Vandellia Cordifolia Regulated Cell Proliferation and Cytokines Production in Human Mononuclear Cells

American Journal of Chinese Medicine, Summer-Fall, 2000 by An-Pang Lin, Wei-Jern Tsai, Chi-Yen Fan, Ming-Jen Lee, Yuh-Chi Kuo

Abstract: Vandellia cordifolia (V. cordifolia) used for treatment inflammation in traditional Chinese medicine was selected for immunopharmacological activity test. The effects of V. cordifolia extracted fractions on human mononuclear cells (HMNC) proliferation were determined by tritiated thymidine uptake. The results indicated that VC-ME fraction suppressed HMNC proliferation activated with phytohemagglutinin (PHA) and stimulated cell cycle progression was arrested at the G0/G1 stage. The inhibitory mechanisms may involve the blocking of interleukin-2 (IL-2) and interferon-[Gamma] (IFN-[Gamma]) production, since VC-ME suppressed IL-2 and IFN-[Gamma] production of HMNC in a dose-dependent manner. Therefore, it is suggested that immunomodulatory agents are contained in V. cordifolia.

Although Chinese herbs have generally been used in traditional Chinese medicine for a long time, there has been a relative scarcity of definitive evidence to prove their pharmacological activity. In the present study, Vandellia cordifolia (Colsm) G. Don (V. cordifolia) that has antiinflammatory activity was selected for immunomodulatory activity determination. Its putative active functions include: (1) treatment of diarrhea and pneumonia, (2) relief of nephritis, (3) reduction of swelling, (4) enhancement of blood circulation, and (5) detoxification (Chang et al., 1985).

The inflammatory response is a nonspecific immune response triggered by pathogenic microorganism infection or tissue injury (Janeway et al., 1997). There are three major events that occur during an inflammatory response: (1) vasodilation, (2) increased capillary permeability, and (3) influx of phagocytic cells (Kuby, 1997). Inflammation is characterized by redness, swelling, heat and pain in damaged tissues or infected sites. The inflammatory response provides early protection in restricting the tissue damage to the site of infection or tissue injury. Several immune cells including lymphocytes, neutrophils, monocytes, eosinophils, and basophils are involved in inflammatory response. They secrete cytokines at the site of inflammation, then participate in clearance of the antigen and healing of the tissue (Arai et al., 1990). Both tumor necrosis factor-[Alpha] (TNF-[alpha]) and interleukin-1 (IL- 1) induce increased expression of adhesion molecules on vascular endothelial cells leading to an increase in vascular permeability (Aggrwal et al., 1985; Giovine and Duffy, 1990). Interleukin-8 (IL-8) and interleukin-6 (IL-6) serve as chemotactic factor for various leukocyte populations (Akira and Kishimoto, 1992; Akuffo, 1992). On the other hand, interferon-[Gamma], (IFN-[Gamma]) is an important inflammatory factor for the attraction of macrophages (Dalton and Pitts-MeeK, 1993). Interleukin-2 (IL-2) stimulates proliferation of T lymphocytes and also acts on natural killer cells and macrophages to induce other cytokines involved in inflammatory responses (Taniguchi and Minami, 1993). Both leukocytes and cytokines play important roles in inflammatory response regulation. Yet the inflammatory response to invasive organisms, if sufficiently intense or inappropriately prolonged, could paradoxically aggravate the injury or even cause death. The use of antiinflammatory medications must therefore be discreet (Beutler and Ceromi, 1989). Blockade of the lymphocyte activation and proliferation is one of antiinflammatory mechanisms (Adams and Hamilton, 1984).

In order to prove the immunomodulatory function of V. cordifolia, human mononuclear cells (HMNC) involving T lymphocytes, B lymphocytes and macrophages isolated from peripheral blood were used as target cells. The V. cordifolia was extracted by ethanol, then separated into five fractions. The effects of these five fractions on HMNC proliferation and cytokines production were determined.

Materials and Methods

The Source of V. Cordifolia

The V. cordifolia was collected and identified by Dr. An-Pan Lin and Dr. Ming-Jen Lee.

Preparation of V. Cordifolia Crude Extracts

300 gm of dried V. cordifolia was extracted with ethanol (EtOH) three times (5 Lx 3). The ethanolic extracts were evaporated with Rotavapor under vacuum. The crude extracts were partitioned successively between hexane and water, followed by methanol, chloroform, and butanol, respectively. All extracted fractions of V. cordifolia were dissolved by dimethylsulfoxide (DMSO) and stored at 4 [degrees] C until for use.

Preparation of HMNC

Heparinized human peripheral blood (20 ml) was obtained from healthy donors. HMNC was isolated by the Ficoll-Hypaque gradient density method as described previously (Yang et al., 1999). The 20 ml peripheral blood was centrifuged at 2000 rpm, 4 [degrees] C for 10 min to remove the plasma. Blood cells were diluted with PBS buffer, then centrifuged in a Ficoll-Hypaque discontinuous gradient at 1500 rpm for 30 min. The HMNC layers were collected and washed with cold distilled water and 10 x Hanks' buffer saline solution (HBSS) to remove red blood cells. The cells were resuspended to a concentration of 2 x [10.sup.6] cells/ml in RPMI-1640 medium supplemented with 2% fetal calf serum (FCS), 100 U/ml penicillin and 100 [micro]g/ml streptomycin.