The gut microbiota confers the glucose-and lipid-lowering effect of Laurolitsine in db/db mice

Modulations on gut microbiota by traditional Chinese medicines (TCMs) and their active components are emerging as potential therapeutic agents on diabetes. Litsea glutinosa is a TCM used in clinic to treat diabetes with alkaloids as the active constituents, and the Laurolitsine is the richest one. Purpose Based on that, this study was designed to identify the potential capability of Laurolitsine on alleviating Type 2 diabetes and the changes of the composition of intestinal ora related to this disease. In present study, Laurolitsine was administered to diabetic mice (db/db) by daily oral gavage at doses of 50, 100 and 200 mg/kg/day for 4 weeks. The body weight, fasting blood glucose, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT), lipid metabolism of serum and liver and liver function were measured to assess the anti-hyperglycemic and anti-hyperlipidemic effects of Laurolitsine. The liver pathological changes were observed by HE staining. Meanwhile, the effects of Laurolitsine on the changes of the composition of gut microbiota in mice were investigated via metagenomic analysis.


Introduction
Type 2 diabetes is a chronic disease caused by genetic and environmental factors (Bysani, Agren et al. 2019), and insulin resistance is the main pathophysiological mechanism. The International Diabetes Federation showed that the number of people aged 20-79 years with diabetes mellitus (DM) worldwide was estimated to be 463 million in 2019. Almost 90% of these people will have type 2 diabetes mellitus (T2DM), and this number may increase to 700 million by 2045 (Caron, Ahmed et al. 2020). Oral antidiabetic agents include sulfonylureas, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, meglitinides and etc (Wu, Huang et al. 2019). However, these treatments have appreciated side effects with unsatisfactory clinical bene ts. Therefore, the discovery and development of safer and more effective hypoglycemic drugs is of great signi cance to overcome this global disease. Intestinal micro ora is a microbial community in the human intestinal tract, which participates in digestion, absorption, metabolism, nutrition, and immunity to infection of in the human body (Ke, Walker et al. 2019). Researches demonstrated that the changes in the structure and function of intestinal micro ora are closely related to diabetic phenotypes such as hyperglycemia and insulin resistance ). Intestinal micro ora and their related metabolites play an important role in the pathophysiological mechanisms of type 2 diabetes such as blood glucose metabolism, insulin resistance and chronic in ammation (Molinaro, Koh et al. 2020, Toda, Soeda et al. 2020). Therefore, regulating the structure of intestinal ora in patients with type 2 diabetes is expected to become a new way to pretreat type 2 diabetes.
Most TCMs play their role through oral administration, and the drugs entering the digestive tract inevitably modulate intestinal microenvironment and the structure of intestinal micro ora ( Litsea glutinosa is a TCM belonging to Lauraceae and is distributed in tropical and subtropical region worldwide. In China, this plant is mainly growed in Hainan Province. Our group has been engaged in structure and function of Litsea glutinosa for a long time. Previous research showed that the extract of L. glutinosa could signi cantly reduce the blood glucose in ob/ob mice ). Moreover, Lignans, alkaloids were the characteristic components of L. glutinosa (Ji et al.;Jin et al., 2018), which of the Laurolitsine (LL) was found at a high concentration in the gastrointestinal tract by our group recently (Tan et al., 2021). Thus, this study aimed to investigate whether LL treatment prevents the development of Type 2 diabetes and modulates gut microbiota dysbiosis in diabetic mice. More importantly, we aimed to identify potential correlations between LL treatment and changes in the gut microbiota, providing solid insight into gut microbiota-related mechanisms underlying the anti-diabetic effects of LL.

Plant material
The stem barks of L. glutinosa were collected in August 2019 in Tongguling, Wenchang City, Hainan Province, and identi ed by Prof. Niankai Zeng from the Department of Pharmacy at Hainan Medical University. A voucher specimen (No. LG201908) was deposited at the herbarium of School of Pharmacy, Hainan Medical University.The study protocol complied with relevant institutional, national, and international guidelines and legislation.

Preparation of LL
The dried stem barks (100.0 Kg) of L. glutinosa were cut into small pieces. Ethanol was then used to extract the plant under re ux for three times, each time for 2 hours. The extract was concentrated under reduced pressure till there is no ethanol to give the water solvent of the extract. Appropriate water was added and then extracted by petroleum ether to remove the lipid solvable constituents. Then the mother liquid was further extracted by ethyl acetate to give the total alkaloids. The total alkaloid was isolated by applying a silica gel chromatography using a mixture of dichloromethane: acetone = 4:1 to give six fractions (Fra1-Fra6). Fra4 was further puri ed by using Sephadex LH-20 using methanol as the eluent to give three subfractions (Subfra1-Subfra3). Subfra2 was puri ed by preparative equipped with a SBphenyl column using a mixture of methanol: water = 65:35 as the eluent to give the LL (8.0 g).

Structural identi cation of LL
The structural identi cation of LL was conducted by interpreting its NMR and MS data. NMR experiments were performed on ECZ400S 400 MHz spectrometers operating at 400 MHz for 1 H and 100 MHz for 13 C, respectively (TMS as an internal standard). Chemical shifts were expressed in δ (ppm), and coupling constants in Hz. MS experiment was conducted on an HPLC-MS/MS system. The HPLC system was equipped a SIL-20AC XR autosampler, two LC-20AD XR pumps, an online degasser, and a CTO-20A column oven, and they were all purchased from Shimadzu (Kyoto, Japan). The chromatographic column was Synergi™ Fusion-RP 80 Å C 18 (4 μm, 2.10 mm i.d × 50 mm, Phenomenex, Torrance, CA, USA), the temperature was maintained at 40 °C during analysis. The aqueous solution containing 0.5% formic acid (A) and acetonitrile with 0.5 % formic acid (B) made up the mobile phase. The gradient elution was 5% B at 0-3 min, 35 % B at 3-3.5 min, 95% B at 3.5-5min, 5% 5-6.5 min. The ow rate was set at 0.3 mL/min and the injection volume was 5μL. An AB Sciex Triple Quad TM 5500 system was operated in the electrospray positive ionization mode (ESI + ). The MS analysis detection was optimized when the collision energy was at 18 volt. The optimized declustering potential was 80 volt, temperature, 55 °C; curtin gas, 55psi; nebulizer gas, 60psi; ion spray voltage, 5500, scan time, 40ms.
The determination of puri cation of LL The purity of LL was determined to be 98.73% by our group using HPLC method. The column was C 18 (4.6 × 250 mm, 5.0 μM), the detection wavelength was set at 282 nm. The ow rate was 1.0 mL/min. The column temperature was set at 30 °C, the mobile phase was phase A (acetonitrile), phase B was triethylamine-phosphoric acid in water (pH = 7.0), and the gradient elution was set at A 15-25% for 20 min.

Animals
Animal experiments were carried out according to the National Institutes of Health guides for the care and use of laboratory animals and were approved by the Medical Ethics Committee of Hainan Medical University (No. SYXK-2017-0013). The study is reported in accordance with ARRIVE guidelines. Ten 8week old male C57BL/KsJ mice with a body weight of 20-25g and sixty db/db mice were obtained from GemPharmatech. Co,. LTD (Nanjing China).

Experimental designs
After 1 week of acclimatization, the db/db mice were randomly divided into 5 groups, including model group (n = 10), LL 50 mg·Kg -1 group (n = 10), LL 100 mg·Kg -1 group (n = 10), LL 200 mg·Kg -1 group (n = 10), and metformin 200 mg·Kg -1 (n = 10). The normal group (C57BL/KsJ group (n = 10)) was given a standard rodent chow diet and db/db mice were fed with high-fat diet (HFD) for 4 weeks. Both LL and metformin were dissolved in normal saline and were given by intragastric administration at 14:00 every day for 4 weeks. The normal and negative model groups were gavaged with equal volume of normal saline. Body weight was recorded every ve day. Water uptake and food intake was recorded every week. After fasting for 12 h every week, blood samples were taken from the tail vein to detect fasting blood glucose.

OGTT and ITT measurements
Oral Glucose Tolerance Test (OGTT) and insulin tolerance test (ITT) was performed one day before the terminal sacri ce. Mice were fasted for 12 h (OGTT) or 6 h (ITT). Next, a solution of 25% glucose (2 g·Kg -1 body weight) was administered into the peritoneal cavity for the OGTT or a solution of insulin (0.5 U·Kg -1 body weight) for the ITT. Blood samples were collected from the tail vein at 0, 30, 60 and 90 min for the determination of blood glucose levels and the area under curve (AUC) of OGTT and ITT was calculated for respective groups and used for statistical analysis.

Measurements of levels of lipid, AST and ALT in serum and liver
After 4 weeks of LL treatment, all mice were fasted for 8 h before sacri ce. The whole blood was obtained from the orbital vascular plexus and blood samples were collected for estimation of serum levels of total triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) by respective kits (Nanjing Jiancheng Bioengineering Institute). Liver tissues were taken for the subsequent histological analysis. Otherwise, liver levels of TG, TC, LDL-c, HDLc, aspartate aminotransferase (AST), and alanine aminotransferase (AST) were measured.

H&E Staining
The fresh liver tissues were xed in 4% paraformaldehyde. Then, the samples were gradually dehydrated and embedded in para n. After that, the samples were cut into 3 μm sections and stained with hematoxylin and eosin for further light microscopy observation. Scores were evaluated by a pathologist based on the lung tissue integrity, alveolar integrity, and mononuclear in ltration (0 = none; 1 = mild; 2 = moderate; 3 = severe).

Metagenomic analysis
Fresh stool were collected from each mouse, snap-frozen in liquid nitrogen, and stored at − 80 °C for subsequent metabolomics analysis. Fecal DNA extraction using a FastDNA® Spin Kit for Stool, 16S rRNA V4-V5 regions ampli cation by barcoded composition primers. The PCR reaction was performed as follows: 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, with a nal extension at 72 °C for 5 min. The amplicons were pooled and puri ed using a QiaQuick PCR puri cation kit (Qiagen, Valencia, USA) and DNA pyrosequencing was performed by Majorbio BioTech Co., Ltd. (Shanghai, China) according to the methods mentioned in the literature ).

Data analysis
For phylotypes analysis, the alpha diversity of the microbiome was calculated based on the OTU level by mothur (version 1.30.1). Principal component analysis (PCA) and principal coordinate analysis (PCoA) were performed using R and visualized by the R package, whose signi cant differences were evaluated by Adonis analysis. All data presented in this paper are shown as mean standard error of the mean (SEM). For pharmacological results, comparisons between groups were assessed by one-way analysis of variance (ANOVA) with Dunnett's test as a post hoc test. A P value of <0.05 was considered statistically signi cant.  Figure S1A). These data indicated that this compound was an aporphine alkaloid. The above elucidation was con rmed by its 13  After carefully comparing its 1D-NMR and 2D NMR data with literature, the compound was determined to be Laurolitsine, and its molecular structure was depicted in Figure 1A. The purity of LL was determined to be over 98% by our research group using HPLC method (Figure 1 B). With this extractive craft, we got 8.0g LL from the 100 kilogram dried stem barks of L. glutinosa.

Modulation of body weight, food intake and water uptake by LL
In order to observe the effects of LL on the body weight, food and water intake of db/db mice, we chose 50, 100 and 200 mg·Kg -1 as the low, medium and high dose of LL in mice. The body weight of mice was measured every ve days, it was found that the body weight of mice in C57BL/KsJ group was almost stable, but the body weight of mice in the model and administration group increased gradually over time.
Besides, the body weight of the model mice was signi cantly higher than that of the C57BL/KsJ group, but there was no signi cant difference between the model group and LL group (Figure 2A). In addition, food intake and drinking water tended to be stable in the control group. However, food intake decreased slightly and drinking water increased slightly in the model and administration group ( Figure 2B and 2C). Therefore, the treatment of LL had a little effect on the body weight, diet and drinking water of mice.

Effects of LL treatment on blood glucose
Then, we evaluated the effect of LL on blood glucose in db/db model mice. Results showed that the fasting blood glucose of mice in the model group was signi cantly higher than that of the control group (P < 0.001). Compared with the model group, LL could reduce the level of fasting blood glucose in a dosedependent manner (P < 0.001). The hypoglycemic effect of 200 mg·Kg -1 was similar to that of metformin group ( Figure 3A). Moreover, oral glucose tolerance test showed that the blood glucose of the model group still remained a high level after glucose administration, indicating that the glucose tolerance and the ability to consume glucose were decreased, while LL could signi cantly reduce the level of blood glucose after 30min (P < 0.001), and the effect of 200mg/kg was similar to that of metformin. In addition, insulin tolerance test showed that LL treatment can also rapidly reduce the level of blood glucose and improve insulin resistance after insulin injection ( Figure 3B and 3C). These data demonstrate that LL has the potential capability to restore the disorder of glucose metabolism in db/db diabetic mice.

The effects of LL on lipid metabolism of serum and liver
At the same time, we detected the effect of LL on lipid metabolism in serum and liver of mice in each group. The results showed that 200mg·Kg -1 LL treatment showed a trend to normalize hyperlipidemia, especially on decreasing the levels of TC (P < 0.05) and TG (P < 0.01) in serum and liver, LDL-c (P < 0.01) and HDL-c (P < 0.01) in serum, but failed to reach statistical signi cance on restoring the level of LDL-c and HDL-c in liver ( Figure 4A-H). Metformin treatment also had no signi cant effect on liver HDL-c ( Figure   4H). Taken together, these results veri ed that LL treatment effectively ameliorated hyperlipidemia in db/db mice.
The effects of LL on liver function Besides, we also examined the effect of LL on liver function. Histopathological pro le of mice in C57BL/KsJ group showed normal hepatocytes with well cytoplasm, prominent nucleus, nucleolus and central vein with no sign of in ammation or necrosis in these mice. In model group, liver sections showed hepatocyte nuclear pyknosis, hepatic cord degeneration, in ammatory in ltration, and marked necrosis.
Treatment with LL at 50, 100 and 200 mg·Kg -1 dose showed reduction of necrosed area and in ammatory in ltrates ( Figure 5A and 5D). These results indicated that LL could ameliorate the severity of liver damage in db/db mice. Serum aminotransferase such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are commonly used as an indicator for liver disease (Yang, Chen et al. 2020). In the present study a signi cant elevation of serum ALT and AST activities (P < 0.05) were observed in model mice. However, LL at 50, 100 and 200 mg·Kg -1 dose prevented these elevations in a dosedependent manner, in which mice treated with 200 mg·Kg -1 LL group showed a signi cant reduction in serum ALT and AST ( Figure 5B and 5C). In addition, it is well-established that the serum lactate level indicates glycolytic status in mice. The liver has a strong ability to remove blood lactic acid, so the level of blood lactic acid will increase in varying degrees when liver function is impaired. Mice in model group showed much higher levels of serum lactate than mice in C57BL/KsJ group (Figure 5E), indicating a lower glycolytic rate in model mice, LL at 100 and 200mg·Kg -1 dose can reduce serum levels of lactate but no signi cant difference. In summary, these results show that LL can somewhat recover liver injury in db/db mice.

The effects of LL on diversity of gut microbiota
The gut microbiotas play essential roles in the incidence and development of many diseases such as obesity, hyperlipidemia and T2DM. To check the in uence of LL on the gut microbes, we rstly assessed the diversity of the gut microbiota by 16S rDNA-based metagenomic analysis. The results showed that the Shannon, Simpson, inverse Simpson, Richness and Evenness in the model group were decreased signi cantly, but there was little effect on these indexes of α-diversity after LL treatment ( Figure 6A-E). In addition, we observed the changes of β-diversity of intestinal ora by PCA ( Figure 6F) and PCoA ( Figure  6G) analysis. Compared with normal group, the intestinal ora of mice in the model group was almost isolated along the PC1 direction in PCA analysis ( Figure 6F) and along the PCo1/2 directions in PCoA analysis (Fig. 6G). In summary, LL had slight effects on the diversity of intestinal ora.

The effects of LL on composition of gut microbiota
Taxonomic analysis also displayed a marked impact of LL on gut microbes (Figure 7). At the phylum level, compared with the normal group, Deferribacteres and Firmicutes increased and Bacteroidetes decreased in the model group, which could be reversed by the LL group. Moreover, LL treatment can also decrease Viruses_noname and increase Fusobacteria in mice feces ( Figure 7A). The genus-level analysis showed that the genera in the model group with a signi cant decrease in abundance were Parabacteroides, Alphapapillomavirus and Clostridium, while the genera with a signi cant increase in abundance were Anaerotruncus, Escherichia and Mucispirillum, the administration of LL reversed these changes markedly (Figure 7B). At the species levels, oral administration of LL enriched Parabacteroides_unclassi ed, yet decreased Anaerotruncus_sp_G3_2012, Mucispirillum_schaedleri and Clostridium sp ASF502 with the same trend. Then we use linear discriminant analysis to more deeply characterize the microbiota alterations in LL-treated diabetic mice ( Figure 7C). Genera with logarithmic LDA scores of >4.0 are plotted in Figure 7D, a circular cladogram based on the LEfSe results demonstrated differentially abundant taxa between three groups ( Figure 7E). There were 15 taxa and 25 taxa with signi cant changes in the model and LL group. Overall, these results shows that Parabacteroides, Parabacteroides_unclassi ed, Mucispirillum, Mucispirillum_schaedleri could be the differential bacteria between model group and LL group.

Discussion
Previous research showed that the extract of L. glutinosa could signi cantly reduce the blood glucose. Intriguing by these ndings, our group isolated and identi ed the chemical constituents of L. glutinosa and results indicated that lignans, alkaloids were the characteristic components(Wu et al., 2017) (Sun et al., 2019). Previously, we obtained the total alkaloid (CG) from L. glutinosa and investigated its antihyperglycemic effects in ob/ob mice ). The results revealed that the alkaloid-rich extract of CG displayed potential anti-hyperglycemic and anti-hyperlipidemic effects. In this research, eight main alkaloids were identi ed in CG and Laurolitsine (LL) was the richest one among them. We speculate that LL is the active ingredient in modulating the anti-hyperglycemic and anti-hyperlipidemic effects of CG, and then we carried out experiment in vivo to con rm the glucose-and lipid-lowering effect, lastly we analyzed the overall and ne regulation of LL on intestinal ora in type 2 diabetic mice by 16sRNA sequencing. It was found that Parabacteroides was decreased signi cantly and Mucispirillum was increased evidently in db/db mice, which can be reversed after LL treatment. Thus, it can be seen that the changes of these key bacteria may be an important reason for the improvement of glucose and lipid metabolism in type 2 diabetic mice. Of course, it is necessary to further con rm the role of key bacteria in improving glucose and lipid metabolism by means of antibiotic treatment and fecal bacteria transplantation.
Although this study adds a new evidence for the relationship between the e cacy of traditional Chinese medicine and intestinal ora, this association needs to be further veri ed, such as whether new compounds are formed after the metabolism of LL through intestinal ora, the bioavailability, bioactivity and toxicity of the new compounds if formed need to be further studied. In addition, the effects of traditional Chinese medicine on speci c bacteria can be divided into direct effects (promotion, inhibition and killing) ( acting as signal molecules, energy and nutritional resources, these molecules can exert extensive effects on physiological processes such as intestinal and immune homeostasis (Nakkarach, Foo et al. 2021), energy metabolism (Wang, Li et al. 2020) and brain behavior (El Aidy, Dinan et al. 2014), and then improve disease. We can use multi-omics analysis to study the interaction between traditional Chinese medicine and intestinal ora. The joint analysis of genomics and metabolomics can avoid the in uence of epigenetic modulation and post-translational modi cation of transcriptome and proteomics directly re ect the functional state of cells and are more likely to be associated with phenotypes. Therefore, metabonomics is an ideal tool to study the interaction between traditional Chinese medicine and intestinal ora. Through the comprehensive study, the complex interaction mechanism between host and intestinal ora can be systematically clari ed, and the microorganisms and active compounds with important value in the treatment of TCM can be screened out.

Conclusions
In conclusion, our results indicate that Laurolitsine, an alkaloid obtained form L. glutinosa, have glucoseand lipid-lowering effects and improve diabetes-related symptoms in db/db mice. Modulation of the gut microbiota may play a role in the anti-diabetic effect of Laurolitsine.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. GraphicAbstract.jpeg