1. Introduction
Sophora tonkinensis Gagnep. is widely used in traditional Chinese medicine and is Guangxi’s “Guishiwei” authentic medicinal material in China. It grows in calcium-rich, arid karst areas. This species is native to a relatively small region, encompassing specific provinces in south-central and southeastern China (e.g., Guangxi, Guizhou, Yunnan) and northern Vietnam [
1]. Historically, it has been extensively employed to alleviate symptoms such as fire toxin accumulation, throat paralysis, sore throat, swollen and painful gums, and oral and tongue sores [
2]. Research indicates that over 170 chemical constituents, including 108 flavonoids and more than 20 alkaloids, have been isolated from
S. tonkinensis [
3,
4]. The primary medicinally active compounds are quinolizidine alkaloids and prenyl flavonoids, with matrine and oxymatine standing out as key quinolizidine alkaloid representatives [
5,
6]. Recently, the escalating demand for
S. tonkinensis has led to varying degrees of degradation in its natural habitat within its native range in China [
6,
7]. Furthermore, rocky desertification severely erodes its natural ranges, resulting in frequent droughts [
7]. Due to habitat destruction and over-exploitation, it is listed as a national class II protected plant species in China. Therefore, investigating effective and practical drought tolerance methods for
S. tonkinensis is crucial for ensuring steady production and sustainable medicinal resource utilization.
In this context, nanotechnology presents a promising, rapid, and non-transgenic strategy for enhancing plant resilience, offering advantages over more complex and time-consuming genetic improvement approaches [
8]. The application of nanoparticles (NPs) has demonstrated faster enhancement of plant tolerance to abiotic stressors [
8]. Numerous studies have shown that NPs positively impact plant growth, development, yield, and abiotic stress resistance. Silicon (Si), the second most abundant element in Earth’s crust after oxygen, accumulates significantly in plants despite not being considered essential for higher plants. Its accumulation aids plants in resisting pests, pathogens, and various biotic and abiotic stresses, including diseases, metal stress, drought, and salt stress, making it a beneficial element [
9,
10]. Nanosilica (SiO
2 NPs), due to their high reactivity and biochemical activity, may be more effective than traditional silicon sources in mitigating diverse stressful conditions [
11,
12]. Notably, emerging studies have begun to reveal the beneficial role of SiO
2 NPs in enhancing growth and stress tolerance in certain medicinal plants [
13,
14], highlighting their potential for agricultural application.
While previous research on
S. tonkinensis has explored strategies to mitigate water stress, such as the application of melatonin [
7], the potential of nanotechnology, particularly SiO
2 NPs, remains completely unexplored for this endangered species. While SiO
2 NPs have shown efficacy in crops like wheat and rice, their role in medicinal plants under drought stress, particularly for enhancing the biosynthetic efficiency and accumulation of key bioactive components in endangered species like
S. tonkinensis, remains underexplored. This study addresses this gap by exploring SiO
2 NPs’ effects on biomass, physio–biochemical traits, and bioactive compound accumulation (e.g., alkaloids and flavonoids) in
S. tonkinensis. The findings aim to provide a novel nano-based strategy to meet rising medicinal demands and foster sustainable utilization of this vital plant resource.
2. Materials and Methods
2.1. Materials
The seedlings were propagated from seeds collected at the S. tonkinensis planting base in Napo, Baise City, Guangxi Zhuang Autonomous Region, China. The plant material was identified as Sophora tonkinensis Gagnep., a leguminous plant, by Wei Fan, an associate researcher at the Guangxi Botanical Garden of Medicinal Plants. The seedlings were cultivated in a growth chamber under the following conditions: a day/night temperature of 28/25 °C, a 14/10 h light/dark photoperiod, a light intensity of 300 μmol·m−2·s−1 provided by LED lamps, and a relative humidity of 60–70%.
Seedlings were planted into plastic pots with a pot height of 10 cm, a pot mouth diameter of 13 cm, and a pot bottom diameter of 9 cm. The pots are filled with a mixture of nutrient soil and vermiculite (2:1, v/v). The nutrient soil had the following properties: pH 6.9 ± 0.3, organic matter content 5.2 ± 3.5%, total nitrogen 0.5 ± 0.1%, available phosphorus 15.5 ± 6.2 mg/kg, and available potassium 120.3 ± 12.4 mg/kg. The vermiculite was used to improve aeration and drainage. Each pot contained approximately 1 kg of growth medium. The experiment was conducted at the Guangxi Key Laboratory of Medicinal Resources Protection and Genetic Improvement from February to March 2024.
2.2. Instruments
Agilent1100 high-performance liquid chromatograph (Agilent Company, Santa Clara, CA, USA); M8800H-C ultrasonic cleaner (Branson Company, Branson, MO, USA); Mikro 220R high-speed refrigerated centrifuge (Hettich Company, Kirchlengern, Germany); Multiskan SkyHigh microplate reader (Thermo Fisher Scientific Company, Waltham, MA, USA); WGLL-125BE drying oven (Tianjin Test Instrument Co., Ltd., Tianjin, China); HH.S11-2 constant temperature water bath (Shanghai Boxun Medical Bioinstrument Co., Ltd., Shanghai, China).
2.3. Experimental Design
Seedlings of S. tonkinensis (6 months old, uniformly developed, and healthy) were selected and divided into six treatment groups. Each treatment was applied to 60 individual pots (one plant per pot), totaling 360 seedlings. Drought stress was simulated by withholding water. Soil moisture was monitored daily by weighing all pots. The soil water content (SWC) was calculated gravimetrically. The initial SWC at field capacity was maintained at approximately 75~80%. For the drought stress groups, irrigation was withheld until the SWC reached 30~35%, a level which was then maintained by replenishing water daily based on weight loss to sustain this stress level throughout the experimental period. The control groups were maintained at 75~80% soil moisture content by daily watering. The drought stress and foliar SiO2 NPs treatments were designed as follows:
T1 (CK): Cultivated under normal moisture (75~80% soil moisture content) without NPs; T2: Cultivated under normal moisture with foliar application of 100 mg/L SiO2 NPs solution; T3: Cultivated under normal moisture with foliar application of 200 mg/L SiO2 NPs solution; T4 (SD): Cultivated under severe drought (30~35% soil moisture content) without NPs; T5: Cultivated under severe drought with foliar application of 100 mg/L SiO2 NPs solution; T6: Cultivated under severe drought with foliar application of 200 mg/L SiO2 NPs solution.
An equal volume of SiO2 NPs solution was applied to all relevant groups every 4 days (5 times total). Control groups (T1 and T4) received corresponding volumes of deionized water. Silica nanoparticle dispersion (<30 nm; catalog #791334, Sigma-Aldrich, St. Louis, MI, USA) was used.
2.4. Determination of Medicinal Material Yield
Fresh weights (FW) of roots, stems, and leaves were measured separately after harvest. Tissues were then fixed at 105 °C for 1 h to terminate enzyme activity and subsequently dried at 50 °C to constant weight to obtain the dry weight (DW). The dry matter content was calculated as (dry weight/fresh weight) × 100%. After drying, the dry materials were ground into powder and stored in a desiccator for subsequent analysis of active ingredients.
2.5. Determination of Physiological and Biochemical Indicators
All biochemical parameters were determined using assay kits and the results were normalized on a fresh weight basis. The detailed methods were as follows: The content of soluble protein adopts coomassie brilliant blue method. The content of soluble sugar adopts anthrone colorimetric method. Super oxidase dimutase (SOD) activity used the WST-8 method [
15], catalase (CAT) activity used the ultraviolet absorption method, peroxidase (POD) activity used the guaiacol method. The content of malonydialdehyde (MDA) was determined by thiobarbituric acid method (TBA), and the content of superoxide anion (O
2−) is determined by the hydroxylamine hydrochloride oxidation method, and the content of hydrogen peroxide (H
2O
2) is determined by the titanium sulfate colorimetric method. The above determination methods refer to the instructions of the kit. Assay kits for the determination of physiological indices (catalog numbers: M1805A (Soluble Protein), M1503A (Soluble Sugar), M0102A (SOD), M0103A (CAT), M0105A (POD), M0106A (MDA), M0114A (O
2−), and M0107A (H
2O
2)) were purchased from Suzhou Mengxi Biomedical Technology Co., Ltd. (Suzhou, China). This analysis was performed with three independent biological replicates per treatment.
2.6. Determination of Matrine and Oxymatrine Content
The high-performance liquid chromatography (HPLC) method under the “Content Determination” of S. tonkinensis in the “Pharmacopoeia of the People’s Republic of China” (2020 Edition) was used to determine the content of matrine and oxymatrine. Standard matrine (item number BWC9032-2016, purity ≥ 98%) and oxymatrine (item number 110780, purity ≥ 98%) were purchased from Northern Weiye Measurement Group Co., Ltd., Beijing, China. This analysis was performed with three independent biological replicates per treatment.
2.7. Determination of Genistein and Genistin Content
The contents of genistein and genistin were determined using a modified high-performance liquid chromatography (HPLC) method based on Xu et al. [
16]. Briefly, approximately 0.1 g of powdered dry sample was accurately weighed and extracted with 1.0 mL of 70% (
v/
v) aqueous methanol using a tissue grinder, followed by ultrasonic extraction for 2 h at room temperature. The extract was centrifuged at 12,000×
g for 10 min. The supernatant was collected, passed through a 0.22 μm syringe filter, and analyzed by HPLC.
HPLC analysis was performed on an Agilent 1260 Infinity II system equipped with a DAD detector. Separation was achieved on a Compass C18(2) reversed-phase column (250 mm × 4.6 mm, 5 μm) maintained at 30 °C. The mobile phase consisted of methanol and 1% acetic acid in water (30:70, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was set at 260 nm. Identification and quantification were performed by comparing the retention times and peak areas with those of authentic standards.
Genistein (catalog number S31565, purity ≥ 98%) and genistin (catalog number S31591, purity ≥ 98%) standard compounds were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Calibration curves were constructed for each standard for quantification. This analysis was performed with three independent biological replicates per treatment.
2.8. Determination of Total Alkaloids and Total Flavonoids Content
Total alkaloids content was determined as follows: Approximately 0.1 g of powdered dry sample was accurately weighed and extracted with 1 mL of 80% (v/v) aqueous ethanol. The mixture was vortexed thoroughly, transferred to a 2.0 mL microcentrifuge tube, and subjected to ultrasonic extraction for 60 min at room temperature. Subsequently, the extract was centrifuged at 12,000× g and 25 °C for 10 min. The supernatant was carefully collected for the subsequent assay.
The total flavonoids content was determined using a different extraction protocol: Approximately 0.2 g of powdered dry sample was accurately weighed and extracted with 5 mL of 60% (v/v) aqueous ethanol. The mixture was shaken and extracted at 60 °C for 2 h in a thermostatic water bath, followed by centrifugation at 12,000× g and 25 °C for 10 min. The resulting supernatant was collected for analysis.
The contents of total alkaloids and total flavonoids in the respective supernatants were quantified spectrophotometrically according to the manufacturer’s instructions of the commercial assay kits (Alkaloid Content Assay Kit, Cat. No. M0122A; Flavonoid Content Assay Kit, Cat. No. M0118A; Suzhou Mengxi Biomedical Technology Co., Ltd., Suzhou, China). The specific assay principles are based on colorimetric reactions with appropriate chromogenic agents, and the absorbance was measured at the wavelengths specified in the kits’ protocols. Calibration curves were established using standard solutions provided in the kits for quantitative calculation.
This analysis was performed with three independent biological replicates per treatment.
2.9. Statistical Analysis
All data are presented as the mean ± standard deviation (SD) of at least three independent biological replicates. The data were processed using Microsoft Excel 2019 and subjected to one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons in SPSS 19.0 software. Differences were considered statistically significant at p < 0.05.
3. Results
3.1. Effect of SiO2 NPs on the Yield of S. tonkinensis Medicinal Materials Under Drought Stress
The impact of SiO
2 NPs spraying on the biomass of
S. tonkinensis subjected to drought stress is illustrated in
Table 1. Under drought stress (T4), there was a significant decrease in both the fresh and dry weights of the roots and leaves compared to normal moisture levels (T1). Specifically, the fresh weight dropped by 44.17% and 49.46%, respectively, while the dry weight decreased by 38.96% and 9.89%. Notably, the stems exhibited a different trend: the fresh weight decreased by 13.4% compared with T1, but the dry weight increased by 19.05%. Additionally, drought stress (T4) markedly elevated dry matter content of the roots, stems, and leaves of
S. tonkinensis by 8.94%, 39.86%, and 78.28%, respectively, in comparison with T1.
When SiO
2 NPs at a concentration of 100 mg/L (T5) were sprayed under drought stress, the dry matter content of the roots, stems, and leaves of
S. tonkinensis peaked, showing increases of 19.11%, 59.67%, and 86.80% compared with normal moisture levels (T1). Furthermore, these dry matter contents were 9.34%, 14.16%, and 4.78% higher, respectively, than those observed under drought stress alone (T4). Simultaneously, the dry weight of the stems and leaves increased under drought stress when 100 mg/L SiO
2 NPs were applied. Conversely, the application of 200 mg/L SiO
2 NPs (T6) resulted in decreased fresh weight, dry weight, and dry matter content of the roots, stems, and leaves of
S. tonkinensis, irrespective of whether the plants were subjected to normal moisture (T3) or drought stress (T6). As summarized in
Table 1, the 100 mg/L dose (T5) under drought stress consistently yielded the most positive effects on biomass accumulation across all plant organs.
3.2. Effects of SiO2 NPs on Physiological and Biochemical Characteristics of S. tonkinensis Seedlings Under Drought Stress
The influence of SiO
2 NPs on the physiological and biochemical attributes of
S. tonkinensis seedlings subjected to drought stress is depicted in
Figure 1. Compared with T1, T4 (drought stress without SiO
2 NPs) exhibited significant increases in the soluble sugar content, superoxide dismutase (SOD) activity, catalase (CAT) activity, malondialdehyde (MDA) content, and superoxide anion (O
2−) content in the roots, stems, and leaves of
S. tonkinensis. Specifically, the roots showed increases of 78.84%, 34.03%, 10.10%, 19.86%, and 6.15%, respectively; the stems increased by 77.83%, 71.03%, 76.05%, 36.05%, and 36.04%, respectively, and the leaves increased by 84.67%, 73.67%, 13.16%, 62.74%, and 173.21%, respectively. Conversely, the soluble protein content, peroxidase (POD) activity, and hydrogen peroxide (H
2O
2) content in the leaves decreased by 47.54%, 44.23%, and 0.42%, respectively, compared with T1.
Under drought stress with SiO2 NPs applications (T5 and T6), the roots of S. tonkinensis exhibited changes in the content of soluble protein, soluble sugar, O2−, H2O2, and the activities of CAT, POD, and MDA ranging from 0.82% to 33.72% compared with T4. Notably, the SOD activity in the roots under T5 treatment increased by 26.10% compared with T4, while the H2O2 content in the roots under T6 treatment decreased by 33.72% compared with T4. The change range of the above physiological and biochemical indicators in the stems of S. tonkinensis ranged from 5.33% to 26.19%, with the most significant change observed in O2− content under T6, which was 26.19% lower than T4. In the leaves, the changes in the above physiological and biochemical indicators of S. tonkinensis ranged from 0.06% to 38.24%, with the soluble protein content under T5 showing the largest increase at 38.24% compared with T4.
3.3. Effect of SiO2 NPs on the Quality of S. tonkinensis Medicinal Materials Under Drought Stress
This study evaluated the effects of foliar application of SiO
2 NPs on the content of bioactive compounds in
S. tonkinensis under both normal moisture conditions (T1) and drought stress (T4) conditions (
Figure 2). A striking finding was the remarkable 244.33% increase in genistein content in the roots under T6 (drought + 200 mg/L SiO
2 NPs) compared to the well-watered control (T1), highlighting the high biotechnological potential of SiO
2 NPs to elicit valuable secondary metabolites under stress. The active ingredients, including matrine, oxymatrine, genistin, genistein, total alkaloids, and total flavonoids, were analyzed in the roots, stems, and leaves of the plant. The distinct patterns of response between different plant organs and compounds are visually summarized in the integrative heatmap (
Figure 3), which illustrates the relative variation in each compound across all treatments.
Under normal moisture (T1), the application of SiO2 NPs resulted in an increase in the contents of several active ingredients in the roots. Specifically, the contents of oxymatrine, genistin, genistein, total alkaloids, and total flavonoids were all enhanced with SiO2 NPs treatment. In contrast, under drought stress, while drought alone reduced these contents, the application of 200 mg/L SiO2 NPs mitigated this effect (T6), leading to higher levels of matrine (214.15 μg/g), genistin (4.06 μg/g), genistein (48.56 μg/g), total alkaloids (9.96 mg/g) and total flavonoids (11.44 mg/g) compared with the drought-stressed control (T4) without SiO2 NPs. Notably, the genistein content in the roots increased significantly by 244.33% compared with T1 and 48.66% compared with T4.
In the stems, the results were mixed. Under normal moisture (T1), an increasing concentration of SiO2 NPs led to higher levels of matrine, oxymatrine, genistin, and total alkaloids. When spraying 200 mg/L SiO2 NPs, the contents of oxymatrine (4289.98 μg/g) and total alkaloids (13.87 mg/g) reached the highest values. However, under drought stress (T4), the contents of matrine, oxymatrine, genistin, and total alkaloids decreased as the SiO2 NPs concentration increased.
In the leaves, under normal moisture (T1), the matrine and oxymatrine contents were largely unaffected by SiO2 NPs treatment. Under drought stress (T4), the genistin and genistein contents showed a slight increase with increasing SiO2 NPs concentration. The contents of total alkaloids and total flavonoids, on the other hand, exhibited a biphasic response, increasing first and then decreasing with higher SiO2 NPs concentrations. When spraying 100 mg/L SiO2 NPs, the contents of total alkaloids (15.11 mg/g) and total flavonoids (9.80 mg/g) reached the highest values.
In summary, SiO2 NPs treatment had beneficial effects on the active ingredient content of S. tonkinensis, particularly under drought stress, where it mitigated the decrease in active ingredients caused by water scarcity. The optimal SiO2 NPs concentration varied depending on the plant part and the specific active ingredient being analyzed.
4. Discussion
The present study demonstrates that foliar application of SiO2 NPs can effectively mitigate the adverse effects of drought stress on S. tonkinensis, with the optimal concentration being highly dependent on the target parameter—growth and physiological resilience or the accumulation of specific bioactive compounds. Our principal findings revealed that 100 mg/L SiO2 NPs was most effective in enhancing biomass and dry matter content under drought stress, whereas 200 mg/L SiO2 NPs was superior in boosting the content of several key medicinal alkaloids and flavonoids, most notably triggering a remarkable 244.33% increase in root genistein compared to the well-watered control.
Among the diverse range of technologies available, the utilization of nanoparticles has emerged as one of the most efficacious and sustainable strategies for augmenting plant growth and development. Numerous studies have validated the beneficial impacts of exogenously applied nanoparticles (NPs) on plant growth, physiology, and the production of secondary metabolites [
17]. In this study, the application of 100 mg/L SiO
2 NPs through spraying significantly enhanced the growth attributes of
S. tonkinensis under drought stress conditions, which coincided with peak dry matter content of roots, stems, and leaves, as well as maximum dry weights of stems and leaves. Comparable results have been obtained in diverse plant species. For instance, the application of Si NPs on corn foliage mitigated the mineral nutrient imbalance induced by drought stress and led to increased corn weight [
18]; similarly, SiO
2 NPs have been found to promote root development in fir seedlings and elevate yields by boosting their biomass [
19]. The facilitation of plant growth and development by SiO
2 NPs may be attributed to its regulatory role in plant hormone balance and sugar metabolism [
20]. We hypothesize that the differential effects of the two concentrations are linked to the degree of stress elicitation. The 100 mg/L concentration likely provides a “moderate stress” signal, sufficient to prime the plant’s defense and growth maintenance mechanisms without overwhelming its resources, thereby optimizing biomass production. In contrast, the higher 200 mg/L concentration may induce a “high stress” state, which, while potentially more detrimental to growth, acts as a stronger elicitor, diverting more resources towards the synthesis of defensive secondary metabolites like alkaloids and flavonoids.
SiO
2 NPs also play a crucial role in enhancing plant drought resistance. Our study revealed that the spraying of SiO
2 NPs not only improved the yield of
S. tonkinensis but also altered its physiological and biochemical characteristics. Specifically, SiO
2 NP application under drought stress increased the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) in the roots, as well as the soluble protein content in the leaves. Concurrently, it reduced the malondialdehyde (MDA), superoxide anion (O
2−), and hydrogen peroxide (H
2O
2) content in the leaves. Generally, severe drought stress triggers oxidative stress, leading to an increase in H
2O
2 and MDA levels [
21]. However, the application of SiO
2 NPs effectively decreased the H
2O
2 content, thereby mitigating the detrimental effects of oxidative stress [
22]. Studies have indicated that drought stress elevates the activity of antioxidant enzymes such as CAT and SOD in the leaves of
S. tonkinensis [
1], and the spraying of SiO
2 NPs further amplifies this activity [
23]. The Si element acts as a free radical scavenger, eliminating the effects of reactive oxygen species (ROS), and serves as an antioxidant that boosts the activity of oxidative enzymes (CAT and SOD), scavenges H
2O
2 and MDA, and thereby enhances the antioxidant capacity of plants [
24]. For example, SiO
2 NPs bolster wheat’s resilience to water deficit conditions by balancing ROS production and fortifying the antioxidant system under drought stress [
25]. The application of SiO
2 NPs curbs the accumulation of MDA and H
2O
2, alleviates the damage caused by drought stress in rough chaff trees, and elevates their drought resistance [
26]. Comparable findings have been reported in other drought-stressed plants when SiO
2 NPs were applied. Specifically, SiO
2 NPs enhance the drought tolerance of faba beans, nigella, wheat, rice, and other plants by reducing ROS production [
27,
28,
29,
30]. These studies reveal that the enhancement of antioxidant defense facilitated by SiO
2 NPs represents a pivotal mechanism in safeguarding plants against drought-induced oxidative stress. The molecular underpinning of this elicitation likely involves ROS signaling itself. The moderate ROS burst triggered by NP-induced stress can act as a signal to activate transcription factors responsible for the upregulation of key genes in the phenylpropanoid and antioxidant pathways [
31,
32], leading to the enhanced biosynthesis of protective flavonoids and alkaloids observed in our study, particularly at the 200 mg/L dose.
In recent times, numerous scholars have explored the potential of NPs as novel elicitors for boosting the biosynthesis of bioactive compounds, suggesting that NPs can elevate the production of high-value secondary metabolites in plants [
33,
34]. In this research, the application of SiO
2 NPs through spraying was found to augment the levels of oxymatrine, genistin, total alkaloids, and total flavonoids in the roots of
S. tonkinensis. Notably, when the plants were sprayed with 200 mg/L SiO
2 NPs under drought stress, the concentrations of matrine, genistin, genistein, total alkaloids, and total flavonoids in the roots surpassed those observed during drought conditions alone. Strikingly, the genistein content in the roots reached its peak value. The findings of this study align with the report by Zahedi et al. [
35], which indicated that SiO
2 NPs (at concentrations of 50 and 100 mg/L) not only activated the strawberry antioxidant system to counteract reactive oxygen species (ROS), but also facilitated the accumulation of total phenolic compounds and anthocyanins, ultimately enhancing the growth efficiency and yield of strawberries under both normal and drought stress conditions.
The suggestion of 100 mg/L SiO2 NPs as a cost-effective solution for mitigating yield loss in karst regions requires careful consideration of scaling and environmental factors. While promising, the field application of NPs necessitates future studies to address potential risks, including the long-term accumulation of NPs in the unique karst soil ecosystem, their impact on soil microbiota, and the overall environmental toxicology. The economic viability of large-scale NP production and application also remains a key factor for practical adoption.