Immunomodulatory effects of crude phenylethanoid glycosides from Ligustrum purpurascens

Journal of Ethnopharmacology 144 (2012) 584–591

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Immunomodulatory effects of crude phenylethanoid glycosides from Ligustrum purpurascens Xun Song a,b,1, Chen-yang Li a,b,e,1, Yong Zeng d, Hai-qiang Wu c, Zhong Huang a,b, Jian Zhang a,b, Rui-sha Hong a, Xian-xiong Chen a, Li-yan Wang c, Xiao-peng Hu a,b, Wei-wei Su e, Yan Li d,nn, Zhen-dan He a,b,n a

Department of Pharmacology, School of Medicine, Shenzhen University, Shenzhen 518060, China Institute of Biotherapy, Shenzhen University, Shenzhen 518060, China c College of Life science, Shenzhen University, Shenzhen 518060, China d The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China e School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 May 2012 Received in revised form 4 September 2012 Accepted 16 September 2012 Available online 11 October 2012

Ethnopharmacological relevance: Ligustrum purpurascens, named as ‘‘Ku ding cha’’, has been used as a kind of functional tea in southern China for about two thousand years, which has the effects on diuresis, anti-hypertension, weight-loss and anti-inflammation. The aim of the study: This study was aimed to investigate the immune enhancement effects of the crude phenylethanoid glycosides (CPGs) from Ligustrum. Purpurascens on mice and analyze the chemical profiles of phenylethanoid glycosides in the CPGs. Materials and methods: The immune functions enhancing potential of CPGs was determined using serum hemolysin antibody, phagocytosis, splenocyte antibody production, and NK cells activity assays. The contents of five major constituents in the crude glycosides of Ligustrum purpurascens were determined by using liquid chromatography, other five glycosides were deduced according to their UV and MS spectra compared with the literature as well. Results: In the immunizing experiment, mice treated with different doses of CPGs showed an increase (p o 0.01) in the haemagglutination titre compared with the control group. The increases (po 0.05) were found to be significant at doses of 440 mg/kg and 1.32 g/kg in the experiments of antibody production of spleen cells, MF phagocytosis of chicken RBCs and NK cell activity. Further chemical characterization yielded 10 constituents from CPGs, five glycosides were quantified by HPLC and the structures of other five compounds were speculated according to their UV and MS spectra. Conclusion: The results suggested that phenylethanoid glycosides from Ligustrum purpurascens have immunomodulatory effects on mice. Crown Copyright & 2012 Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Ligustrum purpurascens Glycosides Immunomodulation LC-MSn

1. Introduction Ku Ding Cha is a folk health beverage in southern China for about two thousand years, which was first documented in the ‘‘Shen Nong’s Herbal Classic’’. Its original materials contain 30

Abbreviations: TCM, Traditional Chinese Medicine; CPGs, Glycosides from Ligustrum purpurascens; HA titre, haemagglutination antibody titre; SRBC, Sheep Red Blood Cell; LDH, lactate dehydrogenase; PI, phagocytic index; PC, conversion value of Percentage of phagocytosis; ESI, electrospray ionization; PFC, Plaque forming cells; MF, macrophage; RBC, red blood cell n Corresponding author at: Shenzhen University, Department of Pharmacology, School of Medicine, Shenzhen 518060, China. Tel.: þ 86 755 8667 1916; fax: þ 86 755 8667 1906. nn Corresponding author. Tel.: þ 86 871 5320841; fax: þ 86 871 5357191. E-mail addresses: [email protected] (Y. Li), [email protected] (Z.-d. He). 1 Xun Song, Chen-yang Li contributed equally to this work.

species in 12 families, and are mainly yielded by fermentation method (He et al., 2010a). Ku Ding Cha has been used for treating a variety of ailments including inflammation, hepatoprotective, hypertension, and also has the effects of antiobesity, diuresis and antioxidant (Huang et al., 2008; Wong et al., 2001; Lau et al., 2002; Chen et al., 2002; He et al., 2011, 2003). In addition, the research proved that Ku Ding Cha could also enhance physical fitness to resist the diseases (He et al., 2010b). Ligustrum purpurascens (Oleaceae) was often used as a kind of famous original plant of Ku Ding Cha. It was reported that Ligustrum purpurascens was rich in glycosides (He and Yang, 1989; He et al., 1992). More and more attention was attracted to the research of immunomodulation to alleviate and cure diseases. Traditional Chinese Medicine (TCM) was reputed to promote physical and mental health, improve defense mechanism of the body and enhance longevity. These attributes were similar to the modern concept of adaptogenic agents, which were known to afford

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X. Song et al. / Journal of Ethnopharmacology 144 (2012) 584–591

protection of the human physiological system against diverse stress (Bhattacharya et al., 2000). A number of medicinal plants have been claimed to possess immunomodulation activity. Some of them were known as immunomodulation agents in China, such as Rehmannia glutinosa and Cistanche deserticola (Chen et al., 1993; Zhang et al., 2011). In addition, the type of glycosides isolated from Ligustrum purpurascens was similar to that of Rehmannia glutinosa and Cistanche deserticola (Ma et al., 2008; Albach et al., 2007). So the glycosides from Ligustrum purpurascens might have the similar immune activity. However, there was paucity of data available on the immune effects of the extract of Ligustrum purpurascens in animals. Therefore, the present study was undertaken to evaluate the immunomodulatory effects of crude glycosides of Ligustrum purpurascens, and elucidation of its chemical constituents by UV and HPLC-MSn.

2. Materials and methods 2.1. Medicinal plant and chemical reagents Ligustrum purpurascens was purchased from Suijiang county, Yunnan province, China, and was authenticated by Peng Hua (The specimen voucher number: NPCR-Lipu-1, deposited with the herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences). Panax ginseng was purchased from Eu Yan Sang in Hongkong, and was authenticated by Dong Tingxia (The specimen voucher number: NPCR-Lipu-2, deposited with School of Medicine, Shenzhen University). Methanol and acetonitrile (chromatographic grade) were purchased from Fisher Scientific (USA). Water was ultra pure grade, and all other chemicals used in the study were of analytical grade at least. RPMI-1640 medium was purchased from Hyclone (Hyclone, USA). 2.2. Preparation of reagents The dried leaves (1.92 kg) of Ligustrum purpurascens were extracted with hot EtOH (20 l  3 l), and the residue obtained by removal of solvent in vacuo was triturated with H2O (3 l  4 l). The insoluble and aqueous phase was parted, and the aqueous phase was directly subjected to chromatography column on the highly porous polymer Diaion (HP20, 1500 ml), eluting with H2O, H2O–EtOH (1:1) and EtOH. The eluates with 50% H2O–EtOH (4 l) were concentrated in vacuo to give residues CPGs (230.4 g). CPGs were suspended in ultra pure grade water for administration to the experimental animals. The root of P. ginseng (200.0 g), crushing, was extracted by circulation reflux twice with hot water (2 l  3 l). Then the extracts were concentrated to specific gravity of 1.3 under reduced pressure concentration. 46.0 g yellow powder of H2O extracts was acquired by spray drying. The powder was suspended in ultra pure grade water for the positive reagent. 2.3. Animals and cells Female Kunming (KM) mice, weighing 18–22 g (8 weeks old), were purchased from the Animal Supply Center of Guangdong Academy of Medical Science. The animals were kept in an environmentally controlled breeding room (temperature: 25 71 1C, humidity of 5575%, and a 12/12 h light/dark cycle) for at least one-week acclimatization before experiment. All mice were fed rodent laboratory chow with sterile water ad libitum. All the procedures were in strict accordance with the China legislation on the use and care of laboratory animals and with the guidelines established by the Institutional Animal Ethics Committee and

585

Committee for the Purpose of Control and Supervision of Experiments on Animals in China. YAC-1 cells were purchased from Cell Bank of Chinese Academy of Science (Cell type: YAC-1 lymphoma cells from mice; Medium: RPMI-1640 with fetal bovine serum to a final concentration of 10%; Subculturing: Cultures can be maintained by addition or replacement of fresh medium. Start cultures at 3  105 cells/ml and maintain between 2  105 and 2  106 cells/ml; every 2 to 3 days to renew medium; 24 h before the experiment, renew the medium; stationary phase of cells used for the experiment; purchased from Shanghai, China). 2.4. The qualitative and quantitative analysis of CPGs The qualitative and quantitative analysis of CPGs was performed using an Agilent Technology 6400 Series Triple Quadrupole LC/MS (1260 Infinity Binary LC, Agilent Technologies, USA). 1260 Infinity Binary LC was equipped with G1312B binary solvent-delivery system, G1379B. Thermostatted column compartment, G1316B vacuum degasser, G1367E autosampler, and G1315C diode array detector (DAD). Chromatographic separation was achieved on an Ultimate XB-C18 column (250 mm  4.6 mm i.d., 5 mm; Welch, MD, USA) coupled with Agilent C18 pre-column (250 mm  4.6 mm i.d., 5 mm; Palo Alto, CA, USA) at temperature of 25 1C. An initial eluting mixture of H2O containing 0.1% formic acid and acetonitrile (82:18, v/v) was used at a flow rate of 1 ml/ min. The initial mobile phase ratio was held for 20 min, and then the percentage of acetonitrile was increased to 20% from 20 min to 80 min. The sample injection volume was 10 ml. The detection wavelength was 227 nm. A MS detector with an electrospray ionization (ESI) interface in positive ion mode (ESI þ ) was used for qualitative analysis, with acquisition in Product Ion Scan mode. All the parent ions and product ions were recorded, respectively. The optimized electrospray conditions were: capillary voltage 4.00 kV; fragementor: 250 V; source temperature: 100 1C; desolvation temperature: 350 1C; collision energy 40 V for all peaks; gas flow: 13 l/min; nebulizer: 55 psi. CPGs, acteoside, ligupurpuroside A, cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B (CPGs, 20 mg/ml in methanol; all the pure compound, 1 mg/ml in methanol) were injected into the C-18 column, respectively for quantitative analysis. All chromatographic data were acquired and analyzed by using Agilent ChemStation software. A typical HPLC chromatogram of CPGs and the spectra of UV and mass are shown in Fig. 1. 2.5. Effects of test extracts on haemagglutination antibody titre (HA titre) The method described previously by Puri et al. (1994) was followed for this experiment. The mice were randomly separated into five groups (n ¼12) as follows: Group I (control group) were daily gavaged with the vehicle for a period of 7 days; Group II (positive group) were daily gavaged with P. ginseng at a dose of 220 mg/kg body weight for 7 days; Groups III–V(treatment groups) were daily gavaged with CPGs at a dose of 220 mg/kg, 440 mg/kg and 1.32 g/kg body weight for 7 days, respectively. The mice were immunized by 200 ml of fresh sheep RBC suspension (2%) via abdominal injection on the 3rd day. Blood samples were taken from the eye socket vein of mice on day 7 and the serum was collected. Hemolysin (SRBC antibody) was determined by haemagglutination technique. Briefly, equal volumes of individual serum samples of each group were pooled. Two fold dilutions of pooled serum samples were made in 50 ml volumes of normal saline in microtitration plate, and added 50 ml of 0.5% suspension

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X. Song et al. / Journal of Ethnopharmacology 144 (2012) 584–591

Fig. 1. The HPLC chemical profile, UV and MS2 spectra of CPGs. a A showed the HPLC chemical profile of glycosides present in CPGs; B showed the UV spectra of ten major peaks detected in the HPLC; C showed the MS2 spectra of ten major peaks detected in the HPLC-MS2. See text for the conditions. The peaks were identified as: peak 2-acteoside; peak 3-ligupurpuroside A; peak 8-osmanthuside B; peak 9-trans-ligupurpuroside B; peak 10-cis-ligupurpuroside B. The peaks labled with * were speculated as: peak 1*-angoroside A; peak 4*-isoacteoside; peak 5*-ligurobustoside D; peak 6*-ligupurpuroside D or ligurobustoside Q; peak 7*-ligurobustoside Q or ligupurpuroside D according to their UV and MS spectra compared with the literature.

of sheep RBC in saline. After mixing, the plates were incubated at 37 1C for 3 h and examined for haemagglutination under microscope. The reciprocal of the highest dilution of the test serum giving agglutination was taken as the antibody titre. 2.6. Antibody produced by spleen cells in mice assay 2.6.1. Animals immunized The blood without fiber of sheep was washed 3 times with saline, centrifuged at 2000 rpm for 10 min each time, and finally resuspended with saline for 2% suspension (v/v). The mouse was injected with 200 ml suspension intraperitoneally each. 2.6.2. Spleen cell suspension After 4 days of immunization, mice were killed by cervical dislocation. The spleen was removed aseptically, and laid on the small culture dish contained Hank’s solution, triturated lightly to cell suspensions. The cell suspension was filtered by 200 mesh strainer. The filtrate were centrifuged at 1000 rpm for 10 min, and

then washed with Hank’s solution twice. Finally, the cells were resuspended with 5 ml RPMI1640 medium. After determination of the spleen cell concentration, the cell suspension was diluted to 5  106 cells/ml.

2.6.3. Hemolytic plaque assay A solution of molten medium was prepared, and equivalent Hank’s solution (PH 7.2–7.4, double concentration) was pipetted into tubes, maintained at 45–50 1C, and divided into tubes, 500 ml per tube. Each tube was added 50 ml of 10% SRBC (V/V, with saline) and kept at 30 1C, followed immediately by 25 ml of the prewarmed spleen cell suspension. The contents of each tube were rapidly mixed and poured onto labeled microscope slides, precoated with 0.1% aqueous agarose. For the determination of optimum temperature, the slides were incubated for 1–1.5 h at 37 1C in carbon dioxide incubator. The slides were then flooded with a 1:8 dilution of complement which was diluted with saline, and incubation continued for a further 1–1.5 h. For direct comparison of the hemolytic plaques formed, slides were prepared

X. Song et al. / Journal of Ethnopharmacology 144 (2012) 584–591

from the same spleen cell suspension and SRBC, but incubated in different incubators set at the desired temperatures. Hemolytic plaques were scored either directly or under indirect illumination at an eightfold magnification. In all experiments, suitable plaque counts were obtained by using 105 cells per slide. Based on these counts, the numbers of PFC per slide were calculated as the mean 7standard error from three experiments, with duplicate determinations per experiment. 2.7. Evaluation of phagocytosis of chicken red blood cells by MFs Mice were injected (i.p.) with a dose of 200 ml 0.2% SRBC 4 days before harvesting the peritoneal cells. Mice were killed by cervical dislocation. Briefly, cells were harvested by injecting 4 ml cold (4 1C) Hank’s solution with 5% Fetal Calf Serum. The abdomen was rubbed lightly 20 times in order to wash out the MFs. Then, cut a microstomia in the abdomen, through which liquid were obtained using straw. 500 ml liquid was added into a tube with 500 ml chicken red blood cells, and the contents of tube were rapidly mixed. 500 ml of the complexes were added to labeled microscope slides, precoated with agarose cultures and maintained at 37 1C for 15–20 min. After this time, the cover slips were washed in saline, and the cells were fixed with methanol for 1 min, stained with Giemsa stain for 15 min and observed by light microscopy to calculate the percentage of phagocytosis or phagocytic index. A total of 100 cells per cover slip were evaluated. All counts were performed at least in duplicate. Phagocytosis was determined and expressed as the percentage of cells that internalized at least one red cell. The maximum phagocytosis index was obtained by determining the percentage of cells showing five or more internalized particles. The Calculation formula was PI¼(The number of being swallowed RBCs/the total number of MFs  100%. 2.8. Activity of NK cell assay 2.8.1. Spleen cell suspensions (effective cells) The whole spleen was aseptically removed and forced through a sterile stainless 200 mesh to obtain a single-cell suspension. The suspension was washed by Hank’s solution 3 times, centrifuged at 1000 rpm for 10 min each time. Then, cells were resuspended in 2 ml complete medium. Typan blue solution was used to count the amount of living cells ( 495%). Finally, cells were resuspended in RPMI 1640 medium. After determination of the spleen cell concentration, the cell suspension was diluted to 1 ml contained 2  107 cells.

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2.8.2. Yac-1 mouse lymphoma cell line (target cells) The Yac-1 mouse lymphoma cell line was used as target cells in the activity assay. 100 ml of 4  105 Yac-1 cells in suspension culture and 100 ml effector spleen cells (in ratio of 50:1) were added to microtiter wells in quadruplicate. Wells containing 100 ml target cells alone and 100 ml medium served as controls for spontaneous release (SR), and 100 ml of target cells and 100 ml of 1% NP40 were added to determine total potential release (TR). And all the groups were set 3 wells. The plates were incubated in a humidified 5% CO2 incubator for 4 h at 37 1C. After centrifugation at 1500 rpm for 5 min at room temperature, 100 ml aliquots of the supernatants (SN) were removed to 96 well microtiter plates, and 100 ml LDH was added to the well that contained SN. 3 min later, 30 ml HCL(1 mol/l) was added into the well. Optical density value was menstruated at 492 nm. 2.9. Statistical analysis Data expressed as means 7standard deviations (SD) were either calculated from at least 10 independent experiments. For statistical comparisons, results were analyzed using analysis of Student–Newman–Keuls Test. Po0.05 was considered a statistically significant difference. All statistical tests were carried out using the computer program SPSS (SPSS Inc., Chicago, IL).

3. Results 3.1. Effects of CPGs on the level of serum hemolysin antibody titre Animals treated with different doses of CPGs showed an increase in the haemagglutination titre compared with the control group. This increase was found to be significant at all doses when compared with the control group (group I). The results showed that different doses of CPGs were equal to the positive group (Table 1). 3.2. Effects of CPGs on the level of antibody production of spleen cells Animals treated with doses of 220 mg/kg showed an increase in the antibody production compared with the control group. This increase was found to be in-significant compared with the control group (group I). However, the animals treated with dose of 440 mg/kg and 1.32 g/kg also showed a significantly increase in the antibody production. The increases were found to be significant at doses of 440 mg/kg and 1.32 g/kg compared with control group. The effect of antibody production for CPGs at dose of

Table 1 Effects of CPGs treatment on the level of serum hemolysin antibody titre and antibody production of spleen cells in the subjects. Treatment

Group I Group II Group III Group IV Group V F value

Dose(g/kg BW)

0.00 0.22 0.22 0.44 1.32

Serum hemolysin antibody titre

Antibody production of spleen cells

n

Antibody volume

P value

n

Numbers of PFC/106 Cells

P value

12 12 12 12 12

108.2 721.4 155.1 7 33.5n 154.3 7 36.8nn 151.2 7 33.8nn 151.2 7 34.9nn 5.105 (Po 0.01)

o0.01 o0.01 o0.01 o0.01

10 10 10 10 10

194.2 7 25.4 212.3 7 36.3n 199.5 7 37.7 232.0 744.3n 235.1 7 28.7 3.232 (Po 0.01)

o 0.05 40.05 o 0.05 o 0.05

Group I (control group): receiving gavage of the vehicle; Group II (positive group): receiving gavage of Panax ginseng; Group III to Group V: receiving gavage of CPGs. All data of treatment on serum hemolysin antibody titre were expressed as mean 7SD (n¼12). All data of treatment on antibody production of spleen cells were expressed as mean 7SD (n ¼10). P value was compared with group I. n

p o 0.05. p o0.01.

nn

Table 3 Content of some purpurascensa. Compound

major

phenlethanoid

glycosides

present

in

Ligustrum

Content (mg/100 mg of Ligustrum purpurascens dried leaves)

Acteoside 2.2560 7 0.1040 Ligupurpuroside A 5.49487 0.3120 trans-ligupurpuroside B 0.9804 7 0.0120 cis-ligupurpuroside B 0.1692 7 0.0060 Osmanthuside B 0.4896 7 0.0522 a

Average of three separate extractions.

220 mg/kg was lower than that of positive group; however, the ability of antibody production of spleen cells was promoted as the increase of the dose of CPGs (Table 1). 3.3. Effects of CPGs on peritoneal MF phagocytosis of chicken RBCs in mouse Animals treated with doses of 220 mg/kg, 440 mg/kg and 1.32 g/kg all showed an increase in the phagocytosis of chicken RBCs which expressed by percentage of phagocytosis and phagocytic index. But the increase at dose of 220 mg/kg showed no significant change compared with control group. The increases at doses of 440 mg/kg and 1.32 g/kg were found to be significant compared with control group (Po0.05, Po0.01, respectively), however, were equal to the positive group (Table 2). 3.4. Effects of CPGs on NK cell activity in mouse The activity of the NK cells was measured using LDH releasing assay at different E: T ratios. The effect of CPGs in vitro on the NK cell activity was examined at an E: T ratios of 50:1(E: T, Effect cells: the target cells). Culturing the splenocytes of mice with different treatments for 7 days resulted in the enhancement of NK cell activity. Two concentrations of CPGs (0.44 g/kg and 1.32 g/kg) increased the NK cell activity by 171% and 157% at E: T ratio of 50:1, respectively. And the increases were significant compared with control group (Po0.01, Po0.05, respectively). Treatment with 220 mg/kg of CPGs increased by 128% of the NK cell activity at E: T ratio of 50:1 (Table 2). However, the increase at dose of 220 mg/kg was found to be no significant compared with control group (P4 0.05). And the results showed group IV and V could promote the NK cell activity. CPGs did not exert any cytotoxic activity on Yac-1 cells by themselves.

nn

P o0.05. Po 0.05.

3.5. The analyzed constituents and the content of major glycosides in CPGs

n

Group I (control group): receiving gavage of the vehicle; Group II (positive group): receiving gavage of Panax ginseng; Group III to Group V: receiving gavage of CPGs. All data of treatment on mouse peritoneal MF phagocytosis of chicken RBCs were expressed as mean 7 SD (n ¼10). nP o0.05, nnP o 0.05. PI and PC represent phagocytic index and conversion value of percentage of phagocytosis, respectively. P value was compared with group I. All data of treatment on NK cell activity were expressed as mean 7 SD (n¼ 10).

0.167 0.09 0.177 0.08 0.197 0.06 0.237 0.05nn 0.227 0.04n 3.158 (Po 0.05) 3.17 7 2.50 3.52 7 1.34 4.08 72.21 5.45 7 1.81nn 4.99 7 1.65n o 0.05 40.05 o 0.01 o 0.05 o 0.05 40.05 o 0.01 o 0.05 0.00 0.22 0.22 0.44 1.32 Group I Group II Group III Group IV Group V F value

14.07 11.4 23.3 7 15.2 21.7 7 12.4 29.1 7 10.6 25.5 7 13.6

0.36 70.15 0.53 70.12n 0.47 70.16 0.56 70.13nn 0.52 70.16n 3.005 (P o 0.05)

0.27 7 0.26 0.61 7 0.25n 0.51 7 0.32 0.69 7 0.26nn 0.59 7 0.36n 3.206 (P o0.05)

conversion value of NK cell activity NK cell activity p-value PI p-value PC Percentage of phagocytosis

NK cell activity mouse peritoneal MF phagocytosis of chicken RBCs Dose(g/kg BW) Treatment

Table 2 Effects of CPGs on mouse peritoneal MF phagocytosis of chicken RBCs and NK cell activity.

40.05 40.05 o0.01 o0.05

X. Song et al. / Journal of Ethnopharmacology 144 (2012) 584–591

p-value

588

The chemical profile of CPGs was analyzed by LC/MS (Fig. 1A). The compounds of peaks 2, 3, 8, 9 and 10 were purified from the dried leaves of Ligustrum purpurascens, and their structures were illustrated by spectrometric methods and comparing with literature (He and Yang, 1989; He et al., 1992). The UV spectra of all the compounds were shown at Fig. 1B. The UV spectra of all the peaks in the HPLC profile suggested the presence of hydroxyl, ester and conjugated aromatic groups. The UV characteristic absorption of compounds 1, 2, 3, 4 and 6 was at 330 nm, while the UV characteristic absorption of compounds 5, 7, 8, 9 and 10 at 310 nm. Five constituents in the HPLC profile of CPGs were presumed by the MS2 spectra (see Fig. 1C). In these spectra, the parent ions and product ions of these five constituents were compared with literature (Li et al., 2010, 2011; She et al., 2008). The structures

X. Song et al. / Journal of Ethnopharmacology 144 (2012) 584–591

of compounds 1, 4, 5, 6 and 7 were reckoned as angoroside A, isoacteoside, ligurobustoside D, ligupurpuroside D and ligurobustoside Q, and Fig. 1 showed all the structures, respectively. The content of compounds 2, 3, 8, 9 and 10 was shown in Table 3, compound 2 and 3 were two major constituents. The content of five compounds in Table 3 accounted for 53.2% of total phenylethanoid glycosides in the fraction according the analysis of HPLC.

4. Discussion A large number of herbal medicines widely recognized in TCM, have been shown to alter immune function and might become a source of clinical relevant therapeutic medicine. For instance, results of clinical researches demonstrated that Panax ginseng could improve psychologic function, immune function, and quality of life (Kiefer, and Pantuso, 2003; Coleman et al., 2003). The functions of traditional herb extracts are usually complex, which might may influence several cellular signal pathways and may play key roles in some disease development (Tang et al., 2010).

589

Recently, some researchers have pointed out that a lot of diseases are relevant to the status of body’s immunity. Improving the immunity has become an important topic. Chen et al. have reported that TCM plays an important role in innate auto immunity to resist virus (Chen et al., 2011). Moreover, inflammatory and autoimmune diseases have a notable history of being treated with herbal formulas. It is considered that these herbs may play function as immunomodulators. In 2004, Park et al. showed for the first time a direct evidence of immunomodulational activity by oral gavage administration of caffeic acid phenethyl ester in vivo (Park et al., 2004). And then in 2006, it is reported that caffeic acid phenethyl ester changed the orientation of immune responses by modulating the cytokine pattern (Gre´my et al., 2006). In the early study, Lau et al. found that the extract CPGs of TCM Ligustrum genus had anti-oxidative, anti-inflammatory and hepato-protective effects (Lau et al., 2002). In this study, therefore, we will investigate the immune effects of Ligustrum genus. First, quantification of hemolysin antibody production in the serum was tested to evaluate the effects of the CPGs on immune stimulation. Our results showed that the hemolysin antibody titre of the three CPGs treatment groups, III–V groups, were

OH OH HO

HO

HO

O O O C OO

O

OH O C OO

O

O

OH H3C O HO HO OH angoroside A (1*) HO HO

O

OH

OH OH

HO

O

OH O C OO

O

H3C OH OH

HO

H3C O O OH OH O OH

O

O OH

O C

H3C HO HO

O C

H3C O O OH H3C OH O OH OH OH

O

O

HOO H3C O O HO OH O OH

O

OH

OH O C OO

HO

O

O

OH

OH

OH OH

OH OH

O

O

OH

OH

osmanthuside B (8) OH OH OH O C OO

OH

trans-ligupurpuroside B (9)

O

OH

ligupurpuroside D (6*)

H3C O OH OH OH

O

O

OH

ligurobustoside Q (7*)

OH O C OO

O

HOO OH H3C O HO HO OH isoacteoside (4*)

HO HO

O

OH H3C O HO HO OH acteoside (2)

HO

OH

OH H3C O HO HO OH ligurobustoside D (5*)

HO

HO

OH

H3C O O OH H3C OH O OH OH OH ligupurpuroside A (3) HO OH

HO O C OO

HO O C OO

HO

H3C OH OH

H3C O O OH OH O OH

O

O

OH

cis-ligupurpuroside B (10)

Fig. 2. The structures of ten major peaks detected in the HPLC profile of CPGs.

OH

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significantly greater than that of the control group. The bioactivities of treatment groups were similar to positive group. Therefore, the results indicate that the CPGs can improve the immune activity in mouse. Antibodies are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. The ability of antibody production is reflected the capability of immunity in body. Secondly, we investigate the level of antibody production of spleen cells. Our results showed that the level of antibody production groups IV and V were significantly greater than that of control group. It suggested that CPGs can improve the function of spleen in mice. Mononuclear MF phagocytosis is one of the key indicators in the body immunity (Van et al., 1972; Aderem and Underhill, 1999). Through interaction with lymphocytes, MFs act as phagocytic, microbicidal and tumoricidal effector cells, play an important role in the initiation and regulation of immune response (Gordon, 1998). Phagocytosis and the killing of invading microorganisms by MFs constitute the body’s primary line of defense against infections (Hawiger, 2001; Hume et al., 2002). Lots of the plant extracts have reported to play important effect on nonspecific immunity, i.e., MFs function (Mehvar, 2003; Ooi and Liu, 2000; Sliva, 2004). Phagocytosis of red cells experiment was used to evaluate the effects of CPGs on the function of MFs. In this study, we found that the mice belonging to groups IV and V exhibited significantly higher phagocytic index as compared to the control group. This indicates that CPGs can enhance the phagocytic activity of MFs. In present study, CPGs of 440 mg/kg and 1.32 g/kg increased both PI (phagocytic index) and PC (conversion value of Percentage of phagocytosis), which were similar to the positive group, indicating a stimulatory effect on MFs and strong phagocytic activity in mice. Thus, CPGs is likely to enhance nonspecific immunity. The study gave a result in that the dose of 220 mg/kg CPGs elicited small increases in PI and CI. However, the increase is found to be no significant compared with control group. Considering that the calculation of PI takes into account not only the number (percentage) of cells that are phagocytic, but also how phagocytic they are (i.e., how many red cells are internalized by each cell), it may be presumed that CPGs does not influence the percentage of phagocytic cell or the number of red cells ingested by each phagocytic cell in a parallel manner. Nevertheless, an exact explanation for this result requires further investigation. Consistently, the first line of defending pathogen in the body is nonspecific immunity which Includes NK cells. NK cells are a type of cytotoxic lymphocyte that is a major component of the innate immune system, because they have the ability to recognise stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. So the activity of NK cells reflects the immunity of the body. Accordingly, the augmentation of NK activity by CPGs treatment had been appeared. In our study, CPGs is a potent immunomodulator that significantly enhances the NK cells activity in vitro. It indicates that CPGs can improve the immunity of the body. In addition, the present results expand the database on the effect of CPGs on the immune system. The CPGs consists of the glycosides (phenylethanoids and monoterpenoids), which always has the activities of anti-inflammation, antibiosis, antioxidant and hepatoprotection. This study revealed that glycosides might play an important role in immune enhancement, especially in the process of prevention and cure of flu. Acteoside, ligupurpuroside A, cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B are the major constituents of CPGs, they might become the immunotherapeutic agent, the further mechanism study and safety research are expected to carry out. As the activity of CPGs, chemical profiles of CPGs were further investigated. The UV characteristic absorption of compound 1, 2,

3, 4 and 6 was observed red shift compared with the five other compounds. The results suggested that compound 1, 2, 3, 4 and 6 contain more phenolic hydroxyl groups than others. The structures of compounds 1–6 contain caffeoyl groups, and compounds 7–10 contain coumaroyl groups. The aglycone of compounds 1, 2, 3, 4, 6 and 7 exist the group of 3, 4-bihydroxyl phenylethyl, compounds 8, 9 and 10 exist the group of p-hydroxyl phenylethyl. All the compounds except 5 belong to phenylethanoid glycosides, while compound 5 was speculated as monoterpenoid glycosides (Fig. 2).

5. Conclusions In conclusion, the present studies with three different dosage groups have proven CPGs to be an effective immunoenhancement. We found that CPGs exhibit multiple modulating effects on immune function and were correspondent with positive drug on mice. They stimulate different immune cells and their function, suggesting that CPGs possess an immunomodulating potential for the immune system. The pharmacokinetics of CPGs will be undertanken on the next step to get more information of CPGs later. Moreover, the chemical profiles of active CPGs were elucidated. Therefore, the CPGs have the potential value as a new kind of immunotherapeutic agent.

6. Funding This work was supported by the National Natural Science Foundation of China (NSFC, 81060077).

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