Chemical Profile, In Vitro Antioxidant, Pancreatic Lipase, and Alpha-Amylase Inhibition Assays of the Aqueous Extract of Elettaria cardamomum L. Fruits

Phytochemical and pharmacological investigations of Elettaria cardamomum L. were mostly focused on its essential oil and organic crude extracts with little attention on its aqueous extracts. ,erefore, the current study aimed to investigate the phytochemical profile, in vitro antiobesity, and antidiabetic activities of the aqueous extract of Elettaria cardamomum L. family Zingiberaceae. UPLC-ESI-MS/MS analysis is used for the aqueous extract characterization in both ionization modes. ,e analysis revealed the tentative identification of forty-seven compounds based on their MS/MS fragmentation pattern and comparison with the reported data. ,e identified compounds include eight flavones, thirteen phenolic and nonphenolic acids, one coumarin, and nineteen anthocyanins. Moreover, the in vitro antiobesity and antidiabetic activities were also studied. ,e results showed that E. cardamomum L. aqueous extract inhibits pancreatic lipase and α-amylase enzyme in a concentration-dependent manner, as 1mg/mL extract was able to inhibit pancreatic lipase and α-amylase by 62.25%± 0.58 (IC50 = 288.75± 1.3 μg/mL) and 70.42%± 1.5 (IC50 = 220.5± 1.3 μg/mL), respectively. Conclusively, the current study indicated that the investigated biological activities of Elettaria cardamomum aqueous extract were attributed to the existence of biologically active metabolites such as flavones, phenolic and nonphenolic acids, coumarins, and anthocyanins. Moreover, it proposed that the aqueous cardamom extract can be used as a natural potential source in different pharmaceutical preparations to protect against variable chronic disorders, especially obesity and diabetes. Deeper in vivo investigations, isolation, purification, and structural elucidation of the major active metabolites from cardamom are recommended.


Introduction
Plants and their secondary metabolites have played a crucial and important role in the human's life particularly those which are used in traditional medicine or as food due to their healing and nutritional properties [1]. Spices are often used as food additives and considered a promising source for finding newer digestive enzyme inhibitors that do not have the same side effects as synthetic ones [2,3].
Spices are well known to improve gastric function by increasing salivary flow and gastric juice secretions and help in digestion [3]. Elettaria cardamomum L. is a member of the family Zingiberaceae. It is known as "Queen of Spices" and considered as the second essential "national spice" of India [4]. Traditionally, cardamom is used to treat different disorders, such as gum infections, asthma, cataracts, and cardiac, digestive, and kidney diseases. It also has antidiabetic, anti-inflammatory, antioxidant, and anticarcinogenic effects [5,6].
Most of the previous biological investigations focused on cardamom volatile oil [7] which has valuable constituents such as terpene, esters, and flavonoids. e major constituents of cardamom volatile oil are 1,8-cineole (36.3%) and α-terpinyl acetate (31.3%). It was also reported that organic

UPLC-ESI-MS/MS Instrument and Separation Technique.
e UPLC-ESI-MS/MS (ultra-performance liquid chromatography with electrospray ionization quadrupole linear ion trap tandem mass spectrometry) analysis performed on ESI-MS positive and negative ionization acquisition modes was carried out on a XEVO TQD triple quadruple instrument mass spectrometer. For the quantitative determination of the phytochemicals, a multiple-reaction monitoring (MRM) mode method was employed. E. cardamomum L. fruits aqueous extract was analyzed by UPLC to get the chromatographic profiles of the highly polar portions of the extracts, which contain polar compounds such as phenolic and flavonoid compounds. HPLC-grade methanol was used to dissolve the sample and filtered through 0.2 μm membrane disc filter and resulting solution concentrations were in 0.2 to 0.5 mg/mL range, depending on each fraction. e UPLC system was a Waters Corporation, Milford, MA01757 U.S.A, mass spectrometer. e reverse-phase separations were performed (ACQUITY UPLC BEH C18 1.7 µm, 2.1 × 50 mm Column) (50 mm × 1.2 mm (inner diameter) and 1.7 µm particle size) at 0.2 mL\min flow rate. e gradient program previously reported in [9] was used for the analysis. e mobile phase consists of acidified water containing 0.1% formic acid (A) and acidified methanol containing 0.1% formic acid (B). e elution conditions were 0-2 min 10% B isocratic; 2-5 min, linear gradient B 10 to 30%; 5-15 min, linear gradient from 30% to 70% B; 15-22 min, linear gradient from 70% to 90% B; 22-25 min, 90% B isocratic and lastly washing and reconditioning of column was carried out. To obtain more data, electrospray ionization (ESI) was performed in both negative and positive ion modes. e analysis parameters were carried out using negative ion mode as follows: source temperature 150°C, cone voltage 30 eV, capillary voltage 3 kV, desolvation temperature 440°C, cone gas flow 50 L/h, and desolvation gas flow 900 L/h. Mass spectra were detected in the ESI between m/z 100-1000 atomic mass units. e identification of the phytochemical constituents was done by their fragmentation patterns and ESI-QqQLIT-MS/MS spectra. Peaks and spectra were processed using the MassLynx 4.1 software and tentatively identified by comparing its retention time (R t ), mass spectrum with reported data, and library search (such as FooDB (http://www.Foodb.ca)).

In Vitro Antidiabetic Assay Using α-Amylase Inhibition
Method. In this method, the solution of the enzyme was prepared by dissolving α-amylase in 20 mM phosphate buffer (6.9) at the concentration of 0.5 mg/mL. 1 mL of the extract of various concentrations (7.81-1000 μg/mL) and 1 mL of enzyme solution were mixed and incubated at 25°C for 10 min. After incubation time, 1 mL of starch (0.5%) solution was added to the mixture and further incubated at 25°C for 10 min. e reaction was stopped by the addition of 2 mL of dinitro salicylic acid (DNS, colour reagent), heating in a boiling water bath (5 min). After cooling, the absorbance was measured using a calorimeter at 565 nm (Spectrumlab S23A, Globe Medical, England). e percentage of inhibition was calculated using the given formula, where As is the absorbance of tested extracts and Ac is the absorbance of control. Acarbose was used as a control [10]. e IC 50 value is the concentration of α-amylase inhibitor to inhibit 50% of its activity under the assay conditions. Nonlinear regression analysis of GraphPad Prism 5 software (GraphPad software, San Diego, California) was used to calculate IC 50 from graphic plots of the dose response curve for each applied concentration. Each experiment was performed in triplicates, and all values are represented as means ± SD.

In Vitro Antiobesity Using Pancreatic Lipase Inhibitory
Assay.
e lipase inhibition activity of E. cardamomum aqueous extract was determined by a method in [11]. In this method, the porcine pancreatic lipase activity was measured using p-nitrophenyl butyrate (NPB) as a substrate. Lipase solution (100 µg/mL) was prepared in a 0.1 mM potassium phosphate buffer (pH 6.0). Samples with different concentrations (7.81-1000 μg/mL) were preincubated with 100 µg/ mL of lipase for 10 min at 37°C. e reaction was then started by adding 0.1 mL NPB substrate. After incubation at 37°C for 15 min, p-nitrophenol amount released in the reaction was measured using Multiplate Reader (BioTek, Synergy HT, VT, USA). Orlistat was used with the same concentrations as a control. e results were expressed as percentage inhibition, which was calculated using the formula, where (Ac) is the absorbance of control and (As) is the absorbance in the presence of test substance. e concentration of pancreatic lipase inhibitor to inhibit 50% of its activity under the assay conditions is the IC 50 value. Its IC 50 was done from dose response curve graphic plots for each concentration by using nonlinear regression analysis of GraphPad Prism 5 software. Each experiment was performed in triplicates, and all values are represented as means ± SD of triplicates.

Characterization of the Phytoconstituents of the Aqueous
Extract of E. cardamomum L. Fruits. e polyphenolic constituents of E. cardamomum L. fruit aqueous extract was investigated for the first time using UPLC-ESI-MS/MS to identify its bioactive constituents related to their in vitro antiobesity and antidiabetic activities. Some of the identified compounds were previously reported in E. cardamomum.

Anthocyanins.
Nine compounds were identified as cyanidin derivatives based on their MS fragmentation, leading to cyanidin aglycone (m/z 287) in the positive mode. Compound 10 was identified as cyanidin-3-O-acetyl rhamnoside with m/z 475 and an MS 2 fragment at 287, a loss of 188 amu from acetyl rhamnoside [12]. Compounds 13 and 44 had a common protonated pseudomolecular ion peak at m/z 595, with fragment ions at m/z 449 and 287 and neutral losses of 146 and 162 amu from coumaroyl and hexose moieties, respectively; they were identified as cyanidin-3-Ocoumaroyl hexoside [24]. Compound 24 had a [M + H] + ion at m/z 449 and MS 2 fragment ion at m/z 287, which is consistent with cyanidin-3-O-hexoside [22].

Antiobesity Activity.
Obesity which is recently known as "new world syndrome" results from the interaction of nutritional, environmental, and genetic factors [1,40]. It is one of the worldwide health problems as it is associated with various chronic disorders such as hypertension, type 2 diabetes, dyslipidaemia, osteoarthritis, and cardiovascular, diseases. Pancreatic lipase enzyme secreted by the pancreas is responsible for 50-70% digestion of fats into monoglyceride and free fatty acids. Orlistat is an antiobesity drug which inhibits the dietary triglycerides hydrolysis and consequently decreases the subsequent intestinal absorption of monoglycerides and free fatty acids (lipolysis products). Despite of being the only drug for long-term control of Journal of Chemistry obesity, it has gastrointestinal side effects [40][41][42]. erefore, natural products and plant-derived compounds have attracted the attention for the obesity management by developing safe and efficient antiobesity drugs [43]. e inhibitory activity of the aqueous extract on pancreatic lipase enzyme is shown in Table 2 and Figures 3(a)  and 3(b). e results demonstrated that E. cardamomum aqueous extract inhibits pancreatic lipase in a concentrationdependent manner; the inhibition % ranged from 24.13% ± 1.2 to 70.42% ± 1.5 from the lowest to the highest concentration (31.25-1000 µg/ml). e IC 50 of the extract was (288.75 ± 1.3 µg/ml) while that of the orlistat standard was (IC 50 � 23.8 ± 0.7 µg/ml). E. cardamomum aqueous extract in vitro inhibition of pancreatic lipase could be attributed to the presence of major biologically active polyphenolic metabolites such as anthocyanins and flavonoid compounds. Our findings are consistent with those previously published studies. Rahman et al. [36] reported that hepatic damage, dyslipidaemia, and oxidative stress can be prevented by cardamom powder supplementation in high carbohydrate and high fat diet fed rats.  3.3. Antidiabetic Activity. Diabetes mellitus (DM) is a longterm metabolic disease characterized by elevated blood sugar levels. It may be attributed to a lack of insulin secretion, resistance to peripheral insulin's effects, or both. It affects around 10% of the population. Moreover, it is expected that diabetic patients may reach 230 million in 2025. DM is increasing all over the world, due to the prevalence of unhealthy habits, such as obesity, sedentarism, dietary patterns, smoking, or alcohol intake [44,45]. Diabetes mellitus is usually accompanied by lipid, carbohydrate, and protein metabolic abnormalities. In patients with diabetes mellitus, chronic hyperglycemia, in combination with other metabolic disorders, can damage various organ systems, resulting in potentially fatal health complications, the most common of which are retinopathy, nephropathy, neuropathy, and cardiovascular disease [45]. Despite the availability of other therapeutic options, herbal remedies (medicinal plants or their extracts) are highly recommended for the treatment of diabetes since ancient times [44,46]. Alpha-amylase and α-glucosidase are digestion enzymes of carbohydrates which can significantly decrease the postprandial elevation of blood glucose after a carbohydrate diet. So, inhibition of these enzymes is considered as essential strategy in blood glucose management [10].
In the present study, the in vitro α-amylase inhibitory activity of E. cardamomumfruits aqueous extract was investigated using variable doses (7.81-1000 µg/mL). e extract showed a good inhibition of carbohydrate-hydrolyzing enzymes (α-amylase) in a dose-dependent manner, as shown in Table 3 and Figures 3(c) and 3(d). e inhibition % ranged from 6.17 ± 1.7 to 62.25 ± 0.58 from the lowest to the highest concentration (15.63-1,000 µg/ml). However, the highest inhibition % of acarbose standard was 40.75 ± 1.5 to 86.32 ± 0.63 from lowest to highest concentration, as shown in Table 3. e IC 50 of the extract was (220.5 ± 1.3 µg/ml) while that of the acarbose standard was (IC 50  Anthocyanin is considered one of the most important classes of polyphenols which play a key role in the treatment and prevention of type 2 diabetes mellitus [47]. It was reported that cyanidin, delphinidin, malvidin, and cyanidin-3-O-glucoside are the most potent anthocyanins for alphaamylase inhibition. Moreover, cyanidin and its glucosides have been shown to inhibit carbohydrate-digestive enzymes by forming covalent and/or noncovalent bonds between their hydroxyl groups and the polar residues of the enzyme active sites [44,45].
Recently, flavonoids' activity has been investigated even up to clinical studies for diabetes management in humans. e findings are compiled in several reviews [48][49][50]. Flavonoids have also been identified as α-amylase inhibitors in other studies [51]. Additionally, flavonoids are considered as natural alternative in diabetes treatment as they can regulate insulin secretions through different mechanisms. For example, taxifolin-3-O-rhamnoside works in a mechanism like sulfonylureas by K ATP channel closure and cell surface Ca 2+ channel signaling pathway opening [52].

Conclusion
In the present study, the aqueous extract of E. cardamomum fruits was investigated for the first time using UPLC-ESI/MS/ MS in both negative and positive ionization modes. e antidiabetic and antiobesity activities of the extract were also evaluated in vitro. e results revealed the tentative identification of forty-seven compounds including (anthocyanins, flavones, and phenolic and nonphenolic acids). E. cardamomum aqueous extract showed a good antidiabetic and antiobesity activities which may be related to the presence of major biologically active metabolites (cyanidin-3-O-coumaroyl hexoside, cyanidin-3-O-hexoside, azelaic acid, and taxifolin quinoyl hexoside) with reported antioxidant, antiobesity, and antidiabetic activities. Finally, future bioavailability and pharmacokinetic studies of E. cardamomum fruit are recommended to identify and isolate its active metabolites and expand its medical application.

Data Availability
e data used to support the findings of this study are included in the manuscript.

Conflicts of Interest
e authors declare that they have no conflicts of interest.