Spectroscopic, Biological, and Topological Insights on Lemonol as a Potential Anticancer Agent

Abstract

A monoterpene alcohol known as lemonol was investigated experimentally as well as theoretically in order to gain insights into its geometrical structure, vibrational frequencies, solvent effects on electronic properties, molecular electrostatic potential, Mulliken atomic charge distribution, natural bond orbital, and Nonlinear Optical properties. The frontier molecular orbital energy gap values of 5.9084 eV (gas), 5.9261 eV (ethanol), 5.9185 eV (chloroform), 5.9253 eV (acetone), and 5.9176 eV (diethyl ether) were predicted, and it shows the kinetic stability and chemical reactivity of lemonol. Topological studies were conducted using Multiwfn software to understand the binding sites and weak interactions in lemonol. The antiproliferative effect of lemonol against the breast cancer cell line Michigan Cancer Foundation (MCF-7) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while nuclear damage, condensation, and reactive oxygen species generation were identified using acridine orange/ethidium bromide, propidium iodide, and dichlorodihydrofluorescein diacetate staining. The theoretical and experimental findings are highly correlated, confirming the structure, and the results of in vitro studies suggest that lemonol acts as a potent inhibitor against the human breast cancer cell line MCF-7, highlighting its strong antiproliferative activity.

1. Introduction

Lemonol, chemically known as 3,7-dimethylocta-2,6-dien-1-ol with a molecular formula of C₁₀H₁₈O and a molecular mass of 154.25 g/mol, is a terpene alcohol and is structurally related to limonene, linalool, and menthol. There are two double bonds in the eight-carbon chain; the first is at position 2, while the second occurs at position 6. The hydroxyl (−OH) group is located at position 1 at the terminus of the chain.^1,2 Lemonol also called geraniol is a colorless liquid oil that is readily soluble in organic solvents including ethanol, chloroform, and diethyl ether and is marginally dissolved in water.³ In addition, the indispensable oils of fragrant herbs such as Cinnamomum tenuipilum Kosterm and Verbena officinalis contain lemonol naturally and are also found in oils of rose, palmarosa, lime, ginger, and orange.^4,5 A genetically modified Escherichia coli strain was used to produce lemonol by knocking out the yjgB gene, which is responsible for geraniol endogenous dehydrogenation and increasing the expression of geraniol synthase activity. Also, through metabolic engineering in E. coli, the enzyme YjgB has been found to be responsible for the process of producing 2-phenylethanol (2-PE) and 2-phenylethyacetate (2-PEAc). Lemonol is also chemically synthesized by the reduction of trans methyl geranate and hydrogenation of alpha-pinene.⁶⁻⁸ Numerous researchers have reported the biological and pharmacological benefits of lemonol including its antibacterial, antiproliferative, antioxidant, and anti-inflammatory properties.⁹⁻¹¹ Due to the increased toxicity of N,N-diethyl-3-methylbenzamide (DEET) is a standard insect repellent, and lemonol is utilized as a natural insect repellent.¹²

Cancer is a group of disorders defined by the uncontrollable growth and spread of abnormal cells to neighboring tissues. According to a recent investigation by the International Agency for Research on Cancer (IARC) and the World Health Organization (WHO), cancer is thought to be the primary cause of one in seven fatalities worldwide. Over time, cancer has moved to less urbanized countries, which now account for about 57% of new cases and 65% of deaths worldwide.¹³ Breast cancer is most common in women aged 40–55. Several studies have shown that postmenopausal women with a higher Body Mass Index (BMI) are more likely to acquire breast cancer. Breast cancer may be caused by genetic, hormonal, behavioral, environmental, and dietary factors. Oral contraceptives and hormone replacement therapy for postmenopausal women may increase the risk. Early breast cancer surgery may remove the tumor mass in the breast or regional lymph nodes. Chemotherapy is the standard short-term treatment for cancer although it kills both malignant and normal cells. Most anticancer drugs work only at higher doses. Naturally occurring chemicals that diminish clinical protest and are less toxic may prevent breast cancer in patients and healthy people.

In view of the above-mentioned research works, a close examination of the scientific literature reveals that a detailed theoretical and experimental analysis of the vibrational assignments, electronic properties, topological properties, and biological activities of lemonol has yet to be reported. In response to this knowledge gap, this present investigation lies in the spectroscopic, electronic, and structural properties of lemonol by employing Density Functional Theory (DFT) to conduct quantum chemical computational calculations, and the results were compared with experimental findings of the Fourier transform infrared (FT-IR), Fourier transform-Raman (FT-Raman), and ultraviolet–visible (UV–vis) analytical techniques. The topological analyses including the Electron Localization Function (ELF), Localized Orbital Locator (LOL), Reduced Density Gradient (RDG), Non-Covalent Interaction (NCI), and Quantum Theory of Atoms in Molecule (QTAIM) have been carried out to distinguish about the distribution of lone pair electrons, bonding orbitals, hydrogen bonding, van der Waals interactions, weak interactions, critical points of the electron density, and chemical reactivity of lemonol. Furthermore, a more profound understanding of the physiochemical properties and in vitro antiproliferative activity of lemonol against human breast cancer cell line Michigan Cancer Foundation-7 (MCF-7) was analyzed.

2. Materials and Methods

2.1. Sample and Experimental Details

The pure sample of lemonol in liquid form was acquired from M/s. Sigma-Aldrich Co., USA, is used as such for experimental measurements. The FT-IR spectrum was recorded in the range 4000–400 cm^–1 using a 0.5 cm^–1 resolution PerkinElmer Spectrum Two FT-IR/ATR Spectrometer. The FT-Raman spectrum was recorded in the range 4000–400 cm^–1 using a 2 cm^–1 resolution Bruker RFS 27 stand-alone FT-Raman spectrometer system. The UV–vis spectrum was recorded in the range of 400–200 nm using a PerkinElmer-Lambda 35 UV Winlab V6.0 Spectrometer.

2.2. Computational Details

All of the quantum chemical calculations were done without any kind of geometric limitation using the DFT/B3LYP level of theory¹⁴⁻¹⁶ that was included in the Gaussian 09 W program package¹⁷ along with the 6-311++G(d,p) basis set. The vibrational frequencies, chemical shifts, electronic transitions, and molecular geometry optimizations were all determined. Vibrational Energy Distribution Analysis (VEDA 4) was used to do the calculations for the Potential Energy Distribution (PED) studies.¹⁸ The vibrational assignments, chemical shifts, and electronic transitions were determined by combining the results of the GaussView 6¹⁹ and the Chemcraft program,²⁰ which provides a visual representation and a high degree of accuracy. In addition, topological parameters like Localized Orbital Locator (LOL), Electron Localization Function (ELF), and Reduced Density Gradient (RDG) analysis were carried out using Multiwfn software.²¹ All the correlation graphs were analyzed using Origin software.²²

2.3. Cell Line and Culture

Michigan Cancer Foundation-7 (MCF-7) cells were purchased from the National Centre for Cell Sciences (NCCS), Pune, and cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and maintained at 37 °C, 5% CO₂, and 95% humidity in a carbon dioxide (CO₂) incubator to avoid contaminations and maintain the cells under standard growth conditions.

2.4. Cell Proliferative Assay

The cytotoxic activity of lemonol on cell viability was obtained using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.^23,24 In this study, the dimethyl sulfoxide (DMSO) solvent was used to dissolve the crystal formazans because it has high solubility, high stability, and low toxicity by comparison with other organic solvents. The cells were cultured in 96-well plates and maintained for 24 h with a complete medium. Then, the cells were treated with lemonol (5, 10, 20, 30, 40, 80, 100, 200, 300, 400, and 500 μM) and incubated for 24 and 48 h in a CO₂ incubator along with blank and control. After the incubation, the medium was withdrawn, and each well plate received 10 μL of MTT stock solution and was then incubated with the cells for 4 h. After that, the MTT solution was discarded and 100 μL of DMSO was added to each well and read at 570 nm.

2.5. Propidium Iodide (PI) Nucleic Acid Staining

3000 cells per well were used to culture the cells on 12-well plates, and they were left to incubate for a whole night. After, the cells were treated with lemonol (300 μM) for 24 and 48 h. Afterward, the cells were rinsed with phosphate buffer saline (PBS), and the treated cells were stained with PI for 5 min. In the end, the intensity of the fluorescence was measured using a fluorescence microscope.

2.6. 2′,7′-Dichlorodihydrofluorescein Diacetate (DCFHDA) Assay

Using the oxidatively sensitive DCFHDA staining technique, the formation of intracellular reactive oxygen species (ROS) was assessed. At a density of 3 × 10³ cells/well, the cells were grown on 12-well plates with sterile coverslips. The plates were incubated with DMEM medium overnight to reach confluence. Then the cells were healed with the half-maximal inhibitory concentration (IC₅₀) value of lemonol for 24 and 48 h, respectively. The culture medium was taken out after incubation, and the wells were gently rinsed with PBS buffer. The culture plates were then maintained in the dark for 20 min with the addition of a nonfluorescent probe (2,7-dichlorofluorescein-diacetate), before being examined under a fluorescence microscope with a green filter at a magnification of 10×.

2.7. Acridine Orange/Ethidium Bromide (Ao/EtBr) Staining

In the culture plates with coverslips, the cells were cultured at a population of 3 × 10³ cells/well in 12-well plates. The plates were incubated with DMEM medium overnight to reach confluence. Then the cells were treated with a half-maximal inhibitory concentration (IC₅₀) value of lemonol for 24 and 48 h. The medium was taken out after incubation, and the wells were gently rinsed with PBS buffer. After 10 min of Ao/EtBr staining, the cells were immediately imaged using a fluorescence microscope.

2.8. Statistical Data Analysis

Using one-way analysis of variance (ANOVA) and least significant difference (LSD), Statistical Package for Social Sciences (SPSS/10) software²⁵ was used to calculate all the data statistically. The quantitative results are presented as mean ± standard deviation (SD) and measured as statistically significant if P 0.05.

3. Results and Discussion

3.1. Optimized Structural Parameters

The optimized structure of lemonol with atomic numbering is shown in Figure 1, and the bond lengths and angles are presented in Table 1. These values are compared with the experimental values of structurally similar derivatives of lemonol downloaded from the Cambridge Crystallographic Data Centre (CCDC), as reported in the literature.²⁶ In the optimized structure of lemonol, there are 4 types of bond lengths such as carbon–carbon (C–C), carbon–hydrogen (C–H), oxygen–hydrogen (O–H), and oxygen–carbon (O–C) as well as 6 types of bond angles carbon–carbon–carbon (C–C–C), carbon–oxygen-hydrogen (C–O–H), oxygen–carbon-hydrogen (O–C–H), carbon–carbon–hydrogen (C–C–H), oxygen–carbon-carbon (O–C–C), and hydrogen–carbon-hydrogen (H–C–H) being identified. The optimized bond length of O₁–C₁₁ is observed as 1.428 Å and calculated as 1.439 Å, while the bond angle of O₁–C₁₁-C₈ is observed as 107.9° and theoretically estimated as 113.9 °. The bond length of O₁–H₂₉ is observed as 0.840 Å and calculated as 0.962 Å. In addition, optimized bond angles of C–O–H and O–C–H were theoretically estimated in the ranges of 107.94 and 104.6–109.7 and experimentally observed as 109.5 and 110.1, respectively. In comparison to other C–C bonds in the structure, the bond length of C₄–C₈ is 1.342 Å (DFT), 1.325 Å (experimental) and that of C₅–C₆ is 1.339 Å (DFT), 1.276 Å (experimental), and these are shorter because of the influence of the methyl group connected to carbon. Additionally, the optimized theoretical bond angles of C–C–C were estimated to vary from 112.1 to 128.6 and experimentally observed to range from 110.4 to 128.5.

Figure 1.

Optimized molecular structure of lemonol.

Table 1. Experimental and Theoretical Geometrical Parameters of Lemonol.

Bond lengths (Å)	B3LYP	Experimentala	Bond lengths (Å)	B3LYP	Experimentala
O1–C11	1.439	1.428	C7–H17	1.095	0.981
O1–H29	0.962	0.840	C7–H18	1.095	0.980
C2–C3	1.549	1.543	C7–H19	1.089	0.979
C2–C4	1.512	1.511	C8–C11	1.507	1.503
C2–H12	1.098	0.988	C8–H20	1.088	0.950
C2–H13	1.093	0.991	C9–H21	1.096	0.980
C3–C5	1.503	1.557	C9–H22	1.091	0.981
C3–H14	1.092	0.990	C9–H23	1.096	0.980
C3–H15	1.096	0.990	C10-H24	1.096	0.980
C4–C7	1.507	1.488	C10-H25	1.089	0.980
C4–C8	1.342	1.325	C10-H26	1.096	0.980
C5–C6	1.339	1.276	C11–H27	1.096	0.990
C5–H16	1.090	0.950	C11–H28	1.092	0.990
C6–C9	1.509	1.481	C6–C10	1.508	1.502

Bond angles (°)	B3LYP	Experimentala	Bond angles (°)	B3LYP	Experimentala
C11–O1-H29	107.9	109.5	C4–C8-H20	117.9	115.8
O1–C11-C8	113.9	107.9	C6–C5-H16	117.0	115.7
O1–C11-H27	109.7	110.1	C5–C6-C9	120.7	121.6
O1–C11-H28	104.6	110.1	C5–C6-C10	124.9	124.1
C3–C2-C4	114.2	114.6	C9–C6-C10	114.2	114.4
C3–C2-H12	108.3	108.6	C6–C9-H21	110.9	109.5
C3–C2-H13	108.7	108.5	C6–C9-H22	111.8	109.4
C2–C3-C5	112.1	110.4	C6–C9-H23	110.9	109.4
C2–C3-H14	108.9	108.1	C6–C10-H24	110.4	109.5
C2–C3-H15	108.9	109.5	C6–C10-H25	113.4	109.4
C4–C2-H12	108.9	108.7	C6–C10-H26	110.4	109.5
C4–C2-H13	109.6	108.6	H17–C7-H18	106.7	109.4
C2–C4-C7	116.2	116.0	H17–C7-H19	108.8	109.6
C2–C4-C8	120.5	119.3	H18–C7-H19	107.0	109.5
H12–C2-H13	106.6	107.6	C11–C8-H20	116.0	115.9
C5–C3-H14	111.5	109.6	C8–C11-H27	109.4	110.1
C5–C3-H15	108.6	109.6	C8–C11-H28	111.6	110.2
C3–C5-C6	128.6	128.5	H21–C9-H22	108.1	109.4
C3–C5-H16	114.3	115.8	H21–C9-H23	106.5	109.5
H14–C3-H15	106.3	108.1	H22–C9-H23	108.1	109.5
C7–C4-C8	123.2	124.7	H24–C10-H25	107.8	109.5
C4–C7-H17	110.4	109.4	H24–C10-H26	106.4	109.6
C4–C7-H18	110.8	109.5	H25–C10-H26	107.9	109.4
C4–C7-H19	112.6	109.5	H27–C11-H28	107.1	108.3
C4–C8-C11	126.0	128.3

^aTaken from ref (26).

In contrast, the bond angles of C–C–H and H–C–H are theoretically predicted in the range of 108.3–117.9° and 106.3–108.9° and experimentally observed as 108.5–115.8° and 107.6–109.6°, respectively. The optimized bond lengths of aliphatic C–H in methyl and methylene groups were calculated in the range of 1.089–1.096 and 1.092–1.098 Å, and experimental results show that they fall in the range of 0.979–0.981 and 0.988–0.991 Å. According to the findings, theoretical values were discovered to be marginally greater than practical values because optimization was carried out in an isolated setting in the gaseous state, but the experimental findings were impacted by the crystal environment. Figure 2 displays the correlation graphs between the optimal calculated and observed bond length and bond angle values. Bond lengths and bond angles have linear coefficient values (R²) of 0.99465 and 0.92554, respectively. Nevertheless, there is good agreement between the calculated and observed data in the literature.²⁶

Figure 2.

Correlation graph for bond lengths and bond angles of lemonol.

3.2. Vibrational Properties

Lemonol contains 29 atoms and has 81 fundamental vibrational modes based on the (3N-6) degrees of freedom expected to be C₁ symmetry. The theoretical vibrational frequencies were determined using the DFT/B3LYP levels with the 6-311++G(d,p) basis set in the gas phase. The vibrational spectra of lemonol revealed six different bands of stretching and bending vibrations, including CH, OH, CC, CH₃, CH₂, and CO. The theoretical along with experimental wavenumbers of lemonol were presented in Table 2, and the theoretical and experimental FT-IR and FT-Raman spectra were shown in Figures 3 and 4. The computed vibrational wavenumbers are scaled with 0.96 for all frequencies.²⁷ The correlation graph between theoretical and experimental wavenumbers is displayed in Figure 5, with a linear coefficient value (R²) of 0.99403, and the optimized calculated values exhibit good agreement with the experimental values.

Table 2. Experimental FT-IR and FT-Raman and Theoretical Wave Numbers of Lemonol.

	Experimental wavenumbers (cm–1)		Theoretical wave numbersb (cm–1)
Modes	FT-IR	FT-Raman	Unscaled	Scaled	IIR	SARaman	Vibrational assignmentsa(≥10% PED)
1		3400	3825	3672	19.79	65.77	υ OH(100)
2	3326		3125	2999	27.67	61.17	υ CH(46), υasCH3(52)
3			3124	2998	19.06	87.68	υ CH(88)
4			3120	2994	32.50	42.35	υ CH(42), υasCH3(55)
5			3106	2981	49.81	111.83	υ CH(91)
6	2967		3096	2972	6.30	33.37	υ CH(16), υasCH3(71)
7			3077	2953	19.81	23.74	υ CH(18), υas CH2(60)
8			3074	2951	16.20	94.16	υas CH2(53), υ CH(46)
9			3062	2940	18.46	79.46	υas CH2 in CH3(97)
10			3058	2936	16.97	34.56	υas CH2(69), υ CH(24)
11	2917		3048	2926	36.52	170.02	υas CH2 in CH3(86)
12		2913	3044	2922	8.42	47.95	υs CH2 in CH3(86)
13			3020	2899	27.46	297.26	υs CH3(27), υs CH2(54)
14			3013	2892	16.82	199.08	υs CH3(64), υs CH2(32)
15			3011	2891	56.60	242.67	υs CH3(49), υs CH2(29)
16			3007	2887	79.43	138.43	υs CH2 (93)
17			3004	2883	32.26	121.80	υs CH3 (75)
18	2856, 2108	2731, 2260	2998	2877	20.89	90.88	υs CH2 (88)
19	1668	1956, 1846, 1668	1727	1658	2.12	96.04	υ C=C(71), δas CH3(11), ω CH2(10)
20			1702	1634	33.21	116.27	υ C=C (72), δasCH3(10), δ CH(11)
21			1520	1459	4.64	8.93	χ CH2(25), δas CH3(56)
22	1442	1442	1502	1442	7.20	18.36	χ CH2(10), δas CH3(44), β CH(10)
23			1496	1436	8.39	23.32	χ CH2(11), δas CH3(12), υ CC(30)
24			1492	1432	12.94	0.66	δas CH3(39), β CH(38)
25			1489	1429	7.03	3.70	δas CH3(32), χ CH2(15)
26			1485	1425	8.56	13.36	δas CH3(48), χ CH2(30)
27			1481	1422	3.54	7.78	χ CH2(35), δas CH3(16)
28			1473	1414	0.88	16.15	δas CH3 (25), χ CH2(43)
29			1471	1412	3.83	8.68	δas CH3(11), χ CH2 (47)
30	1378	1378	1421	1363	2.72	19.21	ω CH3 (38), ω CH(23)
31			1419	1362	11.99	9.92	β CH(12), δs CH3(15), υ CC(35)
32			1412	1355	49.85	10.40	ω CH2(10), δ OH(20), υ CC(43)
33			1411	1353	6.36	5.39	β CH(18), υ CC(43)
34		1327	1385	1328	2.07	8.21	β CH (18), δs CH3(17), ω CH2(23)
35			1381	1324	8.75	9.61	β CH(23), δs CH3(18), δ OH(12), τ CH2(22)
36			1358	1303	3.57	44.23	ω CH2(33), δ OH(42), υ CC(11)
37		1280	1346	1291	2.86	8.64	δ OH(22), τ CH2(11), υ CC(10)
38			1310	1258	0.48	16.21	τ CH2(80)
39	1235	1231	1283	1231	0.78	2.61	ω CH2(60)
40			1246	1195	11.75	7.92	τ CH2(22), δ OH(10)
41	1182		1239	1189	4.94	2.17	δ OH(12), τ CH2(11)
42			1188	1140	10.63	5.61	δ OH(11), τ CH2(28)
43			1172	1125	1.28	7.07	τ CH2(33)
44	1094		1130	1085	12.90	9.71	ω CH2 (34)
45			1102	1057	1.61	1.41	β CH (42)
46			1100	1056	5.16	4.84	δ OH(20), τ CH2 (36)
47			1067	1023	4.84	9.56	β CH(48)
48	995	999	1047	1004	8.34	3.58	τ CH2(11), δ OH(10), υ CO(18)
49			1028	986	0.51	0.92	γ CH(28)
50			1015	973	3.51	1.61	γ CH (27)
51			1005	963	7.97	36.13	δas CH3(58)
52			990	950	90.34	4.97	δ OH (55)
53			973	934	1.86	1.14	δas CH3(10), τ CH2 (12)
54			964	925	33.84	4.82	δ OH(10), δas CH3(25)
55			959	921	0.72	1.25	δas CH3(45)
56	830		863	828	4.90	3.22	δas CH3(33), γ CH(18)
57			856	822	7.32	0.64	γ CH (22)
58	779	781	811	779	1.03	9.32	δs CH3(56)
59	744		789	756	7.90	4.27	δs CH3(45)
60			755	725	1.72	1.20	ρ CH2(44)
61	592		631	605	13.32	0.64	δ OH(10), δas CH3(15), γ CH(11), τ CH2(12)
62			565	541	7.78	1.20	δ OH (12), γCH(15), δas CH3(18)
63			505	485	0.89	2.39	δas CH3(38)
64			481	462	2.22	1.16	δas CH3(38)
65			434	417	12.64	1.57	δ OH(18), δas CH3(11)
66			400	383	0.21	0.76	δas CH3(18), υ CC(26)
67			389	373	2.45	2.06	δs CH3(18), τ CH2(11)
68			314	300	14.68	4.35	δ OH(18), δ CH(22)
69			307	294	40.72	0.73	δ OH(12), δ CH(18), τ CH2(22)
70			290	277	28.35	0.42	δ OH (34), ρ CH2(30)
71			258	247	66.66	0.45	δ OH(30), ρ CH2(34)
72			226	217	10.76	1.31	δas CH3(20), δ OH(20), ρ CH2(18)
73			198	190	1.64	0.46	δas CH3(40), δ OH(18)
74			151	145	0.75	0.28	δas CH3(15), δ OH(10)
75			138	132	0.67	0.27	τ CH3(63)
76			106	102	0.03	0.56	τ CH3(43)
77			70	67	0.13	0.68	τ CH3(66), δ OH(13)
78			60	58	0.20	0.72	δ OH(10), ρ CH2(11), δas CH3(12),δ CH(11)
79			52	49	1.20	1.34	δ OH(11), ρ CH2(18), δas CH3(11),δ CH(12)
80			32	30	0.72	1.76	δ OH(12), ρ CH2(10), δs CH3(10), δ CH(12)
81			30	28	0.10	1.53	δ OH(22), ρ CH2(18), δs CH3(11)

^aυ_s - symmetric stretching; υ_as - asymmetric stretching; δ - bending/deformation; β - in-plane bending; γ - out-of-plane bending; χ - scissoring; ω - wagging; τ - twisting; ρ - rocking.

^bTheoretical wavenumber scaling factor: 0.96 for all vibrations.

Figure 3.

Theoretical and experimental FT-IR vibrational spectra of lemonol.

Figure 4.

Theoretical and experimental FT-Raman vibrational spectra of lemonol.

Figure 5.

Correlation between all experimental and theoretical wavenumbers.

3.2.1. Hydroxyl Group Vibrations

When compared to other groups, the hydroxyl group vibrations are more susceptible to the environment and offer the three common vibrations of stretching in-plane and out-of-plane deformation. The O–H stretching modes are generally expected in the range of 3550–3700 cm^–1.²⁸ The O–H stretching vibration is expected to occur at 3672 cm^–1 theoretically from the current investigation, and a single band was seen in the matching experimental FT-Raman spectra with PED of 100% at 3400 cm^–1. The out-of-plane and in-plane deformation vibrations are expected at 710–517 and 1420–1330 cm^–1,²⁹ respectively. Experimentally, a medium band is observed at 1378 cm^–1 (FT-IR) and a weak intense band is observed at 1378 cm^–1 (FT-Raman) in the spectrum, while the corresponding theoretical bands are simulated at 1363 and 1324 cm^–1. The theoretical assignments of the out-of-plane deformation vibrations at 605 and 541 cm^–1 are in good agreement with the observed FT-IR spectrum at 592 cm^–1.

3.2.2. CH Vibrations

C–H stretching modes are anticipated in the range of 3000–3100 cm^–1, as this range is the defining region for the detection of C–H vibrations.^30,31 From this study, CH vibrations are anticipated to be at 2999, 2998, 2994, 2981, 2972, 2953, 2951, and 2936 cm^–1, with their matching experimental peaks in the FT-IR spectrum at 3326 and 2967 cm^–1. The typical ranges for the in-plane and out-of-plane deformation modes are 1450 to 1000 cm^–1 and 1000 to 750 cm^–1.³² A theoretical simulation of the C–H in-plane deformation modes falls in the range 1442–1023 cm^–1, as observed experimentally ranging from 1442 to 1378 cm^–1 (FT-IR) and 1442–1327 cm^–1 (FT-Raman). The DFT approach assigned the out-of-plane deformation vibrations to wavelengths of 986, 973, 828, and 822 cm^–1, while the equivalent experimental wavenumber is found in the FT-IR spectrum at 830 cm^–1.

3.2.3. CC Vibrations

The frequency ranges of the double-bonded (C=C) and single-bonded (C–C) stretching vibrations are typically in the ranges of 1280–1380 and 1400–1625 cm^–1.³³ In this instance, the C=C bands appeared at 1436, 1362, 1355, 1353, and 1291 cm^–1 theoretically; however, the experimental Raman spectrum at 1280 cm^–1 has been ascribed to these bands. The peaks at 1668 cm^–1 (FT-IR) and 1668 cm^–1 (FT-Raman) correspond to the predicted values at 1658 and 1634 cm^–1 and are ascribed to the C–C stretching modes.

3.2.4. Methyl Group Vibrations

The symmetric and asymmetric stretching vibrations of methyl groups are typically observed between 2870 and 2980 cm^–1.^34,35 Theoretically, the CH₃ symmetric stretching occurs at 2922, 2899, 2892, 2891, and 2883 cm^–1, and experimentally, the corresponding mode is found in the FT-Raman spectrum at 2913 cm^–1. Similarly, the asymmetric stretching occurs at 2999, 2994, and 2972 cm^–1, and the corresponding experimental values are found at 3326 and 2967 cm^–1 in the FT-IR spectrum. The predicted ranges for the asymmetric and symmetric deformation vibrations are 1465–1440 and 1390–1370 cm^–1.³⁶ In contrast to what is experimentally observed, the CH₃ asymmetric bending is anticipated to occur between 1668 and 1442 cm^–1 in the FT-IR and FT-Raman spectrum and the corresponding simulated values at 1658–1412 cm^–1. The bands for CH₃ symmetric deformation seen in this work at 1362, 1328, and 1324 cm^–1 exhibited good harmony with the experimental spectrum at 1327 cm^–1 in the FT-Raman spectrum.

3.2.5. Methylene Group Vibrations

Six fundamental frequencies are displayed by the methylene group, including symmetric and asymmetric, two in-plane deformations (rocking and scissoring), and two out-of-plane deformations (wagging and twisting). In the ranges of 3000–2900 and 3100–3000 cm^–1, the symmetric and asymmetric stretching vibrations of CH₂ are observed in the previous report.³⁷ In the molecular structure of lemonol, the CH₂ symmetric stretching vibrations are theoretically assigned at 2899, 2892, 2891, and 2887 cm^–1, experimentally observed at 2913 cm^–1 in the FT-Raman spectrum. The DFT technique predicts the asymmetric stretching vibrations at 2953, 2951, 2940, 2936, and 2926 cm^–1, and the equivalent experimental band was noticed at 2917 cm^–1 in the FT-IR spectrum. The CH₂ deformation vibrations were anticipated in the range below 1500 cm^–1. The CH₂ scissoring frequencies were observed at 1459, 1442, 1436, 1429, 1425, 1422, 1414, and 1412 cm^–1 as well as the experimental frequencies at 1442 cm^–1 in both FT-IR and FT-Raman spectra. While the CH₂ wagging vibrations were experimentally measured at 1235, 1094 cm^–1 in the FT-IR spectrum and at 1327, 1231 cm^–1 in the FT-Raman spectrum, the corresponding simulated vibrations are at 1355, 1328, 1303, 1231, and 1085 cm^–1. The CH₂ twisting vibrations are measured and assigned to wavenumbers 1280, 999 cm^–1 in the FT-Raman spectrum and to 1182, 995 cm^–1 in the FT-IR spectrum and simulated in the range of 1324–1004 cm^–1. Similarly, the calculated CH₂ rocking vibrations were at 725, 277, 247, and 217 cm^–1.

3.2.6. CO Vibrations

In general, the frequency range of CO stretching vibrations is observed as 1260–1000 cm^–1.^38,39 The CO stretching vibration of lemonol is calculated at 1004 cm^–1, and the corresponding experimental band is found as a strong intensity peak at 995 cm^–1 (FT-IR) and a weak intensity peak at 999 cm^–1 (FT-Raman), which are satisfactorily correlated with the theoretical value.

3.3. Electronic Properties

When the physical and chemical activities of molecules are described, the frontier molecular orbital (FMO) energies play an intriguing role. By using the time-dependent (TD) DFT approach with the B3LYP/6-311++G (d,p) basis set, the theoretically determined oscillator strengths (f), wavelength (λ), and excitation energy (E) of lemonol have been simulated in the gas phase and different solvents (ethanol, chloroform, acetone, and diethyl ether) which are compared to experimental findings. Figure 6 shows the theoretical and experimental spectra, and Table 3 lists the theoretical and experimental values. In the present work, a strong peak was observed at 210 nm experimentally, which shows good agreement with the theoretical spectra at 217 nm (gas), 214 nm (ethanol, acetone), and 215 nm (chloroform, diethyl ether). The second peak observed as a medium band at 278 nm has a good correlation with the calculated spectrum at 232 nm (gas, chloroform) and 231 nm (ethanol, acetone, diethyl ether). The deviation between the calculated and experimental spectra is due to a specific solvent–solute interaction. The data shown in Figure 7 and Table 4 represent the FMO plots of lemonol; the positive phases are shown in red, and the negative phases are shown in green. The charge transfer and interactions that occur within the molecules were revealed by the FMO energy gap, which was found to be 5.9084 eV (gas), 5.9261 eV (ethanol), 5.9185 eV (chloroform), 5.9253 eV (acetone), and 5.9176 eV (diethyl ether). In addition, the energy gap of lemonol was determined as 5.186 eV experimentally with the help of Tauc’s plot as shown in Figure 8, which matches well with the theoretical energy gap values calculated in the gas phase.

Figure 6.

(a) Simulated electronic spectra of lemonol in different solvents. (b) Experimental electronic spectrum of lemonol in ethanol.

Table 3. Experimental and Calculated Wavelengths (λ), Excitation Energies (E), Absorbance Values (A), and Oscillator Strengths (f) of Lemonol.

		TDDFT/B3LYP/6-311++G(d,p)
Experimental(Ethanol)		Gas			Ethanol			Chloroform			Acetone			Diethyl ether
λ (nm)	Abs.	λ (nm)	E (eV)	f	λ (nm)	E (eV)	f	λ (nm)	E (eV)	f	λ (nm)	E (eV)	f	λ (nm)	E (eV)	f
278	0.298	232	5.33	0.0841	231	5.35	0.1355	232	5.34	0.1365	231	5.35	0.1351	231	5.34	0.1299
		229	5.41	0.0199	226	5.47	0.0019	227	5.45	0.0041	226	5.47	0.0019	227	5.45	0.0044
210	2.286	217	5.70	0.0101	214	5.77	0.0227	215	5.75	0.0207	214	5.77	0.0225	215	5.75	0.0194

Figure 7.

FMO plots of lemonol.

Table 4. FMO Energies and Energy Gap of Lemonol.

	TDDFT/B3LYP/6-311++G(d,p)
Parameters(eV)	Gas	Ethanol	Chloroform	Acetone	Diethyl ether
HOMO energy	–6.4542	–6.4368	–6.4319	–6.4363	–6.4316
LUMO energy	–0.5458	–0.5107	–0.5134	–0.5110	–0.5140
HOMO–LUMO energy gap	5.9084	5.9261	5.9185	5.9253	5.9176
HOMO–1 energy	–6.9372	–6.9489	–6.9402	–6.4363	–6.9394
LUMO+1 energy	–0.3322	–0.2277	–0.2487	–0.2288	–0.2514
(HOMO–1)-(LUMO+1) energy gap	6.6050	6.7212	6.6915	6.2075	6.6880

Figure 8.

Tauc’s plot of lemonol.

When substituents within the molecular structure of lemonol can donate or accept electrons, the electronic properties are influenced, resulting in a smaller band gap. The electron-donating groups facilitate electron donation to the system, which stabilizes the energy level of the highest occupied molecular orbital (HOMO). In contrast, the electron-accepting groups can withdraw electrons from the system, thereby lowering the lowest unoccupied molecular orbital (LUMO) energy levels. While the HOMO–1 and LUMO+1 indicate the donor and acceptor energy levels, which lie one energy state lower and higher than their respective levels, the nature of HOMO and LUMO is revealed through band theory, which interprets HOMO as the conduction band and LUMO as the valence band.⁴⁰⁻⁴² The combination of these substituents results in an effective decrease in the band gap of lemonol. Furthermore, the conjugation present in lemonol can also contribute to a smaller band gap by introducing additional conjugated π systems such as double bonds. Lemonol in various phases is characterized by a smaller band gap due to the extended conjugation, which allows electrons to disperse over a larger spatial region. In line with this observation, the minimum energy gap of lemonol was observed in the gas phase at 5.9084 eV exploring the low kinetic stability and high chemical reactivity. Furthermore, the electronic descriptors such as ionization potential (IA), electron affinity (EA), chemical hardness (η), chemical softness (S), chemical potential (μ), electronegativity (χ), and global electrophilicity (ω) are presented in Table 5. It is important to note that the values of chemical hardness (η) and softness (S) are two important characteristics to determine the chemical stability and polarizability of molecules. In the present work, the maximum and minimum chemical hardness were found at 2.9630 (ethanol) and 2.9542 (gas) exploring that the lemonol has more stability and less reactivity in the solvent phase and less stability and more reactivity in the gas phase, respectively.

Table 5. Calculated Quantum Chemical and Physicochemical Parameters of the Lemonol.

Parameters (eV)	Formula	Gas	Ethanol	Chloroform	Acetone	Diethyl ether
EHOMO		–6.4542	–6.4368	–6.4319	–6.4363	–6.4316
ELUMO		–0.5458	–0.5107	–0.5134	–0.5110	–0.5140
Energy gap ΔE	(EHOMO – ELUMO)	5.9084	5.9261	5.9185	5.9253	5.9176
Ionization potential (IP)	–EHOMO	6.4542	6.4368	6.4319	6.4363	6.4316
Electron affinity (EA)	–ELUMO	0.5458	0.5107	0.5134	0.5110	0.5140
Electronegativity (χ)	–1/2 (ELUMO + EHOMO)	3.50	3.4737	3.4726	3.4736	3.4728
Chemical hardness (η)	1/2 (ELUMO – EHOMO)	2.9542	2.9630	2.9592	2.9626	2.9588
Chemical softness (S)	1/2η	0.08462	0.08437	0.08448	0.08438	0.08436
Chemical potential (μ)	–χ	–3.50	–3.4737	–3.4726	–3.4736	–3.4728
Global electrophilicity (ω)	μ2/2η	1.0366	1.0180	1.0186	1.0180	1.0174
Maximum electronic charge (ΔNmax)	–(μ/η)	0.5923	0.5861	0.5867	0.5862	0.5868

3.4. Natural Bond Orbital (NBO) Properties

The chemical nature of orbitals, hyperconjugative relations, and hydrogen bonding can be accomplished with the help of natural bond orbital analysis. To review the donor and acceptor interactions of lemonol, the second-order Fock matrix is used. Table 6 includes the electron density, donor and acceptor stabilization energies, and localized NBO. The hyperconjugation relationships between electron donors and acceptors within the molecule become more intense as E(2) increases. In the lemonol compound, the strongest interactions are associated with electron donation from C₅–H₁₆ to the acceptor C₆–C₁₀ with a stabilizing energy of 8.24 kJ mol^–1 with a transition of σ–σ*. Similarly, significant delocalization was also found at electron donation from C₈–H₂₀ to the acceptor C₄–C₇ with a stabilizing energy of 7.95 kJ mol^–1. Moreover, the electron is donated from lone pair donor O₁ to the acceptor C₁₁–H₂₇ with a stabilizing energy of 6.98 kJ mol^–1 with a transition of L(2)-σ*. The simulated stabilizing energy of charge transfer from donor C₄–C₈ to acceptor O₁–C₁₁ is 6.68 kJ mol^–1 with a π–σ* transition. In the lemonol, the maximum contribution was made by Cieplak interaction from occupied σ orbital to unoccupied σ* including C₂–C₄, C₂–H₁₃, C₃–C₅, C₆–C₉, C₇–H₁₉, and C₈–C₁₁ donated its σ electron to σ* antibonding orbitals C₈–C₁₁, C₄–C₇, C₆–C₉, C₃–C₅, C₂–C₄, and C₂–C₄ with stabilizing energies of 4.09, 4.82, 4.14, 4.69, 4.29, and 4.41 kJ mol^–1.

Table 6. Second-Order Perturbation Theory of the Fork Matrix in NBO Analysis of Lemonol Based on the B3LYP/6-311++G(d,p) Basis Set.

Donor (i)	Type of bond	Acceptor (j)	Type of bond	Type of transition	E(2)a(kJ/mol)	E(j) – E(i)b(a.u.)	F(i,j)c(a.u.)
C2–C4	σ	C8–C11	σ*	σ–σ*	4.09	1.04	0.058
C2–H13	σ	C4–C7	σ*	σ–σ*	4.82	0.91	0.059
C3–C5	σ	C6–C9	σ*	σ–σ*	4.14	1.05	0.059
O1	L(2)	C11–H27	σ*	L(2)-σ*	6.98	0.68	0.062
C4–C8	π	O1–C11	σ*	π–σ*	6.68	0.54	0.054
C5–H16	σ	C6–C10	σ*	σ–σ*	8.24	0.92	0.078
C6–C9	σ	C3–C5	σ*	σ–σ*	4.69	1.06	0.063
C7–H18	σ	C4–C8	π*	σ–π*	4.81	0.56	0.041
C7–H19	σ	C2–C4	σ*	σ–σ*	4.29	0.93	0.057
C8–C11	σ	C2–C4	σ*	σ–σ*	4.41	1.06	0.061
C8–H20	σ	C4–C7	σ*	σ–σ*	7.95	0.93	0.077

^aE(2) – Mean energy of hyper-conjugative interactions (stabilization energy).

^bE(j) – E(i) – The energy difference between the donor (i) and acceptor (j) natural bonding orbitals.

^cF(i,j) – Fock matrix element between i and j natural bonding orbitals.

3.5. Nonlinear Optical (NLO) Properties

Several studies on novel compounds with nonlinear characteristics have attracted a lot of attention in recent years. To find NLO property molecules on an inexpensive basis and to ascertain the molecule’s first-order hyperpolarizability tensor, computational research is performed. The effectiveness of the electron connection between the donor and acceptor groups determines these polarizabilities and hyperpolarizabilities. In the present case, the NLO properties of dipole moment, anisotropy polarizability, and first-order hyperpolarizability of lemonol are presented in Table 7. The dipole moment is a first-order tensor that describes the distribution of electric charge in a molecule expressed in the Debye unit (1 Debye = 3.336 × 10^–30 Coulomb-meter). The dipole moment along the X axis is found to be 1.8978 Debye, which is high when compared to the Y (0.1214 Debye) and Z (0.7879 Debye) axes. The overall dipole moment is calculated to be 2.0584 Debye, which indicates a moderate to relatively strong dipole moment. The ability of the material to be polarized is measured by the second-order polarizability tensor. α_xx, α_yy, and α_zz represent interactions between the X, Y, and Z components of the electric field and induced polarization. The corresponding polarizability values are calculated to be −80.2609, 66.8832, and −73.0347 e.s.u., respectively. The negative values indicate that the induced polarization is opposite in direction to the applied electric field. In this case, the total polarizability is calculated to be −73.39 e.s.u., which is 19.05 times greater than that of urea. Each component of the third-order hyperpolarizability tensor obtained from theoretical calculations provides information about the directional dependence and magnitude of the nonlinear response of a material. Lemonol exhibited the highest NLO property in the β_xxx direction with a positive value of 51.4818 × 10^–31 e.s.u. The next strongest interaction is in β_zxx with 24.8102 e.s.u. followed by β_yzz with 5.1512 e.s.u. Similarly, the component β_xxy = −8.8482 e.s.u. represents the interaction between the x-component of the applied electric field and the y-component of the induced polarization. The negative value indicates that there is an opposite phase or orientation in this interaction. The computed other parameters are given in Table 7. The value of first-order hyperpolarizability was found to be 46.74 e.s.u., which is 12.534 times greater than the value of urea, making it an excellent choice for future research on nonlinear optics.⁴³⁻⁴⁶

Table 7. Electric Dipole Moment μ(D), the Average Polarizability α_tot (×10^–24 e.s.u), and First-Order Hyperpolarizability β_tot (×10^–31 e.s.u) Calculated at B3LYP/6-311++G(d,p) for Lemonol.

Parameters	B3LYP/6-311++G(d,p)	Parameters	B3LYP/6-311++G(d,p)
μx	1.8978	βxxx	51.4818
μy	0.1214	βxxy	–8.8482
μz	0.7879	βxyy	–9.2997
μ(D)	2.0584	βyyy	8.9232
αxx	–80.2609	βzxx	24.8102
αxy	2.7516	βxyz	–6.8277
αyy	–66.8832	βzyy	–1.6729
αxz	–4.3588	βxzz	–2.5006
αyz	1.1042	βyzz	5.1512
αzz	–73.0347	βzzz	–3.6737
αtotal(e.s.u)	–73.39	βtotal(e.s.u)	46.74

3.6. Mulliken Charges

The application of quantum mechanical calculations to molecular systems relies profoundly on the accurate determination of the atomic charges. The Mulliken charge can be thought of as a quantitative representation of how the electronic structure changes in response to atomic displacement.^47,48Table 8 shows the calculated Mulliken charges of lemonol as well as graphically represented in Figure 9. The results of lemonol illustrate that all the hydrogen atoms have positive potential; especially, the hydrogen atom H₂₉ has a higher positive charge (0.2289e) than all other hydrogen atoms due to the interaction of oxygen atom O₁, which is evident for the electronegativity of the oxygen atom. The carbon atoms C₄ (0.8782e) and C₆ (0.4313e) show positive potential which is attached to methyl groups, whereas other carbon atoms C₂, C₃, C₅, C₇, C₈, and C₁₁ show negative potential; especially, the C₁₁ atom exhibits more negativity due to the attachment of oxygen atom. On the whole, carbon atom C₄ exhibits a more positive charge of (0.8782e), and carbon atom C₇ exhibits a more negative charge of (−0.7306e), which is also confirmed with molecular electrostatic potential (MESP) analysis.

Table 8. Mulliken Population Analysis of Lemonol.

Atoms	Charges (e)	Atoms	Charges (e)
O1	–0.231876	H16	0.166465
C2	–0.585991	H17	0.15039
C3	–0.289292	H18	0.172824
C4	0.878218	H19	0.172086
C5	–0.015905	H20	0.162222
C6	0.431364	H21	0.142858
C7	–0.730697	H22	0.150826
C8	–0.232234	H23	0.138774
C9	–0.633561	H24	0.13852
C10	–0.621358	H25	0.139948
C11	–0.707736	H26	0.140575
H12	0.135283	H27	0.152511
H13	0.158713	H28	0.165559
H14	0.09728	H29	0.228919
H15	0.125315

Figure 9.

Mulliken atomic charge distribution of lemonol.

3.7. Molecular Electrostatic Potential Surface Analysis

By using different colors on the map, the molecular electrostatic potential (MESP) surface is utilized to examine the hydrogen bonding and electron-rich and -poor regions in the molecular structure of lemonol.⁴⁹ The mapped color bands illustrated in Figure 10 correspond to specific regions, which are predicted between variations ranging from −4.974 × 10^–2 to 4.974 × 10^–2 e.s.u. for lemonol. From the surface map, the electron-poor regions, which serve as binding sites for electrophilic reactive species, are depicted as white color around the hydrogen atoms, while the electron-rich region, acting as binding sites for nucleophilic reactive species, is depicted as red color around the electronegative oxygen atom in the hydroxyl group of lemonol. The obtained results show an excellent correlation with the Mulliken population analysis.

Figure 10.

Total electron density (left) and the contour map (right) with the molecular electrostatic potential surface of lemonol.

3.8. Electron Localization Function (ELF) and Localized Orbital Locator (LOL)

Figures 11 and 12 describe the two-dimensional description of the electron localization function (ELF) and the localized orbital locator (LOL). The ELF is a tool used in computational chemistry to analyze the electronic structure of molecules. The ELF is a function that measures the degree of electron localization in a molecule. It provides a quantitative measure of the degree to which electrons are localized in a given nucleus. The ELF is useful in the study of chemical bonding, particularly in the analysis of bonding in solid-state materials. It can be used to distinguish between covalent, metallic, and ionic bonding and to identify regions of high electron density where chemical reactions are likely to occur. The electron localization function is a valuable tool in computational chemistry for understanding the electronic structure of molecules and materials. It provides a quantitative measure of the degree of electron localization, which can be used to analyze chemical bonding and predict reactivity.⁵⁰ The LOL is a function that measures the degree of localization of an orbital in a molecule or material. It provides a quantitative measure of the extent to which a given orbital is localized around a given nucleus or group of nuclei. The hydrogen atoms of the lemonol compound with single electrons exhibit the highest Pauli repulsion, which is shown by the red zone, and the carbon and oxygen atoms exhibit the highest Pauli repulsion, which are shown by the blue region. Red spots in the figure represent locations with high LOL values and may be attributed to covalent regions, whereas blue areas can be interpreted as areas where there is electron depletion among the valence and inner shell.⁵¹ The C₅ and C₆ atoms show the active region of the lemonol compound.

Figure 11.

ELF projection and contour map of lemonol.

Figure 12.

LOL projection and contour map of lemonol.

3.9. RDG (Reduced Density Gradient) Analysis and NCI (Noncovalent Interaction) Analysis

RDG stands for reduced density gradient analysis. It is a tool used in computational chemistry to analyze the electronic structures of molecules and determine their reactivity. The reduced density gradient (RDG) is a function that measures the magnitude of the electron density and its gradient. High values of the RDG correspond to regions of high electron density, which are associated with chemical reactivity. RDG analysis is useful in the study of various chemical processes, such as nucleophilic and electrophilic reactions, hydrogen bonding, and charge transfer. In summary, RDG analysis is a useful tool in computational chemistry for studying molecular electronic structure and reactivity. It provides a quantitative measure of the electron density and its gradient, which can be used to identify reactive regions and predict chemical reactivity. Figure 13 demonstrates how the noncovalent interaction (NCI) approach may be used to examine all kinds of interactions, especially in low-density areas or regions.

Figure 13.

NCI of lemonol.

The three-dimensional isosurfaces of the lemonol were plotted in Figure 14, which presents a color code that differentiates the various types of interactions. The color blue signifies a hydrogen bond, the color green signifies the van der Waals interaction, and the color red signifies the strong repulsive force, also known as the steric effect in rings and cages. In strong attractive force ρ > 0, λ2 < 0, in van der Waals interaction ρ > 0, λ2 = 0, and strong repulsive force or steric effect in ring and cage which shows ρ > 0, λ2 > 0. The van der Waals interactions are located at −0.010 to 0.010 a.u. The strong attractive force of the lemonol compound is exhibited at −0.050 to −0.010 a.u., and the corresponding strong repulsive force or steric effect in the ring and cage is localized at 0.010 to 0.050 a.u.

Figure 14.

RDG analysis of lemonol.

3.10. QTAIM Analysis

The QTAIM (Quantum Theory of Atoms in Molecules) analysis is a tool used in computational chemistry to analyze the electronic structure of molecules. The QTAIM analysis is a method that allows the study of the topology of the electron density in a molecule and interaction within the molecule including hydrogen and noncovalent interactions.⁵²⁻⁵⁵ It provides information about the nature of the chemical bonds, the distribution of the electronic charge, and the geometry of the molecule. The definition of the critical points (CPs) of the electron density, which are locations where the gradient of the electron density is zero, serves as the foundation for the QTAIM analysis.

The CPs are classified into four types: nuclear, bond, ring, and cage critical points. The QTAIM analysis allows the outcome of diverse properties of the electron density, such as the electron density at the CPs, Laplacian, and the total energy of the compound.^56,57 Also, the analysis provides information about the electron density distribution and the nature of the chemical bonds, such as the bond path and the bond critical point (BCP). The BCPs are used to classify chemical bonds into covalent, ionic, and hydrogen bonds. The quantum theory of atoms in lemonol is plotted in Figure 15, and the parameters are presented in Table 9. The primary covalent connections between the atoms of the molecule also include a summary of the kinetic energy (G), potential energy (V), total electron density (H), ellipticity, and interaction energies (in kcal/mol) of the principal molecular interactions. The MOD(V/G) values are greater than 1, which means that all of the interactions are covalent.^58,59

Figure 15.

QTAIM analysis of lemonol.

Table 9. Quantum Theory of Atom in Molecules Bond Critical Points (BSP) and Its Parameters.

Interaction	δ(r)a.u.	V2(r)a.u.	H(r)a.u.	G(r)a.u.	V(r)a.u.	λ1a.u.	λ2a.u.	λ3a.u.	ELF	LOL	Ellipticity of electron density
BCP1	0.0110	0.0524	0.00280	0.0102	–0.00748	–0.00938	0.0110	0.0507	0.02259	0.1390	–1.847
BCP2	0.4330	–0.00248	–0.0623	–0.00286	–0.0626	–0.0879	–0.0877	–0.0727	0.9983	0.9614	0.0024

3.11. Cell Proliferative Assay

Lemonol’s ability to inhibit the proliferation of the human breast cancer cell line MCF-7 was examined using the MTT assay; with increasing lemonol concentrations, cell death ensues as illustrated in Figure 16. For 24 and 48 h, the cells were exposed to lemonol at various doses ranging from 5 to 500 μM. The solvent DMSO-treated cells served as a control. From the results, lemonol exhibits potential cytotoxicity against MCF-7 cells with an IC₅₀ value of 300 μM concentration; meanwhile, further experiments were carried out using the IC₅₀ value of the lemonol.

Figure 16.

Effect of lemonol on the cell viability in cultured MCF-7 cells. Cultured MCF-7 cells were treated with lemonol at different concentrations (5, 10, 20, 40, 80, 100, 200, 300, 400, 500 μM) for 24 and 48 h.

3.12. Propidium Iodide Nucleic Acid Staining

The nuclear staining of propidium iodide was used to examine the changes in the nucleus caused by lemonol in MCF-7 cells. Nuclear condensation and nuclear fragmentation are biochemical features of programmed cell death or apoptosis. In comparison with the control, lemonol-treated MCF-7 cells show more intense fluorescence due to condensation and fragmentation of nuclear deoxyribonucleic acid (DNA) shown in Figure 17A.

Figure 17.

(A) Fluorescent microscope images of PI staining of MCF-7 cells controlled and treated with 300 μM of lemonol, blue arrow shows PI-stained cells. (B) DCFHDA Staining of MCF-7 cells, blue arrow shows the generation of ROS (Reactive oxygen species).

3.13. 2′,7′-Dichlorodihydrofluorescein Diacetate (DCFHDA) Assay

To determine the mechanism of the lemonol antiproliferative effect and whether reactive oxygen species-mediated programmed cell death apoptosis occurs, the reactive oxygen species generation was examined using DCFHDA in fluorescence microscopy. The lemonol was treated with a 300 μM concentration for 24 and 48 h, as shown in Figure 17B. MCF-7 cells emit more fluorescence intensity compared with the control MCF-7 cells. This reactive oxygen species generation analysis revealed that lemonol at 300 μM induced increased reactive oxygen species generation in MCF-7 cells after 24 and 48 h of treatment.

3.14. Acridine Orange/Ethidium Bromide (Ao/EtBr) Staining

To detect the morphological evidence of apoptosis, AO/EtBr staining was carried out. The MCF-7 cells were treated with 300 μM concentration for 24 and 48 h as shown in Figure 18. Acridine orange stains live and dead cell components, including the cytoplasm and the nucleus. Ethidium bromide stains the nucleus of the damaged cells. AO/EtBr staining revealed that lemonol-treated MCF-7 cells were yellow and red due to nuclear damage, confirming apoptosis. However, no EtBr was detected in the control cell.

Figure 18.

Fluorescent microscope images of AO/EtBr dual staining blue arrows indicate DNA damage, as evidenced by nuclear staining with ethidium bromide.

4. Conclusions

High-level quantum chemical calculations were employed to determine the structural and spectroscopic characteristics of lemonol, which is a significant class of monoterpenoid. The experimental and theoretical bond lengths and bond angles of lemonol are correlated well with linear coefficient values (R²) of 0.99465 and 0.92554, respectively. Due to the influence of electron donors and acceptors as well as conjugation systems, the minimum energy gap value found theoretically in the gas phase is 5.9084 eV. With the help of Tauc’s plot, the energy gap was determined as 5.186 eV from the experimental UV–vis spectrum. In the NBO, the strongest interactions associated with electron donation were observed from C₅–H₁₆ to the acceptor C₆–C₁₀ with a stabilizing energy of 8.24 kJmol^–1 with a transition of σ–σ*. The reactive sites and distribution of charges and noncovalent bonds of lemonol were found and confirmed by topological, MESP, and Mulliken charge distribution studies. Furthermore, the polarizability and hyperpolarizability values of lemonol are 19.05 and 12.53 times greater than urea, respectively. The in vitro MTT assay revealed that lemonol shows potent antiproliferative activity against the human breast cancer cell line MCF-7 cells with an IC₅₀ value of 300 μM, which resulted in DNA damage, inhibition of DNA synthesis, generation of increased intracellular reactive oxygen species, and activation of the programmed cell death mechanism apoptosis. In conclusion, lemonol exhibits promising characteristics that make it a highly favorable candidate for a wide range of applications to contribute significantly to various scientific and pharmacological fields.

The authors declare no competing financial interest.