Introduction
Polygonatum odoratum, also called yuzhu, waisheng, weirui, or lingdangcai in Chinese, originated in the Southwest region of China and is well distributed in the wild (Lan et al., 2011). Its rhizomes are widely used as an ingredient/supplement (e.g., in functional foods, flavorings, and teas). It is well known in traditional Chinese medicine (TCM) for removing dryness, promoting the secretion of fluid, and quenching thirst (Deng et al., 2012; Yang et al., 2010).
Previous phytochemical studies have shown that P. odoratum contains a variety of chemical constituents, such as steroidal glycosides, dipeptides, flavonoids, and polysaccharides. P. odoratum polysaccharides (POPs) are of primarily functional composition, with formidable anti-tumor, antioxidant, anti-inflammatory, and immunity improvement effects (Bai et al., 2013; Wang et al., 2009; Guo et al., 2013; Qian et al., 2010; Wang et al., 2013). The microbial fermentation is a process of biotransformation, and has been applied for centuries in TCM. More than a thousand years ago, the Chinese people began to use microbial fermentation to enhance efficacy and potency and reduce toxicity. For example, microorganisms such as Saccharomyces cerevisiae, lactic acid bacteria, and particularly medicinal fungi like Poria cocos, Ophiocordyceps sinensis, and Ganoderma lucidum are used to ferment and transform TCMs, producing new prescriptions with a variety of active ingredients (Li et al., 2004; Wu et al., 2013).
Polysaccharides are the main active ingredients in Polygonatum odoratum and have good antioxidant properties (Cui，2018). The main purpose of this article is to determine the best method for extracting antioxidant POP, in order to improve its antioxidant effect in cosmetics and better develop the efficacy of POPs.
Methods
1. Chemicals and reagents
P. odoratum was collected in Hunan province, China. S. cerevisiae (109 colony-forming units [CFU]/mL) was purchased from China General Microbiological Culture Collection Center. Ethylbenzothiazoline-6-sulphonic acid (ABTS; Sigma-Aldrich, USA), 1,1-diphenyl-2-picrylhydrazyl (DPPH; Tokyo Chemical Industry, Japan), potassium peroxydisulfate (Xilong Chemical Co., Ltd., China), TPTz (Adamas Reagent Co., Ltd., China), iron chloride hexahydrate (Shanghai Macklin Biochemical Co., Ltd., China), iron (II) sulfate heptahydrate (Xilong Chemical Co., Ltd., China), hydrochloric acid (Beijing Chemical Works, China), and L-ascorbic acid (J&K Scientific Co., Ltd., China) were all of analytical grade.
2. Extraction methodds
Water extraction: The medicinal material of Polygonatum odoratum is washed, dried in a drying oven (Rotary evaporator; Shanghai Ailang Instrument Co., LTD; China), and crushed by a crusher (Rocking crusher; Guangzhou Xulang machinery Equipment Co., LTD; China) to obtain the medicinal powder. Weigh 10 g powder and add 300mL deionized water at 1:30 (m/V), and extract it twice at 100℃ by hot reflux for 120 min each time. The extract was mixed and filtered through a 0.45 μm filter plate to obtain the polysaccharide extract of Polygonatum odoratum. The extract was concentrated by Rotary evaporator evaporation at 65℃.The flowchart is shown in Figure 1A.
Fermentation process: Polysaccharides were extracted from dried samples using the optimal parameters. The supernatant was collected in a 250 mL vacuum flask with S. cerevisiae medium. The clear liquid was autoclaved at 121℃ for 20 min for sterilization. When the liquid temperature decreased to 30℃, activated S. cerevisiae was inoculated into the liquid and fermentation was initiated in a shaker (Jia et al., 2006). The flowchart is shown in Figure 1B.
Fermentation will be carried out on the polysaccharide extract obtained after optimizing the water extraction conditions. The fermentation method will produce various secondary metabolites, which will significantly change the drug properties and improve its efficacy. By optimizing two extraction methods and comparing the antioxidant effects of polysaccharides obtained, a more suitable extraction method for POPs was determined.
3. Total polysaccharide content
The total polysaccharide content was determined using the improved phenol-sulfuric acid method (Liu et al., 2009). Measurements were acquired at 490 nm using a microplate reader (SpectraMax 190; Molecular Devices LLC). The total polysaccharide content was calculated as glucose/g using a calibration curve. Samples were analyzed in triplicate.
4. Single-factor tests for optimal water extraction
To obtain the appropriate conditions for maximum polysaccharide extraction, temperatures of 60, 70, 80, 90, and 100℃, liquid-solid ratios of 1:20, 1:30, 1:40, 1:50, and 1:60, and durations of 1.0, 1.5, 2.0, 2.5, and 3.0 h were independently tested. The total polysaccharide content was determined by the phenol-sulfuric acid method.
5. Orthogonal test for water extraction
According to the results of the single factor experiment, an L9 (34) orthogonal array with three factors at three levels was used to identify optimum conditions for the maximal extracted polysaccharide yield (Choi & Park, 2002; Wang et al., 2015). The used factors and the levels of orthogonal design are shown in Table 1.
6. Fermentation process
Polysaccharides were extracted from dried samples using the optimal parameters. The supernatant was collected in a 250 mL vacuum flask with S. cerevisiae medium. The clear liquid was autoclaved at 121℃ for 20 min for sterilization. When the liquid temperature decreased to 30℃, activated S. cerevisiae was inoculated into the liquid and fermentation was initiated in a shaker (Jia et al., 2006; Zhu et al., 2012).
7. Single-factor tests for optimal fermentation
To determine the appropriate conditions for maximum S. cerevisiae growth, inoculation volumes of 2, 4, 6, 8, and 10%; rotary speeds of 100, 120, 140, 160, and 180 r/min; temperatures of 26, 28, 30, 32, 34℃; and the effects of time on the yield of S. cerevisiae were independently tested. The optical density of S. cerevisiae was determined at 600 nm by microplate reader.
8. Orthogonal test for fermentation optimization
According to the results of the single factor experiments, an L9 (34) orthogonal array with four factors at three levels was used to determine the optimal conditions for maximal S. cerevisiae yield. The factors used and the levels of orthogonal design are shown in Table 2.
9. Molecular weight distribution assay
The molecular weights of water-and fermentation-extracted polysaccharides were determined using a Waters e2695 Alliance high performance gel permeation chromatography system equipped with a DAWN HELEOS-II Multi-Angle static Light Scattering detector (Wyatt Technologies) and a refractive index detector. All samples (3.0 mg) were dissolved in 0.1 M NaNO3, passed through a 0.45 μm filter, and applied to a Shodex SUGAR KS-805/KS-803 gel-filtration chromatographic column. The column was maintained at 60℃ and eluted with 0.1M NaNO3 at a flow rate of 0.8 mL/min.
10. Determination of DPPH radical scavenging activity
DPPH radical scavenging rates in the extracts were measured according to Brand et al. (1995). Samples (1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mg/mL) were added to 1.0 mL of 0.2 mM ethanolic DPPH and shaken. After 30 min, the decrease in absorbance was measured at 517 nm. Methanol was used as a blank, and ascorbic acid was used as a positive control. Assays were performed in triplicate.
11. Scavenging of ABTS radicals
The radical scavenging activities of the extracts against ABTS radical cations were measured using the method of Li et al. (2012) with some modifications. ABTS was dissolved in water to 7 mmol/L. ABTS radical cations were produced by reacting an ABTS stock solution with 2.45 mmol/L potassium persulfate and allowing the mixture to stand in the dark at 25℃ for 12-16 h before use. The ABTS radical cation solution was diluted in ethanol to an absorbance of 0.70±0.02 at 734 nm and equilibrated at 30℃. Samples (0.1 mL; final concentrations of 0.1, 0.5, 1.0, 2.0, 4.0, and 6.0 mg/mL) were mixed with 3.9 mL of diluted ABTS radical cation solution, and the absorbance at 734 nm was measured after reaction for 6 min.
12. Total antioxidant capacity assay
The total antioxidant capacity of the extracts was measured by the ferric reducing antioxidant power (FRAP) method (Wang et al., 2021; Nilsson et al., 2005). We added 180 μL FRAP working liquid into each well of a 96-well plate, then added 5.0 μL of FeSO4 standard solution at concentrations of 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM to the standard curve detection wells. Sample wells received 5.0 μL of the extracted samples in triplicate, and a 0.15 mM sample containing 1.5 mM Trolox was used as a positive control. The plate was gently shaken and incubated at 37℃ for 5-7 minutes, and then the decrease in absorbance was measured at 593 nm. Distilled water was used as a blank. The total antioxidant capacity of the sample was calculated according to the standard curve.
Results
1. Optimization of extraction conditions for water-soluble polysaccharides
Single-factor and orthogonal tests were used to determine the optimal extraction conditions for water-soluble polysaccharides. The effects of extraction temperature, ratio of raw material to water, and extraction time on the polysaccharide yield are shown in Figure 2. The optimal temperature was 90℃, and the optimal liquid-solid ratio was 1:50. Considering energy and efficiency, the optimal extraction time was 2.0 h.
An L9 (33) orthogonal array was used to optimize the extraction parameters, including nine experiments corresponding to nine rows and three columns. The results of all tests are shown in Table 3. The F-value was used to qualitatively identify effective factors. The liquid-solid ratio was of highest importance for effective polysaccharide extraction, followed by temperature and extraction time. The optimum extraction parameters were a 1:60 liquid-solid ratio, an 80℃ extraction temperature, and a 2.5 h extraction time. Under optimal conditions, the total polysaccharide contents were 83.49%.
2. Optimization of fermentation conditions
Single-factor and orthogonal tests were applied to determine the appropriate condition for maximum S. cerevisiae growth. The optimal fermentation time was 24 h. Effects of inoculation volume, rotary speed, and temperature on the fermentation process are shown in Figure 3, and the growth phase of S. cerevisiae is shown in Figure 3D. The optimal inoculation volume was 6%, and the optimal rotary speed was 160 r/min. The most suitable temperature for fermentation was 28℃.
An L9 (34) orthogonal array was used to further optimize the fermentation conditions. The results are shown in Table 4. The best fermentation conditions were an inoculation volume of 8%, a rotary speed of 180 r/min, a 28 h fermentation time, and a temperature of 28℃. The inoculation volume was of highest importance for effective fermentation, followed by rotary speed, fermentation time, and temperature.
3. Molecular weight distribution
The molecular weight distributions of water and fermentation-extracted polysaccharides were 2.067×104 and 9.475×103 Da, respectively, indicating that the molecular weight after fermentation extraction was lower and more homogeneous than that of water-extracted polysaccharides.
4. DPPH radical scavenging activity
The antioxidant capacities of water- and fermentation-extracted polysaccharides were examined by the DPPH method. Polysaccharides extracted by water and fermentation exhibited DPPH scavenging activity with concentrations showing 50% inhibition (IC50 values) of 4.9 and 1.0 mg/mL, respectively. Fermentation produced better DPPH radical scavenging activity than water extraction (Figure 4A).
5. ABTS radical scavenging activity
ABTS is converted to its radical cation (ABTS•+) by reacting with a strong oxidizing agent (e.g., potassium permanganate, potassium persulfate). This radical cation is blue and absorbs light at 734 nm, and can be converted back to its colorless neutral form by hydrogen-donating antioxidants. The ABTS•+ inhibitive efficiencies of water- and fermentation-extracted polysaccharides are presented in Figure 4B. Water-and fermentation-extracted polysaccharides showed ABTS radical scavenging activity, with IC50 values of 1.4 and 0.3 mg/mL, respectively. Fermentation produced better ABTS radical scavenging activity than water extraction.
6. Total antioxidant capacity
The total antioxidant capacities of water- and fermentation-extracted polysaccharides (0 to 10 mg/mL) were examined by FRAP. The standard curve provided the equation y=0.411x -0.0755 (R2=0.9946, x: concentration of FeSO4 (mM); y: absorbance). The total antioxidant capacity of each sample was expressed as the FeSO4 concentration. Fermentation produced better total antioxidant capacity than water extraction (Figure 4C, 4D).
Discussion
Maceration, heat reflux, ultrasound assistance, and acidic hydrolysis are common methods for extracting polysaccharides from plant tissues, but they require long extraction times, high extraction temperatures, or expensive equipment. In addition, they may pollute the environment (Qian, 2014). Fermentation is one of extraction method highly influenced by various parameters including the nature of the substrate, pH of the medium, nutrient availability, inducer supplementation, fermentation temperature, etc. (Singhania et al., 2010). Compared with traditional solvent extraction method, fermented small molecule has advantage in skin absorption, and the fermentation process is environmental friendly.
P. odoratum is rich in dietary fibers, whose functional properties are closely related to some of its therapeutic effects, such as reducing the risk of coronary heart disease, diabetes, obesity, and some forms of cancer, and lowering cholesterol and fat levels (Liu et al., 2015; Lan et al., 2012). Most dietary fibers are components of plant cell wall polysaccharides, which are resistant to digestion by the alimentary enzymes of humans (Bhat, 2000). S. cerevisiae is commonly used to ferment materials. Fermented foods or traditional medicines are the result of extensive microbial growth, during which microbial metabolism and biosynthesis pathways transform the starting materials to products with distinct organoleptic properties (Wolfe & Dutton, 2015).
In the present study, the antioxidant activities of POPs were obviously increased by fermentation. Molecular weight distribution analysis indicated that polysaccharide hydrolysis during fermentation increased antioxidant activities. Therefore, fermentation is a preferred technique to extract antioxidant POPs. Subsequent research can apply the fermented polysaccharides extracted from Polygonatum odoratum to cosmetics with anti-aging effects, and use human experiments or mouse skin model experiments to test their antioxidant effects, further demonstrating their efficacy in products.
Conclusion
In this study, we developed an effective fermentation-based extraction method for POPs using single-factor and orthogonal tests. The optimal fermentation method used an extraction inoculation volume of 8%, rotary speed of 180 r/min, fermentation time of 28 h, and fermentation temperature of 28℃. During fermentation, the decreased molecular weight of the POPs produced was consistent with increased antioxidant activity. Our results suggest the potential use of fermented POPs as a material in foods, medicines, or cosmetics.
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