HPLC fingerprint analysis of polysaccharides from different accessions of Polygonatum odoratum

Abstract Polysaccharide was one of the considered major active ingredient in Polygonatum odoratum which was crucial for its quality evaluation. In this study, High performance liquid chromatography (HPLC) combined with chemometrics methods were performed to assess the quality of P. odoratum polysaccharide (POP) harvested from different locations. The methodology validation and similarity evaluation results showed that the analysis method was able to meet the requirement of fingerprint analysis, and 10 batches of POPs had a high degree of similarity based on the similarity values were greater than 0.960. The results of hierarchical cluster analysis (HCA) showed that different regions POPs could be classified by clustering analysis based on their nuances. The results of principal component analysis (PCA) showed that the mannose (58.13%∼78.18%) and glucuronic acid (2.36%∼11.72%) could be selected as herb markers for the quality control of P. odoratum. In conclusion, a more quantitative quality control method was established, and could be applied to the identification and quality control of different P. odoratum and their products. Graphical Abstract


Introduction
Polygonatum odoratum (Mill.)Druce was a famous traditional Chinese medicine used as a nutritious tonic and remedy to treat lung disease, upset stomachs, hypoimmunity, and rheumatic heart disease.Many compounds, including quercetin, steroidal saponins, flavonoids, and polysaccharides, had been reported from P. odoratum (Lin et al. 1994;Wang et al. 2009;Yang et al. 2015).Among them, the polysaccharide was one of the main bioactivity components with immunoregulation, anti-osteoporosis, and neuro-protection activity (Chen et al. 2010).
Polysaccharide had been used as a marker for quality control of traditional Chinese medicines (TCMs) as compiled in the pharmacopeias of China and other countries (The United States Pharmacopeia Commission 2015) or reported in literatures (Zhou et al. 2011).For example, according to the quality standards of P. odoratum, Ganoderma, and Lycii fructus in Pharmacopeia of China, glucose was used as the reference standard to determine the content of the total polysaccharides (National Pharmacopoeia Committee 2020).Although the content determination of the total polysaccharide played a key role in the quality evaluation of TCMs (Zhou et al. 2011), it lacks specificity and was inapplicable for the qualitative purpose.Therefore, it is necessary to develop a specific method to characterise the polysaccharides of P. odoratum (POPs).At present, multi-fingerprinting combined with chemometric methods had been applied to control the quality of polysaccharides such as Genus Astragalus polysaccharides (Xia et al. 2020), Sarcandra glabra polysaccharides (Huan et al. 2019), Poria coco and Polyporus umbellatus polysaccharides (Liu et al. 2019).Chemometrics methods applied mathematics, statistics, and other methods and means to maximise the acquisition of the composition, structure, and other relevant information about the material system through the processing and analysis of measurement data.Combined with the HPLC fingerprint, it could provide a reference for quality control and quality evaluation more easily and accurately (Sun et al. 2014).
In our present work, the polysaccharides were extracted from different P. odoratum samples and completely hydrolysed with TFA, and then analysed by RP-HPLC with PMP pre-column derivatisation.Subsequently, the similarity of these fingerprints was calculated and the data were carried out with hierarchical cluster analysis (HCA) and principal component analysis (PCA).

Methodology validation
The precision of chromatographic separation and detection was investigated by injecting samples in succession 6 times.The results showed that the relative standard deviations (RSD) of relative retention time (RT) were less than 0.59% and RSD of relative peak area (RPA) of common peaks were less than 0.63%, indicated that precisions of chromatographic separation and detection were satisfactory.The samples were extracted, hydrolysed, derivatised, and determined by HPLC to test the repeatability of the method.The results revealed that RSD of RT were less than 1.73% and RSD of RPA were less than 1.82%, so the methods of sample preparation and chromatography analysis had good repeatability.The linearity regression equation y ¼ 1045011405x-473531 (y-absorbance, x-concentration of Man) with the correlation coefficient R 2 of 0.9991 demonstrated a good linearity within the tested concentration range of 0.03125-0.5mg/mL.The recovery was 99.65%, and the RSD of 1.86%, indicating a satisfactory accuracy of the established method.The intra-and inter-day variations were 0.63% and 1.80%, respectively, suggesting an acceptable precision of the developed method.Moreover, derivatives of acid hydrolyzates from the sample were stored at room temperature and analysed to evaluate the stability of the derivative solution in 0, 2, 4, 8, 12, and 24 h, respectively.The results indicated that derivatives of hydrolyzates were stable, RSD of RT were less than 1.0% and RSD of RPA were less than 1.71%.All results indicated that the analysis method was able to meet the requirement of fingerprint analysis.

Standard fingerprint profiling and similarity evaluation
The monosaccharide composition of polysaccharides from 10 batches of P. odoratum was analysed by HPLC.The fingerprints (Figure S1a) were obtained and then analysed by software 'Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004 A)'.The standard fingerprint (Figure S1b) of P. odoratum samples was identified as the derivatives of Man (58.13%78.18%),GlcA (2.36%11.72%),Glc (7.86%20.45%),and Gal (3.20%20.87%)by comparing the retention times with those of the reference substances.The similarity of each fingerprint was evaluated by software with the corresponding standard fingerprints.The results showed that the similarity values were almost larger than 0.960, which indicated that the fingerprint characteristics of POPs from different geographical regions were highly similar.

HCA of polysaccharides in P. odoratum
To further analyse the fingerprint characteristics of polysaccharides from different geographic regions, we performed an HCA analysis using IBM SPSS software (version 21.0).The result showed that POPs could be divided into two clusters, I and II.Category I was then subdivided into two categories of A and B, whereas Category II was divided into C and D. Subcategory A only included POP7, and subcategory B consisted of POP2, POP4, and POP6.The C consisted of POP1 and POP3, and samples from POP6, POP8, POP9, and POP10 were clustered into group D. The samples from different geographical regions could not be classified as a different category, which indicated that there was no significant difference in the fingerprint of POPs cultivated in four different geographical regions (Shaoyang of Hunan, Changsha of Hunan, Yiyang of Hunan and Panyu of Guangdong).The results were consistent with the results of similarity evaluation of the fingerprints.Therefore, the fingerprint of polysaccharides by HPLC could reflect common characteristics in P. odoratum from various regions.

PCA of polysaccharides in P. odoratum
To compare the fingerprint characteristics of POPs, and further analyse the main components that affect the identification, IBM SPSS software (version 21.0) was used for principal component analysis.The loading plot was shown in Figure S2a.The two principal components PC1 (58.45%) and PC2 (22.60%) made up 81.05% of the total variance.The plot of PCA scores was presented in Figure S2b.The further away from the origin, the greater the contribution of the component to distinguish between various regions.The points representing Man and GlcA were the farthest from the origin, which hinted that Man and GlcA were the main two kinds of monosaccharides to distinguish polysaccharides in P. odoratum from various regions.The results showed that the polysaccharide fingerprint could be used to distinguish different varieties of P. odoratum, and polysaccharides could be used as one of its quality control and evaluation indicators (Huan et al. 2019).

Identification of confusion
A similar species of Polygonatum sibiricum was purchased to verify whether multiple fingerprints combined with chemometrics could be applied for quality control.The HPLC chromatograms of P. sibiricum polysaccharide (PSP) and POP were shown in Figure S1b.PSP was mainly composed of Man, Gal, and Ara, with a mole ratio of 74.02: 15.40: 10.58.POP and PSP were identified by the monosaccharide composition and molar ratio.Thus the fingerprints combined with chemometrics could identify adulterants successfully.

Conclusions
In conclusion, a chromatographic fingerprint representing the typical chemical characteristics of the analyte was proposed as a strategy for quality control of P. odoratum.The fingerprint profile of POPs was successfully established by PMP-HPLC.The results of similarity evaluation, HCA, and PCA of the fingerprints showed that POPs from different regions had their chromatographic fingerprint characteristics.Chemometric analysis based on the absolute peak areas of monosaccharides derivatives indicated that Man and GlcA were the major discriminatory components.Based on these results, the samples with the value of Man (58.13%78.18%)and GlcA (2.36%11.72%)were defined as P. odoratum.The fingerprint analysis method established in this research had good repeatability and high chromatographic resolution of the hydrolyzates related to unique structural features and chemical compositions of the polysaccharides, so it possesses practical value as a kind of important means of quality monitoring of P. odoratum products.