Phytochemical Screening by LC-ESI-MS/MS and Effect of the Ethyl Acetate Fraction from Leaves and Stems of Jatropha macrantha Müll Arg. on Ketamine-Induced Erectile Dysfunction in Rats

Jatropha macrantha Müll Arg. L is also known as “huanarpo macho” and used in the Peruvian traditional medicine as an aphrodisiac and erectile dysfunction (ED). The aim of this study was to determine the phytochemical constituents in leaves and stems ethyl acetate fraction (LEAF and SEAF) of J. macrantha and to compare the antioxidant activity and the ameliorative effect on ketamine-induced erectile dysfunction in rats. The phytochemical constituents were determined by LC-ESI-MS/MS, the total phenolic compounds and total flavonoids (TPC and TF) by Folin-Ciocalteu and aluminum chloride, respectively. The antioxidant activity was determined by DPPH, ABTS, and FRAP assays. Experimental groups were divided as follows: I: negative control; II: positive control (ketamine at 50 mg/ kg/d); III: sildenafil 5 mg/kg; IV, V, VI: LEAF at 25, 50 and 100 mg/kg, respectively, and VII, VIII, IX: SEAF at 25, 50, and 100 mg/kg, respectively. The phytochemical analysis revealed the presence mainly of coumarins, flavonoids, phenolic acids, and terpenes. TPC of LEAF and SEAF were 359 ± 5.21 mg GAE/g and 306 ± 1.93 mg GAE/g, respectively; TF in LEAF and SEAF were 23.7 ± 0.80 mg EQ/g, and 101 ± 1.42 mg EQ/g, respectively. The DPPH, ABTS, FRAP in SEAF were 647 ± 3.27; 668 ± 2.30; and 575 ± 2.86 μmol TE/g, respectively, whilst LEAF showed 796 ± 3.15; 679 ± 0.85; and 806 ± 3.42 μmol TE/g, respectively. Regarding sexual behavior, LEAF showed a better effect in mount frequency, intromission frequency, ejaculation frequency, mount latency, intromission latency, ejaculatory latency, and post ejaculatory latency than SEAF. As conclusion, LEAF of J. macrantha at 50 mg/kg showed a better effect on sexual behavior in male rats with erectile dysfunction than SEAF but not higher than sildenafil.


Introduction
Erectile dysfunction (ED) is a physiological or pathological condition characterized by the disability to achieve or maintain a penile erection during a sexual activity in men and nowadays is considered the second most frequent problem of sexual dysfunction in men [1]. In Europe the prevalence was of 52% in men aged 40-70 years [2] as well as estimated projections of prevalence around 322 million by 2025 in the world [3]. ED is a common condition associated with older adults and significantly compromises the sexual performance, personal satisfaction, commitment and low self-esteem [4]. On the      The retention times (Rt), adductions, experimental, and theoretical m/z values, ppm error, MS/MS spectrum (m/z: absolute intensity), SMILES (simplified molecular input line entry system) string, InChIKey (IUPAC international chemical identifier), and tentative com-pounds are available in the Supplementary Tables S1 and S2.

Total Phenolic Content, Total Flavonoids and Antioxidant Activity of J. macrantha
Regarding the antioxidant capacities of J. macrantha leaves and stems are observed that the leaves have a higher antioxidant capacity than stems ethyl acetate fraction and were demonstrated by the DPPH, ABTS, and FRAP assays (Table 4), these differences were statistically significant (p < 0.05 in Paired sample t-test) in DPPH and FRAP. Otherwise, TPC and TF in leaves were higher content than stems.

Effect of the Ethyl Acetate Fraction from Leaves and Stems of J. macrantha on Ketamine-Induced Erectile Dysfunction in Rats
Before the pre-copulatory actions of rats treated with J. macrantha and sildenafil citrate, some signs and sexual behaviors were recorded and were shown in Figure 3. Here, it is observed some behaviors such as doing circling around, body-sniffing, anogenital exploration, ear-wiggling, lordosis, hopping, mounting, and grooming. All these findings were recorded using a video camera to avoid any interference or disturbance.
it is observed some behaviors such as doing circling around, body-sniffing, anogenital exploration, ear-wiggling, lordosis, hopping, mounting, and grooming. All these findings were recorded using a video camera to avoid any interference or disturbance.
In Figure 4A is observed that in mount frequency at doses of 50 mg/kg of leaves ethyl acetate fraction (LEAF-50; p < 0.0001) presented 11 mounts, higher than the other concentrations including all groups administrated with J. macrantha stems, but lower than sildenafil, which revealed an average of 13.75 mounts. SEAF-25 did not have a significant difference with the positive control group (p = 0.8722). Regarding Figure 2B, intromission frequency (IF) in rats administrated with LEAF-100 (p < 0.0001) and stems ethyl acetate fraction at 50 mg/Kg (SEAF-50; p < 0.0001) had 11.38 and 7.88 penetrations, respectively, being more than positive control (PC) group, meanwhile sildenafil had 13.50 penetrations. Other contrary, SEAF-25 did not show a significant difference with PC group (p = 0.1555). On the other hand, it is observed an increase in ejaculation frequency with LEAF-25, LEAF-50, and SEAF-50 ( Figure 4C) compared to PC group (p < 0.0001). Treatments with LEAF-50 and SEAF-50 ( Figure 4D, and Figure 4E) showed a decrease in mount latency and intromission latency, which means increased sexual motivation and stimulation in rats, compared with sildenafil which had better effect (p < 0.0001).
In Figure 4F, on ejaculatory latency (EL), there was a decrease in time with all doses from leaves and stems but better at doses of 50 mg/kg (leaves and stems; p < 0.0001). Therefore, decreasing this parameter would improve the ejaculation in rats. The opposite happens with the increase of this indicator EL (p < 0.0001), which is an indicator of ejaculatory difficulty. Figure 4G, on post-ejaculatory latency is a parameter that measures the recovery time between one mating session to another, as is observed the recovery time was between 6 to 10 min in rats treated with LEAF-25 (p < 0.0001), LEAF-50 (p = 0.025), and LEAF-100 (p < 0.0001), respectively, and were higher than sildenafil group. Whilst SEAF-100 did not show a significant difference with PC group (p = 0.1682).  In Figure 4A is observed that in mount frequency at doses of 50 mg/kg of leaves ethyl acetate fraction (LEAF-50; p < 0.0001) presented 11 mounts, higher than the other concentrations including all groups administrated with J. macrantha stems, but lower than sildenafil, which revealed an average of 13.75 mounts. SEAF-25 did not have a significant difference with the positive control group (p = 0.8722). Regarding Figure 2B, intromission frequency (IF) in rats administrated with LEAF-100 (p < 0.0001) and stems ethyl acetate fraction at 50 mg/kg (SEAF-50; p < 0.0001) had 11.38 and 7.88 penetrations, respectively, being more than positive control (PC) group, meanwhile sildenafil had 13.50 penetrations. Other contrary, SEAF-25 did not show a significant difference with PC group (p = 0.1555). On the other hand, it is observed an increase in ejaculation frequency with LEAF-25, LEAF-50, and SEAF-50 ( Figure 4C) compared to PC group (p < 0.0001). Treatments with LEAF-50 and SEAF-50 ( Figure 4D,E) showed a decrease in mount latency and intromission latency, which means increased sexual motivation and stimulation in rats, compared with sildenafil which had better effect (p < 0.0001). In Figure 4F, on ejaculatory latency (EL), there was a decrease in time with all doses from leaves and stems but better at doses of 50 mg/kg (leaves and stems; p < 0.0001). Therefore, decreasing this parameter would improve the ejaculation in rats. The opposite happens with the increase of this indicator EL (p < 0.0001), which is an indicator of ejaculatory difficulty. Figure 4G, on post-ejaculatory latency is a parameter that measures the recovery time between one mating session to another, as is observed the recovery time was between 6 to 10 min in rats treated with LEAF-25 (p < 0.0001), LEAF-50 (p = 0.025), and LEAF-100 (p < 0.0001), respectively, and were higher than sildenafil group. Whilst SEAF-100 did not show a significant difference with PC group (p = 0.1682).

Evaluation of the Vasodilator Effect in Rats Treated with J. macrantha and Sildenafil
The evaluation of the vasodilator effect was measured as the tension expressed in grams on rat's penile tissue. As is shown in Figure 5A, acetylcholine was not inhibited by LEAF, SEAF, and sildenafil citrate. In Figure 5B adrenaline was not inhibited by the different concentrations of SEAF and LEAF but sildenafil had better effect on adrenergic receptors (p = 0.0034). In Figure 5C, Calcium chloride (CaCl 2 ) solution was used as an agonist of calcium channels and was only inhibited by sildenafil citrate (p < 0.0001).

Discussion
For the induction of erectile dysfunction, male rats were administered intraperitoneally with ketamine at a dose of 50 mg/kg/day for a period of 14 days and exposed to a dim light (1-watt fluorescent tube) for 5 days before experiment. As can be observed, ketamine was used to produce erectile dysfunction presenting dissociative, psychotomimetic, cognitive, and peripheral side effects, which are involved in short or long-term administration as well as its dissociative use. Ketamine is an antagonist at N-methyl-D-aspartate receptors, glutamate receptors that are mainly expressed in the hippocampus and prefrontal cortex. In a study, ketamine induced the increase of stress oxidative markers such as malondialdehyde (MDA), catalase (CAT), and reduced the total antioxidant capacity

Discussion
For the induction of erectile dysfunction, male rats were administered intraperitoneally with ketamine at a dose of 50 mg/kg/day for a period of 14 days and exposed to a dim light (1-watt fluorescent tube) for 5 days before experiment. As can be observed, ketamine was used to produce erectile dysfunction presenting dissociative, psychotomimetic, cognitive, and peripheral side effects, which are involved in short or long-term administration as well as its dissociative use. Ketamine is an antagonist at N-methyl-D-aspartate receptors, glutamate receptors that are mainly expressed in the hippocampus and prefrontal cortex. In a study, ketamine induced the increase of stress oxidative markers such as malondialdehyde (MDA), catalase (CAT), and reduced the total antioxidant capacity (TAC) in rats, proposing that the erectile and testicular dysfunction would be through oxidative stress and decreasing the serum testosterone and luteinizing hormones [23]. Other study revealed that ketamine at 100 mg/kg/day in rats induced erectly disfunction activating apoptosis by up-regulating of inducible nitric oxide synthase (iNOS) leading to the loss of corporal smooth muscle content and reducing of neuronal nitric oxide synthase (nNOS) expression on cavernous nerve [24].
Of the phytochemical constituents determined in leaves and stems of J. macrantha ethyl acetate fraction, some of them have demonstrated to ameliorate the ED in experimental animals. Scopoletin showed a positive effect in penile erection of rats through the NO-cGMP and adenylyl cAMP-PKA signaling pathways [25]. Vitexin at doses of 40 mg/kg regulated some endocrine hormones and had an improving in the fertility rate sex performance in diabetic mice, the mechanism involved might be in the modulation of hypothalamuspituitary-gonadal axis [26]. On the other hand, quercetin, naringenin, aromadendrin, taxifolin, and kaempferol determined in the ethanol extract from Anaxagorea luzonensis had an inhibitory effect on phosphodiesterase 5 [27]. In a study, moringa extract containing phenols and flavonoids such as gallic acid, catechin, chlorogenic acid, ellagic acid, quercitrin, isoquercitrina, quercetin, rutin, kaempferol, and epicatechin inhibited enzymes as arginase and angiotensin converting enzyme 2 (ACE-2), which were linked to erectile dysfunction and oxidative stress in rat's penile [28].
Oxidative stress generated during inflammation, damaged tissue or chronical diseases have been associated as main key factors in the pathogenesis of ED. A previous study showed that sildenafil reduced the malondialdehyde (MDA) levels and increased the activity of antioxidant enzymes in rats with ED. Hence, some mechanisms of ED drugs are involved with the decrease of free radical levels and increase the antioxidant capacity, i.e., superoxide dismutase (SOD) and CAT [29]. In other study, paroxetine a selective serotonin reuptake inhibitor antidepressant drug significantly increased malondialdehyde (MDA) levels in rats' penile tissues producing ED. Hereby, free radicals probably generated by antidepressant drugs would attack to the penile tissues producing lipid peroxidation and high MDA levels [30]. Thus, antioxidant compounds found in natural products might ameliorate this condition and protect to penile tissue of stress oxidative. Regarding to the mechanism stablished in our study, it can be demonstrated that SEAF and LEAF did not relax the penile tissue when were exposed to acetylcholine, however, SEAF and LEAF at 0.1 mg/mL had a stimulant effect. It is known that cholinergic stimulation of the cavernous nerve leads to increased blood flow within the penis, on the contrary the adrenergic stimulation reduces blood flow leading to the flaccid state [31], according to our results, a relaxation was observed with sildenafil whilst SEAF and LEAF did not reveal any effect on adrenergic receptors. On the other hand, the exposition to CaCl 2 leads to extracellular calcium influx after the opening of calcium channels [32]. Although, nonsignificant differences were observed in LEAF and SEAF, at 0.1 mg/mL antagonized the Ca 2+ -induced contractions in rat's penile tissue. As main limitation to understand the main mechanism involved in the vasodilator effect, Nitric oxide pathways were not evaluated. However, due to presence of several phytochemicals determined in LEAF and SEAF of J. macrantha, multiple mechanism could be linked to the relaxation of rat's penile tissue and produce erection during the sexual activity.

Preparation of the Ethanolic Extract and Ethyl Acetate Fraction
The leaves and stems were dried at room temperature for 2 weeks, the ground dry samples were macerated for 14 days with 10 L of 80 % ethanol with constant stirring. Then, filtered and concentrated using a R-300 ® rotary evaporator (Buchi, Flawil, Switzerland) until dryness. For obtaining the phenolic fraction of leaves and stems, the hydroalcoholic extracts were suspended in distilled water, then degreased with petroleum ether (in order to eliminate fats, waxes, pigments and other metabolites that may interfere with the extraction of flavonoids). Then a liquid-liquid extraction was carried out with 300 mL of ethyl acetate using a separatory funnel that was left for a period of 24 h, then the ethyl acetate fraction was evaporated until dryness. Each ethyl acetate fraction of leaves and stems (LEAF and SEAF) were refrigerated until further use at 4 • C.

Phytochemical Analysis by LC-ESI-MS/MS of the Main Constituents of the Ethyl Acetate Fraction of Leaves and Stems of J. macrantha 4.3.1. Preparation of the Sample
The ethyl acetate fraction of the leaves and stems of J. macrantha were weighed and diluted with ethyl acetate until a final concentration of 10 mg/mL had been obtained. Next, each sample was vortexed for 1 min and subsequently centrifuged for 10 min at 10,000 rpm. Finally, 800 µL of the 1 mg/mL solution supernatant (methanol:water, 1:1) was removed in vials for LC-MS analysis in a Dionex UltiMate 3000 liquid chromatograph (Thermo Fisher Scientific, San José, CA, USA) coupled to a Thermo QExactiveTM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with an electrospray ionization source.

Chromatographic Conditions
This analysis used a chromatographic column Thermo Scientific Syncronis RP-C18 (50 mm × 2.1 mm × 1.9 µm). Solvent A was 0.1% formic acid in water and 5 mM ammonium formate, and Solvent B was 0.1% formic acid in MeOH. The gradient elution of the method was as follows: 0-0.

Determination of Total Phenolic Compounds (TPC)
In total, 50 µL of the ethyl acetate fraction of leaves and stems (10 mg/mL) were mixed with 1 mL of distilled water, 0.5 mL of 0.2 N Folin-Ciocalteu reagent, and 2.5 mL of 5% sodium carbonate, then the sample was allowed to react in the darkness for 40 min at room temperature (20 • C). The absorbance was read at 725 nm using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA). A standard curve was made with a gallic acid solution (50 µg/mL) at concentrations of 10, 20, 30, 40, and 50 µg/mL. The results are presented in mg gallic acid equivalent per g of extract (mg GAE/g of extract) [30].

Determination of Total Flavonoids
In total, 0.5 mL of ethyl acetate fraction of leaves and stems (10 mg/mL) were mixed with 1 mL with distilled water and 0.15 mL of 5% sodium nitrite; 5 min later, 0.15 mL of 10% aluminum chloride was added, then at 6 min, 2 mL of 4% sodium hydroxide was added. The sample was made up to 5 mL with distilled water, mixed, and allowed to react in the darkness for 15 min at room temperature. The absorbance was read at 510 nm against a blank using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA). A standard curve was made with quercetin (200 µg/mL) at concentrations of 40, 80, 120, 160, and 200 µg/mL. The flavonoid content is presented as mg quercetin equivalent per g of extract (mg QE/g of extract) [31].

Determination of the Antioxidant Capacity by the Free Radical Sequestration Method with 2,2-Diphenyl-1-Picrylhydrazyl
For this assay, 150 µL of ethyl acetate fraction of leaves and stems (10 mg/mL) were mixed with 2850 µL of a methanolic solution of DPPH radicals (20 mg/L) with the absorbance adjusted to 0.6 ± 0.02 nm. After mixing, the sample was incubated in the dark for 30 min and the absorbance was read at 515 nm using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The standard curve was elaborated with Trolox at concentrations of 0 to 800 µmol/mL [32]. The Trolox equivalent antioxidant capacity (TEAC) was expressed as µmol Trolox equivalent per gram of extract (µmol TE/g of extract).

Determination of the Antioxidant Capacity by the Sequestration Method with the Radical Cation of the 2.2 -Azinobis-(3-Ethylbenzothiazoline)-6-Sulfonic Acid
A standard solution (ST) was prepared by mixing 10 mL of ABTS (4.06 mg/mL) with 10 mL of potassium persulfate (0.7 mg/mL) and reacted for 12 h. The working solution (ST) was prepared with 1 mL of each extract and 60 mL of methanol. The absorbance was adjusted to 0.7 ± 0.02 with methanol at a wavelength of 734 nm, then 150 µL of the ethyl acetate fraction of leaves and stems (5 mg/mL) were mixed with 2850 µL of the working solution and incubated in the dark for 7 min, followed by reading the absorbances at 734 nm using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA) [33]. The standard curve was made with Trolox at 0-400 µmol/mL. The Trolox equivalent antioxidant capacity (TEAC) was expressed as µmol Trolox equivalent per gram of extract (µmol TE/g of extract).

Determination of the Antioxidant Capacity by the Ferric Reducing Antioxidant Power (FRAP) Method
For the determination, 150 µL of sample was mixed with 2850 µL of the previously prepared 2,3,5-triphenyltetrazolium chloride (TPTZ) reagent, which was left for 30 min at 37 • C. The absorbance was measured at 593 nm using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The standard curve was made with Trolox from 50 to 800 µmol/mL. The results were expressed as µmol Trolox equivalent per gram of extract (µmol TE/g of extract) [33].

Effect of the Ethyl Acetate Fraction from Leaves and Stems of J. Macrantha on Ketamine-Induced Erectile Dysfunction in Rats Evaluation of Erectly Dysfunction
The animals were prepared and conditioned by the method of Shang et al., [24]. In this study, 100 females and males Holtzman rats weighing 200 ± 50 g were acquired from the Bioterium of the Universidad Nacional Agraria La Molina (UNALM) and transported to the city of Ayacucho and adapted in the Bioterium of the School of Pharmacy and Biochemistry of the Universidad Nacional de San Cristóbal de Huamanga (UNSCH). Then, nine groups of eight animals each were grouped as follows: (a) Group I: distilled water at doses of 10 mL/kg was administered orally, which served as negative control (NC). For the induction of erectile dysfunction, male rats were administered intraperitoneally with ketamine at doses of 50 mg/kg/day for a period of 14 days and were exposed to a dim light (1-watt fluorescent tube) for 5 days prior to experiment. After induction, on day 20, experimental groups were administrated with J. macrantha and sildenafil citrate by oral administration in a single dose, a screening of sexual behavior was performed by evaluating the frequencies of mounting, intrusion and ejaculation, such as the respective latencies of mounting, intrusion, ejaculation and post-ejaculation. For evaluating sexual behavior, a time interval of 15 min per rat was used, in which the respective frequencies and latencies were evaluated. Female rats were administered subcutaneously 48 h and 4 h with estradiol benzoate at 10 µg/100 g of body weight and progesterone at doses of 0.5 mg/100 g of body weight, before putting them together with male rats. Then, a receptive female was introduced to the male cages. Observation of mating was carried out at 8:00 p.m.
The parameters to measure male sexual behavior were: ejaculation. It is usually characterized by a prolonged, deep pelvic thrust and slow dismounting followed by a period of inactivity or reduced activity.
(g) Post ejaculatory latency (PEL): It is the time interval between ejaculation and the first penetration of the following series.
The Local Committee of Ethics on Animal Experimentation approved all experimental procedures, with i.d. 261-2020-FCSA-UNSCH.

Evaluation of the Vasodilator Effect
For the determination of the vasodilator effect, the corpus cavernosum smooth muscle was kept in vital condition, providing the necessary elements for its maintenance and stability. The corpus cavernosum was isolated and poured into the Krebs-Henseleit (KHS) nutrient liquid with oxygenation 95.0% O 2 and 5.0% CO 2 for 30 min. Two strips of the smooth muscle of the corpus cavernosum (10 to 12 mm in length and 1-2 mm in thickness) were isolated by dissecting part of the penis, working with a solution containing KHS (pH 7.3) with the following composition: NaCl = 7.01 g/L, KCl = 0.34 g/L, KH 2 PO 4 = 0.1 g/L, NaHCO 3 = 1.99 g/L, CaCl 2 = 0.2 g/L, MgSO 4 = 0.3 g/L and glucose 1.8 g/L, eight repetitions were performed with the ethyl acetate fractions of Jatropha macrantha leaves and stems at concentrations of 0.1; 0.5 and 1.0 mg/mL and sildenafil 3.2 × 10 −5 mg/mL as a positive control. To achieve maximum contractions, acetylcholine, adrenaline and calcium chloride (CaCl 2 ) were administered at 5 × 10 −2 M, 1.0 µg/mL, and 17.8 mg/mL, respectively. The data were expressed in tension (g) by reading contraction or relaxation.

Data Analysis
The results are presented as the means plus standard deviation of eight animals per group. The differences between the means were analyzed using ANOVA followed by Tukey and Dunnett's Test for erectile dysfunction study and for TPC, TF, and antioxidant capacity, were used paired sample t-test using Graph Pad Prism v6 software. A p-value less than 0.05 is considered significant.

Conclusions
Based on our results, the ethyl acetate fraction of Jatropha macrantha leaves at 50 mg/kg/day by oral administration presented an ameliorative effect on ketamine-induced erectile dysfunction in rats. Furthermore, it showed a high content of total phenols and flavonoids than stems ethyl acetate fraction, which leads to a high antioxidant capacity in DPPH, ABTS and FRAP assays. In the analysis carried out with LC-ESI-MS/MS, leaves and stems contained 77 and 42 phytochemical constituents, respectively. Some chemical groups highlighted were organic acids, phenolic acids, flavonoids, coumarins, fatty acids, lipids, sesquiterpene lactones, terpenoids and anthraquinones. J. macrantha might be useful as a promising herbal medicine in erectile dysfunction or aphrodisiac.
Supplementary Materials: The following are available online, Table S1: List of compounds putatively identified by LC-HRMS/MS in the ethyl acetate fraction of Jatropha macrantha leaves. Table S2  Institutional Review Board Statement: This study was approved by the Universidad Nacional de San Cristóbal de Huamanga con Id. Number 261-2020-FCSA-UNSCH (02-11-2020).

Data Availability Statement:
The data that support the results and findings of this study is available from the corresponding author upon request.