1. Introduction
A number of Indonesian plants have the potential to be explored as traditional medicines, cosmetics, and food sources [
1]. The usage of plant extracts as ingredients in cosmetics manufacturing is increasingly in demand by consumers, who are gradually becoming concerned with environmentally friendly products [
2]. One of the benefits of utilizing herbal plant-based cosmetics is to protect the skin from ultraviolet (UV) exposure. Most customers need cosmetics with high antioxidant properties to protect against UV radiation [
3]. The secondary metabolites derived from plants, generally known as essential compounds for antioxidant agents, are flavonoids [
4]. Cinnamon (
Cinnamomum burmannii) is rich in this type of compound [
5].
Cinnamon, obtained from the dried outer layer of
C. burmannii’s bark, is commonly consumed as a traditional medicine, spice, and flavoring agent and can increase the farmers’ economy in selected Indonesian areas [
6,
7]. This plant is extensively recognized as a potential source of essential oils and phenolic compounds, such as flavonoids, phenolic aldehydes and acids, coumarins, and proanthocyanidins [
7,
8]. A previous study reported that the major compounds in
n-butane extract of Chinese cinnamon from subcritical extraction consisted of (
E)-cinnamaldehyde, eugenol, and coumarin, while its ethanol extract presented procyanidin trimer, (
E)-cinnamaldehyde, and (
Z)-cinnamaldehyde [
9]. In addition, methanol extract of Indonesian cinnamon from Kerinci, Sumatra, showed the presence of catechin, epicatechin, procyanidin B2, quercitrin, 3,4-dihydroxybenzaldehyde, protocatechuic acid, and cinnamic acid by ultrasonic extraction [
10]. Other extracts from different regions of Indonesia, i.e., Karanganyar and Padang, exhibited cinnamaldehyde as a major compound using steam distillation [
11]. Differences in the phytochemical constituents in plants are affected by physiological age, habitat or environmental conditions, and genetic factors [
12].
Cinnamon has a wide range of biological activities, such as antioxidant, anticoagulant, antidiabetic, anti-inflammatory, anti-tumor, anticancer, anti-microbial, anti-fungal, antiviral, and gastro-protective properties, treating dental problems, and lowering blood pressure, cholesterol, and lipids [
13,
14,
15]. Due to its health benefits, this plant captures scholars’ attention to exploring its flavonoid content as a potential candidate to be used in cosmetics [
16,
17,
18]. However, there are still limited studies on cinnamon extract for beauty products. In addition, ascorbic acid as a plant extract stabilizer should be added to prevent significant changes in the physical properties, flavonoid contents, antioxidant activities, and chemical constituents.
Studies using additive compounds have been conducted to enhance the stability of plant extracts. Using sodium carboxymethyl cellulose (CMC-Na) as a gelling agent improved the consistency, stickiness, dispersibility, and antibacterial activity of the hand sanitizer gel based on basil leaf extract for five days [
19]. Moreover, the addition of polyvinyl alcohol (PVA) and guar gum as a cold-swelling agent presented the best performance in the physical and physicochemical stability tests of a peel-off facial mask containing soybean extract for 15 days [
20]. An emulsion stabilizer was also introduced in the cream formulation of
Sueada asparagoides extract and showed the best stability on pH, viscosity, and absorbance during 12 weeks of observation [
21]. Furthermore, the combination of a cinnamon essential oil nano-emulsion as a polyphenol oxidase (PPO) inhibitor and ascorbic acid as a reducing agent could improve the inhibition of enzymatic browning and maintain the quality of apple juice for 54 h [
22].
This research selected the best solvent to extract flavonoids, extracted flavonoids in a large-scale maceration, and assessed the extract’s stability for 16 weeks in the absence and presence of ascorbic acid at room temperature (RT, 27.0 + 1.0 °C) and in an incubator (40 + 0.5 °C). The stability tests included physical stability tests, such as the organoleptic test and pH (every week), flavonoid content and antioxidant activity tests (every four weeks), and volatile and non-volatile chemical compound analyses (every eight weeks). Identifying the effect of ascorbic acid on the stability of physical properties, flavonoid contents, antioxidant activities, and chemical compositions in Indonesian cinnamon extract (C. burmannii) could significantly impact commercializing Indonesian plant extracts for cosmetics.
2. Materials and Methods
2.1. Chemicals and Instrumentations
The solvents used for extraction were ethanol (EtOH), ethyl acetate (EtOAc), and acetone of technical grade (CV. Satya Darmawan). For the qualitative and quantitative phytochemical screening, quercetin was purchased from Sigma Aldrich, while the others, i.e., NaOH, HCl, Mg ribbon, FeCl3, CHCl3, NH4OH, H2SO4, Dragendorff, Meyer, Wagner, AlCl3, and CH3COOK were obtained from Merck. For the antioxidant activity test, ascorbic acid (SmartLab) was used as a stabilizer in the stability test and positive control. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) from SmartLab and EtOH from Merck were also used for the antioxidant analysis. Weekly pH measurements for the stability test were conducted using an ICONIX PC-50 multiparameter with a pH5F and a semisolid probe. The operated instrumentations were a UV-visible (UV-Vis) Shimadzu UV2450, a gas chromatography-mass spectrometry (GC-MS) 5975 Series G 1701 EA 02.02 (Agilent Technologies), and a liquid chromatography-mass spectrometry (LC-MS) Acquity UPLC H-class system.
2.2. Sample Preparation and Identification
Three-year-old cinnamon (Cinnamomum burmannii (Nees and T. Nees) Blume) bark was obtained from the Tropical Biopharmaca Research Center (Trop BRC) garden, Institute for Research and Community Service (LPPM) IPB University, Dramaga, Bogor, West Java, Indonesia, in February 2021. The dark brown outer layer of cinnamon bark was determined by Taopik Ridwan, S.P., M.Si., in the same institution and dried at room temperature for three days. The air-dried sample then was ground using a Honda GX 160 equipped with a Disk Mill FFC-15 (Hong Tong Fang - Shan Dong Ji Mo Disk Machienery The People’s Republic of China) to obtain the 80-mesh powder form.
2.3. Small-Scale Plant Extraction
Into three different glass-stoppered Erlenmeyer flasks, finely ground air-dried cinnamon (50 g) was placed and macerated for 3 × 24 h using various solvents, i.e., EtOH, EtOAc, and acetone. All samples were then filtered using Whatman No. 1 filtration paper and concentrated under reduced pressure with an IKA RV 8 rotary evaporator (40 °C, 90 rpm, 175–556 mbar). Each concentrated extract was weighed and determined for its yield (Equation (1)). All the extracts were then tested for qualitative phytochemical screening, their total flavonoid contents, and their antioxidant activities. The solvent that showed promising results was then selected for large-scale isolation.
2.4. Large-Scale Plant Extraction
A total of 6 kg of dried powdered cinnamon was used in a large-scale extraction and was macerated using EtOH three times at room temperature.
The following procedures to yield the brownish-black extract (788 g, 13.13% w/w) remained the same as the small-scale isolation. The qualitative phytochemical investigation was then also applied for the large-scale extract.
2.5. Qualitative Phytochemical Analysis
A 100 mg extract was diluted in 10 mL of ethanol as the plant extract. The plant extract was then applied for a preliminary investigation of the presence of secondary metabolites following standard procedures [
23] and conducted in two replicates.
2.5.1. Flavonoids
A 2 mL volume of plant extract was placed into two test tubes. Two drops of NaOH 10% (w/v) were added to the first tube, while two drops of HCl and 0.1 g of Mg ribbon were added to the second test tube. The color change of the solution to red, yellow, or orange indicated the presence of flavonoids.
2.5.2. Tannin
A 2 mL volume of plant extract was arranged into a test tube, and 1 mL of FeCl3 5% (w/v) was added. The color change of the solution to blue-black indicated the presence of tannin.
2.5.3. Terpenoid and Steroid
Acetate anhydride acid was added to 500 mg of concentrated extract until soaked for 15 min. The filtrate was then separated into a test tube, and 2–3 drops of H2SO4 conc. were added. The color change of the solution to orange, red, or purple indicated the presence of terpenoids and steroids.
2.5.4. Saponin
A 2 mL volume of plant extract was put into a test tube and shaken until a lather was formed. A stable lather for about 30 min indicated the presence of saponin.
2.5.5. Alkaloid
A 2 mL volume of plant extract was added into a test tube, followed by 2 mL of CHCl3, 2 mL of NH4OH, and 3–5 drops of H2SO4 conc. The mixture’s upper layer was then transferred into three test tubes to undergo Dragendorff, Meyer, and Wagner tests. Brown, orange, and white precipitation observed in the solution indicated the presence of alkaloids, respectively.
2.6. Sample Preparation of Stability Test
A stability test was conducted in the presence and absence of ascorbic acid. Around 600 g of cinnamon extract was divided into six wide-mouth glass bottles with screw caps to receive different treatments (
Table 1). Samples A and B were assigned without the addition of ascorbic acid, while samples C and D were treated using 10% (
w/
w) ascorbic acid, and samples E and F were 15% (
w/
w) ascorbic acid. Samples A, C, and E were stored at RT (27.0 + 1.0 °C), while the others were kept in the incubator (40 + 0.5 °C). In subsequent stages, all samples were then subjected to physical stability tests (every week), followed by an evaluation of flavonoid content and antioxidant activity (every four weeks) and an analysis of volatile and non-volatile chemical compounds using GC-MS and LC-MS (every eight weeks).
2.7. Physical Stability Test
Organoleptic tests were conducted to observe the texture, odor, and color changes. A pH measurement was also assessed for all samples and was measured in two replicates.
2.8. Determination of Total Flavonoid Content
Total flavonoid content was determined by the AlCl
3 colorimetric method using UV-Vis [
24]. Each 0.5 mL sample was treated with 1.5 mL of ethanol, 0.1 mL of AlCl
3 (10%,
w/
v), 0.1 mL of potassium acetate 1 M, and 2.8 mL of distilled water. The mixture was then homogenized with a vortex mixer for about 30 s, allowed to stand at room temperature for 30 min, and the absorbance was measured at λ 435.5 nm. Various concentrations of quercetin were used as a comparison, which was expressed as the quercetin equivalent (QE). Duplicates were maintained, and the experiment was repeated two times. The flavonoid content was presented in mg of quercetin equivalent in g of total extract (QE mg/g) based on Equation (2), where F = total flavonoid content (QE mg/g),
c = quercetin equivalent concentration of extract (mg/mL),
V = volume of extract (mL),
f = dilution factor, and m = mass of extract (g).
2.9. DPPH Radical Scavenging Activity Assay
The DPPH radical scavenging method was used to determine the antioxidant activity following the standardized procedures [
25] with some modifications by mixing 2 mL of an ethanolic solution of 0.1 mM DPPH and 2 mL of EtOH dissolved in the samples at various concentrations. After incubating in the dark for 30 min, the absorbance was measured at λ 510 nm. The experiment was repeated twice in this study. DPPH in EtOH without a sample was used as a negative control, while ascorbic acid was selected as a positive control. The radical scavenging activity percentage was then determined using Equation (3), where
A0 = the absorbance of the negative control and
Ai = the absorbance of the sample. The antioxidant activity in IC
50 (mg/mL) was calculated by substituting 50% RSA in the linear equation obtained by plotting %RSA with the sample’s concentration.
2.10. Chemical Constituent Analysis
The identification of volatile and non-volatile chemical constituents in samples was performed using GC-MS and LC-MS, respectively, in ethanol. GC-MS with He UHP gas was set at a 50 °C column oven temperature for 5 min, then the temperature was gradually increased to 280 °C. The injection temperature was set to 280 °C at a pressure of 101 kPa and a column flow of 0.85 mL/min. The MS detector temperature was also selected for ion source at 200 °C, interface temperature at 280 °C, detector temperature at 280 °C, and pyrolyzer temperature at 300 °C. An ultra-performance LC was used with C-18 as a stationary phase with a 50 °C column temperature and a flow rate of 0.2 mL/min for 23 min. A 5 mM ammonium formate and acetonitrile with 0.05% formic acid were used as a mobile phase. Electrospray ionization with positive mode was used for MS detection. A mass analysis was carried out at 50–1200 m/z with two different energies, 4 and 25–50 V. The chromatogram was then analyzed using MassLynx software.