Antioxidant Activities of Methanol Extracts from Selected Taiwanese Herbaceous Plants

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All plant materials originated from and were grown in Taiwan, and were purchased from different local supermarkets in Taipei City, Taiwan. Leaves of samples were divided into eight individual batches, weighed, lyophilized, ground to powder, and stored at – 80°C until use. Extracts were prepared according to Harnly et al. ^[20]. Briefly, 5 g of powder was refluxed at 75°C for 5 h in 50 mL of acidified methanol (1.2 N HCl) with 0.4 g/L BHT and filtered through Whatman grade no. 1 qualitative filter paper. Extracts were then concentrated in a rotary vacuum evaporator (R205, Buchi, Flawil, Switzerland), re-suspended in acidic methanol to 6 mL, and stored at–20°C until testing for antioxidants with high-performance liquid chromatography (HPLC).

2.3. Determinations of Polyphenols, Total Flavonols, Total Flavonoids, and Anthocyanidins

Polyphenol content was determined according to the method of Taga et al. ^[21]. Briefly, standard gallic acid and an aliquot of the acidic methanolic extract were diluted with acidified methanol solution containing 1% HCl. Two mL of 2% Na₂CO₃were mixed into each sample of 100 𝜇L and allowed to equilibrate for 2 min before adding 50% Folin-Ciocalteu reagent. Absorbance at 750 nm was measured at room temperature using the Varioskan Flash Multimode Reader (Thermo Scientific, Rockford, IL, USA). The standard curve for gallic acid was used to calculate polyphenol levels. Total phenolics were expressed as the mg gallic acid equivalent (GAE)/g of dry weight. Total flavonols in plant extracts were estimated based on Kumaran and Joel Karunakaran ^[22]. Briefly, 80% of ethanol containing 1% HCl of solvent was used to extract the lyophilized vegetable samples in a shaker for 2 h at room temperature, then centrifuged at 1430 g for 15 min at 4°C, and repeated extraction for three times. Two mL of 2% AlCl₃ethanol and 3.0 mL (50 g/L) sodium acetate were added to 2.0 mL acidic ethanolic extracts. Absorption at 440 nm was read after 2.5 h at 25°C Sample extracts were evaluated at a final concentration of 0.1 mg/mL. Total flavonols were calculated as a quercetin equivalent (mg/g) and expressed as the mg quercetin equivalent (QUE) mg/g dry weight. Total flavonoids were determined using the method of Ordonez et al. ^[23]. Briefly, 0.5 mL of 2% AlCl₃-ethanol solution was added to 0.5 mL of acidic ethanolic extract and absorbance was measured at 420 nm after standing for 1 h at room temperature. Extract samples were evaluated at a final concentration of 0.1 mg/mL. Total flavonoids were calculated as a quercetin equivalent (mg/g) and expressed as QUE. Anthocyanidins were determined by the method of Mancinelli et al. ^[24]. Briefly, an acidified methanol solution (contained 1% HCl) was used to extract lyophilized plant samples in a shaker for2 h at room temperature followed by centrifuging at 4°C under 1430 g for 15 min and repeated twice. Supernatants were then measured at absorbances of 657 nm and 530 nm. Briefly, a solution of 99% methanol and 1% HCl was used to extract lyophilized plant samples in a shaker for 1 h at room temperature followed by centrifuging at 4°C under 1430 g for 15 min and repeated three times. Supernatants were then measured at absorbances of 657 nm and 530 nm. Anthocyanidins (unit/g) = (A₅₃₀ - ₆₅₇) x mL of extraction/g DW.

2.4. Flavonols and Anthocyanidins Analysis

One mL of acid hydrolysates methanolic extract was passed through a 0.45 μm filter prior to injecting 20 mL of it into an HPLC. Samples were analyzed with a Hitachi D-2000 containing a Photodiode Array Detector (L-2455) (Hitachi High-Tech, Tokyo, Japan) and an ODS column (250 × 4.6 mm, 5 μm; YMC, ODS-A, Kyoto, Japan). The mobile phase consisted of acetonitrile-water (30%:70%; v/v) containing 1% phosphate acid. The eluent was 100% in 30 min at a flow rate of 1.00 mL/min. The spectrum was recorded at 365 nm for flavonols determination ^[25]. Two running solvents were delivered at a rate of 1 mL/min: solvent A was 90% water and 10% formic acid, and solvent B was 22.5 % acetonitrile, 22.5% methanol, 40% water, and 10% formic acid. The gradient program used was as follows: 0 min, 20% of solvent B; 0.1~30 min, 28% of solvent B; 30.1~40 min, 70% of solvent B; 40.1~45 min, 100% of solvent B ^[19]. The spectrum was recorded at 520 nm for anthocyanidins determination. Flavonols (myricetin, morin, quercetin, and kaempferol) and antocyanidins (delphinidin, cyanidin, pelargonidin, and malvidin) were used as standards

2.5. TEAC, ORAC, and DPPH Radical Assays

The total antioxidant capacity of hydrophilic and lipophilic antioxidants was determined using the horseradish peroxidase catalyzed oxidation of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) ^[26].The reaction mixture contained 0.5 mL of 1000 μM ABTS (in ddH₂O) and 3.5 mL of 100 μM H₂O₂(in 0.1 M PBS). The reaction was started by adding 0.5 mL of 44 U/mL peroxidase (in 0.1 M PBS). After 1 h, 0.05 mL of the sample extract was added to the mixture. Absorbance was measured at 730 nm after 10 min. Trolox was used as a standard, and the total antioxidant capacity of the sample extract was measured. The TEAC value was expressed as μmol Trolox per gram dry weight.

For the lipophilic antioxidant assay, the dried hexane extract was dissolved in 250 μL of acetone and then diluted with 750 μL 7% RMCD solution (50% acetone to 50% water, v/v) ^[27]. Any further dilution was with the 7% RMCD solution. The 7% RMCD solution was also used as a blank and to dissolve Trolox standards for the lipophilic assay. For sample extract lipophilic analysis, 20 μL of this solution was added to each well in a 96-well microplate. Two hundred μL of fluorescein solution was added to the microplate reader followed by 75 μL of 63.4 mM AAPH (17.2 mg/mL and 9.4 μmol/well), and readings were initiated immediately. Any further dilution of the hydrophilic fraction (acetone/water/acetic acid extract) during the ORAC assay was made with the phosphate buffer. A 20-μL portion of the diluted sample extract was added to each well in a 96-well microplate. Fluorescein solution and AAPH were added in the same manner as in the lipophilic assay, except that 75 μL of 31.7 mM AAPH was added to the hydrophilic assay mixture ^[28].

The scavenging activity of the DPPH radical in sample extracts was determined according to Shimada et al. ^[29]. Briefly, an aliquot of 1 mL of a methanolic extract with series dilution was added to 1 mL of 0.8 mM DPPH freshly prepared in methanol, mixed well, and left to stand for 30 min before measuring absorbance at 517 nm. The scavenging effect percentage was calculated as [1 − (OD₅₁₇nm)/(control OD₅₁₇nm)] × 100. The IC₅₀of the scavenging effect percentage was then calculated.

2.6. Statistical Analysis

Data is expressed as the mean±standard deviation, and statistical significance was analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s Range Test at the 0.05 significance level ^[30]. Pearson’s correlation coefficients between antioxidant substances and capacities were also determined ^[30]. The means of three replicates are reported.

3. Results and Discussion

3.1. Antioxidant Content

Table 2 documents the content of various antioxidant substances in the leaves of tested plants. SO and OC contained significantly higher polyphenol levels (29.80 ± 1.65 and 26.24 ± 0.96 mg gallic acid/g DW, respectively) than the other species. Flavonols were abundant in FV, GF, ArP (powder), and AV at levels >40 mg quercetin equivalent/g DW. The leaves of DM (dry), FV, HC, and ArP (fresh) were significantly rich in flavonoids, ranging from 11.54 ± 2.21 to 15.49 ± 2.44 mg quercetin equivalent/g DW. In general, DM (dry), FV, ArP (fresh), and OC contained abundant polyphenols, flavonols, and flavonoids. Anthocyanidins were the principal antioxidants in OC (83.39± 3.63 unit/g DW) and LP (43.94± 1.22 unit/g DW). However, the leaves of FV, CC, PO, TT, CF, AlP, and AV were not rich in anthocyanidins. Table 2 lists the flavonol contents in acidic ethanolic extracts of plant leaves. DM (dry) was rich in quercetin (18009.58 ± 328.91 μg/g DW), while DM (fresh) and SO followed at levels of 10915.12 ± 550.88 and 10315.36 ± 539.84 μg/g DW, respectively). Myricetin was abundant in FV (1283.16 ± 60.13 μg/g DW) compared to other plants. Interestingly, AV and OC only contained quercetin (139.45 ± 9.03 μg/g DW) and myricetin (80.27 ± 18.41 μg/g DW), respectively. However, quercetin and myricetin were not detected in GF, CT, LP, or PA. Samples from ArP (fresh), ArP (powder), and LJ (14,521.52 ± 492.51, 16,517.43 ± 599.75, and 14,764.31 ± 502.53 μg/g DW, respectively) contained significantly higher levels of morin than other samples. CT and PA contained only morin (1218.54 ± 306.18 and 604.99 ± 27.58 μg/g DW, respectively). Kaempferol was present only in ZA (47.96 ± 5.99 μg/g DW, the highest level), GF, and DM (4.29 ± 1.43 μg/g DW, the lowest level), but the other plants did not contain any kaempferol at all. Furthermore, no flavonols were detected in LP. Thus, these Taiwanese landrace plants display a wide variation in flavonol levels. Among the tested 19 species, only LP and OC contained anthocyanidins, namely delphinidin (Del), cyanidin (Cyan), and malvidin (Mal) (data not shown). Both Cyan and Mal were abundant in LP and OC, and OC had the highest levels of Cyan and Mal at 650.44 ± 1.34 μg/g DW and 40.02 ± 2.98 μg/g DW, respectively (data not shown). Interestingly, Del was detected only in OC, and none of the tested plants contained pelargonidin (data not shown).

Table 2. The content of various antioxidant substances in acid methanolic hydrolysates of Taiwanese landrace plants

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Table 3. Flavonols content in acidic methanolic hydrolysates from Taiwanese landrace plants

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Plants are rich in flavonoids and other pigments. All of the tested plants contained polyphenols, flavonols, and flavonoids. Most samples were found to contain anthocyanidins, quercetin, myricetin, and morin (Tables 2 and 3). Quercetin and morin were the dominant flavonols in the samples. However, kaempferol was not detected in most of the plants due to it being sensitive to acid methanolic hydrolysis and because it rarely occurs in vegetables and other herbaceous plants. Quercetin and morin are important for preventing peroxidation and oxidative damage to DNA ^[15]. The high levels of quercetin and morin are believed to account for the high DNA protective potential of HC and MA ^[15]. Anthocyanidins act as strong antioxidants and show activity comparable to antioxidants α-tocopherol and Trolox at a concentration of 10 μM ^[31]. Generally, medicinal herbs contain significantly higher levels of phenolics than common vegetables and fruits ^[32]. Furthermore, quercetin, the most abundant flavonoid in the human diet, is an excellent free radical scavenging antioxidant ^[33]. Previously, we showed that PA, BP, CA, MC, and MA have free radical scavenging activity ^[14]; moreover, BP, MA, HC, CA, PA, and LC were found to be rich in flavonols, especially myricetin in BP, morin in MA, quercetin in HC, and kaemperol in CA ^[15]. Recently, we reported the antioxidant components of purple sweet potato leaf extract (PSPLE), quercetin, and cyanidin may down-regulate intracellular redox-dependent signaling pathways in human aortic endothelial cells (HAECs) upon TNF-α stimulation ^[34]. In the present study, OC contained high levels of polyphenols, flavonols, and anthocyanidins, and also had higher antioxidant activity in term of TEAC and ORAC values. Moreover, SO, ArP (fresh), and OC were abundant in polyphenols while HC, LP, and OC were abundant in anthocyanidins. DM (fresh), DM (dry), FV, and SO had significant levels of quercetin. Xia et al. ^[35] reported that SO is rich in polyphenols (388.4 mg GAE/g DW) and flavonoids (148.5 mg rutin/g DW). In addition, Jimoh et al. ^[36] also showed that SO contains polyphenols (9.72 ± 1.06 mg tannic acid equivalents/g DW) and flavonoids (1.21 ± 0.01 mg quercetin/g DW).

3.2. Antioxidant Activity of Methanol and Ethanol Extracts Measured for TEAC and ORAC Values and DPPH Radical Scavenging Activity

Plant extracts from all species show antioxidant activity, proving their capacity for scavenging ABTS radical cations. The antioxidant activity of acidic methanolic hydrolysate and ethanolic extracts of leaf tissue is expressed in TEAC. Methanolic extracts had higher TEAC values than ethanolic extracts in all tested plant species except SO, ArP (fresh), Arp (powder), LJ, and OC, indicating that methanolic extracts were more effective than ethanolic extracts in scavenging ROS (Table 4). For instance, the methanolic extract of HC had a high TEAC value (124.11 ± 0.32 μmol Trolox/g DW) while the ethanolic extract exhibited only 16.18 ± 2.89 μmol Trolox/g DW. Moreover, the methanolic extract (24.62 ± 1.24 μmol Trolox/g DW) of AV was19-fold higher than the TEAC value of the ethanolic extract (1.24 ± 0.21 μmol Trolox/g DW). However, and interestingly, DM (dry), ArP (fresh), LJ, and OC plants also had higher TEAC values in both extraction solvents.

Cai et al. ^[32] demonstrated that TEAC values and total phenolic levels of 112 traditional Chinese medicinal plants in methanolic extracts ranged from 0.467 to 173.23 μmol Trolox equivalent/g DW and from 2.2 to 503 mg of gallic acid equivalent/g DW, respectively. A significant linear relationship between antioxidant activity and total phenolic content shows that phenolic compounds are the dominant antioxidant components of these112 medicinal herbs, and the major types of phenolic compounds from most of them included mainly phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids ^[32]. In our study, TEAC values and total phenolics in methanolic extracts ranged from 24.62 to 747.48 μmol Trolox equivalent/g DW (Table 4) and from 12.09 to 29.80 mg of gallic acid equivalent/g DW (Table 1), respectively. Our TEAC values were apparently higher than Cai’s data ^[32]. Particularly, FV, PO, ArP, and OC in our study had much higher TEAC-methanolic values and total phenolics in methanolic extracts compared to Cai’s data ^[32]. Part of the antioxidant activity may be due to those phenolic compounds. In addition, antioxidant activity observed in plants could be a synergistic effect of more than two compounds present in the plant ^[37]. Different phytochemicals may exhibit effective antioxidant activity alone or synergistically, and are a likely cause of species differences. Synergisms among antioxidants make antioxidant activity dependent not only on their concentrations but perhaps also on their structures, plus interactions among the antioxidants ^[38].

All extracts exhibited distinct antioxidant capacities, with hydrophilic oxygen radical absorption capacity (ORAC) values from 23599.54 ± 477.01 (DM-dry) to 787.48 ± 70.80 (GF) μmol Trolox/g DW (Table 4), while ORAC-lipophilic values ranged from 6922.42 ± 414.72 μmol Trolox/g DW (HC) to 430.00 ± 36.47 μmol Trolox/g DW (CD). Most of the selected landrace plants had higher ORAC-hydrophilic values than ORAC-lipophilic values, indicating that hydrophilic extracts are more effective than lipophilic extracts in scavenging ROS. Compared to some South Asia herbs ^[39] and Chinese herbs ^[40], the tested herbs in our study showed higher ORAC values. The ability of antioxidant’s strength is to eliminate oxygen free radicals, and both ORAC-hydrophilic and TEAC assays display similar top rankings for extracts, with DM (dry) and OC being the most effective, followed by ArP (fresh). The lowest antioxidant capacities were obtained with GF, CF, and AV.

Plant methanolic extracts exhibited a wide range of DPPH scavenging values from 2413.91 ± 26.19 μg/mL

(AV) to 15.48 ± 0.10 μg/mL (OC). OC had a significantly higher efficacy of DPPH radical scavenging activity than other samples. Hence, each tested sample showed significant differences in scavenging DPPH at IC₅₀(Table 3). The IC₅₀of SO’s DPPH radical scavenging activity was 56.5 μg/mL in the 70% methanolic extract, and SO also contained the highest amounts of phenolics and flavonoids ^[41]. TR was abundant in quercetin, myricetin, and kaempferol, and its DPPH radical scavenging IC₅₀was 87.5 μg/mL ^[42]. Khan et al. ^[43] showed that Morus alba leaf methanolic extract and butylated hydroxytoluene (BHT) DPPH radical scavenging values were 108.69 and 8.5 μg/mL, respectively, while OC leaf methanolic extract DPPH radical scavenging activity was 15.48 μg/mL in our study (Table 4). The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability ^[33], and methanolic extracts of these plants have good free radical scavenging ability and can be used as radical inhibitors or scavengers.

Table 4. TEAC values, ORAC values, and IC₅₀ of DPPH scavenging activity in acid methanolic hydrolysates and ethanolic extracts of Taiwanese landrace plants

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Table 5. Correlation analysis among antioxidant substances and activity versus hydrophilic- and lipophilic-ORAC and TEAC values treated with acid methanolic extracts

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