UPLC-ESI-MS/MS Profile and Antioxidant, Cytotoxic, Antidiabetic, and Antiobesity Activities of the Aqueous Extracts of Three Different Hibiscus Species

)e aqueous extracts of Hibiscus calyphyllus (HcA), Hibiscus micranthus (HmA), and Hibiscus deflersii (HdA) growing in Saudi Arabia did not receive enough attention in phytochemical and biological studies. )is inspired the authors to investigate the phytochemicals of these extracts for the first time using UPLC-ESI-MS/MS in negative and positive ionizationmodes.)e analysis afforded the tentative identification of 103 compounds including phenolic compounds, flavonoids, and anthocyanins. Moreover, in vitro evaluations of their cytotoxic, antioxidant, antidiabetic, and antiobesity activities were carried out.)e results showed that aqueous extract of Hibiscus calyphyllus had the highest activity as an antioxidant agent (SC50 = 111± 1.5 μg/mL) compared with ascorbic acid (SC50 = 14.2± 0.5 μg/mL). MTTassay was used to evaluate cytotoxic activity compared to cisplatin.Hibiscus deflersii showed the most potent cytotoxic effect against A-549 (human lung carcinoma) with IC50 = 50± 5.1 μg/mL, and Hibiscus micranthus showed a close effect with IC50 = 60.4± 1.7 μg/mL. Hibiscus micranthus showed the most potent effect on HCT-116 (human colon carcinoma) with IC50 = 56± 1.9 μg/mL compared with cisplatin (IC50 = 7.53± 3.8 μg/mL). HcA and HdA extracts showed weak cytotoxic activity against A-549 and HCT-116 cell lines compared to the other extracts. Eventually,Hibiscus deflersii showed astonishing antidiabetic (IC50 = 56± 1.9 μg/mL) and antiobesity (IC50 = 95.45± 1.9 μg/mL) activities using in vitro α-amylase inhibitory assay (compared with acarbose (IC50 = 34.71± 0.7 μg/mL)) and pancreatic lipase inhibitory assay (compared with orlistat (IC50 = 23.8± 0.7 μg/mL)), respectively. In conclusion, these findings are regarded as the first vision of the phytochemical constituents and biological activities of differentHibiscus aqueous extracts.Hibiscus deflersii aqueous extract might be a hopeful origin of functional constituents with anticancer (on A-549 cell line), antidiabetic, and antiobesity activities. It might be a natural alternative remedy and nutritional policy for diabetes and obesity treatment without negative side effects. Isolation of the bioactive phytochemicals from the aqueous extracts of aerial parts of Hibiscus calyphyllus, Hibiscus micranthus, and Hibiscus deflersii and estimation of their biological effects are recommended in further studies.


Introduction
Hibiscus (Malvaceae) consists of approximately 200 species widely distributed in tropical and subtropical regions of the world [1]. Hibiscus is a genus of herbs, shrubs, and trees [2]. Phytochemical investigation of Hibiscus has been reported to contain many classes of secondary metabolites including anthocyanins, flavonoids, steroids, terpenoids, alkaloids, quinones, and sesquiterpene [2].
Hibiscus deflersii (HdA), which is native to Ethiopia and grown as ornamental plant worldwide, is 1 m high erect perennial or annual leafy untidy shrub of bright green narrow dentate leaves surrounding bright crimson-red flowers. Many interesting pharmacological activities of HdA had been reported; its leaves extract is used to treat cardiac disorders and diabetes. However, its flower infusion and extract are used as demulcent and emollient, while its decoction is used for the treatment of bronchial catarrh [5].
Hibiscus micranthus (HmA), which is commonly distributed from south to western part of Saudi Arabia, is a 45 cm shrub with heavy leaves, white flowers, and short pedicels with very distinctive capsules of pea-size fruits and is distributed vastly in Saudi Arabia, India, tropical Africa, and Ceylon. Its flowers and fruits exhibited antidiabetic and laxative activities when used orally, and when applied topically it is used as antidandruff agent.
Hibiscus calyphyllus (HcA) is 1 m high leafy shrub characterized by bright yellow flower with dark red center surrounded by simple wide serrate leaves. It is commonly found in Jazan, south-western region of Saudi Arabia. e ethyl acetate fraction of this plant showed potent antioxidant activity [5].
Hibiscus aerial parts are considered as food crops consumed as hot or cold beverages (aqueous extract). Numerous scientific papers have been published discussing the chemical contents of different fractions of HcA, HdA, and HmA, which showed their high biological effectiveness, having antioxidant, antidiabetic, antiobesity, and cytotoxic activities. For our knowledge, the previous literature on Hibiscus species suffers from insufficiency of detailed information on the phytoconstituents of aqueous extracts of Saudi HdA, HcA, and HmA and their biological activities. erefore, the aims of the present research were (i) to perform direct analysis of aqueous extracts, which relies on UPLC coupled with ESI-MS/MS detection, (ii) to detect the antioxidant, antidiabetic, antiobesity, and anticancer activities of aqueous extracts of HdA, HcA, and HmA; and (iii) to anticipate the components responsible for antioxidant, antidiabetic, antiobesity, and anticancer activities.

Plant Material.
Aerial parts of three different species of genus Hibiscus were collected from As-Sarawat mountains, Jabal As-Sahla', and Aseer province, in Saudi Arabia (2, HA-16240) were kept at the herbarium of the department.

Preparation of Extracts.
Air-dried powder aerial parts of selected plant samples (600 g) were individually extracted with distilled water at 100°C with continuously shaking for 3 hrs. e marc of each plant material was extracted thrice under similar conditions by repeating the above-mentioned procedure.
e aqueous extracts were then filtered by centrifugation, and the filtrates were pooled. e obtained filtrates were freed from the solvent by freeze-drying to get dark brown solid masses. e weight of resulted residues was 34.02, 26.25, and 36.41 g for H. calyphyllus (HcA), H. deflersii (HdA), and H. micranthus (HmA), respectively.

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 ion acquisition mode, was carried out on a XEVO TQD triple quadruple instrument. Method in a multiple-reaction monitoring (MRM) mode was employed for the quantitative determination of phytochemicals. e crude Hibiscus extracts were analyzed by UPLC, in order to obtain chromatographic profiles of the more polar portions of the extracts, which contain phenolic and flavonoid compounds. e samples were dissolved in HPLC grade methanol, filtered through 0.2 μm membrane disc filter, and resulting solution concentrations were in the range of 0.2 to 0.5 mg/mL, depending on each crude extract.
e UPLC system was a mass spectrometer, Waters Corporation, Milford, USA. e reverse-phase separations were performed (ACQUITY UPLC BEH C18 Column, 1.7 μm-2.1 × 50 mm; 50 mm × 1.2 mm inner diameter; 1.7 μm particle size) at 0.2 m/mL flow rate. A previously reported gradient program was applied for the analysis [6]. e mobile phase comprised acidified water containing 0.1% formic acid (A) and acidified methanol containing 0.1% formic acid (B). e employed elution conditions were as  follows: 0-2 min, isocratic elution at 10% B; 2-5 min, linear  gradient from 10 to 30% B; 5-15 min, linear gradient from  30% to 70% B; 15-22 min, linear gradient from 70% to 90%  B; and 22-25 min, isocratic elution at 90% B; finally, washing and reconditioning of column were done. Electrospray ionization (ESI) was performed in both negative and positive ion modes to obtain more data. e parameters for analysis were set 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/hr. Mass spectra were detected in the ESI between m/z 100 and 1000 atomic mass units. Chemical constituents were identified by their ESI-QqQLIT-MS/MS spectra and fragmentation patterns. e peaks and spectra were processed using the MassLynx 4.1 software and tentatively identified by comparing their retention time (R t ) and mass spectrum with reported data and library search (such as FooDB (http://www.Foodb.ca)).

Antioxidant
Activity. e antioxidant activity of extracts was determined at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University by the DPPH free radical scavenging assay in triplicate, and average values were considered. [7]. Freshly prepared (0.1 mM) solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and different tested extracts prepared at 5, 10, 20, 40, 80, 160, and 320 μg/mL in methanol were vigorously mixed and allowed to stand for 30 min at room temperature in the dark [8]. e absorbance values of the resulting solution were recorded with a UV-visible spectrophotometer (Milton Roy, Spectronic 1201) at λ max of 517 nm against DPPH radical without antioxidant (control) and the reference compound ascorbic acid (5,10,20,40,80,160, and 320 μg/mL). All the determinations were performed in three replicates and averaged. e percentage inhibition of the DPPH radical was calculated according to the following formula:

DPPH Radical Scavenging Activity
where AC is the absorbance of the control solution and AS is the absorbance of the sample in DPPH solution. e percentage of DPPH radical scavenging was plotted against each extract concentration and ascorbic acid (μg/ mL) to determine scavenging capacity (SC 50 ), which is the concentration required to scavenge DPPH by 50% (i.e., concentration giving 50% reduction in the absorbance of a DPPH solution from its initial absorbance).
For antitumor assay, the tumor cell lines were suspended in medium at concentration of 5 × 10 4 cell/well in Corning ® 96-well tissue culture plates and then incubated for 24 hrs. e tested extracts were then added to 96-well plates (six replicates) to achieve eight concentrations for each extract ranging from 1 μg/mL to 500 μg/mL. Six vehicle controls with media or 0.5% DMSO were run for each 96-well plate as a control. After incubation for 24 h, the numbers of viable cells were determined by the MTT test. Briefly, the media were removed from the 96-well plates and replaced with 100 μL of fresh culture DMEM without phenol red; then, 10 μL of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of phosphate buffered saline (PBS)) was added to each well including the untreated controls. e 96-well plates were then incubated at 37°C and 5% CO 2 for 4 hrs. An 85 μl aliquot of the media was removed from the wells, and 50 μL of DMSO was added to each well, mixed thoroughly with the pipette, and incubated at 37°C for 10 min. en, the optical density was measured at 590 nm with the microplate reader (Sunrise, Tecan, Inc., USA) to determine the number of viable cells, and the percentage of viability was calculated: where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. e relation between surviving cells and each extract concentration (1-500 μg/mL) was plotted to get the survival curve of each tumor cell line after treatment with the tested extract. e 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose-response curve (log extract concentration on x-axis vs. percentage viability from untreated cells on y-axis) for each concentration through nonlinear regression analysis (dose-response inhibition, log inhibitor vs. normalized responsevariable slop) using GraphPad Prism 5 software (GraphPad Software, San Diego, California) [9,10]. All experiments were repeated at least three times. Results are reported as means ± SD.

2.7.
In Vitro Antidiabetic Assay 2.7.1. α-Amylase Inhibition Method. In a-amylase inhibition method, the enzyme solution was prepared by dissolving α-amylase in 20 mM phosphate buffer (6.9) at a concentration of 0.5 mg/mL. One mL of the extract of various concentrations (7.81-1000 μg/mL) and 1 mL of enzyme solution were mixed together and incubated at 25°C for 10 min. After incubation, 1 mL of starch (0.5%) solution was added to the mixture and further incubated at 25°C for 10 min. e reaction was then stopped by adding 2 mL of dinitrosalicylic acid (DNS, colour reagent), heating the reaction mixture in a boiling water bath (5 min). After cooling, the absorbance was measured calorimetrically at 565 nm. e inhibition percentage was calculated using the following formula: % inhibition � (1 − As/Ac) × 100, where Ac is the absorbance of control and As is the absorbance of tested extracts. Acarbose was used as a control [11]. e IC 50 value was defined as the concentration of α-amylase inhibitor needed to inhibit 50% of its activity under the assay conditions.
Nonlinear regression analysis using GraphPad Prism 5 software (GraphPad Software, San Diego, California) was conducted to calculate IC 50 from graphic plots of the doseresponse curve for each applied concentration. Each experiment was performed in triplicate, and all values are represented as means ± SD.

In Vitro Antiobesity Using Pancreatic Lipase Inhibitory
Assay.
e lipase inhibition activity of plant extract was determined by the method in [12]. In this assay, 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. Orlistat was used with the same concentrations as a control. e results were expressed as percentage inhibition, which was calculated using the following formula: inhibitory activity (%) � (1 − As/Ac) × 100, where As is the absorbance in the presence of test substance and Ac is the absorbance of control. e IC 50 value was defined as the concentration of pancreatic lipase inhibitor required to inhibit 50% of its activity under the assay conditions. Estimation of IC 50 was done from dose-response curve graphic plots for each concentration by nonlinear regression analysis using GraphPad Prism 5 software. Each experiment was performed in triplicate, and all values are represented as means ± SD of triplicates.

UPLC-ESI-MS/MS.
Identification of the chemical composition of the aqueous extract of the HdA, HmA, and HcA was carried out by UPLC-ESI-MS/MS in negative and positive ion modes. Totally, 103 secondary metabolites arranged according to retention time (R t ) were identified depending on their MS 2 information given by the precursor ion's mass, their fragments, known fragmentation patterns for the given classes of compounds, and neutral mass loss, as well as comparison with the available literature and searching in an online database [13] as shown in Table 1. Figure 1 shows the base peak chromatograms of the three aqueous extracts.  [17] and its derivative (85) [39] and syringic acid derivative (76) [33] were identified. Compound 33 and its isomer (89) were tentatively identified as 4hydroxybenzoic acid while compound 55 and its isomer (59) were tentatively identified as 3-hydroxybenzoic acid [13].
Tyrosol (4) and its isomers (26, 67, and 94) were characterized by two fragments: m/z 77, corresponding to the aromatic ring; m/z 93, corresponding to the phenol group, respectively [15]. Tyrosol precursor ion at m/z 121 does not refer to the [M + H] + ion, but to the [M + H-H 2 O] + according to [15]; this may be due to in-source fragmentation, even under mild ionization conditions.  [18,25,40].
Oleuropein ( [14]. It was previously reported that oleuropein was identified in Hibiscus [47]. Succinic acid (14) and its isomers (17,52,66,70,73,83,87,90, and 103) were recognized by comparing their MS 2 fragmentation pattern with the reported data [22]. ey showed a deprotonated molecular ion at m/z 117 and an intense fragment at m/z 99, attributed to the loss of water molecule [22], while compound 3 showed a protonated molecular ion at m/z 119 with MS 2 intense fragment ion at m/z 101 [M + H-18] + , attributed to the loss of water molecule (−18 Da); thus, it was tentatively identified as succinic acid.
Hydroxycitric acid derivative (6) and butein chalcone (7) were identified by comparison with published data [13,17], respectively. Hydroxycitric acid is the principal organic acid found in the calyces of Hibiscus according to [48]. Moreover, compound 38 was identified as a sesquiterpenoid derivative, gamma-eudesmol rhamnoside derivative (MS 1 [13]. N-feruloyltyramine (10), its isomer (60), and 22-dehydrocholesterol (77) were recognized by comparing their MS/MS fragmentation pattern with the reported data [13].  (Table 1). In conclusion, combination of accurate mass measurement and LC ability to separate isomeric compounds can be considered a powerful tool in the identification of polyphenol diversity in three species of the Hibiscus genus even in the absence of standards, but the stereochemical differentiation between the large number of isomers that were found in our species, for example, isomers of luteolin C-hexoside-C-pentoside, apigenin C-hexoside-C-pentoside, and cyanidin rutinoside, was not possible with our methodology.

Antioxidant Activity.
It is well known that plant phenols and flavonoids in general are highly effective free radical scavengers and antioxidants. us, they are used for the prevention and cure of various disorders which are mainly associated with free radicals. Series of concentrations ranged from 5 to 320 μg/mL in methanol were used. e DPPH scavenging percentage of different extracts as well as ascorbic acid and SC 50 values (the concentration required to scavenge DPPH by 50%) are shown in Figures 2 and 3, respectively. HcA exhibited the highest antioxidant activity as indicated by its high DPPH scavenging percentage (65%) at 320 μg/mL and low SC 50 values (111 ± 1.5 μg/mL). Its activity can be attributed to its contents of polyphenolic compounds such as phenolic acids, flavonoids, and anthocyanins (i.e., apigenin C-hexoside-C-pentoside, luteolin Chexoside-C-pentoside, luteolin derivative, 4-hydroxybenzoic acid, tyrosol and peonidin derivative, and succinic acid). Unfortunately, both HdA and HmA displayed moderate antioxidant activities with SC 50 � 137.6 ± 0.3 and 135 ± 0.5 μg/mL, respectively, with ascorbic acid SC 50 � 14.2 ± 0.5 μg/mL as standard.
During this work, many major anthocyanins were detected in LC-MS analysis of HcA (such as peonidin dirhamnoside, peonidin derivative, and peonidin-3-(p-coumaroyl-glucoside)). According to [49], anthocyanin's high antioxidant activity was evidenced and is related to its structure, including the type, number, and position of the substituents in the flavylium cation. 3′-Hydroxyl group in cyanidin forms a catechol structure in the B ring, stabilizes the semiquinone radical, and forms a stable quinone that inhibits free radicals, such as DPPH, while 3′,5′-dihydroxyl group of delphinidin forms a pyrogallol structure in the B ring that has more delocalized electrons to stabilize the free radical generated in the medium. Ethyl acetate fraction of HcA, in a previous study, showed more significant antioxidant activity (SC 50 � 17.6 ± 1.8 μg/mL) than the other extracts of HdA and HmA [5]. Many potential pathways for phenolic compounds to act as antioxidants were listed such as inhibiting free radical formation, peroxide decomposition, oxygen radical absorbance, free radical scavenging, suppression of singlet oxygen, increasing the levels of endogenous defenses, chelating of metal ions, and enzymatic inhibition [50,51]. ere is a direct relation between the total polyphenolic content and the antioxidant activity [52] as polyphenolic compounds such as phenolic acids, flavonoids, and anthocyanins may be responsible for the antioxidant activity on a large proportion [51].
Correlation between radical scavenging ability, SC 50 values, and the identified phenolic acids in LC-MS analysis is considerable as extracts with higher flavonoids and/or phenolics contents showed higher antioxidant activity and lower SC 50 value. Salem et al. [53] stated in a previous study that flowers and leaves of H. rosa-sinensis, H. sabdariffa calyx extract, and H. platanifolius leaves extract possessed antioxidant activity that may be attributed to anthocyanins, flavonoids, and ascorbic acid content.

Cytotoxicity
Assay. About 70% of death, in low-and middle-income countries, is caused by cancer [54]. For new effective anticancer drug discovery, screening of the cytotoxic activity of the plant extracts and natural products is necessary [55]. Edible plants are excellent resources of anticancer agents [56].
In our study, in vitro cytotoxic activity of the applied samples against tested cell lines using MTT assay and cisplatin as a positive standard showed a decrease in cell viability in dose-dependent manner as illustrated in Figure 4 and Table 2. Evaluation was based on IC 50 values as follows: IC 50 ≤ 20 μg/mL highly active, IC 50 21-200 μg/mL moderately active, IC 50 201-500 μg/mL weakly active, and IC 50 > 501 μg/mL inactive, which is in a good accordance with the American National Cancer Institute protocol [57].
As indicated by IC 50 values, the cytotoxicity of tested samples against HCT-116 cell is arranged as follow: HmA > HcA > HdA. A close cytotoxic effect on HCT-116 cell line was shown by HdA and HcA (IC 50 � 96 ± 3.2 and 92.9 ± 4.1 μg/mL, respectively). e cytotoxic activity of HmA may be attributed to N-feruloyltyramine as it was reported as a cytotoxic agent in a previous study [60], and this cytotoxic activity may be enhanced by the presence of ascorbic acid according to [61,62].
Our results are in agreement with those reported for the cytotoxicity of flavonoids, phenolic acids, and terpenes content which are major constituents identified in HmA in that study [63][64][65][66]. In a previous study, different extracts (ethyl acetate, chloroform, petroleum ether) of HcA, HdA, and HmA showed strong anticancer property against human hepatocellular carcinoma (HepG2) and human breast carcinoma (MCF-7) cell lines [63]. Leaves   cancer cells such as breast, lung, and human leukemia cells (HL-60) and liver cancer cell lines, and they showed potent cytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoids, tannins, triterpenes, phenols, steroids [67,68], polyphenolic compounds, such as protocatechuic acid, anthocyanins such as delphinidin-3-sambubioside, and myristic acid and uncarinic acid A [45,48,52].

Antidiabetic Assay.
Diabetes mellitus (DM) is a persistent disorder that is incurable due to the deficiency of insulin that affects 10% of the population. It is expected to extend the number of diabetic individuals to 230 million in 2025. ere are many side effects for drugs currently used in DM treatment, so herbal medicines are highly recommended for the treatment of diabetes instead of other synthetic drugs [3]. Since ancient times, DM has been treated orally using folklore medicine with several medicinal plants or their extracts [65].
In the present study, in vitro α-amylase inhibitory activity of the applied samples, evaluated using different doses (7.81-1000 μg/mL), showed significant inhibition of carbohydrate hydrolyzing enzymes (α-amylase) in dose-dependent manner as illustrated in Figures 5 and 6 [69] previously reported that oleuropein prevents some metabolic diseases related to oxidative stress such as diabetes, hypercholesterolemia associated with diabetes, and cardiovascular complications which are very predominant in diabetics, due to its hypoglycemic activity as it enhances peripheral glucose uptake or insulin release and stimulates the synthesis of liver glycogen through its antioxidant power.
In agreement with our results, other studies have reported that aerial parts of HdA were used as potential antidiabetics due to the presence of flavonoids [70]. e flowers and fruits of HmA were found effective in diabetes [71]. e reported hypoglycemic activity of methanol leaf extract of H. sabdariffa and H. rosa-sinensis and flowers extract of H. vitifolius and H. tiliaceus may refer to the presence of flavonoids, phenols, tannins, alkaloids, and saponins [48,65,72].

Antiobesity Activity.
Overweight and obesity are chronic disorders that are considered as a growing issue influencing both adults and children. Obesity is defined as irregular or excessive fat accumulation caused by the imbalance between energy intake and expenditure. e vast majority of metabolic disorders such as cardiovascular disease, dyslipidemia, hypertension, and diabetes may be due to obesity or overweight [45,73]. e inhibition of the digestion and absorption of dietary fats is a promising remedy for obesity. Natural products are preferable to obesity drugs such as orlistat which have many side effects (i.e., development of cardiovascular problems, restlessness, sleeping disorder, and stomach pain) [73].
Results of the antiobesity activity of the three Hibiscus species aqueous extracts grown in Saudi Arabia using in vitro   pancreatic lipase inhibitory assay are shown in Figure 7 and Table 3. HdA exhibited higher inhibitory activity than the HmA and HcA with IC 50 of 95.45 ± 1.9, 107.7 ± 1.5, and >1000 μg/mL, respectively, comparable with orlistat (IC 50 � 23.8 ± 0.7 μg/mL) as standard. Lipase inhibitory activity of HdA may be attributed to the presence of anthocyanins (peonidin-3-(p-coumaroyl-glucoside), peonidin dirhamnoside, peonidin glucoside feruloyl glucuronide, peonidin dipentoside, malvidin derivative, and malvidin-3-O-glucoside derivative) and organic acids (such as succinic, ascorbic, and 4-hydroxybenzoic acid). It was reported that polyphenol compounds such as anthocyanins [74] and organic acids [75] are responsible for the antiobesity activity. Da-Costa-Rocha et al. [48] stated that Hibiscus extract (or tea) may help in weight loss as antiobesity agent due to its effects on fat absorption-excretion, inhibition of the activity of α-amylase, starch absorption, and blocking sugars. Moreover, aqueous extract of Hibiscus species showed a powerful inhibition of triglyceride accumulation as whole extract was more active than isolated polyphenols.
In a previous study, aqueous extract of H. sabdariffa (with anthocyanins being major compounds) exhibited many potential antiobesity mechanisms, including antihyperglycemic activity, reduction in plasma cholesterol level, inhibition of gastric and pancreatic lipase enzymes, thermogenesis stimulation, inhibition of lipid droplet accumulation in fat cells, and fatty acid synthase inhibition [76]. Drinking a cup of Hibiscus tea after meals can reduce the absorption of dietary carbohydrates and assist in weight loss [45].

Conclusion
Phenolic compounds, flavonoids, and anthocyanins were identified in three different Hibiscus species using UPLC-ESI-MS/MS analysis. HcA showed the highest in vitro antioxidant activity compared with other tested extracts, and this activity can be attributed to its contents of polyphenolic compounds such as apigenin C-hexoside-C-pentoside, luteolin C-hexoside-C-pentoside, luteolin derivative, 4hydroxybenzoic acid, and tyrosol and peonidin derivative in addition to presence of anthocyanin contents such as peonidin dirhamnoside, peonidin derivative, and peonidin-3-(p-coumaroyl-glucoside). HdA showed the most potent effect on human lung carcinoma (A-549) cell line, which may be attributed to the presence of major compounds such as butein flavonoid and peonidin dirhamnoside and peonidin dipentoside anthocyanins, with the highest activities as antidiabetic (due to oleuropein presence) and as antiobesity (may be attributed to the presence of major anthocyanins      such as peonidin-3-(p-coumaroyl-glucoside), peonidin dirhamnoside, and peonidin glucoside feruloyl glucuronide in addition to organic acids (such as succinic, ascorbic, and 4-hydroxybenzoic acid)). e results recommend that HdA need further studies for the possible use as anticancer, antidiabetic, and antiobesity agent as it might be a natural alternative remedy and nutritional policy for diabetes and obesity treatment without negative side effects. Isolation of the bioactive phytochemical from the HcA, HmA, and HdA and estimation of their biological effects are recommended in further studies.

Data Availability
e data used to support the findings of this study are included within the article.

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