Do Vermicompost Applications Improve Growth Performance, Pharmaceutically Important Alkaloids, Phenolic Content, Free Radical Scavenging Potency and Defense Enzyme Activities in Summer Snowflake (Leucojum aestivum L.)?

Leucojum aestivum L. contains galanthamine and lycorine, which are two pharmaceutically valuable alkaloids. Vermicompost (VC), an organic waste product created by earthworms enhances soil quality and can improve the medicinal quality of the plant that is crucial to the pharmaceutical industry. The aim of this study was to determine the effects of four different VC concentrations (5 %, 10 %, 25 %, and 50 %) on L. aestivum growth parameters, alkaloid levels (galanthamine and lycorine), total phenol‐flavonoid content, free radical scavenging potential, and defense enzyme activities (SOD and CAT) compared to control (no VC). The width, length, and fresh weight of the leaves were improved by 10 % VC treatment. The highest total phenolic content was found in the bulbs and leaves treated with 50 % VC. HPLC‐DAD analysis of alkaloids showed that 10 % and 50 % VC treatments contained the most galanthamine in the bulb and leaf extracts, respectively. The application of 25 % VC was the most efficient in terms of lycorine content in both extracts. CAT activity was elevated at 10 %, 25 %, and 50 % VC. Based on the growth performance and galanthamine content of the bulbs and leaves, it can be concluded that a 10 % VC application was the most effective in the cultivation of L. aestivum.


Introduction
Leucojum aestivum L. subsp.aestivum, is a bulbous perennial plant in the Amaryllidaceae family.Its common names are Loddon lily, snowflake lily, and summer snowflake.L. aestivum grows naturally in alluvial soils near rivers, lakes, and humid and semi-shaded fields, such as swamps, wetlands, and floodplains, from sea level to 1000 m elevation. [1,2]The flower stalk has two to five umbrella-shaped bell-like flowers placed at equal intervals and blooms between March and June. [3]L. aestivum is a threatened plant because it has been collected from its natural habitat for the pharmaceutical industry.It is a medicinal and ornamental plant native to southern Europe, Turkey, the Balkans, Caucasia, and northern Iran. [1]Galanthamine, an isoquinoline alkaloid, is a long-acting, selective, reversible, and competitive acetylcholinesterase (AChE) inhibitor used to treat neurological disorders, primarily senile dementia of Alzheimer's type, poliomyelitis, and other neurological diseases. [4]Lycorine is a pyrrolophenanthridine alkaloid that exhibits antiretroviral, antimitotic, anticancer, antiplasmodial anti-inflammatory, analgesic, emetic, and cytotoxic activities.In addition, several studies have shown that lycorine has strong antiviral effects against poliovirus, measles, severe acute respiratory syndromeassociated coronavirus, and Middle East respiratory syndromeassociated coronavirus. [4,5]Recent studies have indicated that lycorine is a strong COVID-19 inhibitor. [6]ermicompost (VC) is a biological fertilizer created by the continuous and slow movement of decomposing organic matter in the gastrointestinal systems of certain earthworm species.Before excretion, substances pass through the earthworm's body and are filled with gastrointestinal mucosa, vitamins, and enzymes. [7]Vermicomposting is a useful, environmentally friendly, and economically viable waste recycling method, in which organic waste material is transformed into usable compost through the combined activities of microorganisms and earthworms. [8]VC is often generated by epigeic earthworms that feed on fresh organic materials in the litter layer and the first centimeter of the soil.The most well-known earthworm used in vermicomposting is Eisenia fetida, which has a remarkable ability to change the physical, biological, and chemical characteristics of organic substances, as well as its resilience and wide temperature tolerance.[11][12] Furthermore, VC improves the microbial diversity of the soil and stores nutrients for an extended period without negatively affecting the environment. [13]Plant roots are generally unable to adapt to mineral N unless the carbon: nitrogen (C : N) ratio is � 20 : 1. Earthworms contribute to the decline in the C : N ratio of fresh organic material during respiration. [14]Nutrients are secreted and transformed into soluble and accessible forms throughout vermicomposting, supplying nutrients such as accessible N, soluble K, exchangeable Ca, Mg, and P, as well as microelements such as Fe, Mo, Zn, and Cu that plants can easily absorb. [15]An organism that enhances the ecological and biological state of the soil is known as an eco-biological engineer.Thus, earthworms alter the characteristics of soil that has been contaminated by pesticides or heavy metals and prevent the contamination of these substances caused by humans. [14,16]C inhibits soil-borne diseases and provides microorganisms with antimicrobial activity.Earthworms release plant growth regulators, such as gibberellin, auxin, and cytokinin, during vermicomposting, which work synergistically with microorganisms.It also synthesizes plant growth hormone-like molecules including fulvic acid, humic acid, and humates.These conditions can enhance plant growth and yield. [10,17,18]The mechanism by which VC alters secondary metabolites remains unclear.Investigations on the use of humic compounds in medicinal plants have shown that they can boost the production of secondary metabolites and the activity of bioactive substances, such as flavonoids, coumarins, alkaloids, phenylpropanoids, total phenols, and anthocyanins. [19]This is explained by the chemical composition of VC, which usually includes several types of nonhormonal plant growth stimulants, phytohormones, and soluble phenolic compounds, together with a variety of microorganisms and macronutrients that can affect plant physiology. [20]erharvesting destroys and endangers the natural populations of L. aestivum, which contain the pharmaceutically valuable alkaloid galanthamine.This plant has been cultivated in licensed fields in Turkey to export bulbs to pharmaceutical manufacturers. [1]Although galanthamine in L. aestivum has commercial significance in the pharmaceutical industry, and the impact of VC on secondary metabolite augmentation is wellknown, to our knowledge, no studies have investigated the effectiveness of VC on this pharmaceutically famous alkaloid.In this context, it was hypothesized that (i) various VC applications can enhance growth parameters and alkaloid levels, (ii) the phenolic content and free radical scavenging power can be improved with different VC treatments, and (iii) stress endurance capacity may be supported by VC implementation in L. aestivum.

Results and Discussion
Growth parameters and water accumulation with VC treatments After 2.5 months of culture in VC treatments, the width, length, and weight of L. aestivum bulbs and leaves were determined (Figure 1 and 2; Table 1).Compared with the control, 5 %, 10 %, and 25 % VC applications increased bulb width by 21.6 %, 13.5 %, and 13.5 %, and individual fresh weight by 7.7 %, 22.4 %, and 30 %, respectively.Interestingly, the bulb length was not influenced by VC application.
Application of 10 % VC contributed the most to the development of the width (22 % increase), length (6.3 % increase), and individual fresh weight (5.3 % increase) of the leaves (Table 1) compared to the control.It was evident that VC treatments of 10 %, 25 %, and 50 % caused a slight increase in water accumulation capacity in the bulbs compared to the control, but the water content of all VC-treated leaves was lower than that of the control (Figure 3).
An increase in water accumulation capacity in the bulbs should be related to an increase in bulb width with VC application (Table 1).Due to the hydrophilic groups included in VC, the soil temperature rises, water retention increases, and plant development accelerates, improving yield. [16]By using VC, it is possible to improve soil structure, porosity, air intake, and water holding capacity.Moreover, VC aids in the greater completion of root formation in plants, resulting in plants that take nutrients from the soil more readily and perform better in terms of growth. [21][24][25][26][27][28][29][30] Balmori et al. explained that the foliar application of humic extract from VC at a 1 : 40 (v:v) on Allium sativum was the best treatment for growth parameters.They found that the humic substance increased the fruit quality and improved the bulb diameter. [31]Akram found that the bulbs of Narcissus pseudonarcissus that were treated with humic acid grew faster and were heavier than the control bulbs. [32]According to Babarabie et al. the addition of VC to the soil enhanced flower life, germination rate, and stem diameter of Narcissus flowers. [33]ahraman and Akçal investigated the effects of different nutrient solution recipes containing NPK (nitrogen, phosphorus, and potassium) on L. aestivum growth in soilless agriculture.They found the highest bulb diameter in nutrient solutions with 200 % NPK and 125 % NPK when compared with the control groups. [34]Bakian et al. investigated how L. aestivum bulbs from different zones responded to organic and biological fertilizers.They used both biochar fertilizers and biofertilizers.They discovered that a biochar treatment of 12 tons/h resulted in the maximum root weight and total plant weight. [35]Other findings corroborated the results of this study.For example, El-Attar et al. observed a significant increase in the width of Narcissus tazetta bulbs and their fresh weights with the application of K sources (K-nano or K-humate) and soil treated with VC. [36] Srivastava et al. reported that 50 % VC + 50 % NPK treatment significantly increased the size and fresh weight of Allium cepa bulbs. [37]García et al. indicated that 34 and 46 mg humic acid/L increased the water accumulation in rice seedlings under water stress when compared with the control groups. [26]

Alkaloid accumulation with VC treatments
Quantification of alkaloid content in 10 different methanolic extracts (bulbs and leaves) was carried out using HPLC-DAD analysis, and the extraction yield of the methanol extract is presented in Table 2.The chromatograms of the standards used were indicated in Figure 4A.All tested VC concentrations enhanced the galanthamine content in the bulb extracts in comparison with the control.Additionally, it was found that 5 %, 25 %, and 50 % VC treatments increased lycorine content compared to the control in the bulb extracts.The maximum galanthamine content was detected in the 10 % VC treatment (Figure 4B), while the highest amount of lycorine was identified in the bulb extracts treated with 25 % VC compared to the control (44 % increase in both) (Table 2).All VC treatments increased the amount of galanthamine in the leaves, except for the 5 % VC application.Compared to the control, leaf extracts treated with 50 % VC had the highest galanthamine content (37 % increase) (Table 2; Figure 4C).The application of 10 % VC was the same as that of the control.Treatment with 25 % and 50 % VC increased lycorine content in the leaf extracts, with the 25 % VC treatment providing the greatest increase (84 %).
][40]    production in tomato crops using GC/MS analysis.They found that 20 % VC tea treatment increased the production of lycopersene and β-phellandrene. [20]In addition, Alinejad et al.
VC treatment may mitigate the negative effects of water shortage stress in Datura stramonium.They reported that the leaves with the highest scopolamine content were found in the 15 % water deficit treatment with 15 % VC. [39] Celikcan et al. reported that 2.5 %, 5 %, and 10 % VC treatments increased the caffeic acid content, whereas 2.5 %, 5 %, 10 %, and 20 % VC treatments decreased the estragole and eucalyptol contents in Ocimum basilicum. [40]Rasa et al. indicated that 30 % VC treatment enhanced alkaloids in Fumaria valliantii, such as fumaryn and synaktyn. [41]Gholami et al. determined that the highest caffeic acid content was found in a mixture of humic acid (0.3 kg per hectare) and VC (5 t per hectare) treatments in Cichorium intybus.They reported that the highest ellagic acid content was observed in shoots when only VC (5 t per hectare) was applied. [38]orinova et al. noticed that galanthamine biosynthesis was likely controlled by soil fertility levels.They found that L. aestivum grew best in soils with a pH close to neutral and rich in organic matter, and that galanthamine levels were highest in soils with ample supplies of K, N, B, Mg, Zn, Mo, Cu, and Fe. [42,43]imilarly, Demir et al. reported that soils that were neutral to slightly alkaline with a high organic matter content were effective in increasing the alkaloid content in L. aestivum bulbs and leaves.Moreover, they demonstrated that the amount of galanthamine present in L. aestivum might be correlated with soil K levels. [44]he galanthamine and lycorine contents of L. aestivum were enhanced with different VC concentrations in this study (Table 2), as in previous studies, owing to the organic nature of the VC applied to the soil, which increases soil nutrients. [21]kram reported that higher levels of humic acid resulted in a higher level of alkaloids (galanthamine and haemanthamine) in N. pseudonarcissus. [32]The presence of increased alkaloid in L. aestivum in this study may be due to the humic acid in VC.

Phenolic content and free radical scavenging potency with VC treatments
Phenolics and flavonoids are strong scavengers of free radicals because of their hydroxy groups.The total phenolic and flavonoid contents of L. aestivum bulb and leaf extracts were assessed using gallic acid and quercetin calibration curves, respectively (R 2 = 0.998).The total phenolic and flavonoid contents of all extracts were shown in Table 3.All concentrations of VC enhanced the total phenolic content in the bulbs compared to the control (no VC).
The maximum total phenolic and flavonoid contents were identified in the bulbs treated with 50 % VC, with increases of 30.46 % and 55.8 %, respectively, compared with the control.Although the total phenolic content was the highest with 50 % VC (44.13 % increase), the total flavonoid level was the highest with 5 % VC in the leaves (17.22 % increase) in comparison with the control (Table 3).
Free radical-scavenging activity was demonstrated as IC 50 value (half maximal inhibitory concentration).Quercetin was utilized as the antioxidant standard.The best antioxidant activity was observed with the 50 % VC treatment in both bulbs and leaves (Table 3).The highest antioxidant activity with 50 % VC treatment was related to the highest total phenolic contents in both (Table 3).A strong negative correlation was observed between free radical scavenging potential and total phenolic content of the bulbs and leaves (r = À -0.77 and À 0.92, respectively, P < 0.05), indicating that an increase in the overall phenolic content generated by the impact of VC caused an increase in antioxidant capability.Like this study, Demir et al. reported that high levels of organic matter in the soil were beneficial for enhancing the phenolic constituents and antioxidant capacity of L. aestivum. [44]everal studies have investigated the effects of VC on various plants.Mardani-Talaee et al. reported the highest phenol activity in Capsicum annuum with 30 % VC. [45] Souffront et al. determined that 10 % VC tea treatment resulted in a higher concentration of total phenolic compounds than the control and 20 % VC tea treatments in tomato crops. [20]Omar et al. indicated that VC treatment on cassava tubers had the greatest total phenolic content (10.88 mg GAE/g fw) when compared with the inorganic fertilizer (8.35 mg GAE/g fw). [46]uján-Hidalgo et al. determined the total phenol content in Annona purpurea leaf samples.[50] Wang et al. indicated that mixing VC and soil at a ratio of 4 : 7 (w/w) showed the best DPPH radical-scavenging activity on Chinese cabbage leaves, which was enhanced by 92 % compared with the activity of the full soil treatment (0 : 7 w/w). [25]Omar et al. found that VC treatment resulted in the highest DPPH scavenging activity (67.30%) in cassava tubers. [46]Choirunnisa et al. observed that all VC treatments (40, 60, and 80 g/plant) showed positive antioxidant activity in Echinacea purpurea. [51]

Defense enzyme activities with VC treatments
Superoxide dismutase is an enzyme that catalyzes the dismutation of the superoxide anion (O 2 *À ) into H 2 O 2 and O 2 .When both oxidation and reduction reactions occur in the same reactant (O 2 *À ) in a biological system, dismutation reactions occur and produce two compounds: one with a higher oxidation state (O 2 ) and one with a lower oxidation state (H 2 O 2 ).This enzyme is one of the most important enzymatic systems in plants that scavenges stress-generated free radicals (O 2 *À ). [52]lterations in SOD and CAT activities in the bulbs and leaves were shown in Table 4.All of the VC treatments reduced SOD activity in the bulbs and leaves.
Catalase is a tetrameric heme-containing enzyme that directly dismutates H 2 O 2 into H 2 O and O 2 .They are essential for reactive oxygen species (ROS) detoxification under stress conditions. [53]CAT activity in the bulbs increased 2.17, 2.02, and 2.35-folds, respectively, with 10 %, 25 %, and 50 % VC treatments.The administration of 5 % VC had no effect on CAT activity (Table 4).In the leaves, CAT activity was 1.02, 1.25, and 1.04-folds higher in the 10, 25, and 50 % VC treatments, respectively.As with the bulbs, VC treatment reduced CAT levels in the leaves (Table 4).Antioxidant enzymes have been frequently shown to be elevated in VC-applied trials with various plants.Xu et al.  reported that 15 % VC treatment increased SOD and CAT enzyme activities in the aerial parts of Silybum marianum.In addition, they determined that 15 % VC and 1 % NaCl + 5 % VC treatments enhanced SOD activity in the roots. [54]In Mentha haplocalyx, all VC treatments enhanced SOD activity in the aerial parts.However, all VC treatments decreased CAT activity in the roots. [54]Ahmad et al. determined that under drought conditions, both SOD and CAT activities were enhanced by the application of 8 tons per hectare (t/ha) VC on wheat straw. [55]ahbouki et al. indicated that Opuntia ficus-indica infected with arbuscular mycorrhizal fungi (AMF), treated with VC, and their combination demonstrated higher activities of SOD and CAT than control plants.They revealed that SOD and CAT activities increased when AMF and VC were added to soils. [56]García et al. determined that the association of humic acid with the radicular system in plants stimulates antioxidative enzymatic functions in rice plants. [57]Zuo et al. ( 2018) indicated that the SOD activity of strawberries was significantly enhanced by the application of 20 % and 30 % VC.Lower SOD activity with all VC applications in comparison with the control (no VC application) showed that these treatments did not impose too much stress on L. aestivum.On the other hand, CAT activity was the lowest in the 5 % VC treatment, and activity of this enzyme was elevated in the 10 %, 25 %, and 50 % VC treatments compared with the control.Application of 5 %, 10 %, and 25 % VC increased the bulb width and fresh weight.The lowest level of CAT activity with 5 % VC application (low stress) may be associated with greater bulb enlargement (Table 1).
The accumulation of secondary plant products is strongly influenced by growing conditions such as temperature, light regime, and nutrient availability.Various stress conditions have an impact on the metabolic pathways responsible for the accumulation of these products in plants, [58] such as alkaloid (galanthamine and lycorine) levels and phenolic content. [1,2,44]he highest galanthamine and lycorine levels were observed with 10 % and 25 % VC in the bulbs, and 50 % and 25 % VC in the leaves, respectively.It can be deduced that these VC concentrations (10 %, 25 %, and 50 %) caused moderate stress in L. aestivum (higher CAT activity) and were associated with higher levels of galanthamine and lycorine.
CAT activity and galanthamine levels in the bulbs showed a strong positive correlation (r = 0.90, P < 0.05) in response to various VC applications.Plants treated with VC are resistant to adverse environmental conditions. [40]Enhanced CAT activity was noted in response to oxidative stress with 10 %, 25 %, and 50 % VC administration.These concentrations may improve endurance (resistance) to abiotic stress conditions owing to increased antioxidant capacity.
In this study, we demonstrated for the first time that VC application in L. aestivum cultivation caused remarkable increases in galanthamine and lycorine levels, bulb and leaf expansion, phenolic content, and free radical scavenging power.It was also verified that increased CAT activity with some VC treatments supports the stress endurance capacity of L. aestivum.Overall, it has been shown that increased biomass with much higher galanthamine levels can be obtained from the same field with VC application in the cultivation of L. aestivum.

Conclusions
Augmentation with 5 %, 10 %, and 25 % VC enhanced the width, length, and fresh weight of the bulbs and leaves.Application of 50 % VC increased the total phenol and flavonoid contents and, consequently, the free radical scavenging activity of the bulbs and leaves.Galanthamine levels in the bulbs and leaves were improved by 10 % and 50 % VC application, respectively.Elevated CAT activity (enzymatic antioxidant defense system) with 10 %, 25 %, and 50 % VC applications may provide a greater resistance to abiotic stress conditions in this species.When all outcomes were considered together, this plant can be cultivated successfully in soil amended with 10 % VC to increase bulb galanthamine production with eco-friendly approach.Future research should focus on alleviation of the adverse effects of various abiotic stresses with VC treatments and their influences on alkaloid accumulation in L. aestivum.

Cultivation of L. aestivum with different VC concentrations
L. aestivum was collected from Gölcük-Bolu, Turkey when it reached approximately 5 cm in diameter in March 2021.It was identified by Prof. Dr. Arzu Ucar Turker using "Flora of Turkey and the East Aegean Islands" [3] and voucher specimens (AUT-1974) were deposited in Bolu Abant Izzet Baysal University Herbarium.Nearly the same size of the L. aestivum bulbs was chosen randomly and placed one bulb into each pot (18.5 cm×15.5 cm).Combinations of VC: soil mixture with ratios of 5 : 95, 10 : 90, 25 : 75, and 50 : 50 (w/w), as well as a control (only soil mixture with no VC) were prepared.The soil mixture contained 4 : 1 : 1 (v:v) ratios of peat (Terradena®, 65 % peat, and 35 % soil), sand, and vermiculite (Agrekal®), respectively.All preparations were very well homogenized.The supply of VC (made from cow manure and Eisenia fetida) was Labfarm Inovatif Organik Tarım® and VC analysis was performed by AgrioLabEN®.Characteristics of VC were as follows: pH -7.1; EC -5.1 dS/m; organic matter content -65.04 %; moisture -34.4 %; organic carbon (C) -50.4 %; organic nitrogen (N) -2.1 %; C/N ratio -17.3; total nitrogen -2.9 %; total diphosphorus trioxide (P2O3) -1.7 %; total humic + fulvic acid -31.5 %; water-soluble potassium oxide (K2O) -0.86 %; cadmium (Cd) -0.4 mg/kg; copper (Cu) -50.9 mg/kg; nickel (Ni) -12.4 mg/ kg; lead (Pb) -3.57mg/kg; zinc (Zn) -238.5 mg/kg; mercury (Hg) -< 0.01 mg/kg; chromium (Cr) -8.89 mg/kg; tin (Sn) -4.4 mg/kg.Analysis of VC and soil mixture were also conducted by Bolu Directorate of Provincial Agriculture and Forestry, Turkey.Properties of VC and soil mixture were as follows: soil texture-clay and clay loam; pH -6.71 and 6.99, EC -1.9 dS/m and 1.62 dS/m; total salt -0.21 % and 0.66 %; organic matter content -25.22 % and 4.93 %; total nitrogen -5.04 % and 0.98 %; phosphorus (P) -326 mg/L and 0.02 mg/L; potassium (K) -746.10 mg/L and 53.28 mg/L, respectively.The experimental design was completely randomized in triplicate in pots (a total of 15).The experiments were performed twice.The maximum pot water holding capacity for each treatment was calculated as described by Gutiérrez-Miceli et al. [22] Irrigation of the treatments was adjusted according to the maximum soil moisture using a soil moisture meter (Extech Instruments®, MO750), and the pots were controlled every other day.The experiment was performed under plant room conditions at 22 � 1 °C using a 16/8 h (light/dark) photoperiod (cool-white, fluorescent lights, 22-28 μmol/m 2 /s) with 60 % relative humidity.After collecting L. aestivum bulbs from natural habitats in March at the vegetative stage, they were planted in pots one day after collection for a 1week acclimatization process.At the end of 1 week, the bulbs were planted in pots for 2.5 months and plant samples were harvested separately at the vegetative stage by the end of 2.5 months.After harvesting, all plant samples were lyophilized at À 65 °C (Christ®) and stored at À 20 °C until extraction and biological activity investigations.The length, width, and weight of the bulbs and leaves were individually specified.

Table 1 .
Effect of VC treatments on growth parameters of L. aestivum.

Table 2 .
Extraction yields and HPLC-DAD analysis of alkaloid levels treated with various VC concentrations.
bMeans with the same letter within columns are not significantly different at P > 0.05.* Yield (%) = Extract weight (g)/dried plant sample weight×100.

Table 3 .
Effect of VC treatments on DPPH radical scavenging potency and total phenol-flavonoid content of L. aestivum.Means with the same letter within columns are not significantly different at P > 0.05.

Table 4 .
Effect of VC treatments on SOD and CAT activity of L. aestivum.
abMeans with the same letter within columns are not significantly different at P > 0.05.