Table 2.

Benefits of hesperidin on cutaneous functions.

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; ↑ Filaggrin expression; ↑ Lamellar body secretion.	[31]
Aged mice	Topical applications of 2% hesperidin twice-daily for 9 days.	↑ Acute barrier recovery; ↓ Skin surface pH;	↑ Differentiation; ↑ Lipid production; ↑ NHE1 and sPLA2 expression; ↑ Lamellar body formation and secretion	[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; ↓ Skin surface pH;	↑ Proliferation; ↑ Filaggrin expression; ↑ Lipid processing; ↑ Antioxidation; ↑ Lamellar body formation and secretion; ↑ β-glucocerebrosidase activity.	[33]
Protecting UV Irradiation
Keratinocytes	Keratinocytes first treated with 50 μM hesperidin for 1 hr, followed by UVB irradiation (30 mJ/cm2)	↓ DNA damage ↓ lipid peroxidation ↓ Protein carbonylation ↓ Apoptotic index	↓ Absorb UVB ↓ Reactive oxygen species ↓ Bcl-2 expression ↓ BAX expression	[34]
	Keratinocytes were treated with hesperidin (220μg/ml) for 24 hr, followed by UVA irradiation (10 J/cm2). Cells were incubated with hesperidin for additional 6 or 24 hr	↑ Cell viability ↓ MDA content ↓ TNF-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 expression ↓ β‐galactosidase expression ↑ Collagen biosynthesis	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 ↓ Epidermal proliferation ↓ Lipid peroxidation ↓ Inflammation ↓ DNA damage	↑ 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 ↓ Lipid peroxidation	↑ Nuclear factor erythroid 2-related factor 2; ↑ Glutathione peroxidase-1, glutathione reductase & heme oxygenase-1	[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; ↓ Cytokine expression; ↓ Myeloperoxidase activity; ↓ Lipid peroxidation	↑ 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; ↓ matrix metalloproteinase-9 expression & activity; ↓ Cytokine expression ↓ wrinkle formation ↓ Epidermal proliferation	↓ Phosphorylation of mitogen activated protein kinase & extracellular signal-regulated kinases	[42]
	Single UVB irradiation at dose of 180 mJ/cm2	↓ Cyclobutane pyrimidine dimers; ↑ p53 expression	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 μg/mL of Citrus extracts or 3-50 μM of hesperetin for 48 hr.	↑ Melanin content; ↑ Tyrosinase protein; ↑ Tyrosinase activity.	↑ Melanogenesis-related proteins; ↑ β-Catenin expression; ↑ Phosphorylated glycogen synthase kinase-3β	[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; ↓ Melanin content;	Not determined	[36]
	Cells incubated with 5-20 μM hesperidin for 3 days	↑ Melanin content;	Not determined	[49]
	Cells incubated with 50 μM hesperidin for 3 days	↓ Melanin content; ↓ Tyrosinase protein; ↓ Tyrosinase-related protein 1,2	↓ Melanogenesis-related proteins; ↑ p-Erk1/2; ↑ Proteasome activity	[50]
	Cells incubated with Citrus extracts (12.5, 25.0, and 50.0 µg/mL) for 3 days	↓ Melanin content; ↓ Tyrosinase protein & activity; ↓ Tyrosinase-related protein 1,2	↓ Microphthalmia-associated transcription factor (MITF) proteins	[51]
Human epidermal melanocytes	Cells incubated with 3-50 μM of hesperetin for 48 hr.	↑ Melanin content; ↑ Tyrosinase activity	Not determined	[46]
	Cells incubated with 50 μM hesperidin for 3 days	↓ Melanin content; ↓ Tyrosinase activity	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; ↑ SOD and GSH levels; ↓ MDA and NO levels;	[55]
	After wound, rats were given oral hesperidin (10-80 mg/kg body weight) for 20 days.	↑ Wound closure.	↑ VEGFR1 and VEGFR2 levels; ↓ TNFα, IL-6; ↑ SOD and GSH levels; ↓ MDA levels;	[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; ↓ Hospitalization duration	Not determined	[58]
γ irradiated mice	Mice were given oral hesperidin (100mg/kg body weight) once 1 hr before γ irradiation. Wound was made prior to irradiation.	↑ Wound contraction; ↓ Wound healing time.	↑ NO; ↑ DNA synthesis; ↑ Collagen; ↑ Hexosamine; ↑ Densities of bold vessels and fibroblasts	[59]
	Mice given 1, 2, 5 or 10 % of hesperidin ointment topically covering the whole excision wounds, twice daily after exposure to 6 Gy ${\widehat{\mathrm{I}}}^{\mathrm{3}}$-radiation until complete healing of wounds.	↑ Wound contraction; ↓ Wound healing time.	Anti-oxidative stress	[60]
Inflammation
Mouse RAW 264.7 cell line	Incubated with hesperidin (5-250 μg/ml)	↓ Lipopolysaccharide-induced nitric oxide production	Not determined	[30]
	Incubated with hesperidin or hesperetin (40-100 μM) for 30 min, followed by stimulation with 1 μg/mL of Lipopolysaccharide	↓ Antioxidative stress; ↓ PGE2; ↓ COX‐2 expression; ↓ Nitric oxide production	↓ NF‐κB activation; ↓ JNK1/2 and p38 phosphorylation; ↓ IκBα; ↓ iNOS mRNA; ↓ Antioxidative stress;	[61]
Keratinocytes	Keratinocytes treated with HES (20 µg/mL) for 2 hr, followed by incubation with H2O2 for 48 hr	↓ IL-8 protein & mRNA; ↓ TNF‐α protein & mRNA; ↓ COX‐2 expression	↓ NF‐κB activation, phosphorylated IκBα and phosphorylated p38 MAPK	[62]
	Cells were incubated with both heat-killed Propionibacterium acnes and 5-50 µg/mL of hesperidin for 24 hr.	↓ IL-8 protein & mRNA; ↓ TNF‐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; ↓ Total vessel area; ↓ IL-8 production.	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 μM) for 24 hr	↑ DNA fragmentation; ↑ Apoptotic proteins (p21 and Bax); ↓ levels of cyclin A2, B1, D1, D3 and E1	↑ ERK, JNK, p38, ROS	[68]
	Cells treated with hesperidin (10, 25 and 50 μM) for various times	↓ Cell viability; ↓ Levels of cyclin D, CDK2 and thymidylate synthase; ↑ Apoptosis	↑ ROS ↓ ATP content	[69]
Mice	Subcutaneous injection of 125 μl of 1% hesperidin solution daily 1 week prior to tumor induction.	↓ Incidence of tumor and number of tumor per mouse	Not determined	[70]

Note: SOD: superoxide dismutase; GSH: reduced glutathione: NO: nitric oxide (NO); PGE2: prostaglandin E2; VEGF: vascular endothelial growth factor; MPO: myeloperoxidase; MDA: malondialdehyde; COX2: cyclooxygenase-2, COX-2; SP: substance P; DMBA: 7,12-dimethylbenz[a]anthracene; TPA: 12-O-tetradecanoyl-13-phorbol acetate; ROS: reactive oxygen species; ERK: extracellular signal-regulated kinase; JNK: c-Jun NH2-terminal kinase; CDK2: cyclin-dependent kinase 2.