Evaluating the Efficacy of an Extract for UV Defense and Mitigation of Oxidative Stress, Transitioning from Biomass to Bioprotection

Abstract

This study evaluated natural extracts from plant biomass for UV protection and oxidative stress reduction. Conducted in Bucharest, Romania, it focused on medicinal mushrooms and pomegranate bark. The biotechnological process involved a two-phase extraction: hot water processing of Ganoderma lucidum, Hericium erinaceus, Inonotus obliquus, and Tremella mushrooms, followed by ethanol extraction with pomegranate bark and green tea. The spectrophotometric analysis identified phenolics and flavonoids. The ethanol extract showed higher phenolic content and antioxidant activity, particularly in DPPH radical scavenging. UVB exposure tests demonstrated its protective effect, comparable to vitamin B3, delaying oxidative stress onset by 30 min. This research underscores the potential of using natural biomass extracts in skincare, promoting environmental sustainability and economic viability by converting agricultural waste into valuable bioactive compounds.

1. Introduction

The shift towards a circular economy, which aims to reduce waste and prolong the lifespan of resources, requires the development of creative methods for utilizing biomass. Biomass conversion and valorization methods provide a means to accomplish this goal by converting plant-derived components into valuable goods [1]. This study investigates the possibility of natural extracts obtained from plant biomass as a crucial element in this approach. The utilization of various dietary components found in these extracts to develop sustainable and valuable nutritional products, supporting a circular economy theory, is important to identify new sources of bioactive compounds [2].

In response to many external and internal stressors, the body must maintain a fragile equilibrium to operate effectively. A crucial element of this equilibrium is regulating oxidative stress, which arises from an imbalance between free radical generation and antioxidant availability in the body. Free radicals, which are highly reactive entities, may damage cells, proteins, and DNA, so contributing to the onset of many illnesses and the aging process [3]. Exposure to ultraviolet (UV) light from the sun is a major cause of oxidative stress. This exposure leads to the creation of free radicals in the skin, significantly increasing the likelihood of cellular damage and the development of skin malignancies [4].

Scientists have investigated natural extracts derived from plant and mushroom biomass to determine their potential to safeguard against UV radiation and oxidative stress [5]. The biomasses include abundant bioactive substances such as polyphenols, flavonoids, and carotenoids, which possess antioxidant qualities and might counteract the harmful effects of free radicals caused by UV radiation. Because the extract is derived from a natural source with potential dietary applications, if used as a dietary supplement, it can be used for UV protection and oxidative stress mitigation [6]. Thus, this paper is relevant to the field of nutrition because it explores various aspects of nutrition and its impact on health, and the potential role of nutrition is relevant in disease prevention and management.

These natural extracts play a crucial role in the circular economy by being utilized in the conversion and valorization of biomass. Valuable bioactive chemicals can be isolated from plant and mushroom biomass, commonly regarded as agricultural or industrial waste. This process enhances the worth of what would otherwise be considered useless, diminishes the adverse effects on the environment, and encourages the practice of sustainability [7]. Research conducted on certain extracts, such as green tea and grape seed, demonstrates the potential of these substances. Studies have examined the possibility of green tea extract, which includes potent antioxidants, to counteract detrimental free radicals and minimize oxidative damage to skin cells [8]. Moreover, grape seed extract, which has a high concentration of proanthocyanidins, can shield the skin from oxidative stress caused by UV radiation. This is achieved by effectively neutralizing harmful free radicals and enhancing the skin’s protective mechanisms [9].

Though natural extracts from plant biomass have the potential to protect against oxidative stress caused by UV radiation, they should be used in addition to, rather than as a substitute for, established sun protection measures such as sunscreen [10]. Incorporating these extracts into broader protection methods can improve overall defense against UV damage, which aligns with the principles of the circular economy by maximizing the utilization of biomass resources and reducing waste. This strategy enhances environmental sustainability and fosters economic viability by converting biomass into valuable phytochemical compounds [11].

The relationship between natural extracts derived from plant biomass, such as pomegranate bark and medicinal mushrooms, is a vital subject of study in the circular economy context. They play a crucial role in the economic context of biomass valorization due to their abundance as a source of dietary compounds [12]. Retrieval of bioactive chemicals from these forms of biomass entails comparable processes. There is a direct relationship between optimizing these technologies and maximizing the benefits derived from both forms of biomass [13].

The ideas presented here emphasize the complex and varied connection between extracts from pomegranate bark and medicinal mushrooms. This includes their chemical characteristics, positive effects on health, impact on the environment, and their roles in the growing market for natural health products derived from plant material. The significant issue regarding UV radiation’s impact on skin health is its ability to cause oxidative stress. Botanical extracts, which contain high levels of antioxidants, can mitigate the harmful effects of oxidative stress. While additional research is necessary, incorporating these natural elements into skincare routines may offer a holistic approach to maintaining skin health in the face of environmental challenges.

2. Materials and Methods

2.1. Plant Matter

The plant-based components used in this study mostly come from medicinal mushrooms and pomegranate bark. These materials were selected based on their high concentration of bioactive chemicals recognized for their antioxidant qualities and possible positive effects on health. This study examined the therapeutic mushrooms Ganoderma lucidum, Hericium erinaceus, Inonotus obliquus, and Tremella. These mushrooms were sourced as raw materials from Anoom Laboratories SRL in Romania. The selection of pomegranate (Punica granatum) bark was based on its high concentration of polyphenols. Dried pomegranate bark was utilized for this study [14].

2.2. Extraction Process

Obtaining an extract requires two distinct phases, which are essential for achieving a satisfactory conclusion. The initial stage involves the meticulous selection and preparation of medicinal mushrooms for extraction. This process entails pulverizing and mincing to improve the overall caliber of the ultimate extract. The dual-phase process enhances the extraction yield. Afterward, the specified components (Ganoderma lucidum:Hericium erinaceus:Inonotus obliquus:Tremella, in a ratio of 1:1:1:1) are extracted using hot water (7.5 g per 100 mL; Exract-Phase1). The second phase is dedicated to separating the acquired extract using filtration. After the initial treatment with Viscozyme, the leftover substrate was enriched with dried pomegranate bark (9%) and green tea (1%) and then extracted using ethanol (50%; Exract-Phase2). This stage is crucial for guaranteeing the concentration of the desired chemicals and an effective extraction process. The two extracts were combined following the ultimate separation and concentration process (Final extract) [15].

2.3. Examination of the Bioactive Chemicals

The total phenolic compounds were quantified using spectrophotometry [16,17]. In addition, the spectrophotometric method was used to determine the total flavonoid compounds [17].

2.4. Method for Analyzing Samples in a Controlled Laboratory Environment

The DPPH and ABTS scavenging procedures were conducted using spectrophotometry, with ascorbic acid 1% as the control for both methods [18,19].

2.5. HPLC-PDA Analysis of Major Phenolic Compounds

Phenolic compounds were investigated using a previously published method [20]. The individual phenolic compounds were analyzed using the Shimadzu LC-20AT HPLC system coupled with a diode-array detector (280, 320, and 360 nm). The separation of the compounds was performed on a 150 × 4.6 mm, 5 μm Kinetex C18 column, with 5 μm particles, using mobile phases A and B in the gradient below mode for 45 min at a temperature of 35 °C and a flow rate of 0.8 mL/min. The injection volume of standards/samples was 10 μL. The mobile phase consisted of a gradient mixture of solvent A, represented by a mixture of water and phosphoric acid (pH 2.3), and solvent B (acetonitrile of chromatographic purity). Before starting the HPLC procedure, the mobile phase components were filtered through Macherey–Nagel (MV) filters with a 0.20 μm pore size and sonicated to remove air bubbles. The elution during the analysis assumed a gradient between 5–90% component B in 45 min. The acquisition of data and the interpretation of the results were performed using LCSolution software. In the present study, linearity was studied in the concentration range of 1–50 µg/mL, and the calibration curves were first created to quantify the phenolic compounds in the samples. The results obtained are expressed in µg/100 g dry extract.

2.6. Determination of the In Vivo Antioxidant Potential under Blue Light

This method was based on a modified method [21]. It used the same strain of Saccharomyces boulardii, and the viability analysis involved determining the critical time using the reaction mixture: 0.2 mL sample (extract), 0.1 mL yeast culture, and 0.1 mL culture medium. The intersection between the lines of viability and mortality (at different time intervals of 15, 30, 45, and 60 min) was defined as the critical exposure time at ʎ = 320 nm. It was expressed as the critical point (%). The untreated sample and B3 vitamin (1%) were used as the controls.

2.7. Statistical Data Analysis

The data were processed using standard statistical tests and regression analysis. The tests applied were one-way ANOVA and the post hoc Tukey test. The tests were performed within the R program (version 4.4.1) and its interface, RStudio (2 July 2023), using the R package “agricolae” [22] and “DescTools” [23]. The graphical outputs were produced using R package “ggplot2” [24]. More about using R statistical packages can be found in a textbook [25]. The correlation matrix supplied was computed using the Pearson correlation coefficient. The labels a, b, c, d, and e are used to denote homogeneous groups, where groups sharing the same label indicate no statistically significant difference in their means.

3. Results and Discussion

The ANOVA results show that the “Phase” factor has a highly significant effect on the dependent variable, with a very small p-value (<2 × 10⁻¹⁶) and a large F value (6,879,461), indicating strong differences between phases (Exract-Phase1, Exract-Phase2, and Final extract) and most of the variability being explained by Phase. The total phenols analysis indicates that the Extract-Phase 2 group has the highest phenol content (8759.333), followed by the Final extract (4269.667) and Extract-phase 1 (2681.333), with significant differences between all groups, as indicated by the different letters (a, b, and c) representing different homogeneous groups.

The correlation analysis shows that these were the principal extraction (total phenolic; Figure 1) compounds since the functional compounds could dissolve in ethanol. The disparity in comparison to the initial stage was quadrupled. The correlation coefficient between “Extract-Phase 1” and “Final Extract” for total phenolic compounds was approximately −0.737, indicating a strong negative correlation and an inverse relationship. On the other hand, the correlation coefficient for “Extract-Phase 2” was approximately 0.986, indicating a robust positive correlation compared to “Final Extract”.

The final extract presented various phenolic compounds identified using HPLC. Nine phenolic compounds were identified, mainly specific flavonoid compounds of pomegranates (

Table 1. The identification of major phenolic compounds in the dried form of the final extract.

Compound	Retention Time (Minute)	Concentration (µg/100 g Dry Extract)
Galic acid	3.63	37.41 ± 2.74
Catechin	7.99	373.29 ± 27.65
Epicatechin	9.77	33.39 ± 0.08
Hesperidin	15.00	5.22 ± 1.12
Resveratrol	17.37	2.14 ± 0.36
Rutin	12.73	7.74 ± 3.10
Isoquercetin	13.50	3.74 ± 0.32
Myricetin	15.98	2.38 ± 0.47
Quercetin	18.49	1.32 ± 0.53

). Gallic acid was the predominant phenolic acid, and catechin stood out with a high quantity exceeding the rest of the identifiable compounds. The antioxidant activity of the final formula was likely dependent on the combination of several phenolic compounds. Although catechins contribute significantly, potential interactions between these compounds can influence the antioxidant effect. Additionally, reducing oxidative stress due to UV exposure was a direct response to the bioavailability of each component.

The linearity of calibration curves was calculated by linear regression analysis of the area versus concentration of six-point concentration levels of each standard compound. All the standards were injected in triplicate and each point on the calibration curves was the average of all three peak areas. The relationship parameters between concentrations and peak area are shown by regression equation and correlation coefficient (Table S1 in the Supplementary Material). All the analytes exhibited good linearity (r) over the range tested, with correlation coefficients ranging from 0.9994 to 0.9998. The LOD and LOQ were estimated from the calibration curve and values subsequently ranged from 0.12 to 0.86 µg/mL and 0.38–2.82 µg/mL, respectively. The LOD and LOQ were determined at three and 10 times the baseline noise, respectively.

For DPPH, the ANOVA shows a highly significant effect of Phase (p = 6.42 × 10⁻⁹) with an F value of 370.2, indicating substantial differences between phases. Extract-Phase 2 has the highest DDPH values, followed by Final extract, Ascorbic acid 1%, and Extract-Phase 1, each forming distinct groups (Figure 2).

For ABTS, the ANOVA results show a highly significant effect of the Phase factor on the dependent variable, with an F value of 370.2 and a p-value of 6.42 × 10⁻⁹, proving substantial differences between phases. The ABTS analysis shows that Ascorbic acid 1% and Extract-Phase 2 form a homogeneous group with the highest values, followed by Final extract and then Extract-Phase 1, each with significantly different mean values (Figure 2).

Figure 2 shows overall similar activity, except for the phase 2 extract, which displayed specificity towards the type of radical used. The DPPH radical exhibited twice as much activity as the ABTS radical. The inhibitory activity values of the phase 2 extract remained consistent and were also observed in the final form of the functional extract. The correlation coefficient between extract-phase one and extract-phase 2 was 0.5, indicating a moderately positive correlation. This value was obtained when compared to the final extract. The ANOVA results indicate that the Phase factor has a highly significant effect on the dependent variable, with an F value of 683.3 and a p-value of 3.85 × 10⁻¹², suggesting substantial differences between phases (Figure 3). The critical point analysis shows that Control has the highest value, followed by F1, F, Vitamin B3, and F2, with each group being significantly different. To demonstrate the protective capacity against UV radiation stress, the critical point for each component was determined individually (extract) compared to the two controls. This was expressed in minutes after exposure to a laboratory UV lamp. Figure 3 was irrelevant in this process, except for fraction 2 after alcoholic extraction. Its effect was similar to that of vitamin B3. The critical exposure time determining oxidative stress’s onset is approximately 30 min (Supplementary Figure S1). All of these data were directly influenced by gastrointestinal transit, demonstrating the bioavailability of the bioactive components and the effectiveness of the final formula. The results showed a relative bioavailability of 30% at the gastric level, as demonstrated by two alternative in vitro methods. After gastric passage in the small intestine, absorption was complete, indicating that the bioactive components have a high capacity to reach the skin level. What was important was the relative bioavailability at the intestinal level, which ranged from 70–90%. This indicated the amount of bioactive components (phenolic compounds) that are present in circulation after administration. Although biotransformations may occur during digestion, they only suggest that the effect is determined by components (molecules with smaller molecular masses) that influence protection against free radical formation [26].

The extracts examined in this research are mainly intended for topical treatment; nevertheless, their bioavailability, shown by gastrointestinal transit data, also implies potential efficacy for systemic applications. Future research should provide more explanation about their appropriateness for both topical and oral administration.

Phenols and flavonoids, natural compounds in different plants, have received considerable interest due to their potential to safeguard the skin against the harmful effects of solar radiation. Solar radiation, including ultraviolet (UV) rays, can lead to skin damage, premature aging, and an increased risk of skin cancer. The protective effects of phenols and flavonoids are attributed to their potent antioxidant and anti-inflammatory properties, which are crucial in counteracting the oxidative stress and inflammation caused by UV exposure [27].

Phenols and flavonoids are increasingly being added to sunscreen products. Unlike chemical UV filters, which can sometimes cause irritation or adverse reactions, phenolic and flavonoid compounds are generally well-tolerated and offer a more comprehensive range of protection. They directly absorb UV light and enhance the skin’s antioxidant defenses [10]. Research may also show some negative points, such as natural extracts should not be seen as a complete replacement for traditional UV protection methods (sunscreens) [10]. In addition, the two-phase extraction method, although effective, may require further optimizations to maximize the yield and efficiency of bioactive compounds, and the efficiency of the extracts may vary depending on the biomass source [28].

4. Conclusions

This study indicated that this mixture combines two powerful, natural antioxidant sources innovatively for skincare applications. Total phenols and flavonoids offer a robust, multimodal remedy for UV protection due to their ability to mitigate the adverse effects of sun exposure. These substances provide a natural defense by efficiently absorbing harmful UV radiation and reducing oxidative stress and inflammation, complementing conventional sun protection strategies. Their potential should be viewed not only as an alternative but also as an essential supplement that strengthens the skin’s resilience against the harmful effects of the sun. With the growing demand for natural and holistic methods in dermatology, a broader investigation of these bioactive substances is imperative. Harnessing their full potential could transform sun protection methods, offering safer and more sustainable options for combating UV-induced skin damage.