Comparison of chemical composition in the buds of Aralia elata from different geographical origins of China

Aralia elata buds contain many nutrients and have a pleasant taste with a unique flavour. Previous studies mainly focused on triterpene saponins in the root bark of this species, but little information existed concerning other chemical components, especially in the buds. To better understand the nutritional value of A. elata, we compared total flavonoids, total saponins, phenolic compounds and mineral element contents in the buds of A. elata collected from eight different geographical regions (S1: Benxi; S2: Linjiang; S3: Pingwu; S4: Enshi; S5: Changbaishan; S6: Shangzhi; S7: Xiaoxinganling and S8: Harbin) in China. The results showed that the basic composition in the buds presented a wide variation, with ash (8.76–10.35%), crude fibre (5.38–11.07%), polysaccharides (33.85–46.79 mg g−1), total flavonoid content (TFC, 4.06–48.63 mg g−1) and total saponins (13.62–27.85 mg g−1). UPLC combined with the LC-MS/MS method was used for the phenolic compounds analysis, and 11 phenolic compounds were identified and quantified in the eight samples. The total phenolic content in Enshi (S4) was significantly higher than others, and quercetin was the predominant phenolic compound in this sample. We used ICP-OES to identify and quantify nine mineral elements in the buds. The Fe and Cu contents in S5 were much higher than that of others. We obtained maximum Mg, Mn, Co and Ni contents in S4, and found rich Zn content in S7. Moreover, the maximum estimated quantities of Ca and Sr were found in S8. This study indicated that the chemical composition in the buds of A. elata was obviously affected by geographical origin. Our results provided an essential theoretical basis of quality evaluation of A. elata buds in the food production field.


Chemicals
Standards of protocatechuic acid, caffeic acid, chlorogenic acid, ferulic acid, gallic acid, catechin, epicatechin 4-hydroxybenzoic acid, quercetin, salicylic acid and scopoletin were purchased from National Institutes for Food and Drug Control, 3-Hydroxycinnamic acid was purchased from ChromaDex (USA), sinapic acid and 4-Hydroxycoumarin were purchased from Stanford Chemicals (USA). Methanol, acetonitrile and Folin phenol reagent were obtained from Sigma-Aldrich (USA). The standards of Zn, Cu, Fe, Cr, Ni, Sr, Ca and Mg were purchased from National Nonferrous Metals and Electronic Materials Analysis and Testing Center. In addition, nitric acid was electronic grade, and all other reagents used in this experiment were of analytical grade.

Basic composition analysis
The ash content and crude fibre determinations were measured following GB/T12532-2008 and GB/ T5009. 10-2003. Polysaccharides were extracted by the traditional hot water extraction. The dried buds were ground into fine powder, and extracted with 95% ethanol for 4 h to remove lipid. The degreased powders (0.5 g) were obtained with distilled water (80 ml) under reflux for 2 h. After centrifugation (6000 r.p.m., 15 min), the supernatant was concentrated. The yield was determined by the phenol -sulfuric acid colorimetric method [16,17].
The total flavonoid content (TFC) was determined using a colorimetric method with some modifications as described in [18]. After the powders (0.1 g) were dissolved in 2 ml of 70% ethanol, ultrasonic extraction was performed for 1 h and then centrifugation for 10 min. The supernatant dilute was added with 70% ethanol to a volume of 10 ml. We took 5 ml from it, and 0.3 ml of 5% NaNO 2 was added. The tubes were then allowed to stand for 6 min; subsequently 0.3 ml of 10% Al(NO 3 ) 3 was added to the reaction mixture, which was again allowed to stand for 6 min. Finally, 4 ml of 1 M NaOH and 0.4 ml of 70% ethanol were added and mixed immediately into the mixture, which was allowed to stand for 10 min. The absorbance was measured at 510 nm against a blank prepared similarly by replacing the extract with 70% ethanol. The TFC was then calculated according to the standard curve.
Total saponin (TS) content was determined as follows: The dry bud powders (1.0 g) were treated with 20 ml of petroleum ether for 1 h to remove the lipid. The degreased powders (0.1 g) were extracted with 70% ethanol, and ultrasonic extraction for 1 h. After centrifugation for 10 min, the supernatant was diluted with 70% ethanol to 5 ml. We took 50 ml from it, and 0.2 ml vanillin-glacial acetic acid and 0.8 ml perchloric acid were added. After placing the reaction mixture in a water bath (608C) for 15 min, 4 ml of ethyl acetate was added. The absorbance was measured at 560 nm.

Phenolic compounds analysis 2.4.1. Extraction
Two sets of samples (0.1 g for each) were prepared. One set of samples were extracted with 2 ml of distilled water and ultrasonic extraction for 90 min, and then centrifuged (13 000 r.p.m., 20 min). The supernatant was rotated to dry at 458C, then 200 ml of methanol and water (1 : 1) were added, and the supernatant was used for the analysis of phenolic compounds. Another set of samples was used for the study of the total phenolic content (TPC).

UPLC analysis of phenolic compounds
A Waters ACQUITY UPLC system (Waters, Milford, MA, USA) was used for the phenolic compounds analysis. Separation was performed on the ACQUITY UPLC HSS T3 column (2.1 Â 50 mm, 1.8 mm). The mobile phase consisted of water with 0.5% acetic acid (solvent A; v/v), methanol with 0.5% acetic acid (solvent B; v/v) and acetonitrile with 0.5% acetic acid (solvent C; v/v) at a flow rate of 0.40 ml min 21 . Samples (10 ml) were injected into the UPLC system. The gradient elution conditions started with 95% A, 4% B, 1% C to 4.0 min, adjusted to 85% A, 12% B, 3% C from 4.0 min to 6.0 min, 80% A, 16% B, 4% C from 6.0 min to 10.0 min, 70% A, 25% B, 5% C from 10.0 min to 13.0 min, 20% A, 40% B, 40% C from 13.0 min to 16.0 min, 0% A, 50% B, 50% C from 16.0 min to 19.0 min and back to the initial condition from 19.0 min to 24.0 min. The identification and assignation of each compound were performed by comparing their retention time and standard, and also confirmed by an AB Sciex LC-MS/MS spectrometer equipped with an electrospray ionization source. The electrospray ionization mass spectrometry detection was performed in the negative ion mode with the following optimized parameters: capillary voltage 2.50 kV, cone voltage 75 V, desolvation gas 650 l h 21 , cone gas 50 l h 21 , desolvation temperature 4508C, source temperature 1508C and extractor 3.00 V.

Statistical analysis
The data were analysed by one-way analysis of variance (ANOVA). The significance level was set to p , 0.05. Hierarchical cluster analysis (HCA) was performed based on the Ward method to classify the eight different geographical origins of A. elata, and principal component analysis (PCA) was performed using SPSS v. 17.0.

Basic chemical composition of A. elata buds from different geographical origins
The basic chemical composition such as ash, crude fibre, polysaccharides, total flavonoid and TS contents from the various sites are presented in table 1. The obtained results showed that the ash content ranged from 8.76% to 10.35%, and the sample 7 (S7) had the highest content (10.35%) and S8 had the lowest content (8.76%). The highest crude fibre content was found in S4, which was 11.07% and significantly higher than other samples (p , 0.05). Also, the lowest crude fibre contents were recorded in S3 and S6, which were 5.38% and 5.43%, respectively.
The highest polysaccharide content was in S5 (52.48 mg g 21 ), and the lowest content was in S2 (33.85 mg g 21 ). There was no significant difference in polysaccharide content among other samples (p . 0.05). The TFC was highest in S4 (48.63 mg g 21 ), but only 4.06 mg g 21 in S2. In general, plant polyphenols such as flavonoids and phenolics were biosynthesized through several pathways and formed a heterogeneous group [19]. Flavonoids, as critical secondary metabolites in the plant, exhibited various biological activities, such as anti-cancer and anti-oedema activities, free radical scavenging, prevention of coronary heart disease and inhibition of the arachidonic acid pathway to exert anti-inflammatory activity, etc. [20,21]. Our results indicated that S4 had the highest biological activities related to phenolic compounds.
The results also showed that the TS content in S3 was much higher than the other samples (27.85 mg g 21 ). The lowest contents were found in S5 and S7, which were 13.96 mg g 21 and 13.62 mg g 21 , respectively. The total saponins of A. elata were approved as a plant extract drug by State Food and Drug Administration of China in 2002 (Approval number: B20020945), which was used to reduce enzymes, protect the liver and for anti-tumour effects [22]. A. elata leaves total saponins capsule (H drug, pharmaceutical name Z20131018) is a hospital preparation of the Second Affiliated

Phenolic composition of A. elata buds from different geographical origins
The gradient method was optimized to provide separation of all the bioactive compounds by UPLC combined with LC-MS/MS. We measured 14 phenolic standards in this experiment, and 11 phenolic compounds have been identified ( figure 1a,b).We clearly found that the compounds were unambiguously identified as gallic acid ( peak 1), protocatechuic acid ( peak 2), 4-hydroxybenzoic acid ( peak 3), chlorogenic acid ( peak 4), caffeic acid ( peak 5), catechin ( peak 6), epicatechin ( peak 7), salicylic acid (peak 8), scopoletin ( peak 9), 3-hydroxycinnamic acid ( peak 10), ferulic acid ( peak 11), sinapic acid (peak 12), 4-hydroxycoumarin ( peak 13) and quercetin ( peak 14). The content of each analyte in different geographical origins of A. elata buds was calculated from the corresponding calibration curve and is summarized in table 2. Gallic acid and protocatechuic acid were estimated to be maximum in S1 and S2. S3 was rich in chlorogenic acid (0.96 mg g 21 ), quercetin (0.85 mg g 21 ) and protocatechuic acid (0.45 mg g 21 ). In addition, quercetin content was found to be maximum in S4 (11.71 mg g 21 ), which was 46.8-fold that of S2; chlorogenic acid (1.90 mg g 21 ) and salicylic acid (0.58 mg g 21 ) also had higher contents. Quercetin, gallic acid and chlorogenic acid contents were found as the major constituents in S5. With regard to S6 and S7, they were rich in quercetin and gallic acid contents. The maximum estimated quantities of quercetin, gallic acid and protocatechuic acid were, respectively, 3.4, 2.6 and 0.76 mg g 21 in S8.
The comprehensive quantitative analysis results indicated that quercetin was detected as the major constituent, especially in S4 (11.7 mg g 21 ). Gallic acid was higher in S1, S2, S5, S6, S7 and S8. However, it was not detected in S3 and S4. In addition, the protocatechuic acid content was much higher in S1, S2, S3, S4 and S8. The highest total amount of phenolic compounds was found in S4, which was 14.55 mg g 21 and much higher than other samples (p , 0.05). The lowest content was 2.86 mg g 21 in S6. These compounds showed different contents supposedly because of the location where the samples were collected as well as the dominant climatic and environmental factors [26]. From table 2, gallic acid, protocatechuic acid, quercetin and chlorogenic acid were found to be high in the eight different geographical origins of A. elata. Therefore, it is speculated that this may be related to the anti-tumour, antioxidant and anti-inflammatory effects of A. elata, which need to be further studied.

Hierarchical cluster and principal component analysis of phenolic compounds
To evaluate the possible similarities and relationship of the phenolic compounds in A. elata buds among the eight different geographical origins, HCA was performed based on the phenolic compounds. The HCA results were presented in the form of a dendrogram in figure 2a. Based on this analysis result, eight different geographical origins of A. elata buds were classified into two groups. The first group consisted of seven samples (S1, S2, S3, S5, S6, S7 and S8). The second group only included S4, which is mainly because of its exceptionally high level of quercetin (11.7 mg g 21 ) and TPC (14.55 mg g 21 ).
In addition, PCA classification confirmed the results of cluster analysis (figure 2b). The PCA result showed that the first component explained 76.2% of the total variation, and the second principal component explained 18.5% of the total variation. It clearly showed that S1 and S2 were rich in protocatechuic acid, S6 was rich in gallic acid, and S4 formed a single group characterized by higher quantities of quercetin.

Mineral elements in A. elata buds from different geographical origins
The mineral content in A. elata buds of the eight different geographical origins of A. elata buds is presented in table 3. The obtained results showed that the Ca content ranged from 801.47 to 3496.73 (mg g 21 ), Mg content ranged from 2577.53 to 3593.53 (mg g 21 ), Fe content ranged from 39.58 to 115.01 (mg g 21 ), Sr content ranged from 2.44 to 12.57 (mg g 21 ), Co content ranged from 0 to 0.34 (mg g 21 ), Mn content ranged from 25.66 to 214.32 (mg g 21 ), Ni content ranged from 1.25 to 6.43 (mg g 21 ), Cu content ranged from 20.1 to 46.73 (mg g 21 ) and Zn content ranged from 44.05 to 73.51 (mg g 21 ).
The contents of Fe and Cu in the S5 were higher than others. The Mg, Mn, Co and Ni contents were estimated to be maximum in S4, and S7 was rich in Zn content. In addition, the maximum estimated quantities of Ca and Sr were found in S8. However, Co was not detected in S3, S7 and S8. These minerals are necessary for the regulation of various functions in the human body.  Figure 1. (a) UPLC chromatogram of 14 standard mixtures, peak 1: gallic acid; peak 2: protocatechuic acid; peak 3: 4-hydroxybenzoic acid; peak 4: chlorogenic acid; peak 5: caffeic acid; peak 6: catechin; peak 7: epicatechin; peak 8: salicylic acid; peak 9: scopoletin; peak 10: 3-hydroxycinnamic acid; peak 11: ferulic acid; peak 12: sinapic acid; peak 13: 4-hydroxycoumarin; peak 14:  Gal. Que.

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Sco. In summary, the present study compared the total flavonoids, total saponins, phenolic compounds and mineral elements contents in the buds of A. elata collected from eight different geographical regions in China, and found that the chemical composition in the buds of A. elata was obviously affected by the geographical origin. The results could provide an important theoretical basis of quality evaluation of A. elata buds in the food production field.

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Ethics. Research Ethics. Permission for collection of A. elata buds was granted to Alkali Soil Natural Environmental Science Center, Northeast Forestry University/Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education. Animal Ethics. No experiment was done with animals.
Permission to carry out fieldwork. Permission for collection of A. elata buds was granted to Northeast Forestry University.
These eight sampling sites have cooperation with the university. We did our experiments in the university.