Synergistic Anti-skin Pigmentation Effect Of Cistanche Phenylethanoid Glycosides And Glabridin
Jan 14, 2025
Abstract To investigate the synergistic effect of Cistanche phenyl ethanol side (PhGs) and glabridin (Gla) against skin pigmentation, the mass ratio of PhGs and Gla was screened by in vitro tyrosinase inhibition experiment, 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and 2,2-diazo-bis(3-ethyl-benzothiazole-6-sulfonic acid diammonium cation (ABTS+) free radical scavenging experiments. The UVB-induced hypopigmentation model of B16F10 was established, and the inhibition of melanin production was evaluated using tyrosinase activity and melanin content as indexes.
The inflammatory model of HaCaT cells induced by lipopolysaccharide (LPS) was established, and the anti-inflammatory effect of the drug was evaluated by inhibiting the release of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The oxidative stress model of HaCaT cells induced by azodiisobuamidine hydrochloride (AAPH) was further established, and the enhanced activity of superoxide dismutase (SOD) and catalase (CAT) was used as indexes to evaluate the antioxidant effect of the drug. The optimal mass ratio of PhGs and Gla was determined by the above three cell models. The results showed that aqueous solutions of PhGs/Gla(1∶1), PhGs/Gla(5∶1), and PhGs/Gla(10∶1) prepared with m(PhGs)∶m(Gla)=1∶1, 5∶1, 10∶1 exhibited good inhibitory and synergistic effects on tyrosinase activity and antioxidant activity. The inhibition rates of PhGs/Gla(1∶1),
PhGs/Gla(5∶1) and PhGs/Gla(10∶1) aqueous solutions with a mass concentration of 0.4 g/L were 94.37%, 92.93% and 88.06%, respectively. The scavenging rates of DPPH free radicals were 89.44%, 88.72,% and 88.10%, respectively.
The scavenging rates of ABTS+ free radicals were 100.13%, 100.0,1%, and 99.87%, respectively. When the mass concentration of PhGs/Gla was 25 μg/mL, PhGs/Gla(1∶1), PhGs/Gla(5∶1), and PhGs/Gla(10∶1) aqueous solutions showed no cytotoxicity to B16F10 and HaCaT cells. The inhibition of tyrosinase activity in PhGs/Gla(1 ∶1) aqueous solution was stronger (tyrosinase activity 23.80%), and the melanin content (30.90%) was significantly decreased. PhGs/Gla(1∶1) aqueous solution showed the best inhibition effect on IL-6 and TNF-α release, and the ability to enhance the activity of SOD and CAT. The combination of PhGs and Gla showed good synergistic performance, superior a to single drug and higher than PhGs/ Gla(5∶1) and PhGs/Gla(10∶1) aqueous solution. The combination of PhGs and Gla played a synergistic role in improving skin pigmentation by inhibiting melanin production, and anti-inflammatory and antioxidant effects.
Keywords Cistanche phenylethanoid glycosides; glabridin; synergistic effects; anti-skin pigmentation; anti-oxidation; antiinflammatory; drug materials
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Skin pigmentation is a skin disease caused by factors such as injury, skin inflammation, and ultraviolet radiation [1-2]. These factors may lead to an abnormal increase in melanin, which in turn causes problems such as chloasma, freckles, and post-inflammatory hyperpigmentation (PIH) [3-4], affecting people's mental state and quality of life. At present, the treatment of skin pigmentation mainly focuses on inhibiting tyrosinase, a key enzyme in the formation of melanin [5-6].
The latest research [7-8] shows that skin pigmentation depends on the close connection between keratinocytes and melanocytes in the epidermis. Once the free radicals and inflammatory factors released by keratinocytes come into contact with melanocytes, they will induce an increase in tyrosinase activity, leading to an increase in melanin content. They will also accelerate melanin transport and cause melanin to deposit on the surface of the skin. Therefore, the treatment of skin pigmentation requires a variety of measures such as inhibiting melanin production, anti-inflammatory, and anti-oxidation.
At present, although traditional drug preparations, such as hydroquinone and hydroquinone, have certain effects in the treatment of skin pigmentation, their modes of action are relatively single and may produce adverse reactions [9-10]. In comparison, natural medicines are natural and mild in effect and are increasingly recognized and praised by consumers [11-12]. In particular, the combination of multiple plant extracts [13-14] can not only inhibit the production of melanin but also has multiple effects such as anti-inflammatory and antioxidant effects, providing new ideas and methods for the treatment of skin pigmentation.
Cistanche deserticola Ma has unique medicinal value and is known as the "desert ginseng". Studies have found that the characteristic ingredient phenylethanoid glycoside contained in Cistanche has good antioxidant activity [15] and anti-inflammatory effects [16], and also has a certain tyrosinase inhibitory effect [17]. Glycyrrhizin is a flavonoid component in Glycyrrhiza glabra L., which has a powerful whitening effect and is known as "whitening gold" [18]. Traditional Chinese medicine treatment of skin pigmentation usually starts fbystrengthening the spleen and kidney, clearing away heat and detoxifying, and replenishing qi and blood [19].
Phenylethanolic glycosides of Cistanche deserticola can replenish kidney yang and benefit essence and blood, while Glycyrrhiza glabra can replenish spleen and qi, clear away heat, and detoxify. The two can work together to resist pigmentation. The combination ofphenylethanoidc glycosides of Cistanche deserticola and glabridin may resist skin pigmentation from multiple dimensions and targets, protect against ultraviolet rays in the whole band, synergistically inhibit tyrosinase activity and reduce melanin production, and can also reduce free radicals and inflammatory factors, reduce skin inflammation and oxidative stress, and regulate melanin production and transport. However, there are few reports in the literature on the synergistic inhibition of skin pigmentation by phenylethanoid glycosides of Cistanche deserticola and glabridin.
This paper intends to explore whether there is a synergistic effect between pphenylethanoidglycosides of Cistanche deserticola (PhGs) and glabridin (Gla) through in vitro biochemical experiments and the establishment of 3 cell models, nd obtain the optimal compound mass ratio for a synergistic effect. It is expected to provide theoretical support and practical guidance for the development of natural plant skin care products and to help promote the skin care industry to develop in a more natural, healthy ,and efficient direction.
Where: A reaction is the absorbance of sample solution, L-tyrosine solution, PBS, and tyrosinase solution; A reaction control is the absorbance of sample solution, L-tyrosine solution,n and PBS solution; A blank is the absorbance of L-tyrosine solution, PBS solution and tyrosinase solution; A blank control is the absorbance of L-tyrosine and PBS solutions.
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1.3.2 Determination of DPPH free radical scavenging ability
First, accurately weigh 0.0197 g (0.05 mmol) of DPPH and dissolve it in 250 mL of anhydrous ethanol to prepare a DPPH working solution with a concentration of 0.2 mmol/L. Then, PhGs and Gla were prepared into PhGs ethanol solution and Gla ethanol solution with mass concentrations of 0.001, 0.01, 0.1, 0.4, 0.8, 1.2, 1.6, 2.0 mg/mL respectively using 50% ethanol by volume, and Vc ethanol solution was prepared in the same way. Then, PhGs ethanol solution and Gla ethanol solution were mixed evenly at m(PhGs solution) ∶ m(Gla solution) = 40∶1, 20∶1, 10∶1, 5∶1, 1∶1, 1∶5, 1∶10, 1∶20, 1∶40 to obtain 9 kinds of PhGs/Gla ethanol solutions with different mass ratios, which were recorded as PhGs/Gla (40∶1)~PhGs/Gla (1∶40) ethanol solutions. Finally, add 100 μL of different sample solutions and 100 μL of DPPH working solution to the 96-well plate, mix well, and keep away from light at room temperature for 30 min. Measure the absorbance at 517 nm with an enzyme marker. VC was used as the positive control, and each experiment was repeated 3 times. Calculate the DPPH free radical scavenging rate (%) according to formula (2).
DPPH free radical scavenging rate/% = (1 −𝐴1−𝐴3𝐴2) × 100 (2) Where: A1 is the absorbance of sample solution + DPPH working solution; A2 is the absorbance of 50% ethanol by volume + DPPH working solution; A3 is the absorbance of sample solution + 50% ethanol by volume.
1.3.3 Determination of ABTS+ free radical scavenging ability
First, weigh 1 g (1.82 mmol) of ABTS and dissolve it in deionized water to prepare an ABTS working solution with a final concentration of 7.4 mmol/L; weigh 0.5 g of potassium persulfate and dissolve it in deionized water to prepare a potassium persulfate working solution with a final concentration of 2.6 mmol/L; mix the ABTS working solution and the potassium persulfate working solution in equal volumes, and react for 12 h in the dark to prepare the ABTS+ working solution. Then, according to the method in Section 1.3.2, prepare PhGs ethanol solution, Gla ethanol solution,n and Vc ethanol solution, as well as PhGs/Gla (40∶1)~PhGs/Gla (1∶40) ethanol solution.
Finally, add 40 μL of different sample solutions and 160 μL of ABTS+ working solution to the 96-well plate, mix well, react at room temperature in the dark for 6 min, and measure the absorbance of the solution at 734 nm using an ELISA reader. Calculate the ABTS+ free radical scavenging rate (%) according to formula (3).
Where: A1 is the absorbance of sample solution + ABTS + working solution; A2 is the absorbance of deionized water + ABTS + working solution; A3 is the absorbance of sample solution + deionized water.
1.4 Cell experiment
1.4.1 Cell culture
After B16F10 and HaCaT cells are revived, they are inoculated into DMEM high-glucose medium (containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin by a mass fraction) and cultured in an incubator at 37 °C, 5% CO2 volume fraction, and 70% humidity for 24 h. The cell growth status is observed under a microscope, and the medium is replaced or subcultured according to the cell growth status.
1.4.2 Experimental grouping
B16F10 and HaCaT cells in the logarithmic growth phase are inoculated in 96-well plates with appropriate cell numbers and cultured for 24 h to allow the cells to adhere to the wall. Sample solutions of different mass concentrations are prepared using a DMEM culture medium. The normal group, model group, low-dose PhGs (125 μg/mL) group, medium-dose PhGs (250 μg/mL) group, high-dose PhGs (500 μg/mL) group, low-dose Gla (6.25 μg/mL) group, medium-dose Gla (12.5 μg/mL) group, high-dose Gla (25 μg/mL) group, and PhGs/Gla (1∶1) group, PhGs/Gla (5∶1) group, and PhGs/Gla (10∶1) culture medium group were set up.
In the UVB-induced B16F10 pigmentation model, the model group cells were added with 30 μL of PBS (pH=7.4) and placed 3 cm directly below the UVB irradiator for 110 s; the experimental group was pretreated with 100 μL of sample solutions of different mass concentrations for 1 h, then added with 30 μL of PBS and placed 3 cm directly below the UVB irradiator for 110 s; the normal group always used high-glucose DMEM culture medium; the blank group was not inoculated with cells, but with an equal amount of culture medium instead.
In the LPS-induced HaCaT cell inflammation model, the model group was cultured with PBS solution of LPS at a concentration of 20 μg/mL for 24 h; the experimental group was pretreated with 100 μL sample solutions of different mass concentrations for 24 h and then added with 20 μg/mL LPS solution for 24 h; the normal group always used high-glucose DMEM medium; the blank group was not inoculated with cells, but with an equal amount of culture medium.
In the AAPH-induced HaCaT cell oxidative stress model, the model group was cultured with 100 mL AAPH (2.5 mmol) solution at a concentration of 25 mmol/L for 24 h; the experimental group was pretreated with 100 μL sample solutions of different mass concentrations for 24 h and then added with AAPH solution at a concentration of 25mmol/L for 24 h; the normal group always used high-glucose DMEM medium; the blank group was not inoculated with cells, but with an equal amount of culture medium.
Each group was set up with 3 replicate wells and cultured in an incubator at 37 ℃, 5% CO2 volume fraction, and 70% humidity.
1.4.3 Cytotoxicity assay
The CCK8 method was used to determine cytotoxicity. B16F10 and HaCaT cells in the logarithmic growth phase were digested with 0.25% trypsin for 3 min. After the digestion was terminated, the culture medium was repeatedly blown to make a cell suspension with a density of 1×104 cells/mL. The cells were evenly inoculated in a 96-well plate, 100 μL per well, and PBS was added to the wells around the cells. After the cells adhered to the wall, different mass concentrations of sample solution were added, 3 replicate wells per group, and cultured in an incubator at 37 ℃, 5% CO2 volume fraction, and 70% humidity for 24, 48, and 72 h, and then the solution was discarded. All groups were added with 100 μL of complete culture medium containing 10% CCK8 volume fraction and incubated for 60 min. The absorbance of the solution at 450 nm was measured using an ELISA reader. Cell viability (%) was calculated according to formula (4).
Where: An experimental group is the absorbance of the wells containing cells and sample solution under UVB irradiation; A normal group is the absorbance of the wells containing cells and sample solution without UVB irradiation; A blank group is the absorbance of the wells containing culture medium but no cells.
1.4.5 Determination of melanin content
The melanin content was determined bythe NaOH lysis method. After treating the cells for 72 h according to the method in Section 1.4.2, the supernatant was discarded, and washed twice with PBS, and 100 μL of NaOH aqueous solution containing 10% DMSO by volume (concentration of 1 mol/L) was added. After being placed in a 90 ℃ oven for 1 h, the absorbance of the solution at 490 nm was determined by an enzyme marker. The experiment was repeated 3 times. The melanin content (%) was calculated according to formula (5).
1.5 Determination of inflammatory factor content and oxidative stress index
After treating the cells for 48 h according to the method in Section 1.4.2, the HaCaT cells in each group were collected with a cell scraper, centrifuged, and the supernatant was collected. The content of IL-6 and TNF-α was determined according to the instructions of the ELISA kit.
After treating the cells for 48 h according to the method in Section 1.4.2, the HaCaT cells in each group were collected with a cell scraper, and the cells were broken by repeated freezing and thawing. The cells were centrifuged at 12000 r/min at 4 ℃ for 10 min, and the supernatant was collected. The SOD activity and CAT content were determined according to the instructions of the ELISA kit.
1.6 Determination of the combined index
The combined index (CI) method [20] was used to determine whether PhGs and Gla in different mass ratios had a synergistic effect in inhibiting tyrosinase activity and anti-oxidation. The combination index was calculated according to formula (6):
Wherein: D1 and D2 are the mass concentrations (mg/mL) of the two drugs when the activity reaches x% in the combined treatment; DX is the mass concentration (mg/mL) required for the activity to reach x% when the two substances are used alone. Compusyn software was used for calculation. CI>1 indicates an antagonistic effect, CI=1 indicates an additive effect, and CI<1 indicates a synergistic effect.
1.7 Data analysis
All data are expressed as "arithmetic mean ± standard deviation (𝑥̅± 𝑠)". Graph Prism 9.5.0 software was used for statistical analysis. One-way analysis of variance was used for comparison among multiple groups. P<0.05 was considered statistically significant.
