On the remarkable nonlinear optical properties of natural tomato lycopene

In line with the renewed interest in developing novel Non Linear Optical (NLO) materials, natural Lycopene’s NLO Properties are reported for the first time within the scientific literature. Correlated to its 1-D conjugated π-electrons linear conformation, it is shown that natural Lycopene exhibits a significantly elevated 3rd order nonlinearity χ(3) as high as 2.65 10−6 esu, the largest value of any investigated natural phyto-compound so far, including β-carotene. In addition to a saturable absorption, the corresponding observed self-defocusing effect in Lycopene seems to be the result of a thermal nonlinearity. The nonlinear response coupled to the observed fluorescence in the Visible spectral range points to a potential photodynamic therapy application as well as the possibility of engineering of novel hybrid Lycopene based NLO nano-materials.

www.nature.com/scientificreports/ tomato fruit, possesses such a 1-D π-electrons electronic conformation ( Fig. 1), it should exhibit a χ (3) response too. Indeed, Lycopene from tomato natural extract is a terpene consisting of 8 isoprene molecules with a chemical formulation of C 40 H 56 . Lycopene from tomato Is a π-electrons conjugated carbon chain long molecule (Fig. 1). As in the case of the large carotenoids family, the backbone of such a molecule consists of alternating carbon single and double bonds 12 . More precisely, Lycopene has 11 conjugated carbon double bonds along its backbone, two un-conjugated double bonds, and no end groups (Fig. 1). In addition of tomatoes as a source of Lycopene, this latter is found in several photosynthetic pigment-protein complexes in plants (Watermellon, Wolfberry, Papaya, Seabuckthorn, ….), photosynthetic bacteria (Saccharomyces cerevisiae, …), fungi, and algae [13][14][15] . They are responsible for the bright red-orange color of fruits and vegetables, and perform various functions ( Fig. 1) in photosynthesis, and protect photosynthetic organisms from excessive light damage. Lycopene is a key intermediate in the biosynthesis of carotenoids including b-carotene, and xanthophylls.
Likewise, and from a medical perspective, Lycopene was found to exhibit a significant activity as membrane-protective antioxidants which efficiently scavenge 1 O 2 and trap peroxyl radicals (ROO*) [16][17][18][19] . In addition, anticancer, antiproliferative and pro-differentiation activities have been attributed to Lycopene 20,21 . This set of medicinal properties makes of Lycopene, an appropriate carotenoid to be investigated from nonlinear optical perspective. Furthermore, its chemical and thermal stability within the broad family of carotenoids makes it ideal as a potential NLO in thin films structure, if any.
Henceforth, the novelty of this contribution lies within the fact that it reports for the first time in the scientific literature the NLO responses of Lycopene. It is hoped that the obtained results could firstly, trigger a broader interest within the photonics community and, secondly, be exploited for potential photodynamic therapy or/and skin cancer treatment of lycopene based green photosynthetizer specifically as well as engineering novel NLO Lycopene based materials in view of its chemical and thermal stabilities.

Experiments, results and discussion
Materials and methods and lycopene extraction. Fresh ripe tomatoes identified as Lycopersicon esculentum (Solanaceae), required for the extraction of lycopene were purchased from a local market. After washing, the raw tomatoes were cleaned, homogenized, and stored at 9 °C in a glass bottle until analysis. Before the final sample preparation, the tomato fruits were immersed in boiling water for 2-3 min. The paste of tomato was prepared by a mixer crushing and 100 gm of it was placed in a 250 ml beaker. The sample was submitted to a filtration phase through Whatman n o 1 and n o 42 filter papers. Lycopene was extracted using a sample of ethanol (1:1, v/v), and quantified spectrophotometrically at 472 nm and expressed accordingly in mg/100 g FW according to the established procedure by Periago et al., Fish et al., and Lavecchia et al. [22][23][24] . Additional solvents were used including acetone, hexane, cyclohexane, and ethyl acetate (purchased from E-Merck (99.9%) were used as solvents. Based on their molecular dipoles, the various solvents were tested in view of identifying those extracting the maximum of lycopene. All the chemicals used in the study were of analytical grade. For the various expriments, solution of lycopene dissolved in hexane (0.1 mg of Lycopene in 100 ml hexane) were used.
Sample characterizations. The UV-VIS Absorbance and the fluorescence spectra were acquired using Ocean Optics units within the spectral range of interest of 250-800 nm. The fluorescence measurements were recorded using a fibre-optics linked Ocean Optics system consisting of a UV light-emitting diode source coupled to a high sensitivity QE Pro-FL spectrometer. The excitation wavelength was fixed at 240 nm. The Raman spectroscopy investigations were carried out on a Horiba LABRAM unit with a laser green excitation of 514.5 nm. www.nature.com/scientificreports/ Z-scan experiments. Figure 2 depicts the Z-scan experimental setup used in the present study. It consisted of a CW laser source operating at 650 nm wavelength with total power of 5 mW. The nonlinear index of refraction, n 2 and nonlinear coefficient of absorption, β of lycopene were measured by closed and open aperture Zscan method respectively. In the closed aperture configuration, the aperture was placed before the detector while in the open aperture geometry; a convex lens was used to collect the beam. A convex lens with focal length of f = 5 cm was used to focus the beam on to the sample. The cuvette of thickness 1 mm in thickness was mounted on a translational stage and moved the sample in the Z directions within the − 20-+ 20 mm range. The transmittance of the beam was measured by a photodetector fed to a digital power meter. The thin sample approximation was used as the measured Rayleigh length was greater than the sample's length. Figure 3a reports the absorption spectrum within the spectral range of 250-800 nm for a solution of lycopene dissolved in hexane (0.1 mg of Lycopene in 100 ml hexane). In addition to a low intensity peak located in the UV region at about ~ 300 nm, one observes 3 strong absorbance peaks centered at 440, 475, and, 510 nm respectively (Fig. 3b). These lycopene intrinsic electronic absorptions, relatively intense, are due to the various π-π* and σ-σ* transitions. The ~ 300 nm absorbance peak which is, a priori, a convolution of 2 peaks (Fig. 3c), is likely to be caused by an aggregation of the pigment molecules with participation of the solvent molecules as suggested by Hager 25,26 . This formation of polymers-like would lead to an alteration in the distribution of the electrons in the chromophore system of the lycopene molecule and thereby to a potential change of the light absorption. Figure 4a reports the fluorescence spectrum under a light emitting diode emitting at 240 nm. One observes a broad emission consisting in fact of the convolution of 3 emissions peaking at l Max , 422, 430 and 460 nm with width at half maximum Dl 1/2 of 22.93, 40.64 and 72.94 nm respectively (Fig. 4b). Similar fluorescence patterns were observed by Fujii et al. [27][28][29] . According to Fuji et al. 27,28 , such a set of fluorescence emissions were attributed to 1 Bu to 2Ag − 1Ag transitions via intermediary non radiative 1Bu to 2Ag transitions following an excitation from 1Ag to 1Bu as schematically summarized an inset of Fig. 4c.

Linear optical investigations.
In view of checking the Lycopene's quality & its purity, Raman spectroscopy studies were carried out on the extract. Figure 5 reports the Raman spectrum under an argon laser excitation of 514.5 nm. It happened that this special excitation wavelength coincides with the resonance Raman conditions for Lycopene 30,31 and hence the Raman modes qre expected be intense, if any. Indeed, as one can observe, the Raman response is characterized by two prominent Stokes lines centered approximately at 1158, and 1518 cm −1 , which have nearly similar relative intensities. Such emissions are intrinsic to Lycopene 30,31 , and, originate, from carbon-carbon single-bond and double-bond stretch vibrations of the conjugated backbone of the lycopene molecule. The relatively less intense emission peaking approximately at 1010 cm −1 is attributed to rocking motions of the molecule's methyl components 30,31 . There is an additional vibrational mode located at 1285 cm −1 that was unable to be precisely identified.
Z-scan method and measurement of third order nonlinear optical properties. The nonlinear index of refraction, n 2 and nonlinear coefficient of absorption, β of lycopene are related to the real and imaginary factors of the third-order NLO susceptibility (χ (3) ). Figure 6a illustrates the open aperture Z-scan profile of lycopene. It is observed from such a figure that the transmitted intensity increases at the focus, and forms a fine peak which is an indication of a saturable absorption (SA). The solid line in Fig. 6a is the theoretical fit, which is relatively well-matched with the experimental profile. With the Saturable Absorption phenomenon as the    www.nature.com/scientificreports/ The closed aperture Z-scan measurements have been used to measure the magnitude and the sign of the nonlinear index of refraction of the sample. Figure 6b displays the pure nonlinear refraction curve of the Lycopene sample. A peak followed by a valley normalized transmittance curve is observed (Fig. 6b) indicating that the Lycopene exhibits a self-defocusing behaviour and, consequently, a negative nonlinear index of refraction. The observed self-defocusing effect in the natural pigment Lycopene is likely to be the result of a thermal nonlinearity. The normalized peak-valley difference ΔT p-v as a function of on-axis phase shift |Δφ 0 | is given by: where S = 1 − exp −2r 2 0 /ω 2 0 is the linear aperture transmittance, ω 0 is the beam radius and r 0 denotes the aperture radius. The transmittance of the natural pigment is given by: where X = Z/Z o . The nonlinear index of refraction n 2 of the sample is given by 32 : www.nature.com/scientificreports/ where λ is the laser wavelength, and L eff is the effective length of the sample. The real and imaginary components of third-order NLO susceptibility (χ (3) ) are given by 32 : where ε 0 and c are the permittivity of the vacuum and the velocity of light. The calculated third-order NLO parameters of Lycopene (summarized in Table 1) are − 7.26 × 10 −12 m 2 /W and − 0.20 × 10 −5 m/W respectively. Therefore, the corresponding real and imaginary parts of the third-order NLO susceptibility [Re (χ (3) )] & [Im (χ (3) )] are − 2.45 × 10 −6 and − 1.01 × 10 −6 esu. Hence, the Lycopene third-order NLO susceptibility (χ (3) ) is about 2.65 × 10 −6 esu. It is to be pointed out that according to Fig. 3, Lycopene has a weak absorption at 650 nm yet the open-aperture Z-scan curve in Fig. 6a shows that Lycopene exhibits a saturable absorption (SA). What is the mechanism for this SA response?. It is to be highlighted that the saturable absorption is a nonlinear phenomenon which arises at high light input intensities at the focus. Hence, at sufficient light intensities at the focus, the ground state is excited into an upper energy state at such a rate there is insufficient time to decay back to the ground state before the ground state becomes depleted. The absorption cross-section of ground state is higher than that of excited state is also the mechanism of absorption saturation.
To sustain the thermal nature of the registered NLO response of the lycopene, on should highlight the following considerations. The laser beam, at the spot size was 1.15 mm. The measured Rayleigh length was 1.47 mm. Hence, the intensity at the focus was 1.047 kW/cm 2 . In lycopene pigment, the observed self-defocusing effect is the result of thermal nonlinearity as it is in most if not each and all organic dyes and pigments, thermal nonlinearity is the predominant. Under CW irradiation, the nonlinearity is due to thermal in nature and not because of other effects. This is confirmed from the following reasons; (i) the value of nonlinear refractive index n 2 > 10 −5 esu and (ii) A peak-valley separation of more than 1.7 times the Rayleigh range is the indication of thermal nonlinearity and indicates the observed nonlinear effect is the third-order process. Therefore, the observed nonlinearity in the pigment is due to thermal in nature.  www.nature.com/scientificreports/ Table 2 reports the NLO 3rd order susceptibility χ (3) of various carotenoids and natural extracts published in the last decade. Also it displays the relative enhancement (χ (3) Lyc − χ (3) Pigm )/χ (3) Lyc ). This relative parameter, is a priori an optimal parameter as a comparative measure of χ (3) Lyc relatively to that of a series of natural extracts reported in the scientific literature. As one can notice, it seems that the measured χ (3) Lyc of Lycopene of 2.65 10 −6 esu is, a priori, the highest value relatively to each & all natural compounds and carotenoids with conjugated π-electrons investigated so far. Indeed, the relative increase ((χ (3) Lyc − χ (3) Pigm )/χ (3) Lyc ) is within 42% and 93%.
Computational results: calculation of quantum molecular descriptors. The observed enhanced experimental value of the 3rd NLO susceptibility of Lycopene is likely to originate from a high electronic polarizability of the Lycopene and its 1-D π-electrons electronic conformation (Fig. 1). For such, it is necessary to investigate the Lycopene molecule's LUMO and HOMO. Hence, the structure of Lycopene was optimized at B3LYP level of theory using the 6-311 basis set carried out using Gaussian 09 37 . At the same level of theory, frequency calculation was done on the optimized structure to make sure of the true minima. The orbital energies of HOMO and LUMO were calculated to obtain the quantum molecular descriptors. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), referred to as frontier orbitals, play a significant role in chemical reactivity and molecular interactions 38 . The molecular electrostatic potential (MEP) and the HOMO-LUMO orbitals of the optimized structure of Lycopene are presented in Fig. 7. The reddish area in Fig. 7c indicates the most active sites of Lycopene. The derived energy of HOMO and LUMO orbital is − 4.62 and − 2.34 eV, respectively. The chemical potential (μ) can be used to assess the evasion affinity of a molecule from equilibrium. The chemical hardness (η) is a property that quantifies the charge transfer and chemical reactivity of a molecule while the Electronegativity ( ξ ) determines the ability of the molecule to attract electrons, and finally higher value of electrophilicity index (ω) means higher electrophilic power of the molecule 39,40 . The values of these parameters are reported in Table 3. www.nature.com/scientificreports/ The static dipole moment ( µ x , µ y , µ z ), and polarizabilities (α xx , α xy , α yy , α xz , α yz , α zz and β xxx , β xxy , β xyy , β yyy , β xxz , β xyz , β yyz , β xzz , β yzz , β zzz ) were calculated at the same level of theory analytically using the keyword Polar in Gaussian 41 and reported in Tables 4, 5 and 6. The total static dipole moment µ , the mean polarizability α , the anisotropy of the polarizability �α and the mean first hyperpolarizability β were calculated by utilizing the following equations The values of polarizabilities and first-order hyperpolarizabilities are reported in atomic units (a.u.), which are converted into electrostatic units (esu) using conversion factor of 0.1482 × 10 −24 esu for α and 8.6393 × 10 −33 esu for β. The results in Table 5 indicate that the x component of hyperpolarizability tensor (along the main molecular axis) has a significant contribution on β . The obtained values of static polarizability and first-order  www.nature.com/scientificreports/  Chemical potential: used to assess the evasion affinity of a molecule from equilibrium − 3.48 (eV) Chemical hardness: a property that quantifies the charge transfer and chemical reactivity of a molecule 1.14 (eV)  www.nature.com/scientificreports/ hyperpolarizability are 127.96 × 10 −24 and 101.67 × 10 −30 esu respectively. Since one of the most critical factors of the NLO system is the magnitude of molecular hyperpolarizability, the mean first hyperpolarizability of lycopene was compared with other organic molecules in Table 7, which shows significant NLO activity of lycopene. The substantial π delocalization along the major molecular axis, as seen in Fig. 7, accounts for the relatively large hyperpolarizability of lycopene. The above derived values of the static polarizability ( �α = 209.2711 ×10 −24 esu) & hyperpolarizabitlity ( �β� = 101.6731 × 10 −30 ) of Lycopene , seem pointing to a higher electronic polarizability of the Lycopene molecule corroborating therefore the experimentally observed enhanced third-order NLO response χ (3) .

Conclusion
Linear & NLO properties of Lycopene were investigated. While the Raman & the UV-VIS studies confirmed the significant purity of the studied Lycopene, their nonlinear optical investigations by Z-scan indicated that Lycopene exhibits a significant optical nonlinearity response of a third order in nature under a CW laser excitation. Its corresponding third-order NLO susceptibility (χ (3) ) was found to be substantially elevated reaching the high value of 2.65 X 10 −6 esu. According to the published literature, such a value is likely to be the highest registered NLO susceptibility of any natural extract including Beta-Carotene, Violaxanthin, Xanthophyll, and Chlorophyll. More accurately, the relative increase ((χ (3) Lyc − χ (3) Pigm )/χ (3) Lyc ) lies within the range of 42%-93%. As a follow up of this study, NLO studies on Lycopene embedded in various optically passive or active polymeric membranes as well as in form of spin-coated nanocomposite thin films. This study reveals that natural Lycopene is a promising material for third order nonlinear optical devices application.

Statement.
It is to be mentioned that: (i) All methods were carried out in accordance with relevant guidelines and regulations of international standards.  www.nature.com/scientificreports/