1. Introduction
Polygonatum species are perennial herbs from the Asparagaceae family that are widely distributed in China and have been used as medicine and food sources for more than 2000 years.
Polygonatum cyrtonema Hua, a species from this genus that was introduced in the
Chinese Pharmacopoeia (2020 edition) [
1], is mainly distributed in the middle and lower reaches of the Yangtze River, including the Guizhou, Zhejiang, Hunan, Hubei, and Sichuan provinces [
2], where it is planted in the forest and does not occupy arable land [
3].
Polygonati rhizoma, commonly known as
Huangjing in China, is the dried rhizome from several
Polygonatum species, including
P. cyrtonema Hua (the State Pharmacopoeia Committee of China, 2020), and has been used as a substitute for food since ancient times [
4].
Mingyi Bielu documented that the long-term consumption of
Huangjing can strengthen the body, delay aging, and eradicate hunger [
5].
Baopuzi Neipian recorded that
Huangjing can replace grain to combat hunger during times of famine [
6]. Modern research has revealed that
Huangjing contains abundant active ingredients such as saponins, flavonoids, polysaccharides, and lectins [
7,
8]. However, polysaccharides are the only component specified in the pharmacopoeia [
1].
Huangjing polysaccharides lower blood sugar and blood lipid contents, show antitumor and anti-inflammatory activities, relieve fatigue and aging, improve immunity, and regulate the intestinal microbial community [
9,
10,
11,
12]. With the aging of the population and the shift in dietary needs from eating well to eating nutritiously and healthily,
Polygonatum has gained attention due to its numerous health advantages.
Currently, approximately 80% of
Huangjing produced is consumed as food in China, with an annual demand ranging from 3500 to 4000 tons [
13,
14]. Rhizomes are commonly steamed and sundried nine times to make preserves, and there are multiple
Huangjing health items on the market, including wine, tea, yogurt, biscuits, cream, and tablets [
9]. Due to its active ingredients, health products to resist aging, decrease fatigue, enhance immunity, regulate blood sugar, and promote sleep have been developed [
14].
The nutrient composition of
Huangjing includes 2.23–39.54% polysaccharides [
15,
16,
17], 3.82–11.81% protein [
18,
19], 0.13–4.03% fat [
19,
20], and 1.58–9.80% ash [
19,
21]. The carbohydrate (17.5–68.01%) and vitamin C (0.5–170 mg/100 g) contents of the rhizome vary significantly between species [
18,
21]. In addition,
Huangjing is a rich source of K, Ca, Mg, Fe, and amino acids [
22]. The available literature on the nutrients contained in
Huangjing is limited and mainly focuses on polysaccharides. According to Si and Zhu (2021), polysaccharides, oligofructose, and fructose make up around half of the nutrients in the rhizome [
3]. However, there are few reports concerning monosaccharides and oligofructose. A previous study showed significant variation in the polysaccharide content of 25 wild
P. cyrtonema resources, ranging from 2.234% to 14.094% [
16]. Similarly, a high variation (9.56–17.68%) in the polysaccharide content was observed in 18 wild
P. cyrtonema located in the Hunan experimental forest [
23]. In addition, dietary fiber is an important nutrient that promotes satiation and satiety [
24], but its content and composition remain unclear.
While
Huangjing has been reported to bring considerable benefits to farmers, with yields of 1500 kg of dried product per hectare after four years of cultivation [
25] and a price after processing of USD 55.6/kg, roughly five times the cost of the raw materials (USD 10.43/kg) [
3], people have begun to artificially plant
P. cyrtonema to increase income. Jiao et al. [
26] reported all new
Polygonatum Mill. rhizomes produced by artificial planting had an increased polysaccharide content and met the
Chinese Pharmacopoeia standard limits after. However, few studies have carried out a comprehensive nutritional quality evaluation of different germplasm artificially cultivated
Polygonatum resources.
Thus, in order to explore the nutrient composition and provide a scientific basis for the further processing and utilization of Polygonatum, we carried out a comprehensive evaluation of the nutritional properties of different germplasms. Five P. cyrtonema germplasms introduced from Hubei, Hunan, and Anhui to Zhejiang were selected after three years of cultivation, and their nutrient compositions and nutritional quality were analyzed and evaluated. Then, a correlation analysis (CA) and principal component analysis (PCA) were employed to identify their nutritional differences and evaluate the nutritional quality of the Polygonatum germplasm.
2. Materials and Methods
2.1. Source of Materials and Preparation
Five wild
P. cyrtonema germplasms were collected from different regions and cultivated in Yuhang district, Hangzhou city, Zhejiang province, China. Three were collected from Huanggang (Hubei province), Qingyang (Anhui province), and Chizhou (Anhui province), respectively and planted in the germplasm resource garden (common garden) in Baizhang town. The other two were collected from different districts in Yueyang, Hunan province and planted on two different family farms. The geographic and climatic characteristics of these locations are shown in
Table S1.
Figure 1 shows a map of the provenance and planting location of the five
P. cyrtonema. All samples were authenticated by Prof. Zongsuo Liang (Zhejiang Sci-Tech University), assigned voucher specimen numbers, and deposited at the Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province. The voucher specimen number and specific information on the planting locations of the five
P. cyrtonema types are listed in
Table 1. All samples were harvested in November 2021 after three years of cultivation. The rhizomes were sliced, dried to a constant weight, and ground into a fine powder (100 mesh). The powder was packaged in sealed plastic bags and stored in dry glassware at room temperature until use.
2.2. Proximate and Carbohydrate Composition Analysis
The moisture content was determined using an oven-drying method (105 °C for 8 h), based on Chinese National Standards (CNS) GB 5009.3-2016 [
27]. A macro-Kjeldahl method (N × 6.25) was used to evaluate the protein content, in accordance with CNS GB 5009.5-2016 [
28]. The lipid content was measured using petroleum ether as the extractant in a Soxhlet apparatus (Hangzhou Mick Chemical Instrument Co., Ltd., Hangzhou, China) (CNS GB 5009.6-2016) [
29]. The ash content was determined by weighing the samples before and after heat treatment in a muffle furnace (Jinan PRECISION&SCIENTIFIC Instrument Co., Ltd., Jinan, China) (550 °C for 5 h), in accordance with CNS GB 5009.4-2016 [
30]. The polysaccharide content was determined by anthrone–sulfuric acid colorimetry as follows. First, 0.5 g of powder mixed with 10 mL of water was placed in a 75 °C water bath for 2.5 h; then, 40 mL of anhydrous ethanol was added to the supernatant and left overnight at 4 °C. Finally, the precipitate was dissolved in water for determination [
31]. The carbohydrate content was obtained by combining the polysaccharide content with the glucose, fructose, and sucrose contents. The starch content was quantified using an acid–hydrolyzed starch assay kit (BC0705, Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Each sample was prepared in triplicate.
2.3. Fiber Composition Analysis
The resistant starch (RS) content was determined using the enzyme digestion method, as described in the AOAC methods [
32]. The sample powder (0.1 g) and 4 mL of α-tryptic amylase suspension were added into a centrifuge tube, mixed thoroughly, and shaken at 37 °C for 16 h. Then, 4 mL of anhydrous ethanol and 8 mL of 50% ethanol were added to wash the precipitate, respectively. The precipitate was treated with 2 mL of 2 mol/L KOH, 8 mL of 1.2 mol/L sodium acetate buffer (pH 3.8), and 1 mL of amyl glucosidase (AMG) in a 50 °C water bath for 30 min, and the glucose content was determined using the 3,5-dinitrosalicylic acid (DNS) method. The resistant starch content of the sample was calculated by multiplying its glucose content by 0.9.
The pectin content was analyzed using the spectrophotometric method, in accordance with CNS NY/T 2016-2011 [
33]. The powder (0.1 g) was placed in a centrifuge tube and mixed with anhydrous ethanol in an 85 °C water bath for 10 min. The precipitate was then washed continuously with anhydrous ethanol in a water bath at 85 °C until the Molisch reaction of sugar was no longer present in the supernatant. The precipitate was then mixed with 10 mL of distilled water and 0.5 mL of 40 g/L NaOH and agitated for 15 min before being filtered. Then, 1 mL of the filtrate, 0.25 mL of carbazole ethanol solution, and 5 mL of concentrated sulfuric acid were mixed and placed in an 85 °C water bath for 20 min. After quick cooling, the absorbance was measured at 525 nm. Galacturonic acid was used as the standard substance and quantified by the standard curve.
The cellulose content was determined by the method described by Zhao et al. (2021) [
34]. The sample powder (0.05 g) was treated with a mixture of 5 mL of acetic acid and nitric acid and heated for 25 min; then, 10 mL of 10% sulfuric acid and 10 mL of 0.01 mol/L potassium dichromate were added, and the mixture was heated for 10 min. The precipitate from each treatment was washed with distilled water three times. The cellulose content was calculated by adding 5 mL of 20% potassium iodide and titrating with 1 mL of 0.5% starch as an indicator and 0.2 mol/L of sodium thiosulfate.
The hemicellulose content was determined, as described previously, as follows. First, 0.1 g of powder was boiled in 10 mL 80% calcium nitrate solution, the precipitate was rinsed three times with hot water, hydrolyzed with 2 mol/L of hydrochloric acid, and neutralized with sodium hydroxide solution, and the reducing sugar in the solution was determined with the DNS method [
34].
The lignin content was determined using sulfuric acid hydrolysis methods as follows: First, 0.05 g of powder was treated with 1% acetic acid. Then, a mixture of 70% ethanol and 30% ether (1:1 by volume) was added, and the precipitate was dissolved in 72% sulfuric acid for 16 h and then mixed with distilled water and barium chloride solution. Then, the precipitate was washed twice in distilled water, and the lignin concentration was titrated with 0.2 mol/L of sodium thiosulphate by adding 5 mL of 20% potassium iodide and titrating with 1 mL of 1% starch as an indicator [
34].
The national standard method CNS GB 5009.88-2014 was used to determine the total dietary fiber content [
35]. After desugarizing, drying, and sieving, the duplicate specimens were digested with heat-stable α-amylase, protease, and glucoamylase, precipitated with 95% ethanol, and filtered. The residue was washed with 78% ethanol, 95% ethanol, and acetone and dried, and the residue was weighed. The protein and ash contents of the residue were determined, and the dietary fiber content was calculated using the formula.
where
= mean weight of double sample residues,
= weight of protein in sample residue,
= weight of ash in sample residue,
= weight of empty,
= mean weight of double samples,
= weight of sample before desugarizing, and
= weight of sample after desugarizing.
2.4. Monosaccharide and Oligosaccharide Analysis
The monosaccharide and oligosaccharide concentrations were determined using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD), which were slightly modified from previous studies [
36,
37]. Each sample (0.05 g) was extracted using distilled water (1.8 mL) with ultrasonication (100 W) for 1 h. After that, the extracts were centrifuged (12,000 r/min for 10 min) to separate the supernatant from the residues. The extracts of small-molecule saccharides were passed through a 0.22 µm filter for HPLC-ELSD analysis. Standard solutions of D-glucose, D-fructose, sucrose, nystose, 1-kestose, and 1F-fructofuranosylnystose at appropriate concentrations were prepared for the construction of calibration curves by plotting the extracted chromatogram peak area versus the concentration.
Chromatographic analysis was carried out on a Waters e2695 liquid chromatography system coupled with a 2424 ELS detector (Waters Corporation, Milford, MA, USA). Acetonitrile (containing 0.02% triethylamine) and ultrapure water were used as mobile phases A and B, respectively. The solvent flow rate was 1.0 mL/min, the column (XBridge Amide 5 µm, 250 mm × 4.6 mm) was operated at 35 °C, and the injection volume was 15 µL. The solvent gradient was used as follows. For the first 10 min, the mobile phase composition transitioned from 85% A to 75% A; for 10–50 min, 75% A and 25% B were used as the mobile phases; and for 50–55 min, 75% A was gradually returned to 85% A. The ELSD conditions were as follows: the drift tube was kept at 50 °C, the drying gas (N2) had a flow rate of 40 psi, the nebulizer cooling mode was used, and the gain value was 1.
2.5. Mineral Analysis
The mineral content of each sample was determined by atomic absorption spectroscopy. Each sample (0.2 g) was mixed with nitric acid (10 mL) and hydrogen peroxide (2 mL) in a polytetrafluoroethylene sample cup. A digestion tank was assembled and placed in a microwave digestion instrument (Jinan Hanon Instrument Co., Ltd., Jinan, China) for digestion. Then, the digestion tanks were taken out for acid removal after cooling to 60 °C, and the digested sample was pipetted into a 50 mL volumetric flask and made up to the desired volume with distilled water. The sample solution was filtered through a 0.45 μm microporous membrane prior to analysis [
38].
Manganese, zinc, iron, sodium, potassium, calcium, magnesium, copper, cadmium, lead, and aluminum were determined using an Agilent 240 flame atomic absorption spectrometer with the 120 Graphite Tube Atomizer (Agilent Technologies Inc., Santa Clara, CA, USA) [
39]. A set of standard solutions for each element was prepared using the following concentrations: 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 ppm or 0, 2, 4, 6, 8, 16, and 32 ppb. The standard solutions were tested, and their values were recorded. The appropriate cathode lamp was fixed for each element. The atomizer of the instrument was dipped into the sample solution, and a meter reading was taken. The values obtained from the standards were used to plot the calibration curve for each test element, and the concentrations of the sample element were determined through extrapolation from the graph as ppm or ppb off the curve.
2.6. Vitamin Determination
The vitamin C concentration was determined spectrophotometrically with fast blue B salt (FBSB), as described by Zhou and Lou (2004) [
40]. First, 0.1 g of powder was mixed with 0.5 mL of 2 mol/L acetic acid, 0.2 mL of 0.25 mol/L LEDTA, and 4.3 mL of anhydrous ethanol and sonicated for 15 min (100 W). Then, 5 mL of water was added, and the mixture was sonicated for another 15 min. Next, 600 μL of the supernatant was mixed with 200 μL of water, 50 μL of 2 mol/L acetic acid, 100 μL of 0.25 mol/L LEDTA, and 50 μL of 0.6% FBSB. Finally, the absorbance was measured at 420 nm after centrifugation at 8500 r/min for 5 min. The carotenoid content was determined using the following method. First, 0.2 g of powder was mixed with 5 mL of ethyl acetate in a test tube, the mixture was placed in a 50 °C water bath for 50 min, and then the absorbance value of the supernatant was measured at 450 nm [
41]. The thiamine content was obtained with the fluorescence spectrophotometric method described in CNS GB 5009.84-2016 [
42]. The riboflavin content was detected using a vitamin B2 test kit (fluorescence spectrophotometry), which was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China).
2.7. Amino Acid Analysis
The free amino acid composition was examined by the Tea Quality Inspection and Supervision Centre, Ministry of Agriculture and Rural Affairs, P.R.C., using an automated amino acid analyzer (Sykam S433D, Sykam GmbH, Munich, Germany). First, fine powder (0.2 g) was extracted in a 90 °C water bath for 30 min with 20 mL of boiling water. The supernatant was collected and filtered through 0.22 μm hydrophilic nylon membrane filters, and the volume was made up to 20 mL with distilled water. Afterward, the solution was mixed 1:1 (v:v) with the sample diluent and stored at 4 °C for testing.
2.8. Statistical Analysis
Data represent the mean of three replication analyses, and the results are reported as the mean ± standard deviation. Statistical analysis was performed using IBM SPSS 25.0 Statistics (SPSS Inc., Chicago, IL, USA). A one-way analysis of variance (ANOVA) was used to determine whether there were any statistically significant differences (p < 0.05) between groups. A correlation analysis (CA) and principal components analysis (PCA) were performed using Origin 2021 (OriginLab, Northampton, MA, USA).
4. Conclusions
In this study, the nutritional compositions of five P. cyrtonema germplasms were systematically evaluated and analyzed. There were significant differences in the proximate, carbohydrate, and dietary fiber contents as well as the mineral, vitamin, and amino acid compositions among the different germplasms. Our study demonstrates that the five artificially cultivated P. cyrtonema are excellent sources of protein, carbohydrates, dietary fiber, vitamin C, and minerals such as Ca, Fe, and Mn in comparison with ginger, yam, and potatoes. A correlation analysis revealed significant correlations of the carbohydrate components and dietary fiber fractions with other nutrients; for example, there was a positive correlation between polysaccharides and Zn and between pectin and Ca. Moreover, the PCA analysis showed that the five germplasms were distinguished successfully in terms of their nutritional characteristics, with Huanggang possessing the highest nutritional quality. Finally, ten characteristic indicators were selected from the comprehensive nutrient analysis of the five P. cyrtonema germplasms. In conclusion, this study demonstrates that P. cyrtonema is a nutritious food, and the obtained nutritional indicators can provide valuable guidance for the identification and evaluation of Polygonatum.