Application Of Hibiscus Cannabinus L. (kenaf) Leaves Extract As Skin Whitening And Anti-aging Agents in Natural Cosmetic Prototype
Mar 26, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Yan Yi Sim & Kar Lin Nyam
ABSTRACT
In Malaysia, the industrial wastes from the Hibiscus cannabinus L. (kenaf) industry, especially leave, cause challenges in sustainability. The biological activity of kenaf leaves was reported by a few previous studies, it is, therefore, believable that the kenaf leaves extract (KLE) can be used as a functional ingredient in cosmetic formulations. Thus, the aim of this study was to develop a natural cosmetic formulation containing KLE and evaluate its physicochemical characteristics, microbiological properties, storage stability, biological activities (antioxidant, antityrosinase, and anti-aging), in vitro cytotoxicity (on normal human dermal fibroblast and B16F10 melanoma cells) and melanogenesis assay (intracellular tyrosinase activity and reduction of melanin production). The results showed that KLE lotion (KLEL) prepared by 15 % kenaf seed oil (KSO) (F2) with 0.1 % w/w KLE yields the best physical and microbiological stability, with no toxicity on human cells. The KLEL also presented antioxidant content up to 1.84 ± 0.07 mg caffeic acid equivalent (CAE)/g and 21.62 ± 0.76 mg catechin hydrate equivalent (CHE)/g for total phenolic content (TPC) and total flavonoid content (TFC), respectively. In addition, the KLEL presented antityrosinase capacity on inhibition of mycophenolate (30.28 ± 3.90 %) and diphenols (11.40 ± 0.29 %) formation, and revealed, for the first time, anti-aging properties by inhibiting collagenase (36.41 ± 0.54 %) and elastase (23.13 ± 1.56 %) activities. Concerning melanogenesis inhibitory activity, KLEL presented high efficacy in suppressing cellular tyrosinase activity and melanin content on B16F10 cells. Overall, the results from this study are very promising towards the development of natural cosmetic prototypes using kenaf leaves.
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1. Introduction
Recently, the circular economic model which can enhance the sustainability of waste management by minimizing waste production and maintaining long-term value, while decreasing the negative consequences of resource scarcity and environmental degradation has gained public interest (Morseletto, 2020). In general, a significant amount of by-products with low economic value is generated by industrial crops industries each year. Hibiscus cannabinus L. KR9 (kenaf) industry, is one of the agricultural sectors contributing to Malaysia’s GDP (gross domestic product) with production weight of kenaf dry stem increased from 7.1 (000 ton) in 2013 to 7.6 (000 ton) in 2014, and the global kenaf market projected to cross US$ 854 million by 2025 (Abdelrhman et al., 2016). However, large quantities of by-products are generated, such as kenaf seeds and leaves, which contribute to sustainability problems.
Kenaf leaves, accounting for a significant amount of total by-products, are the least characterized and valued among all by-products generated. Because of the circular approach, it is important to reuse or recover the kenaf leaves into value-added products as it offers multiple benefits in fields of economic, environmental, and social (Coderoni and Perito, 2019). Kenaf leaves consist of rich sources of bioactive compounds such as chlorogenic acid, caffeic aid, kaempferol, and catechin hydrate as proven by studies carried out by Kho et al. (2019); Sim and Nyam (2019), and Haw et al. (2020). However, it is usually destined for the production of low economic value products: dietary fiber and animal feed (Lim et al., 2020).
The term “return to nature” has been extensively used in cosmetic industry research and development, as the use of extracts of botanical origin has resulted in good acceptance by consumers. According to the study carried out by Sim et al. (2019), kenaf leaves extract (KLE) demonstrated promising antioxidant and antityrosinase properties, had the potential to be used as value-added ingredients in the development of cosmetic products. It is important to develop stable and safe formulations containing the KLE as it contains a lot of polyphenol compounds that demonstrated skin whitening and anti-aging properties. There are several requirements to be taken into account when developing new cosmetic formulations, such as formulation type, the intent of use, and potential interaction between the ingredients used in the formulations, which as a whole, contribute to the need for stability and safety studies (Garbossa and Maia Campos, 2016).
Stability studies, which include the study of physical, chemical, and microbial characteristics, are necessary for cosmetic formulations after the development process. In addition, to prevent any adverse effect or allergic reaction, cosmetic products should also be tested by using an in vitro cytotoxicity assay on a normal human skin cell line. Thus, the objective of this work is to develop a natural cosmetic formulation containing KLE (KLE lotion- KLEL). Then, the KLEL was evaluated for its physicochemical characteristics, microbiological properties, storage stability, in vitro cytotoxicity (on normal human dermal fibroblast and B16F10 melanoma cells), and melanogenesis assay (intracellular tyrosinase activity and reduction of melanin production). This study also allowed us to determine, for the first time, not only the antioxidant and anti-tyrosinase activity of KLEL but also their inhibitory activity on collagenase and elastase.
2. Materials and methods
2.1. Plant materials and chemicals
Fresh Hibiscus cannabinus L. KR9 (kenaf) leaves 90 days after sowing and seed were obtained from Lembaga Kenaf & Tembakau Negara (LKTN, Malaysia). Span 20 and Tween 80 were purchased from SigmaAldrich (Munich, Germany), Cosmedia® ACE, Iscaguard® PEG, and Emulgade® SE-PF were sourced from BASF (Malaysia), and glycerine was purchased from Croda International Plc. (United Kingdom). Normal human dermal fibroblast (NHDF) cell line was sourced from the Lonza (Basel, Switzerland) and mouse melanoma (B16F10) was purchased from the ATCC (Manassas, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Nacalai Tesque (Japan). All other chemicals used were of analytical reagent grade (Belgium, Germany, Malaysia). The ultra-pure water (Millipore, USA) was used throughout the analysis.
2.2. Methods
2.2.1. Drying of kenaf leaves
The kenaf leaves were cleaned with ultra-pure water, dried, and then kept at − 80 ◦C overnight. Then, the sample was dried in a freeze dryer (Christ, Germany), under 0.0004 bar for 48 h, ground, vacuum-packed, and stored at − 20℃.
2.2.2. Preparation of partial purified kenaf leaves extract (KLE)
The pulsed ultrasonic-assisted extraction (PUAE) of KLE was carried out according to Sim et al. (2019). Samples were weighed with extraction solvents (ethanol) in the ratio of 1:10. Then, the mixture was subjected to the pulsed ultrasonic-assisted extraction (Sartorius, Germany) at 50 % sonication amplitude, 1 min pulse duration period, and a 1 min pulse interval period with temperature maintained at 18–22 ± 3℃ (three cycles). Then, the extract was filtered and concentrated in a vacuum rotary evaporator (Buchi, Switzerland). The partial purification of KLE was carried out according to Seabra et al. (2010) with slight modification. The crude extract was applied to a column filled with silica gel and eluted with a gradient solvent of n-hexane- ethyl acetate (100:00, 90:10, 80:20, 50:50, 20:80, 10:90, 00:100) and ethyl acetatemethanol (100:00, 90:10, 80:20, 50:50, 20:80, 10:90, 00:100). The partially purified KLE was concentrated to dryness by using a rotary evaporator and stored at − 20℃ for future use.
2.2.3. Solvent extraction of kenaf seed oil (KSO)
KSO was extracted according to Chew et al. (2015). Kenaf seeds were ground into a fine powder using a grinder (Panasonic, Japan) and added with hexane in the ratio of 1:5. The oils were extracted by using a Soxhlet extractor at 60 ◦C for 3 h, and the hexane was removed by using a rotary evaporator. After flushing with nitrogen, the KSO was stored at − 20℃ for future use (Chew et al., 2015).
2.2.4. Experimental development of lotion formulation containing the KLE
At first, the lotion base formulation (without KLE) was optimized by using 4 different concentrations of kenaf seed oil (KSO) (10 %, 15 %, 20 %, and 25 %). The lotion base was then comparatively evaluated for various parameters: physical appearances, odor, homogeneity, pH, viscosity, centrifugation, and freeze-thaw evaluation, to select the best lotion base formulation for further study. Regarding the results from the above parameters, the most suitable lotion base formulation (15 % KSO) was selected for incorporation with the KLE (0.1 % w/w) (KLEL). Briefly, non-polar phase (Emulgade® SEPF, Span 20, and KSO) and polar phase (water, Tween 80, and glycerine) ingredients were heated up to 75 ± 2℃. Then, the non-polar phase was added drop by drop to the polar phase with rapid stirring using a magnetic stirrer (Thermo Fisher Scientific, USA) at 350 rpm to form a primary emulsion, followed by high-shear homogenization using IKA T25 digital ULTRA-TURRAX® (IKA Laboratory Equipment, Germany) at 3200 rpm for 3 min and ultrasonic homogenization at 40 % amplitude for 3 min. The Cosmedia® ACE, Iscaguard® PEG, and KLE were added and mixed at the O/W emulsion at room temperature. The KSO lotion base without KLE serves as a control (KSOL). While the lotion base added with kojic acid (0.1 % w/w) (KAL) served as a positive control for anti-tyrosinase activity and melanogenesis assay. The lotion base added with catechin hydrate (0.1 % w/w) (CHL) served as a positive control for anticollagenase and antielastase activity
2.2.5. Physicochemical analysis
2.2.5.1. Appearances, odor, and homogeneity.
According to Hanifah and Jufri (2018), the lotion formulations were inspected for their color, odor and homogeneity, and phase separation.
2.2.5.2. pH. The standard solutions were used to calibrate the pH meter electrode before measurement.
The pH of the samples was determined with a pH meter (Mettler Toledo, Switzerland) at room temperature.
2.2.5.3. Colour.
The Colour will be evaluated by using a colorimeter (Hunter Lab, United State). The results will be expressed according to the color space L* (lightness), a* (green), b*(yellow), and total color differential (ΔE). The total color differential (ΔE) for all samples were calculated by using the equation as follows: ΔE = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ ( L∗ − L∗ 0 )2 + (a∗ − a∗ 0) 2 + ( b∗ − b∗ 0 )2 √
2.2.5.4. Viscosity.
The viscosity of samples was evaluated in Brookfield viscometer using LV-64 spindle according to Gyawali et al. (2016) with slight modification. The sample was directly immersed into the spindle with a 100 rpm rotation rate and the viscosity (cP) was measured.
2.2.5.5. Spreadability.
The spreadability of samples was determined by the parallel plate method (Gyawali et al., 2016). The sample was weighed precisely (1 g) and placed over one of the glass slides (20 × 20 cm2 ). Then, the other slide was placed upon the top of the sample and 100 g weight was placed upon the upper slide, to ensure that the sample was pressed evenly. After 1 min, the weight was removed, and the spread diameter (cm) was measured.
2.2.5.6. Centrifugation evaluation.
The sample was loaded into a centrifugation tube and then centrifuged at 3750 rpm for 30 min (Eppendorf 5417R, USA). This centrifugation test (equal to 1-year gravity) determines the stability of the sample (Hiola et al., 2018).
2.2.5.7. Freeze-thaw evaluation.
Freeze-thaw studies were carried out according to the previous methods with a few modifications (Krongrawa et al., 2018). Each sample was kept alternately at cold temperature, 4 ± 1 ◦C (24 h) and hot temperature, 45 ± 1 ◦C (24 h) with 75 % ± 2 % RH (relative humidity) for 6 cycles in airtight glass containers. The viscosity of the samples was determined after the temperature stress cycle.
2.2.6. Determinant of antioxidant content
2.2.6.1. Total phenolic content (TPC).
The total phenolic content was determined according to Lim et al. (2007). Sample (10 mg/mL) was mixed with 10 % Folin-Ciocalteu reagent and 7.5 % (w/w) Na2CO3, follow by 30 min incubation in dark. The absorbance was taken at 765 nm by using a UV–vis spectrophotometer (Secoman, France). The calibration equation for caffeic acid was y = 0.0269 + 9.69x ( r 2 = 0.999) (Sim et al., 2019). The results were expressed as mg of caffeic acid equivalent (CAE)/ g sample.
2.2.6.2. Total flavonoid content (TFC).
Total flavonoid content was evaluated according to Ogbunugafo et al. (2011). In general, the sample (10 mg/mL) was mixed with 15 % NaNO2, 10 % AlCl3 solution, ultra-pure water, and 1 M NaOH. The absorbance was measured immediately at 510 nm in comparison with the standard prepared using catechin hydrate (0.02− 0.1 mg/mL). The calibration equation for catechin hydrate was y = 0.004 + 3.1x ( r 2 = 0.999) (Sim et al., 2019). Total flavonoid content (TFC) was expressed as milligrams of catechin hydrate equivalents (CHE)/ g sample.
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2.2.7. Anticollagenase activity
The anti collagenase activity based on the proteolytic degradation between collagenase and synthetic substrate (FALGPA- N-(3-[2-Furyl]- acryloyl)-Leu-Gly-Pro-Ala) at 345 nm in the presence of collagenase inhibitors was carried out according to Barrantes and Guinea (2003) with some modification. The 0.25 units/mL collagenase (40 μL) derived from Clostridium histolyticum was allowed to react with 10 μL test samples and 20 μL 50 mM tricine buffer (pH 7.5, with 100 mM CaCl2 and 5 mM NaCl) in dark at for 15 min. After the pre-incubation, an amount of 40 μL of the 2 mM FALGPA solution was added to each well. Each sample was accompanied by a blank that had all the components except FALGPA and the absorbance was determined after incubated for 20 min.
2.2.8. Bacteriological shelf life analysis of KLEL
Methods used for the total aerobic microbial count (TAMC) and the total yeasts and molds count (TYMC) of the KLEL were modified from the FDA bacteriological analytical manual (BAM) (Huang et al., 2017). The sample (1 g) was mixed with 1 mL sterile Tween 80 and volume was adjusted with sterile peptone water to obtain a complete dilution series from 10− 1 to 10− 3. For the TAMC, the samples were inoculated on nutrient agar using a spread plate method, and then the plates were incubated at 30 ± 2 ◦C for 48 h. In the TYMC case, the samples were inoculated on plates of potato dextrose agar using a spread plate method, and the plates were subsequently incubated at 30 ± 2 ◦C for 7 days. The plates were examined for microbial growth after the incubation period.
2.3. Statistical analysis
All results were analyzed using Minitab 16.2.1 statistical package (Minitab Inc., Pennsylvania, USA), One-way Analysis of variance (ANOVA) was performed, followed by Tukey’s test to determine the significant difference (p < 0.05). Means ± standard deviation (SD) (n = 3) was presented for data analysis.
3. Results and discussions
3.1. Optimization of lotion base formulation
According to Hiola et al. (2018), optimization of lotion base (pre-formulation) has an important role to get good and stable formulation. Therefore, the lotion base was optimized by using 4 different percentages of KSO as shown in Table 1. They were evaluated by appearance, odor, homogeneity, physical stability, pH, spreadability, viscosity, and freeze-thaw analysis (supplementary materials- S1 and S2). All the lotion base formulations prepared by using KSO had light milky yellow to milky yellow color, with good odor (Chu and Nyam, 2020). Based on the results obtained, all the lotion base formulations' pH values were within the range of skin pH (4–6), and this is important to minimize the allergic reaction and ensure the stability of cosmetic formulation over storage time (Chu and Nyam, 2020). All the lotion base formulations showed a drop in viscosity after freeze-thaw analysis, but still within the range for good lotion (500–5000 cP) (Kusuma et al., 2017). For the spreadability, the higher the concentration of KSO, the smaller the spreadability diameter due to high viscosity. The larger the spreadability, the easier the cosmetic formulation can be applied on the skin surface. For the stability test by resistance to centrifugation, F2 and F3 showed no phase separation, suggesting that both formulations were stable for 1 year. However, F3 cannot be poured easily and gave the appearance resembling cream preparation since it had a higher viscosity than F2. Therefore, F2 was selected as the optimized lotion base to incorporate with the KLE.
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3.2. Evaluation of KLE lotion (KLEL) formulation
3.2.1. Physicochemical analysis
Based on the results in Table 2, the lotion base added with KLE showed light milky color with a pleasant smell and no phase separation observed. The KLEL showed a higher pH value than the control, indicating KLE addition into cosmetic formulation may result in increasing pH value. The pH values of samples were all compatible with the skin pH range and safe for skin. There were no significant differences in viscosity and spreadability between KLEL and control, which indicated adding KLE into emulsion did not affect the viscosity and spreadability. Significant changes were observed in the L*, a*, and b* values of KLEL compared with the control, which proved that the color of the lotion was affected by the natural green color of kenaf leaves. KLEL has the greenest for a* value (-2.24 ± 0.03) and the yellowest for b* value (15.54 ± 0.01). The total color difference (ΔE) of the KLEL compared with the control was statistically significant p＜0.05, with the value of 6.82 ± 0.04 for KLEL. Previous studies regarded ΔE = 2 as the threshold for visual discrimination (Zhou et al., 2009). Thus, the results of ΔE proved that the addition of KLE can affect the color of the cosmetic formulation.
3.2.2. Antioxidant content analysis
Table 3 presents the amount of total phenolic (TPC) and flavonoid content (TFC). The obtained results indicated the formulated lotion base with KLE contains significantly higher TPC (1.84 ± 0.07 mgCAE/g) than the control (1.64 ± 0.06 mgCAE/g). While, for TFC, KLEL also showed significant higher in TFC (21.62 ± 0.76 mgCHE/g) in compared with control (19.42 ± 0.27 mgCHE/g). In the same range of some plant by-products, the inclusion of KLE on a dermatological basis still presents high antioxidant contents, highlighting its potential source of polyphenol compounds in the cosmetic industry (Adhikari et al., 2019; Rodrigues et al., 2014). According to the study by Haw et al. (2020), the kenaf leaves are rich in different types of polyphenol compounds such as caffeic acid, tannic acid, catechin, and chlorogenic acid.
3.2.3. Antioxidant activity analysis
The free radical scavenging abilities of the lotions were investigated by using DPPH and ABTS assay. Table 3 indicates that the KLEL demonstrated higher DPPH (1.16 ± 0.18 mgTEAC/g) and ABTS (0.50 ± 0.04 mgTEAC/g) radical scavenging activity compared with the control. DPPH and ABTS assay findings have shown similar patterns as in TPC and TFC, where it can greatly improve antioxidant activity by adding KLE into the lotion base, these findings have suggested that KLE contains free radical scavengers that when applied into cosmetic formulations, may play a significant role as a primary antioxidant in suppressing free radical that caused skin aging. In addition, the KLEL (0.69 ± 0.20 mgTEAC/g) also demonstrated a significant highest in ferric ion reduction ability than the control (0.46 ± 0.09 mgTEAC/g), in agreement with the DPPH and ABTS. There is a need for a constant supply of antioxidant compounds from external sources such as cosmetics to restore the individual skin antioxidant defense system against ROS that caused skin aging (Działo et al., 2016). In addition, lotion formulations with radical scavenging activity and reducing power can be used as a skin whitening agent by down-regulated UV-induced melanin production (Działo et al., 2016).
3.2.4. Enzyme inhibitory activity
The lotions were tested against tyrosinase, a copper-containing metalloprotein enzyme involved in melanin biosynthesis, for the assessment of in vitro antityrosinase activity. Table 5 shows that the KLEL (30.28 ± 3.90 %) exhibited no significant differences in anti-tyrosinase activity with the positive control (34.05 ± 4.60 %) when using L-tyrosine as substrate. However, ALL (11.40 ± 0.29 %) demonstrated lower antityrosinase activity than the positive control (15.01 ± 0.84 %), when using L-DOPA as substrate. This showed that KLEL mainly inhibited mycophenolate rather than diphenols. The in vitro anti-aging properties were evaluated through collagenase and elastase inhibitory activity. An enzyme such as collagenase and elastase can degrade collagen and elastin, which are responsible for skin integrity and elasticity (Jesumani et al., 2019). So, the inhibition of elastase and collagenase activity would reduce the degradation of elastin and collagen thus preventing wrinkle formation, one of the major signs of aging. As shown in Table 5, KLEL demonstrated remarkable anti collagenase (36.41 ± 0.54 %) and antielastase (23.13 ± 1.56 %) activity in comparison with the positive control (CHL). The KSOL also demonstrated antityrosinase (18.82 ± 0.17 % (L-tyrosine as substrate); 8.48 ± 0.29 % (L-DOPA as substrate), anti collagenase (18.84 ± 0.63 %) and antielastase (5.78 ± 0.59 %) activity, but lower than ALL. This might indicate that the combination of KLE and KSO demonstrated a synergistic effect with better enzyme inhibitory activity. This is supported by the studies carried out by Pascoal et al. (2015) and Chew et al. (2016), in which the KSO is rich in tocopherols, while KLE is rich in quercetin and kaempferol derivatives. These compounds demonstrated skin whitening and anti-aging properties, with interest in the reduction of skin hyperpigmentation and wrinkles production (Lin et al., 2007; Keen and Hassan, 2016).
3.2.5. In vitro melanogenesis assay
To further explore the skin whitening properties of the KLEL, the B16F10 melanoma cell model was used to study the inhibitory effect on melanogenesis. For cellular tyrosinase activity, as shown in Fig. 5a, KLEL (32.35 %) at 500 μg/mL demonstrated significantly stronger inhibition on cellular tyrosinase activity than the KAL (14.85 %) and KSOL (13.64 %), when compared with a-MSH treated control cell. For extracellular and intracellular melanin content, KLEL dose-dependently reduced the melanin content of the B16F10 cells (Fig. 5b and c). The ALL (500 μg/mL) was found to have a comparable inhibitory effect on extracellular melanin content (56.13 %) (compared with an MSH-treated control cell) with KAL (54.53 %). In addition, the KLEL (36.52 %) also demonstrated a stronger inhibitory effect on intracellular melanin content than KAL (7.98 %). While, KSOL also showed an inhibitory effect on extracellular melanin content (17.73 %), but no effect on intracellular melanin content. Compared with KAL, the stronger inhibitory impact of KLEL on the inhibition of melanogenesis may be clarified by the synergistic activity between KSO and KLE, which can enhance the KLE skin whitening properties in the lotion base. These results further proved that KLE can be used as an effective skin whitening agent in the natural cosmetic prototype.
3.2.6. In vitro cytotoxicity assay
By assessing the biocompatibility of the KLEL (MTT analysis), the NHEF cells viability (after 24, 48, 72 h) grown in the presence of different KLEL concentrations (0.125− 2 mg/mL) was maintained above 95 % (Fig. 4a). This showed that the KLEL will not cause toxicity to humans. While B1610 melanoma cells are widely used in studies for skin whitening agents (Chatatikun et al., 2019; Lee et al., 2019). Prior to the measurement of the ability of KLEL to suppress melanogenesis in B16F10 melanoma cells, the cytotoxicity of KLEL was assessed. As shown in Fig. 4b, it was confirmed that KLEL was not toxic to B16F10 melanoma cells at a concentration lower than 500 μg/mL. As such, 32.5− 500 μg/mL KLEL was used for the following experiments.
4. Conclusion
This study proved to be of great significance in the fulfilled circular economic model, as it introduces new possible applications for kenaf leaves as high value-added ingredients with skincare properties for the cosmetic industry, namely antioxidant, anti-aging, and anti melanogenic activities. The findings presented in the study also indicated that lotion formulation prepared by 15 % KSO (F2) with 0.1 % KLE yields the best physical and microbiological stability, with no toxicity on human cells. In continuation, further studies should be also focused on the KLEL microbial challenge test and clinical efficacy.
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