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

American Journal of Chinese Medicine, Wntr, 2001 by Jung-Chou Chen, Leih-Der Chen, Wei Tsauer, Chin Chuan Tsai, Bih-Chern Chen, Ying-Jeng 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.