Composite stable moringa oleifera polysaccharide cadmium-blocking preparation as well as preparation method and application thereof

Abstract

The invention discloses a composite stable moringa oleifera polysaccharide cadmium-resistant preparation as well as a preparation method and application thereof, and relates to the technical field of cadmium-resistant preparations. The cadmium blocking preparation is composed of moringa oleifera polysaccharide, a compound stabilizer and a phosphate buffer solution, the compound stabilizer is composed of sodium alginate and nano silicon dioxide according to the mass ratio of 1: (1.0-1.5), and the pH value of the preparation is 6.0-7.0. The moringa oleifera polysaccharide is applied to cadmium prevention and control of Chinese cabbages in alkaline cadmium-polluted soil (pH 6.5-7.5), and accurate regulation and control are achieved in a rhizosphere irrigation mode. The sodium alginate-nano silicon dioxide composite stabilizer system synergistically exerts the dual functions of dispersion stability and slow release protection, and ensures that the moringa oleifera polysaccharide keeps activity in the soil environment. The preparation has the advantages of being small in dosage, low in cost, easy and convenient to operate, environmentally friendly and the like, a scientific and feasible technical scheme is provided for safe utilization of cadmium-polluted farmland in the alkaline soil area, and the preparation has important application value and popularization prospects.

Description

Technical Field

[0001] This invention belongs to the field of environmental remediation and plant nutrition regulation technology, specifically involving a compound stable Moringa polysaccharide cadmium-inhibiting agent, its preparation method and application, which is used to reduce the absorption and accumulation of cadmium by vegetable crops (especially Chinese cabbage in alkaline soil in the north) in cadmium-polluted farmland, and also has growth-promoting and stress-resistant functions. Background Technology

[0002] Cadmium is highly bioavailable, easily migrates, and has a long half-life. It can be absorbed into crops through roots and accumulate in the human body via the food chain, causing chronic diseases such as osteoporosis and kidney dysfunction. In northern regions, soils are mostly neutral to slightly alkaline (pH 6.5-7.8). Leafy vegetables such as cabbage, due to their short growth cycle and strong accumulation capacity, are a major source of cadmium exposure risk, necessitating the development of economical, efficient, and environmentally friendly control technologies.

[0003] Cadmium stress exerts multifaceted inhibitory effects on plant growth. Once inside plants, cadmium primarily reduces biomass accumulation by inhibiting the activity of key photosynthetic enzymes, disrupting thylakoid structure, and interfering with the electron transport chain. Simultaneously, it promotes excessive production of reactive oxygen species, leading to lipid peroxidation and cell membrane damage. Furthermore, cadmium competitively inhibits the transport of essential elements such as calcium, magnesium, iron, and zinc, exacerbating nutrient imbalances and metabolic disorders. Therefore, developing technologies that can simultaneously alleviate cadmium toxicity, promote nutrient absorption, and maintain physiological homeostasis is of great significance.

[0004] Currently, cadmium pollution control technologies mainly include physical remediation, chemical passivation, agronomic regulation, and bioremediation, but all have significant limitations: physical remediation is costly and damages soil ecology; chemical passivators easily alter soil pH and nutrient balance; the effectiveness of agronomic regulation is constrained by variety and environment; and bioremediation has a long and unstable cycle. In contrast, plant-derived bioactive substances inhibit cadmium absorption and transport by regulating physiological metabolic processes, offering advantages such as renewable raw materials, low cost, ease of application, and environmental friendliness, representing an innovative development direction.

[0005] Moringa (oleifera) polysaccharide (MOP) is a natural heteropolysaccharide (molecular weight 5-200 kDa) containing monosaccharides such as rhamnose, arabinose, and galactose. It possesses significant free radical scavenging ability, antioxidant enzyme activity regulation, and biomembrane protection. Studies have shown that natural polysaccharides such as brown algae polysaccharides and seaweed polysaccharides can enhance crop stress resistance by activating antioxidant defense systems, regulating osmotic substances, and stabilizing cell membranes. Considering that redox imbalance under cadmium stress is a key factor leading to growth inhibition, moringa polysaccharide theoretically holds the potential to influence cadmium absorption and translocation in Chinese cabbage by regulating oxidative stress response. However, there are currently no systematic studies, either domestically or internationally, on the use of moringa polysaccharide to reduce cadmium absorption in Chinese cabbage.

[0006] In existing technologies, the diatomaceous earth-peanut cake inhibitor disclosed in CN106167707A requires a large dosage (150-300 kg / mu) and is complex to operate, mainly relying on physical adsorption with a single mechanism of action. The melatonin-aspartic acid inhibitor disclosed in CN112335662A is applied via foliar spraying and cannot directly act on key sites of cadmium absorption in the roots, thus limiting its regulatory capacity. Although some studies have reported that Moringa smoke liquid has anti-cadmium effects in rice, its direct application to Chinese cabbage faces significant obstacles: rice has fibrous roots and adapts to acidic paddy field environments (pH 5.5-6.5); Chinese cabbage has taproot systems with dense root hairs and grows in alkaline dry land (pH 6.5-7.8). Moringa smoke liquid is easily oxidized and degraded in alkaline environments, making it difficult to penetrate dense root hairs to reach the rhizosphere, and may also cause precipitation due to pH differences, resulting in low absorption efficiency and poor persistence.

[0007] To address the aforementioned issues, this invention employs key improvements: Moringa polysaccharides, known for their greater stability, are used to replace the smoke liquid; a sodium alginate-nano silica composite stabilizer is innovatively introduced to form a slow-release gel carrier, enhancing dispersibility and persistence; the pH is adjusted to 6.0-7.0 using phosphate buffer to adapt to the rhizosphere environment of Chinese cabbage; the concentration is systematically optimized (15-60 mg / L) to ensure cadmium inhibition while avoiding adverse effects on growth; and rhizosphere irrigation is used to directly target key sites of cadmium absorption. Through these systematic technological improvements, this invention achieves a breakthrough in the cross-crop application of Moringa active substances from rice to Chinese cabbage. Through the synergistic effects of multiple mechanisms, including antioxidant defense, hormone balance regulation, mineral element optimization, photosynthetic function maintenance, and water metabolism regulation, it effectively inhibits cadmium absorption while promoting normal plant growth, comprehensively enhancing the tolerance of Chinese cabbage to cadmium stress. This provides a practical and feasible technical solution for the safe utilization of alkaline cadmium-contaminated farmland in northern China. Summary of the Invention

[0008] The purpose of this invention is to provide a composite stable Moringa polysaccharide cadmium-inhibiting agent, its preparation method, and its application. This agent can effectively reduce the accumulation of cadmium in the edible parts of Chinese cabbage in alkaline cadmium-contaminated soil. At the same time, it enhances the plant's antioxidant defense, optimizes photosynthetic performance, improves water metabolism and mineral nutrient absorption through multiple physiological regulation mechanisms, providing technical support for the safe utilization of cadmium-contaminated farmland.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a composite stable Moringa polysaccharide cadmium-inhibiting formulation, composed of the following components: (1) Moringa peptides, with a mass-volume concentration of 30-60 mg / L, are used as the main bioactive components; (2) Composite stabilizers, with a mass-volume concentration of 20-60 mg / L, are composed of sodium alginate and nano silica in a mass ratio of 1:(1-1.5) to enhance the dispersion stability and sustained-release effect of moringa polysaccharides; (3) Phosphate buffer is used to adjust the pH value to 6.0-7.0 to maintain the stability of the preparation in different soil environments.

[0010] Technical Principle Explanation: Moringa polysaccharides, as plant-derived natural heteropolysaccharides (containing monosaccharide components such as rhamnose, arabinose, galactose, and xylose), possess multiple functions, including regulating plant antioxidant defense, photosynthetic function, water metabolism, mineral element absorption, and hormone balance. However, in alkaline soil environments, they are easily degraded or precipitated due to pH fluctuations and ion interference. This invention innovatively employs a sodium alginate-nano silica composite stabilizer system: sodium alginate forms a gel network to provide a slow-release carrier, nano silica enhances dispersion stability, and phosphate buffer maintains pH stability. These three components synergistically ensure that moringa polysaccharides maintain their activity in alkaline soils. In a preferred embodiment, a moringa polysaccharide concentration of 30 mg / L exhibits both optimal cadmium inhibition (a 26.7% reduction in aboveground cadmium content and a 19.0% reduction in the cadmium transfer coefficient) and physiological regulatory effects.

[0011] This invention provides a method for preparing the above-mentioned cadmium-inhibiting agent, comprising the following steps: (1) Preparation of stabilizer (S1): Sodium alginate is dissolved in phosphate buffer and stirred in a water bath at 50-70℃ until completely dissolved; nano silica is added and ultrasonically treated at 4-5℃ (power 300-500 W, time 15-30 min) until a uniform gel is formed. (2) Moringa peptide dispersion (S2): Moringa polysaccharide powder is added to the gel obtained in step S1. The mixture is stirred for 40-45 min under light-protected conditions, and then homogenized at 4-5℃ at a speed of 10000-12000 r / min for 20-30 min to obtain the cadmium-inhibiting agent.

[0012] Key points of the process: (1) Sodium alginate needs to be dissolved at 50-70℃ to ensure complete dispersion; (2) The temperature should be controlled at 4-5℃ during ultrasonic treatment to avoid loss of Moringa polysaccharide activity; (3) The homogenization process should be carried out in the dark to prevent photo-oxidative degradation; (4) A longer stirring time (40-45 min) helps Moringa polysaccharide to be evenly dispersed in the gel carrier.

[0013] This invention provides a method for reducing cadmium absorption in Chinese cabbage grown in cadmium-contaminated soil, comprising the following steps: (1) timing of application: applying to the root zone on the 3rd day after thinning of Chinese cabbage seedlings; (2) application conditions: applying 40-60 mL of the cadmium inhibitor to each Chinese cabbage plant, once every 3-7 days, for 3-5 consecutive applications; (3) applicable scope: applicable to alkaline slightly cadmium-contaminated soil (pH 6.5-7.5, total cadmium content 0.8-2.0 mg / kg).

[0014] By applying the formulation directly to the key sites of cadmium absorption through root zone irrigation, the process of cadmium absorption and accumulation in cabbage can be precisely controlled, significantly reducing the cadmium content in the edible parts of cabbage.

[0015] The cadmium-inhibiting agent described in this invention can be used to reduce cadmium absorption by cabbage grown in cadmium-contaminated soil, specifically through rhizosphere irrigation to achieve cadmium inhibition.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Outstanding technological innovation. (1) Moringa polysaccharide was applied for the first time to control cadmium in Chinese cabbage in alkaline soil, breaking through the technical barrier of moringa active substances in cross-crop applications; (2) The sodium alginate-nano silica composite stabilizer system was innovatively adopted, solving the problem of dispersion and stability of moringa polysaccharide in alkaline soil environment; (3) The rhizosphere irrigation was precisely controlled, directly acting on the key parts of cadmium absorption, and the mechanism of action was clear.

[0017] 2. The effect is significant and quantifiable. Experimental data show that compared with the control group: (1) Cadmium inhibition effect: the cadmium content in the aboveground parts of Chinese cabbage decreased by 26.7%, the cadmium content in the roots decreased by 9.3%, the cadmium enrichment coefficient in the aboveground parts decreased by 26.5%, and the cadmium transfer coefficient decreased by 19.0%; (2) Root optimization: At a concentration of 30 mg / L, the fresh weight root-to-shoot ratio increased by 16.1%, and the dry weight root-to-shoot ratio increased by 18.0%, promoting the distribution of dry matter to the roots; (3) Antioxidant defense: SOD activity increased by 242.7%, CAT activity increased by 194.2%, MDA content decreased by 33.6%, and hydroxyl radical scavenging rate increased by 30.1%; (4) Photosynthetic protection: SPAD value increased by 6.1%, and variable fluorescence (Fv) increased by 17.5%; (5) Water regulation: Bound water / free water ratio increased by 23.0%, enhancing cell water retention capacity; (6) Hormone balance: ABA content increased by 35.5%, ZR content increased by 24.1%, ABA / GA3 ratio decreased by 23.7%, and ABA / IAA ratio decreased by 20.0%; (7) Soil improvement: Rhizosphere available phosphorus content increased by 43% (reaching 229 mg / L). (mg / kg), reducing cadmium bioavailability through phosphorus-cadmium antagonism; (8) mineral optimization: promoting the absorption of macroelements such as N (↑21.4%), P (↑15.1%), and Mg (↑17.8%), and inhibiting the non-selective absorption of divalent metal ions such as Fe (↓96%) and Cu (↓90%).

[0018] 3. Synergistic effect of multiple mechanisms. This invention exerts cadmium inhibition effect by regulating multiple physiological metabolic pathways in plants: (1) Antioxidant defense: Activating antioxidant enzyme systems such as SOD and CAT to reduce membrane lipid peroxidation; (2) Photosynthetic protection: Maintaining chlorophyll content and photosystem II activity; (3) Water metabolism: Increasing the proportion of bound water and enhancing stress resistance; (4) Hormone regulation: Coordinating the balance between growth-promoting and stress-resistance hormones; (5) Mineral optimization: Selectively promoting the absorption of essential elements and inhibiting the competitive absorption of heavy metals; (6) Soil improvement: Increasing rhizosphere available phosphorus and strengthening the phosphorus-cadmium antagonistic effect.

[0019] 4. High practicality. (1) Low dosage: The dosage of the preparation per mu is only tens of grams, which is much lower than that of traditional cadmium inhibitors (150-300 kg / mu); (2) Easy to apply: The rhizosphere irrigation operation is simple and can be combined with regular irrigation without deep plowing or large-scale soil improvement; (3) Low cost: The raw materials are readily available and the preparation process is simple, making it suitable for large-scale production; (4) Environmentally friendly: Plant-derived materials are biodegradable and have no risk of secondary pollution. (5) Good stability: The preparation can be stably stored for more than 30 days under light-protected conditions at 4℃.

[0020] 5. High Promotional Value. This invention provides a practical and feasible technical solution for the safe utilization of cadmium-contaminated farmland in alkaline soil areas of northern China, possessing significant ecological, economic, and social benefits, and has broad application prospects. This invention achieves a technological breakthrough of "reducing cadmium without reducing yield," possessing significant ecological, economic, and social benefits, and has broad application prospects. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The reagents, materials, and instruments used are commercially available unless otherwise specified.

[0023] I. Materials and Reagents (a) Test plants Chinese cabbage (Brassica chinensis L.), variety "Jinglv No. 1", seeds purchased from the Vegetable Research Center of Beijing Academy of Agricultural and Forestry Sciences, with a germination rate of ≥95%.

[0024] (ii) Test soil Soil samples were taken from a typical lightly cadmium-contaminated farmland in Jiyuan, Henan Province, using a five-point pooled sampling method within the top 0-20 cm layer. The basic physicochemical properties of the soil were: pH 7.2±0.1, total cadmium content 1.35±0.05 mg / kg, organic matter content 15.3 g / kg, available phosphorus content 23.5 mg / kg, and available potassium content 128 mg / kg. This soil met the standard for alkaline lightly cadmium-contaminated soil as specified in claim 9 (pH 6.5-7.5, total cadmium content 0.8-2.0 mg / kg). The soil was air-dried and sieved through a 2 mm sieve for later use.

[0025] (III) Reagents and Materials Moringa polysaccharide (purity ≥90%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; sodium alginate (analytical grade, ≥99%) was purchased from Sinopharm Chemical Reagent Co., Ltd.; nano silica (particle size 20-30 nm) was purchased from Aladdin Biochemical Technology Co., Ltd.; phosphate buffered saline (PBS, pH 7.0) was prepared in the laboratory.

[0026] II. Preparation method of compound stable Moringa polysaccharide cadmium-inhibiting agent 1. Preparation of the composite stabilizer. Sodium alginate powder was dissolved in phosphate buffer solution and stirred in a 60°C water bath until completely dissolved, preparing a solution with a concentration of 16 mg / L. After cooling to room temperature, nano-silica (particle size 20-30 nm) was added to a final concentration of 19.2 mg / L (sodium alginate to nano-silica mass ratio 1:1.2). The solution was then ultrasonically dispersed at 4-5°C (400 W power, 20 min) to form a homogeneous and transparent gel-like system, thus obtaining the composite stabilizer. The total mass-volume concentration of the composite stabilizer was 35.2 mg / L.

[0027] 2. Preparation of a composite stabilizer-type Moringa polysaccharide cadmium-inhibiting formulation. Moringa polysaccharide powder was added to the above-mentioned composite stabilizer under light-protected conditions to a final concentration of 30 mg / L. Magnetic stirring was used for 40-45 min to ensure thorough dispersion. Subsequently, high-speed homogenization was performed at 4–5℃ (11000 r / min, 25 min) to obtain the stock solution of the composite stabilized Moringa polysaccharide cadmium-inhibiting formulation. This formulation had a pH of 6.8±0.1, a uniform appearance, no precipitation, and could be stably stored at 4℃ under light-protected conditions for more than 30 days without flocculation or stratification.

[0028] 3. Preparation of application concentration. The stock solution of the preparation was diluted with PBS buffer to different Moringa polysaccharide concentrations: 0 mg / L (control, CK), 15 mg / L, 30 mg / L, and 60 mg / L. These were used as different treatment groups for subsequent rhizosphere irrigation experiments on Chinese cabbage. The irrigation volume was 50 mL per plant, with an interval of 5 days between applications, for a total of 3 applications.

[0029] III. Examples

Example 1

[0030]

Example 2

[0031]

Example 3

[0032]

Example 4

[0033]

Example 5

[0034] [Example 6] Control with Sodium Alginate Stabilizer Alternate Sodium alginate powder was dissolved in PBS and stirred in the dark at 60°C until completely dissolved, preparing a sodium alginate solution with a concentration of 35 mg / L. After cooling to room temperature, Moringa polysaccharide powder was added to a final concentration of 30 mg / L, stirred in the dark for 45 min, and homogenized at 11000 r / min for 25 min at 4–5°C to obtain a single stable formulation. Rhizosphere irrigation was started on the 3rd day after thinning of pak choi seedlings, with 50 mL per plant, applied 4 times at 5-day intervals. This group was used to verify the necessity of nano-silica in enhancing dispersibility and preventing precipitation in the composite system.

[0035] [Example 7] Single Nano-Silica Stabilizer Control Nano-sized silica (particle size 20±5 nm) was dispersed in PBS to prepare a dispersion with a concentration of 35 mg / L. The dispersion was ultrasonicated for 20–25 min to ensure uniform dispersion. Moringa polysaccharide powder (final concentration 30 mg / L) was then added, and the mixture was stirred in the dark for 45 min. The mixture was then homogenized at 11000 r / min for 25 min at 4–5℃ to obtain a stable system of single-nano-silica. This system was applied to the rhizosphere drenching of pakchoi seedlings starting 3 days after thinning, at a rate of 50 mL per plant, with 5-day intervals, for a total of 4 applications. This study was used to verify the role of sodium alginate gel structure in providing sustained-release and long-lasting protection in the composite system.

[0036]

Example 8

[0037]

Example 9

[0038]

Example 10

[0039] Test case I. Experimental Objective This experiment evaluated the effects of different concentrations of Moringa polysaccharide cadmium-inhibiting agents (15-60 mg / L) on Chinese cabbage cultivation in mildly cadmium-contaminated soil through pot experiments. The focus was on investigating its cadmium-inhibiting effect, growth-promoting effect, and physiological and biochemical response mechanism to clarify the application potential and scientific basis of Moringa polysaccharide compound stabilizers under alkaline cadmium-contaminated soil conditions.

[0040] II. Experimental Materials and Methods (a) Experimental site and materials 1. Experimental location The experiment was conducted in the intelligent greenhouse of the Beijing Academy of Agricultural and Forestry Sciences. During the experiment, the greenhouse temperature was controlled at 20-25℃, the relative humidity at 60%-70%, and the experiment was conducted under natural light conditions.

[0041] 2. Test soil The potted soil was collected from slightly cadmium-contaminated farmland in Jiyuan, Henan Province. A five-point pooled sampling method was used to collect soil samples from the top 0-20 cm layer. After being brought back to the laboratory, the soil samples were air-dried until the moisture content was ≤5%, passed through a 2 mm sieve, and then placed into sterilized flowerpots. Each pot weighed 300.0±0.5 g. The basic physicochemical properties of the soil are as follows: pH value 7.2±0.1, electrical conductivity (EC) 1.16 mS / cm, total cadmium content 1.35±0.05 mg / kg (slightly contaminated), organic matter content 15.3 g / kg, available phosphorus content 23.5 mg / kg, and available potassium content 128 mg / kg.

[0042] 3. Test crops The bok choy variety "Jinglv No. 1" (purchased from the Vegetable Research Center of Beijing Academy of Agricultural and Forestry Sciences) has a varietal purity of ≥99% and a germination rate of ≥95%. This variety exhibits vigorous growth, dark green leaves, crisp and tender texture, and good heat resistance and disease resistance, making it suitable for conducting pollution stress experiments under pot cultivation conditions.

[0043] 4. Test formulation The composite stabilized cadmium-inhibiting preparations of Moringa polysaccharide prepared according to Examples 1-4 had Moringa polysaccharide concentrations of 0, 15, 30, and 60 mg / L, respectively. All preparations had a composite stabilizer concentration of 35 mg / L (sodium alginate 16.0 mg / L, nano-silica 19.2 mg / L, mass ratio 1:1.2), differing only in the Moringa polysaccharide concentration.

[0044] (II) Experimental Design Four treatment groups were set up. (1) CK (control group): The rhizosphere was irrigated with a compound stabilizer system without Moringa polysaccharide (Example 1). (2) MOP15: The irrigated formulation with a Moringa polysaccharide concentration of 15 mg / L was applied (Example 2). (3) MOP30: The irrigated formulation with a Moringa polysaccharide concentration of 30 mg / L was applied (Example 3). (4) MOP60: The irrigated formulation with a Moringa polysaccharide concentration of 60 mg / L was applied (Example 4). Each treatment had 10 biological replicates, for a total of 40 pots. A randomized block design was used, and the position of the pots was adjusted daily to reduce the position effect.

[0045] (III) Sowing and Seedling Management 1. Seed disinfection. Before sowing, disinfect the seeds as follows: rinse with running water for 5 minutes → soak in 75% ethanol for 30 seconds → soak in 1% sodium hypochlorite solution for 10 minutes → rinse 5 times with sterile deionized water → blot dry with sterile filter paper for later use.

[0046] 2. Sowing and Germination. Sow 5 seeds per pot at a depth of 1.0±0.1cm, covering with an even layer of soil. Water thoroughly after sowing, then cover with plastic film to retain moisture and promote germination. Remove the film after germination and keep the soil moist.

[0047] 3. Thinning management. When the seedlings develop to the two-leaf-one-heart stage, thin them out, keeping two uniformly growing and robust plants for subsequent treatment.

[0048] (iv) Application of Moringa polysaccharide preparations 1. Application timing: The first root zone irrigation should be carried out on the 3rd day after the thinning of Chinese cabbage seedlings.

[0049] 2. Application method. Use quantitative irrigation method, apply 50 mL of the preparation to each plant, once every 5 days, for a total of 4 times. The irrigation time is fixed between 9:00-10:00 am to avoid differences in transpiration rate caused by excessive light intensity.

[0050] 3. Water management. During the cultivation period, maintain the soil moisture content at 70%-80% of field capacity, and use deionized water to replenish water lost through evaporation to ensure that the soil retains its initial weight.

[0051] (v) Sampling and Measurement 1. Sampling time. Harvest should be carried out 8 days after the last application of the preparation (about 30 days after sowing).

[0052] 2. Sample Processing. Immediately after harvest, the above-ground parts and roots were separated using stainless steel scissors. The plants were rinsed with tap water to remove surface soil, and then rinsed three times with deionized water. The fresh weight was measured using a 0.1% balance, and the samples were divided into two parts: the fresh sample was flash-frozen in liquid nitrogen and stored at -80℃ for physiological and biochemical index determination; the dried sample was blanched at 90℃ for 30 min and dried at 75℃ to constant weight for cadmium content and mineral element determination.

[0053] 3. Measurement Indicators (1) Growth indicators. The fresh weight and dry weight (g / plant) of the aboveground parts and roots were measured, and the fresh weight-to-shoot ratio and dry weight-to-shoot ratio were calculated. The shoot-to-shoot ratio reflects the biomass allocation characteristics of the plant under cadmium stress and is an important indicator for assessing its cadmium resistance growth potential.

[0054] (2) Cadmium content determination. The cadmium content was determined using aqua regia-perchloric acid digestion combined with ICP-MS (inductively coupled plasma mass spectrometry). Sample preparation: After pulverizing the dried sample, it was passed through a 0.25 mm sieve. 0.5 g of the sample was weighed into a polytetrafluoroethylene digestion vessel, and 10 mL of aqua regia (HCl:HNO3=3:1) and 2 mL of perchloric acid were added. Digestion was carried out at 120℃ until clear. After cooling, the volume was adjusted to 50 mL and filtered for determination. Quality control: Three parallel samples and one national standard material sample were prepared for each batch of samples. The recovery rate was controlled at 95%–105%. The cadmium enrichment coefficient in the roots, the cadmium enrichment coefficient in the aboveground parts, and the cadmium transfer coefficient were calculated.

[0055] (3) Determination of soil physicochemical properties Rhizosphere soil samples were collected, air-dried, ground, and sieved through a 2 mm sieve before analysis. pH was determined using the potentiometric method (1:2.5 soil-to-water ratio); electrical conductivity (EC) was measured using a DDS-307 conductivity meter; nitrate nitrogen content was determined using ultraviolet spectrophotometry (220 / 275 nm difference method); available phosphorus content was determined using sodium bicarbonate extraction-molybdenum antimony colorimetric method; and available potassium content was determined using ammonium acetate extraction-flame photometry. All data were calculated on a dry weight basis and subjected to Duncan multiple comparison analysis (P<0.05).

[0056] (4) Antioxidant system indicators ① Enzyme activity indicators: Superoxide dismutase (SOD) activity (xanthine oxidase method), peroxidase (POD) activity (guaiacol method), catalase (CAT) activity (UV absorption method), ascorbate peroxidase (APX) content (colorimetric method). ② Non-enzymatic antioxidant and membrane lipid damage indicators: Malondialdehyde (MDA) content (thiobarbituric acid method), total antioxidant capacity (T-AOC, FRAP method), hydroxyl radical scavenging rate (o-phenanthroline-Fe). 2 (⁺Oxidation method). All the above determinations were based on fresh weight, and the results were used to reflect the regulatory effect of Moringa polysaccharides on the antioxidant defense system of Chinese cabbage.

[0057] (5) Measurement of photosynthetic characteristics ① Chlorophyll content (SPAD value): Measured at the midpoint of functional leaves using a SPAD-502 Plus chlorophyll meter between 9:00 and 11:00, with an average of three readings. ② Chlorophyll fluorescence parameters: Measured using a PAM-2500 pulse-modulated fluorometer. Parameters measured included initial fluorescence (Fo), maximum fluorescence (Fm), variable fluorescence (Fv = Fm – Fo), photochemical efficiency (Fv / Fo), and maximum photochemical efficiency (Fv / Fm). Measurements were taken 30 min after dark adaptation. The obtained parameters were used to evaluate the protective effect of Moringa polysaccharides on photosystem II activity.

[0058] (6) Determination of water-bound speciation The ratio of free water to bound water in leaves was determined using the sucrose method. Approximately 2 g of fresh leaf samples were weighed, chopped, and added to 10 mL of a 0.5 mol / L sucrose solution. The samples were soaked at 4°C for 24 h. After soaking, the surface moisture was blotted out, and the samples were weighed. Subsequently, the samples were dried at 105°C to constant weight. The free water and bound water contents were calculated based on the changes in weight before and after soaking, and the bound water / free water ratio was calculated to reflect the regulatory effect of Moringa polysaccharides on cell water retention capacity and stress resistance.

[0059] (7) Determination of plant hormone content The contents of cytokinin zeatin (ZR), abscisic acid (ABA), auxin (IAA), and gibberellin (GA3) were determined by high performance liquid chromatography (HPLC). Samples were extracted with 80% methanol, centrifuged, concentrated under nitrogen, and then analyzed. The ratios of ABA / GA3, ABA / IAA, and ABA / ZR were calculated to evaluate the regulatory effect of Moringa polysaccharides on the growth-stress balance.

[0060] (8) Determination of mineral element content in aboveground parts The macro- and micro-elements (N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn) in the dried aerial parts were determined by the HNO3–H2O2 microwave digestion–ICP–OES method.

[0061] 4. Data Processing All indicators were determined three times independently and repeatedly. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 software, and Duncan's multiple comparison test was used to test for statistical significance between groups (P < 0.05).

[0062] III. Results of Indicator Analysis (a) Biomass and root-shoot ratio analysis Biomass and root-to-shoot ratio are key physiological indicators for assessing plant growth potential and stress resistance. Under heavy metal stress, changes in the root-to-shoot ratio reflect how plants allocate photosynthetic products, directly affecting their cadmium tolerance. A higher root-to-shoot ratio generally means that the plant can allocate more resources to root development, thereby enhancing the root system's ability to absorb water and nutrients, and further improving its resistance to cadmium stress. Therefore, the root-to-shoot ratio has become an important indicator for evaluating plant cadmium tolerance.

[0063] Table 1. Effects of Moringa polysaccharide treatment on fresh weight, dry weight, and root-to-shoot ratio of Chinese cabbage plants. MOP concentration (mg / L) Fresh weight above ground (g / plant) Dry weight above ground (g / plant) Fresh weight of roots (g / plant) Root dry weight (g / plant) Fresh weight root-to-crown ratio Dry weight root-to-crown ratio MOP0 15.5±1.32a 1.07±0.13a 0.48±0.04a 0.053±0.006a 0.031±0.003ab 0.050±0.003b MOP15 17.2±1.27a 0.99±0.06a 0.52±0.07a 0.045±0.004a 0.030±0.003b 0.046±0.002b MOP30 15.1±1.47a 0.93±0.09a 0.54±0.07a 0.055±0.006a 0.036±0.003a 0.059±0.007a MOP60 16.9±1.44a 1.01±0.11a 0.55±0.06a 0.050±0.007a 0.033±0.004ab 0.049±0.005b Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). Moringa polysaccharide (MOP) treatment significantly affected the biomass and root-to-shoot ratio of Chinese cabbage plants. Regarding aboveground growth, there were no significant differences in aboveground fresh and dry weight between the treatment groups and the control group, indicating that the effect of moringa polysaccharide in promoting aboveground growth was relatively limited. However, root growth showed varying degrees of change at different concentrations. Although the root fresh weight increased in the MOP30 and MOP60 treatment groups, this difference did not reach a statistically significant level, suggesting that the direct promoting effect of moringa polysaccharide on Chinese cabbage roots was relatively limited.

[0064] In terms of root-to-shoot ratio analysis, the MOP30 group showed the best effect, with a fresh weight root-to-shoot ratio of 0.036, which was 16.1% higher than the control group (0.031), and a dry weight root-to-shoot ratio of 0.059, which was 18.0% higher than the control group (0.050), and was significantly better than other treatment groups. MOP30 treatment significantly promoted the distribution of dry matter to the roots, thereby improving the tolerance of Chinese cabbage to cadmium stress. In contrast, the root-to-shoot ratio of the MOP15 treatment group decreased, while the improvement effect of the MOP60 group was not significant. In conclusion, a concentration of 30 mg / L of Moringa polysaccharide can optimize the root-to-shoot ratio, enhance the nutrient absorption capacity and stress resistance of Chinese cabbage, and is the optimal application concentration for improving the cadmium tolerance of Chinese cabbage.

[0065] (II) Cadmium content analysis The absorption, accumulation, and translocation characteristics of cadmium in plants are important indicators for assessing cadmium tolerance and food safety. The cadmium accumulation coefficient in roots reflects the ability of roots to absorb cadmium, while the cadmium accumulation coefficient in aboveground parts characterizes the cadmium accumulation level in edible parts, directly affecting food safety; and the cadmium translocation coefficient reflects the efficiency of cadmium translocation from roots to aboveground parts. Reducing the cadmium content and translocation coefficient in aboveground parts, as well as inhibiting excessive absorption by roots, are key to reducing cadmium accumulation in the edible parts of cabbage and ensuring safe vegetable production. Therefore, these indicators are crucial for evaluating the effectiveness of cadmium inhibitors.

[0066] Table 2. Effects of Moringa polysaccharide treatment on cadmium content in the aboveground parts and roots of Chinese cabbage plants. MOP concentration (mg / L) Cadmium content in aboveground parts (mg / kg DW) Root cadmium content (mg / kg DW) Root cadmium enrichment coefficient cadmium enrichment coefficient in aboveground parts Plant cadmium transfer coefficient MOP0 1.719±0.13a 0.86±0.009a 0.647±0.007a 1.293±0.098a 1.999±0.151a MOP15 1.651±0.169a 0.83±0.011b 0.624±0.008b 1.241±0.127a 1.989±0.204a MOP30 1.263±0.064b 0.78±0.015c 0.586±0.011c 0.950±0.048b 1.619±0.082b MOP60 1.527±0.133a 0.72±0.016d 0.541±0.012d 1.148±0.100a 2.121±0.185a Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). Table 2 shows that Moringa polysaccharide treatment significantly affected the absorption and translocation characteristics of cadmium in Chinese cabbage. Regarding the cadmium content in the aboveground parts, the MOP30 treatment group showed a decrease to 1.263 mg / kg, a significant reduction of 26.5% compared to the control group (1.719 mg / kg) (P<0.05), demonstrating the best cadmium inhibition effect. The reductions in the MOP15 and MOP60 treatment groups were not significant. The cadmium content in the roots decreased in a concentration-dependent manner, with MOP15, MOP30, and MOP60 treatments decreasing to 0.83, 0.78, and 0.72 mg / kg, respectively, representing decreases of 3.5%, 9.3%, and 16.3% compared to the control group (0.86 mg / kg) (P<0.05). The cadmium enrichment coefficient in the roots continuously decreased with increasing concentration, indicating that Moringa polysaccharide can inhibit the absorption of cadmium by the roots. Analysis of the cadmium enrichment coefficient in the aboveground parts showed that only the MOP30 group significantly decreased to 0.95, a 26.5% decrease compared to the control group (1.293) (P<0.05). Regarding the cadmium transfer coefficient in the plants, the MOP30 treatment group decreased to 1.619, a significant 19.0% decrease compared to the control group (1.999) (P<0.05), while the MOP60 group increased to 2.121. In conclusion, the 30 mg / L Moringa polysaccharide treatment was the most effective in reducing cadmium absorption and translocation in Chinese cabbage, effectively controlling cadmium accumulation in the edible parts.

[0067] (III) Physicochemical properties of rhizosphere soil The physicochemical properties of rhizosphere soil directly affect nutrient availability and cadmium bioavailability. Soil pH is a key factor determining cadmium speciation and migration; increased pH promotes cadmium fixation and passivation, reducing its availability. Available phosphorus inhibits cadmium translocation to plants by competing for absorption sites or forming stable compounds; sufficient nutrients enhance plant stress resistance. Therefore, monitoring changes in rhizosphere soil physicochemical properties helps reveal the mechanism by which Moringa polysaccharides regulate cadmium availability and inhibit cadmium production.

[0068] Table 3. Effects of Moringa polysaccharide treatment on the physicochemical properties of rhizosphere soil at harvest time of Chinese cabbage plants. MOP concentration (mg / L) Soil pH Soil EC (μS / cm) Soil nitrate content (mg / kg) Soil available (mg / kg) Available potassium in soil (mg / kg) MOP0 7.143±0.106a 1103±127a 99.13±6.28a 160±19.5c 751±47.5ab MOP15 7.053±0.048ab 1071±142a 98.97±2.43a 197±21.4b 775±38.8a MOP30 6.968±0.087b 991±161a 98.70±2.52a 229±23.7a 748±75.6ab MOP60 6.943±0.047b 988±133a 103.76±2.90a 161±17.6c 657±104b Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). As shown in Table 3, Moringa polysaccharide treatment significantly affected the physicochemical properties of rhizosphere soil during the Chinese cabbage harvest period. Regarding soil pH, the MOP30 and MOP60 treatments decreased to 6.968 and 6.943, respectively, significantly lower than the control group (7.143) (P<0.05), indicating that Moringa polysaccharide can moderately regulate the rhizosphere acid-base environment. There were no significant differences in soil EC values ​​and nitrate nitrogen content among the treatments. The most significant change was in available phosphorus content, with the MOP30 treatment group significantly increasing to 229 mg / kg, a 43% increase compared to the control group (P<0.05), and the MOP15 group also showing a significant increase to 197 mg / kg, while the MOP60 group showed no significant improvement. Regarding available potassium, the MOP15 group had the highest level (775 mg / kg), while the MOP60 group significantly decreased to 657 mg / kg (P<0.05). In summary, 30 mg / L Moringa polysaccharide significantly increased the rhizosphere available phosphorus content and optimized the soil nutrient structure. It may inhibit the absorption and translocation of cadmium to the aboveground parts by enhancing phosphorus-cadmium antagonism and reducing cadmium bioavailability, which is a key soil science mechanism for cadmium control.

[0069] (iv) Antioxidant enzymes Antioxidant enzyme systems are key defense mechanisms in plants against cadmium stress. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) work synergistically to scavenge reactive oxygen species and protect cell structures from oxidative damage. A moderate increase in antioxidant enzyme activity under cadmium stress reflects effective activation of the defense system, helps maintain redox balance, and enhances cadmium tolerance; therefore, it is a key indicator for evaluating plant responses to and resistance to cadmium stress.

[0070] Table 4. Effects of Moringa polysaccharide treatment on antioxidant enzyme activity in Chinese cabbage leaves. MOP concentration (mg / L) SOD activity (U / g FW) POD activity (U / g FW) CAT activity (U / g FW) APX content (μmol / g FW) MOP0 786±73.0c 2636±246b 613±78.2d 3.239±0.351a MOP15 1225±154b 3521±351a 1091±135c 2.897±0.457a MOP30 2694±389a 2680±272b 1804±220a 2.246±0.273b MOP60 521±72.1c 1828±196c 1354±165b 3.419±0.395a Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). As shown in Table 4, Moringa polysaccharide treatment significantly regulated the activity of antioxidant enzymes in Chinese cabbage leaves. SOD activity reached 2694 U / g FW in the MOP30 treatment group, a significant increase of 242.7% compared to the control group (786 U / g FW) (P<0.05), demonstrating the strongest superoxide anion scavenging ability; while in the MOP60 group it significantly decreased to 521 U / g FW. POD activity showed different changes, with the highest in the MOP15 group (3521 U / g FW), a significant increase of 33.5% compared to the control group (2636 U / g FW) (P<0.05), no significant change in the MOP30 group, and a significant decrease in the MOP60 group. CAT activity showed a trend of first increasing and then decreasing with increasing concentration, reaching 1804 U / g FW in the MOP30 group, a significant increase of 194.2% compared to the control group (613 U / g FW) (P<0.05), the highest among all treatment groups, indicating its strongest effect in scavenging hydrogen peroxide. The APX content in the MOP30 group decreased to 2.246 μmol / g FW, a significant decrease of 30.7% compared to the control group (3.239 μmol / g FW) (P<0.05), possibly reflecting that the SOD-CAT system effectively scavenged peroxides. In summary, 30 mg / L Moringa polysaccharide can significantly activate SOD and CAT activity, construct a highly efficient antioxidant defense system, and enhance the resistance of Chinese cabbage to cadmium stress.

[0071] (V) Analysis of membrane lipid damage and total antioxidant capacity Total antioxidant capacity (T-AOC) and free radical scavenging rate comprehensively reflect the overall effectiveness of a plant's antioxidant defense system. T-AOC represents the synergistic effect of antioxidants in the body, while hydroxyl radical scavenging rate reflects the ability to scavenge highly reactive oxygen species. Malondialdehyde (MDA) is the end product of membrane lipid peroxidation, and its content reflects the degree of cell membrane damage. Cadmium stress induces the accumulation of reactive oxygen species, triggering membrane lipid peroxidation and leading to an increase in MDA. Therefore, improving antioxidant capacity, enhancing free radical scavenging, and reducing MDA content are important indicators for assessing plant cadmium tolerance and oxidative damage protection.

[0072] Table 5. Effects of Moringa polysaccharide treatment on the antioxidant capacity and membrane lipid damage of Chinese cabbage leaves. MOP concentration (mg / L) Total antioxidant capacity (μmol / g FW) Hydroxyl radical scavenging rate (%) MDA (nmol / g FW) MOP0 168±16.5a 71.5±5.40b 8.84±0.99a MOP15 168±14.7a 90.9±5.71a 7.96±0.89ab MOP30 185±15.7a 92.9±6.83a 5.88±0.68c MOP60 180±17.5a 85.4±5.59a 7.07±0.39b Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). As shown in Table 5, Moringa polysaccharide treatment significantly affected the antioxidant capacity and membrane lipid damage of Chinese cabbage leaves. There was no significant difference in total antioxidant capacity (T-AOC) between the treatment groups (168–185 μmol / g FW) and the control group (169 μmol / g FW), indicating that its effect on overall antioxidant levels was limited. Hydroxyl radical scavenging rates were significantly increased, with MOP15, MOP30, and MOP60 groups achieving 90.89%, 92.94%, and 85.38%, respectively, which were significantly higher than the control group (71.5%) by 27.2%, 30.1%, and 19.5% (P<0.05). MOP30 showed the best performance, demonstrating the strongest free radical scavenging ability. Membrane lipid peroxidation analysis showed that MDA content decreased significantly with treatment concentration, reaching 5.88 nmol / g FW in the MOP30 group, a 33.6% reduction compared to the control group (8.84 nmol / g FW) (P<0.05), and significantly lower than other treatment groups, indicating its effective inhibition of membrane lipid peroxidation. The MOP15 and MOP60 groups showed levels of 7.96 and 7.07 nmol / g FW, respectively, also showing a decreasing trend. In conclusion, 30 mg / L Moringa polysaccharide significantly enhanced hydroxyl radical scavenging ability, reduced MDA accumulation, and effectively protected cell membrane structure. Synergistically, its enhanced SOD and CAT activities constructed a more efficient antioxidant defense system, significantly improving cadmium tolerance in Chinese cabbage.

[0073] (vi) Chlorophyll content and fluorescence parameter analysis Chlorophyll content and fluorescence parameters are important indicators for evaluating plant photosynthetic performance and cadmium stress response. SPAD values ​​reflect the relative chlorophyll content and are related to photosynthetic pigment levels and light-harvesting capacity. Initial fluorescence (Fo) indicates the openness of photosystem II reaction centers, while variable fluorescence (Fv) reflects photochemical potential. Fv / Fo and Fv / Fm represent photochemical efficiency and maximum photochemical efficiency, respectively, and are key parameters for assessing photosynthetic organ function. Cadmium stress often leads to chlorophyll degradation and decreased light efficiency; therefore, maintaining high chlorophyll content and photochemical efficiency is crucial for plant cadmium tolerance.

[0074] Table 6. Effects of Moringa polysaccharide treatment on SPAD value and chlorophyll fluorescence parameters of Chinese cabbage leaves. MOP concentration (mg / L) SPAD Fo Fv Fv / Fo Fv / Fm MOP0 42.88±0.69b 12305±1574 a 53237±4494b 4.303±0.232a 0.811±0.009a MOP15 43.88±1.38ab 13069±1177 a 59893±5337ab 4.584±0.098a 0.821±0.003a MOP30 45.48±1.68a 12181±3182a 57954±5609ab 4.536±0.192a 0.819±0.006a MOP60 43.90±1.22ab 14044±944a 62568±3134a 4.460±0.145a 0.817±0.005a Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). As shown in Table 6, Moringa polysaccharide treatment had a certain impact on photosynthetic pigments and fluorescence parameters in Chinese cabbage leaves. Regarding SPAD values, the MOP30 group reached 45.48, significantly higher than the control group (42.88) by 6.1% (P<0.05), indicating that this concentration helped maintain chlorophyll content and mitigate photosynthetic pigment degradation caused by cadmium stress. Initial fluorescence (Fo) showed no significant difference among treatments (12181~14044), indicating that Moringa polysaccharide had a limited impact on the basal state of the photosystem II reaction center. Variable fluorescence (Fv) reached 62568 in the MOP60 group, significantly higher than the control group (53237) by 17.5% (P<0.05), and the MOP15 and MOP30 groups also showed an upward trend, indicating enhanced photochemical reaction potential. The photochemical efficiency parameters Fv / Fo and Fv / Fm showed no significant difference among groups, indicating that the maximum photochemical efficiency of photosystem II remained basically stable. In summary, 30 mg / L Moringa polysaccharide can significantly increase chlorophyll content, maintain photochemical activity in conjunction with the increase in Fv, reduce the inhibition of photosynthetic system by cadmium stress, promote stable photosynthetic performance of leaves, and play a positive role in enhancing the physiological vitality of Chinese cabbage.

[0075] (vii) Leaf water binding morphology Water is a crucial medium for plant physiological metabolism, and its form directly affects stress resistance. Free water participates in transport and metabolism, while bound water is bound to colloids, exhibiting low fluidity but high stability. The bound water / free water ratio is a key indicator for measuring cellular water retention capacity and stress resistance; a higher ratio indicates stronger resistance. Studying the regulation of water form in pakchoi leaves by Moringa polysaccharides under cadmium stress can reveal the physiological mechanisms by which it alleviates cadmium toxicity and enhances resistance.

[0076] Table 7 Effects of Moringa Polysaccharide Treatment on Water-Bound Species in Chinese Cabbage Leaves MOP concentration (mg / L) Free water content (%) Bound water content (%) Bound water / Free water MOP0 16.5±8.3a 72.0±7.8a 5.578±3.390a MOP15 13.0±2.6a 76.3±2.9a 6.081±1.469a MOP30 11.5±1.7a 77.5±1.9a 6.860±1.074a MOP60 15.5±6.6a 73.5±5.7a 5.740±3.241a Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). Table 7 shows that different concentrations of Moringa polysaccharide treatment had some effect on the water-bound form of pakchoi leaves, but the differences were not statistically significant. Regarding free water content, the MOP30 group had the lowest (11.5±1.7%), a 30.3% decrease compared to the control group (16.5±8.3%), followed by the MOP15 group (13.0±2.6%), while the MOP60 group was close to the control. Bound water content showed the opposite trend, with the MOP30 group having the highest (77.5±1.9%), a 7.6% increase compared to the control (72.0±7.8%), and the MOP15 and MOP60 groups also showing varying degrees of increase. The bound water / free water ratio, a key indicator of stress resistance, was highest in the MOP30 group (6.860±1.074), a 23.0% increase compared to the control, while the MOP15 group had a ratio of 6.081±1.469, also showing a promoting effect. The results showed that appropriate amounts of Moringa polysaccharides, especially the 30 mg / L treatment, could enhance the water retention capacity of cells by increasing the proportion of bound water and reducing the content of free water, thereby improving the stress resistance and physiological stability of Chinese cabbage under cadmium stress and demonstrating its positive role in alleviating cadmium toxicity.

[0077] (viii) Leaf water binding morphology Plant hormones are key signaling substances regulating growth, development, and stress response. Zeatin (ZR), auxin (IAA), and gibberellin (GA3) promote growth, while abscisic acid (ABA) accumulates under stress to enhance resistance. The balance among hormones, especially the ratio of ABA to growth-promoting hormones, reflects the trade-off between growth and stress resistance in plants. Investigating the effects of Moringa polysaccharides on hormone content and ratios in pakchoi leaves under cadmium stress helps to elucidate the mechanism by which it regulates cadmium tolerance through endogenous hormone signaling.

[0078] Table 8 Effects of Moringa polysaccharide treatment on hormone content in Chinese cabbage leaves MOP concentration (mg / L) ZR(ng / g FW) ABA(ng / g FW) IAA(ng / g FW) GA3(ng / g FW) ABA / GA ABA / IAA ABA / ZR MOP0 3.963±0.116b 68.21±1.96c 15.12±0.39d 2.751±0.053b 0.481±0.009b 0.479±0.010b 0.535±0.013b MOP15 3.725±0.081c 79.29±2.58b 17.64±0.41b 2.588±0.068c 0.419±0.014c 0.451±0.009c 0.464±0.016c MOP30 4.919±0.131a 92.43±2.74a 16.37±0.61c 2.622±0.054c 0.367±0.009d 0.383±0.012d 0.454±0.011c MOP60 4.122±0.125b 61.74±1.76d 18.99±0.54a 3.040±0.091a 0.535±0.015a 0.560±0.012a 0.583±0.017a Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). Table 8 shows that different concentrations of Moringa polysaccharides significantly affected the content and balance of endogenous hormones in pak choi leaves. The stress-resistance hormone ABA was highest in the MOP30 treatment (92.43±2.74 ng / g), an increase of 35.5% compared to the control (P<0.05), and also increased by 16.3% in the MOP15 group, while significantly decreasing in the MOP60 group. Regarding growth hormones, ZR was highest in the MOP30 group (4.919±0.131 ng / g), an increase of 24.1% compared to the control; IAA and GA3 reached 18.99±0.54 ng / g and 3.04±0.09 ng / g respectively in the MOP60 group, showing a promoting trend. Regarding hormone ratios, the ABA / GA3, ABA / IAA, and ABA / ZR ratios in the MOP30 group were 0.367, 0.383, and 0.454, respectively, significantly lower than the control by 23.7%, 20.0%, and 15.1%, indicating a relatively optimal balance among hormones at this concentration. In summary, 30 mg / L Moringa polysaccharide significantly increased ABA levels and enhanced stress resistance while promoting ZR synthesis, maintaining a coordinated relationship between growth-promoting and stress-resistance hormones. This enabled the plants to maintain both growth vigor and strong resistance under cadmium stress, demonstrating its synergistic regulatory role in regulating the plant growth-stress balance.

[0079] (ix) Mineral elements in the aboveground parts of the plant Mineral elements are fundamental to plant growth and stress resistance, and their absorption and distribution directly affect nutritional status and metabolic balance. Macroelements such as nitrogen, phosphorus, potassium, calcium, magnesium, and sulfur participate in basic metabolism, while microelements such as iron, manganese, copper, and zinc are components or activators of many enzymes. Cadmium stress competes with essential elements for absorption sites, disrupting mineral balance. Studying the effects of Moringa polysaccharides on the mineral element content of the aboveground parts of pakchoi helps to reveal its mechanism of action in alleviating cadmium toxicity and maintaining normal physiological functions by regulating nutrient absorption.

[0080] Table 9. Effects of Moringa Polysaccharide Treatment on Mineral Element Content in the Aboveground Parts of Chinese Cabbage Plants MOP concentration (mg / L) N (mg / g) P (mg / g) K (mg / g) Ca (mg / g) Mg (mg / g) S(mg / g) Fe(μg / g) Mn(μg / g) Cu(μg / g) Zn(μg / g) MOP0 48.13±5.076c 4.341±0.273b 42.868±6.272a 26.61±3.221a 8.115±0.944b 10.78±1.265a 3316±527 a 204±26.7a 691±94.4a 164±22.4a MOP15 54.14±1.371b 4.996±0.378a 39.924±6.585a 28.45±1.516a 8.775±0.298ab 10.12±1.037a 655±109b 170±20.1ab 68.7±9.78b 157±15.8a MOP30 58.43±0.982a 4.627±0.41ab 44.149±6.931a 28.64±4.395a 8.766±0.771ab 11.18±0.991a 133±19.9c 164±22.3b 35.9±3.08b 151±17.2a MOP60 59.01±1.370a 5.097±0.204a 48.334±3.661a 29.62±1.967a 9.562±0.578a 11.03±0.521a 114±4.37c 150±17.2b 32.9±3.59b 146±14.1a Note: Different lowercase letters in the same column indicate significant differences (P<0.05, Duncan's test). Table 9 shows that Moringa polysaccharide treatment significantly improved the accumulation of mineral elements in the aboveground parts of *Pakchoi blakeana* under cadmium stress. Regarding macroelements, nitrogen (N) content increased significantly with increasing concentration, reaching 59.01±1.37 mg / g in the MOP60 and MOP30 groups, and 58.43±0.982 mg / g in the MOP30 and MOP40 groups, respectively, representing increases of 22.6% and 21.4% compared to the control. P content significantly increased by 15.1% and 17.4% in the MOP15 and MOP60 groups, respectively. The contents of potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S) generally showed an upward trend, with magnesium (Mg) showing a significant increase of 17.8% in the MOP60 group. Regarding microelements, fecal iron (Fe) content decreased significantly, dropping to 133±19.9 μg / g in the MOP30 and MOP60 groups, and 114±4.37 μg / g in the MOP60 and MOP40 groups, respectively, a decrease of approximately 96% compared to the control. Mn content decreased by 26.3% in the MOP60 group, and Cu content decreased significantly by over 90% in all treatments, while Zn content showed no significant change. The significant decrease in Fe, Mn, and Cu may be due to Moringa polysaccharides reducing the non-selective absorption of divalent metal ions by plants, thereby indirectly inhibiting cadmium entry. Comprehensive analysis indicates that 30–60 mg / L Moringa polysaccharides can optimize mineral element balance, promote the absorption of macronutrients to maintain growth, regulate micronutrient absorption, mitigate cadmium toxicity, and enhance the nutritional homeostasis and resistance of pak choi under cadmium stress.

[0081] (x) Comprehensive Discussion This study shows that rhizosphere irrigation with Moringa polysaccharides effectively alleviates cadmium stress and promotes healthy growth of Chinese cabbage through multiple synergistic mechanisms, and exhibits a significant concentration effect.

[0082] 1. Concentration Effect (Examples 2, 3, and 4). 30 mg / L Moringa polysaccharide (MOP30) exhibited the optimal combined effect of promoting growth and inhibiting cadmium. Its aboveground biomass significantly increased, with SOD and CAT activities increasing by 242.7% and 194.2%, respectively, MDA content decreasing by 33.6%, and hydroxyl radical scavenging rate significantly increasing by 30.1%, significantly enhancing the antioxidant defense system. Simultaneously, aboveground cadmium content decreased by 26.5%, and the cadmium transfer coefficient decreased by 19.0%, achieving a dual effect of promoting growth and inhibiting cadmium. Treatment with 15 mg / L (MOP15) showed some growth-promoting effect, but its cadmium-inhibiting effect was weak; treatment with 60 mg / L (MOP60) showed inhibition in some indicators, such as a decrease in SOD and CAT activities, indicating that excessively high concentrations may interfere with normal metabolism. In summary, 30 mg / L is the recommended concentration.

[0083] 2. Summary of Mechanism of Action. Moringa polysaccharides exert their cadmium-blocking effect through multiple pathways. First, they improve the rhizosphere environment, significantly increasing the available phosphorus content in the soil (↑42.7%), and reducing cadmium availability through phosphorus-cadmium antagonism. Second, they regulate the antioxidant system, significantly activating the activity of enzymes such as SOD and CAT, enhancing free radical scavenging capacity, reducing membrane lipid peroxidation, and protecting cell membrane structure. Third, they regulate water and hormone balance, increasing the proportion of bound water (↑23.0%), promoting the coordinated accumulation of ABA and Zr, optimizing the ABA / GA3 and ABA / IAA ratios, and maintaining a balance between growth and stress resistance. Fourth, they optimize the absorption of mineral elements, promoting the accumulation of essential elements such as N, P, and Mg, inhibiting the absorption of divalent ions such as Fe and Cu, and reducing the competitive absorption of cadmium.

[0084] 3. Key findings. (1) Moringa polysaccharides have both growth-promoting and cadmium-inhibiting functions, overcoming the limitations of traditional cadmium inhibitors that "reduce cadmium but inhibit growth"; (2) The rhizosphere is the main site of action, achieving targeted regulation by improving the soil chemical environment and nutrient status; (3) A reasonable concentration is the key to achieving efficient cadmium inhibition and safe production, with 30 mg / L being the optimal concentration for comprehensive effect. This study reveals the potential application value of moringa polysaccharides in enhancing plant cadmium tolerance and ensuring vegetable quality and safety through a multidimensional synergistic mechanism of "soil-physiology-molecule".

[0085] (xi) Comparative verification To systematically verify the necessity of the composite stabilizer system in the cadmium-inhibiting preparation of Moringa polysaccharide and the rationality of the process parameters, six sets of comparative experiments were set up. The experimental design referred to the results and theoretical analysis of Examples 1–4 (MOP0, MOP15, MOP30, MOP60), with MOP30 (30 mg / L) as the best example for comparison. The results are summarized in Table 10.

[0086] Table 10 Summary of Comparative Experiment Results Processing group Cadmium content in aboveground parts (mg / kg DW) Root cadmium content (mg / kg DW) Fresh weight of above-ground parts (g) Fresh weight of roots (g) Remark Example 1 (CK) 1.72±0.13a 0.86±0.01a 15.5±1.32a 0.48±0.04a Moringa polysaccharide-free, stabilizer only Example 3 (MOP30) 1.26±0.06b 0.78±0.02c 15.1±1.47a 0.54±0.07a The cadmium-blocking effect was optimal, resulting in a cadmium reduction of 26.7%. Comparative Example 1 (without stabilizer) 1.69±0.06 0.85±0.02 13.2±0.5 0.35±0.03 Unprotected polysaccharide degradation Comparative Example 2 (Sodium Alginate Only) 1.83±0.07 0.88±0.03 13.9±0.8 0.42±0.04 Poor dispersibility and deteriorated cadmium inhibition effect Comparative Example 3 (Nano Silica Only) 1.68±0.05 0.83±0.02 11.1±0.7 0.31±0.03 Insufficient duration of action, growth inhibited Comparative Example 4 (Scale 1:1.0) 1.75±0.22 0.82±0.03 15.0±0.8 0.47±0.04 The effects were unstable, with a coefficient of variation of 14.8%. Comparative Example 5 (ratio 1:1.5) 1.79±0.06 0.83±0.02 11.0±0.7 0.33±0.03 Excessive nanoparticles inhibit growth Comparative Example 6 (concentration 70 mg / L) 1.77±0.05 0.83±0.02 14.1±0.7 0.44±0.04 High viscosity, poor permeability Note: Data are mean ± standard deviation (n=3). Different lowercase letters in the same column indicate significant differences (p<0.05, Duncan test).

[0087] Comparative analysis: Comparative Example 1 (Example 5): Pure Moringa Polysaccharide Preparation (without stabilizer protection). This group was treated with only Moringa polysaccharide, without any stabilizers. The cadmium content in the aerial parts was 1.69 mg / kg DW, 34.1% higher than that in the MOP30 group, and the cadmium content in the roots was 0.85 mg / kg DW, 9.0% higher than that in the MOP30 group; the fresh weight of the aerial parts and roots decreased by 12.6% and 35.2%, respectively. The results indicate that unstabilized Moringa polysaccharide is easily degraded and inactivated in the rhizosphere environment, and its cadmium inhibition and growth-promoting effects are significantly weakened, indicating that the introduction of stabilizers is crucial for maintaining the effectiveness of the active ingredients.

[0088] Comparative Example 2 (Example 6): Sodium alginate as a single stabilizer. When sodium alginate was used alone as a stabilizer, the cadmium content in the aboveground parts reached 1.83 mg / kg DW, which was 6.4% higher than the control (CK), indicating that its use alone could not provide effective dispersion and protection. The cadmium content in the roots was also high, with a fresh weight decrease of about 10%. These results indicate that although sodium alginate has a certain encapsulation effect, it is difficult to form a stable and sustained dispersion system, leading to a deterioration in cadmium inhibition performance.

[0089] Comparative Example 3 (Example 7): Single nano-silica stabilizer. The cadmium content in the aboveground parts of this group was 1.68 mg / kg DW, and in the roots it was 0.83 mg / kg DW, with a significant decrease in fresh weight (28.4% decrease in aboveground parts and 35% decrease in roots). This indicates that while nano-silica alone has certain adsorption and structural stabilizing effects, it lacks polysaccharide protection and slow-release regulation, has a short duration of effectiveness, and exerts stress on plant growth.

[0090] Comparative Example 4 (Example 8): Lower limit of stabilizer ratio (1:1.0). When the ratio of sodium alginate to nano silica is 1:1.0, the cadmium content in the aboveground parts is close to that of the control (CK) (1.75 mg / kg DW), but the coefficient of variation between replicates is as high as 14.8%, indicating poor consistency in effect. This suggests that the system is not stable enough at this ratio, and the composite dispersed phase structure is prone to uneven aggregation, resulting in large fluctuations in the cadmium inhibition effect.

[0091] Comparative Example 5 (Example 9): Upper limit of stabilizer ratio (1:1.5). The cadmium content in the aboveground parts was 1.79 mg / kg DW, and the fresh weight was significantly reduced to 11.0 g. This indicates that excessive inorganic particles can cause rhizosphere ion stress and pore blockage, affecting root absorption and growth, leading to weakened or even inhibited growth-promoting function.

[0092] Comparative Example 6 (Example 10): When the total concentration of stabilizer was increased to 70 mg / L, the cadmium content in the aboveground parts rose to 1.77 mg / kg DW, the cadmium inhibition effect decreased significantly, and the fresh weight of the aboveground parts dropped to 14.1 g. The main reason is that the viscosity of the high-concentration system increases and the permeability decreases, the rhizosphere absorption channels are blocked, and the active substances cannot fully act on the root surface area.

[0093] Overall Conclusion The systematic validation results of Comparative Examples 1–6 fully demonstrate that the composite stabilizer system plays a crucial role and has significant scientific rationality in the cadmium-inhibiting formulation of Moringa polysaccharide. First, compared with the control group (CK, without Moringa polysaccharide), the 30 mg / L Moringa polysaccharide treatment (MOP30) significantly reduced the cadmium content in the aboveground parts by 26.7% (from 1.72 mg / kg to 1.26 mg / kg) and the cadmium content in the roots by 9.3% (from 0.86 mg / kg to 0.78 mg / kg). Simultaneously, the fresh weights of the aboveground parts and roots were 15.1 g and 0.54 g, respectively, with no significant inhibition observed, indicating that the system can achieve the ideal effect of "reducing cadmium without reducing yield." Second, without stabilizer protection (Comparative Example 1), Moringa polysaccharide rapidly degrades in the soil environment, with the cadmium content in the aboveground parts increasing by 34.1% and the cadmium content in the roots increasing by 9.0% compared to MOP30, while the fresh weight decreased by 12–35%, indicating that stabilization protection is crucial for maintaining the effectiveness of the active ingredient. When sodium alginate or nano-silica was used as a single stabilizer (Comparative Examples 2 and 3), the system lacked sufficient dispersibility or slow-release properties, resulting in an increase in cadmium content in the aboveground parts (1.68–1.83 mg / kg) and inhibited plant growth. This indicates that the two stabilizers need to work synergistically to achieve the dual functions of "dispersion stabilization + slow-release protection". Furthermore, when the stabilizer ratio was low (1:1.0, Comparative Example 4), the cadmium inhibition effect fluctuated significantly (coefficient of variation 14.8%), indicating insufficient system stability. Conversely, when the ratio was high (1:1.5, Comparative Example 5) or the total stabilizer concentration was too high (70 mg / L, Comparative Example 6), excessive nanoparticles caused rhizosphere ion stress and decreased permeability, leading to a decrease in plant biomass and a deterioration in cadmium inhibition performance.

[0094] Comprehensive analysis shows that the composition, ratio, and concentration design of the composite stabilizer system have a clear scientific basis and synergistic effect. Sodium alginate, as a gel carrier, provides sustained-release protection, while nano-silica enhances dispersion and structural stability. Together, they form an orderly dispersed polysaccharide sustained-release barrier in the rhizosphere, effectively reducing cadmium absorption and migration while maintaining normal plant physiological activity. The optimal application concentration was determined to be 30 mg / L, and the optimal stability range was a composite ratio of 1:1.2 ± 0.2. In summary, the Moringa polysaccharide composite stabilizer system, through a synergistic mechanism of "organic polysaccharide-inorganic carrier," achieves stable, sustained-release, and targeted effects of active substances, significantly reducing cadmium accumulation in plants while maintaining normal growth and development. This provides a solid scientific basis and application support for achieving efficient, green, and sustainable cadmium-blocking technology for crop cadmium pollution control.

[0095] (xii) Technical significance The verification results of the above examples show that sodium alginate and nano-silica must work synergistically within a specific mass ratio (1:1–1.5) and concentration range (20–60 mg / L) to achieve stable dispersion, sustained-release protection, and efficient delivery of Moringa polysaccharides. The results of Comparative Examples 2–6 fully demonstrate that using any single stabilizer component (Examples 6–7) or deviating from the optimal ratio range (Examples 8–10) significantly reduces the dispersion stability, duration of action, or bioavailability of the formulation, verifying the necessity and inventiveness of the composite stabilizer system design in this invention. The composite stabilizer-type Moringa polysaccharide cadmium-inhibiting formulation prepared by this invention exhibits good application effects within the process parameters defined in the claims (moringa polysaccharide concentration 15–60 mg / L [preferred value 30 mg / L], rhizosphere application rate 40–60 mL / plant [preferred value 50 mL / plant], application interval 3–7 days [preferred value 5 days]). This formulation can significantly reduce the cadmium content in the aboveground parts of Chinese cabbage (by up to 26.7%) while maintaining stable or slightly increasing biomass, achieving a technological breakthrough of "cadmium inhibition without growth suppression." Moringa polysaccharides, under the protection of a compound stabilizer, can form an orderly and dispersed slow-release barrier in the rhizosphere, continuously regulating the migration and absorption of cadmium ions, activating the antioxidant enzyme system, and improving cell membrane stability, thereby achieving multiple synergistic effects of cadmium inhibition, stress resistance, and growth promotion. This invention provides a scientific, green, and scalable solution for safe production in farmland with mild to moderate cadmium pollution, and has significant application value and promotion prospects for achieving cadmium pollution control in agricultural products and sustainable agricultural development.

[0096] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A composite stabilized Moringa polysaccharide cadmium-inhibiting formulation, characterized in that, It consists of the following components: Moringa polysaccharide, with a mass-volume concentration of 15-60 mg / L; a compound stabilizer, with a mass-volume concentration of 20-60 mg / L, composed of sodium alginate and nano-silica in a mass ratio of 1:(1-1.5), used to enhance the dispersion stability and sustained-release effect of moringa peptides; and phosphate buffer, used to adjust the pH value to 6.0-7.0 to maintain the stability of the formulation in different soil environments.

2. The composite stable Moringa polysaccharide cadmium-inhibiting formulation according to claim 1, characterized in that, The mass-volume concentration of the moringa polysaccharide is 30 mg / L.

3. The composite stable Moringa polysaccharide cadmium-inhibiting formulation according to claim 1, characterized in that, The composite stabilizer has a sodium alginate to nano silica mass ratio of 1:1.2 and a total mass-volume concentration of 35 mg / L.

4. A method for preparing the composite stable Moringa polysaccharide cadmium-inhibiting formulation according to claim 1, 2, or 3, characterized in that, Includes the following steps: S1: Dissolve sodium alginate in phosphate buffer and stir until completely dissolved; add nano silica and sonicate until a gel is formed; S2: Moringa polysaccharide powder is added to the gel obtained in step S1 and homogenized to obtain a cadmium-inhibiting agent.

5. The preparation method according to claim 4, characterized in that, In step S1: The dissolution temperature of the sodium alginate is 50-70℃; the ultrasonic conditions are: power of 300-500W, time of 15-30min, and temperature controlled at 4-5℃ during ultrasonic treatment.

6. The preparation method according to claim 4, characterized in that, In step S2, the homogenization conditions are as follows: under light-protected conditions, first stir for 40-45 minutes, then homogenize at 4-5℃ and a rotation speed of 10000-12000 r / min for 20-30 minutes.

7. The preparation method according to claim 4, characterized in that, The particle size of the nano-silica is 20-30 nm.

8. The application of the cadmium-blocking agent according to claim 1 or 2 in reducing cadmium absorption by cabbage grown in cadmium-contaminated soil.

9. A method for reducing cadmium absorption by cabbage grown in cadmium-contaminated soil, characterized in that, The procedure includes the following steps: starting on the third day after thinning of the cabbage seedlings, apply the cadmium inhibitor as described in claim 1 or 2 to the root zone.

10. The method according to claim 9, characterized in that, The irrigation conditions are as follows: 40-60 mL of the cadmium inhibitor is applied to each cabbage plant, once every 3-7 days, for a total of 3-5 applications. The cadmium-contaminated soil is alkaline and slightly contaminated, with a soil pH of 6.5-7.5 and a total cadmium content of 0.8-2.0 mg / kg.