Bioactive Compounds of Underground Valerian Extracts and Their Effect on Inhibiting Metabolic Syndrome-Related Enzymes Activities

Extractions of the underground parts of valerian were prepared with water and ethanol (25–95%) at 25–75 °C. Extraction yields, bioactive compounds, and the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability of lyophilized extracts were determined. The inhibitory effects of the extracts, valerenic acid derivatives and phenolic acids, on metabolic syndrome (MS)-related enzymes activities were further examined. Both roots and rhizomes extracted with 95% ethanol at 75 °C had the highest levels of bioactive compounds. The antioxidant capacity and inhibition of MS-related enzymes of the roots extract were better than those of the rhizomes. The roots extract more strongly inhibited pancreatic lipase (inhibition of 50% of enzyme activity (IC50), 17.59 mg/mL), angiotensin-converting enzyme (ACE, IC50, 3.75 mg/mL), α-amylase (IC50, 12.53 mg/mL), and α-glucosidase (IC50, 15.40 mg/mL). These four phenolic acids inhibited the activity of MS-related enzymes. Valerenic acid demonstrated more of an inhibitory ability for ACE (IC50, 0.225 mg/mL, except for caffeic acid) and α-glucosidase (IC50, 0.617 mg/mL) than phenolic acids. Valerian extract inhibited key enzyme activities that were associated with obesity (lipase), hypertension (ACE), and type 2 diabetes (α-amylase and α-glucosidase), suggesting that it is a potential candidate for the development of functional supplements.


Introduction
Valeriana officinalis (valerian) is a flowering plant of the Caprifoliaceae family that is commonly used to relieve sleep and anxiety disorders. It is safe for short-term use, but its side effects include headache, stomach upset, etc. [1,2]. Valerian is rich in phytochemicals, such as phenolic compounds, which are related to antioxidant activity. More than 150 different phytochemical compounds have been identified in valerian root, with the main components being sesquiterpenes (e.g., valerenic acid), iridoids, and flavonoids [3]. Among these components, valerenic acid exerts anxiolytic activity by regulating the γ-aminobutyric acid (GABA) receptor, and it is often utilized as a medicinal quality indicator [4][5][6].
Phenolic compounds inhibit free radicals to link with antioxidant activity, limiting nutrient oxidation by inhibiting oxidative chain reactions, which positively affect cardiovascular health and have anti-inflammatory, antioxidant, and anti-cancer properties [7]. Clinicians highly value phenolic compounds in food, and research has confirmed that plant extracts that are rich in phenolic compounds (i.e., phenolic acids) inhibit pancreatic lipase, α-amylase, α-glucosidase, and angiotensin-converting enzyme (ACE) activity [8,9]. Moreover, studies have revealed that valerian roots extract has antioxidant properties, slowing cell damage and aging [10,11].
Moreover, studies have revealed that valerian roots extract has antioxidant properties, slowing cell damage and aging [10,11].
Theoretically, valerian could be utilized to treat metabolic syndrome (MS); however, to date, few studies [3,12,13] have investigated phenolic compounds and the enzymatic activities that are related to the inhibition of MS. In a mouse model [12], it was demonstrated that above-ground valerian extracts inhibited the differentiation of lipid content in 3T3-L1, a cell line used to study the fundamental cellular mechanisms related to MS. Another study [13] used 70% ethanolic valerian root extract and demonstrated that iridoids enhance autophagy to break down lipid droplets, thereby relieving fatty liver.
MS is a group of syndromes that increase the risk of coronary heart disease, diabetes, stroke, and other serious health problems [14]. At least one-quarter of the world's population have MS, with massive annual healthcare costs, causing a loss of productivity in the workforce [15]. A healthy lifestyle to maintain an ideal body weight could prevent MS, and medications are available to treat MS; however, medicines have side effects, which also increases healthcare costs. Therefore, finding natural substances that can modulate MS is important. Systematic review and meta-analysis revealed that green tea, phaseolus vulgaris, garcinia cambogia, nigella sativa, and ferulic acid have an effect on metabolism syndrome-related enzymes [16][17][18]; among them, ferulic acid [18] and garcinia family [16] are phenolic acids. This study investigated the extraction yield and bioactive compounds of lyophilized extracts from the underground parts (roots and rhizomes) of V. officinalis. Further, the inhibitory effects of the extracts on the specific MS-related enzymes activities were examined: pancreatic lipase, ACE, α-amylase, and α-glucosidase.

Plant Materials
Dried underground valerian (V. officinalis) parts, including the roots and rhizomes (Figure 1), were harvested from Guizhou, China (longitude 108.83967, latitude 27.69956). Valerian was donated by KO DA Pharmaceutical Co. Ltd. (Taoyran, Taiwan, China). Foreign matter was removed using a 24-mesh sieve. The valerian samples were divided into four groups of 600 g each, and the roots and rhizomes were separated with scissors, milled, and sieved (<0.7 mm). Then, the samples were packed in laminated bags (PET/Al/LLDPE) and stored in a −25 °C freezer.

General Experimental Procedure
Valerian powder (15 g) was added to 150 mL of water or various concentrations (25%, 50%, 75%, and 95%) of ethanol in a shaking water bath at 150 rpm for 30 min at 25 °C-75 °C, then centrifuged at 6932× g (10,000 rpm, 20 min), and filtered through Advantec No.1 filter paper. The residue was re-extracted with 150 mL portions of the solvent, as described above. The combined filtrate was concentrated under reduced pressure at a 50 °C water bath and freeze-dried. The lyophilized extracts obtained by extracting the samples with

General Experimental Procedure
Valerian powder (15 g) was added to 150 mL of water or various concentrations (25%, 50%, 75%, and 95%) of ethanol in a shaking water bath at 150 rpm for 30 min at 25-75 • C, then centrifuged at 6932× g (10,000 rpm, 20 min), and filtered through Advantec No.1 filter paper. The residue was re-extracted with 150 mL portions of the solvent, as described above. The combined filtrate was concentrated under reduced pressure at a 50 • C water bath and freeze-dried. The lyophilized extracts obtained by extracting the samples with water, and 25%, 50%, 75%, and 95% ethanol in a 25 • C water bath were labeled 25C0E, 25C25E, 25C50E, 25C75E, and 25C95E, respectively. The first set of numbers and letters indicates the bath temperature in Celsius, and the second set of numbers and letters indi- cates the percentage of ethanol. All treatments were randomly generated and performed in quadruplicate.

Determination of Extraction Yields and Bioactive Compounds
The extraction yield of all treatments is calculated as (freeze-dried extract weight/ sample weight) × 100%. Total phenols levels of the extracts were analyzed following the method described by Mau et al. [19]. The total phenols content was calculated based on the calibration curve of gallic acid (absorbance at 760 nm = 0.0008 C gallic acid (µg/mL) + 0.0077, R² = 0.9993). The results were expressed as milligrams of gallic acid equivalent (GAE) per gram of lyophilized extract. The phenolic acid levels in the lyophilized extract were determined based on the method of Sarikurkcu et al. [20], with some modifications. Each extract (100 mg) was dissolved in methanol (4 mL) using an ultrasonic bath at 53 kHz for 15 min, and then the volume was adjusted to 5 mL with methanol, and centrifuged at 1900× g for 5 min. The solution was filtered with a nylon syringe filter (13 mm × 0.45 µm) before injection into an HPLC system consisting of a Hitachi 5110 pump, a Hitachi 5430 Diode array detector (Tokyo, Japan), and a Phenomenex Luna C18 (2) column (250 mm × 4.6 mm, 5 µm particle size; Phenomenex, Torrance, CA, USA). The mobile phase was composed of 0.1% (v/v) formic acid (solvent A) and methanol (solvent B) at a flow rate of 0.4 mL/min. Elution was carried out using a linear gradient as follows: 2% B from 0 min to 6 min, 2-25% B from 6 min to 12 min, 25-50% B from 12 min to 20 min, 50-95% B from 20 min to 28 min, 95% B from 28 min to 34 min, 95-2% B from 34 min to 35 min, and 2% B from 35 min to 45 min. The sample injection volume was 10 µL. Gallic acid and protocatechuic acid were detected at a wavelength of 280 nm. Chlorogenic, caffeic, and rosmarinic acids were detected at 330 nm.
Valerenic acid derivatives in the extracts were determined using a modified method by Donovan et al. [21]. The sample preparation method and HPLC system were the same as for the analysis of phenolic acids. The mobile phase composed of acetonitrile (solvent A) and 0.1% phosphoric acid (solvent B) at a flow rate of 1.5 mL/min, and UV detection was at 218 nm. Elution was carried out using a linear gradient as follows: 60-20% B from 0 min to 20 min, and 60-20% B from 20 min to 25 min. The sample injection volume was 10 µL. The valerenic acid derivative levels of the extract were calculated based on the calibration curves of hydroxyvalerenic, acetoxyvalerenic, and valerenic acids. Each analysis was carried out in quadruplicate.

Antioxidant Properties
The scavenging ability of each extract on DPPH radicals was determined based on the method by Shimada et al. [22]. Each extract was prepared in methanol at 0-1000 µg extract/mL, and 4 mL was mixed with 1 mL of methanolic solution containing DPPH radicals to a final concentration of 0.2 mM DPPH. After the mixture was shaken vigorously, it was left to stand in the dark for 30 min, and then the absorbance of the mixture was measured at 517 nm against a blank. The EC 50 value (mg extract/mL) is the effective concentration at which the DPPH radicals were scavenged by 50%. Ascorbic acid, BHA, α-tocopherol, acetoxyvalerenic acid, and valerenic acid were compared. Each analysis was carried out in quadruplicate.

Lipase, ACE, α-Amylase, and α-Glucosidase Inhibition Assays
The inhibitory abilities of the extracts, phenolic acids, or valerenic acid derivatives; or orlistat, acarbose, or captopril solutions on lipase, α-amylase, α-glucosidase, and ACE were determined according to a previous study [23,24]. The lipase, ACE, α-amylase, and α-glucosidase inhibitory activities were expressed as the percentage of inhibition, and IC 50 value (mg/mL) is the inhibition concentration at which enzyme activity was inhibited by 50%. Each analysis was carried out in quadruplicate. The analytical method is briefly described below. In the lipase inhibitory activity, 25 µL of extracts (roots 0-60 mg/mL and rhizomes 0-100 mg/mL), orlistat (0-125 ng/mL), phenolic acids (gallic, protocatechuic, and caffeic acids at 0-1.25 mg/mL; and chlorogenic acid at 0-2.5 mg/mL), valerenic acid derivatives (0-2 mg/mL) dissolved in 5% DMSO solution, were incubated with 50 µL substrate and 25 µL lipase (150 U/mL) for 30 min at 37 • C. The supernatant of the reacted solutions was read at 405 nm using an ELISA reader. Orlistat was used as a positive control.
In the α-amylase inhibitory activity, a total of 200 µL of extract (0-50 mg/mL), acarbose (0-25 µg/mL), phenolic acids (0-2.5 mg/mL), or valerenic acid derivative (0-2 mg/mL) solutions; and 200 µL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing α-amylase solution (10 U/mL) were incubated in a shaking bath (37 • C) at 100 rpm for 45 min. Then, 400 µL of a 0.5% starch solution was added to each tube and incubated in a shaking bath (37 • C) for 10 min. The reaction was stopped with 1.0 mL of dinitrosalicylic acid color reagent. The test tubes were incubated in a boiling water bath for 10 min and cooled to room temperature. The reaction mixture was then diluted after adding 3 mL of distilled water, and the absorbance was measured at 540 nm using a spectrophotometer. The readings were compared with the controls containing buffer instead of the sample extract. Acarbose was used as a positive control.

Statistical Analysis
All measurements were performed in quadruplicate. The data were subjected to analysis of variance with SAS software (SAS Institute, Cary, NC, USA). When a significant difference was found among the treatment groups, Duncan's multiple range tests were performed to determine the differences among the mean values at a level of α = 0.05.

Extract Yield and Bioactive Compounds
The extraction was performed using solvents (i.e., water and food-grade ethanol) to separate the desired natural products from the raw materials [25]. In this study, the extraction yield of valerian extract products decreased with increasing ethanol concentration, but increased with increasing extraction temperature (Table 1). The total phenols extracted from the roots and rhizomes are detailed in Table 1. Using 95% ethanol in a water bath at 75 • C, the highest amount of total phenols from roots (R75C95E) and rhizomes (Rh75C95E) were extracted at 33.16 and 26.78 mg GAE/g lyophilized extract, respectively. Considering the extraction yield, the total phenols content of the powder sample was used to represent the extraction efficiency. Roots and rhizomes were extracted with 50% and 75% ethanol in a water bath at 75 • C, and the highest levels of total phenols content were 7.56 and 5.55 mg GAE /g powder, respectively  Table 1). The total phenols content extracted was higher than that in previous studies [26,27], indicating that the solvent polarity and heating methods used in this study are the best extraction method. We also determined the phenolic acids content of roots and rhizomes extracts ( Table 2). The HPLC profiles of phenolic acids standards, valerian roots, and rhizomes extracts are shown in Figure S1. In the 75 • C water bath, the contents of four phenolic acids, including gallic, protocatechuic, chlorogenic, and caffeic acids increased with increased ethanol concentration, indicating the solubility of phenolic acids in different solvents [28]. In both the roots and rhizomes extracts, the phenolic acid compounds of 75C95E were mainly protocatechuic acid. The HPLC profiles of the valerenic acid derivatives standards, valerian roots, and rhizomes extracts are shown in Figure S2. The valerenic acid derivatives extracted from roots and rhizomes are detailed in Table 3. The levels of three valerenic acid derivatives increased with increasing ethanol concentration and water bath temperature, which is in line with a previous study [29]. Thus, 75C95E had the highest valerenic acid derivatives, and the level in rhizomes was higher than that in roots. In rhizomes, acetoxyvaleric acid had the highest content (24.2 mg/g) of valeric acid derivatives, accounting for 72.85% of the total valeric acid derivatives.

Antioxidant Properties
Human disease (such as diabetes mellitus and atherosclerosis) progression is linked to free radicals [30]. Naturally occurring antioxidants retard the progress of many chronic human diseases by scavenging free radicals. The ability of extracts to scavenge DPPH radicals displayed a dose-dependent effect (Figure 2). In comparison with rhizomes, the roots extract had a better antioxidant property. When the concentration of the roots extract was 0.2 mg extract/mL, 75C95E had the highest DPPH radical scavenging activity (26.65%); the concentration increased to 1.0 mg extract/mL and the DPPH radical scavenging activity increased to 86.83%. The EC 50 values (mg extract/mL) of roots extracts for scavenging DPPH free radicals were 0.352 (75C95E), 0.374 (75C75E), 0.535 (75C50E), 0.639 (75C25E), and 0.708 (75C0E) ( Table 4). Thus, the higher ethanol concentration as the extraction solvent, the stronger the ability of the obtained extract to scavenge DPPH radicals, and the lower the EC 50 value. The EC 50 value of the antioxidant activity of the valerian roots essential oil was 493.40 µg/mL [31]. In another study on valerian roots extracted with methanol as the extraction solvent, the 50% DPPH radicals scavenging concentration of the extract obtained with ultrasonic-assisted extraction was 54.6 µg/mL [10], which was related to the phenolic compounds content in the extract [10,32].  Roots extracts had better antioxidant properties, which may be attributed to the fact that the levels of phenolic compound in the roots extracts were higher than those in the rhizomes extracts (Tables 2 and 3). Valerenic and acetoxyvaleric acid (0-250 µg/mL) were not able to scavenge the DPPH radicals in this study. The EC 50 values (µg/mL) of ascorbic acid, BHA, and α-tocopherol in scavenging DPPH radicals were 15.67, 16.48, and 16.09, respectively. Valerian extracts were less effective than ascorbic acid, BHA, and α-tocopherol at DPPH scavenging, but these standards are additives that are used in foods or that are present at milligram levels. However, valerian extract can be used as a food supplement at the gram level.   (Tables 2 and 3). Valerenic and acetoxyvaleric acid (0-250 not able to scavenge the DPPH radicals in this study. The EC50 values (μg/m acid, BHA, and α-tocopherol in scavenging DPPH radicals were 15.67, 16. respectively. Valerian extracts were less effective than ascorbic acid, BHA, a erol at DPPH scavenging, but these standards are additives that are used in are present at milligram levels. However, valerian extract can be used as a ment at the gram level.

Inhibitory Effects on Lipase
Pancreatic lipase is a key enzyme that regulates lipid absorption [33]. Pancreatic lipase inhibition by the extracts was dose-dependent ( Figure 3A). At the same dosage, the roots extract had a better inhibitory ability than that of the rhizomes extract. When the dosage of the roots extract was 10 mg extract/mL, 75C95E had the highest inhibitory ability of pancreatic lipase (45.54%); the dosage increased to 60 mg extract/mL and the inhibition of pancreatic lipase increased to 86.00%. The IC 50 values (mg extract/mL) for pancreatic    Orlistat is an anti-obesity agent that potently inhibits pancreatic lipase [34], and it also improves glycemic control in type 2 diabetes [35]. However, it may increase the risk of colon cancer [36] and reduce the absorption of the fat-soluble vitamins A, D, E, and K [35]. With increasing orlistat concentration, the inhibition of pancreatic lipase was better ( Figure 3A). When the concentration was 9.648 ng/mL, IC 50 was achieved (Table 6), which was much lower than the IC 50 values of extracts. When the concentrations of four phenolic acids (gallic, protocatechuic, chlorogenic, and caffeic acid) increased, a more potent inhibition of pancreatic lipase was observed ( Figure 4A). The IC 50 values of gallic, protocatechuic, chlorogenic, and caffeic acid for inhibiting pancreatic lipase were 0.623, 0.673, 1.108, and 0.726 mg/mL, respectively (Table 6). Thus, gallic acid had the best inhibitory ability among the four phenolic acids on lipase activity. Neither the standardized samples of valerenic acid nor acetoxyvalerenic acid effectively inhibited pancreatic lipase activity at 0.5-2 mg/mL, which indirectly indicates that the inhibition of pancreatic lipase activity is related to phenolic acids. Other studies support that phenolic compounds in vegetable and fruit extracts inhibit pancreatic lipase activity [8,37].

Inhibitory Effects on ACE
ACE is a carboxypeptidase, which plays an important role in hypertension management and cardiovascular protection. Thus, ACE inhibition may be a useful treatment for patients with MS [38]. The ACE inhibition by the extract was concentration-dependent ( Figure 3B). The roots extract had a better inhibitory ability on ACE than that of the rhizomes extract at the same dosage. When the concentration was 2.5 mg extract/mL, 75C95E had the highest ACE inhibitory abilities (39.72%); the dosage increased to 20 mg extract/mL, and ACE inhibition was 77.31%. The IC 50 value (mg extract/mL) of the inhibition of ACE activity by the roots extracts (Table 5) shows that their inhibitory abilities were 75C95E (3.75), 75C75E (4.02), 75C50E (4.24), 75C25E (11.22), and 75C0E (13.66).

Inhibitory Effects on ACE
ACE is a carboxypeptidase, which plays an important role in hypertension manag ment and cardiovascular protection. Thus, ACE inhibition may be a useful treatment patients with MS [38]. The ACE inhibition by the extract was concentration-depende ( Figure 3B). The roots extract had a better inhibitory ability on ACE than that of the r zomes extract at the same dosage. When the concentration was 2.5 mg extract/mL, 75C9 At a captopril concentration of 0.33 ng/mL, the inhibition of ACE reached 40%. As the concentration increased, its inhibitory ability against ACE also increased ( Figure 3B). The IC 50 value of captopril on ACE inhibition was 0.498 ng/mL (Table 6), which was much lower than those of valerian extracts ( Table 5). As the concentrations of valerenic, gallic, protocatechuic, chlorogenic, and caffeic acid increased, the inhibition of ACE increased ( Figure 4B). The IC 50 values of valerenic, gallic, protocatechuic, chlorogenic, and caffeic acid for inhibiting ACE activity were 0.225, 2.100, 2.462, 4.803, and 0.094 mg/mL, respectively (Table 6). Caffeic acid demonstrated the most potent inhibition of ACE, followed by valeric, gallic, protocatechuic, and chlorogenic acid. The dosage of acetoxyvaleric acid was developed at 2 mg/mL, and its inhibitory ability on ACE was only 15%, which indirectly confirms that the ability of extracts to inhibit ACE is mainly related to the valerenic acid and phenolic acids contents. Captopril demonstrated the strongest ACE inhibition, but it has side effects such as angioedema [39].
Both the roots and rhizomes extracts inhibited ACE activity, especially 95% ethanol extract. Notably, although the rhizomes extracts prepared with the same extraction solvent contained higher concentrations of valerenic acid than the root extracts, their ability to inhibit ACE showed the reverse, which may be related to the total phenols content of roots extracts being higher than that of rhizomes extracts (Tables 2 and 3). Many studies have confirmed that plant phenolic compounds are the main factors inhibiting ACE. For example, these include Echinacea flower [23], oolong tea [40], and asparagus hard-stem [41].

Inhibitory Effects on α-Amylase
Type 2 diabetes treatment can decrease postprandial hyperglycemia by inhibiting carbohydrate hydrolases in the gastrointestinal tract [42]. Compared with the same dosage, the inhibitory ability of roots extract on α-amylase was better than that of rhizomes extract. When the roots extracts were prepared at 10-50 mg extract/mL, the ability to inhibit α-amylase activity increased with an increasing extract concentration in a dose-dependent manner ( Figure 3C). At low concentrations (10 mg extract/mL), 75C95E had the highest degree of inhibition on α-amylase, at 45.16%. At a high concentration (50 mg extract/mL), the inhibitory ability of roots extracts on α-amylase was 93.80%. As shown in Table 5, the IC 50 value (mg extract/mL) of the inhibition of roots extracts on α-amylase was 75C95E (12.53), 75C75E (20.65), 75C50E (28.23), 75C25E (30.41), and 75C0E (32.32). The R75C95E demonstrated the most strongly inhibitory ability of α-amylase. Compounds that inhibit α-amylase activity in methanol extracts from the upper part of V. officinalis may result from phenolic compounds [17]. Other studies have confirmed that plant phenolic compounds inhibit α-amylase activity. For example, phenolic compounds in the ethanol-water extract of Citrus aurantium (L) peel strongly inhibit α-amylase activity; at 1.0 mg extract/mL, αamylase activity is inhibited by up to 76% [43].
These extracts obtained using 95% ethanol have the best inhibitory effect on α-amylase, and the inhibitory ability of 75C95E on α-amylase activity in roots (IC 50 , 12.53 mg extract/mL) is higher than that in rhizomes (IC 50 , 21.99 mg extract/mL). The total phenols (33.16 mg GAE/g lyophilized extract) and total contents of four phenolic acids (3.08 mg/g lyophilized extract) in roots R75C95E were significantly higher than those in rhizomes 75C95E (Tables 2 and 3). The findings indirectly confirmed that the abilities of roots and rhizomes extracts to inhibit α-amylase activity were related to the content of phenolic acids, which is in line with prior in vitro studies [20,23].
Acarbose is commonly used as an α-amylase and α-glucosidase inhibitor for type-2 diabetes. This study showed that acarbose was more effective in inhibiting α-amylase activ-ity than valerian extract, valeric acid derivatives, and phenolic acids. Nevertheless, it has side effects and leads to the abnormal bacterial fermentation of undigested carbohydrates in the colon [44].

Inhibitory Effects on α-Glucosidase
α-Glucosidase breaks down disaccharides and oligosaccharides, and when it is inhibited, it can reduce postprandial blood glucose [45]. In this study, the inhibition of α-glucosidase by extracts increased as the concentration increased ( Figure 3D). The inhibitory ability of roots extract on α-glucosidase was better than that of rhizomes extract. When the concentration of the roots extract was 5 mg extract/mL, 75C95E had the highest inhibitory ability of α-glucosidase (31.71%); the dosage increased to 30 mg extract/mL and the inhibition of α-glucosidase increased to 72.85%. The extracts from 95% ethanol extraction demonstrated the best inhibitory ability of α-glucosidase, while the water extract showed the lowest. Valerian extract may delay the digestion and absorption of carbohydrates, thereby inhibiting postprandial hyperglycemia.
Based on the IC 50 data in Table 5, 75C95E extracts at 17.59 and 40.92 mg/mL for roots and rhizomes, respectively, exhibited a comparable lipase inhibition effect to orlistat at 9.648 ng/L ( Table 6). ACE inhibition by R75C95E and Rh75C95E was comparable to that of captopril (IC 50 = 0.498 ng/mL) when the added doses were increased to 3.75 and 4.87 mg/mL, respectively (50% inhibition). Additionally, at doses of 12.53 mg/mL (R75C95E) and 21.99 mg/mL (Rh75C95E), these extracts demonstrate an equivalent level of inhibition against α-amylase, compared to that of acarbose applied at a dose of 5.404 µg/mL (50% inhibition). For α-glucosidase, the estimated IC 50 values for R75C95E and Rh75C95E were 15.40 and 17.10 mg/mL, respectively, while the acarbose dose was 95.47 µg/mL. Therefore, theoretically, a combination of 49.27 mg R75C95E or 84.88 mg Rh75C95E may result in a 50% inhibition of lipase, ACE, α-amylase, and α-glucosidase activities. To increase the inhibition of pancreatic lipase, ACE, α-amylase, and α-glucosidase to 70%, the dosage of 75C95E in roots and rhizome extracts should be 104.45 and 141.52 mg extract/mL ( Figure 3). The 75C95E extracts prepared from roots and rhizomes may induce delayed lipid and carbohydrate digestion, thereby inhibiting dyslipidemia, hypertension, and hyperglycemia.

Conclusions
With the rapid development of food processing technologies, researchers have been increasingly interested in developing natural products as dietary supplements to meet consumers' health needs. Valerian extracts prepared with 95% ethanol as the extraction solvent and in a 75 • C water bath produced the highest bioactive component levels. The advantage of the specific extraction method is that ethanol is non-toxic, easy to mix with water, and low-cost; however, disadvantages exist since ethanol is flammable. There are other possible extraction methods, such as ultrasonic-assisted extraction, which is low-cost and high-yield; however, special equipment is required. The total phenols content of the roots extract was higher than that of the rhizomes. The phenolic acids in the roots and rhizomes mainly consisted of protocatechuic acid, but the total valeric acid content of the rhizomes was higher than that of the roots. The inhibition of R75C95E on pancreatic lipase, α-amylase, α-glucosidase, and ACE was higher than that of Rh75C95E. It is important to pay attention to these specific extracts as they have the highest inhibitory effects on MS-related enzymes activities. In conclusion, valerian roots and rhizomes extracts can be considered as a source for functional food development. Additional in vivo studies are required to investigate valerian extract, for preventing and managing MS.