*Article* **A Docosahexaenoic Acid Derivative (***N***-Benzyl Docosahexaenamide) as a Potential Therapeutic Candidate for Treatment of Ovarian Injury in the Mouse Model**

**Lirong Guo 1,2, Qing Gao 1,2, Jieqiong Zhu 1, Xiaobao Jin 1, Hui Yin 1,\* and Tao Liu 1,\***


**Abstract:** Commonly used clinical chemotherapy drugs, such as cyclophosphamide (CTX), may cause injury to the ovaries. Hormone therapies can reduce the ovarian injury risk; however, they do not achieve the desired effect and have obvious side effects. Therefore, it is necessary to find a potential therapeutic candidate for ovarian injury after chemotherapy. *N*-Benzyl docosahexaenamide (NB-DHA) is a docosahexaenoic acid derivative. It was recently identified as the specific macamide with a high degree of unsaturation in maca (*Lepidium meyenii*). In this study, the purified NB-DHA was administered intragastrically to the mice with CTX-induced ovarian injury at three dose levels. Blood and tissue samples were collected to assess the regulation of NB-DHA on ovarian function. The results indicated that NB-DHA was effective in improving the disorder of estrous cycle, and the CTX+NB-H group can be recovered to normal levels. NB-DHA also significantly increased the number of primordial follicles, especially in the CTX+NB-M and CTX+NB-H groups. Folliclestimulating hormone and luteinizing hormone levels in all treatment groups and estradiol levels in the CTX+NB-H group returned to normal. mRNA expression of ovarian development-related genes was positive regulated. The proportion of granulosa cell apoptosis decreased significantly, especially in the CTX+NB-H group. The expression of anti-Müllerian hormone and follicle-stimulating hormone receptor significantly increased in ovarian tissues after NB-DHA treatment. NB-DHA may be a promising agent for treating ovarian injury.

**Keywords:** docosahexaenoic acids; ovary; granulosa cells; cyclophosphamide; macamide

#### **1. Introduction**

Docosahexaenoic acid (DHA) is mainly used in the form of DHA-triglycerides or DHA-ethyl esters (DHA-EE). DHA and its derivatives have attracted considerable attention in various research fields, including those involving cell signaling, photoreceptors, the nervous system, and brain development, and exhibit positive effects on ovarian functions and diseases [1]. Additionally, DHA can migrate from different tissues to the ovary during gonadal development and promote the development of model animal ovaries [2]. The DHA content in the lungs is significantly reduced after removal of the ovaries [3]. The dysregulation of ovarian gene expression induced by a high-fat diet is restored by chronic polyunsaturated fatty acid (PUFA)/DHA supplementation [4]. Considering this progress in DHA research for the prevention and mitigation of ovarian-related diseases and functions, the continued discovery and development of new DHA derivatives for ovarian injury remains imperative.

*N*-benzyl docosahexaenamide (NB-DHA) is characterized by the presence of benzylamineconjugated DHA via an amide bond. Benzylated fatty acids are active ingredients exclusive to maca (*Lepidium meyenii*), named macamides. Notably, NB-DHA had the highest

**Citation:** Guo, L.; Gao, Q.; Zhu, J.; Jin, X.; Yin, H.; Liu, T. A Docosahexaenoic Acid Derivative (*N*-Benzyl Docosahexaenamide) as a Potential Therapeutic Candidate for Treatment of Ovarian Injury in the Mouse Model. *Molecules* **2022**, *27*, 2754. https://doi.org/10.3390/ molecules27092754

Academic Editor: Giovanni Ribaudo

Received: 31 March 2022 Accepted: 23 April 2022 Published: 25 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

degree of unsaturation among all the identified macamides. Benzylamide is a chemical used to enhance drug activity. For example, *N*-benzyl salinomycin has been reported to exhibit anticancer and antibacterial activities [5]. Deoxynojirimycin derivatives have been studied and can be used as α-glucosidase inhibitors to improve type II diabetes; compound 18, containing an *N*-benzyl amide residue, showed the highest activity [6]. A molecule containing an *N*-benzyl amide residue was screened from a library of compounds, and the results revealed that the synthesized compound is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication inhibitor at non-toxic concentrations in vitro and a dual-acting SARS-CoV-2 protease inhibitor against the main protease [7]. Macamides possess various bioactivities, including reproductive health improvement, antioxidation, neuroprotection, anticancer, immunomodulation, and digestive system function-improving activities [8–13]. As a newly identified PUFA, NB-DHA can protect the intestinal epithelial barrier and effectively relieve the symptoms of acute colitis in mice [14], while *N*-benzyl eicosapentaenoamide (NB-EPA) alleviates neurobehavioral disorders in neonatal mice with hypoxic-ischemic brain injury through the p53–PUMA signaling pathway [15].

Chemotherapeutic drugs, such as clinically available cyclophosphamide (CTX), are toxic to dividing and proliferating cells [16,17]. Chemotherapy with CTX can cause ovarian injury, permanent amenorrhea, and increase the risk of premature menopause [18,19]. The maintenance of ovarian reserve function and prevention of infertility have always been considered by physicians as important prognostic factors during chemotherapy [20–22]. Granulosa cells (GCs), as the largest cell group in follicles, play a crucial role in follicle growth and ovarian function regulation. GCs also regulate the development of follicles and are the main functional cells that secrete reproductive hormones [23]. Therefore, it is necessary to find potential therapeutic candidates to relieve and treat ovarian injury, protect GCs, and maintain the growth and development of follicles and ovaries. Although hormone-based treatments for ovarian injury have been adopted in current clinical practice, they do not achieve the desired therapeutic effect, and they also have obvious side effects on the human body [24].

In the present study, NB-DHA were synthesized, purified, and administered intragastrically into mice with CTX-induced ovarian injury to assess the regulation of ovarian function. Frequency of occurrence, the stages of the estrous cycle, follicle numbers after H&E staining, four typical sex hormone levels, mRNA expression of five ovarian development-related genes (FOXL2, GDF9, LIF, OCT4, and SCF), granular cell apoptosis ratios, anti-Müllerian hormone (AMH), and follicle-stimulating hormone receptor (FSHR) expression were obtained and analyzed. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) fluorescence staining was performed to explore the ovarian injury repair mechanism.

#### **2. Results**

#### *2.1. Preparation of DHA-EE and NB-DHA*

Fish oil was hydrolyzed by lipase into free fatty acids, which were used for the synthesis of DHA-EE via the esterification reaction and NB-DHA via the carbodiimide condensation method. The DHA-EE and NB-DHA fractions were collected separately from an HPLC system with elution times of 26.5–30.5 and 26.0–29.0 min, respectively. Both collected fractions were rotary evaporated to dryness and identified by infrared spectroscopy and mass spectrometry (data not shown). Their purities, analyzed by HPLC, were 96.2% (DHA-EE, Figure 1A) and 98.3% (NB-DHA, Figure 1B). The dried samples were used for subsequent animal experiments.

**Figure 1.** Chromatograms and structural formulas of DHA-EE and NB-DHA. (**A**) Chromatograms of synthetic DHA-EE material and purified DHA-EE sample collected using a semipreparative HPLC system (26.5–30.5 min). (**B**) Chromatograms of synthetic NB-DHA material and purified NB-DHA sample collected using a semipreparative HPLC system (26.0–29.0 min). Structural formulas of DHA-EE (**C**) and NB-DHA (**D**).

#### *2.2. Body/Ovarian Weight and Estrous Cycle Analysis*

As shown in Figure 2A, CTX caused a significant decrease in body weight compared with the control group. The body weight of mice in all DHA-treated groups was significantly higher than that of mice in the CTX group at the end of the 21-day experiment, and body weight recovery was ranked as follows: CTX+NB-H > CTX+NB-M > CTX+DHA-EE > CTX+NB-L. Additionally, compared with the control group, CTX significantly decreased the ovarian weight in the CTX group. After modeling with CTX, ovarian atrophy and ovarian weight decreased significantly. The ovarian weight of mice in the CTX+NB-H, CTX+NB-M, and CTX+DHA-EE groups was higher than that in the CTX group (Figure 2B). CTX is an inducer for the ovarian model that involves prolonging or stagnating the female estrous cycle. The statistical results showed that the time of estrus was shortened, and proestrus, metestrus, and diestrus were prolonged in the CTX group, indicating a disordered estrous cycle in CTX-treated mice (Figure 2C). The 21-day complete estrous cycle of mice was plotted, and the results showed that the average estrous cycle of the control group was 5–6 days. In the CTX group, a complete cycle could not be observed after modeling, but a complete estrous cycle could be observed in each DHA group. DHA-EE and NB-DHA were both effective in alleviating the disorder of the estrous cycle and were ranked as follows: CTX+NB-H > CTX+NB-M > CTX+DHA-EE (Figure 2D).

**Figure 2.** Body weight, ovarian weight, frequency of occurrence, and estrous cycle. Weight Change/% (**A**) and ovarian weight (**B**) measured on days 1, 4, 7, 10, 13, 16, 19, and 22 after administration. Initial average weight of mice was set as 100%. Effect of DHA-EE and NB-DHA on estrous cycles. Frequency of occurrence of cycle stages during the 21 days (n = 6) (**C**) and estrous cycle regularity (**D**). Values in all figures are expressed as the mean ± SEM (x ± sem, n = 6), \*\* *p* < 0.01, \*\*\* *p* < 0.005 (the same below).

#### *2.3. Follicle Counting and Morphological Analysis*

The effects of DHA-EE and NB-DHA on follicular development were analyzed by H&E staining of the ovarian sections (Figure 3A). Abundant healthy follicles were observed in the control group, including primordial, primary, secondary, and atretic follicles, while the distribution of the four types of follicles in the other groups was altered (Figure 3B–E). For example, there were fewer primordial, primary, and secondary follicles, but more atretic follicles in the CTX group than in the control group. Compared with the CTX group, both CTX+DHA-EE and CTX+NB-DHA significantly increased the number of primordial follicles, especially in the CTX+NB-M and CTX+NB-H groups. Moreover, there were fewer atretic follicles in all DHA-treated groups than in the CTX group. The groups ranked as follows: CTX+NB-H > CTX+NB-M > CTX+DHA-EE > CTX+NB-L.

**Figure 3.** Effect of DHA-EE and NB-DHA on the development of follicles. Follicles after H&E staining (**A**). Magnification 100× and 400×. Scale bar: 250 and 50 μm. Number of different follicles: primordial follicles (**B**), primary follicles (**C**), secondary follicles (**D**), and atretic follicles (**E**). \*\*\* *p* < 0.005.

#### *2.4. Ovarian Hormone Levels in Serum and mRNA Expression Levels in Ovarian Tissue*

As shown in Figure 4A–D, serum gonadotropin levels, including E2 and AMH, were significantly lower in the CTX group than in the control group, whereas those of serum FSH and LH were significantly higher. Furthermore, the E2 level was significantly higher in all treatment groups compared with the CTX group. FSH and LH levels in all treatment groups and E2 levels in the CTX+NB-H group returned to normal. Additionally, the mRNA expression of five ovarian development-related genes (FOXL2, GDF9, LIF, OCT4, and SCF) were measured; FOXL2, OCT4, GDF9, and LIF were significantly downregulated (Figure 4E–H), whereas that of SCF was significantly upregulated in the CTX group compared with the control group (Figure 4I). Compared with those in the CTX group, the mRNA expression levels of FOXL2 and LIF in CTX+NB-M, FOXL2, GDF9, LIF, and OCT4 in CTX+NB-H, and FOXL2, LIF, and OCT4 in CTX+DHA-EE were significantly upregulated. SCF in CTX+NB-M, CTX+NB-H, and CTX+DHA-EE was significantly downregulated.

**Figure 4.** Effect of model and treatment on sex hormone levels and mRNA expression in mice. Sex hormone levels of E2 (**A**), AMH (**B**), FSH (**C**), and LH (**D**). mRNA expression levels of FOXL2 (**E**), GDF9 (**F**), LIF (**G**), OCT4 (**H**), and SCF (**I**) in mouse ovarian tissues, as determined by real-time PCR. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.005.

#### *2.5. GC Apoptosis*

To analyze ovarian cell apoptosis, the fractured DNA of apoptotic GCs in the antral follicles was observed using an in situ TUNEL assay. The number of apoptotic cells in the CTX group was significantly higher than that in the control group. However, after treatment with DHA-EE and NB-DHA, apoptosis of GCs was significantly decreased (Figure 5A). These results showed that apoptosis of GCs plays a crucial role in ovarian function development and growth in ovarian injured mice, and that DHA-EE and NB-DHA can restore ovarian function by inhibiting apoptosis. The proportion of TUNEL-positive cells decreased significantly after the additional administration of different doses of DHA-EE and NB-DHA (Figure 5C), especially in the CTX+NB-H and CTX+DHA-EE groups, in which the TUNEL-positive cell ratio was similar to that of the control group. This result reveals that high-dose NB-DHA can be considered suitable for the alleviation of CTX-induced ovarian cell apoptosis. DHA-EE also reduced GC apoptosis. The groups ranked as follows: CTX+NB-H > CTX+NB-M > CTX+DHA-EE > CTX+NB-L.

**Figure 5.** TUNEL and immunohistochemical analysis. Apoptosis of granulosa cells in ovarian tissues was measured via TUNEL assay (**A**). Green fluorescence: apoptotic cells; blue fluorescence: nucleus; magnification: 400×. The expression of AMH and FSHR in ovarian tissues were measured by immunohistochemical analysis (**B**). Blue: nucleus; brown: cells expressing AMH and FSHR in the cytoplasm; scale bar: 50 μm; magnification: 400×. TUNEL-positive cell ratios in the treatment groups were analyzed according to the number of TUNEL-positive granular cells among all granulosa cells in the follicle (**C**). Scoring of staining results (**D**,**E**). \*\* *p* < 0.01, \*\*\* *p* < 0.005.

#### *2.6. AMH and FSHR Expression in Ovaries*

FSHR and AMH expression was detected by immunohistochemical analysis. Positive immunohistochemical results were scored. As shown in Figure 5B, the expression of AMH and FSHR in the CTX group was significantly lower than that in the control group. The expression of AMH and FSHR significantly increased after DHA-EE and NB-DHA treatment (Figure 5D,E). Notably, the effect of NB-H was better than that of DHA-EE at the same dosage. The treatments increased the expression of AMH and FSHR in the following order: CTX+NB-H > CTX+NB-M > CTX+DHA-EE > CTX+NB-L.

#### **3. Discussion**

Macamide-rich extract has been reported to stimulate the reproductive system, increase the number of mature follicular cells in female mice, and increase the number of sperm produced by male mice [25]. NB-DHA is a DHA derivative that belongs to the macamide family. It is difficult for higher plants to synthesize long-chain PUFAs, causing the content of NB-DHA in maca to be extremely low [14]. Therefore, in this study, DHA-rich fish oil was used as the starting material, and NB-DHA was efficiently synthesized with the carbodiimide condensation method [26]. Commercially available DHA is mainly in the form of ethyl esters or triglycerides. Theoretically, the degradation of the ester and benzylamide groups is different in vivo; as DHA-EE and NB-DHA are metabolized and absorbed at different rates and via different mechanisms, they might display different bioactive profiles in the human body [27–29].

After modeling with CTX, the mice lost their appetite and caused weight loss. After the experiment, the ovaries atrophied and the weight of the ovaries decreased, causing ovarian injury. Compared with the CTX group, the body and ovarian weights of the mice increased significantly after the administration of CTX+NB-M and CTX+NB-H, and CTX+DHA-EE treatment has just stabilized their body weight. We speculated that CTX caused changes in the ovarian microenvironment in mice, causing ovarian damage and leading to ovarian atrophy, while DHA-EE and NB-DHA alleviated CTX-induced ovarian injury. Our results indicate that both DHA-EE and NB-DHA reverse ovarian injury by increasing the number of normal follicles and decreasing that of atretic follicles. Ovarian injury clinically manifests as an abnormal estrous cycle and ovarian function, and severe injury may lead to premature ovarian failure and infertility [30–32]. CTX-induced ovarian injury mice in the CTX group exhibited an irregular estrous cycle and changes in ovarian function indicators. The primordial follicles, which act as the initial unit of follicle maturation or generation, undergo a series of developmental stages that form primary, secondary, and mature follicles, which then release the oocytes for reproduction. Most primitive follicles eventually become closed follicles, and only a few reach maturity [33,34]. Therefore, the growth of primordial follicles is related to the development of the entire ovary. Ovarian injury can cause a decrease in AMH and E2 levels and an increase in FSH and LH levels, which are considered four important indices for the evaluation of ovarian injury and abnormal follicular maturation. Our results indicated that the irregular estrous cycles of mice in the CTX+DHA-EE, CTX+NB-M, and CTX+NB-H groups were positively regulated to close the regular cycle of mice in the control group with a prolonged estrus period and shortened late estrus and interval. CTX-induced ovarian injury resulted in enhanced serum FSH and LH levels and decreased serum E2 and AMH levels. Meanwhile, the number of primordial, primary, and secondary follicles was reduced, and the number of atresia follicles increased. However, the administration of DHA-EE and different doses of NB-DHA to alleviate this injury increased the number of primordial, primary, and secondary follicles, reduced the number of atretic follicles, increased serum AMH and E2 levels, and reduced FSH and LH levels. These results reveal the regulatory function of both DHA-EE and NB-DHA on the estrous cycle of mice with ovarian injury caused by CTX.

The formation, maturation, and growth of follicles are regulated by many intra- and extra-ovarian factors. FOXL2 is involved in multiple dysfunctional states in the ovary and is essential for GC differentiation and maintenance of ovarian function, and is expressed in GCs with low differentiation in small- and medium-sized follicles [35]. GDF9 encodes a protein secreted into the follicle by oocytes [36] and plays a pivotal role in optimizing the oocyte microenvironment and growth, development, atresia, ovulation, fertilization, and normal reproduction of the follicle [37]. It also has a role in promoting the proliferation and apoptosis of GCs while stimulating the expression of Kit ligands on GCs. LIF is expressed in the ovaries and promotes follicle growth. LIF has also been shown to coordinate follicular growth and ovulation sequences and can locally regulate follicular growth [38]. Oct4 has the potential to recruit mature oocytes. Overexpression in ovarian stem/stromal cells enhances oocyte-like differentiation in vitro and follicle formation in vivo [39]. SCF is essential for the early follicular development. It stimulates stromal cell function and promotes follicular growth through the Erk1/2 pathway, and can be used as a crucial regulator of embryo and ovarian growth to exert its biological effects [40,41]. CTX can cause ovarian injury and disorders in the expression levels of these mRNA [40]. Our results also revealed that the mRNA levels of FOXL2, GDF9, LIF, and OCT4 were decreased, while those of SCF were increased in the CTX group. After high-dose NB-DHA treatment, the levels of FOXL2, GDF9, LIF, and OCT4 increased, whereas those of SCF decreased. The mechanism of action of NB-DHA might be to promote the growth and maturation of follicles via the regulation of the mRNA expression levels of these five genes, thereby reversing ovarian injury caused by CTX and protecting the ovaries. At the same dose, the effect of NB-H was better than that of DHA-EE.

According to previous reports, follicular atresia occurs when more than 10% of GCs undergo apoptosis. Follicular atresia can cause a decline in ovarian function [42,43]. Our results indicated that apoptotic GCs and atretic follicles were significantly increased in the CTX group and reduced after both DHA-EE and NB-DHA treatment, whereas that of other types of follicles increased, suggesting that NB-DHA acts on AMH expression through cytokines secreted by granulocytes. AMH is produced by GCs of early ovarian developing follicles and is expressed at high levels throughout follicle formation. When ovarian GCs undergo apoptosis, DNA is fragmented and 3 -OH is combined with TdT to generate fluorescence. Our results analyzed the ratio of fluorescent cells and showed that CTX induced apoptosis of ovarian cells, which was reversed by DHA-EE and NB-DHA administration. In this case, we infer that DHA-EE and NB-DHA can reduce the apoptosis of ovarian granulosa cells, thereby achieving the effect of protecting the ovary. However, the anti-apoptotic activity of NB-DHA remains to be explored. The serum AMH level may represent the quantity and quality of the follicular pool, which is related to ovarian aging and failure, and reflects the state of the ovaries. Follicles are surrounded by GCs instead of membranous cells, oocytes, and ovarian stromal cells [44,45]. In addition, there is no expression of AMH when the follicle is atresia [43]. FSHR is expressed specifically in the GCs of the ovary and plays a key role in follicular function by interacting with its ligand FSH in the ovaries. When the follicle is atresia, FSHR expression is downregulated [46]. The immunohistochemical analysis results showed that both AMH and FSHR were expressed in secondary follicles, and the serum levels of AMH and FSHR were consistent with those in the ovary. The expression of AMH and FSHR in the CTX group was significantly lower than that in the other groups. The levels of both recovered after NB-DHA treatment, indicating that NB-DHA facilitates the growth of GCs and the expression of AMH and FSHR, thereby promoting follicle growth. The decreased primary follicles in the mouse model of ovarian injury, potentially owing to decreased serum AMH levels, lead to premature depletion of the original follicular pool. After treatment with NB-DHA, the levels of AMH and FSHR increased, indicating that NB-DHA increased the number of primordial follicles, reduced the failure of the primordial follicle pool caused by CTX, restored GC growth, and restored ovarian function. However, DHA-EE at the same dose as high-dose NB-DHA had no significant effect on AMH and FSHR expression in the follicles.

#### **4. Materials and Methods**

#### *4.1. Materials*

Fish oil (DHA content > 80%) was obtained from Shanxi Taike Biotech Co., Ltd. (20200712-002, Xi'an, China). Rhizomucor miehei lipase (L8621) was obtained from Solarbio (Beijing, China). Ethyl dimethylaminopropyl carbodiimide (EDC), benzylamine, dichloromethane, HOBt ·H2O, and triethylamine were obtained from Aladdin Co., Ltd. (Shanghai, China). The 3,3-diaminobenzidine kit (20×) (CW0125) was obtained from Cwbio Co., Ltd. (Beijing, China). Proteinase K (BL104A) and anti-fluorescence quenching mounting fluid (BL701A) were obtained from Biosharp Co., Ltd. (Hefei, China). Anti-FSHR (#40941) and anti-Müllerian-inhibiting factor (#42063) polyclonal antibodies were obtained from SAB Co., Ltd. (Baltimore, MD, USA).

Enzyme-linked immunosorbent assay (ELISA) kits for luteinizing hormone (LH; CSB-E12770m), follicle-stimulating hormone (FSH; CSB-E06871m), estradiol (E2; CSB-E05109m), and anti-Müllerian hormone (AMH; CSB-E13156m) were obtained from Cusabio Biotech Co., Ltd. (Wuhan, China). Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728) and SYBR Green Pro Taq HS premixed qPCR kits (AG11701) were obtained from AgBio Co., Ltd. (Changsha, China). A TUNEL kit (in situ cell death detection; C1086) and DAPI (4 ,6-diamidino-2-phenylindole; C1005) were obtained from Beyotime Biotech Co., Ltd. (Shanghai, China).

#### *4.2. Synthesis and Purification of DHA-EE and NB-DHA*

Twenty milliliters of fish oil were added to 20 mL of 10% (*w*/*v*) lipase solution, mixed homogeneously, and hydrolyzed at 45 ◦C for 24 h. The oil layer was collected, washed alternately with distilled water (40 mL) and n-hexane (40 mL), and the aqueous layer was discarded. The supernatant was concentrated using a rotating vacuum evaporator at 45 ◦C for 30 min. The fatty-acid-rich residues were stored frozen for subsequent synthesis experiments. DHA-EE was synthesized using a transesterification method. In brief, free fatty acids (700 μL) were mixed with 500 μL of NaOH-ethanol solution at 70 ◦C for 30 min, washed twice with saturated NaCl solution, and centrifuged at 5000× *g* for 10 min to collect the oil layer containing DHA-EE. NB-DHA was synthesized using the carbodiimide condensation method. Briefly, 100 mL of dichloromethane, 700 μL of free fatty acids, 528.6 μL of triethylamine, 0.206 g of HOBt H2O, and 0.292 g of EDC were mixed and agitated at 25 ◦C for 20 h, and 166 μL of benzylamine was added and stirred at 25 ◦C for 4 h. Subsequently, 200 mL of 10% HCl was added to the residue after drying, and 200 mL n-hexane was added, mixed homogeneously, and rested for 10 min. The upper layer was collected and washed alternately with 10% HCl and 10% NaOH to remove macroscopic impurities. Finally, DHA-EE and NB-DHA were purified according to our previous method, and their purities were analyzed by HPLC [14].

#### *4.3. Animals and Treatment*

Healthy female mice (20 ± 2 g, 7–8 weeks old, C57BL/6) were obtained from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). The mice were kept under pathogen-free conditions in a temperature (23 ± 2 ◦C) and humidity (55% ± 15%) control system, and all animal facilities were kept in a 12 h light–dark cycle. Food and water were provided free access for one week prior to the experiment. A preliminary experiment was carried out to determine the effective dose range of DHA-EE and NB-DHA. The mice were randomly divided into six independent groups (n = 6): control, ovarian injury model caused by CTX, CTX+DHA-EE (100 mg/kg/day), CTX+low-dose NB-DHA (CTX+NB-L, 25 mg/kg/day), CTX+medium-dose NB-DHA (CTX+NB-M, 50 mg/kg/day), and CTX+high-dose NB-DHA (CTX+NB-H, 100 mg/kg/day) groups. All drugs were administered to the mice after dissolving in Tween 80 solution at a concentration of 1%. Mice in the DHA-EE and NB-DHA groups were gavaged once a day from day 1 to day 21. The CTX, CTX+NB-DHA, and CTX+DHA-EE groups were injected intraperitoneally with CTX (200 mg/kg) on the eighth day after adaptive feeding. The control group was fed

normally without any drugs until the end of the experiment. All treatments were started at the same time and were sustained for 21 days. The survival rate of the mice was 100% during the experiments.

#### *4.4. Ovarian Index and Estrous Cycle Examination*

The mice were weighed prior to euthanasia. The isolated ovaries were repeatedly rinsed with precooled sterile saline, blotted dry with filter paper, and weighed. Nucleated cells, keratinized epithelial cells, and leukocytes in vaginal smears were observed under a light microscope, and the stages of the estrous cycle, including proestrus, estrus, metestrus, and diestrus phases, were determined based on the identification and proportions of cells. The estrous cycle was monitored continuously for 21 d.

#### *4.5. Morphological Analysis and Follicle Counting*

The ovaries were fixed with paraformaldehyde solution (4%) for 12 h and then washed with running water for 12 h. Subsequently, ovaries were dehydrated, embedded in paraffin, and stored at −20 ◦C. The tissues were sliced serially (4 μm thick), and one every five sheets was selected for hematoxylin and eosin (H&E) staining. Filming was performed using a slide scanning system (SQS-40P, Teksqray, Shenzhen, China). The viewing angle was determined under a microscope at low magnification, while primordial, primary, secondary, and atretic follicles were counted at high magnification. Six ovarian samples were randomly selected from each group, and sections were observed in 3 views under 400× to count follicles at all stages.

#### *4.6. ELISA*

The mice were fasted for 8 h after the final administration. Blood samples were collected from the eye veins, placed in anticoagulation tubes, and centrifuged at 4000× *g* for 15 min. The levels of serum FSH, LH, E2, and AMH were measured using an ELISA kit.

#### *4.7. RNA Extraction and Reverse-Transcription qPCR*

Total RNA was extracted from ovarian tissues using an RNA extraction kit. The RNA concentration was 500–1000 ng/μL. Then, 1 μg of RNA was reverse transcribed into cDNA, as required by the reverse transcription kit. The qPCR kit was used to measure the expression levels of GAPDH, FOXL2, GDF9, LIF, OCT4, and SCF. The primer sequences were as follows:

GAPDH-forward primer: 5 -TGTGTCCGTCGTGGATCTGA-3 , GAPDH-reverse primer: 5 -TTGCTGTTGAAGTCGCAGGAG-3 ; FOXL2-forward primer: 5 -CACCTCCAGGCCAGGTCTTTA-3 , FOXL2-reverse primer: 5 -TTTAGCAAACTCCAAGGCCATTAC-3 ; GDF9-forward primer: 5 -GTTCCCAAACCCAGCAGAAGTC-3 , GDF9-reverse primer: 5 -GTCCAGGTTAAACAGCAGGTCCA-3 ; LIF-forward primer: 5 -TTGATCCCGACTCAAGCAACC-3 , LIF-reverse primer: 5 -CTGAAGCCGCTACCATGCAA-3 ; OCT4-forward primer: 5 -CAGACCACCATCTGTCGCTTC-3 , OCT4-reverse primer: 5 -AGACTCCACCTCACACGGTTCTC-3 ; SCF-forward primer: 5 -AGATCTGCGGGAATCCTGTGA-3 , SCF-reverse primer: 5 -CATCCCGGCGACATAGTTGA-3 .

#### *4.8. In Situ Cell Death Detection*

For the in situ TUNEL paraffin staining, a part of each ovarian sample slice was randomly selected (n = 6). The sections were covered with protease K solution (20 mg/mL) and incubated in a wet chamber at 37 ◦C for 30 min. The sections were washed five times with PBS and then covered with Triton X-100 (1%) at 4 ◦C for 10 min. An in situ cell death assay kit was used for the TUNEL assay. The sections were incubated in TUNEL reaction mixture (TdT enzyme and fluorescent-labeled buffer) for 60 min at 37 ◦C under dark and humid conditions to capture the fragmented DNA of apoptotic cells. The sections were then incubated with DAPI at 24 ◦C for 5 min, washed with PBS, dried around the tissues, mounted with an anti-fluorescence quencher, and observed under a fluorescence microscope (BX53, Olympus, Tokyo, Japan). Whether the section was intact was established at 100× magnification, and the apoptosis of atretic follicles was carefully observed at 400× magnification. ImageJ 1.53a software was used to analyze the proportion of TUNELpositive cells in the antral follicles.

#### *4.9. Immunohistochemistry*

Paraffin-embedded tissue sections were dewaxed in a microwave oven for antigen repair. Immunohistochemical staining was performed using an SP immunohistochemistry kit. Rabbit anti-AMH (1:100) and anti-FSHR (1:150) antibodies were incubated with the tissue at 4 ◦C for 12 h. Six areas on each slide were randomly selected for inspection and filmed using the SQS-40P slide scanning system. The German immune response scoring standard (IRS) was used to score the staining results [47].

#### *4.10. Data Analysis*

All data were analyzed using GraphPad Prism 8 software, and the results are shown as the mean ± standard error of the mean (SEM). One-way analysis of variance was used to evaluate statistical significance among the experimental groups. All data were considered statistically significant at \* *p* < 0.05, \*\* *p*< 0.01, and \*\*\* *p*< 0.005.

#### **5. Conclusions**

In summary, these data demonstrate that NB-DHA alleviates ovarian injury in mice. NB-DHA reverses the high levels of gonadotropins and low levels of estrogen in the serum of mice with ovarian injury, promotes follicular development, inhibits follicular atresia and GC apoptosis via the upregulation of AMH and FSHR expression in GCs, and regulates the mRNA expression levels of ovarian-related genes to increase the ovarian reserve capacity. Natural DHA can be used as a beneficial dietary supplement to improve ovarian function, and NB-DHA is a promising compound for the clinical treatment of patients with ovarian injury.

**Author Contributions:** L.G.: Methodology and Writing—Original Draft; Q.G. and J.Z.: Investigation; X.J.: Data Curation; T.L. and H.Y.: Conceptualization, Writing—Review and Editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (81770527 and 82171700) and Shenzhen Longgang District medical and health science and technology plan project (LGKCYLWS2021000022).

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of Guangdong Pharmaceutical University (protocol code: gdpulac2020124, 3 April 2020).

**Data Availability Statement:** Not available.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds *N*-Benzyl Docosahexaenamide are available from the authors.

#### **References**


**Siyuan Peng 1,†, Xiwen Ling 2,†, Wenjing Rui 2, Xiaobao Jin <sup>2</sup> and Fujiang Chu 2,\***


† These authors contributed equally to this work.

**Abstract:** Diarrhea-based Irritable Bowel Syndrome (D-IBS) and diarrhea are both associated with ecological imbalance of the gut microbiota. Low Molecular Weight Peptides (LMWP) from the larvae of *Musca domestica* have been shown to be effective in the treatment of diarrhea and regulation of gut microbiota. Meanwhile, the single polypeptide S3-3 was successfully isolated and identified from LMWP in our previous studies. It remains unclear exactly whether and how LMWP (S3-3) alleviate D-IBS through regulating gut microbiota. We evaluated the gut microbiota and pharmacology to determine the regulation of gut microbiota structure and the alleviating effect on D-IBS through LMWP (S3-3). The rates of loose stools, abdominal withdrawal reflex (AWR) and intestinal tract motility results revealed that LMWP (S3-3) from the larvae of *Musca domestica* had a regulating effect against diarrhea, visceral hypersensitivity and gastrointestinal (GI) dysfunction in D-IBS model mice. Additionally, 16S rRNA gene sequencing was utilized to examine the gut microbiota, which suggests that LMWP induce structural changes in the gut microbiota and alter the levels of the following gut microbiota: *Bacteroidetes*, *Proteobacteria* and *Verrucomicrobia*. LMWP putatively functioned through regulating 5-HT, SERT, 5-HT2AR, 5-HT3AR and 5-HT4R according to the results of ELISA, qRT-PCR and IHC. The findings of this study will contribute to further understanding how LMWP (S3-3) attenuate the effects of D-IBS on diarrhea, visceral hypersensitivity and GI dysfunction.

**Keywords:** larvae of *Musca domestica*; D-IBS; GI dysfunction; 5-HT; gut microbiota

#### **1. Introduction**

Irritable Bowel Syndrome (IBS) is a functional disorder of the GI tract that is characterized by stomach ache, bloating and altered bowel behavior. Notably, the global prevalence of IBS was estimated to be around 7–30% [1]. The latest epidemiological study shows that the global prevalence of IBS is 11.2%, which also showed that D-IBS, C-IBS, M-IBS and U-IBS subtypes accounted for 23.4%, 22.0%, 24.0% and 22.2% of patients with IBS, respectively [2]. Although IBS is not a life-threatening disease, it seriously affects the normal life of patients and also creates economic burden. Among the four types of IBS, D-IBS is the most common. Young and middle-aged groups (18–59 years old) are the main patients who have the disease [3]. Stress in life comes from various sources, which act as predisposing risk factors for the development of irritable bowel syndrome (IBS). Physical stressors can affect visceral events. Additionally, IBS patients are at a greater risk of comorbidities, incur higher overall medical costs and have a reduced quality of life. Existing evidence suggests that the pathogenesis of IBS is multifaceted, including immunological, genetic and environmental influences [1]. Nonetheless, the etiology of IBS remains unclear, although accumulating evidence suggests that visceral hypersensitivity, impaired gastrointestinal motility, disturbance of microbial equilibrium, inflammation and/or intestinal infection may all be biological

**Citation:** Peng, S.; Ling, X.; Rui, W.; Jin, X.; Chu, F. LMWP (S3-3) from the Larvae of *Musca domestica* Alleviate D-IBS by Adjusting the Gut Microbiota. *Molecules* **2022**, *27*, 4517. https://doi.org/10.3390/ molecules27144517

Academic Editors: Masahide Hamaguchi and Giovanni Ribaudo

Received: 9 May 2022 Accepted: 11 July 2022 Published: 15 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

abnormalities associated with the condition [4–6]. Additionally, IBS can be classified into four groups based on clinical symptoms: Diarrhea-based Irritable Bowel Syndrome (D-IBS), Constipation-based Irritable Bowel Syndrome (C-IBS), Mixed Irritable Bowel Syndrome (M-IBS) and Undefined Irritable Bowel Syndrome (U-IBS) [7], with D-IBS being the most common. Notably, antispasmodic drugs, anticholinergics, antidiarrheal agents, visceral analgesics and antipsychotics are currently used to treat D-IBS [8]. Furthermore, treatment of D-IBS is primarily symptomatic relief medication, although it is associated with adverse effects that may have severe psychiatric consequences for patients [9]. Having similar efficacy, natural drugs have safer and less adverse effects as compared to synthetic chemical drugs. Therefore, it is critical to identify new therapeutic approaches capable of changing the composition of gut microbiota, enhancing the metabolism of neuroendocrine transmitters, decreasing visceral vulnerability and having a comprehensive regulatory effect on gut microbiota.

Currently, *Musca domestica* (housefly, Diptera: Muscidae) larvae are regarded as excellent sources of high-quality protein, polyunsaturated fats, saccharides, vitamins, minerals and other nutrients, for both human consumption and animal feed. In China, Li Shizhen demonstrated the role of these larvae in alleviating malnutrition in infants (stool induration or diarrhea) [10].

These peptides from natural sources were identified and had an alleviative effect on disease. A recent study showed that a spider-venom peptide with multi-target activity on sodium and calcium channels alleviates chronic visceral pain in IBS [11]. Bioactive fish collagen peptides weaken intestinal inflammation by orienting colonic macrophages phenotype through mannose receptor activation [12]. *Musca domestica* cecropin, a novel antimicrobial peptide, possessed potential antibacterial, anti-inflammatory, immunological functions and had a protective effect on colonic mucosal barrier injury caused by *Salmonella typhimurium*, which were reported by our laboratory [13].

Moreover, our previous studies reported that LMWP from the larvae of *Musca domestica* had antidiarrheal effects via regulation of the gut microecology and LMWP (S3-3) that were successfully isolated and identified [14]. Notably, the gut microbiota consists of more than 100 trillion microbes residing within the GI tract. Furthermore, extensive research has demonstrated that gut microbiota play a vital role in maintaining human health [15–17]. Additionally, disturbance of microbial equilibrium or dysbiosis has been shown to be closely related to multiple disorders including D-IBS, obesity, hyperlipidemia, atherosclerosis and numerous types of cancer. Therefore, the present study hypothesized that LMWP (S3-3) from the larvae of *Musca domestica* would be effective in alleviating diarrhea and D-IBS.

Therefore, this study aimed to determine the in vivo effect of LMWP (S3-3) from larvae of *Musca domestica* in alleviating D-IBS and gut microbiota imbalance. The psychosocial stress (restraint) model better simulates the pathogenesis of human IBS and gastrointestinal dysfunction [18,19]. Similar studies also verified the high efficacy and sustainability of the model, allowing for a better understanding of the pathological process as well as the vulnerability and triggering factors in D-IBS [20–22].

#### **2. Materials and Methods**

#### *2.1. Preparation of LMWP (S3-3) from the Larvae of Musca domestica*

LMWP (S3-3) from the larvae of *Musca domestica* (purity 94.70%, molecular weight: 1069.4391 Da; the 10 amino acid sequences of S3-3 were Val-Tyr-Arg-Asp-Asn-Val-Leu-Phe-Gln-Ala) were prepared as described in our previous study [14].

The laboratory strain of *Musca domestica* was obtained as a kind gift from the Guangdong Provincial Center for Disease Control and Prevention CDC, China. The larvae of *Musca domestica* were then dried using conventional drying systems. Briefly, third-instar larvae of *Musca domestica* were collected, washed for 3 h with running tap water, frozen for 2 h at −20 ◦C and sun dried for 6 h.

Following that, 1000 g of dried larvae were fried until they turned light yellow and then sifted using 40-mesh sieves. The larvae was fried to modulate the therapeutic properties of treated herbal medicines, i.e., enhancing efficacy, reducing toxicity or side effects, which is a kind of processing of Chinese medicine [23–25]. Following that, the dried powder of larvae of *Musca domestica* (10.0 g) was immersed in deionized water (250 mL) in a beaker for 30 min [14]. The samples were then simmered for 10 min before removing the supernatant and centrifuging at 12,000 r/min for 10 min. In addition, an equal volume of deionized water was added, and the same procedure was repeated. Following that, both supernatants with a molecular weight of <30 kD were collected by ultrafiltration technology (ultrafiltration membrane with a molecular weight cut-off of 30 kD) and lyophilized [15]. Finally, the supernatants were freeze-dried and LMWP were collected from larvae of *Musca domestica*.

A suitable amount (800 mg) of the LMWP powder was accurately weighed and dissolved in 2.0 mL of ultra-pure water, and the mixture was filtered on a 0.22 μm filter. Then, the LMWP powder was fractionated according to their molecular masses by using gel-filtration chromatography (GFC) on a column packed with SuperdexTM 30 and eluted with deionized water at a flow rate of 0.6 mL/min. Each eluate (3 mL) was collected and monitored at 280 nm, and the fractions (1 mL) were collected at a flow of 0.25 mL/min. Based on gel-filtration chromatography, the components of LMWP (S3-3) (t = 3.0 min) were further separated by RP-HPLC. YMC-Pack C4-HG columns (4.6 mm × 250 mm, 10 μm) were separately used for S3-3 separation. The fractions were automatically collected at a flow rate of 1 mL/min and dried by centrifugation under vacuum. The identification of the LMWP (S3-3) from the larvae of *Musca domestica* was performed in our previous study [9]. The purity of the fractions was determined by HPLC, the molecular weight was identified by MALDI-TOF spectrometer and the N-terminal sequences were determined using Edman degradation (Supplementary Figures S1–S3, Supplementary Table S1).

#### *2.2. Animals and Experimental Design*

A total of 32 male SPF C57BL/6J mice were provided by Medical Laboratory Animal Center, Guangdong Province (Guangzhou, China approval number SCXK (Yue) 2013-0002). The 6-week-old male mice were then housed in a specific-pathogen-free facility (room temperature 22 ± 2 ◦C and 12/12 h light/dark cycle) for 7 days (eating and drinking ad libitum). This was conducted in accordance with the guidelines by Care and Use of Experimental Animals. Additionally, the use of animals was approved by the Guangdong Pharmaceutical University and the Guangdong Pharmaceutical University Animal Care and Use Committee, China. Animal grouping design is shown in Figure 1. The 32 C57/BL6J mice (20 ± 2 g) were randomly assigned to four groups (*n* = 8) as shown in Figure 1: the Control, D-IBS, LMWP and LMWP + ampicillin groups. Among these groups, the LMWP + ampicillin group was established to identify the role of gut microbiota in D-IBS. After confirming the successful establishment of the D-IBS model, mice in the Control and D-IBS groups were given 10 mL/kg of Control saline, the LMWP group was intragastrically treated with 0.2 g/kg of LMWP (S3-3) from the larvae of *Musca domestica* (10 mL/kg) and those in the LMWP + ampicillin group received 500 mg/kg of ampicillin (10 mL/kg) and 0.2 g/kg of the LMWP (S3-3) from the larvae of *Musca domestica* (10 mL/kg), through the intragastric route, and all the mice were treated for 7 days. Thereafter, at the end of the therapy cycle, five fecal samples were randomly collected from each group for 16S rDNA gene sequencing.

**Figure 1.** Timeline of experimental procedures.

#### *2.3. Introduction of D-IBS in Mice*

The D-IBS model was then established by chronic restraint stress (24 mice were immobilized using a plastic restrainer for a duration of 1 h daily) for 14 days [18,19], in D-IBS, LMWP and LMWP + ampicillin groups. An abdominal withdrawal reflex (AWR) score ≥ 2 points and a loose stool rate ≥ 0.5 revealed that the D-IBS mice model was successfully established. Physical stressors can affect visceral events. Animals subjected to stress result in abnormal intestinal motility and visceral hypersensitivity. Stress-induced IBS models, e.g., restraint stress, could largely mimic IBS symptoms from intestinal motility to visceral sensitivity [20–22].

#### *2.4. Determination of the Rate of Loose Stool*

The total number of stools and loose stools was determined using the filter paper imprinting method [26,27]. The mice were placed in individual cages and the cage floor was covered with filter paper. The number and morphology of the stools were recorded for 6 h. The loose stools were classified into five grades based on the diameters of stain formed by loose stools on the filter paper: Grade 1 (0 < 1 cm), Grade 2 (1~2 cm), Grade 3 (2~3 cm), Grade 4 (3~4 cm) and Grade 5 (4~5 cm). Therefore, the rate of loose stool (%) = number of loose stools/total number of stools × 100%. Loose stool grade was defined as the calculated mean of the diameters of stain formed by loose stools on the filter paper. Loose stool index was determined as follows: loose stool index = rate of loose stool × loose stool grade.

#### *2.5. The Abdominal Retraction Reflex (AWR) Score*

The mice were subjected to a 24 h fast before inserting the 6F catheter, after paraffin oil lubrication, through the anus. A double-lumen balloon was then placed about 2.0 cm from the anus. Then, mice were placed inside a restraint device. After adapting to the new environment, the mice were gradually injected with water to dilate the balloon. The dilation capacity was 0.25 mL, 0.35 mL and 0.50 mL and each rectal dilation lasted for 30 s. The procedure was repeated thrice and the mean value was calculated. Finally, the AWR scores were determined using the following scale: 0, no behavioral response to Colorectal Distension (CRD); 1, brief head movement followed by immobility; 2, contraction of abdominal

muscles; 3, lifting of the abdomen; 4, body arching and lifting of pelvic structures. These methodologies were performed and modified according to those previously described [28].

#### *2.6. Colonic Bead Expulsion Test*

Under anesthesia, glass beads (2 mm in diameter) were inserted into the rectum (about 3 cm from the anus). The mice were then placed in a cage (1 mouse/cage) with no access to food or water. After the mice were fully awake (the standard was that the mice could turn over freely and climb up), the study began by observing the time to bead ejection. These methodologies were performed and modified as previously described [29,30].

#### *2.7. Upper GI Transit*

The mice in each group were fasted for 24 h and then orally administered with 0.2 mL of a suspension of the charcoal meal (10% charcoal in 5% gum arabic). The mice were sacrificed 20 min after receiving the charcoal meal. The small intestine was removed en bloc and the length of the small bowel and the distance traveled by the charcoal meal were then measured for each mouse. The ratio of the distance traveled by the charcoal meal to the total length of the small bowel was then used as the upper GI transit. These methodologies were performed and modified according to those previously described [31,32].

#### *2.8. Gastric Emptying*

The mice in each group received 0.2 mL of a suspension of the charcoal meal (10% charcoal in 5% gum arabic) and were sacrificed after 20 min. This was followed by abdominal dissection and ligation of the gastric cardia and pylorus. The stomach was then dried using a filter paper and the full weight was obtained. Following that, the stomach was cut along its bend before washing off the stomach contents and drying with filter paper. Gastric emptying (%) was calculated using the following formula: Gastric emptying (%) = (full weight of stomach − dry weight of stomach/weight of suspension of charcoal meal) × 100%. These methodologies were performed and modified according to those previously described [32,33].

#### *2.9. Histological Analysis*

The colonic tissues from 8 mice were examined in each group. Colon sections were excised to assess histological changes in the colon and were gently irrigated with normal saline to dislodge the intestinal contents. They were then fixed immediately at 4 ◦C overnight in 4% paraformaldehyde solution. Following three washes in tap water, they were dehydrated with serial ethanol concentrations. They were rinsed with xylene, paraffinembedded, sliced into 4 μm sections and stained with Hematoxylin and Eosin (H&E). They were then examined under a light microscope and photomicrographs of the sections were taken using a digital camera (DFC495 Digital camera Leica, Leica Microsystems, Wetzlar, Germany).

#### *2.10. Enzyme-Linked Immunoassay (ELISA)*

Blood samples were centrifuged at 3000 rpm for 10 min at 4 ◦C. Following that, serum was collected and immediately frozen in liquid nitrogen before being stored at −80 ◦C for further analysis. Additionally, the distal colon was homogenized in cold PBS. Following this, the frozen colonic tissues were homogenized and lysed in the tissue lysis buffer, followed by centrifugation at 12,000 rpm for 10 min at 4 ◦C. The supernatant was then collected. 5-HT, a critical signaling molecule in the gut, activated both intrinsic and extrinsic primary afferent neurons to initiate peristaltic and secretory reflexes. The levels of 5-HT in serum and colonic tissues were determined using an ELISA kit (Shanghai MLBIO Biotechnology Co., Ltd., Shanghai, China), and the operation steps of the kit were performed following the manufacturer's instructions.

#### *2.11. Real-Time Quantitative PCR Detection*

RNA from the distal colon was extracted. Total RNA isolation and cDNA synthesis were accomplished using the Trizol reagent (Accurate Biology Co., Ltd., Changsha, China) and the PrimeScriptTM RT reagent Kit with a gDNA Eraser (Accurate Biology Co., Ltd., Changsha, China), respectively. The mRNA levels of particular genes were then determined by real-time PCR using SYBR Green Pro Taq HS Premix (Accurate Biology Co., Ltd., Changsha, China) in the CFX Connect fluorescence quantitative PCR detection system (BIO-RAD, Hercules, CA, USA). Notably, the 20 μL PCR reaction mixture comprised 10 μL 2× SYBR Green Pro Taq HS Premix, 0.4 μL Forward Primer (10 μM), 0.4 μL Reverse Primer (10 μM), 2 μL reaction solution (cDNA) and 7.2 μL RNase-free water. Additionally, the following protocol was used for the Shuttle PCR: Stage 1 was the initial denaturation of one cycle at 95 ◦C for 30 s; Stage 2 was the PCR reaction of 40 cycles at 95 ◦C for 5 s, and 60 ◦C for 30 s; Stage 3 was the dissociation step. The data were analyzed using the comparative threshold cycle (Cq) method and normalized to an endogenous reference, Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH). 5-HT2AR, 5-HT3AR and 5-HT4R were 5-HT receptors, and SERT was a 5-HT reuptake transporter, which was involved in the reuptake and inactivity of 5-HT. The relative expression levels of genes associated with gastrointestinal movement (5-H2AR, 5-HT3AR, 5-HT4R, SERT) were then determined in colon tissues and calculated using the 2−ΔΔCT method. The primers used in this experiment are listed in Table 1.

**Table 1.** The primers used in this experiment.


#### *2.12. Immunohistochemistry (IHC)*

Mice were sacrificed at the end of the experiments. Colon tissues were then isolated, embedded on paraffinized blocks and cut into 4 μm-thick sections, individually, using a microtome (Leica, Wetlar, Germany). Next, the sections were incubated with anti-5- HT1AR rabbit polyclonal antibody, anti-5-HT2AR rabbit polyclonal antibody, anti-SERT polyclonal antibody (1:50) (Sangon Biotech, Shanghai, China) and anti-5-HT4AR rabbit polyclonal antibody (1:100) (Bioss, Beijing, China) overnight at 4 ◦C in a dilution ratio of 1:100 using the BondTM Primary Antibody Diluent (Servicebio, Wuhan, China). On the next day, the sections were incubated for 1 h with horseradish peroxidase 4-layered goat anti-rabbit secondary antibodies at 37 ◦C (Sangon Biotech) according to the manufacturer's instructions. Finally, the sections were treated with diaminobenzidine (DAB) solution (Servicebio, Wuhan, China) and visualized under a microscope (NIKON, Eclipse, Ci, Tokyo, Japan). We measured the integrated optical density (IOD) from at least three fields of each slice using the Image pro-plus 6.0 software (Media Cybernetics, Bethesda, MD, USA), which could accurately reflect the complete expression of the proteins in immunohistochemical staining.

#### *2.13. Gut Microbiota Analysis*

The fresh stool was collected from the colons of mice after being sacrificed and immediately frozen at −80 ◦C. Additionally, bacterial genomic DNA was extracted from frozen stool samples using the Qiagen QIAamp DNA stool Mini Kit (Hilden, Germany) according to the manufacturer's instructions. Following that, the 16S rRNA in the V3-V4 region (341F-805R, F: GATCCTACGGGAGGCAGCA; R: GCTTACCGCGGCTGCTGGC) was amplified via thermal cycling consisting of initial denaturation step at 98 ◦C for 1 min, followed by 30 cycles of denaturation at 98 ◦C for 10 s, annealing at 50 ◦C for 30 s and elongation at 72 ◦C for 60 s and a final hold at 72 ◦C for 5 min. Purification was subsequently performed using the MinElute Gel Extraction Kit (Qiagen, Shanghai, China) and samples with 400–450 bps were chosen for further experiments. Sequencing libraries were generated using the NEB Next Ultra DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer's instructions, and index codes were added. The PCR results were then subjected to high-throughput sequencing on an Illumina HiSeq2500 platform (Biomarker Technologies Co., Ltd., Beijing, China). Additionally, in the Greengenes database (13.5 version) (Lawrence Berkeley National Laboratory, Berkeley, CA, USA), USEARCH software (10.0 version) (Robert Edgar, Tiburon, CA, USA) was used to select OTUs and the RDP classifier (2.2 version) was used to annotate taxonomic information for each representative sequence. The alpha diversity indices among groups were compared after the sequences were rarefied to control for depth. The QIIME software package was also used to perform UniFrac distance-based Principal Component Analysis (PCA). Permutational Multivariate Analysis of Variance (PERMANOVA) was also performed. The Z score, the value corresponding to the heatmap, was obtained after the relative abundance of each row of species had been standardized in the heatmap of the gut microbiota at the genus level. Finally, Linear discriminant analysis Effect Size (LEfSe) analysis was performed using Metastats software to identify the biomarker species, Linear Discriminant Analysis (LDA) highlighting significant biomarker species among each group's microbiota, LDA threshold of >4. The relative abundance of significant biomarker species, obtained in gut microbiota from the LEfSe results, was compared in each group.

#### *2.14. Statistical Analysis*

SPSS Statistics 17.0 software (IBM, Armonk, NY, USA) was used to perform statistical analyses. All data were presented as mean ± SD and multiple comparisons were performed using one-way Analysis of Variance (ANOVA). *p* values less than 0.05 were considered statistically significant. Spearman's correlation was measured to demonstrate the relationships between parameters, the correlation coefficient was always in the range of +1 to −1. The correlations between gut microbial biomarkers and the phenotype were corrected by False Discovery Rate (FDR), which was calculated through the Benjamini–Hochberg (BH) method.

#### **3. Results**

#### *3.1. Effects of LMWP (S3-3) from Larvae of Musca domestica on Physiological Conditions and the Frequency of Loose Stools in D-IBS Mice*

After 14 days of D-IBS induction, D-IBS mice had significantly lower body weight and food intakes than the Control group. On day 1 of treatment, there were no statistically significant differences in initial body weight and food intake between the D-IBS, LMWP and LMWP + ampicillin groups. After 7 days of administration of LMWP (S3-3) from larvae of *Musca domestica*, the body weight and food intake in the LMWP group were significantly greater than in the D-IBS group. After seven days of treatment, the body weight and food intake in the LMWP + ampicillin group were significantly lower than in the LMWP group (Figure 2A,B). The D-IBS group had a higher loose stool rate, loose stool grade and loose stool index than the Control group. Additionally, when compared to the D-IBS group, the LMWP group demonstrated significant improvement in loose stool rate, loose stool grade and loose stool index. Additionally, as demonstrated in Figure 2C–E, the

LMWP + ampicillin group had increased loose stools rate, loose stool grade and loose stool index compared to the LMWP group.

**Figure 2.** LMWP (S3-3) from the larvae of *Musca domestica* affects body weight, food intake and diarrhea in D-IBS mice. (**A**) Body weight on day 1 and day 7 in the treatment period. (**B**) Food intake on day 1 and day 7 in the treatment period. (**C**–**E**) Loose stool rate, loose stool grade and loose stool index. Values are presented as means <sup>±</sup> SD (*<sup>n</sup>* = 8). ## *<sup>p</sup>* < 0.01 and ### *<sup>p</sup>* < 0.001 compared with Control, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 compared with D-IBS, \$ *p* < 0.05, \$\$ *p* < 0.01 and \$\$\$ *p* < 0.001 compared with LMWP (S3-3) from the larvae of *Musca domestica*.

#### *3.2. Effects of LMWP (S3-3) from Larvae of Musca domestica on Gastrointestinal Motility and Visceral Sensitivity in Mice*

Intestinal transit in mice was evaluated using the time to bead expulsion, upper GI transit and degree of gastric emptying. There was an increase in upper GI transit and a significant decrease in the efflux time of glass beads as well as in the degree of gastric emptying in mice in the D-IBS group compared to those in the Control group during the modeling process (Figure 3A–C). This indicated that the frequency of GI transport increased significantly after the modeling process. Additionally, mice in the LMWP group exhibited a slower upper GI transit and an increased efflux time for the glass beads, as well as a greater degree of gastric emptying, compared to those in the D-IBS group. However, there were significant differences in upper GI transit, efflux time of glass beads and degree of gastric emptying between the LMWP + ampicillin and LMWP groups. LMWP + ampicillin group had increased upper GI transit and decreased efflux time of glass beads, as well as a greater degree of gastric emptying than the LMWP group.

**Figure 3.** LMWP (S3-3) from the larvae of *Musca domestica* affect intestinal tract motility, visceral hypersensitivity and colonic histology in D-IBS mice. (**A**) Time to bead expulsion. (**B**) Upper gut transit. (**C**) Gastric emptying. (**D**) AWR scores. (**E**) Morphology found in the colon. Values are presented as means <sup>±</sup> SD (*<sup>n</sup>* = 8). ### *<sup>p</sup>* < 0.001 compared with Control, \*\* *<sup>p</sup>* < 0.01 and \*\*\* *<sup>p</sup>* < 0.001 compared with D-IBS, \$ *p* < 0.05 and \$\$\$ *p* < 0.001 compared with LMWP (S3-3) from the larvae of *Musca domestica*.

Additionally, visceral sensitivity was assessed using the abdominal uplift and back arch volume thresholds. Compared to the Control group, there was a significant increase in the AWR scores of the D-IBS group, demonstrating increased visceral sensitivity following the modeling process. However, as compared to the D-IBS group, the AWR scores decreased significantly following treatment with LMWP (S3-3) from larvae of *Musca domestica* (Figure 3D), suggesting meliorative visceral sensitivity following treatment with LMWP (S3-3) from larvae of *Musca domestica*. In comparison to the LMWP group, the LMWP + ampicillin group showed significantly higher AWR scores.

#### *3.3. Effect of LMWP (S3-3) from Larvae of Musca domestica on Colonic Histological Assessment*

Figure 3E showed that the colon tissues of the Control, D-IBS, LMWP and LMWP + ampicillin groups were normal. The mucosa was normal and complete, and neatly arranged villi were observed; the muscle layer was even and moderate, and colonic epithelial cells were arranged regularly in each group. Moreover, no significant pathological changes were observed in any group.

#### *3.4. Effect of LMWP (S3-3) from Larvae of Musca domestica on the Expression of Genes and Proteins Involved in 5-HT-Related Pathways*

The levels of 5-HT in the serum and colon of mice were examined to investigate the effect of LMWP (S3-3) from the larvae of *Musca domestica* by ELISA. The 5-HT concentrations in the serum and colon of mice are shown in Figure 4A,B. Notably, the serum and colon 5-HT concentrations were significantly decreased following treatment with LMWP (S3-3) from the larvae of *Musca domestica*. Additionally, the study used qPCR to examine the expression of the 5-HT2AR, 5-HT3AR, 5-HT4R and SERT genes. The results showed that 5-HT2AR and 5-HT3AR expression in the colon was higher, whereas 5-HT4R and SERT expression was lower in the D-IBS group compared to the Control group (Figure 4C–F). However, LMWP (S3-3) from larvae of *Musca domestica* resulted in a decrease in the expression of both 5-HT2AR and 5-HT3AR (Figure 4C,D). The treatment of larvae of *Musca domestica* with LMWP (S3-3) increased the expression of both 5-HT4R and SERT (Figure 4E,F). However, the LMWP + ampicillin groups increased the 5-HT concentrations in serum and colon tissue, increased 5-HT2AR and 5-HT3AR expression and decreased 5-HT4R and SERT expression (Figure 4). Next, the immunohistological changes in the protein levels of 5-HT2AR, 5- HT3AR, 5-HT4R and SERT were examined in the colon. The results suggested that stress caused an increase in the levels of 5-HT2AR and 5-HT3AR on the membrane surface of the intestinal tissues, which decreased substantially after LMWP (S3-3) treatment (Figure 5A,B). Furthermore, potential immunohistological changes in the protein expressions of SERT in the colon were evaluated. The results showed a significant increase in 5-HT4R and SERT-positive protein expression in the colon in the LMWP (S3-3) group relative to the IBS group (Figure 6A,B). Furthermore, results showed a significant increase in 5-HT2AR, 5-HT3AR protein and a remarkable decrease in 5-HT4R, SERT protein in the colon tissues obtained from mouse in the LMWP (S3-3) + ampicillin group as compared to those in the LMWP (S3-3) group (Figures 5 and 6).

**Figure 4.** LMWP (S3-3) from the larvae of *Musca domestica* affect 5-HT, 5-HT2AR, 5-HT3AR, 5-HT4R and SERT in D-IBS mice. (**A**,**B**) 5-HT levels in serum and colon. (**C**–**F**) The relative expression of 5-HT2AR, 5-HT3AR, 5-HT4R and SERT mRNA in the colon. Values are presented as means ± SD (*n* = 8). ### *p* < 0.001 compared with Control, \*\*\* *p* < 0.001 compared with D-IBS, \$\$ *p* < 0.01 and \$\$\$ *p* < 0.001 compared with LMWP (S3-3) from the larvae of *Musca domestica*.

**Figure 5.** Immunohistochemical staining in the colon. (**A**,**B**) 5-HT2AR and 5-HT3AR. Values are presented as the means <sup>±</sup> SD (*<sup>n</sup>* = 8). ### *<sup>p</sup>* < 0.001 compared to Control, \*\*\* *<sup>p</sup>* < 0.001 compared to D-IBS and \$\$\$ *p* < 0.001 compared to LMWP (S3-3) from the larvae of *Musca domestica*.

**Figure 6.** Immunohistochemical staining in the colon. (**A**,**B**) 5-HT4R and SERT. Values are presented as the means <sup>±</sup> SD (*<sup>n</sup>* = 8). ### *<sup>p</sup>* < 0.001 compared to Control, \*\* *<sup>p</sup>* < 0.01 compared to D-IBS, \*\*\* *p* < 0.001 compared to D-IBS, \$ *p* < 0.05 compared to LMWP (S3-3) from the larvae of *Musca domestica* and \$\$\$ *p* < 0.001 compared to LMWP (S3-3) from the larvae of *Musca domestica*.

#### *3.5. Effects of LMWP (S3-3) from Larvae of Musca domestica on Gut Microbiota in D-IBS Mice*

In this study, 16S rDNA sequencing was conducted to determine whether LMWP (S3-3) from larvae of *Musca domestica* influenced the gut microbiota and to define the changes in the composition of gut microbiota. It is noteworthy that the interaction between LMWP (S3-3) from larvae of *Musca domestica* feeding and the gut microbiota has been linked to D-IBS-related metabolic disorder. Therefore, the present study examined the effects of LMWP (S3-3) from the larvae of *Musca domestica* on the composition of gut microbiota by sequencing the V3 + V4 region of bacterial 16S rRNA. The samples were analyzed using high-throughput sequencing, which produced 1,600,995 pairs of raw reads. However, pair-end read alignment and filtering resulted in 1,551,510 clean tags which were subjected to subsequent analysis. All of the effective reads were then clustered into Operational Taxonomic Units (OTUs) based on a 97% similarity level. The dilution curve showed an inflection point at about 1000 and then leveled off, indicating that the sequencing amount of this study was enough to cover almost all bacterial species, indicating that the sample sequence was sufficient. The graded abundance curve indicates that the abundance and evenness of this study are both high, which supports the following data analysis (Supplementary Figures S4 and S5). The functional role of LMWP (S3-3) from the Larvae of *Musca domestica* in improving species richness and diversity of gut microbiota was further supported by increased numbers of OTUs

(Figure 7A), ACE (Figure 7B), Shannon (Figure 7D), Chao1 (Figure 7E) index and a lower Simpson index (Figure 7C). However, the LMWP + ampicillin group showed a decrease in species richness and diversity compared to the LMWP group.

**Figure 7.** The diversity, richness and structure of the gut microbiota in response to LMWP (S3-3) from the larvae of *Musca domestica* in D-IBS mice. (**A**) The number of OTUs in the gut microbiota. (**B**–**E**) The ACE index, Simpson index, Shannon index and Chao1 index. Values are presented as means ± SD (*n* = 5). ## *p* < 0.01 and ### *p* < 0.001 compared with Control, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 compared with D-IBS, \$ *p* < 0.05, \$\$ *p* < 0.01 and \$\$\$ *p* < 0.001 compared with LMWP (S3-3) from the larvae of *Musca domestica*.

A deeper examination of the microbial community revealed that LMWP (S3-3) from larvae of *Musca domestica* had a significant positive effect on the phylum and genus levels. According to the findings, the phylum level, the 10 most abundant bacteria in the level of phylum could be found and compared in all samples, and the most abundant phyla in all samples were *Firmicutes*, *Bacteroidetes, Proteobacteria* and *Verrucomicrobia* (Figure 8A). At the genus level (Figure 8B), the 10 most abundant bacteria were found and listed in all samples. Additionally, unsupervised multivariate statistical methods such as Principal Component Analysis (PCA) were used to assess structural changes in the gut microbiota. All four groups presented distinct clustering of microbiota composition, and the LMWP group had a similar structure to that of the Control group (Figure 8C). The results of PCA were confirmed through PERMANOVA test, detecting significant differences between groups (Supplementary Figure S7).

**Figure 8.** Gut microbiota diversity at the phylum and genus level. (**A**,**B**) Relative abundances of the gut microbiota at the phylum and genus levels. (**C**) Weighted UniFrac-based PCA.

Additionally, an overview of the heatmap (Figure 9) suggested a significant effect of LMWP (S3-3) from the larvae of *Musca domestica* on the profile of the gut microbiota. As seen in Figure 9, the abundance increased with the color changing from blue to red; genuslevel species clustering analysis was performed according to the distance between each genus-level species. The clustering indicated the similarity of the abundance of different species between samples. The closer the distance between two species was, the shorter the branch length was, indicating that the abundance of these two species was more similar between samples. The clustering revealed the similarity of community composition at each classification level. Therefore, all effective sequences were evaluated using the LEfSe approach to determine the major phenotypes that were significantly altered in response to LMWP (S3-3) of larvae from *Musca domestica* treatment. The findings of the LEfSe analysis revealed the presence of high-dimensional biomarkers in the gut microbiota in each group (Supplementary Figure S8).

**Figure 9.** Heatmap of the gut microbiota at the genus level.

Collectively, these findings indicated that treatment with LMWP (S3-3) from larvae of *Musca domestica* reversed D-IBS-induced dysbiosis of the gut microbiota. In comparison to the LMWP group, the LMWP + ampicillin group induced a decrease in *Bacteroidetes* and Verrucomicrobia but increased *Proteobacteria* at the phylum level (Figure 10A–C). The LMWP + ampicillin group induced a decrease in *g\_uncultured\_bacterium\_f\_Muribaculaceae*, *Akkermansia*, *Lachnospiraceae\_NK4A136\_group* and *Lachnoclostridium* but an increase in *Acinetobacter* at the genus level in comparison to the LMWP group (Figure 11A–F).

**Figure 10.** Relative abundances of the gut microbiota at the phylum level. (**A**–**C**) Relative abundances of *Proteobacteria*, *Bacteroidetes* and *Verrucomicrobia*.

**Figure 11.** Relative abundances of the gut microbiota at the genus level. (**A**–**F**) Relative abundances of Uncultured\_bacterium\_f\_Muribaculaceae, *Akkermansia*, *Lactobacillus*, *Acinetobacter*, *Lachnospiraceae\_NK4A136\_group* and *Lachnoclostridium* at the genus level.

#### *3.6. Potential Correlations among Phenotypes, Molecular Biology Indicators and Gut Microbiota*

To further investigate the potential role of the gut microbiota in D-IBS, correlation analyses were performed between diarrhea, gastrointestinal motility, visceral sensitivity, 5-HT, 5-HT2AR, 5-HT3AR, 5-HT4R, SERT and changes in the microbiota (Figure 12). 5-HT, 5-HT2AR and 5-HT3AR were positively correlated with the genus *Acinetobacter*. Negative correlations between 5-HT4R and SERT with *Acinetobacter* were also observed. Additionally, 5-HT, 5-HT2AR and 5-HT3AR were negatively correlated with *Akkermansia* and *Lachnoclostridium*, while 5-HT4R and SERT were positively correlated. *Lactobacillus* was also negatively correlated with 5-HT2AR and 5-HT3AR. *g\_uncultured\_bacterium\_f\_Muribaculaceae* and *Lactobacillus* were positively correlated with 5-HT4R and SERT, respectively.

**Figure 12.** Association map for three-tiered analyses integrating the gut microbiome, D-IBS phenotypes and molecular biology indicator. Scale indicates the level of positive (red) or negative (blue) correlation, \* *p* < 0.05 and \*\* *p* < 0.01.

#### **4. Discussion**

IBS is a chronic relapsing functional gastrointestinal disorder that is characterized by diarrhea and abdominal pain, both of which significantly decrease patients' quality of life. Currently, available management options mainly focus on symptom alleviation and control of the disease course.

Moreover, our previous study proved intestinal microbiological regulation might be one of the potential antidiarrheal mechanisms of LMWP from the larvae of *Musca domestica* and LMWP (S3-3) successfully isolated and identified from LMWP from the larvae of *Musca domestica* [14]. Therefore, this study focused on determining whether LMWP (S3-3) alleviated D-IBS through regulating gut microbiota. The results suggested that LMWP (S3-3) could alleviate D-IBS by impacting the gut microbiota.

Therefore, the present study established a restraint stress model of D-IBS in mice. After 4 weeks, mice in the D-IBS group had a lower body weight and a higher diarrheal index, indicating that they were experiencing diarrhea. Interestingly, LMWP (S3-3) from the larvae of *Musca domestica* decreased diarrhea caused by D-IBS, indicating that LMWP (S3-3) from the larvae of *Musca domestica* was capable of relieving diarrhea in these mice. Additionally, LMWP (S3-3) from the larvae of *Musca domestica* reduced the AWR scores in D-IBS mice, implying that it might be able to alleviate intestinal visceral hypersensitivity in

the animals. LMWP (S3-3) from the larvae of *Musca domestica* also inhibited colonic motility and prolonged gastrointestinal transit in mice. These results demonstrated that LMWP (S3-3) from the larvae of *Musca domestica* were beneficial for gastrointestinal motility and possessed antinociceptive properties. Nonetheless, cotreatment with antibiotics (ampicillin + LMWP (S3-3) from the larvae of *Musca domestica*) significantly reduced the beneficial effects of LMWP (S3-3) from larvae of *Musca domestica* against D-IBS. Metabolic products from gastrointestinal microbiota fermentation, such as SCFAs, or peptides can act on the ENS and affect gut transit [34]. The neuroendocrine system of the gut has also been shown to interact with microbiota [35] via 5-HT [36]. 5-HT is produced in both the ENS and CNS and is a key neurotransmitter that plays a pivotal role in mediating motor and secretory responses in the ENS [37]. 5-HT stimulates local enteric nervous reflexes to initiate secretion and propulsive motility and acts on vagal afferents to modulate contractile activities [37].

IBS is a complex disorder characterized by changes in sensation, secretion and gastrointestinal motility. 5-HT is a critical signaling molecule in the gut that targets enterocytes, smooth muscles and enteric neurons, activating both intrinsic and extrinsic primary afferent neurons to initiate peristaltic and secretory reflexes, as well as transmitting information to the central nervous system. Therefore, the serum and colon 5-HT concentrations were determined in this study by ELISA. Additionally, the data revealed that LMWP (S3-3) from the larvae of *Musca domestica* were capable of lowering 5-HT levels in the serum and colon. 5-HT has been shown to play an important role in regulating intestinal motility [38]. However, excessive 5-HT production induces high visceral sensitivity, and this is an important mechanism of D-IBS [38,39]. 5-HT has a remarkable range of effects that are attributable to the existence of multiple receptor subtypes on enteric neurons, enterochromaffin cells (EC cells), GI smooth muscle and probably enterocytes and immune tissue. 5-HT receptors are now classified into seven families and subtypes, with 5-HT2AR, 5-HT3AR and 5-HT4R known to affect gut motor functions. Additionally, 5-HT is inactivated by the SERT-mediated uptake into enterocytes or neurons. Therefore, the genes and proteins of the 5-HT-related pathway were detected by qPCR and IHC in this study. The present study demonstrated that LMWP (S3-3) from the larvae of *Musca domestica* could decrease the levels of 5-HT in the colon of D-IBS model mice by down-regulating the expression of 5-HT2AR, 5-HT3AR and up-regulating the expression of 5-HT4R and SERT. Furthermore, the binding of 5-HT and 5-HT2AR was reported to block voltage-gated K+ channels and increase visceral sensitivity by generating enteric neuron excitation [40]. Moreover, 5-HT3R are found in a variety of locations, including peripheral primary sensory nerve endings, autonomic preganglionic and postganglionic neurons, the central nervous system and the lower brainstem, among other areas. Notably, activation of the 5-HT3R, which has excitatory effects, mediates the rapid activation of sensory afferents, hence enhancing nervemediated gastrointestinal motility and secretion. Additionally, it generates visceral pain stimuli, which results in abdominal pain [41]. Furthermore, 5-HT4R is positively associated with adenylate cyclase, which is located on the mesenteric plexus neurons [42]. 5-HT4R has also been identified in the intestinal primary afferent neurons [43] and was shown to be involved in the peristaltic reflex [38,39]. On the other hand, Serotonin Reuptake Transporter (SERT) is a highly regulated protein, located on the membrane of intestinal epithelial cells and is involved in the reuptake of 5-HT [44]. Moreover, excess 5-HT is often transported into epithelial cells via SERT and inactivated there. Therefore, inhibiting the expression of SERT can induce the sensitivity of primary neurons to 5-HT, and this can, in turn, enhance visceral sensitivity [44,45].

The findings indicated that the levels of 5-HT in the serum and colon were elevated in D-IBS mice and that LMWP (S3-3) from the larvae of *Musca domestica* could decrease the levels of 5-HT in the serum and colon of D-IBS mice. 5-HT, which is primarily produced in the gut, regulates intrinsic reflexes (e.g., stimulates motility, secretion and vasodilation) and may contribute to the development of diarrhea by promoting inflammation [46,47]. The EC cells are mucosal sensory cells that release mediators (5-HT, among others) in response to chemical or mechanical stimulation [48]. It is hypothesized that excessive release of 5-HT from EC cells may contribute to diarrhea in IBS patients [48]. Rapid intake of 5-HT occurs via a selective SERT transporter that regulates 5-HT in the gut [48]. The enteric nervous system (ENS) is also involved in intestinal absorption and secretion, and diarrhea has been associated with decreased absorption of ions and/or solutes and water [49]. Numerous studies indicate that 5-HT and the ENS may play an important role in the pathophysiology of IBS and perhaps in diarrhea [48]. The findings also demonstrated that the SERT levels in the colon of D-IBS mice were decreased and LMWP (S3-3) from the larvae of *Musca domestica* could enhance the levels of SERT in the colon of D-IBS mice SERT decrease can affect motility and thus contribute to diarrhea [48,50]. Therefore, LMWP (S3-3) from the larvae of *Musca domestica* may be used to treat diarrhea in D-IBS mice by regulating 5-HT and SERT levels.

In our view, LMWP (S3-3) supplemented intestinal nutrition and produced prebiotics, then regulated gut microbiota through prebiotics, and then regulated SCFAs, which affect release of 5-HT, through gut microbiota [51–53]. In this study, changes in the composition of the microbiota were examined using high-throughput sequencing. The results demonstrated that the alpha diversity of the gut microbiota was reduced in the D-IBS group, which had a lower Shannon index, ACE index and Chao1 index and a higher Simpson index than the Control group. Nevertheless, treatment with LMWP (S3-3) from the larvae of *Musca domestica* was able to restore diversity. Additionally, PCoA indicated significant distances between each group, indicating that the beta diversity of gut microbiota was different in D-IBS model mice and LMWP (S3-3)-treated mice. According to the findings, the relative abundance of *Lactobacillus* was decreased in D-IBS model mice compared to Control mice. Moreover, treatment with LMWP (S3-3) from the larvae of *Musca domestica* increased the relative abundance of *Lactobacillus*, which has previously been shown to have positive therapeutic benefits on IBS [54,55]. Short-chain Fatty Acids (SCFAs) are the primary metabolites of the gut microbiota and can boost the growth of *Lactobacillus*. They are also critical signaling molecules that affect intestinal function, and abnormal changes in SCFA levels have been associated with IBS. Additionally, it was demonstrated that intestinal microbiota imbalances in IBS patients have a direct effect on the normal signaling interactions between intestinal microbiota, SCFAs and intestinal epithelial cells, resulting in a low inflammatory response, increased permeability of the intestinal epithelial barrier and hypermotility [56]. We also found that LMWP (S3-3) increased related SCFA concentration, such as propionate and butyrate (Supplementary Figure S6, Supplementary Methods). On the contrary, the current study found a significant increase in the abundance of *Akkermansia* after treatment with LMWP (S3-3) from the larvae of *Musca domestica*. *Akkermansia* is a probiotic belonging to the *Verrucomicrobia* phylum and is involved in nutrition metabolism. Recent studies also indicate that *Akkermansia* improves metabolic health and protects against obesity, diabetes and inflammation in the intestinal tract of rodents by interacting with intestinal epithelial cells [56,57]. Additionally, *Akkermansia muciniphila* is the type species of the genus *Akkermansia*, which was first proposed in 2004 as a mucin-degrading, anaerobic Gram-negative bacterium that resides in the mucus layer [58]. Notably, the mucus layer lining the intestinal tract serves as a lubricant and physiological barrier between the luminal contents and mucosal surface. Furthermore, the presence of *A. muciniphila* in IBS mice may play an important role in preserving the integrity of the mucin layer. However, it is unclear whether LMWP (S3-3) from the larvae of *Musca domestica* increases the abundance of *A. muciniphila* by providing the primary source of energy for this bacterium, thereby favoring its growth. Additionally, it is unknown if an increase in *A. muciniphila* increases mucus production and degradation. However, it was discovered that treatment with LMWP (S3-3) from the larvae of *Musca domestica* inhibited the proliferation of *Acinetobacter*, which are mostly opportunistic microbes whose population increased significantly in mice with diarrhea [59]. According to the findings, the relative abundance of *g\_uncultured\_bacterium\_f\_Muribaculaceae* was decreased in D-IBS model mice compared to Control mice, which belong to *Muribaculaceae*. LMWP (S3-3) also increased the relative abundance of *g\_uncultured\_bacterium\_f\_Muribaculaceae*. Schmidt et al. also found that the

abundance of *Muribaculaceae* was strongly correlated with the concentration of propionate belonging to SCFAs [60].

The findings also showed that gut microbiota play a key role in D-IBS by enhancing the function of LMWP (S3-3) from the larvae of *Musca domestica*. This is because when LMWP (S3-3) from larvae of *Musca domestica* were combined with an antibiotic, the regulatory effect on physiological conditions, diarrhea, gastrointestinal motility, visceral sensitivity, colon histology, levels of 5-HT, expression of associated pathway genes and proteins, and gut microbiota were significantly reduced. Ampicillin is a β-lactam antibiotic that has the potential to disrupt gut microbiota and cause diarrhea [61,62]. The mechanism of ampicillin-induced diarrhea may be related to disruption to the normal composition and functional attributes of the gut microbiota [63], where 5-HT was related to diarrhea [64]. Compared with the LMWP group, for LMWP (S3-3) from larvae of *Musca domestica* that were combined with ampicillin, the regulatory effect on physiological conditions, diarrhea, gastrointestinal motility, visceral sensitivity, colon histology, levels of 5-HT, expression of associated pathway genes and proteins, and gut microbiota were significantly reduced, which suggested that the changes in gut microbiota composition might alter colonic motility. Gut microbiota regulated SCFAs, which affect release of 5-HT [51,52]. 5-HT has been shown to play an important role in regulating GI motility [38]. Additionally, several studies have revealed that the germ-free condition is characterized by increased plasma 5-HT concentrations. Plasma 5-HT levels are thought to be mostly derived from intestinal EC cells of the gut [65,66].

In this study, these data suggested that LMWP (S3-3) from the larvae of *Musca domestica* had an obvious protective effect on D-IBS through regulating 5-HT-pathway-related genes and proteins and adjusting gut microbiota.

#### **5. Conclusions**

According to the current study's findings, treatment with LMWP (S3-3) from the larvae of *Musca domestica* regulates gut microbiota by increasing the relative abundance of *Akkermansia* and *Lactobacillus*. Additionally, LMWP (S3-3) treatment decreases *Acinetobacter* levels, resulting in favorable benefits against diarrhea, increased visceral sensitivity and excessive gastrointestinal motility. This also regulates the 5-HT levels in serum and colon as well as the expression of 5-HT-pathway-related genes and proteins (Figure 13). The data of this study suggested that LMWP (S3-3) from larvae of *Musca domestica* had an obvious protective effect on D-IBS, potentially by adjusting gut microbiota, down-regulating 5-HT, 5-HT2AR and 5-HT3AR, up-regulating 5-HT4R and SERT, relieving diarrhea, decelerating the gastrointestinal motility and alleviating intestinal visceral hypersensitivity. These findings could enhance our understanding of the effect and mechanism of LMWP (S3-3) from larvae of *Musca domestica* on D-IBS and contribute to developing effective therapies in the future.

**Figure 13.** A schematic presentation of the therapeutic effect of LMWP (S3-3) from the larvae of *Musca domestica* on D-IBS.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27144517/s1, Figure S1: The retention time in the RP-HPLC; Figure S2: The molecular weight of LMWP(S3-3) was detected by MALDI-TOF spectromete; Figure S3: *N*-terminal sequence of LMWP(S3-3) were determined by Edman degradation; Figure S4: OTU Rarefaction Curve; Figure S5: Rank Abundance Curve; Figure S6: SCFA variations; Figure S7: Permanova test; Figure S8: LEfSe analysis results; Table S1: LMWP(S3-3) HPLC peak area integral results. Table S2: Association map data.

**Author Contributions:** F.C. and S.P. conceived and designed the research; S.P., X.L. and W.R. performed the experiments; S.P. analyzed the data; S.P. interpreted the results of the experiments; S.P. prepared the figures; S.P. and X.L. drafted the manuscript; F.C. and X.J. edited and revised the manuscript; S.P., X.L., W.R., X.J. and F.C. approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (Grant No. 81373960 and 81573600); Science and Technology Planning Project of Guangdong Province, China (Grant No. 2014A020212416); and Guangzhou Municipal Science and Technology Project, China (Grant No. 201707010132).

**Institutional Review Board Statement:** The animal study was reviewed and approved by the Medical Ethics Committee of Guangdong Pharmaceutical University (approval code: gdpulacspf2017134, 11 June 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The original contributions presented in the study are publicly available. These data can be found here: https://www.ncbi.nlm.nih.gov/bioproject/900466 (accessed on 23 February 2022), BioProject ID is PRJNA900466.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

**Sample Availability:** Samples of the compounds LMWP(S3-3) are available from the authors.

#### **References**


## *Review* **Current Progress in the Chemoenzymatic Synthesis of Natural Products**

**Evan P. Vanable 1, Laurel G. Habgood <sup>2</sup> and James D. Patrone 2,\***


**Abstract:** Natural products, with their array of structural complexity, diversity, and biological activity, have inspired generations of chemists and driven the advancement of techniques in their total syntheses. The field of natural product synthesis continuously evolves through the development of methodologies to improve stereoselectivity, yield, scalability, substrate scope, late-stage functionalization, and/or enable novel reactions. One of the more interesting and unique techniques to emerge in the last thirty years is the use of chemoenzymatic reactions in the synthesis of natural products. This review highlights some of the recent examples and progress in the chemoenzymatic synthesis of natural products from 2019–2022.

**Keywords:** chemoenzymatic; natural product synthesis; biocatalysis

#### **1. Introduction**

The biodiversity of organisms from plants to microbes to mammals on Earth has led to a vast wealth of natural products. Throughout history from ancient civilizations to our contemporary one, these natural products have been an invaluable source of bioactive molecules capable of improving their quality of life. Natural products and their derivatives found success in modern drug discovery for a wide range of disease states ranging from diabetes and cardiovascular disease to viral infections and inflammatory diseases with notably high success as antibiotic and anticancer agents [1]. Despite the continued success of natural products in the clinical setting, the pharmaceutical industry divested resources from their discovery in the 1990s due to challenges associated with the rediscovery of known chemical entities, target deconvolution, and resources being allocated to alternative methods of drug discovery [2,3]. More recently there has been a resurgence in natural product discovery, structure elucidation, and progression of natural products to the clinic as a consequence of increased resources and advances in methodologies.

The field of natural product synthesis dates back to 1828, fascinating and inspiring generations of chemists [2,4]. Natural products are often characterized for their high structural complexity stemming from an enriched number of stereocenters, sp<sup>3</sup> carbons, oxygen atoms, and rigid carbon skeletons as compared to synthetically designed molecules [1]. The combination of the rich, diverse, and structurally complex structures of natural products and the drive, creativity, and talent within the synthetic community makes the synthesis of natural products one of if not the most important fields for both training chemists and developing novel synthetic methods [4]. The pursuit of these diverse targets has seen the field of organic chemistry expand its capabilities in leaps and bounds in areas such as but not limited to retrosynthetic analysis, stereoselective and regiospecific C-C bond formations, cascade reactions, orthogonal protecting groups, protecting group free synthesis, organometallic catalysis, convergent synthesis, atom efficiency, and green chemistry [4]. To this point, many modern organic techniques have been applied to natural product synthesis. For example, organometallic mediated C-H activation bond activation chemistry (directed and non-directed) such as in the synthesis of (−)-epicoccin G and artemisinin [5]. The

**Citation:** Vanable, E.P.; Habgood, L.G.; Patrone, J.D. Current Progress in the Chemoenzymatic Synthesis of Natural Products. *Molecules* **2022**, *27*, 6373. https://doi.org/10.3390/ molecules27196373

Academic Editor: Giovanni Ribaudo

Received: 9 September 2022 Accepted: 23 September 2022 Published: 27 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

boundaries and application of electrochemical reactions such as decarboxylative couplings have been extended into to the synthesis of *R*-(Z)- nerolidol [6]. Photochemical reactions such as cycloadditions, arene couplings, and C-N bond formations are an emerging methodology in the synthesis of natural products such as (−)-pavidolide B, (+)-flavisiamine F, and (+)-iosocorynantheol [7].

Over the past twenty years, chemists have been going back to nature and its biosynthetic pathways to develop new advantageous bond forming methodologies through chemoenzymatic syntheses [8–14]. These pioneering scientists have enriched our synthetic landscape across numerous reaction types such as chiral resolutions (many of the first applications of chemoenzymatic processes), saponifications, hemiacetal formations, oxidations and reductions, and C–C bond forming reactions as well as classes of molecules including glycans, peptides or derivatized amino acids, polyketides, and terpenoids. The benign nature, stereospecificity, and potential of chemoenzymatic processes has led researchers to invest heavily in their development.

As chemoenzymatic methods became more widely available and applicable, their benefits to the synthetic communities are greater than just expanded methodologies. Enzymecatalyzed reactions incorporate the majority of the twelve principles of green chemistry that seek to reduce our impact on human health and the environment [15]. Enzymes are inherently non-toxic and natural (less hazardous chemical synthesis and use of renewable feedstocks). Their catalytic nature affords reactions that can be run at ambient to slightly elevated temperatures in biphasic or completely aqueous media (catalysis, design for energy efficiency, safer solvents and auxiliaries) and impart regio- and stereoselectivity (atom economy, waste prevention) [16]. Since chemoenzymatic methods combine high regioselectivity and stereoselectivity with environmental and cost benefits, they are attractive method for large scale synthesis and as such have been adopted for the synthesis of several high value pharmaceutical agents such as sitagliptin, simvastatin, and darunavir [16,17].

The continued application and success of chemoenzymatic syntheses in these settings has continued to fuel the diversity and pace of research into biocatalytic approaches. This research has produced advances in the variety and number of chemoenzymatic processes and increased their capabilities through scalability, multiple enzyme cascades, and flow processes. The importance of the chemoenzymatic synthesis of natural products can be seen in the explosion of recent syntheses and review articles highlighting their accomplishments [11,18–30]. This report is organized by classification of molecule and aims to highlight the diversity and power of this field through selected chemoenzymatic syntheses of natural products from 2019–2022.

#### **2. Selected Natural Product Syntheses Incorporating Chemoenzymatic Methods** *2.1. Terpenoids*

One of the principal scientists featured throughout this review, Hans Renata, pushes the boundaries of the utility and elegance of chemoenzymatic synthesis across multiple complex classes of molecules. The work of the Renata group is often impressive in its nuanced design which is integrated within traditional synthetic sequences [20,22,23,31–33]. In a recent paper they disclosed the synthesis of chrodrimanin C (**3**), verruculide A, and polysin using multiple chemoenzymatic steps (**Scheme 1**) [33]. A key step featured in these syntheses is an enzymatic hydroxylation of a 6,6,5 or 6,6,6, steroid core, intermediate 1 in the case of chrodrimanin C (**3**). These reactions were performed on gram scale, 67 & 83% yields, depending on starting material, selectivity for oxidation of a single methylene despite the presence of 6 or 7 other oxidizable methylene groups, and with enantioselectivity of course. This scale is an impressive feature for chemoenzymatic methods, considering the importance of this feature for transformations in total synthesis.

**Scheme 1.** Selective chemoenzymatic hydroxylation towards the synthesis of chrodrimanin C (**3**) [32].

Tang and co-workers' synthesis of the bicyclic terpenoid nepetalactolone, the active molecule in catnip and a natural insect repellent, features a one-pot multienzyme (OPME) system that is stereoselective setting three contiguous stereocenters while utilizing geraniol (**4**) as a precursor (**Scheme 2**). [34]. This synthesis features a ten-enzyme cascade, half of which are necessary to perform the requisite biosynthetic steps, and half of which are required for auxiliary needs or cofactor regeneration. The chemical steps performed by the enzymes are allylic hydroxylation, alcohol oxidation, aldehyde reduction, cyclization, and a hemiacetal oxidation. One of the more elegant aspects of this system is the ability to perform oxidative and reductive steps in the same pot, with the same NAD/NADH system. Although the experiments were run on a small scale, the yields are excellent (93%) with potential to produce approximately 1 g nepetalactone per liter of solution at a reasonable cost (<\$120/g).

**Scheme 2.** One-pot multienzyme cascade synthesis of nepetalactone (**5**) from geraniol (**4**) [34].

A novel method using an OPME cascade of enzymatic reactions to synthesize triterpenes of highly varied structures, including cyclized variants was recently reported by Allemann and coworkers [35]. Noteworthy is that the scope of starting material, enzymatic variance, and enzymatic combinations, as many as four enzymes total, all within a OPME framework to generate simple but highly varied triterpenoids. The enzymatic transformations utilized include monophosphorylation by *Ec*THIM, diphosphorylation by *Mj*IPK, synthesis of natural and unnatural farnesyl diphosphosphates by *Gs*FDPS, and cyclization and/or bicyclization using a variety of enzymes. Pyruvate kinase (PK) acts as a supplementary enzyme to replenish the ATP substrate pool throughout the phosphorylation reactions. Seven sesquiterpenoid compounds, many first reported in this study, and the antibacterial/antifungal (*S*)-germacrene D (**8**) are synthesized. Prenol (**6**) and isoprenol (**7**) were mixed in a 1:2 ratio with *Ec*THIM, *Mj*IPK, PK, *Gs*FDPS, and ScGDS to yield germacrene D (**Scheme 3**). Advantages of their methodology include using less expensive 4- or 5-carbon starting materials and producing both natural and unnatural products in a modular fashion on a milligram scale.

**Scheme 3.** OPME system utilized in the activation, condensation, and cyclization of prenol (**6**) and isoprenol (**7**) for the synthesis of (*S*)-germacrene D **(8**) [35].

#### *2.2. Polyketides*

The area of chemoenzymatic synthesis to produce polyketide natural product targets is so critical that it can be said that it is the driver of advancements in the field as a whole. Alison Narayan and David Sherman have been and will continue to build on their pioneering work [14,18,26,28,36–40]. The importance of these two scientists to the field is evidenced by the previous coverage in the literature, including other reviews. Therefore, this work will not include it but allow for interested readers to explore it within these references.

Stereoselective reductions of simple organic moieties are an easy way to introduce stereocenters: if it can be done. To afford the desired diol products selectively, Husain et al. have applied the use of T4HNR to reduce ketones and enols selectively in naphthol systems (**Scheme 4a**) [41]. Intriguingly this process reacts very differently with 2-hydroxy and 3-hydroxyjuglone starting materials. The phenol orients the molecule within the enzyme active site to provide the selectivity for the adjacent ketone to be reduced. While exhibiting a high level of selectivity, the reduction of 3-hydroxyjuglone affords an 82:18 d.r. for **10a** and **10b** which is comparatively modest for an enzymatic transformation. Building off this initial strategy, the Husain group recently reported the small-scale synthesis *(R*)-scytalone (**12**) from simple accessible starting materials using the anthrole reductase ARti-2 and a NADPH cofactor (**Scheme 4b**) [42]. Notable about this chemoenzymatic transformation is that scytalone, generated by the desymmetrization of a perfectly flat tetrahydroxynaphtalene in a stereoselective fashion, also includes another phenol, which is oxidized to a ketone. Despite a small scale and modest yield (23%), the selectivity was >99% for the observed stereoisomer is exceptional.

**Scheme 4.** (**a**) Stereoselective chemoenzymatic reductions using T4HNR to form polyketide metabolites **10a**, **10b**. (**b**) Stereoselective chemoenzymatic reductions using Arti-2 to synthesize (*R*)-scytalone (**12**) [41,42].

Husain and coworkers continued studies utilizing a system of T4HNR, NADPH, and glucose with GDH to synthesize polyketide natural products in the nodulone family (**Scheme 5**) [43]. The synthesis of both nodulone C (**14**) and an unnatural diastereomer of nodulone D are featured. In the case of nodulone D, two stereocenters were set with near perfect d.r. Their ability to doubly hydrogenate the hydroxynapthoquinone selectively, while leaving a benzylic ketone untouched, would be difficult to duplicate using traditional synthetic organic techniques as overreduction would be facile. In nodulone C they once more selectively reduced a hydroxynaphthalene to a phenol, enacting a single enol reduction in a naphthalene with three hydroxy groups selectively in an excellent 90% yield.

**Scheme 5.** High yielding stereoselective reduction in the synthesis of nodulone C (**14**) [43].

A recent synthesis of fasamycin A (**6**) from the precursor naphthacemycin B1, utilizing a highly unusual enzymatic halogenation, was recently reported by the Renata group (**Scheme 6**) [33]. The report involved a convergent synthesis that culminated with a halogenation via a chemoenzymatic system that contained a flavin-dependent halogenase, CtcQ as a reductase, Opt13 to regenerate NADH, and NADH/NADPH. The success of the synthesis hinges on a single halogenation of a polyphenol (**15**), at a specific site, with regioselectivity to afford the product in 5% yield. There are 4 rings in precursor (**15**) which could be halogenated, two of which are almost identical electronically and sterically making the regioselectivity achieved even more impressive. The author notes that low yield has been previously reported with halogenases and that enzyme engineering may assist with the issue. Progress in the area of halogenases as a whole will allow this methodology to be used by the broader synthetic community.

**Scheme 6.** Regioselective halogenation for the synthesis of fasamycin A (**16**) [33].

#### *2.3. Glycans*

Glycans are a diverse set of natural products whose size and purpose vary greatly. The range in size from small monosaccharides to enormous polysaccharides possessing hundreds of glycan units correlates with their variety of biological targets and purposes of sugars. Given their versatility, they are used in multiple fields such as food chemistry, medicinal chemistry, and investigations of fundamental biological processes [44–53].

The Chen group has continued their focused efforts to improve synthetic routes to create structurally diverse libraries of gangliosides, specifically GM3 (**19**) [54]. Comprised of glycan and lipid moieties, GM3 has been implicated as a risk factor in metabolic diseases as well as placed on a prioritized cancer antigen list. An OPME strategy was employed to install sialic acid variants on lactosyl sphingosine (LacbSph) followed by subsequent acylation of a fatty acyl chain to form multiple GM3bSph gangliosides (**Scheme 7**). The six sialic acid variants (ManNAc) were attached to LacbSph forming the GM3 sphingosines in high yields (85–95%) utilizing a OPME approach containing three enzymes, including PmNanA (*P. multocida* sialic acid aldolase), NmCSS (*N. meningitis* CMP-sialic acid synthetase), and PmST3 (*P. multocida* a2-3 sialyltransferase). Subsequent acylation with stearoyl chloride (98–100%) or alternate fatty acyl chains (98–100%) produced ten GM3 gangliosides. Advantages of the synthetic strategy include gram-scale production of LacbSph from an L-serine derivative with minimal purification and efficient mg scale (average 25 mg) production of diverse GM3 gangliosides with fluorine, azide, and diazirine sialic acid derivatives.

**Scheme 7.** Chemoenzymatic installation of sialic acid in the synthesis of GM3 (**19**) [54].

Glycosphingolipids (GSLs) comprised of a glycan and ceramide component are a major component of the cell membrane and are notable signaling molecules essential to numerous biological processes and diseases. Future studies related to mechanisms of these processes, diseases, and applications are contingent on the ready availability of pure and structurally characterized GSLs. To meet this need, the Guo group envisioned a diversity-oriented strategy involving chemoenzymatic glycan synthesis in conjunction with the chemoselective modification of the sphingolipid chain [55]. A series of eight natural and non-natural GSLs were synthesized including Gb3 (**22**), Gb4 (**24**), GM3, and GD3, all of which are known cancer biomarkers. The synthesis of Gb 3 starts with the core intermediate of the strategy being diversified enzymatically by adding Gal using an α-1,4-galactosyltransferase to form the trisaccharide (**21**). The trisaccharide is the chemically modified via a Grubbs-Hoveyda-II catalyzed cross metathesis, Boc removal, and amide formation via an acyl chloride to cleanly yield the fully elaborated GSL Gb3 (**Scheme 8**). The strength of this strategy is its readily amenable to other targets with the same core intermediate and route/steps being utilized with an extra enzymatic step to further diversify the glycan with GalNAc to a tetrasaccharide (**23**) before the chemoselective transformations to yield Gb4 (**24**).

**Scheme 8.** Variable chemoenzymatic glycosylation strategy for the synthesis of Gb3 (**23**) and Gb4 (**24**) [55].

Glycopeptides are another class of glycan-based molecules that have implications in normal cellular signaling and disease progression. Again, a major issue with conducting proper studies to understand the biological underpinnings of these molecules is the difficulty of obtain sufficient quantities of pure homogeneous samples. The Li group devised a robust streamlined chemoenzymatic approach to the synthesis of 16 well-defined SARS-CoV-2 O-glycopeptides, 4 complex MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like peptide-1 [56]. Using the SARS-CoV-2 O-glycopeptides as an example, the authors utilized a combination of liquid-phase peptide synthesis (LPPS) and chemoenzymatic glycan synthesis (**Scheme 9**). First the authors used LPPS to build the core 9mer peptide on a 105 mg scale. This was an efficient process using only 1.2 equivalents of amino acid and coupling reagents and leveraging a hydrophobic tag for quick purification by centrifugation and removal of supernatant liquid. Once the 9mer was constructed with the first glycan unit (GalNAc) attached to the T residue a 2-step global deprotection of all sugar, amino acid protecting groups, and the hydrophobic tag yielded the clean core glycosylated peptide. Enzymatic diversification of the GalNAc moiety through the use of varying combinations and orders of glycosyltransferases including C1GalT1, ST6GalNAc1, ST6Gal1, Pd2, 6ST, ST3Gal1, ST3Gal4, GCNT1, B4GalT1 allowed for the formation a and b glycosidic bonds at varying positions with varying substrates to quickly form highly complex glycans highlights the power of this technique.

**Scheme 9.** Enzymatic diversification of core peptide for the synthesis of SARS-CoV-2 O-glycopeptides **26** and **27** [56].

#### *2.4. Peptides and Amino Acids*

Peptide and amino acid-based natural products have been some of the most versatile and important natural products used in the clinical setting including molecules such as Vancomycin and Insulin [57]. As such, there is a rich library of literature involving their syntheses and specifically their chemoenzymatic syntheses [58].

An area which has been developing recently in chemoenzymatic synthesis is the use of enzymes to create stereocenters in small molecules which can be used as a new "chiral pool" to work from towards natural product synthesis. Commonly this is done by dynamic kinetic resolution (DYKAT) or by enzymatic reductions to make enantiomerically enriched alcohols. A recent Renata publication in this area showcases this trend by performing a DYKAT, completed by an enantioselective reductive amination to set two stereocenters: one which was epimerized, one which was generated by the reduction [31]. This reductive amination is actually a transamination from sacrificial glutamine. The scope of this DYKAT was shown through 25 molecules with varying aryl substitutions, one of which was elaborated over four steps to complete the first synthesis of jomthonic acid (**Scheme 10**) (**30**). Significantly, a scaleup to a half gram with >20:1 d.r. was shown by the authors.

**Scheme 10.** Biocatalyzed DYKAT within the synthesis of jomthonic acid (**30**) [31].

Bruner and coworkers disclosed a recent strategy to synthesize deacetylated microviridin J (**32**) and explore the activity of engineered enzymes MdnB and MdnC, which perform the tricyclization of the 13mer MdnA core peptide sequence (**Scheme 11**) [59]. Fusion expression constructs were engineered with the MdnA leader peptides crosslinked to both MdnB and MdnC, using varying lengths of glycine/serine linkers (GSn, *n* = 5, 10 & 15). This strategy allows for cyclizing just the synthetically produced core 13mer MdnA since the 36 AA leader sequence is already in place on MdnB and C rendering them

constitutively active. Upon incubation of these various engineered enzymes with the core peptide, it was found that GSn *n* = 10 & 15 provided the necessary length and flexibility for efficient tricyclization to deacetylated microviridin J. This strategy is an excellent example of engineering and expressing the necessary enzymes for complex macrocyclizations that allowed for a much simpler synthesis of the 13mer core protein versus the endogenously expressed 39 AA leader and core peptide.

**Scheme 11.** Chemoenzymatic lactonization and lactamization for the synthesis of deacylated microviridin J (**32**) [59].

*In planta* syntheses of moroidin (**33**, previously unsynthesized), and celogentin C (**34**, previously synthesized in 23 steps) were recently reported by the Weng group (**Figure 1**) [60]. Intriguingly, they did this by cloning a gene from *K. Japonica*, the predicted precursor gene for Moroidin, and then expressing it in tobacco. They were able to then grow the tobacco with this newly inserted gene, and modified versions thereof, to produce different extractable natural products on the ~10 mg scale. The only synthetic organic chemistry performed during this synthesis was by the plant itself—enforced by the cloned gene.

**Figure 1.** Structures of the peptide-based moroidin (**33**) and celogentin C (**34**) synthesized *in planta* [60].

#### *2.5. Alkaloids*

Alkaloid natural products have a rich history as both biologically active molecules and synthetic targets. This class of molecules has also proven to be a remarkable boon for chemoenzymatic syntheses [61,62]. Several syntheses are highlighted here to give exemplars of the diversity of molecule structure and enzymatic reaction. However, as there is not enough space in this report for a thorough coverage of the breadth of the syntheses, an alkaloid specific review can be found in by Cigan et al. [27].

Taday et al. published a hybrid bio-organocatalytic approach to the synthesis of the small piperidine-based natural product pelletierene (**Scheme 12**) (**37**) [63]. This work built upon a previously reported elegant one-pot 2-biocatalytic step approach to norsedaminone that utilized cadaverine, a transaminase, CalB, and a decarboxylative Mannich reaction to synthesize 14 different alkaloids but was unable to synthesize pelletierene [64]. The authors developed a system where transaminase ATA256 generated the reactive imine intermediate (**36**) with acetone playing the dual role as the nitrogen acceptor in this biocatalytic step as well as the nucleophile in the subsequent organocatalyzed Mannich reaction to yield the desired pelletierene. This system was optimized to produce pelletierene in 60% yield with 85 mg isolated. The only weakness of the system is the natural product was isolated as the racemate despite using D- or L-proline in the system. Based upon the lack of difference in ee for the proline isomers, the authors conclude this was most likely due the piperidine racemizing after the reaction [65]. The authors have established a sound system and now are looking to expand the scope of hybrid bio-organocatalytic approaches and further optimize their system to an in vivo model.

**Scheme 12.** ATA256 biocatalyzed transamination reaction for the synthesis of pelletierene (**37**) [63].

Indole containing alkaloids are abundant throughout nature and often serve as biologically relevant scaffolds. As such there has been an exciting recent push into the utilization of Pictet-Spangelrases for the synthesis of natural products. The Kroutil group published a concise 2-step chemoenzymatic synthesis of (*R*)-harmicine (**Scheme 13**) (**41**) [66]. The authors were exploring the substrate scope for non-natural substrates for strictosidinesynthases (STRs), an important class of Pictect-Spangelerases that could be leveraged for natural product synthesis. Four STRs from different organisms were cloned and expressed in *E. coli*. The best result was obtained by deleting the signal peptide and adding an N-terminal His-tag. Utilizing the STR from *Rauvolfia serpentina*, tryptamine (**38**) and methyl-4-oxobuta-noate (**39**) were enzymatically condensed with concomitant cyclization to form product (**40**) in 67% yield with >98% ee on 75 mg scale. Smooth reduction of the carbonyl yielded the desired (*R*)-harmicine in a total yield of 62% with >98% ee. This report highlights the power of the enzyme via the concise high yielding synthesis as well the potential for a broad applicability for the future of other targets.

**Scheme 13.** Synthesis of (*R*)-harmicine (**41**) via chemoenzymatic Pictet-Spangler reaction [66].

A 2020 report from the Andrade lab details the first synthesis of the complex bisindole (−)-melodinine K (**45**) via a convergent chemoenzymatic synthesis (**Scheme 12**) [67]. The authors were cognizant of both the efficiency and sustainability of this synthesis and thoughtfully devised their scheme based on the isolation of 1.6 g complex biosynthetic precursor (−)-tabersonine (**42**) from *V. africana* seeds (**Scheme 14**). Beyond the isolation of the carbon skeleton, a critical biotransformation of (−)-tabersonine (**42**) was employed

utilizing the cytochrome P450 monooxygenase tabersonine 16-hydroxylase (T16H) [68,69]. A modified yeast strain, *Saccharomyces cerevisiae* (WAT11 strain) was engineered, and the reaction conditions optimized to allow the site-selective oxidation of (−)-tabersonine (**42**) to (−)-16-hydroxytabersonine (**43**) in 64% yield on the gram scale. (−)-Tabersonine (**42**) is converted to activated epoxide (**44**) in four steps, followed by dimerization with a modified (−)-16-hydroxytabersonine intermediate, which underwent two more synthetic steps to obtain the final product (−)-melodinine K. This synthesis highlights both the power and efficiency of isolating a complex precursor and the selective and efficient site selective chemistry of chemoenzymatic syntheses.

**Scheme 14.** Convergent synthesis of (−)-melodinine K (**45**) featuring chemoenzymatic oxidation of isolated biosynthetic precursor, (−)-tabersonine (**42**) [67].

#### *2.6. Miscellaneous*

As chemoenzymatic synthesis has expanded, there are many interesting natural products and syntheses that fall into molecule classes outside of those listed above that are noteworthy and deserve highlighted in this report.

Prostaglandins (PGs) are lipid-based hormone-like signaling molecules that play multiple functions in humans and several such as cloprostenol (**50**) and bimatoprost (**51**) are marketed drugs for veterinary purposes and antiglaucoma treatment, respectively. The Chen lab devised a divergent flow-based chemoenzymatic synthesis capable of producing both cloprostenol and bimatoprost and three other PGs [70]. This synthesis a powerful combination of synthesis, biocatalaysis and flow chemistry that utilizes 11–12 steps from a common starting material to synthesize five high value PGs (**Scheme 15**). The strategy is highlighted chemoenzymatically by a novel stereoselective oxidation to lactone 47 in 99% ee by a Baeyer-Villager monooxygenase (BVMO) and a diastereoselective reduction in 87:13 to 99:1 d.r. by a ketoreductase (KRED) to alcohol 49. From here three synthetic transformations yield the desired prostaglandins. The authors have demonstrated two unique biotransformations that are responsible for setting stereocenters with high ee and d.r., respectively.

The synthesis of sorbicillins requires a dearomatization to afford a sensitive, cyclohexadienone diol. This challenging transformation has been implemented by Gulder and coworkers, using a SorbC monooxygenase enzyme, in order to afford sorbicillinoids which could then be elaborated to natural products including Saturnispol C (**54**), D, and Trichosorbicillin A (**Scheme 16**) [71]. Interestingly enough, the only requisite reaction to afford these three natural products was a Diels-Alder reaction, which was facile using the electron rich cyclic diene afforded by the dearomative hydroxylation of the enzyme under atmospheric conditions. One limitation of this report is potential scalability; reactions were below 0.15 mmol scale, though it is not clear whether this due to cost or a true limitation.

**Scheme 15.** Synthesis of cloroprostenol (**50**) and bimatoprost (**51**) via a combination of synthetic chemistry, flow chemistry, and two stereochemical chemoenzymatic steps [70].

**Scheme 16.** Stereoselective chemoenzymatic oxidation towards the synthesis of saturnispol C (**54**) [71].

The conversion of abundant natural compounds to other high-value natural products is a valuable path towards synthesizing them. Hydroxytyrosol (**56**) is a sought-after antioxidant with a high scale of demand and a deceptively simple chemical structure. Recently several patents and papers have been published for the synthesis of this compound among others, many of which are chemoenzymatic syntheses [72–74]. One such report by Pinto et al. leverages 10–20% of the mass of dry olive leaves isolated as intermediate 55 to form hydroxytyrosol (**56**), a potentially useful antioxidant compound (**Scheme 17a**) [75]. This is performed by sequential enzymatic hydrolysis of a hemiacetal moiety and an ester moiety using a glucosidase and an acyl transferase acting as an esterase.

As an alternative strategy, Pinto et al. published a constant-flow chemoenzymatic synthesis of hydroxytyrosol. Their method was to oxidize tyrosol (**57**) aerobically in the presence of a tyrosinase from *Agaricus bisporus*, in an ascorbic acid/phosphate buffer (**Scheme 17b**) [76]. Although unable to obtain complete conversions, they were able to design a facile flow-based separation method to afford pure hydroxytyrosol. The authors also demonstrated a flow-based chemoenzymatic acylation of tyrosol and hydroxytyrosol using sacrificial ethyl acetate, catalyzed by an immobilized acyl transferase MsAcT. A current limitation of this is scale: the maximum 0.25 mL/min flow rates were limited the yields obtainable in a 24 h period. This marriage of two frontier tactics in organic synthesis, flow and chemoenzymatic synthesis, is impressive. It is also an elegant solution to one of the classic issues of chemoenzymatic syntheses: low concentrations are common, which means it is difficult to make large amounts of material. Automated flow syntheses mostly sidestep this issue as the product is made without human involvement, and generally at a rate exceeding that of simply scaling batches.

**Scheme 17.** (**a**) Two-step chemoenzymatic hydrolysis in the synthesis of high demand antioxidant hydroxytyrosol (**56**). (**b**) One-step chemoenzymatic oxidation to yield hydroxytyrosol (**56**). [75,76].

#### **3. Conclusions**

Natural products continue to fascinate and inspire isolation, synthetic, and bioorganic chemists with their rich library of molecular complexity and biological applications. Pushing the boundaries of synthetic chemistry and biochemistry by using chemoenzymatic syntheses to create these molecules has become a field on to itself. As is the case in reaction methodology-based fields of synthetic chemistry, progress is achieved in incremental steps through the pioneering work of many scientists. Often the first efforts are accomplishments that have limitation in yield, scale, or substrate scope, but the ingenuity and persistence of researchers continues to advance the field. The discovery of new enzymes/reactions, improvement of yields and stereospecificity, and engineering of systems that utilize multiple enzymes, flow chemistry, and other emerging technologies is a testament to the talented scientists working in the field of chemoenzymatic synthesis of natural products. The molecular diversity and breadth of molecule classes to which chemoenzymatic synthesis is applied, as highlighted in this report, is truly remarkable and we look forward to the evolution and expansion of work in this area in the coming years.

**Author Contributions:** All authors shared equally in the data gathering, analysis, writing, and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The APC was funded by the School of Science, Technology & Mathematics at Elmhurst University and the DJ & JM Cram Endowed Chair at Rollins College.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

