1. Introduction
Advanced glycation end products (AGEs) are final products generated by glycation, which is a non-enzymatic browning process between reducing sugars and proteins, lipids or nucleic acids[1]. AGEs have been implicated in the pathogenesis of many diseases such as diabetes, arteriosclerosis, age-related cardiovascular diseases, Alzheimer’s disease and aging[2,3].
Also, AGEs are known to trigger skin aging through various mechanisms. AGEs are cross-linked with extracellular matrix (ECM) such as collagen and elastic fibers during aging[3]. This non-enzymatic cross-linking contributes to skin stiffening and abnormal structural changes of ECM, causing decrease in skin elasticity[5,6]. Also, AGEs induce oxidative stress and cellular senescence by interacting with their cellular receptor (RAGE). In our previous study, we confirmed that AGEs are causative of the generation of reactive oxygen species (ROS) in human dermal fibroblasts (HDFs)[7]. Thus, it is a strategic approach to develop cosmetic material against AGEs-derived skin aging, which is able to scavenge oxidative stress and inhibit formation of AGEs and cross-linking of AGEs to collagen as well as break existing cross-linking of AGEs to collagen[8].
There are already identified AGEs inhibitor and breaker such as aminoguanidine (AG) and alagebrium (ALT-711). However, the development of these agents was stopped due to the problem of safety and cost[9].
Torreya nucifera is an evergreen coniferous tree, mainly found in areas of Korea and Japan. It has known to exhibit pharmacological effects such as antioxidant, antiproliferative, neuroprotective and anthelmintic activities[10-13]. While most studies have focused on various activities of its fruits, leaves and seeds, there are no studies about its defatted seed cakes. Therefore, the aim of this study was to investigate biological activities of defatted seed cakes of T. nucifera and to demonstrate its value of use in the cosmetic industry.
2. Materials and Methods
2.1. Materials
The seeds of T. nucifera were purchased from Jeju island (Korea). 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), L-ascorbic acid, ethanol, bovine serum albumin (BSA), glycolaldehyde, tetramethylbenzidine (TMB) substrate and sulfuric acid (H2SO4) were purchased from Sigma (USA). Phosphate-buffered saline (PBS) was purchased form Welgene (Korea).
2.2. Preparation of Defatted Torreya nucifera seed cake extracts
The seeds of T. nucifera were pressed to remove oil and dried under the shade to pulverize into fine powders. 200 g of powders were extracted with 2 L of 70% ethanol by ultrasound sonication for 48 h. After filtering supernatants, the residues were evaporated to remove the ethanol by a rotary vacuum evaporator and freeze-dried to obtain final extracts of Defatted Torreya nucifera seed cakes (DTSE).
2.3. Determination of Antioxidant Activity
Total polyphenol contents
The content of total polyphenol was measured by the modified previous method[14]. The absorbance was read at 725 nm by a Gen5TM UV-Vis spectrophotometer (BioTek, USA). The contents of total polyphenol were expressed as gallic acid equivalents (GAE). DM indicates dried materials.
Total flavonoid contents
The content of total flavonoid was measured by the modified previous method[15]. The absorbance was read at 420 nm by a spectrophotometer. The contents of flavonoid were expressed as rutin equivalents (RE).
DPPH radical scavenging activity
DPPH radical scavenging activity was measured by the modified previous method[16]. The scavenging activity was calculated by the following formula, % scavenging activity = [(Acontrol - Asample)/Acontrol × 100]. Acontrol and Asample indicate the absorbance of only DPPH reagent and DTSE-treated groups, respectively. SC50 refer to the concentration of DTSE required to reduce 50% of DPPH and ABTS radicals. L-ascorbic acid was used as a positive control.
ABTS radical scavenging activity
ABTS radical scavenging activity was measured by the modified previous method[17]. It was calculated by the same method as the DPPH radical scavenging activity.
2.4. Anti-glycation assay
Anti-glycation activity was determined by the modified previous method[18]. Briefly, 10 mg/mL BSA was incubated with 10 mM glycolaldehyde at 37 °C with or without DTSE. After incubation, fluorescence intensity was measured at 340 nm excitation and 430 nm emission by an Infinite F200 PRO (Tecan, Switzerland). Aminoguanidine (AG) was used as a positive control.
2.5. Enzyme-linked immunosorbent assay (ELISA)
Inhibitory and breaking activity of DTSE on the cross-linking of AGEs to collagen were determined by the modified previous method[19]. Briefly, AGEs labeled with horseradish peroxidase (AGEs-HRP) were prepared using a peroxidase labeling kit-NH2 Unit (Dojindo, Japan). To measure inhibitory activity on the cross-linking, AGEs-HRP were incubated in collagen-coated 96 well plate with or without DTSE. After washing with PBS containing 0.05% Tween 20 (PBST), AGEs cross-linked with collagen was detected using TMB substrate. This reaction was stopped by adding 1 N H2SO4. The absorbance was read at 450 nm by a spectrophotometer. In the case of breaking activity on the cross-linking, AGEs-HRP were incubated in collagen-coated 96 well plate. After washing with PBST, DTSE was treated. The subsequent procedures to detect AGEs cross-linked with collagen were equal as above.
2.6. Anti-elastase activity assay
Anti-elastase activity was measured by EnzChek Elastase Assay Kit (Thermo Scientific, USA) according to the manufacturer’s instructions. N-methoxysuccinyl-Ala-Ala-Pro- Val-chloromethyl ketone (PC) was used as a positive control.
2.7. Statistical analysis
Statistical significance of data was determined by a Student’s t-test. All results were expressed as the means ± standard deviation (n = 3). * p < 0.05 and ** p < 0.01 were considered to be significant.
3. Results and Discussion
3.1. Total polyphenol, total flavonoid contents and antioxidant activity of DTSE
AGEs are related to increased production of free radicals[20]. These free radicals such as reactive oxygen species (ROS) play a major role in the process of skin aging by causing deoxyribo nucleic acid (DNA) damage, the generation of matrix metalloproteinases (MMPs) and the expression of inflammatory genes[21]. Therefore, it is important to find agents with antioxidant activity because it could prevent skin aging by inhibiting formation of AGEs and oxidative stress. In a previous study, it was reported that green tea shows strong anti-glycation activity in addition to antioxidant activity[22]. We measured total polyphenol, flavonoid contents and free radical scavenging activity to confirm the antioxidant activity of DTSE. As shown in Table 1, total polyphenol and total flavonoid contents of DTSE was 264.8 ± 0.5 μg GAE/mg DM and 5.6 ± 0.2 μg RE/mg DM, respectively. Also, DTSE scavenged 50% of DPPH and ABTS radicals at the concentration of 16.4 ± 1.7 μg DM/mL and 16.7 ± 0.4 μg DM/mL, respectively. It was low antioxidant activity, compared with ascorbic acid used as positive control. However, DTSE could be considered an agent with strong antioxidant activity when it is considered a crude extract, which has not undergone the purified process.
Table 1. Determination of total polyphenol, flavonoid contents and free radical scavenging activities of Defatted Torreya nucifera seed extract (DTSE). The contents of total polyphenol and flavonoid contents were expressed as gallic acid equivalents (GAE) and rutin equivalents (RE). DM indicates dried materials. The results are mean ± standard deviation (SD) (n=3)
3.2. Anti-glycation activity of DTSE
Glycation is a reaction between reducing sugar and proteins. This reaction lead to formation of AGEs that trigger pathogenic signalling pathways and cross-link extracellular matrix proteins[23]. Especially, accumulation of AGEs in dermal tissue not only lead to oxidative stress in the skin, but also turns skin colors yellow, which is not visually appealing[7]. In this study, we performed anti-glycation assay to confirm inhibitory activity of DTSE on formation of AGEs. As shown in Figure 1, DTSE inhibited formation of AGEs by 5.5, 12.9, 32.9 and 54.3% at concentrations of 100, 200, 500 and 1000 μg/mL, respectively. On the other hand, 500 μg/mL of AG inhibited formation of AGEs by 71.6%. AG is a representative positive control that could prevent formation of AGEs as well as cross-linking of AGEs to collagen. However, clinical application of AG is limited by reason of its side effects such as gastrointestinal problems, anaemia and hepatotoxicity[20,23]. There is a great demand for alternative candidate of AG, which can show less toxicity. DTSE can be considered as an alternative candidate of AG but need to be confirmed its safety in various condition.
Figure 1. Anti-glycation activity of Defatted Torreya nucifera seed extract (DTSE). Aminoguanidin (AG) was used as a positive control. The results are mean ± standard deviation (SD) (n = 3). * p < 0.05 and ** p < 0.01 vs. Control.
3.3. Inhibitory and breaking activity of DTSE on the cross-linking of AGEs to collagen
Collagen, a major component of human skin, maintains skin elasticity in combination with elastin. However, accumulation of AGEs in the skin could induce loss of skin elasticity by cross-linking with collagen[24]. Previous study reported that non-enzymatic cross-linking of skin collagen via AGEs contributed to age-related skin stiffening[3]. Therefore, ability to inhibit or break the cross-linking of AGEs to collagen is important activity as an anti-aging agent. ALT-711 (4,5-dimethyl-3-phenacylthiazolium chloride), known as a drug candidate developed for AGE-breaker, has enzymatic characteristics and breaks the covalent bonds formed in cross-linked proteins. According to a previous study, application of ALT-711 increases skin elasticity of aged rats[3]. In this study, we confirmed inhibitory and breaking activity of DTSE on the cross-linking of AGEs to collagen by ELISA. Inhibitory activity of DTSE on the cross-linking was shown in Figure 2. As a result, DTSE prevented the cross-linking of AGEs to collagen by 48.6. 80.3, 91.6 and 93.7% at concentrations of 5, 10, 20 and 50 μg/mL, respectively. On the other hand, AG inhibited the cross-linking of AGEs to collagen by 16.7, 31.9, 45.7 and 51.6% at concentrations of 5, 10, 20 and 50 mM, respectively. Breaking activity of DTSE on the cross-linking of AGEs to collagen was shown in Figure 3. As a result, DTSE broke the cross-linking of AGEs to collagen by 22.1, 30.8, 48.5 and 85.9% at concentrations of 5, 10, 20 and 50 μg/mL, while ALT-711 reduced the cross-linking of AGEs to collagen by 27.2, 37.3, 56.0 and 67.4% at concentrations of 1, 2, 5 and 10 mM, respectively. It was reported that some extracts such as Cornus officinalis and Trapa bispinosa Roxb. have the activity as an inhibitor or breaker of cross-linking AGEs to collagen[25,26]. DTSE showed similar inhibitory effect on the cross-linking of AGEs to collagen. Taken together, it is expected that DTSE will be effective in skin aging as an inhibitor and breaker of collagen-AGEs cross-linking.
Figure 2. Inhibitory activity of Defatted Torreya nucifera seed extract (DTSE) on the collagen-AGEs cross-linking. Aminoguanidin (AG) was used as a positive control. The results are mean ± standard deviation (SD) (n = 3). * p < 0.05 and ** p < 0.01 vs. Control.
Figure 3. Breaking activity of Defatted Torreya nucifera seed extract (DTSE) on the collagen-AGEs cross-linking. ALT-711 was used as a positive control. The results are mean ± standard deviation (SD) (n = 3). ** p < 0.01 vs. Control.
3.4. Inhibitory effect of DTSE on the elastase activity
Elastin is a major component of elastic fibers, which are responsible for elasticity of skin. However, elastin is degraded by elastase, which is degrading enzyme, leading to skin aging[27,28]. For this reason, inhibitor of elastase activity could prevent loss of skin elasticity. As shown in Figure 4, DTSE had an inhibitory effect on the elastase activity in a dose-dependent manner. DTSE inhibited elastase activity by 6.4, 20.3, 38.3 and 86.1% at concentrations of 5, 10, 20 and 50 μg/mL, while 1 μg/mL of PC used as a positive control had inhibited by 32.0%. These result s indicate that DTSE could be used as cosmetic material capable of inhibiting loss of skin elasticity.
Figure 4. Inhibitory effect of Defatted Torreya nucifera seed extract (DTSE) on the elastase activity. Selective inhibitor of elastase (SIE), N-methoxysuccinyl-Ala-Ala-Pro-Val -chloromethyl ketone, was used as a positive control. The results are mean ± standard deviation (SD) (n = 3). ** p < 0.01 vs. Control.
4. Conclusion
In this study, we demonstrated that DTSE had anti-aging activity through various in vitro assay. DTSE had significant amount of polyphenol and flavonoid as well as strong free radical scavenging activity and showed anti-glycation activity, anti-elastase activity as well as inhibitory and breaking activity on the cross-linking of AGEs to collagen. In addition, DTSE is easy to acquire and affordable as the wastes of seed after removing oil. Consequently, these results suggest that DTSE could be used as an attractive cosmetic material for improving skin aging.
References
- N. Ahmed, Advanced glycation endproducts-Role in pathology of diabetic complications, Diabetes Res. Clin. Pract., 67(1), 3 (2005). https://doi.org/10.1016/j.diabres.2004.09.004
- X. Xu, Y. Zheng, Y. Huang, J. Chen, Z. Gong, Y. Li, C. Lu, W. Lai, and Q. Xu, Cathepsin D contributes to the accumulation of advanced glycation end products during photoaging, J. Dermatol. Sci., 90(3), 263 (2018). https://doi.org/10.1016/j.jdermsci.2018.02.009
- S. Vasan, P. Foiles, and H. Founds, Therapeutic potential of breakers of advanced glycation end product-protein crosslinks, Arch. Biochem. Biophys., 419(1), 89 (2003). https://doi.org/10.1016/j.abb.2003.08.016
- K. M. Glynn, P. Anderson, D. J. Fast, J. Koedam, J. F. Rebhun, and R. A. Velliquette, Gromwell (Lithospermum erythrorhizon) root extract protects against glycation and related inflammatory and oxidative stress while offering UV absorption capability, Exp. Dermatol., 27(9), 1043 (2018). https://doi.org/10.1111/exd.13706
- S. Daniel, M. Reto, Z. Fred, Cosmetics: Collagen glycation and skin aging, Mibelle AG Cosmetics, Cosmetic and Toiletries Manufactrue Worldwide, 1, Switzerland (2001).
- S. Yang, J. E. Litchfield, and J. W. Baynes, AGE-breakers cleave model compounds, but do not break Maillard crosslinks in skin and tail collagen from diabetic rats, Arch. Biochem. Biophys., 412(1), 42 (2003). https://doi.org/10.1016/S0003-9861(03)00015-8
- S. W. Shin, D. H. Son, M. K. Kim, S. J. Lee, K. B. Roh, D. H. Ryu, J. S. Lee, E. S. Jung, and D. H. Park, Ameliorating effect of Akebia quinata fruit extracts on skin aging induced by advanced glycation end products, Nutrients, 7(11), 9337 (2015). https://doi.org/10.3390/nu7115478
- S. Vasan, P. G. Foiles, and H. W. Founds, Therapeutic potential of AGE inhibitors and breakers of AGE protein cross-links, Expert Opin Investig Drugs, 10(11), 1977 (2001). https://doi.org/10.1517/13543784.10.11.1977
- J. W. L. Hartog, A. A. Voors, S. J. L. Bakker, A. J. Smit, and D. J. van Veldhuisen, Advanced glycation end-products (AGEs) and heart failure: pathophysiology and clinical implications, Eur. J. Heart Fail., 9(12), 1146 (2007). https://doi.org/10.1016/j.ejheart.2007.09.009
- M. Yu, M. Zeng, F. Qin, Z. He, and J. Chen, Physicochemical and functional properties of protein extracts from Torreya grandis seeds, Food Chem, 227(15), 453 (2017). https://doi.org/10.1016/j.foodchem.2017.01.114
- S. P. Chen, M. Dong, K. Kita, Q. W. Shi, B. Cong, W. Z. Guo, S. Sugaya, K. Sugita, and N. Suzuki, Anti-proliferative and apoptosis-inducible activity of labdane and abietane diterpenoids from the pulp of Torreya nucifera in HeLa cells, Mol Med Rep, 3(4), 673 (2010). https://doi.org/10.3892/mmr_00000315
- H. S. Jeon, Y. S. Lee, and N. W. Kim, The antioxidative activities of Torreya nucifera seed extract, J Korean Soc Food Sci Nutr., 38(1), 1 (2009). https://doi.org/10.3746/jkfn.2009.38.1.001
- Y. P. Jang, S. R. Kim, and Y. C. Kim, Neuroprotective dibenzylbutyrolactone lignans of Torreya nucifera, Planta med., 67(5), 470 (2001). https://doi.org/10.1055/s-2001-15804
- Z. Maksimovic, D. Malencic, and N. Kovacevic, Polyphenol contents and antioxidant activity of Maydis stigma extracts, Bioresour. Technol., 96(8), 873 (2005). https://doi.org/10.1016/j.biortech.2004.09.006
- D. C. Abeysinghe, X. Li, C. Sun, W. Zhang, C. Zhou, and K. Chen, Bioactive compounds and antioxidant capacities in different edible tissues of citrus fruit of four species, Food Chem., 104(4), 1338 (2007). https://doi.org/10.1016/j.foodchem.2007.01.047
- K. J. Wang, Y. J. Zhang, and C. R. Yang, Antioxidant phenolic compounds from rhizomes of Polygonum paleaceum, J Ethnopharmacol., 96(3), 483 (2005). https://doi.org/10.1016/j.jep.2004.09.036
- R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, and C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic. Biol. Med., 26(9-10), 1231 (1999). https://doi.org/10.1016/S0891-5849(98)00315-3
- M. H. Nam, H. S. Lee, C. O. Hong, Y. C. Koo, Y. S. Mun, and K. W. Lee, Preventive effects of Rosa rugosa root extract on advanced glycation end product-induced endothelial dysfunction, KOREAN J. FOOD SCI. TECHNOL., 42(2), 210 (2010).
- J. Y. Lee, J. G. Oh, J. S. Kim, and K. W. Lee, Effects of chebulic acid on advanced glycation endproducts-induced collagen cross-links, Biol. Pharm. Bull., 37(7), 1162 (2014). https://doi.org/10.1248/bpb.b14-00034
- I. Grzegorczyk-Karolak, K. Golab, J. Gburek, H. Wysokinska, and A. Matkowski, Inhibition of advanced glycation end-product formation and antioxidant activity by extracts and polyphenols from Scutellaria alpina L. and S. altissima L., Molecules, 21(6), 739 (2016). https://doi.org/10.3390/molecules21060739
- H. W. Kim, B. J. Kim, S. H. Lim, H. Y. Kim, S. Y. Lee, S. I. Cho, and Y. K. Kim, Anti-oxidative effects of Taraxaci Herba and protective effects on human HaCaT keratinocyte, Kor. J. Herbology., 24(3), 103 (2009).
- T. Nakagawa, T. Yokozawa, K. Terasawa, S. Shu, and L. R. Juneja, Protective activity of green tea against free radical- and glucose-mediated protein damage, J. Agric. Food Chem., 50(8), 2418 (2002). https://doi.org/10.1021/jf011339n
- H. Ghelani, V. Razmovski-Naumovski, R. R. Pragada, and S. Nammi, (R)-alpha-Lipoic acid inhibits fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro, BMC Complement Altern Med, 18(1), 13 (2018). https://doi.org/10.1186/s12906-017-2076-6
- M. Ichihashi, M. Yagi, K. Nomoto, and Y. Yonei, Glycation stress and photo-aging in skin, ANTI-AGING Med., 8(3), 23 (2011). https://doi.org/10.3793/jaam.8.23
- C. S. Kim, D. S. Jang, J. Kim, G. Y. Lee, Y. M. Lee, Y. S. Kim, and J. S. Kim, Inhibitory effects of the seeds of Cornus officinalis on AGEs formation and AGEs-induced protein cross-linking, Korean Journal of Pharmacognosy, 39(3), 249 (2008).
- S. Takeshita, Y. Ishioka, M. Yagi, T. Uemura, M. Yamada, and Y. Yonei, The effects of water chestnut (Trapa bispinosa Roxb.) on the inhibition of glycometabolism and the improvement in postprandial blood glucose levels in humans, Glycative Stress Research, 3(3), 124 (2016).
- O. K. Popoola, J. L. Marnewick, F. Rautenbach, F. Ameer, E. I. Iwuoha, and A. A. Hussein, Inhibition of oxidative stress and skin aging-related enzymes by prenylated chalcones and other flavonoids from Helichrysum teretifolium, Molecules, 20(4), 7143 (2015). https://doi.org/10.3390/molecules20047143
- G. Ndlovu, G. Fouche, M. Tselanyane, W. Cordier, and V. Steenkamp, In vitro determination of the anti-aging potential of four southern African medicinal plants, BMC Complement. Altern. Med., 13(1), 304 (2013). https://doi.org/10.1186/1472-6882-13-304