The Effect of P₂O₅ Fertilizer, Zeolite, and Volcanic Soil Media from Different Altitudes on the Soil Mineral, Growth, Yield, and Asiaticoside Content of Centella asiatica L.

Abstract

Centella asiatica is an herbal plant with many health benefits due to the content of asiaticoside compounds. Factors affecting asiaticoside content are altitude, soil texture, and soil nutrient status. This research aimed to identify the effect of zeolite, P₂O₅ fertilizer, and soil media from different altitudes on C. asiatica. The research was conducted in a greenhouse from August 2017–June 2018. The experimental design was a factorial, completely randomized design with three factors and four replications. The first factor was soil media that originated from 100, 450, and 900 m above sea level (asl), the second factor was the dose of P₂O₅ fertilizer (0, 27, 54, and 81 kg ha⁻¹), and the third was the dose of zeolite (0, 3, and 6 t ha⁻¹). The results showed that applying zeolite minerals at all altitudes increased nutrient availability and soil cation exchange capacity (CEC) by up to 70%. The novelty of this study is that the soil from an altitude of 900 m asl, with a P₂O₅ fertilizer dose of 54 kg ha⁻¹, has a loamy sand soil texture and produces the highest asiaticoside content (3.61%) and the largest plant dry weight (19.24 g). These results did not significantly differ from those obtained from the soil 450 m asl with a sandy loam soil texture (the most suitable soil texture for C. asiatica), that is 3.37% asiaticoside and 19.87 g plant dry weight. This study concluded that C. asiatica could develop in loamy sand soil by giving it 54 kg ha⁻¹ P₂O₅ fertilizer.

1. Introduction

Centella asiatica is one of the important herbal plants developed in the Yogyakarta Special Region of Indonesia. C. asiatica contains the active ingredients asiaticoside, madecassoside, brahmoside, meso-inositol, centelloside, carotenoids, tannins, and mineral salts such as potassium (K), sodium (Na), magnesium (Mg), calcium (Ca), and iron (Fe) [1]. Asiaticoside functions as a free radical scavenger and revitalizes blood vessels [2]; functions as an antidepressant, antifungal, antioxidant, and anti-inflammatory [3]; repairs skin cells and stimulates the growth of nails, hair, and connective tissue [4]; and has a hepatoprotective effect on liver damage [5].

C. asiatica is a stoloniferous annual herb that is widely grown in several tropical countries. This herbal plant grows on slightly moist soil and gets sunlight in places such as in meadows, edges of ditches, and rice fields [6]. The production of C. asiatica was measured by the quantity of biomass and the quality of the raw materials produced. The quality of C. asiatica is determined by the content of active ingredients and the content of harmful heavy metals. The content of active ingredients is influenced by plant genetic factors, growing conditions, climatic variations, harvest age, and processed plant parts [7]. The C. asiatica leaves are rich sources of nutrients such as carbohydrates, crude fiber, ash, and proteins [8]. The results of Kunle et al. [9] showed that asiaticoside contains a lot of phosphate (P), carbon (C), and K. The NPK-Organo-Zeolite (NPKOZs) treatments enhance the K content of the C. asiatica leaves [10]. This indicates the potential to increase the yield and content of asiaticoside through the provision of nutrients for optimal plant growth.

Zeolite is a chemical compound that has potencies to be used for agriculture, e.g., increases fertilization efficiency, land quality, and agricultural yields [11]. The application of zeolite increases the availability of K, Ca, Mg, Na, and P, enhances the soil cation exchange capacity (CEC), prevents the leaching of fertilizers, and promotes the nutrient uptake of P in the plants [12,13]. Moreover, zeolite has three main functions for agricultural purposes: high water-holding capacity in the free channels both in soil and pots, high CEC, and high adsorption capacity [14]. Furthermore, Sepaskhah and Yousefi [15] described that the application of zeolite on the texture of sandy loams and dusty loams inhibited the conversion of NH₄⁺ to NO₃⁻ by 30–40%. Zeolite mixed with 100–299 kg ha⁻¹ urea fertilizer reduced the N loss by 19–20% and 19–22% through leaching and volatilization, respectively. Zeolite application at a rate of 6 t ha⁻¹ in Ukrainian sandy soils boosted the yields of potatoes, barley, and clover [12]. Zeolite is abundantly available in the hilly area of Batur Agung, Gedangsari, and Ngawen, Gunungkidul Regency, Yogyakarta Special Region. The number of zeolite reserves for the entire area is recorded at about 55,000,000 m³ [16].

Meanwhile, in Yogyakarta, there is an active volcano, Merapi, which erupts frequently. One of the largest eruptions was estimated to be 150 × 106 m³ of eruptive materials in 2010 [17]. The volcanic material of Merapi, which spread over various elevations, has weathered and developed into soil rich in nutrient minerals such as andisols and inceptisols. The mineralogical composition of Merapi lavas consisted of feldspars and pyroxene minerals which are easily weathered and have a high potential as nutrient reserves in the soil [18]. The feldspar mineral contains large amounts of PO₄³⁺, Na⁺, Ca²⁺, and K⁺ [19]. In addition, K-feldspar and Mica minerals are sources of K, and they also contain other minerals, such as Mg, Fe, Ca, Na, and Si, and some micronutrients, such as Fe, copper (Cu), manganese (Mn), and zinc (Zn), as a follow-up to silicate minerals [20]. Volcanic ash from Merapi were composed predominantly of silica (>50%) and smaller amounts of the aluminum oxide (Al₂O₃), Fe, Ca, and Na [21]. However, the utilization of eruptive materials, i.e., mineral resources from rock or soil that develops from the Merapi volcanic ash, has not been widely used as a source of nutrients for herbal plants. To our knowledge, C. asiatica are generally collected from the wild and there is still no serious effort to cultivate C. asiatica in Indonesia in response to high market demand. Moreover, there is still a lack of research that focus on the soil management of sandy soil at different altitudes. Hence, this study was undertaken to determine the effects of zeolite minerals, P₂O₅ fertilizer, and volcanic soil media from different altitudes on the growth performance, yield, and asiaticoside content of C. asiatica.

2. Materials and Methods

2.1. Study and Soil Collection Site

The research was conducted in the greenhouse of the agriculture faculty of Gadjah Mada University during August 2017–June 2018. Determination of the location for taking volcanic soil media on the southern slopes of Merapi was achieved used the semidetailed soil map based on the results of the second land resource evaluation and planning project (LREP II) survey. The Sleman district was presented on a scale of 1:50,000 and combined (overlapping) with topographic maps and maps of the earth to check land use and verify soil types in the studied locations (Figure 1). The soil media to cultivate the C. asiatica plants in experimental pots was collected from three places with different elevations, namely: Hargobinangun 900 m above sea level (asl), Wukirsari 450 m asl, and Kalitirto 100 m asl.

2.2. Experimental Design

This study used a factorial completely randomized design (FCRD) with 3 factors so that 36 treatment combinations were obtained (3 × 4 × 3 treatments) and were repeated 4 times. This research used P₂O₅ fertilizer and zeolite minerals in order to obtain the optimal P and K nutrient uptake for increasing the asiaticoside in C. asiatica plants. The soils were developed from volcanic ash, such as inceptisols, which are andic and can bind very strongly with P, K, and several other microelements in clay-silicate adsorption complexes, even though the P potential is very high, making it unavailable to plants. Provision of P fertilizer in the form of P₂O₅ to increase the P available to plants. Generally, a P fertilizer dose of more than 27 kg ha⁻¹ is applied to soils containing allophane compounds with pH NaF > 10.0 [23]. The characteristics of zeolite consisted of 65% SiO₂, 13.27% of Al₂O₃, 2.78% of Fe₂O₃, 3.65% of CaO, 1.54% of MgO, 1,83% of K₂O, 1.36% of NaO, 8.49% of moisture content, and 117,53 meq 100 g⁻¹ [24]. Zeolite can bind P strongly to the clay-silicate adsorption complex. Generally, the dose of zeolite for biopharmaceutical plants on andosols or inceptisols, which are andic, is 3 t ha⁻¹ to increase CEC and reduce P retention [25]. The factors applied in the pot experiment were:

• Factor 1: volcanic soil media from 3 different locations on the hilly of Merapi:
  • A1: Inceptisol from Kalitirto (representing an altitude of 100 m asl).
  • A2: Inceptisol from Wukirsari (representing an altitude of 450 m asl).
  • A3: Inceptisol from Hargobinangun (representing an altitude of 900 m asl).
• Factor 2: The dose of inorganic P₂O₅ fertilizer with 4 levels:
  • P0: P₂O₅ fertilizer with a dose of 0 kg ha⁻¹.
  • P1: P₂O₅ fertilizer with a dose of 27 kg ha⁻¹ or the equivalent of 0.15 g 10 kg⁻¹ of soil.
  • P2: P₂O₅ fertilizer with a dose of 54 kg ha⁻¹ or the equivalent of 0.30 g 10 kg⁻¹ of soil.
  • P3: P₂O₅ fertilizer with a dose of 81 kg ha⁻¹ or the equivalent of 0.45 g 10 kg⁻¹ of soil.
• Factor 3: The dose of zeolite mineral application with 3 levels:
  • Z0: Without zeolite (0 t ha⁻¹ of zeolite).
  • Z3: Zeolite mineral application 3 t ha⁻¹ or equivalent to 6 g 10 kg⁻¹ of soil.
  • Z6: Zeolite mineral application 6 t ha⁻¹ or equivalent to 12 g 10 kg⁻¹ of soil.

All treatments were given 150 kg ha⁻¹ KCl (equivalent to 0.30 g 10 kg⁻¹ of soil). C. asiatica was fertilized with one-third part of urea at a dose of 300 kg ha⁻¹ (equivalent to 0.60 g 10 kg⁻¹ soil) when plants reached 1, 2, and 3 months after planting. Each experimental pot was filled by 10 kg of air-dried soil. After the volcanic soil media was put into the pot, then SP-36 inorganic fertilizer was added according to the treatment and added 0.30 g of KCl fertilizer (equivalent to 150 kg ha⁻¹) and 0.30 g of urea (equivalent to 150 kg ha⁻¹) then mixed until homogeneous. The C. asiatica plant material was already planted in stolon and accompanied by at least 2 budding candidates. The plant material comes from parents who are at least a year old. Propagation was achieved through the stolons (vegetative), which were planted in advance for 2–4 weeks. Each pot contained one clump. Plant maintenance during the pot experiment (greenhouse research) included replanting, watering, weeding, and controlling pests and diseases by spraying biological pesticides. Embroidery was carried out 5–7 days after planting (DAP) using the same age of plant material. Watering was carried out regularly at field capacity conditions. Harvesting was conducted when the plant reached 4 months old (16 weeks) by cutting the top of the plant.

2.3. The Mineralogy and Soil Analysis

A preliminary observation was conducted by soil mineralogical analysis using thin slices of coarse sand particles and observed under a polarizing microscope method [26]. Analysis of the physical and chemical properties of the soil were carried out before the experiment to determine the level of soil fertility and after harvested C. asiatica plants, which included: soil texture (pipette method), particle and bulk density (soil sample ring method) [27], C-organic content (Walkley and Black method), N-total (Kjeldahl method), P available (Olsen method), K available (based on saturation of NH₄OAc-pH 7.0 method), potential P and K (15% HCl extraction method), CEC (based on saturation of NH₄OAc-pH 7.0 method), soil pH (ratio 1:5), pH NaF (Blakemore method, ratio 1:50), Ca and Mg (based on saturation of NH₄OAc-pH 7.0 method), and P and K uptake (washing with H₂SO₄ method) [28]. In terms of agronomic parameter aspects, there were the number of leaves, the number of stolons, the root length, the weight of wet plant, the dry weight of plant, and asiaticoside content on the plant leaves, which were analyzed by using the HPCL method [29].

2.4. Statistical Analysis

The experimental data were analyzed by analysis of variance (ANOVA) using SAS Program, version 9.0 and followed the agricultural data analyzing guidance [30]. When a significant difference between treatments was detected, the analysis continued with Duncan’s multiple-range test (DMRT). Correlation and nonlinear regression methods were used to determine the close relationship (correlation) between the plant-growth components of C. asiatica and the soil P and K nutrient uptake on the increase of asiaticoside.

3. Results

3.1. Characteristic of Soil Minerals and Soil Properties of Volcanic Soil Media

The soil type in study area was classified as inceptisols, which has distinctive and very complex properties depending on the level of its development intensity. This soil is relatively young age but is more developed than the soil type of entisol and has shown a significant horizon formation. The soil texture of the Hargobinangun, is dominated by sand by around 86.20% (classified as loamy sand). Sand and clay of the Wukirsari range from 58.94 to 67.44% and 7.87 to 12.10%, respectively. Then, the texture class of the Wukirsari is classified as sandy loam. The soil of Kalitirto is lower in the sand particle content, with the sand and clay content ranging from 60.03 to 75.90% and 5.25 to 9.93%, respectively. The particle density in the three locations was in the range from 2.42 to 2.56 g cm⁻³ (low-medium), while bulk density ranged from 0.93 to 1.25 g cm⁻³ (low-medium) (

Table 1. Physical soil properties of volcanic soil media.

Altitude (m asl)	Soil Particles			Soil Texture Class	PD	BD
					(g cm−3)
	Clay (%)	Silt (%)	Sand (%)
100	9.72 ± 0.58	25.38 ± 1.54	64.90 ± 0.89	Sandy loam	2.56 ± 0.08	1.25 ± 0.04
450	12.10 ± 1.2	28.97 ± 1.52	58.93 ± 1.50	Sandy loam	2.42 ± 0.02	0.95 ± 0.06
900	2.51 ± 0.08	11.29 ± 0.68	86.20 ± 0.61	Loamy sand	2.51 ± 0.02	0.93 ± 0.08

Noted: PD = Particle Density; BD = Bulk Density.

).

The pH (H₂O) value at the altitude 900 m asl was 5.18 (acid condition), while at 450 m asl and 100 m asl were 6.12 and 6.34 (slightly acid). The pH NaF value at the altitude 900 m asl obtained the highest, which was 11.48, then it decreased to 9.43 at the altitude 100 m asl. The pH NaF indicates that the soil sample has a pH NaF value > 10.0, meaning the soil still has andic properties and the allophane content is quite high. The highest organic matter content in the study area was found at 900 m asl, which reached 3.16%, while the lowest was found at 100 m asl, which was approximately 1.15% (

Table 2. The chemical properties of volcanic soil media before treatments application.

Altitude (m asl)	Soil Depth (cm)	Soil pH			C-Org.	Base Cation		N Total	C/N	K Avail.	P Avail (Olsen)	Pot. P	CEC
		H2O	KCl	NaF		Ca	Mg	%		cmol kg−1	µg g−1	mg kg−1	cmol kg−1
					%	cmol kg−1
100	0–21	6.34 (SA)	5.88 (N)	9.43	1.15 (L)	11.46 (H)	2.83 (H)	0.14 (L)	8.21 (L)	0.95 (H)	35.85 (H)	92.06 (VH)	10.94 (L)
450	0–24	6.12 (SA)	5.63 (N)	10.89	2.52 (M)	8.06 (M)	1.79 (M)	0.19 (L)	13.26 (H)	0.26 (L)	26.42 (M)	185.84 (VH)	15.45 (L)
900	0–22	5.18 (A)	5.43 (N)	11.48	3.16 (H)	2.76 (L)	0.49 (L)	0.25 (M)	12.64 (M)	0.34 (L)	16.06 (L)	272.93 (VH)	22.43 (M)

Noted: SA = Slightly Acid; A = Acid; V = Very High; H = High; M = Medium; L = Low.

). The low levels of organic matter at the 100 m asl altitude due to intensive management of the soil that developed from volcanic ash (used as paddy field) resulted in faster decomposition of organic matter in each growing season.

The content of alkaline elements in the tillage layer; especially Ca and Mg at 100 m asl was classified as medium-high. Ca and Mg at the 100 m asl reached 11.46 cmol kg⁻¹ and 2.83 cmol kg⁻¹, respectively. The content of Ca was 8.06 cmol kg⁻¹ and Mg was 1.79 cmol kg⁻¹ at the at 450 m asl, while the lowest levels of Ca and Mg were found at 900 m asl, which were around 2.76 cmol kg⁻¹ and 0.49 cmol kg⁻¹, respectively. This indicates that either the volcanic ash soil at 900 m asl has not further developed or the elements have not much been decomposed.

The P potential at the altitude 900 m asl reached 272.93 mg kg⁻¹ (very high). P and K available were the lowest ones, and were 16.06 µg g⁻¹ and 0.34 cmol kg⁻¹, respectively. On the other hand, at an altitude of 100 m asl, the P potential obtained the lowest value, which was 92.06 mg kg⁻¹, while the P and K available reached 35.85 µg g⁻¹ and 0.95 cmol kg⁻¹ (high), respectively.

The CEC value at 100 and 900 m asl were included as medium level. The CEC at the 100 m asl was 10.94 cmol kg⁻¹, while at 900 m asl, CEC was higher (22.43 cmol kg⁻¹). This is most likely because at the altitude 900 m asl has a higher organic matter content than at the altitude 450 m asl, although the content of clay particles in the upper horizon is low (<15%). Carbon content in the soil reflects the organic matter content in the soil, which is an important benchmark in soil management.

Figure 2, Figure 3 and Figure 4 present the sand and clay fractions analysis by the thin slice of loose material method, measuring with a microscope electron 0.05–1.8 mm. The sample of the sand fraction is indicated as a primary mineral from volcanic material which is characterized by the amount of weathered mineral reserves (lithic andesite, plagioclase, and hornblende). The number of mineral reserves ranges from 80–85%, especially at altitudes of 450 m asl and 900 m asl. These minerals are very rich in sufficient nutrients available in a long term, while resistant primary minerals, such as opaque, is still relatively low (9–12%). The sand fraction samples from 100 m asl, 450 m asl, and 900 m asl contain 4, 5, and 6 types of primary minerals, respectively.

The mineralogy analysis showed that the volcanic ash soil at the altitude 900 m asl is dominated by primary minerals lithic andesite (50%), plagioclase (16%), hornblende (19%), pyroxene (6%), opaque minerals (9%), and amphiboles, which are rich in alkaline elements (Ca, Mg, and Na) (Figure 5a). However, they are still strongly bound in the amorphous clay mineral adsorption complex. These elements have not been decomposed and are altitude-available for the plants, so the content of alkaline elements at an altitude of 900 m asl is lower compared to 450 m asl and 100 m asl. The dominant primary mineral types at 450 m asl are similar to at 900 m asl, namely lithic andesite (40%), plagioclase (25%), hornblende (15%), and pyroxene (10%), which are rich in alkaline and other micronutrients. The composition of primary minerals at 900 m asl and 450 m asl is more quantity; however, on the lower slopes (altitude 100 m asl), lithic andesite and mica minerals containing sufficient P and K nutrients, respectively, were found, so the availability of these two elements are higher compared to the upper slopes (Figure 5b).

3.2. The Effect of Volcanic Soil Media, P₂O₅ Fertilizer, and Zeolite Minerals on C. asiatica Growth

The provisions of P fertilizers and zeolite minerals are expected to provide nutrients in volcanic soil media but in reality, the available nutrients are limited because they are bound in clay-silica mineral adsorption complexes. The results of the analysis are presented in

Table 3. The recapitulation of analysis of variance on the factorial completely randomized design (FCRD) with 3 factors as an experimental treatment.

Observation Data	Soil Altitude	P2O5 Fertilizer	Zeolite Mineral	A × P	A × Z	P × Z	A × P × Z
The number of leaves	**	**	*	*	ns	ns	ns
Root dry weight	**	**	*	**	ns	ns	ns
Biomass Plant weight	**	**	**	**	ns	ns	ns
The Length of roots	**	**	*	**	ns	ns	ns
Ca2 ± nutrient content	**	**	**	**	ns	ns	ns
Mg2 ± nutrient content	**	*	**	*	ns	ns	ns
P Uptake on plant	**	**	**	**	ns	ns	ns
K Uptake on plant	**	*	**	**	ns	ns	ns
P Available	**	*	ns	ns	ns	ns	ns
K Available	*	**	ns	ns	ns	ns	ns
Plant dry weight	**	**	**	**	ns	ns	ns
Asiaticoside content	**	**	**	**	ns	ns	ns

Noted: * = significant on level of 0.05; ** = significant on level of 0.01; ns = nonsignificant; A = Altitude factor; P = P₂O₅ fertilizer factor; Z = Zeolite mineral factor.

.

Based on the results of the statistical analysis (analysis of variance) in Table 3 for A × Z, P × Z, and A × P × Z, the plant-growth performance is nonsignificant, while the results of A × P are very significantly different. Therefore, it can be compiled in

Table 4. The agronomic parameters on harvesting time (16 weeks DAP).

Treatments Code	The Number of Leaves	The Root Dry Weight (g)	Length of the Roots (cm)	Plant Wet Weight (g)	Plant Dry Weight (g)
A1P0	78.85 ± 1.1 f	2.43 ± 0.18 bc	31.25 ± 1.37 e	85.32 ±1.71 f	12.43 ± 0.95 e
A1P1	79.14 ± 0.85 f	2.38± 0.1 bc	36.73 ± 0.95 b	90.67 ± 0.67 f	12.16 ± 1.44 e
A1P2	83.42 ± 0.68 ef	2.26 ± 0.13 bc	36.07 ± 0.39 bc	99.47 ± 0.73 ef	15.23 ± 1.13 c
A1P3	89.73 ± 1.04 de	2.5 ± 0.12 b	36.17 ± 0.28 bc	117.52 ± 0.87 d	14.82 ± 0.99 c
A2P0	79.53 ± 0.91 f	2.46 ± 0.14 bc	35.44 ± 0.26 bc	135.64 ± 1.56 c	15.97 ± 0.94 bc
A2P1	78.11 ± 0.77 f	2.86 ± 0.09 ab	37.25 ± 0.86 ab	165.07 ± 1.06 b	16.94 ± 0.84 b
A2P2	95.23 ± 1.52 d	3.47 ± 0.17 a	38.72 ± 1.02 a	170.68 ± 1.31 a	19.87 ± 0.86 a
A2P3	90.55 ± 0.91 de	3.28 ± 0.08 a	34.03 ± 1.0 c	174.16 ± 1.36 a	16.64 ± 1.01 c
A3P0	113.2 ± 1.76 b	2.53 ± 0.06 b	31.57 ± 0.84 de	110.96 ± 1.46 de	13.86 ± 0.56 d
A3P1	104.11 ± 3.27 c	2.62 ± 0.1 b	36.48 ± 0.72 b	113.64 ± 0.95 d	13.04 ± 1.49 de
A3P2	120.88 ± 1.09 a	3.10 ± 0.12 a	38.58 ± 0.97 a	168.76 ± 1.47 ab	19.24 ± 1.16 a
A3P3	110.44 ± 1.58 bc	2.02 ± 0.13 c	34.18 ± 1.29 c	143.75 ± 0.96 bc	15.58 ± 0.03 c
CV (%)	10.12	9.65	9.72	10.68	10.72

Notes: The numbers in the same column followed by the same letters indicate there is no significant difference by DMRT at 5% significance level.

as follows.

3.3. The P and K Nutrient Uptake of C. asiatica

Plant growth and development largely depend on the combination and concentration of mineral nutrients available in the soil. Plants often face significant challenges in obtaining an adequate supply of these nutrients to meet the demands of basic cellular processes due to their relative immobility. Nutrient uptake refers to the process of nutrient movement from an external environment into a plant. The value of the Ca and Mg content, as well as the P and K nutrient uptake of C. asiatica, are presented in

Table 5. The amount of Ca²⁺ and Mg² content, P and K uptake, P available, and asiaticoside content.

Treatments Code	Ca2+ Nutrient	Mg2 Nutrient	P Nutrient Uptake	K Nutrient Uptake	P nutrient Available	Asiaticoside Content
	(cmol kg−1)	(cmol kg−1)	(g·tan−1)	(g·tan−1)	(µg g−1)	(% Dry Weight)
A1P0	9.48 ± 0.12 c	2.52 ± 0.06 d	0.387 ± 0.05 f	0.469 ± 0.05 d	29.62 ± 0.84 d	1.63 ±0.10 e
A1P1	9.93 ± 0.48 c	2.47 ± 0.07 d	0.405 ± 0.03 e	0.482 ± 0.09 c	31.57 ± 0.64 d	1.56 ± 0.06 e
A1P2	10.78 ± 0.15 d	2.86 ± 0.12 d	0.551 ± 0.07 cd	0.698 ± 0.07 a	37.93 ± 0.48 e	1.89 ± 0.10 de
A1P3	10.65 ± 0.09 d	2.91 ± 0.07 d	0.514 ± 0.04 d	0.607 ± 0.06 ab	38.53 ± 0.88 e	1.99 ± 0.12 d
A2P0	7.29 ± 0.13 b	1.66 ± 0.07 b	0.488 ± 0.04 de	0.541 ± 0.06 bc	21.36 ± 0.77 c	2.04 ± 0.13 bc
A2P1	7.48 ± 0.12 b	1.53 ± 0.08 a	0.578 ± 0.03 c	0.577 ± 0.07 b	24.38 ± 0.93 cd	2.68 ± 0.12 c
A2P2	8.15 ± 0.1 bc	1.79 ± 0.2 c	0.793 ± 0.08 a	0.572 ± 0.09 b	28.45 ± 0.85 d	3.37 ± 0.07 ab
A2P3	7.94 ± 0.37 b	1.73 ± 0.1 bc	0.692 ± 0.05 b	0.614 ± 0.06 ab	30.18 ± 0.90 d	3.14 ± 0.13 b
A3P0	2.57 ± 0.15 a	0.49 ± 0.06 a	0.419 ± 0.04 e	0.489 ± 0.04 c	10.74 ± 0.57 a	2.03 ± 0.17 d
A3P1	2.29 ± 0.09 a	0.46 ± 0.04 a	0.518 ± 0.07 d	0.546 ± 0.09 bc	13.81 ± 1.35 ab	2.85 ± 0.09 c
A3P2	2.83 ± 0.07 a	0.59 ± 0.05 ab	0.702 ± 0.05 b	0.590 ± 0.04 b	16.67 ± 0.69 b	3.61 ± 0.18 a
A3P3	2.72 ± 0.09 a	0.53 ± 0.07 a	0.687 ± 0.04 b	0.505 ± 0.03 bc	17.92 ± 0.49 b	3.36 ± 0.09 ab
CV (%)	11.47	10.32	9.98	11.24	10.73	11.43

Notes: The numbers in the same column followed by the same letters indicate there is no significant difference by the DMRT at 5% significance level.

as follows.

The Ca²⁺ and Mg²⁺ nutrient content in the soil after harvesting at the altitude of 100 m asl was still high, especially with the application of 54 kg ha⁻¹ P₂O₅ inorganic fertilizer and was significantly different from the soil at altitudes of 450 m asl and 900 m asl (Table 5). This is because weathering on the lower slopes is more intensive than on the upper slopes, so the availability of exchangeable base elements is relatively higher. Moreover, at the altitude of 900 m asl, the Ca²⁺ and Mg²⁺ elements are still strongly bound to the clay-silica adsorption in the amorphous clay mineral complex. Finally, the availability of their nutrients becomes lower in the soil.

The combination treatment dose of P₂O₅ fertilizer and volcanic soil as planting media had a positive effect on increasing P and K nutrient uptake, while N uptake had no significant effect. Nutrient uptake is the amount of nutrients that enter the plant tissue. This can be calculated based on the results of an elemental analysis in plant tissue multiplied by the dry weight of the plant.

3.4. Asiaticoside Content of C. asiatica

The combination dose of P₂O₅ fertilizer and volcanic soil media from different altitudes affected the dry weight of C. asiatica plants at 16 weeks. The 54 kg ha⁻¹ of SP-36 application gave the highest yield at 450 m asl and 900 m asl (Table 5), and it was significantly different from 100 m asl. The increase in dry weight of up to 24.42% and 38.84% was achieved using soil media from 450 m asl and 900 m asl, respectively.

The increased dose of P fertilizer in soil from 100 m asl indicated a significant difference at a dose from 54 to 81 kg ha⁻¹. On the other hand, the use of soil media from 450 m and 900 m asl at the dose of 54 kg ha⁻¹ P₂O₅ fertilizer gave a significant difference from the dose of 27 kg ha⁻¹. This indicates that the total of P content in the soil from 100 m asl is relatively lower, thus requiring a higher dose of P₂O₅ fertilization than the soil from the 450 m and 900 m asl locations.

The availability of P in the soil and the provision of P through the application of P fertilizer greatly affect the increase in the active ingredient asiaticoside. The results of the analysis of the average content of asiaticoside at maximum vegetative growth is presented in

Table 6. Effect of zeolite minerals on plant dry weight, P, K uptake, Ca²⁺ and Mg²⁺ nutrients, and the asiaticoside content of C. asiatica.

Dose of Zeolite (t ha−1)	Plant Dry Weight (g)	Nutrients Uptake (g plant−1)		Ca 2+ Mg 2+ Nutrient Content (cmol kg−1)		Asiaticoside Content (%)
		P	K
0.0	13.49 b	0.516 b	0.622 c	5.48 b	1.43 b	1.15 b
3.0	16.72 a	0.823 a	0.968 a	8.64 a	2.37 a	3.28 a
6.0	16.47 a	0.804 a	0.795 b	8.29 a	2.58 a	3.32 a

Notes: The numbers in the same column followed by the same letters indicate there is no significant difference in the DMRT on 5% significance level.

The highest increase in asiaticoside was obtained with the application of 54 kg ha⁻¹ P₂O₅ fertilizer in soil media from Hargomulyo (900 m asl). However, this value was not significantly different from Wukirsari location (400 m asl) with the same dose of P fertilizer, while at Kalitirto location (100 m asl) the asiaticoside content was lower.

Increasing the dose of P₂O₅ fertilizer in volcanic soil media from 100 m asl significantly increased the content of asiaticoside of C. asiatica. While soil media from 450 m asl had a significant effect up to a dose of 54 kg ha⁻¹, increasing the dose above that was not significantly different. Meanwhile, for the soil media that originated from 900 m asl, increasing the dose of fertilizer up to 27 kg ha⁻¹ had a significant effect compared to no P₂O₅ fertilizer. When given P₂O₅ fertilizer up to 81 kg ha⁻¹, the asiaticoside content would be a decreased. This indicates that the volcanic soil media from 900 m asl has not responded to the increase in P dose fertilizer, because the potential P nutrient content is already quite enough in the soil colloid (Figure 6).

3.5. The Dry Weight of Plant and Asiaticoside Content of C. asiatica

The application of zeolite minerals and P₂O₅ fertilizer as well as the use of volcanic soil media as planting media had no significant interaction with the parameters of nutrient uptake P, K, plant dry weight, and asiaticoside content (Table 5). The single factor for the effect of zeolite mineral dose on several observed parameters is presented in Table 6.

The application of zeolite minerals at the level of 6 t ha⁻¹ to the content of asiaticoside of C. asiatica gave the highest yield of 3.32 % and was significantly different from the treatment without the application of zeolite (0 kg ha⁻¹). However, the increasing content of active ingredients was not significantly different with the application of zeolite minerals at a dose of 3 t ha⁻¹. Therefore, based on the results of this study, the recommended optimal dose of zeolite minerals for further research on volcanic soil media, which was dominated by sand particles, is a dose of 3 t ha⁻¹. This is based on considerations of efficiency of the farming system on giving zeolite minerals to C. asiatica plants.

3.6. The Correlation of Plant-Growth Components and Nutrient Uptake of the Asiaticoside Content

The characteristics of the growth and yield components greatly affect the content of the active ingredients of C. asiatica plants. Parameters including the number of leaves, plant dry weight, and nutrient uptake were analyzed. Nutrient uptake, which had a positive correlation with the levels of asiaticoside content at each location, had different values, as presented in

Table 7. Correlation of dry weight, nutrient P and K uptake, and on level of asiaticoside content of C. asiatica.

Variables	Correlation of the Asiaticoside Content on the Different Altitude (r)
	900	450	100
		m asl
The number of leaves	0.516 *	0.497 *	0.435
Plant dry weight (g)	0.779 **	0.505	0.318
P nutrient uptake (kg ha−1)	0.878 **	0.656 *	0.587 *
K nutrient uptake (kg ha−1)	0.238	0.349	0.559 *
C-organic content of plant (%)	0.623 **	0.509 *	0.478 *

Noted: H = Hargobinangun; W = Wukirsari; K = Kalitirto; (*) = significant positive correlation at 5% level; (**) = very significant positive correlation at 5% level.

. The correlation of plant dry weight to the asiaticoside content at the altitude 900 m asl (r = 0.779 **) obtained the highest value, followed by the soil from 450 m asl (r = 0.505) and 100 m asl altitudes (r = 0.318).

4. Discussion

4.1. Environmental Condition

The main obstacle to planting biopharmaceuticals crops of C. asiatica on the slopes of Merapi is that the soil is dominated by sand particles with low organic matter content, and the distribution of nutrient contents is uneven, especially P and K levels. Devkota and Pramod [31] suggested that the growth and yield of C. asiatica is affected by the soil type, soil texture, and the compactness of soil particles. Maximum growth of C. asiatica will be expressed in habitats with sandy loam type soil rather than sandy or clay soil.

The different altitude showed the different nutrient availability of the soil. On the upper slope (900 m asl), the potential P content is very high, while the available P is low. On the middle slope (450 m asl), the potential P content is high and the available P is medium, while on the lower slope (100 m asl), the potential P content is classified as high and the available P is quite high, as is the available K. The different nutrient conditions of the different altitudes of the trial site will give affects to the growth and yield of C. asiatica as well.

The low levels of organic matter shown in the lower slope occur because the environmental temperature condition is higher, so the decomposition process is faster than on the upper slopes. The average temperature at 100 m asl was 31.3 °C during the dry season and 29.1 °C during the rainy season, while the temperatures at 900 m asl during the dry season and rainy season were 25.2 °C and 22.4 °C, respectively [32]. Soil organic matter content in the upper position is higher than on the lower and middle slopes because the lower slope of land used is more intensive than the upper slope of Merapi Mount. This is because the amount of litter on the lower slopes is less than on the upper slope. In addition, the soil density at a 0–25 cm depth in the lower slope (100 m asl) tends to be higher than the upper slope (900 m asl). Therefore, the more intensive soil management has a low organic matter content, causing a higher bulk density along with the lower total porosity and soil permeability value.

4.2. Performance Growth and Productivity of C. asiatica

The dose of P fertilizer and altitude determine the growth and performance of C. asiatica plants. A dose of 81 kg ha−1 at an altitude of 450 m asl gave the highest fresh weight yield, but it was not significantly different than a dose of 54 at an altitude of 900 m asl. Halimi [33] stated that was an indication that the leaves of the plants grown in mid- and highland regions were larger in size with shorter petiole compared to plants in lower regions. The increase in P fertilizer would increase the dry weight of C. asiatica and the content of asiaticoside [34,35]. This indicated that C. asiatica plants grown in the midland and highland were more productive than lowland plants. The leaf is considered as an important organ of the plant in which the vital metabolism process of photosynthesis occurs.

4.3. The effect of Volcanic Soil Media, P₂O₅ fertilizer, and Zeolite Minerals on Nutrient Uptake and Asiaticoside Content

The highest P uptake in this experiment was achieved by volcanic soil media from 450 m asl, while soil media from 900 m asl and 100 m asl had a lower P uptake in the P₂O₅ fertilizer at the dose of 54 kg ha⁻¹. This condition can happen because at 900 m asl the potential P content is very high, but the available P is very low, while at 100 m asl the amount of P potential content is low, so the absorbed P element without additional nutrients from P₂O₅ fertilizer is lower. The results of Dahono et al. [36] showed that by increasing the dose of NPK fertilizer from 0.25 of the recommended standard dosage to 1.0 of the recommended standard, they were able to increase the P and K nutrient uptake by 45.77% and 28,68%, respectively. The high nutrient uptake of these two elements was thought to be due to the addition of 30 t ha⁻¹ of manure, which was able to improve the chemical, physical, and biological properties of the soil and contribute the nutrients of N, P, and K. Moreover, Ghulamahdi et al. [37] stated that planting C. asiatica in the highlands yielded higher levels of active ingredients than planting in the lowlands and increasing the application of P fertilizer could increase the weight of the petiole, leaf tendrils, harvest weight, and asiaticoside content. The highest harvest weight was obtained at the treatment of 72 kg ha⁻¹ P₂O₅, while the highest asiaticoside content was obtained at the treatment of 36 kg ha⁻¹ P₂O₅.

On the soil media from the upper slope location (900 m asl), the P retention from the clay-organic complex is very high compared to the lower slope location (450 m and 100 m asl). Therefore, P nutrient available on the upper slope of Merapi is also very low, and the soil is more dominated by sandy particles. Mulugeta and Sheleme [38] stated that that P retention can reach >80% in young to moderate andic soils. At lower slopes, available P is lower because the decomposition process is faster with increasing temperature. To increase the available P, it can be done by giving fertilizers containing P, organic matter, and mineral ameliorants, which can increase the negative charge exchange capacity.

4.4. Correlation of Plant Growth and Nutrient Uptake of the Asiaticoside Content

The high uptake of P nutrients in the soil from 900 m asl was due to the addition of P fertilizer, which already contains available P nutrient. This is because the soil from 900 m asl has more potential nutrient reserves. With the addition of inorganic fertilizer, which enriched with P nutrient plus zeolite minerals as soil improvement material, at the optimal dose (3 ton/ha), the volcanic soil can effectively absorb soil nutrients, including P element, from amorphous clay adsorption so that it is more efficient. It is also widely absorbed by the roots of C. asiatica. The high uptake of P nutrients in the soil from 900 m asl also affected the absorption of alkaline soil nutrients, especially K nutrients. The results of Sari et al. [39] showed that the addition of phosphorus by as much as 54 kg ha⁻¹ was able to increase the dry weight of C. asiatica leaves by 18.86% higher than without P fertilization. The dry weight of this plant significantly affected the levels of the active ingredients produced by C. asiatica.

Moreover, P fertilization had no significant effect on the number of primary vines, secondars, fresh weight and dried biomass, and tissue P content in C. asiatica [40]. The P fertilizer application generally did not have a significant effect but there was a tendency for an upward trend to increase the yield of P fertilizer doses given to the plants. The tendency is that fertilized plants give higher yields than plants without fertilization. Parameters of the observation were wet weight and dry weight of leaves and petioles or roots and tendrils [41].

P nutrient uptake at 900 m asl gave the highest correlation value (r = 0.878 **) compared to the soil from an altitude of 450 m asl (r = 0.656 *) and an altitude of 100 m asl (r = 0.587 *). This is because the potential P content at 900 m asl is very high and most of it has been bound with mineral zeolite which contains a lot of negative charge in the form of available P elements so that the amount absorbed by the root increases. These results are in accordance with Musyarofah et al. [42], who found that the role of fertilizers in the cultivation of biopharmaceutical crops greatly affects the quality of production; improper application of fertilizers will have an impact on low yields and the content of active ingredients. Various medicinal plants are the target of research through different approaches. The results of this study revealed that chemical fertilizers positively affected the mineral conditions in the soil, plant growth, and increased the active ingredient asiaticoside content in C. asiatica. The utilization of volcanic soil from an altitude of 900 m asl combined with a dose of P₂O₅ fertilizer and zeolite minerals is a reference for C. asiatica plant developers in increasing crop yields and quality of raw materials.

5. Conclusions

Zeolite mineral treatment at all altitudes increased nutrient availability and soil cation exchange capacity (CEC) by up to 70%. This means that zeolite could indeed enhance the chemical properties of sandy soil. The novelty of this study is that the soil from an altitude of 900 m asl and a P₂O₅ fertilizer dose of 54 kg ha⁻¹ has a loamy sand soil texture and produces the highest asiaticoside content (3.61%) and the largest plant dry weight (19.24 g). These results did not significantly differ from those obtained from the soil of 450 m asl with sandy loam soil texture (the most suitable soil texture for C. asiatica), that is, 3.37% asiaticoside and 19.87 g plant dry weight. This study concluded that C. asiatica could develop in loamy sand soil by giving 54 kg ha P₂O₅ fertilizer.