Quantitative assessment of secondary metabolites and cancer cell inhibiting activity by high performance liquid chromatography fingerprinting in Dendrobium nobile

https://doi.org/10.1016/j.jchromb.2020.122017Get rights and content

Highlights

  • First report of HPLC fingerprints for relative quantitative analysis in D. nobile.

  • Eleven less-polar metabolites corresponding to cancer cell inhibiting activity.

  • Beneficial effects of spring frost on imitative-wildly cultivated medicinal plants.

  • Applicability of HPLC fingerprinting methods on complex agricultural products.

Abstract

Dendrobium nobile is an important medicinal food beneficial for human health, well known for polysaccharides and dendrobine. For fast, accurate, and comprehensive comparison of its quality, high performance liquid chromatography (HPLC) fingerprinting method was constructed. Firstly, spring frost stressed D. nobile herb was observed for assessment. Decreased leaf thickness, chlorophyll, and drying rate, and increased free-proline indicated heavy damages on growth. But, the content of polysaccharides increased significantly in during-frost (DF), and dropped significantly in after-frost (AF). The content of dendrobine accumulated significantly in AF. Then, low similarity among HPLC fingerprints of before-frost (BF), DF, and AF, and 75.82% of significantly variant peaks indicated the changing of much more components. Especially, some less-polar components increased significantly in DF, but not in AF. Moreover, the highest suppression rates (SRs) to A549 lung cancer cells were up to 33.08% in DF, but only 15.63% and 12.12% in BF and AF. After association analysis, eleven less-polar components were found to be significantly and positively correlated to SRs under relatively high concentration. The result shows that frost stress not only causes damages to plant growth, but also promotes the accumulation of some health-beneficial bioactive metabolites. HPLC based fingerprinting method shows good applicability on quality evaluation and bioactivity correlation analysis of complexed agricultural products.

Introduction

Dendrobium nobile is one of the en-dangerous species in Orchidaceae. It is famous as ornamental and medicinal plant in worldwide. D. nobile had been used as medicinal food for a long history in some countries [1], [2]. In Chinese Phamacopoeia, D. nobile and some other Dendrobium species were listed as the traditional herbs [3]. It showed bioactivity in inhibiting invasion and inducing apoptosis and autophagy against cancer cells [4], [5].

At present, more than 100 chemical compounds have been found and identified from Dendrobium, including polysaccharides, alkaloids, phenanthrenes, bibenzyls, lactones, flavonoids, phenols, terpenoids, and sterols [6], [7]. Polysaccharides and dendrobine were the two mostly researched components in D. nobile [8], [9]. And they were used for annual evaluation of herb quality on Dendrobium [10]. More, for fast and high-throughput comparison of the abundant and complex medicinal substances, HPLC based methods were constructed [11], [12]. The consistency and stability of HPLC fingerprints made it suitable for relative quantitative analysis [13], [14]. The HPLC based fingerprinting methods had been applied on quality control, metabolites evaluation, and correlation analysis with some bioactivities [15], [16], [17].

Various kinds of biotic and abiotic stresses affected the metabolism of field cultivated plants. Under similar genetic backgrounds, the product quality depends a lot on growth environments [2], [18], [19].The contents of dendrobine [8], [10], polysaccharides [7], [9], flavonoids [20], [21], and some other indexes were reported to be varied with seasons and locations in D. nobile, D. huoshanense, D. catenatum, and D. candidum [10], [14], [18], [22]. Mycorrhizal fungus, light quality and intensity, salt, osmotic, and injury stresses were also reported to influence the accumulation of dendrobine, polysaccharides and some other medicinal components in D. nobile, D. denneanum, and D. officinale [23], [24], [25], [26]. Temperature changing also played an important role in quality formation [27]. Cold acclimation was thought to be suitable for biosynthesis of some pharmaceutical compounds in a certain level [28]. Frost is an episodic event in worldwide. It was usually thought to cause damages on plant growth [29], [30]. But its effects on quality of medicinal plant product were poorly studied.

Here, to put some new insights into the above problem, HPLC fingerprints, and cancer cell suppressing activity were co-analyzed to evaluate the changes of medicinal substances and bioactivity in imitative-wild-cultivated D. nobile went through a frost event.

Section snippets

Cell lines, chemicals, and biochemicals

A549 lung cancer cell lines were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cell was cultured in RPMI-1640 medium (Wisent, Canada) plus 10% fetal calf serum (Lanzhou Bailing, China) and 1% streptomycin and penicillin (Wisent, Canada) at 37 °C within 5% CO2. Proline standard (purity greater than 99%) was employed from National Institutes for Food and Drug Control (China). Dendrobine standard (purity greater than 98%) was purchased from Yuannuo Tiancheng (China). Dimethyl

Annual indexes indicating frost damages on D. Nobile

A typical frost event occurred at Baichaoxiang in January 9, 2018 to January 12, 2018. During this time, the lowest temperature was less than −4°C, and the highest temperature was more than 10 °C (Fig. 1A). The growth of D. nobile was influenced visibly (Fig. 1B). The length and width of leaves, as well as length, width, thickness, fresh weight, and dry weight of pseudobulbs didn’t show significant variances among the three groups. But, the thickness of leaves decreased linearly (Fig. 2A). The

Rapid relative quantification of metabolites and association analysis with bioactivity by HPLC fingerprints

HPLC is a basic chromatographic method for chemical analysis in many food products [11], [14], [19], [20], [21]. It becomes the standard method for quality assessment in more and more medicinal food [3], [15], [16]. Here, it is the first time to use HPLC fingerprints for relatively quantitative comparison in D. nobile. Its stability and repeatability was confirmed again in this study. The correlation coefficient was more than 0.9 between independent HPLC chromatograms in constructing of

Conclusion

In conclusion, this work enriches the records on relative quantification by HPLC fingerprints and response of secondary metabolites in Dendrobium genus under abiotic stress. Less-polar components showing strong suppressing efficacy to A549 cancer cells are firstly reported on Dendrobium. The results also underline that increasing bioactive compounds by abiotic stress might provide a more attractive functional herb for the market in agricultural production.

CRediT authorship contribution statement

Shigang Zheng: Conceptualization, Writing - original draft. Yadong Hu: Visualization, Methodology. Ruoxi Zhao: Software, Supervision. Tingmei Zhao: Investigation. hongjie Li: Investigation. Dan Rao: Validation. Ze Chun: Writing - review & editing, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Sichuan Provincial 13th Five-Year breeding program of traditional Chinese medicine (2016NYZ0036), and Science and technology support program of Sichuan province (2017SZ0020, 2017SZ0022, 2018SZ0096, 2018NZ0062) from Department of Science and Technology of Sichuan Province in China.

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