UPLC-ESI/MS/MS Profiling and Anti-Inflammatory Activity of Gleditsia caspica

The Caspian Locust is the common name of Gleditsia caspica, a tree cultivated in the southern and western Caspian Sea in Russia and Iran. Herein, a qualitative characterization of the chemical constituents of Gleditsia caspica leaves extract using ultra-performance liquid chromatography coupled to electrospray ionization tandem mass fragmentation (UPLC-ESI/MS/MS) analysis was performed revealing the presence of nine compounds: six flavonoids, one triterpenoid, one phytosterol, and one long chain ester. Six compounds were newly reported from Gleditsia caspica and two for the first time from genus Gleditsia . Moreover, the in-vivo anti-inflammatory activities of two different doses; 50 and 100 mg/kg of the aqueous-alcoholic extract of Gleditsia caspica leaves were evaluated in the carrageenan-induced paw edema model. The percentage of edema inhibition was calculated after 1, 2, 3, and 4 h of the administration showing a significant reduction in edema thickness in a dose and time-dependent manner compared to the control drug, indomethacin. These results put forward Gleditsia caspica extract as a potential natural anti-inflammatory agent with minimal side effects.


INTRODUCTION
More than 70% of the anti-inflammatory drugs are synthetic despite having potentially adverse effects [29]. Non-steroidal antiinflammatory drugs (NSAIDS) as salicylates, pyrazolones, indole derivatives, and quinolone derivatives are the most widely used antiinflammatory molecules [30]. Recently, many NSAIDs have been used as topical preparations in the treatment of some inflammatory disorders. Photoallergic contact dermatitis has been reported from propionic acid derivatives and photosensitivity from piroxicam [31,32]. Moreover, contact urticaria has been reported from oxyphenbutazone [33]. On the other hand, topical anti-inflammatory synthetic corticosteroids possess a variety of adverse effects such as skin atrophy and telangiectasia, that's why long-term use of topical steroids is limited [34]. Topical synthetic retinoids are a class of anti-inflammatory drugs used mainly in acne treatment and can lead to toxic contact dermatitis [35].
Medicinal herbs have great potential to act as therapeutic agents against inflammation mainly because of their antioxidant and cytoprotective properties [36][37][38]. Biological investigations of Gleditsia species revealed that both thorns and fruits extracts of Gleditsia sinensis possessed anti-inflammatory activities with different mechanisms of actions [18, 22,27,28]. Herein, we aimed to characterize for the first time the chemical profile of Gleditsia caspica leaves using UPLC-ESI/MS/MS; moreover, the in-vivo anti-inflammatory activity in carrageenaninduced paw edema model was probed to present a potential new natural topical anti-inflammatory agent with the least side effects.

Plant material
Fresh leaves of Gleditsia caspica (Fabaceae) were collected from the plant growing in El-Orman botanical garden, Ministry of Agriculture, Giza, in December (2016). They were kindly authenticated by Ms. Therese Labib, a botanical specialist and consultant at Orman and Qubba Botanical Gardens. A voucher specimen of the authenticated plant was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University under the registration code (PHG-P-GC-212).

Extract preparation
The intact air-dried plant material was ground into powder, macerated in distilled methanol 70% for 3 days, and filtered. The process was repeated four times before the hydro-alcoholic extract was concentrated using a rotary evaporator. The filtrate was completely evaporated in vacuo at 45 ºC till dryness to yield a solid dark brown residue (250 g).

UPLC-ESI/MS/MS profiling
Ultra-performance liquid chromatographic (UPLC) analysis coupled with an ESI mass spectrometer detector allowed simultaneous isolation of the compounds and determination of the isolated peaks molecular weights. Samples (100 μg/mL) were dissolved in high-performance liquid chromatography (HPLC) analytical grade methanol, filtered using a membrane disc filter (0.2 μm), and subjected to UPLC-ESI-MS/MS analysis. Samples (10 μL) were injected into the UPLC instrument, Waters® equipped with a reversed-phase C-18 column (ACQUITY UPLC -BEH C18 1.7 µm particle size -2.1×50 mm). The mobile phase was filtered using a 0.2 μm membrane disc filter and sonicated before injection. The Elution flow rate was adjusted to 0.2 mL/min; the gradient was composed of two eluents: water acidified with 0.1% formic acid and methanol acidified with 0.1% formic acid. Both negative and positive ion modes were used on a triquadrupole (XEVO) mass spectrometry, Waters® Corporation, Milford, MA01757 U.S.A. 30 eV cone voltage, 3 kV capillary voltage at 150 °C, 440 °C desolvation temperature were applied. Detection of mass spectra was in the ESI range m/z 100-1000 using the software Maslynx 4.1 and tentatively identified by comparing the retention time (Rt) peaks in the mass spectrum and their fragmentation pattern with reported data.

. In-vivo anti-inflammatory evaluation
The anti-inflammatory activity of the hydro-alcoholic extract of Gleditsia caspica leaves was evaluated in the carrageenan-induced paw edema model according to the method described before [39]. Twenty four adult male albino rats, weighing between 130-150 g were maintained under normal laboratory conditions at room temperature of 25-30 °C, 60-65% relative humidity, and provided with standard diet and water. The study was conducted according to regulations of the Institutional Animal Ethical Committee of the National Research Centre which gave its consent following the National Regulations on Animal Welfare and Institutional Animal Ethical Committee (registration no. 90123) [40]. Four groups, each of six animals, were treated as follow: group І: received 1 mL of saline serving as control, group ІI: received 50 mg/kg of the Gleditsia caspica extract, group III: received 100 mg/kg of the Gleditsia caspica extract, and group ІV: received 20 mg/kg of the reference indomethacin. One hour later, paw swelling was induced by sub plantar injection of 0.1 mL of 1% carrageenan solution in saline into the right hind paw of all groups. The edema thickness was calculated by measuring the hind paw thickness immediately before sub plantar injection and at 1, 2, 3, and 4 h after drug administration. The percentage of inhibitory activity at each time point was calculated according to the following equation [41].

Percent of inhibition= X-Y/X ×100
Where "X" represents the animal's paw diameter in the control group and "Y" represents the diameter of the animal's paw administered with the tested materials.

. Statistical analysis
Anti-inflammatory data were expressed as mean ± standard deviation and statistical analysis was performed using the one-way analysis of variance (ANOVA), followed by posthoc Tukey's test for multiple comparisons. Values with P<0.05 were considered statistically significant.

Metabolites profiling of Gleditsia caspica extract
This study aimed to characterize the Phytoconstituents (Table 1) within the bioactive Gleditsia caspica extract using the UPLC-ESI/MS/MS approach, which revealed the presence of six flavonoids, one triterpenoid, one phytosterol, and one long chain ester.   Data represented as mean ± SD of six independent experiments. Analysis was done using the one way analysis of variance (ANOVA), followed by post-hoc Tukey's test. *The difference is significant at P<0.05.

. Evaluation of the in-vivo antiinflammatory activities
One of the most frequently used methods to evaluate the anti-inflammatory effects of natural products is Carrageenan-induced paw edema. Carrageenan was injected and induced inflammation in rat hind paw and increased paw diameter to 4.75 mm. Gleditsia caspica extract reduced hind paw inflammation time and dosedependently. Results were summarized in (table 2 and Fig. 1).

DISCUSSION
In this study, the characterization of the Phyto-constituents of Gleditsia caspica extract using the UPLC-ESI-LC/MS/MS approach was carried out to identify the chemical components within the plant associated with this biological activity. Kaempferol This study was also conducted to evaluate the in-vivo anti-inflammatory activity of Gleditsia caspica extract. As aforementioned, the carrageenan-induced paw edema model is one of the major models used in the assessment of the anti-inflammatory effects of natural products. After one hour of Gleditsia caspica administration, there wasn't a marked reduction in the rat hind paw edema compared to the reference group. During the follow-up period, an increase in the percentage of the edema inhibition was noticed. Paw diameter reduction was increased by increasing the dose from 50 mg/kg to 100 mg/kg, and the effect of the extract at 100 mg/kg dose was comparable to that of the reference drug indomethacin. The reduction in paw diameter recorded 18.32% and 22.11% for the extract (100 mg/kg) and reference drug (indomethacin), respectively, after four hours of carrageenan administration. Accordingly, it could be concluded that the hydro-alcoholic extract of Gleditsia caspica exhibited time and dosedependent anti-inflammatory activity. Flavonoids are key constituents involved in the antiinflammatory activities within the plants. In  and their glycosides. It could be concluded that Gleditsia caspica extract is a potential bioactive candidate that could be incorporated in topical anti-inflammatory preparations owing to its flavonoids components.

CONCLUSION
UPLC-ESI/MS/MS analysis of Gleditsia caspica extract revealed its richness in flavonoids, newly reported compounds from this genus, and from this species. Antiinflammatory assays revealed that the hydro-alcoholic extract of Gleditsia caspica possessed dose and time dependant activity. These results suggested the possible use of Gleditsia caspica extract in topical anti-inflammatory preparations with minimal side effects.

Ethics approval and consent to participate
The protocol of the experiment was approved by the Institutional Animal Ethical Committee in the National Research Center (registration no. 90123) [40].

Consent to publish
Not applicable.

Availability of data and materials
All data generated or analyzed during this study were included in the main manuscript.

Competing interests
The authors declare that no competing interests exist.

Funding Statement
No funding source was received.

Authors' contributions
The manuscript was drafted and written by Hagar Ashraf. Dr. Ashaimaa Y. Moussa, Prof. Omayma A. Eldahshan, and Prof. Abdel Nasser B. Singab have provided comments and revised the manuscript. All authors have read and approved the final manuscript.

Acknowledgment
The authors would like to acknowledge all colleagues in the Pharmacognosy Department; Ain shams University for their support.