Animal Models/Human Diseases/Cells Treatment Benefits Mechanisms Ref. Epidermal Permeability Barrier Function Young mice Topical applications of 2% hesperidin twice-daily for 6 days. ↑Acute barrier recovery ↑ Proliferation; [31 ] Aged mice Topical applications of 2% hesperidin twice-daily for 9 days. ↑ Acute barrier recovery; ↑ Differentiation; [32 ] Glucocorticoid-treated mice One hour after each topical application of 0.05% clobetasol propionate, 2% hesperidin was applied. Treatments were twice-daily for 9 days ↑ Acute barrier recovery; ↑ Proliferation;β -glucocerebrosidase activity. [33 ] Protecting UV Irradiation Keratinocytes Keratinocytes first treated with 50 μ M hesperidin for 1 hr, followed by UVB irradiation (30 mJ/cm2 ) ↓ DNA damage ↓ Absorb UVB [34 ] Keratinocytes were treated with hesperidin (220μ g/ml) for 24 hr, followed by UVA irradiation (10 J/cm2 ). Cells were incubated with hesperidin for additional 6 or 24 hr ↑ Cell viability α , IL-1β and IL-6 mRNA Levels ↑ SOD activity [35 ] Keratinocytes were treated with hesperidin (50, 100, 200, 400, 600, 1 000mg/L) for 24 hr, followed by UVB irradiation (15 mJ/cm2 ). ↓ CXCR2 expression Not determined [36 ] Dermal fibroblasts Fibroblasts irradiated with UVA at dose 10 J/cm2 and treated with hesperetin containing extract at various concentration for 72 hr ↓ Matrix metalloproteinase expressionβ ‐galactosidase expression Not determined [37 ] Pretreated fibroblasts with 3 and 30 µM hesperetin glucuronide, followed by UVA irradiation at 500 kJm−2 ↓ Necrotic cell death Not determined [38 ] Mice Mice treated topically with hesperidin (3 mg/ml) daily for 10 days, 30 min after each application of hesperidin, mice were irradiated with 180 mJ/cm2 UVB. ↓ Skin erythema & edema ↑ Catalase and superoxide dismutase activity [39 ] Mice were treated topically 1% hesperidin methyl chalcone before and after one irradiation with 4.14 mJ/cm2 UVB ↓ Cytokine expression ↑ Nuclear factor erythroid 2-related factor 2; [40 ] Hesperidin methyl chalcone at the dose of 300 mg/kg was intraperitoneally given 1 hr before and 7 hr after, irradiation with 4.14 mJ/cm2 UVB ↓ Skin edema, neutrophil recruitment and matrix metalloproteinase-9 activity; ↑ Glutathione levels and catalase activity. [41 ] Orally administered 0.1 mL of water containing 100 mg/kg body weight hesperidin daily, while mice were irradiated 3 times at 48 h intervals per week for 12 weeks. Does of UVB were increased 60 mJ/cm2 per exposure at week 1 to 90 mJ/cm2 at week 7. ↓ Transepidermal water loss; ↓ Phosphorylation of mitogen activated protein kinase & extracellular signal-regulated kinases [42 ] Single UVB irradiation at dose of 180 mJ/cm2 ↓ Cyclobutane pyrimidine dimers; Not determined [43 ] Guinea pigs The dorsal skin was exposed to UVB 3 times a week (every other day) for 2 consecutive weeks. The total energy dose of UVB was 1 J/cm2 per exposure. One week later, 1% hesperetin was topically applied daily to the hyperpigmented areas (2 mg/cm2 ) for 4 successive weeks. ↓ Transepidermal water loss; Not determined [44 ] Pigmentation B16 mouse melanoma cells Cells incubated with 20 μ g/mL of Citrus extracts or 3-50 μ M of hesperetin for 48 hr. ↑ Melanin content; ↑ Melanogenesis-related proteins;β -Catenin expression;β [45 , 46 ] Cells incubated with hesperidin (32.25mg/mL) for 3 days Minimum inhibition of melanogenesis Not determined [47 , 48 ] Cells were treated with hesperidin (50, 100, 200, 400, 600, 1000mg/L) for 24 hr, followed by UVB irradiation (15 mJ/cm2 ). ↓ Tyrosinase activity; Not determined [36 ] Cells incubated with 5-20 μ M hesperidin for 3 days ↑ Melanin content; Not determined [49 ] Cells incubated with 50 μ M hesperidin for 3 days ↓ Melanin content; ↓ Melanogenesis-related proteins; [50 ] Cells incubated with Citrus extracts (12.5, 25.0, and 50.0 µ g/mL) for 3 days ↓ Melanin content; ↓ Microphthalmia-associated transcription factor (MITF) proteins [51 ] Human epidermal melanocytes Cells incubated with 3-50 μ M of hesperetin for 48 hr. ↑ Melanin content; Not determined [46 ] Cells incubated with 50 μ M hesperidin for 3 days ↓ Melanin content; Not determined [50 ] Cells incubated with 0.4mg/ml Citrus extracts for 3 days ↓ Tyrosinase activity Not determined [52 ] Enzymatic assay of mushroom tyrosinase ↓ Tyrosinase activity Not determined [49 ] 1mg/ml of Citrus extracts induced over 40% inhibition of tyrosinase activity Not determined [52 ] 1.75 mg/ml of calamondin peel extract,containing hesperidin, induced 90% inhibition of tyrosinase activity Not determined [53 ] Guinea pigs The dorsal skin was exposed to UVB 3 times a week (every other day) for 2 consecutive weeks. The total energy dose of UVB was 1 J/cm2 per exposure. One week later, 1% hesperetin was topically applied daily to the hyperpigmented areas (2 mg/cm2 ) for 4 successive weeks. ↓ Pigmentation Not determined [44 ] Reconstructed human epidermis The epidermis was treated topically with 0.2% hesperidin for 14 days ↓ Pigmentation Not determined [49 ] Humans Topical applications of cream containing 0.4 mg/ml Citrus extracts for 56 days >8% increase in skin brightening Not determined [52 ] Cutaneous Wound Healing Dermal fibroblast Fibroblasts were incubated with mixture containing 0·05 mg/ml hesperidin for 24 or 96 hr. ↑ Wound closure. ↑ Collagen synthesis [54 ] Diabetic rats After wound, rats were given oral hesperidin (25-100 mg/kg body weight) for 21 days. ↓ Wound closure. ↑ VEGF-c, Ang-1/Tie-2, TGF-β and Smad-2/3 mRNA expression; [55 ] After wound, rats were given oral hesperidin (10-80 mg/kg body weight) for 20 days. ↑ Wound closure. ↑ VEGFR1 and VEGFR2 levels;α , IL-6; [56 ] Humans with venous ulcers Fifteen patients were treated orally with diosmin/hesperidin (450/50 mg, twice daily) for 90 days. Another 15 patients treated with pycnogenol (50 mg orally, 3 times daily) served as controls No differences in wound healing time between two groups Not determined [57 ] Fifty-three patients received Daflon 500 mg, and 52 received placebo for 2 months ↓ Wound healing time; Not determined [58 ] γ irradiated miceMice were given oral hesperidin (100mg/kg body weight) once 1 hr before γ irradiation. Wound was made prior to irradiation. ↑ Wound contraction; ↑ NO; [59 ] Mice given 1, 2, 5 or 10 % of hesperidin ointment topically covering the whole excision wounds, twice daily after exposure to 6 Gy ↑ Wound contraction; Anti-oxidative stress [60 ] Inflammation Mouse RAW 264.7 cell line Incubated with hesperidin (5-250 μ g/ml) ↓ Lipopolysaccharide-induced nitric oxide production Not determined [30 ] Incubated with hesperidin or hesperetin (40-100 μ M) for 30 min, followed by stimulation with 1 μ g/mL of Lipopolysaccharide ↓ Antioxidative stress; ↓ NF‐κ B activation; κ Bα ; [61 ] Keratinocytes Keratinocytes treated with HES (20 µ g/mL) for 2 hr, followed by incubation with H2 O2 for 48 hr ↓ IL-8 protein & mRNA;α protein & mRNA; ↓ NF‐κ B activation, phosphorylated Iκ Bα and phosphorylated p38 MAPK [62 ] Cells were incubated with both heat-killed Propionibacterium acnes and 5-50 µ g/mL of hesperidin for 24 hr. ↓ IL-8 protein & mRNA;α protein & mRNA; Not determined [63 ] Human skin explants Human skin explants were pre-incubated with hesperidin methyl chalcone (0.2 mg/ml) and then stimulated with SP for 24 hours. ↓ Proportion of dilated vessels; Not determined [64 ] Rats Thirty min prior to carrageenan or dextran injection, hesperidin (50 or 100 mg/kg body weight) was subcutaneous injected ↓ Edema Not determined [65 ] Mice Intraperitoneal injection of hesperidin (75 mg/kg), following by subcutaneous injection of carrageenan ↓ Edema Not determined [66 ] Guinea pigs Hesperidin (40mg/kg) was orally given 1 hr prior to injection of carrageenan. ↓ Edema Not determined [67 ] Skin Cancers A431 human skin carcinoma cells Incubation of cells with hesperetin (10,100,500 μ M) for 24 hr ↑ DNA fragmentation; ↑ ERK, JNK, p38, ROS [68 ] Cells treated with hesperidin (10, 25 and 50 μ M) for various times ↓ Cell viability; ↑ ROS [69 ] Mice Subcutaneous injection of 125 μ l of 1% hesperidin solution daily 1 week prior to tumor induction. ↓ Incidence of tumor and number of tumor per mouse Not determined [70 ]
