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American Journal of Chinese Medicine

Ginseng Flowers Stimulate Progesterone Production from Bovine Luteal Cells

Ginseng Flowers Stimulate Progesterone Production from Bovine Luteal Cells

Leang-Shin Wu

Abstract: Our previous report first showed evidence that polysaccharides isolated from ginseng leaves obtained from Jilin, China possess luteotropic activities. In this study, we made further investigations on the root and flowers of Korean ginseng by means of the same bioassay system described briefly as follows. Frozen-thawed bovine luteal cells (1 x [10.sup.5] cells/ml/well) in M199 were incubated in 24-well culture plates at 37 [degrees] C in a 5% [CO.sub.2] incubator. Ten [micro]l of tested drugs with 1, 10 and 100 [micro]g/ml were added into each well. After 4- and 24-hr incubation, the media were harvested and assayed for progesterone by an enzyme immunoassay. The production of progesterone from cells is the indicator for evaluating the action of tested drugs. Results showed that hot water extracts of ginseng flowers (GF-1) with 10 to 100 [micro]g/ml significantly increased progesterone production, whereas those from ginseng root (GR-1) could not. Crude polysaccharides (GF-2) isolated from GF-1 is the active component and the small molecules (mw [is less than] 10,000 dalton) are excluded, indicating that the ginseng root has no luteotropic activities, but the polysaccharides of ginseng flowers have.

The root of Panax ginseng has been used clinically in Chinese herbal medicine to treat many kinds of chronic diseases including hepatitis and nephritis, to enhance immune-system activities, to increase hypothalamo-pitiutary adrenocortical functions and sexual ability (Wang, 1991; Fu et al., 1981; Lin et al., 1995; 1998a, b). Such diversity of pharmacological activities was also approved in research works (Saito, 1974; Rim, 1979; Gao et al., 1989; Hu et al., 1995; Lin et al., 1995; 1998a; Huh et al., 1998; But, 1999). For this reason ginseng has been called the `king of tonic medicine’ in the Orient and a panacea in the West. Brekhman and Dardymov (1969) even introduced a concept of adaptogen to describe it. Adaptogen means something that can keep human beings or animals in a state of nonspecifically increased resistance (SNIR), which not only maintains homeostasis, but overcomes stress. Many Chinese medicinal herbs categorized in the tonic drugs, e.g. ginseng, san-qi (P. notoginseng), tsu-wu-jia (Ancanthopana senticosus) etc. have such actions (Lin et al., 1998b).

In recent years, some scientists, especially Japanese groups, have further investigated the functions of ginseng leaves and found that the leaves also possess some properties, as do the roots, such as anti-ulcerative, immunity enhancing activities etc. (Gao et al., 1984; Sun et al., 1992, 1994; Yamada, 1994). This suggests ginseng leaves may replace roots in some aspects of clinical use. Even more recently, the ginseng flower is also being focused on to develop its use, especially on the nutraceutical sector. Some commercial products have already appeared on the market in Korea, Japan, China and Taiwan. Meanwhile, very few papers concerning research works and clinical use of ginseng flower have been published in the scientific media.

In the present study, the luteotropic activities of ginseng flower and root were examined to evaluate their bioactivities.

Materials and Methods

Materials

Roots and flowers of Panax ginseng C. A. Meyer were cultivated and harvested in Korea and their dried products bought in Taipei, Taiwan. The culture media were purchased from Pharmacia Co. Ltd. Ovine luteinizing hormone (NIDDK-oLH-1-3, Bethesda, MD) was kindly provided by the National Hormone and Pituitary Program, USA. The EIA kits for progesterone were made in our laboratory (Wu et al., 1997). The bovine luteal tissues were obtained from Heng-Chun Station, Taiwan Livestock Research Institute. Other chemical compounds such as bovine serum albumin, collagenase, desoxyribonuclease I etc. were obtained from Sigma Chemical Co.

Preparation of Crude Hot Water Extracts and Polysaccharides from Roots or Flowers of P. Ginseng

One hundred grams of dried roots or flowers of P. ginseng were decocted with water (1 L) until the volume was reduced by half. The residue was then reboiled further after adding water as above. The two extracts were combined and lyophilized to obtain the water-soluble extracts. The extracts were named GR-1 for root extracts and GF-1 for flower extracts, respectively. Ten grams of GF-1 was refluxed with 500 ml MeOH for 1 hr and then centrifuged to get a MeOH-insoluble precipitate. This precipitate was dissolved in water and dialyzed against running water for three days. After the non-dialyzable portion was centrifuged at 8,000 rpm for 30 min, the supernatant was lyophilized to obtain crude polysaccharides (GF-2, yield 1.2 g). The MeOH-soluble fraction was evaporated to dryness. After that, the residue was redissolved in water and lyophilized to obtain GF-MeOH extract. This process was modified from Sun et al. (1992) for isolating crude soluble polysaccharides of ginseng leaves.

A Frozen-thawed Bovine Luteal Cell System to Screen Luteotropic Activity in Drugs

The frozen-thawed bovine luteal cell system established since 1994 has become a useful system for studying the steroidogenesis of chemicals due to its simplicity and reliability (Yuan et al., 1994). Once a good batch of frozen cells is obtained the data from each test between cryopreservative tubes are quite similar. The dispersed luteal cells were obtained enzymatically from corpus luteum enucleated aseptically from Holstein or beef cow between days 9 and 11 of the estrus cycle (estrus = day 0) by vaginal approach (Hickey and Hansel, 1987). The cryotubes each containing 1 x [10.sup.7] cells/ml and freezing media (10% DMSO; Gibco 320-1101PF) were frozen in liquid nitrogen for storage. Before the drug test, the frozen cells in the cryotube were thawed rapidly in water batch at 37 [degrees] C. The thawed cells were washed twice and resuspended in medium 199.1 ml of media and put in each well of 24-well culture plates (Coaster) containing 1 x [10.sup.5] viable cells/mi. Test drugs i.e. oLH, extracts were added in volumes of 10 [micro]l. The plates were incubated at 37 [degrees] C and 5% [CO.sub.2] for 24 hrs. During the incubation period 50 [micro]1 of media were collected periodically depending on the experimental design and frozen at -20 [degrees] C until progesterone assay. Each drug test was done in triplicate and each experiment was performed at least three times. The data shown were either selected from the best set of results with corresponding response tendency or from the average of three experiments.

Enzyme Immunoassay for Progesterone

A valid competitive enzyme immunoassay for progesterone was established in our laboratory using monoclonal antibody G7 (Wu et al., 1997). In brief a 50 [micro]l of diluted tested medium together with 150 [micro]l of progesterone-horseradish peroxidase were added to a 96-well microtiter plate (Costar 3590) coated with G7 monoclonal antibody. After incubation at room temperature for 15 min and washing with PBS twice, the color was developed in 200 [micro]l of 2.2 mM o-phenylene-diamine in 0.003% [H.sub.2][O.sub.2] at room temperature for 30 min. The reaction was stopped by 50 [micro]l of 8 N sulphuric acid. Absorbance rate at 490 nm was compared with a progesterone standard curve. The variation coefficients within and between assays were approximately 7 and 10% respectively. The sensitivity was 0.3 pg/well.

Statistical Analysis

Data obtained from pharmacological experiments were expressed as mean [+ or -] S.D. The differences between the control and the treatment groups in these studies were tested for statistical significance by using Student’s t-test and Duncan’s multiple range test. A value of P [is less than] 0.05 was considered a statistically significant difference.

Results and Discussion

Although most Chinese herbs are used by formulas, it is possible to use ginseng root as an exception, as a single herb for treatment (Wang, 1991; Hub et al., 1998; Lin and Wu, 1999). Ginseng is used to enhance the immune system, to stimulate appetite and weight gain, to control blood pressure, to protect the liver from toxins, to restore the iatrogenic Cushing syndrome, to increase the sexual ability, etc. (Saito et al., 1974; Rim, 1979; Lin et al., 1995; Hu et al., 1995).

In previous reports, scientists found that the leaves of ginseng show similar biological activities to the roots such as an increasing effect on immune reactions, anti-ulcerative abilities (Gao et al., 1989; Sun et al., 1992) and enhancing influence on steroidogenic activity (Rim, 1979; Lin et al., 1998a) which may improve reproduction performance. Unlike the roots, which can be obtained only every 5-6 yrs, the leaves and flowers can be harvested every year, which provides a great advantage for the use of ginseng leaves instead of roots if they have the same properties.

Flowers are the reproductive organs of plants. For humans, flowers present themselves in many ways: we can enjoy their beauty and elegance, add them to our food for nutritious reasons and even use them as medicinal herbs to treat diseases.

The ginseng flower blossoms once a year. Recently, it has been used commercially in the nutraceutical sector for tonifying body functions. Still very few scientific papers concerning the clinical use of ginseng flower have yet been published. Therefore we evaluated its possible abilities by testing its luteotropic activity, which is an indicator for enhancing reproductive functions.

Table 1 shows that hot-water extracts of ginseng flower (GF-1) could significantly increase the progesterone secretion of bovine luteal cells in a dose response manner (1-100 [micro]g) during 4-hr incubation whereas the ginseng root (GR-1) extract could not. After 24-hr incubation the stimulation effect of GF-1 was reduced in comparison to o-LH treatment. These results implied that the mechanism of ginseng flowers and ginseng leaves may both involve the acute steroidogenic synthesis only (Lin et al., 1998a).

Table 1. Effect of GF-1 and GR-1 on the Progesterone Production from Bovine Luteal Cells in Vitro

Progesterone

(ng/ml/[10.sup.5] cells

4 hr 24 hr

Basal 5.4 [+ or -] 1.0 15.6 [+ or -] 2.8

oLH 1 ng 12.6 [+ or -] 2.3(*) 47.8 [+ or -] 5.8(**)

GF-1

1 [micro]g 6.7 [+ or -] 1.2 14.6 [+ or -] 3.0

10 [micro]g 8.8 [+ or -] 1.5(*) 28.7 [+ or -] 4.5(*)

100 [micro]g 9.9 [+ or -] 1.0(*) 25.6 [+ or -] 2.4(*)

GR-1

1 [micro]g 5.6 [+ or -] 1.4 12.3 [+ or -] 1.5

10 [micro]g 6.2 [+ or -] 1.5 10.4 [+ or -] 4.9

100 [micro]g 4.7 [+ or -] 1.2 12.2 [+ or -] 6.0

Data are expressed as mean [+ or -] S.D. (n = 3),

(*) P < 0.05,

(**) P < 0.01. GF-1 and GR-1 were the hot-water extracts from flowers and roots of ginseng, respectively.

Surprisingly, the roots of ginseng did not show any enhancing effect on the steroidogenesis in luteal cells in the present study. The question now is: how does it work in the sexual or adrenal system (Rim 1979; Lin et. al., 1995). One of the possible ways of ginseng root actions may involve the pituitary system. Odani et al. (1986) demonstrated that the ginsenosides enhanced the ACTH secretion of rat pituitary cells in vitro. Or the active compounds in ginseng root are not enough to express in this examining system. Further studies must be done soon to answer these questions.

The next step in our study was to have the GF-1 fraction undergo molecular cutting at 10,000 daltons using molecular sieving technology and then perform further extraction for the lipid soluble substances. Those extracts from GF-1 were also examined for their luteotropic activities (Hickey and Hansel, 1987). Only the fractions of a molecular weight larger than 10,000 daltons possessed luteotropic activities indicating that crude polysaccharides of ginseng flower may involve this action like those from ginseng leaves (Figure 1). Therefore, further purification of crude polysaccharides from ginseng flower (GF-2) was done and, additionally, the residue of methanol soluble fraction (GF-MeOH) was collected (Sun et al., 1992). Data also demonstrated that only GF-2 could stimulate the progesterone production on bovine luteal cells whereas the GF-MeOH did not (Table 2). The stimulation potential of GF-2 is slightly higher than that of GF-1, but not as significantly as expected. This may be due to the bioassay or the cellular mechanism of polysaccharides of ginseng flower.

[Figure 1 ILLUSTRATION OMITTED]

Table 2. Effect of GF-2 and GF MeOH on the Progesterone Production from Bovine Luteal Cells in Vitro

Progesterone

(ng/ml/[10.sup.5] cells

4 hr 24 hr

Basal 7.7 [+ or -] 1.2 19.3 [+ or -] 2.4

oLH 1 ng 20.3 [+ or -] 2.5(*) 61.8 [+ or -] 8.0(**)

GF-1

10 [micro]g 13.3 [+ or -] 1.6(*) 30.5 [+ or -] 4.1

GF-2

1 [micro]g 8.9 [+ or -] 1.3 16.7 [+ or -] 2.7

10 [micro]g 15.7 [+ or -] 2.0(*) 38.9 [+ or -] 4.4(*)

100 [micro]g 16.3 [+ or -] 2.6(*) 32.6 [+ or -] 3.7(*)

GF-MeOH

1 [micro]g 5.8 [+ or -] 2.0 21.3 [+ or -] 3.3

10 [micro]g 6.9 [+ or -] 1.6 18.9 [+ or -] 2.5

100 [micro]g 5.2 [+ or -] 1.7 16.6 [+ or -] 2.7

Data are expressed as mean [+ or -] S.D. (n = 3),

(*) P < 0.05,

(**) P < 0.01. GF-2, a crude polysaccharide fraction, and GF-MeOH, a methanol fraction, were the two further extracts from GF-1.

In conclusion, it is a very interesting fact that, like ginseng leaves, ginseng flowers possess significant luteotropic activity especially in regard to the possible application for reproductive clinical uses in the future. The big advantage in being able to harvest leaves and flowers every year is that the availability for drug use increases immensely in comparison to harvesting ginseng roots. Also, ginseng flowers are already distributed commercially in the nutraceutical sector. Further scientific works are requested on these findings to analyze the exact mechanism and the active structure of the polysaccharides.

Acknowledgments

We thank the National Science Council, Republic of China, for financial support (NSC 88-2313-B-002-115).

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Leang-Shin Wu(1), Shiow-Yunn Sheu(2), Cheng-Chun Huang(2), Chih-Hsien Chiu(1), Jan-Chi Huang(1), Jenn-Rong Yang(1), Wai-Xiong Lian(1), Chih-Hao Lai(1), Yuh-Pan Chen(3) and Jen-Hsou Lin(1*)

(1) Department of Animal Science, National Taiwan University, Taipei 106, Taiwan,

(2) School of Pharmacy, Taipei Medical College, Taipei, Taiwan,

(3) Brion Research Institute of Taiwan, Taipei, Taiwan

(*) Corresponding author

(Accepted for publication June 12, 2000)

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