Chemical constituents of Pedicularis longiflora var. tubiformis (Orobanchaceae), a common hemiparasitic medicinal herb from the Qinghai Lake Basin, China

Background: Pedicularis longiflora var. tubiformis (Orobanchaceae) is an abundant parasitic herb mainly found in the Xiaopohu wetland of the Qinghai Lake Basin in Northwestern China. The species has an important local medicinal value, and in this study, we evaluated the chemical profile of its stems, leaves and seeds using mass spectrometry. Methods: Dried samples of stems, leaves and seeds were grinded, weighted, and used for a series of extractions with an ultrasonic device at room temperature. The chemical profiles for each tissue were determined using Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS). Results: Twenty-seven amino acids and organic acids were identified and quantified from stems, leaves and seeds. The content of amino acids detected in leaves and seeds was higher than the amount found in stems. Eight flavonoids were also detected, including isoorientin, orientin, luteolin-7-O-glucoside, verbascoside, scopoletin, luteolin, apigenin and tricin. The concentrations of verbascoside, luteolin and tricin were the highest and more concentrated in leaves, while that of orientin and scopoletin were the lowest and mainly found in stems. Soluble monosaccharides and oligosaccharides below tetramer were also examined, and our analyses detected the presence of arabitol, fructose, galacturonic acid, glucose, glucuronic acid, inositol, sucrose, and trehalose. Conclusions: This is the first study to identify and quantify the main components of amino acids, organic acids, flavonoids and soluble sugars from stems, leaves and seeds of P. longiflora var. tubiformis . Eight of the amino acids detected are essential for humans, highlighting the medicinal importance of this species. Results shown here can be used as a reference case to develop future studies on the chemical constituents of Pedicularis herbs and other medicinal plants from the Tibetan region.

Yunnan, and Southern Gansu) found in alpine meadows, marshes, lakes, valleys, streams, and spruce margins from 2700 to 5300 m. 1 This plant is considered as one of the most important herbs used in the traditional medicine from Tibet, where it has been used to cool, hydrate and detoxify the body. strengthen tendons, and as a fixing essence [1,2].
The objective of this study was to identify and quantify the chemical constituents present in stems, leaves, and seeds of P. longiflora var. tubiformis using Gas Chromatography-Mass 3 Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS). The use of mass spectrometry will allow us to compare the biochemical profile from each tissue and test if there are differences in the biochemical compounds produced by different parts of the plant.

Plant samples
Samples of P. longiflora var. tubiformis were collected from the Xiaopohu wetland

Sample preparation
Fresh samples of P. longiflora var. tubiformis were air-dried in the shade for several days to preserve the integrity of their biochemical compounds. Then, stems, leaves and seeds were separated and grinded into powder, which was stored at -20 °C. Powdered tissue from each of the three tissues were treated under analytical analyses modified from previous studies (described below) to identify their chemical compounds [5,[10][11][12][13].
For the extraction of amino acids (AA) and organic acids (OA), 15 mg of frozen powder from each tissue was weighted and transferred it into 2 ml microtubes containing 800 µl of an 80% methanol-distilled water solution. After adding the internal standard "Norvaline", the extractions were performed with the assistance of an ultrasonic device (TGCXZ-2B, Hongxianglong Technology Company, Beijing, China) at 4 C for about 1 h, and centrifugated for 10 min at 12000 rpm. The supernatant was transferred into a 2 ml microtube, and a second extraction was conducted using the precipitate. The supernatants from the first and second extractions were combined and its pH was adjusted to 2.0 using 1 mol·L -1 HCl.
The combined supernatants were purified three times using an equal volume of diethyl ether-4 petroleum ether (1:1), and the soluble phase was retained. The soluble phase was then centrifugated and concentrated using a vacuum chamber, transferred to microtubes, dried with the vacuum chamber, and stored at -20 °C [12].
To extract soluble sugars from samples, 15 mg of dried powder from the three tissues were weighted and transferred into 2 ml microtubes. Then, the extraction was performed using 800 µL of 80% methanol-distilled water in the ultrasonic device at room temperature for 1 h, and preserved overnight at -4 °C. The next day, residues were extracted again using 800 µL of 80% methanol-distilled water, and both extractions were combined and analyzed for soluble monosaccharides and oligosaccharides.
To isolate total flavonoids, 80 mg of dried tissue powder from each tissue were weighed and put it into 2 ml microtubes with 1 ml methanol containing 0.1% ethylic acid. The extraction was performed using an ultrasonic device at 4C for about 1 h, centrifuging samples for 10 min at 12000 rpm, and transferring the supernatant into 2 ml microtubes. The extraction was repeated a second time using the precipitate. Both supernatants were combined and centrifuged until dried, and resuspended in 400 µL methanol. Finally, the experimental samples were filtered using microporous membranes (0.22 µm) and used for the mass spectrometry analyses [5,10,12,13].

Gas Chromatography-Mass Spectrometry analysis
The extractions of AA, OA, and soluble sugars described above were evaporated until completely dried, and treated with 50 L (20 mg/mL) of methoxylamine hydrochloride
Regarding seeds, the most common AA were L-asparagine (3108.85 ± 273.93), glutamic (2528.78 ± 167.34) and aspartic (1352.68 ± 41.21), whereas the lowest were glycine (21.36 ± 1.41), methionine (13.32 ± 0.28) and cysteine (10.26 ± 0.27), following a similar pattern similar to the one observed for leaves (Table 1). Data for the AA content in stems showed that 6 the three most abundant AA were arginine, tyrosine and histidine, while the three less represented were glycine, cysteine and L-alanine ( In the case of flavonoids, eight compounds were detected ( Table 3). The most concentrated flavonoids found in stems were orientin and scopoletin, while luteolin-7-O-glucoside, verbascoside, isoorientin, luteolin, apigenin and tricin showed high levels in leaves. It is interesting that we did not detect traces of orientin in leaves (Table 3). In seeds, the highest concentrations of flavonoids were for verbascoside, tricin and apigenin, while the lowest levels were found in Scopoletin and Orientin (Table 3).
About the types of monosaccharides and oligosaccharides present per tissue type, a multiple comparative analysis detected significant differences in the content of eight of them (arabitol, fructose, glucose, inositol, sucrose, trehalose, glucuronic acid and galacturonic acid) among stems, leaves and seeds, except for fructose (P < 0.05; Table 4). For instance, the concentration of glucose was the highest in stems and leaves with 6.7993 ± 0.1296 and 8.5927 ± 0.4144, respectively, and contrasting with sucrose, which was the highest in seeds ( Table 4).
The monosaccharides and oligosaccharides with lowest concentrations were glucuronic acid in stems and galacturonic acid in leaves, while inositol was not even detected in seeds.

Discussion
The chemical profile reported here for P. longiflora var. tubiformis is congruent with the findings reported by previous authors using the whole plant. For example, Deng et al (2017) studied the chemical constituents of a population of P. longiflora var. tubiformis located in Deqin County (Yunnan Province, ca. 900 km SW from our sampling site) and found that the content of luteolin there was 12.55 µg/g, while the content of tricin was 9.85 µg/g, which is close to the values we obtained from stems [14]. Zhang et al (2012) found that the population of P. longiflora var. tubiformis from Gangcha County, Qinghai Province (in the Qinghai Basin within 100 km from our sampling site) showed 31 µg/g of luteolin, 20.5 µg/g of apigenin, and 74.5 µg/g of verbascoside, which are closer to the values we obtained from seeds except verbascoside, that our values were much higher ( µg/g of verbascoside [5]. These values resemble the data we obtained for stems, except for verbascoside whose concentrations were considerably higher in our sampling (Table 3).
In spite of having a substantial number of earlier studies exploring the chemical constituents of P. longiflora var. tubiformis, none of them measured the compounds and concentrations present in individual parts of the plant. Therefore, we cannot draw effective comparisons between our results and the ones presented in previous studies. Regarding the discrepancies observed in the type and concentrations of AA identified in each of the three tissues investigated, we speculate that these might be related to growth stages and metabolic activities of sampled plants, environmental and/or genetic factors [16]. For example, we found that the content of flavonoids in leaves was relatively higher than the one measured in seeds and stems (except for orientin and scopoletin). A possible explanation for this trend is that, as over 2% of the carbon fixed during photosynthesis is eventually converted into flavonoids, concentrations are expected to be higher in leaves than in stems and seeds, as reported for other plants [17]. In humans, flavonoids have antioxidant effects, eliminating free radicals in the body [18]. For instance, luteolins can inhibit the proliferation of cancer cells, resist inflammation and oxidation, and reduce the damage caused by excessive reactive oxygen [19]. 11 The differences found in the chemical constituents present in stems, leaves and seeds of P.
longiflora var. tubiformis have complex mechanisms for regulation, and further research is needed on how these processes operate. However, it is possible that some of the chemical compounds here reported show similar biological activities in humans as those described in prior studies using other plant species. In fact, a number of the constituents identified in this study have important medical applications such as the OA GABA, which can be used as an anticancer drug, the flavonoid luteolin-7-O-glucoside, that can increase anti-oxidant and antiinflammatory activity, and the soluble sugar D-chiro-inositol, reported to improve insulin resistance and menstrual cycle in women with polycystic ovary syndrome [20][21][22].

Conclusions
In conclusion, we identified and quantified 27 AA and OA, and eight flavonoids and soluble sugars from the stems, leaves, and seeds of P. longiflora var.