Chromatographic-grade solvents, namely, methanol and acetonitrile, were obtained from Tedia High-purity Solvent Co., Ltd. (Fairfield, OH, USA), and formic acid was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China) The double-distilled water was prepared in the laboratory by using a Milli-Q system (Millipore, Bedford, MA, USA). Schaftoside (1), isoschaftoside (2), 2,4,7-trihydroxy-9,10-dihydrophenanthrene (3), dihydroresveratrol (4), coelonin (5), nudol (6), gigantol (7), 4-methoxy-2,5- phenanthrenediol (8), 3-hydroxy-3,4,5-trimethoxybibenzy (9), erianin (10), and tristin (11) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan province, Chengdu, China). The purity of all the reference standards was above 98%. The structures of these standards are shown in Figure 1.

Dendrobium huoshanense was collected on November 14, 2020, from Huoshan County, Anhui Province, China. D. nobile, D. chrysotoxum, and D. fimbriatum were collected on November 14, 2020, from Wenshan, Yunnan Province, China. All the plants were identified by Prof. Nai-Fu Chen, West Anhui University, China. Six replicates of each Dendrobium species were collected and analyzed. The samples were prepared as follows: the fresh stems were freeze-dried to constant weight at −50°C, separately powdered in a blender, and screened using a 200-mesh sieve. The Dendrobium powder was weighed (2.0 g) and treated by condensation reflux three times by using 120 ml of 90% methanol at 80°C, with each treatment lasting for 1 h. The methanol extracts of the four Dendrobium species were obtained by decompressing and concentrating the filtrate to dryness.

Solid-phase extraction was performed using a CNWBOND LC-C18 solid-phase extraction (SPE) cartridge (2.0 g/10 ml; ANPEL Laboratory Technologies Inc., Shanghai, China) as follows: the cartridge was activated using 50 ml of methanol followed by 25 ml of Milli-Q water. Next, 100 mg of methanol extract was precisely weighed, dissolved in 20 ml of distilled water, and filtered through a 0.22-μm microporous membrane. The sample solution was then injected into the activated SPE cartridge. The solid-phase extraction procedure was performed as follows: first, elution was performed with 45 ml of Milli-Q water, followed by elution with 45 ml of 20% methanol solution, and a final elution with 60 ml of 60% methanol solution. The eluate obtained after the 60% methanol elution was decompressed and concentrated to dryness for further analyses.

Standard stock solutions of the reference standards were prepared by mixing and dissolving appropriate amounts of the pure substances in methanol. The initial concentrations of compounds 1–11 were 1.38, 1.55, 1.0, 1.05, 1.23, 1.0, 2, 1.1, 1.0, 1.55, and 1.04 mg/ml, respectively. Then, the mixed standard solution was gradient-diluted to obtain the working solutions. All the solutions were stored in a refrigerator at 4°C.

Chromatographic analyses were performed using the Agilent1260 HPLC system (Santa Clara, CA, USA) equipped with a quaternary pump (G1311B), automatic injector (G7129A), a column temperature controller, and a diode array detector (G4212B), all controlled by an Agilent ChemStation. Chromatographic separation was performed on a Zorbax Eclipse XDB-C18 (150 × 4.6 mm, 5 μm) at a column temperature of 35°C. The mobile phase was composed of acetonitrile (A) and Milli-Q water (B) with the following gradient program: 0–6 min, 14.7–16.2% A; 6–25 min, 16.2–60% A; 25–27 min, 60–100% A; 27–30 min, 100% A; 30–32 min, 100–14.7% A; and 32–35 min, 14.7% A. The flow rate was 1.0 ml/min, and the injection volume was 10.0 μl. The DAD spectra were acquired over a scan range of 200–400 nm. Subsequently, HPLC chromatography of the 11 mixed standards was performed (Figure 2).

In this paper, compounds 1–11 in the Dendrobium samples were identified by the comparison of the HPLC retention times (RTs), UV spectra, and mass spectrometry between the standards and the possible polyphenol peaks.

As shown in Table 1, in our established HPLC chromatogram conditions, the RTs of the possible polyphenol peaks in the HPLC chromatograms of the four investigated Dendrobium species were very close to those of the standards for compounds 1–11, and the UV spectra of the possible polyphenol peaks (all showing peak purities above 94%) were very similar to those of the standards. Subsequently, schaftoside (1), isoschaftoside (2), 2,4,7-trihydroxy-9,10-dihydrophenanthrene (3), dihydroresveratrol (4), nudol (6), gigantol (7), and tristin (11) were identified from D. huoshanense; schaftoside (1), isoschaftoside (2), 2,4,7-trihydroxy-9,10-dihydro-phenanthrene, dihydroresveratrol (4), nudol (6), erianin (10), and tristin (11) were identified from D. nobile; schaftoside (1), isoschaftoside (2), 2,4,7-trihydroxy-9,10-dihydrophenan-hrene (3), dihydroresveratrol (4), nudol (6), and tristin (11) were identified from D. chrysotoxum; and schaftoside (1), isoschaftoside (2), 2,4,7-trihydroxy-9,10-dihydrophenanthrene (3), dihydroresveratrol (4), coelonin (5), and tristin (11) were identified from D. fimbriatum. Thus, the 11 polyphenols were not uniformly distributed in the four Dendrobium species used for preparing Shihu.

In our study, samples of four different Dendrobium species were analyzed by the established Ultra High Performance Liquid Chromatography Quadrupole-Time of Flight Mass Spectrometry/Mass Spectrometry (UPLC-Q-TOF-MS/MS) system. The target compounds in samples were identified by comparing the observed MS and MS/MS spectrum with those of standards. In our experiments, a total of nine polyphenols were detected and identified from the four forms of four different original plants of Shihu by comparing their accurate mass and fragmentation pattern with their corresponding standard compounds.

As it was shown in Tables 2, 3, in our established UPLC-Q-TOF-MS/MS conditions, compound schaftoside (1), isoschaftoside (2), 2,4,7-trihydroxy-9,10-dihydrophenanthrene (3), dihydroresveratrol (4), coelonin (5), nudol (6), gigantol (7), erianin (10), and tristin (11) were identified in the four Dendrobium plants. Nine polyphenols in the four Dendrobium plants demonstrated quasi-molecular ions [M + H] ⁺ in positive ion mode. Compounds 1 and 2 exhibited the same molecular ion at m/z 565.1552 [M + H] ⁺. It is suggested that these two components may be isomers. Their fragment ions were different, where compound 1 MS2 ions at 177.0180([M + H] ⁺), 121.0281 ([M + H] ⁺), 191.0326 ([M + H] ⁺), compound 2 MS² ions at 122.0281([M + H] ⁺), 177.0193 ([M + H] ⁺), 189.0879([M + H] ⁺).

The developed HPLC method was applied for quantitative analysis of the 11 investigated compounds in the four Dendrobium species used for preparing Shihu, and the results were summarized in Table 5. Four polyphenols, namely, compounds 1, 2, 4, and 11, were found in all the four Dendrobium species, and the contents of compounds 4 and 11 were very similar in the samples. This finding indicated chemical consistency among the four Dendrobium species. However, the samples also showed substantial variations for other compounds. For example, the content of compound 1 was 33.21 ± 9.34 mg/kg in D. chrysotoxum, about 8–10 times that in D. huoshanense, D. nobile, and D. fimbriatum (Table 5). The content of compound 2 was 67.89 ± 5.54 mg/kg in D. nobile, which was obviously higher than that in D. huoshanense, D. chrysotoxum, and D. fimbriatum. In addition, the total contents of the 11 investigated polyphenols were also quite different (Table 5). Compound 8, which has been reported in D. nobile (18), and compound 9, which has been reported in D. nobile (19) and D. chrysotoxum (20), were not detected in any of the four Dendrobium species in our experiments. Thus, the levels of 8 and 9 might be very low in the four Dendrobium species and may vary with the locality and harvest time of D. nobile and D. chrysotoxum.