Effect of Total Flavonoids from Phyllanthus emblica L. on Tumor Proliferation and Immune Function

Guangxi University of Chinese Medicine, Nanning 530001, China

Abstract [Objectives] To explore the effect of total flavonoids from Phyllanthus emblica L. on tumor proliferation and immune function. [Methods] The effects of total flavonoids of P. emblica L. on the proliferation of 6 different tumor cell lines (human hepatoma cell line HepG-2, human cervical cancer cell line Hela, human gastric cancer cell line SGC7901, human nasopharyngeal carcinoma cell line CNE-2, human lung cancer cell line H460 and human ovarian cancer cell line A2780) were compared by SRB method. The effect of total flavonoids from P. emblica L. on the proliferation of mouse lymphocytes induced by concanavalin (ConA) or lipopolysaccharide (LPS) in vitro was detected by CCK-8 method. [Results] The results of SRB assay showed that compared with the normal group, the total flavonoids of P. emblica L. had obvious inhibitory effect on 6 kinds of tumor cells. Among them, the inhibitory effect on H460 cells and CNE-2 cells was the most significant, and the IC50 was (471.36±50.66), (463.26±40.75) μg/mL, respectively. The high dose group of total flavonoids from P. emblica L. had the same inhibitory effect as the positive drug 5-Fu. The results of CCK-8 assay showed that compared with the blank group, the total flavonoids of P. emblica L. significantly inhibited the proliferation of mouse lymphocytes induced by ConA or LPS (*P<0.05，**P<0.01). [Conclusions] The total flavonoids of P. emblica L. had significant anti-tumor activity.

Key words Phyllanthus emblica L., Total flavonoids, Anti-tumor, Immune function

1 Introduction

Malignant tumor is now one of the major health problems facing the world. Among all the causes of death in China, tumor accounts for 25%[1]. Local infiltration and distant metastasis is the most important feature of malignant tumor, and it is the main cause of death caused by malignant tumor. The treatment methods of tumor include surgical resection, radiation and drug control therapy, and most tumor drugs are chemical synthetic drugs, and they also have great lethal effect on normal cells in the process of killing tumor cells.PhyllanthusemblicaL. is a kind of plant for both medicine and food use, and its roots, fruits and leaves are often used in diseases such as rheumatism, diabetes, and bronchitis. And studies have shown that the extract ofP.emblicaL. has anti-tumor and antibacterial effects[3]. In this study, the effects of total flavonoids ofP.emblicaL. on the proliferation of many kinds of tumor cells and mouse lymphocytes in vitro were studied to explore the effects of total flavonoids ofP.emblicaL. on tumor proliferation and immune function.

2 Materials and methods

2.1Materials

2.1.1Instruments. DMI3000B inverted fluorescence microscope (LEICA); 1312004047 full-wavelength enzyme labeling instrument (TECAN); 5430R freezing low speed centrifuge (EPPENDORF); MCO-18AIC constant temperature carbon dioxide incubator (SANYO); SQP analytical balance (Sartorius Scientific Instruments Beijing Co., Ltd.); PYX-190H-B constant temperature incubator (Shaoguan Keli Experimental Instrument Co., Ltd.).

2.1.2Reagents. 0.25%-EDTA pancreatic digestive enzyme (Solarbio, batch No.20160420); Cell Counting Kit-8,CCK-8 (Solarbio, batch No.20160422); fetal bovine serum (FBS,MULTICELL, batch No.086110037); RPMI-1640 complete medium (HyClone, batch No.AAK-208939); dimethyl sulfoxide (DMSO,AMRESCO, batch No.302A0337); fluorouracil (5-Fu), Shanghai Xudong Haipu Pharmaceutical Co., Ltd.; CCK-8 (Solarbio, batch No.20160422).

2.1.3Cell line. Human gastric adenocarcinoma cells (SGC-7901) and human nasopharyngeal cancer cells (CNE-2) were purchased from KeyGen. Human lung cancer cells (H460) were purchased from Cell Resource Center of Shanghai Academy of Life Sciences, Chinese Academy of Sciences. Human hepatoma cell line (HepG 2) was purchased from Xiangya Central Laboratory. Human ovarian cancer cells (A2780) and human cervical cancer cells (Hela) were purchased from the Basic Medical Cell Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences.

2.2Methods

2.2.1Preparation of drug solution. 0.1 g of powder of total flavonoids fromP.emblicaL. was accurately weighed, and 100 μL of DMSO and 900 μL of complete culture medium were used to dissolve the total flavonoids. It was filtered by 0.22 μm microporous membrane and put into the 0.5 mL aseptic centrifuge tube. It was used as the mother liquid of the drug used in the experiment, and the concentration was 100 mg/mL.

2.2.2Detection of the effect of total flavonoids fromP.emblicaL. on tumor cell proliferation by SRB. The cells in the logarithmic growth phase were selected, and the cell density was adjusted to 5×104-8×104mL[A]. They were cultured in a 96-well plate, 100 μL/well. After 24 hours of culture, the old culture medium was discarded and cleaned with PBS. The drugs were given in groups with 5 wells in each group. New complete culture medium was added in the blank group, and 150 μL/well complete culture medium containing total flavonoids was added in the experimental group. The final concentrations were 32.15, 62.5, 125, 250, 500, 1 000 μg/mL, respectively. The culture medium containing 5-Fu was added to the positive group, and the final concentration was 25 μg/mL. The culture plate was put in a 5% CO2constant temperature incubator at 37 ℃ for 24 h. The old culture medium was discarded and 50μL of 10% trichloroacetic acid solution (TCA) was added to each well and fixed at 4 ℃ for 1 h. The fixed liquid was discarded and it was washed repeatedly with distilled water for 5 times. After natural drying in the air, 100 μL of SRB was added to each well for 15-30 min staining and it was washed with 1% acetic acid solution for 5 times. After drying in the air, 150 μL of Tris solution was added. After shaking and mixing, theODvalue was determined by enzyme labeling instrument (wavelength of 550 nm).

The inhibition rate of drugs on the growth of tumor cells was calculated according to the following formula:

Tumor cell growth inhibition rate (%)=(1-ODaverage value of experimental group/ODaverage value of control group)×100%.

2.2.3Determination of the direct effects of total flavonoids fromP.emblicaL. on mouse lymphocytes and proliferation induced by LPS or ConA using CCK-8. (i) Extraction of lymphocytes from mice. Three SD mice were killed by cervical vertebra dislocation and were soaked in 75% ethanol for 5 min. Under aseptic condition, the spleen was removed, washed in RPM1640 medium, and the surrounding tissue was removed. It was cut into small pieces with eye scissors, about the size of 1 mm3. A single suspension cell was made by gently pressing the aseptic syringe core on a 200-mesh cell sieve. The cells were washed twice with aseptic PBS and centrifuged with 1 500 rmp/min for 5 min. Then 2 mL of sterile ultrapure water was used to slowly break red blood cells. After 20 s, 0.9% of the same volume of normal saline was added to restore the cells to the isotonic state. The cells were washed twice with aseptic PBS and centrifuged with 1 500 rmp/min for 5 min. The RPM1640 culture medium containing 10% FBS was transferred to the culture flask. After being cultured in a 5% CO2incubator at 37 ℃ for 4 hours, the suspension cells were collected to be mouse lymphocytes.

(ii) Detection of the effect on lymphocyte proliferation in mice by CCK-8. The above extracted mouse lymphocytes were taken and their concentration was adjusted to 2×106mL. The cells were cultured in 96-well plate with 92.5 μL cell suspension per well. In the blank control group, 7.5 μL of complete RPM1640 culture medium was added. 5 μL of total flavonoids fromP.emblicaL. (the final concentration of 31.25, 62.5, 125, 250, 500, 1 000 μg/mL) and 2.5 μL of complete RPM1640 medium were added in the treatment group. After incubated in 5% CO2incubator at 37 ℃ for 24 h, 5 μL of cck-8 was added to each well at 37 ℃ for further culture for 1 h. TheODvalue was measured by enzyme labeling instrument (wavelength of 450 nm), and the degree of cell proliferation was indicated byODvalue.

(iii) Effects of total flavonoids fromP.emblicaL. on T lymphocyte proliferation induced by Con A. 92.5 μL/well cell suspension in Section2.2.3(ii) was added to the 96-well plate. At the same time, 2.5 μL/well Con A (final concentration of 5 μg/mL) was added. In the blank control group, 7.5 μL of complete RPM1640 culture medium was added. 5 μL of total flavonoids fromP.emblicaL. was added to each group (the final concentration of 31.25, 62.5, 125, 250, 500, 1 000 μg/mL, respectively). It was incubated in a 5% CO2incubator at 37 ℃ for 24 h, and then 5 μL of cck-8 was added to continue incubating at 37 ℃ for 1 h. TheODvalue was measured by enzyme labeling instrument (wavelength of 450 nm), and the degree of cell proliferation was expressed byODvalue.

(iv) Effect of total flavonoids on proliferation of B lymphocytes induced by LPS. Except for replacing ConA with LPS (the final concentration of 10 μg/mL), the grouping, sample addition and detection were the same as in Section2.2.3(iii).

2.2.4Statistical analysis. TheIC50values of total flavonoids fromP.emblicaL. on various tumor cells were calculated and analyzed by the statistical analysis software SPSS Statistics 17.0. The effect of total flavonoids inP.emblicaL. was analyzed.T-test was used to compare the mean between the two groups (P<0.05).

3 Results and analysis

3.1DeterminationoftheinhibitoryeffectoftotalflavonoidsfromP.emblicaL.onsixkindsoftumorcellsbySRBstainingThe results are shown in Table 1-6.

GroupConcentrationμg/mLOD valueInhibitionrate∥%Blank control-0.527±0.021∗∗-5-Fu250.291±0.021∗∗44.70Total flavonoids1 0000.225±0.016∗∗57.075000.296±0.016∗∗43.752500.344±0.016∗34.511250.378±0.020∗∗28.0862.50.433±0.019∗∗17.5831.250.489±0.013∗∗7.14

Note:*means comparison with the blank group,P<0.05;**means comparison with the blank group,P<0.01.

GroupConcentrationμg/mLOD valueInhibitionrate∥%Blank control-0.618±0.055∗∗-5-Fu250.359±0.043∗∗41.59Total flavonoids1 0000.284±0.021∗∗53.975000.343±0.021∗∗44.262500.406±0.022∗34.031250.453±0.039∗∗26.6562.50.487±0.028∗∗20.8831.250.531±0.037∗∗13.86

Note:*means comparison with the blank group,P<0.05;**means comparison with the blank group,P<0.01.

GroupConcentrationμg/mLOD valueInhibitionrate∥%Blank control-0.519±0.036∗∗-5-Fu250.241±0.030∗∗53.63Total flavonoids1 0000.195±0.024∗∗62.255000.275±0.017∗∗46.972500.311±0.020∗∗40.001250.346±0.029∗∗33.1062.50.429±0.016∗17.0831.250.490±0.029∗5.50

Note:*means comparison with the blank group,P<0.05;**means comparison with the blank group,P<0.01.

GroupConcentrationμg/mLOD valueInhibitionrate∥%Blank control-0.637±0.043∗∗-5-Fu250.360±0.038∗∗43.37Total flavonoids1 0000.268±0.017∗∗58.135000.347±0.023∗∗45.642500.395±0.019∗∗38.001250.450±0.034∗∗29.4162.50.510±0.033∗∗20.1831.250.564±0.047∗∗11.73

Note:**means comparison with the blank group,P<0.01.

GroupConcentrationμg/mLOD valueInhibitionrate∥%Blank control-0.757±0.073∗∗-5-Fu250.396±0.047∗∗47.20Total flavonoids1 0000.300±0.009∗60.185000.392±0.021∗∗47.942500.449±0.028∗∗40.461250.500±0.029∗∗33.7262.50.585±0.034∗∗22.2831.250.689±0.025∗∗8.44

Note:*means comparison with the blank group,P<0.05;**means comparison with the blank group,P<0.01.

Table6InhibitoryeffectoftotalflavonoidsfromPhyllanthusemblicaL.onhumannasopharyngealcarcinomacelllineCNE-2

GroupConcentrationμg/mLOD valueInhibitionrate∥%Blank control-0.848±0.096∗∗-5-Fu250.413±0.050∗∗50.52Total flavonoids1 0000.335±0.024∗∗60.155000.434±0.040∗∗48.722500.488±0.022∗∗41.971250.555±0.040∗∗34.0362.50.630±0.067∗∗25.4031.250.761±0.043∗∗9.79

Note:**means comparison with the blank group,P<0.01.

From Table 1 to 6, 24 h after administration, compared with the blank group, with the increase of the concentration of total flavonoids inP.emblicaL., its inhibitory effect on tumor cells was enhanced, showing an obvious dose-response relationship (P<0.05,P<0.01). When the concentration of total flavonoids was 500-1 000 μg/mL, the inhibitory effect of total flavonoids on six kinds of tumor cells was similar to that of positive drug 5-Fu. Among them, theIC50on lung cancer cell line H460 was the smallest, within (471.36±50.66) μg/mL, and the inhibitory effect was the least. CNE-2, Hela, A2780, SGC-7901 and HEPG-2 were also inhibited to varying degrees, and the larger theIC50was, the weaker the inhibitory effect was (Fig.1).

Fig.1IC50oftotalflavonoidsfromPhyllanthusemblicaL.on6kindsofhumantumorcells

3.2DeterminationofthedirecteffectoftotalflavonoidsfromPhyllanthusemblicaL.onmouselymphocytesandtheproliferationinducedbyLPSorConAbyCCK-8As shown in Table 7 and Fig.2, ConA (5 μg/mL) and LPS (10 μg/mL) could significantly induce the proliferation of mouse lymphocytes in vitro, while under the action of total flavonoids fromP.emblicaL., all the inhibitory effects of ConA induction group were greater than those of direct administration group. When the concentration of total flavonoids was 31.25-125.00 μg/mL, the inhibitory effect of LPS group was similar to that of direct administration group. When the concentration of total flavonoids was more than 125 μg/mL, the inhibitory effect of LPS group was higher than that of direct administration group.

Concentrationμg/mLDirect administrationOD valueInhibition rate∥%ConAOD valueInhibition rate∥%LPSOD valueInhibition rate∥%Blank control0.245±0.079-0.311±0.025-0.256±0.067-ConA--0.321±0.007---Lps----0.264±0.015-1 0000.117±0.027∗∗51.150.124±0.009∗∗61.360.112±0.0378∗∗54.235000.153±0.033∗39.610.157±0.007∗∗51.050.131±0.013∗∗41.152500.168±0.063∗32.280.182±0.027∗43.140.170±0.026∗∗35.271250.172±0.042∗28.400.211±0.007∗∗34.240.188±0.020∗∗28.4062.50.185±0.023∗∗18.380.217±0.011∗32.500.204±0.050∗18.3831.250.212±0.059∗12.670.267±0.021∗16.680.211±0.025∗∗12.67

Note:*means comparison with the blank group,P<0.05;**means comparison with the blank group,P<0.01.

Fig.2InhibitoryeffectoftotalflavonoidsfromPhyllanthusemblicaL.onlymphocytesinmice

4 Discussion

At present, a lot of literature has reported the inhibitory effect of traditional Chinese medicine on tumor proliferation[4-6], and it has been reported thatP.emblicaL. extract has broad-spectrum tumor effect[4], but the specific anti-tumor effective part ofP.emblicaL. extract has not been studied. In this experiment, we confirmed for the first time that the total flavonoids ofP.emblicaL. had broad-spectrum anti-tumor effect.

The main purpose of this experiment was to study the effects of total flavonoids fromP.emblicaL. on the proliferation of many kinds of tumor cells including H460, CNE-2, Hela, A2780 and HEPG-2. The inhibitory effect of total flavonoids fromP.emblicaL. on the proliferation of these seven kinds of tumor cells was detected by SRB method, and the inhibitory effect on tumor cells was enhanced with the increase of drug concentration and action time. The results showed that the total flavonoids ofP.emblicaL. could inhibit the growth of many kinds of tumor cells, and showed a dose-response relationship.

Zhong Zhenguoetal. reported that the extract ofP.emblicaL. could improve the non-specific immune function of normal mice. In our experiment, the total flavonoids ofP.emblicaL. significantly inhibited the activation and proliferation of mouse lymphocytes induced by ConA and LPSinvitro, which showed that the total flavonoids ofP.emblicaL. were a potential immunosuppressant. The immunosuppressant is a kind of drug which can improve the immunity of the body in the process of tumor chemotherapy, reduce the symptoms of low immune function caused by radiotherapy and chemotherapy, cause fewer complications, and improve the effect of tumor treatment.