In silico molecular docking analysis of potential antilipidemic bioactive constituents of Coriandrum sativum root extract using GC-MS

2.1. Plant specimen acquisition and authenticity

C. sativum roots were confidently gathered from the local spice garden of Paikaramau, Kursi Road in Lucknow, in December 2018. These roots were subsequently authenticated by the National Botanical Research Institute (NBRI) in Lucknow (NBRI/CIF/668/2018).

2.2. Drugs and chemicals

Biochemical kits for evaluation of total cholesterol (TC), high-density lipoprotein (HDL), triglycerides (TG), and low-density lipoprotein (LDL) were procured from Agappe Diagnostic Ltd., Kerala, INDIA. Further chemicals and analytical-grade solvents, methanol, ethanol, and chloroform, were purchased from SD Fine Chemicals Ltd. The Standard Orlistat approved by the US-FDA is purchased from Quantum In. 64 C, Virat Khand Rd, Kamta, Lucknow, U.P., was used for anti-obesity and anti-adiposity drugs [10].

2.3. Preparation of extract

Coriandrum sativum roots (250 g) were thoroughly rinsed with tap water, and the earthy stuff was scraped out with a sharp knife. The roots are dried in the shade at ambient temperature for 4 weeks before being pulverized into a coarse powder (88.5 g). The Soxhlet apparatus was used for the extraction of powdered root (50 g) with 250 mL of ethanol. A rotary evaporator was used to evaporate the filtrate at 40°C under reduced pressure until it became dry, yielding 1.85 g (3.7% w/w) of ethanol extract [11]. Chemical tests and GC-MS analysis were employed to qualitatively and quantitatively assess the bioactive phytochemicals present in the ethanolic extract of C. sativum roots (ECSR) [12].

2.4. GC-MS analysis

The GC-MS analysis of the ECSR was carried out using the PerkinElmer GC Clarus 500 system. This system included an AOC-20i autosampler and a gas chromatograph-mass spectrometer. The GC column used was an Elite 5MS, which is a specific type of column with certain characteristics. Helium gas, with a very high level of purity, was used as the carrier gas at a steady flow rate. The process involved maintaining the column at a steady temperature while the sample was analyzed. The ionization generator was set to electron bombardment mode at 70 eV. Throughout the analysis, 250°C was the oven temperature, whereas 200°C and 250°C were designated as the ion and reaction temperatures, respectively. Mass spectra were attained by using Turbo-Mass ver-5.2, with the mass instrument being Turbo-Mass Gold-Perkin-Elmer. The revenue percentage for each component was determined based on the maximum average across the entire region. The GC-MS analysis lasted for 30 min, including a weighing delay of 0 to 2 min. A GC Clarus 500 system with an AOC-20i autosampler and GC-MS was utilized for analysis. The system utilized an Elite 5MS coupling with a capillary line measuring 30 x 0.25 µm ID x 0.25 µm df. The carrier gas was helium with a 99.999% purity, flowing at a steady rate of 1 mL/min and 2 µL (comparative ratio 10:1). The ionization generator functioned in electron bombardment mode with an ionization energy of 70 eV. The oven temperature was established at 250°C, the ion temperature at 200°C, and the reaction temperature was sustained at 250°C. Mass spectra were acquired using Turbo-Mass version 5.2, with the mass spectrometer being Turbo-Mass Gold-Perkin-Elmer [13].

2.5. Identification of phytocomponents

Examining the GC-MS mass spectrum means tapping into the extensive NIST (National Institute of Standards and Technology) database, which holds around 62,000 samples. Researchers recognized the components’ names, molecular weights, and structures by comparing the unknown sample’s spectrum to the NIST library’s [14].

2.6. Molecular docking of identified compounds with 4qho

2.7. Animals

Sprague Dawley rats (30 female, 140-160 g) purchased from Central Drug Research Institute, Lucknow, Uttar Pradesh, India, were kept in standard laboratory conditions. The rats were fed a commercial meal and unrestricted access to water. For the rats to get used to their new environment, they were allowed to spend a week in the lab before the experiment started. The experiments were done under the IAEC approval number (IU/IAEC/19/13).

2.8. Schedule for the experiment

Following 7 days of familiarization, the animals were randomly allocated to either the normal or overweight group and fed either a normal pellet diet (NPD) or a high-fat diet (HFD), correspondingly, and permitted to consume whatever they pleased for another week [17]. The HFD (Harlan Teklad TD93075) contains fat (54.8%), carbs (24.0%), protein (21.2%), and several vitamins and minerals. The rats were categorized into five groups, each consisting of six animals. The control group was fed a regular diet and normal saline, group II was fed an HFD, and groups III and IV were given 100 and 200 mg/kg per day orally of CSRE, while group V received the standard drug 5 mg/kg. Each group, except the control group, was given the HFD for 10 days, and then III, IV, and V groups were given the tested drug simultaneously with HFD on the last day of the experiment (28 days). On the 29^th day, the rats were evaluated for the physical parameters, and anesthetized with ketamine hydrochloride to determine their biochemical and enzymatic parameters [18].

2.9. Assessment of physical parameters

2.10. Determination of lipid serum profile and biochemical parameters

On day 29, experimental rats were anesthetized with ketamine hydrochloride, blood was withdrawn through the tail vein, and the collected blood sample was centrifuged at 3000 rpm, blood serum was separated. Biochemical markers, including cholesterol, TG, very low-density lipoprotein (VLDL), LDL & HDL, and (AI) atherogenic index, were measured [21].

2.11. Antioxidant parameters

2.12. Statistical analysis

The findings are provided as the mean ± Standard Error of the Mean (S.E.M.). and ± SD for six rats. Graph Pad Prism 2.01 was used to do a one-way analysis of variance (ANOVA) followed by Dunnett’s test. If the p-value < 0.05, the results were deemed significant.

3.1. Identification of phytocomponents

The crucial step of isolating and assessing plant components is a vital aspect of formulating herbal remedies to ensure consistency and quality assurance. To achieve this objective. The study used GC-MS to detect and validate bioactive components in C. sativum root ethanolic extract (Figure 2). Twelve phytocomponents were present in the ethanol extract of C. sativum roots (Table 1 and Figure 3). The identified compounds comprising phenolic compounds, volatile oils, steroids, and terpenoid compounds namely 1,3-propanediol,2-ethyl-2-(hydroxymethyl) (72.54%), 3-tridecanol (3.94%), (E)-tetradec-2-enal (6.97%), 3-tridecanol (7.58%), cyclohexane,1,4-diethoxy, trans (1.52%), (E)-hexadec-2-enal (0.85%), Hexadecanoic acid, methyl ester (0.75%), cyclohexane, 1,4-diethoxy, cis (0.42%), 11,14-eicosadienoic acid, methyl ester (1.30%), squalene (0.20 %), stigmasta-5,23-dien-3-ol, (3.beta.) (2.53%) and stigmast-5-en-3-ol, (3. Beta., 24S) (1.39%).

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Figure 2.

Phytocomponents recognized in the methanol extract of C. sativum root.

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Figure 3.

Phytocomponents identified in the methanolic ECSR by GC-MS.

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3.2. Molecular docking (MD): The chemicals’ binding interactions with the fat mass and obesity associated protein (FTO)

To examine the coupling interactions with the FTO receptor, MD was performed using the identified compounds from F1 (Code for sample or extract etc.) viz. (1), (2), (3), (5), (6), (7), (8), (9), (10), (11), and (12). The results of the binding affinity (docking scores) and hydrogen bonding catalytic residue for the chosen compounds against FTO (PDB identification: 4qho) have been provided in Table 2. The analysis revealed that 1,3-propanediol-2-ethyl-2-(hydroxymethyl) (1) was the most active and powerful compound, with its binding mode showing that the hydroxyl groups of the 1,3-propanediol moiety formed hydrogen bonds with ASP233, HIS307, and HIS231, while the oxygen atom bonded with ARG322. Additionally, the 2-(hydroxymethyl) group of 1 formed a hydrogen bond with ASN205 and displayed an alkyl interaction with VAL309 on the active site of the FTO receptor. The binding connections of 3-Tridecanol (2) with the FTO receptor displayed a hydrogen bond with SER318, and hydrophobic interactions, including alkyl and π-alkyl interactions, were also formed with LEU203, VAL228, HIS231, TYR108, and VAL309. Similarly, the binding affinity of cyclohexane-1, 4-diethoxy-trans (5), (E)-hexadec-2-enal (6), and cyclohexane-1, 4-diethoxy, cis (8) demonstrated formation of a hydrogen bond with ARG316 and TYR295. Moreover, the ethoxy group attached to the cyclohexane ring of 5 and 8 exhibited a hydrophobic bond interaction with VAL94, VAL228, VAL244, HIS307, and MET297. Also, the cyclohexane ring of the above isomers illustrates a strong π-σ interaction with VAL309. Furthermore, binding interactions of (E)-tetradec-2-enal (3), hexadecanoic acid-methyl ester (7), and 11, 14-eicosadienoic acid-methyl ester (9) demonstrated hydrogen bonding with ARG322, ARG96, and TYR106, and the methyl ester group of compounds 7 and 9 also revealed hydrogen bond interactions with TYR295 and HIS231 residues of the FTO protein. Additionally, these compounds displayed σ-πinteraction with HIS231 and hydrophobic alkyl and π-alkyl interactions with PRO93, VAL228, LEU109, TYR108, TYR106, and VAL309 on active sites of the FTO receptor. In the case of compounds 10, 11, and 12, no binding interactions were observed with the FTO receptor (Figures 4a-g).

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Figure 4.

Docking analysis (a) Compound 1 with FTO, (b) Compound 2 with FTO, (c) Compound 3 with FTO, (d) Compound 5 with FTO, (e) Compound 6 with FTO, (f) Compound 7 with FTO, (g) Compound 8 with FTO.

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3.3. BW and food intake

For each group body weight (BW) was measured on the initial day before given diet and after 10 days of the feeding, and final weight was recorded at the end the experiments (28 days) after administration of the test drug, as shown in the Table 3, as well as the calculate food intake (g), nutritional status, and BMI at the end of 28 days and showed in Table 4.

3.4. Relative organ weight

Table 5 displays the relative organ weights of experimental rats. Group II rats had considerably (p<0.05) higher heart, liver, and spleen weights compared to group I. After 28 days, ECSR at 100 and 200 mg/kg orally significantly reduced liver, spleen, and heart weights (p<0.05 and p<0.01) compared to the standard group III.

3.5. Blood lipid profile

Table 6 revealed that the lipid biomarker levels (TC, TG, LDL, and VLDL) in experimental rats were significantly elevated, while the levels of HDL biomarker were significantly decreased in the HFD-treated group II compared to the control group I. Management with orlistat at a dose of 5 mg/kg in Group III, and ECSR 100 and 200 mg/kg orally in Groups IV and V, resulted a significant (p<0.05 and **p<0.01) decrease the level of TC, TG, LDL and VLDL as well as HDL level was significant (**p<0.01) increase in Sprague Dawley (SD) rats when compared with group II. Additionally, the test results showed an increase in serum AI levels (p<0.01) in HFD-induced obese animals, which was lower than that of normal controls.

3.6. Oxidative Stress Parameters

Table 7 demonstrates that participants in the HFD (Group II) group exhibited lower levels of oxidative stress parameters, including SOD, GSH, GPx, and TBARS, when compared to the normal control group (Group I). Treatment with ECSR 100 and 200 mg/kg and standard 5 mg/kg Orlistat in Groups IV, V, and III resulted in a significant increase (p<0.05 and **p<0.01) in SOD, GSH, GPx levels, and a decrease in TBARS levels.

3.7. Discussion

Ethanol ECSR contains various types of bioactive compounds that play an important role in different pharmacological activities (1). 1, 3-propanediol, 2-ethyl-2-(hydroxymethyl) is a new volatile oil compound that may be utilized as an anti-microbial activity, not reported in the root of this plant. (2) 3-tridecanol and (3) (E)-tetradec-2-enal were previously quantified in the root and reported in leaves used as an adjuvant and flavoring agent. (5) cyclohexane,1,4- diethoxy- trans and (8) cyclohexane,1,4-diethoxy-, cis are the new compounds identified in the root as not reported in other parts of this plant. (5) (E)-hexadec-2-enal is present in the root and previously reported in aerial parts used as a flavoring agent and as an adjuvant. (6) hexadecanoic acid, methyl ester, also found in leaves, possesses antiandrogenic, antioxidant, and nematocidal activities. It has been found to have hemolytic hypocholesterolemic activity and as a flavoring agent [26]. (9) 11,14-eicosadienoic acid, methyl ester is a fatty acid methyl ester and a polyunsaturated fatty ester, a new compound, not reported in the aerial part of this plant. (9) squalene, (10) stigmasta-5,23-dien-3-ol, (3. Beta.), (11) stigmast-5-en-3-ol, (3. Beta.,24S) and (12) stigmast-5-en-3-ol, (3. Beta.,24S) are also present in seed oil. Squalene may initiate the lowering of cholesterol if added to some drugs, as it decreases TG and TC levels. It is also used as an adjuvant in the treatment of cancer [27]. (10) stigmasta-5,23-dien-3-ol, (3.Beta.), (11) stigmast-5-en-3-ol, (3.Beta.,24S), and (12) stigmast-5-en-3-ol, (3. Beta.,24S) are supports in the governing and tissue rebuilding mechanisms associated with estrogen effects. It is used as an intermediate in the biosynthesis of androgens, estrogens, and corticoids, and correspondingly used as a precursor of vitamin D3 (VIT-D3) [28]. They enhance the antioxidant activity and are used as a potential anti-inflammatory, anti-apoptotic, and anticancer agent in pharmaceuticals. It also neutralizes the venom of snakes such as vipers and cobras and inhibits the progression of tumors by acting on protein kinase C (PKC) and in the sphingomyelin cycle [29]. Increased plasma LDL cholesterol (LDL-C) acts as a considerable risk for cardiovascular disorders. Plant sterols decrease the levels of LDL and are cardio-protective, as they lower the cholesterol level [30].

The HFD-based model is rich in calories and fat content, and it is typically given to rats to induce obesity [31]. Numerous studies have shown that rats nourished with HFD serve as an ideal prototype for an obesity model, and their diet is comparable to the diet consumed by humans with obesity [32]. The HFD model examined BW, food intake, nutritional quality, BMI, and biochemical markers like AI, TC, TG, HDL, LDL, and VLDL levels [33]. When rats are fed high-fat foods, their body weight increases, resulting in increased liver weight and increased TC and TG levels. The drug C. sativum prevents weight gain in rats by preventing body fat deposits.

Obesity can significantly alter biochemical markers, particularly the lipid profile. In obese individuals, an increase in TC, TG, LDL, and VLDL levels, accompanied by a decrease in HDL (beneficial cholesterol), is commonly observed. The elevated levels of TC, TG, and LDL are significant risk factors for cardiovascular disorders. HFD raises TC and TG levels, which are delivered to tissues via the bloodstream. The animals fed with ECSR prevent the increase in TC, TG, LDL, and VLDL levels compared to HFD-fed rats. Additionally, the levels of oxidative stress markers such as SOD, GSH, GPx, and TBARS increased significantly in rats given ECSR. C. sativum protects rats by affecting changes in the body’s lipid profile and signs of oxidative stress. Chithra and Leelamma’s predicted hypolipidemic process for coriander seeds may involve a considerable increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) activity, which is a critical enzyme in cholesterol synthesis [34]. ECSR-fed rats have lower liver cholesterol because its breakdown into bile acids is greater than its synthesis. Instead, the decrease in TC levels was attributed to the addition of fiber to the experimental diet of hypertriglyceridemic animals, which increases plasma lecithin cholesterol acyltransferase (LCAT), thereby enhancing hepatic bile acid synthesis and increasing blood cholesterol levels. It removes bile acids and cholesterol from feces and reduces LDL production in the liver [21].

The coriander ethanol root extract is rich in sterols, particularly stigmast-5-en-3-ol, (3. Beta.,24S), stigmasta-5,23-dien-3-ol, (3.Beta.), and squalene, which may inhibit cholesterol absorption [35]. The subtle structural variation between stigmasterol and cholesterol enables stigmasterol to be incorporated into intestinal micelles, effectively displacing cholesterol and consequently diminishing its absorption. Moreover, phytosterols, especially stigmasterol, enhance the activity of the enzyme HMG-CoA reductase, which inhibits cholesterol absorption and promotes the production of hepatic bile acids, resulting in increased cholesterol excretion. Unsaturated fatty acids, including 11,14-eicosadienoic acid, can reduce LDL-C levels. Mono and polyunsaturated fatty acids can also elevate LDL-C levels.