Feeding Aquilaria sinensis Leaves Modulates Lipid Metabolism and Improves the Meat Quality of Goats

Aquilaria (A.) sinensis is a medicinal plant widely grown in tropical South China. Given the abundant pruning waste of its leaves, the use of A. sinensis leaves is valuable. In this study, goats were fed a diet containing 20% A. sinensis leaves. Compared with the basal diet, feeding A. sinensis leaves to goats did not affect growth performance but considerably reduced the feeding cost. Strikingly, feeding A. sinensis leaves resulted in a significant decrease in the blood cholesterol levels (2.11 vs. 1.49 mmol/L, p = 0.01) along with a significant increase in the high-density lipoprotein levels (1.42 vs. 1.82 mmol/L, p = 0.01). There was also a tendency to lower the content of low-density lipoprotein levels in goats (0.78 vs. 0.45 mmol/L, p = 0.09). Furthermore, metabolomics analysis demonstrated that the reduction in cholesterol levels occurred in both the serum (0.387-fold change) and muscle (0.382-fold change) of goats during A. sinensis leaf feeding. The metabolic responses to feeding A. sinensis leaves suggest that the activation of lipolysis metabolism might happen in goats. These observed changes would be conducive to improving animal health and meat quality, ultimately benefiting human health.


Introduction
Aquilaria (A.) sinensis is a highly valuable medicinal plant belonging to the genus Aquilaria in the family Thymelaeaceae; it is native to China and widely grown in tropical South China [1]. Notably, A. sinensis has already been demonstrated to be rich in bioactive compounds (over 249 compounds have been isolated and identified) that exhibit a wide range of biological effects [2]. The major chemical constituents of A. sinensis leaves identified so far are amino acids, flavonoids, phenolic acids, lipids, and xanthones [3]. In order to maximize the commercial value of the A. sinensis industry, apart from its medicinal value, the development of A. sinensis leaves has been conducted in recent years [4]. The safety evaluation of the A. sinensis leaves was performed for toxicology in animal and clinical trials [5], and both led to guaranteed safety. Because of the considerable production and pruning waste, efforts are increasingly being made to maximize the use of the prodigal In the last decade, the sources of feedstuff have become increasingly more expensive and difficult to access. Thus, there is a growing number of experimental investigations in promising non-conventional feed resources for improving goat production, such as pterocarpus lucens leaves [6], cassava leaves [7], vachellia karroo leaves [8], barberry leaves [9], moringa leaves [10], orange leaves [11], larrea divaricata and acacia aroma leaves [12], fenugreek leaves [13], paulownia leaves [14], etc. In general, farmers feed goats with relatively low-quality straw and crop residues, which might not satisfy adequate levels of performance. Partially substituting these ingredients with leaves will reduce the feeding cost and improve production performance. In this study, we first evaluated and confirmed that goats like to ingest A. sinensis leaves ( Figure S1) and then analyzed the leaves' nutritional value. We found A. sinensis leaves to be a promising non-conventional feed resource for goat production. We also identified the specific bioactive compounds from A. sinensis leaves using UPLC-MS/MS analysis to illustrate their potential function in goats.
Therefore, the objective of this experiment was to investigate the effects of feeding A. sinensis leaves on production performance and the corresponding metabolite profile changes in the serum and muscles of goats.

Animals and Experimental Design
All animal procedures performed in this study were approved and supervised by the Animal Care and Use Committee of the Guangdong Academy of Agricultural Sciences (IAS2020001, Guangzhou, China).
Twelve three-month-old healthy, castrated male Chinese native growing goat kids with the same genetic background and similar weight (16.7 ± 1.6 kg) were randomly assigned to two different total mixed ration (TMR) diets: (1) the control group (CON, n = 6) with a normal diet, and (2) the A. sinensis leaves feeding group (ASL, n = 6) with a diet containing 20% A. sinensis leaves (dry matter basis). The diets and the dosage of A. sinensis leaves were formulated based on the nutrient requirements of goats in this phase ( Table 1). The experiment lasted three months. Animals in both groups were housed in individual units with ad libitum access to diets and water. At the end of the experiment, the goats were weighed after fasting for 12 h with free access to water. Serum samples were obtained via jugular venipuncture and centrifuged at 3000× g for 10 min (4 • C). Subsequently, all goats were slaughtered according to the related provisions of animal welfare. The longissimus muscle from the lumbar region was collected from the right side of each carcass and culled out of the connective and fat tissues. Muscle samples were cut into 2 cm 3 cubes. All samples were frozen in liquid nitrogen and stored at −80 • C until analyzed.

Nutritional Ingredients Analysis of A. sinensis Leaves
The fresh A. sinensis leaves were dried in a forced-draft oven at 65 • C for 48 h. The dry matter was calculated by the weight change before and after the drying. The dried samples were ground with a cutting mill and then passed through a 1 mm sieve for further analysis. Organic matter, crude protein, and ether extract were determined using Association of Official Analytical Chemists (AOAC) methods. Neutral detergent fiber and acid detergent fiber were analyzed using an Ankom fiber analyzer according to the previous study [15].

Bioactive Compounds Analysis of A. sinensis Leaves
The freeze-dried A. sinensis leaves were crushed using a mixer mill with a zirconia bead for 1.5 min at 30 Hz. The powder was weighted at 100 mg and extracted with 0.6 mL of 70% aqueous methanol at 4 • C overnight. The extracts were centrifugated at 10,000× g for 10 min, and then they were absorbed and filtrated (0.22 µm pore size, ANPEL, Shanghai, China). The sample extracts were analyzed using a UPLC-MS/MS system (UPLC, Shimpack UFLC SHIMADZU CBM30A system coupled with a triple quadrupole-linear ion trap mass spectrometer, Applied Biosystems 4500 Q TRAP). Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 2.1 mm × 100 mm). The mobile phase consisted of (A) pure water with 0.04% acetic acid and (B) the solvent acetonitrile with 0.04% acetic acid. The gradient program started with 5% B at 350 µL/min, followed by a 10 min ramping to 95% B, a 1 min hold at 95% B, and a quick switch to 5% B within 0.1 min. The column was re-equilibrated with 5% B for 2.9 min prior to the next run.
The detection of metabolites was performed by MetWare Biotechnology Co., Ltd. (Wuhan, China). Metabolites were distinguished by comparing the m/z values of precursor ions, retention times, and fragmentation patterns with the standards in a database compiled by MetWare Biotechnology Co., Ltd., and quantitative detection was performed according to a multiple reaction monitoring pattern [16,17].
The total flavonoid, isoflavone, and anthocyanin contents in A. sinensis leaves were detected by spectrophotometry. The composition of fatty acids in A. sinensis leaves was analyzed by a trace GC ultra gas chromatograph. The contents of hydrolyzed amino acids in A. sinensis leaves were determined by the amino acid analyzer.

Serum Biochemistry Analysis of Goats
The concentrations of total protein, albumin, glutamic pyruvic transaminase, glutamic oxalacetic transaminase, glucose, blood urea nitrogen, uric acid, total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein in serum were analyzed and determined by a semiautomatic biochemistry analyzer using commercial kits (Nanjing Jiancheng Biological Engineering Institute, Nanjing, China).

UPLC-MS/MS Metabolomics Analysis of Serum and Muscle Samples
Serum and muscle samples were thawed on ice. To each 50 µL serum sample, 150 µL of ice-cold methanol was added; the mixture was whirled for 3 min, and then centrifuged with 10,000× g at 4 • C for 15 min. Each muscle sample containing 50 mg of tissue was homogenized in 1 mL of ice-cold methanol/water (70% v/v) using a superfine homogenizer (30 Hz for 3 min). After whirling the mixture for 1 min, samples were centrifuged with 10,000× g at 4 • C for 10 min. The supernatant collected from each serum and muscle sample was used for UPLC-MS/MS analysis as described above.

Statistical Analysis
Data for growth performance, serum biochemical indexes, and quantitatively determined cholesterol contents by HPLC were analyzed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). The Student's t test was used for the comparison of sample data. Standard errors of the mean (SEM) were represented. Differences were considered significant at p < 0.05, and significantly different trends were defined at 0.05 < p < 0.1.
For metabolomics analysis, the relative relevance of each metabolite to the orthogonal partial least squares discriminant analysis (OPLS-DA) model was determined. The variable importance in projection (VIP) parameters were extracted from the OPLS-DA results. VIP > 1 and fold change > 2 or <0.5 were used for determining the significantly different metabolites.

Nutritional Ingredients and Bioactive Compounds of A. sinensis Leaves
As shown in Table 2, A. sinensis leaves could be utilized as a potential source of feedstock for ruminants. UPLC-MS/MS analysis identified a total of 418 bioactive compounds from A. sinensis leaves (Table S1). Of these, we identified 81 flavonoids, 78 amino acids and derivatives, 63 lipids, 50 phenolic acids, and other bioactive compounds ( Figure 1). A. sinensis leaves are particularly rich in flavonoids; the contents of total flavonoids, isoflavones, and anthocyanin were 8.14 mg/g, 4.02 mg/g, and 188.32 nmol/g, respectively. The contents of 17 amino acids and fatty acids in A. sinensis leaves are presented in Figure 2. Results indicated that A. sinensis leaves contained high levels of unsaturated fatty acids.

Effects of Feeding A. sinensis Leaves on Growth Performance and Serum Biochemical Indexes of Goats
Compared with the control group, no effect was observed on the growth performance of goats after feeding A. sinensis leaves. There were no significant differences between the two groups on the indexes of initial weight, final weight, average daily intake, and average daily gain (Table 3). Additionally, no significant differences were observed in the serum concentrations of total protein, albumin, glutamic pyruvic transaminase, glutamic oxyacetic transaminase, glucose, blood urea nitrogen, and triglyceride between the two groups (Table 3). Intriguingly, we found that feeding A. sinensis leaves significantly decreased blood cholesterol levels while significantly increasing high-density lipoprotein levels in goats (p < 0.05, Table 3). Meanwhile, there was a consistent decreasing tendency in the content of uric acid and low-density lipoprotein in goats' blood after feeding A. sinensis leaves (p < 0.1, Table 3).

Changes of the Metabolites and the Corresponding Pathways of Serum and Muscle in Goats
To further elucidate the metabolic responses of feeding A. sinensis leaves to goats, serum and muscle metabolomics data were obtained in parallel (Tables S2 and S3). The results of the principal component analysis (PCA) indicated obvious discrimination between the two groups ( Figures 3A and 4A). The supported information of PCA scores plot and an orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot of serum and muscle metabolome distribution according to the diet of goats were also presented in Tables S4 and S5 and Figure S2. differential metabolites revealed that significant changes occurred in the key metabolic pathways involving unsaturated fatty acid biosynthesis, linoleic acid metabolism, and αlinolenic acid metabolism ( Figure 3D).       After feeding A. sinensis leaves, 29 serum metabolites (also including cholesterol) were significantly increased, while five metabolites declined ( Figure 3B and Table 4). Therein, the contents of unsaturated fatty acids, such as α-linolenic acid and γ-linolenic acid, were found to be higher in serum from goats fed A. sinensis leaves. The details of the top 20 differential metabolites between the two groups are shown in Figure 3C, based on linear discriminant analysis and effect size analysis. The KEGG enrichment analysis of differential metabolites revealed that significant changes occurred in the key metabolic pathways involving unsaturated fatty acid biosynthesis, linoleic acid metabolism, and α-linolenic acid metabolism ( Figure 3D). Meanwhile, a total of 26 differential compounds in the longissimus muscle of goats in response to feeding of A. sinensis leaves, have been identified using metabolomics analysis. Of these, 14 metabolites were significantly increased, while 12 metabolites were significantly decreased in the A. sinensis leaves feeding group ( Figure 4B and Table 5). In this process, it is worth noting that the contents of cholesterol in muscle were reduced, and the contents of flavor substances in muscle, such as succinic acid and γ-glutamyl-leucine, were increased. The top 20 differential metabolites in muscle are displayed in Figure 4C. The KEGG enrichment analysis indicated that the differential metabolites in muscle were associated with phenylalanine metabolism, lysine degradation, cysteine, and methionine metabolism pathways ( Figure 4D).

Discussion
Previous studies have reported A. sinensis leaves' analgesic, antiarthritic, antidiabetic, anti-inflammatory, antimicrobial, antioxidant, and hepatoprotective roles [18,19]. Given the abundance of bioactive compounds in A. sinensis leaves, there could potentially be several pharmacological effects for goats. The analysis of serum biochemical indexes after feeding A. sinensis leaves points to marked differences in the metabolic state by providing useful insights on the evaluation of metabolites associated with body fluid distribution in goats [20]. In the present study, we found that feeding A. sinensis leaves might most likely to play a role in hypolipidemic in goats, as indicated by decreased cholesterol and low-density lipoprotein and increased high-density lipoprotein. Moreover, the decrease in cholesterol has also been verified by serum metabolomics analysis. Alam et al. [18] demonstrated that administering 200 and 400 mg/kg of an extract of A. sinensis leaves would significantly reduce the cholesterol levels in rats. Consistent with this study, goats treated with the extract of A. sinensis plants exhibited significantly increased activity of the liver X receptor in HepG2 cells [21]. It is well known that the liver X receptor could correct sterol overload by promoting cholesterol disposal from the cell [22]. Furthermore, HepG2 cells treated with the extract of A. sinensis plants also resulted in a significant increase in PPARα [21], which is known for its hypolipidemic effect. As shown in Table S1, A. sinensis leaves are rich in flavonoids and phenolic acids (especially genkwanin, kaempferol, and mangiferin), which have already been reported to bind well with PPARs or act as PPAR agonists [23,24]. On the basis of a previous experiment with rats and cells and this study (bioactive compounds of A. sinensis leaves and serum biochemical indexes of goats), we suspect that the A. sinensis leaves' hypolipidemic role in goats occurs through the modulation of a lipid metabolism signaling pathway that reduces cholesterol and low-density lipoprotein and enhances high-density lipoprotein. This inference merits further research and verification at the cellular level.
The compounds present in A. sinensis leaves and shown as serum differential metabolites in goats include 3-hydroxybutyrate, α-linolenic acid, γ-linolenic acid, palmitoleic acid, and N-acetylneuraminic acid (Tables S1 and 4). As shown in the present study, A. sinensis leaves contained high levels of unsaturated fatty acids (Table 4). Ahmed et al. [25] demonstrated that a diet rich in linolenic acids would decrease the cholesterol contents in the blood and muscles of goats, while linolenic acids would induce the upregulation of PPARα and PPARγ expression [26,27]. Integrated with the above research findings, Liang et al. [28] and Liu et al. [29] concluded that linolenic acids could reduce lipid accumulation and blood lipid, as indicated by the decrease in cholesterol, low-density lipoprotein, and adipose tissue weight and the increase in the gene and protein expressions of lipid catabolism. In the current study, our data support the notion that supplementation of A. sinensis leaves (enriched with linolenic acids, flavonoids, and phenolic acids) promoted lipolysis in goats and ultimately reduced the contents of cholesterol in muscle.
It is curious that the increase in methylparaben concentration was observed in both serum and muscle tissue of goats fed with A. sinensis leaves ( Figures 3C and 4C). As we know, methylparaben is a conjugate between 4-hydoxybenzoic acid and methanol synthesized by two enzymatic reactions [30]. We speculated that the methylparaben was derived from 4-hydroxybenzoic acid in the leaves of A. sinensis (Table S1). This biosynthetic process might occur in the rumen.
Methylmalonic acid and succinic acid appeared in the list of the muscle differential metabolites in goats and were also derived from A. sinensis leaves (Tables S1 and 5). Succinic acid, a natural flavor enhancer, has been reported to confer an umami taste for meat [31] and plays a significant role in determining meat color stability [32]. Additionally, succinic acid can induce muscle fiber remodeling [33] and positively correlate with meat shear force (toughness) [34]. We observed that feeding A. sinensis leaves can give rise to an increasing level of succinic acid in the muscle of goats, suggesting the enhancement of the flavor and texture of the meat. To obtain a comprehensive insight into the flavor and texture, a sensory evaluation should be conducted by the potential consumers of this product in the future.
According to our findings, the amount of cholesterol in muscle was reduced during this process. Consumption of dietary cholesterol is a potential cause of human disease, including atherosclerosis and coronary heart disease. The World Health Organization advised that dietary cholesterol intake should not exceed 300 mg daily [35]. It is generally known that the sources of dietary cholesterol are primarily derived from foods of animal origin, such as meat, eggs, fish roe, and animal viscera [36]. In order to validate the decreasing level of cholesterol in meat, we further quantitatively determined the cholesterol contents by HPLC, as referred to in the previous study [37]. The results confirmed that feeding A. sinensis leaves dramatically lowered cholesterol levels in the longissimus muscle (CON: 889.02 mg/kg vs. ASL: 366.28 mg/kg, p < 0.01), thus improving the meat quality of goats. In consequence, we suspect that feeding A. sinensis leaves to goats could open an effective alternative strategy for humans to reduce cholesterol intake from mutton and benefit their health.

Conclusions
Overall, from the analysis of nutritional ingredients and the feeding experiment, it can be concluded that A. sinensis leaves can be used as a non-conventional feedstuff for goat production. Despite feeding A. sinensis leaves to goats, it did not improve their growth performance but could considerably reduce the feeding cost.
Furthermore, this study demonstrated that A. sinensis leaves contained a broad array of 418 bioactive compounds, mainly belonging to flavonoids, amino acids, lipids, and phenolic acids. The diet effects of feeding A. sinensis leaves to goats are proposed and summarized in Figure 5. When A. sinensis leaves are ingested via the diet of goats, the most important responsive metabolic pathways in serum are focused on fatty acid metabolism, while those in muscle are reflected in amino acid metabolism. Further research is warranted to illustrate the different metabolic pathways that occur in the different parts of goats. A. sinensis leaves are rich in linolenic acids, flavonoids, and phenolic acids that play the role of hypolipidemic in goats via the modulation of the lipid metabolism signaling pathway. This inference needs further research and verification. The reduction in blood cholesterol and low-density lipoprotein levels and an increase in high-density lipoprotein levels in goats in response to the diet strongly implied the activation of lipolysis metabolism during feeding with A. sinensis leaves. One of the noteworthy biomarkers found in this study is cholesterol, which changes consistently based not only on serum biochemical analysis and quantitative determination in the muscle but also on untargeted metabolomics analysis of serum and muscle tissue. warranted to illustrate the different metabolic pathways that occur in the different parts of goats. A. sinensis leaves are rich in linolenic acids, flavonoids, and phenolic acids that play the role of hypolipidemic in goats via the modulation of the lipid metabolism signaling pathway. This inference needs further research and verification. The reduction in blood cholesterol and low-density lipoprotein levels and an increase in high-density lipoprotein levels in goats in response to the diet strongly implied the activation of lipolysis metabolism during feeding with A. sinensis leaves. One of the noteworthy biomarkers found in this study is cholesterol, which changes consistently based not only on serum biochemical analysis and quantitative determination in the muscle but also on untargeted metabolomics analysis of serum and muscle tissue. The decrease in cholesterol content in serum and muscle suggested that the diet of A. sinensis leaves benefits goat health and improves goat meat quality for the benefit of human health. Figure 5. A schematic diagram of responses to feeding A. sinensis leaves to goats. A. sinensis leaves are rich in linolenic acids, flavonoids, and phenolic acids that play the role of hypolipidemic in goats via the modulation of the lipid metabolism signaling pathway. The reduction in blood cholesterol and low-density lipoprotein levels and the increase in high-density lipoprotein levels in goats in response to the diet strongly implied the activation of lipolysis metabolism during feeding A. sinensis leaves.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: The goats would like to ingest A. sinensis leaves; Figure S2: An orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot of serum and muscle metabolome distribution according to the diet of goats. Table S1: Bioactive compounds identified from A. sinensis leaves; Table S2: UPLC-MS/MS metabolomics analysis of serum and muscle samples from goats fed A. sinensis leaves; Table S3: The supported information of PCA scores plot of serum and muscle metabolome distribution according to the diet of goats.  A schematic diagram of responses to feeding A. sinensis leaves to goats. A. sinensis leaves are rich in linolenic acids, flavonoids, and phenolic acids that play the role of hypolipidemic in goats via the modulation of the lipid metabolism signaling pathway. The reduction in blood cholesterol and low-density lipoprotein levels and the increase in high-density lipoprotein levels in goats in response to the diet strongly implied the activation of lipolysis metabolism during feeding A. sinensis leaves.
The decrease in cholesterol content in serum and muscle suggested that the diet of A. sinensis leaves benefits goat health and improves goat meat quality for the benefit of human health.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods12030560/s1, Figure S1: The goats would like to ingest A. sinensis leaves; Figure S2: An orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot of serum and muscle metabolome distribution according to the diet of goats; Table S1: Bioactive compounds identified from A. sinensis leaves; Table S2: UPLC-MS/MS metabolomics analysis of serum samples from goats fed A. sinensis leaves; Table S3: UPLC-MS/MS metabolomics analysis of muscle samples from goats fed A. sinensis leaves; Table S4. The supported information of PCA scores plot of serum metabolome distribution according to the diet of goats; Table S5. The supported information of PCA scores plot of muscle metabolome distribution according to the diet of goats.