Introduction
Sarcandra glabra is a valuable medicinal plant belonging to the family Chloranthaceae, mainly distributed south of the Yangtze River in China. Phytochemical analysis has shown that Sarcandra glabra is rich in coumarins, flavonoids, sesquiterpenes, and phenols, which are reported to have diverse biological activities, including hepatoprotective,1,2 antitumor,3 antiviral,4 anti-inflammatory, and immunomodulatory actions.5,6 Isofraxidin, a major coumarin compound derived from Sarcandra glabra, is considered an index component due to its remarkable anti-inflammatory, anti-cancer, and antioxidative properties, and multi-target effects.7 The advantages of a definitive clinical effect, good safety profile, and abundant resources have led to the development of numerous Chinese patent medicines containing Sarcandra glabra as a primary ingredient in modern times. Of these, 38 have been approved for marketing by the State Food and Drug Administration of China.1
Isofraxidin can attenuate a variety of destructive signaling mediators and therapeutic targets in certain diseases. Mechanistically, isofraxidin has been reported to regulate signaling pathways, exerting anti-inflammatory and anti-cancer effects.8–10 Additionally, isofraxidin can affect the activity of various enzymes involved in the pathogenesis of certain cardiac and neurological diseases.11–13 Although chemical synthesis of isofraxidin has been applied due to its wide application, the process is limited by the number of synthesis steps and the unmanageable nature of the process.14
The term “endophyte” refers to “microbes that colonize living, internal tissues of plants” without causing any immediate, overt negative effects.15 Endophytes can stimulate the growth and production of secondary metabolites in host plants. Due to the possibility of genetic recombination between the endophyte and the host,16 rare compounds that can be produced by the host can also be produced by endophytes, thus reducing the need for harvesting slow-growing and rare plants. Previous studies have indicated that the endophytes of Taxus spp. (e.g., Taxomyces andreanae and Pestalotiopsis microspore) are capable of producing the antileukemic and antitumor drug taxol.17 Currently, the wild resources of Sarcandra glabra plants are decreasing, and cultivated ones contain lower concentrations of isofraxidin. The demand for such important chemical constituents far exceeds what is available from existing natural resources. Endophytes, a less-studied class of microorganisms, have great potential for exploitation in the medical, agricultural, and industrial fields.
Fermentation technology is a new method for obtaining the active ingredients of functional health foods. It uses the powerful enzyme systems of microorganisms to achieve directional biotransformation of certain active ingredients in natural food, thereby improving the yield of these active ingredients. In the present study, to produce isofraxidin in a targeted and efficient way, we isolated and identified endophytes from Sarcandra glabra. The isolated endophytes were then co-fermented with Sarcandra glabra to induce the production of active metabolites.18
Materials and methods
Sampling of Sarcandra glabra
The Sarcandra glabra samples were collected from Changsha, Hunan Province, and identified by Professor E-Hu Liu (Department of Pharmacognosy at China Pharmaceutical University). The stems, roots, and leaves were separated using a sterile scalpel, placed in sterile plastic bags, and stored at −20°C.
Isolations of endophytes from Sarcandra glabra
For the isolation of endophytes from Sarcandra glabra, portions of lesion-free roots, stems, and leaves were washed in tap water.19 They were surface sterilized by immersion in 75% ethanol for 3 m under sterile conditions. After allowing the alcohol to evaporate, the roots, stems, and leaves were surface sterilized with 0.1% mercury chloride for 2 m. The samples were rinsed with autoclaved water three to five times and dried with sterile paper towels. The roots, stems, and leaves were then cut into small pieces and inoculated onto potato dextrose agar (PDA) plates containing potato (20%), dextrose (2%), and agar (1.5–2%) (Qingdao Hope Bio-Technology, China) using a sterile scalpel. The inoculated plates were incubated at 28 ± 2°C for seven to ten days, during which the emergence of bacteria and fungi from the tissues was observed. The nascent bacteria were transferred to beef extract peptone agar plates containing peptone (1%), beef extract (0.3%), NaCl (0.5%), and agar (1.5–2.5%) (Qingdao Hope Bio-Technology, China) and incubated at 37°C for one to two days. Individual mycelia were inoculated on each PDA plate supplemented with streptomycin at 100 µg/mL and incubated for seven to ten days at 28 ± 2°C under dark conditions. All cultures were periodically transferred to fresh plates until pure endophyte cultures were obtained. The same procedure was used to blot sterilized roots, stems, and leaves on PDA plates, while a plate coated with 200 µL of sterile water from the last rinse served as a negative control.
Fermentation sample preparation
The bacteria isolated in this study were grown in beef extract peptone medium (Qingdao Hope Bio-Technology, China), and the fungi were grown in potato dextrose water (Qingdao Hope Bio-Technology, China) liquid medium supplemented with streptomycin at 100 µg/mL. The endophyte suspensions were prepared by shaking the cultures at 37 °C and 150 rpm for 24 h.
Five grams of Sarcandra glabra root, stem, and leaf powder (sieved through a four-mesh sieve) were accurately weighed respectively and placed in a conical flask, each with 30 mL of sealed growth medium. The samples were sterilized at 121°C for 20 m and then used as substrates. Four milliliters of each bacterial suspension was added to the corresponding substrate containing the respective medium (n = 3) (endophytes isolated from different tissues were only added to the corresponding substrates), respectively, and the samples were lyophilized after fermentation to obtain fermentation samples. Another 4 mL of sterile water was added to the substrate, and the same culture method was used to prepare a blank control sample.
Screening of endophytes to increase isofraxidin content
The endophytic isolates obtained were screened for strains capable of increasing the isofraxidin content. Chromatographic separation was performed using a Thermo BDS Hypersil C18 column (4.6 × 250 mm, 5 µm, Thermo Fisher Scientific, Waltham, MA, USA) at a column temperature of 40°C. The mobile phase consisted of ultra-pure water with 0.1% phosphoric acid (eluent A) and acetonitrile (eluent B), using isocratic conditions of 80% A and 20% B. The compounds were monitored at 344 nm, with a sample injection volume of 10 µL. The flow rate was set at 1.0 mL/m.20
A standard solution of isofraxidin was prepared by accurately weighing 10 mg of the standard, dissolving it in methanol to achieve a concentration of 100 µg/mL. This standard solution was further diluted with 50% methanol to obtain final concentrations of 5, 10, 20, 40, and 80 µg/mL, respectively.
Fermentation samples weighing 0.5 g were extracted with 10 mL of 70% methanol using ultrasonic extraction for 30 m. The extract was then centrifuged at 4,000 rpm for 10 m, and the supernatant was passed through a 0.22 µm microporous filtration membrane to obtain the working solution for high-performance liquid chromatography (HPLC) analysis. Each extraction was performed in triplicate.
Validation of methodology
Based on the established HPLC conditions, standard curves were prepared using different concentrations of isofraxidin standard solutions. Precision was assessed by analyzing six replicate sample solutions. The repeatability was determined by evaluating six replicates of a single sample. A stability study was conducted by analyzing a sample at different time points over a day (0, 2, 4, 6, 8, 12, and 24 h). Each sample solution was tested three times in parallel.
Extraction of fungal genomic DNA
Total genomic DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The DNA concentration was determined using a Nano-100 micro-spectrophotometer (AllSheng Instruments, Hangzhou, China) according to the manufacturer’s instructions for polymerase chain reaction (PCR) amplification.
Amplification of gene
The universal internal transcribed spacer (ITS) region was amplified using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) (all primers from Sangon Biotech, Shanghai, China) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) for fungal strain identification. The total PCR mixture volume was 50 µL, consisting of 21 µL dd water, 1 µL of each primer, 25 µL Taq PCR Mastermix (TIANGEN Biotech, Beijing, China), and 2 µL of template DNA. The PCR was programmed with an initial denaturation at 95°C for 5 m, followed by 35 cycles of denaturation at 94°C for 1 m, annealing at 55°C for 30 s, and extension at 72°C for 2 m. The final extension was performed at 72°C for 10 m.21
Taxonomic identification of the endophytic bacterial strains was carried out using the universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′). The total PCR mixture volume was 25 µL, comprising 9.5 µL dd water, 1 µL of each primer, 12.5 µL Premix Taq™ (Takara Bio, Beijing, China), and 1 µL of endophytic bacterial solution. The PCR was programmed with an initial denaturation at 94°C for 5 m, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 59°C for 45 s, and extension at 72°C for 1 m. The final extension was performed at 72°C for 10 m.22
Quality and integrity detection for PCR products
To prepare a 1% (w/v) agarose gel, dilute 50 × TAE Buffer (Sangon Biotech, Shanghai, China) to 1 × with purified water and mix it with the appropriate amount of agarose powder (Baygene, Shanghai, China).
Add 5 µL of GelRed (Sangon Biotech, Shanghai, China) to 1 mL of 10 × Loading Buffer (Vazyme, Nanjing, China). Then, add 1 µL of GelRed to 200 µL of DNA Marker (Takara Bio, Beijing, China), mix well, and store at 4°C. In each well, add 10 µL of PCR products and 1 µL of 10 × Loading Buffer, and place 5 µL of DL 3000 Marker in wells on both sides of the gel electrophoresis tank to indicate the molecular weight. Electrophoresis was conducted at 120 mV for 10 m. Successfully amplified PCR products (single, bright bands) were sequenced by Sangon Biotech Ltd Co.
Sequencing and construction of phylogenetic tree
The sequences were submitted to the GenBank database and compared with existing sequences in GenBank using Blast analysis (MEGA 6.0 software). A phylogenetic tree was constructed using MEGA 6.0 software to determine the relationships and conduct phylogenetic analysis.
Statistical analysis
The means and standard deviations were calculated using MS Excel 2010. Statistical analyses were conducted with GraphPad PRISM version 8.0 (GraphPad Software, La Jolla, CA, USA). Data were presented as mean ± SD. The significance of differences among multiple groups was analyzed by one-way analysis of variance with Tukey’s multiple comparison test, and p < 0.05 indicated a significant difference.
Results
Isolation of the endophytes
The endophytes were placed on a medium for continuous inoculation passaging to obtain pure cultures. A total of 15 strains of endophytic bacteria and 6 strains of endophytic fungi were isolated from the roots, stems, and leaves of Sarcandra glabra. Five endophytic bacteria [Enterobacter (BR 1), Leclercia adecarboxylata (BR 2), Burkholderia stabilis (BR 3), Bacillus mobilis (BR 4), and Bacillus wiedmannii (BR 5)] and two endophytic fungi [Trametes versicolor (FR 1) and Penicillium rubens (FR 2)] were found in the roots. Five endophytic bacteria [Enterobacter (BS 1), Lelliottia amnigena (BS 2), Priestia aryabhattai (BS 3), Paenibacillus humicus (BS 4), and Lysinibacillus macrolides (BS 5)] and one endophytic fungus Aspergillus aflatoxiformans (FS 1) were isolated from the stems. Five endophytic bacteria [Enterobacter (BL 1), Pantoea rodasii (BL 2), Kluyvera intermedia (BL 3), Priestia aryabhattai (BL 4), and Lysinibacillus pakistanensis (BL 5)] and three endophytic fungi [Diaporthe celeris (FL 1), Diaporthe hongkongensis (FL 2), and Nodulisporium indicum (FL 3)] were isolated from the leaves.
Estimation of isofraxidin contents in Sarcandra glabra after the fermentation of endophytes
HPLC-based analysis revealed absorption peaks at 344 nm for the samples. The fermented samples displayed the same peak at the same retention time as the raw samples and the isofraxidin standard (Figs. 1 and 2). Compared with the root blank samples, the content of isofraxidin in Sarcandra glabra fermented by endophytes BR 1, BR 5, and FR 1 was 1.37, 1.27, and 1.11 times higher than the control, respectively. The content of isofraxidin in Sarcandra glabra fermented by endophytes FL 1 and FL 2 was 1.40 and 1.16 times higher than the blank samples, respectively (Tables 1 and 2). These results indicate that two strains of endophytic bacteria (BR 1 and BR 5) and three strains of endophytic fungi (FR 1, FL 1, and FL 2) were able to increase the isofraxidin content in the corresponding fermentation samples, with the endophytic fungus FL 1 having the most significant effect (Fig. 3).
Table 1Determination of isofraxidin in the fermentation samples of Sarcandra glabra
| Endophytic bacteria | Content of isofraxidin (mg/g)
|
|---|
| Fermentation samples of roots | Fermentation samples of stems | Fermentation samples of leaves |
|---|
| BR 1 | 0.8895 ± 0.0033**** | | |
| BR 2 | 0.6306 ± 0.0158 | | |
| BR 3 | 0.5273 ± 0.0088 | | |
| BR 4 | 0.5704 ± 0.0127 | | |
| BR 5 | 0.8190 ± 0.0045**** | | |
| BS 1 | | 0.3746 ± 0.0021 | |
| BS 2 | | 0.3317 ± 0.0026 | |
| BS 3 | | 0.2811 ± 0.0064 | |
| BS 4 | | 0.3764 ± 0.0021 | |
| BS 5 | | 0.2423 ± 0.0050 | |
| BL 1 | | | 0.0329 ± 0.0090 |
| BL 2 | | | 0.0116 ± 0.0006 |
| BL 3 | | | 0.0033 ± 0.0001 |
| BL 4 | | | 0.0270 ± 0.0007 |
| BL 5 | | | 0.0161 ± 0.0045 |
| Sterilized water | 0.6470 ± 0.0036 | 0.4838 ± 0.0037 | 0.1128 ± 0.0030 |
| Fermentation solution control | —— |
Table 2Determination of isofraxidin in the fermentation samples of Sarcandra glabra
| Endophytic fugus | Content of isofraxidin (mg/g)
|
|---|
| Fermentation samples of roots | Fermentation samples of stems | Fermentation samples of leaves |
|---|
| FR 1 | 0.7395 ± 0.0003**** | | |
| FR 2 | 0.6578 ± 0.0034 | | |
| FS 1 | | 0.5322 ± 0.0096 | |
| FL 1 | | | 0.5751 ± 0.0047**** |
| FL 2 | | | 0.4754 ± 0.0029**** |
| FL 3 | | | 0.3953 ± 0.0021 |
| Sterilized water | 0.6639 ± 0.0108 | 0.5510 ± 0.0010 | 0.4102 ± 0.0023 |
| Fermentation solution control | —— |
Validation of methodology
The standard curve was plotted with the sample volume (µg) of the standard on the horizontal axis and the peak area on the vertical axis. The regression equation for isofraxidin is y = 5.9826x + 13.479, with an r-value of 0.9998. The relative standard deviations for precision, repeatability, and sample stability were 1.82%, 2.34%, and 2.51%, respectively (all less than 3%), indicating that the developed HPLC method is valid and suitable for sample analysis (Table 3).
Table 3Regression equation, precision, repeatability, and stability of isofraxidin
| Sample | Regression equation | R2 | Precision RSD(%) | Repeatability RSD(%) | Stability RSD(%) |
|---|
| n = 6 | n = 6 | 24 h |
|---|
| Isofraxidin | y = 5.9826x + 13.479 | 0.9998 | 1.82 | 2.34 | 2.51 |
Identification of the yield-enhancing endophytes
The bacterial fluid of the endophytic bacterium was amplified to obtain a target fragment of approximately 1,500 bp. The total DNA of the endophytic fungi was extracted, and the ITS region was amplified to obtain a target fragment of approximately 550 bp (Fig. 4). The yield-enhancing endophytes (BR 1, BR 5, FR 1, FL 1, and FL 2) were characterized using gene sequencing technology. The nucleotide sequences obtained from the endophytes were submitted to the NCBI database, and their corresponding accession numbers are available in GenBank. Based on the results of sequence alignment conducted on the NCBI BLAST server, two yield-enhancing endophytic bacteria were identified as BR 1 and BR 5). The three yield-enhancing endophytic fungi were identified as FR 1, FL 1, and FL 2) (Figs. 5–7).
Discussion
Sarcandra glabra was chosen as the source plant for isolating endophytes because it has been reported to be the most vital natural source of isofraxidin. The complex pathophysiological mechanisms underlying diseases such as cancer, heart failure, and neurodegenerative disorders have prompted the need for multi-targeted compounds. Given these complexities, isofraxidin might be introduced as an effective multi-target drug. We aimed to isolate endophytes from Sarcandra glabra and screen them for their potential to promote isofraxidin production. Isofraxidin, present in herbal sources, has a unique pharmacokinetic profile and low levels of side effects.23 However, the isolation and purification of isofraxidin are slow and low-yielding, and the chemical synthesis involves multiple steps.24 To our knowledge, the main pathway of isofraxidin biosynthesis has not been revealed.25 Thus, there is a need to use biotechnological methods to enhance the yield of isofraxidin. Previous studies have shown that interactions between fungi and plants can enhance secondary metabolites in vivo and make plants more resistant to harsh environments.26 Among the methods to enhance secondary metabolites in plants, microbial fermentation techniques are the most cost-effective. Endophytes have been reported to effectively increase the yield of secondary metabolites.27
In this study, five strains of endophytes (BR 1, BR 5, FR 1, FL 1, and FL 2) were isolated, which can increase the content of isofraxidin. The results of the HPLC analysis demonstrated the presence of unidentified compounds in the raw Sarcandra glabra. However, these compounds disappeared after fermentation with the production-enhancing endophytes. This suggests that the increased levels of isofraxidin may result from biotransformation by the endophytes. For example, C. globosum CGMCC 6882, as reported by Wang, could produce extracellular polysaccharides with excellent inhibitory effects against both E. coli and S. aureus.28 Endophytic fungi of Aspergillus aculeatus, Penicillium georgiense, and Aspergillus flavus isolated from Polygonum cuspidatum could convert polydatin into resveratrol, thus promoting plant growth and development.29 Our findings align with these studies, suggesting the application of endophytes to enhance the yield of secondary metabolites. Notably, FL 1 exhibited the greatest capacity to enhance yield. Although we have not confirmed the production of isofraxidin by the endophytes themselves, most studies indicate that endophytic fungi can produce host compounds. For example, a macrolide antibiotic, lasiodiplodin, was isolated from the endophytic fungus Lasiodiplodia pseudotheobromae J-10 associated with the medicinal plant Sarcandra glabra.30 However, the specific mechanisms remain poorly understood.
Future research directions
The limitation of this study is that the optimal fermentation process was not identified, despite discovering that five strains increased the content of isofraxidin. The fermentation conditions need to be further optimized to obtain a higher amount of isofraxidin. Meanwhile, the separation of isofraxidin in fermented samples by column chromatography is straightforward and effective, making it suitable for obtaining purified isofraxidin products. Additionally, isolating isofraxidin from fungi and bacteria is a future research goal. Further studies are needed to reveal the mechanisms of interaction among these endophytes and to discover novel active metabolites, which could serve as a foundation for developing new drugs.
Conclusions
The results indicate that five bioactive endophytes (BR 1, BR 5, FR 1, FL 1, and FL 2) were isolated and identified. We found that the effect of the endophytic fungus FL 1 was the most significant, with the isofraxidin content in Sarcandra glabra treated with FL 1 increasing by 1.40 times. Therefore, the biotransformation function of endophytes can be utilized to enhance the yield of active ingredients.
Declarations
Data sharing statement
No additional data are available.
Funding
The authors greatly appreciate the financial support from the National Natural Science Foundation of China (82274074) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
Conflict of interest
The authors declare that they have no conflict of interest that could appear to influence the work reported in this paper.
Authors’ contributions
Conceptualization (YC, FL, EL), methodology (YC, TH, EL), data curation (YC, TH), writing (YC, EL), reviewing and editing (TH, EL), project administration (FL), and funding acquisition (EL).