Glutathione as a photo-stabilizer of avobenzone: an evaluation under glass-filtered sunlight using UV-spectroscopy

Abstract

Avobenzone is the most widely used UVA filter in sunscreen lotion and it is prone to degradation in the presence of sunlight/UV radiation. To overcome the photo-instability of avobenzone, various photostabilizers have been used as additives, including antioxidants such as vitamin C, vitamin E, and ubiquinone. In the present study, the well known antioxidant, glutathione, was evaluated for protecting avobenzone from photodegradation in the presence of glass-filtered sunlight. The features of glutathione as a skin whitener and a radical scavenger in cells have prompted the assessment of the photostabilzing activity of glutathione on avobenzone. Glutathione significantly attenuated the glass-filtered sunlight-induced degradation of avobenzone at equimolar or higher ratios of glutathione and avobenzone. Mutational studies have been undertaken to investigate the role of the thiol group and the isopeptide bond of glutathione on its photoprotection activity towards avobenzone. The thiol group of glutathione plays a vital role in exhibiting the photoprotection activity, which was further supported by the studies on photodegradation of avobonzone in the presence of β-mercaptoethanol. The dual role of glutathione as a skin whitening agent and a photostabilizer of avobenzone may be useful for the development of multipurpose cosmetic lotions.

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Introduction

Sunscreens have been evolved not only to combat sunburn, but also to reduce the harmful effects of UVA (320–400 nm) radiation, such as photocarcinogenesis and photoaging.^1–4 An important ingredient in sunscreen lotion is the UVA filter that prevents the UVA radiation from reaching deep into the skin.^5–8 Avobenzone is one of the most widely used UVA filters in a majority of sunscreen formulations.^9,10 It is still an extensively used UVA filter because of the cost effectiveness and the broad spectrum absorbance of UVA radiation.^11,12 The major drawback associated with avobenzone is its photo-instability, i.e., it undergoes photodegradation upon exposure to sunlight/radiation.^13–16 The photodegradation products of avobenzone cause deleterious side effects to the skin, including the oxidative damage by the radicals.^17–19 In order to overcome the photodegradation of avobenzone and its associated side effects on the skin, various stabilizers were employed, including triplet quenchers such as octocrylene, and methylbenzylidine camphor.^20–25 A triazine derivative was also employed as a stabilizer to block the diketo form of avobenzone.²⁶ Recently, antioxidants such as vitamin C, vitamin E and ubiquinone were demonstrated as photostabilizers of avobenzone.^13,27–29 Glutathione is a well-known peptidic antioxidant in the cellular environment (Fig. 1a) and is widely distributed across the phyla.^30–34 The present study demonstrates the utility of glutathione as a photostabilzer of avobenzone. Even though peptides have proven applications in the cosmetic field,^35,36 they were not tested thus far to improve the photostability of avobenzone.

Fig. 1 Chemical structures of the molecules investigated in the present study for their effect on the photodegradation of avobenzone. (a) Glutathione of cytosol, (b) synthetic analogous peptides of glutathione, and (c) β-mercaptoethanol.

Glutathione is widely used in the cosmetic field as a skin whitening or skin lightening agent.^37,38 Interestingly, it is associated with the modulation of skin complexation and is indeed present in the skin. It has been well documented in the literature that glutathione protects the cellular system from oxidative damage by directly reacting with reactive oxygen species (ROS), such as hydroxyl radicals, hydrogen peroxide and nitroso radicals.^39–41 The feature of radical scavenging activity of glutathione and the radical-mediated degradation of avobenzone have prompted us to assess the ability of glutathione to protect avobenzone from sunlight-induced photodegradation. The present report evaluates the photostabilizing activity of glutathione towards avobenzone in ethanol/acetonitrile under glass-filtered sunlight. Synthetic mutant peptides of glutathione (Fig. 1b) and β-mercaptoethanol (Fig. 1c) were also evaluated to confirm the involvement of the sulfur atom of glutathione in the photostabilization activity.

Results and discussion

Sunlight induced degradation of avobenzone

The UV spectrum of avobenzone shows a predominant peak (λ_max) at 356 nm. The absorbance value corresponding to the λ_max has been widely used to estimate the extent of photodegradation of avobenzone.^13,42,43 The present study also adopted a similar approach and used UV spectrophotometry as a tool to measure the extent of photodegradation of avobenzone under glass-filtered sunlight. Fig. 2a shows the UV spectra of avobenzone in ethanol drawn at regular intervals of time from a solution constantly exposed to glass-filtered sunlight. It is apparent from Fig. 2a that the degradation of avobenzone increases with the increase in time of exposure to glass-filtered sunlight. Higher the exposure to glass-filtered sunlight, higher will be the extent of degradation. Similar results were obtained with the polar aprotic solvent, acetonitrile (Fig. 2b). Approximately 50% of photodegradation of avobenzone (Fig. 2c) was observed in ethanol as well as in acetonitrile upon 5 h of exposure to glass-filtered sunlight. However, complete photodegradation of avobenzone was observed during the peak summer season at Central University of Karnataka (Fig. S1a and S1b†). Even though avobenzone degraded completely in ethanol (Fig. S1a†), ∼15% of avobenzone still remained non-degraded in the acetonitrile solvent system (Fig. S1b†) under the same conditions of glass-filtered sunlight. These observations confirm the role of proticity of the solvent on the extent of photodegradation of avobenzone. This result was consistent with a previous report on the effect of polarity and proticity of the solvent on avobenzone degradation using a high pressure-mercury lamp as the UV source.⁴² These results confirm that the photoprotection activity of glutathione on avobenzone can be assessed in the presence of glass-filtered sunlight.

Fig. 2 Glass-filtered sunlight-induced photodegradation of avobenzone. (a) UV spectra of avobenzone in ethanol drawn at regular intervals of time from a solution constantly exposed to the glass-filtered sunlight. (b) UV spectra of avobenzone in acetonitrile drawn at regular intervals of time from a solution constantly exposed to the glass-filtered sunlight. (c) Extent of photodegradation of avobenzone in polar protic/aprotic solvents under glass-filtered sunlight. Error bars represent five independent experiments on different days of glass-filtered sunlight.

Solar and UV radiation measurements

The dose of solar and ultraviolet (UV) radiation reaching the test center/test solution was measured using solar and UV power meters, respectively, as described in the Experimental section. The solar radiation predominantly contains UV, visible, and infrared (IR) radiation.^44–46 Visible and IR radiation constitute a major portion of the solar spectrum. The ozone layer present in the stratosphere allows only UVA (320–400 nm) and UVB (285–320 nm) to reach the earth surface. Hence, the intensity of UV radiation was recorded from 290–390 nm. In order to estimate the actual solar/UV radiations reaching the test solution, the intensity of solar/UV radiation was measured by the transmission of sunlight through samples of the experimental glassware. The glass plate was found to be opaque to radiation <325 nm, and the conical flask glass was opaque to radiation <308 nm, indicating that the glass filtration predominantly produced a UVA source.

Table 1 shows the average intensity of solar and UV radiation at selected time points that caused ≥50% photodegradation of avobenzone upon constant exposure to sunlight for 6 h. Table 1 also shows the corresponding altered intensity of solar and UV radiation in the test solution when passed through the glass plate and the conical flask glass. The following inference can be drawn from the data in Table 1: (i) ∼85% and ∼94% solar radiation reached the test solution through the glass plate and the conical flask, respectively; (ii) ∼ 62% and ∼88% UV radiation reached the test solution through the glass plate and the conical flask, respectively; (iii) ∼3.2% UV radiation was present in the sunlight reaching the test center; and (iv) difference in the intensity of UV radiation before and after passing through the conical flask (≤308 nm) was ∼12%, which corresponded to UVB radiation in the sunlight. The intensity of solar and UV radiation mentioned in Table 1 correspond to the conditions of glass-filtered sunlight (hereafter, sunlight indicates glass-filtered sunlight) used to assess the effect of the additives on the photodegradation of avobenzone.

Table 1 Average intensity of solar and UV radiations at selected time points which caused ≥50% photodegradation of avobenzone over 6 h of constant exposure to sunlight

Time point	Solar radiation (W m^−2)			UV radiation (W m^−2)
	Normal	Glass filter		Normal	Glass filter
		Glass plate	Conical glass		Glass plate	Conical glass
Note: Average values are from four independent readings on different days of sunlight.
10:00 AM–12:00 PM	1141	976	1082	38.23	23.91	34.13
12:00 PM–2:00 PM	1222	1050	1161	41.84	25.46	36.67
2:00 PM–4:00 PM	964	824	909	27.20	16.98	24.37

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Assessing the photoprotection activity of glutathione on avobenzone under sunlight

To assess the photoprotection activity of glutathione on avobenzone, 1 : 2 ratio and various molar ratios (as seen in Fig. 4d) of avobenzone and glutathione in ethanol/acetonitrile were exposed to sunlight. Avobenzone alone was also exposed to sunlight under identical conditions to serve as the control. The extent of photodegradation of avobenzone in the presence of glutathione was monitored by recording the UV spectra of the avobenzone and glutathione mixture drawn at regular intervals of time from a solution constantly exposed to sunlight. It should be noted that the assessment of the photoprotection activity of glutathione on avobenzone was considered only if the degradation of the control avobenzone alone was >25% under identical sunlight. Fig. 3 shows the UV spectra of avobenzone and the extent of its degradation in the absence as well as in the presence of glutathione under identical conditions of sunlight. Interestingly, a significant time-dependent attenuation of the photodegradation of avobenzone was observed in the presence of glutathione. The prominent degradation of avobenzone in the absence of the additive (Fig. 3a) and the near non-degradation in the presence of glutathione (Fig. 3b) under identical conditions of sunlight confirm that glutathione protects avobenzone from photodegradation. Fig. 3c shows the percentage of degradation of avobenzone in the absence as well as in the presence of glutathione in ethanol/acetonitrile. It is evident from Fig. 3c that glutathione acts as a photostabilizer of avobenzone under sunlight. The extent of photodegradation of avobenzone in the presence of glutathione was also investigated during the peak summer season at Central University of Karnataka (Fig. S1c and S1d†). The complete photoprotection of avobenzone was observed in the ethanol solvent system (Fig. S1c†), whereas in acetonitrile, a slight initial degradation of avobenzone occurred before its photoprotection by glutathione (Fig. S1d†).

Fig. 3 Assessing the ability of glutathione to protect avobenzone from the sunlight induced photodegradation in ethanol/acetonitrile. UV spectra of (a) avobenzone alone and (b) avobenzone in the presence of glutathione at different intervals of time under identical conditions of sunlight. (c) Extent of photodegradation of avobenzone in the presence of glutathione in ethanol/acetonitrile. (d) Dependence of the photoprotection of avobenzone on the concentration of glutathione. Bar graph represents the results of five/three independent experiments conducted on different days of sunlight.

Fig. 4 Comparison of photodegradation of avobenzone using UV spectroscopy and RP-HPLC. (a) RP-HPLC elution profiles of avobenzone alone and (b) RP-HPLC elution profiles of avobenzone with glutathione at different intervals of time upon exposure to identical conditions of sunlight. Inset shows the overlapping of the corresponding elution profiles. (c) Extent of photodegradation of avobenzone alone and avobenzone with glutathione as per UV spectroscopy and RP-HPLC methods. Asterisk indicates the P < 0.05 by two sample t-test between percents derived from statistic calculator (StatPac, Inc.).

To identify the minimal concentration of glutathione required for exhibiting the photoprotection activity on avobenzone, the sunlight-induced degradation of avobenzone was monitored in the presence of varied concentrations of glutathione in ethanol. The percentage of the protection offered by glutathione was calculated, as shown in the Experimental section. Fig. 3d shows the dependence of the extent of protection of avobenzone on the concentration of glutathione. As the concentration of glutathione increases, the photoprotection of avobenzone by glutathione also increases gradually. At the 0.2 : 1 ratio of glutathione and avobenzone, 69% photoprotection was conferred by glutathione. As the concentration of glutathione increases, the extent of photoprotection also increases (Fig. 3d) and the maximum protection of 90% is observed at the 2 : 1 ratio of glutathione and avobenzone. A similar extent of photoprotection of avobenzone was also observed with the further increase in the concentration of glutathione.

Comparison of percentage degradation of avobenzone using UV spectroscopy and RP-HPLC

To further authenticate the UV-based estimation of photodegradation of avobenzone, RP-HPLC was employed to analyse the samples drawn at regular intervals of time from the avobenzone solutions constantly exposed to sunlight. Avobenzone alone and avobenzone with glutathione in ethanol, upon exposure to sunlight, were subjected to UV spectroscopy, followed by RP-HPLC, as described in the Experimental section. Fig. 4 shows the RP-HPLC elution profiles of avobenzone alone and avobenzone with glutathione drawn at regular intervals of time from a solution exposed to identical conditions of sunlight. The photodegradation of avobenzone was evident from the RP-HPLC elution profile (Fig. 4a). Similarly, the photoprotection of avobenzone by glutathione was also evident from the RP-HPLC elution profiles (Fig. 4b). Table 2 shows the estimated concentrations of avobenzone using the UV spectra and the RP-HPLC elution profiles at given time intervals of exposure of solutions to sunlight. The deduced concentrations of avobenzone in the samples exposed to sunlight were similar according to both UV spectroscopy and RP-HPLC methods. Fig. 4c shows the percentage degradation of avobenzone upon exposure to sunlight for avobenzone alone and avobenzone with glutathione, deduced using both UV spectroscopy and RP-HPLC methods. No significant differences in the estimation of concentrations of avobenzone were observed between two methods according to the statistical analysis of data derived from three independent experiments conducted on different days. These results further confirm glutathione as a photostabilizer of avobenzone.

Table 2 Comparison of estimated concentrations of avobenzone by UV spectroscopy and RP-HPLC at regular time intervals during the constant exposure to sunlight

Time (Hours)	Avobenzone alone				Avobenzone with glutathione
	Concentration (μM)		% Degradation		Concentration (μM)		% Degradation
	UV	RP-HPLC	UV	RP-HPLC	UV	RP-HPLC	UV	RP-HPLC
0	36.18	—	0.00	—	35.29	—	0.00	—
1	31.47	31.02	13.02	14.26	33.82	33.74	4.16	4.39
2	29.70	29.42	17.91	18.68	33.53	33.22	4.99	5.86
3	26.18	25.64	27.64	29.13	34.12	33.86	3.31	4.05
4	21.76	22.12	39.86	38.86	34.12	33.82	3.31	4.16
5	13.53	13.34	62.60	63.13	34.41	34.14	2.50	3.26
6	12.65	11.98	65.03	66.89	34.70	34.54	1.67	2.12

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Characterization of synthetic _mutglutathione, _isoglutathione and (Ala)₃-tripeptide

Analogous peptides of glutathione were synthesized to assess the role of thiol and iso-peptide bonds on the photoprotection activity of glutathione. Table 3 shows the list of synthetic peptides. Fig. 5a shows the ESI-MS/MS data of _mutglutathione. The observed y₂ ion at m/z 163 and the concomitant loss of 18 Da to yield the [y₂-H₂O] ion correspond to the Ser-Gly dipeptide fragment. The difference between the mass of the molecular ion peak and the mass of the y₂ ion is 129 Da, which corresponds to the residual mass of glutamic acid. These observations were further supported by the presence of the b₂ ion at m/z 217. These results are consistent with _mutglutathione having the sequence Glu^γ-Ser-Gly. Fig. 5b shows the ESI-MS/MS data of _isoglutathione. The observed mass at m/z 233, 215, and 187 correspond to the b₂ ion, [b₂-H₂O], and [b₂-H₂O-CO], respectively. These fragments confirm the presence of Glu-Cys dipeptide fragments in _isoglutathione. The presence of b₃ ion at m/z 290 and [b₃-H₂O] at m/z 272 confirm the sequence of _isoglutathione as Glu^α-Cys-Gly. Fig. S2† shows the ESI-MS/MS data of glutathione. Distinct differences in the ESI fragmentation patterns of _isoglutathione and glutathione further authenticate the sequence of _isoglutathione. Fig. 5c shows the ESI-MS/MS data of (Ala)₃-tripeptide. The observed b₂ ion at m/z 143 and the concomitant loss of 28 Da to yield the [b₂-CO] ion correspond to the alanine dipeptide fragment. The difference between the mass of the molecular ion peak and the mass of the b₂ ion is 89 Da. This corresponds to the residual mass of alanine and the mass of a water molecule. These observed fragment ions are consistent with (Ala)₃-tripeptide, confirming the sequence of synthetic (Ala)₃-tripeptide. The synthetic peptides helped to draw the following inferences. (1) Glutathione and _mutglutathione differ by the mutation of cysteine to serine residue, i.e., a switch from sulfur to oxygen atom. Thus, _mutglutathione helps to dissect the role of the sulfur atom of glutathione on the photoprotection activity. (2) Glutathione and _isoglutathione differed by the shift in the Glu^γ-Cys amide bond to the Glu^α-Cys amide bond. _isoGlutathione helps to reveal the role of the unique feature of the γ-peptide bond on the photoprotection activity of glutathione on avobenzone. (3) (Ala)₃-tripeptide and glutathione share similar chain lengths; therefore, it may be used as a control to assess the photoprotection activity of analogous peptides of glutathione.

Fig. 5 ESI-MS/MS data of the synthetic analogous peptides of glutathione. (a) _mutGlutathione, (b) _isoglutathione and (c) (Ala)₃-tripeptide. Inset shows the expected fragment ions. Observed fragment ions are indicated.

Table 3 Characterization of analogous synthetic peptides of glutathione

Sl. no.	Peptide name	Sequence	Calculated mass [M + H]^+ (Da)	Observed mass [M + H]^+ (Da)
1	mutGlutathione	Glu^γ-Ser-Gly	292.1	291.7
2	isoGlutathione	Glu^α-Cys-Gly	308.0	307.7
3	(Ala)3-tripeptide	Ala-Ala-Ala	232.1	231.7

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Assessing the photoprotection activity of analogous peptides of glutathione on avobenzone

Fig. 6 shows the sunlight-induced degradation of avobenzone in the presence of synthetic peptides under identical conditions of sunlight. As revealed from Fig. 6b and c, attenuation of the photodegradation of avobenzone is less in the presence of _mutglutathione compared with glutathione under identical conditions of sunlight. This observation confirms the involvement of the sulfur atom of glutathione on its photoprotection activity. Interestingly, a similar extent of photoprotection of avobenzone was observed in the cases of both glutathione (Fig. 6c) and _isoglutathione (Fig. 6d), indicating that the γ-peptidic nature of glutathione may not have an influence on the photoprotection activity of glutathione. Surprisingly, ∼20% more avobenzone degradation was observed in the presence of (Ala)₃-tripeptide (Fig. 6e). This anomaly may be ascribed to the hydrophobicity of the peptide. Vallejo et al. reported that ∼90% degradation of avobenzone was observed in hexane, while only 20% degradation was observed in ethanol/acetonitrile.⁴³ These observations confirm that the hydrophobicity of the medium including both solvent and substrate may play a vital role on the extent of radiation-induced degradation of avobenzone.

Fig. 6 Assessing the photoprotection activity of glutathione and its analogous peptides on avobenzone under identical conditions of sunlight. UV spectra of (a) avobenzone alone, (b) avobenzone with _mutglutathione, (c) avobenzone with glutathione, (d) avobenzone with _isoglutathione and (e) avobenzone with (Ala)₃-tripeptide at regular intervals of time. (f) Extent of photodegradation of avobenzone in the presence of various peptide additives. Error bars represent data of three independent experiments conducted on different days of sunlight.

Assessing the photoprotection activity of β-mercaptoethanol on avobenzone

To further support the involvement of the thiol group of glutathione in its photoprotection activity on avobenzone, the sunlight-induced degradation of avobenzone was monitored in the presence of β-mercaptoethanol. The structural difference between the solvent ethanol and β-mercaptoethanol is the thiol group. Fig. 7 shows the UV spectra of avobenzone and the extent of its degradation in the presence of β-mercaptoethanol in ethanol. It is evident from Fig. 7 that fair attenuation of photodegradation of avobenzone is observed in the presence of β-mercaptoethanol.

Fig. 7 Assessing the ability of β-mercaptoethanol to protect avobenzone from the sunlight-induced degradation under identical conditions of sunlight. UV spectra of (a) avobenzone and (b) avobenzone in the presence of β-mercaptoethanol in ethanol at regular intervals of time. (c) Extent of photodegradation of avobenzone in the presence of β-mercaptoethanol in ethanol/acetonitrile. Error bars represent data of three independent experiments conducted on different days of sunlight.

Similar results were also observed in acetonitrile (Fig. S3†). These observations further support the involvement of the thiol group in the photoprotection activity on avobenzone. It should be noted that the extent of attenuation of photodegradation of avobenzone was more in the presence of glutathione than β-mercaptoethanol. Mturi and Martincigh have reported that polarity and proticity of the medium have a direct influence on the extent of photodegradation of avobenzone.⁴² Thus, apart from the thiol group, the backbone skeleton of glutathione may also influence the photoprotection activity.

Conclusions

Avobenzone is one of the most commonly used UVA filters in sunscreen lotions. An important drawback of avobenzone is its photo-instability, i.e., it undergoes degradation in the presence of radiations/sunlight. Various stabilizers, including antioxidants, were evaluated to impart stability to avobenzone against photodegradation. The present report has demonstrated the ability of glutathione to protect avobenzone from sunlight-induced photodegradation. Analysis of avobenzone photoprotection in the presence of glutathione using both UV spectrophotometry and RP-HPLC has clearly established glutathione as a photo-stabilizer of avobenzone. Equimolar concentrations of glutathione and avobenzone significantly attenuated the photodegradation of avobenzone under sunlight. Analogous peptides of glutathione were chemically synthesized and assessed for photoprotection activity. These results clearly indicated that the sulfur atom of glutathione plays a vital role in exhibiting the photoprotection activity. The dual role of glutathione as a skin whitening agent and a photo-stabilizer of avobenzone may find applications in the design of multi-purpose sunscreen lotion. The peptidic nature of glutathione has an advantage in developing novel cosmetic peptides to protect skin from harmful radiations.

Experimental

Materials

Avobenzone was obtained from Tokyo Chemical Industry (TCI), China. Ethanol was obtained from Changshu Hongsheng Fine Chemical Co., Ltd (CS), China. Acetonitrile was obtained from Merck, India. Glutathione was obtained from HiMedia, India. Trifluoroacetic acid was obtained from S.D. Fine Chemicals, India. Fmoc-Gly-OH and Fmoc-Cys(Trt)-OH were obtained from GL Biochem, USA. Fmoc-Glu(OH)-OtBu was obtained from Avra, India. Chromatographic grade water was obtained from Merck, India. Strata C18-columns were purchased from Phenomenex, India.

Measurement of intensity of solar and UV radiations of sunlight

Irradiance of solar radiation was measured using a Tenmars TM-207 solar power meter, which covers ultraviolet, visible, and infrared radiations of sunlight. The intensity of the UV radiation in sunlight was measured using a Leutron UV-340A light metre, which registers UV radiation within the spectral range of 290–390 nm. The photodegradation of avobenzone was also conducted in parallel during the measurements of intensity of solar and UV radiations. The directions of the meters were regularly changed in accordance to the direction of the sun during the course of exposure. Radiation intensity was also recorded through the glassware used in the experiments, namely, glass plate and piece of conical flask. Averaged radiation intensity corresponds to that obtained on four different sunny days.

Glass-filtered sunlight irradiation

Initially, 25 μM avobenzone, 50 μM glutathione, 50 μM β-mercaptoethanol, and 50 μM of peptide were prepared in 40 mL of ethanol/acetonitrile. Various concentrations of glutathione were prepared from a 1 mM stock solution by serial dilution. Note: 200 μL solution of glutathione/peptide in water was suspended in a total 40 mL of ethanol or acetonitrile, which yielded a homogeneous solution. The samples were taken in 250 mL conical flasks and covered with glass plates to avoid solvent evaporation during the exposure to glass-filtered sunlight. The samples were exposed to the peak period of 10 AM–4 PM for glass-filtered sunlight at Central University of Karnataka, Kalaburagi. The samples were occasionally stirred during the course of exposure to sunlight. The samples were drawn to record the UV spectra and the RP-HPLC elution profiles at regular intervals of time from solution constantly exposed to the sunlight. Since UV content in the sunlight varies, avobenzone alone was used as a control in all the experiments. The judgment on the photodegradation/photoprotection of avobenzone in the presence of an additive was considered only if the degradation of avobenzone alone was >25%.

UV spectroscopy

The spectra of the solutions were recorded using a PerkinElmer Lambda 25 double beam UV-visible spectrophotometer. The spectra were recorded over the wavelength range of 200–500 nm. The measurements were made immediately after the samples were withdrawn from sunlight irradiation at regular intervals during the 0–5 h time period. The absorbance intensity at 356 nm was used as a read-out to estimate the extent of photodegradation of avobenzone under sunlight. The percentage of photodegradation of avobenzone was calculated as follows:
where C_I is the concentration of avobenzone before the irradiation. C_F is the concentration of avobenzone after irradiation.

The percentage of photoprotection on avobenzone by glutathione was calculated as follows:

where P_A is the percentage degradation of avobenzone alone. P_B is the percentage degradation of avobenzone in the presence of glutathione under identical conditions of sunlight.

The error bars were derived from the independent experiments conducted during different days of sunlight. The statistical analysis of the data was performed on the Origin Pro 8.5 graphical platform.

Reverse phase-high performance liquid chromatography

The samples, namely, avobenzone alone and avobenzone with glutathione in ethanol, drawn at regular intervals of time were quantified using both UV spectroscopy and RP-HPLC. RP-HPLC was performed using an UFLC Shimadzu HPLC system equipped with binary LC-20AD pumps, an SIL-20AC autosampler and an SPD-20A detector. In brief, 500 μL of a sample was injected using the autosampler, eluted from a Luna phenomenix C-18 column (250 mm × 4.6 mm, 5 μm), and monitored at 356 nm. The linear gradient of acetonitrile containing 0.1% TFA was used as the mobile phase: 0 min-0%; 5 min-0%; 10 min-65%; 40 min-100%. Area under the curve was estimated using the LabSolutions software provided by the manufacturer. With respect to the area under the curve of a known concentration of avobenzone, the concentration of avobenzone in test solutions was estimated. Three independent experiments on different days of sunlight were conducted (six hours of exposure to identical sunlight) and quantified by performing both UV spectroscopy and RP-HPLC. The statistical difference between the percentages of degradation of avobenzone estimated by UV spectroscopy and RP-HPLC was analysed using the Stat. Pac Software.

Solid phase peptide synthesis

_mutGlutathione, _isoglutathione and (Ala)₃-tripeptide were synthesized manually by the standard solid phase peptide synthesis methodology using Fmoc-(9-fluorenylmethyloxycarbonyl) chemistry and the activation of the C-terminus by HBTU. After the synthesis, the peptides were cleaved from the resin using reagent K and precipitated using ether. The precipitated peptides were washed thoroughly multiple times with cold ether. The peptide was dissolved and purified using a phenomenex solid phase extraction cartridge using the protocol provided by the manufacturer. The purified peptides were dried using a vacuum concentrator and subjected to mass spectrometry. The dried purified peptides were used to assess the photoprotection activity.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This article is dedicated to Prof. S. S. Murthy and Prof. M. N. Sudheendra Rao for their contribution to the Department of Chemistry, Central University of Karnataka, Kalaburagi. PChVG is supported by the award of a Senior Research Fellowship (SRF) from the University Grant Commission (UGC), India. B. H. acknowledges DST-SERB-NPDF grant. V. D. acknowledges IAS-INSA-NIAS summer fellowship. We acknowledge the help of Ms. Priyanka Nelogi. This research work is supported by the DST-INSPIRE faculty research grant. We acknowledge proteomic facility, MBU, IISc, Bengalure.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8pp00343b

‡ These two authors are equally contributed to the work.

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