Introduction
Allium microdictyon is a perennial plant that belongs to the Liliaceae family and is a type of wild edible greens that smells of garlic all over (Kim, 2018). In South Korea, it is known to grow naturally in forests of Jirisan Mountain, Seoraksan Mountain, and Ulleungdo Island, or in the northern alpine regions (Lee, 1985). Having been used as a type of wild herbs and vegetables, the importance of Allium microdictyon as a functional food is increasing as its pharmaceutical efficacy has been recently recognized, such as the effect of its extracts on hyperlipidemia and obesity (Choi, 2005), the pharmaceutical efficacy of its extracts for white rats (Choi et al., 2003), and neuroprotective effect (Chung et al., 2015).
Accordingly, studies have been conducted on the cultivation method of wild herbs and vegetables (Kim, 2018; Choi et al., 1998; Ryu et al., 1997; Kim et al., 2010) as well as economic analysis (Park et al., 2014), but most are limited to production through the establishment of cultivation methods such as proper shipping timing and yield improvement. In particular, unlike field cultivation, forest cultivation has a great impact on the generation and growth of understory plants due to light conditions affected by upper-layer stands, and thus research on this is necessary (Choi, 2001).
Growing environments affect the formation of plants including growth, organ differentiation, and primary and secondary metabolites (Shohael et al., 2006), and they also serve as factors of environmental stress in plant growth. Plants must endure stress by using the least amount of energy available through the antioxidant-related mechanism in the body in order to properly resist and defend themselves from stress (Han et al., 2006). In this process, superoxide dismutase (SOD) serves as a catalyst for disproportionation reaction that converts superoxide ion (O2−) to hydrogen peroxide (H2O2) (Bowler et al., 1992), and hydrogen peroxide is converted to water as it is transferred into peroxisomes by catalase (Bowler et al., 1994).
This study was conducted to obtain data on the production quality and relationship of Allium microdictyon leaves depending on the slopes of the cultivation site by investigating the change in antioxidant enzyme activity according to the light conditions of the cultivation site in forest cultivation of Allium microdictyon.
Research Methods
Sites
One-year seedlings of Allium microdictyon were planted in April 2015 in test sites with different elevations and slopes in a forest in Inje-gun, Gangwon-do. Each test site was selected on the south-facing slope and north-facing slope each at 700 m and 500 m above sea level, and each test site was created within a straight-line distance of 200m (Table 1). The maximum intensity for each test site was described after measuring the intensity from noon to 2 p.m. during planting. Artificial thinning was not performed in order to cultivate in an environment similar to natural state, and only parts of the herbaceous layer in the understory of the planting site were removed. Leaf length (cm), leaf width (cm), leaf area (cm2), and fresh weight (g) were investigated from April to June the following year after planting. Leaf area was measured using WinFOLIA (Regent Instruments Inc., Canada).
Soil analysis
To analyze the chemical properties of the soil with focus on nitrogen (N), phosphoric acid (P), and potassium (K), the organic matter of the O layer was removed from the test sites, after which samples were collected repeatedly three times in the test sites using a soil can. The collected samples were air-dried indoors, pulverized, and passed through a 2 mm sieve. For sample analysis, the total nitrogen content was analyzed by the Kjeldahl method using Foss Kjeltec 2200. The organic content was measured by the Tyurin method, available phosphate content by the Lancaster method, and exchangeable cation content by mixing and shaking 1N-ammonium acetic acid, then filtering and measuring with an atomic absorption spectrophotometer (Analytik Jena AG Nov AA-300, Germany)
Measurement of antioxidant enzyme activity
0.5 g of the sample was homogenized by adding 10 ml of a mixed solution of 50 mM phosphate buffer (pH 7.0), 10 mM ascorbic acid, and 1.0% (w/v) polyvinylpyrrolidone (PVP), and then centrifuged at 4 °C in 20,000 × g for 30 minutes, after which the supernatant was used to analyze enzyme activity.
Superoxide dismutase (SOD) activity was measured using the nitroblue tetrazolium (NBT) reduction method, and the absorbance increase was measured at 530 nm for 120 seconds with a spectrophotometer (Beauchamp and Fridovich, 1971), after which it was calculated using the method by Asada et al. (1974).
For catalase activity analysis, 30ul of enzyme extract and 500 ul of 10 mM H2O2 were mixed and reacted at room temperature for 1 minute, after which the reaction was stopped with 500ul sodium acid. 20 ul of the reaction solution and 2 ml of HRP/Chromogen were added again, and absorbance was measured at 520 nm after 10 minutes (Fossati et al., 1980).
Measurement of MDA content
Malondialdehyde (MDA) content was measured using the method by Heath and Parker (1968). 10 ml of the mixture containing 6.25 mM of phosphate buffer (pH 7.0) was added to 0.5g of the sample, which was homogenized and then centrifuged at 4 °C in 12,500 rpm (12,000 × g) for 20 minutes, after which the supernatant was used for analysis. 1 ml of the extract and 1 ml of 0.5% 2-thiobarbituric acid were added, heated for 15 minutes, and rapidly cooled. The absorbance was measured at 532 nm to determine the MDA content.
DPPH free radical scavenging activity
10 ml of methanol was added to 1g of leaves collected according to the method by Biois (1958) and extracted at room temperature for 24 hours, after which it was centrifuged and 2.5 ml of the supernatant was collected and mixed uniformly with 1ml of 0.3mM DPPH (α,α-diphenyl-β-picrylhydrazyl), and then it was left at room temperature for 30 minutes and measured at 525 nm. The antioxidant effect was expressed as electron donating capacity (%).
Results and Discussion
The leaf growth of Allium microdictyon reached its maximum between April 13 and April 22, after which there was almost no growth (Table 2). Allium microdictyon is shipped from farms with a length of 20 cm and a width of 10 cm at the least, and the leaf area is 140–150 cm2. The point of time when the length and width appeared to be similar to the time of shipment from the farms by test site was April 22 for the south-facing slope in Jeongja-ri, May 22 for the north-facing slope in Jeongja-ri, and April 13 for the south-facing slope in Misan-ri. The south-facing slope showed earlier growth than the north-facing slope. For the north-facing slope in Misan-ri, the root part survived, but shipping was impossible due to the stems damaged by wild animals. Forest cultivation requires more management measures than field cultivation. The reason why the south-facing slope showed relatively faster leaf growth than the north-facing slope is because the south-facing slope was affected by the amount of light (Table 1). Thus, the growth was faster due to physiological factors such as temperature difference between slopes and photosynthesis. Like garlic, an increase in temperature leads to summer decline of Allium microdictyon (RDA, 1999). Thus, the growth stops and the fresh weight decreases as the temperature rises, with the leaves losing vitality, withering, and dropping, thereby showing that fresh weight has decreased over time. There was almost no growth in all test sites after this period. Unlike Ligularia fischeri and Ligularia stenocephala, if the leaves of Allium microdictyon are picked once a year, the leaves to not come out again that same year. Thus, to adjust the harvest time and increase the harvest period, methods such as differentiating the planting sites in consideration of the slope directions can be applied.
Adjusting the cultivation and harvest times of forest products is a key factor of success. Field cultivation is mostly done on bare land, and thus the harvest times are determined mostly by temperature changes. However, in forest cultivation, light conditions can be controlled according to the conditions of the stands, which greatly affects the occurrence and physiological state of understory plants (Choi, 2001; Park and Bae, 2012). High light conditions lead to increased temperature of the cultivation sites. Allium microdictyon is a plant that grows naturally in the alpine area of 1,000m above sea level and in cool areas of Ulleungdo, and the temperature of cultivation is about 8–20 °C in July, thereby preventing summer decline in a cool place (RDA, 1999). Therefore, forest cultivation can provide an environment that is closer to native land compared to field cultivation.
Soils by test site showed weak acidity (pH 5.59–6.52) regardless of region (Table 3). The acidity was higher than that of general forest soil, which was higher than the pH of the soil where Aster scaber Thunb and Ligularia fischeri are cultivated (pH 5.2) (Choi et al., 2009). The major components shown in the study results of upland soil of cultivation sites for Aster scaber Thunb and Ligularia fischeri are as follows (Choi et al. 2009). Due to phosphoric manure fertilization, the available phosphate in upland soil was 6–20 times higher than that in forest land. Calcium, potassium, and magnesium were generally higher in Misan-ri, which showed a fast appearance and growth rate of vegetation. These are all major elements in soil and are directly related to plant growth, thereby showing the result above. Previous studies also revealed that growth such as leaf length and width increased as nitrogen fertilization amount increased when planting Ligularia fischeri (Choi et al. 2009). This study also revealed that total nitrogen content increased in the soil of Misan-ri where leaf growth appeared quickly. It has also been reported that the growth of perilla increases as the amount of phosphatic fertilization increases, and that the aboveground growth is severely inhibited when it is deficient (Choi and Park, 2008). Cation exchange capacity, which activates nutrient absorption by converting anions in soil into cations, was higher than upland soil in all test sites.
Superoxide dismutase (SOD) showed the highest level overall in the south-facing slope in Misan-ri. The south-facing slope in Jeongja-ri and the north-facing slope in Jeongja-ri generally showed similar values and barely showed any change over time (Fig. 1). The south-facing slope in Misan-ri showed a somewhat high value on April 22, but the difference was not much. Catalase showed a relatively large difference between test sites (Fig. 2). The south-facing slope in Jeongja-ri showed a continuous decline, and the south-facing slope in Misan-ri and north-facing slope in Jeongja-ri showed an increase between April 18 and April 22 and then a decrease after that. Accordingly, the values were similar in all three sites on April 30, after which they all decreased and showed almost no activity by May 5. Plants produce reactive oxygen species under various stress conditions and cope with this stress by producing antioxidant enzymes such as SOD and catalase as a self-defense response (Asada, 1999). SOD plays an important role in the antioxidant system as a catalyst for the disproportionation reaction that converts superoxide ion (O2−) into hydrogen peroxide (H2O2) and oxygen (O2) (Bowler et al., 1992). Hydrogen peroxide is known to reduce damage caused by excessive reactive oxygen species by being decomposed into water molecules and oxygen molecules by peroxidase (POD) or catalase (Anderson et al., 2005; Salin, 1991). Catalase, which showed a large difference in this experiment, continued to decrease on the south-facing slope in Jeongja-ri, which implies that the point at which leaf growth reached its maximum was earlier than in other sites. Both the south-facing slope in Misan-ri and the north-facing slope in Jeongja-ri showed an increase and then a decrease. As the plant grew, its self-defense function also increased, but it decreased at the peak of the growth period, leading to decline and death. Regarding DPPH free radical scavenging activity, Sedum acre showed a huge difference of 61.3%–82.4% depending on the regional species when lyophilization was extracted (Kim et al., 2008), but in this study, almost no difference was observed depending on the test site and period (Fig. 3). As a result of measuring the malondialdehyde (MDA) content to find out the degree of lipid peroxidation, the south-facing slope in Misan-ri showed a big change, showing a significant decrease from April 18 to April 22 when there was a great change in growth, thereby reaching its minimum (Fig. 4). The south-facing slope in Jeongja-ri also showed a minimum value on April 22, but the change was smaller than that of Misan-ri. On May 5 when the harvest time was the latest, MDA content was almost nonexistent compared to other regions. Plants show different activities depending on the growth period. Alnus firma leaves that grow naturally in abandoned mine areas showed lowest activity in April and highest activity in June (Han et al., 2006). Ligularia fischeri showed more effective harvest in May compared to early harvest (April) (Suh et al., 2020). It is believed that there is a difference in the change of the active period depending on plant species.
Conclusion
Based on the results of this study, it is possible to control the harvest date up to 30 days by adjusting the location environment such as the slope direction of the sites for forest cultivation of Allium microdictyon. Catalase and MDA tended to be proportional or inversely proportional depending on the harvest date, serving as most active indicators of change in physiological activity during harvest.
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