Insecticidal Activity and Insecticidal Mechanism of Tea Saponins from Camellia oleifera

Chemical pesticides are commonly used during the cultivation of agricultural products to control pests and diseases. Excessive use of traditional pesticides can cause environmental and human health risks. There are ongoing searches for new plant-derived pesticides to reduce the use of chemical pesticides. In this study, tea saponin extracts of different purities were extracted from Camellia oleifera seeds using AB-8 macroporous resin and gradient elution with ethanol. The insecticidal effects of the tea saponin extracts were evaluated by contact toxicity tests and stomach toxicity tests using the lepidopteran pest of tea plantation, Ectropis obliqua. The total saponins extracted using 70% ethanol showed strong contact toxicity (LC50 = 8.459 mg/L) and stomach toxicity (LC50 = 22.395 mg/L). In-depth mechanistic studies demonstrated that tea saponins can disrupt the waxy layer of the epidermis, causing serious loss of water, and can penetrate the inside of the intestine of E. obliqua. After consumption of the tea saponins, the intestinal villi were shortened and the cavities of the intestinal wall were disrupted, which resulted in larval death. This study highlights the potential of tea saponins as a natural, plant-derived pesticide for the management of plant pests.

Tea is one of the most popular non-alcoholic beverages in the world today, favored for its unique aroma and taste [1]. Ectropis obliqua Prout (Lepidoptera:Geometridae) is a chewing defoliator in tea plantations, causing devastating effects on the quality, yield, and growth of tea plants [2,3]. Because of its fast breeding, rapid spatial spread, and large appetite, outbreaks of this insect have resulted in losses of more than 60% in tea production [4]. E. obliqua is the primary target pest in tea cultivation [5].

At present, E. obliqua is mainly controlled by chemical pesticide spraying [6]. Tea is a direct receptor of aerial pesticides, and pesticide residues may cause food safety and other issues. At the same time, studies have shown that only a small amount (about 0.3%) of pesticides can enter the target organism and that most pesticides (99.7%) will eventually enter the environment, posing environmental safety hazards [7]. International trade standards demand higher quality agricultural products and set maximum residual levels of pesticides in tea products. Plant-derived products can be more environmentally friendly and can represent cost-effective alternatives to control phytophagous insects and plant-pathogenic microorganisms. Therefore, biological control of this pest has become the method of choice [5].

Tea saponins (TS) are a promising bio-pesticide with good dispersibility, permeability and wetting effect [8]. Tea saponin content in the seeds of Camellia oleifera is greater than 10%. The annual output of C. oleifera has exceeded 2 million tons, and about 200,000 tons of saponins can be supplied for use [9]. Previous studies have found that tea saponins have a strong insecticidal effect on the diamondback moth, Plutella xylostella, and aphid, Aphis craccivora [10]. In a study on saponin repellent activity, saponins at 4000 mg/L highly repel (48.57%) the third instar of P. xylostella. However, the feeding preference index (PI) of the third instar for saponins was less at higher concentrations (0.63). Treatment of diamondback moth larvae with LC20 and LC50 doses of TS led to lower growth rates, decreased feed consumption, reduced frass production, lower pupal weights, reduced percentage pupation, slower adult emergence, and diminished fecundity, but prolonged durations of the larval and pupal periods [10].

Most current research indicates that the insecticidal mechanism of tea saponins is related to effects on the detoxification enzymes of insects. Tea saponins can reduce superoxide dismutase (SOD), catalase (CAT), acetylcholinesterase (AChE), and carboxylesterase (CES) activities [11,12]. Some studies suggest that the insecticidal activity of saponins is due to their interaction with cholesterol, which leads to interference with the synthesis of ecdysteroids. These substances are also protease inhibitors or cytotoxic to certain insects [13]. However, the specific insecticidal mechanism has not been reported.

Tea saponins were extracted from Camellia oleifera seeds using ethanol and then purified using AB-8 macroporous resin and a gradient ethanol elution. The obtained eluates were subjected to thin-layer chromatography in combination with a sulfuric-acid–ethanol color reaction (Figure S1). The purple color indicating saponins was observed in different eluents. Of all the eluates, 70% ethanol eluate (EE) did not have a chromogenic reaction of flavonoids, which demonstrates the higher purity of the saponin in the 70% ethanol eluate.

The extracted tea saponins were analyzed by HPLC (Figure S2). Since there are no commercial Camellia saponin standards, the total saponin content was quantified using an individual oleiferasaponin C1 standard, as described previously [14]. The oleiferasaponin C1 standard curve was expressed as y = 0.6938x − 1.5247 (R2 = 0.9999), showing a linear correlation over the concentration range of 0.005 to 1.0 mg/mL.

The purity of the saponins extracted using different ethanol–water eluents was tested using a standard multitest method. The purity of the saponins in the C. oleifera seed cake ethanol extract (CSCEE) was 39.5% ± 3.46%; in the 30% EE, the purity was 35.9% ± 2.31%; in the 50% EE, the purity was 55.6% ± 1.69%; in the 70% EE, the purity was 99% ± 0.71%.

UPLC-Q-TOF/MS analysis was used to characterize the saponins in the 70% EE. The eluate contained 29 triterpenoid saponins (Figure S3). The molecular formulas and molecular weights of these saponins were confirmed by high-resolution mass measurements, deprotonated molecular ions [M − H]−, isotope abundance for each pseudomolecular ion, and fragment ions (Tables S1 and S2). The identification of each saponin was based on published fragmentation data and nominal masses calculated from known structures [8,14].

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