Sesquiterpenoids from the Inflorescence of Ambrosia artemisiifolia

Abstract

The successful invasion of Ambrosia artemisiifolia is largely due to allelopathy. As an invasive alien plant, A. artemisiifolia has spread rapidly in Asia and Europe. Studies have shown that sesquiterpenoids play an important role in plant allelopathy. However, it is unclear whether the inflorescence of A. artemisiifolia also contains allelopathic components. In this paper, our phytochemical research focuses on the inflorescence of A. artemisiifolia. Twenty sesquiterpenoids, including four new ones (1–4) were isolated through successive chromatographic columns and identified by spectroscopic methods. At a concentration of 200 μg/mL, all the compounds tested were evaluated for their allelopathic activities on seedling growth of wheat. Our results indicate that nine compounds inhibited both the root and shoot growth of seedlings. Compounds 14, 15, 17, and 20 significantly inhibited root length, which was more than 50% shorter than the control. This study identified the chemical profile of the sesquiterpenoids occurring in the inflorescence of A. artemisiifolia. The bioactivity screening results provide further understanding of the chemical basis of allelopathy in A. artemisiifolia.

1. Introduction

Ambrosia artemisiifolia L. (Asteraceae), which is native to North America, is an invasive weed widely distributed throughout temperate regions of the world [1]. This invasive plant has the potential to destroy native ecosystems and reduce agricultural yields [2]. Allelopathy describes the chemicals exuded from roots, rhizomes, leaves, stems, and/or litter of an individual and the resultant beneficial or deleterious effects on other organisms and is an important factor in the successful invasion of alien plants [3]. Meanwhile, because allelopathic components generally have strong phytotoxic activities against many weeds, the allelochemicals have the potential to develop new herbicides, which have the advantages of safety, easy degradation, and no resistance in weed control [4]. Previous studies reported that different solvent extracts of A. artemisiifolia had significant inhibitory effects on crop growth and seed germination, weeds, and other plants [5,6]. Studies have shown that sesquiterpenoids are the main chemical components of Ambrosia and play an important role in plant allelopathy [7,8,9,10]. Every year between July and August, clusters of columnar inflorescence from A. artemisiifolia produce large amounts of pollen causing a series of allergic reactions and affecting human health [11]. However, it is unclear whether the inflorescence of A. artemisiifolia also contains allelopathic components. Therefore, our phytochemical research focuses primarily on the inflorescence of A. artemisiifolia, from which we obtained twenty sesquiterpenoids, including four new ones (1–4). Moreover, all the compounds were evaluated for their allelopathic activities on the seedling growth of wheat.

2. Results and Discussions

Air-dried and powdered inflorescence of A. artemisiifolia was extracted with petroleum ether and then further extracted with ethanol. A total of 20 sesquiterpenoids (Figure 1), including four new ones (1–4), were isolated from the petroleum ether extract and ethanol extracts using chromatographic methods.

2.1. Chemical Characterization of the Compounds

Compound 1 was obtained as a yellowish syrup. The molecular formula was C₁₅H₂₄O₄ as determined by the HRESIMS peak at m/z 267.1598 ([M − H]⁻, calcd. for 267.1596), with four degrees of unsaturation. The ¹H NMR data of 1 (

Table 1. ¹H NMR spectrum (600 MHz) data of compounds 1–4.

NO.	1	2	3	4
	δH, mult (J in Hz)	δH, mult (J in Hz)	δH, mult (J in Hz)	δH, mult (J in Hz)
1	3.14, td (10.3, 8.6)	2.79, ddd (12.1, 10.7, 8.1)	1.90, m	2.21, m
			1.79, m	/
2	2.16, m	1.84, m	2.05, m	2.12, dddd (14.0, 10.1, 8.6, 5.5)
	1.82, m	1.55, m		1.90, m
3	2.05, dt (12.0, 8.8)	1.76, m	/	2.58, ddd (15.8, 8.8, 5.5)
	1.90, ddd (11.8, 8.5, 2.7)	1.77, m		2.49, ddd (15.7, 8.6, 7.0)
4	/	/	2.56, dd (9.5, 4.4)	/
5	2.69, ddd (13.4, 9.9, 3.6)	2.23, t (12.1)	0.44, t (9.5)	1.88, m
6	2.19, m	4.23, dd (11.6, 10.1)	0.91, ddd (12.1, 9.5, 5.6)	1.52, m
	1.79, m			1.36, m
7	3.33, td (10.8, 3.9)	1.81, m	2.26, m	1.15, m
	/		1.93, m	1.01, td (13.3, 11.5)
8	2.22, m	1.96, m	1.89, m	2.40, m
	1.80, m	1.32, m
9	2.26, m	1.94, m	/	4.92, m
	1.96, ddd (12.6, 9.5, 3.7)	1.63, td (14.0, 13.4, 4.6)
10	/	/	3.36, ddd (12.3, 7.2, 4.5)	/
11	/	2.20, m	/	2.90, m
12	/	/	4.15, d (11.1)	/
			4.06, d (11.1)
13	6.48, d (1.6)	1.22, d (7.0)	1.42, s	1.11, d (7.4)
	5.68, d (1.6)
14	1.50, s	1.15, s	1.43, s	5.64, d (1.5)
				4.93 d, (1.5)
15	1.44, s	1.33, s	1.73, s	0.75, d (7.0)
1′		3.37, m
2′		1.11, t (7.0)

Compounds 1, 3, and 4 were measured in C₅D₅N, and 2 was measured in CDCl₃.

) show two singlet methyls at δ_H 1.50 (H₃-14) and 1.44 (H₃-15) and a terminal alkyne at δ_H 6.48 and 5.68 (H₂-13). The ¹³C NMR (

Table 2. ¹³C NMR spectrum (150 MHz) data of compounds 1–4.

NO.	1	2	3	4
	δc, DEPT	δc, DEPT	δc, DEPT	δc, DEPT
1	53.1 CH	46.5 CH	25.7 CH2	43.9 CH
2	26.5 CH2	25.6 CH2	38.4 CH2	29.2 CH2
3	40.8 CH2	39.3 CH2	81.2 C	33.0 CH2
4	80.8 C	80.3 C	47.8 CH	176.2 C
5	53.8 CH	55.1 CH	26.7 CH	34.7 CH
6	33.2 CH2	83.0 CH	29.4 CH	37.4 CH2
7	41.6 CH	51.1 CH	19.5 CH2	18.5 CH2
8	31.7 CH2	25.9 CH2	39.2 CH2	44.8 CH
9	41.7 CH2	37.9 CH2	73.2 C	83.1 CH
10	73.9 C	78.0 C	54.5 CH	143.9 C
11	150.3 C	41.4 CH	24.9 C	39.7 CH
12	170.1 C	178.0 C	63.0 CH2	178.6 C
13	121.4 CH2	12.9 CH3	24.7 CH3	10.9 CH3
14	28.0 CH3	22.5 CH3	32.6 CH3	111.5 CH2
15	24.9 CH3	24.0 CH3	26.4 CH3	14.0 CH3
1′		55.4 CH2
2′		16.2 CH3

Compounds 1, 3, and 4 were measured in C₅D₅N, and 2 was measured in CDCl₃.

) indicated 15 resonances ascribed to two methyls, a terminal alkyne, five methylene, three methines, two oxygenated tertiary carbons, and one α,β-unsaturated carboxylic carbon. The ¹H-¹H COSY correlations enabled the establishment of a long coupling carbon chain from C-3 to C-1 that extended via C-5 sequentially to C-9 as shown in bold bonds (Figure 2). The HMBC correlations from H₃-15 (δ_H 1.44) to C-3 (δ_C 40.8), C-4 (80.8), and C-5 (53.8) and from H₃-14 (δ_H 1.50) to C-1 (δ_C 53.1), C-9 (41.7), and C-10 (73.9) established 1 as a 4,10-dihydroxylguaiane-type sesquiterpene (Figure 2). While HMBC correlations from H₂-13 (δ_H 6.48 and 5.68) to C-11 (δ_C 150.3), C-12 (170.1), and C-7 (41.6) indicated that a conjugated acrylic acid moiety was attached at C-7. Accordingly, the planar structure of compound 1 was established as 4,10-dihydroxyguaia-11(13)-en-12-acid. The strong NOESY correlation between H-1 and H-5 indicated cis-fused framework between the five-carbon ring and the seven-carbon ring. NOEs, H-5 with H-7 show that all of those protons (H-1, H-5, and H-7) were cofacial and were assigned to be α-oriented [12]. In addition, NOEs, H₃-14 with H₃-15 and H₃-15 with Hβ-6 were observed in the cis-fused framework suggesting CH₃-14 and CH₃-15 were β-oriented (Figure 2). Thus compound 1 was determined to be 4β,10β-dihydroxy-1α,5α,7αH-guaia-11(13)-en-12-acid.

Compound 2 was obtained as a colorless syrup. The molecular formula of 2 was C₁₇H₂₈O₄ as determined by the HRESIMS peak at m/z 615.3872 ([2M +Na]⁺, calcd. for 615.3873) with four degrees of unsaturation. The ¹H NMR data (Table 1) show signals of four tertiary methyls at δ_H 1.22 (d, H₃-13), 1.15 (s, H₃-14), 1.33 (s, H₃-15), and 1.11 (t, H₃-2’) and an oxygenated methine at δ_H 4.23 (H-6) and an oxygenated methylene at 3.37 (H₂-1’). The ¹³C NMR data of 2 (Table 2) identify 17 resonances ascribed to four methyls, five methylenes (one oxygenated), five methines (one oxygenated), two oxygenated tertiary carbons, and one ester carbonyl carbon. The ¹H and ¹³C NMR data of 2 (Table 1 and Table 2) display characteristic signals of a guaiane-type sesquiterpenoid, which quite resembled that of α-Hydroxy-10α-methoxyguaian-12,6-olide [13]. A careful comparison of ¹H and ¹³C NMR data and molecular formula showed that ethoxyl, rather than methoxyl, existed at C-10 in 2. The relative configuration of 2 was deduced from the NOE correlations (Figure 3). The cross peaks of H-1/H-5, H-5/H-7, and H-7/H₃-13 indicated that these protons were cofacial and α-oriented. The cross peaks of H-6/H₃-16, H-6/H₃-14, and H-6/H-11 proved that they were on the opposite face and were assigned to be β-oriented. Accordingly, compound 2 was determined structurally as 4α-Hydroxy-10α-ethoxyguaian-12,6-olide.

Compound 3 was obtained as a colorless crystal plate The molecular formula of 3 was C₁₅H₂₆O₃ as determined by the HRESIMS peak at m/z 253.1803 ([M − H]⁻, calcd. for 253.1804). The ¹H NMR data (Table 1) show signals of three tertiary methyls at δ_H 1.42 (H₃-13), 1.43 (H₃-14), and 1.73 (H₃-15). The ¹³C NMR data of 3 (Table 2) identify 15 resonances ascribed to three terminal methyls, five methylenes (one oxygenated), four methines, and three tertiary carbon (two oxygenated). Taking into account the molecular formula, a tricyclic skeleton was indicated. The presence of one characteristic quaternary carbon at high field (C-11, δ_C 24.9) displayed it is an aromadendrane-type sesquiterpene [14,15]. The HMBC correlations from H₂-12 (δ_H 4.06 and 4.15) to C-11, C-13, C-6, and C-7 and from H₃-13 (δ_H 1.42) to C-11, C-12, C-6, and C-7 verified the existence of a cyclopropyl unit at C-6 and C-7. Moreover, The HMBC correlations from H₃-14 (δ_H 1.43) to C-1, C-9, and C-10 and from H₃-15 (δ_H 1.73) to C-3, C-4, and C-5 established the planar structure of compound 165 as aromadendrane-4,10,12-triol. The relative configurations of the six chiral centers at C-1, C-4, C-5, C-6, C-7, and C-9 in 3 were similar to those in (−)-alloaromadendrane-4β,10β-diol [16], which was confirmed by the ROESY correlations of H-1with H-5 and H₂-12; H-6 with H₃-11; and H-7 with H-8 in its ROESY spectrum (Figure 4). Therefore, compound 3 was characterized as (−)-alloaromadendrane-4β,10β,12-triol.

Compound 4 was obtained as a colorless viscous oil. The molecular formula of 4 was C₁₅H₂₂O₄ as determined by the HRESIMS peak at m/z 265.1441 ([M − H]⁺, calcd. for 265.1440). In ¹H NMR, it can be recognized that there are two methyl groups at δ_H 1.11 and 0.75 (each d, 3H) and an exocyclic methylene at δ_H 5.64 and 4.93. The ¹³C NMR spectrum indicated 15 carbon resonances (Table 2), which contained two carbonyl carbons, two downfield alkene carbons, an oxygenated carbon, two methyls, two methylenes, and five methines. A long coupling carbon chain from C-3 to C-9 that extended from C-8 to C-13 was deduced by the ¹H–¹H COSY correlations (Figure 5). The HMBC correlations from H₃-13 to C-8, C-11, and C-12 and from H-9 to C-12, as well as from H₃-14 to C-1, C-9, and C-10, established a preguaiane-type sesquiterpene containing α-methyl-γ-lactone. The relative configuration of 4 was concluded from the NOESY experiment (Figure 5). The cross peaks of H-1/H₃-15, H-7α/ H₃-15, and H-7α/ H₃-13 indicated that they were cofacial and α-oriented. The cross peaks of H-8/H-9 and H-8/H-11 sustained that these protons were on the other face and assigned to be β-oriented. Therefore, compound 4 was characterized as depicted and named preambrosia A.

Known compounds were identified by comparison with the experimental and reported spectroscopic data as follows: (-)-compressanolide (5) [17], magnograndiolide (6) [18], 1β,7β,9β,10β,13αH-guaia-4(5)-en-12,6β-olide-9-O-β-d-glucoside (7) [19], tetrahydrocoropilin (8) [8], psilostachyin C (9) [20], psilostachyin B (10) [21], 11α,13-dihydroperuvin (11) [22], 3α,4β-dihydrocumanin (12) [23], dihydrocumanin diacetate (13) [23], dihydrocumanin acetonide (14) [23], damsinic acid (15) [24], ambrosic acid (16) [25], 11,13-dihydroparthenolide (17) [26], 11β,13-dihydro-14-hydroxyparthenolide (18) [26], (-)-9α-hydroxydihydroparthenolid (19) [26], and (+)-alloaromadendrane-4β,10α-diol (20) [27]. The stucture of these isolated sesquiterpeniods can be divided into guaiane-, pseudoguaiane-, secopseudoguaiane-, germacrene-, and aromadendrane-type carbon skeletons. Among them, guaiane- and pseudoguaiane-type sesquiterpeniods are the most characteristic and diverse subclass in the inflorescence of A. artemisiifolia. Aromadendrane-type sesquiterpenes are rare in the genus Ambrosia [28]; however, aromadendraniols 3 and 20 were isolated from the inflorescence of A. artemisiifolia.

2.2. Allelopathy Activity

Compounds 1–20 were evaluated for their allelopathic activities on the seedling growth of wheat (T. Aestivum) at a concentration of 200 μg/mL. Our results show that fourteen compounds (1–4, 5, 9–12, 14–15, 17, and 19–20) inhibited the root growth of the tested seedlings to varying degrees. Compounds 14, 15, 17, and 20 significantly inhibited root length, which was more than 50% shorter than that of the control (Figure 6). In contrast, the inhibitory effect of these active compounds on shoot growth was reasonably weak, with relative shoot length being no less than 60% of the control (Figure 7). It is notable that compound 14, a pseudoguaiane-type sesquiterpene, showed the most potent allelopathic activity both on root and shoot length. Comparing the structure–activity relationships of compounds 11–14 showed that 14 had an acetonide group on the adjacent hydroxyl of C-3/C-4, which may be related to a high allelopathic effect. Previous research has demonstrated that parthenolides, germacrene-type sesquiterpenes, have strong allelopathic inhibitory activity [29]. The structural analogue of 17 also showed good inhibitory activity, but compounds 18 and 19 with an additional hydroxyl at C-14 and C-9, respectively, exhibited very weak allelopathic activities.

Sesquiterpenoids, which play an important role in plant allelopathy, are abundant in many invasive plants and can significantly inhibit the growth of neighboring plants [9,10]. On the other hand, allelopathic components generally have strong phytotoxic activities against many weeds [30,31]. Our findings reveal the chemical profile of the sesquiterpenoids occurring in the inflorescence of A. artemisiifolia, and our bioassay results are useful in understanding the chemical basis of allelopathy in A. artemisiifolia.

3. Materials and Methods

3.1. General

Melting points were obtained on a SGW X-4 micromelting point apparatus (INESA Co., Shanghai, China). Optical rotations were measured on SGW-533 automatic polarimeter (INESA Co., Shanghai, China). HRESIMS spectra were taken on an API QSTAR mass spectrometer (Applied Biosystem/MSD Sciex, Concord, ON, Canada). The 1D- and 2D-NMR spectra were recorded on a Bruker Avance III 600MHz NMR spectrometer using TMS as an internal standard. Column chromatography was performed on silica gel (100–200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). Preparative TLC plates (HSGF254, Jiangyou silicone Development Co., Ltd., Yantai, China), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), and Develosil ODS (50 μm, Nomura Chemical Co. Ltd., Osaka, Japan) were used for the isolation experiments. Preparative HPLC was performed on a Waters 1525 Binary HPLC pump and a Waters 2414 refractive index detector (Waters Corp, Milford, MA, USA) using a YMC-Pack ODS-A column (250 mm ×10 mm I.D.; S-5 μm, 12 nm).

3.2. Plant Material

The inflorescence of A. artemisiifolia was collected from Cangwu County, Guangxi province, China, in August 2019, which was identified by Prof. Dai-gui Zhang (Key laboratory of Plant Resources Conservation and Utilization, Jishou University). A voucher specimen (zdg20190801) was deposited in Hunan Agricultural University. Seeds of Triticum aestivum L. were purchased from a seed company (Jiangsu Dingsheng Seed Co., Jiangsu, China).

3.3. Extraction and Isolation

The air-dried and powdered inflorescence of A. artemisiifolia (9.0 kg) was extracted with petroleum ether (10 L × 4) and then extracted with ethanol (95%, 10 L × 4)). The ethanol extract was suspended in water and partitioned with EtOAc and n-butanol successively, as described in our previous research [32].

The petroleum ether extract (254.9 g) was subjected to silica gel column chromatography (CC, 100–200 mesh, 2000 g) eluted successively with gradient petroleum ether–acetone mixtures of increasing polarity (100:0 → 60:40, v/v) to separate fifteen fractions (PE.A1–PE.A15). A large number of crystals precipitated in PE.A8, and compound 17 (1100.4 mg) was obtained by recrystallization.

The EtOAc extract (370.0 g) was separated into twelve fractions (ET.A1–ET.A12) by a silica gel column (100–200 mesh, 1000 g) eluted with an increasing gradient of CHCl₃–MeOH mixtures.

Fraction ET.A2 (165.7 g) was decolorized on an MCI gel column to obtain a yellowish syrup subfraction A2B (133.0 g) from the 90% MeOH eluent. A2B was then subjected to silica gel CC (200–300 mesh, 1000 g) eluted carefully with a gradient of petroleum ether–acetone mixtures, yielding nine fractions Fr. B1–B9. These fractions (B1–B9) were first chromatographed on an ODS-C18 column eluting with MeOH-H₂O (30:70→90:10, v/v) to produce seven subfractions. Each of the subfractions was further purified by a sephadex LH-20 column eluting with MeOH to collect the points with color features of terpenoids on TLC. Finally, three major points were obtained from subfraction B2 (10.3 g), of which compound 15 (157.4 mg) was directly crystallized after sephadex LH-20 CC, and compounds 5 (15.3 mg, Rt = 17 min) and 3 (103.2 mg, Rt = 28 min) were yielded by semipreparative HPLC (55% MeOH /H₂O, 3 mL/min). Two major points were obtained from subfraction B3 (12.8 g), yielding compounds 13 (73.1 mg, Rf = 0.3) and 2 (12.6 mg, Rf = 0.5) by TLC preparation with CHCl₃-MeOH (90:10) as the developing solvent. Four major points were obtained from subfraction B4 (20.4 g), and compounds 16 (571.8 mg) and 9 (43.6 mg) were crystallized after sephadex LH-20 CC; compounds 8 (22.5 mg, Rf = 0.4) and 11 (10.2 mg, Rf = 0.6) were yielded by TLC preparation using CHCl₃-MeOH (95:5) as the developing solvent. Two major points were obtained from subfraction B5 (12.8 g), which obtained compounds 4 (24.8 mg, Rf = 0.2) and 20 (12.2 mg, Rf = 0.5) by TLC preparation with petroleum ether–acetone (90:10) as the developing solvent. Three major points were obtained from subfraction B6 (11.8 g), and compound 12 (238 mg) was crystallized after sephadex LH-20 CC; compounds 1 (15.8 mg, Rt = 36 min) and 14 (20.0 mg, Rt = 45 min) were yielded by semipreparative HPLC (45% MeOH /H₂O, 3 mL/min). Three major points were obtained from subfraction B7 (10.5 g), and compounds 6 (13.0 mg, Rt = 18 min), 18 (15.1 mg, Rt = 22 min), and 19 (28.2 mg, Rt = 30 min) were yielded by semipreparative HPLC (30% MeOH/H₂O, 3 mL/min).

Similarly, fractions ET.A9 (6.3 g) and ET.A10 (6.8 g) were chromatographed on ODS-C18 followed by Sephadex LH-20 (MeOH) to collect clear spots using TLC detection. Compound 10 (5.8 mg) was obtained from ET.A9 by TLC preparation with ether–acetone (90:15) as the developing solvent, while compound 7 (15.3 mg) was obtained from ET.A10 by TLC preparation using ether–acetone (80:20) as the developing solvent.

3.4. Physical and Chemical Data of 1–4

Compound 1: yellowish syrup, [α]${}_{\mathrm{D}}^{25}$ − 62.0 (c 0.6, MeOH); ¹H NMR (600 MHz, C₅D₅N) and ¹³C NMR (150 MHz, C₅D₅N) spectroscopic data, see Table 1 and Table 2; positive ion ESIMS m/z: 291 [M + Na]⁺; negative ESIMS m/z: 267 [M − H]⁻; HRESIMS m/z: 267.1598 [M − H]⁻ (calcd. for C₁₅H₂₃O₄, 267.1596). (Data from Figures S1–S7).

Compound 2: colorless syrup, [α]${}_{\mathrm{D}}^{25}$ − 43.0 (c 0.8, MeOH); ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) spectroscopic data, see Table 1 and Table 2; positive ion ESIMS m/z: 319 [M + Na]⁺, 615 [2M + Na]⁺; HRESIMS m/z: 615.3872 [2M + Na]⁺ (calcd. for C₃₄H₅₆O₈Na, 615.3873). (Data from Figures S8–S14).

Compound 3: colorless crystal plate, mp 305–307 °C, [α]${}_{\mathrm{D}}^{25}$ + 14.0 (c 0.1, MeOH); ¹H NMR (600 MHz, C₅D₅N) and ¹³C NMR (150 MHz, C₅D₅N) spectroscopic data, see Table 1 and Table 2; negative ESIMS m/z: 253 [M − H]⁻, 289 [M + Cl]⁻; HRESIMS m/z: 253.1803 ([M − H]⁻ (calcd. for C₁₅H₂₅O₃, 253.1804). (Data from Figures S15–S21).

Compound 4: colorless viscous oil, [α]${}_{\mathrm{D}}^{25}$ − 26.0 (c 0.6, MeOH); ¹H NMR (600 MHz, C₅D₅N) and ¹³C NMR (150 MHz, C₅D₅N) spectroscopic data, see Table 1 and Table 2; positive ion ESIMS m/z: 267 [M + H]⁺, 289 [M + Na]⁺; negative ESIMS m/z: 265 [M − H]⁻, 301 [M + Cl]⁻; HRESIMS m/z: 265.1441 ([M − H]⁻ (calcd. for C₁₅H₂₁O₄, 265.1440). (Data from Figures S22–S28).

3.5. Bioassays

Inhibitory activities on the growth of T. aestivum seedlings were evaluated using the plate culture method [33]. To enhance the germination rate, T. aestivum seeds were submerged in a 0.3% potassium permanganate solution for 15 min before being thoroughly rinsed with distilled water. After skin breaking, T. aestivum seeds were germinated on filter paper in the dark at 25 °C for 24 h. After being dissolved in acetone or DMSO, each compound (1–20) was prepared as a stock solution of 2 mg/mL. The stock solutions were diluted with distilled water (containing 1% Tween 80) to obtain concentrations of 200 μg/mL. The same volume of acetone or DMSO was added to distilled water (containing 1% Tween 80) as a control. Following germination, uniformly growing seedlings (10 seeds) were transferred to 9 cm diameter Petri dish lined with filter paper. Each dish was treated with 7 mL of the prepared corresponding concentration of test solution (or control solution). Each treatment was replicated 3 times. The seedlings were then incubated in a constant temperature humidity chamber in the dark at 25 °C for 72 h. At the end of the experiment, root and stem lengths were measured.

3.6. Statistical Analysis

All data were subjected to analysis of variance by use of SPSS 18.0. Significant differences in seedling growth between treatment and control were calculated by one-way analysis of variance (ANOVA). Relative length (percent) was determined by the formula [treated length)/control length] × 100.