Introduction

Codonopsis lanceolata Trautv is a perennial vine plant of the Campanulaceae family and is widely distributed throughout East Asia, including Japan, China, and Korea. In addition, C. lanceolata is used for natural, traditional medicines.

Recently, various pharmacological efficacies have been identified in C. lanceolata, promoting its usage as a raw material in food. C. lanceolata’s benefits include anti-obesity effects (Choi et al., 2013), increased antioxidant activity (Song et al., 2012), anti-inflammation (Joh, 2014), antitussive, expectorant, antidote (Ichikawa et al., 2009), promotion of enhanced serum lipid contents (Park et al., 2012), amending colitis in mice (Joh et al., 2009), improving memory and learning deficits (Jung et al., 2012), and remediating androgen hormone deficiency that causes male climacterium syndrome (Ushijima et al., 2007).

As described above, the pharmacological efficacy in C. lanceolata extracts are reportedly caused by lancemasides, which are a major saponin that include lancemaside A, lancemaside B, lancemaside C, lancemaside E, lancemaside G, foetidissimoside A, and aster saponin Hb (Ichikawa et al., 2009). In addition, C. lanceolata contains medicinal components such as inulin, phytoderin, leoithin, and pentosan (Hong et al., 2006).

Codonopsis lanceolata plants are divided into mountain C. lanceolata, which grows naturally in mountains, and cultivation C. lanceolate, which is cultivated using an artificial method (MAFRA, 2015). Kim et al. (1985) reported that mountain C. lanceolata is superior to cultivation C. lanceolata in all aspects excluding carbohydrate components. In addition, Lee et al. (1995) reported that aroma components are higher in mountain than in cultivation C. lanceolata. Therefore, mountain C. lanceolata was determined more valuable compared to cultivation C. lanceolata. However, as a result of the short supply of mountain C. lanceolata, cultivation C. lancelata is preferentially consumed.

After three years of sowing, cultivation C. lanceolata is grown, harvested, and sold. As it resides in the same plot for three years, this plant requires extensive cultivation management, including fertilizer, weed control, and disease and pest control. Thus, mulching cultivation is required for C. lanceolata as weeds inhibit its growth similar to water, nutrient competition, and photosynthesis inhibition. A vinyl material of black color has been generally used for C. lanceolata mulching cultivation, not only for its ability to control weeds, but also for water and temperature control in the soil. In addition, rice husks and rice straw have often been used as mulching materials (Oh et al., 1998). Vinyl mulching cultivation of medicinal crops such as C. lanceolata must be effective for 2-3 years; however, vinyl mulching needs improvement as it is susceptible to tears from strong wind and wild animals.

Some crops like Ginseng and Astragalus membranaceus are widely known to increase saponin content during mulching cultivation. However, little research has been done on C. lanceolata mulching cultivation and most of the limited findings focus on preventing weed growth (Oh et al., 1998). Consequently, it is not known how saponin content in C. lanceolata changes during mulching cultivation.

Thus, this study was conducted to identify optimum mulching materials that increase C. lanceolate saponin content and growth.

Materials and Methods

Plant Materials and Mulching Treatments

Two-year-old C. lanceolata plants cultivated in Jeju were planted at the Gyeongsang National University farm and later harvested when they were 3 years old. The mulching materials non-woven fabric (black, 3 m × 200 m × 0.45 mm), biodegradable film (green, 90 cm × 500 m × 15 μm), and rice husk were covered after the application of 100% basal fertilization based on the recommended rate (N-P-K, 6-6-6 kg). Hand weeding (controlling weeds by hand) and nontreatment (no weed removal intervention) served as experimental controls.

Soil Characterization

The soil pH (Radiometer M-92, Copenhagen, Denmark) was measured at a soil: water ratio of 1:5 (w/v). Total N (T-N) and phosphoric acid content were measured by the Kjeldahl and Lancaster methods (Varian Cary-50, Mulgrave, Australia), respectively. Soil cations was determined in accordance with Gillman (1979).

Shoot and Root Growth Characteristics

The shoot and root growth were measured in three replicates with five plants from each replicate. The plant height and stem diameter were measured for the shoot growth. The root length, root diameter, number of rootlets and fine roots, root fresh weight, and root dry weight were examined for root growth.

Sample Preparation

The roots of cultivated C. lanceolata were harvested and dried at 60°C for 48 h after washing, ground into fine powder using a mill, and stored at -20°C until analysis.

Preparation of the Crude Saponin

The dried root (5 g) was successively extracted over three times for 3 h using a Soxhelt extractor with 80% MeOH equivalent to 20 times the dry root weight. The extracts were filtered, combined, and concentrated using a rotary evaporator (R-520, Ilsin, Daejeon, Korea) under vacuum. The residue was dissolved in 60 mL of distilled water and extracted three times with an equal volume of ethyl ether, based on the method described by Shibata et al. (1966). The ethyl ether layer was removed and an equivalent volume of n-butanol was added to the water layer twice. The obtained n-butanol layer was evaporated under vacuum. This was considered the crude saponin. The extracts were stored at -20°C until LC-MS/MS analysis. Crude Saponin (mg·g^-1) = A-B/S; A [flask weight concentrated on n-butanol layer (mg)], B [vacant flask weight (mg)], S [dry weight (g)].

Water Content and Temperature in Soil, Electrical Conductivity (EC), and Weed Occurrence

The soil temperature, soil water content, and electrical conductivity (EC) were measured using reflectometry (WT100N, RF sensor, Co., Seoul, Korea) on three replicates every week for four months after planting. The weed occurrence was investigated exclusively for weeds over 3 cm high with respect to area (m²).

LC-MS/MS Analysis

Liquid chromatography-mass spectrometry / mass spectrometry was performed by coupling a HPLC system (Agilent 1100, Agilent Technologies, CA, USA) to a Qtrap mass spectrometer (Qtrap, AB Sciex CO, CA, USA) equipped with an electrospray ionization (ESI) source. LC separation was performed on a YMC-Pack Pro C₁₈ RS Column (150 × 2.0 mm × I.D., 5 μm). The mobile phase consisted of H₂O (0.1% formic acid) and acetonitrile (60:40, v/v) delivered at a flow rate of 0.2 mL·min^-1. The column temperature was maintained at 40°C, and the injection volume was 10 μL (Ichikawa et al., 2009). Ginsenoside Rb1 was used as the internal standard for quantitative analysis of lancemasides (Fig. 1). The mass spectrometer was operated under negative ion and selected ion monitoring (SIM) modes. ESI was conducted using a spray voltage of 4.5 kV. The capillary voltage and the tube lens offset were fixed at -40 and -130 V, respectively. The heated capillary temperature was fixed at 400°C. The nitrogen sheath and the auxiliary gas were set to 35 and 5 arbitrary units, respectively.

Fig. 1.

LC-MS/MS base peak chromatograms of lancemaside A, B, D obtained from extract of Codonopsis lanceolata roots in the negative-ion mode. Ginsenoside Rb1 (1,000 ng·mL^-1) was used as the internal standard.

Statistical Analysis

The data were analyzed using one-way analysis of variance (ANOVA) by the SPSS Statistics 21 program. Statistical assessments of the differences between mean values were compared using Duncan’s multiple range test (DMRT) at p < 0.05.

Results and Discussion

The Influence of Various Mulching Materials on Physical and Chemical Soil Properties

This study examined how biodegradable film, non-woven fabric, and rice husks control weed growth compared to the handweeding and non-treatment controls. It was observed that the soil pH was about 7.1-7.4, and there was no significant difference before and after the different treatments (Tables 1 and 2). In addition, although the content of P2O5 has no significance with regards to treatments, its content was highest in the biodegradable film and non-woven fabric treatments. Yoon et al. (2000) reported that the phosphoric acid content increased in soil cultivated with blank and transparent film mulching for Angelica gigias. This result shows the physicochemical components in the soil are influenced by mulching treatment. The contents of Ca²⁺ among other cations in the soil were higher in all treatments compared to K⁺ and Mg²⁺, although there was a slight difference among the treatments. The total nitrogen content in the soil was higher in the biodegradable film and non- woven fabric treatments compared to the other treatments (Table 2). Specifically, the non-woven fabric treatment had the highest amount of both NH4 and NO3. In addition, the content of NO3 was higher in the biodegradable film compared to the other treatments. Generally, NO3 nitrogen inflow into groundwater occurs by eluviation or is lost into the atmosphere by denitrification (Fenn and Hossnet, 1985). However, in this study, it seems there was no occurrence of eluviation and denitrification in the mulching treatments. While the content of NH4 was higher in the non-woven fabric than in the biodegradable film, it seems that the biodegradable film treatment accelerated the denitrification of NH4, as to be greatly maintained in soil temperature and soil temperature than other treatments. However, the non-woven fabric treatment did not inhibit the denitrification of NH4 as to be relatively maintained appropriate for soil temperature and soil water compared to the biodegradable film.

Table 1. Soil physical and chemical properties of treatments before and after application of mulching material during Codonopsis lanceolata cultivation

zDuncan's multiple range test at the p < 0.05.

Table 2. Growth characteristics of Codonopsis lanceolata by mulching material

zDuncan's multiple range test at the p < 0.05.

We investigated the seasonal change of soil temperature, soil water content, and EC during the mulching treatments. Both soil temperature and soil water content were higher in the biodegradable film than in the other treatments (Fig. 2). This result complies with previous reports where soil moisture and temperature was higher in vinyl mulching cultivation than in non-mulching cultivation for Chinese cabbage and garlic (Yun et al., 2011; Lee et al., 2015). Electrical conductivity (EC) of the soil was higher in the biodegradable film treatment than in the other treatments. In addition, EC has been used as an indirect indicator to identify salt content in the soil. EC patterns have indicated salts move from deeper soil layers to the surface soil, promoted by rising capillary fringe water as well as the soil temperature (Lee et al., 2007). In this study, it seems the soil EC was increased by salt concentration in the surface soil, rising soil temperature, and water content by mulching with biodegradable film. This result corresponds with reports from Lee et al. (1985), which demonstrated EC concentration was higher in vinyl mulching than in non-mulching during sweet corn cultivation.

Fig. 2.

Change of soil temperature, soil moisture content, and electrical conductivity with respect to mulching materials in Codonopsis lanceolata field. NF (Non-woven fabric), RH (Rice husk), BF (Biodegradable film), HW(Hand weeding), Cont (Control, non-treatment). Vertical bars represent standard error of the means (n = 3).

Growth Characteristics by Mulching Treatments

For the growth characteristics of C. lanceolata, plant height and stem dimeter were best in the biodegradable film and non- woven fabric film treatments (Table 3). Lee et al. (2015) reported that the best growth in mulching treatment was biodegradable film during garlic cultivation. It seems it was caused by the higher soil temperature, water content, and eluviation inhibition sponsored by the biodegradable film and non-woven fabric film treatments. Root fresh and dry weight were superior in the biodegradable film and non-woven fabric compared to the other treatments (Table 3). This result seems to suggest the increase of root fresh and dry weight was maintained by extended duration of soil water content from the biodegradable film and non-woven fabric treatments. Some research has reported a positive correlation between soil water content and yield and growth in ginseng cultivation (Mog et al., 1981; Lee et al., 2007).

Table 3. Characteristics of Codonopsis lanceolata root by mulching material

zDuncan's multiple range test at the p < 0.05.

Saponin Content of C. lanceolata Roots by Mulching Materials

Although the content of crude saponin was higher in the non-woven fabric treatment than in the biodegradable film treatment, there were no significant differences among the treatments except for the non-treatments, which had the lowest saponin content (Table 3). In addition, while there were no differences among treatments in lancemaside A content excluding the non-treatment, Lancemaside A content was highest in the biodegradable film and non-woven fabric treatments. The content of lancemaside B and D was the same among the treatments and had a relatively lower content than the lancemaside A content (Fig. 3). A similar result was reported where the saponin content was the highest in non-woven fabric compared to other various mulching materials during ginseng cultivation (Kim et al., 2010). Therefore, it appears that saponin content can be increased with mulching treatment during C. lanceolata cultivation.

Fig. 3.

Lancemasides content in Codonopsis lanceolata root by mulching materials. NF (Non-woven fabric), RH (Rice husk), BF (Biodegrable film), HW (Hand weeding), Cont. (Control, non-treatment). Vertical bars represent standard error of the means (n = 3). Different letters are significantly different at p < 0.05 by Duncan’s multiple range test (DMRT).

The dominant weeds that appeared in the no-mulching treatment were Digitaria ciliaris, Equisetum arvense, and Artemisia princeps. They seem to disrupt the growth of C. lanceolata by nutrient competition. In addition, it seems that the difference in lancemasides content between the hand-weeding and non-treatment controls is derived from stress induced by hand weeding, such as cutting the fine roots and soil inversion.

Fig. 4.

Occurrence of weeds with respect to mulching materials during Codonopsis lanceolata cultivation NF (Non-woven fabric), RH (Rice husk), BF (Biodegrable film), HW (Hand weeding), Cont. (Control, non-treatment). Vertical bars represent standard error of the means (n = 3). Different letters are significantly different at p < 0.05 by Duncan’s multiple range test (DMRT).

Mulching treatments with non-woven fabric and biodegradable film were confirmed to inhibit the occurrence of weeds (Fig. 4). Nevertheless, there were no significant differences in the weed occurrence between biodegradable film and rice husk treatments. Weeds may have been able to grow in the biodegradable film treatment as it is thin and easily tears during cultivation. Rice husk was not a suitable mulching material compared to the biodegradable and the non-woven films due to its poor growth performance and difficult operation. In addition, the biodegradable film and non-woven fabric film treatments were the best treatments for growth and crude saponin content, and they effectively inhibited the occurrence of weeds. However, the non-woven fabric film and the biodegradable film have trouble during mulching operation and can easily tear, respectively. Therefore, future work should remedy these defects and generate better non-woven fabric film and biodegradable film mulching materials.