Study on the Polar Extracts of Dendrobium nobile, D. officinale, D. loddigesii, and Flickingeria fimbriata: Metabolite Identification, Content Evaluation, and Bioactivity Assay

The polar extract of the Dendrobium species or F. fimbriata (a substitute of Dendrobium), between the fat-soluble extract and polysaccharide has barely been researched. This report worked on the qualitative and quantitative studies of polar extracts from D. nobile, D. officinale, D. loddigesii, and F. fimbriata. Eight water-soluble metabolites containing a new diglucoside, flifimdioside A (1), and a rare imidazolium-type alkaloid, anosmine (4), were identified using chromatography as well as spectroscopic techniques. Their contents in the four herbs were high, approximately 0.9–3.7 mg/g based on the analysis of quantitative nuclear magnetic resonance (qNMR) spectroscopy. Biological activity evaluation showed that the polar extract of F. fimbriata or its pure component had good antioxidant and neuroprotective activity; compounds 1‒4 and shihunine (8) showed weak α-glucosidase inhibitory activity; 4 and 8 had weak anti-inflammatory activity. Under trial conditions, all samples had no cytotoxic activity.


Introduction
"Shi Hu" is the general name for Dendrobium Sw. (Orchidaceae) and F. fimbriata (Orchidaceae; a substitute of Dendrobium) in China; it has been used as a tonic in both medicine and food since ancient times [1,2]. The most notable health benefits of "Shi Hu" are the effects of supplementing the stomach, nourishing the lungs to arrest cough, promoting the production of body fluids, clearing heat, enhancing the body's immunity, anti-aging, and reducing blood sugar levels [3,4]. The chemical components of "Shi Hu" have been widely researched. Diverse metabolites such as alkaloids, bibenzyls, phenanthrenes, sesquiterpenoids, flavonoids, fluorenones, coumarins, and polysaccharides have been isolated [5][6][7]. The chemical profile and quality assessments of "Shi Hu" have also been studied with HPLC (high performance liquid chromatography) and hyphenated analytical techniques [8,9]. The biological activities of "Shi Hu" vary with effect due to the content of the active ingredients [10]. D. nobile, D. officinale, D. loddigesii, and F. fimbriata are four of the most frequently used "Shi Hu". The dried stems of D. loddigesii or F. fimbriata are usually used in clinical medicine. The fresh or dried

Results and Discussion
This work provided a new perspective to further understand the chemical composition of "Shi Hu".

Content and Bioactivity
qNMR has been applied more frequently in natural product areas for its unique advantages: it does not need the references of the determined components, and it exhibits easy operation, non-destructiveness for the determined sample, high accuracy, and repeatability [32][33][34]. These advantages made it possible for us to evaluate the content of water-soluble metabolites using qNMR.
The 1 H-NMR profiles of the four polar extracts are shown in Figure 3. Several differences in the qualitative/quantitative composition of the four plant extracts were evident from the spectral analysis. Comparing the spectra of F. fimbriata ( Figure 3A) and D. officinale ( Figure 3C), similar signals of carbohydrates were observed in the spectral region from 3.0 to 5.5 ppm. F. fimbriata and D. officinale showed some metabolite similarities, with their molecules containing carbohydrate elements; the metabolites of F. fimbriata were glycosides, while the metabolites of D. officinale were fructose. Also, compound 6, a disaccharide, was one common metabolite between F. fimbriata and D. officinale. Comparing Figure 3B,D, it seems that there were no similarities; the major signals of D. nobile were the signals of anosmine (4), and the major signals of D. loddigesii were those of shihunine (8). However, the similarity between D. nobile and D. loddigesii may be that both of them contain a large number of water-soluble alkaloids.  The signals of protons from 1-a to 8-g were chosen as the target peaks for content determination, since they were quite well separated from the others (Figures S19-S23) and were not exchangeable protons [33]; another important reason was that the target peaks had smooth baselines in the 1 H-NMR spectra, which could decrease the measuring error. The chemical shifts and splitting patterns of the target peaks are listed in Table 2. A solution of 0.18 M salicylic acid was used as the calibration for the electronic reference signal ( Figure S24) [35]. The concentrations of water-soluble metabolites in the four crude herbs were determined by comparing the signal intensities of the target proton against the intensity of the reference signal of salicylic acid using 1 H-NMR, and the results are shown in Table 2. The content of 3 could not be evaluated as it was too low to show a suitable signal-to-noise ratio in the 1 H-NMR spectrum of PEF. Compound 7 could not be evaluated by 1 H-qNMR as its proton signals could not be well resolved. In the 13 C-NMR spectrum of PEO, the anomeric carbon signals of three fructose isomers were well resolved ( Figure S22).
The result of the quantitative analysis showed that the content of water-soluble metabolites was high, approximately 0.10-0.38% of the weight of crude drugs. Compared with the literature [8][9][10]15,36], the analysis showed that the water-soluble metabolites may be the most abundant metabolites. Table 3 shows the results of the biological activities evaluation of the polar extracts as well as the water-soluble metabolites including DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging activity, OH radical scavenging activity, inhibitory activity against α-glucosidase, and inhibitory activity against NO production. The result showed that the four polar extracts and their main components had different biological activities. Three polar extracts had DPPH radical and OH radical scavenging effects, except for that of D. nobile. Compounds 1, 4 and 8 had no antioxidant activity. PEO and PEF had moderate α-glucosidase inhibitory activity; compounds 1-4 and 8 had weak α-glucosidase inhibitory activity. Compounds 4 and 8 exhibited weak inhibitory activity against NO production in LPS (lipopolysaccharide)-activated RAW264.7 cells. The protective effect of the extracts and isolated metabolites against glutamate-induced HT22 cell apoptosis was evaluated. PEF and 3 showed good neuroprotection in a concentration-dependent manner ( Figure 4A,B); their EC 50 were 78.5 and 39.5 µg/mL, respectively. All samples had no cytotoxic activity against HeLa cells, HepG2 cells, MDA-MB-231 human breast cancer cells, HT22 cells, and RAW 264.7 cells.
Solvents were of analytical grade (Guangzhou Chemical Reagent Factory, Guangzhou, China); methanol-d 4 (99.9%) was purchased from Aldrich (Milwaukee, WI, USA). Analytical-grade salicylic acid was purchased from Nanjing Reagent (Nanjing, China) and purified by recrystallization with ethanol-H 2 O.

Extraction
The dried stems of F. fimbriata (50 g), dried stems of D. nobile (17.2 g), fresh stems of D. officinale (200 g), and dried stems of D. loddigesii (30 g) were used in the study. The extraction of each sample was performed as follows. Plant sample was ground, and then extracted with a mixture of acetone-water (80:20, v/v) using ultrasonication for 80 min at room temperature. The extraction was repeated three times. The extract was filtered, combined, and evaporated under vacuum to generate the crude extract. Four crude extracts, namely, crude-extract-f (4.30 g) from F. fimbriata, crude-extract-n (1.88 g) from D. nobile, crude-extract-o (4.95 g) from D. officinale, and crude-extract-l (2.77 g) from D. loddigesii, were obtained.

Polar Extract Preparation
The polar extract was enriched from the corresponding crude extract using C-18 SPE, which was performed as follows. The crude extract obtained was dissolved with methanol (about 3 mL) and deposited on a RP-18 column (130 mm × 20 mm Φ). The column that adsorbed the sample was eluted with five bed volumes of H 2 O (20% MeOH-H 2 O for F. fimbriata). The H 2 O eluate was collected and evaporated to generate the polar extract. Four polar extracts, polar-extracts-f (1.80 g) from crude-extract-f, polar-extracts-n (0.78 g) from crude-extract-n, polar-extracts-o (3.65 g) from crude-extract-o, and polar-extracts-l (0.95 g) from crude-extract-l, were obtained.

qNMR Analysis
Samples for qNMR analysis were prepared as follows: 30 mg dried polar extract was weighed out precisely, re-dissolved in 1 mL of methanol-d 4 , and the container of the sample was sealed. Subsequently, the compounds were dissolved using vortex-shaking and centrifuged for 3 min (3000 r/min) to remove the suspended matter. A 600-µL aliquot of the supernatant solution was transferred into an NMR tube and analyzed immediately by 1 H-NMR. The same operation was performed for salicylic acid, which was used as an external standard. All experiments were performed in triplicate. 1 H-NMR spectra were recorded on a Bruker Avance 600 NMR spectrometer. Topspin 3.5 software (Bruker, Germany) was used. The 1 H-NMR quantitative experimental parameters were set based on those described by Tapiolas et al. [32]. Quantification was performed using the magnetic resonance quantitative tool-ERETIC2 (Electronic Reference To access In vivo Concentrations) based on Topspin 3.5 software (Bruker Biospin, Rheinstetten, German) [35].

Biological Assay
The biological activities of the four polar extracts and isolated water-soluble metabolites were evaluated using the following protocol. The methods of DPPH radical scavenging activity and OH radical scavenging activity were used to evaluate the antioxidant activities [37][38][39]. BHT and VC were used as positive controls. Evaluation of the α-glucosidase inhibitory activity was carried out according to the method in the literature [40]. Trans-resveratrol was used as a positive control. The anti-inflammatory activity was investigated using the method of inhibition of nitric oxide (NO) production in RAW 264.7 cells activated by LPS, which were operated as per the literature [41,42]. L-NMMA was used as a positive control. The protective effect against glutamate-induced HT22 cell death was evaluated according to the literature [43]. NAC was used as a positive control. Cytotoxic bioassay was performed using the MTT assay as per the literature [44]. Paclitaxel was used as a positive control. Five cell lines, HeLa cells, HepG2 cells, MDA-MB-231 human breast cancer cells, HT22 cells, and RAW 264.7 cells, were used for the cytotoxic evaluation of all samples.