[0090] It is obvious from FIG. 3 that more than one methyl jasmonate application has a beneficial influence on phyllodulcin content in the dry matter of the leaves. These beneficial effects could be validated in another experiment in 2020 by applying MeJ a total of nine times as shown in FIG. 4, using 90 plants in each group.

[0091] The aforementioned saturation point for MeJ-applications is between a total of three to five applications, as can be seen in FIG. 4.

[0092] Once again, these results were successfully confirmed in 2021 using 15 plants in each group, as shown in FIG. 5.

[0093] The beneficial effects of MeJ-applications on the phyllodulcin content in the dry matter of the leaves were also shown using another genotype (Amagi Amacha) in 2020 (sampling of 50 individual plants of each group) as shown in FIG. 6.

[0094] Once again, in 2021 by sampling 15 individual plants of each group, the results shown in FIG. 6 were replicated as can be seen from the results shown in FIG. 7.

Example 5: Phyllodulcin Accumulation After Combined UV-A Treatment and Methyl Jasmonate Application.

[0095] In the same manner as already described, 64 plants (genotype Oamacha) were cultivated in a greenhouse. Four groups were randomly composed, comprising 16 plants each. One group was cultivated under normal conditions (no artificial light, no MeJ-applications). Another group was cultivated under additional artificial light (PPF: 100-130 mol/s) using Sanlight M30 modules. The other two groups were cultivated under modified Sanlight M30 modules, which provide an additional 5.49% UV-A light-faction (UV-A peak at 375 nm, PFF 300-400 nm: 3,5 mol/s; total PPF measured at the height of the plants: 100-130 mol/s).

[0096] One of those two groups was also treated four times with MeJ in the same manner as already described. The cultivation of all four groups under these conditions lasted seven days, before the first samples were taken.

[0097] From the results shown in FIG. 8 it is concluded, that especially UV+MeJ has a beneficial effect on the phyllodulcin content in dry leaves and that the combination of UV and MeJ treatment reduces the transient nature of a MeJ-induced elevated phyllodulcin content before harvest (FIG. 9). For comparison, the spectra of the respective UV-VIS and VIS LED panels used are shown in FIG. 10.