Antioxidant and UV Protection Activities of Squid (Loligo sp.) Ink Powder Lotions

Skin is the largest part of the human body located at the outermost extent, protecting the body from external environments1. One of the possible environmental factors for skin damaged is ultraviolet radiation (UVR). In continuous exposure, UVR leads to some molecular damage (DNA photodamage) and clinical damage (erythema, tanning, skin cancer, photoaging) to the skin. UVR activates matrix metalloproteinases (MMPs), which are implicated in photoaging and collagen breakdown2,3. The topical formulation that contains filtering or scattering UVR is called sunscreen, and the efficacy can examine by measuring the sun protection factor (SPF)4,5. One of the body's photoprotective parts, which role as a broadband UV-absorbing agent, is melanin. Melanin is a human skin pigment that acts as a protection from UVA, UVB, and visible blue light, which has radical scavenging and antioxidant properties6. However, endogenous melanin does not adequately protect the skin, especially in tropical climate areas like Indonesia. Thus, it is necessary to use sunscreens containing exogenous melanin or melanin-related compounds or mimic endogenous melanin7. One of the melanins from natural sources known to have potential as free radical scavengers are squid (Loligo sp.) ink melanin8. Squid ink plays a significant role in eliminating intracellular excessive reactive oxygen Antioxidant and UV Protection Activities of Squid (Loligo sp.) Ink Powder Lotions


INTRODUCTION
Skin is the largest part of the human body located at the outermost extent, protecting the body from external environments 1 . One of the possible environmental factors for skin damaged is ultraviolet radiation (UVR). In continuous exposure, UVR leads to some molecular damage (DNA photodamage) and clinical damage (erythema, tanning, skin cancer, photoaging) to the skin. UVR activates matrix metalloproteinases (MMPs), which are implicated in photoaging and collagen breakdown 2, 3 .
The topical formulation that contains filtering or scattering UVR is called sunscreen, and the efficacy can examine by measuring the sun protection factor (SPF) 4,5 .
One of the body's photoprotective parts, which role as a broadband UV-absorbing agent, is melanin. Melanin is a human skin pigment that acts as a protection from UVA, UVB, and visible blue light, which has radical scavenging and antioxidant properties 6 . However, endogenous melanin does not adequately protect the skin, especially in tropical climate areas like Indonesia. Thus, it is necessary to use sunscreens containing exogenous melanin or melanin-related compounds or mimic endogenous melanin 7 .
One of the melanins from natural sources known to have potential as free radical scavengers are squid (Loligo sp.) ink melanin 8 . Squid ink plays a significant role in eliminating intracellular excessive reactive oxygen species (ROS) and improve its antioxidant ability 9, 10 . It also has anti-retroviral, anti-inflammatory, antimicrobial activity, and other traditional uses 11 . Antioxidants can protect against photo-induced radical reactions, thereby helping sunscreens in inorganic and organic UV filters 12 .
Also, there was a positive relation and linear correlation between sunscreen and antioxidants 13 .
There is currently no research on using squid ink as an active ingredient in sunscreen products because of its limitation in the low solubility of organic solvent and water 8 . UV-absorbing and antioxidant activity within squid ink may be used as the active compound in sunscreen lotion to increase its benefit and utilization.
Lotions are emulsion dosage forms for external application to the skin, which has many characteristics like creams. Lotions consist of an oil-in-water emulsion, water washable, and widely acceptable cosmetically 14 .
Therefore, this study investigates the physicochemical characteristics, stability, antioxidant activity, and UV protection effectivity of squid ink powder in lotion preparations.

Materials
Squid from Sendang Biru beach was purchased from the local market in Malang city, East Java, then dried to a dry squid ink powder. The squid used was determined in the

Squid ink powder preparations
Freshly obtained squids were dissected, and ink glands were manually removed from the viscera. The 50 g of ink squid was added with 100 mL HCl 0.5 M in a tight light condition. The solution was then stirred using a magnetic stirrer for 30 minutes then stored for 24 hours at 10°C 9 .
After 24 hours, the solution was centrifuged for 15 minutes and dried and stored in the climatic chamber at 60°C 15 .

Squid ink powder lotion preparation
The oil-in-water lotion was prepared with the composition as shown in Table I 19 .

Antioxidant DPPH scavenging activity test
This method was adapted and modified from Fatimah Zaharah and Rabeta 20 as well as Saputri et al. 21

Physicochemical evaluation of squid ink powder lotion
The squid ink powder lotions had a soft texture, black in color, and citrus scent. The black color of these lotions follows the squid ink powder's color, and formula III had sharper color than formula II and I, as shown in Figure 1.
Visually, the squid ink powder lotions had no coarse particle, which indicated that the preparations were homogenous. The measurement of pH value, viscosity, and lotion spreadability was shown in Table II   Stability testing of squid ink powder lotion

Real-time method
The results of real-time stability testing of squid ink powder lotions showed no changes in color, odor, and phase separation after storage at 4°, 30°, and 40°C.
Significantly, the pH values were affected by the addition of squid ink powder and the storage temperature. Table III showed that the pH value of squid ink powder lotions was decreased over time, but the preparations were most stable at 30°C storage.    26 . The freeze-thaw cycling test implies that the antioxidant DPPH scavenging activity of squid ink powder lotions was decreased but not significantly.

UV protection effectivity test
Erythema is induced by UV-B radiation causes cellular immunologic changes that lead to blood vessel dilation 22 .
As shown in Table VII, this study showed that the squid ink powder lotions significantly decrease in the erythema area compared to the negative control group. These lotions were able to inhibit the effects of acute UV exposure. The presence of antioxidant activity could explain this finding. Antioxidant activity had been shown to enhance protection against UV-induced DNA damage by reducing oxidative stress and inhibiting NF-kB. It also neutralizes the UV-induced free radicals. It made this agent could play a role as potential "nonsunscreen" agents 27 . and also inhibited UV exposure. It indicates that these squid ink powder lotions had the potential as a sunscreen product.