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In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root
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In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root Kejuan Li, Xi Yang, Xuansheng Hu, Chao Han, Zhongfang Lei, Zhenya Zhang∗ Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
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Article history: Received 14 September 2015 Revised 18 December 2015 Accepted 23 December 2015 Available online xxx Keywords: Immunomodulatory Macrophages Cytotoxic Anticancer Antioxidant
a b s t r a c t Helicteres angustifolia L. (H. angustifolia) has been widely used in traditional Chinese medicine system to treat a variety of diseases including cancer. In order to characterize the bioactivities of H. angustifolia, we first obtained the aqueous root extract (ARE) which was then further partitioned into two fractions, namely ethanol fraction (EF) and water fraction (WF), and their antioxidant, immunomodulatory and anticancer activities were evaluated respectively. Results indicated that both EF and WF possessed strong antioxidant activities, and EF was the major component to exhibit the cytotoxic activity in ARE with IC50 values of (33.98 ± 1.58) and (35.56 ± 0.42) μg/ml against human lung cancer cell lines A549 and H1299 for 72 h treatment, respectively. As for immunomodulatory activities, WF was shown to stimulate the proliferation of macrophages (292.76 ± 31.42%) and phagocytic activity at 12.5 μg/ml, and increase the production of nitric oxide. Furthermore, WF was also observed to significantly mitigate doxorubicin (DOX) induced toxicity (from 18.42% to 63.63%) and apoptosis (from 90.8% to 57.5%) at 100 μg/ml, respectively. The above findings highlight the functional characteristics of WF and EF in their contribution to the anticancer activities of ARE, which also provides important scientific evidence for developing ARE as a potent anticancer reagent. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Cancer is a worldwide health problem due to the lack of comprehensive early detection methods and effective treatment. It is well known that immunomodulation in host system may bring inhibition to tumor growth [1], and it has also been demonstrated that the excessive production of reactive oxygen species (ROS) may contribute to cancer [2]. Therefore, searching for medicinal plants or functional foods with immunomodulatory potential and high antioxidant activities as alternative anticancer resources has been attracting more and more attention [3]. Helicteres angustifolia L. (H. angustifolia) is a common shrub widely distributed in China, Japan, and many Southeast Asian countries. Its dry root has been widely used in traditional medicine system of oriental countries, such as China and Laos, to treat a variety of ailments ranging from influenza fever, headache, carbuncle, hemorrhoid, tonsillitis, pharyngitis, parotitis to
Abbreviations: H. angustifolia, Helicteres angustifolia L.; ARE, aqueous root extract of H. angustifolia; EF, ethanolic fraction; WF, water fraction. ∗ Corresponding author. Tel. +81 29 853 4712; fax: +81 29 853 4712. E-mail address: [email protected] (Z. Zhang).
inflammatory and cancer [4]. Although many benefits have been gained from this medical plant, scientific evidence of its promoting health is still scarce. Previous phytochemical investigations mainly contributed to the identification of naphthoquinone [5], sesquiterpenoid quinones [6], flavonoids [7], lignans [8], triterpenoids [9], steroids [10], alkaloids [11], cucurbitacins and their derivatives [12] from this plant. Up to now, H. angustifolia root has been claimed to have anti-hepatic fibrosis and antiviral activities [13,14]. In addition, constituents from chloroform extract [12,9] and ethyl acetate extract [11] of H. angustifolia root bark have been found to have cytotoxic effect against several cancer cell lines. Most recently, our research work indicated that aqueous root extract of H. angustifolia (i.e. ARE) possessed significant high antioxidant and anticancer activities, showing cytotoxic effect on human colon (DLD-1), lung (A549) and hepato (HepG2) carcinoma cells in vitro, and xenograft tumor suppression effect in vivo as well [15]. In order to shed light on the mechanisms involved in the above mentioned bioactivities, especially its anticancer activity, ARE was further partitioned into two fractions, namely ethanol fraction (EF) and water fraction (WF), and their antioxidant, immunomodulatory, and anticancer activities were investigated in vitro, respectively.
http://dx.doi.org/10.1016/j.jtice.2015.12.022 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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2. Materials and methods 2.1. Sample collection and preparation H. angustifolia roots were collected in August 2013 from Vientiane, Laos (18.069609, 102.828844). Botanical identification was done at Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. A voucher specimen (No. 090155) was deposited at the same Herbarium. Samples were prepared according to the following procedure. The dry root of H. angustifolia was ground into fine powders and then extracted twice with 1:10 (w/v) of distilled water for 24 h each time. The obtained extracts were freeze-dried and labeled as aqueous root extract (ARE). ARE was then dissolved in deionized water and precipitated by adding eight volumes of ethanol to a final concentration of 80% (v/v) and stored at 4 °C overnight. The precipitate, labeled as water fraction (WF), was collected by centrifuging at 8000 rpm for 30 min and lyophilized. The supernatant were then collected, freeze dried, and labeled as ethanol fraction (EF). Both fractions were stored at –20 °C till the following experiments. 2.2. Determination of total phenol and crude polysaccharides The total phenolic content was determined by using Folin– Ciocalteu reagent according to a previously described method [16]. The crude polysaccharides were quantified with the phenolsulfuric acid method [17]. 2.3. Antioxidant activities assessment DPPH and ABTS radical scavenging activity, reducing power, and ferrous metal ion-chelating activity of EF and WF were estimated according to our previous work [15]. 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Toxlox) and ethylenediaminetetraacetic acid (EDTA) were used as the positive control. 2.4. Anticancer activities assessment in vitro 2.4.1. Cell lines and culture Human lung cancer cell lines A549 and H1299 were used for in vitro anticancer assays. Both cell lines were obtained from the Cell Resource Center for Biomedical Research of Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 100 U/ml penicillin and streptomycin in a humidified 5% CO2 atmosphere incubator at 37 °C. 2.4.2. Cytotoxicity effect (MTT assay) The cytotoxic effects of EF and WF against A549 and H1299 cells were determined by using 3-(4, 5-diethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) assay. Cells were seeded at 5 × 103 cells/well in 96-well plates 24 h prior to treatment. Afterward, cells were treated with various concentrations (6.25– 100 μg/ml) of EF or WF for 24 h, 48 h, and 72 h, respectively. Then MTT solution (0.5 mg/ml) was added to each well, followed by further incubation at 37 °C for another 4 h and then by the replacement with 100 μl DMSO to each well for complete dissolution of formazan crystals. Absorbance was measured at 570 nm using a Model 550 microplate reader (BIO-RAD, Tokyo, Japan). 2.4.3. Morphological observation Cells were seeded at 3 × 105 cells/well in a 6-well plate and treated with different concentrations of EF or WF for 24 h after
attachment. Cell morphological changes were directly recorded using a phase-contrast inverse microscope fitted with digital camera (Digital sight DS-L1, Nikon, Japan). Later, the plates were stained with 0.5% of crystal violet and scanned for records. 2.4.4. Colony forming assay Cells were seeded in 6-well plates at a density of 500 cells/well in triplicate. After 24 h of adherence, the cells were treated with or without 50 μg/ml of EF/WF for 4 h. Then the cells were maintained in normal medium until the appearance of colonies with regular change of medium. Colonies were fixed with pre-chilled methanol and stained with a 0.5% crystal violet, photographed and counted. 2.5. Immunomodulation activities assessment 2.5.1. Cell line and proliferation assay The murine macrophage cell line RAW 264.7 was obtained from the Riken Cell Bank (Tsukuba, Japan) and maintained in DMEM in the same condition as described above. The proliferation effect of EF or WF on RAW 264.7 cells for 24 h of treatment was estimated by using MTT assay. 2.5.2. Measurement of the production of nitric oxide (NO) and phagocytosis ability Nitrite accumulation and phagocytic ability of WF were measured by using the Griess reagent method and a neutral red uptake assay [18]. 1 μg/ml of lipopolysaccharide (LPS) was used as the positive control. 2.5.3. Protective activity on doxorubicin (DOX)-induced damage RAW 264.7 cells were seeded in a 96-well plate at 5 × 103 cells/well. After 24 h of adherence, cells were treated with DOX (0.5 or 1 μmol/L, final concentration) in the absence or presence of various concentrations (3.125–100 μg/ml) of WF for 24 h. Cell viability was determined using MTT method. 2.5.4. Apoptosis assay RAW 264.7 cells were seeded at a density of 3 × 105 cells/well in 6-well plates and incubated with DOX (2 or 4 μmol/L) in the absence or presence of WF (6.25, 25 and 100 μg/ml) after adherence. After 24 h of treatment, cells were harvested and apoptosis assay was conducted by using Guava Nexin Assay (Guava PCA flow cytometer, Millipore). Percentage apoptotic cells were determined by Flow Jo software. 2.6. Statistical analysis All experiments were carried out in triplicates, and data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, USA). Differences among samples were evaluated by using analysis of variance (ANOVA) and Duncan’s multiple comparison method. Significant difference was assumed at p < 0.05. 3. Results and discussion 3.1. Phenolic and crude polysaccharide contents in the two fractions of ARE In this study, ARE was fractionated by 80% ethanol. The yield of water fraction (WF) and ethanol fraction (EF) were 2.86% and 4.67% of the dry root powder (d.w., Table 1). EF contained a total phenolic content of (90.5 ± 1.69) mg GAE/g as obtained from the calibration curve, y = – 0.01 + 0.012 x (R2 = 0.999, y is the absorbance, and x is the concentration of gallic acid solution, μg/ml).
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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Table 1 Extraction yield, total phenolic content (TPC), crude polysaccharide (CPS) and the EC50 values of antioxidant activities of EF and WF. Sample
EF WF Trolox EDTA
Yield (%)
4.67 2.86
TPC (mg GAE/g)
90.5 ± 1.69 8.63 ± 2.56
CPS (mg DGE /g)
35.9 ± 3.33 127.8 ± 1.72
EC50 values (mg/ml)∗ DPPH assay
ABTS assay
Reducing power
Ferrous chelating ability
0.71 ± 0.01 0.78 ± 0.01 0.16 ± 0.02
0.24 ± 0.01 0.13 ± 0.02 0.06 ± 0.01
0.16 ± 0.01 0.11 ± 0.01 0.07 ± 0.01
>10 7.42 ± 0.84 1.05 ± 0.20
TPC was calculated as gallic acid equivalent (GAE) μg/mg of dry basis; CPS was calculated as D-glucose equivalent (DGE) μg/mg of dry basis; ∗ EC50 value (mg/ml) for free radicals obtained by interpolation from linear regression analysis was the effective concentration at which radicals were scavenged by 50%; for reducing power, the effective concentration at which the absorbance at 700 nm reached 0.5 by interpolation from linear regression analysis; for chelating ability, the effective concentration at which ferrous ions were chelated by 50%.
On the other hand, WF was determined to have a crude polysaccharide content of (127.8 ± 1.72) mg DGE/g of the dry powder according to the calibration curve, y = 0.015 + 0.014 x (R2 = 0.990, y is the absorbance, and x is the concentration of D-glucose solution, μg/ml). Data are expressed as mean ± SD from three independent experiments.
3.2. Evaluation of antioxidant activities As shown in Figs. 1A and B, both fractions exhibited a concentration-dependent scavenging effect on DPPH and ABTS radicals. EF displayed a slightly significantly stronger DPPH scavenging activity (EC50 = 0.71 mg/ml, Table 1) than WF (EC50 = 0.78 mg/ml, Table 1, p < 0.05). However, WF possessed a significantly lower
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Fig. 1. Antioxidant activities of WF and EF indicated by DPPH radical scavenging ability (A), ABTS radical scavenging ability (B), reducing power (C) and ferrous chelating ability (D). Results are expressed as mean ± SD from three independent experiments.
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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EC50 value of 0.13 mg/ml than EF (EC50 = 0.24 mg/ml, Table 1, p < 0.05). A medium strong positive correlation (r2 = 0.521, p < 0.05) was found between the DPPH and ABTS tests. Regarding DPPH and ABTS assays, a similar antioxidant relationship between ethanol extract and water extract was also reported in previous works [16,19]. The antioxidant capacities of EF and WF were further explored by using the reducing power and ferrous chelating assays. As shown in Fig. 1C, both fractions showed a linear increase trend in reducing power with the increase in their concentration. The EC50 values of reducing power in WF, EF and Trolox followed a descending order of Trolox (0.07 mg/ml) > WF (0.11 mg/ml) > EF (0.16 mg/ml) (Table 1). As shown in Fig. 1D, WF was found to exhibit a relatively higher chelating ability than EF. The EC50 values were determined to be 1.05, 7.42 and > 10 mg/ml for EDTA, WF and EF, respectively, which to some extent agrees with the finding of Andjelkovic´ et al. [20], who claimed that the ability of phenolic compounds (the major component of EF) to chelate iron was far lower than that of EDTA. The correlation analysis revealed that the reducing power of EF had the strongest correlation with ABTS assay (r2 = 0.995, p < 0.05) and a medium strong correlation with DPPH assay (r2 = 0.548, p < 0.05). 3.3. Evaluation of anticancer activity of WF and EF in vitro Cytotoxic effects of EF and WF were examined by using MTT assay against human lung cancer cell lines A549 and H1299 for daily treatment of 24, 48 and 72 h, respectively. As shown in Figs. 2A and B, EF exhibited stronger cytotoxic effect than WF, with IC50 values of 68.01 ± 2.69, 55.53 ± 3.23, and 33.98 ± 1.58 μg/ml against A549; and 70.47 ± 1.22, 59.30 ± 3.22, and 35.56 ± 0.42 μg/ml against H1299 for 24 h, 48 h and 72 h, respectively. Cell morphological changes also indicated that the treatment of lung cancer cells at various doses of EF reflected its significant cytotoxicity to human lung cancer cells at doses of 25 μg/ml and above, and the cells treated with WF revealed less cytotoxic effect (Figs. 2C and D). We also performed the long-term inhibition effect of EF and WF on the colony formation abilities of A549 and H1299 cells. As shown in Figs. 3A and B, the untreated A549 cells produced 145.67 ± 5.51 colonies, whereas the colony numbers were significantly (p < 0.05) suppressed to 110 ± 4.36 and 25.67 ± 4.73 after being treated with 50 μg/ml of WF and EF, respectively. Meanwhile, treatment with 50 μg/ml of WF and EF reduced the colony number of H1299 cells from 84.33 ± 3.06 to 64 ± 7.94 and 40.33 ± 2.89, respectively. This observation also suggested that EF showed higher proliferation inhibition effect on cancer cells than WF. Results from this study suggests that EF, with a higher phenolic content, possesses stronger anticancer activity than WF, which is in agreement with the previous finding of Zou and Chang [21] who discovered that poly-phenolic extracts from various plants exhibited high efficiency in antitumor activity. Interestingly, our previous 24 h treatment tests revealed that ARE possessed cytotoxic effect against A549 with an IC50 value of 62.50 ± 6.99 μg/ml [15], which is slightly lower than either EF (68.01 ± 2.69 μg/ml) or WF (> 100 μg/ml). In order to examine their functional contributions to the anticancer activities of ARE, we investigated immunomodulatory activities of WF and EF in the followed-up experiments.
Table 2 Effect of water fraction (WF) on nitric oxide (NO) production and the phagocytic activity of macrophages RAW 264.7. Concentrations of WF (μg/ml)
Production of nitric oxide (μM)
0 3.125 6.25 12.5 25 50 100 LPS (1 μg/ml)a
0.56 7.00 9.68 10.00 13.00 14.89 11.56 15.00
± ± ± ± ± ± ± ±
0.69∗∗∗ 2.00∗∗∗ 2.68∗∗ 1.33∗∗ 1.15 1.02 0.38∗ 1.86
Phagocytosis (O.D.540 nm) 0.255 ± 0.020∗ 0.343 ± 0.005∗∗ 0.351 ± 0.014∗∗∗ 0.382 ± 0.024∗∗∗ 0.370 ± 0.023∗∗∗ 0.353 ± 0.017∗∗∗ 0.347 ± 0.015∗∗ 0.291 ± 0.011
Results are represented as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 indicate statistically significant difference compared with LPS group. a LPS was used as the positive control.
latory effect reached to maximum with a cell viability rate of 249.76 ± 31.42%, while WF had a proliferation effect on macrophage with a cell viability rate of 292.76 ± 31.42% at 12.5 μg/ml. However, the proliferation effect decreased as WF concentration further increased from 12.5 to 100 μg/ml, probably due to the “immunologic paralysis” [22]. On the other hand, EF showed a relatively mild proliferation effect with a maximum proliferation rate of 156.73% (p < 0.001) at the concentration of 100 μg/ml. As pointed out by Schepetkin and Quinn [23], compared with bacteria derived immunomodulatory polysaccharides and synthetic compounds, most higher plants derived polysaccharides are relatively nontoxic and do not have significant side effects. Indeed, a variety of plant polysaccharides have been found to have beneficial therapeutic effects by modulating monocyte/macrophage immune functions in previous studies [24,25]. In this study, the strong stimulatory effect of WF on macrophage proliferation is most probably attributable to the high content of polysaccharides in WF. Since WF exhibited considerable proliferation effect on macrophages, we anticipated that WF has potent immunomodulatory activity. Thus WF was further assessed by testing its stimulation effect on NO production in macrophages, and then its phagocytic ability and stress-protection effect.
3.4. Evaluation of immunomodulation activities on RAW 264.7 macrophages
3.4.2. Effect of WF on NO production A low amount of NO (0.56 μmol/L) was detected in the nonactivated macrophages as shown in Table 2, whereas incubation of the cells with increasing amount of WF (3.125–50 μg/ml) resulted in a significant stimulation effect on NO production in a dose-dependent manner. Treatment with 50 μg/ml of WF significantly stimulated NO production to 14.89 μmol/L. NO is produced in response to stimulation by a variety of agents to inhibit the growth of a wide variety of microorganisms, parasites and tumor cells [23], thus its production could be used as a quantitative index of macrophage activation [26]. In this work, WF was found to stimulate macrophages to produce NO, which is reminiscent of similar properties possessed by 1 μg/ml of LPS (15 μmol/L). Interestingly, it has been claimed that NO has dual effects in the immune system that a low concentration of NO may have macrophage-activating activity while an over-production of NO could induce an inflammatory response [27]. Results from this study revealed an increase of NO production in the supernatants of the WF treated macrophages cells, while this increased level is lower than the non-harmful one, 21.16 μmol/L as reported by Shi et al. [18].
3.4.1. Effect of EF and WF on the proliferation of macrophages As shown in Fig. 4, exposure to ARE stimulated macrophage proliferation. At a concentration of 12.5 μg/ml, the stimu-
3.4.3. Effect of WF on the phagocytic activity of macrophages WF treatment significantly increased (p < 0.05) the phagocytosis of macrophages when compared to the control cells (Table 2).
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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Fig. 2. Cytotoxic effect of EF (A) and WF (B) against human lung cancer lines A549 cells and H1299 cells. Image (C) and cell morphology (D) changes for 24 h treatment of EF and WF in A549 and H1299 cells. Results are expressed as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 statistically indicate the significant difference from control group.
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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Fig. 3. Inhibition on colony formation of A549 and H1299 cells after EF (50 μg/ml) and WF (50 μg/ml) treatment for 4 h.
It is well known that macrophages play an important role in host defense system that phagocytizes the pathogens and tumor cells [28]. This work suggests that administration of WF may enhance the immune response of macrophage by stimulating its phagocytosis ability thus contribute to the anticancer activities of ARE in vivo.
Fig. 4. Effects of ARE, EF and WF on proliferation of macrophages RAW 264.7 cells. Results are expressed as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 statistically indicate the significant difference from the control group.
The resultant optical density value of 3.125 μg/ml for WF treatment (0.34) was significantly higher (p < 0.01) than that of the positive control (0.29) in the cells treated with 1 μg/ml of LPS. Moreover, the absorption value reached a maximum of 0.38 at the concentration of 12.5 μg/ml, which is partially in agreement with its proliferation effect observed in Fig. 4.
3.4.4. Protective effect of WF on DOX-induced toxicity and apoptosis in macrophages RAW 264.7 As shown in Fig. 5A, treatment with 0.5 μmol/L DOX decreased the survival rate of RAW 264.7 cells to 41.65%. While in the presence of WF, the viability of the RAW 264.7 cells was significantly increased, reaching to the maximum (143.19%, p < 0.001) at the concentration of 25 μg/ml. Considering the outstanding protective effect of WF on DOX-induced toxicity to macrophages, additional tests were further carried out at a higher level of DOX (1 μmol/L). As shown in Fig. 5B, results showed that only a survival rate of 18.42% was obtained in the control macrophages cells. However, when incubated with various concentrations (3.125–100 μg/ml) of WF, the macrophages viability increased with the increase in the concentration of WF tested. Incubation with 100 μg/ml of WF could elevate the survival rate of macrophages to a significantly higher extent of 63.63% (p < 0.001). DOX is a commonly used anticancer drug in chemotherapy, and it has been demonstrated to exert apoptosis effect by causing DNA damage [29]. In order to confirm the protection effect of WF in DOX-induced macrophages, a flow cytometric analysis was
Fig. 5. Protective effect of WF on 0.5 μmol/L (A) or 1 μmol/L (B) DOX-induced viability of macrophage RAW 264.7 cells. Results are expressed as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 statistically indicate the significant difference from the negative control group (DOX).
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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Fig. 6. Protective effect of WF on apoptosis induction by 2 or 4 μmol/L DOX in macrophage RAW 264.7 cells observed by cell morphology changes (A) and cell populations analysis (B and C).
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carried out to evaluate apoptosis populations (Annexin V positive) in control, DOX treated, and DOX+WF treated cells, respectively. As shown in Fig. 6, the apoptotic population of macrophage RAW 264.7 cells increased from 11.4% (control) to 63.8% and 90.8% after being treated with 2 and 4 μM DOX, respectively. However, WF significantly reduced the apoptosis rates. For example, incubation with 6.25, 25 and 100 μg/ml of WF mitigated the apoptosis rate to 72.40%, 59.9% and 57.5% in 4 μM DOX treated macrophages. This observation implies that WF may contribute to the mitigation of DOX-induced toxicity and apoptosis due to its high proliferation effect on macrophages and strong antioxidant activities. 4. Conclusion This work was the first report on the strong immunomodulatory effect of H. angustifolia root, and disclosed the functional relationship among its antioxidant, immunomodulatory activity and anticancer activity in ARE fractions. Findings from this study suggest that EF is the main cytotoxic factor of ARE, and the remarkable immunomodulatory activities of WF also contribute to the anticancer activities of ARE to a great extent, hence provides important scientific evidence for developing ARE as potent anticancer reagents. References [1] Ehrke MJ. Immunomodulation in cancer therapeutics. Int Immunopharmacol 2003;3:1105–19. [2] Liu W, Wang H, Pang X, Yao W, Gao X. Characterization and antioxidant activity of two low-molecular-weight polysaccharides purified from the fruiting bodies of Ganoderma lucidum. Int J Biol Macromol 2010;46:451–7. [3] Aziz MH, Kumar R, Ahmad N. Cancer chemoprevention by resveratrol: In vitro and in vivo studies and the underlying mechanisms (Review). Int J Oncol 2003;23:17–28. [4] N-Y Chiu, K-H Chang. The illustrated medicinal plants of Taiwan. Taipei: Southern Materials Center Inc; 1995. (in Chinese) p. 104. [5] Wang M, Liu W. A naphthoquinone from Helicteres angustifolia. Phytochemistry 1987;26:578–9. [6] Chen C-M, Chen Z-T, Hong Y-L. A mansonone from Helicteres angustifolia. Phytochemistry 1990;29:980–2. [7] Chen Z-T, Lee S-W, Chen C-M. New flavoid glycosides of Helicteres angustifolia. Heterocycles 1994;38:1399–406. [8] Chin Y-W, Jones WP, Rachman I, Riswan S, Kardono LBS, Chai H-B, et al. Cytotoxic lignans from the stems of Helicteres hirsuta collected in Indonesia. Phytother Res 2006;20:62–5. [9] Pan M-H, Chen C-M, Lee S-W, Chen Z-T. Cytotoxic triterpenoids from the root bark of Helicteres angustifolia. Chem Biodivers 2008;5:565–74. [10] Chen W, Tang W, Lou L, Zhao W. Pregnane, coumarin and lupane derivatives and cytotoxic constituents from Helicteres angustifolia. Phytochemistry 2006;67:1041–7. [11] Wang G-C, Li T, Wei Y-R, Zhang Y-B, Li Y-L, Sze SCW, et al. Two pregnane derivatives and a quinolone alkaloid from Helicteres angustifolia. Fitoterapia 2012;83:1643–7.
[12] Chen Z-T, Lee S-W, Chen C-M. Cucurbitacin B 2-sulfate and cucurbitacin glucosides from the root bark of Helicteres angustifolia. Chem Pharm Bull 2006;54:1605–7. [13] Huang Q, Li Y, Zhang S, Huang R, Zheng L, Wei L, et al. Effect and mechanism of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) on hepatic fibrosis induced by carbon tetrachloride in rats. J Ethnopharmacol 2012;143:889–95. [14] Huang Q, Huang R, Wei L, Chen Y, Lv S, Liang C, et al. Antiviral activity of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) against hepatitis B virus. Antivir Res 2013;100:373–81. [15] Li K, Lei Z, Hu X, Sun S, Li S, Zhang Z. In vitro and in vivo bioactivities of aqueous and ethanol extracts from Helicteres angustifolia L. root. J Ethnopharmacol 2015;172:61–9. [16] Li F, Wang F, Yu F, Fang Y, Xin Z, Yang F, et al. In vitro antioxidant and anticancer activities of ethanolic extract of selenium-enriched green tea. Food Chem 2008;111:165–70. [17] Li S, Wang L, Song C, Hu X, Sun H, Yang Y, et al. Utilization of soybean curd residue for polysaccharides by Wolfiporia extensa (Peck) Ginns and the antioxidant activities in vitro. J Taiwan Inst Chem Eng 2014;45:6–11. [18] Shi M, Yang Y, Hu X, Zhang Z. Effect of ultrasonic extraction conditions on antioxidative and immunomodulatory activities of a Ganoderma lucidum polysaccharide originated from fermented soybean curd residue. Food Chem 2014;155:50–6. [19] Chen T, He J, Zhang J, Li X, Zhang H, Hao J, et al. The isolation and identification of two compounds with predominant radical scavenging activity in hempseed (seed of Cannabis sativa L). Food Chem 2012;134:1030–7. [20] Andjelkovic´ M, Van Camp J, De Meulenaer B, Depaemelaere G, Socaciu C, Verloo M, et al. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chem 2006;98:23–31. [21] Zou Y, Chang SKC. Effect of black soybean extract on the suppression of the proliferation of human AGS gastric cancer cells via the induction of apoptosis. J Agri Food Chem 2011;59:4597–605. [22] Li J, Shan L, Liu Y, Fan L, Ai L. Screening of a functional polysaccharide from Zizyphus Jujuba cv. Jinsixiaozao and its property. Int J Biol Macromol 2011;49:255–9. [23] Schepetkin IA, Quinn MT. Botanical polysaccharides: Macrophage immunomodulation and therapeutic potential. Int Immunopharmacol 2006;6:317–33. [24] Berner MD, Sura ME, Alves BN, Hunter KW Jr. IFN-γ primes macrophages for enhanced TNF-α expression in response to stimulatory and non-stimulatory amounts of microparticulate β -glucan. Immunol Lett 2005;98:115–22. [25] Naeini A, Khosravi A, Tadjbakhsh H, Ghazanfari T, Yaraee R, Shokri H. Evaluation of the immunostimulatory activity of Ziziphora tenuior extracts. Comp Clin Pathol 2010;19:459–63. [26] Lorsbach RB, Murphy WJ, Lowenstein CJ, Snyder SH, Russell SW. Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide. J Biol Chem. 1993;268:1908–13. [27] Cheng A, Wan F, Wang J, Jin Z, Xu X. Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish. Int Immunopharmacol 2008;8:43–50. [28] Yu M, Xu X, Qing Y, Luo X, Yang Z, Zheng L. Isolation of an anti-tumor polysaccharide from Auricularia polytricha (jew’s ear) and its effects on macrophage activation. Eur Food Res Technol 2009;228:477–85. [29] Mizutani H. Mechanism of DNA damage and apoptosis induced by anticancer drugs through generation of reactive oxygen species. J Pharm Soc Jpn 2007;127:1837–42 (in Japanese with English abstract).
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In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root Kejuan Li, Xi Yang, Xuansheng Hu, Chao Han, Zhongfang Lei, Zhenya Zhang∗ Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e
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Article history: Received 14 September 2015 Revised 18 December 2015 Accepted 23 December 2015 Available online xxx Keywords: Immunomodulatory Macrophages Cytotoxic Anticancer Antioxidant
a b s t r a c t Helicteres angustifolia L. (H. angustifolia) has been widely used in traditional Chinese medicine system to treat a variety of diseases including cancer. In order to characterize the bioactivities of H. angustifolia, we first obtained the aqueous root extract (ARE) which was then further partitioned into two fractions, namely ethanol fraction (EF) and water fraction (WF), and their antioxidant, immunomodulatory and anticancer activities were evaluated respectively. Results indicated that both EF and WF possessed strong antioxidant activities, and EF was the major component to exhibit the cytotoxic activity in ARE with IC50 values of (33.98 ± 1.58) and (35.56 ± 0.42) μg/ml against human lung cancer cell lines A549 and H1299 for 72 h treatment, respectively. As for immunomodulatory activities, WF was shown to stimulate the proliferation of macrophages (292.76 ± 31.42%) and phagocytic activity at 12.5 μg/ml, and increase the production of nitric oxide. Furthermore, WF was also observed to significantly mitigate doxorubicin (DOX) induced toxicity (from 18.42% to 63.63%) and apoptosis (from 90.8% to 57.5%) at 100 μg/ml, respectively. The above findings highlight the functional characteristics of WF and EF in their contribution to the anticancer activities of ARE, which also provides important scientific evidence for developing ARE as a potent anticancer reagent. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Cancer is a worldwide health problem due to the lack of comprehensive early detection methods and effective treatment. It is well known that immunomodulation in host system may bring inhibition to tumor growth [1], and it has also been demonstrated that the excessive production of reactive oxygen species (ROS) may contribute to cancer [2]. Therefore, searching for medicinal plants or functional foods with immunomodulatory potential and high antioxidant activities as alternative anticancer resources has been attracting more and more attention [3]. Helicteres angustifolia L. (H. angustifolia) is a common shrub widely distributed in China, Japan, and many Southeast Asian countries. Its dry root has been widely used in traditional medicine system of oriental countries, such as China and Laos, to treat a variety of ailments ranging from influenza fever, headache, carbuncle, hemorrhoid, tonsillitis, pharyngitis, parotitis to
Abbreviations: H. angustifolia, Helicteres angustifolia L.; ARE, aqueous root extract of H. angustifolia; EF, ethanolic fraction; WF, water fraction. ∗ Corresponding author. Tel. +81 29 853 4712; fax: +81 29 853 4712. E-mail address: [email protected] (Z. Zhang).
inflammatory and cancer [4]. Although many benefits have been gained from this medical plant, scientific evidence of its promoting health is still scarce. Previous phytochemical investigations mainly contributed to the identification of naphthoquinone [5], sesquiterpenoid quinones [6], flavonoids [7], lignans [8], triterpenoids [9], steroids [10], alkaloids [11], cucurbitacins and their derivatives [12] from this plant. Up to now, H. angustifolia root has been claimed to have anti-hepatic fibrosis and antiviral activities [13,14]. In addition, constituents from chloroform extract [12,9] and ethyl acetate extract [11] of H. angustifolia root bark have been found to have cytotoxic effect against several cancer cell lines. Most recently, our research work indicated that aqueous root extract of H. angustifolia (i.e. ARE) possessed significant high antioxidant and anticancer activities, showing cytotoxic effect on human colon (DLD-1), lung (A549) and hepato (HepG2) carcinoma cells in vitro, and xenograft tumor suppression effect in vivo as well [15]. In order to shed light on the mechanisms involved in the above mentioned bioactivities, especially its anticancer activity, ARE was further partitioned into two fractions, namely ethanol fraction (EF) and water fraction (WF), and their antioxidant, immunomodulatory, and anticancer activities were investigated in vitro, respectively.
http://dx.doi.org/10.1016/j.jtice.2015.12.022 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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2. Materials and methods 2.1. Sample collection and preparation H. angustifolia roots were collected in August 2013 from Vientiane, Laos (18.069609, 102.828844). Botanical identification was done at Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. A voucher specimen (No. 090155) was deposited at the same Herbarium. Samples were prepared according to the following procedure. The dry root of H. angustifolia was ground into fine powders and then extracted twice with 1:10 (w/v) of distilled water for 24 h each time. The obtained extracts were freeze-dried and labeled as aqueous root extract (ARE). ARE was then dissolved in deionized water and precipitated by adding eight volumes of ethanol to a final concentration of 80% (v/v) and stored at 4 °C overnight. The precipitate, labeled as water fraction (WF), was collected by centrifuging at 8000 rpm for 30 min and lyophilized. The supernatant were then collected, freeze dried, and labeled as ethanol fraction (EF). Both fractions were stored at –20 °C till the following experiments. 2.2. Determination of total phenol and crude polysaccharides The total phenolic content was determined by using Folin– Ciocalteu reagent according to a previously described method [16]. The crude polysaccharides were quantified with the phenolsulfuric acid method [17]. 2.3. Antioxidant activities assessment DPPH and ABTS radical scavenging activity, reducing power, and ferrous metal ion-chelating activity of EF and WF were estimated according to our previous work [15]. 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Toxlox) and ethylenediaminetetraacetic acid (EDTA) were used as the positive control. 2.4. Anticancer activities assessment in vitro 2.4.1. Cell lines and culture Human lung cancer cell lines A549 and H1299 were used for in vitro anticancer assays. Both cell lines were obtained from the Cell Resource Center for Biomedical Research of Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 100 U/ml penicillin and streptomycin in a humidified 5% CO2 atmosphere incubator at 37 °C. 2.4.2. Cytotoxicity effect (MTT assay) The cytotoxic effects of EF and WF against A549 and H1299 cells were determined by using 3-(4, 5-diethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) assay. Cells were seeded at 5 × 103 cells/well in 96-well plates 24 h prior to treatment. Afterward, cells were treated with various concentrations (6.25– 100 μg/ml) of EF or WF for 24 h, 48 h, and 72 h, respectively. Then MTT solution (0.5 mg/ml) was added to each well, followed by further incubation at 37 °C for another 4 h and then by the replacement with 100 μl DMSO to each well for complete dissolution of formazan crystals. Absorbance was measured at 570 nm using a Model 550 microplate reader (BIO-RAD, Tokyo, Japan). 2.4.3. Morphological observation Cells were seeded at 3 × 105 cells/well in a 6-well plate and treated with different concentrations of EF or WF for 24 h after
attachment. Cell morphological changes were directly recorded using a phase-contrast inverse microscope fitted with digital camera (Digital sight DS-L1, Nikon, Japan). Later, the plates were stained with 0.5% of crystal violet and scanned for records. 2.4.4. Colony forming assay Cells were seeded in 6-well plates at a density of 500 cells/well in triplicate. After 24 h of adherence, the cells were treated with or without 50 μg/ml of EF/WF for 4 h. Then the cells were maintained in normal medium until the appearance of colonies with regular change of medium. Colonies were fixed with pre-chilled methanol and stained with a 0.5% crystal violet, photographed and counted. 2.5. Immunomodulation activities assessment 2.5.1. Cell line and proliferation assay The murine macrophage cell line RAW 264.7 was obtained from the Riken Cell Bank (Tsukuba, Japan) and maintained in DMEM in the same condition as described above. The proliferation effect of EF or WF on RAW 264.7 cells for 24 h of treatment was estimated by using MTT assay. 2.5.2. Measurement of the production of nitric oxide (NO) and phagocytosis ability Nitrite accumulation and phagocytic ability of WF were measured by using the Griess reagent method and a neutral red uptake assay [18]. 1 μg/ml of lipopolysaccharide (LPS) was used as the positive control. 2.5.3. Protective activity on doxorubicin (DOX)-induced damage RAW 264.7 cells were seeded in a 96-well plate at 5 × 103 cells/well. After 24 h of adherence, cells were treated with DOX (0.5 or 1 μmol/L, final concentration) in the absence or presence of various concentrations (3.125–100 μg/ml) of WF for 24 h. Cell viability was determined using MTT method. 2.5.4. Apoptosis assay RAW 264.7 cells were seeded at a density of 3 × 105 cells/well in 6-well plates and incubated with DOX (2 or 4 μmol/L) in the absence or presence of WF (6.25, 25 and 100 μg/ml) after adherence. After 24 h of treatment, cells were harvested and apoptosis assay was conducted by using Guava Nexin Assay (Guava PCA flow cytometer, Millipore). Percentage apoptotic cells were determined by Flow Jo software. 2.6. Statistical analysis All experiments were carried out in triplicates, and data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, USA). Differences among samples were evaluated by using analysis of variance (ANOVA) and Duncan’s multiple comparison method. Significant difference was assumed at p < 0.05. 3. Results and discussion 3.1. Phenolic and crude polysaccharide contents in the two fractions of ARE In this study, ARE was fractionated by 80% ethanol. The yield of water fraction (WF) and ethanol fraction (EF) were 2.86% and 4.67% of the dry root powder (d.w., Table 1). EF contained a total phenolic content of (90.5 ± 1.69) mg GAE/g as obtained from the calibration curve, y = – 0.01 + 0.012 x (R2 = 0.999, y is the absorbance, and x is the concentration of gallic acid solution, μg/ml).
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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Table 1 Extraction yield, total phenolic content (TPC), crude polysaccharide (CPS) and the EC50 values of antioxidant activities of EF and WF. Sample
EF WF Trolox EDTA
Yield (%)
4.67 2.86
TPC (mg GAE/g)
90.5 ± 1.69 8.63 ± 2.56
CPS (mg DGE /g)
35.9 ± 3.33 127.8 ± 1.72
EC50 values (mg/ml)∗ DPPH assay
ABTS assay
Reducing power
Ferrous chelating ability
0.71 ± 0.01 0.78 ± 0.01 0.16 ± 0.02
0.24 ± 0.01 0.13 ± 0.02 0.06 ± 0.01
0.16 ± 0.01 0.11 ± 0.01 0.07 ± 0.01
>10 7.42 ± 0.84 1.05 ± 0.20
TPC was calculated as gallic acid equivalent (GAE) μg/mg of dry basis; CPS was calculated as D-glucose equivalent (DGE) μg/mg of dry basis; ∗ EC50 value (mg/ml) for free radicals obtained by interpolation from linear regression analysis was the effective concentration at which radicals were scavenged by 50%; for reducing power, the effective concentration at which the absorbance at 700 nm reached 0.5 by interpolation from linear regression analysis; for chelating ability, the effective concentration at which ferrous ions were chelated by 50%.
On the other hand, WF was determined to have a crude polysaccharide content of (127.8 ± 1.72) mg DGE/g of the dry powder according to the calibration curve, y = 0.015 + 0.014 x (R2 = 0.990, y is the absorbance, and x is the concentration of D-glucose solution, μg/ml). Data are expressed as mean ± SD from three independent experiments.
3.2. Evaluation of antioxidant activities As shown in Figs. 1A and B, both fractions exhibited a concentration-dependent scavenging effect on DPPH and ABTS radicals. EF displayed a slightly significantly stronger DPPH scavenging activity (EC50 = 0.71 mg/ml, Table 1) than WF (EC50 = 0.78 mg/ml, Table 1, p < 0.05). However, WF possessed a significantly lower
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Fig. 1. Antioxidant activities of WF and EF indicated by DPPH radical scavenging ability (A), ABTS radical scavenging ability (B), reducing power (C) and ferrous chelating ability (D). Results are expressed as mean ± SD from three independent experiments.
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EC50 value of 0.13 mg/ml than EF (EC50 = 0.24 mg/ml, Table 1, p < 0.05). A medium strong positive correlation (r2 = 0.521, p < 0.05) was found between the DPPH and ABTS tests. Regarding DPPH and ABTS assays, a similar antioxidant relationship between ethanol extract and water extract was also reported in previous works [16,19]. The antioxidant capacities of EF and WF were further explored by using the reducing power and ferrous chelating assays. As shown in Fig. 1C, both fractions showed a linear increase trend in reducing power with the increase in their concentration. The EC50 values of reducing power in WF, EF and Trolox followed a descending order of Trolox (0.07 mg/ml) > WF (0.11 mg/ml) > EF (0.16 mg/ml) (Table 1). As shown in Fig. 1D, WF was found to exhibit a relatively higher chelating ability than EF. The EC50 values were determined to be 1.05, 7.42 and > 10 mg/ml for EDTA, WF and EF, respectively, which to some extent agrees with the finding of Andjelkovic´ et al. [20], who claimed that the ability of phenolic compounds (the major component of EF) to chelate iron was far lower than that of EDTA. The correlation analysis revealed that the reducing power of EF had the strongest correlation with ABTS assay (r2 = 0.995, p < 0.05) and a medium strong correlation with DPPH assay (r2 = 0.548, p < 0.05). 3.3. Evaluation of anticancer activity of WF and EF in vitro Cytotoxic effects of EF and WF were examined by using MTT assay against human lung cancer cell lines A549 and H1299 for daily treatment of 24, 48 and 72 h, respectively. As shown in Figs. 2A and B, EF exhibited stronger cytotoxic effect than WF, with IC50 values of 68.01 ± 2.69, 55.53 ± 3.23, and 33.98 ± 1.58 μg/ml against A549; and 70.47 ± 1.22, 59.30 ± 3.22, and 35.56 ± 0.42 μg/ml against H1299 for 24 h, 48 h and 72 h, respectively. Cell morphological changes also indicated that the treatment of lung cancer cells at various doses of EF reflected its significant cytotoxicity to human lung cancer cells at doses of 25 μg/ml and above, and the cells treated with WF revealed less cytotoxic effect (Figs. 2C and D). We also performed the long-term inhibition effect of EF and WF on the colony formation abilities of A549 and H1299 cells. As shown in Figs. 3A and B, the untreated A549 cells produced 145.67 ± 5.51 colonies, whereas the colony numbers were significantly (p < 0.05) suppressed to 110 ± 4.36 and 25.67 ± 4.73 after being treated with 50 μg/ml of WF and EF, respectively. Meanwhile, treatment with 50 μg/ml of WF and EF reduced the colony number of H1299 cells from 84.33 ± 3.06 to 64 ± 7.94 and 40.33 ± 2.89, respectively. This observation also suggested that EF showed higher proliferation inhibition effect on cancer cells than WF. Results from this study suggests that EF, with a higher phenolic content, possesses stronger anticancer activity than WF, which is in agreement with the previous finding of Zou and Chang [21] who discovered that poly-phenolic extracts from various plants exhibited high efficiency in antitumor activity. Interestingly, our previous 24 h treatment tests revealed that ARE possessed cytotoxic effect against A549 with an IC50 value of 62.50 ± 6.99 μg/ml [15], which is slightly lower than either EF (68.01 ± 2.69 μg/ml) or WF (> 100 μg/ml). In order to examine their functional contributions to the anticancer activities of ARE, we investigated immunomodulatory activities of WF and EF in the followed-up experiments.
Table 2 Effect of water fraction (WF) on nitric oxide (NO) production and the phagocytic activity of macrophages RAW 264.7. Concentrations of WF (μg/ml)
Production of nitric oxide (μM)
0 3.125 6.25 12.5 25 50 100 LPS (1 μg/ml)a
0.56 7.00 9.68 10.00 13.00 14.89 11.56 15.00
± ± ± ± ± ± ± ±
0.69∗∗∗ 2.00∗∗∗ 2.68∗∗ 1.33∗∗ 1.15 1.02 0.38∗ 1.86
Phagocytosis (O.D.540 nm) 0.255 ± 0.020∗ 0.343 ± 0.005∗∗ 0.351 ± 0.014∗∗∗ 0.382 ± 0.024∗∗∗ 0.370 ± 0.023∗∗∗ 0.353 ± 0.017∗∗∗ 0.347 ± 0.015∗∗ 0.291 ± 0.011
Results are represented as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 indicate statistically significant difference compared with LPS group. a LPS was used as the positive control.
latory effect reached to maximum with a cell viability rate of 249.76 ± 31.42%, while WF had a proliferation effect on macrophage with a cell viability rate of 292.76 ± 31.42% at 12.5 μg/ml. However, the proliferation effect decreased as WF concentration further increased from 12.5 to 100 μg/ml, probably due to the “immunologic paralysis” [22]. On the other hand, EF showed a relatively mild proliferation effect with a maximum proliferation rate of 156.73% (p < 0.001) at the concentration of 100 μg/ml. As pointed out by Schepetkin and Quinn [23], compared with bacteria derived immunomodulatory polysaccharides and synthetic compounds, most higher plants derived polysaccharides are relatively nontoxic and do not have significant side effects. Indeed, a variety of plant polysaccharides have been found to have beneficial therapeutic effects by modulating monocyte/macrophage immune functions in previous studies [24,25]. In this study, the strong stimulatory effect of WF on macrophage proliferation is most probably attributable to the high content of polysaccharides in WF. Since WF exhibited considerable proliferation effect on macrophages, we anticipated that WF has potent immunomodulatory activity. Thus WF was further assessed by testing its stimulation effect on NO production in macrophages, and then its phagocytic ability and stress-protection effect.
3.4. Evaluation of immunomodulation activities on RAW 264.7 macrophages
3.4.2. Effect of WF on NO production A low amount of NO (0.56 μmol/L) was detected in the nonactivated macrophages as shown in Table 2, whereas incubation of the cells with increasing amount of WF (3.125–50 μg/ml) resulted in a significant stimulation effect on NO production in a dose-dependent manner. Treatment with 50 μg/ml of WF significantly stimulated NO production to 14.89 μmol/L. NO is produced in response to stimulation by a variety of agents to inhibit the growth of a wide variety of microorganisms, parasites and tumor cells [23], thus its production could be used as a quantitative index of macrophage activation [26]. In this work, WF was found to stimulate macrophages to produce NO, which is reminiscent of similar properties possessed by 1 μg/ml of LPS (15 μmol/L). Interestingly, it has been claimed that NO has dual effects in the immune system that a low concentration of NO may have macrophage-activating activity while an over-production of NO could induce an inflammatory response [27]. Results from this study revealed an increase of NO production in the supernatants of the WF treated macrophages cells, while this increased level is lower than the non-harmful one, 21.16 μmol/L as reported by Shi et al. [18].
3.4.1. Effect of EF and WF on the proliferation of macrophages As shown in Fig. 4, exposure to ARE stimulated macrophage proliferation. At a concentration of 12.5 μg/ml, the stimu-
3.4.3. Effect of WF on the phagocytic activity of macrophages WF treatment significantly increased (p < 0.05) the phagocytosis of macrophages when compared to the control cells (Table 2).
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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Fig. 2. Cytotoxic effect of EF (A) and WF (B) against human lung cancer lines A549 cells and H1299 cells. Image (C) and cell morphology (D) changes for 24 h treatment of EF and WF in A549 and H1299 cells. Results are expressed as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 statistically indicate the significant difference from control group.
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Fig. 3. Inhibition on colony formation of A549 and H1299 cells after EF (50 μg/ml) and WF (50 μg/ml) treatment for 4 h.
It is well known that macrophages play an important role in host defense system that phagocytizes the pathogens and tumor cells [28]. This work suggests that administration of WF may enhance the immune response of macrophage by stimulating its phagocytosis ability thus contribute to the anticancer activities of ARE in vivo.
Fig. 4. Effects of ARE, EF and WF on proliferation of macrophages RAW 264.7 cells. Results are expressed as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 statistically indicate the significant difference from the control group.
The resultant optical density value of 3.125 μg/ml for WF treatment (0.34) was significantly higher (p < 0.01) than that of the positive control (0.29) in the cells treated with 1 μg/ml of LPS. Moreover, the absorption value reached a maximum of 0.38 at the concentration of 12.5 μg/ml, which is partially in agreement with its proliferation effect observed in Fig. 4.
3.4.4. Protective effect of WF on DOX-induced toxicity and apoptosis in macrophages RAW 264.7 As shown in Fig. 5A, treatment with 0.5 μmol/L DOX decreased the survival rate of RAW 264.7 cells to 41.65%. While in the presence of WF, the viability of the RAW 264.7 cells was significantly increased, reaching to the maximum (143.19%, p < 0.001) at the concentration of 25 μg/ml. Considering the outstanding protective effect of WF on DOX-induced toxicity to macrophages, additional tests were further carried out at a higher level of DOX (1 μmol/L). As shown in Fig. 5B, results showed that only a survival rate of 18.42% was obtained in the control macrophages cells. However, when incubated with various concentrations (3.125–100 μg/ml) of WF, the macrophages viability increased with the increase in the concentration of WF tested. Incubation with 100 μg/ml of WF could elevate the survival rate of macrophages to a significantly higher extent of 63.63% (p < 0.001). DOX is a commonly used anticancer drug in chemotherapy, and it has been demonstrated to exert apoptosis effect by causing DNA damage [29]. In order to confirm the protection effect of WF in DOX-induced macrophages, a flow cytometric analysis was
Fig. 5. Protective effect of WF on 0.5 μmol/L (A) or 1 μmol/L (B) DOX-induced viability of macrophage RAW 264.7 cells. Results are expressed as mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 statistically indicate the significant difference from the negative control group (DOX).
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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Fig. 6. Protective effect of WF on apoptosis induction by 2 or 4 μmol/L DOX in macrophage RAW 264.7 cells observed by cell morphology changes (A) and cell populations analysis (B and C).
Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022
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carried out to evaluate apoptosis populations (Annexin V positive) in control, DOX treated, and DOX+WF treated cells, respectively. As shown in Fig. 6, the apoptotic population of macrophage RAW 264.7 cells increased from 11.4% (control) to 63.8% and 90.8% after being treated with 2 and 4 μM DOX, respectively. However, WF significantly reduced the apoptosis rates. For example, incubation with 6.25, 25 and 100 μg/ml of WF mitigated the apoptosis rate to 72.40%, 59.9% and 57.5% in 4 μM DOX treated macrophages. This observation implies that WF may contribute to the mitigation of DOX-induced toxicity and apoptosis due to its high proliferation effect on macrophages and strong antioxidant activities. 4. Conclusion This work was the first report on the strong immunomodulatory effect of H. angustifolia root, and disclosed the functional relationship among its antioxidant, immunomodulatory activity and anticancer activity in ARE fractions. Findings from this study suggest that EF is the main cytotoxic factor of ARE, and the remarkable immunomodulatory activities of WF also contribute to the anticancer activities of ARE to a great extent, hence provides important scientific evidence for developing ARE as potent anticancer reagents. References [1] Ehrke MJ. Immunomodulation in cancer therapeutics. Int Immunopharmacol 2003;3:1105–19. [2] Liu W, Wang H, Pang X, Yao W, Gao X. Characterization and antioxidant activity of two low-molecular-weight polysaccharides purified from the fruiting bodies of Ganoderma lucidum. Int J Biol Macromol 2010;46:451–7. [3] Aziz MH, Kumar R, Ahmad N. Cancer chemoprevention by resveratrol: In vitro and in vivo studies and the underlying mechanisms (Review). Int J Oncol 2003;23:17–28. [4] N-Y Chiu, K-H Chang. The illustrated medicinal plants of Taiwan. Taipei: Southern Materials Center Inc; 1995. (in Chinese) p. 104. [5] Wang M, Liu W. A naphthoquinone from Helicteres angustifolia. Phytochemistry 1987;26:578–9. [6] Chen C-M, Chen Z-T, Hong Y-L. A mansonone from Helicteres angustifolia. Phytochemistry 1990;29:980–2. [7] Chen Z-T, Lee S-W, Chen C-M. New flavoid glycosides of Helicteres angustifolia. Heterocycles 1994;38:1399–406. [8] Chin Y-W, Jones WP, Rachman I, Riswan S, Kardono LBS, Chai H-B, et al. Cytotoxic lignans from the stems of Helicteres hirsuta collected in Indonesia. Phytother Res 2006;20:62–5. [9] Pan M-H, Chen C-M, Lee S-W, Chen Z-T. Cytotoxic triterpenoids from the root bark of Helicteres angustifolia. Chem Biodivers 2008;5:565–74. [10] Chen W, Tang W, Lou L, Zhao W. Pregnane, coumarin and lupane derivatives and cytotoxic constituents from Helicteres angustifolia. Phytochemistry 2006;67:1041–7. [11] Wang G-C, Li T, Wei Y-R, Zhang Y-B, Li Y-L, Sze SCW, et al. Two pregnane derivatives and a quinolone alkaloid from Helicteres angustifolia. Fitoterapia 2012;83:1643–7.
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Please cite this article as: K. Li et al., In vitro antioxidant, immunomodulatory and anticancer activities of two fractions of aqueous extract from Helicteres angustifolia L. root, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2015.12.022