Nutricosmetic effects of Asparagus officinalis: a potent matrix metalloproteinase-1 inhibitor

This study aimed to investigate the nutricosmetic effect of Asparagus officinalis extracts. The tip and spear of A. officinalis were successively extracted with 95% ethanol. The rutin, phenolic, and flavonoid contents of A. officinalis extracts were investigated. The antioxidant activities were determined by 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) and a ferric reducing antioxidant power assay. Matrix metalloproteinase-1 (MMP-1), elastase, and hyaluronidase inhibition were determined by in vitro enzyme reaction assay. The cytotoxicity was analyzed on peripheral blood mononuclear cellss. Findings revealed that drying temperature and drying duration had significant effects on the chemical composition and biological activity of A. officinalis extract. A. officinalis tips dried at 50 °C for 24 h contained the (significantly) highest flavonoid and rutin content. The most potent extract was from A. officinalis spears since it possessed the (significantly) highest MMP-1, elastase, and hyaluronidase inhibition rates of 83.4 ± 1.5%, 70.4 ± 4.1%, and 75.2 ± 1.0%, respectively. Interestingly, at the same concentration, the A. officinalis spear extract was more potent in MMP-1 inhibition than oleanolic acid and epigallocatechin gallate, the well-known natural MMP-1 inhibitors. The results show that A. officinalis extract is an attractive source of natural anti-skin-wrinkle ingredients.

www.nature.com/scientificreports/ On the other hand, drying temperature had no effect on the total flavonoid content of A. officinalis tips. Although the flavonoid content in spear extracts was lower at 80 °C than at 50 °C, in the tip extracts the content was equivalent between 80 and 50 °C (Fig. 1B). A likely explanation might be due to the more delicate structure of the A. officinalis tip compared to the spear, for which the temperature affected their chemical compositions. Therefore, the flavonoids could be degraded at only 50 °C, and the total flavonoid content of the A. officinalis tip dried at 80 °C and 50 °C was not significantly different. Conversely, due to the tougher structure of the spear, only some flavonoids were degraded at 50 °C. A higher temperature (80 °C) was needed to degrade the flavonoids inside the A. officinalis spear. Hence, flavonoid content in the spear extracts was lower at 80 °C than at 50 °C.
In contrast, a higher drying temperature led to significantly higher phenolic content but lower total flavonoid content ( Fig. 1) (p < 0.05). Therefore, it might be assumed that phenolic compounds are more stable than flavonoids at high temperature. These findings were well supported by Lu et al. 15 , who reported that the nutritional value of A. officinalis decreased significantly as a function of blanching time and temperature. At blanching temperatures below 70 °C, the total phenolic content slightly decreased by 3.93% and 14.25% after 80 and 160 min, whereas total flavonoid content remarkably reduced by 13.34% and 23.02% after blanching for 80 and 160 min, respectively 15 . Additionally, a previous study reported that thermal processing caused a significant reduction in total soluble phenolic content but an increase in total insoluble-bound phenolics 16 . Therefore, alteration of phenolic compounds after drying in a hot-air oven depended on the type of phenolic compound.
In addition to drying temperature, the drying duration also had an effect on both phenolic and flavonoid content of A. officinalis ethanolic extracts (Fig. 1). A longer drying duration led to significantly lower phenolic and flavonoid content (p < 0.05). A likely explanation is due to the degradation of some phenolics and flavonoids during the drying process 15,16 . Drying methods, especially hot air drying, had great effect on phenolic content 17 .
Since rutin was reported as the main flavonoid present in the tip and spear of A. officinalis 18 , the content of rutin was also investigated in the present study. HPLC chromatograms of standard gallic acid, standard rutin, and A. officinalis extracts are shown in Fig. 2. There were several peaks detected in the extracts of A. officinalis. The main peaks detected at the retention times of 3.255 and 3.362 min in HPLC chromatograms of A. officinalis tip extracts (50 °C, 24 h) and spear extracts (50 °C, 24 h), respectively. These peaks correlated well with the peak of standard gallic acid (retention time = 3.248 min). Therefore, gallic acid was remarked as one of the main components in both A. officinalis tip and spear extracts. On the other hand, there are also the peak at 5.603 and 5.580 min with similar peak area and peak height that could be also the main component. The retention times of these peaks correlated well with the peak of standard rutin (retention time = 5.603 min). Therefore, both gallic acid and rutin were remarked as the main components in both A. officinalis tip and spear extracts.
The gallic acid and rutin content of A. officinalis extracts showed a similar pattern (Fig. 3). A. officinalis tips contained a significantly higher gallic acid and rutin content than the spear (p < 0.05). The results were in accordance with previous studies reporting that the A. officinalis tip was a rich source of gallic acid and rutin 18,19 . In addition, the present study revealed that both drying temperature and drying duration significantly affected the gallic acid and rutin content in both A. officinalis tip and spear. However, gallic acid contents were not found to be significantly different in A. officinalis spear drying under different conditions. The (significantly) highest gallic acid (0.42 ± 0.01% w/w) and rutin (1.52 ± 0.02% w/w) content was detected in an extract from the A. officinalis tip, which was dried at 50 °C for 24 h.
In addition to gallic acid and rutin, A. officinalis has been reported to contain several chemical constituents, such as asparagusic acid, ketone vanillin, thiazole, thiophene, and their methyl and ethyl esters 20 . The combination of these chemical constituents could lead to the synergistic effects and confer superior biological activity to A. officinalis. Although other vegetables also contained some similar chemical components, lower level of these compounds would result in a higher dose requirement. A. officinalis has been reported to contain higher flavonoids level and possess higher antioxidant than Brassica oleracea 21 , while some mineral elements, especially organic selenium, of A. officinalis were much higher in A. officinalis than in other vegetables, pork, eggs, and even mushrooms 22 . Although there was some evidence that A. officinalis contains an abundance of biologically active compounds and is referred to as the "king of vegetables" 22 , the present study investigated its antioxidant and anti-wrinkle properties to confirm its potential use in the nutraceutical and/or cosmeceutical industry.  Table 2. Since at least two different test methods were suggested to determine the antioxidant activity, the ABTS assay (radical scavenging or antiradical activity) and FRAP assay (reducing capacity of ferric ions in the compounds) were performed in the present study 23,24 . The results showed that the extract from A. officinalis tips, which were dried at 50 °C for 24 h, was the most potent antioxidant, with the (significantly) highest EC 1 value of 9.6 ± 0.8 μM FeSO 4 /g extract and the (significantly) highest TEAC value of 32.6 ± 2.1 mg Trolox/g extract. The antioxidant activities were related to their chemical composition profile since the extract from A. officinalis tips that contained the highest amount of phenolic, flavonoids, and rutin possessed the (significantly) highest antioxidant activity.

Antioxidant activitiess of
Gallic acid had the most potent radical scavenging and reducing capacity. Therefore, the antioxidant activities should be related to the total phenolic content. However, A. officinalis tips dried at 80 °C for 24 h, which contained the significantly highest total phenolic content, did not possess the highest antioxidant activity (i.e., radical scavenging and reducing capacity). Hence, it could be summarized that gallic acid was not the major phenolic component of A. officinalis. Apart from gallic acid, there are other abundant phenolic compounds in A. officinalis such as 3-O-feruloylquinic acid, asparanin, asparoffin, asparenyol, gobicusin, etc. 25,26 Therefore, although the phenolic content in spear and tip extracts was higher at 80 °C than at 50 °C (Fig. 1A), the antioxidant activities in spear and tip extracts were lower at 80 °C than at 50 °C ( www.nature.com/scientificreports/ www.nature.com/scientificreports/ In contrast, the antioxidant activity of A. officinalis extracts was related to their flavonoid content. Both flavonoids in the present study, including quercetin and rutin, exhibited strong reducing capacity, which was comparable to that of gallic acid and ascorbic acid. Therefore, both quercetin and rutin are suggested as the biological compounds responsible for the antioxidant activities of A. officinalis extracts. On the other hand, both drying temperature and drying duration affected the antioxidant activity of A. officinalis extracts. A higher drying temperature and longer drying duration led to lower antioxidant activities via both radical scavenging and reducing capacity. Flavonoids, including rutin and quercetin, were suggested as the compounds responsible for the different activities between 80 and 50 °C since the antioxidant activity of A. officinalis extracts related well with the content of flavonoids ( Fig. 1) and rutin (Fig. 3). However, it was noted that the antioxidant activities of A. officinalis ethanolic extracts were not potent enough since they were much lower than that of ascorbic acid, a well-known natural antioxidant compound.
Anti-wrinkle activities of A. officinalis extracts. Anti-wrinkle activities of A. officinalis extracts are shown in Table 3. Quercetin was noted as a major component in A. officinalis extracts, which possessed the most potent inhibitory activities against MMP-1 and elastase, whereas gallic acid was the most potent hyaluronidase inhibitor. However, the anti-wrinkle activities of these pure compounds were not as potent as the positive control (oleanolic acid and EGCG), except for MMP-1 inhibition of quercetin, which was comparable to that of EGCG.
Although the contents of phenolics, flavonoids, and rutin are higher in tip extract than in the spear extract at 50 °C, these compounds did not play an important role in the anti-wrinkle activities of A. officinalis extracts. Therefore, the anti-wrinkle activities of A. officinalis extracts were not related to their phenolic and flavonoid content. Furthermore, the anti-wrinkle activities were not related to their antioxidant activities. Although the antioxidant activity was higher in the tip extract than in the spear extract at 50 °C (Table 2), in contrast, the antiwrinkle activity was lower in the tip extract than in the spear extract at 50 °C (Table 3).
Interestingly, the extract from A. officinalis spears dried at 50 °C for 24 h had the most potent anti-wrinkle activity since it possessed the (significantly) highest MMP-1, elastase, and hyaluronidase inhibition. This extract was more potent in MMP-1 inhibition than oleanolic acid and EGCG, the well-known natural MMP-1    Table 4. Although the results showed that the spear of A. officinalis was safer than the tip, the effective concentration of A. officinalis spears dried at 50 °C for 24 h (100 µg/mL), which showed the most potent anti-ageing activity, was less than the IC 20 value (163.9 ± 26.3 µg/mL), i.e., more than 80% of human PBMCs were viable.

Stability of A. officinalis extracts.
Since the ethanolic extract from A. officinalis spears dried at 50 °C for 24 h possessed the most potent inhibitory activities against MMP-1, elastase, and hyaluronidase, it was suggested for use as a natural anti-wrinkle agent. However, the temperature affected both the chemical composition and biological activity of A. officinalis ethanolic extracts. Therefore, the stability of selected A. officinalis spear extracts was investigated after storage at various temperatures for 3 months. The results as shown in Fig. 4 denote that the A. officinalis extract was stable after storage at low temperature (4 °C) since the gallic acid content (105 ± 3.5%) and rutin content (102 ± 3.4%) did not change from the initial level. However, degradation was detected after storage at room temperature and high temperature (45 °C), with the remaining gallic acid contents of 94 ± 0.4% and 86 ± 1.5%, respectively and the remaining rutin contents of 92 ± 2.7% and 81 ± 1.5%, respectively. Therefore, A. officinalis spear ethanolic extract should be kept at low temperature. Moreover, further developments of the nano delivery systems that could protect a compound from degradation, such as lipid nanoparticles, polymeric nanoparticles, vesicle systems, etc., are suggested.

Materials and methods
Plant material. The aerial part of CH105 A. officinalis, which was an in-house breed cultivated for con-

Preparation of A. officinalis extract.
The dried tip and spear powders of A. officinalis from different drying procedures were extracted by a maceration method. Briefly, 100 g of the A. officinalis dried powder was macerated in 500 mL of 95% ethanol for 3 days. After that, the resulting mixture was filtered through a Whatman no. 1 filter paper (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The solvent was then removed under reduced pressure using a rotary evaporator (Tokyo Rikakikai, Co., Ltd., Tokyo, Japan) until dryness.

Chemical composition determination
Determination of rutin content using high-performance liquid chromatography (HPLC). The chemical compositions of A. officinalis extracts were investigated using HPLC according to the previously described method with some modifications 31,32 . Briefly, gallic acid and rutin was used as a standard marker in the quantitative determination of A. officinalis extract. The HP1100 series HPLC was equipped with a UV/Vis spectrophotometric detector set at 210 nm (Hewlett Packard, Palo Alto, CA, USA). All samples and standard solutions were dissolved in HPLC-grade methanol and filtered through a 0.45 µm membrane filter (ANPEL Laboratory Technologies, Inc., Shanghai, China) before analysis. The injection volume of each sample was 20 µL. Chromatographic separation was performed on a Luna C18 column (5 µm, 4.6 × 250 mm) (Phenomenex, Torrance, CA, USA) using acetonitrile with 0.5% w/v phosphoric acid solution (A) and 0.5% w/v phosphoric acid aqueous solution (B) as a mobile phase, which was set at a flow rate of 1 mL/min. The gradient program was as follows: 0-2 min, 15-20% A; 2-8 min, 20-35% A; 8-10 min, 35-15% A; and 10-12, 15% A. All experiments were performed in triplicate.
Determination of total phenolic content. The total phenolic contents of A. officinalis extracts were determined by the Folin-Ciocalteu method as previously described 33,34 . Briefly, 20 μL of the sample solution in DMSO at the concentration of 1 mg/mL was mixed with 180 μL of 1:10 diluted Folin-Ciocalteu reagent and incubated at room temperature for 4 min. Then, 80 μL of saturated sodium carbonate solution (0.7 M) was added and incubated at room temperature for another 2 h. The UV absorbance was measured at 750 nm using a multimode detector (Beckman Coulter DTX880, Fullerton, CA, USA). Gallic acid was used as a standard compound, and the total phenolic content is expressed as milligrams per gram of gallic acid equivalents (GAE).
Three independent experiments repeated in triplicate were performed. www.nature.com/scientificreports/ Determination of total flavonoid content. The total flavonoid content of A. officinalis extracts was determined using an aluminum chloride colorimetric method as previously described [35][36][37]

Determination of anti-ageing activities of A. officinalis extracts. Matrix metalloproteinase-1
(MMP-1) inhibitory activity determination. The MMP-1 inhibitory activity of A. officinalis extracts was determined by spectrophotometric methods according to the previous studies 35,41 . Firstly, the enzyme activity of MMP-1 was determined before the experiment. Only enzyme activity of MMP-1 above 90% was used in further experiments. Briefly, the sample solution was added to 5 units/mL of MMP-1 solution and incubated for 15 min. Then, 2.0 M FALGPA in tricine buffer was added. The final concentration of A. officinalis extracts was 0.1 mg/ mL. Immediately after the enzyme reaction, the absorbance of the resulting mixture was continuously measured for 20 min at a wavelength of 335 nm, using a multimode detector (BMG Labtech, Offenburg, Germany). Inhibitory activity against MMP-1 of each sample was calculated using the following equation: % inhibition = (1a/b) × 100, where a is the reaction rate of the mixture with A. officinalis extracts, and b is the reaction rate of the mixture without A. officinalis extracts. Oleanolic acid and EGCG were used as positive controls in the present study. Three independent experiments repeated in triplicate were performed.
Elastase inhibitory activity determination. The elastase inhibitory activity of A. officinalis extracts was determined by spectrophotometric methods according to the previous studies 41,42 . Firstly, the enzyme activity of elastase was determined before the experiment. Only elastase enzyme activity above 90% was used in further experiments. Briefly, the sample solution was incubated with 4.5 unit/L of elastase for 15 min. Thereafter, 1.6 mM AAAVPN in tris HCI buffer (pH 8.0) was added. The final concentration of A. officinalis extracts was 0.1 mg/ mL. Immediately after the reaction started, the absorbance of the resulting mixture was continuously measured for 20 min at a wavelength of 410 nm using a multimode detector (BMG Labtech, Offenburg, Germany). The inhibitory activity of each sample against elastase was calculated using the following equation: % inhibition = (1-a/b) × 100, where a is the reaction rate of the mixture with A. officinalis extracts, and b is the reaction rate of the mixture without A. officinalis extracts. EGCG was used as positive control in the present study. Three independent experiments repeated in triplicate were performed.
Hyaluronidase inhibitory activity determination. The inhibitory activity of A. officinalis extracts against hyaluronidase was determined by spectrophotometric methods according to the previous studies 41,42 . Firstly, the enzyme activity of hyaluronidase was determined before each experiment. Only enzyme activity of hyaluronidase above 90% was used in the further experiments. Briefly, the sample solution was incubated with 15 unit/ mL hyaluronidase for 10 min in an incubator (BMG Labtech, Offenburg, Germany) at 37 °C. Thereafter, 0.03% w/v hyaluronic acid in phosphate buffer (pH 5.35) was added and incubated again in the same conditions for 45 min. The final concentration of A. officinalis extracts was 0.1 mg/mL. The precipitate of hyaluronic acid after addition of acid bovine serum albumin solution, composed of sodium acetate, acetic acid, and bovine serum albumin, was measured at 600 nm using a multimode detector (BMG Labtech, Offenburg, Germany). The inhibitory activity of each sample against hyaluronidase was calculated using the following equation: % inhibition = (1 − a/b) × 100, where a is the reaction rate of the mixture with A. officinalis extracts, and b is the reaction