Introduction

Beauveria bassiana is a widely distributed fungal species within the phylum Ascomycota1. Due to its ability to parasitize a broad range of insects—including those from the orders Lepidoptera, Coleoptera, and Hymenoptera—it has been extensively utilized in biological pest control2. When the fourth-instar larvae to fifth-instar larvae of Bombyx mori Linnaeus are infected (either naturally or artificially) with Beauveria bassiana (Bals.) Vuillant and subsequently dried after death, the resulting product is referred to as Bombyx batryticatus3. The pathogenic mechanism of Beauveria bassiana involves the secretion of multiple hydrolytic enzymes—such as lipases, proteases, and chitinases—which enable the fungus to actively penetrate the host integument and proliferate within the hemocoel. This leads to host death through nutrient depletion, metabolic disruption, and toxin production4. As a traditional Chinese medicinal product formed by fungal infection, Bombyx batryticatus contains a variety of chemical constituents, including proteins and peptides, fatty acids, flavonoids, nucleosides, steroids, coumarins, and polysaccharides. Proteins are the dominant components, with peptides receiving research attention. One example is BB octapeptide, a novel platelet aggregation inhibitory peptide isolated from Bombyx batryticatus, which has a molecular mass of 885.0 Da and an amino acid sequence of Asp-Pro-Asp-Ala-Asp-Ile-Leu-Gln5. Five fatty acids have also been isolated from Bombyx batryticatus, including meso-erythritol, citric acid, decanoic acid, stearic acid, and palmitic acid6. Flavonoids, commonly found in many herbal medicines, have also been identified. In particular, quercetin and kaempferol were detected using RP-HPLC methods7. Among the nucleoside compounds identified through HPLC analysis are uracil, uridine, hypoxanthine, and xanthine, with uracil present at the highest concentration8. To date, eight steroid compounds have been isolated from Bombyx batryticatus6. Additionally, one coumarin compound—6-methoxy-7-O-β-D-(4′-methoxy)-glucopyranosyl coumarin—has been reported9. The polysaccharide content of Bombyx batryticatus is approximately 4.4%, and these polysaccharides have demonstrated significant antioxidant activity10. The rich chemical composition of Bombyx batryticatus underpins its pharmacological potential. Variations in the levels of these constituents can serve as important indicators for assessing the quality of the medicinal product.

One of the key pharmacological features of Bombyx batryticatus is its action on the nervous system, including anticonvulsant, antiepileptic, sedative, and neurotrophic effects6. Ethanol extracts of Bombyx batryticatus have shown significant anticonvulsant activity in mouse models11. Additionally, aqueous extracts followed by ethanol precipitation have been found to produce sedative effects by suppressing spontaneous locomotor activity in mice12. In vitro studies have identified compounds isolated from Bombyx batryticatus that exhibit marked neurotrophic effects by stimulating the synthesis of nerve growth factor (NGF) in astrocytes8,13. Another important pharmacological property of Bombyx batryticatus is its anticoagulant activity. Water extracts have demonstrated the ability to inhibit blood coagulation14. Injection of Bombyx batryticatus was reported that can also inhibit venous thrombosis through increasing tPA activity and decreasing PAI-1 activity15. In recent years, extensive research has focused on the anti-timor potential of Bombyx batryticatus. It has shown significant antiproliferative effects against various human cancer cell lines, including cervical, liver, and gastric cancers16. Notably, ethanol extracts were found to induce apoptosis in human gastric cancer cells (SGC-7901) by upregulating pro-apoptotic proteins Bax and P21, and downregulating the anti-apoptotic protein Bcl-217. Bombyx batryticatus also exhibits antimicrobial activity. Ethanol extracts have shown antibacterial effects against Escherichia coli, with a minimum inhibitory concentration (MIC) of 0.6 mg/mL18. In addition, notable antifungal activity has been observed against Colletotrichum gloeosporioides, Valsa mali, and the leaf cast pathogen of Pericarpium Zanthoxyli19. An interesting study further reported that the supernatant of Bombyx batryticatus, obtained after ethanol extraction and water precipitation, demonstrated antiviral activity against respiratory syncytial virus (RSV)20. Reactive oxygen species (ROS) are implicated in the pathogenesis of many diseases. Flavonoids isolated from Bombyx batryticatus have shown strong free radical scavenging abilities, particularly against DPPH and hydroxyl radicals21. In summary, Bombyx batryticatus exhibits a broad range of pharmacological activities, including effects on the nervous system, anticoagulant, antitumor, antibacterial, antifungal, antiviral, and antioxidant actions. These findings support its potential use as a traditional animal-derived medicine for the prevention or treatment of various conditions, particularly convulsions, epilepsy, thrombosis, and cancer.

As an animal-derived traditional Chinese medicine (TCM) formed through infection of Bombyx mori by Beauveria bassiana, Bombyx batryticatus is widely used in medical and pharmaceutical applications both in China and globally. One notable morphological feature of Bombyx batryticatus is the presence of Silk Gland Rings (SGRs), which become visible when the specimen is broken crosswise. According to the 2020 edition of the Pharmacopoeia of the People’s Republic of China, Bombyx batryticatus is characterized by “four bright brown or dark brown silk gland rings at the center”3. Despite their prominence in quality descriptions, the relationship between SGRs and the overall quality of Bombyx batryticatus remains unexplored in both domestic and international research. Several studies have demonstrated a correlation between cross-sectional features and the quality of Chinese medicinal materials. For example, a study on the correlation between the number of concentric rings in Spatholobus suberectus and flavonoid content revealed that the xylem and phloem of this plant are arranged in concentric or eccentric rings. Traditional evaluation criteria consider that the presence of three to eight eccentric resin rings indicates higher quality. Modern analysis confirmed this observation by showing that the content of five pharmacologically active components increased proportionally with the number of rings, thereby establishing a clear link between external morphological traits and internal quality22. Similarly, multiple studies have shown that the color of the cross-section of Polygonum multiflorum is significantly positively correlated with the levels of stilbene glycosides and bound anthraquinones23. This finding supports the traditional view that “darker cross-sections indicate superior quality” and provides quantifiable morphological criteria for quality grading of P. multiflorum. According to the Chinese Association of Traditional Chinese Medicine (Standards for Commercial Specifications and Grades of Bombyx Batryticatus), first-grade samples are required to have four distinct black silk gland rings in the cross-section. In contrast, second-grade and mixed-grade products are only required to show the presence of silk gland rings without specifying their number. This indicates that the number of silk gland rings in the cross-section of Bombyx batryticatus is positively correlated with its medicinal quality to a certain extent.

If a correlation between the number of SGRs and medicinal quality can be established, it would offer a rapid, cost-effective method for preliminary quality assessment, significantly reducing the need for more expensive laboratory testing. In this study, 34 batches of Bombyx batryticatus samples from various sources across China were collected. We aimed to investigate the correlation between the number of SGRs and the quality of the samples, and to evaluate quality differences among samples from different geographic or production origins.

Materials and methods

Preparation of Bombyx Batryticatus and characterization of the cross-sectional silk gland rings

In the Bombyx batryticatus production industry, Beauveria bassiana (Bals.) Vuillant is sprayed onto the surface of silkworms (Bombyx mori), where it proliferates and metabolizes, ultimately leading to the silkworm’s death. Due to the fungal hyphae breaching the host’s cuticle and growing outward, authentic Bombyx batryticatus specimens are characteristically coated with a layer of white mycelial powder. The specific procedures involved in this process and the resulting external features are illustrated in Fig. 1.

Fig. 1
figure 1

Schematic illustration of the formation process of Bombyx Batryticatus.

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The silk gland is a highly specialized organ in Bombyx mori responsible for the synthesis and secretion of silk proteins. Its development reflects the silkworm’s nutritional status and overall health. During the early phase of the fifth instar (up to day 3), silkworms primarily use ingested nutrients for bodily growth. After the third day, physiological focus shifts toward the synthesis of silk proteins and the development of the silk glands24. When silkworms are well-fed on high-quality mulberry leaves and free from disease, their silk glands develop robustly, as shown in Fig. 2. However, poor nutrition or developmental stress can significantly impair silk gland growth. Upon infection with Beauveria bassiana, infected silkworms typically show reduced feeding behavior compared to healthy ones. Consequently, even in otherwise healthy larvae, fungal infection may lead to partial underdevelopment of the silk glands. As a result, the dried cross-sectional view of Bombyx batryticatus may reveal between two to four visible Silk Gland Rings (SGRs). The presence of hollow or white SGRs is generally associated with infection by other pathogens or exposure to toxic substances rather than B. bassiana alone. Anatomical observation of the internal silk gland distribution, combined with empirical analysis, indicates that cutting the specimen at the level of the first abdominal proleg provides the clearest and most consistent view of SGRs. To date, no samples have been observed with fewer than two or more than four distinct SGRs.

Fig. 2
figure 2

Anatomical Diagram of the Silk Gland Rings.

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As shown in Fig. 3, Bombyx batryticatus specimens were transected between the first and second pairs of abdominal prolegs to observe the cross-sectional features of the Silk Gland Rings (SGRs). Samples exhibiting four, three, or two distinct bright brown or dark brown rings were classified as SG4, SG3, and SG2, respectively. When no silk gland rings were visible or the gland structures appeared hollow, the samples were categorized as SG0. In some specimens, silk gland rings were present but appeared white or partially degraded; these were classified as type W.

Fig. 3
figure 3

Anatomical micrographs of the five types of Silk Gland Rings.

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Collection of Bombyx Batryticatus samples from different sources

In total, 34 batches of Bombyx batryticatus were collected from diverse sources across China to reflect a range of production practices (Table 1). Ten batches originated from standardized breeding bases characterized by large-scale farming operations and formal protocols. Seven batches were sourced from individual farmers operating at a smaller scale without standardized procedures. The remaining seventeen batches were obtained from major traditional Chinese medicine markets in Sichuan, Guangxi, Hebei, and Anhui provinces. This diverse sampling provided a foundation for examining the relationship between SGR characteristics and the quality of Bombyx batryticatus, as well as assessing quality differences among samples from various production origins.

Table 1 Information Table of 34 Bombyx Batryticatus Samples.
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Determination of quality indicators for different Bombyx Batryticatus samples

Determination of total ash, extract, and total protein

The total ash and ethanol-soluble extract content were determined according to the methods specified for Bombyx batryticatus in the Pharmacopoeia of the People’s Republic of China (2020 edition)3. Total protein content was measured following the instructions of a commercial assay kit and based on established literature methods25. After method optimization, the following procedure was adopted: 0.1 g of Bombyx batryticatus powder (passed through a No. 2 sieve) was extracted with 5 mL of ultrapure water using ultrasonic extraction at room temperature for 30 min. The mixture was then centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. The residue was re-extracted with an additional 5 mL of the same solvent under identical conditions. The two supernatants were combined and diluted to a final volume of 50 mL with ultrapure water, yielding the sample solution. For protein quantification, 20 μL of the sample solution was added to each well of a 96-well microplate. Then, 200 μL of diluted 1 × G250 dye reagent was added to each well. The plate was incubated at a constant temperature for 3 to 5 min, and absorbance was measured at 595 nm using a microplate reader.

Determination of beauvericin and nucleoside contents

The content of beauvericin was determined following the method described in the literature26, while the quantification of nucleoside components was performed according to a previously established method developed by our research group27. Chromatographic analysis was conducted under the following conditions: an AQ-C8 column (4.6 mm × 250 mm, 5 μm) was used for separation. The mobile phase consisted of water (A) and methanol (B), with gradient elution as follows: 0–4 min, 1% B; 4–6 min, 1–2% B; 6–6.1 min, 2.0–6.5% B; 6.1–14 min, 6.5–14% B; 14–14.1 min, 14.0–14.5% B; 14.1–18 min, 14.5% B; 18–18.1 min, 14.5–20% B; 18.1–24 min, 20% B; 24–28 min, 20.0–1.0% B; 28–30 min, 1.0% B. The detection wavelength was set at 260 nm. Column temperature was maintained at 25 °C, and the flow rate was 0.8 mL/min.

Determination of flavonoid contents

Chromatographic analysis of flavonoid components was conducted using a Waters C18 column (4.6 mm × 50 mm, 1.7 μm). The mobile phase consisted of 0.1% phosphoric acid in water (A) and acetonitrile (B), with gradient elution as follows: 0–2 min, 15–20% B; 2–3.5 min, 20–50% B; 3.5–4 min, 50–55% B; 4–5 min, 55% B; 5–6 min, 55–90% B; 6–7 min, 90% B; 7–7.5 min, 90–15% B; and 7.5–10 min, 15% B. Detection was performed at a wavelength of 365 nm. The column temperature was maintained at 30 °C, the flow rate was set at 0.3 mL/min, and the injection volume was 3 μL. Reference standards of rutin, astragalin, quercetin, and kaempferol were accurately weighed and dissolved in 80% methanol in 20 mL volumetric flasks to prepare stock solutions for each flavonoid. Appropriate volumes of each stock solution were combined in a 10 mL volumetric flask and diluted with 80% methanol to obtain a mixed standard solution.

For sample preparation, 2.0 g of Bombyx batryticatus powder was accurately weighed and extracted with 20 mL of 80% methanol using ultrasonic extraction for 30 min. The extract was filtered, and 10 mL of the filtrate was concentrated to dryness under reduced pressure. The residue was redissolved in 2 mL of 80% methanol, filtered through a 0.22 μm microporous membrane, and the filtration was used for analysis. Standard curves were constructed by precisely transferring 1000, 500, 200, 100, and 50 μL of the mixed standard solution into 1 mL volumetric flasks, diluting to volume with 80% methanol, and mixing thoroughly. Peak areas were recorded under the chromatographic conditions described above. Standard curves were generated by plotting peak area (Y) versus concentration of each standard (X, μg/mL).

Statistical analysis

All data were processed using Microsoft Excel. The distribution of different Silk Gland Ring (SGR) types in Bombyx batryticatus samples was visualized using bar plots generated with the ‘ggplot2’ package in RStudio 202528. Quality indicators were analyzed using the ‘raincloudplots’ package. Principal Component Analysis (PCA) was performed using the ‘FactoMineR’ package. Heatmaps were constructed with the ‘ComplexHeatmap’ package, and circular representations were achieved with the integration of the ‘Circlize’ package. Pearson correlation coefficients among variables were calculated and visualized in a matrix format using the ‘Corrplot’ package29. All R-based visualizations were created using the online platform www.chiplot.online (accessed on April 28, 2025). One-way ANOVA was conducted using SPSS version 26.0, and Duncan’s multiple range test was applied for post hoc comparisons.

Results

Proportional analysis of silk gland rings in 34 batches of Bombyx Batryticatus

The cross-sectional traits of Bombyx batryticatus samples from different sources are presented in Fig. 3. According to the 2020 edition of the Chinese Pharmacopoeia, Bombyx batryticatus is expected to exhibit four distinct bright brown or dark brown Silk Gland Rings (SGRs) in cross-section. However, under this criterion, the average compliance rate for the Base group was 45.16%, while the Farmer group showed a slightly higher average compliance rate of 55.29% (Fig. 4). Among regional samples, the average compliance rate in Sichuan was 37.96%, in Guangxi 45.94%, in Hebei 62.01%, and in Anhui 54.09%. These results suggest that Bombyx batryticatus sourced from Hebei demonstrated the highest conformity with pharmacopoeial standards. Across the 34 batches of Bombyx batryticatus examined, the overall proportion of samples showing four SGRs averaged 47.90%. Only 26 batches exhibited a combined proportion of SGRs (S4 + S3 + S2) greater than 60%, while the remaining 8 batches had a total SGR ratio below 60%. These findings indicate considerable variation in SGR counts among Bombyx batryticatus from different sources. Moreover, the number of visible SGRs appears to be closely associated with the overall quality of the medicinal material.

Fig. 4
figure 4

Statistical Chart of Silk Gland Ring Proportions in 34 Batches of Bombyx Batryticatus.

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Methodological study on the quality indicators of Bombyx Batryticatus

The mixed standard solution and sample solutions were injected and analyzed by HPLC, and the chromatograms were recorded. As shown in Fig. 5, the resolution between each target compound and its adjacent peaks exceeded 1.5, the theoretical plate number (calculated based on the quercetin peak) was no less than 6000, and the symmetry factors for all chromatographic peaks ranged from 0.95 to 1.05, indicating good peak shape and column efficiency. To assess instrument precision, the standard solution was injected six consecutive times. The relative standard deviations (RSDs) of the peak areas for rutin, astragalin, quercetin, and kaempferol were 0.40%, 0.62%, 0.71%, and 2.09%, respectively, all within acceptable limits, indicating excellent instrument precision.

Fig. 5
figure 5

Chromatograms of Rutin, Astragalin, Quercetin, and Kaempferol. (A) represents the mixed reference standards, (B) represents the test sample. Peaks: 1 for Rutin, 2 for Astragalin, 3 for Quercetin, and 4 for Kaempferol.

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To evaluate method repeatability, six replicate sample solutions were prepared and analyzed. The RSDs of the peak areas for rutin, astragalin, quercetin, and kaempferol were 2.82%, 2.16%, 1.24%, and 2.50% (Table 2), respectively, meeting the required criteria and confirming good reproducibility of the method. To assess method accuracy, six portions of Bombyx batryticatus powder (1.0 g each) were precisely spiked with known quantities of each standard, and sample solutions were prepared and analyzed. The recovery rates for rutin, astragalin, quercetin, and kaempferol ranged from 95 to 110%, with RSDs meeting the acceptance criteria, indicating that the method has good accuracy. For stability testing, the sample solution was stored at room temperature and injected at 0, 2, 4, 6, 8, 12, and 24 h. The RSDs of the peak areas for rutin, astragalin, quercetin, and kaempferol were 0.92%, 1.64%, 0.84%, and 2.58%, respectively, demonstrating that the sample solution remained stable for up to 24 h.

Table 2 Linear relationships of the four flavonoid components.
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Quality analysis of Bombyx Batryticatus under different silk gland ring quantities

The results for Total Ash, Extract, Total Protein, and Beauvericin in Bombyx Batryticatus samples with varying numbers of silk gland rings (SGRs) are presented in Fig. 6. Total Ash content across all samples ranged from 5.01 to 10.15%, with a coefficient variation of 13.15%. Significant differences were observed among groups with different SGR counts. Specifically, the Total Ash in samples with no SGRs (SG0) and wild-type (W) were significantly higher than those with four (SG4), three (SG3), or two (SG2) SGRs. The average Total Ash content of SG4 was 6.33%, significantly lower than that of SG0 (18.71%) and W (18.21%). These findings indicate that Total Ash content increases as the number of SGRs decreases, likely due to a higher proportion of mulberry residue within samples with fewer SGRs, resulting in elevated ash content. According to the 2020 edition of the Chinese Pharmacopoeia (Volume I), the Total Ash content of Bombyx Batryticatus must not exceed 7.0%. Among 34 batches tested, two batches in SG4 exceeded this limit (5.88%), compared to 12 batches in SG3 (35.29%), 13 batches in SG2 (38.24%), 27 batches in SG0 (79.41%), and 28 batches in W (82.35%). This suggests a strong negative correlation between the number of SGRs and Total Ash content: higher SGR counts are associated with lower Total Ash content and lower rates of exceedance.

Fig. 6
figure 6

Determination of Bombyx Batryticatus quality indicators based on the number of Silk Gland Rings (SGRs). (A) Total ash, (B) Extract, (C) Total protein, and (D) Beauvericin.

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In Fig. 6B, Extract content ranged from 20.31 to 39.39%, with a coefficient variation of 13.48%. Extract levels increased as SGR numbers decreased, with significant differences observed among the groups. The average Extract content in SG2, SG3, and SG4 was 27.49%, 26.64%, and 25.33%, respectively, with SG4 being significantly lower than SG0 and W (18.07% and 18.32%, respectively). Figure 6C shows Total Protein content varying from 0.66 to 5.91%, with a coefficient variation of 26.65%. Average Total Protein values for SG4, SG3, SG2, SG0, and W were 3.56%, 3.76%, 3.70%, 3.98%, and 3.97%, respectively, with no significant differences between groups. Figure 6D presents Beauvericin levels, ranging from 0.036 to 0.391 mg/g, with a high coefficient of variation of 49.89%. As an important quality marker unique to Bombyx Batryticatus, Beauvericin content showed a clear positive correlation with SGR number. While no significant difference was found between SG4 and SG3, SG4 contained 42.10% more Beauvericin than SG0 and 47.59% more than W, both significant differences.

Overall, these results demonstrate that among the four key quality indicators (Total Ash, Extract, Total Protein, and Beauvericin), significant differences across SGR groups were observed in Total Ash, Extract, and Beauvericin contents. Total Ash and Extract increased with decreasing SGR numbers, whereas Beauvericin decreased. This clearly indicates that the quantity of SGRs influences the quality of Bombyx Batryticatus, with higher SGR counts correlating with better quality.

The nucleoside components identified in Bombyx Batryticatus include hypoxanthine, guanosine, uracil, adenine, and uridine (Fig. 7). Hypoxanthine content ranged from 5.13 to 328.02 μg/g, with a coefficient of variation (CV) of 48.03%. Significant differences were observed among groups with different SGR counts, showing a clear decrease in hypoxanthine content as the number of SGRs declined. The average hypoxanthine concentration in SG4 samples was 128.29 μg/g, significantly higher than SG0 (32.62%) and W (62.52%). Guanosine content varied between 80.57 and 1177.2 μg/g (CV = 45.52%), but differences among SGR groups were not significant. Uracil content ranged from 85.45 to 674.36 μg/g (CV = 40.46%), with significant differences detected across SGR groups. Overall, uracil levels increased as SGR counts decreased, although the trend was less pronounced compared to other indicators. Specifically, SG4 levels were significantly lower than those of SG0 (24.47%) and W (26.31%). Adenine content ranged from 93.07 to 674.25 μg/g (CV = 42.17%) and uridine from 26.20 to 944.31 μg/g (CV = 73.38%), with no significant differences observed between SGR groups.

Fig. 7
figure 7

Determination of nucleoside and flavonoid contents in Bombyx Batryticatus based on the number of Silk Gland Rings (SGRs). (A) Hypoxanthine, (B) Guanosine, (C) Uracil, (D) Adenine, (E) Uridine, (F) Astragalin, (G) Quercetin, (H) Kaempferol, (I) Rutin.

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Regarding flavonoid content, four compounds were detected: astragalin, quercetin, kaempferol, and rutin. Astragalin ranged from 3.22 to 39.63 μg/g (CV = 38.55%), showing significant differences among SGR groups and a decreasing trend with fewer SGRs. The average astragalin concentration in SG4 was 24.04 μg/g, significantly higher than in SG0 (54.92%) and W (67.24%). Quercetin content varied from 2.90 to 71.22 μg/g (CV = 54.04%) but did not differ significantly among groups. Kaempferol ranged from 1.35 to 43.97 μg/g (CV = 84.79%), with significant differences observed; SG0 had the highest concentration (12.33 μg/g), significantly greater than SG4 and SG3. Rutin content ranged from 3.42 to 84.09 μg/g (CV = 61.86%) without significant variation among groups.

In summary, hypoxanthine, uracil, astragalin, and kaempferol showed significant variation with differing SGR counts. Hypoxanthine and astragalin decreased as SGR numbers declined, while uracil increased with fewer SGRs.

Quality evaluation of Bombyx Batryticatus from different sources

We applied PCA to evaluate quality differences among Bombyx Batryticatus samples from various sources across different SGR grades. The PCA results are shown in Fig. 8, with the corresponding loading plots in Supplementary Fig. S1 and eigenvalues/eigenvectors in Supplementary Table S2.

Fig. 8
figure 8

Principal component analysis (PCA) of Bombyx Batryticatus quality from different sources under varying numbers of Silk Gland Rings (SGRs). (A) SG4, (B) SG3, (C) SG2, (D) SG0, (E) W.

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For SG4, PC1 (25.18%, eigenvalue = 3.273) and PC2 (19.39%, eigenvalue = 2.521) together explained 44.57% of the variance. Both eigenvalues exceeded 1, indicating that these components effectively captured the major variation in this dataset. The loading plot showed that adenine and rutin were positively associated with PC1, while total ash and beauvericin were negatively associated with PC1. Uracil correlated positively with PC2, whereas uridine correlated negatively. The score plot indicated that Farmer-derived samples were mainly located in the first quadrant, suggesting higher levels of adenine, rutin, and uracil. Base samples clustered in the second quadrant, reflecting higher uracil together with elevated total ash and beauvericin. Other samples positioned along the negative axis of PC1, also indicating relatively high total ash and beauvericin.

For SG3, PC1 (25.58%, eigenvalue = 3.325) and PC2 (21.39%, eigenvalue = 2.780) explained 46.97% of the variance. Loadings indicated that Echinacea purpurea and beauvericin correlated positively with PC1, whereas adenine and uridine correlated negatively. Kaempferol and quercetin correlated positively with PC2, while beauvericin correlated negatively. Farmer samples clustered along the positive axis of PC2, reflecting higher kaempferol and quercetin. Base samples were positioned on the negative axis of PC1, suggesting higher adenine and uridine. Sichuan samples clustered along the positive axis of PC1, associated with higher Echinacea purpurea and beauvericin contents. For SG2, PC1 (24.48%, eigenvalue = 3.183) and PC2 (18.34%, eigenvalue = 2.384) explained 42.82% of the variance. Adenine and uridine were positively correlated with PC1, while beauvericin and Echinacea purpurea were negatively correlated. Kaempferol and quercetin correlated positively with PC2. Farmer and Base samples were mainly located on the positive side of PC1, suggesting higher adenine and uridine, while other samples clustered along the negative side of PC2, reflecting higher beauvericin and Echinacea purpurea.

For SG0, PC1 (21.70%, eigenvalue = 2.820) and PC2 (15.27%, eigenvalue = 1.985) explained 36.97% of the variance. Echinacea purpurea and Extract correlated positively with PC1, whereas adenine and uridine correlated negatively. Kaempferol was negatively correlated with PC2. Sichuan and Hebei samples clustered in the first quadrant, indicating higher Echinacea purpurea and Extract, while Farmer samples were mainly positioned on the negative axis of PC2, suggesting higher kaempferol levels. For the wild-type group (W), PC1 (21.57%, eigenvalue = 2.804) and PC2 (16.12%, eigenvalue = 2.095) explained 37.69% of the variance. Echinacea purpurea correlated positively with PC1, while adenine and uridine correlated negatively. Guanosine was positively correlated with PC2, while total ash correlated negatively. No clear separation was observed among different sources, indicating little quality differentiation in W samples.

Notably, across SG4, SG3, and SG2, the loading plots consistently highlighted beauvericin, adenine, and uridine as high-loading variables on PC1. This suggests that these three compounds serve as core quality evaluation indicators for Bombyx Batryticatus under these SGR grades.

After standardizing all quality indicators, cluster analysis was performed to explore the grouping patterns of Bombyx Batryticatus samples from various sources. The results are shown in Fig. 9. In the SG4 group, samples were divided into three main clusters: the first cluster comprised Base samples; the second included Guangxi, Anhui, and Hebei samples; and the third grouped Farmer samples together with some Base samples. Sichuan samples exhibited poor clustering consistency. For SG3, three major clusters were also identified: the first combined Anhui and some Sichuan samples; the second included Hebei, parts of Sichuan, and some Farmer samples; the third cluster contained five batches from Base, two batches from Guangxi, and three batches from Sichuan. In SG2, samples were primarily split into two clusters, with Base and Sichuan samples forming separate major groups. In SG0, samples from Base and Farmer are predominantly clustered together. In the wild-type group (W), the clustering of samples from different sources was poor, consistent with the PCA results.

Fig. 9
figure 9

Cluster dendrograms of Bombyx Batryticatus quality from different sources under varying numbers of Silk Gland Rings (SGRs). (A) SG4, (B) SG3, (C) SG2, (D) SG0, (E) W.

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Our findings indicate that, except for the wild-type group, the distinction among samples from different sources was limited across SGR grades. The main differentiation occurred between Farmer and Base samples compared to other sources.

We conducted a correlation analysis of all quality indicators in Bombyx Batryticatus, revealing strong relationships between various parameters (Fig. 10). Notably, the number of Silk Gland Rings (SGRs) showed a significant negative correlation with Total Ash, Extract, Total Protein, Uracil, and Kaempferol, and a significant positive correlation with Beauvericin, Hypoxanthine, Uridine, and Astragalin. These findings are crucial, as large-scale quality measurements confirm that a higher number of SGRs corresponds to superior quality in key indicators. This suggests that SGR count can serve as a simple yet effective marker for assessing the quality of Bombyx Batryticatus herbal material.

Fig. 10
figure 10

Correlation analysis of different quality indicators in Bombyx Batryticatus. *P < 0.05; **P < 0.01; ***P < 0.001 indicate significant correlation levels.

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Total Ash was positively correlated with Extract, Uracil, and Adenine, and negatively correlated with Beauvericin, Hypoxanthine, and Astragalin. Extract showed significant positive correlations with Guanosine, Uracil, and Kaempferol, and negative correlations with Beauvericin and Astragalin. Beauvericin was positively correlated with Astragalin and negatively correlated with Uracil, Adenine, Uridine, Kaempferol, and Rutin. Total Protein correlated positively with Guanosine and Uracil, and negatively with Adenine, Uridine, and Rutin. Hypoxanthine demonstrated positive correlations with Guanosine and Astragalin. Guanosine was positively correlated with Astragalin and negatively with Quercetin. Uracil correlated positively with Quercetin, Kaempferol, and Rutin. Adenine was positively associated with Uridine and Rutin, and negatively with Astragalin. Uridine correlated positively with Rutin and negatively with Astragalin. Astragalin showed a positive correlation with Quercetin, which in turn was positively correlated with Kaempferol and Rutin.

Among the quality indicators in Bombyx Batryticatus herbal material, Total Ash, Extract, and Beauvericin are considered the most important. The regression analysis results are presented in Fig. 11. Total Ash and Extract exhibited a significant positive linear relationship, with R2 = 0.0818, P < 0.001, and the regression equation Y = 1.17X + 20. Conversely, Total Ash and Beauvericin showed a significant negative linear correlation, with R2 = 0.225, P < 0.001, and the regression equation Y =  − 0.0437X + 0.481. These findings indicate that as Total Ash decreases, both Extract content decreases and Beauvericin content increases.

Fig. 11
figure 11

Linear regression analysis between Total ash & Extract, and Beauvericin. The gray area represents the 95% confidence interval.

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Discussion

Since ancient times, Bombyx Batryticatus quality has been traditionally evaluated by its cross-sectional appearance, with “a glossy black cross-section” regarded as superior. The various editions of the Chinese Pharmacopoeia that include Bombyx Batryticatus uniformly require “four bright brown or black silk gland rings in the cross-section”3. This highlights that the silk gland rings (SGRs) are a critical factor reflecting the quality of this medicinal material, and the number of SGRs in the cross-section positively correlates with its quality to some extent. Chemical studies have identified key components of Bombyx Batryticatus, including beauvericin, flavonoids, nucleosides, amino acids, peptides, and proteins. In this study, we established methods to quantify total protein, nucleoside content, and flavonoids in the material, and performed correlation analyses between the number of SGRs and all measured quality indicators. Results showed that SGRs were significantly negatively correlated with Total Ash, Extract, Total Protein, Uracil, and Kaempferol, and significantly positively correlated with Beauvericin, Hypoxanthine, Uridine, and Astragalin. Beauvericin, an endogenous metabolite produced in silkworms (Bombyx mori) infected by Beauveria bassiana, has been reported to exert strong anticonvulsant effects in both in vivo and in vitro models. In addition, it demonstrates notable antibacterial and antitumor activities. As one of the core bioactive constituents of Bombyx batryticatus, beauvericin not only aligns well with the traditional medicinal functions of this material but also possesses favorable pharmacokinetic properties and controllable safety. These attributes highlight its considerable potential for development as an antiepileptic agent, as well as in the discovery of novel antibacterial and anticancer therapeutics30. Beauvericin is known for its sedative and anticonvulsant pharmacological effects, and many researchers have adopted it as an efficacy evaluation and quality control marker for Bombyx Batryticatus due to its specificity in this material31,32,33. In our study, Beauvericin content ranged from 0.036 to 0.391 mg/g, with a variation coefficient of 49.89%, indicating a relatively high and specific content as a single quality marker. Furthermore, Beauvericin levels in samples with four (SG4) and three (SG3) silk gland rings were significantly higher than those with fewer rings. Beauvericin has also been shown to exert potent anticancer activities: it inhibits the proliferation of human breast cancer cells by modulating the estrogen receptor-α (ERα)/p38 pathway, acting as a full ER antagonist34. Additionally, it induces apoptosis in hepatocellular carcinoma (HCC) cells via the PI3K/AKT pathway, thereby promoting tumor cell death35. These findings underscore Beauvericin’s promising therapeutic potential in cancer treatment.

The nucleoside components of Bombyx Batryticatus include five compounds: hypoxanthine, guanosine, uracil, adenine, and uridine. Nucleosides, composed of purines and pyrimidines, are fundamental building blocks of nucleic acids and exhibit significant pharmacological activities. These effects extend beyond their traditional roles in cellular energy metabolism and nucleic acid synthesis, encompassing a wide range of therapeutic applications. Research has explored the neuroprotective, antiviral, anticancer, and immunomodulatory properties of nucleosides, highlighting their value in treating various diseases. Nucleosides such as adenosine, guanosine, and inosine have demonstrated potential for treating neurodegenerative disorders by regulating excitotoxicity and interacting with adenosine receptor subtypes. They exhibit neurotrophic and neuroprotective effects, including seizure suppression, alleviation of anxiety and depression, and slowing of neurodegenerative progression36. Novel nucleoside analogs, including halogen-substituted derivatives, have shown potent antiviral and anticancer activities in vitro37. Such nucleoside analogs are incorporated into pharmaceutical formulations for treating infections, tumors, and autoimmune diseases. They are particularly effective in modulating type 1 and type 2 cytokine expression, thereby enhancing immunoregulatory effects38. Hypoxanthine, often considered one of the active components in earthworm-based antitussive and asthma-relieving treatments, also contributes to expectorant and anti-congestive effects. Given that Bombyx Batryticatus is frequently used in combination with earthworm in traditional prescriptions, it may share some of these therapeutic properties39. It is important to note that the pharmacological effects of nucleosides vary depending on their specific chemical structures.

The flavonoid components in Bombyx Batryticatus include four compounds: astragalin, quercetin, kaempferol, and rutin. Astragalin is a metabolite derived from Bombyx Batryticatus consuming mulberry leaves and possesses pharmacological effects such as anti-inflammatory and analgesic activities40. It exhibits antioxidant, anti-inflammatory, and anti-necrotic properties, protecting against spinal cord ischemia/reperfusion injury by alleviating oxidative stress and inflammation, thereby improving motor function and histopathological outcomes in affected mice41. Astragalin can inhibit palmitic acid-induced insulin resistance and oxidative stress in HepG2 cells through the NF-κB and JNK signaling pathways42. Quercetin, a natural flavonoid, demonstrates diverse pharmacological effects including anti-inflammatory, antioxidant, antibacterial, anticancer, antiviral, antidiabetic, cardioprotective, and neuroprotective activities. Its anticancer mechanisms involve anti-angiogenesis, anti-metastasis, and anti-proliferation effects, highlighting its therapeutic potential43. Quercetin also protects low-density lipoprotein (LDL) from oxidation and inhibits angiogenesis, contributing to its potential health benefits44. Kaempferol has been shown to alleviate hypertension and cardiac hypertrophy in rats by inhibiting the tumor necrosis factor-α (TNF-α) pathway, which plays a key role in inflammatory responses45. Additionally, kaempferol exerts multipotent neuroprotective effects in central nervous system disorders by modulating pro-inflammatory pathways, suppressing oxidative stress, and increasing brain-derived neurotrophic factor (BDNF) levels, thus offering therapeutic benefits for diseases such as Alzheimer’s, Parkinson’s, and ischemic stroke46. Rutin possesses a variety of pharmacological activities including antioxidant, anti-inflammatory, antidiabetic, antihyperlipidemic, renoprotective, hepatoprotective, cardioprotective, and anticancer effects. It modulates signaling pathways such as PI3K/AKT, β-catenin, JAK-STAT, and apoptosis pathways, enhancing its therapeutic potential47. The potential therapeutic effects of rutin on coronary heart disease have been demonstrated in porcine models through regulation of ERK1/2 and Akt signaling pathways48 At the same time, the links between the nucleoside and flavonoid components of Bombyx Batryticatus and their pharmacological functions warrant further investigation.

These findings collectively demonstrate the broad global medicinal applications of Bombyx Batryticatus as an animal-derived medicine. Our study primarily focuses on the correlation between the number of silk gland rings (SGRs) and these quality indicators; this research and its results represent the first such discovery worldwide. Our findings provide a foundation for assessing the quality of Bombyx Batryticatus herbal material through simple quantification of SGRs.

Conclusions

This study systematically investigated the quality characteristics of 34 batches of Bombyx Batryticatus herbal materials from different geographic origins and markets. By measuring 13 quality indicators, we comprehensively analyzed the correlation between Silk Gland Rings (SGRs) and the quality of Bombyx Batryticatus. The results showed that samples with four Silk Gland Rings exhibited the highest quality.