Comparative nutritional characteristics of the three major Chinese Dendrobium species with different growth years

Dendrobium, an important medicinal plant, is a source of widely used herbal medicine to nourish the stomach and treat throat inflammation. The present study is aimed at distinguishing and evaluating three major Dendrobium species by comparing physiochemical characteristics and understanding differences between different growth years in the Ta-pieh Mountains. Polysaccharides and total alkaloids of Dendrobium were determined, and the amino acids and trace elements were determined by UPLC (Ultra High-Performance Liquid Chromatography) and ICP-MS (Inductively coupled plasma mass spectrometry). It can be seen from the results that the polysaccharide content of these three kinds of Dendrobium in different growth years ranges from 249.31 mg·g-1 to 547.66 mg·g-1, and the highest content is in the 3-year-old Dendrobium huoshanense. The total alkaloid content ranges from 0.21 mg·g-1 to 0.54 mg·g-1, and the highest content is also the 3-year-old Dendrobium huoshanense. We determined the amino acid content of these three Dendrobium in different growth years, and we can see that each of the three kinds of Dendrobium contain seven kinds of amino acids required by the human body. We conducted a safety evaluation of the essential trace elements of Dendrobium, and the results showed that the dosage of 12g·d-1 Dendrobium prescribed in China Pharmacopoeia is in accordance with the recommended daily intake of trace elements recommended by the Food and Drug Administration of the United States, and will not cause trace element poisoning. Linear discriminant analysis was carried out on the basis of amino acids and trace elements and confirmed the applicability of multi-elemental analysis for identifying different Dendrobium species.


Introduction
Dendrobium is an important edible-medicinal plant belonging to orchid family, with more than 1400 species in the world. In China, there are 74 species and two varieties of Dendrobium  [1], more than 50 of which can be used as 'Shihu', a popular tonic and a traditional Chinese medicine [2]. Dendrobium is a perennial herb, which contains polysaccharides, alkaloids, essential amino acids and some trace mineral elements [3,4]. As a high-grade drug, it had been documented in Shennong's Herbal, as the stems of Dendrobium have been used to make a tonic or a functional medicine to nourish stomach, relieve throat inflammation, improve eyesight and promote body fluid production for thousands of years in Chinese medicine [5,6]. Furthermore, Chinese people use Dendrobium as supplements in their daily life. In recent years, the market demand has been constantly expanding.
Essential trace elements are very important nutrients for human body functions and growth [7]. A deficiency of trace elements could bring on structural and physiological abnormalities, while high levels of those essential trace elements may be harmful to human health [8,9]. There are many studies reporting on trace elements in medicinal herbs, such as Panax ginseng, Salvia miltiorrhiza, Houttuynia cordata and so on [10][11][12][13]. In Dendrobium species, there is one report about the determination of trace elements levels for identifying the cultivation technique of Dendrobium officinale [14]. In this study, we attempted to distinguish three different Dendrobium species using the determination of trace elements and amino acids.
Dendrobium huoshanense is named after Huoshan County of Anhui Province and is endemic to the Ta-pieh Mountains. Due to its special geographical location and climatic environment, the Ta-pieh Mountains have become the main area in which of medicinal plants of Dendrobium are distributed in Anhui Province [15,16]. The Ta-pieh Mountains are located in south-eastern China, at the border of Anhui, Hubei and Henan provinces (Fig 1A). Lu'an City is in the heart of the Ta huoshanense [17]. Due to the extremely low germination rate of natural Dendrobium seeds and the scarcity of Dendrobium in the wild, as well as excessive collection, existing resources cannot meet the current market demand.
In the traditional Chinese medicine market, tissue-cultured Dendrobium have already become the major resource of pharmaceutical Dendrobium [18]. Plantlets were usually regenerated through protocorm-like bodies (PLBs) or direct shoot organogenesis [19]. In Dendrobium cultivation, different tissue culture systems for micropropagation have been established, such as protocorms [20], shoot tips [21], axenic shoot nodal segments [22], which have been used as explants. Furthermore, many other studies including plantlet strengthening culture [23] and suspension culture of PLBs derived from seed embryos [24], have also been reported. Altogether, these studies have made great progress in propagating and exploiting Dendrobium.
Driven by huge economic interests, many fake species referred to as "Dendrobium" are circulating in the market [25]. This unhealthy market-oriented process is detrimental to the industrial development of Dendrobium and is not conducive to the safety of its clinical applications. Hence, there is an urgent need for a comprehensive method of germplasm identification and quality control in Dendrobium species. At present, there is no detailed report on a comprehensive analysis of the differences among different growth stages of three major Dendrobium species in the Ta-pieh Mountains (Fig 1A). Because the characteristics of D. huoshanense are different from the two other Dendrobium species, including its long nutrient accumulation time, we chose the three-year and four-year-old D. huoshanense to compare, and we also made a comparison of three Dendrobium species in three years. The main aim of this study was to evaluate the medicinal value of three kinds of major Dendrobium species and provide information for these three kinds of Dendrobium in the Ta-pieh Mountains area, China on the best age

Chemicals and reagents
All chemicals used in this study were of analytical grade. All the water used in the study was ultrapure, obtained from a Milli-Q Plus ultrapure purification system (Millipore, Bedford, MA, USA). The amino acids standards were purchased from National Institutes for Food and Drug Control (China). UPLC grade acetonitrile and methanol, and formic acid, were supplied from Merck (Darmstadt, Germany). Concentrated HNO 3 (70%) was purchased from Merck (Darmstadt, Germany). The ICP-MS multi-elemental standard stock solution (10 mg�L -1 ) was obtained from Agilent (Santa Clara, USA). The mercury standard (1000 mg�L -1 ) was obtained from National Standard Material Center (China). All glass containers were soaked in 10% HNO 3 for 12 h, cleaned with ultrapure water and air dried before use. All plastic containers, polypropylene flasks, pipette tips, polytetrafluoroethylene digestion tubes, and reagents that came into contact with samples and standards were checked for contamination.

Instrumentation
All samples that needed to be dried were treated with an electric drying oven (Humgine, China). Amino acids were analyzed using Agilent 1290 Ultra High-Performance Liquid Chromatography (UPLC) (Agilent, USA), equipped with an automatic liquid sampler, a fluorescence detector and a diode array detector. Trace elements were determined by NexION 300X (PerkinElmer, USA) Inductively coupled plasma mass spectrometry (ICP-MS), equipped with a cyclonic spray chamber and a Meinhard nebulizer. The MARS microwave digestion instrument (CEM, USA) was used for acid digestion of samples. The polysaccharides and total alkaloids were measured using an ultraviolet-visible spectrophotometer (Thermo Scientific GENESYS, USA).

Sample collection and preparation
Dendrobium plants were artificially cultivated in the greenhouse of Anhui Tongjisheng Biotechnology Company, Lu'an, China. Seed germination was cultured on half-strength Murashige and Skoog (MS) medium. Seeds were germinated to form protocorms, then growth of protocorm-like bodies were cultured on half-strength MS medium containing adding 6-BA 0.1 mg�L −1 , NAA 0.5 mg�L −1 and 1% additives (30 g�L −1 sucrose + 4 g�L −1 agar + 20% potato) under a 12/12 h light-dark cycle (approx. 30 μmol�m−2 S-1) at 25 ± 2˚C, the seedlings were finally formed. After 18 months of age, the plants were transplanted into pots and placed in the greenhouse at a temperature of 25-27˚C with a light/dark cycle of 12/12 h and 60-70% relative humidity. The planting substrate was selected to be breathable and moisturizing: bark + sawdust and other mixed fermentation (1:1), and then stone chippings added. From the grown plants of the respective seedlings of the three species of Dendrobium, three-year-old and four-year-old D. huoshanense, and two-year-old and three-year-old D. moniliforme and D. officinale were selected to provide 5 replicates of each sample ( Fig 1B). The voucher specimens were authenticated by the authors and deposited at the College of Forestry in Nanjing Forestry University, Nanjing, China.
After removal of roots and leaves, we obtained stem samples. All stem samples were washed with pure water, placed in a 60˚C and oven dried to constant weight, and then crushed and sieved for the determination.

Extraction procedure and analysis of polysaccharide
Polysaccharides were extracted according to the method described by Chinese Pharmacopoeia (version 2010). Initially, samples (0.3g) were precisely weighed, 200 ml of water was added, and the samples heated and refluxed for 2 h, filtered, then diluted to 250 ml. We accurately measured 5 ml of the solution into a 50 ml centrifuge tube, added 25 ml of ethanol, shook and refrigerated for 1 h, centrifuged at 4000 r�min -1 for 20 min, discarded the supernatant, and centrifuged again with 20 ml of 80% ethanol (same as above). We repeated these 2 times, poured off the supernatant, and dissolved precipitate in heated water to a constant volume of 50 ml.
The polysaccharide content was determined by the phenol-sulphuric acid method [26,27]. 1 ml of sample solution was transferred to the test tube, then 1 ml of 5% phenol solution was added. The solution was mixed thoroughly and 5 ml of concentrated sulfuric acid was added, shaken and placed in a 75˚C water bath for 20 mins. Afterwards, it was cooled until it was at room temperature, the absorbance was measured at 490 nm using UV-visible spectrophotometer with 1 ml of water as a blank, and the test was performed in parallel three times. The calibration curve was prepared from the glucose reference, and the equation of regression was: Y = 7.3717X-0.0025, R 2 = 0.9998, where Y is the absorbance and X is the concentration. Quantification of polysaccharides was performed on the basis of linear calibration plots of the absorbance versus the corresponding concentration.

Extraction procedure and analysis of total alkaloid
The method of Bush et al. was modified to analyze the alkaloid content [28]. Briefly, 0.5g of samples were precisely weighed, and soaked 1 ml of 25% ammonia solution for 30 mins and then extracted with 10 ml of chloroform in a 100 ml conical flask. The flasks were weighed and placed in a 75˚C water bath for 2 h and then allowed to cool to room temperature. Chloroform was added to their original weight. Afterward, 2 ml of the above filtrate was mixed with 8 ml of chloroform as sample solution.
The total alkaloid content was determined using the method described as follows [29]. 2 ml of the sample solution was precisely transferred to a 15 ml centrifuge tube, diluted to 10 ml with chloroform. 5 ml of pH 4.5 buffer and 1 ml of 0.04% bromocresol green solution were added, after which the mixture was left to stand for 30 mins after strongly shaking for 3 mins.
Subsequently, 1 ml of 0.01 mol�L -1 NaOH was added to 5 ml of the lower fractions for analysis. The alkaloid was determined by using a UV-visible spectrophotometer at 620 nm with dendrobine as a reference standard. The calibration curve was prepared from the dendrobine reference, and the equation of regression was: Y = 9.5429X-0.038, R 2 = 0.9998, where Y is the absorbance and X is the concentration. Quantification of total alkaloids was performed on the basis of linear calibration plots of the absorbance versus the corresponding concentration.

Analysis of amino acids by UPLC
The amino acid test solution was obtained as follows [30]. 3g of Dendrobium samples were precisely weighed, ground, and put into a plug test tube. Then 10 ml of 6 mol�L -1 hydrochloric acid was added, and the samples were hydrolyzed in a 110˚C oven for 24 h. They were taken out and let to cool to room temperature. After filtering and the filtrate was transferred to evaporating dish, evaporated in a water bath dissolved with 0.1 mol�L -1 of hydrochloric acid and diluted to 10 ml.
2.0 ml of test solution was transferred to a 10 ml centrifuge tube, and then 1.0 mol�L -1 triethylamine acetonitrile solution and 0.1 mol�L -1 PITC acetonitrile solution 2.0 ml were added respectively. Vortex blending was applied for 1 min and then the samples were left at room temperature for 60 mins. After adding 4.0 ml n-hexane, vortex blending was applied for 1 min and then they were left at room temperature for 10 min. We discarded the upper n-hexane layer, extracting it 3 times and removed the lower solution and placed it in 5ml volumetric flask, with 0.05 mol�L -1 sodium acetate solution (pH value of 6.5) to volume. The extract was then filtered by a 0.22 μm cellulose membrane syringe filter before UPLC analysis.

Analysis of trace elements by ICP-MS
The trace elements content of Dendrobium was determined using the method described as follows [14]. Powdered samples (0.500 ± 0.005g) were weighed accurately into the digestion tank, and 5.0 ml concentrated HNO 3 was added for pre-digestion. After 1 h, 2 mL of 30% (w/w) H 2 O 2 was added and the mixture was subjected to a microwave digestion furnace. Then we set the maximum power of 100 W, so that the temperature rose to 100˚C within 10 mins, and was held at the temperature for 20 mins, and then the digestion tank cooled to room temperature. After microwave digestion, the digested solutions were diluted to 25 mL with ultra-pure water. Then the ICP-MS instrument was used to determine the contents of the samples. The optimized operating conditions of the ICP-MS used in this work are summarized in Table 1. All ICP-MS parameters were optimized to improve the sensitivity. Sensitivity was measured before each set of measurements.

Validation of the methods
The UPLC method for quantitation of amino acids was validated by determining the linearity, precision, repeatability, stability and recovery. The results are shown in Table 2. All of the amino acids contents showed good linearity with the determination coefficients (R 2 ) ranging from 0.9992 to 0.9999 over a relatively wide concentration range. The results of precision test RSDs (relative standard deviation) of the amino acids were less than 3.47%, repeatability RSDs were less than 2.3% and stability RSDs were less than 1.76%. The overall recoveries were between 98.17 and 101.8% for the reference compounds, with RSDs less than 3.8%. Hence, the UPLC method is accurate, precise, and sensitive enough for quantitative evaluation of the amino acids in these samples.
Validation of the ICP-MS analytical method was performed using certified reference material: GBW-10016 tea (National Analysis Centre, China). Table 3 shows that the results obtained were in good agreement with certified values, which demonstrates the excellent accuracy of the selected method. The lowest concentration of the working solution for calibration use was diluted with the corresponding solvent to a series of concentrations. The limit of detection and limit of quantification for each analyte was acquired while the signal-noise ratios were respectively 3 and 10.

Statistical analysis
Differences in three kinds of Dendrobium of physicochemical characteristics were evaluated by Student's t-tests and ANOVA one-way analysis of variance, and differences were considered

Polysaccharide contents variation in different Dendrobium with different growth years
Polysaccharides are present naturally in food ingredients and provide many benefits to the body. It has been well documented that polysaccharides from natural sources (mushrooms, algae, lichens and higher plants) are very potent macrophage immunomodulators [31,32]. Polysaccharide is one of the main medicinal components of Dendrobium. We determined the polysaccharide of Dendrobium in different species with different growth years. The results are shown in Fig 2. This indicates that the accumulation of Dendrobium polysaccharides will not be at their maximum levels if harvesting is delayed beyond a certain period of time, as the polysaccharide will be slowly consumed. According to statistical analysis, there are significant differences between the two-year-old D. officinale and the three-year-old D. officinale. At the same time, in comparing all the three-year-old Dendrobium species, there are also significant differences between D. huoshanense and the other two Dendrobium species. The average content of polysaccharides in the three-year-old D. huoshanense reached 547.66 mg�g -1 . In view of the medicinal efficacy of polysaccharides, D. huoshanense has higher value than other two species of Dendrobium. However, in general, the polysaccharides of these three kinds of Dendrobium are in accordance with the 25.0% specified in the 2015 edition of the Chinese Pharmacopoeia.

Total alkaloid contents variation in different Dendrobium with different growth years
According to reports, alkaloids from genus Dendrobium, such as dendrobine, increase antioxidant capacity and show anti-cancer activity through improvement of human immunity [33]. It can be seen from the results of our measurements that the total alkaloids of the three species of Dendrobium are between 0.2 mg�g -1 and 0.4 mg�g -1 (Fig 3). It has also been reported in the literature that the alkaloid content of D. officinale is about 0.2 mg�g -1 , which is consistent with our results [34]. Analysis of our results and showed that there are significant differences between the two-year-old D. officinale and the three-year-old D. officinale. Furthermore, from comparing the three-year-old Dendrobium, there are significant differences between D. moniliforme and the other two Dendrobium species. Among the three species of Dendrobium, the alkaloid content of D. huoshanense is still slightly higher than that of the two other Dendrobium species.

Amino acids contents variation in different Dendrobium with different growth years
Amino acids have important functions in both nutrition and health [35,36]. The human body has eight kinds of essential amino acids. Due to the instability of tryptophan, we selected for study the seven other essential amino acids in the human body. The body cannot synthesize these amino acids by itself, but they must be provided in other ways. Without these essential amino acids, the normal growth and development of the human body can be affected, leading to serious illness. Therefore, the amino acids contained in traditional Chinese medicine can be used as an auxiliary component or the main component of the treatment of certain diseases. In this study, the results of amino acids are shown in  Among seven essential amino acids, threonine (9.10 mg�g -1 in D. huoshanense) has the highest concentration, while valine (0.28 mg�g -1 in D. huoshanense) has the minimum value. Valine, methionine, leucine, isoleucine, phenylalanine and lysine in the three species of Dendrobium showed concentrations similar to the values reported by Zhang et al. [37], whereas threonine showed higher concentrations. As can be seen from Fig 4A-4C, the amino acids contents of three-year-old D. huoshanense are higher than those of four-year-old D. huoshanense. The two-year-old amino acids contents of D. moniliforme and D. officinale are higher than those of the three-year-old D. moniliforme and D. officinale. This difference may be related to factors such as the function of amino acids and the physiological and biochemical effects of Dendrobium. Amino acids are essential substrates for protein synthesis and play an indispensable role in plant growth and development regulation [38]. However, no previous studies have reported the differences between these essential amino acids in three species of Dendrobium. We compared amino acids contents of three-year-old plants of different Dendrobium species. As shown in Fig 4D, amino acids contents of D. huoshanense were significantly higher than the other two Dendrobium species. Also, by analysis of variance, threonine, methionine and isoleucine, of D. huoshanense and of the other two species of Dendrobium showed significant differences.
Amino acids not only play central roles as building blocks of proteins but also participate in metabolism as intermediates. Amino acid disorders can lead to clinical signs and symptoms, even serious diseases, in metabolism, immunity and the cardiovascular and nervous systems [39][40][41][42]. Among the seven essential amino acids, the content of threonine is the highest in the three Dendrobium species, and isoleucine and lysine are the second highest. Many studies have shown that these seven essential amino acids are contained in the Dendrobium species studied, but the highest values of these seven amino acids are different due to the different origins and different varieties of Dendrobium [37,43].

Trace elements contents variation in different Dendrobium with different growth years
The results of the elemental analysis of samples are shown in Fig 5. As shown in Fig 5A-  three-year-old Dendrobium, D. officinale is higher than the other two Dendrobium species in Fe, Mg, Mn and Zn, this being especially significant in Mg.
Attention has been given to the harm of heavy metal elements to the human body, and the safety evaluation of essential trace elements is very necessary. A safety evaluation of the trace element level can be carried out by analyzing the dosage. Scientific recommendations were made for the dosage of the tested samples by comparing the contents of trace elements in the tested medicinal materials with the US Food and Drug Administration recommended daily intake of trace elements (RDI) (https://ods.od.nih.gov/factsheets/Iodine-HealthProfessional/) [44]. The 2010 edition of the Chinese Pharmacopoeia recorded that the daily dosage of Dendrobium was 6-12g dry product. According to this, we can calculate whether the elements obtained by taking 12g Dendrobium per day exceed the daily nutrient supply recommended by the Food and Drug Administration of the United States. Taking Fe in the three-year-old D. huoshanense as an example, the amount of Fe per day is 0.0669 mg�gg -1 × 12 g, which is 0.803 mg, without considering the loss of absorption. In RDI, Fe is in the range of 0.27 mg to 27 mg, and therefore can be taken safely according to the dosage prescribed by the Pharmacopoeia. The results of the calculation results are shown in Table 4. The Dendrobium studied in this study, when taken according to the dosage prescribed by the Pharmacopoeia, does not exceed RDI and does not causes adverse reactions or trace element poisoning.

Linear discriminant analysis
Linear discriminant analysis (LDA) is a classical statistical approach for dimensionality reduction and classification. It can maximize the variance between groups and minimize the variance within the same group. For achieving better classification and identification of different Dendrobium species, LDA was carried out on the basis of 13 elements. The stepwise discriminant procedure was carried out to extract the best discriminant variable separating different Dendrobium species, which enters or removes variables by analyzing their effects on the discrimination of the groups based on the Wilks' lambda criterion.
The separation of different groups in the discriminant space was obtained by plotting the first two discriminant functions. As shown in Fig 6, function 1 explained 61.4% of the variance and function 2 explained 38.6% of the variance; therefore, the total of variance explained by these two functions was 100%. Different Dendrobium species were clumped in different regions of the space. The D. huoshanense samples were entirely separated from D. officinale and D. moniliforme. D. officinale samples and D. moniliforme samples were a little closer but there was no overlap among them. These results confirmed the applicability of multi-elemental analysis for identifying different Dendrobium species.

Conclusions
This study provides a detailed report on polysaccharide, total alkaloid, amino acids and trace elements in different growth years of three kinds of Dendrobium species and evaluated the safety of trace elements in Dendrobium. According to the results, the quality of D. huoshanense is the best among the three Dendrobium species, followed by D. officinale and D. moniliforme. Discriminant analysis was carried out on data showing the concentration of elements found in Dendrobium in order to classify them according to several factors. This information can be useful to address the nutritional value and safety concerns related to the use of this plant as food or medicine. This developed method may be useful to identify substitution or adulteration in commercial Dendrobium samples.

Author Contributions
Data curation: Bo Zhang.