Plant extract of raphanus sativus seeds for protection from damage following cryopre se rvat i on and in vitro incubation of human spermatozoa, usable in in-vitro fertilization procedures

Abstract

The present invention relates to the ability of an aqueous plant extract in dried form of Raphanus sa ti vus seeds (common radish, Brassicaceae family) to exert a protective action against the damage induced by the cryopreservation of human spermatozoa, safeguarding the motility, the viability and the DNA integrity thereof. A similar protective effect is exerted against the damage induced by in vitro incubations of spermatozoa. The subj ect of the invention is also the method for preparing such an extract and the active components representative of the biological properties thereof identified therein, sinapine and sinapic acid derivatives, together with the use thereof in treatment methods for protecting from the cryodamage deriving from the semen cryopreservation, even in the veterinary context, and in the in vitro incubation procedures.

Description

[0001] PLANT EXTRACT OF RAPHANUS SATIVUS SEEDS FOR PROTECTION FROM DAMAGE FOLLOWING CRYOPRESERVATION AND IN VITRO INCUBATION OF HUMAN SPERMATOZOA, USABLE IN IN-VITRO FERTILIZATION PROCEDURES

[0002] DESCRIPTION

[0003] Summary of the invention

[0004] The present invention relates to the ability of an aqueous plant extract in dried form of Raphanus sa ti vus seeds ( common radish, Brassicaceae family) to exert a protective action against the damage induced by the cryopreservation of human spermato zoa, safeguarding the motility, the viability and the DNA integrity thereof . A similar protective ef fect is exerted against the damage induced by in vitro incubations of spermatozoa . Both cryopreservation and in vitro incubation are fundamental procedures for Medically Assisted Procreation (MAP ) techniques .

[0005] The cryopreservation procedure , i . e . , freezing biological material in liquid nitrogen, ~ 196 ° C, is aimed at preserving the biological properties of a sample . However, the cryopreservation procedure at low temperatures also generates damage to the sample itsel f , whereby it is essential to implement strategies to reduce such a damage as much as possible . The preparation used to protect human spermatozoa from cryodamage consists of an aqueous extract from Raphanus sa ti vus seeds . Speci fically, in accordance with the preparation and extraction method of the present invention, the compound sinapine and sinapic acid ( as a representative molecule of the di f ferent glycosidic derivatives of this acid present in the extract ) have been identi fied as the main components of the extract , which in thawed samples demonstrated an improvement in the parameters of viability, motility and DNA integrity of spermatozoa, as presented by the extract . Moreover, the plant extract and the two molecules mentioned above , i . e . , sinapine and sinapic acid, have been shown to protect spermatozoa even during in vitro incubations , a condition known to induce loss of sperm motility and DNA integrity per se . In fact , the addition of this extract , and of the identi fied active ingredients , sinapine and sinapic acid, during the in vitro incubation of spermatozoa improves the two parameters indicated with respect to the control .

[0006] Scope of the invention

[0007] Human semen cryopreservation is currently the only strategy to preserve fertility in subj ects who must undergo gonadotoxic therapies , such as cancer patients . Semen cryopreservation is also indicated for other categories of patients , such as those with oligospermia, and is used for semen donors in heterologous fertili zation . Despite the obvious usefulness of semen cryopreservation, it causes considerable damage to sperm structure and function . In particular, spermatozoa motility and viability are reduced, oxidative stress is induced as well as damage to the acrosome structure and DNA integrity, the latter typically measured as sperm DNA fragmentation ( sDF) . sDF takes particular importance in view of the fact that frozen semen samples are intended for DMA, in particular ICS I ( intraCytoplasmic Sperm Inj ection) , on the outcome of which sperm DNA damage has a known negative effect (Cissen et al, 2016) . The extent of the induced damage basically depends on two aspects: i) the quality of the starting sample and ii) the cryopreservation procedure used.

[0008] Cryopreservation procedures differ from one another in the speed of freezing, the freezing medium (and related cryoprotectants) and the carrier used, all aspects strongly affecting the extent of the damage induced. Another important parameter is the thawing protocol. The inventors referred to the following procedures in carrying out the project: i) procedure A: that used by the CRR - Cryopreservation of the Seminal Liquid - Andrology Laboratory of the Careggi Hospital, which consists of a Vapor Fast Freezing (VFF) procedure which uses the Test Yolk Buffer (cryoprotectants: egg yolk and glycerol) as a freezing medium and 500 pl capillaries as a carrier; and ii) procedure B: a variant of the previous one which uses SpermFreeze (cryoprotectants: glycerol, sucrose and human serum albumin) as a freezing medium in place of the Test Yolk Buffer.

[0009] VFF is a freezing procedure which is carried out in two stages, in the first the sample is placed in nitrogen vapors for 8 minutes (at a freezing rate of about 15.6°C / min) and in the second it is immersed directly in liquid nitrogen. The main difference between Test Yolk Buffer Test and SpermFreeze is that SpermFreeze lacks animal-derived components and is therefore quite widespread, although, in the experimental tradition of Careggi ' s cryobank, it performs worse than Test Yolk Buffer in terms of recovery of sperm motility and viability, after thawing. For thawing the samples, the capillaries were placed at 37°C for 15 min and then the freezing medium was removed by centrifuging and washing with fresh medium.

[0010] As already mentioned, the cryopreservation techniques currently used induce variable damage to the sperm cell depending on the procedure used and the subject from whom the biological sample comes. The results of the damage induced by one of the two procedures (procedure A) used as a reference by the inventors were recently published in a study which used semen samples from normozoospermic subjects, i.e., subjects with all sperm parameters above the reference values (Arciero et al, 2022) . This study showed that, with respect to the fresh sample values, the procedure reduces progressive motility to 38.951 20.12%, total motility to 45.011 19.90%, and viability to 49.011 19.90% of the initial value. Moreover, the aforesaid procedure causes an increase in sDF of more than 50% with respect to the initial value (Arciero et al, 2022) . The procedure also induces high levels of oxidative stress (Arciero et al, 2022) to which much of the damage listed above can be traced. The performance of the same procedure, applied however in a group of subjects more representative of patients who actually resort to semen cryopreservation than the normozoospermic subjects are, is reported in the literature (Degl ' Innocenti et al, 2013) . The other scope of the present invention relates to in vitro incubations, another procedure of the PMA technique. In in-vitro fertilization, the oocytes are inseminated with the semen of the partner, after selection and capacitation of the spermatozoa. In particular, in the IVF technique (In Vitro Fertilization with Embryo Transfer) , the selected spermatozoa are then incubated in vitro together with the oocyte until the latter is fertilized. In all these steps, the spermatozoa are incubated in special culture media. It is known that even in vitro incubation induces damage to the spermatozoa motility and the DNA integrity thereof (Muratori et al, 2003) .

[0011] It is the object of the present invention to reduce the damage induced by both cryopreservation and in vitro incubation, providing a product capable of protecting spermatozoa: said product consists of an aqueous extract obtained from seeds of the Raphanus sativus plant (radish) , obtained from local markets located in Tunisia and Italy, purchased in 2021 and 2022, for a total of five different batches of seeds.

[0012] The invention also relates to the method of preparing such an extract, as described below, and the identified active components, sinapine and sinapic acid derivatives .

[0013] BACKGROUND ART

[0014] The chemical characterization of the plant extract of Raphanus sativus , carried out with chromatographic and mass spectrometry techniques, highlighted, as the main components of the same extract, sinapine and several glycoside derivatives of sinapic acid. Therefore, the inventors considered it necessary to carry out appropriate prior research on the specific use of such components aimed at improving the viability, motility and DNA integrity of cryopreserved spermatozoa and the motility and DNA integrity of incubated spermatozoa.

[0015] The results are as follows:

[0016] Patent document CN110963918 (A) describes specific structures of sinapic acid derivatives and the compound sinapine, as well as a preparation method and the use thereof. The document claims the aqueous extraction of the molecule from radish seeds and the antioxidant action in vitro thereof, without defining a specific pharmaceutical field of use.

[0017] In the article entitled: " Raphanus sativus extract protects against Zearalenone induced reproductive toxicity, oxidative stress and mutagenic alterations in male Balb / c mice." (Ben-Salah-Abbes et al, 2009) , the protective effect of an aqueous extract of Raphanus sativus developed in Tunisia against reproductive toxicity induced by ZEN (Zearalenone) , a non-steroidal estrogenic mycotoxin produced by many Fusarium species in agricultural products and grains, is evaluated.

[0018] In the article "Sinapic acid ameliorates cisplatin-induced disruptions in testicular steroidogenesis and spermatogenesis by modulating androgen receptor, proliferating cell nuclear antigen and apoptosis in male rats," Demir M et al (2022) discuss the ameliorative effects of pure sinapic acid on testicular toxicity induced by cisplatin, a chemotherapy drug used in many types of cancer. Finally, in the article by Rezaei et al ( 2021 ) entitled : " Protective ef fects of sinapic acid against cyclophosphamide-induced testicular toxicity via inhibiting oxidative stress , caspase-3 and NF-kB activity in BALB / c mice , " the improvement of pure sinapic acid against testicular inj ury induced by another chemotherapeutic agent , cyclophosphamide , is evaluated .

[0019] As can be seen, none of the documents highlighted by this research describes or suggests the use of Raphanus sa ti vus extract and the molecules sinapine and sinapic acid in the prevention of spermatozoa cryodamage during semen cryopreservation, nor the protective use of the same extract during in vitro incubation carried out under conditions similar to those involved in the PMA procedures .

[0020] Further features and advantages of the present invention will become apparent from the following detailed description, with reference to the accompanying drawings and tables , which show the results of the various analyses carried out on the aqueous extracts of Raphanus sa ti vus seeds , and the ef fects thereof in cryopreservation and in vitro incubation procedures . LIST OF FIGURES :

[0021] Figures la and lb show the profiles resulting from the chemical analyses of two aqueous extracts obtained by HPLC-DAD-MS a ) from top to bottom, the chromatographic profile at 330nm, to highlight the sinapic acid derivatives (numbered from 1 to 8 in Figure la ) , the corresponding total ion current (TIC) of extract 2 and the same for extract 5, in negative ionization with 180V fragment applied; b) from top to bottom, only for extract 3: chromatographic profile at 330 nm, to highlight the sinapic acid derivatives, the TIC in positive ionization, and Extract Ion (El) at 207 m / z in positive ionization, useful to identify the presence of synapoyl groups in the compounds quantified at 330nm.

[0022] Figure 2 shows the1H-NMR spectra of five dried extracts, where: Radish 6,7 and 8 = Extracts 1, 2 and 3; Radish 9 and 10, Extracts 4 and 5. The main signals over 6 ppm are related to the phenolic protons of the sinapic acid derivatives, those between 3 and 4 ppm correspond to the signals of the sugar units of the synapoyl glycosides;

[0023] Figure 3 shows the calibration curves of the two selected standards, sinapine and sinapic acid. The curves are constructed by HPLC-DAD analysis (the areas in mAu are obtained from the profile at 330 nm) and both show an excellent correlation in the concentration range studied with a correlation factor (R )> 0.999;

[0024] Figure 4a shows the chemical structure of the two standards used for the quantification of the extract;

[0025] Figure 4b shows the typical Ultraviolet-Visible (UV-Vis) absorption spectra of the glycosidic derivatives of sinapic acid (right) and sinapine (left) obtained during HPLC-DAD analysis of the extracts;

[0026] Figure 5 shows the amount of sinapine and the total amount of phenolic compounds (glycosylated synapoyl derivatives) in the five extracts. The data are expressed as mg / g of dried extract (mean ± standard deviation of triplicate measurements) , using the calibration curve of sinapine at 330 nm;

[0027] Figure 6 shows the sum of the glycosylated synapoyl derivatives in the five extracts. The data are expressed as mg / g of dried extract (mean ± standard deviations of measurement in triplicate, using the sinapic acid curve.

[0028] Figure 7 refers to the selection of the biologically active dose of extract to be used for in vitro studies, and shows the histograms of progressive (A) and total (B) motility at time 0 and after 2h of incubation with, and without, the plant extract used at the indicated concentrations: A. a,p (vs 2h control, C2h)<0.01; B. a,p (vs 0 time control, COh) <0.05; b,p (vs 2h control) <0.05. C, control. N=8.

[0029] Figure 8 shows typical SCD test images (used for determination of sperm DNA fragmentation) in the baseline sample (A) and after 24h incubation, with (C) and without (B) the extract. The arrows indicate nonfragmented (NF) and fragmented (F) spermatozoa;

[0030] Figure 9 shows typical TUNEL / DAPI dot plots obtained with the cytof luorimetric technique for the evaluation of sDF referred to as liveTUNEL, capable of evaluating DNA damage in the vital fraction of native semen samples incubated for 24 h with and without the extract. A, baseline sample; B, sample after 24h of incubation; C, sample treated with the extract after 24h of incubation;

[0031] Figure 10 shows the effect of extract (R) , sinapine (S) and sinapic acid (AS) on progressive (A) and total (B) motility after 2h of incubation. A, a,p (vs control at 0h)<0.001; b,p(vs control at 2h) <0.05.

[0032] B, a,p (vs Oh control ) <0.001 ; b,p (vs 2h control) <0.05. N= 6 ;

[0033] Figure 11 shows the effect of extract (R) , sinapine (S) and sinapic acid (AS) on the sDF evaluated with the SCD test, after 24h of incubation; a,p (vs Oh control) <0.01; b, p (vs 24h control ) <0.01. N=6.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] Preparation of the extract

[0036] The seeds were first ground with a coffee grinder (Moulinex Coffee grinder, Two Stainless Steel Blades, 180W , 50G, MC300161) and the powder obtained was immersed in water under stirring overnight. Specifically, 5 grams of seeds were extracted with 200 mL of water and the extraction was carried out at the temperature of 25°C, under magnetic stirring.

[0037] Then the aqueous extract was filtered with 0.45 pm filters (PVDF syringe filter 32 mm 0.45 pm, Rephile Bioscience, French) and frozen at -80°C. The frozen sample was then subjected to lyophilization. The lyophilized extract was kept at -18-20°C in a hermetically sealed container.

[0038] With reference to the method followed, one gram of dried seeds of Raphanus sativus produced 54.25 mg ± 4.35 mg (mean and standard deviation, N=3) of dried extract (DE) . The dry extracts were dissolved in water for chromatographic analyses carried out by HPLC-DAD- MS, or in D2O (deuterated water) to record the proton spectra (1H-NMR experiments) . The mass spectra were acquired in both positive and negative ionization, with a mass range between 100- 1000 Daltons, at a gas temperature of 350°C, nitrogen flow of 12 L / min, pressure of 35 psi, capillary voltage of 3500 V, fragmentation energy between 150 V and 250 V.

[0039] For the biological experiments, the dried extract was dissolved in freezing medium (Test Yolk Buffer or SpermFreeze) or in HTF (Human Tubal Fluid, a culture medium for human spermatozoa) at a concentration of 25 pg / ml .

[0040] The choice of such a concentration was made by testing the effect of the following extract concentrations: 100 pg / mL, 50 pg / mL, 25 pg / mL, 10 pg / mL, 5 pg / mL and 1 pg / mL on sperm motility after 2h of incubation. These experiments did not show a doseresponse trend with the increase of concentrations; however, the concentration of 25 pg / mL was found to be the one most capable of preventing motility loss, both progressive and total, during incubation (Figure 7) .

[0041] The procedure for preparing the aqueous extract was adapted from the procedure published in Salaweh et al (2015) , which reports the effect of an extract of Ci ssampelos capensis on human spermatozoa in vitro.

[0042] Effects on cryodamage

[0043] For the cryopreservation experiments, the extract is added to the freezing medium (Test Yolk Buffer or SpermFreeze) at the concentration of 25 pg / ml (established as described above, Figure 7) .

[0044] The preparation was tested in 32 normozoospermic patients with procedure A, i.e., with Test Yolk Buf fer . Of these patients , 17 were also tested with procedure B, i . e . , with SpermFreeze . The semen features of such patients are shown in Table 1 . In all the patients tested, the protective ef fect of the 5 extract on motility, viability and DNA integrity was found .

[0045] Table 1 . Age , abstinence and main semen parameters of the subjects recruited in the cryopreservation study

[0046] 10

[0047] Effects on incubation-induced damage

[0048] The extract was also tested for the protective ef fects against the damage generated by incubating spermatozoa in HTF in vitro , under conditions similar 15 to those used in PMA procedures ( 37 ° C, atmosphere with 5% CO 2 ) and at the extract concentration of 25 gg / ml ( established as described above , Figure 7 ) . In these experiments , the extract was capable of attenuating, in all the patients tested, the loss of motility 20 (progressive and total ) after 2h of incubation and of preserving much of the integrity of the sperm DNA after 24h of incubation . To evaluate these parameters , unless otherwise indicated, selected sperm preparations were used with swim up and a total of 33 25 semen samples were collected, using a baseline progressive motility >30% as an inclusion criterion . The semen features of these samples are shown in Table 2.

[0049] Table 2 . Age , abstinence and main semen parameters of the subjects recruited in the incubation study

[0050] Methods used .

[0051] Motility was measured under an optical microscope on 10 pl of sample , observing at least 200 spermatozoa

[0052] 10 and distinguishing between progressive , in situ and immobile motility . Total motility is derived from the sum of progressive motility and in situ motility .

[0053] Sperm vitality was evaluated using eosin labeling, with a sample : eosin ratio of 1 : 1 and quanti fied by

[0054] 15 optical microscopy, counting at least 200 spermatozoa per sample . Eos in labeling reveals only non-vital spermatozoa, which appear dark pink, while vital spermatozoa appear white . sDF was evaluated with the SCD ( Sperm Chromatin

[0055] 20 Dispersion) test , a microscopic assay which distinguishes fragmented and non- f ragmented spermatozoa in DNA, based on the absence and presence , respectively, of a halo around the nuclear core ( Figure 8 ) .

[0056] 25 In some incubation experiments , sDF was also evaluated with TUNEL, an assay which adds labeled nucleotides at DNA breaks . In this proj ect , the version of TUNEL which discriminates between vital and non- vital cells was used (LiveTUNEL, Figure 9, Muratori et al, 2020) . By virtue of the ability thereof to focus the vital sperm fraction, liveTUNEL made it possible to use native semen samples, obviating the presence of non-vital spermatozoa in unselected samples. LiveTUNEL includes triple staining with three fluorochromes (DAPI, FITC and LIVE / DEAD Fixable Far Red stain) then quantified with a FACSCanto II cytof luorimeter (Muratori et al, 2020) .

[0057] RESULTS AND DISCUSSION

[0058] Chemical characterization of the extract

[0059] The chemical characterization of the aqueous extract of Raphanus sativus seeds was done following the evaluation of both data reported in the literature (Sham et al, 2013; Kim et al, 2015; Dong et al, 2020; Gao et al, 2022) and data acquired in the laboratory after HPLC-DAD and HPLC-DAD-MS analysis and1H-NMR data (proton spectra) . Following the results of the qualitative chemical analysis, the standards of sinapine and sinapic acid were selected, which were then used for the quantitative analysis of sinapine and synapoyl glycosides in the extracts, respectively. The results obtained by HPLC-DAD-MS and by1H-NMR are discussed separately below.

[0060] Specifically, the chromatographic analyses were carried out on reverse phase columns (Poroshell, EC-C1, 120 A, 150 mm x 3.0 mm id, 2.7 pm, Agilent Technologies, with a flow of 0.37 mL min-1) , coupled to a diode spectrophotometric detector and a single quadrupole mass spectrometer (HPLC-DAD-MS) . To carry out the quantitative analyses, the dried extract of 5 different samples was dissolved in an ethanol / acidif led water mixture (ratio 7:3) , and the sample was then centrifuged at the rate of 14,000xg before HPLC analysis. The HPLC analysis was carried out using a mobile phase of acetonitrile (phase A) and water at pH 3.2 with formic acid (phase B) . Below is the applied gradient: A 5% at tO A 40% at t40 min with 5 min of plateau, followed by A 70% at t50 min with a plateau of 10 min, and to finish A 100% at t65 min with a last plateau step of 3 min; the return to the starting conditions A 5% occurred in 2 min. The chromatograms were acquired at the following wavelengths of 220, 280, 330 and 350 nm.

[0061] HPLC-DAD-MS analysis (see Figure 1) was used for both qualitative and quantitative purposes and allowed determining the amount of the individual components of the extract. The chromatographic profile recorded at 330 nm followed by the superposition of the corresponding UV-Vis spectra of each analyte with that of the sinapic acid standard, were used to identify all the synapoyl derivatives having the maximum absorption thereof at 330 nm. As can be seen from Figure 1, the same components are identified in the five different extracts analyzed, even if present in different quantities. Extracts 1, 2 and 3 show a compositional profile very similar to the different wavelengths, while some differences are detected in extracts 4 and 5, which show a higher content of some sinapic acid glycosides, in particular of the compounds with retention time of 21.8 min and 24.5 min. The integration of the information obtained from the analysis of the spectra acquired with the DAD and those acquired in mass spectrometry at di f ferent ioni zation energies , in both positive and negative ioni zation, together with the literature data allowed obtaining the li st of the main glycosylated synapoyl derivatives , identi fied in the five extracts and shown in Table 3 . Each of the identi fied compounds is associated with a number as shown in Figure 1 .

[0062] Table 3. List of analytes identified in the extracts and spectral features thereof (the numbering is in accordance with Figure 1 ) The data in Table 3 are in accordance with the results of the1H-NMR spectra (Figure 2) . The spectra of the 5 samples included in the study indicate that the pattern of signals between 6 and 7.5 ppm can be attributed to the aromatic protons of the synapoyl groups present in both sinapine and in all the other compounds listed in Table 3. No other appreciable intensity signals were detected in the same ppm range. The signal observed at 4.7 ppm corresponds to that of water, while the other signals, between 3 and 4.5 ppm, confirmed the presence of various sugar fractions, corresponding to the glycosidic derivatives of sinapic acid (Table 3) . A further confirmation of the presence of glycosidic groups is given by signals in the range between 5 and 5.5 ppm, which are related to the anomeric protons of the glycosidic groups. As further confirmation of the correct interpretation of the proton spectra of the extracts, the proton NMR spectra of pure standards of sinapine and sinapic acid were also used. Sinapine is characterized by an intense signal at 3.1 ppm attributed to the methyls linked to the quaternary nitrogen of the molecule, while the signal at 3.7 ppm corresponds to the methoxyl group of the synaptic derivatives.

[0063] Based on the analyses carried out, first qualitative and then quantitative, it can be concluded that the aqueous extract of Raphanus sativus seeds was rich in sinapine and variously glycosylated derivatives of sinapic acid. The main chemical structures of the components of the extract have already been described previously (Gao et al, 2022) . A peculiar feature of the invention is the remarkable reproducibility of the aqueous seed extract, as confirmed by the analysis of extracts from 5 different batches.

[0064] In view of the considerable variability of phytocomplexes often observed in many other plant matrices, in this case it should be highlighted that the extraction procedure of different batches of seeds allows obtaining aqueous extracts with a very reproducible compositional profile.

[0065] As mentioned above, the chemical analyses allowed selecting two pure standards used as external standards to determine the active ingredient titer of the dried aqueous extracts. The two chosen standards representative of the phytocomplex of the extract are: sinapine and sinapic acid. The quantitative analysis of sinapine and glycosidic derivatives of sinapic acid was carried out by HPLC-DAD. The quantitative data of the total sinapic acid glycosides were expressed in two ways using the two calibration curves shown in Figure 3. The curves were constructed with aqueous solutions of 0.51 mg / mL and 0.051 mg / mL for sinapine (sinapine chloride, code 84156, HPLC purity 99.3%) and 0.0976 pg / ml of sinapic acid in the trans configuration (code 80007, HPLC purity degree 100%) . The two commercial compounds were provided by Phytolab (GmbH & Co. KG Dutendorfer, 91487 Vestenbergsgreuth-Germany) (Figure 4) .

[0066] The quantitative data for the 5 dry extracts analyzed are shown in Figures 5 and 6 and are each the mean of triplicates (N=3) . In accordance with such analyses, 1 g of lyophilized extract of Raphanus sativus contains sinapine in a range between 15.910.05 mg and 24.611.9 mg (Extr 2 and 4 repetitively, Figure 5) . The total of synapoyl glycosides in 1 g of dry 5 extract, expressed as sinapic acid, oscillates between 16.810.98 mg and 21.0 + 0.02 mg (Extr. 4 and Extr. 3, respectively, Figure 6) .

[0067] As shown in Figures 5 and 6, respectively, the sinapine and glycoside content of sinapic acid of the 10 five extracts have comparable concentrations. The chromatographic resolution achieved allowed individually identifying and quantifying the main components in the five extracts, and knowing in detail the concentration of each of these in the dry extract dose used for the 15 biological tests (25 pg / ml of dried extract) during the cryopreservation procedures and in vitro incubations of the spermatozoa. Table 4 shows the concentrations in 25 mg of dried extract of the main compounds present. The data are expressed in pg / 25 mg dry extract and were 20 obtained as mean triplicates (N=3) using the standard sinapine curve at 330 nm.

[0068] Table 4. Concentrations of the different sinapic acid- derived analytes (numbers as in Table 1) in 25 mg of dried 25 extract (data obtained as mean triplicates using the standard sinapine curve at 330 nm) .

[0069] From the data previously reported, it can be concluded that : i ) the analyses carried out by HPLC-DAD-MS and1H- NMR of the dried aqueous extracts of Raphanus sa ti vus and the comparison with the scienti fic literature , confirm that the main components of the extract correspond to sinapine and glycosidic derivatives of sinapic acid; ii ) the concentration of sinapine and glycosidic derivatives of sinapic acid is very similar in the five analyzed extract samples , confirming the possibility of obtaining aqueous extracts with well reproducible phytocomplex composition .

[0070] Biological tests

[0071] Based on the quantitative data of the extracts , the average-representative concentrations were chosen for the two standards of sinapine and sinapic acid to be tested individually, regarding the ability thereof to reproduce the ef fects of the extract , in particular protection against the loss of motility, viability and integrity of sperm DNA after cryopreservation and motility and integrity of sperm DNA during the in vitro incubation of sperm . The semen samples were treated with a concentration equivalent to 25 pg / ml of the extract , i . e . , 0 . 376 pg / ml for sinapine and 0 . 282 pg / ml for sinapic acid . The samples treated with sinapine and sinapic acid subj ected to cryopreservation (n=5 for each of the two freezing procedures ) come from the group of normozoospermic subj ects having the semen features shown in Table 1 . The samples treated with sinapine and sinapic acid subj ected to incubation (n=6 ) come from the group of subj ects having the semen features shown in Table 2 .

[0072] USE OF RAPHANUS SATIVUS PLANT EXTRACT DURING THE CRYOPRESERVATION OF HUMAN SPERMATOZOA

[0073] Tables 5A and 5B show the means ± standard deviations ( SD) of the motility, viability, and sDF values obtained with and without the addition of the extract , after thawing the cryopreserved samples with two di f ferent freezing media : Test Yolk Buf fer, procedure A ( Table 5A) and SpermFreeze, procedure B ( Table 5B ) . As shown in Tables 5A and 5B, in both procedures , the extract is capable of improving the recovery of both the motility and viability parameters . Moreover, there is also a protective action for DNA integrity . Such ef fects were highlighted in all patients analyzed .

[0074] Table 5A. Effect of the extract on motility (progressive and total ) , viability and spermatozoa DNA fragmentation levels following cryopreservation with procedure A.

[0075]

[0076] TABLE 5B. Effect of the extract on motility (progressive and total) , viability and spermatozoa DNA fragmentation levels following cryopreservation with procedure B

[0077] The following Tables 6A and 6B show the mean ± standard deviations (SD) of the motility, viability and sDF values obtained with and without the addition of the extract, or of sinapine or sinapic acid after thawing the samples cryopreserved with the two different freezing media, procedure A (Table 6A) and B (Table 6B) . The concentrations of sinapine and sinapic acid used are 0.376 pg / mL and 0.282 pg / mL, respectively. The samples come from the subjects in Table 1. TABLE 6A. Effect of extract , sinapine and sinapic acid on motility (progressive and total ) , viability and spermatozoa DNA fragmentation levels following cryopreservation with procedure A

[0078] TABLE 6B . Effect of extract , sinapine and sinapic acid on motility (progressive and total ) , viability and spermatozoa DNA fragmentation levels following cryopreservation with procedure B

[0079] As shown in Tables 6A and 6B, there is an almost complete superposition of the actions of each of the two molecules with those of the whole extract . This data indicates that the main compounds identi fied in the extract , namely sinapine and the glycoside group of sinapic acid, are indeed the biologically active components of the extract representative of the biological properties of the extract itsel f .

[0080] USE OF RAPHANUS SATIVUS PLANT EXTRACT IN IN-VITRO INCUBATIONS UNDER CONDITIONS S IMILAR TO THOSE USED IN ASS ISTED REPRODUCTION PROCEDURES .

[0081] Another application of the present invention is in in-vitro incubations , another PMA procedure . In in-vitro fertili zation, the oocytes are inseminated with the semen, after spermatozoa selection and capacitation ( carried out with various methods including the so- called " swim up" , in which spermatozoa with better motility are recovered) . In the IVF technique (In Vitro

[0082] Fertilization with Embryo Transfer) , the selected spermatozoa are then incubated in vitro together with the oocyte until the latter is fertilized. Given that even during in vitro incubation, spermatozoa lose DNA motility and integrity, the effect of the plant extract was evaluated by incubating spermatozoa in Human Tubal Fluid (HTF, one of the culture media used in vitro in the assisted reproduction procedures) , at 37°C, at 5% C02.

[0083] To carry out this study, 33 patients were recruited, their mean values of age, abstinence and main semen parameters being shown in Table 2.

[0084] The patients were recruited using the inclusion criterion of >30% motility. The experiments were carried out on spermatozoa populations selected with swim up, unless otherwise indicated. The choice of the optimal concentration of extract to be used was carried out as already described (Figure 7) . The results obtained on spermatozoa motility parameters (n=22) indicated that incubation with the extract for 2h had a significant effect on total spermatozoa motility (87.36115.33% vs 83.41117.37%; p<0.001) and on progressive motility

[0085] (75.86 117.56% vs 65.91120.14%; p<0.001) . At the same time, the extract caused a decrease in the percentage of in situ motility (p<0.01) , indicating that a greater fraction of spermatozoa maintained progression in the presence thereof. These data are summarized in Table 7.

[0086] Table 7. Effect of the extract on motility (progressive, in situ, and total) during in vitro incubation

[0087] A protective effect of the extract on sperm DNA integrity was also shown. In fact, the results of the

[0088] SCD test, for the evaluation of sDF, indicated, after 24h of incubation, a decrease in DNA-induced damage in the treated samples with respect to the controls: 16.38114.48% vs 25.92119.23%, p<0.001, n= 22 (Table 8) .

[0089] Table 8. Effect of the extract on sDF, measured with the SCD test, during a 24h in vitro incubation.

[0090] The protective effect of the extract on sperm DNA integrity was also confirmed using, alternatively to the SCD test, the LiveTUNEL method in native semen samples (Figure 9) . In fact, with this technique, after 24h of incubation, the level of DNA fragmentation in the fraction of viable spermatozoa treated with the extract was 20.00116.15%, while in the control samples it was 24.46115.16% (p<0.01, n=ll, Table 9) .

[0091] Table 9. Effect of the extract on sDF, measured with LiveTUNEL, during a 24h in vitro incubation.

[0092] The protective effect on loss of motility (2h incubation) and sperm DNA integrity (24h incubation) is also obtained with the addition of sinapine (0.376 pg / mL) and sinapic acid (0.282 pg / mL) , as shown in Figure 10 and Figure 11. Thus, the addition of this extract, and of the active ingredients identified, during the in vitro incubation of spermatozoa in PMA procedures is confirmed to be beneficial in maintaining spermatozoa motility and the DNA integrity thereof.

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[0098] Demir M, Altindag F. Sinapic acid ameliorates cisplatin-induced disruptions in testicular steroidogenesis and spermatogenesis by modulating androgen receptor, proliferating cell nuclear antigen and apoptosis in male rats. Andrologia. 2022 May ; 54 ( 4 ) : el4369. doi: 10.1111 / and.14369. Epub 2022 Jan 8. PMID: 34997775.

[0099] Dong, M., Tian, Z . , Zhou, Q. , Liu, Y., Wang, X., Ren, W., Li, Y., Jiang, H., Chemical constituents from the seeds of Raphanus sativus L. and their chemotaxonomic significance. Biochem. Systemat. Ecol. 93, (2020) ; https : / / doi . org / 10.1016 / j . bse .2020.104138.

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[0102] Muratori M, Pellegrino G, Mangone G, Azzari C, Lotti F, Tarozzi N, Boni L, Borini A, Maggi M, Baldi E. DNA Fragmentation in Viable and Non-Viable Spermatozoa Discriminates Fertile and Subfertile Subjects with Similar Accuracy. J Clin Med. 2020 May 4; 9 (5) :1341. doi: 10 .3390 / cm9051341. PMID: 32375389; PMCID: PMC7290809 .

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Claims

CLAIMS1 ) Use of an aqueous plant extract , in dried form, of Raphanus sa ti vus ( common radish, Brassi caceae family,) seeds for the cryopreservation of human spermatozoa, and protection of the motility parameters of the spermatozoa themselves and the integrity of the DNA thereof , during in vitro incubation, in medically assisted procreation (MAP ) procedures .2 ) A method for producing the aqueous plant extract according to claim 1 , characteri zed in that in consideration of the signi ficant variability o f the phytocomplexes in many plant matrices , the extraction procedure occurs on a mixture of Raphanus sa ti vus seeds of di f ferent origin so as to obtain aqueous extracts with a very reproducible compositional profile .3 ) Use of the aqueous plant extract , in dried form, of Raphanus sativus seeds for the cryopreservation of spermatozoa according to claim 1 , characteri zed in that it includes adding said dry extract to the spermatozoa freezing medium at a concentration of 20-30 pg / mL, preferably 25 pg / mL, considering a maximum percentage error of 10% .4 ) Use of the aqueous plant extract , in dried form, of Raphanus sativus seeds to maintain the sperm motility and the integrity of the DNA thereof in vitro , wherein the aqueous plant extract according to claim 1 is added to the spermatozoa culture medium, at a concentration of 20-30 pg / mL, preferably in an amount of 25 pg / mL, considering a maximum percentage error of 10% .5 ) Use of the aqueous plant extract in driedform of Raphanus sativus seeds according to claim 1, characterized in that said extract comprises, as active components representative of the biological properties thereof, sinapine and sinapic acid, the latter as representative of all glycosylated sinapoyl derivatives of the extract.6) Use of the plant extract of Raphanus sativus seeds according to claim 1, characterized in that one gram of dry extract contains sinapine in the range of 15.9-24.6 mg and a sum of sinapic acid glycosides in the range of 16.8-21.0 mg, considering a maximum percentage error of 10%.7) Use of a composition comprising sinapine, sinapic acid and derivatives of the two molecules for the cryopreservation of human spermatozoa, and protection of the motility parameters of the spermatozoa themselves and the integrity of the DNA thereof, during in vitro incubation in the context of medically assisted procreation procedures.8) Use of a composition according to claim 7, characterized in that one gram contains at least 15.9 mg of sinapine and 16.8 mg of sinapic acid glycosides expressed as sinapic acid considering a maximum percentage error of 10%) .9) Use of solid or liquid formulations containing sinapine, synaptic acid and / or derivatives thereof, for the cryopreservation of human spermatozoa, and protection of the motility parameters of the spermatozoa themselves and the integrity of the DNA thereof, during in vitro incubation in the context of medically assisted procreation procedures.10 ) Use of an aqueous plant extract, in dried form, of Raphanus sativus seeds for semen cryopreservation in veterinary procedures .11 ) Use of solid or liquid formulations containing sinapine, synaptic acid and / or derivatives thereof for semen cryopreservation in veterinary procedures .

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