Abstract
Intrinsic and extrinsic aging affect the health of human skin. Extracellular matrix protein degradation, DNA damage and oxidative stress are known to disturb skin architecture and skin homeostasis leading to skin aging. Traditional Chinese Medicine (TCM) delivers a large amount of knowledge regarding the phytotherapeutic power of diverse plants. Panax ginseng, Polygonatum cyrtonema, Epiphyllum oxypetalum, Nelumbo nucifera and Osmanthus fragrans are five plants used in TCM for their protective effect. In this study, several combinations of these TCM plants were explored: first, an in silico analysis was performed to predict their potential to target biological activities in the skin and then, some predictions were verified with in vitro studies to underline the synergistic effect of plant extracts. The results showed a stronger anti-aging activity for the combination with the five plants compared to the combination with Panax ginseng, Polygonatum cyrtonema, Epiphyllum oxypetalum and, compared to Panax ginseng alone.
Keywords: Phytochemicals, Plant extract combination, In silico analysis, skin anti-aging
1. Introduction
Skin aging is the consequence of both intrinsic and extrinsic factors leading to the degradation of skin integrity and skin physiological functions [1,2]. The skin is daily exposed to extrinsic factors responsible for aging such as ultraviolet irradiation, pollution, mechanical and bacterial stresses. In parallel, genetic, ethnic variabilities and hormonal changes lead to intrinsic aging with functional decrease of internal machinery.
Nine hallmarks of aging were described by Lopez and collaborators: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, dysregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication; and completed by Schmauck- Medina and collaborators with other hallmarks such as compromised autophagy, microbiome disturbance, altered mechanical properties, splicing dysregulation, and inflammation [2,3]. These multiple hallmarks characterize the aging phenotype and can be studied using specific markers. Indeed senescence-associated beta-galactosidase (SA-beta-gal) activity is a main marker to study the switch mechanism used by cells to enter in senescence [4]. The γH2AX marker, which is a sensor of DNA double-strand breaks [5], allows to follow genomic instability. Moreover, as skin ages, the protein synthesis and the mRNA expression of ECM markers, as collagen I and collagen III, decrease in senescent cells [6,7].
Global well-being is essential in our life. The first sign of this state is the health and the beauty of our skin. To improve the appearance and the condition of our skin, it is necessary to fight against the characteristics of the aging phenotype. The Traditional Chinese Medicine (TCM) is a tradition belonging to a 2200-year-old ancient civilization and includes various forms of therapy such as acupuncture, massage, exercise, dietary therapy, and herbal medicine. This ancestral treasure is at the origin of the derived use of plants related to Chinese medicine in cosmetic applications. Indeed, the use of plant extracts plays a key role in skin anti-aging and the mix of different plant extracts is an interesting approach to pool their different anti-aging potential to target cosmetic claims.
Panax ginseng, one of the most popular TCM plants, is known for its beneficial effects such as anti-inflammatory, improvement of lipid and glucose metabolism, antioxidation, inhibition of cell apoptosis [8]. Other effects of Panax ginseng are also described in skin such as the decrease of hyperpigmentation [9], the protection against photodamage [10] and the repair of skin barrier [11]. Several of these anti-aging effects are due to the main bioactive compounds of Panax ginseng: the ginsenosides (also named panaxosides). Among the thirty ginsenosides of Panax ginseng, Rb1, Rb2, Rc, Re and Rg1 are the most represented (70–80% of total ginsenosides in fresh Ginseng).
Polygonatum cyrtonema is an important plant in TCM through its different abilities to fight against age-related disorders induced by inflammation and oxidation [12].
Epiphyllum oxypetalum, also called “Queen of the Night” and “Night-blooming cactus” is a less well known traditional medicinal plant, described to improve wound healing. Other pharmacological activities were also identified such as antioxidant, anti-inflammatory, and anti-microbial effects [13,14].
Nelumbo nucifera, also called Sacred Lotus, is an important and potent plant in traditional medicines revered in Asia as a divine symbol. Recent molecular research has shown that lotus could be a “living fossil” that already existed more than 138 million years ago. Under favorable circumstances, the lotus seeds may remain viable for hundreds of years [15]; with the oldest recorded lotus germination being from that of seeds 1300 years old recovered from a dry lakebed in northeastern China. This survival potential could confer a strong disposition to Nelumbo nucifera to fight against aging process through its secondary metabolites. Nelumbo nucifera is also associated with good health and healing. It was described as targeting markers associated with visible signs of skin aging, including skin hydration, barrier function, skin wrinkles, lipolysis, and skin pigmentation [16].
Finally, Osmanthus fragrans has been used as folk medicine for thousands of years. The extracts of Osmanthus fragrans flowers were reported to have various bioactivities including free radical scavenging and anti-inflammation [17]. Skin whitening properties were also reported in cosmetic use [18].
Research related to the synergy of different plant extracts was rarely conducted and described in the literature. In this study, we evaluated different combinations of plant extracts (Panax ginseng root extract, Polygonatum cyrtonema rhizome/root extract, Epiphyllum oxypetalum flower extract, Nelumbo nucifera extract and Osmanthus fragrans flower extract). First, an in silico approach was conducted using the phytochemical composition and some validated proteinic targets of each extract to predict potential biological activities. In a second time, in silico predictions were assessed through in vitro studies. Thus, the anti-aging potential of plant extract combinations were evidenced focusing on different markers with significant role in maintenance of skin structure and youth and related to senescence and DNA damage.
2. Materials and methods
2.1. Plant extracts
Panax ginseng root extract, Polygonatum cyrtonema rhizome/root extract and, Epiphyllum oxypetalum flower extract were prepared by Shanghai Jahwa United Co., Ltd. They were extracted following the preparation procedure reported in Ref. [19].
The Nelumbo nucifera extract was obtained using Ashland proprietary Zeta Fraction technology free solvent process described in Ref. [20].
Osmanthus fragrans extract was developed using Ashland proprietary PSR™ technology with some adjustments described in Ref. [21].
2.2. Phytochemical compounds of interest
Phytochemical compounds were identified from previous analytical study on 4 TCM extracts (Table 1). The main phytochemical compounds of Epiphyllum oxypetalum flower extract identified using UV spectrometer, High Performance Liquid Chromatography coupled with Mass spectrometer (HPLC-MS) and Nuclear Magnetic Resonance (NMR), are quercetin 3-O -β- d-galactoside and isorhamnetin (Table 1). Panax ginseng root extract contains panaxoside Rg1, Rb1 and Re, identified by HPLC coupled with UV and fluorescence detector (Table 1). Kaempferol, liriodendrin and myricetin are the main compounds of Polygonatum cyrtonema rhizome/root extract, determined by UV spectrometer, IR spectrometer, Fast Atom Bombardment Mass Spectrometry and NMR (Table 1). Osmanthus fragrans flower extract contains caffeic, p-coumaric acid and syringic acid identified by HPLC-MS (Table 1). From these data, the simplified molecular-input line-entry system (SMILES) files were retrieved using Pubchem database (PubChem (nih.gov)) for each compound of each extract. The quercetin and hyperoside corresponding to the associated sugar of quercetin was used as SMILES files for the quercetin 3-O-β-d-galactoside, d-galactoside.
Table 1.
Phytochemical compounds of interest in TCM extracts.
| Plant extracts | Phytochemical compounds |
|---|---|
| Epiphyllum oxypetalum flower extract | quercetin 3-O -β- d-galactoside |
| isorhamnetin | |
| Panax ginseng root extract | panaxoside Rg1 |
| panaxoside Rb1 | |
| panaxoside Re | |
| Polygonatum cyrtonema rhizome/root extract | kaempferol |
| liriodendrin | |
| myricetin | |
| Osmanthus fragrans flower extract | caffeic acid |
| p-coumaric acid | |
| syringic acid |
2.3. Plant extracts combinations
Different combinations of these extracts were used for in silico and in vitro analysis: (i) 0.5% Panax ginseng root extract only (1 TCM), (ii) the combination of 3 TCM plant extracts: 0.167% Panax ginseng root extract, 0.166% Polygonatum cyrtonema rhizome/root extract and 0.167% Epiphyllum oxypetalum flower extract (3 TCM), (iii) the combination of 5 TCM plant extracts: 0.1% Panax ginseng root extract, 0.1% Polygonatum cyrtonema rhizome/root extract, 0.1% Epiphyllum oxypetalum flower extract, 0.1% Osmanthus fragrans flower extract and 0.1% Nelumbo nucifera extract (5 TCM) (Table 2).
Table 2.
Composition of TCM combinations.
| TCM plant extracts: | 1 TCM | 3 TCM | 5 TCM |
|---|---|---|---|
| Panax ginseng root extract | 0.5% | 0.167% | 0.1% |
| Polygonatum cyrtonema rhizome/root extract | / | 0.166% | 0.1% |
| Epiphyllum oxypetalum flower extract | / | 0.167% | 0.1% |
| Osmanthus fragrans flower extract | / | / | 0.1% |
| Nelumbo nucifera extract | / | / | 0.1% |
| Total | 0.5% | 0.5% | 0.5% |
2.4. Validated targets
Some of the TCM plant extracts have been tested individually in previous biological in vitro analysis. Epiphyllum oxypetalum flower extract had 7 validated targets. Polygonatum cyrtonema rhizome/root extract had 6 validated targets. Osmanthus fragrans flower extract and Nelumbo nucifera extract modulated 2 and 15 targets respectively [[16], [22]] (Table 3).
Table 3.
in vitro validated targets for TCM plant extracts.
| TCM plant extracts: | Uniprot ID: |
|---|---|
| Epiphyllum oxypetalum | P02452, P02461,P02462, Q02388, P15502, P07585, P13611 |
| Polygonatum cyrtonema | P05231, P10145, P13500, P03956, P16104, P38936 |
| Nelumbo nucifera | P08246, Q92839, Q92819, O00219, P20930, Q92482, P02452, P35354, P10145, P08254, P14679, P17643, P40126, Q05469, Q99685 |
| Osmanthus fragrans | P20930, P14679 |
2.5. Target predictions
Target predictions were performed by the input of SMILES files of each phytocompounds on a published database SwissTargetPrediction [23] and processed based on the target/ligand similarity principle. Then, predicted targets corresponding to each TCM plant extract were listed by Uniprot ID, pooled and duplicates were discarded.
2.6. Gene enrichment study
Target enrichment was performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) [24,25]. The gene enrichment was conducted in 3 parts: (i) on predicted target genes of 1 TCM, (ii) on predicted target genes and additional in vitro validated targets for 3 TCM, (iii) on predicted target genes and additional in vitro validated targets for 5 TCM. The DAVID application provided functional annotation to understand the biological significance behind the established lists of genes, based on a modified Fisher's exact test (EASE score) [25]. The level threshold of the p-Value for selection of functional annotations was set to 0.05. The DAVID database gives enriched biological themes, particularly Gene Ontology (GO) terms, terms associated with KEGG pathway, WikiPathways and Reactome data sources. Then, a comparison of annotation terms was performed between the result for 1 TCM, 3 TCM and 5 TCM combinations.
2.7. Cell culture
For collagen I and III detections, normal human dermal fibroblasts (NHDF) (Guangdong Boxi Biotechnology Co., Ltd, Lot#Fb19052002) were seeded at 2 × 105 cells in 6-well plates in low-sugar DMEM culture medium (Gibco) and incubated overnight at 37 °C in a humidified atmosphere containing 5% of CO2. At 40%∼60% confluence, cells were treated with the different TCM plant combinations (Table 2) or with 100 ng/ml TGFβ1 as positive control for 24h. After 24h, each well was washed twice with PBS 1X. Three independent experiments were performed.
For evaluation of SA-β-galactosidase activity and γH2AX detection, NHDF were seeded at 1 × 105 cells in 6-well plates in low-sugar DMEM culture medium and incubated overnight at 37 °C in a humidified atmosphere containing 5% of CO2. To induce DNA damage and senescence, cells were stressed with H202 and submitted to replicative senescence. Indeed, cells were treated with 400 μM H202 in serum free medium for 2h and washed three times with PBS 1X. In the next step, cells were treated with the different TCM combinations (Table 2) and with 100 ng/ml TGFβ1 for 24h. After 24h, the cells were washed again with PBS 1X three times. These two steps were repeated three times. After the last treatments with TCM combinations and TFGβ1, the cells were sub-cultured for 5 generations.
2.8. Detection of collagen I and collagen III mRNA by Real-Time PCR
Total RNAs were extracted by using RNA iso Plus (Takara). cDNA was synthesized from the isolated RNAs using reverse transcription kit (Takara). Quantitative PCR was performed using COL1A1 and COL3A1 specific primers (SYBR Green). The relative quantification of target expression was determined by using the comparative Ct method. In the comparative Ct method, the Ct values obtained from two different samples are directly normalized to a housekeeping gene (Actin) and then compared. Finally, the results were expressed as mean standard deviation (SD) of 3 independent experiments.
2.9. Detection of SA-β-galactosidase activity
For SA-β-galactosidase activity detection, the previously obtained H2O2-stressed senescent cells were seeded into 6-well plates at 80%–90% confluence and incubated at 37 °C in a humidified atmosphere containing 5% of CO2 for 24h. Cells were washed once with PBS 1X, and SA-β-galactosidase activity was detected using the “cell aging β-Galactosidase staining” kit (Biyuntian) as described in the supplier protocol. Pictures were acquired with an optical microscope (Olympus CKX41) and blue-stained cells were counted in ten fields. The results were expressed as the percentage of positive cells.
2.10. Western blotting for detection of γH2AX
To investigate the level of senescence-related protein γH2AX, Western Blotting analysis was performed. Cells were lysed in RIPA lysis buffer, and proteins were separated using SDS-12% polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto Immobilon P membrane (Millipore). Then, γH2AX (Abcam) and β-actin antibodies were used at a 1:200 and 1:10000 dilution respectively and secondary goat anti-rabbit IgG HRP (Bio-Rad) was used at a 1:2000 and 1:10000 dilution. The chemiluminescence was detected with Tanon 5200 Multi Chemiluminescence Image System. Quantification of protein bands was established by Band-Scan software (PROZYME, San Leandro, California).
2.11. Statistics
Data was graphed using GraphPad Prism (PraphPad Software), expressed as Mean ± SD. The student t-test statistical analysis was used for comparison between groups, and the statistical analysis was double-tailed with the Dunnett's test. It was considered p ≤ 0.05 as significant, p ≤ 0.01 as very significant and p ≤ 0.005 as highly significant.
3. Results
3.1. Target prediction
The in silico analysis was performed through the modified workflow from Wang article [26] (Fig. 1). The SwissTarget prediction based on phytochemical compounds allowed to identify 120 potential targets for Epiphyllum oxypetalum flower extract, 33 targets for Panax ginseng root extract, 120 for Polygonatum cyrtonema rhizome/root extract and 72 for Osmanthus fragrans flower extract. The compilation of predictive targets with the additional in vitro validated targets (Table 1) for each TCM plant combination allowed to establish a list of 225 potential targets for the 5 TCM, 173 potential targets for the 3 TCM and 33 potential targets for the 1 TCM (Table 2_supplementary material).
Fig. 1.
In silico analysis workflow to determine predicted targets and potential predictive biological activities impacted by the 3 combinations of plant extracts.
3.2. Gene enrichment
The lists of genes identified for each TCM combination were analyzed with DAVID to determine the terms associated with the most relevant potential biological activities. This study highlighted anti-aging and anti-senescence as the main predictive potential biological activities for each combination (supplementary data_Table 1), with the better significance for the 5 TCM. Indeed, the following terms were identified: “cellular senescence”, “FoxO signaling pathway”, “longevity regulating pathway”, “aging”, “mTOR signaling pathway”. Other terms associated with hallmarks of aging were also listed. Thus, the autophagy regulation, mitochondrial homeostasis, proteolysis, ubiquitination, and proteasome functions were characterized as predictive potential biological activities targeted by the combination of plant extracts with the better significance for the 5 TCM (pValue 5 TCM < pValue 3 TCM < pValue 1 TCM). The anti-glycation predictive potential was also revealed with the potential regulation of the AGE/RAGE pathway impacting skin aging [27].
In addition, the 5 TCM and 3 TCM blends could potentially regulate the senescence-associated secretory phenotype (SASP) involving protein secretion induced during senescence process including inflammatory cytokines, growth factors and matrix remodeling factors [28]. No prediction suggested a role of Panax ginseng root extract alone in that process.
The analysis highlighted other potential biological activities related to anti-aging for the 5 TCM and 3 TCM such as "telomere protection”, "Protein folding integrity/homeostasis”, "Chromosome organization” and "DNA damage response” mainly with "Base excision repair” process, with a better significance for the 5 TCM.
The predictive impact on histone modification was identified only for the 5 TCM combination. Interestingly, the term "histone demethylase activity” could correspond to the potential regulation of H3K9 and H3K36 methylation involved in the epigenetic regulation of SASP [29].
Moreover, other potential biological activities have been identified for all combinations: epidermal homeostasis, cell to cell adhesion and junctions. The establishment of skin barrier and the water transport are predictive characteristics identified for the 5 TCM combination only. The in silico analysis showed the potential impact of each combination on the dermal regeneration, on the extracellular matrix and hyaluronan homeostasis with a better significance potential for the 5 TCM combination (pValue 5 TCM < pValue 3 TCM < pValue 1 TCM). The 5 TCM combination could potentially target the metabolic process of global skin glycosaminoglycans known as chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, and hyaluronic acid [30]. Other potential activities were identified and attributed to all TCM combinations such as regulation of cellular proliferation and differentiation, immune response and anti-inflammation, and stress response specifically on the oxidative stress response. The response to xenobiotics or pollutants was highlighted through the potential regulation of the aryl hydrocarbon receptor pathway known to be involved in this response [31].
The in silico analysis allowed to predict strong ability to TCM combinations to play on anti-aging and thus orient the following in vitro studies on dermal proteins, senescence and DNA damage.
Other domains of the skin biology were also predicted such as adipocyte homeostasis, lipid metabolism, vitamins homeostasis and metabolism, skin tone modulation, melatonin metabolism, HSP90 and cannabinoid pathways with a better significance for the 5 TCM combination. Finally, a predictive regulation of the hair development was determined for the 5 TCM combination only.
3.3. Collagen mRNA expressions
The COL1A1 mRNA expression was significantly increased in cells treated with 1 TCM, 3 TCM and 5 TCM combinations by 29%, 44%, 92% respectively compared to control condition (Fig. 2), meaning that the 5 TCM application has a better efficacy than the 3 TCM application which has a better efficacy than the 1 TCM application. The positive control, TGFβ1, induced an increase of COL1A1 mRNA expression equivalent to the 5 TCM condition (+93%).
Fig. 2.
Evaluation of COL1A1 mRNA expression in fibroblasts treated with TCM combinations for 24h using Real-Time PCR. Statistical analysis was carried out with the Student's t-test compared to the control condition. (n = 3, **: very significant, ***: highly significant).
The COL3A1 mRNA expression was significatively increased in cells treated with 1 TCM, 3 TCM and 5 TCM combinations by 55%, 72%, 113%, respectively compared to control condition (Fig. 3). In line with results obtained for COL1A1, the 5 TCM application has a better efficacy than the 3 TCM application which has a better efficacy than the 1 TCM application on COL3A1 expression. The positive control, TGFβ1, induced an increase of COL3A1 mRNA expression equivalent to the 3 TCM condition (+87%).
Fig. 3.
Evaluation of COL3A1 mRNA expression in fibroblasts treated by TCM combinations for 24h using Real-Time PCR. Statistical analysis was carried out with the Student's t-test compared with the control condition. (n = 3, ***: highly significant).
3.4. SA-β-galactosidase activity
Results showed that H2O2 stress increased by 262% the number of positive senescent cells compared to the control condition, moving from 5.97% of SA-β-gal positive cells in the control to 21.6% in the H2O2 stress condition (Fig. 4 (a)(b)). The application of 1 TCM decreased by 50% the SA-β-gal positive cells, while the application of the 3 TCM decreased by 90% and the application of the 5 TCM decreased by 96% compared to stress condition. Thus, the 5 TCM exhibited again a better efficacy than the 3 TCM which exhibited a better efficacy than the 1 TCM. Interestingly, the TGFβ1 condition had an equivalent efficacy than the 3 TCM by reducing the SA-β-gal positive cells (−88%).
Fig. 4.
(a) SA-β-gal positive cells (x100) in the H2O2 -stressed fibroblasts after 24h of incubation with TCM combinations or TGFβ1. (b) The percentage of SA-β-gal positive cells in each condition. Statistical analysis was carried out with the Student's t-test compared to control condition for H2O2/senescent condition and to the H2O2/senescent condition for the other conditions. (n = 3, ***: highly significant).
3.5. γH2AX sensor of DNA damage increasing with aging
To further investigate the anti-aging properties of the plant extract combinations, the γH2AX detection in H2O2-senescent cells was performed. A highly significant increase of γH2AX protein was observed in H2O2-senescent cells compared to the control condition (Fig. 5(a)(b)). The application of 1 TCM, 3 TCM and 5 TCM combinations induced a decrease of γH2AX protein level in H2O2-senescent cells (−87%, −32%, −94% respectively). The 5 TCM combination allowed to retrieve the basal level of γH2AX level. Again, TGFβ1 had an equivalent efficacy than the 3 TCM (−39%)
Fig. 5.
(a) Evaluation of γH2AX expression in H2O2-senescent cells treated with TCM combinations using Western blot analysis. (b) γH2AX expression level in H2O2-senescent cells treated with TCM combinations. Statistical analysis was carried out with the Student's t-test compared to control condition for H2O2/senescent condition and to the H2O2/senescent condition for the other conditions. (n = 3,***: highly significant).
4. Discussion & conclusions
TCM is a Chinese practice that has existed for centuries and is a reference for using Chinese herbal medicines to restore the yin yang balance for a healthy and harmonious human body. In TCM, herbal formulas are used considering the specific properties of each herb to improve the state of the human body. In this study, different plants with known potential related to skin improvement, were used in different combinations. The in silico study allowed us to predict a strong anti-aging and anti-senescence potential, for the three different plant extracts combinations, revealed by the following terms: “cellular senescence”, “longevity regulating pathway” and “aging”. The antioxidant response was also predicted with the term “oxidative stress response” for all TCM combinations. A better predictive impact of the 5 TCM combination was shown. These predicted activities were confirmed in vitro during the assessment of the effect of 1TCM, 3 TCM and 5 TCM combinations for their capabilities to reverse H2O2 induced senescence in fibroblasts. Moreover, the level of SA-β-gal positive cells decreased in H2O2/senescent cells when treated with 1 TCM, 3 TCM and 5 TCM combinations, with a better significance for the 5 TCM. Additional activities were also predicted with in silico analysis for the 3 TCM and 5 TCM combinations: the protection of DNA, with a better significance for the 5 TCM. In vitro study of γH2AX protein in H2O2/senescent cells showed an improvement of the induced DNA damage, through a decrease of γH2AX protein, mainly with the 5 TCM combination. These results confirmed the predicted performance of the 5 and 3 TCM combinations in the protection against DNA damage. Surprisingly, Panax ginseng alone showed good efficacy too while no evidence was predicted in the in silico study. In this case, the limitation of in silico predictions was highlighted. The in silico was performed based on some compounds of interest present in the plant extract but other non-identified compounds could explain its biological activity. Predictive anti-aging effect of the 3 and 5 TCM combinations, regarding anti-senescence and genomic stability, was thus accurate. Moreover, bioinformatic prediction showed a potential impact of each combination on dermal regeneration and extracellular matrix homeostasis with a better significance for the 5 TCM. This prediction was confirmed with the increase of collagen I and III in fibroblasts with also a better effect of the 5 TCM. The wide range of predictions revealed other potential axes of study in the domain of aging and skin biology: response to xenobiotic and pollutants, autophagy regulation, mitochondrial homeostasis, proteolysis, ubiquitination, proteasome functions, histone demethylase activity, epidermal homeostasis, cell adhesion and junction, skin barrier and water transport, hyaluronan homeostasis, glycosaminoglycans, adipocyte homeostasis, lipid metabolism, vitamins homeostasis and metabolism, skin tone modulation, melatonin metabolism, HSP90 and cannabinoid pathways. The global accuracy of in silico predictions allowed us to hypothesize that the 5 TCM combination is the best formula to fight against skin aging and improve skin homeostasis and, finally, the in vitro results confirmed this potential.
Target prediction is a key tool for understanding the mechanism of action of phytochemical compounds present in TCM plants. In this study, the robustness of the prediction was demonstrated by confirming the biological effects described in the literature for the evaluated TCM plants and by validating the effects identified in the presented in vitro analysis. In addition, target prediction is a predictive approach with low computational cost, robustness, and efficiency compared to experimental screening and it continues to progress.
However, in silico analysis has limitations that depend on the availability of chemical and biological data in the bioinformatics tool. In this study, we limited the in vitro evaluation to a few biological processes identified by the in silico prediction. Further experiments are required to validate the other predicted effects.
To conclude, the in silico analysis is a relevant tool to help define the better plant extracts combination to enhance the anti-aging activities and finally providing a large source of data regarding biological activities potentially targeted. Finally, in vitro studies of the five plant extracts showed a real impact in the fighting of skin aging and the improvement of skin homeostasis, with the best results obtained with the 5 TCM combination.
Data availability
Data included in article/supplementary material/referenced in article.
CRediT authorship contribution statement
Chunbo Feng: Validation, Resources, Project administration, Methodology, Investigation, Conceptualization. Catherine Serre: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation. Weimiao Chen: Writing – original draft, Investigation. Isabelle Imbert: Validation, Supervision. Le Zhu: Investigation, Conceptualization. Feng Ling: Methodology, Investigation.
Declaration of Generative AI and AI-assisted technologies in the writing process
During the preparation of this work, authors used [SwissTargetPrediction, DAVID] in order to predict biological targets of phytochemical compounds and to predict biological activities of several plant extracts. After using this tool, authors reviewed and edited the content as needed and take full responsibility for the content of the publication.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank Audrey LE MESTR and Christophe CAPALLERE for their valuable discussions; Billy TANG and Michael XIAN for their involvement in communication and translation between the two collaborating entities; Jean-Marie Botto, Elodie Oger, Imane Garcia and Haidong JIA for their support in this work.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e26131.
ABBREVIATIONS
- TCM
Traditional Chinese Medicine
- DNA
Deoxyribonucleic acid
- HPLC
High Performance Liquid Chromatography
- MS
Mass spectrometer
- NMR
Nuclear Magnetic Resonance
- UV
Ultra Violet
- IR
Infrared
- 1 TCM
Panax ginseng root extract
- 3 TCM
Panax ginseng root extract, Polygonatum cyrtonema rhizome/root extract and Epiphyllum oxypetalum flower extract combination
- 5 TCM
Panax ginseng root extract, Polygonatum cyrtonema rhizome/root extract, Epiphyllum oxypetalum flower extract, Osmanthus fragrans flower extract and Nelumbo nucifera extract combination
- SMILES
simplified molecular-input line-entry system
- DAVID
Database for Annotation, Visualization, and Integrated Discovery
- GO
Gene Ontology
- KEGG
Kyoto Encyclopedia of Genes and Genomes
- NHDF
normal human dermal fibroblast
- DMEM
Dulbecco's Modified Eagle Medium
- PBS
Phosphate-buffered saline
- TFGβ1
Transforming Growth Factor Beta 1
- PCR
Polymerase chain reaction
- COL1A1
Collagen Type III Alpha 1 Chain
- COL3A1
Collagen Type I Alpha 1 Chain
- SA-β-gal
Senescence-associated beta-galactosidase
- SDS-PAGE
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- HRP
Horseradish peroxidase
- AGE
advanced glycation endproducts
- RAGE
receptor for advanced glycation endproducts
- SASP
Senescence-associated secretory phenotype
- H3K9
histone H3 lysine 9
- H3K36
histone H3 lysine 36
- mRNA
messenger ribonucleic acid
- RNA
ribonucleic acid
- H2O2
Hydrogen Peroxide
Appendix A. Supplementary data
The following are the Supplementary data to this article.
figs1.
figs2.
figs3.
figs4.
figs5.
figs6.
figs7.
figs8.
References
- 1.Farage M.A., Miller K.W., Elsner P., Maibach H.I. Intrinsic and extrinsic factors in skin ageing: a review. Int. J. Cosmet. Sci. 2008;30(2):87–95. doi: 10.1111/j.1468-2494.2007.00415.x. [DOI] [PubMed] [Google Scholar]
- 2.López-Otín C., Blasco M.A., Partridge L., Serrano M., Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194–1217. doi: 10.1016/j.cell.2013.05.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Schmauck-Medina T., Molière A., Lautrup S., Zhang J., Chlopicki S., Borland Madsen H., Cao S., Soendenbroe C., Mansell E., Bitsch Vestergaard M., Li Z., Shiloh Y., Opresko P.L., Egly J.M., Kirkwood T., Verdin E., Bohr V.A., Cox L.S., Stevnsner T., Rasmussen L.J., Fang E.F. New hallmarks of ageing: a 2022 Copenhagen ageing meeting summary. Aging (Albany NY) 2022;14(16):6829–6839. doi: 10.18632/aging.204248. 31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Dimri G.P., Lee X., Basile G., Acosta M., Scott G., Roskelley C., Medrano E.E., Linskens M., Rubelj I., Pereira-Smith O., et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92(20):9363–9367. doi: 10.1073/pnas.92.20.9363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mah L.J., El-Osta A., Karagiannis T. γH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010;24:679–686. doi: 10.1038/leu.2010.6. [DOI] [PubMed] [Google Scholar]
- 6.Levi N., Papismadov N., Solomonov I., Sagi I., Krizhanovsky V. The ECM path of senescence in aging: components and modifiers. FEBS J. 2020;287(13):2636–2646. doi: 10.1111/febs.15282. [DOI] [PubMed] [Google Scholar]
- 7.Lago J.C., Puzzi M.B. The effect of aging in primary human dermal fibroblasts. PLoS One. 2019;14(7) doi: 10.1371/journal.pone.0219165. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Mancusoa C., Santangelob R. Panax ginseng and Panax quinquefolius: from pharmacology to toxicology. Food Chem. Toxicol. 2017;107:362–372. doi: 10.1016/j.fct.2017.07.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Liu J., Jiang R., Zhou J., Xu X., Sun Z., Li J., Chen X., Li Z., Yan X., Zhao D., Zheng Z., Sun L. Salicylic acid in ginseng root alleviates skin hyperpigmentation disorders by inhibiting melanogenesis and melanosome transport. Eur. J. Pharmacol. 2021;910 doi: 10.1016/j.ejphar.2021.174458. 10.1016/j.ejphar.2021.174458 Full text linksCite. [DOI] [PubMed] [Google Scholar]
- 10.Li Z., Jiang R., Liu J., Xu X., Sun L., Zhao D. Panax ginseng C. A. Meyer Phenolic acid extract alleviates ultraviolet B-Irradiation-Induced Photoaging in a hairless Mouse skin photodamage Model. Evid Based Complement Alternat Med, eCollection. 2021 doi: 10.1155/2021/9962007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Li Z., Jiang R., Jing C., Liu J., Xu X., Sun L., Zhao D. Protective effect of oligosaccharides isolated from Panax ginseng C. A. Meyer against UVB-induced skin barrier damage in BALB/c hairless mice and human keratinocytes. J. Ethnopharmacol. 2022;30(283) doi: 10.1016/j.jep.2021.114677. [DOI] [PubMed] [Google Scholar]
- 12.Chen Z., Zhu B., Chen Z., Cao W., Wang J., Li S., Zhao J. Effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping analysis and pharmacological activity assays. Chin. Med. 2022;17(1):97. doi: 10.1186/s13020-022-00650-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Prajitha P., Suresh A., Deepak V.S., Faslu H. A review on Epiphyllum oxypetalum (DC) Haw. Asian Journal of Research in Chemistry and Pharmaceutical Sciences. 2019;7(3):824–830. [Google Scholar]
- 14.Mahmad A., Shaharun M.S., Saad B., Dash G.K. Epiphyllum oxypetalum haw.: a lesser-known medicinal plant. IAJPS. 2017;4(10):3670–3672. [Google Scholar]
- 15.Shen-Miller J., Mudgett M.B., Schopf J.W., Clarke S., Berger R. Exceptional seed longevity and robust growth: ancient Sacred Lotus from China. Am. J. Bot. 1995;82:1367–1380. [Google Scholar]
- 16.Lebleu A., Bressier G., Gondran C., Imbert I., Cucumel K., Dueva-Koganov O., Zhang L., Duev A., Koganov M., Domloge N. Powerful comprehensive benefits of Nelumbo nucifera (lotus) extract targeting attributes of skin appearance associated with skin aging. Poster IFSCC. 2017;2017 [Google Scholar]
- 17.Lee H.H., Lin C.T., Yang L.L. Neuroprotection and free radical scavenging effects of Osmanthus fragrans. J. Biomed. Sci. 2007;14(6):819–827. doi: 10.1007/s11373-007-9179-x. [DOI] [PubMed] [Google Scholar]
- 18.Le D.D., Lee Y.E., Lee M. Triterpenoids from the leaves of Osmanthus fragrans var. aurantiacus with their anti-melanogenesis and anti-tyrosinase activities. Nat. Prod. Res. 2022;36(24):6414–6420. doi: 10.1080/14786419.2022.2035384. [DOI] [PubMed] [Google Scholar]
- 19.Zhao Y.F., Zhu L., Yang L., Chen M., Sun P., Ma Y., Zhao Y., Jia H.D. In vitro and in vivo anti-eczema effect of Artemisia annua aqueous extract and its component profiling. J. Ethnopharmacol. 2024;318 doi: 10.1016/j.jep.2023.117065. [DOI] [PubMed] [Google Scholar]
- 20.Koganov M. Publication; 2008. U.S. Patent No. 7,442,391 B2, Bioactive Botanical Cosmetic Compositions and Processes for Their Production. Date Oct 28. [Google Scholar]
- 21.Oger E., Mur L., Lebleu A., Bergeron L., Gondran C., Cucumel K. Plant small RNAs: a new technology for skin Care. J. Cosmet. Sci. 2019;70(3):115–126. [PubMed] [Google Scholar]
- 22.Koganov M., Menon G., Chakraborty A., Dueva-Koganov O., Zhang L., Duev A. Targeting key Enzymes of the Melanogenic pathway with a Novel Lotus Ingredient. IFSCC magazine. 2016;3:119–123. [Google Scholar]
- 23.Daina A., Michielin O., Zoete V. SwissTargetPrediction: updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res. 2019;47:W357–W364. doi: 10.1093/nar/gkz382. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Sherman B.T., Hao M., Qiu J., Jiao X., Baseler M.W., Lane H.C., Imamichi T., Chang W. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update) Nucleic Acids Res. 2022;50(W1):W216–W221. doi: 10.1093/nar/gkac194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Huang D.W., Sherman B.T., Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009;4(1):44–57. doi: 10.1038/nprot.2008.211. [DOI] [PubMed] [Google Scholar]
- 26.Wang Z.Z., Jia Y., Srivastava K.D., Huang W., Tiwari R., Nowak-Wegrzyn A., Geliebter J., Miao M., Li X.M. Systems pharmacology and in silico Docking analysis Uncover association of CA2, PPARG, RXRA, and VDR with the mechanisms Underlying the Shi Zhen Tea formula effect on eczema. Evid Based Complement Alternat Med. 2021 doi: 10.1155/2021/8406127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Gkogkolou P., Böhm M. Advanced glycation end products: key players in skin aging? Dermatoendocrinol. 2012;4(3):259–270. doi: 10.4161/derm.22028. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Coppe J.P., Patil C.K., Rodier F., Sun Y., Muñoz D.P., Goldstein J., Nelson P.S., Desprez P.Y., Campisi J. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 2008;6:2853–2868. doi: 10.1371/journal.pbio.0060301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Desbats M.A., Sara Zumerle S., Alimonti A. Epiregulation of the SASP makes good neighbors. Nature Aging. 2021;1:420–421. doi: 10.1038/s43587-021-00068-w. [DOI] [PubMed] [Google Scholar]
- 30.Lee Dong Hun, Oh Jang-Hee, Chung Jin Ho. Glycosaminoglycan and proteoglycan in skin aging. J. Dermatol. Sci. 2016;83(3):174–181. doi: 10.1016/j.jdermsci.2016.05.016. [DOI] [PubMed] [Google Scholar]
- 31.Vogel C.F.A., Van Winkle L.S., Esser C., Haarmann-Stemmann T. The aryl hydrocarbon receptor as a target of environmental stressors - Implications for pollution mediated stress and inflammatory responses. Redox Biol. 2020;34 doi: 10.1016/j.redox.2020.101530. [DOI] [PMC free article] [PubMed] [Google Scholar]
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