Abstract
In this study, the potential of lemon peel extracts as cosmetic raw materials was explored, with focus on their composition, aroma, antimicrobial, and other bioactivities. Lemon peel essential oil (LPEO), extract (LPE) and absolute oil (LPAO) were prepared by hydrodistillation and organic solvent extraction, respectively. GC/MS analysis indicated that LPEO, LPE, and LPAO contained 22, 39, and 9 components, respectively, with terpenoids being the predominant component. LPE had the highest total flavonoid content and exceeded the total phenolic content. LPEO demonstrated the strongest aroma intensity and persistence, as measured by electronic nose. All three lemon peel extracts showed good antioxidant, anti-tyrosinase, and antimicrobial properties, while inhibition rates exceeded 90% in the experimental concentration range in a dose-dependent manner. Although the antioxidant and antibacterial effects of LPAO were stronger than those of LPEO, the latter had better anti-tyrosinase action. LPEO also demonstrated superior anti-inflammatory effects, with inhibitory rates of 87.79 ± 3.86% and 80.75 ± 2.33% on TNF-α and IL-6 at 1 × 10-2 mg/ml. Moreover, LPEO promoted HaCaT cell migration better than LPE and LPAO, and the healing rate of scratched HaCaT cells treated with LPEO at 1 × 10-2 mg/ml for 12 h was 95.29 ± 3.41%. In addition to its antioxidant and antibacterial properties, the overall performance of LPEO was superior by comparison with LPE and LPAO. In summary, the three extracts can be combined to expand their application as skincare and cosmetic additives with aroma-improving, antioxidant, whitening, antibacterial, anti-inflammatory, and wound-healing activities.
Keywords: Lemon peel extracts, ingredient, aroma, antimicrobial, bioactivity, cosmetic raw material
Introduction
Current trends in cosmetic formulations are developing towards a more natural and chemical-free approach [1, 2]. Along with active ingredients, cosmetic products often include artificial flavors to enhance fragrance, and preservatives to extend the shelf life. However, synthetic fragrances generally lack biological activity, and some can also cause skin allergies. Meanwhile, preservatives such as methylchloroisothiazolinone/methylisothiazlinone and formaldehyde-releasing agents can result in contact dermatitis [3]. The overuse of these harmful substances can even lead to the emergence of drug-resistant strains, posing a serious risk to consumers [4].
The essential oil extracted from natural plants has a series of biological activities, including aroma, antioxidant, anti-aging, antibacterial, anti-acne and anti-inflammatory properties [5]. Lemon, as the third largest variety of citrus in Rutaceae, is famous for its dual application in both medicine and food. Lemon peel accounts for about 20% of the whole lemon, which is rich in terpenes, alkaloids, phenolic acids and other active compounds, and has a pleasant aroma and unique biological activities [6]. Scientific research has verified the benefits of lemon essential oil in promoting microvascular circulation, promoting subcutaneous fat combustion, stimulating collagen synthesis, restoring the skin’s oil-water balance, and inhibiting melanin production [7]. Furthermore, lemon essential oil could modulate cell permeability and demonstrate potent inhibitory effects against Aeromonas aeruginosa. The terpenoids in lemon peel extract had strong antioxidant activity and had obvious scavenging effect on DPPH and ABTS free radicals [8]. The (Z)-citral found in lemon extract demonstrated a potent inhibitory effect on tyrosinase, thereby promoting skin whitening [9]. Furthermore, (Z)-citral has been shown to alleviate LPS-induced cell damage by inhibiting inflammation, oxidative stress, and epithelial-mesenchymal transition in THLE-2 cells. It might also influence the brain via the olfactory system, potentially regulating anxiety and depression [10]. Lemon peel is rich in antifungal compounds such as limonene, β-pinene, and γ-terpene, which could disrupt the balance of organelles and inhibit the metabolism of C. albicans [11]. Neryl acetate and limonene in lemon essential oil are the key components of advanced aromatic agents. The flavonoids and polyphenols contained in lemon peel extract had effectively scavenged free radicals, inhibited tyrosinase activity, and reduced the production of NO by macrophage RAW264.7 cells [12], while also exhibiting strong antibacterial effects against Staphylococcus aureus and Escherichia coli [13].
The current research on lemon peel mainly focuses on the extraction and application of lemon essential oil, highlighting its antioxidant and antibacterial properties, and its application in the field of food industry. However, with the growing preference for natural ingredients in the cosmetics industry, there is a high demand for plant extracts with natural aroma, antibacterial properties, and biological activities. In this paper, the lemon peel extracts obtained by three different extraction methods were compared from the aspects of composition, aroma, antioxidant, anti-inflammatory, antibacterial, whitening potential, cytotoxicity, and cell repair ability. The aim is to develop a lemon peel extract with enhanced flavor and antibacterial and antiseptic properties, as well as a variety of biological activities, so as to provide the basis for its application as a cosmetic raw material.
Materials and Methods
Reagents
Rutin, gallic acid, arbutin, ascorbic acid, folin phenol, polyphenol oxidase, L-tyrosine, aluminum chloride hexahydrate, potassium persulfate, sodium nitrite, phosphate - buffered saline (PBS, pH6.8), 2,2’-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazide (DPPH), as well as solvents (ethanol, dimethylsulfoxide (DMSO), petroleum ether, propylene glycol, sodium chloride) were analytical grade and purchased from Sinopharm (China). HaCaT cell lines (No.KMCC-001-0256), Luria-Bertani medium (LB medium), recombinant broth, fetal bovine serum (FBS), cell counting kit-8 (CCK-8), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) ELISA kits were purchased from Beina Biological Co. (China). E. coli (No. CMCC(B)44102), S. aureus (No. CMCC(B)26003) and Candida albicans (C. albicans, No. CMCC(B)98001) were gifted by Zhang Jie (Zhejiang Wanli University). Lemon was purchased in Anyue, Sichuan, China and identified as Citrus × Limon 'Eureka' by Professor Liu Liping.
Instruments and Equipment
Electronic balance (BSA124S, Sartorius, Germany); rotary evaporator (RV10, Ika, Country); gas chromatography-mass spectrometry (8890-5977B, Agilent, Country); electronic nose (PEN3.5, Airsense, Country); spectrophotometer (TU-1810, Beijing general instruments, Country); high-speed centrifuge (5417R, Sigma); microplate reader (FC, Thermo Fisher Scientific, Country); incubator (THZ-D, Ningbo Southeast Instruments); carbon dioxide incubator (4111FO, Thermo Fisher Scientific).
Preparation of Lemon Peel Extracts
The lemon peel was first washed and then dried at 40°C for 2~4 h before being pulverized and passed through a 10-mesh sieve for later use.
Lemon peel essential oil (LPEO) was extracted using the steam distillation method. Lemon peel and distilled water (with 2% w/v NaCl) at a solid-liquid ratio of 1:10 were placed in a distillation flask for a micro-boiling reflux extraction process, continuing until the distillate was free of oil droplets. The resulting oily liquid was separated using an oil-water separator, dried with anhydrous Na2SO4, and stored in a brown bottle.
Lemon peel extract (LPE) was prepared using the ethanol extraction method. A total of 100 g of lemon peel was extracted twice with 500 ml of 90% v/v ethanol containing 1% v/v propylene glycol at 40°C for durations of 4 h and 2 h, respectively. The supernatants were combined, filtered, and evaporated to obtain the extract, which was then sealed in a brown bottle.
Lemon peel absolute oil (LPAO) was obtained using the petroleum ether extraction method. A total of 100 g of lemon peel was extracted to two reflux extractions with 500 ml of petroleum ether at 40°C for durations of 4 h and 2 h, respectively. The supernatants from both extractions were combined, filtered, and evaporated into a paste. Anhydrous ethanol was added to the paste at a volume ratio of 10:1, stirred well, and allowed to stand at -20°C for 4 h. The resulting supernatant was filtered, and evaporated, leading to the acquisition of the absolute oil, which was stored in a brown bottle.
Gas Chromatography-Mass Spectrometry (GC/MS) Detection of Components
All three samples were diluted with n-hexane.
GC conditions: The HP-5MS capillary column used in this study was 30 m × 0.25 mm × 0.25 μm. Helium served as the carrier gas, with a flow rate of 1 ml/min. The injection was performed with a split ratio of 200:1 and an injection sample volume of 1 μl at 220°C. The initial temperature was set at 50°C and maintained for 3 min, then increased by a rate of 2°C/min up to 140°C for 12 min, 10°C/min up to 250°C for 5 min. The total operating time for this procedure was 84 min.
MS conditions: An electron ionization source (EI) was utilized with an electron energy of 70 eV. The temperature of the ion source, the quadrupole, the transmission line was 230°C, 150°C, and 270°C, respectively. The full scanning range covered a mass-to-charge ratio (m/z) of 35~550. The identification of each volatile substance was performed using the National Institute of Standards and Technology (NIST) MS spectrometry library, while the relative content was calculated employing the area normalization method.
Determination of Total Flavonoid and Phenolic Content
Total flavonoid content was determined in the samples by spectrophotometry using rutin as a control, with reference to source [15]. Five ml of rutin standard solution was prepared with concentrations ranging from 0 to 100 μg/ml using dimethyl sulfoxide (DMSO) as the solvent. To this solution, 0.3 ml of 5% NaNO2, 0.3 ml of 10% w/v AlCl3·6H2O and 4 ml of 4% w/v NaOH were added, and the mixture was allowed to react at room temperature for 15 min. The absorbance at 510 nm (A510nm) was measured with deionized water as a blank. The standard curve for rutin was described by the equation A = 0.0067C + 0.0098 (r = 0.9978). Three lemon peel extracts were diluted with DMSO and analyzed using the same method.
Total phenolic content was determined using the Folin-Ciocalteu colorimetric method [16]. Standard solutions of gallic acid, ranging from 0 to 120 μg/ml, were prepared in DMSO. Then, 4 ml of distilled water, 0.5 ml of Folin reagent, and 4 ml of 8% w/v Na2CO3 solution were added to each standard solution. The reaction was conducted at room temperature in the dark for 30 min. The absorbance at 740 nm (A740nm) was measured with deionized water as a blank. The standard curve for gallic acid was established as A = 0.0104C + 0.0021 (r = 0.9998). Subsequently, the sample solutions of lemon peel extracts were prepared in DMSO and 1 ml of the dilution solution was used to determine the total phenolic content following the same procedure.
E-Nose Detection of Odor
Odor was detected in headspace aspiration with PEN3 e-nose [17]. The experimental parameters were set as follows: a collection time of 300 s, with a data collection cycle of 1 s, and a collection delay of 120 s. The injection volume was 2.0 ml with the flow rate of 150 ml/min. Each sample underwent an average incubation time of 2 min at a temperature of 35°C, with a stirring speed of 500 r/min. The sensor was programmed for a self-cleaning time of 150 s, followed by a zeroing period of 5 s. Data analysis was conducted using WinMuster software in conjunction with the PEN 3 e-nose.
Determination of Free Radical Scavenging Rate
The antioxidant activity of lemon peel extracts was assessed using the DPPH and ABTS methods as described by Patricia [18]. Half-scavenging concentration (IC50) refers to the concentration of lemon peel extract required to eliminate 50% of DPPH and ABTS free radicals.
Determination of Anti-Tyrosinase Activity
The experiment involved four groups: Group A, which contained 1.5 ml of 50 U/ml tyrosinase, Group B, which did not contain it, Group C, which contained 1.5 ml of 50 U/ml tyrosinase and 1 ml of sample solution, and Group D, which contained only 1 ml of sample solution. All groups were supplemented with PBS buffer to a total volume of 5 ml. The samples were then incubated for 10 min at 37°C, and immediately 2.5 mmol/l tyrosine solution was added to initiate the reaction in the dark for 30 min at 37°C. The absorbance at 475 nm was determined for each set of samples with deionized water as a blank. Arbutin was used as a positive control. The inhibition rate of tyrosinase was calculated as follows [19]:
Inhibition rate/%=[(AA-AB)-(AC-AD)]×100/(AA-AB).
Determination of Antibacterial Activity
Preparation of bacterial suspension [20]: E. coli and S. aureus were activated and cultured at 37°C and C. albicans at 28°C to logarithmic growth phase. The bacteria were centrifuged and washed twice with 0.9% w/v normal saline, resuspended in LB medium, and the bacterial concentration was adjusted to 1 × 108 ~5×108 CFU/ml.
Preparation of sample solution: The sample was first dissolved in sterile water containing 8% DMSO, and then diluted with LB medium to concentrations of 0.5, 2.5, 5, 12.5, and 25 mg/ml, respectively, and filtered through a 0.22 μm filter for later use.
Bacteria grouping: Experiments were performed in sterile 96-well plates. The control group was 30 μl bacterial suspension and 170 μl LB medium; the blank group was 200 μl LB medium; the sample group was 30 μl bacterial suspension and 170 μl sample solutions; the background group was 30 μl LB medium and 170 μl sample solutions. After incubating for 24 h at 37°C, the absorbance at 600 nm was measured using a microplate reader. The inhibition rate was the following equation:
Inhibition rate/%=[(Acontrol-Ablank)-(Asample-Abackground)]×100/(Acontrol-Ablank).
Determination of Cytotoxicity and Anti-Inflammatory Properties
The cytotoxicity on HaCaT cells was determined using the CCK-8 method. Logarithmically grown HaCaT cells (5×104 cells/ml) were seeded at 100 μl/well in 96-well plates and incubated in a CO2 incubator overnight at 37°C. The three lemon peel extracts were dissolved in 0.05% DMSO and subsequently diluted to six concentrations (1×10-4 ~ 10 mg/ml) with DMEM medium containing 10% FBS. Adherent cells and 100 μl of medium were added to the control group; only 100 μl of medium was added to the blank group; the sample group included adherent cells and 100 μl of the sample solution; and the background group was supplemented with 100 μl of medium. After a 24 h cell culture period, 10 μl of CCK-8 reagent was added to each well, followed by an additional 4 h incubation. The absorbance of each well was measured at 450 nm using a microplate reader, with six replicates set for each group. The cell survival rate was calculated according to the following formula:
Cell survival rate/%=(Asample-Abackground)×100/(Acontrol-Ablank).
The content of inflammatory factors in cells was measured using an ELISA assay kit. Then, 100 μl of 5 × 104 cells/ml logarithmic growth phase HaCaT cells were seeded into a 96-well plate and cultured in an incubator overnight. After discarding the original culture medium, 100 μl of culture medium containing 1 ng/ml TNF-α and 1 ng/ml IFN-γ was added to the model group and sample group to stimulate the cells for 6 h to induce inflammation. The induction medium was then discarded and the cells were washed three times with PBS. Next, 100 μl of culture medium was added to the model group, and 100 μl of culture medium containing samples was added to the sample group. After cultivation in the incubator at 37°C for 24 h, the contents of TNF-α and IL-6 in the supernatant were determined according to the instructions of the ELISA kit, with six replicates set for each group. Meanwhile, the control group was established. The inhibition rate was on the following equation:
Inhibition rate/%=(Amodel-Asample)×100/Amodel.
Repair Experiment on Scratched HaCaT Cells
The samples were prepared using DMEM medium without FBS. Then, 2 ml/well of 3 × 105 cells/ml of HaCaT cells in the logarithmic growth phase were added into a 6-well plate. When the cells were cultured to almost cover the bottom of the well, the cells were scratched by the sterile pipette head and washed multiple times with PBS solution to remove the suspended cells. Following that, 2 ml of DMEM medium containing samples was added into the cells of test groups. Then, 2 ml of DMEM medium without FBS was added into the control group. After the scratch cells were further cultured for 0, 6, 12, and 24 h, the cultures were observed and photos were taken. The scratch area was calculated by ImageJ software. Healing rate/% = (A0 - Ai) × 100/A0, where A0 was the scratch area at 0 h and Ai was the scratch area at 6, 12 or 24 h.
Statistical Analysis
The data were expressed as x ± sd. GraphPad Prism was used to make all the graphs. One-way analysis of variance (ANOVA) was used to determine the statistical differences among experimental groups, the value of p < 0.05 indicated a significant difference.
Results and Discussion
Comparison of Sensory Properties of Lemon Peel Extracts
Due to different extraction methods, the three extracts had differences in color, state, and aroma, as shown in Table 1.
Table 1.
Sensory comparison of three types of lemon peel extracts.
| Index | LPEO | LPE | LPAO |
|---|---|---|---|
| Color | Colorless transparent | Yellow-brown | Yellow-bright |
| State | Liquid | Semi-paste | Liquid |
| Fragrance | Pure and fragrant aroma | Aroma plump | Aroma clear and lasting |
| Extraction rate/% | 4.67 ± 0.28 | 31.54 ± 1.85 | 20.42 ± 1.03 |
GC/MS Results of Lemon Peel Extracts
According to the total ion chromatograms (Fig. 1) of the three extracts, the detected peaks were shown in Tables 2 and 3.
Fig. 1. Total ion chromatograms of three lemon peel extracts.
Table 2.
Chemical composition and relative contents of the three lemon peel extracts.
| No. | Compound | Molecular formula | CAS | Relative percentage content/% | ||
|---|---|---|---|---|---|---|
| LPEO | LPE | LPAO | ||||
| 1 | m-Cymene | C10H14 | 535-77-3 | 2.93 | 0.14 | - |
| 2 | β-Phellandrene | C10H16 | 555-10-2 | 0.67 | 0.16 | - |
| 3 | β-Pinene | C10H16 | 18172-67-3 | 14.28 | 1.16 | 5.13 |
| 4 | Myrcene | C10H16 | 123-35-3 | 2.31 | 0.43 | 2.47 |
| 5 | (+)-Camphene | C10H16 | 79-92-5 | 0.1 | - | - |
| 6 | D-Limonene | C10H16 | 5989-27-5 | 58.05 | 22.95 | 63.79 |
| 7 | (R)-Isocarvestrene | C10H16 | 1461-27-4 | 2.20 | 1.87 | - |
| 8 | γ-Terpinene | C10H16 | 99-85-4 | 11.98 | 5.74 | 18.82 |
| 9 | Terpinolene | C10H16 | 586-62-9 | 0.87 | - | 2.8 |
| 10 | Carene | C10H16 | 13466-78-9 | 2.91 | 0.29 | - |
| 11 | Tetradecane | C14H30 | 629-59-4 | - | 0.19 | - |
| 12 | trans-Caryophyllene | C15H24 | 87-44-5 | 0.14 | 7.76 | - |
| 13 | Santalene | C15H24 | 512-61-8 | - | 0.23 | - |
| 14 | α-Limonene | C15H24 | 17699-05-7 | 0.12 | 11.82 | - |
| 15 | 1, 5, 9, 9-tetramethyl-1, 4, 7-cyclodecatriene | C15H24 | 1000062-61-9 | - | 0.57 | - |
| 16 | β-Sandalene | C15H24 | 25532-78-9 | - | 0.48 | - |
| 17 | β-Fucoidin | C15H24 | 28973-97-9 | - | 1.15 | - |
| 18 | α-Terpinene | C15H24 | 451-55-8 | - | 0.4 | - |
| 19 | β-Sesquiphellandrene | C15H24 | 20307-83-9 | - | 0.62 | - |
| 20 | Valencia Orangerene | C15H24 | 4630-07-3 | - | 1.42 | - |
| 21 | Bicyclogermacrene | C15H24 | 24703-35-3 | - | 2.66 | - |
| 22 | α-Himachalene | C15H24 | 3853-83-6 | - | 1.62 | - |
| 23 | β-Bisabolene | C15H24 | 495-61-4 | 0.08 | 19.39 | - |
| 24 | β-Curcumene | C15H24 | 28976-67-2 | - | 0.15 | - |
| 25 | (Z)-Gamma-bisabolene | C15H24 | 13062-00-5 | - | 0.18 | - |
| 26 | (+)-δ-Cadinene | C15H24 | 483-76-1 | - | 0.28 | - |
| 27 | (E)-α-Bisabolene | C15H24 | 25532-79-0 | - | 0.3 | - |
| 28 | Phenyl isothiocyanate | C7H5NS | 103-72-0 | - | 0.22 | - |
| 29 | Nonanal | C9H18O | 124-19-6 | 0.07 | 0.23 | - |
| 30 | (Z)-Citral | C10H16O | 106-26-3 | 0.58 | 1.04 | 2.49 |
| 31 | Citral | C10H16O | 5392-40-5 | 0.05 | 1.9 | - |
| 32 | (E)-Citral | C10H16O | 141-27-5 | 0.62 | - | 2.97 |
| 33 | Linalool | C10H18O | 78-70-6 | 0.33 | 0.76 | 0.64 |
| 34 | (-)-Terpinen-4-ol | C10H18O | 20126-76-5 | 0.65 | 0.37 | - |
| 35 | α-Terpineol | C10H18O | 10482-56-1 | 0.72 | 2.05 | 0.89 |
| 36 | Undecanal | C11H22O | 112-44-7 | - | 0.17 | - |
| 37 | 4-Terpinyl acetate | C12H20O2 | 4821/4/9 | - | 0.18 | - |
| 38 | dl-Citronellol acetate | C12H22O2 | 150-84-5 | - | 0.29 | - |
| 39 | Nerylacetate | C12H20O2 | 141-12-8 | 0.25 | 6.76 | - |
| 40 | Acetic acid geranyl ester | C12H20O2 | 105-87-3 | 0.09 | 3.67 | - |
| 41 | 1,1-Diethoxynonane | C13H28O2 | 54815-13-3 | - | 0.27 | - |
| 42 | 3,5,5-Trimethylnonyl Hexanoic acid | C18H36O2 | 1000406-06-0 | - | 0.13 | - |
| Total detected components/each | 22 | 39 | 9 | |||
“-” indicates that the content was less than 0.01 or not detected.
Table 3.
Components and proportion of the three lemon peel extracts.
| Items | LPEO | LPE | LPAO |
|---|---|---|---|
| Terpenes/% | 96.64 | 81.96 | 93.01 |
| Alcohols/% | 1.70 | 3.18 | 1.53 |
| Aldehydes/% | 1.32 | 3.34 | 5.46 |
| Esters/% | 0.34 | 11.12 | - |
| Other compounds/% | 0.00 | 0.42 | - |
| Total | 100.00 | 100.00 | 100.00 |
Chromatographic analysis revealed 22, 39, and 9 distinct peaks in LPEO, LPE and LPAO, respectively. The main components were terpenes, which constituted 81.96% to 96.64% of the extracts, as well as oxygen-containing compounds such as alcohols, aldehydes, and esters. These components are the principal contributors to the aroma of lemon peel extracts. Among them, β-pinene, myrcene, D-limonene, γ-terpinene, (Z)-citral, linalool and α-terpineol were identified in all three extracts. The content of D-limonene was the highest in the three extracts, and it has typical orange aroma characteristics [21], antibacterial properties, certain anti-cancer properties [22], as well as effects such as relieving nerve tension and pain, and detoxifying, beautifying and enhancing memory [23]. Terpenoids are the most important aromatic components, with β-terpenes serving as precursors for various terpene synthetic fragrances and possessing anti-inflammatory and anti-allergic effects. γ-Terpenes had strong antibacterial and antioxidant capabilities, while (Z)-citral was noted for its anti-inflammatory, antioxidant and apoptosis inducing properties. Additionally, α-terpineol exhibited considerable antioxidant activity [24].
Total Flavonoid and Phenolic Content in Lemon Peel Extracts
The total flavonoid contents in the three lemon peel extracts were more than that of total polyphenols, with LPE having the highest content, as shown in Table 4.
Table 4.
Content of total flavonoids and phenolic in three lemon peel extracts (n = 3).
| No. | Total flavonoid/μg/ml | Total phenolic/μg/ml |
|---|---|---|
| LPEO | 42.57 ± 0.05 | 32.06 ± 0.08 |
| LPE | 77.94 ± 0.03 | 61.24 ± 0.04 |
| LPAO | 62.72 ± 0.06 | 48.02 ± 0.07 |
Flavonoids and polyphenols possess potent antioxidant properties that effectively eliminate free radicals in the body. These compounds can reduce skin pigmentation caused by reactive free radicals and fatty acids, prevent cellular aging, inhibit the exudation of inflammatory factors [25] and inhibit microbial growth [26].
E-Nose Results of the Odor of Lemon Peel Extracts
The PEN3 e-nose consists of 10 kinds of sensors. As shown in Fig. 2, the response values were mainly concentrated on the probes of S2 (sensitive to nitrogen oxides), S7 (sensitive to terpenoids and sulfur-containing compounds), and S9 (sensitive to aromatic compounds and organic sulfides). The maximum odor response peaks of three lemon peel extracts and the corresponding changes in response values after reaching the peaks of 60, 100, and 140 s were shown in Table 5.
Fig. 2. Response values of the odors of three lemon peel extracts.
Table 5.
The peak odor and retention values of three lemon peel extracts.
| NO. | 0 s | 60 s | 100 s | 140 s | |||
|---|---|---|---|---|---|---|---|
| Peak value/G0 | Peak value/G | Aroma retention value/% | Peak value/G | Aroma retention value/% | Peak value/G | Aroma retention value/% | |
| LPEO | 43.4134 | 42.3630 | 97.58 | 41.7211 | 96.10 | 40.9111 | 94.24 |
| LPE | 20.3461 | 18.4422 | 90.64 | 17.0511 | 80.81 | 15.5736 | 76.54 |
| LPAO | 16.6896 | 16.3119 | 97.74 | 15.9618 | 95.64 | 15.3355 | 91.89 |
According to the maximum response peak, the intensity of the odor was: LPEO>LPE>LPAO. According to the retention value in peak values within the same time period, the persistence of odor was: LPEO and LPAO > LPE. In addition to terpenoids and aromatic components, LPE also contains some impurities such as plant wax, which affects the odor response values.
Results of Lemon Peel Extracts in Scavenging Free Radicals
The scavenging rate of three lemon peel extracts against DPPH and ABTS radicals increased with concentration, as shown in Fig. 3.
Fig. 3. Scavenging rate of DPPH (A) and ABTS (B) by three lemon peel extracts (n = 3).
The IC50 values of LPEO, LPE, and LPAO were 237.30, 1.53 and 3.15 mg/ml, respectively, and the clearance rate of 4 mg/ml LPE to DPPH was 86.64 ± 1.96%. The IC50 values of LPEO, LPE, and LPAO were 20.76, 0.49 and 0.31 mg/ml, respectively, and the clearance rate of 0.7 mg/ml LPAO to ABTS was 91.80 ± 2.46%. Research found that D-limonene had a good antioxidant effect. The contents of (Z)-citral, α-terpinene and α-terpineol were relatively high in LPE, which had strong scavenging effects on DPPH. LPAO contained a higher amount of γ-terpinene and (Z)-citral, which had strong scavenging effects on ABTS [28].
Anti-Tyrosinase Activity of Lemon Peel Extracts
The inhibitory effect of three lemon peel extracts on tyrosinase increased with concentration, as shown in Fig. 4. The inhibition of tyrosinase from the three extracts was LPEO ( IC50 3.16 mg/ml) > LPE (IC50 16.02 mg/ml) > LPAO (IC50 21.73 mg/ml), and the IC50 values of the three extracts were lower than those of the positive control arbutin (26.62 mg/ml). This indicated that their anti-tyrosinase activities were superior to arbutin. The inhibition rate of tyrosinase by LPEO at a concentration of 20 mg/ml was 93.82 ± 1.06%. Studies have shown that the bioactive substances such as the flavonoids and polyphenols rich in lemon peel had a similar structure to the substrate of tyrosinase, and acted as substrate analogues to bind with tyrosinase, thereby inhibiting the production of melanin [29]. Meanwhile, the compounds such as (Z)-citral, neryl acetate, and β-pinene had a significant impact on the activity of tyrosinase [30].
Fig. 4. Inhibition effect of lemon peel extract on tyrosinase (n = 3).
**p ≤ 0.01, ***p ≤ 0.001,****p ≤ 0.0001
Antibacterial Activity of Lemon Peel Extracts
The antibacterial ability of three lemon peel extracts increased with increasing concentration, as shown in Fig. 5. The inhibitory effect of three lemon peel extracts on E. coli was more sensitive than that on S. aureus and C. albicans. The minimum inhibitory concentration (MIC) of the three extracts against E. coli was as follows: LPAO (4.38 mg/ml) > LPEO (5.79 mg/ml) > LPE (11.89 mg/ml). When the concentrations of LPEO, LPE, and LPAO were 12.53, 23.33, and 11.78 mg/ml, respectively, the antibacterial rates of the three extracts against the three microorganisms exceeded 90%. The antibacterial activity of essential oils is closely related to their chemical composition. Studies have shown that the content of D-limonene in pomelo peel essential oil was positively correlated with its antibacterial activity (r = 0.811~0.923) [31]. Turpentine could damage the biofilms of S. aureus and E .coli, leading to increased membrane permeability and inhibited microbial growth. Neryl acetate could damage the ultrastructure of fungi, disrupting their membrane regulatory function and deformability. The content of these compounds in lemon peel extracts showed a change of “LPAO > LPEO > LPE” in the GC/MS analysis.
Fig. 5. Antibacterial activity of three lemon peel extracts against E. coli (A) S. aureus (B) and C. albicans (C) (n = 6).
*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
Cytotoxicity and Anti-inflammatory Effect of Lemon Peel Extracts
The toxicity result of lemon peel extracts on HaCaT cells was shown in Fig. 6.
Fig. 6. Comparison of toxicity test results of three lemon peel extracts on HaCaT cells (n = 6).
Comparing the sample with the control, a represents p ≤ 0.05, and b represents p ≤ 0.0001.
According to Fig. 6, it can be seen that when the concentrations of LPEO, LPE, and LPAO were below 0.01 mg/ml, 1 mg/ml, and 0.001 mg/ml, respectively, the activity of HaCaT cells was greater than 90%. That was to say, the safety sequence of the three extracts for HaCaT cells was: LPE > LPEO > LPAO.
Subsequent experiments will be conducted within the safe concentration range mentioned above. The results of anti-inflammation (TNF-α, IL-6) were shown in Fig. 7.
Fig. 7. Effects of three lemon peel extracts on contents of intracellular TNF-α (A) and IL-6 (B) (n = 6).
Letters represent the comparison between model group and control group, between sample group and model group. The asterisks represent the comparison between different sample groups of the same concentration. a and * p ≤ 0.05, b and ** p ≤ 0.01, c and *** p ≤ 0.001, **** p ≤ 0.001
There was a significant difference between the model group and the blank group, indicating successful induction of cellular inflammation. Compared with the model group, all three lemon peel extracts could effectively inhibit the production of TNF-α and IL-6, and the inhibitory rate increased with dose, but the inflammatory level was not reduced to the cellular state of the blank group. LPEO had the strongest anti-inflammatory effect among the three extracts. The inhibition rates of 1 × 10-2 mg/ml LPEO for TNF-α and IL-6 were 87.79 ± 3.86% and 80.75 ± 2.33%, respectively.
The anti-inflammatory properties of essential oils are related to their cascade reactions to cytokines and regulatory transcription factors during signal transduction [32]. Research has confirmed that components such as D-limonene, carene, and undecanal had anti-inflammatory effects. For example, D-limonene effectively reduced the over expression of inflammatory factors induced by doxorubicin by restoring antioxidant enzyme levels and alleviating oxidative stress [33]. D-lemonene, δ-3-carene and β-pinene in the essential oil of Chinese torreya effectively inhibit the generation of pro-inflammatory factors and achieve anti-inflammatory effects [34]. Thymol in thyme essential oil had significant inhibitory effects on aging-induced brain inflammation and blood telomere wear in mice [35]. The experimental results were consistent with the GC/MS detection data trends of these substances in three lemon peel extracts.
Repair Results on Scratched HaCat Cells
The cell scratch test is commonly used to simulate wound healing in vitro [36], and the results were shown in Figs. 8 and 9.
Fig. 8. Imaging of repair of scratched HaCaT cells using LPEO, LPE, and LPAO.
Fig. 9. Effects of LPEO, LPE, and LPAO on the healing rate of scratched HaCaT cells.
Compared with control group, 6 h: *p <0.05,** p <0.01, *** p <0.001; 12 h: #p <0.05, ## p <0.01, ### p <0.001; 24 h: +p <0.05, ++ p <0.01, +++p <0.001.
The HaCaT cell healing rate was significantly higher than that of the control group after 24 h of treatment with lemon peel extract. With the increase of lemon peel extract concentration and the extension of action time, the overall cell healing rate showed an upward trend. Among them, LPEO had a better healing effect on cells than LPE and LPAO. After treating scratched cells with 0.01 mg/ml LPEO for 12 h, the cell healing rate was 95.29 ± 3.41%. Wound repair involves processes such as coagulation, inflammation, cell proliferation, and tissue remodeling. The migration of fibroblasts plays a crucial role in wound healing. Lavender essential oil could promote the transfer of growth factors-β and fibroblast growth factors to accelerate wound healing [37]. Eucalyptus globulus Labill essential oil also had a good wound-healing effect on rat wound models [38].
Conclusion
There were differences in the composition of three lemon peel extracts, namely LPE (39 kinds) > LPEO (22 kinds) > LPAO (9 kinds), with the main components being terpenes. The content of total flavonoids in these extracts was higher than that of total polyphenols, and the content of flavonoids in LPE was higher than that in LPAO and LPEO.
The differences in the composition of the three extracts led to the differences in their aroma and biological functions. The aroma intensity and persistence of LPEO were the best among the three extracts.
Among the three lemon peel extracts, LPE and LPAO showed superior scavenging abilities against DPPH and ABTS compared to LPEO. LPEO showed a stronger inhibitory effect on tyrosinase than LPE and LPAO, with an inhibition rate of 93.82 ± 1.06% at a concentration of 20 mg/ml, which was higher than that of arbutin. The inhibitory effect of three lemon peel extracts on E. coli was more sensitive than that on S. aureus and C. albicans. Among them, compared with MIC values, LPAO had a stronger inhibitory rate on E. coli than LPEO and LPE. The safety sequence of the three extracts for HaCaT cells was: LPE (1 mg/ml)> LPEO (0.01 mg/ml) > LPAO (0.001 mg/ml). LPEO showed a stronger inhibitory effect on inflammatory factors compared to LPAO and LPE, with inhibitory rates of 87.79 ± 3.86% for TNF-α and 80.75 ± 2.33% for IL-6 at 1 × 10-2 mg/ml. Additionally, LPEO enhanced the migration and healing of HaCaT cells more effectively than LPE and LPAO. Following a 12 h treatment with 1×10-2 mg/ml LPEO, the healing rate of scratched cells reached 95.29 ± 3.41%.
In summary, each of the three extracts has its own characteristics. Overall, LPEO demonstrated superior performance compared to LPE and LPAO. The three extracts can also be combined together to expand their application as cosmetic additives, providing benefits such as enhancing aroma, antioxidant, whitening, antibacterial, anti-inflammatory, and skin wound-healing effects.
Acknowledgments
This work was supported by a key R&D plan project in Ningbo City (No. 2023Z158).
Footnotes
Abbreviations
LPEO, Lemon peel essential oil, LPE, Lemon peel extract, LPAO, Lemon peel absolute oil
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- 1.Lili Z, Al Mamun A, Hayat N, Salamah AA, Yang Q, Ali MH. Celebrity endorsement, brand equity, and green cosmetics purchase intention among Chinese youth. Front. Psychol. 2022;13:806177. doi: 10.3389/fpsyg.2022.860177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Gong JR, Qiu JW, Lin CJ. Development and production of green cosmetics guided by dialectics of nature. Deterg. Cosmet. 2024;47:54–58. [Google Scholar]
- 3.Li MR, Zheng CX, Qi JH. Research status and development of "hurdle technology" as an alternative preservation technique for cosmetics. Deterg. Cosmet. 2021;44:46–54. [Google Scholar]
- 4.Wen X, Yang XJ, Xie XB, Zhou SL, Sun TL, Huang XM. Resistance analysis of spoilage microorganisms in cosmetics. Industrial Microbiology. 2015;45:37–41. [Google Scholar]
- 5.Infante PHV, Darvin EM, Campos MGBMP. Characterization and efficacy of essential oil-based cosmetic formulations for acne-prone skin. Cosmetics. 2023;10:158. doi: 10.3390/cosmetics10060158. [DOI] [Google Scholar]
- 6.Cai SH, Zeng XF, Han Z, Feng WH, Chen HQ, Bai WD. Effect of lemon essential oil and its application in aromatic physiotherapy. Farm Products Processing. 2018;14:56–59. [Google Scholar]
- 7.Perpetua IO, Shadrack UUC. Essential oil from lemon peel (Citrus lumion) and application in skin-care lotion. Chem. Sci. Int. J. 2021;30:30–38. doi: 10.9734/CSJI/2021/v30i930252. [DOI] [Google Scholar]
- 8.Bertuzzi G, Tirillini B, Angelini P, Venanzoni R. Antioxidative action of citrus limonum essential oil on skin. Eur. J. Med. Plants. 2013;3:1–9. doi: 10.9734/EJMP/2013/1987. [DOI] [Google Scholar]
- 9.El Hachlafi N, Fikri-Benbrahim K, Al-Mijalli SH, Elbouzidi A, Jeddi M, Abdallah EM, et al. Tetraclinis articulata (Vahl) mast. essential oil as a promising source of bioactive compounds with antimicrobial, antioxidant, anti-inflammatory and dermatoprotective properties: In vitro and in silico evidence. Heliyon. 2024;10:e23084. doi: 10.1016/j.heliyon.2023.e23084. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 10.Zhang YL, Long Y, Yu S, Li D, Yang M, Guan YM, et al. Natural volatile oils derived from herbal medicines: A promising therapy way for treating depressive disorder. Pharmacol. Res. 2020;164:105376. doi: 10.1016/j.phrs.2020.105376. [DOI] [PubMed] [Google Scholar]
- 11.Rina P, Iwan H, Tuti HH. Effects of citrus limon essential oil (Citrus limon L.) on cytomorphometric changes of Candida albicans. Dental J. (Majalah Kedokteran Gigi) 2017;50:43–48. doi: 10.20473/j.djmkg.v50.i1.p43-48. [DOI] [Google Scholar]
- 12.Yu KQ, Li J, Chen YQ, Zhang M, Li JX, Li S, et al. The chemical profiling and identification of Abelia macrotera (Graebn. et Buchw.) Rehd. based on UHPLC-Q-Exactive Orbitrap MS and study their antioxidant, anti-tyrosinase and anti-inflammatory activities. Arabian J. Chem. 2024;17:105575. doi: 10.1016/j.arabjc.2023.105575. [DOI] [Google Scholar]
- 13.Liao N, Wei LB, Jin W, Wei QQ, Chen J, Wu SG. Extraction technology and antioxidant and antibacterial activities of polyphenols from Semiliquidambar cathayensis. Guihaia. 2019;39:523–530. [Google Scholar]
- 14.Yang XY, Wen GH, Zou Y, Jin YF. Analysis of volatile components in pericarpium citri reticulatae extract and its application in electronic cigarette. Flavour Fragrance Cosmet. 2018;6:13–19+32. [Google Scholar]
- 15.Sun ZH, Zhang S, Wang LW, Gong HY, Tian SG. Determination of total flavonoids and total polyphenols in Abelmoschi corolla extracts and their in vitro antibacterial and antioxidant activities. Chem. Bioeng. 2024;41:26–32. [Google Scholar]
- 16.Sahena F, Kashif G, Islam ZMS. Total phenolic content and antioxidant activity of date palm (Phoenix dactylifera L.) seed extracts using soxhlet method. Anal. Chem. Lett. 2023;13:257–266. doi: 10.1080/22297928.2023.2242862. [DOI] [Google Scholar]
- 17.Yan TY, Lin JX, Zhu JX, YE N, Jin S, Huang J, et al. Analysis of E-nose and GC-MS combined with chemometrics applied to the aroma characteristics of high aroma black tea and traditional congou black tea. Sci. Technol. Food Ind. 2022;43:252–261. [Google Scholar]
- 18.Patricia FA, Teresa DPS, Natividad AC, Elena MGM, Pilar DMTN, Juanita HP. Antibacterial and anti-inflammatory activity of extracts and fractions from Agave cupreata. Int. J. Pharmacol. 2017;13:1063–1070. doi: 10.3923/ijp.2017.1063.1070. [DOI] [Google Scholar]
- 19.Lin YY, Yang T, Shen LY, Zhabg J, Liu L. Study on the properties of dendrobium officinale fermentation broth as functional raw material of cosmetics. J. Cosmet. Dermatol. 2021;21:1216–1223. doi: 10.1111/jocd.14197. [DOI] [PubMed] [Google Scholar]
- 20.Chen C, Xue QL, Hu YJ, Wei M, Chen Z. Study on the bacteriostatic activity and stability of ethanolic extract from Yunnan Litsea cubeba. Sci. Technol. Food Ind. 2023;44:147–154. [Google Scholar]
- 21.Wan WJ. Recent advances on limonene, a natural and active monoterpene. China Food Additives. 2005;1:33–37. [Google Scholar]
- 22.Zhao C, Zhang Z, Nie DC, Li YL. Protective effect of lemon essential oil and its major active component, d-limonene, on intestinal injury and inflammation of E. coli. challenged mice. Front. Nutr. 2022;9:843096. doi: 10.3389/fnut.2022.843096. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Xiao J. 2019. Study of extraction essential oil from lemon peel and its antioxidant and scavenging effect of nitrite. Sichuan Agricultural University. DOI: 10.27345/d.cnki.gsnyu. 10.27345/d.cnki.gsnyu [DOI]
- 24.Ouyang QL, Liu YM, Chen Y, Tao NG. Antifungal action of α-terpineol on imazalil-resistant Penicillium digitatum Pdw03. Food Sci. 2022;36:1–9. [Google Scholar]
- 25.Kang EJ, Lee JK, Park HR, Kim H, Kim HS, Park J. Antioxidant and anti-inflammatory activities of phenolic compounds extracted from lemon myrtle (Backhousia citriodora) leaves at various extraction conditions. Food Sci. Biotechnol. 2020;29:1425–1432. doi: 10.1007/s10068-020-00795-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Wang W, Cao W. Study on bacteriostasis of polyphenols, polysaccharides and flavonoids from sea buckthorn pomace and their preservative effects on grapes. China Food Additives. 2023;34:12–18. [Google Scholar]
- 27.Song KY, Li JM, Yan F, Wu XD, Liu YH, Zheng HL. Research progress on extraction of d-limonene from citrus fruit peel and its application in food preservation. Food Ind. 2023;44:217–221. [Google Scholar]
- 28.Darwisy HAA, Qistina NRA, Hasnun MYM, Chin KH, Lias MK, Hafizah NU, et al. Antioxidant and antimicrobial ctivities of essential oils extracted from bamboo (Bambusa vulgaris) leaves and its application in skincare products: A review. Biocatal. Agric. Biotechnol. 2023;54:102930. doi: 10.1016/j.bcab.2023.102930. [DOI] [Google Scholar]
- 29.Wu H, Luo ZS, Luo ZQ, Yang XS, Yao YR, Qin RG. Analysis of antioxidant activity and antioxidant profile of rosa roxburghii tratt from 17 origins in Guizhou province. Northern Hortic. 2023;20:94–101. [Google Scholar]
- 30.Hu JJ, Li X, Liu XH, Zhang WP. Inhibitory effect of lemon essential oil on mushroom tyrosinase activity in vitro. Modern Food Science and Technology. 2015;31:97–105. [Google Scholar]
- 31.Kong QC, Zhang Y, Chen QS, Gong SZ. Antimicrobial activities and application of four essential oils in cosmetics. Flavour Fragrance Cosmetics. 2019;2:33–39. [Google Scholar]
- 32.Zhang L, Chen SX, Fan GR, Liao SL, Wang ZD. Research progress in application and functions of Litsea cubeba essential oil components. Acta Agric. Univ. Jiangxiensis. 2021;43:355–363. [Google Scholar]
- 33.Rehman MU, Tahir M, Khan AQ, Khan R, Oday-O-Hamiza, Lateef A, et al. D-limonene suppresses doxorubicin-induced oxidative stress and inflammation via repression of COX-2, i NOS, and NF Bin kidneys of wistar rats. Exper. Biol. Med. 2014;239:465–476. doi: 10.1177/1535370213520112. [DOI] [PubMed] [Google Scholar]
- 34.Yoon WJ, Kim SS, Oh TH, Lee NH, Hyun CG. Torreya nucifera essential oil inhibits skin pathogen growth and lipopolysaccharide-induced inflammatory effects. Int. J. Pharmacol. 2009;5:37. doi: 10.3923/ijp.2009.37.43. [DOI] [Google Scholar]
- 35.Juliana DW, Huijuan J, Hisanori K. Effects of thyme (Thymus vulgaris L.) essential oil on aging-induced brain inflammation and blood telomere attrition in chronologically aged C57BL/6J mice. Antioxidants. 2023;12:1178–1195. doi: 10.3390/antiox12061178. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Han J, Besheli NH, Deng D, van Oirschot BAJA, Leeuwenburgh SCG, Yang F. Tailoring copper-doped bioactive glass/chitosan coatings with angiogenic and antibacterial properties. Tissue Eng. Part C. 2022;28:314–324. doi: 10.1089/ten.tec.2022.0014. [DOI] [PubMed] [Google Scholar]
- 37.Elahe S, Mohammad RF, Zohreh GT. Lavender officinalis essential oil conjugated carboxymethyl cellulose: as boosters of antibacterial and bio enhancers to accelerate the repair of full-thickness infected wound. Polym. Bull. 2023;81:6091–6113. doi: 10.1007/s00289-023-04984-2. [DOI] [Google Scholar]
- 38.Braga NSDM, Silva MDC, Cunha AL, Cunha AL, Sant'Ana AEG. Chemical characterization and biological potential of the essential oil of eucalyptus globulus labill. J. Pharm. Pharmacol. 2018;6:10. doi: 10.17265/2328-2150/2018.12.001. [DOI] [Google Scholar]