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1. Introduction

Photoaging due to exposure to ultraviolet (UV) radiation results in the appearance of rough skin, wrinkles, xerosis, hyperpigmentation and epidermal thickening owing to the progressive loss of function and regenerative potential of the skin tissue (1). Collagen is the main supporting skin structure; its excessive degradation is directly related to wrinkle formation (2). Matrix metalloproteinase (MMP) is a matrix-degrading enzyme that degrades and modifies some components of the extracellular matrix (ECM). UV radiation can increase MMP-1 production, which in turn increases collagen degradation in human skin (3).

An increase in the ROS content caused by UV exposure can cause oxidative stress accumulation, which activates MMP and increases collagen damage (4). Antioxidants are chemical substances that neutralize or interfere with the action of free radicals (5); these agents are often used to prevent or slow down the oxidation of macromolecules that cause oxidative stress. One natural ingredient that has antioxidant properties by reducing ROS production is black cumin or Nigella sativa (N. sativa) seeds (6).

N. sativa is a small shrub, 20-90 cm in height, which belongs to the Ranunculaceae family; it is known for its antioxidant, antiviral, antifungal, antiparasitic and anti-inflammatory effects (7). The effects of these agents against free radicals are related to their activities of free radical scavenging and inhibiting 5-lipooxygenase during the inflammatory phase (8). This ability is attributed to several important components, such as thymoquinone (TQ), flavonoids, essential fatty acids and various mineral components, which have been widely studied (9). N. sativa has a therapeutic effect by reducing cytotoxicity, oxidative stress, inflammatory response and cell death caused by mitochondrial dysfunction in keratinocyte cells irradiated with ultraviolet A (UVA) light (10). Another study found that N. sativa extract inhibits the formation of advanced glycation end products, collagen cross-linking, collagenase activity and elastase activity (2). An animal study with rats exposed to UVB light showed a significant increase in collagen levels after being given topical N. sativa extract (11).

2. Photoaging

Photoaging is a group of clinical and pathological manifestations caused by skin exposure to UV radiation (12). UVA and UVB both play a role in photoaging, although UVA rays have longer wavelengths because UVA-1 rays (340-400 nm) can be absorbed deeply into the human skin and exert effects directly through the dermal fibroblast level (13); however, UVC is fully reflected by the ozone layer (14). Photoaging involves all skin layers, with primary effects on the dermis and paracrine effects on other skin layers. This condition comprises acute and chronic cellular processes characterized by rough wrinkling, decreased skin elasticity, pigmentation, xerosis, keratosis and telangiectasis (15).

The clinical manifestations of normal aging and photoaging often appear simultaneously; however, there are several differences (12). Normal aging is characterized by sagging skin and an excessive expression of frown lines. Aging skin has decreased hydration and elasticity, permeability and susceptibility to irritation owing to skin barrier damage (16). The manifestations of photoaging include deep wrinkles, rough skin texture, dyspigmentation and skin elasticity disturbance (17). The distribution of photoaging is mostly limited to the face, neck and hands and rarely to the arms and lower legs. Of photoaging cases ~80% are caused by chronic low-level UV exposure, although exposure to high levels of acute UV light can also cause skin burning, darkening and skin structure disorders (18). Microscopic examination showed an increase in the epidermal thickness, dystrophic elastin accumulation and irregular collagen in the dermal layer. Skin susceptibility to UV damage depends on the melanin content and Fitzpatrick skin types I, II and III are more susceptible to photoaging than skin types IV, V and VI (19).

There are three main causes of photoaging: i) Dysfunction of keratinocytes in the basal layer, which inhibits their ability to regenerate and repair the skin; ii) loss of the ability of fibroblasts to synthesize and produce collagen; iii) changes in intracellular homeostasis through certain paracrine mechanisms (20). These changes are related to UV exposure on the skin, which causes oxidative stress (21).

Photoaging and UV exposure

Large amounts of UVB can reach the skin due to direct sunlight exposure during the day (22). UVA is rarely absorbed when passing through the atmosphere (23). High UVA radiation throughout the day causes greater UVA exposure on the skin than UVB exposure under normal conditions (24). UVB exposure can cause erythema, burning, skin damage and skin cancer, whereas intense UVA exposure can cause erythema and blood vessel damage (25). To produce this effect, UVA requires an amount 1,000 times greater than UVB. However, UVA can penetrate deeper than UVB, causing damage to collagen and elastic fibers in the dermal layer (24,26).

UV radiation as photons needs to be absorbed by chromophores, followed by various photochemical events to produce photoaging skin manifestations (19); this process involves fibroblasts, keratinocytes and neutrophils (27). Therefore, the skin is a notable part of the immune system and consists of numerous molecular mechanisms that protect the skin from UV radiation (28). The epidermal layer provides the first line of defense against hazardous external agents consisting of keratinocytes, antigen-presenting cells, Langerhans cells, T lymphocytes and melanocytes (29).

Keratinocytes have an immune function against UV by producing cytokines, hormones, chemokines, neuropeptides and antimicrobial peptides (30). Melanin is produced by melanocytes; it works by inhibiting and absorbing UV rays that enter the epidermis layer. As UV exposure increases, the melanin formation rate increases, causing the skin to appear darker and providing additional protection against further radiation injury (22). Furthermore, DNA damage caused by UV rays can be repaired by nucleotide repair and base excision mechanisms, apoptosis and cell cycle checkpoint system activation (31).

Photoaging and oxidative stress

Humans are constantly exposed to oxidants daily; oxidative stress occurs because of a disturbance in the oxidant-antioxidant balance (32,33). Excessive UV radiation can induce ROS production while downregulating peroxidase and glutathione reductase production, which depends on the amount of UV radiation absorbed (34). UV rays can not only directly damage the skin by destroying lipids, proteins and DNA cells but also indirectly through the increased oxidation of various substances, especially lipids and DNA (24).

The effects of UVB radiation in human keratinocyte cells are characterized by enhanced ROS production, release of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, cyclooxygenase (COX) and nuclear factor-κB (NF-κB) (24). The aging process of fibroblasts during photoaging owing to UVB exposure is associated with ROS accumulation, which escalates proteasomes inhabitation and autophagy initiation (35). Autophagy suppression is required to switch the cell pathway from senescence to the apoptotic process (36). Inactivation of the proteasome system in fibroblast cells is related to the generation of singlet oxygen, protein oxidation and transcriptional agents that regulate MMP-1 expression activation (31).

Molecular impact of ROS on photoaging

Human bodies produce ROS consisting of superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH), singlet oxygen (1O2), lipid peroxide and nitrogen oxide (37). O2 molecules gain a single unpaired electron to establish O2- and thus become the early stage of ROS in cells (38). O2- molecules will form H2O2 through antioxidant enzyme catalysis (39). O2- can release iron ions under stress conditions from the iron-sulfur of proteins, participating in the Fenton reaction and converting H2O2 to OH (40). Hydroxyl radicals are easily reacted with other molecules with a half-life of 10-9 sec (41).

Normal skin aging and photoaging both involve similar signaling pathways, although they are induced differently (42,43). In photoaging, ROS can increase MMP synthesis by stimulating mitogen-activated protein kinase (MAPK) and protein-1 (AP-1) heterodimers (44). MMP-1 is capable of breaking down the integrated fibrillar collagen, whereas MMP-2, MMP-3 and MMP-9 break down the fragments and degraded forms (44-46). ROS and activated MAPK signaling pathways stimulate NF-κB and induce the expression of several proinflammatory cytokines (46,47). NF-κB organizes the expression of hemeoxygenase-1 and increases free iron levels in cells, thereby encouraging ROS execution through the Fenton reaction (48,49). NF-κB also excites MMP-8 to accelerate collagen breakdown; at the same time, AP-1 downregulates the expression of transforming growth factor-β (TGF-β) receptor type II, which disrupts the downstream Smad/TGF-β signal pathway, thereby reducing the transcription of the COL1A1 and COL3A1 genes encoding type I and type III collagen (41,50-52). Photoaging is caused not only by increased degradation of collagen by various MMPs, serine and other proteases but also by decreased collagen production by fibroblasts (1).

Photoaging and MMP activities

The main function of MMPs is to degrade the ECM, glycoproteins, cytokines, receptors and growth factors (52). Impaired regulation of MMP activity causes various pathologies and is divided into the following: i) Tissue damage; ii) fibrosis; and iii) matrix weakness (53). UV exposure can increase MMPs levels, which are responsible for ECM damage, including collagen, elastin, fibronectin and proteoglycans (54). This exaggerated degradation can cause clinical signs of photoaging, resulting in deep wrinkles and sagging skin through photodestruction, phototransformation and photo-oxidation of collagen and elastin fiber processes (31).

DNA damage following UVB exposure induces MMP-1 production by increasing MMP-1 gene expression in keratinocytes (31,54,55). Protein expression changes have a substantial role in photoaging, including the transformation of TGF-β, MMP-1, MMP-3 and MMP-9(56). ROS stimulates the MAPK signal pathway, which is responsible for AP-1 formation (57). AP-1 plays a crucial role in the transcription regulation of MMP-1, MMP-3 and MMP-9, which progressively damage collagen (58). In addition, AP-1 can inhibit TGF-β, which regulates type I procollagen production and subsequently results in decreased collagen synthesis (59). Another notable transcription factor involved in the UV response is NF-κB (60). It can regulate the production of MMPs, such as MMP-1 and MMP-3, by fibroblasts (31).

Photoaging and collagen levels

Collagen is a structural protein responsible for maintaining skin elasticity (61). Collagen produced by fibroblasts varies according to the amino acid primer sequence of the polypeptide chain (62). Type I collagen is found in 90% of the human body (63); it is notably discovered in bones, tendons, ligaments, corneas and various interstitial connective tissues (64).

Type I collagen production is regulated by two genes, COL1A1 and COL1A2, which structurally consist of α-1 chain and α-2 chain (62). The COL1A1 gene is located in chromosome 17q21.33(65); its synthesis and maturation are arranged at various levels: Epigenetic and transcriptional, posttranscriptional and posttranslational (66). In photoaged skin, COL1A1 mRNA expression levels are reduced, whereas MMP-1 mRNA expression levels are increased in association with collagen metabolism (67).

Collagen fibers are thicker and coarser in the inner dermal layer than in the outer layer, with thinner and more tenuous collagen fibers (68). Collagen fibers in younger skin are intact, dehydrated, elastic and damage-resistant (69). Collagen levels in aging skin begin to decrease, fade, stretch and lose their elasticity (70). The destruction of collagen and damage of other structural components, including elastic and reticular fibers, underlie the characteristic changes in aging skin and photoaging (64). Significant reductions in the amount and collagen structure can weaken the flexibility of the dermal layer, causing wrinkles and xerosis (71).

UV radiation exposure indirectly initiates MMP activation without upregulating collagen production, causing decreased collagen levels and ECM fragmentation (72). MMP activation is influenced by photochemical reactions that change the UV energy absorbed by cellular macromolecules into ROS (73). Over time, ROS levels exceed antioxidant defense, which promotes increased collagen-degrading MMP production. ROS inhibits the activation of protein tyrosine phosphatase, causing receptor tyrosine kinase phosphorylation and activating the MAPK signaling pathway that forms AP-1(74). AP-1 is important for upregulating the transcription of MMP-1, MMP-3 and MMP-9, where excessive MMP expression can cause increased dermal ECM fragmentation (56,58).

Apart from collagen degradation owing to excessive MMP expression, ROS also triggers a decrease in collagen production by inhibiting the TGF-β signal pathway (75). ROS-induced AP-1 prevents the TGF-β/Smad signal pathway in fibroblasts that control collagen synthesis to prevent a decrease in collagen production (76). TGF-β interacts with cell surface receptors and triggers the Smad2/3 transcription factor. The phosphorylated Smad2/3 then associates with, and moves to, Smad4, thereby triggering the transcription of TGF-responsive genes (71); this leads to decreased phosphorylation and production of the Smad 2/3 transcription factor required for type I procollagen transcription (75).

3. N. sativa seed (black cumin)

N. sativa is a member of the Ranunculaceae family and is known as Black Seed or Black Cumin (77). N. sativa seeds originate from Africa and Southwest Asia (78). High-quality seeds come from Egypt, where a suitable environment exists for this plant (77). N. sativa is an annual herbal plant, 20-30 cm tall, with linear leaves, 5-10 pale bluish-white flower petals and tiny black seeds similar to cumin; therefore, it is called black cumin (79). This plant has green branching stems and green leaves that turn red as they grow (80); it starts flowering between April and August and its fruit consists of 3-6 carpels, each of which contains seeds (81). The seeds are oval and 2-3.5 mm in size, composed of three to four fine-grained corners; the color turns black after being cooked and exposed to air (77,79).

The nutritional value of N. sativa seeds includes protein, fat, fiber and total carbohydrates (82). N. sativa seeds also contain significant amounts of iron, copper and zinc (83). N. sativa has phytoconstituents such as alkaloids, sterols, saponins and essential oils (84). Their seeds comprise primary fatty acids, linoleic acid, palmitic acid and phenolics and quinones (TQ, thymol, dithymoquinone and thymohydroquinone) (85,86). TQ (C10H12O2) has a basic quinone structure with methyl and isopropyl side chain groups added at 2 and 5 positions (87). TQ has COX and 5-lipooxygenase activities, which can inhibit eicosanoid formation during inflammatory phases and lipid membrane peroxidation (88).

Quercetin and kaempferol are the main flavonoid glycosides in N. sativa (86). N. sativa also contains phenolic and triterpene compounds such as saponins (89). Saponin is a group of glycosides comprising one or more hydrophilic groups combined with lipophilic triterpenes or steroid derivatives. Triterpene saponins are typical compounds found in N. sativa seeds (85).

N. sativa extract has been accepted and recognized as safe by the Food and Drug Association in the United States (90). N. sativa is available in dietary supplements, oils, topical creams and powders (91). The acceptable and effective dose is equivalent to 1-3 g of N. sativa extract powder once daily, 40 mg/Kgbw of N. sativa extract once daily, or 1 ml of N. sativa extract cream three times daily, based on clinical trial data on humans (92). However, there is considerable variability in doses and extract preparation, including oil-based formulations, methanol extracts and advanced nanoformulations, which might affect bioavailability and clinical efficacy.

The in vivo TQ pharmacokinetic dose study showed a maximum concentration (Tmax) of 3.96±0.19 h reaching 4811.33±55.52 ng/ml as the maximum blood concentration (Cmax), while the elimination half-life (T1/2) was 4.4933±0.015 h. This indicates the suitability of TQ for extravascular administration through nanoparticle formulation, which has been shown to increase bioavailability (93). The seed extract and its constituents appear to have low levels of toxicity (94). The lethal dose 50 (LD50) of TQ is 2.4 g/kg bw in Swiss albino mice (95). Giving 6.4 g/kg bw of N. sativa extract orally every day for 6 weeks to mice caused the death of one mouse following 2 weeks of therapy, while the second and third mice died in the third and fifth weeks after being given 21 and 60 g/kg bw of N. sativa extract, respectively (83). Toxicity in mice after using fixed oil content from N. sativa extract administered intraperitoneally and intraorally showed that the LD50 was found to be 89.7-119. 7 mg/Kgbw and 647.1-1,094.8 mg/Kgbw, respectively (96). Additional studies are needed to confirm the refined dosage recommendations and evaluate N. sativa's long-term safety in other animals and also in clinical studies.

In recent decades, a number of extraction methods, such as cold pressing, supercritical fluid extraction, Soxhlet extraction, hydro distillation method, microwave-assisted extraction, ultrasonic-assisted extraction, steam distillation and accelerated solvent extraction, have been used to extract oil from black seeds under optimal conditions (97). The cold pressing method using hexane as a solvent does not involve heating or chemical treatment during oil extraction but gives low yields, whereas the Soxhlet extraction method using methanol as a solvent has low costs; however, solvent residues remain in the extracted oil. Thymoquinone in black cumin oil is encapsulated to enhance its oxidative stability. Microencapsulation was achieved through emulsification, spray drying and nanoprecipitation. These processes are more time consuming and involve hot gas flow; therefore, they are not suitable for heat-sensitive bioactives. For sensitive compounds, alternative techniques are required to overcome the drawbacks of conventional encapsulation methods. A recently developed encapsulation technology is electrospray; this process is cost-effective and simple for producing a wide range of nanoparticles. No heat is required during drying and the formation of smaller encapsulations of 1-5 µm is increasingly important for the encapsulation of temperature-sensitive bioactives (97). Continued investigation is required to assess the formulation-dependent differences in bioavailability to ensure consistent therapeutic outcomes, optimal absorption and effective systemic distribution.

No severe side effects have been reported after using the N. sativa extract, either orally or topically. Three women were reported to have acute allergic contact dermatitis after using the topical pure oil of N. sativa extract (90). Other studies have reported the side effects of gastrointestinal disorders, gastric irritation and stomach cramps (98). Gastrointestinal disturbances occurred in 9 of 49 children who orally consumed N. sativa extract at a dose of 80 mg/kg twice daily on an empty stomach (99). Further clinical research under controlled conditions is necessary to comprehensively examine the safety profile of N. sativa in dermatological applications.

4. N. sativa seed (black cumin) mechanism against photoaging

N. sativa mechanism is mainly because of its active ingredients, especially TQ (91,100). TQ has a quinone molecular structure for redox activity and the infinite capability to cover all physiological barriers so that it has easy access to subcellular compartments, which affects radical scavenging (101). TQ is known to have anti-inflammatory and antioxidant effects that can protect cells from free radicals. In addition, TQ stimulates the proliferation and migration of cells involved in tissue repair, encouraging the formation of new tissue (102). TQ reduces NF-κB transcription factor activity so that MMP-1 expression can be suppressed (103). Decreased expression of MMP-1 can further reduce collagen degradation. TQ also acts directly on type I collagen synthesis by preserving the physiological state of the interstitial system (104). Currently, there is no recommended dose of TQ for its role in preventing photoaging. Research by Ghorbanibirgani et al (105) using a topical extract of N. sativa twice a day for 6 months in vitiligo cases found significant improvement in patient scores. Unfortunately, this study did not clearly state the manufacture of the topical cream and its concentration. Research by Yousefi et al (106) using a topical extract of N. sativa at a dose of 2% twice a day for 4 weeks in hand eczema cases showed equally effective results as the use of topical betamethasone. The two studies mentioned the anti-inflammatory and antioxidant effects of N. sativa and TQ as their working mechanisms.

Flavonoids, especially quercetin and kaempferol, play a role as strong antioxidants by interrupting free radical chain reactions and protecting against UV radiation (107). Several studies have reported that quercetin has a photoaging defense mechanism by inhibiting MMP-1 in a dose-dependent manner (108-111). Quercetin shows strong antioxidant activity through in vitro chemical tests, showing an increase in quercetin activity following efficient formulation at the cellular level, as quercetin shows cell-protective action on keratinocytes and in vivo on animal skin. This antioxidant effect is also supported by the ability of quercetin to exert anti-inflammatory actions, such as the inhibition of NF-κB and IL-6 induction by UV irradiation. The combination of antioxidant and anti-inflammatory actions and their cross-linking mechanism highlight quercetin as a novel sunscreen. Furthermore, as quercetin has antioxidant and anti-inflammatory activities, it could be beneficial for wound healing; here, fibroblasts are the main target in contrast to keratinocytes in sunscreens. Furthermore, quercetin has a promising rejuvenating effect on keratinocytes with a supportive whitening effect (112). Kaempferol protects against UVB-induced photoaging by preventing p38 MAPK and c-Jun N-terminal kinase activation (113). These agents prevent the action and amount of MMP-1 and increase collagen levels in the dermal layer. Flavonoids can also stimulate macrophage activity, trigger the epithelialization process and escalate ECM production, growth factors, cytokines and angiogenesis by releasing keratinocyte growth factor (10).

Saponin is a type of glycoside from pentacyclic triterpenoids that has pharmaceutical, nutritional and cosmetic potentials with anti-inflammatory and antioxidant properties. Thus, it has the potential to protect against the detrimental effects of photoaging (114). Saponin modulates the inflammatory response, protects against ECM degradation, enhances collagen production and improves wrinkle appearance by preventing UVB-induced MAPK signaling, AP-1 and NF-κB activation (111,115).

Linoleic acid comprises >50% of the total fatty acids and can preserve water permeability and skin integrity (116,117). Linoleic acid, which is known for its percutaneous absorption, can increase drug absorption, whereas the oil emulsion of the substance can reduce skin irritation and improve the skin's moisturizing function (118). Topical application rich in linoleic and oleic acids can suppress transepidermal water loss changes, increase skin hydration, reduce skin erythema and protect against histological changes caused by UVR exposure (119).

N. sativa seeds contain various elements, such as Cu, Zn and Fe (100). Cu is a common cofactor for a number of enzymes and in the skin acts by provoking fibroblast proliferation, increasing collagen (types I, II and V) synthesis and acting as a cofactor for superoxide dismutase (SOD) to interfere with cellular oxidative effects (100,120). Zn also inhibits the accumulation of oxidative stress markers and proinflammatory cytokines. Zn ties up to the sulfhydryl groups of oxidation-protecting biomolecules and increases the activation of glutathione (GSH), catalase and SOD and decreases the activity of enzymes that increase oxidant levels, such as inducible nitric acid synthase (iNOS) and NADPH oxidase and slows the formation of peroxidation products, lipids (121). Iron plays a role in oxygen transport and oxidation-reduction reactions as well as in various oxygenases, including procollagen-proline dioxygenase, which is associated with the skin (122). Table I lists the active ingredients of N. sativa seeds and their mechanism against photoaging.

Table I Nigella sativa contents and their possible mechanism against photoaging.

Table I

Nigella sativa contents and their possible mechanism against photoaging.

Ingredients	Role against photoaging	Mechanism	(Refs.)
Thymoquinone	Prevents oxidative injury and prevent membrane lipid peroxidation	• Inhibits the activation of NF-κB	(97-99)
		• Works directly on the fibrillogenesis of type I collagen
Flavonoids (quercetin and kaempferol)	Interrupts free radical chain reactions and protect against UV radiation	• Inhibits MMP-1 mRNA levels	(101-105)
		• Inhibits ERK and p38 MAPK activation
		• Reduction in NF-κB
Saponins	Modulates inflammatory response, protects against ECM degradation, enhances collagen production and improves wrinkle appearance	• Inhibits MAPK signaling, AP-1, and NF-κB activation	(35,104)
Linoleic and oleic acids	Maintains water permeability barrier and epidermal integrity of the skin	• Suppresses UV-induced changes in TEWL, skin hydration, and erythema	(105,108,111)
Minerals (Cu, Zn and Fe)	Decreases oxidative stress biomarkers and inflammatory cytokines	• Cofactor for many enzymes against oxidants	(96,112,114)

[i] NF-κB, nuclear factor-kappa B; UV, ultraviolet; MMP-1, matrix metalloproteinase-1; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; ECM, extracellular matrix; AP-1, activator protein-1; TEWL, transepidermal waterloss; Cu, cuprum; Zn, zinc; Fe, iron.

Sunlight contains UVA and UVB, which reach the skin surface, triggering oxidative reactions that result in ROS (13). Excessive ROS accumulation in the skin activates the MAPK signaling pathway and the NF-κB pathway (123). The activated MAPK pathway triggers AP-1 production. ROS-induced AP-1 inhibits the TGF-β/Smad signaling pathway in dermal fibroblasts, which controls collagen synthesis to prevent a decrease in collagen production. TGF-β interacts with cell surface receptors and triggers the transcription factor Smad2/3. The phosphorylated Smad2/3 then connects and moves to Smad4, thereby triggering the transcription of genes responsive to TGF-ß. This causes a decrease in the phosphorylation and activation of the transcription factor Smad 2/3, which is required for the transcription of type I procollagen, a precursor of type I collagen, namely the COL3A1 and COL1A1 genes (1,55,57).

ROS and activated MAPK signaling pathways can also activate NF-κB; this activation can increase the expression of proinflammatory cytokines such as IL-1β, IL-6, TNF-α and COX-2 to regulate the inflammatory response and unbalance the MMPs/tissue inhibitors of metalloproteinases (TIMPs) ratio by activating MMPs and reducing TIMPs expression, thereby decompressing the ECM (6,31,41). NF-κB also increases the formation of the MMP1 enzyme to accelerate collagen degradation, especially type 1 collagen (31).

N. sativa contains an active ingredient, TQ, which is a potent antioxidant (6,9,10). The active substance TQ has a therapeutic effect by reducing oxidative stress and inflammatory responses, thereby preventing the activation of the MAPK and NF-κB pathways. Kaymak et al (124) reported that intraperitoneal administration of TQ 10 mg/kg bw in rats for 10 days showed a significant decrease in various inflammatory mediator levels, such as P38 MAPK and NF-kb, in the testes of rats induced by methotrexate. Activating NF-κB is directly associated with an increase in MMP-1(125). Chen et al (103) investigated the effect of TQ on MMP expression in an animal model of osteoarthritis. Accordingly, the downregulation of MMP-1, MMP-3 and MMP-13 expressions and the upregulation of TIMP-1 expression were reported due to the use of TQ in chondrocytes and cartilage in the animal model. The authors also showed that TQ could inhibit the NF-κB p65 protein level. MMP-1 can degrade fibrillar collagen in its three-helical domain, which causes the molecules to be thermally unstable so that they break down to form gelatin, which can then be degraded by other members of the MMP family (126).

Several photoaging therapies are often used with different working mechanisms. Topical retinoids during photoaging are mediated by their interaction with specific cellular and nucleic acid receptors. They improve photoaging by modifying cellular differentiation programs by increasing epidermal proliferation and thickening, compacting the stratum corneum and improving the deposition of glycosaminoglycans in the stratum corneum and intercellular spaces of the epidermis (127). Vitamin C is a naturally occurring antioxidant that has been shown to be effective for preventing and treating sun-damaged skin; it not only has an antioxidant, anti-inflammatory and photoprotective effect against UVA and UVB but also enhances collagen synthesis (128). The major adverse effects of topical retinoids and topical vitamin C are local skin irritation, including erythema, peeling, dryness, burning and itching, which depend on the concentration and formulation of the product (129,130). In addition, the use of retinoid agents is not recommended in pregnant women and topical vitamin C preparations are occasionally unstable, limiting their clinical use.

The summarized mechanism of N. sativa seed extract therapy in combating photoaging is shown in Fig. 1. N. sativa plays a protective role in inhibiting photoaging manifestations by modulating oxidative stress and inflammatory pathways. UV exposure induces the production of ROS, which subsequently activate the NF-κB and MAPK signaling pathways, leading to increased transcription of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6 and COX-2) and MMP-1, both of which contribute to inflammation and collagen degradation. However, N. sativa inhibits ROS generation, thereby downregulating NF-κB and AP-1 activity. These result in reduced inflammatory cytokine expression and decreased MMP-1 levels, preserving collagen density. Additionally, N. sativa enhances TIMPs and TGF-β receptor Smad signaling, which promotes the expression of collagen-related genes (COL1A1 and COL3A1). Collectively, these actions reduce inflammation, prevent collagen breakdown and promote skin integrity, ultimately inhibiting the visible manifestations of photoaging.

Figure 1 Possible mechanism of N. sativa and thymoquinone against photoaging. UV, ultraviolet; ROS, reactive oxygen species; NF-κB, nuclear factor-kappa B; MAPK, mitogen activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; AP-1, activator protein-1; TIMP, tissue inhibitor of metallopreoteinase; MMP-1, matrix metalloproteinase-1; IL-1ß, interleukin 1ß; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; COX-2, cyclooxygenase-2; TGF-ß, tumor growth factor-ß.

While multiple studies highlight the anti-photoaging effects of N. sativa, inconsistencies exist in its reported efficacy. Some studies emphasize strong antioxidant and anti-inflammatory benefits, while others suggest limited or unclear results (2,10,131-134). The concentration of TQ in different extracts varies widely, which may contribute to the heterogeneity in study outcomes. Furthermore, variations in study design, including animal models compared with human clinical trials, need further standardization to ensure comparability.

Despite promising preliminary findings, several research gaps remain. The optimal dosage of TQ for anti-photoaging effects has not been established and most studies lack standardized extraction methods. Future research should focus on well-controlled, long-term clinical trials to assess both efficacy and safety. Additionally, the interaction of N. sativa with other dermatological treatments (such as retinoids, vitamin C, or chemical peels) warrants further investigation to determine possible synergistic or antagonistic effects. A number of studies on N. sativa and photoaging have been conducted with small sample sizes, often in preclinical settings, making it difficult to generalize findings. Additionally, industry-funded research may introduce bias in reporting only positive results and there is a lack of large-scale, double-blind, placebo-controlled clinical trials. This raises concerns about publication bias, which may interfere the perceived efficacy of N. sativa in photoaging treatment.

5. N. sativa seed (black cumin) potential role against photoaging

The use of N. sativa, both orally and topically, has been widely studied and its use has been shown to have a potential role against photoaging. The potential antioxidant and anti-inflammatory effects are possibly caused by the active composition of N. sativa, TQ (6). There are two in vitro studies regarding antiaging and photoaging effects of TQ. Li et al (2) analyzed Thymocid®, a standardized TQ extract, which can inhibit the formation of advanced glycation end products, collagen cross-linking, collagenase and elastase activities in vitro. Thymocid® has potential as an antiaging property by maintaining its protein structure against glycation (135). In addition, Thymocid® can also protect the type I collagen structure from glycation-induced cross-linking; there is a possibility that its antiglycation effect may be because of the antioxidant effect of Thymocid® (136). Unfortunately, this study states that other phytochemical compounds besides TQ in Thymocid® are present, such as alipathic compounds, so there could be a bias that the effect is caused purely by the TQ compound, not the other compounds. In addition, further research on the effects of Thymocid® on elastase in human dermal fibroblast cells is needed to confirm its antiwrinkle effects.

Liang et al (10) then conducted research on TQ for its photoprotective action on human skin keratinocytes irradiated with UVA, finding that the use of pure TQ extract at doses of 6 and 12 µM could improve inflammation, oxidative stress, cytotoxicity and mitochondrial dysregulation caused by UVA radiation. From this research, it was concluded that TQ mechanism for skin damage caused by UVA radiation inhibits COX-2 expression by activating the NrF2/ARE pathway (10). The NrF2/ARE pathway is an important signaling pathway that reduces skin damage caused by UVB radiation (137). In addition, COX-2 is the main trigger of oxidative and inflammatory responses that are often detected in photoaging and other stress/inflammatory disorders (138). Nevertheless, this study suggested that high doses of TQ (>16 µM) may exhibit cytotoxicity to keratinocytes and the most effective protective concentration of TQ should be explored in further studies.

Pandey et al (131) investigated the methanol extract of N. sativa given orally at a dose of 200 mg/100 g bw/day in mice to determine its protective effect against radiation exposure. Oxidative stress caused by free radicals following ionizing radiation can damage normal cells; therefore, protecting normal tissue against radiation injury is important (139). This study showed that N. sativa can prevent radiation-induced increases in lipid peroxide levels in intestinal tissue homogenates. In addition, histological examination showed the prevention of villous loss, shortening of villous height and collagen deposition owing to radiation exposure (131). In this study, it was found that the use of N. sativa extract markedly increased the antioxidant properties of the irradiated rats compared with the control group. This study also stated that repeated oral administration of N. sativa extract for 14 days resulted in a survival rate of 100% up to 200 mg/100 g bw, so that this dose was considered the optimum dose. The antiaging effect of the oral administration of N. sativa extract was also supported by Khalaf and Mostafa (140), who administered N. sativa at a dose of 10 ml/kg bw/day to adult male rats exposed to cigarette smoke. Photoaging and tobacco smoke-induced aging are similar and include the adverse effects of oxidative stress (141). This study also found that N. sativa oil can prevent smoking-induced skin changes because of its antioxidant effects (140). In both studies, the antioxidant effect of N. sativa on organs other than the skin was due to free radicals originating from ionizing radiation and smoking. Furthermore, oral administration is not uniform, so further research is needed to assess the optimal dose of N. sativa extract.

Several studies have investigated the topical use of N. sativa extract and TQ as antiaging agents. Çanakci et al (132) used cold-pressed N. sativa oil extract at 100% concentration topically on the nasal mucosa of mice following exposure to radiotherapy radiation and found that its use could lower inflammatory cell infiltration, superficial erosion, vascular dilatation and exudates compared with the control. This study demonstrated the antioxidant and anti-inflammatory effects of N. sativa extract, even with topical use. In fact, in this study, histological images showed the protective role of N. sativa through its antioxidant, cytoprotective, anti-inflammatory and antineoplastic activities. Although this study did not review further the immunohistochemical and biochemical features, it is sufficient to confirm the role of N. sativa in preventing the detrimental effects of radiation.

Increased collagen density and accelerated wound repair in mice following topical application of N. sativa extract was studied by Turhan et al (133) who compared the use of N. sativa extract, nano-silver solution gold as the standard agent and a combination of the two agents applied twice daily for 15 days on 20 albino rats. The use of N. sativa extract alone resulted in improved collagen density and wound repair than that of the control group, although combined use had the best effect (133). In conclusion, N. sativa can play a synergistic role with other topical agents for wound healing. This research also suggests that N. sativa extract can be used as an agent that protects against sunburn and wounds caused by exposure to sunlight. In addition, the small sample size is a limitation of this study.

This wound healing enhancer effect was also supported by Han et al (142) regarding the topical use of 50% N. sativa oil cream twice daily for 14 days on the skin of mice with skin defects. The use of N. sativa extract reduced oxidant parameters, such as malondialdehyde (MDA), glutathione (GSH) and skin catalase (CAT) and increased antioxidant-supporting enzyme parameters, such as glutathione peroxidase (GPx) and SOD levels, markedly compared with controls. It can be concluded that N. sativa extract exerts wound healing effects through its antioxidant properties. Topical application of N. sativa in the form of oil cream at a concentration of 50% enhances the healing of open wounds in a reliable manner.

Algahtani et al (134) proved the effect of nanoemulsion-based hydrogel of TQ 0.5% (w/w) on collagen fibers in mice with skin defects. Nanoemulsion-based hydrogel systems provide more efficient drug delivery to increase the biopharmaceutical attributes of drugs that are difficult to dissolve. TQ, which is difficult to dissolve in water, can be encapsulated into the lipophilic environment of nano-dimensional oil droplets and stabilized to be more optimal (143). In this study, it was found that the use of nanoemulsion-based hydrogel systems provided higher and more regular collagen density compared with the use of the standard gel form TQ and the gold standard therapy, 1% silver sulfadiazine. All research summaries supporting the potential of N. sativa in fighting photoaging are summarized in Table II.

Table II Studies that support the potential role of Nigella sativa against photoaging.

Table II

Studies that support the potential role of Nigella sativa against photoaging.

First author, year	Subjects	Methods	Results	(Refs.)
Li et al, 2020	In vitro	Analysis of molecular process within glycation, collagen cross-linking, collagenase and elastase activities in murine melanoma B16F10 cells measured with collagenase activity assay kit.	Thymocid® (50, 100 and 300 µg/ml) interfered with the production of advanced glycation end-products, collagen cross-linking, collagenase activity, and elastase activities (type I and III)	(2)
Liang et al, 2021	In vitro	Analysis of oxidative stress and mitochondrial function on HaCaT cells irradiated by UVA using colorimetry, spectrophotometry, bioluminescence and dual-luciferase reporter assay. Cell viability was also evaluated using MTT and ELISA assay.	TQ pure extract (6 and 12 µM) not only significantly improved the UVA-induced cytotoxicity, oxidative stress and inflammation but also promoted mitochondrial dysregulation in HaCaT cells.	(10)
Pandey et al, 2015	In vivo, in rats	Radio-protective effect of oral Nigella sativa methanolic extract (200 mg/100 g bw/day) in albino rats 2 h before Total Body Irradiation at 4, 6, 10 Gy and continued until 7 consecutive days. They evaluated for histology and antioxidant parameters	Oral Nigella sativa methanolic extract (200 mg/100 g bw/day) significantly increased superoxide dismutase and catalase enzymes and inhibited lipid peroxidation activities.	(131)
Khalaf and Mostafa, 2014	In vivo, in rats	Twenty adult male rats were exposed to side and main stream smoke for 10 min twice daily for 4 weeks and treated orally with Nigella sativa oil 10 ml/Kgbw. Histological examination was performed.	Oral Nigella sativa extract 10 ml/kg bw/day had a protective role against tobacco smoking with collagen fiber content almost the same as the nonsmoking group (negative control).	(140)
Canakci et al, 2017	In vivo, in rats	Radio-protective effect of topical cold pressed Nigella sativa oil (0.05 ml at 100% concentration) given on days 1 to 3 to each nostril of female rats (n=18) after radiotherapy with a single dose of 40 Gy. Histopathological evaluation was measured.	Decrease of inflammatory cell infiltration, vascular dilatation, superficial erosion, and exudates in Nigella sativa group compared with the control.	(132)
Turhan et al, 2019	In vivo, in rats	Collagen density and wound closure were measured with histological morphometric analysis on skin defects in 20 albino rats. The wounds were treated with 2 ml cold-pressed Nigella sativa extracts twice daily for 15 days.	Higher collagen density was achieved in Nigella sativa group compared with the nano silver solution and control group. In addition, the combination of Nigella sativa and nano silver solution (1:1) had the highest collagen density among the groups.	(133)
Han et al, 2017	In vivo, in rats	A total of 42 female albino Wistar rats with skin defects were randomly treated with 50% Nigella sativa oil cream, 50% Hypericum perforatum oil cream, and placebo cream twice daily for 14 days. Histological and biochemical evaluations were performed.	A significant decrease in the MDA, GSH and skin CAT levels and an increase in GPx and SOD levels were found in the Nigella sativa group when compared with the other two groups on the 14th day of treatment.	(142)
Algahtani et al, 2021	In vivo, in rats	Collagen fiber was measured with histopathological analysis on 16 Wistar rats with skin defects treated with 0.5% TQ nanoemulsion-based hydrogel twice a day for 20 consecutive days.	Nanoemulsion-based hydrogel of TQ 0.5% (w/w) group had significantly more extensive and organized collagen fiber than the 1% silver sulfadiazine group.	(134)

[i] HaCat, human adult low calcium temperature; UVA, ultraviolet A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ELISA, enzyme linked immunosorbent assay; TQ, thymoquinone; Gpx, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, glutathione; CAT, catalase.

Various studies, both in vitro and in vivo, have shown that the use of N. sativa extract and its active substance, TQ, has a potential role against photoaging because of their antioxidant and anti-inflammatory effects (10,135,139,141). The antioxidant effect was demonstrated by improvements in parameters such as MDA, CAT and GSH levels and increased levels of the SOD enzyme (135,141). In several studies, the anti-inflammatory effect improved inflammatory parameters and histopathological features in irradiated mice (2,10,139). These two effects are thought to modulate collagenase and elastase activity, which in turn improves collagen density and skin hydration, especially in skin exposed to sunlight (2,137,140,142). A summary of the protective effects of N. sativa against photoaging is presented in Fig. 2.

Figure 2 Summary of potential role of N. sativa against photoaging. MDA, malondialdehyde; GSH, glutathione; CAT, catalase; TQ, thymoquinone; UVA, ultraviolet A; HaCat, human adult cutaneous tumor cell line.

These studies have several limitations that require evaluation. First, the use of N. sativa extract has not been standardized in terms of ingredients, extraction methods, ingredient content, oral and topical preparation and concentration of active ingredients contained therein; second, research on the effects of N. sativa against UV rays and photoaging is limited; third, the outcome assessment criteria are not yet standardized; and fourth, a number of studies use relatively small sample sizes, animal studies and have a high potential for bias. Further studies are needed, especially using human samples with large sample sizes and randomized control trial methods, to clarify the role of N. sativa as a photoaging protective agent. Nevertheless, from all available sources, it can be summarized that N. sativa extract and its active components may have some potential to counteract the detrimental effects of UV radiation.

6. Conclusion

N. sativa seeds contain a number of active ingredients and play a potential role in photoaging therapy. The mechanism underlying the accumulation of free radicals caused by UV rays is unclear. Essential active ingredients, including TQ, flavonoids, saponins, fatty acids and minerals, can participate in this effect. Their mechanism possibly not only inhibits the MAPK signaling and NF-κB pathways but also decreases the expression of proinflammatory cytokines in the inflammatory state. Blocking this pathway will reduce MMP1 production and collagen degradation. Several studies have shown that N. sativa seed extract and TQ have potential as antiaging agents. However, further research is needed to demonstrate its protective ability against photoaging. The use of N. sativa extract is still limited because of its preventive effects on photoaging, so further research is needed, especially in clinical trials, to determine the optimal dose or its use as a combination therapy.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by Maranatha Christian University, Bandung, Indonesia.

Availability of data and materials

Not applicable.

Authors' contributions

AE collected articles and wrote the first draft of manuscript, JWG initiated the study and revised the manuscript and TLW finalized the manuscript. Data authentication is not applicable. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References