Effects of Ginsenoside Rb2 and Rc on Inferior Human Sperm Motility In Vitro

Effects of Ginsenoside Rb2 and Rc on Inferior Human Sperm Motility In Vitro

Jung-Chou Chen

(Accepted for publication June 6, 2000)

Abstract: The purpose of the study was to investigate the effects of two constituents of Panax notoginseng flower extract, Ginsenoside Rb2 and Rc, on human sperm motility and progression in vitro. Semen samples were collected from 20 patients with sperm motility between 20% and 40% of normal. All samples had sperm counts of over 20 million per milliliter, in accordance with the World Health Organization standard. Sperm were separated by a Percoll discontinuous gradient technique, and divided into a Percoll sperm control group, and three Ginsenoside Rb2 experimental groups (0.1, 0.01 and 0.001 mg/ml) and three Ginsenoside Rc experimental groups (0.1, 0.01 and 0.001 mg/ml). The results showed that at concentrations of 0.01 mg/ml and 0.001 mg/ml, Ginsenoside Rc enhanced both sperm motility and sperm progression significantly at the end of the 1st and 2nd hour. However, the three concentrations of Ginsenoside Rb2 did not increase sperm motility at the 1st or 2nd hour, but promoted sperm progression at the 2nd hour, when compared to the Percoll group.

Poor sperm motility is an important factor causing male infertility (Hong et al., 1991). The quality and quantity of sperm can be improved by treatment with drugs or by better semen washing. Pentoxyfylline, a methylxanthine derivative, was shown to stimulate motility, path velocity, and percent hyperactivation of human sperm (Rees et al., 1990; Sikka and Hellstrom, 1991; Tesarik et al., 1992; Pang et al., 1993; Wang et al., 1993). Pentoxyfylline was also reported to improve fertilization rates and decrease fertilization failure in in vitro studies on subfertile males (Yovich et al., 1990). Of the commonly used semen washing methods, the swim-up separation method, the two-step wash method and the Percoll discontinuous gradient separation technique (Machelle, 1990), the last is the one mostly employed in male infertility clinics for artificial fertilization in Taiwan.

The effect of ginseng saponins on sperm mobility was first observed in our laboratory. Our previous study (Chen et al., 1998) revealed that crude extracts of Panax notoginseng root can increase human sperm motility in vitro. The present study was designed to investigate the effects of two constituents of Panax notoginseng flower extract, Ginsenoside Rb2 and Rc, on inferior human sperm treated with the Percoll discontinuous gradient separation technique.

Materials and Methods

Semen Samples

Semen samples were collected from 20 male patients (Mean age = 33.20 [+ or -] 3.45 years) with sperm motility between 20% and 40% of normal at the Department of Chinese Medicine, China Medical College Hospital. All samples had a sperm count of over 20 million per mililliter in accordance with the World Health Organization standard (Sharon and Raphael, 1991). The mean semen pH value of the 20 subjects was 7.35 [+ or -] 0.15.

Preparation of Ginsenoside Rb2 and Rc Solution

Ginsenoside Rb2 and Rc powder with high purity were purchased from Extrasynthese, France. Ginsenoside Rb2 and Rc were dissolved in Human Tubal Fluid (HTF) media to make final concentrations of 0.1 mg/ml, 0.01 mg/ml and 0.001 mg/ml, respectively. One hundred ml HTF media contain 10 ml stock A solution (5.931 gm NaCl, 0.35 gm KCl, 0.024 gm Mg[SO.sub.4], 0.05 gm [KH.sub.2][PO.sub.4], 3.7 ml Na lactate, 0.5 gm glucose, 0.06 gm penicillin/100 ml), 1.6 ml stock B solution (1.05 gm Na[HCO.sub.3], 0.005 gm phenol red/50 ml), 0.71 ml stock C solution (0.051 gm Na pyruvate/10 ml), 1.15 ml stock D solution (0.262 gm Ca[Cl.sub.2] [multiplied by] 2[H.sub.2]O/10 ml), 8.4 ml stock E solution (3.254 gm Hepes-Na salt, 0.005 gm phenol red/50 ml), and 78.14 ml [H.sub.2]O. The pH value and osmolarity of HTF media were 7.50 and 280 [+ or -] 2 mOsm/kg respectively.

Preparation of Isotonic Percoll (Isopercoll)

Isotonic percoll was made up with 45 ml Percoll, 5 ml stock A medium and 0.105 gm NaHCO3. Percoll was purchased from the Pharmacia Biotech AB, Uppsala, Sweden.

Measurement of Sperm Counts, Motility (%) and Progression (%)

After each semen sample became liquefied at room temperature, semen viscosity and pH value were measured. To determine the initial sperm count, motility, and progression, 10 ul liquefied semen were taken up with a digital micropipette (R880/A Volac), placed into a Makler Counting Chamber (Sefi-Medical Instruments, Haifa, Israel), loaded into a Hamilton Thron Research computer-auxiliary sperm analyzer (HTM-2030), and then calculated by the HTM IVOS Motility Analyzer Version 8.1 software.

The remaining liquefied semen was separated with a Percoll discontinuous gradient method. The Percoll gradient from top to bottom of the tube was 50% isotonic Percoll (1.0 ml isopercoll + 1.0 ml HTF), 70% isotonic Percoll (2.3 ml isopercoll + 1.0 ml HTF), and 95% isotonic Percoll (1.9 ml isopercoll + 0.1 ml HTF), respectively. Semen was gently placed on top of the gradient, then centrifuged at 2000 rpm for 15 min. Sperm should be most concentrated in the 95% Percoll fraction. The upper two layers were discarded, and the bottom layer was washed twice with HTF media then adjusted to an optimal concentration of 10-15 million/ml.

The Percoll treated sperm were divided into a control group and six experimental groups. The six experimental groups had concentrations of 0.1, 0.01 and 0.001 mg/ml Ginsenoside Rb2 and Rc, respectively. One hundred ul of Ginsenoside solution measured with a digital micropipette (R880/B, Volac) and equal volume of the Percoll treated sperm were mixed and placed into a 37 [degrees] C incubator with 5% [CO.sub.2] and 95% O2 (Napco Model 5410 USA). At the end of the 1st and 2nd hour, 10 ul of the mixture was withdrawn and placed in a Makler Counting Chamber. Sperm motility and progression were determined as described above.

The results described below are based on 20 repetitions of this experiment, one for each of the 20 subjects.

Statistical Analysis

Statistical differences between the Ginsenoside groups and the Percoll control group were analyzed by ANOVA with an IBM compatible software SAS statistical package (SAS Institute, Cary, NC).

Results

Sperm Counts

Sperm counts of the 20 subjects were between 22.20 to 120.50 million/ml with a mean value of 57.80 [+ or -] 35.50 million/ml.

Effects of Ginsenoside Rb2 and Rc on Sperm Motility

Mean values of sperm motility of the Percoll control and the Ginsenoside Rb2 and Rc treated groups are shown in Table 1. The results demonstrated that the three different concentrations of Ginsenoside Rb2 did not increase sperm motility at the 1st or 2nd hour, while 0.01 mg/ml and 0.001 mg/ml Ginsenoside Rc significantly enhanced sperm motility at the end of the 1st and 2nd hour.

Table 1. Mean Values of Sperm Motility (%) by Ginsenoside Rb2 and Rc

Items Percoll group 0.1 mg/ml

Rb2 (1h) 75.40 [+ or -] 13.79 79.63 [+ or -] 19.73

Rc (1h) 75.40 [+ or -] 13.79 81.65 [+ or -] 14.71

Rb2 (2h) 77.55 [+ or -] 21.52 78.35 [+ or -] 15.94

Rc (2h) 77.55 [+ or -] 21.52 78.95 [+ or -] 12.68

Items 0.01 mg/ml 0.001 mg/ml

Rb2 (1h) 76.80 [+ or -] 18.93 74.33 [+ or -] 18.74

Rc (1h) 87.75 [+ or -] 11.40(a) 89.88 [+ or -] 10.32(a)

Rb2 (2h) 81.20 [+ or -] 12.42 83.92 [+ or -] 15.94

Rc (2h) 84.15 [+ or -] 12.56(a) 87.86 [+ or -] 12.74(a)

Each value represents mean [+ or -] S.D.(n=20).

(a): compared with Percoll group, p < 0.001

Repeated measurement ANOVA was performed.

Effects of Ginsenoside Rb2 and Rc on Sperm Progression

Table 2 shows the mean values of sperm progression for the Percoll control group and the Ginsenoside Rb2 and Rc treated groups. The 0.01 mg/ml and 0.001 mg/ml Ginsenoside Rc treated groups enhanced sperm progression, and the increase was statistically significant at the end of the 1st and 2nd hour. The three different concentrations of Ginsenoside Rb2 also increased sperm progression when compared to the Percoll group at the 2nd hour.

Table 2. Mean Values of Sperm Progression (%)

by Ginsenoside Rb2 and Rc

Items Percoll group 0.1 mg/ml

Rb2 (1h) 42.85 [+ or -] 15.05 55.21 [+ or -] 18.39(a)

Rc (1h) 42.85 [+ or -] 15.05 58.40 [+ or -] 18.26(a)

Rb2 (2h) 51.30 [+ or -] 21.57 61.40 [+ or -] 20.93(a)

Rc (2h) 51.30 [+ or -] 21.57 55.55 [+ or -] 20.75

Items 0.01 mg/ml 0.001 mg/ml

Rb2 (1h) 49.10 [+ or -] 17.51 48.27 [+ or -] 16.52

Rc (1h) 60.20 [+ or -] 18.78(a) 61.23 [+ or -] 17.71(a)

Rb2 (2h) 60.65 [+ or -] 21.84(a) 59.65 [+ or -] 19.84(a)

Rc (2h) 61.20 [+ or -] 17.76(a) 63.24 [+ or -] 19.93(a)

Each value represents mean [+ or -] S.D.(n = 20). Repeated measurement

ANOVA was performed.

(a): compared with Percoll group, p < 0.001

Discussion

Sperm motility is one of the major determinants of male fertility (Hartman, 1965). Sperm motility can be improved by treatment with Pentoxyfylline (Rees et al., 1990; Sikka and Hellstrom, 1991; Tesarik et al., 1992; Pang et al., 1993; Wang et al., 1993) and better semen washing. The Percoll discontinuous gradient separation technique is the most frequently employed semen washing method in Taiwan. The swim-up separation method allows a highly motile fraction of sperm to be collected. When increased numbers of abnormal or immature forms are present in the ejaculate or while blood cell and ejaculate debris concentrations are high, swim-up techniques provide a reasonable mechanism for isolating normal motile spermatozoa; however, the motile sperm may carry ejaculate debris into the swim-up suspension. The two-step washing procedure provides the highest sperm recovery rate, but nonviable sperm and debris are collected as well. The Percoll discontinuous gradient separation technique is used most commonly for males with normal sperm density and very low motility. Because ejaculates with increased concentrations of white blood cells or immature forms can benefit from Percoll separation, this is the method of choice to facilitate the collection of a highly motile sperm fraction (Machelle, 1990).

In our previous study (Chen et al., 1999), we found ginseng saponins extracted from the root of Panax notoginseng could enhance inferior sperm motility. So we were interested in investigating the effects of different saponins from Panax notoginseng on sperm motility and progression. We selected the flower ginseng saponins (Ginsenoside Rb2 and Rc) of Panax notoginseng because they are absent from the root of Panax notoginseng also because the root can only be harvested once every five years, but the flower can be collected every year. The main saponins in the root of Panax notoginseng are Ginsenoside Rb1 and Rg1 (Yang and Cheng, 1994). Hence, in this study we wished to observe whether the flower saponins Rb2 and Rc could enhance sperm function. Rather than extracting individual saponin in our laboratory, it was convenient to purchase Ginsenoside Rb2 and Rc from Extra-synthese in France. We chose the three concentrations of 0.1, 0.01, 0.001 mg/ml of Ginsenoside Rb2 and Rc according to our pretest experience. These concentrations were not equivalent to the concentrations of Rb1 and Rg1 in Panax notoginseng root crude extract used in our previous study.

In the present study, the spermatozoa were fractionated by a modified discontinuous Percoll gradients method described by Aitken and Clarkson (1988). Our results showed that 0.01 and 0.001 mg/ml Ginsenoside Rc has a significant stimulating effect on sperm motility and progression at the end of the 1st and the 2nd hour. Although the effect of Ginsenoside Rb2 on sperm motility was not statistically significant, it promoted sperm progression at the 2nd hour when compared to the Percoll control. Sperm motility (%) is calculated as the mobile cell velocity, including rapid (velocity [is greater than] 25 micron/sec), medium (velocity 10~25 micron/sec), and slow (velocity [is less than] 10 micron/sec) total cells of sperm. Sperm progression (%) is calculated as progressive sperm cells/total cells. Progressive sperm cell is to have Cell Straightness over 80% and Path Velocity over 25 micron/sec. Whether Ginsenoside Rb2 and Rc increased sperm progression through Cell Straightness or Path Velocity is worthy of further investigation.

In our previous study (Chen et al., 1999), the stimulating effects of a crude 1.0 mg/ml n-butanol extract of Panax notoginseng root saponins on sperm motility and progression were found to be 1.8 and 2.2 fold, respectively, at the end of the 2nd hour. The present result showed 1.1 and 1.2 fold stimulation of sperm motility and progression, respectively, by 0.01 mg/ml Ginsenoside Rc. The different results found in our two studies may be due to the fact that different ginseng saponins and concentrations were tested in the two separate studies. Pentoxyfylline is known to stimulate the motility of human sperm. Whether Ginsenoside Rb2 and Rc, like pentoxyfylline, can improve the fertilization rate in subfertile males warrants further study.

Acknowledgment

The authors are thankful to the Department of Gynecology and Obstetrics, China Medical College Hospital, for offering HTF media and technical assistance of Percoll discontinuous gradient separation.

References

[1.] Aitken, R.J. and J.S. Clarkson. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques, J. Androl. 9:367-376, 1988.

[2.] Chen, J.C., M.X. Xu, L.D. Chen, Y.N. Chen and T.H. Chiu. Effect of Panax notoginseng saponins on sperm motility and progression in vitro. Phytomedicine 5(4): 289-292, 1998.

[3.] Chen, J.C., M.X. Xu, L.D. Chen, Y.N. Chen and T.H. Chiu. Effect of Panax notoginseng extracts on inferior sperm motility in vitro. Am. J. Chin. Med. 27: 123-128, 1999.

[4.] Cheng, K.C. and C.R. Yang. Biology and application of Panax notoginseng. Science publishing house, Beijin, pp. 10-17, 1994.

[5.] Hamilton-Thron Motility IVOS Operations Manual. In: progressive criteria. Beverly: Hamilton-Thron Research, pp. 37-56, 1992.

[6.] Hartman, H.G. Correlation among criteria of semen quality. Fertil. Steril. 16: 532-644, 1965.

[7.] Hong, C.Y., H.T. Chao and Y.H. Wei. Pharmacological stimulation of sperm motility in vitro. In: Hafez ESE (ed.), Assisted Human Reproduction Technology. Washington, DC: Hemisphere Publishing Co., pp. 201-209, 1991.

[8.] Machelle, M. S. Infertility: A Comprehensive Text. Prentice Hall, U.S.A., pp. 142-192, 1990.

[9.] Pang, S.C., P.J. Chan and A. Lu. Effects of pentoxyfylline on sperm motility and hyperactivation in normozoospermic and normokinetic semen. Fertil. Steril. 60: 336-343, 1993.

[10.] Rees, J.M., W.C.L. Ford and M.G.R. Hull. Effect of caffeine and pentoxyfylline on the motility and metabolism of human spermatozoa, J. Reprod. Fertil. 90: 147-156, 1990.

[11.] Sikka, S.C. and W.J.G. Hellstrom. The application of pentoxyfylline in the stimulation of sperm motion in men undergoing electroejaculation, J. Androl. 12: 165-170, 1991.

[12.] Tesarik, J., A. Thebault and J. Testart. Effect of pentoxyfylline on sperm movement characteristics in normozoospermic and asthenozoospermic specimens. Hum. Reprod. 7: 1257-1263, 1992.

[13.] Wang, R., S.C. Sikka, K. Veeraragavan, M. Bell and W.J.D. Hellstrom. Platelet activating facter (PAF) and pentoxyfylline (PTX) as human sperm cryoprotectants. Fertil. Steril. 60: 711-715, 1993.

[14.] Yovich, J.M., W.R. Edirisinghe, J.M. Cimmins and J.K.L. Yovich. Influence of pentoxyfylline in severe male factor infertility. Fertil. Steril. 53: 715-722, 1990.

[15.] Sharon, B.J. and J. Raphael. The basic infertility investigation. Fertil. Steril. 4: 602, 1991.

Jung-Chou Chen (1,2,3)(*), Leih-Der Chen(4), Wei Tsauer(4), Chin Chuan Tsai(4), Bih-Chern Chen(4) and Ying-Jeng Chen(1) (1)Research Institute of Chinese Medicine, China Medical College, 91 Hsueh Shih Road, Taichung, Taiwan, (2)School of Chinese Medicine, China Medical College, Taiwan, (3)Show Chwan Memorial Hospital, Chunghua, Taiwan, (4)School of Post-baccalaureate Chinese Medicine, China Medical College (*) Corresponding address: Dr. Jung-Chou Chen, 50, Yeong-Shing Street, Taichung 404, Taiwan

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