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Effect of Ten Ingredients from Chinese Herbal Medi

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    RESEARCH ARTICELS

    Effect of Ten Ingredients from Chinese Herbal Medicine

    on Proliferation of Chicken Embryo Fibroblast*

    Yuanliang Hu**, Xiangfeng Kong, Deyun Wang, Jiaguo Liu, Baokang Zhang

    Abstract: Ten Chinese herbal medicinal ingredients, astragalus polysaccharide (APS), Chinese angelica polysaccharide (CAPS), epimedium polysaccharide (EPS), isatis root polysaccharide (IRPS), propolis polysaccharide (PPS), astragalus flavone (AF), epimedium flavone (EF), propolis flavone (PF), astragalus saponin (AS) and ginsenoside (GS), were extracted and diluted into five concentrations which were incubated respectively with chicken embryo fibroblast (CEF) together in 96-well plate. At 24, 36, 48, 60 and 72 hours after cultivation, the proliferation of CEF was determined by MTT methods. The results showed that all of the ten could stimulate multiplication of the cell at suitable concentration and certain time points and the effects have relations with the dose and time.

    Key words: Chinese herbal medicinal ingredient (CHMI); chicken embryo fibroblast (CEF); cell proliferation

    Ginseng, Astragalus root, Chinese angelica root, Epimedium and Isatis rootPropolis are ordinary traditional Chinese medicine (TCM) respectively belonging to tonics and heat-clearing remedies [1]. Many researches have confirmed that many components from TCM possess immuno-enhancement and antiviral effects [2-5]. In the previous researches, the author extracted ten kinds of Chinese herbal medicinal ingredients (CHMI), astragalus polysaccharide (APS), Chinese angelica polysaccharide (CAPS), epimedium polysaccharide (EPS), isatis root polysaccharide (IRPS), propolis polysaccharide (PPS), astragalus flavone (AF), epimedium flavone (EF), propolis flavone (PF), astragalus saponin (AS) and ginsenoside (GS), from above-mentioned TCM and determined the safe concentration for each ingredient on chicken embryo fibroblast (CEF)[6]. In this research their effects on proliferation of CEF were further determined in order to evaluate them comprehensively and select better ones for development of new immuno-potentiator.

    MATERIALS AND METHODS

    1. Preparation of CHMI

    APS, CAPS, EPS, IRPS and PPS, whose extractions were by the means of water-decocting and alcohol-sedimentation and these contents determination were by sulfuric acid anthrone method. AF and EF were extracted by polyamide chromatography, PF, by acid-base precipitation, and the three contents were determined by rutin method. AS and GS, whose extractions were by wide absorbed resin chromatography and content determinations were by TLC spectrophotometry. According to the determining results of safe concentration on CEF [6], the ten CHMI were diluted into five different concentrations, autoclaved and stored at 4.

    2. Reagents

    MEM medium, Gibco BRL, was supplemented with L-glutamine up to 0.03%, penicillin, 100IU/ml, streptomycin, 100g/ml, and fetal calf serum, 5% (as growth solution) or 2% (as maintain solution). Trypase, imported by Kewei Scientific and Technical Company, was diluted into 0.25% with calcium and magnesium free phosphate-buffered saline (CMF-PBS PH 7.4), filtered through 0.22M millipore membrane filter, dispensed into small aliquots and reserved at -20. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, thiazolyl blue), Sigma, was diluted into 2% with CMF-PBS, filtered through 0.22M millipore membrane filter and reserved at 4 in dark bottle. Dimethyl sulfoxide (DMSO), AR, was as cleavage solution.

    3. Main instruments

    CO2 incubator, come from Revco Company, USA. Inverted microscope was made by Chongqing Optics Instrument Company in China. Enzyme-linked immunosorbent assay reader (DG3022) was made by East China Electron Tube Factory.

    4. Experimental methods

    According to the method described in the 7, 8th, references, CEF was prepared into cellular suspension contained 5105 cells/ml and added into 96-well plate, 100l/well. Then all wells were divided into experimental and control groups. In experimental groups, each 4 wells was respectively added with 100l of one concentration CHMI. In control group, each 4 wells was respectively added with 100l MEM growth solution. In addition, the control well without cell was arranged and added with MEM maintain solution only as zeroing well for determination. For each CHMI, 5 plates were repeated. After finishing sample-adding, the plates were put into incubator and the cells were cultured at 37 under 5% CO2 humid atmosphere. At 24, 36, 48, 60, 72h after incubation, one plate of per CHMI was taken out for determined of cell proliferation.

    5. Determination of CEF Proliferation

    MTT assay was applied [7, 8]. For 4h before each determination, 20l of MTT was added into each well and the plates were put back incubator. When determination, the plates were turned over to pour out the fluid in the well and 150l DMSO was added to each well. Then the plates were shaken on micro-plate oscillator for 10 minutes to dissolve the formazan crystals. The absorbance (optical density, OD, as index of cellular proliferation) of each well was read by enzyme-linked immunosorbent assay reader at wavelength of 570 nm. OD data were statistically compared by SPSS in Windows for the differences among different concentration groups at same time point.

    RESULTS

    1. Effect of APS on proliferation of CEF

    The OD data of every concentration groups at 60h were significantly larger than that of control group. At other time point, the OD data in high concentration groups were smaller or strikingly smaller than that in control group. The OD data in last two low concentration groups at 24 and 36h were slightly larger than that in control group. This showed that APS could remarkably promote multiplication of CEF at 60h. While in the early stage, high concentration inhibited and low concentration promoted CEF proliferation (Table 1).

    Table 1. Effect of APS on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h16000.3930.040bc0.4230.062b0.4980.013c0.9050.030c0.7000.063b12000.3550.087b0.4700.058ab0.5580.048b1.0830.074ab0.7330.038bcd6000.3630.028bc0.4880.038ab0.5650.033b1.0250.130ab0.7980.074ac3000.4130.026abc0.5050.057a0.6050.048ab0.9780.062bc0.8300.072a150

    control0.4580.010a

    0.4230.017ac0.5100.071a

    0.4480.010ab0.6100.037ab

    0.6450.026a1.1180.111a

    0.7800.059d0.8050.059ad

    0.8350.051a a, b, c and d shows descending order of column data, which with different alphabet indicate their difference is significant

    (p<0.05). The following tables are the same.

    2. Effect of CAPS on proliferation of CEF

    The OD data of first two high concentration (1,600 and 1,200) groups at 24, 48 and 72h were smaller or striking smaller than that of control group. The OD data in last two concentration (300 and 150) groups at 36 and 60h were strikingly larger than that in control group. The results showed that CAPS in high concentration inhibited and in low concentration promoted multiplication of CEF (Table 2).

    Table 2. Effect of CAPS on proliferation of CEF

    Concentration (g/ml)24h36h48h60h72h16000.3450.006b0.3950.035c0.3730.039d0.6930.064b0.4330.067c12000.3700.029b0.4180.026c0.4530.036c0.8050.066b0.7130.040b6000.4150.017a0.4500.045bc0.4930.057c0.9600.065a0.6480.047b3000.4280.022a0.4980.057ab0.5800.024b1.1280.038a0.7950.073ab150

    control0.4200.014a

    0.4230.017a0.5350.060a

    0.4480.010bc0.5580.053b

    0.6450.026a0.9280.076a

    0.7800.059b0.8400.093a

    0.8350.051a3. Effect of EPS on proliferation of CEF

    The OD data of all groups, except the first high concentration group, were larger than that of control group. After 24hr, the data of most low concentration groups were significantly larger than that of control group. The result indicated that EPS could promote multiplication of CEF, especially at lower concentration (Table 3).

    Table 3. Effect of EPS on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h200.5300.027bc0.5600.024c0.6250.053c0.7150.026c0.7200.089d100.5480.025bc0.5830.021c0.6750.050bc0.7800.047b0.8330.073cd50.5630.013ab0.6000.022bc0.7500.059ab0.8130.022ab0.8980.030bc2.50.6000.050a0.6380.051ab0.8030.087a0.7830.029b0.9480.046ab1.25

    control0.5800.024ab

    0.5730.021ab0.6580.050a

    0.5730.022c0.7350.077ab

    0.6680.017bc0.8480.038a

    0.6750.053c1.0050.031a

    0.7600.014d4. Effect of IRPS on proliferation of CEF

    Comparing with control group, most of OD data in every concentration group at 60, 72h and in the low concentration (120 and 80) groups at 24h were strikingly larger and in first two high concentration (600 and 300) groups at 48h were strikingly smaller. These results showed that IRPS in late stage and low concentration in early stage could notably promote, while high concentration in middle stage inhibit, multiplication of CEF (Table 4).

    Table 4. Effect of IRPS on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h6000.3730.017b0.4550.070a0.5200.018c1.0900.047a0.8450.072ab3000.3950.024ab0.4730.062a0.5700.016b1.0450.044a0.8730.034a1500.3730.010b0.4650.039a0.5730.013ab1.1450.039a0.8080.098bc1200.4080.017a0.4780.036a0.5900.044ab1.1080.059a0.8550.045ab80

    control0.4180.017a

    0.3780.010b0.4330.035a

    0.4480.010a0.5750.033ab

    0.6200.050a0.9150.128b

    0.5950.054c0.7850.037bc

    0.7480.005c5. Effect of PPS on proliferation of CEF

    The OD data of every concentration group were almost greater than that of control group, especially at 36, 60 and 72h, most differences were significant. The results showed that PPS could notably promote multiplication of CEF, especially at lower concentration (Table 5).

    Table 5. Effect of PPS on proliferation of CEF

    Concentration

    (g/ml) 24h36h48h60h72h200.5580.043b0.5850.054b0.6180.061b0.7530.078b0.8780.072bc100.6430.053a0.6800.057a0.6980.048a0.8180.057ab0.9231.101ac50.5950.051ab0.6930.039a0.6650.029ab0.8580.013a0.9530.057ac2.50.6180.051ab0.6900.042a0.6830.036ab0.8380.026a0.9580.108ac1.25

    control0.6380.045ac

    0.5730.021bc0.7380.056a

    0.5730.022b0.7080.070a

    0.6680.017ab0.8400.022a

    0.6750.053c1.0080.092a

    0.7600.014b6. Effect of AF on proliferation of CEF

    The OD data in the highest concentration group at 48 and 60h were strikingly smaller than that in control group. In last two low concentration groups at 36h and late four concentration groups at 72h, the OD data were significantly larger than that in control group. This result displayed that AF in late stage and low concentration in early period could obviously promote the cell multiplication (Table 6), while high concentrations in middle period inhibit it.

    Table 6. Effect of AF on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h50.5500.079b0.6100.067b0.5750.029c0.6100.062b0.7630.066b2.50.6130.043ab0.6550.070bc0.6480.043b0.7280.049a0.9050.049a1.250.5900.029ab0.6950.044ab0.6830.046b0.7480.041a0.9350.026a0.6250.6400.070a0.7150.065ac0.7030.039b0.7750.048a0.9250.042a0.3125

    control0.6250.044ab

    0.5730.021ab0.7330.076a

    0.6180.022b0.7700.054a

    0.6680.017b0.8080.071a

    0.7830.057a0.9050.065a

    0.7600.014b7. Effect of EF on proliferation of CEF

    Almost OD data in experimental groups were larger, at 48 and 72h strikingly higher, than that in control group, especially in low concentration groups. This showed that EF could prominently promote multiplication of CEF, which low concentration was more effective (Table 7).

    Table 7. Effect of EF on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h200.5530.032b0.6680.056a0.7400.045a0.8300.041ab0.9800.092a100.5830.033ab0.6380.035a0.7300.068a0.8400.049ab0.9580.100a50.5800.008ab0.6750.047a0.7600.036a0.8330.025ab0.9500.024a2.50.6100.029a0.6430.038a0.7900.022a0.8830.045a1.0050.085a1.25

    Control0.5830.017ab

    0.5730.021b0.6680.071a

    0.6180.022a0.7880.033a

    0.6680.017b0.8130.040ab

    0.7830.057b1.0450.103a

    0.7600.014b8. Effect of PF on proliferation of CEF

    The OD data of every concentration groups at 36, 60 and 72h were significantly greater than that in control group. While the highest concentration group at 48h were obviously smaller. The result showed PF could promote remarkably multiplication of CEF except for high concentration (Table 8).

    Table 8. Effect of PF on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h200.5530.043a0.6650.070b0.6180.041b0.7500.064b0.8280.055b100.5830.037a0.7480.050a0.6930.056ab0.7900.029ab0.8980.069a50.5930.024a0.6750.026b0.6280.041ab0.8180.026a0.8330.040b2.50.5830.050a0.6530.036b0.6500.059ab0.8100.047a0.8700.061ab1.25

    Control0.5850.069a

    0.5400.018a0.6300.042b

    0.4650.013c0.6650.025ab

    0.6830.041a0.7830.024ab

    0.6630.025c0.8930.046ab

    0.6880.029c9. Effect of AS on proliferation of CEF

    All OD data in experimental groups at 60 and 72h were markedly larger than that in control group except for the highest concentration (300) group, which was strikingly smaller than the control at 48h. The result showed that in late stage AS could promote the cell multiplication, but its effect was contrary at high concentration in middle period (Table 9).

    Table 9. Effect of AS on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h3000.4450.037a0.4780.053a0.4550.072b0.7230.057b0.6000.052cd1500.4900.042a0.4980.040a0.5400.014a0.7550.060b0.6100.059bc1200.4700.052a0.5150.040a0.5480.032a0.9230.108a0.6950.100ab800.4450.013a0.5150.030a0.5680.042a0.9380.090a0.7080.041ab40

    control0.4550.024a

    0.4480.015a0.5030.032a

    0.4750.019a0.5750.021a

    0.5480.045a0.9800.112a

    0.5600.050c0.7500.022a

    0.5300.014d10. Effect of GS on proliferation of CEF

    Comparing with control group, most OD data in late three groups at all time points were larger or remarkably larger and in the highest concentrations were smaller and at 48, 72h was strikingly smaller. These results showed that GS could obviously promote multiplication, especially at low concentration in early stage, but its effect was contrary at high concentration (Table 10).

    Table 10. Effect of GS on proliferation of CEF

    Concentration

    (g/ml)24h36h48h60h72h1200.5950.044ab0.4530.060c0.5180.022d0.6600.026cd0.6030.028d800.6500.022a0.4880.032c0.5880.049c0.7130.036bc0.7030.057c400.6000.042ab0.5580.033ab0.6280.046bc0.7300.047ab0.7880.039b200.5900.081ab0.5430.024b0.7250.044a0.7400.036ab0.8030.066b10

    control0.6080.024a

    0.5400.018b0.6030.036a

    0.4650.013c0.6700.022ab

    0.6830.041ab0.7700.037a

    0.6630.025c0.8830.046a

    0.6880.029cDISCUSSION

    1. All of ten CHMI could promote CEF proliferation

    OD data directly reflects the quantity of active cell and there is a direct ratio related with the cell multiplication. The experimental results demonstrated that most OD data in experimental groups at certain concentrations and time points were strikingly larger than that in control group in the same stage, which showed that all of ten CHMI could stimulate CEF proliferation at suitable concentrations and at certain time points. The CHMI with promotion effect in most groups were PPS, EPS, PF, EF and GS and so on. The CHMI at high concentration with inhibitive effect and at low concentration with promoting effect was APS, CAPS, AF and GS and so on. The order of total number that OD data in experimental groups were strikingly larger than that in control in the same stage were PF (15) > PPS and GS (14) > EF (12) > EPS and IRPS (10) > AS (9) > AF (7). This indicated that their proliferation-promoting effect were better. Liu F reported that EPS could increase marrow cell multiplication rate and DNA synthesis rate [9]. But when the time of administration was changed, the effect also changed (the data will be reported separately). Therefore, an accurate evaluation for effect of each CHMI must combine other experiments to consider comprehensively.

    2. The effects have relations with the dose and time in proliferation-promoting of CHMI

    From the experimental results it could be seen that the proliferation effect of CHMI was relative with the remedy dose and incubation time. In dose-effect aspect, most CHMI at low concentration promoted and at high concentration inhibited CEF multiplication. Some CHMI at high concentration also promoted the cell multiplication in the middle and late stage, such as APS, IRPS, PPS and AS at 60h, EF at 48 and 72h and PF at 36, 48, 60 and 72h. This was also confirmed by other researchers [10].

    As far as time-effect was concerned, the proliferation-promoting effects of most CHMI were unobvious in early stage, while in late stage they become obvious, such as IRPS, EPS, EF and AS. Some CHMI in early period (at 24 and 36h) also had better effects, such as GS, EF, IRPS, PF, and AF. A few CHMI presented obvious proliferation-promoting effect only in one or two time points, such as AS at 60h and CAPS at 36 and 60h. These results provided important message for next research.

    3. About Mechanism of CHMI promoting cell proliferation

    Generally, multiplication of fibroblast is related to fibroblast growth factor (FGF). FGF can promote multiplication of many kinds of cells, such as fibroblast, endotheliocyte, osteoblast, myoblast, neurogliocyte and so on, which is by receptor-signal-transduction system and finally as a result of gene expression. FGF takes effect only after FGF receptor is jointed by osamine polysaccharide with special structure to form stable triarch compound [11]. These growth factor and compound formed by saccharide combination in intercellular substance are considered as extracellular growth factor storage [12]. Their effects are adjusted by the concentration of growth factor. This experiment confirmed that ten kinds of CHMI possessed the action of promoting CEF proliferation, which was likely by affecting FGF and the formation of its receptor compound. Because action of CHMI required certain time, there was a time-effect relationship that obvious proliferation-promoting effect occurred almost in middle or late stage of incubation. The reasons of abnormal dose-effect relationship, which promoting effect was presented at low concentration and inhibiting effect displayed at high concentration, was likely that the cell cultured was sensitive to condition [10] which was affected by CHMI. This remains to be confirmed by experiment.

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    (Edited by Tiebo Zhou, Lian Hu and Xia Gao)

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    (Edited by Yaqin Pang, Zhen Li, Xia Gao and Lian Hu)

    * Supported by National Natural Science Foundation of China, Grant No. 30070566

    ** Corresponding to Yuanliang Hu, male, PhD, professor of Institute of Traditional Chinese Veterinary Medicine, Nanjing Agricultural University, Nanjing; Main research field: Traditional Chinese Veterinary Medicine; Address: Nanjing Agricultural University, Jiangsu, China, Postcode: 210095; Tel: 025-84395203(O), 025-84396471(H); E-mail: ylhu@njau.edu.cn

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