RO136073A0 - Process for preparing an emulsion-type natural antifungal preparation for systemic treatment of crop plants - Google Patents

Abstract

The invention relates to a process for preparing a natural antifungal preparation for the systemic treatment of crop plants. According to the invention, the process consists of the following steps: preparation of floral waters from plants of the Lamiaceae family, as a solution with a minimum 1% dry matter content, preparation of an adjuvant as a stable oil-in-water emulsion from a mixture of vegetable oils chosen from sunflower, olives and rapeseed, glycerol, ethyl alcohol and an emulsifier such as soy lecithin or phosphatidylcholine, addition of the solutions over the emulsion-type adjuvant, as drops, while stirring, homogenization of the emulsion in the presence of 0.1...1.5% nonionic surfactant and addition of water up to 1000 g, to result in stable emulsions with 2...10% dry matter content, pH of 6...7.5, having an antimicrobial effect on diseases such as plant rust, root rot, in organic plant crops.

Description

PROCEDURE FOR OBTAINING AN EMULSION-TYPE ANTIFUNGAL NATURAL PREPARATION FOR THE SYSTEMIC TREATMENT OF PLANTS IN CULTURE

Adriana Florina Popescu, Cristina Mihaela Luntraru, Alexandra Gaspar-Pintiliescu, Ana-Maria Prelipcean, Lucia Moldovan, Oana Crăciunescu

The present invention refers to a process for obtaining a natural preparation with antifungal properties in the form of a water-in-oil emulsion, based on floral waters from aromatic plants and an oily adjuvant, intended for the systemic treatment of cultivated plants by extending the contact period with plant tissue and increased absorption capacity and acting in an environmentally friendly way.

It is known that floral waters are obtained following the process of distilling aromatic plants and separating them from the volatile oils that remain on their surface. They are considered by-products, used in particular in aromatherapy, cosmetic skin care and industrial preparation of cakes and beverages (Paolini J. et al., Journal of Chromatography Part A, 2008, 1193, 37-49; Aazza S. & Miguel MG, Journal of Medicinal Plant Research, 2011, 5, 6688-6696; Acimovic M.G. et al., Advances in Technology, 2020, 9, 54-70). Floral waters contain bioactive plant compounds such as water-soluble polyphenols and the aqueous fraction of volatile oils, which give them a pleasant smell. The most famous floral waters are those from rose and orange, used especially in the Mediterranean area.

It is known that agriculture based on synthetic fungicide products leads to environmental pollution (soil, water), as well as agricultural products with residual toxicity, which can affect human health. in addition, their handling is dangerous and requires special conditions of packaging, transport, storage and use. That is why it is recommended to use products made from natural compounds with antimicrobial action and low toxicity for the development of ecological and sustainable agriculture.

Various fungicidal products based on natural plant extracts are known for the protection and treatment of cultivated plants. The product called Bioprotekt is obtained from neem volatile oil (Azadirachta indica), organic matter, microorganisms, volatile oils, humic and humic acids, which act as an organic insecto-fungicide in the control of some pathogenic fungi in plant crops, such as Puccinia recondite, Puccinia striiformis, Fusarium spp. and Erysiphe graminis (www.biohumussol.ro/produs/bioprotekt). Nimbidin and gedunin sense toxic bioactive compounds isolated from volatile neem seed oil that provide an activity

STATE ORGANIZATION FOR INVENTIONS AND MAR» ¹

Deposit date......j, 2022 biofungicide (Kumar R. et al., Bioactive constituents of neem, In: Synthesis of Medicinal Agents from Plants, pp. 75-103, 2018).

It was shown that the extracts of Curcuma longa rhizomes obtained in hexane and ethyl acetate contain the bioactive compound curcumin and showed fungicidal activity against the phytopathogens Rhizoctonia solani and Phytophthora infestans, ensuring the protection of cultivated plants against diseases such as rice sheath rot and respectively, the gray rot of tomatoes (Kim M.K. et al., Journal of Agricultural and Food Chemistry, 2003, 51, 1578-1581; Han J.W. et al., Journal of Environmental Science and Health Part B, 2018, 53, 135-140 ). The volatile oils extracted from the plants Hyptis marrubioides, Aloysia gratissima and Cordia verbenacea were effective in controlling the plant rust disease caused by the pathogen Phakopsora pachyrhizi by applying preventive treatments (da Silva A.C. et al., European Journal of Plant Pathology, 2012, 134, 865-871).

There are indications that floral waters have an antioxidant and antimicrobial action suitable for applications in organic agriculture (D'Amato S. et al., Food Control, 2018, 86, 126-137). Thus, the in vitro experimental models showed that floral waters obtained from plant species belonging to the Lamiaceae family, namely from Mexican oregano (Poliomintha longiflora), field thyme (Thymus vulgaris), rosemary (Rosmarinus officinalis), garden thyme (Satureja hortensis ), oregano (Origanum vulgare) and basil (Ocimum basilicum) showed antioxidant and bacteriostatic properties against species of Bacillus sp., Enterobacter sp., Staphylococcus sp. and E. coli, as well as fungistatic activity on strains of Rhizoctonia solani, Fusarium oxysporum, Botyris cinerea and Alternaria citri (Cid-Perez T.S. et al., Plants, 2019, 8, 22; Hay Y.O.M. et al., International Journal of Phytocosmetics and Natural Ingredients, 2015, 2, 7; Boyraz N. & Ozcan M, Fitoterapia, 2005, 76, 661-665; Sagdic O. & Ozcan M., Food Control, 2003, 14, 141-143). As a result of these studies, the floral waters obtained from these species were recommended as natural disinfectants for food, prolonging the quality of fresh fruits and vegetables during their storage (Tornuk F. et al., International Journal of Food Microbiology, 2011, 148, 30 -35; Tornuk F. et al., International Journal of Food Properties, 2014, 17, 587-598). instead, lavender floral water (Lavandula augustifolia) showed significant antibacterial activity only after a 50-fold concentration of the extract or its formulation as a nanoemulsion (Prusinowska R. et al., Natural Product Research, 2016, 30, 386- 393; Silha D. et al., Molecules, 2020, 25, 5654; Garzoli S. et al., Industrial Crop Production, 2020, 145, 112068; Kunicka-Styczynska A. et al., Letters in Applied Microbiology, 2015, 60, 27-32).

Antimicrobial properties exerted by hydrophobic and/or hydrophilic interaction with the microbial cell membrane vary depending on the chemical structure of the constituent compounds. Thus, compounds with alcohol and alkyl functional groups (menthol, thymol, linalool) or epoxide groups (eucalyptol) show a higher antimicrobial activity, and those with alkene (terpinene, limonene) and benzene ring (carvacrol) groups have a moderate activity (Basavegowda N. & Baek K.H., Biomolecules, 2021, 11, 1267).

It was also shown that a combination of floral water and volatile oil of garden thyme (Thymus vulgaris) or Spanish oregano (Coridothymus capitatus) floral water and the antifungal drug itraconazole showed a synergistic effect, resulting in an antimicrobial effect higher, compared to individual floral waters (Hay Y.O. et al, Journal of Pharmacy & Pharmacognosy Research, 2018, 6, 216-230; Marino A. et al., BMC Complementary Medicine Therapy, 2020, 20, 1-11) .

However, in order to obtain fungicidal products useful in the treatment of rice plant crops, CN patent 107950597 (A)/24.04.2018 described a mixture of polysaccharides extracted from Astragalus root combined with cinnamon volatile oil. Also, patent CZ 2016819 (A3)/l 1.07.2018 refers to a fungicidal product for plant protection consisting of a mixture of volatile oil of Thymus vulgaris and rapeseed oil encapsulated in a biopolymer of gelatin-chitosan and polyethylene glycol and its preparation method.

The disadvantage of contact fungicides is that they act only on the surface of the plant and do not penetrate the plant tissue, and products based on essential oils have a high volatility, which reduces the effectiveness of treatment by foliar application. in addition, the insolubility in water causes a slow absorption in the plant tissues, and the treatment must be used preventively because, after the disease has set in, the effectiveness is minimal. These preparations ensure the protection of the plants for short periods, as long as they are in contact with the plant, and the treatment must be repeated according to the weather conditions, after the last rain or after the last 7-14 days. That is why it is necessary to improve the preparation and reduce the phytotoxicity of fungicidal products, to optimize the applied treatment scheme and their production costs.

The present invention aims to solve these technical problems and proposes a process for obtaining a natural preparation with antifungal properties in the form of a stable water-in-oil emulsion, based on floral waters from aromatic plants associated with transesterified vegetable oil with mono-alcohols and trihydroxyl, emulsifiers and nonionic surfactants, in well-defined proportions. This combination leads to obtaining a preparation with high absorption due to the synergistic effect of the components, the ability to penetrate the plant cell membrane and high stability, thus extending the period of contact with plant tissue for 3 establishing an effective systemic treatment scheme for plants in culture. In addition, the process allows the recovery of floral waters resulting as by-products, soluble in water, following the process of preparing volatile oils from aromatic plants.

The process of obtaining a stable emulsion-type antifungal natural preparation for the systemic treatment of cultivated plants, according to the invention, consists of the following steps:

(a) preparation of floral waters from aromatic plants, preferably from the Lamiaceae family, using Clevenger-type reflux equipment, at 100 °C, for 1.5...3 hours, finally obtaining solutions with a dry substance of minimum 1%;

(b) preparation of an adjuvant in the form of an oil-water emulsion based on vegetable oil, chosen from sunflower, olive and rapeseed, glycerol and ethyl alcohol, the emulsion being stabilized in the presence of an emulsifier, chosen from lecithin or phosphatidylcholine, in well-defined proportions, as follows: (i) the oily phase is initially obtained based on a mixture of vegetable oil, glycerol and ethyl alcohol in a combination ratio of 2:1:1...3:2:3 through the oil transesterification reaction vegetable with glycerol and ethyl alcohol, at a temperature of 45...65 °C, by stirring on a magnetic plate, for 1...3 hours; (ii) separately, prepare the aqueous phase based on water, phosphate buffer and emulsifier, in a combination ratio of 1:30:5...4:50:10 by dissolving lecithin or phosphatidylcholine in a mixture of water with buffer phosphate, by stirring on a magnetic hotplate with heating, at a temperature of 45...65 °C, for 1...3 hours. finally, the oily phase heated to 50...60 °C is added dropwise over the aqueous phase, under continuous stirring. The oil-water pre-emulsion thus formed is homogenized with the help of a manual stirrer at a speed of 6000...8000 rpm, for 3...5 minutes, thus resulting in an oil-water emulsion type additive with a pH of 7, 0...8.5, which can be used immediately or stored in brown bottles at room temperature;

(c) obtaining a natural antifungal preparation in the form of a water-oil emulsion by adding the floral waters obtained in point (a) over the oily adjuvant obtained in point (b) and heated to 45...65 °C, in relation to weight floral water: adjuvant of 1:2...1:3, dropwise, under stirring on a magnetic plate;

(d) homogenizing the emulsion in the presence of 0.1...1.5% nonionic surfactant, chosen between Tween 20 and Tween 80, and adding water up to 1000 g, continuing the homogenization at 60 °C for 1.. .3 hours;

(e) checking and adjusting the pH to values between 6.0...7.5 using a 0.5 M sodium hydroxide solution, finally obtaining emulsions with a dry substance of 2...10% , which is stored in dark bottles. before use for the systemic treatment of cultivated plants by foliar application in the form of a spray, the emulsions are diluted to predetermined concentrations.

The process of obtaining a natural antifungal preparation of the emulsion type for the systemic treatment of cultivated plants, according to the invention, has the following advantages:

- It is feasible, has low cost, does not require complex equipment and has a short duration.

It is based on a simple, high-yield technology for the recovery of some by-products of the volatile oil extraction process.

- It results in a natural product with antimicrobial effect for disease control (plant rust, root rot) in organic plant crops, as an alternative to existing fungicide products.

improves the systemic action of floral waters by combining with an oily adjuvant in the form of a stable emulsion, which ensures penetration of plant tissue after foliar application and long-lasting protection.

It utilizes natural products, biodegradable in the environment, free of cytotoxicity towards plants and consumers, even if they penetrate the tissues of the plant, for the development of an ecological, durable and sustainable agriculture.

- The treatment is applied only 2-3 times per season and is not affected by weather conditions.

The present invention proposal is highlighted by the following examples:

Example 1.

in a 3000 mL glass vessel, prepare 1000 mL of rosemary floral water using Clevenger reflux equipment at 100 °C for 2 hours. The solution obtained at the end had a dry substance of 2%.

Separately, in another reaction vessel, the adjuvant is prepared as a stable oil-in-water emulsion from a mixture of sunflower oil, glycerol, ethyl alcohol and soy lecithin in two steps. in the first stage, the oily phase based on vegetable oil, glycerol and ethyl alcohol is prepared by the transesterification reaction of 8.5 g of sunflower oil in the presence of an amount of 2 g of glycerol and 4 g of ethyl alcohol, at a temperature of 60 °C, by stirring on a magnetic plate, for 1.5 hours. in the second stage, the aqueous phase is prepared based on lecithin with an emulsifying role and a phosphate-water buffer mixture, by dissolving 2.5 g of lecithin in 3 g of 0.1 M phosphate buffer, pH 7.4 and 110 g of water, by stirring on a magnetic hotplate at 60 °C for 1.5 hours. finally, the oil phase heated to 60 °C is added dropwise over the aqueous phase under stirring. The formed oil-in-water pre-emulsion is mixed using a hand stirrer at 8000 rpm for 3 minutes, finally resulting in a stable emulsion oil adjuvant with a pH of 8.2, which can be used immediately or stored in brown bottles at room temperature.

Rosemary floral water is then added dropwise over the adjuvant heated to 60°C with stirring on a heated magnetic hotplate. Then add 3 g of Tween 20 as surfactant and make up to 1000 g with water, continuing homogenization at 60 °C for 2 hours. The pH is checked and adjusted to a value of 6.3 with the help of a 0.5 M sodium hydroxide solution. The natural antifungal preparation of the emulsion type is packaged in dark colored bottles and is used for the systemic treatment of plants in culture after dilution and packaging in spray tubes.

Example 2.

in a 5000 ml glass vessel, prepare 2000 ml of floral water from a mixture of sage and rosemary using Clevenger reflux equipment at 100 °C for 2 hours. The solution obtained at the end had a dry substance of 2.5%.

Separately, in another reaction vessel, the adjuvant is prepared in the form of an oil-in-water emulsion, under the conditions shown in example 1, with the difference that the oily phase is based on 10 g of olive oil, 4 g of glycerol and 3 g of alcohol ethyl. The aqueous phase is obtained by dissolving 3 g of phosphatidylcholine in a mixture of 5 g of 0.1 M phosphate buffer, pH 7.4 and 120 g of water, with continuous stirring on a heated magnetic plate at 50 °C , for 2 hours. The oil-water pre-emulsion is formed after heating the oil phase to 50 °C and adding it over the aqueous phase, dropwise and under stirring, followed by homogenizing the mixture at 7000 rpm for 5 minutes.

The natural antifungal preparation of the emulsion type is prepared by adding the floral water of sage and rosemary over the oily adjuvant heated to 50 °C. Then add 3 g of Tween 80 as surfactant and make up to 1000 g with water, continuing homogenization at 50 °C for 2 hours. Adjust the pH to 7.0 with 0.5 M sodium hydroxide solution and store in dark bottles until use.

In order to evaluate the in vitro effectiveness of the formulations of the natural emulsion preparation based on floral waters and oily adjuvant, the antimicrobial activity was determined on bacterial cultures of Staphylococcus aureus and Pseudomonas aeruginosa and, respectively, on fungal cultures of Rhizoctonia solani by the microdilution method. Thus, the bacterial suspensions were obtained by cultivation on brain-heart broth, at 35 °C, overnight. The inoculum was adjusted to a concentration of 10 ⁸ cells/ml using a 0.5 McFarland standard. The culture of pathogenic fungi was maintained on potato-glucose-agar medium at 35 °C for 4 days and then subcultured in phosphate buffered saline by vortexing until a homogeneous suspension was obtained. Method 6 of microdilution was carried out by serial dilutions of the emulsion preparation in the culture medium at concentrations between 0.1-10 mg/ml, prepared in the wells of a culture microplate. Plate incubation was performed at 35 °C and bacterial population growth was measured by reading the absorbance at 600 nm using a SPECTROstar Nano plate reader (BMG Labtech) after 24 hours of cultivation.

The results obtained on both bacterial strains indicated an antibacterial effect of the analyzed natural preparations, which varied between Gram-positive and Gram-negative species and was dependent on the sample concentration (Table 1). Thus, the natural preparations more effectively inhibited the growth of the Gram-positive strain of X. aureus, interfering with the multiplication of bacterial cells starting from the minimum concentration of 1.25 mg/ml, while the inhibition of microbial growth in the case of the Gram-negative culture of P. aeruginosa occurred from a concentration of 5 mg/ml. Gram-negative bacteria are known to be more resistant than Gram-positive bacteria to the action of antibacterial compounds, due to their additional lipopolysaccharide-rich outer membrane. The tests carried out were useful because they indicated the antimicrobial effect of natural preparations, both in Gram positive and Gram negative bacterial cultures.

Table 1. Antimicrobial activity of natural antifungal preparations on strains of pathogenic bacteria P. aeruginosa and S. aureus, as well as on the strain of pathogenic fungi R. solani, compared to the positive control of bacitracin and bifonazole, respectively, determined as a minimum inhibitory concentration.

No. sample Natural antifungal preparation (PNA) Minimum inhibitory concentration (mg/ml) X. aureus P. aeruginosa R. solani 1 NAP 1 1.25 5 1.25 2 NAP 2 1.25 5 1.25 3 Bacitracin (antibacterial control) 0.15 0.15 4 Bifonazole (antifungal control) 0.15

The results obtained on the strain of pathogenic fungi indicated an antifungal activity of the analyzed natural preparation variants starting with the minimum concentration of 1.25 mg/ml, after 24 hours after treatment, comparable to that of the standard compound bifonazole. Thus, the usefulness of these natural fungicidal preparations for treating plants against R. solani infection has been demonstrated.

in order to evaluate the in vivo functionality of the natural product variants, their fungistatic and fungicidal activity was determined on experimental batches of young tomato plants obtained from seeds grown in growth chambers with controlled conditions, for 5 weeks, and infected with spores of Mycospharella sp., known to trigger plant rust disease. Thus, 12 young tomato plants were divided into 3 batches of 4 plants each and were sprayed with 1.5 ml of the natural preparation PNA 1, PNA 2, and respectively with a solution of copper sulfate (control) concentration 5 mg/ml. Another 2 lots served as untreated and uninfected plants and untreated but infected plants, respectively. Batches were maintained overnight in the growth chamber under controlled light and humidity conditions. Plants were then infected by foliar application of an inoculum of 5x10 ⁵ spores/ml, suspended in distilled water containing Tween 20. Plants were covered with transparent plastic bags overnight to maintain humidity. Then, the plants were grown under temperature and humidity controlled conditions for 7 days and the occurrence of specific plant rust spots was monitored daily. Measurements were made of the damaged area on treated and untreated leaves and the efficiency of the natural preparation was calculated using the formula:

Efficiency (%) = 100 x (1 - average lesion area (%) on treated leaves / average lesion area (%) on untreated leaves)

The results demonstrated that the natural preparations PNA 1 and PNA 2 had a high efficiency of protection of young tomato plants against Mycospharella sp. and the appearance of spots specific to plant rust disease (Table 2).

Table 2. Efficacy of natural preparations PNA 1 and PNA 2 against infection with spores of

Mycospharella sp. and the appearance of spots specific to the plant rust disease on young tomato plants, compared to a CuSO4 solution and the untreated, infected control.

No. sample Natural antifungal preparation (PNA) Preparation efficiency (%) 1 NAP 1 94.21 2 NAP 2 88.13 3 CuSO4 55.02 4 Untreated, infected control 37.15

in order to establish the treatment scheme with natural antifungal preparations of plants in culture, different variants were applied with a randomized block design with 5 replicates and a split plot over time, on young tomato plants infected with an inoculum of Mycospharella sp. by foliar application as described above. The treatment schemes varied by: 1) the type of preparation, between PNA 1, PNA 2; 2) preparation concentration, between 1.25, 2.5 and 5 mg/ml; 3) the time period between treatment and inoculation, of one hour, 6 hours or 18 hours. after calculating the effectiveness of the preparations, an optimal recommended treatment protocol was established, consisting of the use of a natural antifungal preparation, chosen between PNA 1 and PNA 2, with a concentration of 5 mg/ml, with an interval of 6 hours until exposure to the spores.

Finally, testing the cytotoxicity of young tomato plants treated with the variants of the natural antifungal preparation on a stabilized Caco-2 intestinal cell line by the extract method, at intervals of 24 and 48 hours of cultivation showed that the cell viability was high for all the concentrations tested, the values being between 80-100% (Table 3). Thus, it has been demonstrated that the preparations are biocompatible and do not endanger human health, even if they penetrate the plant tissue.

Table 3. Cell viability of Caco-2 intestinal cell culture grown in the presence of natural antifungal preparation extracts PNA 1, PNA 2 and CuSO ₄ for 48 h, determined by the Neutral Red method. Untreated infected plants served as control.

No. sample Natural antifungal preparation (PNA) Concentration (mg/ml) Cell viability (%) Degree of biocompatibility 1 NAP 1 0.1 105.26 picked up 0.25 97.38 picked up 0.5 82.89 picked up 2 NAP 2 0.1 91.56 picked up 0.25 86.19 picked up 0.5 80.75 picked up 3 CuSO ₄ 0.1 94.54 picked up 0.25 93.31 picked up 0.5 69.77 moderate 4 Untreated, infected control 0.1 99.93 picked up 0.25 97.04 picked up 0.5 91.54 picked up

Claims (1)

CLAIM The process of obtaining a stable emulsion-type antifungal natural preparation for the systemic treatment of cultivated plants, according to the invention, characterized by the fact that it is based on the following stages: (a) the preparation of floral waters from aromatic plants, preferably from the Lamiaceae family, with the help in a Clevenger-type reflux equipment, at 100 °C, for 1.5...3 hours, finally obtaining solutions with a dry substance of at least 1%; (b) preparation of an adjuvant in the form of an oil-water emulsion based on vegetable oil, chosen between sunflower, olive and rapeseed, glycerol and ethyl alcohol, the emulsion being stabilized in the presence of an emulsifier, chosen between lecithin or phosphatidylcholine, in well-defined proportions, as follows: (i) the oily phase is initially obtained based on a mixture of vegetable oil, glycerol and ethyl alcohol in a combination ratio of 2:1:1...3:2:3 through the oil transesterification reaction vegetable with glycerol and ethyl alcohol, at a temperature of 45...65 °C, by stirring on a magnetic plate, for 1...3 hours; (ii) separately, prepare the aqueous phase based on water, phosphate buffer and emulsifier, in a combination ratio of 1:30:5...4:50:10 by dissolving lecithin or phosphatidylcholine in a mixture of water with buffer phosphate, by stirring on a magnetic hotplate with heating, at a temperature of 45...65 °C, for 1...3 hours. finally, the oily phase heated to 50...60 °C is added dropwise over the aqueous phase, under continuous stirring. The oil-water pre-emulsion thus formed is homogenized with the help of a manual stirrer at a speed of 6000...8000 rpm, for 3...5 minutes, thus resulting in an oil-water emulsion type adjuvant with a pH of 7.0. ..8.5, which can be used immediately or stored in brown bottles at room temperature; (c) obtaining a natural antifungal preparation in the form of a water-oil emulsion by adding the floral waters obtained in point (a) over the oily adjuvant obtained in point (b) and heated to 45...65 °C by weight floral water: adjuvant of 1:2...1:3, dropwise, under stirring on a magnetic plate; (d) homogenizing the emulsion in the presence of 0.1...1.5% nonionic surfactant, chosen between Tween 20 and Tween 80, and adding water up to 1000 g, continuing the homogenization at 60 °C for 1.. .3 hours; (e) checking and adjusting the pH to values between 6.0...7.5 using a 0.5 M sodium hydroxide solution, finally obtaining emulsions with a dry substance of 2...10% , which is stored in dark bottles. before use for the systemic treatment of cultivated plants by foliar application in the form of a spray, the emulsions are diluted to pre-established concentrations.