337 Photoprotective Effects of Carotenoid
PIGMENT EXTRACTION
Extraction of the carotenoid was done by a solvent extraction method using ethanol as
a solvent.19 Briefly, R kroppenstedtii was grown in Tryptic soy broth at 37°C for 96 hours
at 110 rpm. The biomass was collected by centrifugation at 4,500 rpm for 10 minutes.
The cell pellet was washed twice with distilled water, and carotenoid (0.38 µg/g) was
extracted using ethanol. The UV–Vis absorption spectrum of the orange-colored
extract displayed absorbance maxima of 475 nm, indicating the presence of carotenoids
(Figure 1). Furthermore, the carotenoid extract was partially purified by silica gel column
chromatography using petroleum ether:acetone (9:1) as a mobile phase. The fraction
obtained was characterized by high-resolution liquid chromatography and in silico analysis
and identified to be 1’OH-4-keto-ϒ-carotene.20
PREFORMULATION STUDIES
Assessment of antioxidant activity of carotenoid extract. The DPPH radical scavenging
activity (RSA) of the varying concentrations of the carotenoid extract (100–1,000 µg/mL)
was assessed by the method of Devyani et al.21 The carotenoid extract was mixed with
equal volumes of DPPH reagent and incubated at 37 ± 2°C in darkness for 30 minutes.
Absorbance was measured at 517 nm using methanol as a blank. A mixture of DPPH and
methanol (1:1) was used as a control, and standard ascorbic acid (0.25–8 µg/mL) was used as
positive control. The percent inhibition of DPPH RSA was calculated using the following
formula (eq. 1):
RSA /A (()×100 %)=-A A
0 1 0 (1)
where A
0 is the absorbance of control and A
1 is the absorbance of the test sample. Calibration
curves of RSA (%)against the standard ascorbic acid and carotenoid extract were plotted.
The IC
50 values of carotenoid extract and standard ascorbic acid were determined from their
respective calibration curves.
Figure 1. Absorption spectrum of the carotenoid extract of R kroppenstedtii.

338 JOURNAL OF COSMETIC SCIENCE
To determine the ferric ion reducing antioxidant power (FRAP) of the carotenoid extract,22
1.0 mL of extract (100–1,000 µg/mL) was mixed with 1 mL of FRAP reagent, and the
mixture was incubated at 37 ± 2°C for 30 minutes. The increase in absorbance of the
blue-colored complex was measured at 593 nm. Ascorbic acid (10–100 µg/mL) was used
as a positive control. A linear equation obtained from a standard ascorbic acid calibration
curve was used to determine the FRAP (μg/mL) of carotenoid extract in terms of ascorbic
acid equivalence.
Cytotoxicity studies of carotenoid extract on human epidermal keratinocyte cell line. Human
epidermal keratinocyte (HaCaT) cells were grown in Dulbecco’s Modified Eagle Medium
containing 10% fetal bovine serum supplemented with penicillin-streptomycin solution
at 100 U/L.23 Briefly, HaCaT cells were plated in 96-well plates with a seeding density
of 3.0 × 103 cells/100 µL of complete media, from a running culture. The plates were
incubated in a CO
2 incubator at 5% CO
2 ,37 ± 1°C for 24 hours. Carotenoid extracted
from R kroppenstedtii biomass was dried to completely remove ethanol and reconstituted
in DMSO (vehicle) to obtain a stock solution (1,000 µM). The stock solution of carotenoid
extract was further diluted (0.5, 1, 2, 5, 10, 20, 50, 75, and 100 µM) in complete media.
To assess the safety of extract, the carotenoid extract (0.5–100 µM) was tested on HaCaT
cells for 24 and 48 hours. At the end of the incubation period, the cell medium was
removed, and 20 µl of MTT solution (5 mg/mL in PBS) was added to each well. Cells were
further incubated at 37 ± 1°C for 3 to 4 hours in a light-protected manner. At the end
of the incubation, MTT solution was removed, and 50 µL/well of DMSO was added for
solubilization of the formazan crystals. Absorbance was recorded at 570 nm using a Bio-
Rad microplate reader, version 680 (Bio-Rad Laboratories, Hercules, CA).
In silico absorption, distribution, metabolism, excretion, and toxicity prediction. To understand
the likely effects of active compounds on human health as well as on the environment,
animal testing becomes imperative during product development. However, experiments
using animals involve challenges with respect to animal procurement, along with stringent
laws and ethical concerns over their use.24 Therefore, computer simulations can be used as
tools to predict the toxicity of a potential therapeutic prospect without sacrificing animals
at the level of screening.24 In vivo testing can then be confined to the most promising
active compounds identified after primary screening using in silico tools. A more realistic
forecast of a chemical’s ability to elicit an action in vivo is based on the combination of
data on absorption, distribution, metabolism, excretion, and toxicity (ADMET). In this
investigation, the ADMETlab 2.0 (Xiangya School of Pharmaceutical Sciences, Central
South University, Changsha, China) prediction tool25 was used to determine the safety of
1’OH-4-keto-ϒ-carotene, which was found to be the major carotenoid of R kroppenstedtii.
SUNSCREEN CREAM FORMULATION AND ITS PREPARATION
The cream base was prepared by emulsifying the oil and aqueous phases. Table I represents
the formula used for preparation of the sunscreen cream. In brief, the two phases (oil and
water) were individually heated in a water bath at 60°C, respectively. The aqueous phase
was mixed with the oil phase with continuous agitation,26 and 10% carotenoid extract
was added and stirred constantly until a thick cream was obtained. A base cream (Bc)
(without carotenoid extract) was used as control. The Bc (labeled as Bc) and the cream with
10% carotenoid extract (labeled as BcCE) were evaluated for quality parameters, and all