Physicochemical and Antioxidant Properties of the Degradations of Polysaccharides from Dendrobium officinale and Their Suitable Molecular Weight Range on Inducing HeLa Cell Apoptosis

Different molecular weight polysaccharides of Dendrobium officinale (DOPs) have gradually attracted attention because of their broad biological activities. They, however, remain poorly defined whether their antitumor activity is associated with molecular weight. In this study, the physicochemical, antioxidant, and antitumor properties of DOPs, including the crude polysaccharide (DOP) and its six degradation fractions (DOP1–DOP6) extracted from Dendrobium officinale, were determined. Consequently, DOPs were mainly composed of different ratios of mannose and glucose as follows: 5.15 : 1, 4.62 : 1, 4.19 : 1, 4.46 : 1, 4.32 : 1, 4.29 : 1, and 4.23 : 1, and their molecular weights were significantly different ranging from 652.29 kDa to 11.10 kDa. With the concentration increase of DOPs, the scavenging capacity against OH and DPPH free radicals increased. The antitumor ability of DOPs was different that DOP1–DOP5 (Mw: 176.29 kDa–28.48 kDa) exhibited the best antiproliferation activity than DOP (Mw: 652.29 kDa) and DOP6 (Mw: 11.10 kDa) in HeLa cells rather than PC9, A549, and HepG2 cells. Moreover, it is worth mentioning that DOP1 and DOP5 showed stronger capability on inducing apoptosis of HeLa cells than DOP and DOP6 via the mitochondrial pathway by upregulating the ratio of the Bax/Bal-2 mRNA expression. The results demonstrated that DOPs can be used as the potential natural antioxidant and antitumor products in pharmaceutical industries, and the molecular weight is a crucial influential factor of their antitumor activity that 28.48 kDa–176.29 kDa is a suitable range we may refer to.


Introduction
Malignancy is one of the major causes of death, accounting for approximately 8 million deaths annually worldwide [1]. In addition, it presents the rising trend in morbidity, especially in developing countries [2]. Nevertheless, almost all chemotherapy drugs in the current market lead to serious side effects so that looking for a novel natural compound without side effects is crucial for the treatment of cancer [3].
Dendrobium officinale is one of the most valuable herbal plants and has been used for centuries as a herbal medicine in China and many other countries of South Asia [4]. In medicinal practice, it is used to nourish yin and enhance the production of body fluid, as well as to provide beneficial effects to the stomach and liver, moisten the lung, modulate the body's immune functions, and so on [5]. Polysaccharide is one of the main active ingredients of Dendrobium officinale. Several lines of evidence demonstrated that Dendrobium officinale polysaccharides (DOPs) exhibit antiinflammatory activities, regulate immunity and antioxidant and antitumor activities, etc. [6,7]. Gradually, polysaccharides in herbal medicines, including DOPs, have attracted much attention because of their antitumor activities with no significant side effects [8][9][10]. Furthermore, many studies have shown that the molecular weight of polysaccharides plays an important role in their biological activities [11][12][13][14]. According to reports in the literature, the lower-molecular-weight (Mw: 6.53 kDa) polysaccharide fragment from Porphyridium cruentum showed stronger immunoenhancing and antitumor activities [15]. Zhao et al. degraded polysaccharides from Porphyra haitanensis using the H 2 O 2 system to obtain fragments of different molecular weights and proved that the antioxidant activity of them was closely related to the molecular weight [16]. However, in terms of DOPs, some studies applied polysaccharides by fractional precipitation or the total polysaccharides to investigate the antitumor activity [17,18], and fewer reports have published concerning polysaccharides that degraded into a series of molecular weights but lacking comparison of molecular weights [18].
In this paper, we extracted and compared the antioxidant and antitumor activities in vitro of seven polysaccharide fractions from Dendrobium officinale with different molecular weights, providing the natural novel antioxidant and antitumor compounds, as well as a molecular weight reference for polysaccharides on antitumor activity in other medicinal materials.

Plant Materials.
e artificially cultivated stems of Dendrobium officinale Kimura et Migo were collected from Yunnan Province in China and identified by Prof. Gang Wei, Guangzhou University of Chinese Medicine, Guangdong, China. ese fresh samples were cut into segments (0.5 cm) and ground into powder after drying in an oven at 60°C.

Extraction and Degradation of DOPs with Different Molecular Weights.
e crude polysaccharide of Dendrobium officinale (DOP) was extracted as described previously [19]. In brief, 50 g of Dendrobium officinale powder was refluxed with 500 mL of petroleum ether at 70°C for 2 h to remove lipids. After the removal of the supernatant by filtration, the residue was dried at room temperature and extracted with 500 mL of 80% ethanol at 90°C for 2 h to discard small molecule compounds. e filtration residue was collected and extracted with 1500 mL of distilled water three times at 100°C for 2 h. All the precipitates were combined and centrifuged at 4000 rpm for 10 min. e supernatant was then collected and concentrated. Four times the volume of ethanol was slowly added and lyophilized at 4°C overnight to obtain the polysaccharide. e protein in the polysaccharides was removed using the Sevage method [20] three to five times until no white floccules were evident at the border between the water and chloroform to obtain DOP. e degradation of DOP with different molecular weights was evaluated by the method our group used for the long term [18] but with slight differences. 3.6 g of DOP was dissolved in 314.7 mL of distilled water in an ultrasonic bath. After dissolving Vitamin C (Vc), iron chloride tetrahydrate (FeCl 2 ·4H 2 O), and 30% H 2 O 2 with distilled water, six 50 mL conical flasks of the reaction solution were prepared according to the method in Table 1. e mixture was shaken and reacted in the dark for 2 h at room temperature (25°C). e reaction product was transferred to a 300 Da dialysis bag and dialysed for 3 days against flowing distilled water. Followed by centrifugation at 6000 rpm for 10 min, the supernatant was concentrated and freeze-dried. Six different polysaccharide fragments (DOP1-DOP6) were obtained.  [21] taking the glucose as the standard sample. e concentration was measured using the standard curve based on the anhydrous glucose standard references.

Monosaccharide Composition Analysis.
e polysaccharide sample DOPs were dissolved with distilled water to the concentration of 1 mg/mL. And the pH of the samples was adjusted to 7.0 using NaOH solution after the 3 M HCl was added to the sample solution, while the concentration of mixed monosaccharide standards, galactose (Gal), mannose (Man), glucose (Glu), and rhamnose monohydrate (Rha), was adjusted to 0.45 mg/mL. e solutions of samples and mixed monosaccharide standards were converted to their derivatives with 0.5 M PMP. All the solutions were allowed to react at 70°C in an oven for 100 min and then cooled to ambient temperature (25°C) and added with 0.3 M HCl. Last, chloroform was added and mixed via vortexing, followed by centrifugation at 3000 rpm for 10 min to discard chloroform.
is procedure was repeated 3 times until no colour was evident in the chloroform layer. e lower water-phase layers were the sample solution and mixed monosaccharide standard solution.

Detection of the Molecular Weight.
e concentration of DOP samples was adjusted to 2 mg/mL with distilled water to obtain the sample solutions. T-series dextrans (Mw: 1270, 5220, 11600, 48600, 80900, 273000, and 409800 Da) were used as standard references. High-performance gel permeation chromatography (HPGPC) was applied in the experiment. After the standard curve was drawn, the molecular weight and purity of the DOPs were determined by an Agilent 1100 series HPLC system (Palo Alto, CA, USA) equipped with an RI-101SHDEX RID detector (Tosoh, Tokyo, Japan). e mobile phase was 20 mM CH 3 COONH 4 with a flow rate of 0.8 mL/min.

Analysis of Antioxidant Activities
e free-radical-scavenging capacity of DOPs was analysed using DPPH according to a previously reported method with slight modifications [22]. Generally, DPPH was dissolved with methanol in an ultrasonic bath, followed by shaking vigorously and storage away from light. 150 μL of DOP solutions at different concentrations (0.5, 1.0, 2.0, 3.0, and 5.0 mg/mL) was fixed by 50 μL of DPPH solution in the 96-well plates and kept at room temperature for 30 min under lightproof condition. ereafter, the reaction was stopped by cooling at room temperature, and the absorption at 510 nm was determined by an iMark microplate absorbance reader (Bio-Rad Inc., CA, USA), while the solution of butylated hydroxytoluene (BHT) was used as a positive control. e test was performed in triplicate, and the DPPH radical-scavenging rate was calculated as follows: where A 1 refers to the absorption of DOP samples with DPPH solution, A 2 refers to the absorption of DOP samples without DPPH solution, and A 0 refers to the absorption of control (DPPH solution without DOP samples).

Hydroxyl (OH) Radical-Scavenging Assay.
OH radical-scavenging activity was measured according to the method described but with slight innovation [23]. In brief, 2 mL of DOP solutions at different concentrations (0.5, 1.0, 2.0, 3.0, and 5.0 mg/mL) was transferred to a test tube, and 2 mL of 6 mM·FeSO 4 solution and 2.4 mM·H 2 O 2 solution was added to the test tube for 10 min. ereafter, 2 mL of 6 mM salicylate was added and incubated at 37°C for 30 min. e mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was applied to measure the absorption at 510 nm using an iMark microplate absorbance reader. e solution of Vc was used as a positive control, and the hydroxyl radical-scavenging rate was expressed as follows: where A i refers to the absorption of the DOP samples with salicylate, A j refers to the background absorption of the DOP samples using distilled water to replace salicylate, and A 0 refers to the blank absorption using distilled water to replace DOP samples.
2.6. Antitumor Activity Analysis 2.6.1. Growth Inhibition Activity. e growth inhibition activity of DOPs in PC9 cells, A549 cells, HepG2 cells, and HeLa cells was measured using the MTT-based colorimetric method. e cells in the logarithmic phase were seeded at a density of 3 × 10 4 cells/well in 96-well plates and incubated at 37°C in 5% CO 2 . 24 h later, the medium was removed, and 100 μL of DOP solutions with concentrations of 25, 50, 100, 200, and 400 μg/mL in the fresh FBS-free medium was added to HepG2 cells and HeLa cells. While evaluated by the preexercise, there existed little difference about the concentration of DOPs in PC9 cells and A549 cells that 50, 100, 200, 400, and 800 μg/mL were added to incubate cells. e FBS-free medium was used as the blank control, and five biological repeats were performed in each experiment. After incubation for 24 h, 20 μL of the MTT reagent (5.0 mg/mL) was added to further incubate for 4 h for producing the purple formazan crystals. en, the medium was removed carefully, and 100 μL of DMSO was added to each well. e absorbance at a wavelength of 490 nm was measured using an iMark microplate absorbance reader. And the cell viability rate was calculated according to the following formula: where A 1 and A 0 represent the absorbance of the DOP sample and the blank control, respectively.

Flow Cytometric Analysis of Cell Apoptosis.
HeLa cells were seeded in 6-well plates (2 × 10 4 cells/well) and incubated at 37°C in 5% CO 2 for 24 h. e medium was removed, and DOP, DOP1, DOP5, and DOP6 were added at a concentration of 100 μg/mL for 24 h. According to the manufacturer's instructions, the HeLa cells digested with trypsin (non-EDTA) were collected into a centrifuge tube and centrifuged at 1000 rpm for 5 min. e supernatant was removed, and the cells were washed with 800 μL of PBS 3 times. 500 μL of the binding buffer was added to resuspend the cells, followed by staining with 5 μL of Annexin V-FITC and PI. After reaction of the solution for 10 min at room temperature in the dark, the samples were immediately analysed using a FACSCanto II flow cytometer (BD Biosciences, USA).

Real-Time Quantitative PCR (RT-qPCR) Assay.
HeLa cells were seeded in 6-well plates (1 × 10 5 cells/well) and incubated in a CO 2 incubator. 24 h later, the cells were exposed to DOP, DOP1, DOP5, and DOP6 with a concentration of 100 μg/mL for another 24 h. According to the manufacturer's protocol, the total RNA was extracted using e target gene expression levels were calculated using the 2 − ΔΔCt method.

Data Analysis.
All statistical data were expressed as mean ± SD, and the results from each group were obtained using triplicate samples independently using the Prism 5 software package (GraphPad Software Inc., La Jolla, CA, USA). Statistical differences among the groups were assessed using a one-way ANOVA, and Fisher's least significant difference (LSD) test was used to determine significant differences for different samples. * P < 0.05 was considered to be statistically significant, while * * P < 0.01 was considered to be highly statistically significant.
e HPLC maps of DOPs are shown in Figure 1, and the purity of the DOPs were greater than 98% as determined by HPLC. e monosaccharide composition of DOPs could be determined after comparing the HPLC results of DOP samples with those of monosaccharide standards. As shown in Figure 2, all DOP samples mainly consisted of mannose and glucose. In addition, the content of mannose was higher than that of glucose in all DOP samples. e mannose-toglucose ratio of DOPs can be obtained by calculating the ratio of the peak areas as follows: 5.15 : 1, 4.62 : 1, 4.19 : 1, 4.46 : 1, 4.32 : 1, 4.29 : 1, and 4.23 : 1, respectively. As shown in Table 3, the relative content of mannose in DOPs was decreased after degradation.

Antioxidant Activity of DOPs In Vitro.
Polysaccharides extracted from Dendrobium Sw. and some other plants exhibited significant antioxidant activity in vitro, including scavenging abilities on DPPH, OH, ABTS + , and Fe 2+ radicals [25,26]. DPPH is a free-radical compound that has been widely used to evaluate the free-radical-scavenging ability of natural materials. In this study, the antioxidant activities of DOPs were compared. e scavenging ability of DOPs on DPPH radicals is shown in Figure 3(a), compared with BHT as a positive standard. At the concentrations of 0.5-5.0 mg/mL, the DPPH radical-scavenging rates of DOPs and BHT were in the range of 48.8%-68.1%, 18.5%-48.6%, 24.0%-43.8%, 22.7%-50.8%, 14.3%-36.5%, 8.6%-27.9%, 18.5%-52.9%, and 91.9%-92.2%, respectively. Furthermore, when comparing with DOP1-DOP6 with lower molecular weights, DOP with a higher molecular weight of 652.29 kDa showed the best DPPH radicalscavenging ability gradually close to that of BHT. e results indicated that all DOPs exerted effective DPPH radical-scavenging ability in a dose-dependent manner and be influenced by molecular weight.
OH radical is regarded to be the most harmful reactive oxygen species and cause severe damage to adjacent biomolecules, resulting in cancer in vivo. e scavenging ability of DOPs against OH radicals is shown in Figure 3(b). In the concentration range of 0.5-5.0 mg/mL, the scavenging rate of DOPs and Vc, used as the positive standard, was 29.7%-57.8%, 28.3%-52.1%, 29.8%-53.1%, 31.5%-50.8%, 14.2%-54.4%, 13.7%-47.0%, 13.5%-47.0%, and 99.0%-100.3%, respectively. Similar to the DPPH radical-scavenging assay, the antioxidant activity of DOP with a molecular weight of 652.29 kDa against OH radicals was obviously higher than that of DOP4-DOP6 with smaller molecular weights in a dose-dependent manner, and the results were the same when comparing DOP1-DOP3 to DOP4-DOP6. e results indicated that the OH radical-scavenging abilities of DOPs have a certain relationship with their molecular weights, and DOPs may be beneficial to protect from oxidative damage.
Above all, DOPs exhibited noticeable antioxidant activity against DPPH and OH radicals in a dose-dependent manner, and molecular weight was one of the influence factors.
us, HeLa cells and DOP, DOP1, DOP5, and DOP6 were selected to be performed in the subsequent experiments for exploring the relationship between different molecular weights of DOPs and their antitumor activity.

Cell Apoptosis Analysis of DOP, DOP1, DOP5, and DOP6 in HeLa Cells.
e antitumor activities of DOP, DOP1, DOP5, and DOP6, measured by the spontaneous apoptosis rate of HeLa cells, were tested using the Annexin V and PI double-staining assay with flow cytometry. As depicted in Figure 5(a), the apoptosis rate of the blank group was 6.6%. After incubation with DOP, DOP1, DOP5, and DOP6 (100 μg/mL) for 24 h, the apoptosis rates were increased to 13.2%, 17.8%, 21.6%, and 14.8% (Figures 5(b)-5(e)), respectively. e values enabled the conclusion that DOP, DOP1, DOP5, and DOP6 could significantly induce cell apoptosis (P < 0.01). Meanwhile, the apoptosis rates of DOP1 and DOP5 were obviously higher than those of DOP and DOP6, suggesting that the apoptosis ability of DOPs might be influenced by the molecular weight similar to the results of the MTT assay, and 28.48 kDa-176.29 kDa is a suitable molecular weight range of DOPs on inducing apoptosis capability in HeLa cells.

Expression of Bcl-2 and Bax Treated with DOP, DOP1, DOP5, and DOP6 in HeLa Cells.
To further explore the relationship between DOPs with different molecular weights and antitumor activity, the RT-qPCR assay was applied to assess the expression level changes of the antiapoptotic gene Bcl-2 and proapoptotic gene Bax after induction by DOP, DOP1, DOP5, and DOP6 (100 μg/mL) for 24 h. Compared with the blank group (Figure 6(a)), the expression level of Bcl-2 and Bax mRNA in HeLa cells was downregulated and upregulated, respectively. e ratio of Bax/Bcl-2 is an index to indicate apoptosis regulation in cells, and the effect of inducing apoptosis would be enhanced as this ratio increased. In this study, the ratio of Bax/Bcl-2 in HeLa cells (Figure 6(b)) was increased after the induction of DOP, DOP1, DOP5, and DOP6. In addition, after mathematical analysis, this ratio was related to the molecular weight of the polysaccharide fractions. When we compared DOP1 and

Discussion
Previous studies have proven that Dendrobium officinale has broad bioactivities, including immunomodulation, antifatigue, antitumor, antioxidation, digestion promotion, stimulation of salivary secretion, hypoglycemic, and antihypertension [27]. However, as one of the major active components in Dendrobium officinale, few reports have described the antitumor effect of DOPs, particularly the comparison of different molecular weights of polysaccharides. In addition, accumulating evidence suggested that the bioactivity of the polysaccharide is related to its molecular weight, viscosity, solubility, and primary and fine structures [28,29]. Hence, we obtained seven polysaccharide fractions with significantly different molecular weights ranging from 11.10 kDa to 652. 29  Molecular weight is one of the important indexes for the quality control of polysaccharide products and medicines. It will be difficult for polysaccharide with a very high molecular weight to be transferred to the membrane in vivo, consequently preventing the polysaccharide from exerting its bioactivity. In our study, DOP (Mw: 652.29 kDa) showed no significant antitumor activities. It is reasonable to consider that this result may be because of high molecular weight. By contrast, polysaccharides with a lower molecular weight will be easy to bind on the bioactive binding sites. However, it will be inappropriate if the molecular weight is too low because it would be impossible to form the aggregate structure for an effect, which we can refer to DOP6 (Mw: 11.10 kDa).
us, an appropriate molecular weight is a critical factor for the biological activity of the polysaccharide. For example, a polysaccharide DOPA-1 from Dendrobium officinale in Huoshan has an average molecular weight of 229 kDa close to the upper limit of our suitable molecular weight range (176.29 kDa), resulting in induction of apoptosis in tumor cells through altered mitochondrial function, ROS production, and altered apoptosis-related protein expression [30]. Similarly, the polysaccharide DCPP1a-1 isolated from the protocorms of Dendrobium officinale with a molecular weight of 189 kDa showed significant antitumor effects on H22-bearing mice and significantly raised the thymus and spleen indexes [31]. Moreover, we speculated that the molecular weight of DOPA-1 and DCPP1a-1 may decrease closer to the lower limit of our suitable molecular weight range (28.48 kDa) after degradation by the gastrointestinal in vivo. e polysaccharide DOP-70 (Mw: 50.3 kDa, within our suitable molecular weight range) from Dendrobium officinale, obtained by our group before [17], also exhibited a better anti-HepG2 cell activity in vitro. In addition, through preexperiments, we have investigated that the antitumor activity of DOPs isolated by our group before (Mw: 776 kDa) [32] and monosaccharide (Mw < 10 kDa), whose molecular weight was all outside our suitable molecular weight range, was not significant. us, we consider that the antitumor activity of DOPs is related to their molecular weights, and 28.48 kDa-176.29 kDa is a suitable molecular weight range. However, the structure analysis of DOPs has not yet been done in this experiment, and whether there is a link between different polysaccharide structures and their antitumor activities needs to be further studied.

Conclusions
In summary, our results provide the promising evidence that molecular weight plays a critical role in the antitumor activity of DOPs isolated from Dendrobium officinale and initially find a suitable weight range for the first time.
Notably, DOPs displayed a good antioxidant activity against DPPH and OH free radicals, as well as the antitumor activity on growth inhibition in several tumor cells. However, DOP1 and DOP5 had the better significant capability of inducting apoptosis and upregulating the ratio of Bax/Bcl-2 expression in HeLa cells owning to different molecular weights. To conclude, polysaccharides from Dendrobium officinale, with a molecular weight range of 28.48 kDa-176.29 kDa, could be better used as the natural novel antioxidant and antitumor compounds, providing the antitumor activity of polysaccharides from other medicinal materials as a reference of molecular weight.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All authors of this study declare no conflicts of interest.