bioengineering

Article
Eco-Friendly Sustainable Nanocarriers to Treat Oxidative
Stresses and Skin Aging-Related Ailments, Valorization of
a By-Product
Zaheer Ullah Khan 1 , Taous Khan 1 , Hira Khan 2 , Naveed Ullah Khan 3 , Yang Ding 4 , Atif Ali 1, * and Jiang Ni 5, *

1 Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus,

Abbottabad 22060, Pakistan; zaheerkhan2092@[Link] (Z.U.K.); taouskhan@[Link] (T.K.)
2 Department of Pharmacy, Abbottabad University of Science and Technology, Havelian,
Abbottabad 22500, Pakistan; hirakhan@[Link]
3 Department of Pharmacy, CECOS University of Engineering and Emerging Sciences,
Peshawar 25000, Pakistan; naveedkhan1676@[Link]
4 College of Pharmacy, Pharmaceutical Series, China Pharmaceutical University, Nanjing 210000, China;
dydszyzf@[Link]
5 Department of Pharmacy, Affiliated Hospital of Jiangnan University, Wuxi 214000, China
* Correspondence: atifali@[Link] (A.A.); nj1876348@[Link] (J.N.); Tel.: +92-0992-383591-6 (A.A.);
+86-13511646862 (J.N.)

Abstract: The peel from Citrus-sinensis L. is a medicinally significant food waste, and its extract (O-
Ext) could be significant against oxidative stresses and skin aging, However, the penetration barriers,
instability in formulation, undefined toxicities, and enzymatic activities make the O-Ext difficult
to formulate and commercialize. The goal of this study was to evaluate O-Ext against oxidative
stress, prepare O-Ext-loaded nano-lipid carriers (O-NLCs), and load them into topical O/W-emulsion
(O-NLC-E) to improve O-Ext permeation and its in vivo antiaging effects. TPC, TFC, DPPH activity,
and mineral/metal contents of O-Ext were determined via atomic-absorption spectroscopy. For
bioactive compounds profiling, GC-MS analysis was carried out. O-NLCs were prepared and tested
Citation: Khan, Z.U.; Khan, T.; Khan, for physicochemical attributes, while HaCaT and fibroblast cells were used to study permeation
H.; Khan, N.U.; Ding, Y.; Ali, A.; Ni, J.
and cytotoxicity. The kinetic characteristics of ex vivo permeation through rat skin were established,
Eco-Friendly Sustainable
following the Higuchi model. Following written consent, safety investigations were conducted
Nanocarriers to Treat Oxidative
on human volunteers for three months, where optimized O-NLC-E and B-NLC-E were regularly
Stresses and Skin Aging-Related
Ailments, Valorization of a
applied on cheeks. Non-invasive procedures were used to assess the volunteer’s skin erythema,
By-Product. Bioengineering 2023, 10, TEWL, sebum level, melanin, hydration, pH, elasticity, and pore sizes after specified intervals.
798. [Link] The results demonstrated that applying O-NLC-E formulation to the skin of volunteers directed
bioengineering10070798 significant antiaging benefits. The study offers nanotechnology-based sustainability approach against
skin ageing.
Academic Editors: Ali Zarrabi and
Noe T Alvarez
Keywords: Citrus sinensis L. peels; NLCs; oxidative stress; skin aging; non-invasive skin investigations
Received: 21 May 2023
Revised: 18 June 2023
Accepted: 28 June 2023
Published: 3 July 2023 1. Introduction
Skin aging is a natural process and major cause of many degenerative diseases. The
utmost instinctual fallout of the aging is exteriorized on the skin, which can be debilitating
Copyright: © 2023 by the authors.
for the structure, function, and appearance of skin. It is directly related to ugly appearance,
Licensee MDPI, Basel, Switzerland. increased number of wrinkles, laxity, emotional discomfort, elastosis, and telangiectasia.
This article is an open access article Various intrinsic and extrinsic factors can accelerate the skin aging by promoting oxidative
distributed under the terms and stresses via enhancing the free radical concentration than normal. The overproduction of
conditions of the Creative Commons free radicals’ and oxidative stresses may be controlled via antioxidants and anti-oxidative
Attribution (CC BY) license (https:// enzymes such as superoxide dismutases (SOD), GST, GSH, POD, and catalase. Enriched
[Link]/licenses/by/ extracts from natural compounds demonstrate good antiaging activity by reducing free
4.0/). radicles and oxidative stress, and mostly exhibit high safety profile. Due to excellent

Bioengineering 2023, 10, 798. [Link] [Link]

Bioengineering 2023, 10, 798 2 of 22

biosafety, natural compounds based pharmacotherapeutics are in large demand, and this
demand is increasing day-by-day throughout the globe [1,2]. Natural bioactive compounds
can be found in the food wastes, also called biowastes, and their effectual use for human
health will be an excellent sustainability approach. It has been reported that guava, grapes,
and citrus fruits produce 10, 20, and 30–50% of waste, respectively, compared to their total
mass [3]. The utilization of these biowastes’ extraction as antiaging agents can lessen the
environmental load, as well as the stress on medicinal plants, which are already 100-fold
greater than the extinction rate [4]. It is a great alternative to costly imported natural
antiaging agents and a step towards the use of sustainable and green resources.
Orange (Citrus sinensis L.) peel is a major industrial waste [5] containing many
flavonoids, such as hesperetin, hesperidin, neodiosmin, nobiletin, kaempferide, naringenin,
vanillin, rutin, and other phenolic acids, such as chrologenic, caffeic, ferulic, cinnamic,
ascorbic acid, vanillic, and p-coumaric [6,7]. Moreover, orange peels extract (OPE) shows
promise within the dermatology arena [5] as an antioxidant, as well as demonstrating
anti-carcinogenic, anti-inflammatory, and anti-aging agents [7,8]. It has been also reported
to demonstrate tyrosinase inhibitory activity, which regulates melanin formation in the skin
and, thus, could be used as skin whitening agent [6,9]. In conventional topical formulations,
OPE have some limitations such as low permeation across stratum corneum, enzymatic
degradation, and lower chemical stability [10].
Nanotechnology is an emerging drug delivery tool and can be effectually utilized
for the delivery of natural bioactive compounds. Nanostructured lipid carriers (NLCs)
represent an innovative delivery system made up of lipids materials, surfactants, and
cosurfactants that may be administered topically, dermally, or trans-dermally. In topical
applications they have some unique properties such as increased skin occlusion, increased
skin hydration and elasticity, improved skin permeation, drug targeting, improved bene-
fit/risk ratio, improved UV blocking activity, and improved chemical stability of chemically
labile compounds [11,12]. The formulation components are safe and fall under the Food
and Drug Administration’s “generally recognized as safe” (GRAS) [13]. The use of green
surfactants in the preparation of NLC containing extract converts them into green formula-
tions with fewer side effects and low cost. The NLCs formulations with green surfactants
can be the most suitable delivery system for bioactive compounds and will be an impactful
sustainability approach.
In this study, a sustainability approach was introduced, where orange peels extract
was evaluated for antiaging activity and heavy metals concentration (safety). They were
then loaded into NLCs (O-NLC) based on green surfactants (rhamnolipids) and character-
ized before loading into a secondary emulsion-based formulation (topical O/W emulsion)
(O-NLC-E) as explained in Figure 1. For further assessments, physico-chemical characteri-
zation of O-NLC-E was performed and their ex vivo permeation was analyzed. For the
in vivo evaluation, human volunteers were considered after their written consent were
given and safety studies were conducted. The results showed a good safety profile and
high antiaging efficacy. This sustainability approach was introduced; 1 for the shelter of
substantial bioactive compounds when applied to skin, 2 to overcome the penetration bar-
riers and improve the bioavailability of O-Ext, 3 to avert the enzymatic interaction, 4 to
keep smooth antioxidant activities against oxidative stresses, 5 to improve economic and
social acquiescence, and 6 providing the topical treatment with ease to fend off oxidative
skin problems and early skin aging. Here, we offered a unique sustainability approach to
defend the skin aging with naturally existed potential and superb biosafety.
Bioengineering 10, 798
2023,2023,
Bioengineering 10, x FOR PEER REVIEW 3 of324of 22

Figure
Figure 1. [Link]
Citrussinensis
sinensispeels
peels extract
extract was
was evaluated
evaluatedfor
forphytochemicals
phytochemicals (GC‐MS,
(GC-MS, TPC, TFC).
TPC, TheThe
TFC).
extract was then loaded into nanocarriers and delineated for in vitro and ex vivo evaluations.
extract was then loaded into nanocarriers and delineated for in vitro and ex vivo evaluations. The The
in vivo efficacy was confirmed via human volunteers for non‐invasive skin anti‐aging parameters.
in vivo efficacy was confirmed via human volunteers for non-invasive skin anti-aging parameters.

2. 2. Materialsand
Materials andMethods
Methods
2.1.
2.1. Materials
Materials
Dimethyl‐sulfoxide (DMSO),
Dimethyl-sulfoxide (DMSO),HH 2O,O,
2
HCl, NaOH,
HCl, DPPH,
NaOH, folin‐ciocalteu
DPPH, reagentreagent
folin-ciocalteu gallic acid
gallic
(GA),
acid AlCl
(GA), 3, ascorbic
AlCl 3 , acid
ascorbic (AA),
acid FeCl
(AA), , sodium
3FeCl
3 , phosphate,
sodium C2H
phosphate, 3OC2H
2K, HNO
O
3 2
3, HClO
K, HNO 4, ,phos‐
3 HClO 4,
phate buffer,
phosphate Cetyl
buffer, alcohol,
Cetyl methanol,
alcohol, and Rhamnolipid
methanol, 90 (Rh‐90)
and Rhamnolipid were purchased
90 (Rh-90) from
were purchased
Sigma‐Aldrich,
from whilewhile
Sigma-Aldrich, Folin–Ciocalteu reagentreagent
Folin–Ciocalteu (FCR) and Na2and
(FCR) CO3Na were acquired from Merck
2 CO3 were acquired from
(Dam‐stadt, Germany). Analytical‐grade pyragallol, dTNB, H 2O2, EDTA, Hank’s balanced salt
Merck (Dam-stadt, Germany). Analytical-grade pyragallol, dTNB, H2 O2 , EDTA, Hank’s
solutionsalt
balanced (HBSS), lipopolysaccharide
solution (LPS), and cDNB(LPS),
(HBSS), lipopolysaccharide were purchased.
and cDNB were purchased.
2.2.
2.2. Methods
Methods
2.2.1.
2.2.1. PreparationofofCitrus
Preparation Citrussinensus
sinensus L.
L. Peels
Peels Extract
Extract
Orangepeels
Orange peelswere
werecollected
collected and
and shade
shadedried
driedafter
afterwashing
washing byby
distilled water;
distilled then,
water; then,
dry peels were ground into a fine powder which was then extracted using methanol
dry peels were ground into a fine powder which was then extracted using methanol (10:1, (10:1,
v/w)via
v/w) viastirring.
stirring. Extract
Extract was
was filtered
filteredout
outand
andconcerted
concerted forfor
drying viavia
drying a rotary‐evaporator.
a rotary-evaporator.
The percentage yield was calculated via Equation
The percentage yield was calculated via Equation (1). (1).
𝑊𝑂𝐸
𝑊𝑂 % WOE 100 (1)
WO (%) = 𝑊𝑂𝑃 × 100 (1)
WOP
where WO% is the percent yield of orange peels extract (O‐Ext), WOP is the weight of
orange
where peelsispowder,
WO% and WOE
the percent yieldisof
theorange
weightpeels
of dryextract
extract.(O-Ext), WOP is the weight of
orange peels powder, and WOE is the weight of dry extract.
2.2.2. Total Phenolic Contents (TPC)
2.2.2. Total Phenolic
The TPC Contents
of O‐Ext (TPC) via the FCR [14,15]. A volume of 110 μL of extract
was ascertained
andTheFCR wasof
TPC shifted
O-Extintowasthe 96‐wells plate
ascertained viaand
the at
FCR37 °C incubated
[14,15]. for 5 min.
A volume Then,
of 110 of2CO
µLNa extract
3

(7%
and FCRw/v)was
wasshifted
added into
for athe
final96-wells
volume plate
of 200and
μL. at 37 ◦the
Then, mixture incubated
C incubated for 5 [Link] were
Then, Na 2 CO 3
taken
(7% w/v)at was
765 nm (Biotech
added for aUSA,
finalHungry,
volumemicroplate
of 200 µ[Link]
Then,Elx
the800). The TPC
mixture were stated
incubated as
readings
milligrams
were taken atof765
gallic
nm acid (standard)
(Biotech USA,equivalents
Hungry, per gram of dry
microplate O‐Ext
reader [14].
Elx 800). The TPC were
stated as milligrams of gallic acid (standard) equivalents per gram of dry O-Ext [14].

2.2.3. Total Flavonoid Contents (TFC)

The TFC of O-Ext was ascertained via the calorimetric method [14,15]. An amount of
5 mg of O-Ext was dissolved in 5 mL of methanol to prepare stock solution. Next, 40 µL
of O-Ext, 10% aluminium chloride, and 1M C2 H3 O2 K solution were shifted into a 96-well
Bioengineering 2023, 10, 798 4 of 22

microplate, followed by the addition of 160 µL of distilled water to bring the finalvolume up
to 0.2 mL, which was then incubated for 30 min. A microplate reader was used to measure
absorbance at 415 nm (Biotech USA, Elx 800). The TFC were expressed as milligrams of
quercetin equivalent per gram of dry O-Ext [14].

2.2.4. Antioxidant Assay

The O-Ext antioxidant capacity was determined using the DPPH-assay [16]. Then,
100 mM solution of DPPH (10% DMSO) was prepared and 90 µL of the solution was
mixed with O-Ext in a 96-well plate and at 37 degrees Celsius and incubated for 0.5 h [17].
The absorbance drop was assessed at 517 nm using a microplate reader (Synergy HT
BioTek® USA, Hungry). Ascorbic acid (AA) was used as a standard. Percent inhibition was
calculated by Equation (2).

( Abs o f − ve control ) − ( Abs o f + ve control )

Inhibition (%) = × 100 (2)
( Abs o f − ve control )

2.2.5. Atomic Absorption Spectroscopy

The extract was analyzed via Atomic Absorption Spectrometer AAnalyst (PerkinElmer,
Waltham, MA, USA) for different elements examination. 500 mg of O-Ext was added to
a conc. HNO3 and HCL mixture (3:1). The mixture was heated up to 85 ◦ C and 1 mL of
HCLO4 added. Then, distilled H2 O was mixed to attain a final volume of 50 mL, which
was injected into the sample for analysis [14,18].

2.2.6. Identification of Compounds in O-Ext via GC-MS

Utilizing GC-MS analysis, compounds in the O-Ext were analyzed and screened.
The GC-MS experiment was carried out according to protocol [14]. Identification was
confirmed using calculated fragments, molecular mass, and molecular structure. Mass-
spectrum analysis was performed using a database from the NIST-library, containing more
than 62,000 molecular patterns [14].

2.2.7. Effect of Extract on Oxidative Stress; Superoxide Dismutases (SOD), GST, GSH, POD,
and Catalase
Peritoneal macrophages of mouse were activated with LPS to examine the O-Ext effect
on oxidative-stress indicators such as GST, GSH, SOD, POD and catalase, as published
previously [14]. GSH was calculated by adding the cDNB as previously published, and
quantification was carried out at 340 nm. While using dTNB, the GST was quantified
350 nm. Catalase was analyzed at 240 using H2 O2 buffer. The SOD concentration was
assessed by mixing Tris-EDTA (50 mM and pH 8.5) and pyragallol (24 mM), and the
absorbance was noted at 420 nm. The SOD contents were determined using pyragallol
(24 mM) in 50 mM tris-EDTA buffer (pH 8.5) at 420 nm. The POD contents were calculated
as previously published [19].

2.2.8. Preparation of NLCs

Selection of Solid Lipid and Liquid Lipid
The O-Ext solubility in different solid lipids (cetyl alcohol, precirol ATO-5, Compri-
tol888ATO, Glycerin monostearate, bee wax and stearic acid) was determined by dissolving
it, in ascending order in defined quantity with stirring (100 rpm) till saturation after 24 h of
stirring at 85 ◦ C. In the case of oils (olive oil, oleic acid, MCT, sesame oil, avocado oil and
black seed oil), the solubility was determined and oil with high solubility were assigned
for further process [20].

Solid Lipid and Oils Compatibility

To analyze the compatibility of the selected solid lipids (SL) and oils, a screening test
was carried out. SL were melted and mixed with oils at high temperature and then cooled
Bioengineering 2023, 10, 798 5 of 22

down in a vial with horizontal rotation to form a film on the wall of vial. The crystals
formation due to the incompatibility and phase-separation (PS) were observed [21].

Surfactant and Cosurfactant Selection

To select most suitable surfactant, 300 mg of tween20, tween80, Pluronic F68, Polox-
amer188, and rhamnolipids 90 were added to the lipid mixture (300 mg) consisting of 85%
solid lipids and 15% oil, and gently heated. Then, 50 mg of the mixture was diluted via
H2 Od up to 50 mL; the stability of the prepared emulsion was observed up to 2 h. The
transmittance (clarity) was observed at 638 nm and surfactant giving high transmittance
was used for further experiment [22,23]. Soya lecithin was selected as a cosurfactant as
reported previously in our lab [24].

Development of NLCs
The high shear rate homogenization approach was employed to generate NLCs as
described previously [14]. The organic phase (OP) was formed by fully dissolving lipids
mixture, soya lecithin in ethanol, and extract. The OP was heated and added drop by drop
to the aqueous-phase (AP) containing surfactant during homogenization at 10 k rpm. The
resulting dispersion, containing O-Ext loaded NLCs (O-NLCs), was sonicated and cooled
to room temperature. The dispersion was lyophilized to obtain pure O-NLCs. Unloaded
NLCs (B-NLCs) were synthesized without extract [10,25]. To avert damage, 3.0 percent
mannitol was used as a lyoprotectant [26].

Optimization of NLCs
To obtain optimized NLCs, the Box–Behnken model was used; 15 Formulations were
produced to find out the effect of independent factors (amount of lipids mixture, solid lipid
(cetyl alcohol) amount in lipids mixture, surfactant amount) on dependent factors (Size
(nm), zetapotential (mV) and polydispersity index) as shown in Table S2. The composition
of produced formulations and their results are shown in Table S1. Suggested optimized
formulation with predicted and actual results are shown in Table S3.

2.2.9. O-NLCs Characterization

Zetapotential, Size and Polydispersity Index (PDI)
B-NLCs and O-NLCs were diluted with H2 Odd (X20). The physicochemical properties
were found out via Nano-ZS90 (Malvern Instruments Ltd., Malvern, UK) at 25 ◦ C [26].

Entrapped Phenolic Contents

Free or unentrapped amount of O-Ext was determined in O-NLCs dispersion via
centrifugation (5000 rpm) of dispersion in 15 mL MILLIPORE tube of 30,000 MWCO. The
PC was determined in collected solution at 765 nm using UV visible spectroscopy [27]. The
percent EE was calculated using the given Equation (3):

PC total − PC f ree
EP (%) = × 100 (3)
PC total

where, PCtotal = total amount of phenolic compounds, PCfree = amount of lipids.

Fourier Transmission Infra-Red Spectroscopy

To confirm the entrapment of O-Ext in O-NLCs and the interaction between B-NLCs
components and extract, FTIR-spectroscopy (Tensor 27, Bruker, Nijmegen, The Netherlands)
was performed [26,28].

Morphological Observation
Transmission-electron-microscopy (JEM-1200EX, Jeol, Groningen, The Netherland)
was utilized to examine the morphology of O-NLCs [29].
Bioengineering 2023, 10, 798 6 of 22

Cytotoxicity Studies via MTT Assay

For cytotoxicity experiments, human-keratinocytes (HaCaT) and fibroblast cells were
utilized [14]. These were grown in DMEM and RPMI 1640, respectively. In incubation,
both cultures were supplemented with FBS, streptomycin, and penicillin [30]. Following
incubation, the medium was sucked out, and replenished with new medium. Test samples
were added and incubated for the appropriate duration. The MTT solution was added
before incubation and gently shaken for 15 min. The absorbance was measured at 570 nm,
and viability of cells was computed using Equation (4) [31].

OD values o f dosing group − blank

Cell viability (%) = × 100 (4)
Control group − blank

Cells Permeation Studies

HaCaT cells were cultivated into a 24-well Transwell insert at a concentration of
105/cm2 to discover O-NLCs’ permeation, as described in our previous publications [14,24].
Later, between 17 and 21 days, the transepithelial-electrical-resistance was assessed using
an EVOM-ohmmeter (Sarasota, FL, USA) to examine the stability of the cells. Cells were
washed off three times before adding 60 µg of O-Ext-E (Emulsion containing O-Ext), and
0.2 mL HBSS (300 µL/mL) O-NLCs to the top of the Transwell and 1.0 mL HBBS to the
lower chamber. Cells were then kept at 37 degrees with horizontal shaking at 50 to 60 rpm
per min. A volume of 500 µL of HBSS was collected at various intervals (1–6 h), and
equivalent quantity of HBSS was replenished in the inferior chamber. The materials were
freeze-dried. As previously stated, the PC was evaluated [27]. The carriage mass (Q) was
computed via Equation (5):

Qm = Ci × V1 + ∑ Ci − 1 × 0.5 (5)

where, “Qm” signifies the carriage transport-mass (µg); “V1” is the mass of solution in
inferior-chamber; and Ci is the PC (µg/mL) [24,32].

2.2.10. Preparation of O-NLCs Loaded O/W Emulsion

An emulsion is a colloidal system of a minimum of two non-miscible liquids where one
is dispersed in another one [33]. To prepare the o/w emulsion, water phase was heated on
a hot plate above 70 ◦ C, containing sodium behenoyl lactylate 3.00% (emulsifier), vegetable
glycerin 5.00% (humectant), acacia senegal gum 2.00% (polymer), sodium benzoate 1.00%
(preservative), glyceryl stearate 2.00% (emulsifier), and liquid paraffin 5.00% (emollient and
moisturizer). Then, oil phase was added drop wise while stirring; 2.0% silica was added as
a sensory modifier. The system was then homogenized by high shear homogenizer (Ultra
Turax) for 10 min at 15,000 rpm. During homogenization, O-NLCs and O-Ext (maintaining
2% O-Ext concentration) were added to produce O-NLCs loaded emulsion (O-NLC-E) and
O-Ext loaded emulsion (O-E); while in production of B-NLCs loaded emulsion, B-NLC-E
unloaded NLCs (B-NLCs) were added. At the end, the homogenizer speed gradually
reduced to avoid the formation of bubbles, and the temperature of the colloidal system was
reduced to room temperature [34].

Characterization of O-NLCs-E
• Dilution test and microscopic evaluation
Emulsion was diluted with distilled water to confirm the emulsion type. The micro-
scopic studies of diluted O-NLC-E were performed using optika microscope and images
were taken with an attached camera [35].

Stability Studies
• Accelerated Thermodynamic stability studies
Bioengineering 2023, 10, 798 7 of 22

Accelerated thermodynamic stability tests are known worldwide as suitable for guess-
ing the shelf-life of formulations [36]. In thermodynamic stability studies, 3 cycles of
centrifugation (C) at 5000× g rpm for 5 min and heating–cooling (HC) cycle, and 4 ◦ C
for 48 h, followed by 45 ◦ C for 48 h, were conducted. In freeze–thaw (FT) cycles, the
formulation was kept [37].

pH
The pH is regarded as an eminence criterion for topical formulations, especially emul-
sions [38]. The pH of O-NLC-E was assessed directly with a pH-meter (HANNA EDGEPH,
HANNA, Padua, Italy). Readings were taken in triplicate over a 90-day period to assess
pH fluctuation at various storage temperatures, namely, 8, 25, 40, and 40 ◦ C + 75Rh [27].

Chemical Stability
Chemical stabilities of O-NLC-E were studied via the finding of PC, comprising
its chief compounds by UV spectroscopy. PC (%) drop after storing was estimated via
Equation (6):
Mpc − Mo
Percentage reduction (%) = × 100 (6)
Mo
where Mpc is PC amount of afterward storage (mg/L) and Mo before storage (mg/L) [37].

Ex Vivo Diffusion Studies

For ex vivo diffusion analysis of O-E and O-NLC loaded emulsions, the 11 mL of
PBS (pH 5.5) was filled in a receptor-compartment of Franz’s diffusion cell and 1 g testing
emulsion in a donor-compartment. To avoid evaporation, the formulation was wrapped
with parafilm. The PBS in the receptor-compartment was stirred at 37 ◦ C at 210 rpm.
At 0.5, 1, 2, 4, 8, 16, 24, 36, and 48 h, 1.0 mL of sample was replaced by fresh PBS for
analysis. To determine the phenolic contents of the samples, a Shimadzu 1800 UV-visible
spectrophotometer was utilized at 765 nm [27].

Rheological Studies of O-NLCs-E

The viscosity of the formulation was measured with a rheometer (RM200 TOUCH,
LAMY RHEOLOGY, Champagne-au-Mont-d’Or, France) and Rheomatic-P software. A spin-
dle R-III measuring-system twelve was employed. The viscosity was evaluated at 25 ± 5 ◦ C
for 60 s at a speed of 100 rpm. The measurements were as follows: Viscosity = f (time).
The viscosity = f (Time) equation enables for measuring at a stationary shear rate over a
specified time period [39]. The following measurement protocols were used: pre-shearing
time of 5 s, pre-shearing rate of 10 s, time of 60 s, shear rate of 50 s, and an immediate start.

Non-Invasive Skin Investigation of O-NLCs-E

• Safety evaluation of O-NLCs-E in human volunteers
Patch testing was used to perform safety testing (B-NLC-E versus O-NLC-E) on the
forearms of 11 participants under ethical committee approval CIIT/ATD/BSC/17-07 [40].
An area of 5 cm × 4 cm on both forearms was blotted. Baseline erythema levels were
determined using a probe fitted to the Multi-Skin-Test Center MC 1000 (Kazak, Gebraucht,
Germany). Self-application by B-NLC-E and O-NLC-E volunteers was used. Each was
applied to the blotting regions on each forearm in separate tests (B-NLC-E versus O-NLC-E).
Following treatment of the B-NLC-E and O-NLC-E, the areas were covered with surgical
dressing. After 48 h, the dressings were removed and the amount of erythema on both
forearms was measured again [41–43].

Effect of O-NLCs-E on Different Skin Aging Parameters via Long Term Use
Face studies were conducted using single-blinded non-invasive procedures, with a
three-month of recommendation. The project procedures were permitted by the ethical
committee of the Institution under the code CIIT/ATD/BSC/17-07. After receiving written
Bioengineering 2023, 10, 798 8 of 22

agreement, preparations for left and right cheeks were given to the 11 volunteers for
consistent usage until the end of the research period. Complete Skin Investigation (CSI)
was utilized to examine skin melanin level, erythema, TEWL, moisture contents, sebum
level, and wrinkles [44,45].

Panel Test of O-NLCs-E

Volunteers evaluated the sensory characteristics of the formulations. They were
instructed to apply a standardized amount, 200 mg/cm2 , of the formulations and answered
a sensory questionnaire with values from −5 to +5 representing very harmful to very
beneficial, respectively, to evaluate sense of softness, spreadability, sense in long-term,
sense just subsequently application, easiness of application, shine, and irritation of the
formulations [46,47].

2.3. Statistical Analysis

All assays were in triplicates (n = 3), and statistical analysis was carried out using
t-test and two-way ANOVA (p < 0.05) using excel and GraphPad prism v8 software.

3. Results
3.1. Preparation and Evaluation of O-Extract
The fine powder of orange dried peels methanolic extraction (O-Ext), concentrated
via rotary evaporator giving 24.7% yield, was obtained. The MeOH was selected for
extraction as it has amphoteric-nature, due to which it is able to extract maximum bioac-
tive compounds. The yield was compared with the reported study conducted by Liew
et al. on Citrus sinensis peels [48]. The procedure was revised thrice to obtain maximum
extraction [14].

3.1.1. Total Phenolic and Flavonoids Contents

TPC of O-Ext was 170.5 mg GAE/g of dry-weight. Phenolic compounds are secondary
metabolites and have been used to treat premature skin aging [49] because they act as
inhibitors of pro-inflammatory-mediators’ (PIMs), which induce “inflame-aging” [50]. The
flavonoids content in O-Ext were determined via a calorimetric method, and reported
31.5 mg QE/g as dry weight of O-Ext. Flavonoids assist in maintaining mitochondrial func-
tion, postponing aging, and extending healthier life [51]. They can condense lipo-protein
damages on cytomembrane, and modify numerous signaling-pathways, e.g., inhibiting
xanthine-oxidase a cause of oxidative-stress which accelerates skin aging [52].

3.1.2. Antioxidant Assay

The IC50 of O-Ext found via DPPH assay was 24.70 µg/mL, and for ascorbic acid
(AA) was 5.50 µg/mL. Free radical scavenging activity has strong association with phenolic
contents, and the high value of DPPH indicates the potency of extract towards scaveng-
ing of free radicals [53]. Free radicals’ scavenging property decrease damaging during
oxidative stress and makes it suitable as an antiaging agent to use in cosmeceutical and
pharmaceutical formulations [54,55].

3.1.3. Atomic Absorption Spectroscopy

To create a suitable antiaging natural-based sustainable delivery system, metals as-
sessment is essential for O-Ext [56]. For this purpose, analysis of the heavy metals, i.e., As,
Cr, Co, Cd, Fe, and Pb, was carried out and results are shown in Table 1. It was noticed
that levels of heavy metals were lower than permitted ranges [14]. In O-Ext, the “As”
was undetectable and under EU, Canada, and Germany laws, topical medication delivery
cannot use any of its salts [57]. It is causative agent of cancer, hair loss, and age spots [58].
In O-Ext, Cr was present at 0.032 ppm, and according to US-FDA, a maximum of 50.0 ppm
concentration is allowed [59]. Co was found to be present at 0.083 ppm in O-Ext, as is a
part of important vitamins, e.g., B12, but repeated contact could cause contact intoxication,
Bioengineering 2023, 10, 798 9 of 22

which leads to irritation and rashes [60]. The level of lead in O-Ext was 0.897 ppm. Lead as
a contaminant in cosmetology is permitted at a maximum amount of 20.0 ppm, according
to the US-FDA. O-Ext had a Cd of 0.028 ppm and is allowed in pharmaceutical preparations
from 0.03 to 0.10 ppm; it is utilized as a coloring agent in formulations [61]. Fe was found
at 0.332 ppm, which is within a suitable range [61,62]. According to the statistics, which
are in line with US-FDA reports and guidelines, O-Ext is appropriate to use in topical
preparations as an antiaging agent.

Table 1. Concentration of different metals (As, Cr, Co, Cd, Pb, and Fe) in O-Ext.

S. No Element Symbols Concentration Found (ppm)

1 Arsenic As 0.000
2 Chromium Cr 0.033 ± 0.001
3 Cobalt Co 0.083 ± 0.0012
4 Cadmium Cd 0.028 ± 0.0043
5 Lead Pb 0.897 ± 0.011
6 Iron Fe 0.332 ± 0.023

3.1.4. Identification of Compounds in O-Ext via GC-MS

The bioactive compounds in O-Ext were identified via GC-MS (Figure 2). The molecu-
lar weight (M.W), molecular formula (M.F), name, and CAS number of compounds were
found using NIST library given in Supporting Table S1. The O-Ext showed different com-
pounds such as Dodecanoic acid; Linalool; Alpha.-terpineol; Hentriacontane; Cyclohexas-
iloxane, dodecamethyl; Heptadecane, 1-bromo; L-norvaline, n-(2-methoxyethoxycarbonyl);
2,4-di-tert-butylphenol; 2,6-octadien-1-ol, 3,7-dimethyl-, propan; 5,5-dimethyl-cyclohex-
3-en-1-ol; Benzoic acid, 2-ethylhexyl ester; Methyl 2-hydroxy-pentacosanoate; Methyl 11-
methyl-dodecanoate; Tetradecanoic acid, 10,13-dimethyl-, met; Ethyl 14-methyl-
hexadecanoate; N-hexadecanoic acid; Oleyl oleate; Isopropyl linoleate; Hentriacontane; and
2,6,11-dodecatrienal, 2,6-dimethyl-10-me. Mostly, detected compounds given in Supporting
Bioengineering 2023, 10, x FOR PEER REVIEW
Table S1 are reported with good antioxidant and antiaging activities, mostly used in topical
anti-aging and skin care formulations.

Figure 2. GC-MS Chromatogram

Figure of O-Ext.
2. GC‐MS Chromatogram of O‐Ext.
3.1.5. Effect on Antioxidant Enzymes
3.1.5. Effect on Antioxidant Enzymes
Oxidative stress is the cause of many pathological conditions and disturbances, where
antioxidant enzymes Oxidative stress is therole.
play a homeostatic cause of many
It speeds up pathological conditions
the natural aging [Link]
In disturb
where antioxidant enzymes play a homeostatic role. It speeds up the
human dermis, different external and internal factors intervene and lead to overproduction natural agin
of FRs which cess.
leadsInto human dermis,
the oxidative different
stress external and
and suppression ofinternal factors intervene
AOE, resulting and lead to
in premature
production of FRs which leads to the oxidative stress and suppression
skin aging involving in skin hyperpigmentation, sagging, wrinkling, roughness, andAOE, res of
in premature skin aging involving in skin hyperpigmentation, sagging, wrinkling, r
ness, and dryness. In the present study, the peritoneal macrophages of mouse were
to evaluate the O‐Ext effect on antioxidant enzymes such as GST, GSH, catalase, POD
SOD [14]. The GSTs are called for biosynthesis and transport of endogenous mol
Bioengineering 2023, 10, 798 10 of 22

dryness. In the present study, the peritoneal macrophages of mouse were used to evaluate
the O-Ext effect on antioxidant enzymes such as GST, GSH, catalase, POD, and SOD [14].
The GSTs are called for biosynthesis and transport of endogenous molecules and cell
defense by catalyzing reduced glutathione conjugation through the cysteine thiol [5]. The
O-Ext noticeably made the glutathione-s-transferase (GST) concentration comparatively
to the negative control, as demonstrated in Figure 3A. Glutathione (GSH) plays a serious
part in redox homeostasis sustaining [6]. O-Ext raised the GSH concentration compared
to the negative control group, Figure 3B. GSH in cytosol (1–10 mM) plays a major role
in removal of ROS which are catalyzed by GSTs [6]. Catalase breaks down H2 O2 to H2 O
and O2 , lowering the oxidative stress inside cells [7]. The extract displayed a significant
surge compared to the negative control in the catalase analysis, Figure 3C. In Figure 3D, the
SOD results that are presented indicate that compared to the negative control, the O-Ext
significantly raises the concentration of SODs. It catalyzes the transformation of superoxide
(O2 ) into less hazardous H2 O2 [7]. Protein carbonylation, DNA damage, and membrane
lipid-peroxidation are all associated with decreased SOD activity. A well-known method
used to detect oxidative stress in cells is lipid peroxidation (POD). In the LPS-treated
negative control group, the POD content was significantly higher, as seen in Figure 3E; the
O-Ext significantly reduced the amount of the POD (GPx) in comparison to the LPS-treated
group. The quantity of POD was, likewise, significantly lowered by the positive control.
Overall
Bioengineering 2023, results
10, x FOR given in Figure 3 reveal that O-Ext could lower oxidative stress, which is
PEER REVIEW 11 of 2
why it is a suitable natural agent against aging. It could be used in topical formulation as a
natural antiaging agent.

Figure
Figure 3. Effect of 3. on
O-Ext Effect of O‐Ext on
antioxidant antioxidant
enzymes enzymes
using using macrophages
macrophages (A) GST, (B)(A) GST,
GSH, (B)
(C) GSH, (C) Catalase
Catalase,
(D) SOD, Data
(D) SOD, (E) Peroxidase. (E) Peroxidase. Data as
are presented aremean
presented
± SDas(nmean
= 3). ±Where
SD (n =**3).
p< Where
0.01, **
***pp< <0.01, *** p < 0.001.
0.001.

3.2. Fabrication3.2.
andFabrication
Evaluationand
of O-Ext Loaded
Evaluation NLCsLoaded NLCs
of O‐Ext
3.2.1. Components Selection and Their Compatibility for NLCs Preparation
3.2.1. Components Selection and Their Compatibility for NLCs Preparation
Among the solid Amonglipids
thebased
solid on solubility
lipids based on cetyl alcohol,
solubility beeswax
cetyl alcohol,and stearicand
beeswax acidstearic acid
were selected, were
and olive oil, oleic acid, and avocado oils were selected based on solubility
selected, and olive oil, oleic acid, and avocado oils were selected based on solubility
for further compatibility tests. The cetyl
for further compatibility alcohol
tests. and alcohol
The cetyl oleic acid
andcombination showed more
oleic acid combination showed mor
compatibility than other mixtures, as shown in Figure S1. For selection of a
compatibility than other mixtures, as shown in Figure S1. For selection of suitable ratio of
a suitable ratio
cetyl alcohol and oleicalcohol
of cetyl acid, they
andwere
oleic mixed in different
acid, they were mixed ratios (70:30 toratios
in different 99.9:0.1), with
(70:30 the
to 99.9:0.1), with
most suitable ratio having
the most solidratio
suitable lipids ~90%,
having which
solid was
lipids selected
~90%, whichforwasfurther evaluation.
selected for furtherIn
evaluation
In surfactant screening, the Rh 90 gives most clear emulsion with minimum inversion and
high intensity.

3.2.2. Optimization of NLC

Bioengineering 2023, 10, 798 11 of 22

surfactant screening, the Rh 90 gives most clear emulsion with minimum inversion and
high intensity.

3.2.2. Optimization of NLC

The effect of B: solid lipid (SL) and A: lipid mixture maintaining constant C: surfactant
amount is shown in Figure 4A–C. The graph shows that increasing and decreasing the SL
(B) from 90% has a negative effect, i.e., increasing the size and selected range 600 mg lipid
mixture (A) gives lower size then 400 mg lipids mixture. The effect of B: solid lipid (SL)
and A: lipid mixture maintaining constant C: surfactant amount is shown in Figure 3. The
graph shows minimum zetapotential at 600 mg lipids (A) and 90% (SL), while maximum
potential (negative effect) was shown at 99.9% solid lipids. The effect of solid lipid (B:
Bioengineering 2023, 10, x FOR PEER REVIEW 12 of 2
SL) amount in lipid mixture and total lipid mixture amount (A: lipids) in an optimized
formulation maintains the constant surfactant concentration.

Figure 4. Effect of dependent factor on NLCs. Effect of solid lipid (B: SL) amount in lipid mixtur
Figure 4. Effect of dependent factor on NLCs. Effect of solid lipid (B: SL) amount in lipid mixture
and total lipid mixture amount (A: lipids) in optimized formulation keeping surfactant concentra
and total lipid mixture amount (A: lipids) in optimized formulation keeping surfactant concentration
tion constant (A). Effect of solid lipid (B: SL) amount in lipid mixture and total lipid mixture amoun
constant (A). Effect
(A: of solidinlipid
lipids) (B: SL)formulation
optimized amount in lipid mixture
keeping and total
surfactant lipid mixture
concentration amount
constant (A: of solid
(B). Effect
lipids) in optimized
lipidformulation
(B: SL) amountkeeping
in lipidsurfactant
mixture andconcentration constant
total lipid mixture (B). Effect
amount of solid
(A: lipids) lipid
in optimized formu
(B: SL) amount inlation
lipidkeeping
mixturesurfactant
and totalconcentration
lipid mixtureconstant
amount(C).(A: lipids) in optimized formulation
keeping surfactant concentration constant (C).
3.2.3. Physico‐Chemical Properties
3.2.3. Physico-Chemical Properties
O‐NLCs demonstrated a size of 185.6 ± 13.5 nm, Figure 5A, 0.21 PDI, and 92.4 ± 1.8%
O-NLCs demonstrated a size of 185.6a ±
EE. B‐NLCs demonstrated 13.5
size of nm,
164.5Figure
± 8.6 5A, 0.21 PDI,
nm (Figure and
5B) 92.40.3±PDI,
with 1.8%signifying
EE. B-NLCs demonstrated a size of 164.5 ± 8.6 nm (Figure 5B) with 0.3 PDI, signifying
homogeneity. The rise in O‐NLCs PS was observed, and may have occurred due to th
homogeneity. loading
The riseofinO‐Ext.
O-NLCsThe PS
sizewas observed,
distribution of and maywas
O‐NLCs have occurred
also augmentedduefromto the
0.12 to 0.21
loading of O-Ext. The size distribution of O-NLCs was also augmented from 0.12
The PDI shows O‐NLCs uniformity [63]. Yichao et al. (2020) reported the blank to 0.21. nanopar
The PDI showsticles
O-NLCs uniformity
based [63]. Yichao
on rhamnolipids et al. (2020)
with (118.7 reported
nm) quite the blank
consistent with nanoparti-
B‐NLCs reported in
this study [64].with
cles based on rhamnolipids Long Ba etnm)
(118.7 al. (2016)
quitealso produced
consistent a nanoemulsion
with B-NLCs reportedwithin same
thissurfactan
of about 130 nm [65]. The surface charge of unloaded NLCs (B‐NLCs) was −77.70 ± 5.5
mV (Figure 4D). Rhamnolipids produce negative surface charge [64]. The high surfac
charge of NLCs is due to the RH, ascribed to its carboxylic groups [66,67]. Long Ba et a
(2016) described nanoemulsion (Rh‐based) having −78.0 mV surface charge [65]. The O
NLCs showed −61.4 ± 6.18 mV (Figure 4C) surface charge, i.e., a small reduction in surfac
Bioengineering 2023, 10, 798 12 of 22

study [64]. Long Ba et al. (2016) also produced a nanoemulsion with same surfactant of
about 130 nm [65]. The surface charge of unloaded NLCs (B-NLCs) was −77.70 ± 5.57 mV
(Figure 4D). Rhamnolipids produce negative surface charge [64]. The high surface charge
of NLCs is due to the RH, ascribed to its carboxylic groups [66,67]. Long Ba et al. (2016)
described nanoemulsion (Rh-based) having −78.0 mV surface charge [65]. The O-NLCs
showed −61.4 ± 6.18 mV (Figure 4C) surface charge, i.e., a small reduction in surface charge
has occurred. The formulated O-NLCs bear the surface charge required for stable NLCs,
i.e., <−30.0 mV, which is essential for stability of NP against aggregates formation [26].
The % EE of PC was 92.4 ± 1.8%. Park et al. (2018) reported turmeric nanostructured lipid
Bioengineering 2023, 10, x FOR PEERcarriers
REVIEW with 282 ± 7.19 nm diameter, 22.75 ± 1.20 mV zeta potential (-), and 93.3 13 ±
of 24
0.01%
encapsulation efficiency [68].

Figure5.
Figure 5. Physicochemical
Physicochemicalcharacterization, B‐NLC
characterization, size (C)
B-NLC sizeand
(C)zeta‐potential (D), and(D),
and zeta-potential O‐NLCs size
and O-NLCs
(A) and zeta‐potential (B).
size (A) and zeta-potential (B).

[Link]
3.2.4. Fourier Transmission
Transmission Infra‐Red
Infra-Red(FTIR)
(FTIR)Spectroscopy
Spectroscopy
FTIR analysis
FTIR analysis isisaapractical approach
practical approachfor for
quickly identifying
quickly loadedloaded
identifying extractsextracts
and es‐ and
tablishing the interface between the lipid‐matrix and the O‐Ext during the production
establishing the interface between the lipid-matrix and the O-Ext during the production of of
the O‐NLCs [26]. The FTIR evaluations of O‐Ext, O‐NLCs, and B‐NLCs
the O-NLCs [26]. The FTIR evaluations of O-Ext, O-NLCs, and B-NLCs are provided are provided in in
Figure 6A. At 1050 (S=O stretch), 1125 (C‐N stretch), 1425 (C‐H bend), 1620 (C=C
Figure 6A. At 1050 (S=O stretch), 1125 (C-N stretch), 1425 (C-H bend), 1620 (C=C stretch), stretch),
2875(O-H
2875 (O‐Hstretch),
stretch),2910
2910(C-H
(C‐Hstretch),
stretch),and
and3400
3400cmcm (O‐Hstretch);
−1−1(O-H stretch);the
theB-NLCs
B‐NLCsbandwidth
band‐
width spiked as reported previously [14]. The FTIR spectrum of O‐Ext showed peaks at
spiked as reported previously [14]. The FTIR spectrum of O-Ext showed peaks at 800 (C-H
800 (C‐H »«/bending), 1000 (CO‐O‐CO «»/stretching), 1600 (O‐H »«), and 3300 (C‐H «»).
»«/bending), 1000 (CO-O-CO «»/stretching), 1600 (O-H »«), and 3300 (C-H «»). The FTIR
The FTIR spectrum of O‐NLCs (O‐Ext loaded NLCs) revealed characteristic peaks at‐
spectrum of O-NLCs (O-Ext loaded NLCs) revealed characteristic peaks attributed at 700,
tributed at 700, 1050 (CO‐O‐CO «»), 1125 (C‐N «»), 1425 (C‐H »«), 1450 (O‐H »«), 1620 (N‐
1050 (CO-O-CO «»), 1125 (C-N «»), 1425 (C-H »«), 1450 (O-H »«), 1620 (N-H »«), 1730
H »«), 1730 (C=O «»), 2830 (O‐H «»), 2910 (C‐H «») cm−1, and 3400 (O‐H «»). The O‐NLCs
(C=O «»), 2830 (O-H «»), 2910 (C-H «») cm−1, and 3400 (O-H «»). The O-NLCs spectrum
spectrum confirmed O‐Ext encapsulation in O‐NLCs. Similar results were also observed
for turmeric loaded into the lipid‐matrix [26].
Bioengineering 2023, 10, 798 13 of 22

Bioengineering 2023, 10, x FOR PEER REVIEW 14 of 2

confirmed O-Ext encapsulation in O-NLCs. Similar results were also observed for turmeric
loaded into the lipid-matrix [26].

Figureof
Figure 6. FTIR spectra 6. B-NLC,
FTIR spectra
O-Extof and
B‐NLC, O‐Ext
O-NLC andTEM
(A), O‐NLC (A), TEM
of O-NLC ofCytotoxicity
(B), O‐NLC (B), Cytotoxicity
of O-Ext of O‐Ex
and O‐NLC (C), and permeation of O‐Ext and O‐NLC across HaCaT cells (D). Data are presente
and O-NLC (C), and permeation of O-Ext and O-NLC across HaCaT cells (D). Data are presented as
as mean ± SD. n = 3. Where ** p < 0.01, *** p < 0.001, and ns = no significance.
mean ± SD. n = 3. Where ** p < 0.01, *** p < 0.001, and ns = no significance.
[Link],
3.2.5. Morphology, Morphology, Safety, andofPermeation
and Permeation NLCs of NLCs
The external geomorphology
The external geomorphology of O-NLCs of is O‐NLCs
given way is given way in6B.
in Figure Figure
The6B. The O‐NLCs con
O-NLCs
struction was found via TEM. The figures of O‐NLCs were portrayed as rounded‐shaped
construction was found via TEM. The figures of O-NLCs were portrayed as rounded-
The mean PS of O‐NLCs was ascertained to be 101.2 ± 2.13 nm (Figure 6B). This imag
shaped. The mean PS of O-NLCs was ascertained to be 101.2 ± 2.13 nm (Figure 6B). This
confirmed the nano size of O‐NLCs holding O‐Ext attained by DLS [26]. NLCs are lipids
image confirmed the nano size of O-NLCs holding O-Ext attained by DLS [26]. NLCs
based nano‐formulations that can efficiently deliver dermal, transdermal, and vesicula
are lipids-based nano-formulations that can efficiently deliver dermal, transdermal, and
drugs. The particle size and surface morphology are important physical characters tha
vesicular drugs. The particle size and surface morphology are important physical characters
determine the rate of delivery. Small particles and spherical particles can penetrate th
that determine skin
the rate of delivery.
barriers Smallthe
easily, where particles
NLCs, and spherical
having diameter particles
aroundcan100 penetrate the throug
nm, can diffuse
skin barriers easily, where the NLCs, having diameter around 100 nm, can diffuse through
the hair follicles and penetrate the skin. A promising effect of using NLCs is the ability t
the hair folliclesimprove
and penetrate the skin.
skin hydration byAcreating
promising effect offilm
a protective using NLCs
in the is the
SC and ability to water los
preventing
improve skin hydration by creating
through the skin [2]. a protective film in the SC and preventing water loss
through the skin [2].The HaCaT cells viability dealt for 48.0 h, through a series of O‐NLCs and O‐Ext con
The HaCaT cells viability
centrations (0–250dealt
μg/mL)forvia
48.0
MTTh, through a series
assay, given of O-NLCs
in Figure and O-Ext to be con
6C and ascertained
concentrationscentration‐independent.
(0–250 µg/mL) via MTT Theassay, givenanalysis
statistical in Figure 6C and
showed ascertained
significant (p *) to be
changes. Thes
concentration-independent. The statistical
outcomes authorize the O‐NLCsanalysis showed
as a safe significant
carriers of O‐Ext.(p *) changes. These
outcomes authorizeThe the monolayer
O-NLCs asconstruction
a safe carriers of O-Ext.
of HaCaT presented the similar absorption possession
The monolayer
such as construction
dermis [49] of
andHaCaT presented
was suitable the similar
for diffusion absorptionlater
experiments, possessions
17 to 21 days incu
such as dermisbation. Thewas
[49] and mass transport
suitable forofdiffusion
O‐Ext was evaluated across
experiments, laterHaCaT
17 to 21after different
days incu- interval
Bioengineering 2023, 10, 798 14 of 22
Bioengineering 2023, 10, x FOR PEER REVIEW 15

bation. The mass transport of O-Ext was evaluated across HaCaT after different intervals,
i.e., 1, 2, 3, and 6 hrs, after stabilization of cells monolayer, as shown in Figure 6D.
i.e., 1, 2, 3, and 6diffusion
h, after stabilization of cells monolayer, as shown in Figure 6D. The
of O‐Ext was considerably (p **) lower than O‐NLCs. The outcomes dem
diffusion of O-Extstrated
was considerably (p **)
that the permeationlower than O-NLCs.
of O‐NLCs The outcomes
was more demonstrated
than that of O‐Ext. This consider
that the permeation of O-NLCs was more than that of O-Ext. This considerable
upsurge in permeation reveals that O‐NLCs are more efficient for upsurge in
O‐Ext permeation
permeation reveals that O-NLCs are more efficient
provement in topical formulations. for O-Ext permeation improvement in
topical formulations.
3.3. Formulation of O‐NLC‐E
3.3. Formulation of O-NLC-E
A dilution test was conducted by the addition of distilled water in O‐NLC‐E w
A dilution test was conducted
stirring; the emulsion bywas
thediluted
addition of distilled
successfully, water
which in O-NLC-E
shows with emul
that the prepared
stirring; the emulsion was diluted successfully, which shows that the prepared emulsion
was O/W. The microscopic image of the diluted emulsion shows the spherical shape
was O/W. The microscopic
homogeneity image ofemulsion
of the the diluted emulsion
(Figure 7D). shows the spherical shape and
homogeneity of the emulsion (Figure 7D).

Figure 7. pH studies at different temperatures conditions (A), accelerated stability studies (B),
Figure 7. pH studies at different temperatures conditions (A), accelerated stability studies (B),
phenolic contents stability studiesstability
nolic contents (C), and microscopic
studies (C), andstudies (D) ofstudies
microscopic O-NLC-E. Data
(D) of are presented
O‐NLC‐E. Data are presente
as mean ± SD. n = 3.
mean ± SD. n = 3.

3.3.1. Stability Tests

• Accelerated Thermodynamic Stability Studies
Accelerated stability studies were successfully carried out by centrifugation, heating
cooling, and freeze–thaw cycles, indicating that formulation could stand the thermal, as
well as mechanical, stresses.
 Bioengineering 2023, 10, 798 15 of 22

pH of O-NLCs-E at Different Storage Conditions

pH is a quantifiable measure of the basicity or acidity of topical formulations. The pH
variation can cause skin irritation [69]. The pH ranged from 4.8 to 5.3 (Figure 6A) and did
not noticeably change during the first, second, and third months of storage under various
settings. The O-NLCs-E buffer-capacity sustained the pH invariant. pH changes (PC) were
measured at different storage conditions at different time intervals shown in Figure 6B,C;
the changes in PC were nonsignificant, calculated by applying two-way ANOVA with post
hoc Tukey test. PC of freshly prepared formulation were kept as a standard for comparison.
The results show that O-NLC-E demonstrates good buffer capacity during the mentioned
shelf life [38].

3.4. Ex Vivo Analysis

3.4.1. Ex Vivo Diffusion and Safety Evaluation
The ex vivo releases percentage was obtained via modified-Franz-diffusion utilizing
Bioengineering 2023, 10, x FOR PEER rat skin as a membrane; results are given in Figure 8A. O-Ext-E release was over 80 of TPC 17 of 24
REVIEW
in 8 h, while O-NLCs-E issued 50% of PC gradually. O-NLC-E displayed a slow-release of
PC compared to O-Ext-E Figure 8A.

[Link]
Figure PCrelease
release studies
studies of O‐NLC‐E
of O-NLC-E (A), safety
(A), safety studiesstudies
of blankofformulations
blank formulations (B), and rheolog‐
(B), and rheological
ical studies
studies of O‐NLC‐E
of O-NLC-E (C). ns
(C). where where
= no ns = no significance.
significance.

3.4.2. Rheological Behavior of O‐NLCs‐E

The most popular manifestation of topical formulations is viscosity which effects the
incorporated actives to penetrate the skin, along with skin feel and spreadability [72]. Rhe‐
ograms in Figure 8C showed viscosity of 0.100 Pa.s with 0.014 std, presenting shear‐thin‐
ning behavior reflecting pseudo‐plastic‐tendency. Rheology studies the materials flow
Bioengineering 2023, 10, 798 16 of 22

The release data of O-Ext from O-NLC-E and O-Ext-E were tailored by release-kinetic
equations. The release with n ≤ 0.43 is governed via the Fickian-diffusion, n ≥ 0.85 by
dissolution, and 0.43 < n < 0.8585 via a combination of both [70]. In this study, the Higuchi
model (R2 = 0.9764) was suitable equation for O-NLC-E. This behavior is premised on
the following assumptions: (a) the extract solubility is lesser than the matrix’s initial
concentration; (b) there is only one-dimensional diffusion of biomaterials; (c) the system-
thickness exceeds bioactive particles in size; (d) there is very little matrix swelling or
disintegration; (e) the bioactive component has constant (continuous) diffusivity; and
(f) the release system consistently achieves entirely sinking conditions [71]. Patch tests of
volunteers for both B-NLC-E, and O-NLC-E was performed (Figure 8B). The results of the
safety tests were evaluated via instrument and found safe.

3.4.2. Rheological Behavior of O-NLCs-E

The most popular manifestation of topical formulations is viscosity which effects
the incorporated actives to penetrate the skin, along with skin feel and spreadability [72].
Rheograms in Figure 8C showed viscosity of 0.100 Pa.s with 0.014 std, presenting shear-
thinning behavior reflecting pseudo-plastic-tendency. Rheology studies the materials flow
and deformation under stress [42], and the suitable pseudo-plastic flow enables the thin
coating formation that covers the skin [35]. The presented rheological behavior shows
smooth-flow afterward tension which leads to create a thin film on the surface of skin.

3.5. Antiaging Efficacy of O-NLC-E

3.5.1. Melanin and Erythema
Tyrosinase is an enzyme responsible for the assembly of melanin in the skin layers.
Skin melanin gives coloring effects of white to dark skin which is regulated by tyrosinase.
In the case of B-NLC-E and O-NLC-E, it was observed that there were slight variations
in skin melanin on the B-NLC-E-treated side, while there was a gradual decrease in melanin
values of ONLC-E treated side-up to the 3rd M (Figure 9). An ANOVA test showed
that effects produced by O-NLC-E were significant and effects produced by B-NLC-E
were insignificant, with respect to time. Through Tukey’s multiple comparisons test,
it was ascertained that the melanin of B-NLC-E-treated side at baseline visit vs. 1 M,
2 M, and 3 M were found to be insignificant. Skin Melanin of the O-NLC-E-treated
side at baseline visit vs. 1 M and 2 M was insignificant, while baseline visit vs. 3 M
was significant. Hesperidin, naringin, eriocitrin, poly-methoxylated flavones (PMF), O-
glycosylated flavanone, nobiletin, gallic acid, ferulic acid, and para-coumaric acid 170–174
are found in peel extract. Nobiletin, naringin, and neo-hesperidin were a few of the
flavonoids that were discovered as tyrosinase inhibitors, but their inhibiting power was
shown to be inferior to kojic acid against mushroom tyrosinase.
Human skin is continuously exposed to environmental stressors such as solar radia-
tions, oxides of nitrogen, ozone, and transition metal ions, as well as some other factors.
Oxidative stress with subsequent oxidative damage is more likely to disrupt the normal to-
cotrienols function in the skin. This skin damage due to the UV is known as skin erythema.
In the case of B-NLC-E and O-NLC-E, there was a minor increase in erythema of
B-NLC-E-treated cheek-side, while there was a steady decrease in O-NLC-E treated side-up
to the 3rd month (3 M). With the aid of an ANOVA test on effects in erythema values,
O-NLC-E produced significant results whilst B-NLC-E produced insignificant values, with
respect to time. By applying Tukey’s multiple comparisons test, it was found that skin
erythema of BNLC-E-treated side at baseline visit vs. 1 M, 2 M, and 3 M were found to be
insignificant, and skin erythema of O-NLC-E-treated side showed a significant decrease
compared to baseline vs. 2 M and 3 M. The decline in erythema level by O-NLC-E provided
the evidence that it is attributed to gallic acid and hesperidin via downregulation of
inflammatory-cascades, MMP1, and oxidative stress.
 respect to time. By applying Tukey’s multiple comparisons test, it was found that skin
erythema of BNLC‐E‐treated side at baseline visit vs. 1 M, 2 M, and 3 M were found to be
insignificant, and skin erythema of O‐NLC‐E‐treated side showed a significant decrease
compared to baseline vs. 2 M and 3 M. The decline in erythema level by O‐NLC‐E pro‐
Bioengineering 2023, 10, 798 vided the evidence that it is attributed to gallic acid and hesperidin via downregulation
17 of 22
of inflammatory‐cascades, MMP1, and oxidative stress.

Figure 9. Melanin contents after

after 1st,
1st, 2nd,
2nd, and
and3rd
3rdmonth
month(A);
(A);erythema
erythemalevel
levelafter
after1st,
1st,2nd,
2nd,andand3rd
3rd
after application
month (B) after applicationof
ofO-NLC-E.
O‐NLC‐[Link]
Percentchange
changeininskin
skinmelanin
melaninand
anderythema
erythemaafterafter1st
1stmonth
month
(1 M),
(1 M), 2nd
2nd month
month (2(2 M),
M), and
and 3rd
3rd month
month (3 (3 M)
M) by
by applying
applying O‐NLC‐E,
O-NLC-E,along
alongwith
withtheir
theirrespective
respectiveB‐
B-NLC-E. # shows significance difference between O-NLC-E readings taken at different timeintervals
NLC‐E. # shows significance difference between O‐NLC‐E readings taken at different time intervals(1
M: standard) and * shows significance difference between O‐NLC‐E and their respective
(1 M: standard) and * shows significance difference between O-NLC-E and their respective B-NLC-E. B‐NLC‐E.
#### and **** indicates p < 0.0001, and ns means no significance. n = 11.
#### and **** indicates p < 0.0001, and ns means no significance. n = 11.

and Moisture
3.5.2. TEWL and Moisture
A correlation
correlation between
betweenstratum-corneum
stratum‐corneum(SC) (SC)hydration
hydrationand andTEWL
TEWLvalues
valuesexist;
exist;both
both
are involved
involved in in skin
skin water
watercapacities.
[Link]
Thecombined
combinedskinskininvestigation
investigationofoftrans-epidermal
trans‐epidermal
water
water loss
lossand
andhydration
hydrationisisappropriate
appropriate to to
assess skin
assess responsiveness
skin responsiveness as shown in Figure
as shown 10.
in Figure
In addition, seeing the way that skin interacts with the climate reveals a dire
10. In addition, seeing the way that skin interacts with the climate reveals a dire role in role in the
protection of the
the protection ofskin against
the skin microbes
against and increased
microbes loss. In
and increased theIncase
loss. theof B-NLC-E,
case it wasit
of B‐NLC‐E,
found that there
was found was awas
that there minor decrease
a minor in TEWL
decrease values,
in TEWL whilewhile
values, a gradual and prominent
a gradual and promi‐
increase in TEWL
nent increase values
in TEWL was reported
values for O-NLC-E
was reported for O‐NLC‐Eup toup theto3rd
theM. [Link]
3rd An ANOVA test
Bioengineering 2023, 10, x FOR PEER showed
REVIEW that the O-NLC-E effect was more significant compared to B-NLC-E. The hydration 19 of 24
test showed that the O‐NLC‐E effect was more significant compared to B‐NLC‐E. The hy‐
level of O-NLC-E
dration was significantly
level of O‐NLC‐E increasedincreased
was significantly compared to B-NLC-E.
compared In both results,
to B‐NLC‐E. In both the
re‐
difference between blank and loaded was due to the bioactive compounds
sults, the difference between blank and loaded was due to the bioactive compounds en‐ enriched O-Ext.
riched O‐Ext.

Figure10.
Figure [Link]
TEWLcontents
contentsafter
after1st,
1st,2nd,
2nd,and
and3rd
3rdmonth
month(A);
(A);moisture
moisturelevel
levelafter
after1st,
1st,2nd,
2nd, and
and 3rd
3rd
month(B)
month (B)after
afterapplication
applicationofofO-NLC-E.
O‐NLC‐[Link]
Percentchange
changeininskin
skinTEWL
TEWLand andmoisture
moisture after
after 1st1st month
month
(1 M),
(1 M), 2nd
2nd month
month (2 (2 M),
M), and
and 3rd
3rd month
month (3(3 M)
M) by
by applying
applying O‐NLC‐E,
O-NLC-E, along
alongwith
withtheir
theirrespective
respectiveB‐
NLC‐E. # shows significance difference between O‐NLC‐E readings taken at different
B-NLC-E. # shows significance difference between O-NLC-E readings taken at different time intervalstime intervals
(1 M: standard) and * shows significance difference between O‐NLC‐E and their respective B‐NLC‐
(1 M: standard) and * shows significance difference between O-NLC-E and their respective B-NLC-E.
E. #### and **** indicates p < 0.0001, ### indicates p < 0.001. n = 11.
#### and **** indicates p < 0.0001, ### indicates p < 0.001. n = 11.

3.5.3. Sebum
3.5.3. Sebum and
andElasticity
Elasticity
The skin
The skin sebum is produced by sebaceous
sebaceous glands
glands andand harmonized
harmonizedby byhormones
hormonesof
thethe
of adrenal cortex
adrenal (corticosteroids),
cortex sex hormones
(corticosteroids), sex hormones(androgens and estrogens),
(androgens and others.
and estrogens), and
others.
Moreover,Moreover, 5-α-reductase
5‐α‐reductase and androgens
and androgens receptors
receptors are responsible
are responsible for converting
for converting testos‐
testosterone into dihydrotestosterone
terone into dihydrotestosterone (the (the
mostmost potent
potent typetype enhancing
enhancing sebum
sebum secretion).
secretion). An
An increase in the sebum secretion in humans is due to: (1) A testosterone metabolite,
increase in the sebum secretion in humans is due to: (1) A testosterone metabolite, dihy‐
dihydrotestosterone
drotestosterone (DHT), (DHT), is produced
is produced by 5-α-reductase
by 5‐α‐reductase type-I
type‐I (2) progesterone,
(2) progesterone, 5α-
5α‐reduc‐
reductase inhibitor.
tase inhibitor. These These hormones
hormones upsurge upsurge skin sebum
skin sebum by motivating
by motivating the division
the division of sebo‐
cytes. In case of sebum, the value was increased for B‐NLC‐E, while O‐NLC‐E decreased
the sebum level (Figure 11A), as shown by applying statistical analysis. The decrease in
skin sebum is related to inhibit 5α‐reductase in the O‐NLC‐E due to bioactive compounds
alpha glucosyl hesperidin, gallic acid, vitamin A, and vitamin C, which can sustain the
 the sebum level (Figure 11A), as shown by applying statistical analysis. The decrease in
skin sebum is related to inhibit 5α‐reductase in the O‐NLC‐E due to bioactive compounds
alpha glucosyl hesperidin, gallic acid, vitamin A, and vitamin C, which can sustain the
natural equilibrium of skin oil secretion and enhances the elasticity of the skin by gripping
Bioengineering 2023, 10, 798 additional oils and eliminating deceased skin cells. 18 of 22
Elastase, on the other hand, is an MMPs enzyme that degrades the elastin fibers. Alt‐
hough elastin fiber can withstand various proteolytic degradations, prolonged exposure
of elastase
to [Link] theofdeterioration
In case of elastin
sebum, the value wasfiber, whichfor
increased causes the skin
B-NLC-E, to lose
while its in‐
O-NLC-E
tegrity, and wrinkle formation initiates. In the case of elasticity measurements,
decreased the sebum level (Figure 11A), as shown by applying statistical analysis. The O‐NLC‐E
increases
decrease inelasticity, while
skin sebum is in B‐NLC‐E,
related slight5α-reductase
to inhibit reduction occurred (Figure 11B).
in the O-NLC-E due toAnbioactive
increase
in elasticity was
compounds alphadue to the bioactive
glucosyl hesperidin,compounds
gallic acid,ofvitamin
O‐Ext which
A, andact via downregulation
vitamin C, which can
of MMP1.
sustain theElastase
natural is responsibleoffor
equilibrium elastin
skin and collagen
oil secretion deprivation,
and enhances and stimulated
the elasticity via
of the skin
MMP1 and [Link] oils and eliminating deceased skin cells.
by gripping

Figure
Figure 11. Sebum contents
11. Sebum contents after
after 1st,
1st, 2nd,
2nd, and
and 3rd month (A); elasticity level after 1st, 2nd, and 3rd
month
month (B)
(B) after
after application
application of
of O‐NLC‐E.
O-NLC-E. Percent change in skin sebum and elasticity after 1st month
month
(1
(1 M),
M),2nd
2ndmonth
month(2(2M),
M),and
and3rd
3rdmonth
month(3(3M)M)bybyap‐plying
ap-plyingO‐NLC‐E,
O-NLC-E, along with
along their
with respective
their B‐
respective
B-NLC-E. # shows significance difference between O-NLC-E readings taken at different time intervals
(1M: standard) and * shows significance difference between O-NLC-E and their respective B-NLC-E.
#### and **** indicates p < 0.0001, n = 11.

Elastase, on the other hand, is an MMPs enzyme that degrades the elastin fibers.
Although elastin fiber can withstand various proteolytic degradations, prolonged exposure
to elastase initiates the deterioration of elastin fiber, which causes the skin to lose its
integrity, and wrinkle formation initiates. In the case of elasticity measurements, O-NLC-E
increases elasticity, while in B-NLC-E, slight reduction occurred (Figure 11B). An increase
in elasticity was due to the bioactive compounds of O-Ext which act via downregulation
of MMP1. Elastase is responsible for elastin and collagen deprivation, and stimulated via
MMP1 and 2.

3.5.4. Panel Test of O-NLCs-E

To analyze the effectiveness of O-NLC-E and their respective B-NLC-E, a questionnaire
was prepared. B-NLC-E received average points for questions relating to the application,
spreadability, sensation just after application, long-term sense, irritation, shine on the skin,
and softness, represented in Figure 12. We applied a paired sample t-test and found a
negligible pairing difference between the average points of sensorial questioners regarding
formulations O-NLC-E, and their respective base (B-NLC-E) at different time intervals.
Therefore, we concluded that there were no huge changes between formulations O-NLC-E
and their respective B-NLC-E regarding the sensory evaluation. Loaded formulations and
their respective bases demonstrate similar performance after sensorial analysis. However,
silica used as a sensory modifier with surfactants in formulations has played well in
addition to sensory attributes of formulations [73]. It is reported from the analysis that
formulations are admissible and suitable for topical use.
 regarding formulations O‐NLC‐E, and their respective base (B‐NLC‐E) at different time
intervals. Therefore, we concluded that there were no huge changes between formulations
O‐NLC‐E and their respective B‐NLC‐E regarding the sensory evaluation. Loaded formu‐
lations and their respective bases demonstrate similar performance after sensorial analy‐
sis. However, silica used as a sensory modifier with surfactants in formulations has played
Bioengineering 2023, 10, 798
well in addition to sensory attributes of formulations [73]. It is reported from the 19 of 22
analysis
that formulations are admissible and suitable for topical use.

Figure
Figure 12.
12. Panel
Panel for
for ease
ease of
of application, spreadability,sensation
application, spreadability, sensation just
just after
after application,
application, sensation
sensation after
after
long‐term
long-term sense, irritation,shine
sense, irritation, shineon
onthe
theskin,
skin,and
andsoftness.
softness.

4. Conclusions
4. Conclusions
The aim
The aim of
of the
thestudy
studywas
wastotoevaluate
evaluateorange
orangepeels
peelsextract
extractforfor antiaging
antiaging activity
activity and
heavy metals concentration (safety). For this purpose, the extract was loaded intointo
and heavy metals concentration (safety). For this purpose, the extract was loaded NLCs
NLCs based
based on green
on green surfactants
surfactants (rhamnolipids)
(rhamnolipids) and and characterized
characterized before
before loading
loading into
into a
a sec‐
secondary formulation (topical O/W emulsion). The results showed good safety profile
ondary formulation (topical O/W emulsion). The results showed good safety profile and
and high antiaging efficacy. This sustainable approach can mitigate the effects on the skin
high antiaging efficacy. This sustainable approach can mitigate the effects on the skin of
of the application of substantial bioactive compounds, which successfully overcome the
the application of substantial bioactive compounds, which successfully overcome the pen‐
penetration barriers and improve the antioxidant activities against oxidative stresses. This
etration barriers and improve the antioxidant activities against oxidative stresses. This
formulation will improve the economic and social compliance, and the topical treatment
formulation will improve
will fend off oxidative the economic
skin problems andskin
and early social compliance,
aging with ease. and the topical
In short, treatment
the sustainable
will fend off
approach mayoxidative
lessen theskin problems
skin and early
related social skin aging with ease. In short, the sustain‐
anxiety.
able approach may lessen the skin related social anxiety.
Supplementary Materials: The following supporting information can be downloaded at: https://
Supplementary Materials: The following supporting information
[Link]/article/10.3390/bioengineering10070798/s1, Figure S1: can be downloaded
Selection of Surfactant, at:
[Link]/xxx/s1, Figure
Liquid and Solid-lipids and theirS1: Selection of for
Compatibility Surfactant, Liquid andFigure
NLCs Preparation; Solid‐lipids and Consent
S2: Written their Com‐
patibility for NLCs
Sample; Table Preparation;
S1: Bioactive Figure found
compounds S2: Written Consent
in GC-MS Sample;
analysis Table
by Nist S1: Bioactive
library in O-Ext;compounds
Table S2:
found in GC‐MS
Composition analysis by Nist
of independent library
factors in O‐Ext; Table
in formulations S2: Composition
produced of independent
via box-benken factors
model; Table S3: in
Suggested independent and dependent factors by BB model.
Author Contributions: Conceptualization, Z.U.K.; methodology, Z.U.K.; software, Z.U.K.; validation,
Z.U.K., N.U.K. and H.K.; formal analysis, N.U.K., Y.D. and H.K.; investigation, T.K. and J.N.; resources,
A.A.; data curation, A.A.; writing—original draft preparation, Z.U.K.; writing—review and editing,
A.A. and N.U.K.; visualization, Z.U.K.; supervision, A.A., T.K. and J.N.; project administration, A.A.;
funding acquisition, J.N. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by The HEC Pakistan (7741/Federal/NRPU/R&D/HEC/2017),
and Wuxi Municipal Health Commission Youth Research Project (Q202230).
Institutional Review Board Statement: Ethical Standard Biosafety Committee COMSATS University
Islamabad, Abbottabad Campus (Reference No. CIIT/ATD/BSC/17-07) approved this project.
Informed Consent Statement: Attached in Supplementary File.
Data Availability Statement: Data are contained within the article.
Bioengineering 2023, 10, 798 20 of 22

Acknowledgments: We are thankful to Higher Education Commision of Pakistan (HEC) for pro-
viding funding of this work under project No. 7741/Federal/NRPU/R&D/HEC/2017. We would
like to extend our gratitude towards DSM, The Netherlands, for providing Valvance Touch 210,
and Corbion-Chemicals Company, the Netherlands, for providing Sodium Behenoyl Lactylate and
Glyceryl Stearate.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Zhong, R.; Zhou, D. Oxidative Stress and Role of Natural Plant Derived Antioxidants in Animal Reproduction. J. Integr. Agric.
2013, 12, 1826–1838. [CrossRef]
2. Raszewska-Famielec, M.; Flieger, J. Nanoparticles for Topical Application in the Treatment of Skin Dysfunctions—An Overview
of Dermo-Cosmetic and Dermatological Products. Int. J. Mol. Sci. 2022, 23, 15980. [CrossRef] [PubMed]
3. Lee, J.K.; Patel, S.K.S.; Sung, B.H.; Kalia, V.C. Biomolecules from Municipal and Food Industry Wastes: An Overview. Bioresour.
Technol. 2019, 298, 122346. [CrossRef]
4. Chen, S.L.; Yu, H.; Luo, H.M.; Wu, Q.; Li, C.F.; Steinmetz, A. Conservation and Sustainable Use of Medicinal Plants: Problems,
Progress, and Prospects. Chin. Med. 2016, 11, 37. [CrossRef]
5. Mukherjee, P.K.; Bahadur, S.; Chaudhary, S.K.; Harwansh, R.K.; Nema, N.K. Validation of Medicinal Herbs for Skin Aging; Elsevier
Inc.: Amsterdam, The Netherlands, 2015; ISBN 9780128009963.
6. Li, S.; Yu, H.; Ho, C.T. Nobiletin: Efficient and Large Quantity Isolation from Orange Peel Extract. Biomed. Chromatogr. 2006,
20, 133–138. [CrossRef] [PubMed]
7. Doha, H.A.B.; Ibrahim, B.M.M.; Abdel-Latif, Y.; Nabila, S.H.; Emad, M.H.; Souad, E.G. Biochemical and pharmacological
prospects of Citrus-sinensis peels. Heliyon 2022, 8, e09979.
8. Goulas, V.; Manganaris, G.A. Exploring the Phytochemical Content and the Antioxidant Potential of Citrus Fruits Grown in
Cyprus. Food Chem. 2012, 131, 39–47. [CrossRef]
9. Lante, A.; Tinello, F. Citrus Hydrosols as Useful by-Products for Tyrosinase Inhibition. Innov. Food Sci. Emerg. Technol. 2015,
27, 154–159. [CrossRef]
10. Shah, P.R.; Desai, P.; Channer, D.; Singh, M. Enhanced Skin Permeation Using Polyarginine Modified Nanostructured Lipid
Carriers. J. Control. Release 2012, 161, 735–775. [CrossRef]
11. Dubey, A.; Prabhu, P.; Kamath, J.V. Nano Structured Lipid Carriers: A Novel Topical Drug Delivery System. Int. J. PharmTech. Res.
2012, 4, 705–714.
12. Salvi, V.R.; Pawar, P. Nanostructured Lipid Carriers (NLC) System: A Novel Drug Targeting Carrier. J. Drug Deliv. Sci. Technol.
2019, 51, 255–267. [CrossRef]
13. Borges, A.; Freitas, V.; de Mateus, N.; Fernandes, I.; Oliveira, J. Solid Lipid Nanoparticles as Carriers of Natural Phenolic
Compounds. Antioxidants 2020, 9, 998. [CrossRef] [PubMed]
14. Khan, Z.U.; Khan, T.; Mannan, A.; Ali, A.; Ni, J. In Vitro and Ex Vivo Evaluation of Mangifera Indica L. Extract-Loaded Green
Nanoparticles in Topical Emulsion against Oxidative Stress and Aging. Biomedicines 2022, 10, 2266. [CrossRef]
15. Baba, S.A.; Malik, S.A. Determination of Total Phenolic and Flavonoid Content, Antimicrobial and Antioxidant Activity of a Root
Extract of Arisaema Jacquemontii Blume. J. Taibah Univ. Sci. 2015, 9, 449–454. [CrossRef]
16. Song, J.H.; Bae, E.Y.; Choi, G.; Hyun, J.W.; Lee, M.Y.; Lee, H.W.; Chae, S. Protective Effect of Mango (Mangifera Indica L.) against
UVB-Induced Skin Aging in Hairless Mice. Photodermatol. Photoimmunol. Photomed. 2013, 29, 84–89. [CrossRef]
17. Baylac, S.; Racine, P. Inhibition of Human Leukocyte Elastase by Natural Fragrant Extracts of Aromatic Plants. Int. J. Aromather.
2004, 14, 179–182. [CrossRef]
18. Anal, J.M.H.; Chase, P. Trace Elements Analysis in Some Medicinal Plants Using Graphite Furnace-Atomic Absorption Spec-
troscopy. Environ. Eng. Res. 2016, 21, 247–255. [CrossRef]
19. Khan, A.M.A.U.; Ali, H.; Islam, S.U.; Seo, E.K.; Khan, S. Continentalic Acid Exhibited Nephroprotective Activity against the LPS
and E. Coli-Induced Kidney Injury through Inhibition of the Oxidative Stress and Inflammation. Int. Immunopharmacol. 2020,
80, 106209. [CrossRef]
20. Walker, R.B. The Use of Quantitative Analysis and Hansen Solubility Parameter Predictions for the Selection of Excipients for
Lipid Nanocarriers to Be Loaded with Water Soluble and Insoluble Compounds. Saudi Pharm. J. 2020, 28, 308–315. [CrossRef]
21. Dragicevic, N.; Maibach, H.I. (Eds.) Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement; Springer:
Berlin/Heidelberg, Germany, 2016. [CrossRef]
22. Goyal, U.; Arora, R.; Aggarwal, G. Formulation Design and Evaluation of a Self-Microemulsifying Drug Delivery System of
Lovastatin. Acta Pharm. 2012, 62, 357–370. [CrossRef]
23. Pezeshki, A.; Ghanbarzadeh, B.; Mohammadi, M.; Fathollahi, I.; Hamishehkar, H. Encapsulation of Vitamin A Palmitate in
Nanostructured Lipid Carrier (NLC)-Effect of Surfactant Concentration on the Formulation Properties. Adv. Pharm. Bull. 2014,
4, 563–568. [CrossRef]
Bioengineering 2023, 10, 798 21 of 22

24. Khan, Z.U.; Razzaq, A.; Khan, A.; Rehman, N.U.; Khan, H.; Khan, T.; Khan, A.U.; Menaa, F.; Iqbal, H.; Khan, N.U. Physicochemical
Characterizations and Pharmacokinetic Evaluation of Pentazocine Solid Lipid Nanoparticles against Inflammatory Pain Model.
Pharmaceutics 2022, 14, 409. [CrossRef]
25. Rodrigues, F.; Alves, A.C.; Nunes, C.; Sarmento, B.; Amaral, M.H.; Reis, S.; Oliveira, M.B.P.P. Permeation of Topically Applied
Caffeine from a Food by—Product in Cosmetic Formulations: Is Nanoscale in Vitro Approach an Option? Int. J. Pharm. 2016,
513, 496–503. [CrossRef]
26. Karimi, N.; Ghanbarzadeh, B.; Hamishehkar, H.; Mehramuz, B.; Kafil, H.S. Antioxidant, Antimicrobial and Physicochemical
Properties of Turmeric Extract-Loaded Nanostructured Lipid Carrier (NLC). Colloids Interface Sci. Commun. 2018, 22, 18–24.
[CrossRef]
27. Damle, M.; Mallya, R. Development and Evaluation of a Novel Delivery System Containing Phytophospholipid Complex for
Skin Aging. AAPS PharmSciTech 2016, 17, 607–617. [CrossRef] [PubMed]
28. Iqbal, H.; Khan, Z.U.; Razzaq, A.; Khan, N.U.; Khan, B.A.; Menaa, F. Fabrication, Physical Characterizations and In Vitro
Antibacterial Activity of Cefadroxil-Loaded Chitosan/Poly (Vinyl Alcohol) Nanofibers against Staphylococcus aureus Clinical
Isolates. Int. J. Biol. Macromol. 2019, 144, 921–931. [CrossRef] [PubMed]
29. Luo, Q.; Zhao, J.; Zhang, X.; Pan, W. Nanostructured Lipid Carrier (NLC) Coated with Chitosan Oligosaccharides and Its Potential
Use in Ocular Drug Delivery System. Int. J. Pharm. 2011, 403, 185–191. [CrossRef] [PubMed]
30. Wang, J.J.; Shi, Q.H.; Zhang, W.; Sanderson, B.J.S. Anti-Skin Cancer Properties of Phenolic-Rich Extract from the Pericarp of
Mangosteen (Garcinia mangostana Linn.). Food Chem. Toxicol. 2012, 50, 3004–3013. [CrossRef] [PubMed]
31. Alnuqaydan, A. Toxicity and Genotoxicity of Beauty Products on Human Skin Cells In Vitro Journal of Clinical Toxicology
Toxicity and Genotoxicity of Beauty Products on Human Skin Cells In Vitro. J. Clin. Toxicol. 2016, 6, 1000315. [CrossRef]
32. Grafe, F.; Wohlrab, W.; Neubert, R.H.; Brandsch, M. Transport of Biotin in Human Keratinocytes. J. Invest. Dermatol. 2003,
120, 428–433. [CrossRef] [PubMed]
33. Khan, N.U.; Ali, A.; Khan, H.; Khan, Z.U.; Zia, A. Stability Studies and Characterization of Glutathione-Loaded Nanoemulsion. J.
Cosm. Sci. 2018, 69, 161–168.
34. Carlotti, M.E.; Sapino, S.; Trotta, M.; Battaglia, L.; Vione, D.; Pelizzetti, E. Photostability and Stability over Time of Retinyl
Palmitate in an O/W Emulsion and in SLN Introduced in the Emulsion. J. Dispers. Sci. Technol. 2005, 26, 125–138. [CrossRef]
35. Ali, A.; Iqbal, S.; Ilyas, A.; Khan, H.; Asad, M.H.H.; Fatima, N.; Akhtar, N. Anti-Pollution Cosmetic-Based One-Step Formation of
w/o/w Multiple Emulsion Containing D-Biotin for Skin Protection: Fabrication and in Vitro and in Vivo Evaluation. Drug Deliv.
Transl. Res. 2019, 9, 1117–1132. [CrossRef] [PubMed]
36. European Cosmetics Association. Cosmetics Europe Guidelines on Stability Testing of Cosmetic Products; Cosmetics Europe: Brussels,
Belgium, 2004; pp. 1–8.
37. Poomanee, W.; Khunkitti, W.; Chaiyana, W.; Leelapornpisid, P. Optimization of Mangifera Indica L. Kernel Extract-Loaded
Nanoemulsions via Response Surface Methodology, Characterization, Stability, and Skin Permeation for Anti-Acne Cosmeceutical
Application. Pharmaceutics 2020, 12, 454. [CrossRef] [PubMed]
38. Wohlrab, J.; Gebert, A. PH and Buffer Capacity of Topical Formulations. Curr. Probl. Dermatol. 2018, 54, 123–131. [CrossRef]
[PubMed]
39. Jaworska, M.; Sikora, E.; Ogonowski, J. Rheological Properties of Nanoemulsions Stabilized by Polysorbate 80. Chem. Eng. Technol.
2015, 38, 1469–1476. [CrossRef]
40. Ali, A.; Akhtar, N.; Khan, M.S. In Vivo Evaluation: The Effects of a Cream Containing Acacia Bark Extract on Skin Melanin and
Erythema Content. Adv. Dermatol. Allergol. 2012, 5, 369–372. [CrossRef]
41. Ali, A.; Akhtar, N. The Safety and Efficacy of 3% Cannabis Seeds Extract Cream for Reduction of Human Cheek Skin Sebum and
Erythema Content. Pak. J. Pharm. Sci. 2015, 28, 1389–1395.
42. Ali, A.; Akhtar, N.; Khan, M.S.; Khan, M.T.; Ullah, A.; Shah, M.I. Effect of Moringa Oleifera on Undesireble Skin Sebum Secretions
of Sebaceous Glands Observed during Winter Season in Humans. Biomed. Res. 2013, 24, 127–130.
43. Ali, A.; Akhtar, N.; Chowdhary, F. Enhancement of Human Skin Facial Revitalization by Moringa Leaf Extract Cream. Adv.
Dermatol. Allergol. Post˛epy Dermatol. Alergol. 2014, 31, 71–76. [CrossRef]
44. Fossa Shirata, M.M.; Maia Campos, P.M.B.G. Sunscreens and Cosmetic Formulations Containing Ascorbyl Tetraisopalmitate
and Rice Peptides for the Improvement of Skin Photoaging: A Double-Blind, Randomized Placebo-Controlled Clinical Study.
Photochem. Photobiol. 2021, 97, 805–815. [CrossRef]
45. Gianeti, M.D.; Mercurio, D.G.; Maia Campos, P.M.B.G. The Use of Green Tea Extract in Cosmetic Formulations: Not Only an
Antioxidant Active Ingredient. Dermatol. Ther. 2013, 26, 267–271. [CrossRef] [PubMed]
46. Palareti, G.; Legnani, C.; Cosmi, B.; Antonucci, E.; Erba, N.; Poli, D.; Testa, S.; Tosetto, A. Comparison between Different D-Dimer
Cutoff Values to Assess the Individual Risk of Recurrent Venous Thromboembolism: Analysis of Results Obtained in the DULCIS
Study. Int. J. Lab. Hematol. 2016, 38, 42–49. [CrossRef] [PubMed]
47. Ali, A.; Akhtar, N.; Khan, H.M.S.; Muhammad, H.; Khan, S. Cream Containing Acacia Bark Extract Significantly Reduces Skin
Sebum Content in Healthy Volunteers. J. Sci. Ind. Res. 2012, 71, 678–681.
48. Liew, S.S.; Ho, W.Y.; Yeap, S.K.; Sharifudin, S.A.B. Phytochemical composition and in vitro antioxidant activities of Citrus sinensis
peel extracts. PeerJ 2018, 6, e5331. [CrossRef]
Bioengineering 2023, 10, 798 22 of 22

49. Rahman, M.; Rahaman, S.; Islam, R.; Rahman, F.; Mithi, F.M.; Alqahtani, T.; Almikhlafi, M.A.; Alghamdi, S.Q.; Alruwaili, A.S.;
Hossain, S.; et al. Role of Phenolic Compounds in Human Disease: Current Knowledge and Future Prospects. Molecules 2022,
27, 233. [CrossRef]
50. Rea, I.M.; Gibson, D.S.; McGilligan, V.; McNerlan, S.E.; Denis Alexander, H.; Ross, O.A. Age and Age-Related Diseases: Role of
Inflammation Triggers and Cytokines. Front. Immunol. 2018, 9, 586. [CrossRef]
51. Fan, X.; Fan, Z.; Yang, Z.; Huang, T.; Tong, Y.; Yang, D.; Mao, X.; Yang, M. Flavonoids—Natural Gifts to Promote Health and
Longevity. Int. J. Mol. Sci. 2022, 23, 2176. [CrossRef]
52. Tungmunnithum, D.; Thongboonyou, A.; Pholboon, A.; Yangsabai, A. Flavonoids and Other Phenolic Compounds from Medicinal
Plants for Pharmaceutical and Medical Aspects: An Overview. Medicines 2018, 5, 93. [CrossRef]
53. Sreelatha, S.; Padma, P.R. Antioxidant Activity and Total Phenolic Content of Moringa Oleifera Leaves in Two Stages of Maturity.
Plant Foods Hum. Nutr. 2009, 64, 303–311. [CrossRef]
54. Khogta, S.; Patel, J.; Barve, K.; Londhe, V. Herbal Nano-Formulations for Topical Delivery. J. Herb. Med. 2020, 20, 100300.
[CrossRef]
55. Manosroi, A.; Chutoprapat, R.; Abe, M.; Manosroi, W.; Manosroi, J. Anti-Aging Efficacy of Topical Formulations Containing
Niosomes Entrapped with Rice Bran Bioactive Compounds. Pharm. Biol. 2012, 50, 208–224. [CrossRef] [PubMed]
56. Cheng, W.H.; Yap, C.K.; Zakaria, M.P.; Aris, A.Z.; Guan, T.S. Lithium Levels in Peninsular Malaysian Coastal Areas: An
Assessment Based on Mangrove Snail Nerita Lineata and Surface Sediments. Pertanika J. Trop. Agric. Sci. 2015, 38, 93–101.
57. Bocca, B.; Pino, A.; Alimonti, A.; Forte, G. Toxic Metals Contained in Cosmetics: A Status Report. Regul. Toxicol. Pharmacol. 2014,
68, 447–467. [CrossRef]
58. Wu, M.L.; Deng, J.F.; Lin, K.P.; Tsai, W.J. Lead, Mercury, and Arsenic Poisoning Due to Topical Use of Traditional Chinese
Medicines. Am. J. Med. 2013, 126, 451–454. [CrossRef] [PubMed]
59. U.S. FDA. FDA’s Testing of Cosmetics for Arsenic, Cadmium, Chromium, Cobalt, Lead, Mercury, and Nickel Content. Available on-
line: [Link]
cobalt-lead-mercury-and-nickel-content (accessed on 23 February 2022).
60. Brügel, M. Global Organic Textile Standard. Zertif. Als Erfolgsfaktor Nachhalt. Wirtsch. Mit Vertrauen Transpar. 2016, 2020, 153–163.
[CrossRef]
61. Massadeh, A.M.; El-khateeb, M.Y.; Ibrahim, S.M. Evaluation of Cd, Cr, Cu, Ni, and Pb in Selected Cosmetic Products from
Jordanian, Sudanese, and Syrian Markets. Public Health 2017, 149, 130–137. [CrossRef]
62. Arshad, H.; Mehmood, M.Z.; Shah, M.H.; Abbasi, A.M. Evaluation of Heavy Metals in Cosmetic Products and Their Health Risk
Assessment. Saudi Pharm. J. 2020, 28, 779–790. [CrossRef]
63. Schwarz, J.C.; Baisaeng, N.; Hoppel, M.; Löw, M.; Keck, C.M.; Valenta, C. Ultra-Small NLC for Improved Dermal Delivery of
Coenyzme Q10. Int. J. Pharm. 2013, 447, 213–217. [CrossRef]
64. Ma, Y.; Chen, S.; Liao, W.; Zhang, L.; Liu, J.; Gao, Y. Formation, Physicochemical Stability, and Redispersibility of Curcumin-
Loaded Rhamnolipid Nanoparticles Using the PH-Driven Method. J. Agric. Food Chem. 2020, 68, 7103–7111. [CrossRef]
65. Bai, L.; McClements, D.J. Formation and Stabilization of Nanoemulsions Using Biosurfactants: Rhamnolipids. J. Colloid Interface
Sci. 2016, 479, 71–79. [CrossRef]
66. Dai, L.; Li, R.; Wei, Y.; Sun, C.; Mao, L.; Gao, Y. Fabrication of Zein and Rhamnolipid Complex Nanoparticles to Enhance the
Stability and in Vitro Release of Curcumin. Food Hydrocoll. 2018, 77, 617–628. [CrossRef]
67. Yi, G.; Son, J.; Yoo, J.; Park, C.; Koo, H. Rhamnolipid Nanoparticles for in Vivo Drug Delivery and Photodynamic Therapy.
Nanomed. Nanotechnol. Biol. Med. 2019, 19, 12–21. [CrossRef] [PubMed]
68. Park, S.J.; Garcia, C.V.; Shin, G.H.; Kim, J.T. Improvement of Curcuminoid Bioaccessibility from Turmeric by a Nanostructured
Lipid Carrier System. Food Chem. 2018, 251, 51–57. [CrossRef]
69. Jose, J.; Netto, G. Role of Solid Lipid Nanoparticles as Photoprotective Agents in Cosmetics. J. Cosmet. Dermatol. 2018, 18, 315–321.
[CrossRef] [PubMed]
70. Fathi, M.; Varshosaz, J.; Mohebbi, M.; Shahidi, F. Hesperetin-Loaded Solid Lipid Nanoparticles and Nanostructure Lipid Carriers
for Food Fortification: Preparation, Characterization, and Modeling. Food Bioprocess Technol. 2013, 6, 1464–1475. [CrossRef]
71. Dash, S.; Murthy, P.N.; Nath, L.; Chowdhury, P. Kinetic Modeling on Drug Release from Controlled Drug Delivery Systems. Acta
Pol. Pharm. Drug Res. 2010, 67, 217–223.
72. Marcon, A.F.; Wagemaker, T.; Maia, M.B.; Campos, P. Rheology, Clinical Efficacy and Sensorial of a Silicone-Based Formulation
Containing Pearl Extract. J. Biomed. Biopharm. Res. 2014, 11, 247–255. [CrossRef]
73. Alison, L.; Rühs, P.A.; Tervoort, E.; Teleki, A.; Zanini, M.; Isa, L.; Studart, A.R. Pickering and Network Stabilization of Biocompati-
ble Emulsions Using Chitosan-Modified Silica Nanoparticles. Langmuir 2016, 32, 13446–13457. [CrossRef]

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