Polysaccharide extraction from Myxopyrum smilacifolium trunk and its antioxidant capacity

. As a traditional medicinal plant in Vietnam, Myxopyrum smilacifolium has been used for a long history to treat cough, nervous disorders, numbness, rheumatism, and cephalalgia. Nevertheless, reports on the antioxidant activity and extraction of M. smilacifolium polysaccharides are still very rare. This study was designed to extract a high yield of polysaccharides from the M. smilacifolium trunk and characterize it . As a result, the maximum yield of the polysaccharides of 5.13 ± 0.05% was obtained with an extraction time of three hours, extraction temperature of 100 o C, the ratio of water to sample of 1:50 as extraction solution, extraction number of times 3, and the ratio of ethanol to extract volume 5:1 (v/v). Polysaccharides was characterized by Fourier transform infrared spectroscopy (FT-IR) and high - performance gel-permeation chromatography (HPGPC) . The average molecular weight of extracted polysaccharides was around 3.78 × 10 5 Da. In vitro assays dan impressive antioxidant activities of the extracted polysaccharides, containing 0.2423 ± 0.0028 mg GA/g or 0.2142 ± 0.0007 μmol AS/g. The IC 50 values of polysaccharides in the DPPH and ABTS methods were 0.89 mg/mL and 3.85 mg/mL, respectively. These findings exhibited the potential for application or further research and development of polysaccharides from Myxopyrum smilacifolium .


Introduction
Polysaccharides (PS) have been appealing great attention over the past decades [1,2].They play a wide variety of important roles in nature, and have been found to be extremely valuable sources of food and cosmetic ingredients and in nutraceutical and pharmacological applications [3][4][5].The polysaccharide extraction process makes their structural diversity and potential biological functions offer unique properties [6][7][8].
For this reason, the extraction of polysaccharides has been concerned as a hot-spot-research topic in the field of biomedicine.In addition, polysaccharides were biological macromolecules that exhibit a variety of physicochemical and pharmacological properties that are important processes for their application or further research and development.

Myxopyrum smilacifolium belongs to the
Myxopyrum genus of the Oleaceae family growing as part of native vegetation in Southeast Asian countries such as Vietnam, Laos, Cambodia, Indonesia Thailand, and India [9][10][11].The plant was used in traditional medicine to treat cough, rheumatism, nerve complaints, asthma, fever, neuropathy, and asthma treatments [11,12].
Studies confirmed that the extracts from M. smilacifolium exert antimicrobial, cytotoxic, antiinflammatory, and antioxidant activities.
Nevertheless, the reason for inducing the bioactivities of M. smilacifolium has been ambiguous.Given that polysaccharides could be the primary component of M. smilacifolium extracts, causing the antioxidant activity.In this context, the extraction and antioxidant activity of polysaccharides can be considered the imperative piece of information explaining their outstanding bioactivities [9][10][11][12].However, to our knowledge,

Qualitative and quantitative analysis of polysaccharides
Polysaccharides were examined by phenolsulphuric acid -colorimetric method using D-Glucose as standard at a wavelength of 490 nm [14].Data were calculated as percentages in the dry weight of the sample.

Structural characterizations of polysaccharide
The FT-IR spectra of polysaccharides were

Total antioxidant capacity (TAC)
The total antioxidant capacity of the samples was determined according to the method described by Nair et al [16].The total antioxidant capacity is displayed as the number of equivalents of gallic acid (GA) [17] or ascorbic acid [18] (the standard curve equation for gallic acid and ascorbic acid is Abs = 2.172 × CGA + 0.1056; R = 0.9995 and Abs = 4.209 × CAS -0.0463; R = 0.9993).

DPPH radical-scavenging activity
The DPPH free radical scavenging activity is used to perform the screening effects of antioxidation of the testing substances.Two mL of sample (in dimethyl sulfoxide (DMSO) with different concentrations (from 0.4 mg/mL to 2 mg/mL)) were added to 1 mL of 100 µM DPPH (in methanol) for 30 min at room temperature.The absorbance is measured by the UV-Vis method at 517 nm.The antioxidative activity was shown by color reduction.Ascorbic acid was used as a reference substance.DMSO was used as a control.
The radical scavenging activity was evaluated using the IC50 value [13].

ABTH•+ radical-scavenging activity
The scavenging capacity on the ABTS radical of polysaccharide was determined according to the method described by Roberta Re et al with several modifications [19].An amount of 0.1 mL sample with different concentrations (from 1 mg/mL to 5 mg/mL) were mixed with 3.9 mL of ABTS+• solution.The absorbance was then measured at 734 nm.Ascorbic acid was used as a positive control.The radical scavenging activity was evaluated using the IC50 value.

Statistical analysis
Unless otherwise stated, all the experiments were performed three times (n = 3 The extraction yields ranged from 3.26 ± 0.05% to 5.10 ± 0.03% with the increasing temperature from After step 1, the effect of the ratio of ethanol to extract volume was studied.Data in Table 1 revealed that the larger ratio of EtOH 96 °: extract volume, the higher percentage of pure polysaccharide.The highest yield of polysaccharides was 5.13 ± 0.05% obtained with a ratio of ethanol 96° to extract volume 5:1.glucopyranoside, whereas the peaks located at 1024 cm -1 could be indicated α-configurations and the presence of fructose residues, respectively [22].To this end, it can be said that the extracted polysaccharide from M. smilacifolium trunk possesses the typical absorption groups of polysaccharides, glucopyranoside, and fructose in the structure.The IC50 values of polysaccharides in the DPPH and ABTS methods are found to be 0.89 mg/mL and 3.85 mg/mL, respectively, which is found to be considerably higher than that of the reported polysaccharide, as depicted in Table 2 (without PS from Bletilla striata in ABTS assay).

In vitro
Overall, the results suggest that polysaccharide from M. smilacifolium trunk is a promising source of natural antioxidants and can be used as an additive in food, pharmaceutical, and cosmetic preparations.

Conclusion
Generally, an increased yield of crude , Japan).Samples were dried at 55 °C in a vacuum drying oven for 48 h prior to tableting with KBr powder.Spectra were scanned in the 4000-400 cm -1 region.The average molecular weight of polysaccharides was determined by gel permeation chromatography (GPC -Agilent 1100 Series coupled to MS detector, microTOF-QII Bruker, USA, an Ultrahydrogen 500 column (7.8 mm x 300 mm, 10 µm)) as described by Wang et al. with some modifications [15].The purified polysaccharides were dissolved in NaNO3 0.1M and injected in the system by maintaining the same flow rate (1 mL/min) and column temperature (40 °C).The molecular mass was estimated by the standard curve which was calibrated using the pullulans (from 50 kDa to 800 kDa).
60 0 C to 100 0 C. The highest yield of polysaccharide was 5.10 ± 0.03% obtained at 100 0 C.As the temperature increased, the diffusion of polysaccharides from the cells into the extraction solvent increased [20].As the solvent volume increased, the polysaccharide yield increased rapidly in the sample: solvent ratio range of 1:30 to 1:50 mg/mL.However, with a further increase in solvent volume, the extracted polysaccharide yield did not increase further.The sample: water ratio of 1:50 mg/mL was selected for polysaccharide extraction.The results indicated that extraction time was proportional to the yield of the PS when extraction time was between 2 and 3 h.The yield of the PS was 5.10 ± 0.03 % when the extraction time was 3 h.After this point, the yield of the polysaccharides started to decrease with increasing the extraction time.In our opinion that increasing the extraction time may enhance the yield of the polysaccharides.However, excessively lengthening extraction time will also induce a change in polysaccharides structure (maybe, the long extraction time induced the degradation of polysaccharides), as a result of which the extraction yield of polysaccharides instead decreased.The effect of extraction number of times on the yield of the polysaccharides was investigated.The results indicated that the extraction number of times was proportional to the yield of the polysaccharides when the extraction number of times was between 2 and 4. The yield of the polysaccharides was 5.11 ± 0.02% when the extraction number of times was 4.However, the yield increased slowly when the extraction number of times increased from 3 to 4 times.To avoid the wasting consumption of solvents and bulky handling in the subsequent processes, the extraction number of times 3 was chosen.
Figure 1 shows a chromatogram of polysaccharide obtained by gel permeation highperformance liquid chromatography.The polysaccharide from Myxopyrum smilacifolium trunk had a molecular weight of 3.78 × 10 5 Da.The ratio Mw/Mn, which represents the polydispersity
Antioxidant activity assay of polysaccharides One of the most commonly used methods to evaluate antioxidant activity in chemical and biological samples is the total antioxidant capacity (TAC) method.The TAC method is based on the redox potential of metal ions.In this study, we investigated the total antioxidant content of the sample based on the redox potential of Mo(VI)/Mo(V).TAC was expressed as the number of equivalents of gallic acid or ascorbic acid.The study revealed that the antioxidant capacity of polysaccharides was observed at the concentration of 1.5 mg/mL where the total antioxidant capacity of polysaccharides from M. smilacifolium trunk showed contained 0.2423 ± 0.0028 mg GA/g or 0.2142 ± 0.0007 μmol AS/g.The total antioxidant capacity of polysaccharides from M. smilacifolium trunk is higher than that of Ophiocordyceps sobolifera (from 0.1460 ± 0.0011 mg GA/g or 0.1222 ± 0.0003 μmol AS/g) [13].This result suggests that this plant is a potent antioxidant.On the other hand, free radical scavenging is a mechanism inhibiting lipid oxidation effectively, commonly used to estimate antioxidant activity.The ABTS radical scavenging activity and DPPH radical scavenging activity are splendid methods for determining the activity of substances according to their capacity to donate a hydrogen atom or electron, which is measured as the capacity of samples to degrade the colour by reacting directly with ABTS radical and DPPH radical.The electron-withdrawing group of polysaccharides and the specific structures activate the hydrogen atoms on sugar residues.The antioxidant activity of the polysaccharide from M. smilacifolium trunk was assessed by the pISSN 1859-1388 eISSN 2615-9678 DOI: 10.26459/hueunijns.v132i1D.701269 DPPH and ABTS methods, and the outcomes are displayed in Figure 3.

Figure 3
Figure3shows the free radical scavenging capacity of the polysaccharide increases with concentration.The DPPH radical scavenging activity at the concentration of 2.0 mg/mL of polysaccharides was over 75%, and the percentage of the ABTS radical scavenging activity rose remarkably and reached a peak at 5 mg/ mL concentration with over 60% inhibition, however, the activity of the polysaccharide was lower than that of ascorbic acid at the same concentration.

Fig. 3 .
Fig. 3.In vitro antioxidant activity of polysaccharide from Myxopyrum smilacifolium trunk polysaccharides was observed when the extraction time, extraction temperature, ratio of water to raw material, and extraction number were raised.The results from this study indicated the appropriate yield of polysaccharides from the Myxopyrum smilacifolium trunk.were as follows: an extraction time of 3 hrs, extraction temperature of 100 °C, the ratio of water to sample 1:50, extraction number times of 3, and the ratio of ethanol to extract volume 5:1.Under these conditions, a crude polysaccharide yield of around 5.13 ± 0.05% of dry weight.Fourier transform infrared spectroscopy indicated that polysaccharide from M. smilacifolium trunk possesses the typical absorption groups of polysaccharides, glucopyranoside, and fructose in the structure.The average molecular weight of extracted polysaccharides was around 3.78 × 10 5 Da.Impressively, the achieved polysaccharide from Myxopyrum smilacifolium trunk exhibits significant in vitro antioxidant activity.The total antioxidant capacity of polysaccharides from Myxopyrum smilacifolium trunk showed contained 0.2423 ± 0.0028 mg GA/g or 0.2142 ± 0.0007 μmol AS/g.The IC50 values of polysaccharides in the DPPH and ABTS methods were 0.89 mg/mL and 3.85 mg/mL, respectively.Thus, polysaccharides from Myxopyrum smilacifolium trunk may be developed as a potential natural antioxidant activity agent and functional food.

Table 1 .
Extraction yield of polysaccharides from Myxopyrum smilacifolium trunk under investigation conditions

The effect of extraction time on the yield of the polysaccharides
Other conditions were as follows: extraction temperature 100 0 C, extraction number times of 3, ratio of ethanol 96 0 to extract volume 4:1, and the ratio of sample: water volume 1:50.

Table 2 .
IC50 values obtained from DPPH and ABTS radical scavenging activity of reported