Saffron is an expensive spice derived from the stigma of the
The scarcity of resources and high cost of saffron lead to the frequent occurrence of saffron adulteration in the market, such as plant-derived materials like
Adulteration of saffron in the market brings about attention of the quality control of saffron. The international standard ISO 3632-2011 for grading saffron reports a standard UV-Vis spectrophotometric method, which tests the strength of aroma, color, and flavour of saffron by determining the concentrations of safranal, crocin, and picrocrocin [
Many published studies have focused on the quality standard of saffron, and various analytical techniques were applied to the quality control of saffron, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), near-infrared spectroscopy (NIRS), ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), and electronic nose (E-Nose) [
Spectrum–effect relationships have been widely used to screen the active compounds of TCMs by combining chromatographic fingerprint of TCMs with their biological activity. Chromatographic fingerprints of traditional Chinese medicines (TCMs) contain a large number of information and could express the chemical characteristics of samples integrally [
In this study, HPLC was used to establish the fingerprints of 21 batches of saffron. Then, similarity analysis (SA), hierarchical clustering analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were applied to distinguish differences among the 21 batches of saffron. Subsequently, the antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay and hydroxyl (∙OH) radical-scavenging assay. The spectrum-effect relationship between HPLC fingerprints and antioxidant activities were elucidated by grey relational analysis (GRA) and partial least square (PLS) analysis. The potential active compounds of saffron were discovered. Finally, a quantitative method for the determination of the potential active compounds, crocin I, crocin II, and picrocrocin, was developed.
Twenty-one batches of saffron were collected from different regions, as shown in Table
The origin and collection sites of 21 batches of saffron used in this study.
ID | Origin | Collection site | ID | Origin | Collection site |
---|---|---|---|---|---|
S1 | Zhejiang, China | Jiande Saffron Agricultural Cooperative | S12 | Tibet, China | Bozhou Yonggang Slices Factory co. LTD |
S2 | Zhejiang, China | Jiande Saffron Agricultural Cooperative | S13 | Shanghai, China | Bozhou Kangmei TCMs Market |
S3 | Zhejiang, China | Jiande Saffron Agricultural Cooperative | S14 | Iran | Bozhou Kangmei TCMs Market |
S4 | Henan, China | Dancheng Saffron Agricultural Cooperative | S15 | Iran | Bozhou Kangmei TCMs Market |
S5 | Zhejiang, China | Jiande Saffron Agricultural Cooperative | S16 | Iran | Bozhou Kangmei TCMs Market |
S6 | Anhui, China | Bozhou Kangmei TCMs Market | S17 | Tibet, China | Bozhou Kangmei TCMs Market |
S7 | Anhui, China | Bozhou Kangmei TCMs Market | S18 | Tibet, China | Bozhou Kangmei TCMs Market |
S8 | Anhui, China | Bozhou Kangmei TCMs Market | S19 | Iran | Bozhou Kangmei TCMs Market |
S9 | Anhui, China | Bozhou Kangmei TCMs Market | S20 | Iran | Bozhou yonggang Slices Factory co. LTD |
S10 | Anhui, China | Bozhou yonggang Slices Factory co. LTD | S21 | Henan, China | Dancheng Saffron Agricultural Cooperative |
S11 | Dubai | Bozhou Kangmei TCMs Market |
Picrocrocin (97.6% purity) standard reference substance was purchased from the National Institutes for Food and Drug Control (Beijing, China). Crocin I (≥98.0% purity) and crocin II (≥98.0% purity) standard reference substances were purchased from Shanghai Standards Technology Co., Ltd. (Shanghai, China). 2,2′-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Purified water (Wahaha Purified Water) was purchased from Hangzhou Wahaha group Co., Ltd. (Hangzhou, China). All other reagents were of analytical grade.
All the saffron samples used in this study were dried samples. The sample drying method was in accordance with the low temperature drying (<60°C) stipulated in technical regulation for production of saffron crocus (
All reference substances were weighed to obtain 1 mg/mL picrocrocin, crocin I, and crocin II stock solutions. An appropriate amount of stock solution was taken and diluted to an appropriate concentration with 50% ethanol. The solution was filtered using 0.45-
An Agilent 1260 HPLC system (Agilent Technologies Inc., California, USA) was used to detect the analytes. Chromatographic separation was carried out with an Eclipse XDB-C18 column (4.6 mm × 150 mm, 5
3 mL of different concentrations of sample solutions and 70
2 mL of different concentrations sample solutions, 2 mL of 3 mmol/L salicylic acid solution, and 2 mL of 3 mmol/L FeSO4 solution were mixed in the test tube. Subsequently, 2 mL of 3 mmol/L H2O2 was added to start the Fenton reaction, and the mixture was incubated for 30 min at 37°C. 2 mL of 50% ethanol was used to instead of sample solution as a control, and 2 mL of H2O was used instead of 3 mmol/L H2O2 as a blank. The absorbance of each reaction mixture was measured at 510 nm. The ·OH radical-scavenging capacity and IC50 were calculated of each sample.
Data analysis was performed by the software Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2012). HCA was performed by SPSS (Version 24). PCA, PLS, and OPLS-DA were performed by SIMCA-P (Version 14.0). Grey relational degree was performed by DPS. Quantitation of three marker components were calculated based on the calibration curves, and the results were expressed as mean ± SD.
The preparation methods of the sample were first studied according to the stirring method in ISO-3632-2011 and the ultrasonic extraction method in our previous study [
Effect of extraction solvent (a, b), extraction time (c, d), light (e, f), and solid-liquid ratio (g, h) on the HPLC fingerprints of saffron at 257 nm (a, c, e, g) and 440 nm (b, d, f, h).
Recently, fingerprints combined with multivariate statistical analysis have been used to classify and discriminate different TCMs sources successfully [
HPLC fingerprints of 21 batches of saffron at 257 nm (a) and 440 nm (b) as well as representative fingerprint of saffron at 257 nm (c) and 440 nm (d).
In this study, the similarity calculation was carried out by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software (Version 2012) issued by Chinese Pharmacopoeia Committee. Time window width was set as 0.1, average mode was used, and then, all the samples had multipoint correction and automatic match to generate a representative fingerprint that represented the characteristic mode. The similarity was calculated by comparing chromatograms of saffron with the representative fingerprints [
The method validation results of the HPLC fingerprint showed that the relative standard deviations (RSDs) of precision, repeatability, and stability of the retention time (RT) and the peak area (PA) met the prescribed requirements. The variations in the RT of the characteristic peaks were less than 0.5%, and the variations in the PA are less than 3.0% (
The method of quantitative analysis was validated in terms of linearity of calibration curves, precision, stability, repeatability, and recovery. Taking the sample concentration as the abscissa (
The regression equation,
Name | Regression equation | Linear range ( | Standard errors | ||
---|---|---|---|---|---|
Slope | Intercept | ||||
Crocin I | 0.9999 | 18.92∼302.70 | 0.12 | 19.24 | |
Crocin II | 0.9999 | 6.28∼100.50 | 0.16 | 8.51 | |
Picrocrocin | 0.9999 | 17.78∼284.40 | 0.02 | 2.57 |
The precision, stability, and repeatability were assessed by the PA of P3, P7, and P10, respectively. The RSDs of precision of crocin I, crocin II, and picrocrocin were 1.11%, 0.10%, and 0.10%, respectively, the RSDs of stability were 1.31%, 0.47%, and 0.21%, respectively, and the RSDs of repeatability were 1.60%, 1.26%, and 0.21%, respectively. Recovery was measured by the standard addition method. The sample (S4) was added with high, medium, and low levels of a mixed standard solution of the three compounds in triplicate. The average recovery rates of crocin I;, crocin II;, and picrocrocin were 99.58%, 98.18%, and 100.04%, respectively. All the results of the method validation tests demonstrated that the proposed method was reliable and valid.
In this study, SPSS statistical software (Version 24.0) was used to perform HCA on the fingerprint of 21 batches of saffron, using the square Euclidean distance as the interval and using the intragroup linkage method, as shown in Figure
Heat map of the HCA of 21 batches of saffron.
PCA is a multivariate statistical method which converts multiple variables into a few unrelated comprehensive variables. The purpose of PCA is to remove overlapping information among numerous of information by dimensionality reduction [
PCA scores plot of 21 batches of saffron (a), OPLS-DA diagram of 21 batches of saffron (b), and VIP plot of OPLS-DA of 13 characteristic peaks (c).
Eigenvalue and variance cumulative contribution rate.
Principal component | Eigenvalue | Variance contribution rate (%) | Cumulative variance contribute rate (%) |
---|---|---|---|
1 | 6.231 | 47.932 | 47.932 |
2 | 2.330 | 17.924 | 65.856 |
3 | 1.516 | 11.659 | 77.515 |
4 | 0.979 | 7.534 | 85.049 |
5 | 0.822 | 6.321 | 91.369 |
6 | 0.550 | 4.228 | 95.597 |
7 | 0.243 | 1.872 | 97.469 |
8 | 0.158 | 1.212 | 98.681 |
9 | 0.095 | 0.729 | 99.410 |
10 | 0.057 | 0.441 | 99.851 |
11 | 0.010 | 0.074 | 99.925 |
12 | 0.007 | 0.052 | 99.977 |
13 | 0.003 | 0.023 | 100.000 |
Component matrixes.
Peak number | Principal component | ||
---|---|---|---|
1 | 2 | 3 | |
1 | −0.392 | 0.397 | 0.743 |
2 | 0.798 | −0.202 | 0.400 |
3 | 0.897 | −0.350 | −0.129 |
4 | 0.181 | 0.804 | −0.289 |
5 | −0.043 | −0.271 | 0.551 |
6 | 0.940 | −0.064 | 0.181 |
7 | 0.981 | −0.119 | 0.012 |
8 | 0.558 | 0.307 | −0.311 |
9 | 0.702 | 0.461 | 0.169 |
10 | 0.930 | −0.289 | −0.049 |
11 | 0.887 | −0.098 | −0.009 |
12 | 0.393 | 0.694 | −0.202 |
13 | 0.381 | 0.625 | 0.445 |
The predictive ability parameter
In OPLS-DA, the corresponding model was obtained by using the PA of 13 common peaks of saffron as input variable. The results were shown in Figures
Permutation test, a computer-based resampling method for remodeling and predicting, was widely used in the computation of variable importance and confidence intervals [
Permutation test plots of OPLS-DA model.
In Iranian pharmacopoeia and European pharmacopoeia, the quality control of saffron does not include quantitative analysis of chemical composition, while Japanese pharmacopoeia and Korean pharmacopoeia list the sum of crocin I and crocin II for the quality control of saffron for only qualitative analysis. Many literatures have shown that the crocins play a critical role in quality control of saffron. Additionally, a variety of components, including crocins, picrocrocin, crocetin, and safranal, show unique pharmacological activity [
The proposed HPLC method was successfully used for simultaneous determination of crocin I, crocin II, and picrocrocin. As shown in Figure
Contents of three compounds in 21 batches of saffron. Light blue, yellow, and dark blue colors represent picrocrocin, crocin I, and crocin II, respectively (a), and heat map of the HCA of three component of 21 batches of saffron (b).
Based on the contents of the three major components, the 21 batches of saffron could be divided into two categories by HCA (Figure
As the results of quantitative analysis show, the contents of three major components of four batches of saffron from Zhejiang province were significantly different from each other. However, one batch of saffron from Zhejiang province was very similar to those from Tibet and Iran et al., indicating that the differences between samples had little correlation with origin. To sum up, origin was not the key factor of the difference that affects the quality of saffron. The difference on quality may be caused by cultivation methods, nutrition and quality of bulb, growth environment, climate, and other factors.
Oxidation is an important process for the energy productive of many biological organs [
As shown in Figure
DPPH and ∙OH scavenging activity of 21 batches of saffron of different concentration (a, b) and IC50 of the antioxidant activity of 21 batches of saffron (c).
As shown in Figure
As shown in Figure
The correlation between 21 batches of saffron and the IC50 values of DPPH radical-scavenging capacity and ∙OH radical-scavenging capacity were analyzed by grey correlation degree analysis (Table
Results of correlation of HPLC fingerprint with antioxidant activities of saffron.
Peak number | Correlation | |
---|---|---|
DPPH | ∙OH | |
1 | 0.602 | 0.690 |
2 | 0.736 | 0.682 |
3 | 0.756 | 0.759 |
4 | 0.663 | 0.727 |
5 | 0.632 | 0.695 |
6 | 0.760 | 0.724 |
7 | 0.772 | 0.804 |
8 | 0.671 | 0.753 |
9 | 0.714 | 0.722 |
10 | 0.772 | 0.775 |
11 | 0.731 | 0.780 |
12 | 0.665 | 0.715 |
13 | 0.651 | 0.627 |
The PA of 13 common peaks of 21 batches of saffron was inputted as the independent variable, while the IC50 of DPPH and ∙OH radical-scavenging capacity were inputted as the dependent variable, and the partial least squares method was used to analyze the variables. The correlation coefficient and variable projection importance value (VIP) were obtained to evaluate the correlation between 13 chromatographic peaks and drug efficacy and their contribution to drug efficacy. It is generally believed that when VIP>1, the independent variable has significant importance on explaining the dependent variable. In this study, the VIP value of each chromatographic peak of DPPH radical-scavenging capacity is ranked as follows: P7 > P10 > P3 > P6 > P13 > P11 > P9 > P2 > P12 > P5 > P8 > P1 > P4 (Figures
The coefficients of the partial least squares regression analysis of DPPH and ∙OH radical-scavenging capacity (a, c) and the VIP values of the partial least squares regression analysis of DPPH and ∙OH radical-scavenging capacity (b, d).
Limited relevant research showed that polysaccharide and ethanol extracts of saffron from seven different productions had remarkable antioxidant activities [
The chemical structure of crocin I (a), crocin II (b), and picrocrocin (c).
Previous studies have focused on mainly the analysis of its chemical composition or only bioactivities, limiting the development and utilization of saffron as a commercial medicine [
The spectrum-effect relationships of HPLC fingerprints and scavenging capacity for DPPH and ∙OH were established to analyze the active components of saffron. The spectrum-effect relationships on the basis of grey correlation degree and OPLS-DA analysis revealed that crocin I, crocin II, and picrocrocin were the main components contributing to the antioxidant activities of saffron and these compounds had synergistic antioxidant effects. Through this study, the main antioxidant components of saffron were further determined, which could provide clue for establishing reliable and reasonable quality standards for saffron and its products.
The data used to support the findings of this study are included within the article.
Ya You and Zijin Xu are co-first authors.
The authors have no conflicts of interest to declare.
Ya You, Zijin Xu, Suhong Chen, and Ping Wang contributed equally to this work. Ping Wang, Suhong Chen, and Yifeng Cao conceived of and designed the experiments; Ya You, Zijin Xu, Qingrou Zhong, and Lin Zhu performed the experiments; YaYou, Yi Tao, Susu Lin, and Qiaoqiao Li analyzed the data; Ya You wrote the paper.
Thanks are due to the Special Project of International Technology Cooperation of One Belt and One Road (grant/award number: 2017C04009) and the Key Projects of International Scientific and Technological Innovation Cooperation between Governments (grant/award number: 2017YFE0130100).