Bioactive composition analysis
The main bioactive components in the three products are listed in Table 1. The main chemical constitutes of DL and LT were quite similar; although significant differences were noted in indicators such as protein (DL > LT, difference = 8.22, P < 0.01), ESE (LT > DL, difference = 1.79, P < 0.01) and WSE (DL > LT, difference = 4.07, P < 0.05). Significant differences were not found in the TPC, TFC and POL contents (P > 0.05) of the samples. Among the bioactive constitutes, only POL contents in LT and DL were significantly lower (P < 0.01) while the other components in LT and DL were all significantly higher than those in DR (P < 0.01). Two major anti-inflammatory active fractions that may enhance wound healing were isolated from dry roots of A. membranaceus28. The extract from A. membranaceus var mongholicus roots has shown significant anti-viral and immune stimulate activities, from which a new saponin and iso-astragaloside (I) were separated3 Leaf products exhibited substantial total flavonoid with contents in the order DL (9.68%) > LT (8.11%) > DR (1.90%). Compared with DL and LT, the DR exhibited significantly higher POL, increased by 50.21% (P < 0.01) and 57.53% (P < 0.01), respectively. Compared with DR, DL and LT showed other higher nutrition components, leading to increased WSE of 14.10% and 10.03%, increased ESE of 11.27% and 13.05%, increased TFC of 7.78% and 6.21%, increased TPC of 3.99% and 3.46%, and increased Protein of 13.70% and 5.48%, respectively. The abundant TPC in LT and DL detected in this study were in accordance with the results of study examining the fleshy roots of Raphanus raphanistrum21. In addition to abundant TPC, increased TFC and protein in LT and DL was also detected in this study; these results were in accordance with studies of leaf tea and dry leaf from C. pilosula20. Differing from C. pilosula products, the contents of WSE and ESE were reversed in LT and DR from A. membranaceus, resulting in a maximum amount of WSE in DL and a maximum amount of ESE in LT20.
Amino acids are essential for human growth and development as well as reproduction and health29. Table 2 shows the amino acids (AA) in DL, LT and DR from A. membranaceus. The results showed that the 17 amino acid contents in DL and LT were all very similar leading to a non-significant difference in the total value (P > 0.01). Compared with DL (24.18%) and LT (28.96%), the total AA content in DR was significantly lower 8.89% (P < 0.01), which were nearly one third times lower than the formers. This indicates that all the three products are rich in amino acids (Table 2), because their total amino acid contents are far higher than those of famous Chinese teas including Maojian (slightly more than 0.02%), Biluochun (close to 0.03%), Longjing (slightly more than 0.03%)30. DR, DL and LT also presented more abundant amino acids than their respective products of dry root (5.36%), dry leaves (19.01%) and leaf tea (21.11%) from C. pilosula20.
The average data are presented in the table. The total described the sum of contents for all the amino acids in each product. DL, the abbreviations are the same as presented in Table 1.
Low ash content is desirable for Chinese Pharmacopoeia1. The crude ash content showed a minimum value in medicinal DR, but in the by-products of LT and DL the values increased by 3.98% and 3.38% respectively greater than that of DR. However, this does not affect the utilization value of leaf products, because these products are consumed by soaking rather than eating leaves as a whole, while ash is insoluble in water. The total ash content is mainly constituted by the mineral element, so the mineral element contents in leaves and leaf tea were higher than those in roots2. Similar results were demonstrated in the products of medicinal plant C. pilosula20.
The above results show that in addition to being rich in amino acids, dry leaf and leaf tea also have higher TFC, a natural antibiotic, providing an important basis for the development and utilization of LT products in the future.
Antioxidant activity analysis
The antioxidant activities of the solutions extracted from the three products were measured using ABTS, DPPH⋅ and FRAP, using VC as a positive control. Figure 2 shows inhibitions of the samples to ABTS radicals. This could suggest that DR also presented lower scavenging activities than those in WSE and ESE from samples of DL and LT.
Inhibition of 2,2-azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) radical for ethanol water-soluble extracts samples (A) and ethanol-soluble extracts samples (B) from DL (filled triangle), processed LT (open triangle) and medicinal DR (open circle) of A. membranaceus, compared with Vc (closed circle); DL, dry leaves; LT, leaf tea ; DR, dry roots; Vc, vitamin C.
When 20,000 μg/mL was reached, ESE from the DR product showed a 51.89% inhibition rate to ABTS free radical, while the value of WSE was 56.52%. WSE and ESE of LT and DL had very similar scavenging ability to free radicals, and both have much stronger scavenging ability to free radicals than the respective root extracts. Once LT and DL concentration reached a certain level, the abilities noticeably reached that of VC. The highest inhibition rate of 99.94% in VC appeared at concentration of 300 μg/mL (Fig. 2). The highest value was 99.49% at concentration of 15,000 μg/mL in DL, and 99.10% at concentration of 20,000 μg/mL in LT for WSE samples (Fig. 2A).
However, for ESE samples, the highest inhibition rates of 89.62% and 94.28% in DL and LT respectively all appeared at a concentration of 20,000 μg/mL (Fig. 2B). Therefore, we concluded that LT and DL could inhibit ABTS radical efficiently at lower concentrations than DR. Furthermore, the inhibition effect of DL and LT were quite similar, and the highest inhibitions reached as high as VC at certain concentrations. The same inhibitions result also appeared in the corresponsive products of another medicinal and edible C. pilosula20.
The DPPH⋅ radical inhibition ability of various products is presented in Fig. 3. The highest inhibition rate of VC was 95.75% at 30 μg/mL. The highest DPPH⋅ radical inhibition rates of DL and LT were 90.78% and 91.47% for ESE samples as well as 93.19% and 80.25% for WSE samples at 1000 μg/mL. The maximum inhibition rates of DR for ESE and WSE samples were only 70.94% and 69.24% at 1000 μg/mL. Therefore, we can draw the conclusion that DR presented much lower scavenging vigor than DL and LT for both water-soluble and ethanol-soluble extracts. At a certain concentration, the highest inhibition of DL and LT to free radicals was approximately equal to the level of VC. These active functions occurred in the leaf products of medicinal plants A. membranaceus and C. pilosula20, both of which were added to the list of Chinese medicine and food materials of the same origin.
Inhibition of 2,2-diphenyl-1-picrylhy-drazyl (DPPH) radical for water-soluble extracts samples (A) and ethanol-soluble extracts samples (B) from DL (filled triangle), processed LT (open triangle) and medicinal DR (open circle) of A. membranaceus, compared with Vc (closed circle)); DL, dry leaves; LT, leaf tea ; DR, dry roots; Vc, vitamin C.
The IC50 of ABTS and DPPH⋅ inhibition is shown in Table 3. In terms of the IC50 values of ABTS, those in DL and LT were higher than that of DR (P < 0.01). The maximum IC50 of WSE appeared in the DL sample (1101.69 μg/mL), but the maximum IC50 of ESE was noted in LT product (2163.60 μg/mL). The WSE of DR exhibited the highest IC50 values of DPPH⋅ than other two leaf products (P < 0.01), reaching a value of 40.97 μg/mL. Compared with IC50 of DR to DPPH⋅ (32.20 μg/mL), the values in ESE exhibited 64.41 μg/mL from LT and 53.90 μg/mL from DL, resulting in no significant difference from each other (P < 0.01), but the LT is significantly higher (P < 0.01). However, the IC50 to DPPH⋅ in ESE from the roots of C. pilosula was up to 417.91 μg/mL, and the IC50 was not detected within the experiment concentration extent20. From the above it could be deduced that the leaf of A. membranaceus have much more potential utilization value in developing antioxidant bio-products than the DR.
FRAP test was to determine whether the samples have efficient reducing power, thus further revealing their authentic antioxidant ability. The reducing power was calculated according to the absorbance of reaction mixture at 593 nm by VC standard curve (y = − 0.013 + 0.006x, r2 = 0.998). The FRAP activity was expressed by x value calculated from above equation, finally shown in Table 4. When the concentration was at 20.0 mg/mL, we observed that the reducing powers of DL and LT to FRAP were very close both for WSE (546.66 μg/mL, 645.03 μg/mL) and for ESE (612.40 μg/mL, 645.33 μg/mL), and these trends also appeared the same to ABTS and DPPH⋅ inhibition. However, when the concentration of DR was at 20.0 mg/mL, the FRAP value for WSE and ESE were 51.33 μg/mL and 86.44 μg/mL, respectively. Obviously, DR showed much lower reducing power than DL and LT. The reducing powers of DL and LT were very similar, even approximated up to the level of VC when getting at a certain concentration. These same trends of reducing power to FRAP also existed in products of C. pilosula as observed in a recent study20.
Regarding the scavenging activities of ABTS, DPPH⋅ and FRAP, the results indicate that DL and LT have similarly very high antioxidant activities, being able to reach the level of VC at a certain concentration. DR antioxidant activities were significantly lower (P < 0.01)than the other two, and the inhibition capacities of ABTS, DPPH⋅ and FRAP were in accordance with the results of Coronopus didymus31. Therefore, DL and LT can be more widely and comprehensively developed and utilized in the future.
Antimicrobial activity analysis
The extracts were diluted to gradient concentrations at 0.02–20.00 mg/mL based on 20.00 mg/mL concentration prepared by LT, DL and DR20,27. The results showed that the different concentrations of extracts from three (3) products had different inhibitory effects on six (6) strains. Table 5 showed the diameters of antibacterial zones determined in vitro experiment. The extracts of some samples showed inhibition to some strains at high concentration, but did not at lower concentration (Table 5). However, the extracts of other samples showed inhibition to some strains at low concentration, but did not at high concentration. DL and DR showed no antimicrobial activity both to S. aureus and A. niger but for A. niger. LT could play antibacterial activity to other bacteria and yeast, showing the varied antibacterial activities with the concentrations (Tables 5, 6).
The MAX and MIN of tested concentration extents are listed in Table 6. The extracts of DL, LT and DR all had antibacterial activities against Salmonella at a concentration of 20.00 mg/mL, however, their MIN antibacterial concentrations appeared at 0.63 mg/mL, 2.50 mg/mL, and 2.50 mg/mL, respectively, indicating DL has stronger bacteriostatic effect. The sensitivities of the extracts from DL, LT and DR to B. subtilis were quite similar, having 0.63 mg/mL for the MIN and at least 20.00 mg/mL for the MAX. All the extracts of DL, LT and DR have antibacterial effects on E. coli at low concentrations of 0.02 mg/mL. Differing from the MIN, the MAX concentration varied with the products. It was 0.63 mg/mL for DL, 0.31 mg/mL both for LT and DR products. The extracts of DL, LT and DR had antimicrobial effect on yeast, but the effects varied with the concentrations, and the MIN and MAX concentrations appeared at 0.02 mg/mL and 0.63 mg/mL for DL, 0.04 mg/mL and 1.25 mg/mL for LT, and 0.08 mg/mL and 10.00 mg/mL for DR. Among the extracts, only the extracts of LT showed antibacterial activity on S. aureus, and the effective concentrations ranged 0.63 mg/mL to 10.00 mg/mL. Therefore, DL and LT were more sensitive to some bacteria and yeast; this proved that they have stronger and efficient antibacterial activity (Table 6). On the basis of exploiting bioactive potential of wild radish, Raphanus raphanistrum using hydroethanolic and decoction extracts, Iyda found both the samples proved to inhibit several Gram-positive and Gram-negative bacteria and revealed antioxidant activity21, while cytotoxicity against non-tumour cell was not observed. In our study, a systematic toxicological test was carried out on the three products from medicinal plant A. membranaceus in vivo and the results reveal no toxicity.
Source: Ecology - nature.com