Ginseng Flowers Stimulate Progesterone Production from Bovine Luteal Cells

American Journal of Chinese Medicine, Summer-Fall, 2000 by Leang-Shin Wu, Shiow-Yunn Sheu, Cheng-Chun Huang, Chih-Hsien Chiu, Jan-Chi Huang, Jenn-Rong Yang, Wai-Xiong Lian, Chih-Hao Lai, Yuh-Pan Chen, Jen-Hsou Lin

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.