Solvent Fractionation and LC-MS Profiling, Antioxidant Properties, and α-Glucosidase Inhibitory Activity of Bombyx batryticatus

Abstract

Bombyx batryticatus is the dried body of silkworm (Bombyx mori Linnaeus) larvae infected with Beauveria bassiana. It is widely used in traditional Chinese medicine for treating convulsions, epilepsy, and hyperglycemia. In this study, Bombyx batryticatus and its extract were prepared. The total reducing power, hydroxyl radical scavenging and superoxide anion radical scavenging activities, as well as the α-glucosidase inhibitory activity of Bombyx batryticatus extract were superior to those of normal silkworm larvae extract. Among them, the IC₅₀ value of Bombyx batryticatus extract for α-glucosidase was 5.76 mg/mL, while that of normal silkworm larvae extract was 7.0 mg/mL. Untargeted metabolomic analysis was employed to compare the material composition of normal silkworm larvae and Bombyx batryticatus. The results revealed 101 metabolic differences between the two groups, including a significant increase in fatty acids and their derivatives in the Bombyx batryticatus extract. Further separation and purification of the Bombyx batryticatus extract were performed using solvents of varying polarity. The chloroform fraction exhibited the highest inhibitory activity against α-glucosidase, with an IC₅₀ value of 0.217 mg/mL. LC-MS further identified compounds in the chloroform fraction, suggesting that those alkaloids, fatty acids, and their derivatives may be responsible for its strong α-glucosidase inhibitory activity. This study elucidates the material basis underlying the pharmacological effects of Bombyx batryticatus, particularly its hypoglycemic components, thereby providing critical experimental support for its future development and application in medicine.

1. Introduction

In recent years, the rapid development of the global economy has led to significant changes in diet and lifestyle, contributing to a sharp rise in the global incidence of diabetes. Diabetes mellitus (DM) is a chronic metabolic disease primarily characterized by hyperglycemia, which results from either defective insulin secretion or insulin resistance [1]. According to the International Diabetes Federation (2021), the number of adults with diabetes worldwide reached 537 million in 2021, and this number is expected to rise to 783 million in 2045. Diabetes is often associated with hyperlipidemia, which significantly increases the risk of cardiovascular disease, neurodegenerative disease, kidney injury, and cancer [2]. Therefore, diabetes has become a critical global public health issue that requires urgent attention and management.

The search for anti-diabetic drugs derived from local natural resources, especially traditional drugs, has been pursued in many regions around the world, yielding achieved promising results. These natural resources typically contain bioactive substances with good antioxidant capacity, which can exert beneficial effects by neutralizing reactive oxygen species (ROS), enhancing endogenous antioxidant defense, and regulating key signaling pathways involved in glucose and lipid metabolism [3,4]. Ighodaro et al. [5] reported that extracts of Sapium ellipticum Pax leaf from Africa were rich in antioxidant compounds such as amentoflavone, lupeol, and luteolin-7-O-glucoside and exhibited hypoglycemic effects in rats. Acalypha hispida leaves from Indonesia, rich in antioxidant polyphenols, also showed hypoglycemic effects in rats [6]. Additionally, these natural resources may contain compounds that inhibit the activity of α-glucosidase. α-Glucosidase is one of the target enzymes of clinical hypoglycemic drugs and can delay the digestion and absorption of carbohydrates, thereby lowering postprandial blood glucose levels. For example, 1-deoxynojirimycin (1-DNJ) was screened from mulberry leaves due to its strong α-glucosidase inhibitory activity, which is now recognized as a hypoglycemic functional agent. Therefore, screening for anti-diabetic drugs from natural resources based on antioxidant and α-glucosidase inhibition activity has become a promising strategy [7,8]. These traditional medicines or natural resources are becoming supplements or substitutes for daily hypoglycemic drugs and also an important source of developing new anti-diabetes drugs.

Bombyx batryticatus (B. batryticatus), also known as stiff silkworm, is a traditional medicine used in China, South Korea, and Japan. According to the Compendium of Materia Medica from the Ming Dynasty in China, B. batryticatus was used as a remedy to treat conditions such as convulsions, epilepsy, sore throats, persistent headaches, and traumatic infections. B. batryticatus is the dried body of the silkworm (Bombyx mori Linnaeus) that forms following infection with Beauveria bassiana (B. bassiana) in the fourth–fifth instar larvae stages of the silkworm. B. bassiana secretes various hydrolytic enzymes, including lipases, proteases, and chitinases, which actively invade the silkworm’s body and proliferate within the larvae’s body cavities. Through competition for nutrients, interference with metabolism, and the secretion of toxins, the infection ultimately results in the death of the host [9]. Once the body of the dead silkworm is covered with white mycelium and conidia, the body is dried and processed into the final B. batryticatus product. Silkworm larvae are recognized as unique anti-diabetic agents and hypoglycemic health foods in Asia [10,11]. It is widely believed that silkworms exhibit significant hypoglycemic activity through the enrichment of 1-DNJ, flavonoids, and phenolic compounds through the consumption of mulberry leaves [10,12]. Compared to silkworms, the phenolic compound and fatty acid profiles of B. batryticatus have undergone alterations [13], contributing to its demonstrated anticonvulsant, antithrombotic, antiepileptic, and neuroprotective effects [14,15,16]. Recently, in the clinical applications of traditional Chinese medicine, B. batryticatus has been regarded as a valuable ingredient in the treatment of diabetes, dyslipidemia, and nephrotic syndrome. However, the material basis underlying the hypoglycemic effect of B. batryticatus remains unclear. Therefore, this study evaluated the anti-diabetic potential of B. batryticatus by assessing its antioxidant capacity and α-glucosidase inhibitory activity and further attempted to identify the bioactive components contributing to this potential. The results of this study will provide experimental evidence for the practical application of B. batryticatus in hypoglycemic agents and support its future use in preventing and treating diseases associated with oxidative stress, including anti-aging and neuroprotection.

2. Results and Discussion

2.1. Morphological Changes During Silkworm Infection with B. bassiana

Healthy fifth instar silkworm larvae were inoculated with the conidiospores of B. bassiana, and, by the third day, the infected larvae exhibited a loss of appetite and slow movement. The surface color of the infected larvae darkened compared to that of healthy larvae, and oil spots gradually appeared on the epidermis (Figure 1A). The observation of hemolymph samples under an inverted microscope (CKX-31, Olympus Co., Ltd., Tokyo, Japan) revealed the presence of hyphae and blastospores in the hemolymph of the infected larvae (Figure 1B), while the hemolymph of healthy larvae remained clear (Figure 1C). These findings indicate that the conidiospores of B. bassiana successfully infected and formed hyphae in the hemolymph of the infected larvae, and the rapid growth of the hyphae consumed water and nutrients in the hemolymph, which is a key factor contributing to the color changes in the epidermis [17]. On the fifth day of infection, a large number of infected larvae died, and the deceased larvae were collected and kept at room temperature for further observation (Figure 1D). Initially, the bodies of the just deceased larvae were soft with oil stains on the surface. Over time, the bodies gradually stiffened, and mycelia emerged from the cuticle junctions. On the third day after death, the surface of the stiffened silkworm was completely covered by white mycelium. Spores of B. bassiana then gradually formed. As storage time progressed, the silkworm bodies gradually became dry and hard, and their weight continuously decreased until the ninth day, reaching 18% of the original fresh weight (Figure 1E). Eventually, the bodies became rigid structures with a grayish-white or grayish-yellow surface, exhibiting a curved and wrinkled appearance.

2.2. The Antioxidant Activity of B. batryticatus Extract

To comprehensively evaluate the antioxidant activities of B. batryticatus extracts (80% methanol), various in vitro testing systems were used in this study (Figure 2). The value of total reducing power indicates the antioxidant potential to participate in free radical reactions and combine with free radicals to generate stable substances. As the extract concentration increased, the total reducing power of B. batryticatus also increased. The total reducing power reached 0.556 when the extract concentration was 25 mg/mL. Compared with the extract from healthy larvae, the total reducing power of B. batryticatus extract was 10% higher. At a concentration of 50 mg/mL, the total reducing power of B. batryticatus extract was 38% higher than that of healthy larvae extract (Figure 2A). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a substance containing stable nitrogen radicals, and its ethanol solution is dark purple with a strong absorption peak at 517 nm. Free radical scavengers donate hydrogen atoms to react with DPPH free radicals, reducing them to colorless 2,2-diphenyl-1-pyridylhydrazine, thereby decreasing the absorbance value at 517 nm. The EC₅₀ (half maximal effective concentration) of B. batryticatus extract was 1.73 mg/mL, while that of the healthy larvae extract was 0.91 mg/mL (Figure 2B). Hydroxyl radicals and superoxide anions are both highly harmful reactive oxygen species to living organisms. In the human body, they attack biomolecules, leading to diseases caused by oxidative stress. The inhibitory ability of B. batryticatus extract on hydroxyl radicals and superoxide anions was much higher than that of healthy larvae extract. The EC₅₀ of B. batryticatus extract for hydroxyl radials was 3.28 mg/mL, while the value for the healthy larvae extract was 11.66 mg/mL. The EC₅₀ of B. batryticatus extract for superoxide anions was 29.40 mg/mL, while that of the healthy larvae extract was 47.25 mg/mL (Figure 2C,D).

It was reported that the 80% methanol extract of freeze-dried powder from the fifth instar silkworm larvae had a high DPPH radical scavenging capacity (137.22 μM of α-tocopherol/g), with the total reducing power of 19.55 μM of Fe(II)/g [18]. The DPPH free radical scavenging ability of silkworm larvae powder from different strains ranges from 20.97% to 58.96%, and the Ferric ion reducing antioxidant power (FRAP) ranges from 1.52 to 2.03 mg Fe(II)/g [19]. The antioxidant capacity of silkworm larvae powder is primarily attributed to the presence of flavonoids [19]. Additionally, silkworm larvae contain unsaturated fatty acids [20] such as oleic acid, linolenic acid, arachidonic acid, docosahexaenoic acid, and bioactive polysaccharides [21], which is also the reason why silkworm powder has antioxidant capacity.

In this study, the antioxidant capacity of B. batryticatus extract was higher than that of healthy larvae extract. This may be attributed to the secretion of hydrolytic enzymes by B. bassiana in silkworm larvae, which produces more free fatty acids, free amino acids, and peptides. Cermeño reported that the FRAP values of silkworm pupae increased after enzymatic hydrolysis [22]. Bae et al. treated silkworm powder with proteolytic enzymes, resulting in an increase in the content of free amino acids and flavonoid, and an increase in the DPPH free radicals scavenging abilities [23]. On the other hand, B. bassiana is an entomopathogenic fungus. The interaction between B. bassiana and its host produces a series of bioactive compounds, including benzene ring derivatives (e.g., salicylaldehyde), terpenes (e.g., myrcene), benzoquinones (e.g., oosporein), unsaturated fatty acids, amides, lactones, and antimicrobial peptides [24]. These compounds interact with free amino acids, fatty acids, carbohydrates, and other biomolecules to form more complex secondary metabolites. Chemical structure analyses have shown that compounds containing unsaturated bonds and phenyl groups typically exhibit antioxidant activity [25,26]. These compounds can eliminate free radicals, regulate antioxidant enzyme activity, or reduce inflammatory mediators, thereby mitigating oxidative stress. Additionally, the biotransformation of flavonoids may also influence the antioxidant capacity of the extracts. It has been reported that B. bassiana can transform quercetin into quercetin-7-O-β-d-4-O-methylglucoside and can also transform kaempferol into kaempferol-7-O-β-d-4-O-methylglucoside [13]. The derivatization of quercetin (and kaempferol) hydroxyl groups reduced the antioxidant activity of their derivatives [27]. The fermentation by B. bassiana produces quercetin 7-O-β-D-(4″-O-methyl) glucopyranoside, which can effectively protect lipid membranes against peroxidation while exhibiting slightly lower antioxidant activity than quercetin [28]. However, the combination of flavonoid glycosides with methyl and sugar groups increases the absorption of flavonoids, leading to an increase in their bioavailability and biological activity [29]. In summary, when healthy silkworm larvae were transformed into B. batryticatus, the metabolic profiles changed, leading to a significant increase in antioxidant components and enhanced antioxidant capacity. Abdel-Daim et al. reported that compounds with antioxidant capacity provide multiple health benefits in preventing and treating disease [30]. Compared with healthy silkworm larvae, B. batryticatus demonstrated potential for more effective oxidative stress resistance and the prevention of metabolic and neurodegenerative disorders, as revealed by our findings.

2.3. The Inhibitory Activities of B. batryticatus Extract on α-Glucosidase and Pancreatic Lipase

α-Glucosidase is a key enzyme that regulates postprandial blood glucose levels. Inhibiting this enzyme reduces carbohydrate absorption in the upper small intestine, delaying glucose uptake and thereby lowering postprandial blood glucose levels. Therefore, α-glucosidase inhibition is a therapeutic target for diabetes treatment. Extracts from healthy silkworm larvae were used as control samples in this study, while the clinical drugs acarbose (an α-glucosidase inhibitor) and orlistat (a lipase inhibitor) served as positive controls for in vitro hypoglycemic and lipid-lowering effects. As shown in Figure 3A, acarbose, B. batryticatus extract, and healthy silkworm larvae extract all showed dose-dependent inhibitory effects on α-glucosidase in vitro. As the concentration of B. batryticatus extract increased, α-glucosidase inhibition rose rapidly before gradually stabilizing. The IC₅₀ (the concentration of the inhibitor required to provide 50% inhibitory activity) value of B. batryticatus extract for α-glucosidase was 5.76 mg/mL, which was lower than that of healthy silkworm larvae extract (7.0 mg/mL). In the same enzymatic reaction system, the IC₅₀ value of acarbose for α-glucosidase was 4.9 μg/mL. The results indicate that the extract of B. batryticatus exhibits stronger inhibitory activity against α-glucosidase. The highest levels of 1-deoxynojirimycin (1-DNJ), flavonoids, phenolics, and other compounds were found in Thai silkworm races on the 3rd day of the fifth instar, with an IC₅₀ value of 2 mg (silkworm powder)/mL for α-glucosidase [10]. So, the further screening of different silkworm strains remains highly significant for the preparation of B. batryticatus with medicinal value.

Pancreatic lipase plays a crucial role in triglyceride digestion by converting dietary fats into glycerol and fatty acids. Therefore, inhibiting the activity of pancreatic lipase in the intestine can reduce fat digestion and absorption, thereby lowering blood lipid levels. As shown in Figure 3B, pancreatic lipase inhibition was positively correlated with the concentration of B. batryticatus extract or healthy larvae extract. The IC₅₀ value of B. batryticatus extract for pancreatic lipase was 147.9 mg/mL, which was slightly better than that of the healthy larvae extract (176 mg/mL). However, the inhibitory activities of the extracts were both significantly lower than that of Osalide (IC₅₀ = 10.6 µg/mL).

Natural materials rich in flavonoids have been reported to exhibit good inhibitory activity against α-glucosidase and pancreatic lipase [31]. Hasnat H. concluded that flavonoids, such as quercetin and kaempferol, possess antioxidant, antidiabetic, and lipid-lowering activities [32]. In most cases, flavonoid aglycones exhibit stronger antioxidant activity, lipid peroxidation inhibition, and other biological effects in vitro than glycosides, including α-glucosidase inhibition, α-amylase inhibition, anti-inflammatory activity, and the ability to inhibit advanced glycation end products (AGEs) [33].

Silkworm larvae are processed into health foods and drugs due to their ability to inhibit α-glucosidase activity [34], lower blood sugar levels [11], regulate liver lipid metabolism, delay aging, and enhance resistance to Parkinson’s disease [18,35]. In this study, the α-glucosidase and pancreatic lipase inhibitory activities of B. batryticatus extract were higher than those of healthy silkworm extract, indicating its potential to combat hyperglycemia and hyperlipidemia. During the infection process of B. bassiana, various hydrolytic enzymes are produced, including chitinase, lipases, and proteases [36]. The hydrolysis of the enzymes produces additional bioactive components. For example, proteases generate peptides and free amino acids, which may contribute to the enhanced α-glucosidase and pancreatic lipase inhibitory activities of B. batryticatus extract. In addition, B. bassiana has been reported to produce lactones, quinolines, piperidines and other secondary metabolites that inhibit α-glucosidase and lipase activity [37]. In recent years, these numerous compounds with such functional groups have been used in the treatment of diabetes and its complications due to their inhibition on α-glucosidase or dipeptidyl peptidase IV [38].

2.4. Nontargeted Analysis of Samples by UPLC-TQ-LIT-MS

To investigate the chemical composition of B. batryticatus, the metabolic profiles of B. batryticatus and healthy silkworm larvae were compared. In the positive ion mode, 87 differential metabolites were detected, while 73 were identified in the negative ion mode. A principal component analysis (PCA) model was constructed using data from both ion modes as an unsupervised analytical method. As shown in Figure 4A1,B1, the B. batryticatus samples clustered distinctly from healthy larvae samples, with the quality control (QC) samples positioned between the two groups. The PCA plots revealed significant differences in the metabolic profiles between B. batryticatus and healthy larvae. In the positive ion mode, the PCA model yielded R² and Q² values of 0.942 and 0.89, respectively, while, in the negative ion mode, the values were 0.92 and 0.78, respectively. These results indicate that the PCA models in both ion modes have strong explanatory and predictive capabilities.

A supervised orthogonal partial least squares discriminant analysis (OPLS-DA) model was constructed using positive ion mode data, yielding an R²Y of 0.999 and a Q² of 0.999. These values indicate that the model can effectively explain the differences between groups and demonstrates strong predictive capability. As shown in Figure 4A2, after 500 permutation tests, both R² and Q² values were lower than the original values. The intercept of the Q² regression line was −0.852 (<0.05), confirming that the model did not overfit and was successfully constructed. Using negative ion mode data, another OPLS-DA model was constructed, yielding an R²Y of 0.865 and a Q² of 0.978, with a difference of 0.113 (<0.3) between the two. Similarly, after 500 permutation tests, both R² and Q² values were lower than the original values, and the intercept of the regression line for Q² was −0.624 (<0.05), confirming that the model did not overfit and that the diagnostic model was successfully constructed (Figure 4B2). These results indicated that the established OPLS-DA models perform well in interpreting and predicting data and effectively identified and discriminated between B. batryticatus and healthy larvae samples.

Variable importance in projection (VIP) reflects the contribution of each variable to the overall model fit and classification ability. According to a VIP score > 1.5 and p < 0.05, differential metabolites between healthy larvae and B. batryticatus were screened, as shown in

Table 1. Significant metabolite differences between B. batryticatus and healthy larvae samples in positive and negative ion modes. Compounds selected by OPLS-DA and VIP analysis (VIP > 1.5, p < 0.05) and univariate statistics (log₂ FC > 1 or <−1, p < 0.05).

NO	RT (min)	Proposed Compound	Molecular Formula	Mass Error (ppm)	Theoretical Exact Mass (m/z)	Assigned Adduct	p-Values	Log2 (FC)	VIP Score
1	0.7	Ornithine	C5H12N2O2	0.11	132.08989	[M + H]+	5.19 × 10−5	−1.27	0.26
2	0.78	Carnitine	C7H15NO3	−0.01	161.10519	[M + H]+	1.30 × 10−4	1.95	0.88
3	0.78	α,α-Trehalose	C12H22O11	−4.06	342.11482	[M − H]−	3.71 × 10−4	−2.42	0.50
4	0.79	Methionine sulfoxide	C5H11NO3S	0.21	165.04600	[M + H]+	5.50 × 10−6	−5.53	0.36
5	0.79	2-Hydroxymethylpiperidine-3,4,5-triol	C6H13NO4	−0.53	163.08437	[M − H]−	5.21 × 10−4	4.28	0.58
6	0.79	Trigonelline	C7H7NO2	−0.01	137.04768	[M + H]+	1.08 × 10−4	2.19	0.43
7	0.81	Serine	C3H7NO3	0.23	105.04262	[M + CAN + H]+	1.77 × 10−2	−1.75	0.44
8	0.85	4-Nitrobenzoic acid	C7H5NO4	−2.55	167.02143	[M − H]−	1.24 × 10−3	5.14	0.65
9	0.88	Nicotinic acid	C6H5NO2	−0.23	123.03200	[M + H]+	9.98 × 10−4	−2.15	0.50
10	0.9	Arginine	C6H14N4O2	0.21	174.11171	[M + H]+	3.22 × 10−02	−2.63	0.48
11	1.11	7-Methylguanine	C6H7N5O	0.91	165.06521	[M + H]+	6.99 × 10−4	3.02	0.35
12	1.11	Acetyl-carnitine	C9H17NO4	0.38	203.11584	[M + H]+	1.05 × 10−4	3.03	0.58
13	1.17	Prolylleucine	C11H20N2O3	0.34	228.14747	[M + H]+	6.18×10−4	1.79	0.45
14	1.19	3-Methyladenine	C6H7N5	0.01	149.07015	[M + H]+	3.39 × 10−4	4.06	0.33
15	1.2	Acrylic acid	C3H4O2	−3.00	72.02091	[M − H]−	6.63 × 10−6	1.73	0.52
16	1.27	3-Butene-1,2,3-tricarboxylic acid	C7H8O6	−2.26	188.03166	[M − H]−	1.59 × 10−6	3.24	1.22
17	1.29	Proline	C5H9NO2	−0.93	115.06322	[M + H]+	5.07 × 10−4	1.56	0.50
18	1.33	2′-Deoxyadenosine	C10H13N5O3	0.14	251.10187	[M + H]+	2.93 × 10−4	−3.22	0.38
19	1.42	3-Carboxy-4-hydroxyphenyl-β-D-glucopyranoside	C13H16O9	−1.51	316.07895	[M − H]−	4.65 × 10−3	−4.47	0.59
20	1.45	Isoleucine	C6H13NO2	−0.67	131.09454	[M + H]+	9.85 × 10−4	−2.52	4.57
21	1.51	Gallic acid	C7H6O5	−2.65	170.02107	[M − H]−	1.48 × 10−4	3.57	0.84
22	1.58	Citric acid	C6H8O7	−2.28	192.02656	[M − H]−	1.24 × 10−2	1.39	2.23
23	1.84	Propionylcarnitine	C10H19NO4	0.01	217.13141	[M + H]+	1.96 × 10−5	3.34	0.37
24	2	Glutaric acid	C5H8O4	−1.44	132.04207	[M + H − H2O]+	3.02 × 10−5	−1.77	0.28
25	2.08	3-Hydroxy-3-(methoxycarbonyl) pentanedioic acid	C7H10O7	−2.35	206.04217	[M − H]−	9.86 × 10−3	2.71	0.43
26	2.39	N6-Me-Adenosine	C11H15N5O4	0.51	281.11255	[M + H]+	1.06 × 10−4	−2.19	0.16
27	2.4	Kynurenine	C10H12N2O3	0.88	208.08498	[M + H]+	6.57 × 10−4	−4.28	0.51
28	2.44	Unknown 1	C18H26O11	−2.07	418.14665	[M + FA − H]−	5.47 × 10−4	−1.68	0.42
29	2.45	Methylimidazoleacetic acid	C6H8N2O2	0.13	140.05860	[M + H]+	2.99 × 10−2	4.23	0.34
30	2.52	Methylsuccinic acid	C5H8O4	−2.01	132.04199	[M − H]−	4.54 × 10−2	1.12	0.80
31	2.65	Unknown 2	C11H15N5O3S	−0.35	297.08946	[M + H]+	1.34 × 10−6	3.58	0.50
32	2.66	Adipic acid	C6H10O4	−1.69	146.05766	[M − H]−	8.98 × 10−6	1.75	0.75
33	2.69	Unknown 3	C17H34N4O4	−0.26	358.25791	[M + 2H]+	1.57 × 10−4	4.4	0.22
34	2.79	Unknown 4	C18H36N4O4	−0.32	372.27354	[M + 2H]+	1.70 × 10−6	3.93	0.31
35	2.8	Sorbic acid	C6H8O2	−0.88	112.05233	[M + H]+	1.60 × 10−4	2.59	0.29
36	2.83	3-Hydroxybenzoic acid	C7H6O3	−1.95	138.03142	[M − H]−	5.32 × 10−3	−2.58	1.10
37	2.85	2-Naphthylamine	C10H9N	−1.62	143.07327	[M + H]+	1.06 × 10−3	−2.81	0.15
38	2.86	7-hydroxy-6-methoxy-2H-chromen-2-one	C10H8O4	−0.04	192.04225	[M + H]+	1.48 × 10−6	4.45	0.21
39	2.87	3-(2,5-Dihydroxyphenyl)-2-propenoic acid	C9H8O4	−2.21	180.04186	[M − H]−	5.10 × 10−4	−2.76	1.10
40	2.88	3-Hydroxy-3,5,5-trimethyl-4-(3-oxo-1-buten-1-ylidene) cyclohexyl β-D-glucopyranoside	C19H30O8	−0.17	386.19400	[M + H]+	2.93 × 10−5	−4.91	0.30
41	2.92	Unknown 5	C15H22O5	−1.87	282.14620	[M − H]−	7.21 × 10−3	1.25	0.28
42	2.97	Tryptophan	C11H12N2O2	−2.06	204.08946	[M − H]−	4.44 × 10−4	2.28	0.32
43	3	4-Acetyl-4-phenylpiperidine	C13H17NO	0.29	203.13107	[M + H]+	1.35 × 10−4	4.33	1.11
44	3.01	N-Acetyl-leucine	C8H15NO3	−1.62	173.10491	[M − H]−	4.77 × 10−3	−1.82	0.57
45	3.02	7,7-dimethyl-3-spiro (4,4,-dimethyl-2,6-dioxocyclohexyl)-1,2,3,4,5,6,7,8-octahydro-5-quinolinone	C18H25NO3	0.25	303.18352	[M + H]+	4.22 × 10−4	3.13	0.19
46	3.02	Unknown 6	C18H30O10	−2.88	406.18273	[M − H]−	1.02 × 10−7	−2.50	0.53
47	3.04	Trifolin	C21H20O11	−1.75	448.09978	[M − H]−	6.33 × 10−8	−1.06	0.69
48	3.05	4-Hydroxybenzaldehyde	C7H6O2	−1.89	122.03655	[M − H]−	3.83 × 10−4	−1.44	0.64
49	3.06	4-[4-(4-Hydroxy-3-methoxyphenyl) tetrahydro-1H,3H-furo [3,4-c] furan-1-yl]-2-methoxyphenyl hexopyranoside	C26H32O11	−1.78	520.19353	[M + FA − H]−	1.34 × 10−3	−5.27	0.85
50	3.06	Suberic acid	C8H14O4	−2.34	174.08880	[M − H]−	2.77 × 10−6	−2.46	1.60
51	3.1	2-(acetylamino)-3-(1H-indol-3-yl) propanoic acid	C13H14N2O3	−1.78	246.10000	[M − H]−	3.65 × 10−4	3.69	0.34
52	3.1	Phenylalanine	C9H11NO2	−0.33	165.07892	[M + H]+	3.58 × 10−3	−3.24	0.34
53	3.25	3-amino-2-phenyl-2H-pyrazolo [4,3-c] pyridine-4,6-diol	C12H10N4O2	0.16	242.08042	[M + H]+	4.78 × 10−6	5.55	0.68
54	3.33	2-(6-Hydroxyhexyl)-3-methylenesuccinic acid	C11H18O5	−2	230.11496	[M − H]−	8.89 × 10−6	−4.27	0.64
55	3.42	Indole-3-acetic acid	C10H9NO2	−0.45	175.06325	[M + H]+	1.11 × 10−3	−1.28	0.14
56	3.5	1-(5-chloro-2-methoxyphenyl)-3-phenyl-2,5-dihydro-1H-pyrrole-2,5-dione	C17H12ClNO3	−0.04	313.05056	[M + NH4]+	2.44 × 10−4	5.19	0.13
57	3.63	7-methoxy-1-methyl-3H,4H,9H-pyrido [3,4-b] indole	C13H14N2O	0.12	214.11064	[M + H]+	9.60 × 10−7	−6.69	0.22
58	3.63	3-Hydroxy-Caprylic acid	C8H16O3	−2.33	160.10957	[M − H]−	1.38 × 10−3	−1.59	0.26
59	3.63	Phenmetrazine	C11H15NO	−0.4	177.11529	[M + H]+	1.70 × 10−4	3.44	0.39
60	3.66	5-[4-(3-hydroxy-4-methoxyphenyl)-hexahydrofuro [3,4-c] furan-1-yl]-2-methoxyphenol	C20H22O6	−0.2	358.14157	[M + H − H2O]+	3.52 × 10−4	3.27	0.16
61	3.84	Corchorifatty acid F	C18H32O5	−1.89	328.22435	[M − H]−	1.36 × 10−6	−1.09	3.20
62	3.92	6-(7-methyloctyl)-1H,3H,4H,6H-furo [3,4-c] furan-1-one	C15H24O3	−0.04	252.17254	[M + H − H2O]+	1.42 × 10−4	−1.24	0.19
63	4.63	6,8-Dihydroxy-9,12-octadecadienoic acid	C18H32O4	−1.58	312.22957	[M − H]−	2.29 × 10−4	−1.26	0.61
64	5	Unknown 7	C13H20O3	0.21	224.14129	[M + H]+	3.48 × 10−6	−2.29	0.18
65	5.79	13-Hydroxy-9,11-octadecadienoic acid	C18H30O3	−2.25	294.21883	[M − H]−	8.12 × 10−8	−1.74	2.20
66	5.79	1-Linoleoyl glycerol	C21H38O4	−1.13	354.27661	[M + H − H2O]+	3.60 × 10−3	3.23	0.19
67	6.23	Docosapentaenoic acid	C22H34O2	0.19	330.25594	[M + H]+	8.59 × 10−4	−4.37	0.16
68	6.39	Sphingosine (d18:1)	C18H37NO2	−0.1	299.28240	[M + H]+	1.19 × 10−3	4.57	0.23
69	6.41	10-Hydroxy-2-decenoic acid	C10H18O3	−3.37	186.12497	[M − H]−	1.79 × 10−4	5.57	0.63
70	6.42	17α-Methyl-androstan-3-hydroxyimine-17β-ol	C20H33NO2	−4.67	319.24964	[M + H]+	1.01 × 10−2	3.12	0.33
71	6.66	12(13)-DiHOME	C18H34O4	−1	314.24540	[M − H]−	1.80 × 10−5	−1.86	0.56
72	6.73	Arachidonic acid	C20H32O2	0.38	304.24035	[M + H]+	3.03 × 10−4	−5.26	0.22
73	6.86	Ergothioneine	C9H15N3O2S	−0.02	229.08849	[M + H]+	3.51 × 10−4	5.77	0.25
74	6.92	3-Hydroxy myristic acid	C14H28O3	−2.67	244.20319	[M − H]−	4.99 × 10−4	3.14	1.34
75	6.99	5-[-5-(Hydroxymethyl)-1,2,4a-trimethyl-1,2,3,4,4a,7,8,8a-octahydro-1-naphthalenyl]-3-methylpentanoic acid	C20H34O3	−2.24	322.25007	[M − H]−	1.87 × 10−3	−1.48	0.59
76	7.19	6-Hydroxy-4-Octadecenoic Acid	C18H34O3	−2.5	298.25005	[M − H]−	7.17 × 10−3	−1.34	3.21
77	7.32	9,11-Conjugated linoleic acid	C18H32O2	−2.44	280.23955	[M − H]−	9.30 × 10−6	1.47	0.54
78	7.39	2-Hydroxymyristic acid	C14H28O3	−2.45	244.20325	[M − H]−	5.00 × 10−3	5.96	0.86
79	7.46	Labdanolic acid	C20H36O3	−2.04	324.26578	[M − H]−	5.17 × 10−4	−2.78	0.83
80	7.53	Ethyl palmitoleate	C18H34O2	−0.63	282.25570	[M + H]+	2.21 × 10−3	6.84	2.98
81	7.53	Oleoyl ethanolamide	C20H39NO2	−0.05	325.29806	[M + H]+	2.77 × 10−3	−4.39	0.67
82	8.03	Dihomo-γ-Linolenoyl Ethanolamide	C22H39NO2	0.02	349.29809	[M + H]+	6.48 × 10−4	−4.51	0.51
83	8.09	Unknown 8	C20H35NO	−0.27	305.27178	[M + H]+	1.25 × 10−3	−6.69	1.01
84	8.19	Eicosapentaenoic acid	C20H30O2	−2.19	302.22392	[M − H]−	4.53 × 10−4	−2.55	1.62
85	8.19	Linolenic acid ethyl ester	C20H34O2	−0.51	306.25573	[M + H]+	1.55 × 10−4	3.66	0.19
86	8.22	Palmitoyl ethanolamide	C18H37NO2	−0.57	299.28226	[M + H]+	6.96 × 10−4	−3.32	3.17
87	8.25	9-Hydroxy-10,11-octadecadienoic acid	C18H32O3	−2.8	296.23432	[M − H]−	4.96 × 10−5	−1.47	0.87
88	8.54	Tomatidine	C27H45NO2	−0.47	415.34483	[M + H]+	3.21 × 10−3	4.82	0.27
89	8.85	Palmitoleic acid	C16H30O2	−2.54	254.22393	[M − H]−	4.29 × 10−4	−2.02	0.35
90	8.88	Oleamide	C18H35NO	−0.21	281.27181	[M + H]+	8.79 × 10−7	1.05	5.08
91	8.92	Octadecenoic acid methyl ester	C19H36O2	0.06	296.27155	[M + H]+	3.04 × 10−3	4.83	0.67
92	9.25	Stearoyl ethanolamide	C20H41NO2	−0.41	327.31359	[M + H]+	4.00 × 10−4	−5.13	2.51
93	9.5	12-Oxo phytodienoic acid	C18H28O3	−0.41	292.20372	[M + H]+	7.96 × 10−6	−1.2	0.20
94	10.02	Cholecalciferol	C27H44O	0	384.33922	[M + H − H2O]+	1.46 × 10−5	−2.07	0.36
95	10.05	10-hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-2-carboxylic acid	C30H46O4	−4.03	470.33771	[M + H]+	1.92 × 10−5	3.81	0.18
96	10.19	Unknown 9	C30H48O2	−0.37	440.36527	[M + H]+	2.77 × 10−3	−2.29	0.17
97	10.41	Maslinic acid	C30H48O4	−0.3	472.35512	[M + NH4]+	2.08 × 10−4	4.73	0.22
98	10.46	Methanandamide	C23H39NO2	−0.09	361.29805	[M + H]+	1.06 × 10−3	−3.28	0.14
99	12.13	17-Hydroxykauran-19-oic acid	C20H32O3	−0.18	320.23509	[M + H]+	1.20 × 10−3	3.59	0.42
100	12.43	Octadecatrienoic acid methyl ester	C19H32O2	−0.08	292.24021	[M + H]+	8.35 × 10−3	1.69	0.09
101	12.56	Cholest-4-en-3-one	C27H44O	−0.51	384.33902	[M + H]+	7.52 × 10−5	−2.02	0.94

p-Values obtained from analysis of t-test using a p-value threshold <0.05 for significant differences. Log fold change (Log₂ FC) values were calculated to compare the healthy larvae and B. batryticatus samples.

. According to the VIP score, the differences are as follows: oleamide (VIP = 5.08, log₂ fold change (FC) = 1.05), isoleucine (VIP = 4.57, log₂FC = −2.52), 6-hydroxy-4-octadecenoic acid (VIP = 3.21, log₂FC = −1.34), corchorifatty acid F (VIP = 3.20, log₂FC = −1.09), palmitoyl ethanolamide (VIP = 3.17, log₂FC = −3.32), ethyl palmitoleate (VIP = 2.98, log₂FC = 6.84), stearoyl ethanolamide (VIP = 2.51, log₂FC = −5.13), citric acid (VIP = 2.23, log₂FC = 1.39), 13-hydroxy-9,11-octadecadienoic acid (VIP = 2.20, log₂FC = −1.74), eicosapentaenoic acid (VIP = 1.62, log₂FC = −2.55), and suberic acid (VIP = 1.60, log₂FC = −2.46). Among them, eight metabolites were significantly upregulated in the B. batryticatus, primarily fatty acids and their derivatives. In contrast, ethyl palmitoleate was significantly downregulated. This is the result of the infection of silkworms by B. bassiana, which affected lipid metabolism.

Meanwhile, univariate statistical analysis (log₂ FC > 1 or < −1, p < 0.05) was used to identify differential metabolites, as shown in Table 1. The results indicated a total of 101 metabolic differences between B. batryticatus extract and healthy larvae extract. Among them, 48 significantly upregulated metabolites were identified in healthy larvae extract, primarily esters, carboxylic acids, as well as some phenolic substances, purine substances, and carnitine substances. Certain phenolic compounds, such as gallic acid, may originate from mulberry leaves. The aforementioned compounds, for example, linolenic acid ethyl ester, cis-12-octadecenoic acid methyl ester, 1-linoleoyl glycerol, 10-hydroxy-2-decenoic acid, gallic acid, and acetyl-carnitine all exhibit antioxidant capacity in vitro or in vivo [39,40,41]. In B. batryticatus extract, 53 significantly upregulated metabolites were identified, primarily fatty acids and derivatives, as well as amino acids. Fatty acids are important energy storage substances in the body of silkworms. As shown in Table 1, the abundance of fatty acids and their derivatives in B. batryticatus extract significantly increased, including arachidonic acid, docosapentaenoic acid, eicosapentaenoic acid, palmitoleic acid, 13-hydroxy-9,11-octadecadienoic acid, 9-hydroxy-10,11-octadecadienoic acid, stearoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, dihomo-γ-Linolenoyl, ethanolamide, 2-naphthylamine. It has been reported that B. bassiana-infected silkworms showed an upregulation of fatty acid metabolites [13]. In addition, a similar phenomenon was observed in the infection of black fly larvae by B. bassiana [42]. These results suggest that B. bassiana infection triggers an energy-intensive immune response in silkworms. These unsaturated fatty acids have been reported to exhibit strong antioxidant capacity, α-glucosidase inhibition ability, and hypoglycemic potential [43,44]. Amide compounds also have good antioxidant capacity [45] and physiological functions, including anti-anxiety, anti-depressive, and sleep-regulating effects [46]. Especially, it exhibits strong α-glucosidase inhibitory activity and can reduce serum triglycerides (TGs) and total cholesterol (TC) [47]. In this study, as shown in Table 1, various amino acids and amino acid oxides were upregulated following B. bassiana infection, such as methionine sulfoxide (log₂FC = −5.53), kynurenine (log₂FC = −4.28), phenylalanine (log₂FC = −3.24), arginine (log₂FC = −2.63), isoleucine (log₂FC = −2.52), N-acetyl-leucine (log₂FC = −1.82), glutaric acid (log₂FC = −1.77), serine (log₂FC = −1.75), and ornithine (log₂FC = −1.27). This phenomenon is due to proteases secreted by B. bassiana, which degrade host proteins, and the need for infected silkworms to accelerate the production of amino acids to cope with energy deficiency and synthesize antimicrobial peptides [48]. The similar phenomenon was reported in the studies of Xu (2015) and Huang (2009) [48,49]. Liu et al. confirmed that amino acids exhibit antioxidant capacity [50]. Additionally, some adenosine and glycosides are characteristic metabolites of B. batryticatus. In summary, B. bassiana infection significantly impacts the energy metabolism, nutrient metabolism, and immune defense mechanisms in the silkworm, leading to metabolic alterations in B. batryticatus and altering its antioxidant capacity, α-glucosidase, and lipase inhibitory capacity. It is worth noting that the compounds upregulated in B. bassiana (as shown in Table 1) contain some valuable bioactive molecules such as 13-hydroxy-9,11-octadecadienoic acid, trehalose, 3-carboxy-4-hydroxyphenyl-β- D-glucopyranoside, etc. 13-hydroxy-9,11-octadecadienoic acid has been reported to target breast cancer stem cells and inhibit their self-renewal and induce apoptosis [51]. Trehalose is associated with oxidative stress modulation, anti-aging, neuroprotection, and the regulation of cancer cell metabolism [52]. 3-Carboxy-4-hydroxyphenyl- β-D-glucopyranoside is a derivative of arbutin and therefore may have potential anti-inflammatory and pigment deposition prevention effects.

2.5. The Antioxidant Activity, Total Flavonoid Content, and α-Glucosidase Inhibitory Activity of Several Extracts of B. batryticatus

By sequentially extracting B. batryticatus with solvents of increasing polarity, the chemical constituents were selectively distributed among different extraction fractions based on their polarity. There were significant differences in the antioxidant capacity (total reducing power shown as Figure 5A, DPPH radical scavenging capacity shown as Figure 5B) of the three polar fractions of the B. batryticatus extract, which were as follows: ethyl acetate fraction concentrate (F3) > chloroform fraction concentrate (F2) > petroleum ether fraction concentrate (F1). This result was attributed to the composition and concentration of antioxidant compounds in extracts from different polar solvents. Notably, although F2 exhibited lower antioxidant capacity than F3, it showed stronger inhibitory activity against α-glucosidase (IC₅₀ = 0.217 mg/mL) (shown in Figure 5C). In accordance with the principles of polarity and solvent affinity, flavonoids were mainly concentrated in the ethyl acetate fraction, followed by the chloroform fraction (shown in Figure 5D).

2.6. Component Profiling of Chloroform Extract Concentrate (F2) by LC-MS

To identify the bioactive compounds in F2, LC-MS analysis was performed on the metabolites of the chloroform extract of B. batryticatus. The identified mass spectrometry data are shown in Table S1 (Supplementary Material). Since the peak area reflects the content of the corresponding substance in the sample, compounds with peak areas in the top 20 of F2 were prioritized for analysis. These compounds were classified and compared, and the results are shown in

Table 2. The top 20 compounds based on their concentrations in the chloroform fraction (F2).

Classification	UHPLC-MS
	Compound Name	RT (min)	m/z	Mode +/−
Amino acids, amino acid analogs and amino acid derivatives	Proline (F2-1)	1.06	116.07	+
	Threo-3-Phenylserine (F2-2)	8.27	182.08	+
Fatty acids and fatty acid esters	Chorifatty acid F (F2-3)	9.10	327.22	−
	9,12,13-Trihydroxy-15-octadecenoic acid (F2-4)	9.54	329.23	−
	9,10-Dihydroxy-12-octadecenoic acid (F2-5)	12.15	313.24	−
	HOTrE (F2-6)	12.78	293.21	−
	HpODE (F2-7)	13.32	311.22	−
	1,2-dihydroxyheptadec-16-yn-4-yl acetate (F2-8)	14.28	349.23	−
Phenolic and flavonoid compounds	Salicylic acid (F2-9)	2.53	137.02	−
	α-Cyano-3-hydroxycinnamic acid (F2-10)	7.8 0	188.04	−
	Quercetin (F2-11)	9.55	301.04	−
Amide compounds	Stearamide (F2-12)	14.19	284.29	+
	Oleamide (F2-13)	15.50	282.28	+
Ketone compounds	3-(1-Hydroxyethyl)-2,3,6,7,8,8a-hexahydropyrrolo [1,2-a] pyrazine-1,4-dione (F2-14)	4.34	199.11	+
	7-Hydroxy-6-methoxy-2H-chromen-2-one (F2-15)	6.74	193.05	+
	4-Hydroxy-6-[2-(2-methyl-1,2,4a,5,6,7,8,8a-octahydronaphthalen-1-yl) ethyl] oxan-2-one (F2-16)	13.21	275.20	+
	1-(2,3-dihydro-2,3-dimethyl-1H-pyrrolo [3,4-b] quinolin-1-yl)-3,5-dimethyl-2,6-dimethoxyphenyl)-3-(2,4-dimethoxyphenyl) propan-1-one (F2-17)	13.92	611.29	+
	1′-Ethylspiro [6,7-dihydro-2H-furo [2,3-f] indole-3,4′-piperidine]-5-yl)-[4-[2-methyl-4-(5-methyl-1,3,4-oxadiazol-2-yl) phenyl] phenyl] methanone (F2-18)	14.54	535.27	+
Other compounds	Senkyunolide H (F2-19)	9.34	247.09	+
	Unknown (C18H32O4) (F2-20)	12.27	335.22	+

. Among these compounds, unsaturated fatty acids, phenolic acids, and flavonoids all exhibit good antioxidant activity. According to the chemical structure, threo-3-phenylserine with benzene rings, some ketones, and amides with unsaturated bonds also have the potential for antioxidant activity.

As shown in Table 2, many heterocyclic compounds containing oxygen or nitrogen were present in the chloroform composition. Substances with this chemical structure often have enzyme inhibitory activity, which has anti-inflammatory, hypoglycemic, and neuroprotective effects in vivo. F2-18 contains a fused structure consisting of furan and indole, which is an analog of benzofuran. Many compounds with a benzofuran structure have activities against diabetes, tumors, hyperlipidemia, convulsions, and Alzheimer’s disease [53]. It has been reported that spiroindolone analogs exhibited excellent dual inhibitory activity against α-amylase and α-glucosidase [54]. Therefore, the compound in F2 has good potential for anti-diabetic effects and may possess other pharmacological activities. F2-15 is a derivative of coumarin. Substances with a benzopyran structure (coumarin scaffolds) are very important in pharmaceutical chemistry, possessing antibacterial, antioxidant, and anti-cancer properties, and also have the potential to develop potent anti-diabetes drugs due to their α-glucosidase inhibitory activity [55]. F2-14 is a biologically active compound with a pyrrolopyrazine skeleton. Substances with this structure often exhibit anti-inflammatory, antiviral, antidepressant, cardiovascular protective, antifungal, antioxidant, antitumor, and kinase inhibitory properties [56]. Similarly, F2-17 is a biologically active compound with a pyrroloquinoline ring skeleton. Substances with this structure often exhibit neuroprotective and cardiovascular protective effects [57]. Moreover, F2-14 and F2-17 are both nitrogen-containing alkaloids. Therefore, they may also have the potential to inhibit α-glucosidase activity [58]. F2-16 contains a lactone group and naphthalene, so it may have pharmacological value.

In addition, the α-glucosidase inhibitory activity of quercetin, fatty acids, and their amide derivatives is well recognized. Salicylic acid and hydroxycinnamic acid both contain a phenolic hydroxyl structure and have been shown to exhibit α-glucosidase inhibitory activity [59,60]. It is noteworthy that, in this study, senkyunolide H was found in F2. Senkyunolide H is a natural phthalide compound with antioxidant properties, as well as anti-inflammatory and antithrombotic effects [61]. It also contains both phthalide and benzofuran characteristics in its structure. Senkyunolide has also been used in the treatment of Parkinson’s disease and thromboembolic disorders due to its ability to inhibit human monoamine oxidase B and thrombin [62,63]. Although there are currently no reports on the α-glucosidase inhibitory activity of senkyunolide, its unique chemical structure makes it a potential candidate for the treatment of diabetes, warranting further attention. In summary, due to the chemical structure of benzene rings, oxygen-containing rings, nitrogen-containing rings, and long fatty chains, these compounds are more enriched in F2 than in F3. Their synergistic effect provides the F2 with antioxidant capacity and strong α-glucosidase inhibitory activity.

3. Materials and Methods

3.1. Chemicals and Reagents

Analytical grade reagents for solvent extraction, total flavonoids, and antioxidant capacity determination experiments were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). α-Glucosidase (activity: 50 U/mg, CAS: 9001-42-7) and pancreatic lipase (activity: 30 U/mg, CAS: 9001-62-1) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Acarbose was purchased from Bayer Biotechnology (China) Co., Ltd. (Shanghai, China). Orlistat was purchased from Hunan Dinuo Pharmaceutical Co., Ltd. (Changsha, China). p-Nitrophenyl α-D-glucopyranoside (pNPG, ≥98.0%, CAS: 3767-28-0) was purchased from Shanghai Huicheng Biotechnology Co., Ltd. (Shanghai, China). p-Nitrophenyl palmitate (pNPP, ≥98.0%, CAS: 1492-30-4) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Methanol (for LC-MS, CAS: 67-56-1) and acetonitrile (for UHPLC-MS, CAS: 75-05-8) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA), and formic acid (for LC-MS, CAS: 64-18-6) was purchased from LiChropur™ (Darmstadt, Germany).

3.2. Preparation of B. batryticatus

The silkworms (Jingsong × Haoyue strains) and B. bassiana used in this study were provided by the Silkworm Pathology and Physiology Laboratory of the Chinese Academy of Agricultural Sciences, Zhenjiang, China. A suspension of 1 × 10⁶ conidia/mL was sprayed onto newly exuviated fifth-instar silkworm larvae. The silkworms were then fed normally and began to die on the fifth day. Dead larvae were collected and placed in a humid environment until their bodies were gradually covered with white mycelium and became stiff. The cadavers were freeze-dried on the ninth day post-mortem using an LGJ-12D freeze dryer (Sihuan Scientific Instrument (Tianjin) Technology Co., Ltd., Tianjing, China). Healthy larvae were freeze-dried on the fifth day of their fifth instar. The freeze-dried samples, with a moisture content of less than 5%, were ground using a grinder (RS-FS1411, Royalstar Electronic Appliance Co., Ltd., Hefei, China), passed through an 80-mesh sieve, and then stored in a desiccator. The rearing of silkworms and the preparation of B. batryticatus were both conducted at the Sericulture Research Institute of Jiangsu University of Science and Technology.

3.3. Sample Extraction and Preparation

The freeze-dried sample (0.5 g) was extracted by vibration with 5 mL of 80% methanol for 60 min at room temperature. The extraction was repeated, and the extraction solutions were combined. The resulting solution was centrifuged at 6791× g for 10 min, and the supernatant was collected for testing antioxidant capacity, α-glucosidase inhibitory activity, pancreatic lipase inhibitory activity, and the analysis of extracts by UPLC-TQ-LIT-MS. As shown in Figure 6, the B. batryticatus powder (150.00 g) was repeatedly extracted with 80% ethanol until the extract became colorless. After filtration through Whatman No. 1 filter paper twice, the extract was concentrated using a rotary evaporator (RV 8, IKA Works GmbH & Co., Staufen, Germany) and then further concentrated in a vacuum dryer (DZF 6032, Shanghai Everone Precision Instruments Co., Ltd., Shanghai, China). The extract concentrate was suspended in a small amount of distilled water and successively partitioned with petroleum ether, chloroform, and ethyl acetate. The petroleum ether-concentrated extract (petroleum ether fraction, PEF: 23.341 g), chloroform-concentrated extract (chloroform fraction, CF: 0.258 g), and ethyl acetate-concentrated extract (ethyl acetate fraction, EAF: 0.096 g) were prepared. After dissolving each fraction in a trace amount of dimethyl sulfoxide, the solution was mixed with 80% methanol and used to measure antioxidant activity, total flavonoid content, and α-glucosidase inhibitory activity.

3.4. Total Flavonoid Determination

The total flavonoid content was determined using the colorimetric method [64]. It was calculated as rutin equivalents (REs) based on the standard curve (absorbance at 510 nm = 10.994x + 0.0173, R² = 0.994), using five concentrations in methanol ranging from 0.05 to 0.4 mg/mL. The final results were expressed as milligrams of rutin equivalents per gram of dry extract weight (mg RE/g).

3.5. Evaluation of Antioxidant Capacity

3.5.1. Total Reducing Power Assay

The total reducing power was determined using the method described by Tang [65]. Briefly, 1.00 mL of extraction solution with different concentrations (5, 10, 25, 50 mg/mL) was taken, followed by the addition of 2.50 mL of 0.2 M, pH 6.6 phosphate buffer, and, then, 2.50 mL of 1% potassium ferrocyanide (K₃Fe(CN)₆) was added. After 30 min in a water bath at 50 °C, 2.50 mL of 10% trichloroacetic acid was added, the mixture was mixed well, and, then, it was centrifuged at 4000 rpm for 10 min. In total, 2 mL of the centrifuged supernatant was mixed with 2 mL of distilled water and 0.4 mL of 0.1% ferric chloride, and colorimetric analysis was performed at 700 nm.

3.5.2. DPPH Radical Scavenging Activity Assay

The DPPH radical scavenging activity was determined using the method described by Tang et al. [65]. Briefly, aliquots (150 μL) of extraction solutions at different concentrations (0, 1, 2.5, 5, 7.5, 10 mg/mL) were taken, followed by the addition of 250 μL of methanol DPPH solution (100 μmol/L). The mixture was left in the dark for 30 min at room temperature before measuring the absorbance (A_i) at 517 nm. The absorbance measured using methanol solvent instead of the extraction solution was recorded as A₀, and the absorbance measured using methanol solvent instead of the DPPH solution was recorded as A_j. The result was calculated using the following formula:

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} (\mathrm{\%})=(1-{\displaystyle \frac{{\mathrm{A}}_{\mathrm{i}}-{\mathrm{A}}_{\mathrm{j}}}{{\mathrm{A}}_0}})\times 100,$$

(1)

the EC₅₀ value is the quantity of antioxidant required to eliminate half of all free radicals.

3.5.3. Hydroxyl Radical Scavenging Activity Assay

The hydroxyl radical scavenging activity was determined using the method described by Jiang et al. [66]. Briefly, aliquots (200 μL) of extraction solutions at different concentrations (0, 2.5, 5, 7.5, 10, 12.5 mg/mL) were taken, followed by the addition of 200 μL of 5 mmol/L FeSO₄ and then 200 μL of 5 mmol/L salicylic acid ethanol solution. Subsequently, 200 μL of 5 mmol/L H₂O₂ was immediately added to initiate the reaction. After shaking well, the mixture was incubated at 37 °C for 30 min and then centrifuged at 7000 rpm for 3 min. The absorbance of the supernatant at 510 nm (A₅₁₀) was measured. The absorbance measured by replacing the extraction solution with methanol was recorded as A_max, while the absorbance obtained by replacing the corresponding H₂O₂ solution with distilled water was recorded as A_b. The result was calculated using the following formula:

$$\mathrm{H}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{y}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} (\mathrm{\%})=\left(1-{\displaystyle \frac{{\mathrm{A}}_{510}-{\mathrm{A}}_{\mathrm{b}}}{{\mathrm{A}}_{\max }}}\right)\times 100$$

(2)

3.5.4. Superoxide Radical Scavenging Activity Assay

The measurement method was slightly modified from that described by Tang et al. [65]. Briefly, aliquots (100 μL) of extraction solutions at different concentrations (2.5, 5, 7.5, 12.5, 25, 50 mg/mL) were taken, followed by the addition of 450 μL of 50 mmol/L Tris-HCl buffer (pH 8.2) and 400 μL of 25 mmol/L pyrogallol solution. The mixture was thoroughly mixed and reacted at 25 °C for 5 min. Then, 100 μL of 8 mmol/L HCl was added to terminate the reaction, the mixture was shaken well, left to stand for 3 min, and the absorbance (A₄₂₀) was measured at 420 nm. The absorbance measured by replacing the extraction solution with methanol was recorded as A_c, while the absorbance measured by using distilled water instead of the pyrogallol solution was recorded as A_d. The result was calculated using the following formula:

$$\mathrm{S}\mathrm{u}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} (\mathrm{\%})=\left(1-{\displaystyle \frac{{\mathrm{A}}_{420}-{\mathrm{A}}_{\mathrm{d}}}{{\mathrm{A}}_{\mathrm{c}}}}\right)\times 100$$

(3)

3.6. α-Glucosidase Inhibition Assay

The measurement method was slightly modified from that described by Tu et al. [67]. Briefly, the enzyme reaction system consisted of 200 µL of a 0.05 mol/L phosphate buffer (pH 6.8), 400 µL of a 0.2 U/mL α-glucosidase solution, 20 µL of extraction solution (0.5–20 mg/mL) or acarbose solution (0.5–25 µg/mL), and 40 µL of 0.02 mol/L substrate solution (pNPG). The reaction mixture was incubated at 37 °C for 20 min, and the reaction was terminated by adding 160 µL of 1 mol/L sodium carbonate solution. The absorbance of the mixed solution was measured at 405 nm. The experiment was repeated three times, and the average value was used to calculate the inhibitory activity and determine the IC₅₀ value from the regression curve.

$$\mathsf{\alpha }-\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{y} (\mathrm{\%})=\left(1-{\displaystyle \frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{blank}}}{{\mathrm{A}}_{\mathrm{test}}-{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}}\right)\times 100,$$

(4)

where A_sample is the absorbance value of a mixture of extraction solution, enzyme solution, and pNPG solution; A_blank is the absorbance value of a mixture of extraction solution, enzyme-free buffer solution, and pNPG solution; A_test is the absorbance value of a mixture of buffer solution, enzyme solution, and pNPG solution; and A_control is the absorbance value of a mixture of buffer solution and pNPG solution.

3.7. Pancreatic Lipase Inhibition Assay

The measurement method was slightly modified from that described by Aloo et al. [68]. Briefly, the enzyme reaction system consisted of 100 µL of 50 mmol/L Tris-HCl buffer (pH 7.5) containing sodium cholate and arabic gum, 40 µL of extraction solution (25–200 mg/mL) or orlistat solution (1–20 µg/mL), 100 µL of 10 mg/mL pancreatic lipase solution, and 160 µL of 16 mmol/L substrate solution (pNPP). After incubation at 37 °C for 20 min, the absorbance was measured at 405 nm, and the inhibitory activity was calculated using the following formula. The experiment was repeated three times, and the average value was used to create a regression curve and calculate the IC50 value.

$$\mathrm{P}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{c} \mathrm{l}\mathrm{i}\mathrm{p}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{y} (\mathrm{\%})=\left(1-{\displaystyle \frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{blank}}}{{\mathrm{A}}_{\mathrm{test}}-{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}}\right)\times 100,$$

(5)

where A_sample is the absorbance value of a mixture of an extraction solution, enzyme solution, and pNPP solution; A_blank is the absorbance value of a mixture of an extraction solution, enzyme-free buffer solution, and pNPP solution; A_test is the absorbance value of a mixture of a buffer solution, enzyme solution, and pNPP solution; and A_control is the absorbance value of a mixture of a buffer solution and pNPP solution.

3.8. UHPLC-TQ-LIT-MS Analysis

The detection procedure was conducted using an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Scientific, Waltham, MA, USA), coupled with an ultra-performance liquid chromatography (UHPLC) system (Thermo Scientific, Waltham, MA, USA) and operated with Xcalibur data acquisition software (version 4.1, Thermo Scientific, USA). Data processing involved spectral library retrieval from MzCloud, ChemSpider, and Mass List databases, using Compound Discoverer software (version 3.2, Thermo Scientific, USA). Chromatographic separation was carried out on a Hypersil Gold C18 column (1.9 µm, 100 mm× 2.1 mm; Thermo Scientific, USA), with an injection volume of 3 µL and a column temperature of 50 °C. The mobile phase comprised solvent A (0.1% formic acid in acetonitrile) and solvent B (0.1% formic acid in aqueous solution). The column was eluted at a flow rate of 0.38 mL/min, following the gradient program: the initial conditions started at 98% solvent B, held for 0–0.5 min; from 0.5 to 1.5 min, solvent B was reduced from 98% to 60%; from 1.5 to 6.5 min, solvent B decreased from 60% to 20%; from 6.5 to 9.5 min, solvent B was reduced to 0%; from 9.5 to 13 min, solvent B was maintained at 0%; from 13 to 13.4 min, solvent B increased from 0% to 98%; and, from 13.4 to 17 min, solvent B was maintained at 98%.

Ionization was performed in both positive and negative electrospray ionization (ESI) modes. The mass spectrometry operating parameters in positive ion mode were as follows: spray voltage, 3500 V; sheath gas flow rate, 40 Arb; auxiliary gas flow rate, 10 Arb; purge gas flow rate, 1 Arb; ion transfer temperature, 275 °C; and heating temperature, 320 °C. The full scan settings were as follows: resolution, 60,000; scanning range, 70–1050 Da; RF lens, 70%. For data-dependent MS² (ddMS²) analysis, the top N value was set to 5, with a resolution of 15,000 and the scanning range mode set to auto. The operating parameters in negative ion mode were as follows: spray voltage, 2800 V; sheath gas flow rate, 40 Arb; auxiliary gas flow rate, 10 Arb; purge gas flow rate, 1 Arb; ion transfer temperature, 275 °C; and heating temperature, 320 °C. The full scan settings were identical to those used in positive ion mode: resolution, 60,000; scanning range, 70–1050 Da; and RF lens, 70%. For ddMS² analysis, the top N value was set to 5, with a resolution of 15,000 and the scanning range mode set to auto.

3.9. Liquid Chromatography/Mass Spectrometry

LC/MS analyses of F2 and F3 were performed with the same reagents and instrument parameters in Section 3.8. Chromatographic separation was performed on an HSS T3 column (1.8 μm, 100 mm × 2.1 mm; Waters, Milford, MA, USA). The liquid chromatography conditions in positive ion mode were as follows: column temperature, 40 °C, and injection volume, 2 μL. The mobile phase consisted of solvent A (0.1% formic acid in acetonitrile) and solvent B (0.1% formic acid in aqueous solution). The column was eluted at a flow rate of 0.25 mL/min, following the gradient program: the initial conditions started at 98% solvent B, held for 0–1 min; from 1 to 9 min, solvent B was reduced from 98% to 50%; from 9 to 12 min, solvent B decreased from 50% to 2%; from 12 to 13.5 min, solvent B was maintained at 2%; from 13.5 to 14 min, solvent B increased from 2% to 98%; and, from 14 to 20 min, solvent B was maintained at 98%. The liquid chromatography conditions in negative ion mode were as follows: column temperature, 40 °C, and injection volume, 2 μL. The mobile phase comprised solvent A (acetonitrile solution) and solvent B (5 mM of ammonium formate in aqueous solution). The column was eluted at a flow rate of 0.25 mL/min, following the gradient program: the initial conditions started at 98% solvent B, held for 0–1 min; from 1 to 9 min, solvent B was reduced from 98% to 50%; from 9 to 12 min, solvent B decreased from 50% to 2%; from 12 to 13.5 min, solvent B was maintained at 2%; from 13.5 to 14 min, solvent B increased from 2% to 98%; and, from 14 to 17 min, solvent B was maintained at 98%.

The mass spectrometry operating parameters in positive ion mode were as follows: spray voltage, 3500 V; sheath gas flow rate, 40 Arb; auxiliary gas flow rate, 10 Arb; ion transfer temperature, 325 °C; and heating temperature, 350 °C. The full scan settings were as follows: resolution, 60,000; scanning range, 100–1500 Da; RF lens, 70%. For data-dependent MS² (ddMS²) analysis, the top N value was set to 5, with a resolution of 15,000 and the scanning range mode set to auto. The operating parameters in negative ion mode were as follows: spray voltage, 2500 V; sheath gas flow rate, 40 Arb; auxiliary gas flow rate, 10 Arb; ion transfer temperature, 325 °C; and heating temperature, 350 °C. The full scan settings were identical to those used in positive ion mode: resolution, 60,000; scanning range, 70–1050 Da; and RF lens, 70%. For ddMS² analysis, the top N value was set to 5, with a resolution of 15,000 and the scanning range mode set to auto.

3.10. Statistical Analysis

The measurements were performed in triplicate. Statistical analysis was performed using SPSS 19.0 software (IBM Corporation, Armonk, NY, USA, 2010), and the results were expressed as the mean ± standard deviation. The data were analyzed using one-way ANOVA, followed by the S-N-K test, with a significance level of p < 0.05. Origin 2021 software (OriginLab Corporation, Northampton, MA, USA, 2021) and SIMCA 14.1 software were used.

4. Conclusions

Silkworm is a widely accepted hypoglycemic food in Asia, and B. batryticatus is a dried silkworm body, which is derived by the infection (or artificial inoculation) of B. bassiana. Nowadays, B. batryticatus is widely used in clinical practice for antiepilepsy, anticonvulsion, antidiabetes and blood lipid regulation, but there is still a lack of functional compound data. In this study, B. batryticatus was prepared, extracted, and separated using solvents of different polarities. Compared with the extracts of healthy silkworm larvae, the total reducing power, hydroxyl radical scavenging capacity, superoxide anion radical scavenging capacity, α-glucosidase inhibitory activity of the methanol extract of B. batryticatus demonstrated comprehensive superiority. Non-targeted LC-MS was further used to compare the differences in characteristic metabolites between B. batryticatus and healthy silkworm larvae. The results showed that fatty acids and their derivatives in B. batryticatus extract were significantly increased. Furthermore, the B. batryticatus extract was fractionated and separated according to polarity using solvents of different polarities, and the chloroform fraction of B. batryticatus exhibited the strongest α-glucosidase inhibitory activity. LC-MS was used to identify the compound composition of chloroform fraction. The results showed that nitrogen-containing heterocyclic compounds and fatty acid derivatives may contribute to the strong α-glucosidase inhibitory ability of chloroform fraction. The results provide a theoretical basis for the clinical application of B. batryticatus, and the active molecules in chloroform fraction offer potential for the development of hypoglycemic drugs. However, the hypoglycemic ability and mechanism of specific active substances in chloroform fraction of B. batryticatus still require further experiments such as molecular dynamic simulations or in vivo experiments of specific active substances.