Quisqualis indica Linn.: HRLCMS/MS profiling and anti-asthma activity of leaf extracts

Background Asthma is a chronic inflammatory disorder of the airways, involving various cells and cellular elements precisely eosinophils, mast cells, neutrophils, T lymphocytes, epithelial cells, and macrophages. Worldwide, about 300 million people are affected by asthma, and is expected that 100 million people may get affected, in 2025. Quisqualis indica is commonly planted as an ornamental plant in India; and has medicinal uses. Therefore, the petroleum ether (60–80 °C) LPE and methanolic extract LME of the Quisqualis indica leaves were analyzed for anti-asthmatic activity by mast cell degranulation and Milk induced eosinophilia and leukocytosis in mice. Results It was found that Quisqualis indica leaf extracts exhibited protection against the degranulation of mast cells and a reduction in the difference count of leucocytes and eosinophils. LPE and LME (400 mg/kg) have shown 33% and 63% of mast cell protection. LME has shown the most significant mast cell stabilizing action comparable with the standard drug. The extracts decreased the difference count of leucocytes and eosinophils. LME (400 mg/kg) has shown a difference in eosinophil count and a decrease in leukocyte count most comparable with the standard Dexamethasone. Methanolic extract analyzed for phytochemicals by High Resolution Liquid Chromatography Mass Spectroscopy /Mass Spectroscopy method showed the presence of various Phyto-compounds. Conclusion From the analysis of methanolic leaf extract of Quisualis indica revealed the presence of phytocom-pounds such as Apigenin7-glucoside, Gallic acid, Quercetin, Quercitrin, Kaempferol, etc. The significant decrease in eosinophil and leukocyte count in animals might be due to the higher content of Tannin and flavonoids. A reduction in leukocyte and eosinophil is regulated by type 1 hypersensitivity and adaptogenic factors hence Quisqualis indica is effectively helpful in allergy conditions like asthma.


Background
Asthma is a chronic inflammatory disorder of the airways, involving various cells and cellular elements, precisely eosinophils, mast cells, neutrophils, T lymphocytes, epithelial cells, and macrophages.Asthma is characterized by hindrance of airflow due to inflammation, bronchospasm, and increased airway secretions.[1] About 300 million people worldwide suffer from asthma, and by 2025, it's predicted that 100 million more will likely develop the condition.The prevalence, severity, and mortality of asthma are greatly affected by geographical variation.Asthma incidence is greater in high revenue countries, and maximum death caused by asthma happens in low middle revenue countries [2].
The multifaceted condition known as asthma is brought on by complex relationships between the environment and heredity.The pathophysiology of this condition includes intermittent airflow restriction, bronchial hyperresponsiveness (BHR), inflammation of the airways and bronchial remodeling.Depending on the severity of the condition, bronchodilators and anti-inflammatory medications can be used to treat asthma.Then, additional treatments are still required to better control asthma, and herbal remedies may be a viable substitute for current synthetic medications [3].Additionally, plant polyphenols are potent components of natural foods and herbal medicine that have anti-inflammatory, antioxidant, and anti-allergic properties.Previous studies have demonstrated the anti-inflammatory effects of resveratrol, genistin, luteolin, and quercetin, as well as their multiple targets, multiple links, and comprehensive coordination [4].
The plant Quisqualis indica Linn.(Combretaceae) is an evergreen plant planted in homes and gardens of countries like India, China, Australia, the Amazonian region in Peru, and Pakistan.It is a large sub-woody climber shrub with 3-8 m long branches.Leaves are simple, oppositely having an oblong-elliptic to elliptic shape.It bears the inflorescence with white to pink-colored flowers.Flowers appear in constant succession in sagging clusters with a sweet scent [5,6].
The leaves of Quisqualis indica were used to isolate four crystalline components, which were later identified as nicotinic acid and methylbetaine (trigonelline), L -proline, L -asparagines and potassium quisqualate [7].Triterpenoids, flavonoids and tannins were isolated from the petroleum ether and methanolic extract of leaf and flower, respectively [8].Ethno-pharmacologically, the flowers, fruit, seeds, leaves, stem and roots of the plant are used.Leaves are used to relieve pain caused by fever.Leaves decoction is given for flatulent distension and pain in the abdomen.Leaf juice is used to heal boils and ulcers [9].Fruits and seeds are used in folk medicine as anthelmintic, anti-emetic, and anti-diarrhoea [10].

Graphical abstract
The methanolic leaf extract shown most significant anti-asthma activity, might be due to the presence of flavonoids and tannins.Quisqualis indica plant immunomodulatory [17], anti-diarrheal [18], etc. due to the existence of various phytoconstituents.The stem bark and flowers were evaluated for the total tannin content and anti-oxidant activity [19].Roots are used to treat rheumatism, cough and hiccup by Philippines [3,20,21].

QUISQUALIS
As the whole plant is used as cough cure [10]; in the current work, we have looked into the phytoconstituents and anti-asthma activity of the Quisqualis indica leaves.The leaf petroleum ether extract (LPE) and leaf methanolic extract (LME) of Quisqualis indica were screened for phytochemicals by High Resolution Liquid Chromatography Mass Spectroscopy /Mass Spectroscopy (HRLCMS/MS) and anti-asthma activity.In the case of Quisqualis indica extract, which is high in flavonoids and phenolic compounds, reduces inflammation [14] and act as an immunomodulatory.[17]

Collection and preparation of plant material
The fresh leaves of Quisqualis indica Linn.plant were collected from the residential region in the Indian state of Maharashtra near Nashik.The plant was verified by the India Botanical Survey, which included Pune and Maharashtra [22,23].The allotted authentication No. was BSI/WRC/IDEN.CER/2016/403 (A).The pulverized and sieved dried leaf powder was processed.The petroleum ether (60-80 °C) and methanol solvents were used to extract the phytochemicals such as steroids, flavonoids, tannins; alkaloids, etc.The petroleum ether (60-80 °C) and methanol solvents were used in a continuous hot percolation process (Soxhlet extraction) to extract the coarse powder successively for 48 h.A rotating vacuum evaporator (Evator) was used to concentrate the liquid extracts to produce a semisolid extract [24,25].

Animals
The healthy adult Wistar albino mice (20-25 g) of either sex were housed in polypropylene cages, under 12 h light: 12 h dark cycle and temperature 22 ± 2 °C and humidity 55 ± 5% maintained in the animal house.The animals had free access to food and water.All the animals were deprived of food but not of water 4 h before the experiment [26].The Institutional Animal Ethical Committee of the institute (Registration No. 121/1999/CPCSEA) has approved all the protocols of the study (Registration No. IAEC/Jan 2020/09).

Acute oral toxicity study
The acute oral toxicity study OECD guideline (2001) [26,27] was followed to carry out acute toxicity study for the extracts of Quisqualis indica plant.

Mast cell degranulation
There were six groups of mice in total, with six mice in each group.A three-day treatment for drugs program has been followed properly.Group I Control groupwas given the treatment with 1% tween 80 solutions (5 ml/ kg, i.p.); Group II received standard drug Sodium chromoglycate (50 mg/kg, i.p) and all test groups Group III to Group VI received LPE and LME at doses of 200 and 400 mg/kg.On the fourth day, 10 mL/kg of 0.9% saline solution was gently massaged into the peritoneal cavity of each mouse before being injected.Five minutes later, the peritoneal fluid was collected.The peritoneal fluid was then put into a test tube containing 7-10 mL of RPMI-1640 buffer medium (pH 7.2-7.3),which is made up of L-glutamine and 25 mM HEPES buffer but not sodium bicarbonate.At 400-500 rpm, this solution was then centrifuged.Centrifugation was used to rinse the mast cells pellets twice with RPMI-1640 buffer media, and the supernatant was rejected.Egg albumin was used to challenge the cell suspension produced from both the treated and control groups of mice, which was then incubated at 37 °C for 10 min.After being stained with 1% toluidine blue, the cells were examined under a microscope.Mast cells that had degranulated were seen as rupture cells rather than whole.In total of 100 cells from various visual regions were counted, and the percentage of protection against degranulation was calculated [28,29].

Milk induced eosinophilia and leukocytosis
There were six groups of mice, six animals in every group.Blood samples (0.5 ml) were collected from the retro-orbital plexus.Group I Control group received vehicle, tween 80 (1%) solution (5 ml/kg, i.p.); Group II received Standard drug Dexamethasone (50 mg/kg, i.p) and test groups Group III to Group VI received LPE and LME at doses 200 and 400 mg/kg.Amul's Fresh milk was procured from the local market.All animals were treated with an injection of freshly cooked and chilled milk (4 mL/kg, s.c.) for 30 min following treatments.Total leukocyte and eosinophil count were carried out in each group before drug administration and 24 h after the milk injection.Difference in total leucocytes count before and after 24 h of drug administration was calculated [30,31].

Statistical analysis
All the results of various studies were expressed as mean ± Standard Error of Mean.Data was analyzed using a one-way ANOVA, which was followed by Dunnet's Multiple Comparison Test.P < 0.05 was considered statistically significant.The graphs were calculated with the GraphPad Prism 5.

HRLCMS/MS profiling
The phytochemicals of the bioactive extract (LME) were analysed by HRLCMS/MS method.High-resolution liquid chromatography combined with the Q-Exactive Plus Mass Spectrometer TOF/Q-TOF mass spectrometer with ion source dual AJS electrospray ionization (ESI) was used [32].The plant extract (50 mg) was dissolved in 1 mL of water: acetonitrile (1:1) solvent mixture, ultrasonically processed for 10 min, and then centrifuged at 10,000 rpm for 10 min.The ZORBX Eclipse Plus C18, narrow bore 2.1 150 mm with 5 microns, was used for the LC/ESI-QTOF-MS/MS analysis.An amount of 5µL was delivered in both positive as well as negative mode.The Mass Spectra scan range was in 120 to 1200, with 1.00 scans rate spectra/second and 1.00 as MS/MS scan rate spectra/second.The gradient elution procedure was done by changing the proportion of water and acetonitrile from 95 to 5% within 30 min, with flow rate of 0.3 mL/ min as a constant flow rate.Then, this section was linked to the TOF/Q-TOF Mass Spectrometer for MS/MS fragmentation spectra.Phytochemicals were recognized by comparing their m/z and MS/MS transitions with those documented in reference databases.Further, the molecular formula, retention time, and adduct formula were detected.

Acute oral toxicity study
Oral administration of plant extracts shows no significant body weight variation, neither any sign of toxicity nor mortality of mice at the dose of 2000 mg/kg, body weight.

Mast cell degranulation
In the pathogenesis of allergic asthma, mast cells also play a crucial role.Inhaled antigens enter the lower respiratory tract and produce localised mast cell degranulation and inflammation that trigger an inflammatory response in the airways.These circumstances cause edema, fluid build up, and increased vascular permeability, which can block the airways.Asthma-related airway obstruction can result from bronchial constriction, which can happen as a result of smooth muscle contraction [33].Novel medications or treatments that help lessen asthma symptoms by lowering the production of these inflammatory mediators can be found by researching the inhibition of mast cell degranulation.In the present study, the effect of LPE and LME at doses of 200 and 400 mg/kg, body weight was examined on mast cell degranulation.The control group showed (83.4 ± 0.34) degranulation of mast cells while groups pre-treated with extracts and disodium chromoglycate significantly protect degranulation of mast cells.[34,35] LME at dose (400 mg/kg) showed (31.6 ± 3.67) and disodium chromoglycate (26.86 ± 2.34) protection against degranulation as shown in Figs. 1 and  2.
Values are in Mean ± SEM, * P < 0.05 when compared against control, n = 6.One Way ANOVA was used to analyze all the data, and then Dunnett's test was performed.The graph was calculated by using PRISM 5 Software.

Milk induced eosinophilia and leukocytosis
Airway hyperresponsiveness (AHR), reversible airflow restriction, and airway inflammation are all symptoms of bronchial asthma, a chronic condition.Eosinophilic asthma and non-eosinophilic asthma are two types of asthma's pathophysiological mechanisms.According to recent studies, eosinophils are crucial to the emergence of asthma exacerbations.Immune-modulatory reactions brought on by a high blood eosinophil count include airway inflammation, hyperresponsiveness, epithelial lining damage, and increased mucus secretion, Eosinophilic inflammation affects nearly fifty percent of asthma sufferers.Studies have proven that eosinophilia has been associated with higher disease severity, exacerbation frequency, and symptom burden, as well as decreased lung functions [36].Therefore, eosinophilic inflammation must be suppressed and eosinophilic and non-eosinophilic asthma must be distinguished in order to treat or avoid asthma aggravation [37].The maximum increase in difference of eosinophil (578.00 ± 11.10) and leucocytes (11,266.16± 149.63) counts was found in the control group which has been treated with milk (4 mL/kg, s.c.) before 24 h Quisqualis indica extracts at doses of 200 and 400 mg/kg significantly inhibited milk-induced eosinophilia and leucocytosis in a manner dependent on dose.Like Dexamethasone [38], the LME (400 mg/kg) exhibits substantial inhibition as shown Figs. 3 and 4.

Effect of LPE and LME extracts on mast cell degranulation
Values are in Mean ± SEM, *** P < 0.05 compared against control, n = 6.One Way ANOVA was used to analyze all the data, and then Dunnett's test was performed.The graph was calculated by using PRISM 5 Software.
Values are in Mean ± SEM, *** P < 0.05 when compared against control, n = 6.One Way ANOVA was used to analyze all the data, and then Dunnett's test was performed.The graph was calculated by using PRISM 5 Software.

LCMS/MS study of the bioactive extract
The LC-MS chromatogram of the methanolic extract of Quisqualis indica is shown in Figs. 5 and 6, and the important identified compounds are given in Tables 1  And 2.

Discussion
A persistent inflammatory illness of the airways is allergic asthma.Leukocyte infiltration into the lung and airway is the primary characteristic of asthma [39].In the current study, mice with milk-induced eosinophilia and leukocytosis were used to test the anti-asthmatic activity of LPE and LME at doses of 200 and 400 mg/kg.According to reports, giving milk subcutaneously causes a noticeable rise in the number of leukocytes and eosinophils 24 h later.Inflammatory mediators such as cytokines, histamine, and major basic proteins are released by leucocytes during asthmatic inflammation, promoting the continued inflammation.A peripheral eosinophil count that has abnormally increased to over 4% of the total leukocyte count is referred to as eosinophilia.The eosinophil count rises in asthmatic patients [40,41].
While mice treated with various doses of plant extracts show a decrease in the difference in leukocyte and eosinophil counts, mice treated with 1% Tween-80 in the control group exhibit an increase in these two cell types.Quisqualis indica leaf extracts may be helpful in allergy conditions because adaptogenic and type I hypersensitivity mediate the decrease in leucocytes and eosinophils.
Mast cells degranulate in response to immunological stimuli where antigen antibody responses are prevalent.In a dose-dependent way, Quisqualis indica leaf extracts at doses of 200 and 400 mg/kg effectively prevent egg albumin-induced mast cell degranulation.At 400 mg/kg, LPE and LME protect mast cells similarly to disodium chromoglycate.This demonstrates the effectiveness of Quisqualis indica leaf extracts in type I hypersensitivity reactions and in mast cell stabilization.The anaphylactic allergic reaction, which can be triggered by a variety of triggers, is a potentially fatal reaction that releases mediators like histamine and pro-inflammatory cytokines.The methanolic extract was subjected to a phytochemical analysis using the HRLCMS/MS technique, which revealed the presence of a number of phytocompounds including apigenin 7-glucoside, 3-Oxo-12, 18-ursadien-28-oic acid, 1-O-Caffeoyl-(b-D-glucose 6-O-sulfate), gallic acid, kurigalin, punicacortein B, etc. Gallic acid has shown a good amount of abundance in LME extract and acts by inhibiting mast cell activation, preventing the release of histamine, and the production of proinflammatory cytokines [42].The highly galloylated compounds inhibits secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in a concentration-dependent manner, acting on the transcriptional activity of NF-κB [43].According to the pharmacological effects of flavonoids like kaempferol, quercetin, and rutin, they may be able to treat allergic inflammatory disorders by inhibiting mast  cell activation [44].Apigenin has been found to be effective in allergic asthma through the decreased activation of epithelial cells, T cells, and eosinophils [45].As per HRLCMS data of LME extract the flavonoids like kaempferol, quercetin, apigenin, and luteolin and few types of tannin like gallic acid and punicacortein B are present in a notable amount.As a result, Quisqualis indica's leaf methanolic extract has shown significant mast cell degranulation and Milk-induced eosinophilia and leukocytosis action.Hence, Quisqualis indica leaves may be helpful in allergic conditions like asthma.

Conclusion
The plant extract is found to comprise phytochemicals such as apigenin 7-glucoside, 3-Oxo-12, 18-ursadien-28-oic acid, 1-O-Caffeoyl-(b-D-glucose 6-O-sulfate), gallic acid, kurigalin, punicacortein B, apigenin, kaempferol, quercetin, rutin, nicotinic acid methylbetaine (trigonelline), L-proline, L-asparagine, potassium quisqualate, etc.Thus, this plant is a plentiful source of phytochemicals with therapeutic value, and the findings of this study offer some scientific support for the use of the plant in traditional medicine for anthelmintic, anti-emetic, analgesic, antiulcer, and anti-diarrheal purposes.The results of the current investigation showed that milk-induced leucocytosis and eosinophilia reduced significantly by Quisqualis indica leaf extracts at 400 mg/kg dose.In anaphylactic reactions, extracts suppress the release of histamine and stabilize antigen-induced mast cells.They also have antiallergic properties.As a result of its anti-allergic and mast cell-stabilizing properties, which might be due to the presence of flavonoids, tannins, and triterpenoids, Quisqualis indica leaf extracts are beneficial in treating asthma.
INDICA LINN.: HRLCMS/MS PROFILING AND ANTI-ASTHMA ACTIVITY OF LEAF EXTRACTS Charulata T. Nemade et.al

Fig. 1 Fig. 2
Fig. 1 Effect of Quisqualis indica leaf extracts on egg albumin induced degranulation of mast cell

Fig. 4 Fig. 5 Fig. 6
Fig. 4 Effect of Quisqualis indica leaf extracts on milk induced leukocytosis Effect of Quisqualis indica leaf extracts on Milk induced eosinophilia

Table 1
Important compounds identified in LME extract of Quisqualis indica by HRLCMS/MS positive ESI

Table 2
Important compounds identified in LME extract of Quisqualis indica by HRLCMS/MS negative ESI