In vitro antioxidant activity and in vivo aphrodisiac activity in male Cavia porcellus of Albizia adianthifolia (Schum.) W. Wight and Pericopsis angolensis (Baker) Meeuwen

This study was carried out to evaluate the aphrodisiac and antioxidant activities of aqueous and methanolic extracts of two plants used as aphrodisiac in traditional medicine in Kampemba municipality, in Lubumbashi DR Congo. Aqueous and methanolic root bark extracts of both Albizia adianthifolia and Pericopsis angolensis were administered orally to male Cavia porcellus before evaluating their physical behavior in the presence of females and their testosterone, cholesterol and triglyceride contents. The antioxidant activity was determined by the DPPH method. At 200 and 250 mg kg-1 body weight (bw), the extracts exhibited a decrease in rise time and intromission time and an increase in ejaculation time, ascending frequency, intromission frequency, ejaculation frequency and testosterone compared to the control group that received sildenafil (10 mg kg-1 bw) used as a reference. A positive relationship between testosterone level and antioxidant activity has also been observed. The aphrodisiac and antioxidant activity observed in the two species justify their uses in traditional medicine as aphrodisiacs. Further work is needed in order to proceed to the isolation and the identification of the active compounds.


Introduction
Erectile dysfunction (ED) refers to the constant inability of a man to achieve or maintain an erection for satisfactory sexual activity [1]. The prevalence and severity of ED increases with age [2] and not only affects the life expectancy, but also the well-being and quality of life of exposed people [3]. In the past, plants called aphrodisiacs, in memory of Aphrodite, the Greek god of love, such as Pausinystalia johimbe (K. Schum.) Pierre, Rubiaceae [4] or Zingiber officinalis L, Zingiberaceae [5], have been used to improve sexual pleasure. Several plants are still used today as aphrodisiacs in several cultures. Albizia adianthifolia is also used as a purgative and antidiabetic as well as in eye, gastrointestinal, hemorrhoidal, cephalic, neurodegenerative diseases [6,7]. A part from ED, Pericopsis angolensis is also used in traditional medicine in respiratory and eye conditions and in gastrointestinal disorders [8].
This study aimed to evaluate the aphrodisiac and antioxidant activities of these two plants, thus contributing to the scientific validation of this aphrodisiac use which they enjoy in the city of Lubumbashi.

Experimental animals
A local strain of Cavia porcellus L. on average weight 380.38 ± 10.9 g of both sexes was obtained from the Faculty of Agronomic Sciences of the University of Lubumbashi. These animals were fed with a standard diet (MIDEMA / RD Congo) and acclimatized for 10 days under the experimental environmental conditions (28 °C, alternating light dark every 12 h, meal and water at will).

Preparation of extracts and determination of experimental doses
The methanolic extract (ME) was prepared by maceration of 350 g of crushed dried plant material in 1.5 L of methanol (67-56-1 Sigma Aldrich, Belgium) for 48 h. The aqueous extracts (EA) were prepared according to the protocol used in traditional medicine by decoction for 30 minutes of 600 g of plant material in 4 L of water for A. adianthifolia and by decoction of 750 g of plant material in 2.5 L of water for 45 minutes for P. angolensis. The macerated or decocted solutions were filtrated under filter paper (ø = 0.25 μm, No. 1172.000002.05 Whatman, USA), and were evaporated under a rotary evaporator (Büchi R-210, Switzerland). The doses administered in Guinea pigs (Table 1)

Aphrodisiac activity assessment
The aphrodisiac activity evaluation test was inspired by a previous protocol [10]. Briefly, fifty male Cavia porcellus were randomized into 10 groups (GA) of 5 animals each. The animals of group A, which served as control, received per 2 mL of 0.9% saline solution and those of group B, used as positive control, received 10 mg.kg -1 of weight of sildenafil (MFCD09026931; Sigma-Aldrich, UK). Animals of groups C, D, (aqueous extract) E, F (methanolic extracts), for A. adianthifolia, and G, H (aqueous extract), I, J (methanolic extract) for P. angiogenesis, were treated with 200 and 250 mg.

Antioxidant activity evaluation
Antioxidant activity was evaluated using the DPPH method developed previously [11]. Briefly, 50 µL of extract or ascorbic acid, positive control, prepared at different dilutions in methanol from a 100 µg / mL solution were interacted with 1950 µL of DPPH ( 0.002% in a 96-well plate (Nunc WVR, Germany). After mixing and incubating in the darkness for 30 minutes, the solution was read in a spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA) at 492 nm.
The tests were performed in triplicate and the 0.002% DPPH solution was used as a negative control. The percentage of antioxidant activity was calculated by the formula: I (%) = (Ab-Ae) x100x Ab -1 with Ab = absorbance measured in the presence of the negative control; Ae = absorbance measured in the presence of the extract, I (%) = Percentage of inhibition. This percentage of activity allowed to generate IC50.

Statistical analysis of data
Statistical analysis of the data was made possible using GraphPad Prism version 6 software (GraphPad Software, La Jolla, USA). The comparison between two means was carried out by Student's t test and that of several variables by oneway ANOVA, the significance level was set at 95%.

Physical parameters of sexual behavior
The different physical parameters vary significantly compared to the negative control group (with a p <0.01). Declines are recorded for RT, IT and PEI unlike ET, ND, NI and NE which increased. At equal doses, Albizia adianthifolia (A. adianthifolia) exhibits more expressive performance indicators than Pericopsis angolensis (P. angolensis). The EM of A. adianthifolia seem more efficient than the EA unlike P. angolensis where the performances between the two extracts (EA and EM) seem to be of the same amplitude. A. adianthifolia at 200 mg.kg -1 and P. angolensis at 250 mg.kg -1 exhibit better physical parameters than Sildenafil, used as a positive control ( Table 2). The rise time (RT) of the extracts varies between 9.3 ± 0.13 (EMAA 250 mg. kg -1 ) and 27.1 ± 1.2 min (EAPA200 mg.kg -1 ), that of intromission (IT) varies between 0.38 ± 0.13 (EMAA250 mg. kg -1 ) and 5, 1 ± 0.13 (EMPA 200 mg. kg -1 ) min and that of ejaculation (ET) varies between 1.5 ± 0.11 (EAPA200 mg. kg -1 ) and 3.6 ± 0.13 (EAAA200 mg. kg -1 ). The post-ejaculatory interval (PEI) varies between 0.30 ± 0.10 (EM AA 200 mg. kg -1 ) and 11.5 ± 2.13 (EMPA 200 mg. kg -1 ). Except for PA 250 mg. kg -1 , RT is lower in the methanolic extract than the aqueous extract and overall a very significant drop in TM is observed in comparison with the negative control group. With the exception of P. angolensis 200 mg. kg -1 , the extracts show a lower RT compared to the positive control group (p <0.01) ( Table 2). The frequencies of ascension (ND) and intromission (NI) vary between 0.03 ± 0.01 (EM PA 200 mg.kg -1 ) and 0.07 ± 0.01 (EM AA 250 mg. kg -1 ) as well as ejaculation frequency (NE) ( Table 3).

Biochemical parameters of libido
Overall, we observe a decrease in triglyceride levels (Figure 1a), compared to the negative control (p <0.00001) as well as an increase in cholesterol ( Figure 1b) and testosterone (Figure 1c) with p <0.001, except for AP at 200 mg. kg -1 . The aphrodisiac index (Figure 1d), calculated for the testosterone levels and by contribution to sildenafil, considered as a positive control, is <1 for AP at 200 mg. kg -1 and> 1 in the rest of the extracts (Figure 1). The triglyceride levels vary between 83.1 ± 1.1 and 98.3 ± 2.8 mg. dL -1 , those of cholesterol between 31.2 ± 11.6 mg. dL -1 and those of testosterol between 3.32 ± 0, 34 and 5.11 ± 0.19 ng. mL -1 (Figure 1).

Antioxidant activity
The antioxidant activity of the extracts, evaluated as a function of the anti-free radical activity on DPPH and expressed as 1 / IC50 (µg.mL -1 ) varies between 0.079 ± 0.001 and 0.204 ± 0.001 (µg mL -1 ) (Figure 2a), but when it is expressed in the form of ascorbic acid equivalent per 100 g of extract, it varies between 15.36 ± 0.06 and 41.27 ± 0.12 mg E vit C / 100 g of extract (Figure 2b). In both forms of expression, AA extracts are the more antioxidant than sildenafil (p <0.001) ( Figure 2).

Correlation between antioxidant activity and testosterone level
A positive linear correlation with high amplitude (R = 0.9996) is observed between the antioxidant activity and the testosterone level unlike the totals of triglycerides for which a negative and low amplitude correlation (R = 0.251) is observed with antioxidant activity (Figure 2c and 2d).

Discussion
During this study, we evaluated the aphrodisiac properties of aqueous and methanolic extracts of 2 plants used in traditional medicine in the commune of Kampemba in Lubumbashi-DRC, Albizia adianthifolia (Schum.) W. Wight (Fabaceae) and Pericopsis angolensis (Baker) Meeuwen (Fabaceae) [12], by examining sexual behavior and hormonal variation. The study also evaluated the antioxidant activity of the same extracts before establishing a correlation between the antioxidant activity and the aphrodisiac index.
The various observations recorded during this study reveal that the administration of doses of 200 mg.kg -1 bw of extract of A. adianthifolia modifies the physical parameters, indicators of libido on Cavia porcellus and that these effects appear to be dose dependent. "Thus", we observed a significant variation between oral administration of the doses of 200 and 250 mg.kg -1 bw (p <0.01). The performances induced by the oral administration of the extracts of A. adianthifolia consist of a decrease in time of RT, IT, PEI and an increase of ND, NI and NE frequencies, as during the oral administration of Sildenafil at 10 mg. kg -1 bw. Furthermore, the oral administration of P. angolensis shows a significant difference between the doses of 200 and 250 mg. kg -1 bw, however, these extracts behave in the same way as sildenafil. A. adianthifolia at 200 mg. kg -1 bw and, P. angolensis at 250 mg. kg -1 bw exhibit very similar aphrodisiac activity. On the other hand, the methanolic extracts exhibit a lower activity (p <0.01) than their aqueous extracts for A. adianthifolia unlike P. angolensis for which, no significant difference is observed. These data make it possible to understand not only why traditional medicine uses A. adianthifolia at doses equivalent to 200 mg. kg -1 bw and, P. angolensis at doses of 250 mg. kg -1 bw but also the need to prefer the aqueous extract of the species A. adianthifolia to the detriment of alcoholic extract [12]. On the other hand, for P. angolensis, these results suggest that the alcoholic extract is also used in the same way as the aqueous extract insofar as they could produce similar results from the point of view of physical activity the methanolic extracts exhibit a lower activity (p <0.01) than their aqueous extracts for A. adianthifolia unlike P. angolensis for which, no significant difference is observed. The methanolic extracts exhibit a lower activity (p <0.01) than their aqueous extracts for A. adianthifolia unlike P. angolensis for which, no significant difference is observed.
The literature reports that TG levels vary inversely with cholesterol and testosterone levels and serum testosterol levels increase during sexual intercourse [13][14][15][16] as a result of an olfactory stimuli leading to the activation of the hypothalamic-pituitary axis [17]. These variations were observed in the same order during this study. In addition, it has been reported that testosterone acts on the periphery by facilitating nitrergic neurotransmission, accentuating the release of nitric oxide in the cavernous bodies thus contributing to penile erection [18,19]. For this purpose, it constitutes a biomarker of aphrodisiac activity as observed during this study in the groups treated with sildenafil and the test groups ( Figure 1). It is in this context that we calculated the aphrodisiac index as the ratio between the testosterone levels of the trial group and sildenafil ( Figure 1c); moreover, the increase in serum cholesterol levels may be justified by the increase in serum testosterone levels since cholesterol constitutes a synthetic precursor [20] and that it increases in vitro in proportion to the levels of testosterones [21,22]. Some studies have even found a positive correlation between increased serum cholesterol levels and increased erectile function [23,24]. It could within certain limits constitute another biomarker of aphrodisiac activity in the absence of the possibility of resorting to conventional biomarkers.
A positive correlation was observed between antioxidant activity and testosterone level (Figure 2c). Previous work [25,26] reported that antioxidant plants increase libido and at the same time it has been argued that flavonoids and other polyphenols have an aphrodisiac property due to their androgenic properties [27] and estrogenic [28] as well as their inhibitory action of phosphodiesterases [29]. These observations can justify the positive correlation observed during this study between aphrodisiac activity and the level of testosterones which would be due to the presence of the natural polyphenols present in the various extracts examined.
Among the compounds identified or isolated in A. adianthifolia [6], some are also found in other species known to be aphrodisiacs. This is notably the case with Aurantiamide acetate isolated from Hybanthus enneaspermus (L.) F. Muell, Violaceae [30], Octadecanoic acid identified from Sida cordata (Burm.f.) Borss.Waalk., Malvaceae [31] and Prosapogenin isolated from Tribulus terrestris L., Zygophyllaceae [32]. However, no study has so far evaluated the contribution of these compounds in the expression of the aphrodisiac activity of the aforementioned plants. It would be desirable for a further study to do so. For Pericopsis angolensis, no sophisticated phytochemical studies are reported. However, preliminary phytochemical screens performed on the species revealed flavonoids, saponins and tannins [8]. Of all these phytochemical groups, only the tannins, through protodioscin and protogracillin, isolated from Tribulus terrestris L., Zygophyllaceae are responsible of the aphrodisiac activity [33]. It would be desirable to evaluate the activity of the major groups of secondary metabolites and to determine among them the phytochemical groups with aphrodisiac property.

Conclusion
A. adianthifolia and P. angolensis at 200 mg. kg -1 bw improve the physical parameters of libido as well as the testosterone level and to this end exhibit an aphrodisiac effect similar to their antioxidant activity. These results constitute a scientific basis likely to justify the use of these two plant species as aphrodisiacs in traditional medicine of Lubumbashi. Further studies should focus on the isolation of these antioxidant and aphrodisiac compounds preferably for P. angolensis whose phytochemistry is not reported.