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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 49  |  Page : 13-20  

Using UHPLC and UV-vis fingerprint method to evaluate substitutes for Swertia mileensis: An endangered medicinal plant


1 College of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming; Institute of Medicine Plants, Yunnan Academy of Agricultural Sciences, Kunming, China
2 Institute of Medicine Plants, Yunnan Academy of Agricultural Sciences, Kunming; Yunnan Technical Center for Quality of Chinese Materia Medica, Kunming, China
3 College of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming; Institute of Medicine Plants, Yunnan Academy of Agricultural Sciences, Kunming; Yunnan Technical Center for Quality of Chinese Materia Medica, Kunming, China
4 College of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming, China

Date of Submission13-Oct-2015
Date of Acceptance22-Jul-2016
Date of Web Publication06-Jan-2017

Correspondence Address:
Yuan-Zhong Wang
Institute of Medicine Plants, Yunnan Academy of Agricultural Sciences,Beijing Road, Panlong District, Kunming
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1296.197655

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   Abstract 

Background: Millions of people are killed by viral hepatitis every year in the world, whereas many relevant medicines are too expensive to purchase. Swertia mileensis, a medicinal plant for hepatitis in the system of traditional Chinese medicine, has been vanishing gradually because of overexploitation. Objective: To find substitutes of S. mileensis and reduce the cost of purchasing drugs for hepatitis patients, the similarity of phytochemical constituents between S. mileensis and other three Swertia species was compared. Materials and Methods: Both ultra high performance liquid chromatographies and ultraviolet-vis fingerprints of four Swertia species were developed. Methanol extracts of the stems and leaves were used as samples to establish the fingerprint. The calibration curve was drawn for quantitative analysis of swertiamarin. The data of ultra high performance liquid chromatographies were evaluated statistically using similarity analysis and principal component analysis. Results: The result shows a significant difference at area of 204–290 nm in the ultraviolet fingerprint. Swertiamarin, the only one common peak, was defined in chromatographic fingerprints of four Swertia species. The quantitative analysis suggested that the highest concentration of swertiamarin is in S. davidii. The similarity indexes between different samples were almost under 0.60. In the principal component analysis, separate points not only represent the distinction among different species, but also perform chemical discrepancies in content between stems and leaves of one same species. Conclusions: S. angustifolia, S. davidii, and S. punicea are not suitable as substitutes of S. mileensis because of their remarkable differences in entirety and local part. In order to address issues about substitutes and high cost of purchasing drugs, more studies need to undertake.
Abbreviation used: UHPLC: Ultra high performance liquid chromatography, UV-vis: Ultraviolet-vis, HBV: Anti-hepatitis virus, DNA: Deoxyribonucleic acid, PCA: Principal component analysis, D-GaIN: D-Galactosamine, BCG: Bacille Calmette-Guerin, LPS: Lipopolysaccharide

Keywords: Fingerprint, Swertia mileensis, ultra high performance liquid chromatographies, ultraviolet-vis


How to cite this article:
Li J, Zhang J, Jin H, Wang YZ, Huang HY. Using UHPLC and UV-vis fingerprint method to evaluate substitutes for Swertia mileensis: An endangered medicinal plant. Phcog Mag 2017;13:13-20

How to cite this URL:
Li J, Zhang J, Jin H, Wang YZ, Huang HY. Using UHPLC and UV-vis fingerprint method to evaluate substitutes for Swertia mileensis: An endangered medicinal plant. Phcog Mag [serial online] 2017 [cited 2017 Jun 28];13:13-20. Available from: http://www.phcog.com/text.asp?2017/13/49/13/197655



Summary

  • The UHPLC fingerprint method indicated the significant difference on chemical ingredients in four plants from Swertia.
  • Swertiamarin is the unique common compounds for four plants, which exist are in leaves of S. davidii with the highest content.
  • The obvious diversity in four plants was displayed from comprehensive point of view though similarity assay and PCA analysis.
  • The UV fingerprint method offsets the defect that the UHPLC fingerprint reflected messages of secoiridoid glycosides only.



   Introduction Top


According to World Health Organization, about two million people are infected with viral hepatitis from unsafe injection every year, especially hepatitis B and C, which together cause roughly 80% of all liver cancer deaths and kill close to 1.4 million people.[1] Viral hepatitis, featured on silent or benign nature in early stages, is induced by one of the five hepatitis viruses involved in types A, B, C, D, and E.[2] African region is considered as the highest prevalence of viral hepatitis in the world, as well as some areas shrouded in the shadow, such as regions of Latin America and the Caribbean, Eastern Mediterranean, Europe, South-East Asia, and Western Pacific.[3] The spread of hepatitis disease has been decreased, which is attributed to persistent improvement of sanitary and living conditions, whereas it causes reductions of acquisitive immunity and universal vaccination. Furthermore, although the medicines for treatment of viral hepatitis efficiently are sold in markets, few patients can afford them because of their high prices. Compared with those famous medicines, some inexpensive and effective herbal drugs, such as Swertia mileensis, may be better choices for hepatitis patients living in developing countries.

S. mileensis T. N. Ho et W. L. Shi (Qingyedan in Chinese), belonging to Gentianaceae family, is distributed in the southern part of Yunnan province in China. It is available in thick patch of grass on versants at an altitude of 1300–1650 m.[4] S. mileensis was documented in the Chinese Pharmacopoeia due to its potent hepatoprotective activity.[5] Geng et al.[6],[7] found that swerilactones in S. mileensis exhibit potent anti-hepatitis B virus (HBV) activity through inhibiting HBV deoxyribonucleic acid (DNA) replication or against the secretion of hepatitis B and e surface antigen, which show the potential to treat viral hepatitis. So far, researchers have extracted and isolated many types of constituents from S. mileensis, mainly including secoiridoids, xanthones, monoterpens, and lactones, which are responsible for therapeutic properties.[7],[8],[9],[10] Despite all this, S. mileensis has not received the attention that it deserves from national community. In past years, the resources of S. mileensis have vanished gradually because of destructive collection, for which Yang et al.[11] explored the sustainable utilization of this plant through investigating its phenological phase, biological characteristics, and ecological environment. What is really exciting is that modern pharmacological studies have demonstrated that certain species of the genus Swertia also exhibit a variety of bioactivities, such as anti-HBV, antidiabetic, antimalarial, and antibacterial activities.[12],[13],[14],[15],[16] Coefficient of similarity is an indicator to measure the level of similarity in different samples, which can reflect similarity degree between two members directly.[17] The traditional Chinese medicine, the precious of people on China, characterized by the Theory of Whole View[18] that means, for drug applications, synergistic effects and multiple targets of many compounds or dissociating products of these compounds may happen in the therapeutic procedure rather than a monomer constituent.[19],[20] In other words the holistic similarity degree of phytochemical constituents was related positively with the therapeutic reactions in Swertia Species when they were used to cure same ailments. Hence, in order to find substitutes of S. mileensis and reduce the cost of purchasing drugs, it is urgent to assess integrally the similarity degree of phytochemical constituents between S. mileensis and other members of the genus Swertia through the fingerprint method.

During the past few years, researchers have developed some works for researches of chemical components in the genus Swertia.[21] Geng et al.[22] identified the structures of four secoiridoids in S. mileensis using diverse spectroscopy methods. Eight compounds were isolated from aerial parts of S. angustifolia by Zhu et al.,[23] four of which were gained firstly. While there are a number of peer-reviewed publications reporting content determination of chemical compounds in a single species,[24],[25] the information about contrasts in different Swertia species is limited.[26] Therefore, to evaluate the overall similarity of the genus Swertia, more interspecific studies are still necessary to be carried out.

A comprehensive and quantifiable identification method of chromatographic fingerprint which been regarded as the first choice by World Health Organization,[27] is able to display chemical information of herbal medicines with chromatogram, spectrograms, and other graphs by analytical and chemical techniques.[28] The ultra high performance liquid chromatographies (UHPLCs), a common instrument for quantification of components, possess two merits which are short total run time and simultaneous determination of compounds. Inoue et al.[29] successfully evaluate gardenia yellow based on UHPLC. The goal of identifying crude drugs could be achieved with the ultraviolet-visible absorption spectrometry (UV-vis), because chemical constituents from different materials have different unsaturated degree, which causes different peak positions, peak intensities, and shapes of curves.[30] In this paper, in order to obtain holistic similarity evaluation of different species (S. mileensis, S. angustifolia, S. davidii, and S. punicea) from Swertia genus, an UHPLC combined with the UV-vis fingerprint method will be established. To the best of our knowledge, this study could be considered as the first report on fingerprints development of these four species. This research may become a base stone to study plants of Sweria genus further in the future.


   Materials and Methods Top


Reagents and materials

The swertiamarin reference standard (purity ≥ 98%) was purchased from Shanghai Shifeng Biological Technology Co., Ltd (Shanghai, China). Methanol and formic acid of HPLC grade were obtained from Thermo Fisher Scientific (USA) and Dikmapure (USA), respectively. Purified water was purchased from Hangzhou Wahaha Group (Hangzhou, China). All other chemicals and reagents were analytical grade. Four batches of plants from the genus Swertia were collected in three provinces of China. All of the samples were identified by Dr. Heng-Yu Huang (College of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming, China). The information of materials is shown in [Table 1].
Table 1: Information of four batches of plants from the genus Swertia

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Instrumentation and analytical conditions

A Shimadzu UPLC system equipped with a degasser, binary gradient pumps, a column oven, an auto sampler and a UV detector, was employed for the chromatography analysis. The UV-vis analysis was built using the binary channel UV-Vis spectrophotometer (Shimadzu, Japan) with the UV spectra of 190–900 nm. Sample solutions of 3 mL were poured into cuvettes for this analysis. Under the UHPLC condition, all of analytes were detected on Shim-pack XR-ODS III (150×2.0 mm, 2.2 µm) C18 column by gradient elution with the mobile phase of 0.1% formic acid (A) and methanol (B) at the flow rate of 0.35 mL·min-1, which the detective wavelength was set at 240 nm. The gradient program was as follow: 0–1 min, 0–18% B; 1–4.5 min, 18–28% B; 4.5–5.7 min, 28–30% B; 5.7–11.5 min, 30–40% B; 11.5–15.2 min, 40–54% B; 15.2–19.5 min, 54–90% B; 19.5–22.0 min, 90% B; 22.0–25.0 min, 90–18% B. The column temperature and injection volume were set at 40°C and 1μL, respectively. The extracts were stored at 4°C.

Preparation of samples

Dried stems and leaves of S. angustifolia, S. davidii, S. punicea, and S. mileensis were powdered respectively as homogeneous size and sifted through a 60 mesh sieve. About 0.025 g of powder was weighed accurately and processed by ultrasonic extraction with 1.5 mL 80% methanol for 30 min. Extraction solutions were filtered through a 0.22 μm membrane filter. The filtrate collected in auto sampler vials was analyzed directly by UHPLC. Methanol and water supplemented 0.1% formic acid were degassed by ultrasonication for 30 min before use to avoid micro bubbles in solutions.

Dried stems and leaves of four species mentioned above were ground into fine power. 0.01 g sample dissolved in 10 mL 100% methanol was extracted ultrasonically for 30 min. The extract was diluted after filtering through a filter paper for UV-vis analysis.

Optimization of UV-vis

Various solvents including methanol, ethanol, petroleum ether, and water were tested. 0.01 g fine power of samples was weighed accurately to dissolve into these different solvents separately. Then, the ultrasonic extraction of sample solutions maintained 30 min. Subsequently, each 0.5 mL sample supplemented with the 3 mL corresponding solvent was used for UV analysis.

Under the extract condition in the part of solvent optimization, filtrates of samples were obtained. Samples mixed by 0.5 mL filtrates and 3 mL methanol (7-fold) were investigated, as well as samples mixed by 0.5 mL filtrates and 5 mL methanol (11-fold). Then, mixtures were employed for UV analysis.


   Results Top


Method validation and optimization of UV-vis

The standard solution containing swertiamarin was diluted to different concentrations to plot the calibration curve. The calibration curve was gained by plotting peak area (y) versus the concentrations (x, μg·mL-1) of the standard solution. The limit of detection and quantification, signal-to-noise of 3 and 10 were determined by serial dilution of the standard solution using the described conditions. The result is shown in [Table 2].
Table 2: The calibration curve, related coefficient, linear range, limit of detection, and limit of quantification of swertiamarin

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The precision was investigated for the developed method and instruments. The standard solution of swertiamarin was analyzed six times successively for UHPLC and the methanol extract of S. davidii for UV. To investigate the repeatability of developed method, six independent samples prepared from S. davidii were tested for UHPLC and UV. The prepared sample solutions were stored at room temperature for the stability assay, which were analyzed at 0, 2, 4, 8, 12, and 24 h for UHPLC and UV, respectively. Satisfactory values of RSD% were obtained. The result is listed in [Table 3]. Recovery tests were chosen to determine the accuracy of the method. Three different amounts (low, medium, and high spike) of the standard solution of swertiamarin were added into the leave extracts of S. mileensis. The recovery rate was calculated using the amount added of standard, and the actual amount (measured amount–original amount) obtained by UHPLC analysis. The result is shown in [Table 4].
Table 3: Values of RSD% in verification of methods

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Table 4: Recovery study of swertiamarin by the developed method

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%R = [(measured amount original amount) / amount added] × 100%

The extraction solvents and dilution multiple were optimized. The result showed that methanol produced the best yield for the four species. Then, the dilution of 7-fold was enough to obtain an available concentration for running process of UV-vis. On the basis of above findings, the best pretreatment was found to be optimal with dilution of 7-fold following methanol extraction. The result of pretreatment is showed in [Figure 1].
Figure 1: UV-Vis diagrams of optimization of extraction solvents (A) and dilution multiple (B).

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UV-Vis fingerprint analysis

The UV data were processed by UVProbe software (Shimadzu, Japan). Absorption peak curves of all samples were addressed with 4-point smoothing to suppress the interference from baseline drift and noise.

UV-vis fingerprints of stems and leaves of four different Swertia species are showed in [Figure 2], respectively. According to two UV fingerprint graphs, the diversity is significant among these four species. Compared with stems, the UV absorbance is remarkably higher in leaves. Both UV fingerprints of stems and leaves could be divided into two parts, one of which is 204–290 nm and another is 290–450 nm. It is clear in two UV spectrum graphs that characteristic absorption peaks of these four plants were concentrated on the area of 204–290 nm, while their absorption tendencies in the area of 290–450 nm were similar approximately. Comparing two fingerprints of stems and leaves, two organs of one same sample were very close on number and wavelength of peaks but obviously distinct on absorbance. The highest absorbance was seized by S. angustifolia in stems at 204–290 nm and in leaves was by S. davidii. Following the elaborative observation, some details were found that S. angustifolia, S. davidii, and S. punicea have a common peak at 240 nm, and S. davidii, S. punicea, and S. mileensis have a common peak at 274 nm. Interestingly, the peak at 211 nm involved in stems of S. punicea was deficient in its leaves. The only common point between S. punicea and S. mileensis is the peak at 211 nm. Therefore, the inference we conjectured is that there are diverse prominently in aspects of content and type of chemical compositions because of different UV absorbance and the amount of absorption peaks in part of 204–290 nm.
Figure 2: UV fingerprints of stems (A) and leaves (B) from four Swertia species.

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UHPLC fingerprint analysis

UHPLC fingerprints of stems and leaves of different Swertia species have shown their characteristics, which is exhibited in [Figure 3]. Every sample had some characteristic peaks and showed their fingerprint features. Whatever the species we observed, the amount and type of characteristic peak were consistent in its stems and leaves, except for the intensity. According to the compared analysis of different samples, only one characteristic peak (peak 2) exceeded the baseline obviously in S. mileensis, which was defined as swertiamarin through comparing samples with corresponding reference standard. On contrary, eight characteristic peaks (peaks 1–8) were placed above the baseline in S. davidii. In addition, 3 (peak 2 and 7) and 5 (peak 2–5 and 7) peaks appeared in S. angustifolia and S. punicea, respectively. In S. angustifolia, intensities of three characteristic peaks were extremely weak so that fused with the baseline almost. So, there were different kinds of characteristic peaks in different species. To sum up, stems and leaves belonged to one species contain similar chemical compounds with different contents. Then, different contents and types of chemical were involved in different species. The exclusive resemblance between these four species is that they all possessed peak 2. Therefore, the unique common characteristic peak of swertiamarin could be considered as the common constituent of S. angustifolia, S. davidii, S. punicea, and S. mileensis.
Figure 3: The UHPLC fingerprint of different tissues of four species from the genus Swertia

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In order to understand difference among four species further, particularly in the slightly similar part, we chose swertiamarin, an indicator in quality evaluation of S. mileensis.[5] to analyze continually. Peak areas were substituted into the regression equation gained through the calibration curves to calculate contents of each sample. The calculation result is shown in [Figure 4]. On the basis of quantitative analysis of swertiamarin, the highest content of 159.8212 mg·g-1 is in leaves of S. davidii and the lowest of 0.3701 mg·g-1 is in stems of S. angustifolia, whereas the swertiamarin was not detected in leaves of the latter. Both contents of S. davidii and S. punicea are higher in leaves than in stems, but S. mileensis and S. angustifolia is contrary. The order of swertiamarin content is S. davidii > S. mileensis > S. punicea > S. angustifolia.
Figure 4: Concentrations of four Swertia species calculated with the calibration curve

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Similarity assay

Compared with the reference chromatogram generated through mean method, the similarity indexes of stems and leaves from S. angustifolia, S. davidii, S. punicea, and S. mileensis were almost under 0.60. This indicated that the differentiation among our eight samples was significant. Nevertheless, through careful observations, there were some unobvious resemblances between some species. For example, the similarity index from leaves of S. davidii and S. mileensis was 0.93, and the similarity index between leaves of the former and stems of the latter was 0.926. In addition, similarity indexes of stems and leaves from the one species were all above 0.90, which means that the qualities of them were moderately similar. Even so, similarities between them were covered by their vast differences. The result is shown in [Table 5].
Table 5: The calculate result of similarity of four species from the genus Swertia

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PCA analysis

The principal component analysis (PCA) analysis of these four plants was employed to show their imparity intuitively. The PCA scatter plot is shown in [Figure 5]. What we could obtain from the picture is that there is the holistic difference between eight samples. From the figure, the obvious difference existed between stems and leaves of S. davidii because they were divided with a far distance at the horizontal negative half axis. Likewise, leaves of S. angustifolia and S. punicea were distributed closely, which probably implied that they were parallel in certain aspects. However, S. mileensis was disparate with other species because its stems and leaves were scattered in the fourth quadrant alone. Furthermore, the fissures between stems and leaves of each species in the scatter plot displayed their differences.
Figure 5: The PCA scatter plot of four species from the genus Swertia

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   Discussion Top


Two UV mark peaks of secoiridoids should appear at 240 nm and 270 nm because of the present of additional conjugated double bond.[31] Secoiridoids ingredients, the main principle in our four species, are widespread in the Gentianaceae family, [32] the Swertia genus was involved certainly. What is similar with the previous study is that both UV spectra of stems and leaves showed absorption peaks at ca. 240 nm, which implied the existence of abundant secoiridoids in the Swertia genus. Nevertheless, it is different with that study that the absorption peaks at 270 nm of S. davidii, S. mileensis, and S. punicea removed to 274 nm nearly. This is probably because of the strong resonating structure consisted of C=O and benzene ring results in red shift in xanthonoids. More than a hundred xanthonoids compounds and its derivatives were also found in members of the genus Swertia,[33] which means that xanthonoids is another big compounds class in this genus.

In the Swertia, most secoiridoids and their glycosides derivatives, kinds of big polar ingredient, are distributed widely. Moreover, many positions on benzene rings of most xanthonoids compounds are substituted by hydroxides in this genus. As everyone knows, the more places of benzene rings hydroxides to occupy, the stronger water solubility of xanthonoids is. This is suggested that small polar solutions like petroleum ether were not appropriate to extract compounds in our eight samples.

Swertiamarin, a constituent with the most extensive distribution and the highest content in the genus Swertia,[34] possesses significant hepatoprotective properties against D-Galactosamine (D-GaIN)-induced hepatotoxicity.[35] Mangiferin and amarogentin, according to previous researches, possess effects of antioxidant and antidyslipidemic, respectively.[36] As the main constituents to cure liver diseases in S. mileensis, swertiamarin is a potential marker to discuss the replacement of the crude drug. Furthermore, the dominant constituent, [Figure 3], is swertiamarin in S. mileensis according our experiment. Therefore, it is reasonable that to choose swertiamarin as the standard to find substitutes for S.mileensis. In order to evaluate similarity of four species deeply, swertiamarin, sweroside, isoorientin, isovitexin, loganic acid, gentiopicrin, and 6'-O-β-D glucopyranosylgentiopicroside were quantified in our experiment for evaluating difference of major active ingredients. Unfortunately, their contents were too low to detect, except for swertiamarin. Therefore, the result of quantification listed swertiamarin only, which suggested the different content of swertiamarin in these four species.

From the UHPLC fingerprint graph of the four species, there are significant differences in type and content of chemical compounds among S. davidii and S. mileensis. Whether on type or content of chemical compounds, S. davidii is richer than S. mileensis. The accumulation of metabolites was affected with multiple reasons, such as nature of themselves and environmental factors of light, temperature, soil, and water.[37] Photosynthesis is the trace to gain carbon sources for synthesis of metabolites. Light could control translations of photosynthetic genes, even increase the translation rate.[38] Certainly, the light condition in different altitude is different more or less, so that S. davidii collected at lowland (341 ± 5 m above sea level) is significantly incongruent with other three species collected at high elevation (1300–2200 m above sea level) where quantum flux areal density >2000 μmol·m-2·s-1 occurred more frequently.[39] The variation between Swertia species was verified from DNA level by Chassot et al.[40] They revealed that nuclear and chloroplast DNA sequence among some Swertia species and species was markedly different, even the chloroplast DNA sequence divergence between different species of Swertia was higher than between Swertia species and other genera. It is interesting that S. angustifolia and S. punicea is relatively close compared with others in the PCA analysis, which probably results from one collection place.

Previous studies indicated that all of species we selected show the effect of liver protection. S. angustifolia showed significant activities inhibiting the secretion of hepatitis B and e surface antigen.[13] S. davidii performed the inhibition of hepatoma carcinoma cell.[41] The total extract of S. punicea revealed the function that prevents alaniaetransaminase and aspartate aminotransferase from increasing induced by CCl4 and BCG / LPS, which is weaker in the latter compared with S. mileensis.[42] Through the fingerprint of total constituents, these four species could be evaluated totally to undertake verification of pharmacology if the similarity of constituents exists in theses four species. On the basis of previous researches and our result, although the differences existed among these four species are obvious, S. davidii is worth studying further due to its high content of swertiamarin. Swertiamarin possessed potent hepatoprotective properties is the primary constituent to against hepatitis diseases in plants of the genus Swertia.[43] In future, S. davidii could play a potential role for the resource plant of swertiamarin. Otherwise, many other Swertia species and other genus from the family Gentianaceae contain swertiamarin, such as S. japonica, Enicostemma axillare, and Centaurium erythraea,[44],[45],[46] possibly are new directions to solve problems about substitutes of S. mileensis and high price of purchasing drugs in subsequent study.


   Conclusion Top


UV and UHPLC methods were developed for the similarity evaluation of four Swertia species. Eight samples were analyzed by those two methods, and the chromatographic data were addressed by similarity analysis and PCA. On one hand, results of PCA and similarity analysis proved holistically that a tremendous interspecific distinction exists in these four plants. On the other hand, the quantitative analysis showed the weakly similarity between our samples from local part of swetiamarin, a main bioactive constituent. So, whatever sides we considered, those four Swertia members are very different. According to this result, we do not agree with that one of these four species could be considered as the substitute of S. mileensis. In order to address issues about substitutes and high cost of purchasing drugs, more studies need to undertake.

Acknowledgement

This work was supported by the National Natural Science Foundation of China (31260077 and 81260608) and the Key Project of Yunnan Provincial Natural Science Foundation (2013FC006).

LJ conducted all laboratory experiments, data analysis, and partial interpretation of results under the guidance of HH-Y, WY-Z and JH, who designed the study. ZJ is responsible for the grammar checking of the paper and finalized the manuscript. All authors have read and approved the final manuscript

Financial support and sponsorship

No support available in the form of grants or aid

Conflicts of interest

There are no conflicts of interest

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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