Profiling of chemical constituents of Matricarla chamomilla L. by UHPLC-Q-Orbitrap-HRMS and in vivo evaluation its anti-asthmatic activity

Matricarla chamomilla L. is native to European countries and widely cultivated in China, especially in Xinjiang. It has been used in Uygur medicine for the treatment of cough caused by asthma. In this study, UHPLC-Q-Orbitrap-MS was used to detect and identify the components from the active fraction of M. Chamomile, 64 compounds were identified by combining the standards, related literatures and mass spectrometry fragments, including 10 caffeoyl quinic acids, 38 flavonoids, 8 coumarins, 5 alkaloids and 3 other compounds. Furtherly, the anti-asthma activity of active fraction of M. Chamomile was investigated in OVA-induced allergic asthma rat model. The results showed that the number of EOS in Penh and bronchoalveolar lavage fluid (BALF) in the group of the active fraction of M. Chamomile was significantly lower than that in the model group. Besides, the active fraction of M. Chamomile can significantly reduce the IgE level and increased glutathione peroxidase (GSH-Px) in the serum of OVA-induced rats, and ameliorated OVA-induced lung injury. Hence, M. Chamomile could be used to treat asthma through their in vivo antioxidant and anti-inflammatory effects. This study explored the potential material basis of M. Chamomile for the treatment of asthma.


Introduction
Asthma is a heterogeneous disease characterized by chronic airway inflammation and is currently among the most common noninfectious diseases in children and adults, affecting approximately 334 million people worldwide [1]. The pathogenesis of asthma is complex, and well-recognized clinical mechanisms are the interaction of chronic airway inflammation and airway hyperreactivity [2]. These lead to variable respiratory symptoms such as wheezing, dyspnea, chest tightness and cough, which can reduced quality of life and even death prematurely [3]. Inflammation plays an important role in the pathophysiology of asthma. Currently, steroidal anti-inflammatory agents combined to bronchodilators are considered as common treatment for controlling asthma [4]. However, its efficacy is limited due to adverse effects and drug dependence or drug resistance. Recently, natural herbal plants and their active ingredients is gaining a lot of attention in the treatment of asthma due to their advantages of high efficiency, few side effects, and low cost. For instance, the extract of Mandevilla longiflora improves airway inflammation in amurine model of allergic asthma, which is rich in phenolic compounds and flavonoids [5]. Besides, plant polyphenols are active ingredients from Chinese herbal medicine or natural food, which have antioxidant, anti-allergic, anti-inflammatory; and previous experiments proved that resveratrol, genistin, luteolin and quercetin have anti-inflammatory effect with the characteristics of multiple targets, multiple links and comprehensive coordination [6]. Hence, many researchers have focused on polyphenol components derived from natural plants as complementary and alternative medicine to against asthma [7].
Matricarla chamomilla L., belong to Asteraceae family, is a native to European countries and and widely cultivated in China, especially in Xinjiang [8]. It has a long history as a spice and as a medical plant to be extensively served in food, cosmetics, and pharmaceutical industries. As a traditional Chinese medicine, M. chamomilla has been reported to have anti-inflammatory, warming the stomach and appetite, promoting digestion, reducing swelling, dispersing knots, antioxidant and anticancer activities, these effects may be associated with natural ingredients, such as flavonoids and phenolic acids [9][10][11][12]. Besides, M. chamomilla is also one of the important components of Zukamu Granule, a prescription recordeded in the Pharmaceutical Quality Standard of the Ministry of Health of the People's Republic of China (Uyghur Medicine Sub-volume), which has been proved to be used for the treatment of common cold or upper respiratory infection in clinical practice [13]. The anti-inflammatory effects of M. chamomilla are well established, and they are broadly used in the management of several respiratory diseases [10,11,14,15]. Thus, M. chamomilla, as a natural resource, are considered to have the pharmaceutical benefit of anti-asthma.
Based on the above, this study aimed to identify the potential active ingredients of M. chamomilla using liquid chromatographymass spectrometry (UHPLC-Q-Orbitrap-HRMS) technology and evaluated for anti-asthmatic activity by using in vivo rat models.

Materials and reagents
Sulfoxide (DMSO) were purchased from Sigma-Aldrich (USA). Formic acid, methanol and acetonitrile for HPLC were obtained from Merck (Darmstadt, Germany). Neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, 1,5-di-O-caffeoylquinic acid, isoquercetin were obtained from Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences. Luteolin, apigenin, hispidulin, galangin, isorhamnetin, narcissin, and isorhamnetin-3-O-glucose were purchased from Shanghai Pureone Biotechnology Co., Ltd (China). Chlorogenic acid, rutin, quercetin and kaempferol were purchased from China National Institute for the Control of Pharmaceutical and Biological Products.

Plant material
M. Chamomile was harvested in early July from Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China and identified as Chamomile (Matricarla chamomilla L.) by Dr. Yonghe Li, Xinjiang Uygur Autonomous Region Hospital of Traditional Chinese Medicine. Fresh chamomile is stored at room temperature and naturally air-dried.

Active fraction preparation
The extract of M. Chamomile was prepared by reflux extraction method [16], and the ethanol concentration, time, and solid-liquid ratio were 70%, 1 h, and 1:10 (g/mL), respectively. The active fraction of M. Chamomile was enriched by AB-8 type macroporous adsorption resin, resin column diameter to height ratio of 1:6, drug solution mass concentration of 0.20 g/mL (equivalent to native drug), the velocity of absorption was 2 BV/h, after absorbed for 7BV, the column was washed by 1 BV water, 3BV 50% ethanol and 1BV 70% ethanol were used to elute the active components.

UHPLC-Q-Orbitrap-HRMS analysis
Chemical constituents of the active fraction of M. Chamomile were analyzed by UHPLC-Q-Orbitrap-HRMS. The dry active fraction (10 mg) was dissolved in methanol and filtered through 0.22 μm microporous membrane to obtain the sample solution (10 mg/mL).

Animal
Forty clean grade SD (Sprague Dawley) rats, body mass 220 ± 20 g, male and female, were provided by the Experimental Animal Centre of Xinjiang Uygur Autonomous Region. During the experiments, rats were fed and watered freely and were housed in a standard clean grade animal laboratory at a room temperature of 20-25 • C and a 12 h light/dark cycle. The experimental procedures of the animal studies were permitted by the Institutional Animal Care and Use Committee of Xinjiang Medical University (approval number: IACUC-20210301-23).

Establishment of asthma rat model
The asthmatic models of rats were established by ovalbumin (OVA). 40 SD rat animals were randomly divided into five groups: Group N (normal control group), Group M (OVA sensitization group), Group D (OVA + chamomile anti-asthmatic active site low dose group), Group Z (OVA + chamomile anti-asthmatic active site medium dose group) and Group G (OVA + chamomile anti-asthmatic active site high dose group), 8 animals in each group. OVA sensitization: Except for the normal control group, each rat was injected intraperitoneally with 1 mL OVA suspension (containing 1 mg OVA and 200 mg aluminium hydroxide gel) on days 1 and 8, and the normal control group was injected with the same dose of saline as a control.
OVA excitation: One week after the second sensitization, rats were placed in an ultrasonic nebulizer from 15th day, and stimulated by spraying saline aerosol containing 1% OVA with an ultrasonic nebulizer for 28 days, once a day and 30 min each time. The rats in the normal control group were nebulized with the same amount of saline. After stimulated, the rats were observed for signs of asthma attack such as irritability, sneezing, coughing, nasal scratching, incontinence, deepening of respiratory amplitude, wheezing and cyanosis, etc. Asthma modelling was considered successful.

Pharmacological interventions
From day 43 to day 72, rats in each group received the following interventions: Group N: saline gavage; Group M: saline gavage; Group D: low dose gavage (0.06 g/kg dose) of the anti-asthmatic active site of chamomile; Group Z: medium dose gavage (0.12 g/kg dose) of the anti-asthmatic active site of chamomile; Group G: high dose gavage (0.18 g/kg dose) of the anti-asthmatic active site of chamomile. (0.18 g/kg dose).

Behavioural observation
The OVA-induced allergic asthma rat model was established, and its behaviour was scored according to the "Asthma Attack Scale" to evaluate the presence or absence of symptoms of cyanosis, wheezing, shortness of breath, slow movement or twisting, agitation, etc. The asthma behavioural scale [17]: 0 points for normal or mild shortness of breath; 1 point for trembling or nodding; 2 points for coughing, marked shortness of breath, restlessness and cyanosis; 3 points for rhythmic retracted wheezing; 6 points for extreme respiratory distress with prostration or fall.

Pulmonary function and level of total Ig E and glutathione peroxidase
Pulmonary function was measured by the Buxco Whole Volume Tracing System, USA (USA), according to reference [17]. 10% chloral hydrate intraperitoneal anesthesia, blood was taken from the abdominal aorta, centrifuged at 3500 r/min for 15 min, serum was taken and stored at − 80 • C. The total IgE content in the serum of each group of rats was determined by rat ELISA kit, and the GSH-Px content was determined by Glutathione peroxidase (GSH-Px) kit.

Collection bronchoalveolar lavage fluid (BALF) and eosinophil count
After blood taken from the abdominal aorta of the rat, the skin of the neck was cut longitudinally, the trachea was exposed, the chest cavity was opened to expose both lungs, the right main bronchus was ligated first; then a trocar needle was inserted into the trachea and ligated and fixed, 4 mL of sterile saline was instilled into the left lung, BALF was recovered after gentle massage of the lungs for 30 s, and the procedure was repeated three times (recovery rate 80%-90%). The supernatant was centrifuged at 3500 rmp for 5 min and stored at − 80 • C. The sediment was resuspended in 0.5 mL Hanks' solution, and 0.2 mL suspension was used to evaluate eosinophil count in a small animal haemocytometer.

Histopathological observation of lung
After collection of blood and BALF, the right lung was cut out and fixed in 10% neutral formaldehyde; the tissues were paraffin embedded and cut into sections at a thickness of 3 μm, then these were stained with hematoxylin-eosin (HE) to observe histopathology and inflammatory cell infiltration.

Statistical analysis
SPSS® statistical software (version 11.0 for Windows) was used for analysis, and experimental data were expressed as mean ± standard deviation (mean ± SD). The behavioral observation scores of the rats in each group were compared by rank sum test; the enhanced respiratory intervals, eosinophil counts, serum IgE and GSH-PX levels of the rats were compared by one-way analysis of variance (ANOVA). P < 0.05 indicated that the differences were statistically significant.

Optimization of Q-Orbitrap-HRMS conditions
The mass spectra were evaluated in positive and negative ionization modes. The result showed that phenolic acids and flavonoids ionized well in the negative ionization mode, while alkaloids generated better signal intensities in positive ionization mode. In addition, there was little difference between positive and negative ion scanning of lignin compounds (Fig. 1). In the secondary mass spectrometry acquisition, the NCE was set to low, medium and high modes, which were 20, 40, 60 eV, respectively. Hence, rich fragment ion information of known and unknown compounds was collected widely.

Identification of chemical constituents
The active fractions of M. Chamomile were analyzed by UHPLC-Q-Orbitrap-HRMS. Considering the retention time (t R ), precise mass-to-charge ratio (m/z), characteristic fragment ions, as well as available standards and previous literatures, Considering the retention time (t R ), precise mass-to-charge ratio (m/z), characteristic fragment ions, as well as available standards and previous literature, 64 compounds were identified (Table 1), mainly including caffeoylquinic acid, flavonoids, coumarins and alkaloids.    Fig. 2C, [25]. The base fragment ion peak at m/z 285 was shown in the secondary mass spectra of compounds 11, 25, 26 and 28, which was the characteristic fragment ion of luteolin. Therefore, Therefore, this confirmed the mother nucleus of these compounds was lignocerebrosides. Based on the retention times of compounds 11 and 26 and the fragment ions with the standards, these two compounds were identified as lignocerebroside-8-C-glucoside and lignocerebroside (Cynaroside, Fig. 2D   The fragment ions at m/z 255, 227, and 151 indicate that the parent nucleus was a quercetin glycogen. Therefore, these compounds are presumed to be quercetin-O-glucoside and its isomers. Based on the above mass spectral information and the agreement of retention times with the standards, compounds 12, 13 and 15 were deduced to be quercetin-3-O-glucoside (isoquercitrin), chrysin, and quercetin-7-O-glucoside, respectively [24]. The position of the sugar was probably 3 ′ or 4 ′ glucoside, but this could not be confirmed based on the current mass spectrometry information and was tentatively identified as quercetin-O-glucoside and its isomers.  /z 255, 227, 163, and 151. Therefore, these compounds presumed to be kaempferol-O-glucoside and its isomers based on the above mass spectral information and reference [30]. According to retention time with the standard, compound 21 was identified as kaempferol-3-O-glucoside (astragalin).
Compounds 31 (t R = 33.38 min), 32 (t R = 34.97 min) and 43 (t R = 55.12 min) presented the molecular ion at m/z 491, 491, and 315, respectively. There were more abundant fragment ions at m/z 315 and 300 in the secondary mass spectra. However, the retention time of compound 43 was not consistent with that of isorhamnetin. Therefore, compounds 31, 32 and 43 were inferred to be methylquercetin-O-glucuronide, methylquercetin-O-glucuronide isomer and methylquercetin, respectively, based on the available mass H-CH3OH-CO] + . Based on the above mass spectrometric information and previous literature, the compound was identified as a hydroxy-methoxy-coumarin and its isomers [34].
Compounds 53 (t R = 16.13 min), 54 (t R = 48.51 min), 55 (t R = 49.97 min) and 56 (t R = 68.30 min) showed the molecular ion m/z 147, 163, 325 and 177 [M+H] + , respectively. The fragment ion at m/z 119 and 91 as well as retention time were consistent with the standard, hence compound 53 was identified as coumarins. The MS 2 spectra of compounds 54, 55 and 56 all had fragment ions at m/z 145, suggesting that the parent nucleus was coumarin. Based on the mass spectral information and literature reports, compounds 54, 55 and 56 were deduced to be hydroxycoumarin, hydroxycoumarin (inocyanin) and methoxycoumarin, respectively [35].

(4) Alkaloids
Five indole alkaloids were identified in the active fraction of M. Chamomile, and fragment ions of MS 2 spectra at m/z 117 or 118, representing the parent nucleus was the indole alkaloids. Under higher collision energies, the parent nucleus of the indole alkaloids produce characteristic fragment ions at m/z 91 and 77. These characteristic fragment information can be used for the determination of indole-like alkaloids [36]. Compound 57 (t R = 4.36 min) represented molecular ion m/z 176 [M+H] + , and the structural formula was C 10 H 10 O 2 N. The fragment ion at m/z 117 [M + H-59] + was observed in the MS 2 spectrum, indicating the loss of the acetic acid. Therefore, the compound was deduced to be indole-acetic acid.
Compounds 58 (t R = 4.60 min) and 60 (t R = 5.37 min) had molecular ion peaks [M+H] + at m/z 146, and the structural formula was C 9 H 6 ON. The fragment ion at m/z 118 [M + H-28] + was observed in the MS 2 spectra, indicating the loss of a formyl group. The fragment ions at m/z 91 and 77 were the characteristic fragment information of the indole-like parent nucleus. Therefore, compounds 58 and 60 are inferred to be indole-carbaldehyde and its isomers.
Compounds 59 (t R = 4.16 min) and 61 (t R = 5.09 min) showed same molecular ion peak at m/z 188 [M+H] + , with a structural formula of C 11 H 10 O 2 N. The fragment ion at m/z 146 [M + H-42] + was observed in the MS 2 spectrum, suggesting that the structure contained an acetyl group; and the fragment ion at m/z 118 [M + H-42-28] + , suggesting further loss of the formyl group. Also, indolelike characteristic fragment ions such as m/z 91, 77 were observed. Based on the above information, compounds 59 and 61 were inferred to be acetylindole formaldehyde and its isomers, respectively.

(5) Other compounds
Compounds 62 (t R = 1.05 min) and 64 (t R = 8.36 min) showed same molecular ion peaks at m/z 191 [M − H] -, and molecular formulas were C 6 H 9 O 7 and C 7 H 13 O 6 , respectively. Based on the retention time and fragmentation information with the standard, compound 62 and 64 were dentified as citric acid and quinic acid [37,38].
Compound 63 (t R = 8.12 min) showed a molecular ion peak [M − H]at m/z 179, and fragment ions were observed in the secondary mass spectrum at m/z 135, indicating the loss of CO 2 . Based on retention time and mass spectral information consistent with the standard, it was confirmed as caffeic acid [39].

Behavioural observations of the rats in each group
The results are shown in Table 2 and Fig. 3 that the behavioural scores of the model group were significantly higher than those of the normal control (P < 0.005); administration group was reduced in turn compared with the model group in a dose-dependent manner (P < 0.005).

Effect of the active fraction of M. Chamomile on the enhanced respiratory intervals (Penh) in asthmatic rats
The results were shown in Table 3 that Penh was significantly higher in the model group compared to the normal control group (P < 0.01); compared with the model group, Penh of administration groups was reduced significantly in rats (P < 0.01).

The effect of the active fraction of M. Chamomile on serum IgE in rats with bronchial asthma
The results were shown in Table 4 and Fig. 4 that the serum IgE level was significantly increased in the model group (P < 0.005), compared with the normal control group; and, compared with the model group, the middle and high dose groups of the active fraction of M. Chamomile could significantly reduce the IgE level in rats (P < 0.01, P < 0.005).

Effect of the active fraction of M. Chamomile on eosinophil counts in BALF of rats with bronchial asthma
The results were shown in Table 5 and Fig. 5 that the eosinophil count in BALF of the model group was significantly higher than that of the normal control group (P < 0.01); the eosinophil count was reduced significantly in BALF of rats in the middle and high dose groups of the active fraction of M. Chamomile, compared with the model group (P < 0.01).

Effect of the active fraction of M. Chamomile on serum glutathione peroxidase (GSH-Px) in rats with bronchial asthma
The results were shown in Table 6 and Fig. 6 that the serum GSH-Px levels in model group were significantly lower than that of the normal control group (P < 0.005); and compared with the model group, the serum GSH-Px levels of administration group was increased significantly in a dose-dependent manner (P < 0.005).

Histopathological analysis of the lungs
The results were shown in Fig. 7. The lung tissue of the rats in the blank group was basically normal in morphology, with no inflammatory cell infiltration around the bronchi and blood vessels, while occasional secretions and exfoliated cells in the lumen of the bronchi. In the model group, inflammatory cells were seen around the bronchi and blood vessels, and blood vessels were congested, and eosinophilic infiltration was seen in the lung tissue of rats. In the low dose group, inflammatory cell infiltration and vascular congestion were seen around the bronchi and blood vessels of the lung tissue of rats, and a small amount of eosinophil infiltration was seen in the lung tissue of rats. The degree and scope of inflammatory cell infiltration such as lymphocytes and eosinophils around the bronchi and blood vessels of lung tissue in the middle and high dose groups of Chamomile Anti-Asthma Active Part were significantly reduced compared with the model group.

Discussion and conclusion
Asthma is a life-threatening chronic airway disease. There are about 300 million people suffering from asthma in the world, and this number is increasing at an alarming level, reaching an annual asthma mortality rate of more than 250,000. T cells associated with asthma are considered to be the key pathogenic factors of inflammatory response in asthma, which release various Inflammatory  1  0  3  2  1  1  2  1  3  1  0  1  3  1  2  1  2  0  4  1  2  2  1  2  5  0  2  1  1  0  6  1  3  1  2  1  7  0  3  2  1  2  8  0  3 Fig. 3. Behavioral score of rats (means, n = 8). Compared with the normal control group, ***P < 0.005; Compared with the model group, ### P < 0.005.  cytokine, such as interleukin (IL-2, IL-4, IL-5, IL-10, IL-13, etc.), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and specific allergens produced by activated B lymphocytes [40]. Goblet cells [41] produce excessive mucus, bronchospasm, reversible airway obstruction and airway inflammation caused by eosinophil infiltration, in addition, it will increase airway smooth muscle contraction and mast cell degranulation to release histamine, causing a series of asthma symptoms. In addition, these inflammatory cells secrete reactive oxygen species (ROS) and induce oxidative stress and inflammatory process, which is considered as the main potential cause of asthma [42]. In this study, UHPLC-Q-Orbitrap-HRMS coupling technique was used to the separation and identification of the components in the active fraction of M. Chamomile. A rapid and effective systematic identification method of chemical components of chamomile was established the separation and identification of the components in the active fraction of M. Chamomile. The chromatographic peaks of quasi-molecular ions were obtained, 64 compounds were identified by combining the standards, related literatures and secondary mass spectrometry fragments, including 10 caffeoyl quinic acid compounds, 38 flavonoids, 8 coumarins, 5 alkaloids and 3 other compounds. This study demonstrated that the active fraction of M. Chamomile contained abundant polyphenol compounds, especially flavonoids, which have been said to account for most of the antioxidant activities and anti-inflammatory effects of plant extracts [6,43,44]. Previous studies found that luteolin, apigenin, quercetin, kaempferol and their related compounds had significant inhibitory effect on IL-4 synthesis and TNF-α [45]; luteolin, quercetin and baicalein can respond to high affinity IgE receptor through inhibit the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) [46]. Flavonoids are antioxidants and anti-allergic nutrients that inhibit the release of chemical mediators and synthesis of Th2 type cytokines, several epidemiological studies suggest that it is beneficial for asthma to increase in flavonoid intake [47]. Chlorogenic acid can inhibite pulmonary eosinophilia, IgE production, and Th2-type cytokine production in the lung of mice with allergic asthma induced by ovalbumin [48]. Coumarin and coumarin-related Table 5 Effect of active fraction of M. Chamomile on eosinophils count in bronchoalveolar lavage fluid (BALF) in rats (mean ± SD, n = 8).  Compared with the normal control group, **P < 0.01; Compared with the model group, ## P < 0.01.

Table 6
Effect of active fraction of M. Chamomile on serum GSH-Px in bronchial asthma rats (mean ± SD, n = 8). compounds are also potential anti-inflammatory agents due to inhibition of cyclic nucleotide phosphodiesterases that generate cAMP and cGMP increasing [49]. These previous researchs indicated that polyphenol compounds from the active fraction of M. Chamomile were effective components against asthma, especially flavonoids were the important bioactive components, mainly including luteolin, quercetin, kaempferol and their related compounds. Hence, the identification results provide a basis for clarifying the active fraction of M. Chamomile and the quality control of related new drug of anti-asthma development process. Furtherly, the OVA-induced allergic asthma rat model was established for the purpose of investigating the anti-asthma effect of the active fraction of M. Chamomile. The results showed that the number of EOS in Penh and bronchoalveolar lavage fluid (BALF) in the active fraction of M. Chamomile was significantly lower than that in the model group. IgE has been the immunoglobulin traditionally linked to an allergic response in conditions such as asthma; and enzyme-linked immunosorbent assay (ELISA) was used to detect the serum IgE level. The active fraction of M. Chamomile can significantly reduce the serum IgE level of asthmatic rats induced by OVA. In addition, compared with the asthma model group, the active fraction of M. Chamomile significantly increased glutathione peroxidase (GSH-Px) in the serum and ameliorated lung injury significantly. The results suggested that the active fraction of M. Chamomile had  inhibitory effects on airway inflammation, hyperresponsiveness and oxidative stress in asthmatic rat.
Overall, combing with retention time, mass data, standards and information reported previously, 64 compounds were identified from the active fraction of M. Chamomile by UHPLC-Q-Orbitrap-HRMS technology, including 10 caffeoylquinic acids, 38 flavonoids, 8 coumarins, 5 alkaloids and 3 other compounds. Flavonoids were the important bioactive components of M. Chamomile against asthma, especially luteolin, quercetin, kaempferol and their related compounds. And, the active fraction of M. Chamomile possessed potent antiinflammatory and anti-oxidative effects to exerts protective effects against OVA-induced asthmatic symptoms. Hence, M. Chamomile could be used in the future treatment of asthma, and future research is required to explore the underlying mechanisms.