Immobilization of Human Sperms by Gossypol Incorporated into l-Ascorbyl Palmitate Coagel

Gossypol and its derivatives have interestingly showed potential contraceptive effects by acting as spermicidal agents concomitantly having other versatile biological activities against numerous human pathogenic infections, to get rid of its systemic toxicity, gossypol was incorporated into l-Ascorbyl Palmitate (l-AP) coagel as topical formulation. The in vitro immobilizing effect of gossypol incorporated into l-Ascorbyl Palmitate coagel on human sperms was investigated. The spermatozoal motility and straight line velocity (VSL) analysis was conducted by using Computer Assisted/Aided Sperms Analyzer-motility (CASA-Mot.) system on 30 human semen samples after exposure to 0.1%, 0.2%, 0.3%, 0.4% and 0.5% (w/w) gossypol-containing coagels. There appeared a lag time up to around 5minutes without any detectable effect followed by a non-immediate and quick immobilization effect through which a great number of progressively motile spermatozoa were completely seen immobilized after an average minimum period of 10minutes. Two out of thirty subjects, few sperms with weak tail shake-like motions were seen but without forward progression even after 30 minutes of exposure. The deep decrease in percentage of motile sperms was dependent to gossypol concentration and duration of exposure, spermatozoal progressive straight line velocity decreased rapidly and remained low until the motility ceased. The 0.2% gossypol coagel showed the best immobilizing effects at reasonable activity time promising a better spermicidal activity required for multipurpose contraceptive formulations for future use for control of both pregnancy and some sexually transmitted infections.


Introduction
Gossypol, a natural complex polyphenolic extract ( Figure 1) from cotton plants (Gossypium ssp.); it is a highly reactive organic compound due to its carbonyl and polar groups (6 hydroxyls and 2 aldehydes) as well as to its bulky binaphtalene structure [1]. Its phenolic groups readily react to form esters and ethers; whereas the aldehyde groups react with amines to form Schiff's bases and with organic acids to form heat labile compounds or it may either also react with other products of the plant to form bound gossypol or remains as free gossypol which is attributed to be its most toxic form [2,3] through the clarifications of the multiple reactivity of gossypol, it is proposed that gossypol exists in three tautomeric (aldehyde, ketonoid, and the hemiacetal) forms ( Figure 2) through which it may interchange into many different reactive sites [4].
Therefore, through many researches concerning the polyvalent reactivity of gossypol and its derivatives; it was interestingly discovered its versatile biological activities including: anticancer [5][6][7]; antioxidant [8]; antimicrobial against main human pathogenic microorganisms such as bacteria, fungi, protozoa and yeasts [9]; antiviral activities against herpes simplex virus [10] human immunodeficiency virus-1 [11][12], some arboviruses and influenza type viruses; insecticidal [13][14][15] and antifertility activities [16][17][18].  In fact, even though gossypol exerts such potential biological activities ; its inescapable systemic toxicity is still a big limit from its clinical uses per any usual route of administration; gossypol has a high binding affinity with albumin and iron of hemoglobin of red blood cells thereby forming a strong complex leading to generalized anemia, lungs edema or hemorrhages and respiratory distress, mostly death depends on ingested amount and time of exposure [19][20][21]; gossypol acts as Prostaglandins biosynthesis stimulating agent and Na-K-ATPase inhibitor leading to renal potassium loss thereby causing chronic hypokalemia-causing cycle in some subjects [22,23]; it was also reported to be associated with reduction in number and early degeneration of testicular leyding cells thereby probably causing drug-induced sterility through negative interferences with spermatogenesis cycle [24][25][26].
Therefore, the systemic toxicity of gossypol and its derivatives is mainly species and dose-dependent but also time of exposure and route of administration may play an important role.
Through some series of clinical trials, no side effects were enhanced by small doses, even before humans have used gossypolcontaining drugs (bronchitis and cough) and foods but few and negligible related adverse effects were reported [27][28][29], the general accepted standards of gossypol consumption have been set to be no more than 0.8mg/kg/day for 6weeks as the maximum safety dose human species can tolerate [30,31].
Lately, after understanding the potentials of gossypol and its derivatives and after studying the mechanisms of action of severity of its adverse effects; many studies have been conducted trying to counteract its toxicity and maintaining its pharmacological activi-ties either by co-administration with selenium and potassium supplements or by suppressing aldehyde groups on which lies its toxicity [32][33][34]. In the framework of minimizing hematological side effects caused by gossypol toxicity; the development of gossypol gel was successful achieved and reported neither side effects nor skin irritations on monkeys and female humans as an effective spermicidal contraceptive in vitro and in vivo [35][36][37]; this was a promising step proving that gossypol used topically is safer than other usual routes of administration. In this way; our laboratory successfully produced two kinds of liposoluble topical formulations by incorporating gossypol into l-ascorbyl ibuprofenate (l-AI) coagel [38] and incorporating gossypol into l-ascorbyl palmitate (l-AP) coagel [39].
Recently, the studies of rheological and viscoelastic properties of these coagels were conducted and revealed that these formulations are the best carriers of hydrophobic drugs like gossypol through the skin and they exert a limited gossypol release either from the drug formulation or skin reservoir systems through the skin but without passing deeper through stratum corneum to the system circulation and it was found that gossypol released from the coagel formulation remained entrapped on and within the outermost skin layer where reside most human pathogenic microorganisms before their distribution into systemic living systems [40] and this is a proof of a trustworthy topical dug formulation promising to restrict the systemic toxicity of gossypol once used in the future as a new and effective drug having potential abilities of acting on most human pathogenic microorganisms and spermicide. The evaluation of this gossypol-containing formulation refers to the facts that gossypol was previously reported to have not only contraceptive properties of acting as the most powerful male antifertility agent possibly effective at 99.9% [41] and sperms motility barrier, spermicidal [42][43][44] and spermatogenesis inhibitor [44], inhibitor of ovum implantation and early pregnancy [45][46] in different animals and human both in vitro and in vivo studies but, also its ability to prevent some sexual transmitted infections (STIs) based on previously reported polyvalent biological activities of gossypol and its derivatives [47][48][49].

Materials and Methods
Incorporation of gossypol into l-ascorbyl palmitate (l-AP) coagel started by dissolving l-ascorbyl palmitate powder (purchased from Hebei Xing Run Biological Technology Co. Ltd.) to make 4% l-AP coagel and successfully achieved by dissolving gossypol powder into l-AP coagel as previously described [26] with controlled mixing and temperature to make 0.1%, 0.2% 0.3%, 0.4% and 0.5% (w/w) gossypol-containing coagel formulations, a small concentration of glycerol less than 3% (v/v) was also used during coagel formulation as recommended in previous studies [50][51][52], immobilization activity was assessed following modified DP. Waller method [53].
The statistical descriptive data were analyzed by SPSS packet (one way ANOVA) and the variables were accepted to be defined as significant at 95% of confidence mean interval with p˂0.05.
The human semen samples used in this study were voluntarily parameters; this is a reliable analysis system recommended for accurate sperm concentration and motility levels analysis [55].

Results and Discussion
The data recorded to study the changes in number of total progressively motile spermatozoa percentage following an exposure to different concentrations of gossypol-containing coagel over time intervals for deep analysis of decrease in percentage of total motile sperms (Figure 3), illustrative figures were taken and minimum immobilization time was studied (Table 1)      With 0.2% and 0.3% c.
And then after 10min. with 0.1% e.
0.2% the same as 0.3% f. 0.4% the same as 0.5% g. And after 30minutes with 0.1%, 0.2%, 0.3%, 0.4% 0.5% of gossypol, h. the system showing complete immobilization of spermatozoa. The volume ratio of semen /coagel was1:1 The study of spermatozoa immobilization effects on progressively motile spermatozoal percentage counts ( Table 2) and progressive straight line velocity (PRᶲVSL) ( Table 3) Table 2) concomitantly with a quite decrease in mean spermatozoal progressive straight line velocity (Table 3) at 10minutes until a complete motility cessation in 28 out of 30 semen samples was observed. The immobilizing effect of different forms of gossypol and its derivatives on human sperms motility has also been reported in many previous studies [9,32,37], but the main mechanism of action of this immobilization is still not clearly known. Note: The progressively motile sperms percentage counts were recorded after 0, 10,15, 20, 25 and 30min of incubation without and with 0.2% gossypol and 0.4% l-Ascorbyl Palmitate coagel without gossypol was used as control. The system considered 4 visual fields during video tracking and these data are expressed as mean ± standard deviation after conducting experience ssin triplicate. Gossypol may interfere with spermatozoa energy metabolism cycle either by causing uncoupled respiratory chain and oxidative phosphorylation [1,47] or by any other way of reducing sperm cell ATP content [32,46] and thus lead to decreased motility of spermatozoa of different animals including human in vitro; but if it is orally or parenterally taken leading to decreased sperms counts [30]. The immobilization of human sperms by gossypol incorporated into l-Ascorbyl Palmitate coagel seen in this study, has been shown to be not immediate but rapid after 10 minutes, there has been a 5minutes lag time before a detectable effect is seen, and this may be the fact that the decrease in motility might be quite linked to the perturbation of spermatozoa energy metabolism and may be another unknown mechanism.
The results obtained from our in vitro experiment showed that the first significant sperms immobilization was clearly achieved to produce contraceptive effects in vivo rabbits model study [52]; taking into considerations of immobilization time, another study reported a complete immobilization of all spermatozoa by gossypol polyvinyl pyrrolidone co-precipitate at concentrations of 5mg/ml, 40 mg/ml within 3minutes and 20 seconds respectively [9]. More interestingly, human post-coital tests with gossypol-containing vaginal gel application showed a significant spermatozoa number decrease in cervical mucus followed by an immobilization after a period of time [21]. Unfortunately, these data contradict with the results reported by Sukumal et al. [51] where only monkey sperms were completely immobilized within 15 minutes by 50µM of gossypol, but motility of human spermatozoa persisted even at 500µM within 2hours of exposure [43].

Conclusion
In the present case, gossypol incorporated into l-AP showed a strong immobilization effect on human sperms after a reasonable