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Chemical Constituents of Pedicularis longiflora var. tubiformis (Orobanchaceae),
a Common Hemiparasitic Medicinal Herb from the Qinghai Lake Basin, China
Feng Liu
1,2,3
, Zilan Ma
1,2,3
, Marcos A. Caraballo-Ortiz
4
, Hui Zhang
5
,XuSu
1,2,3
and Yuping Liu
1,2,3,*
1
Key Laboratory of Medicinal Animal and Plant Resources of the Qinghai-Tibet Plateau in Qinghai Province, School of Life
Science, Qinghai Normal University, Xining, 810008, China
2
Key Laboratory of Physical Geography and Environmental Process in Qinghai Province, Qinghai Normal University, Xining,
810008, China
3
Key Laboratory of Education Ministry of Earth Surface Processes and Ecological Conservation of the Qinghai-Tibet Plateau,
Qinghai Normal University, Xining, 810008, China
4
Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington DC, 200013-7012, USA
5
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China
Corresponding Author: Yuping Liu. Email: lyp8527970@126.com
Received: 28 April 2020; Accepted: 23 June 2020
Abstract: 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. 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). 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. Six fla-
vonoids were also detected, including isoorientin, orientin, luteolin-7-O-gluco-
side, luteolin, apigenin and tricin. The concentrations of luteolin-7-O-glucoside,
luteolin and tricin were the highest and more concentrated in leaves, while that
of orientin was 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. 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.
This work is licensed under a Creative Commons Attribution 4.0 International License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
Phyton-International Journal of Experimental Botany
DOI:10.32604/phyton.2020.011239
Article
ech
T
PressScience
Keywords: Amino acid content; flavonoids; gas chromatography-mass
spectrometry (GC-MS); liquid chromatography-mass spectrometry (LC-MS);
Qinghai-Tibet Plateau; Tibetan medicine
1 Introduction
Pedicularis longiflora Rudolph var. tubiformis (Klotzsch) Tsoong (Orobanchaceae) is a hemi-parasitic
herb native to southwestern China (Qinghai, Xizang, Western Sichuan, Northwest Yunnan, and Southern
Gansu) found in alpine meadows, marshes, lakes, valleys, streams, and spruce margins from 2700 to
5300 m. 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].
In recent years, there have been several studies focused on identifying the chemical constituents and
pharmacology of P. longiflora var. tubiformis [3–9]. The most relevant advances to date include the
identification of four phenylpropanoid glycosides (echinocoside, pedicularioside A, pedicularioside M and
verbascoside) using capillary electrophoresis [3]. In addition, both Zhang et al. [10] and Deng et al. [11]
purified samples using a method involving silica-gel, macroporous resin, sephadex LH-20 and reversed
phase C
18
columns, and identified seven chemical constituents (apigenin, chrysoeriol, isoverbascoside,
luteolin, luteolin-4’-O-β-D-glucoside, verbascoside, 3,5,7-trihydroxy-3’, and 5’-dimethoxyl flavone) and
10 compounds (echinacoside, iridotrial glucoside, jionoside A1, kankanoside 1, kankanoside H1, luteolin,
pedieularioside I, β-sitosterol, (E)-3-(β-D-glucopyranosyloxy)-5-hydroxy-4’-methoxystilbene, and 5,7,4’-
trihydroxy-3’,5’-dimethoxyflavone) [10,11]. Ma et al. [5] reported 20 additional compounds, while Duo
[7], Zhao et al. [8] and Ma et al. [9] found significant anticancer and antioxidation effects. In spite of all
these findings, we still lack quantitative information on the chemical profiles and concentrations of the
compounds present in different organs of this plant species.
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 Spectrometry (GC-
MS) and Liquid Chromatography-Mass Spectrometry (LC-MS). The use of mass spectrometry will allow
us to compare the biochemical profiles from each tissue and test if there are differences in the compounds
produced by different parts of the plant.
2 Materials and Methods
2.1 Plant Samples
Samples of P. longiflora var. tubiformis were collected from the Xiaopohu wetland (36°42’15”N, 100°
47’07”E; 3210 m) in the Qinghai Lake Basin at Qinghai Province, China, where the species is locally
abundant. The plants in this population had both open flowers and ripe fruits at the time of collection in
July, 2018. A voucher specimen for the species was collected by X.S. (SX-2018-01) and deposited in the
Herbarium of the Northwest Institute of Plateau Biology (HNWP) from the Chinese Academy of
Sciences, Xining at the Qinghai Province.
2.2 Sample Preparation
Fresh samples of P. longiflora var. tubiformis were air-dried in shade for several days to preserve the
integrity of their biochemical compounds. Then, stems, leaves and seeds were separated, grinded into
powder, and stored at –20°C. To identify their chemical profiles, the three powdered tissues were treated
under customized analytical procedures modified from previous studies, which are described below [5,10–14].
For the extraction of amino acids (AA) and organic acids (OA), 15 mg of frozen powder from each tissue
was weighted and transferred 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
1084 Phyton, 2020, vol.89, no.4
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 adjusted to 2.0 using 1 mol·L
–1
HCl. The combined
supernatants were purified three times using an equal volume of diethyl ether-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 [13].
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 each dried tissue powder was weighed and transferred 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. This procedure was repeated a second time using the precipitate of the
first extraction. Both supernatants were combined, centrifuged until dried, and resuspended in 400 µL of
methanol. Finally, the experimental samples were filtered using microporous membranes (0.22 µm) and
used for the mass spectrometry analyses [5,10,13,14]. The relative content of anthocyanins and flavonoids
was calculated from peak areas of samples based on the intensity of the corresponding standard
compounds, which include apigenin, cyanidin, cyanidin-3-rutinoside, isoorientin, kaempferol, kaempferol-
3-rutinoside, luteolin, luteolin-7-O-D-glucopyranoside, orientin, pelargonidin, pelargonidin-3,5-
diglucoside, proanthocyanidin B1, proanthocyanidins, quercetin, quercetin-3-glucoside, rutin, scopoletin,
tricin and verbascoside. For compounds lacking corresponding standards, their quantification was carried
out using similar compounds [15].
2.3 Gas Chromatography-Mass Spectrometry Analysis
The extractions of AA, OA, and soluble sugars described above were evaporated until completely dried
and treated with 50 μL (20 mg/mL) of methoxylamine hydrochloride (Sigma-Aldrich, Saint Louis, Missouri,
USA) in anhydrous pyridine at 30°C for 1.5 h, and then with 80 μL of N-methyl-N-(trimethylsilyl)-
rifluoroacetamide at 37°C for 30 min.
Samples to be analyzed for AA and OA content were dissolved in hexane before the procedure, while
samples for soluble sugar components were dissolved in 200 µl of EtOAc and hexane (1:1). All samples were
processed in a gas chromatography-mass spectrometer (GC-QqQ MS 7890/7000C, Agilent Technologies,
Santa Clara, California, USA) with a fused silica glass capillary (Agilent J&W DB-5 column, 30 m × 250
µm × 0.1 µm film thickness, Agilent Technologies, Santa Clara, California, USA). Injection and interface
temperatures were 260°C and 280°C, respectively. The column temperature gradient was maintained at
80°C for 5 min; 80–210°C for 22 min; 210–300°C for 1 min; and at 300°C for 5 min. The settings used
in the GC-MS equipment for the analyses were ion source of EI and scan mode of TIC.
2.4 Liquid Chromatography-Mass Spectrometry Analysis
For the identification and quantification of flavonoids, 10 mg of frozen powder from each tissue was
treated with a solution of 1 ml 0.1% acetic acid-methanol at 4°C overnight, and then centrifuged for
10 min at 10000 rpm. The supernatant was collected and dried using a vacuum centrifuge concentrator
(CV100-DNA, Aijimu, Beijing, China) and stored at –20°C. The dried extracts were resuspended in
MeOH immediately before being analyzed with an ultra-high-performance liquid chromatography-mass
spectrometry (UPLC-MS/MS, Milford, MA, USA) coupled to a triple-Quadrupole Mass Spectrometry
Phyton, 2020, vol.89, no.4 1085
(XEVO
®
-TQ) with electrospray ionization (ESI). The separation was carried out with a ZORBAX Eclipse
plus C
18
(150 mm × 3.0 mm; Agilent Technologies, Santa Clara, California, USA) with a particle size of
1.8 µm at 40°C. The gradient was prepared using 0.1% formic acid (A) and acetonitrile (B) as the mobile
phases, with time intervals of 0–1 min (5% B), 1–8min(5–30% B), 8–12 min (40–95% B), 16–17 min (95–
100% B), 17–21min(100%B),and21–25 min (5% B). The operating conditions included a flow rate of
1.0 ml·min
–1
, positive ion ESI modes, capillary voltages at 3.0 kV, and 16 L·h
–1
of nebulization nitrogen flow.
3 Results
Our analyses detected 27 AA and OA present in the stems, leaves and seeds of P. longiflora var.
tubiformis. The highest concentrations of AA were found on leaves, while the lowest were detected on
stems. The three AA with higher concentration on leaves were L-asparagine (2933.69 ± 462.41 µg/g),
valine (307.93 ± 30.40 µg/g) and L-alanine (285.44 ± 16.23 µg/g), while the lowest were glycine (19.97
± 0.50 µg/g), cysteine (13.80 ± 1.04 µg/g) and methionine (13.57 ± 0.31 µg/g) (Tab. 1). Regarding seeds,
the most common AA were L-asparagine (3108.85 ± 273.93 µg/g), glutamic (2528.78 ± 167.34 µg/g) and
aspartic (1352.68 ± 41.21 µg/g), whereas the lowest were glycine (21.36 ± 1.41 µg/g), methionine (13.32
± 0.28 µg/g) and cysteine (10.26 ± 0.27 µg/g), following a similar pattern similar to the one observed for
leaves (Tab. 1). Data for the AA content in stems showed that the three most abundant compounds were
arginine (27.92 ± 0.29 µg/g), tyrosine (21.38 ± 0.17 µg/g) and histidine (18.91 ± 0.19 µg/g), while the
Table 1: Comparison of the content of 19 amino acids in the stems, leaves and seeds of Pedicularis longiflora
var. tubiformis
Tissue Stems (µg/g) Leaves (µg/g) Seeds (µg/g)
L-alanine 0.76 ± 0.06c 285.44 ± 16.23a 223.49 ± 8.94b
Glycine 3.65 ± 0.31b 19.97 ± 0.50ab 21.36 ± 1.41a
β-alanine 5.72 ± 0.14b 171.35 ± 21.41a 148.03 ± 4.68ab
Valine 9.00 ± 0.18c 307.93 ± 30.40a 202.35 ± 5.99b
Leucine 6.28 ± 0.13c 61.80 ± 3.27a 41.97 ± 1.26b
Isoleucine 10.54 ± 0.12c 214.87 ± 9.93a 43.49 ± 1.30b
Proline 5.69 ± 0.14c 55.18 ± 5.10a 44.81 ± 1.03b
Methionine 12.13 ± 0.12a 13.57 ± 0.31a 13.32 ± 0.28a
Serine 13.60 ± 0.65c 204.57 ± 6.84a 80.87 ± 1.98b
Threonine 12.61 ± 0.13c 88.17 ± 1.75a 65.06 ± 0.67b
Phenylalanine 15.40 ± 0.12c 120.96 ± 3.02a 32.14 ± 0.30b
Aspartic 8.12 ± 0.03c 138.24 ± 10.21b 1352.68 ± 41.21a
Cysteine 3.07 ± 0.03c 13.80 ± 1.04a 10.26 ± 0.27b
Glutamic 12.52 ± 0.29c 157.05 ± 12.03b 2528.78 ± 167.34a
L-asparagine 12.75 ± 0.24b 2933.69 ± 462.41a 3108.85 ± 273.93a
Lysine 9.68 ± 0.17c 65.82 ± 2.77a 33.64 ± 1.93b
Arginine 27.92 ± 0.29c 162.02 ± 18.86b 621.50 ± 23.03a
Histidine 18.91 ± 0.19c 29.51 ± 4.20b 48.68 ± 2.98a
Tyrosine 21.38 ± 0.17c 61.74 ± 1.88a 48.93 ± 1.88b
Data is expressed following the format “Mean ± Standard Deviation”. Small-case letters along same columns indicate
significant differences among groups (p< 0.05; one-way ANOVA).
1086 Phyton, 2020, vol.89, no.4
three less represented were glycine (3.65 ± 0.31 µg/g), cysteine (3.07 ± 0.03 µg/g) and L-alanine (0.76 ± 0.06
µg/g) (Tab. 1). As for OA in stems, the highest concentrations measured were for fumaric acid (24674.50 ±
795.46 µg/g), malic acid (5467.64 ± 339.42 µg/g) and citric acid (4188.51 ± 158.42 µg/g), while the lowest
were GABA (0 µg/g) and L-pyroglutamic acid (13.80 ± 0.42 µg/g). In leaves, our results indicate that the OA
with highest concentrations detected were again fumaric acid (6136.05 ± 553.03 µg/g), malic acid (2877.79 ±
269.49 µg/g) and citric acid (1658.29 ± 179.29 µg/g), while the lowest were oxalic acid (116.59 ± 0.83 µg/g),
GABA (120.54 ± 5.75 µg/g) and succinic acid (94.87 ± 2.62 µg/g). Last, our measurements of OA in seeds
indicate that the malic acid (1604.00 ± 158.86 µg/g) and citric acid (1092.31 ± 81.33 µg/g) were the most
concentrated compounds detected, while the lowest were oxalic acid (45.68 ± 3.25 µg/g) and GABA
(26.50 ± 2.97 µg/g) (Tab. 2).
In the case of flavonoids, six compounds were detected (Tab. 3). The most concentrated flavonoids
found in stems were tricin (10.18 µg/g) and luteolin (8.85 µg/g), while luteolin-7-O-glucoside (1699.62
µg/g), isoorientin (2.82 µg/g), luteolin (487.74 µg/g), apigenin (188.01 µg/g) and tricin (299.67 µg/g)
showed high levels in leaves. It is interesting that we did not detect traces of orientin in leaves (Tab. 3).
In seeds, the highest concentrations of flavonoids were for tricin (41.39 µg/g) and apigenin (26.29 µg/g),
while the lowest levels were for orientin (0.13 µg/g) (Tab. 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,
galacturonic acid, glucose, glucuronic acid, inositol, sucrose, and trehalose) among stems, leaves and
seeds, except for fructose (p< 0.05; Tab. 4). For instance, the concentration of glucose was the highest in
stems and leaves with 6.7993 ± 0.1296 mg/g and 8.5927 ± 0.4144 mg/g, respectively, and contrasting
with sucrose (25.5663 ± 1.3915 mg/g), which was the highest in seeds (Tab. 4). The monosaccharides
Table 2: Comparison of the content of eight organic acids related to amino acids in the stems, leaves and
seeds of Pedicularis longiflora var. tubiformis
Tissue Citric
acid
Fumaric
acid
GABA L-Pyroglutamic acid Malic acid Oxalic
acid
Oxaloacetic
acid
Succinic
acid
Stems (µg/g) 4188.51 ± 158.42a 24674.50 ± 795.46a 0 13.80 ± 0.42c 5467.64 ± 339.42a 77.97 ± 2.17b 317.65 ± 24.73b 362.40 ± 17.81a
Leaves (µg/g) 1658.29 ± 179.29b 6136.05 ± 553.03b 120.54 ± 5.75a 777.59 ± 29.62a 2877.79 ± 269.49b 116.59 ± 0.83a 729.66 ± 29.93a 94.87 ± 2.62b
Seeds (µg/g) 1092.31 ± 81.33c 416.52 ± 33.52c 26.50 ± 2.97b 237.91 ± 11.79b 1604.00 ± 158.86c 45.68 ± 3.25c 202.12 ± 10.70c 50.97 ± 5.32c
Data is expressed following the format “Mean ± Standard Deviation”. Small-case letters along same columns indicate significant differences among
groups (p< 0.05; one-way ANOVA).
Table 3: Comparison of the flavonoid contents in the stems, leaves and seeds of Pedicularis longiflora var.
tubiformis
Peak
No.
Retention
time/min
λmax/nm Parent
ion
MS
2
Identification
name
Stems
(µg/g)
Leaves
(µg/g)
Seeds
(µg/g)
Reference
1 7.64 347.8, 268.8 449.3 –Isoorientin 0.75 2.82 0.81 Standards
2 7.94 347.8, 255.8 449.3 –Orientin 0.49 Undetected 0.13 Standards
3 8.74 347.8, 253.8 449.2 287.2 Luteolin-7-O-
glucoside
5.33 1699.62 22.30 Standards
4 12.18 347.8, 248.8 287.2 –Luteolin 8.85 487.74 17.87 Standards
5 13.85 336.8, 266.8 271.2 –Apigenin 2.36 188.01 26.29 Standards
6 14.02 347.8, 245.8 331.3 –Tricin 10.18 299.67 41.39 Standards
Phyton, 2020, vol.89, no.4 1087
and oligosaccharides with lowest concentrations were glucuronic acid in seeds (0.0133 ± 0.0009 mg/g) and
galacturonic acid (0.0050 ± 0.0006 mg/g) in leaves, while inositol was not even detected in seeds.
4 Discussion
The chemical profile reported here for AA, OA, flavonoids, and soluble sugars in P. longiflora var.
tubiformis is congruent with the findings reported by previous authors when examined whole plants. In
contrast to previous studies, we have dissected the concentrations and biochemical compounds per tissue,
providing a valuable reference for future studies on the organs with the most medicinal value in this
species. A recent study conducted by Deng et al. [11] on the chemical constituents of a population of P.
longiflora var. tubiformis from Deqin County (Yunnan Province, ca. 900 km SW from our sampling site)
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 [11]. Zhang et al. [10] found that the population of the
same taxon from Gangcha County, Qinghai Province (in the Qinghai Basin, within 100 km from our
sampling site) showed 31 µg/g of luteolin and 20.5 µg/g of apigenin, which resemble the values we
obtained from seeds (Tab. 3)[10]. In the Huzhu Northern Mountain National Forest Park at Qinghai (ca.
100 km NE from our sampling site), Ma et al. [5] reported 10.67 µg/g of luteolin, 8.33 µg/g of apigenin,
and 8 µg/g of tricin. These values are similar to the data we obtained for stems, whose concentrations
were considerably higher in our sampling (Tab. 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). A possible explanation for this trend is that
concentrations are expected to be higher in leaves than in stems and seeds because over 2% of the carbon
fixed during photosynthesis is eventually converted into flavonoids [17]. In humans, flavonoids have
antioxidant effects which eliminate 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].
The differences found in the chemical constituents present among stems, leaves and seeds of P.
longiflora var. tubiformis have complex mechanisms for regulation, and further research is needed on the
specifics on how these processes operate. Interestingly, the chemical compounds identified and quantified
in this study have been reported with biological activities in previous chemical surveys [20–24]. In fact, a
number of the constituents identified we report for P. longiflora var. tubiformis 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 anti-inflammatory activities and attenuate the side effects of
Table 4: Comparison of the content of eight soluble monosaccharides and oligosaccharides in the stems,
leaves and seeds of Pedicularis longiflora var. tubiformis
Tissue Arabitol Fructose Galacturonic acid Glucose Glucuronic acid Inositol Sucrose Trehalose
Stems (mg/g) 0.6580 ± 0.0648b 0.7603 ± 0.0116a 0.1023 ± 0.0020a 6.7993 ± 0.1296b 0.0440 ± 0.0015b 0.1100 ± 0.0069b 0.1937 ± 0.0091c 0.1953 ± 0.0145b
Leaves (mg/g) 1.3323 ± 0.0482a 0.7450 ± 0.0431ab 0.0050 ± 0.0006c 8.5927 ± 0.4144a 0.1363 ± 0.0067a 1.0383 ± 0.0500a 2.3117 ± 0.2631b 0.5910 ± 0.0595a
Seeds (mg/g) 0.2263 ± 0.0069c 0.0733 ± 0.0013b 0.0127 ± 0.0003b 0.4697 ± 0.0320c 0.0133 ± 0.0009c 0 25.5663 ± 1.3915a 0.0767 ± 0.0009c
Data is expressed following the format “Mean ± Standard Deviation”. Small-case letters along same columns indicate significant differences among
groups (p< 0.05; one-way ANOVA).
1088 Phyton, 2020, vol.89, no.4
anticancer drugs; and the soluble sugar D-chiro-inositol, reported to improve insulin resistance and menstrual
cycle in women with polycystic ovary syndrome [20–24].
5 Conclusions
In conclusion, we identified and quantified 27 AA and OA, and six flavonoids and eight soluble sugars
from the stems, leaves, and seeds of P. l o n g i flora var. tubiformis using GC-MS and LC-MS techniques. To
our knowledge, this is the first study exploring the chemical profiles per tissue using mass spectrometry in
this species. Eight of the AA detected here are essential for humans, highlighting the nutritional importance
of this plant and the need to explore in more depth its medicinal properties. This study can also serve as a
reference for further studies on the bioactive compounds of Pedicularis herbs, and to design future studies
applying GC-MS and LC-MS to other important medicinal plants used in the traditional Tibetan medicine.
Author Contribution: Feng Liu and Zilan Ma made the experiment and analyzed the experimental data. Xu
Su and Yuping Liu designed this research as well as drafted the manuscript. Marcos A. Caraballo-Ortiz and
Hui Zhang polished the manuscript.
Acknowledgement: We thank the anonymous reviewers for their helpful comments.
Funding Statement: This work was financially supported by the National Natural Science Foundation of
China (41761009), the Natural Science Foundation of Qinghai Province (2017-ZJ-904), the Scientific
Research Fund of Ministry of Education “Chunhui Plan”(Z2015074, Z2016111), the Key Laboratory of
Medicinal Animal and Plant Resources of the Qinghai-Tibetan Plateau in Qinghai Province (2020-ZJ-
Y40) and the Young and Middle-Aged Research Foundation of Qinghai Normal University (2017-33).
Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the
present study.
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