Metabolite Profiling of Methanolic Extract of Gardenia jaminoides by LC-MS/MS and GC-MS and Its Anti-Diabetic, and Anti-Oxidant Activities

In this study, the methanolic extract from seeds of Gardenia jasminoides exhibited strong antioxidant and enzyme inhibition activities with less toxicity to NIH3T3 and HepG2 cells at the concentration of 100 µg/mL. The antioxidant activities (DPPH and ABTS), α-amylase, and α-glucosidase inhibition activities were found higher in methanolic extract (MeOH-E) than H2O extract. Besides, 9.82 ± 0.62 µg and 6.42 ± 0.26 µg of MeOH-E were equivalent to 1 µg ascorbic acid for ABTS and DPPH scavenging, respectively while 9.02 ± 0.25 µg and 6.52 ± 0.15 µg of MeOH-E were equivalent to 1 µg of acarbose for inhibition of α-amylase and α-glucosidase respectively. Moreover, the cell assay revealed that the addition of MeOH-E (12.5 µg/mL) increased about 37% of glucose uptake in insulin resistant (IR) HepG2 as compared to untreated IR HepG2 cells. The LC- MS/MS and GC-MS analysis of MeOH-E revealed a total of 54 compounds including terpenoids, glycosides, fatty acid, phenolic acid derivatives. Among the identified compounds, chlorogenic acid and jasminoside A were found promising for anti-diabetic activity revealed by molecular docking study and these molecules are deserving further purification and molecular analysis.


Introduction
Diabetes mellitus (DM) is a commonly detected chronic disorder causing major mortality worldwide. The progression of diabetes in the global population was reported as 9.3% by 2019 and projected to increase about 10.2% by 2030 and 10.9% by 2045 [1]. Metabolic malfunctions such as high elevation of the blood sugar (glucose) levels, oxidative stress and abnormal protein and lipid metabolism all lead to DM [2]. DM is categorized into two types: insulin-dependent type-1 diabetes (T1DM) and non-insulin-dependent type-2 diabetes (T2DM) [3]. Diabetic patients who are not able to secrete insulin are characterized as T1DM [4], while patients with insulin deficiency or insulin resistance in the human metabolic system, less insulin sensitivity or signaling in the liver, skeletal muscles, and adipose tissue are characterized as T2DM [5,6]. The prolonged diabetic symptoms (hyperglycemia, polyphagia, polydipsia, and insulin resistance) trigger multiple disorders such as cardiovascular diseases, renal failure, coronary artery, neurological complications, premature death, and limb amputation [7,8]. The diabetes incidence is higher in urban areas than in rural areas. Up 50% of people do not know that they are affected by diabetes [1].
Enzymes such as α-amylase and α-glucosidase play a vital role in carbohydrate metabolism. α-Amylase catalyzes the conversion of starch into glucose, while α-glucosidase

Yield, Total Phenol and Total Flavonoids Contents
The yield of different solvent extracts of seed powder of G. jasminoides was found to be 2.45% (w/w) and 1.58% (w/w) for methanol extract (MeOH-E) and water extract (H 2 O-E), respectively ( Table 1). The total phenol and flavonoids are major constituents in secondary metabolites of the plant extracts and they play a vital role in the biological properties of plants [22]. The bioactivities of the plant extracts are strongly correlated with the content of total flavonoids and phenolic substances. The G. jasminoides-derived pigments are shown to have anti-inflammatory, antioxidant, antibacterial activities with bio-health promoting properties by preventing various disorders [14]. Therefore, the content of total phenol (TPC) and total flavonoids (TFC) in MeOH-E and H 2 O-E was determined and the results are expressed as tannic acid equivalents (TAEs) for TPC while the TFC is presented as quercetin equivalents (QEs). For TPC, 769.47 ± 3.74 µg and 632. 15

Antioxidant Activities
Oxidative stress is a major primary cause of various health disorders. Therefore, screening of antioxidants from plant extracts can be a prime way to isolate novel compound against various chronic and metabolic disorders. 1,2-Diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical known to have a purple color with a strong absorption peak at 517 nm. Antioxidants can scavenge the DPPH by donating electrons [23]. (2,2 -Azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS + ) is a commonly used free radical for antioxidant assays. Mixing of ABTS and potassium persulfate produces the free radical form of the ABTS + which can be scavenged by the addition of synthetic or natural antioxidants [23]. The antioxidant activities of the DPPH and ABTS + varied significantly between the H 2 O-E and MeOH-E (p < 0.05). Among the samples, the free radical scavenging activity was found higher in MeOH-E than H 2 O-E in a dose-dependent manner. The free radical scavenging activity of these extracts was compared with a standard to obtain the ascorbic acid equivalents (AAEs). The results revealed that 9.82 ± 0.62 µg of MeOH-E and 13.20 ± 1.25 µg of H 2 O-E were equivalent to 1 µg AAEs for ABTS scavenging. It also varied for the DPPH scavenging with the values of 6.42 ± 0.26 µg for MeOH-E and 9.22 ± 0.81 µg for H 2 O-E, which were equivalent to 1 µg of ascorbic acid (Table 2). Further, the IC 50 concentration was found to be 120.5 ± 1.09 µg/mL and 262.5 ± 0.18 µg/mL for MeOH-E and H 2 O-E, respectively, for the ABTS + radical scavenging (Table 2). In the case of DPPH radical scavenging, the IC 50 was found to be 274.9 ± 1.42 µg/mL and 573.1 ± 0.85 µg/mL for MeOH-E and H 2 O-E, respectively (Table 2). Similarly, the methanol extract of G. volkensii reportedly shows a moderate DPPH scavenging activity [23]. Moreover, an earlier work reported that the water extract of G. jasminoides shows a higher DPPH and ABTS + scavenging activity than the ethanol extract. It is also observed from earlier study that the water extract of G. jasminoides exhibited the IC 50 values of 0.14 and 0.21 mg/mL for DPPH and ABTS + scavenging activities respectively [24]. This result indicates a variation between the present work and earlier work for IC 50 of H 2 O-E, probably due to the differences in the extraction method and sample collection location. The present results indicated that the antioxidant activity was higher in MeOH-E than that in H 2 O-E due to a higher total phenolic and flavonoids content [25]. The present work also found a similar relationship between antioxidant activity and total phenol content of MeOH-E and H 2 O-E, which is in accordance with earlier works [23,25].

Enzyme Inhibitory Activities
The enzymes α-amylase and α-glucosidase are involved in carbohydrate metabolism in the conversion of simple sugars from polysaccharides or disaccharides and also in catalyzing the blood glucose level that results in T2DM hyperglycemia [26]. Therefore, inhibition of these enzymes can control the prevalence of T2DM. Moreover, several studies also reported that screening of these enzyme inhibitors is crucial for the discovery of novel diabetes drugs [27,28]. The present work showed the enzyme (α-amylase and α-glucosidase) inhibitory activity of MeOH-E and H 2 O-E of seed powder of G. jasminoides (Table 2). Among the two samples, MeOH-E exhibited higher α-amylase and α-glucosidase inhibition activities than H 2 O-E. The 9.02 ± 0.25 µg of MeOH-E and 15.22 ± 0.55 µg of H 2 O-E were equivalent to 1 µg of acarbose for α-amylase inhibition activity (Table 2). In the case of α-glucosidase inhibition, 6.52 ± 0.15 µg of MeOH-E and 12.52 ± 0.61 µg of H 2 O-E were found to equivalent to 1 µg of acarbose ( Table 2). The IC50 of MeOH-E were found to be 432.05 ± 0.51 µg/mL and 798.25 ± 0.84 µg/mL for α-amylase and αglucosidase inhibition activity respectively ( Table 2). Among the two samples, MeOH-E showed promising activities of antioxidant and α-amylase and α-glucosidase inhibition. Therefore, MeOH-E was selected further for cell culture experiments.

Cytotoxicity
The cytotoxic effects of MeOH-E in a mouse fibroblast (NIH3T3) cell line was determined using a WST assay. The results revealed that MeOH-E at the concentration of ≤12.5 µg/mL did not show any cytotoxicity, while that at >25-100 µg/mL exhibited moderate cytotoxicity in the NIH3T3 cell line (Figure 1a). Similarly, the extract of G. jasminoides is reportedly non-toxic to the normal human MCF-10A cell line [29]. Another mouse model experiment confirmed that the pigments derived from G. jasminoides are less toxic [30]. Meanwhile, different solvent extracts of G. jasminoides have been reported to have promising cytotoxicity towards various cancer cells, including cervical cancer cell line (HeLa), skin malignancy cell line (A375), human non-small cell lung carcinoma cell line (H1299), and breast cancer cell line (MCF-7) [29,31]. However, to ensure the non-cytotoxicity of the MeOH-E in the NIH3T3 cell line the present study applied an acridine orange/ethidium bromide (AO/EB) fluorescent staining assay. This fluorescent method is used to determine the apoptosis-associated changes in cells based on the nucleus damage [32]. The AO/EB staining results indicated no apoptosis cells in the control group, and in the cells treated with 12.5 µg/mL; however, early stage apoptosis cells were observed at 50 µg/mL and 100 µg/mL (Figure 1b). Similarly, the early apoptosis in the osteosarcoma cells was detected by AO/EB staining as indicated by yellow-green and crescent-shaped cells [32].  (Table 2). In the case of αglucosidase inhibition, 6.52 ± 0.15µ g of MeOH-E and 12.52 ± 0.61 µ g of H2O-E were found to equivalent to 1 µ g of acarbose ( Table 2). The IC50 of MeOH-E were found to be 432.05 ± 0.51 µ g/mL and 798.25 ± 0.84 µ g/mL for α-amylase and α-glucosidase inhibition activity respectively (

Cytotoxicity
The cytotoxic effects of MeOH-E in a mouse fibroblast (NIH3T3) cell line was determined using a WST assay. The results revealed that MeOH-E at the concentration of ≤12.5 µ g/mL did not show any cytotoxicity, while that at >25-100 µ g/ mL exhibited moderate cytotoxicity in the NIH3T3 cell line (Figure 1a). Similarly, the extract of G. jasminoides is reportedly non-toxic to the normal human MCF-10A cell line [29]. Another mouse model experiment confirmed that the pigments derived from G. jasminoides are less toxic [30]. Meanwhile, different solvent extracts of G. jasminoides have been reported to have promising cytotoxicity towards various cancer cells, including cervical cancer cell line (HeLa), skin malignancy cell line (A375), human non-small cell lung carcinoma cell line (H1299), and breast cancer cell line (MCF-7) [29,31]. However, to ensure the non-cytotoxicity of the MeOH-E in the NIH3T3 cell line the present study applied an acridine orange/ethidium bromide (AO/EB) fluorescent staining assay. This fluorescent method is used to determine the apoptosis-associated changes in cells based on the nucleus damage [32]. The AO/EB staining results indicated no apoptosis cells in the control group, and in the cells treated with 12.5 µ g/mL; however, early stage apoptosis cells were observed at 50 µ g/mL and 100 µ g/mL (Figure 1b). Similarly, the early apoptosis in the osteosarcoma cells was detected by AO/EB staining as indicated by yellow-green and crescent-shaped cells [32].

Effect of MeOH-E on Cell Viability and Glucose Uptake in HepG2 Cell Line
MeOH-E did not display significant cytotoxicity on the Hep2 cell line at the concentration of ≤25 µg/mL and only at 100 µg/mL was significant cytotoxicity exhibited ( Figure 2a). This revealed the non-toxicity of MeOH-E in the HepG2 cell line at ≤25 µg/mL. Therefore, the effect of MeOH-E treatment in the glucose metabolism was tested by glucose uptake assay in non-insulin resistant and insulin resistant (IR)-HepG2 cell lines. The glucose uptake was found to be higher in the non-IR HepG2 cell line than that in the IR-HepG2 cell line. However, the addition of MeOH-E (12.5 µg/mL) increased~37% of glucose uptake in IR-HepG2 as compared to untreated IR HepG2 cell line (Figure 2b). This experiment also led to the interesting observation that the treatment above 25 µg/mL of MeOH-E to IR-Hep2 cell line significantly decreased the glucose uptake due to toxicity of the extract (Figure 2b). This is in accordance with an earlier report on ethyl acetate extract of Physalis alkekengi in glucose uptake in HepG2 cells [33]. The present work revealed that the treatment of 12.5 µg/mL was optimal for the increased glucose uptake by the IR-HepG2 cell line.

Effect of MeOH-E on Cell Viability and Glucose Uptake in HepG2 Cell Line
MeOH-E did not display significant cytotoxicity on the Hep2 cell line at the concentration of ≤25 µg/mL and only at 100 µg/mL was significant cytotoxicity exhibited ( Figure 2a). This revealed the non-toxicity of MeOH-E in the HepG2 cell line at ≤25 µ g/mL. Therefore, the effect of MeOH-E treatment in the glucose metabolism was tested by glucose uptake assay in non-insulin resistant and insulin resistant (IR)-HepG2 cell lines. The glucose uptake was found to be higher in the non-IR HepG2 cell line than that in the IR-HepG2 cell line. However, the addition of MeOH-E (12.5 µ g/mL) increased ~37% of glucose uptake in IR-HepG2 as compared to untreated IR HepG2 cell line (Figure 2b). This experiment also led to the interesting observation that the treatment above 25 µ g/mL of MeOH-E to IR-Hep2 cell line significantly decreased the glucose uptake due to toxicity of the extract ( Figure  2b). This is in accordance with an earlier report on ethyl acetate extract of Physalis alkekengi in glucose uptake in HepG2 cells [33]. The present work revealed that the treatment of 12.5 µ g/mL was optimal for the increased glucose uptake by the IR-HepG2 cell line.

Fluorescent Assay
The cytotoxicity of MeOH-E in the HepG2 cell line was measured by fluorescent AO/EB, rhodamine 123 (Rh123), propidium iodide (PI), and 2 -7 dichlorofluorescin diacetate (DCFH-DA) staining assays (Figure 2c-f). The cells were grouped as live cells (light green), apoptosis cells (fluorescent or yellowish, orange), necrosis cells (red) [34]. The MeOH-E at 25 µg/mL and 100 µg/mL) caused slight cytotoxicity for IR-HepG2 cell line as evident by pyknosis and congregated chromatin emitting green or yellow and some red fluorescence while the untreated control cells emitted uniform green fluorescence ( Figure 2c). Rh123 staining is adopted to measure the mitochondrial membrane potential (MMP) loss in the HepG2 cell line. Rh123 dye effectively stains with rich mmP and loss of mmP is indicated with the decrease of dye emission [35]. Similarly, the present study observed that the Rh123 was highly emitted in the HepG2 cell line treated with different concentrations of MeOH-E and it indicated less toxicity of extracts ( Figure 2d). The PI is an impaired nucleic acid membrane stain used for the detection of dead cells in a cell population [36,37]. The present study observed no PI-stained cells in the untreated control group while the treatment of 25 µg/mL and 100 µg/mL of MeOH-E displayed the dead cells as red-colored ( Figure 2e). DCFH-DA staining results indicated that the treatment of MeOH-E (25 µg/mL) did not cause the ROS mediated cytotoxicity while it exhibited slight cytotoxicity in the HepG2 cell line ( Figure 2f).

Metabolite Profiling of the MeOH-E of G. jasminoides
To identify the components of the MeOH-E of G. jasminoides, we tentatively identified them using two major hyphenated techniques: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS), which cover quite different subsets of metabolites. For instance, GC-MS has a preference for volatile metabolites covering primary metabolism including organic and amino acids, sugars, sugar alcohols, and phosphorylated intermediates. In contrast, LC-MS/MS covers mostly polar compounds predominant in secondary metabolites such as phenolics and terpenoids [38,39].

Tentative Identification of Compounds by LC-MS/MS
The compounds present in the MeOH-E were tentatively identified using LC-MS/MS and the TIC chromatogram of metabolic profile of the MeOH-E is shown in the Supplementary Figure S1. The LC-MS/MS analysis revealed the presence of 39 phytochemicals that belonging to various subclasses such as phenolic, flavonoids, terpenes, iridoid glycosides, organic acids, and gardenia carotenoids (Table 3) The compounds were identified using the in-house phytochemical library (UNIFI 1.8; Waters) [40,41] and previously reported literature [14,42]. Structures of the selected compounds are presented in Figure 3.

Monoterpenoids
The monoterpenes, whether linear (acyclic) or containing rings (bicyclic and monocyclic), belons to a class of terpenes that possess remarkable applications in the food and pharmaceutical industries [55]. G. jasminoides was reported to be a rich source of monoterpenoids and a total of 26 monoterpenoids have been reported from G. jasminoides [56][57][58][59][60]. The present study identified a total of 13 monoterpenoids from MeOH-E of G. jasminoides based on the deprotonated molecular ions observed in the LC-MS/MS analysis.  Table 3.

Flavonoids
Flavonoids are a major group of molecules present in the plants with rich bioactivities including antioxidant, anti-diabetes, and anticancer properties. According to the earlier literature, a total of 22 flavonoids has been reported from the various extracts of G. jasminoides [14]. Similarly, the present study had identified compounds such as rutin

Monoterpenoids
The monoterpenes, whether linear (acyclic) or containing rings (bicyclic and monocyclic), belons to a class of terpenes that possess remarkable applications in the food and pharmaceutical industries [55]. G. jasminoides was reported to be a rich source of monoterpenoids and a total of 26 monoterpenoids have been reported from G. jasminoides [56][57][58][59][60]. The present study identified a total of 13 monoterpenoids from MeOH-E of G. jasminoides based on the deprotonated molecular ions observed in the LC-MS/MS analysis. The formulas of identified compounds were as follows: C 10 Table 3.

Flavonoids
Flavonoids are a major group of molecules present in the plants with rich bioactivities including antioxidant, anti-diabetes, and anticancer properties. According to the earlier literature, a total of 22 flavonoids has been reported from the various extracts of G. jasminoides [14]. Similarly, the present study had identified compounds such as rutin (C 27 Figure 4). Similarly, these compounds were reported from the flower and fruit of this plant [62,63].

Carotenoids
The carotenoids are a major constituent of the G. jasminoides, which is composed of carotenoids and similar compounds [61]. These compounds are used as food colorants as well as bioactive food additives.  Figure 4). Similarly, these compounds were reported from the flower and fruit of this plant [62,63].

Organic Acids and Others
According to the earlier research reports, a total of 30 organic acids with various bioactive properties, including phenolic acids and fatty acids can be isolated from G. jasminoides [14]. Similarly, the present study has identified a total of 13 organic acids and others from MeOH-E of G. jasminoides based on LC-MS/MS of deprotonated observed mass and its MS/MS fragmentation. The compounds were identified as chlorogenic acid (C 16 Table 3.

Protein and Ligand Preparation
The protein and ligand were prepared according to the methods described earlier [12]. The protein molecular dock preparation was done using the AutoDock vina after the removal of the water molecules. Further, the ligand was selected for the molecular docking study based on Lipinski's drug-likeness rules (Supplementary Table S2). The Lipinski's indicated five rules, which is favor to select a compound as an orally active agent such as (i) the molecular weight of the compounds < 500 Da, (ii) hydrogen bond donor < 5, (iii) hydrogen bond acceptor < 10, (iv) miLogP < 5 and molar refractivity (40-130) [66]. Out of 33 unique compounds identified from MeOH-E of G. jasminoides by LC-MS/MS ( Figure 3) and GC-MS (Supplementary Figure S2), a total of the 26 compounds were selected for the molecular docking study based on Lipinski's rules satisfactory (Supplementary Table S2).

Molecular Docking Molecular Interaction with α-Amylase
Molecular docking results revealed that all the selected compounds could interact with α-amylase. Among the compounds screened, jasminoside F, chlorogenic acid, jasminoside A, and thymine showed a higher docking score against α amylase (Table 4; Figure 5). The jasminoside F exhibited the binding affinity score of −8.5 kcal/mol with two hydrogen bond interactions with amino acid residues of His 299 and Gln63 in α amylase (Figure 5a). Chlorogenic acid showed the binding affinity score of -8.7 kcal/mol by interacting with amino acid residues of Arg421, Gly403, Arg398, Ser289 through six hydrogen bond interactions in α amylase (Figure 5b). Jasminoside A displayed a strong binding affinity score of −8.7 kcal/mol on α-amylase through interacting its amino acid residues of Arg195, His299 via two hydrogen bonds (Figure 5c). The organic compound thymine showed a binding affinity score of −5.3 kcal/mol against α-amylase by interacting its residues of Gly403, Arg398, Arg421 by six hydrogen bonds (Figure 5d). Moreover, the positive control of the acarbose derived trisaccharide exhibited higher hydrogen bonds of 11 and amino acids (Thr6, Arg10, Gly9, Gln7, Gly334, Arg421, Gln404) interaction with α-amylase with binding affinity score of -8.3 kcal/mol (Figure 5e) while another control acarbose showed only three hydrogen bonds and amino acids (His299, gln63, Thr163) interactions with binding affinity score of −8.3 kcal/mol (Figure 5f). Overall, the results revealed that among the compounds tested, jasminoside A and chlorogenic acid were found to have the potential to interact with α-amylase with high binding affinity score than other molecules including positive controls. Similarly, the compound jasminoside is known for tyrosinase inhibition [56] while the phenolic compound chlorogenic acid exhibits anti-oxidative and anti-diabetic activities [67,68].

Molecular Interaction with α-Glucosidase
The in silico docking study revealed that jasminoside F, jasminoside B, chlorogenic acid and jasminoside A displayed a higher binding affinity with α-glucosidase than other compounds studied in this study (Table 4; Figure 6). The interaction between jasminoside F and α-glucosidase showed the binding affinity score of −7.8 kcal/mol through the formation of five hydrogen bonds with amino acid residues such as Thr473, Asn476, Arg102 (Figure 6a). Jasminoside B established an interaction with α-glucosidase via six hydrogen bonds interacting with amino acids residues (Arg102, Tyr104, Gly241, Arg103, Asn476) of α-glucosidase with the binding affinity of 7.3 kcal/mol (Figure 6b). The chlorogenic acid exhibited the binding affinity score of -8.2 kcal/mol with five hydrogen bond interactions with amino acid residues of Met269, Glu759, Val760, Tyr266 in α-glucosidase (Figure 6c). The molecular interaction between jasminoside A and α-glucosidase exhibited a binding affinity of 7.8 kcal/mol by forming four hydrogen bonds with the amino acid residues Val760, Leu761, Glu762 (Figure 6d). However, the positive controls such as acarbose-derived trisaccharide and acarbose showed the promising dock binding affinity of 8.7 kcal/mol for interaction with α-glucosidase ( Table 4). The acarbose-derived trisaccharide was found to establish an interaction with α-glucosidase through eight hydrogen bonds with amino acid residues of Trp39, Cys40, Ala13, Pro14, Asp11, Arg237, Trp179 ( Figure  6e) while the acarbose established the interaction with α-glucosidase through six hydrogen bonds with amino acid residues of Trp39, Cys40, Pro14, Ala13, Arg237, Asp11 ( Figure  6f). Overall, the docking study revealed that interactions with α-glucosidase of chlorogenic acid and jasminoside A were promising as compared to other compounds screened, and we hypothesize that these interactions might inhibit the activity of α-glucosidase. This finds the support of earlier works on the antidiabetic and enzyme inhibitory activities of these compounds [56,67,68].

Molecular Interaction with α-Glucosidase
The in silico docking study revealed that jasminoside F, jasminoside B, chlorogenic acid and jasminoside A displayed a higher binding affinity with α-glucosidase than other compounds studied in this study (Table 4; Figure 6). The interaction between jasminoside F and α-glucosidase showed the binding affinity score of −7.8 kcal/mol through the formation of five hydrogen bonds with amino acid residues such as Thr473, Asn476, Arg102 (Figure 6a). Jasminoside B established an interaction with α-glucosidase via six hydrogen bonds interacting with amino acids residues (Arg102, Tyr104, Gly241, Arg103, Asn476) of α-glucosidase with the binding affinity of 7.3 kcal/mol (Figure 6b). The chlorogenic acid exhibited the binding affinity score of −8.2 kcal/mol with five hydrogen bond interactions with amino acid residues of Met269, Glu759, Val760, Tyr266 in α-glucosidase (Figure 6c). The molecular interaction between jasminoside A and α-glucosidase exhibited a binding affinity of 7.8 kcal/mol by forming four hydrogen bonds with the amino acid residues Val760, Leu761, Glu762 (Figure 6d). However, the positive controls such as acarbose-derived trisaccharide and acarbose showed the promising dock binding affinity of 8.7 kcal/mol for interaction with α-glucosidase ( Table 4). The acarbose-derived trisaccharide was found to establish an interaction with α-glucosidase through eight hydrogen bonds with amino acid residues of Trp39, Cys40, Ala13, Pro14, Asp11, Arg237, Trp179 (Figure 6e) while the acarbose established the interaction with α-glucosidase through six hydrogen bonds with amino acid residues of Trp39, Cys40, Pro14, Ala13, Arg237, Asp11 (Figure 6f). Overall, the docking study revealed that interactions with α-glucosidase of chlorogenic acid and jasminoside A were promising as compared to other compounds screened, and we hypothesize that these interactions might inhibit the activity of α-glucosidase. This finds the support of earlier works on the antidiabetic and enzyme inhibitory activities of these compounds [56,67,68].

Preparation of Desiccative Ripe Fruits Extract
One hundred gram of seed powder (desiccative ripe fruits) of the G. jasminoides was extracted with methanol (1:5 ratio) for 24 h agitation in a magnetic stirrer. The methanol extract (MeOH-E) was filtered through Whatman no 1 filter paper and then concentrated using a rotary evaporator at 40 °C. Besides the water extraction was done according to the protocols described earlier [23]. The yield of MeOH-E and H2O-E was quantified using a weighing balance and then stored at 4 °C for further analytical experiments. The contents of total phenol and total flavonoids in MeOH-E were measured according to methods described earlier [69][70][71].

Antioxidant Activities
MeOH-E was analyzed for free radicals (DPPH and ABTS) scavenging activity according to the protocols reported earlier [72,73]. For DPPH inhibition assay, 100 µ L of

Preparation of Desiccative Ripe Fruits Extract
One hundred gram of seed powder (desiccative ripe fruits) of the G. jasminoides was extracted with methanol (1:5 ratio) for 24 h agitation in a magnetic stirrer. The methanol extract (MeOH-E) was filtered through Whatman no 1 filter paper and then concentrated using a rotary evaporator at 40 • C. Besides the water extraction was done according to the protocols described earlier [23]. The yield of MeOH-E and H2O-E was quantified using a weighing balance and then stored at 4 • C for further analytical experiments. The contents of total phenol and total flavonoids in MeOH-E were measured according to methods described earlier [69][70][71].

Antioxidant Activities
MeOH-E was analyzed for free radicals (DPPH and ABTS) scavenging activity according to the protocols reported earlier [72,73]. For DPPH inhibition assay, 100 µL of MeOH-E (1.95-1000 µg/mL) and 100 µL of DPPH (100 µM) were mixed and incubated at 27 • C for 10 min. Later the reaction mixture was observed at 517 nm using a UV spectrophotometer. The percentage of the DPPH scavenging was determined by adopting the formula reported earlier [74]. For the ABTS inhibition assay, firstly, the oxidative form of the ABTS + was generated by mixing the potassium persulfate (2.45 mm) and ABTS (7 mm) at the ratio of the 0.5:1 ratio in dark conditions at 27 • C for 24 h. For the reaction, the 100 µL of ABTS + and 100 µL of MeOH-E (1.95-1000 µg/mL ) were mixed and incubated at 27 • C for 10 min. Afterward, the reaction mixture was measured at 734 nm using a UV spectrophotometer. The percentage of ABTS scavenging = ((Control-sample)/control) × 100). The control is ABTS+solution alone.

Enzyme Inhibition Activities
The inhibition of α-glucosidase and α-amylase was measured according to previously reported methods [75][76][77]. Acarbose was used as a positive control for this experiment. For the α-glucosidase inhibition assay, 50 µL of MeOH-E (1.95-1000 µg/mL) was added to 20 µL of α-glucosidase (1 U) and this, 25 µL of p-nitrophenyl glucopyranoside (pNPG; 5 M) was added and incubated at 37 • C for 30 min. Later, the 100 µL of Na 2 CO 3 (0.1 M) was added to stop the reaction and measured at 405 nm using a UV spectrophotometer. For the α-amylase inhibition assay, 50 µL of MeOH-E (1.95-1000 µg/mL), 150 µL of starch (0.5%), 10 µL of α-amylase (2 U) were mixed and incubated at 37 • C for 30 min. Later 20 µL of NaOH (2 M) was added to stop the reaction. Then 20 µL DNS of (3,5-dinitrosalicylic acid) was added to the reaction solution and boiled for 20 min at 100 • C. Finally, the reaction mixture was cooled at room temperature and read at 540 nm using a UV spectrophotometer. The percentage of enzyme inhibition was determined by following the formula reported elsewhere [74].

Cytotoxicity
The cytotoxicity of MeOH-E was tested in the normal NIH3T3 cells and HepG2 cells (1 × 10 4 cells/well) cultured in DMEM composed of FBS (10%), antibiotic solution (1%) for 24 h at 37 • C in 5% of a CO 2 incubator. Later, the cells were treated with MeOH-E (0-100 µg/mL) for 24 h. After the treatment period, WST reagent (10 µL) was added, kept in a CO 2 incubator for 1 h, and then OD was measured at 450 nm using UV spectrophotometer (SpectraMax ® Plus Microplate Reader, Molecular Devices, San Jose, CA, USA). The percentage of cell toxicity was calculated by adopting the formula reported previously [78].

Determination of Glucose Uptake
To assess the MeOH-E induced glucose uptake in the HepG2 cells, an insulin-resistant model cell line (IR-HepG2) was firstly generated according to the protocol reported elsewhere [79,80]. The well-established IR-HepG2 cells (1 × 10 4 cells/well) were cultured in high glucose DMEM incorporated with FBS (10%) and antibiotic solution (1%) in a 5% CO 2 incubator for 24 h. For the treatment, various concentrations of MeOH-E (0-100 µg/mL) were added to cells and incubated for 24 h in the above-mentioned conditions. Besides, the positive control (HepG2) cells were maintained. After the incubation, the cells including the culture media were harvested and centrifuged at 440 g for 5 min, and the supernatant was used for glucose assay by DNS method. Glucose uptake (%) was estimated using the formula:(OD of high glucose DMEM media-IR-HepG2 cultured supernatant OD)/OD of high glucose DMEM media) × 100. Followed by the prevention of oxidative stress, mitochondrial membrane loss, and nucleus damage in IR-HepG2 by treatment of MeOH-E was observed using various staining assay as reported in earlier studies [81][82][83].

UHPLC-QTOF-MS/MS Analysis
For the UHPLC-QTOF-MS/MS analysis, MeOH-E was dissolved in 70% methanol, filtered with PTFE syringe filter (0.2 µm), and finalized in 20 ppm of MeOH-E. The LC/MS systems consisted of a Waters Acquity UPLC I-Class system (Waters Corp., Milford, MA, USA) coupled to Waters Xevo G2 QTOF mass spectrometer (Waters MS Technologies, Manchester, UK) equipped with an electrospray ionization (ESI) interface. The chromatographic separation was done with LC/MS equipped Waters Acquity UPLC BEH C18 (150 mm × 2.1 mm, 1.7 µm) (Waters Corp.). For the UHPLC, 2 µL of the sample was injected with a flow rate of 300 µL/min with a temperature of auto-sampler (10 • C) and column oven (40 • C). The mobile phases were 0.1% formic acid in H 2 O (A) and 0.1% formic acid in acetonitrile (B), and the following gradient was used: 10-90% B (0-12 min) and 100% B (12.1-16.0 min). The MS/MS data were obtained using a collision energy ramp from 15 to 45 eV in MS E mode. The ESI parameters were set as follows: in negative ion mode in Continuum format, a capillary voltage of 2.5 kV, cone voltage of 45 V, source temperature of 120 • C, desolvation temperature of 350 • C, cone gas flow of 50 L/h, and desolvation gas flow of 800 L/h. The ion acquisition rate was 0.25 s with the mass range from m/z 100 to 1600. The instrument was calibrated using a sodium formate solution as the calibration standard. Leucine enkephalin (m/z 554.2615 in negative mode) was used as the reference lock mass at a concentration of 200 pg/µL and a flow rate of 5 µL/min and was sprayed into the MS instrument every 10 s to ensure accuracy and reproducibility. The data acquisition was measured by MassLynx V4.1 (Waters Corp.). The compounds were identified using the in-house phytochemical library (UNIFI 1.8; Waters Corp.) [40,41].

Gas Chromatography Analysis
The organic compounds present in MeOH-E was determined using a gas chromatography (Agilent 789A, Agilent, Santa Clara, CA, USA) mass spectrophotometry (Agilent 5975C; GC-MSD) system in the scan range of m/z 50-500 according to the detailed operation conditions described elsewhere [64,84]. The GS-MS used in this study was equipped with DB-5MS (30 m length × 0.25 mm inner diameter × 0.25 µm thickness of film) column and performed under operation condition as the flow rate of 1 mL/min, injection mode (5:1) with an inlet temperature of 250 • C, interface temperature of 280 • C, ion source of EI, 70 eV, with the temperature of 280 • C. The compounds present in the MeOH-E were tentatively identified by matching the GC-MS data with the electronic library of W8N05ST.L.

Molecular Docking
The compounds with enzyme inhibitory activity identified from MeOH-E were virtually analyzed against human lysosomal acid-α-glucosidase (PDB: 5NN8) and human pancreatic α-amylase (PDB: 5E0F) by molecular docking. The structure files of ligands were prepared using ChemBioDraw 15.0 (PerkinElmer, Waltham, MA, USA) and then saved as mol. These mol files of ligands were used for energy minimization according to the principle of gasteiger [85]. The 3D structure of PDB of 5NN8 and 5E0F were retrieved from RSCB (https://www.rcsb.org/) and before the docking experiment the water residue was removed and the binding packet size was prepared as reported earlier [12]. Finally, the molecular docking between various ligand and targeted protein was carried out using Autodock Vina 1.1.2. Finally, the interactions between the protein and compounds were observed using LIGPLOT+(v.2.2).

Statistical Analysis
All the experiments were executed in triplicate and the results are presented with mean ± standard error (SE). The descriptive statistics, student 't' test, and analysis of various (ANOVA), line diagrams, Duncan's multiple range test (DMRT) were made using excel. 2010 and SPSS (Ver 2016, IBM, Armonk, NY, USA). The difference at p < 0.05 was considered as significant among the factors.

Conclusions
In summary, this work analyzed the enzyme inhibition, anti-diabetic activities and metabolites present in the MeOH-E of G. jasminoides by using LC-MS/MS and GC-MS. The MeOH-E showed higher enzyme inhibition, antioxidant and anti-diabetic activities in IR-HepG2 cells. Metabolic profiling studies tentatively identified a total of 54 compounds including iridoids, terpenoids, fatty acid, phenolic acid derivatives from MeOH-E of G. jasminoides based on the observed m/z molecular ions in LC-MS/MS and GC-MS. The compounds identified were nine iridoid glycosides, 13 monoterpenoides, two each of flavonoids and carotenoids. Among the compounds identified chlorogenic acid and jasminoside A were found promising in interacting with α-glucosidase and αamylase, as evidenced by molecular docking studies. Therefore, the present work concluded that bioactivity of the MeOH-E of G. jasminoides was the synergistic effect of various compounds present in the extract. According to the molecular screening, it is recommended that chlorogenic acid and jasminoside A be considered as candidate molecules for anti-diabetic activity. However, further studies are required for the purification and characterization of these two molecules and to determine their molecular mechanism of anti-diabetic activity for the development of future therapeutics.  Table S1. GC-MS based analysis of alkaloids and low molecular weight molecules from methanolic extract (MeOH-E) of G. jasminoides. Supplementary Table S2. Assessment of the Drug-likeness through Lipinski's strategies for methanolic extract (MeOH-E) of G. jasminoides by web tool (SwissADME).