WO2017091096A2

WO2017091096A2 - Process for essential oils encapsulation into mesoporous silica systems and for their application as plant biostimulants

Info

Publication number: WO2017091096A2
Application number: PCT/RO2016/000025
Authority: WO
Inventors: Cristina Lavinia NISTOR; Raluca IANCHIŞ; Florin Oancea; Maria-Luiza Jecu; lulia RAUT; Dan DONESCU
Original assignee: Institutul National De Cercetare-Dezvoltare Pentru Chimie Şi Petrochimie
Priority date: 2015-11-27
Filing date: 2016-11-18
Publication date: 2017-06-01
Also published as: WO2017091096A3; RO131823A2; RO131823B1

Abstract

The present invention refers to a process for encapsulation, into mesoporous silica based systems, of essential oils intendent to be used as (bio)products with agricultural applications, and to a process for using these resulted encapsulated essential oils as plant biostimulants, especially for the cultivated medicinal / nutraceutical plants treatments. Process for essential oils encapsulation into mesoporous silica systems, consist of the following steps: preparation of a solution of sodium silicate and distilled water, in a ratio of 0.7 parts of silicate to 88,62-89,6 parts of water, under stirring for 30 min, at 500 revolutions per minute and at 40°C temperature; bringing over the sodium silicate solution of a 9.7-10.68 parts of ethanol solution, that contains 7.1- 7.16 parts of ethanol, 1.88-1.86 parts oleic acid, 0.14-0.86 parts 3- aminopropyltriethoxysilane, 0.52-0.86 parts thyme, lavender, basil or cinnamon essential oils, the parts being expressed in mass units; keeping the mixture under stirring for another 4 hours, at 40°C, and transferring the 100 parts of homogeneous and opaque formed dispersion in tightly sealed ampoules. Process of using essential oils encapsulated in mesoporous silica systems for the biostimuiation of the cultivated nutraceutical plant, Passiflora incarnata L. and Momordica charantia L., in order to promote additional accumulation of bioactive compounds, involve the application of doses of 2-4 kg/ha of microcapsules dispersion, with 0,86% thyme oil, in two treatments, at the beginning and at the end of blooming.

Description

PROCESS FOR ESSENTIAL OILS ENCAPSULATION INTO MESOPOROUS SILICA SYSTEMS AND FOR THEIR APPLICATION AS PLANT BIOSTIMULANTS

The present invention refers to a process for encapsulation, into mesoporous silica based systems, of essential oils intendent to be used as (bio)products with agricultural applications, and to a process for using these resulted encapsulated essential oils as plant biostimulants, especially for the cultivated medicinal / nutraceutical plants treatments.

There are known various processes for encapsulation of essential oils which are used as inputs into plant cultivation technologies. Such processes intent to obtain a controlled / gradual release of the respective essential oils active ingredients and thus to extend their biological activity. The patent US 9101 143 B1 claims the production of microcapsules loaded with essential oils, through an interracial polymerization of an aqueous emulsion, of essential oils solubilised in non-volatile hydrophobic carriers - nonvolatile essential oils, vegetable oils or a combination between those. By the interfacial polymerization, a polyurea and/or polyurethane film is formed around the essential oil drops, dissolved in the non-volatile hydrophobic carrier. The monomers involved in the interfacial polymerization/ polycondensation are distributed in the aqueous phase.

The patent application US2015264921 A1 describes a process that includes the dissolution of a di- or poly-isocyanate into essential oil, the emulsification of the resulted mixture in an aqueous solution containing a di- or poly-amine and/or a di- or poly- hydroxylic compound, and realization of the essential oils encapsulation through an interfacial polymerization, which results in the formation of a polyurea and/or polyurethane film around the essential oil drops. The formed film improves the stability of the essential oil, reduces the evaporation rate and controls the release rate when is applied on a substrate.

The patent application WO2007094000 A2 refers to a process of producing microcapsules that contain essential oils in an aqueous medium. The respective microcapsules were prepared by mixing at least one alkanoic acid, selected from decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, tetracosanoic acid, 11- octadecenoic acid, 5,8,1 1 ,14-eicosatetraenoic acid and omega-3 fatty acids, with at least one essential oil, followed by the addition of an aqueous alkaline solution, for obtaining an emulsion, and by mixing in the resulted emulsion of an aqueous solution of a salt containing a multivalent cation, that complexes with the acid groups of the alkanoic acid, stabilizing the microcapsules. In the patent application US2011268780 A1 the same inventors, from the previously mentioned patent, reports the production of microcapsules that contain a solid core and at least one essential oils mixed with a solid porous material, wherein this solid core is coated with a polyurea and/or polyurethane film layer or an amphipathic shell composed of a multivalent salt form of at least one alkanoic acid. Further are presented composition including these microcapsules loaded with essential oils and agricultural and environmental applications.

The patent application EP2737799 A1 protects a biopesticide which contains encapsulated essential oils and fatty acids potassium salts. In the first step, the vegetable oil is treated at ambient temperature with a potassium hydroxide solution in the presence of a couple of surfactants and a solvent, resulting in fatty acid potassium salts with a concentration of 30-40%. In the second step, the hydrogenated vegetable oil or the wax, the essential oil, a pair of surfactants and water are heated under stirring. The mixture is cooled in a controlled manner, resulting in a suspension of solid lipid nanoparticles, that have a shell made of hydrogenated vegetable oil or wax and a liquid core consisting of essential oils. The final product is a suspension (with fluid / suspending medium represented by the potassium soap), wherein exists nanoparticles formed by volatile liquid oil (inside the core) and a shell formed by solid hydrogenated oil. The process for nano-particle formation is based on the property of the hydrogenated oils and waxes to solidify, when their melted forms is cool-down to an ambient temperature. The resulted product is sprayed on plant leaves, after being diluted with water in accordance to required dose.

The patent GR 1008453 B discloses a process for a slow release of the volatile components from essential oils for a period of 24 days, after the formation of "oil in water" microcapsules, that contains different essential oils (orange oil, lemon oil, mixture of orange and lemon oil), as well as the utilization of the respective essential oils for the control of various insect populations.

The patent application EP2684457 A1 presents a process for obtaining a natural herbicide that contains essential oils, alone or in mixtures. The composition of this natural herbicide is characterized by a key component (essential oil or a mixture of essential oils), in combination with a nano-carrier, that can be a temperature resistant starch, different types of maltodextrins, proteins, polysaccharides, gelatine, pullulan, polyethylene oxide or a combination of these.

The disadvantages of the known encapsulation procedures are determined by the variation in time of the essential oil release rate. Initially, the release of the essential oils from the intact structures in which they are encapsulated is slow, but subsequently, as these structures are degrading, the release rate increases.

The mesoporous materials belong to another type of structure, more stable, from which the release of volatile components from essential oils is performed at a more constant rate, for a longer period. The patent application EP2662069A2 refers to a mesoporous material in which at least some of the pores are loaded with essential oils. However, the finalisation of the encapsulation process is made by covering the pores with a biodegradable polymer film, and the degradation in time of the covering film of the mesopores determines in the end the same technical problem of time variation of the essential oil release rate.

In the case of the mesoporous materials, a derived technical problem is the costs of obtaining them, that involves either expensive reagents, either equipment that needs big investments costs.

Another disadvantage of the microcapsules obtained through different procedures that involves the utilization of oil-water emulsions is determined by the difficulty of stabilizing them at a subsequent dilution into water, required for treatment application. This stability issue is accentuated in hard water, which is often used in agriculture.

Therefore, are necessary encapsulation procedures of the essential oils in structures where the release rate, of efficient essential oil concentrations, is more constant, for a longer period. The technical problem that this invention is solving is to develop a process wherein the encapsulation of the essential oils is done rapidly, in structures made from easily accessible components, that exhibit a high stability and could ensure a constant release rate of the volatile components from the essential oils, for a long-time, of at least 6 months.

Another objective of this invention is to describe a process to obtain a final composition wherein the components are in optimal ratio, that allows the dispersion and the stabilization of the essential oils in aqueous medium, including after a subsequent dilution, due to the presence of a biocompatible stabilizer.

Is another objective of this invention to disclose a process through which the encapsulated essential oils are used as plant biostimulants, especially of nutraceutical plants, for the activation of the secondary metabolism and the accumulation of active biological compounds / phyto-nutrients.

The essential oils are known to have actions for plant protection / as„green" pesticides (see for ex. highly cited review, Isman, 2000, Crop Protection, 19: 603-608). However only recently it was proven the activation of the defence mechanisms from plants as a result of the foliar treatment with essential oils (Ben-Jabeur et al. 2015, Plant Physiology and Biochemistry, 94:35-40), without claiming the biostimulant action, which also promote the accumulation of active biological compounds / phytonutrients.

The process for encapsulation of various essential oils into mesoporous silica systems, according to the invention, consists in the following steps:

Preparation of a solution of sodium silicate and distilled water, in a ratio of 0.7 parts of silicate to 88,62-89,6 parts of water, under stirring for 30 min, at 500 revolutions per minute and at 40°C temperature;

Bringing over the sodium silicate solution of a 9.7-10.68 parts of ethanol solution, that contains 7.1-7.16 parts of ethanol, 1.88-1.86 parts oleic acid, 0.14-0.86 parts 3-aminopropyltriethoxysilane, 0.52-0.86 parts thyme, lavender, basil or cinnamon essential oils, the parts being expressed in mass units; Keeping the mixture under stirring for another 4 hours, at 40°C, and transferring the 100 parts of homogeneous and opaque formed dispersion in tightly sealed ampoules.

The technical sodium silicate used during the above process contains 14.2% Na₂0 and 27.58% Si0₂.

The process of using essential oils encapsulated in mesoporous silica systems for the bio-stimulation of the cultivated nutraceutical plant, Passiflora incarnata L. and Momordica charantia L, in order to promote additional accumulation of bio-active compounds, consists in applying a dose of 2-4 kg/ha of microcapsule dispersion, with 0,86% thyme oil, in two treatments, at the beginning and at the end of blooming, and involves the following steps: dilution of the opaque and homogeneous dispersion of thyme essential oil encapsulated in mesoporous silica, 1 part of homogeneous dispersion to 99 parts of water, spray application of the resulted emulsion / suspension on the leaves, with a spraying volumes of 200 litres per ha at the beginning of blooming and of 400 litres per ha at the end of blooming.

The use of thyme essential oil encapsulated in mesoporous silica systems leads to an increase of the accumulation of antioxidant compounds in the Passiflora incarnata leafs of at least 10% and an increase of the accumulation of anti-diabetic compounds in the Momordica charantia leaves of at least 15%.

The invention presents the following advantages:

Allow the production of ecological agro-chemical products, based on natural essential oils (thyme oil, lavender oil, basil oil or cinnamon oil), as bio-active ingredients, encapsulated into mesoporous silica microparticles, stabilised by the oleic acid - sodium oleate vesicular complex, and formulated as homogeneous aqueous dispersions, stable for at least 6 months, even in the case of additional dilution with water, including the standard hard water;

Ensures the in-situ production of the mesoporous silica particles, wherein the essential oil encapsulation in done, in the presence of the essential oils dissolved in ethanol, of oleic acid and a co-structuring agent, 3-aminopropyltriethoxysilane;

s Allows the formation of mesoporous silica particles, the non-volatile matrix in which it takes place the encapsulation of the volatile active ingredient, and their stabilization by the vesicular complex formed of oleic acid and its sodium salt, resulted after the neutralization of sodium silicate;

Establishes the optimal ratio between sodium silicate and oleic acid, so that the sodium ions from the silicate solution are neutralized by half of the oleic acid quantity existing in the synthesis system; the remaining quantity of oleic acid together with the sodium oleate will form the resulted stabilizing vesicular complex oleic acid - sodium oleate;

Uses together with the sodium silicate, applied as ingredient in the synthesis of mesoporous silica particles, a co-structuring agent, 3-aminopropyltriethoxysilane, in a ratio between 20% and 100% towards the essential oil subjected for encapsulation, with the purpose of increasing the capacity of the mesoporous silica particles to encapsulate essential oil, with no phase separation taking place in the final aqueous formulations;

Uses optimal concentrations of essential oils, between 0,5 and 1 ,5%, preferably between 0,8 and 1.2% towards the water present in the reaction system, so that the obtained aqueous dispersions are homogeneous and time stable (at least 6 months), and, in the same time, biological efficient;

Enlarge the spectrum of essential oils application as agricultural inputs, beside their known disinfectant, antifungal, insecticidal and herbicide action, describing a process wherein the essential oils, respectively thyme oil, are used as biostimulant for the nutraceutical plant cultures Passiflora incarnata L. and Momordica charantia L, to accumulate additional bioactive compounds.

Further are presented some examples of the embodiment of the invention, that illustrates it without limiting it.

Example 1. In a reaction vessel, which include a magnetic stirrer (-500 rotations/minute), were introduced 0.7% technical sodium silicate (14.2% Na₂0; 27.58% S1O2) and 89.6% distilled water. The mixture was homogenized for 30 minutes under stirring, at 40°C. Over this mixture is added another solution consisting of 7.16% absolute ethanol, 1.88% oleic acid, 0.14% 3-aminopropyltriethoxysilane and 0.52% thyme essential oil. The obtained mixture become rapidly opaque and is kept under stirring for another 4 hours, at 40°C. Finally, the resulted homogeneous dispersion is transfused in tightly closed ampoules. The dispersion remained stable (without phase separations) within the 6-months examination.

Example 2. The process is as in example 1 , with the only difference that in this case is used lavender oil.

Example 3. The process is as in example 1 , with the only difference that in this case is used basil essential oil.

Example 4. The process is as in example 1 , with the only difference that in this case is used cinnamon essential oil.

Example 5. In a reaction vessel, which include a magnetic stirrer (-500 rotations/minute) were introduced 0.7% technical sodium silicate (14.2% Na₂0; 27.58% Si0₂) and 88.62% distilled water. The mixture was homogenized for 30 minutes under stirring, at 40°C. Over this mixture is added another solution consisting of 7.1% absolute ethanol, 1.86% oleic acid, 0.86% 3-aminopropyltriethoxysilane and 0.86% thyme essential oil. The obtained mixture become rapidly opaque and is kept under stirring for another 4 hours, at 40°C. Finally, the resulted homogeneous dispersion is transfused in tightly closed ampoules. The dispersion remained stable (without phase separations) within the 6-months examination.

Example 6. The process is as in example 1 , with the only difference that in this case is used lavender oil.

Example 7. The process is as in example 1 , with the only difference that in this case is used basil essential oil.

Example 8. The process is as in example 1 , with the only difference that in this case is used cinnamon essential oil.

Example 9. Were performed determinations for the dimension of the mesoporous silica particles, Zeta potential values and pore diameters, in sample obtained per Example 1- Example 8.

Prior to perform particle size measurements (hydrodynamic average diameters) and Zeta potential, the samples were diluted as follows: 0.4 mL sample were introduced in a 25 mL graduated flask and subsequently was brought to the mark with distilled water. The diluted samples were homogenized by ultrasonication for 10 minutes at 50°C, using an ultrasonic bath Bandelin Sonorex Digitec. Measurements were conducted using a Zetasizer Nano ZS 3600 instrument (Malvern Instruments Ltd. UK). The results are expressed as an average of 6 successive measurements. For the determination of hydrodynamic average diameters, was used the DLS technique (Dynamic Light Scattering), and for the determination of Zeta potential average values was used the LDV technique (Laser Doppler Velocimetry).

The determinations regarding pore diameters were performed using a Quantachrome NOVA 2200e instrument (Quantachrome Instruments USA), through absorption-desorption of nitrogen. Before analyses were carried out, the samples were prepared as follows: the oleic acid was removed from the final dispersions through 2 successive washes with a pH=9 sodium hydroxide solution and afterwards, centrifuging at 9000 revolutions per minute, using a Hettich Universal 320 centrifuge. Then, the samples were washed again with distilled water, centrifuged, and dried in air at room temperature for 3 days. Next, they were sintered in a calcining furnace at 600°C for 6 h. The calcined samples were degassed at 150°C for 12 h inside the NOVA 2200e instrument.

The obtained mesoporous silica particles have preferable dimensions between 50 and 200 nm and negative Zeta potential values, ideally between -60 and -90 mV. The pore dimensions of the silica particles are between 2 and 30 nm, ideally between 3 and 10 nm.

Example 10. The stable and homogeneous dispersions, obtained according to Example 1 - Example 8, were tested regarding their antifungal action against phytopathogenic fungi such as Botrytis allii, Rhizoctonia solani, Fusarium graminearum, Macrophomina phaseolina, Sclerotinia sclerotiorum, by using the method described in Manso et al. 2015, Food Control, 47, 20-26. For every phytopathogenic agent were performed three trials, by using some blanks represented by the pristine essential oils, distributed in the same concentration in the dispersion medium. The minimum antifungal concentration (MFC) was determined, and was expressed as being the lowest concentration of essential oil were no increase of micro-organisms was observed, after 5 days of incubation at 25°C. It was observed no differences of MFC values between the pristine essential oils and those included in the mesoporous silica microparticles, obtained per Example 1 - Example 8. Example 11. Was tested the stability of the resulted suspo-emulsions after 1 % dilution in the standard (hard) water of the dispersions obtained per Example 1- Example 8, by using the MT 180 Dispersion stability of suspo-emulsions method (CIPAC Handbook, Ashwort et al. eds, 2005). The standard water was prepared using M29 WHO method (http://www.who.int/whopes/quality/en/MethodM29.pdf). All the tested dispersions corresponded regarding the stability, without phase separation at every 2 hours and with a re-suspension after 24 hours.

Example 12. The homogeneous and stable dispersion obtained according to Example 5 was tested regarding its biostimulant action for nutraceutical plants, determining the influence of the foliar treatments on the accumulation of active biological compounds in Passiflora incarnata L. and Momordica charantia L The nutraceutical plants were cultivated on a reddish molic preluvosol, well and balanced fertilized, according to agrochemical recommendations. The foliar treatments were applied in the second decade of May and on the beginning of July, when the plants were early blooming, and respectively, at the end of blooming. Two equivalent doses were applied, consisting of 2 kg/ha microcapsules dispersion with 0.86% thyme oil at early blooming, and respectively, 4 kg/ha at the end of blooming. Was applied a 1% solution of homogeneous and stable dispersion obtained according to Example 5, in a spraying norm equivalent to 200 Uha at the beginning of blooming, and 400 L/ha at the end of blooming. The solutions were applied using a SG20 back pump (Stihl AG, Waiblingen, Germania), spraying from a 40 cm distance, with a spraying pressure set at 275 kPa, using a flat spray nozzle and limited drift (TeeJett^® flat-fan TT1 1002 model, Spraying Systems Co., Wheaton, IL, SUA). The treatments were performed in an experiment that included also a control non-treated with essential oils, randomly placed in 4 repetitions.

Sample were collected after 2 weeks from each treatment and on these sample were determined the main biological active ingredients.

The vegetable material (P. incarnata leaves, M. charantia fruits) was dried at 50°C and further extracted in 70% (v/v) ethanol, in a 1.5:10 (m/v) ratio, at room temperature, for 10 days. The extracts were filtered, and the samples were stored at 4°C until further utilization. The dry mass was determined using a moisture analyzer (Radwag, Radom, Poland).

In the extract of P. incarnata vegetable material was determined the antioxidant activity, by measuring the extracts' capacity for scavenging the radical cations produced by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) and of the stable radicals generated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH). The results were expressed as Trolox (TEAC) equivalent per g of dry matter and, respectively, as % of DDPH inhibition (Gaspar et al. 2014, Romanian Biotechnological Letters, 19: 9353- 9365).

Tab. 1. The antioxidant activity in the vegetable material extract from Passiflora incarnata plants.

The results presented in Table 1 proves that the foliar application of dispersions of thyme essential oil encapsulated into mesoporous silica, obtained according to Example 5, determines an increase of the antioxidant activity with over 10% in P. Incarnate leaves. The antioxidant activity is directly related with the phyto - therapeutically utilization of Passiflora plants (Sarris et al. 2013, CNS Drugs, 27: 301- 319).

In the extract of M. charantia vegetable material was determined the inhibiting activity of protein-tyrosinephosphatase 1 B, a trans-membrane protein, with a major role in type II diabetes, non-insulin-dependent, which is inhibited by cucurbitan type sapogenin tripertenes from M. charantia. (Zeng et al. 2014. European Journal of Medicinal Chemistry, 81 : 176-180). It was used the method described by Lund et al. 2004 (Journal of Biological Chemistry, 279: 24226-24235), using pNPP (para- nitrophenylphosphate) as a substrate. The assay buffer (pH=7.4) consisting of 50 mM 3,3-dimethylglutarate, 1 mM EDTA, 1 mM dithiothreitol, was adjusted to an ionic strength of 0.15 M by adding NaCI. The tests were performed in a microtiter plate with 96 wells, made of polypropylene, with a working volume of 250 μΙ_ (Nunc™ 96-Well Polypropylene MicroWell™ Plates, Thermo Scientific, Waltham, MA, USA). The corresponding extract concentrations (0 and 30 pL) were added to the assay buffer containing 0 or 2.5 mM pNPP (final concentration) with a total volume of 200 pL. The reaction was initiated by adding 20 pl_, containing 10 proteine-tyrosinephosphatase units (PTP1 B, Prospec, Rehovot, Israel). It was incubated for 30 minutes at 37°C. The reaction was ended by the addition of a 30 μΙ_ 0.5 M NaOH solution. The absorbance was measured in the microtiter plate at 405 nm, using a plate reader (FluoroStar Omega, BMG LabTech, Offenburg, Germania), having the possibility to correct the absorbance caused by the substrate when the enzyme and the compounds are not present. As a positive blank, the activity of PTP1 B was determined in the presence of sodium vanadate, Na₃VO₄, a known inhibitor of protein-tyrosinephosphatasel B activity. The results were expressed as inhibiting % and are presented in Table 2.

Tab. 2. Inhibiting activity of proteine-tyrosinephosphatasel B, PTP1 B, in the vegetable material extract from Momordicacharantia plants.

The results presented in Table 2 proves that the foliar application of dispersions of thyme essential oil encapsulated in mesoporous silica, obtained according to Example 5, determines an increase of the inhibiting activity of the enzyme involved in type II diabetes, proteine-tyrosinephosphatasel B, with over 15% in Momordica charantia vegetable material.

The application of thyme essential oil as bio-stimulant for nutraceutical plants, with a promoting action on the accumulation of the bioactive compounds, represents a new application for essential oils, and particularly for the thyme oil, which was not yet claimed until now. The controlled release of thyme essential oil components, from mesoporous silica particles, in-situ synthesized through the process disclosed in the invention, allows a better expression of this plant biostimulant action, du to time extension of the exposure time.

Claims

1. Process for essential oils encapsulation into mesoporous silica systems, according to invention, characterized in that consist of the following steps: preparation of a solution of sodium silicate and distilled water, in a ratio of 0.7 parts of silicate to 88,62-89,6 parts of water, under stirring for 30 min, at 500 revolutions per minute and at 40°C temperature; bringing over the sodium silicate solution of a 9.7- 10.68 parts of ethanol solution, that contains 7.1-7.16 parts of ethanol, 1 .88-1 .86 parts oleic acid, 0.14-0.86 parts 3-aminopropyltriethoxysilane, 0.52-0.86 parts thyme, lavender, basil or cinnamon essential oils, the parts being expressed in mass units; keeping the mixture under stirring for another 4 hours, at 40°C, and transferring the 100 parts of homogeneous and opaque formed dispersion in tightly sealed ampoules.

2. Process for essential oils encapsulation into mesoporous silica systems, according to claim 1 , characterized in that the technical sodium silicate used during the above process contains 14.2% Na₂0 and 27.58% S1O₂.

3. Process of using essential oils encapsulated in mesoporous silica systems for the biostimulation of the cultivated nutraceutical plant, Passiflora incarnata L. and Momordica charantia L, in order to promote additional accumulation of bioactive compounds, according to invention, characterized in that consists in applying a dose of 2-4 kg/ha of microcapsule dispersion, with 0,86% thyme oil, in two treatments, at the beginning and at the end of blooming, and involves the following steps: dilution of the opaque and homogeneous dispersion of thyme essential oil encapsulated in mesoporous silica, 1 part of homogeneous dispersion to 99 parts of water, spray application of the resulted emulsion / suspension on the leaves, with a spraying volumes of 200 litres per ha at the beginning of blooming and of 400 litres per ha at the end of blooming.

4. Process of using essential oils encapsulated in mesoporous silica systems for the biostimulation of the cultivated nutraceutical plant according to claim 3, characterized in that its application leads to an increase of the accumulation of antioxidant compounds in the Passiflora incarnata leaves of at least 10% and an increase of the accumulation of anti-diabetic compounds in the Momordica charantia fruits of at least 15%.

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