Astragalus membranaceus and Panax notoginseng saponins improves intestinal l-arginine absorption and protects against intestinal disorder in vivo

Abstract

Nutritional supplementation with l-arginine has been shown to promote cell growth and exercise performance, and is helpful to various disorders. Astragalus membranaceus and Panax notoginseng are traditional herbal medicines that display a wide range of beneficial activities. To understand their effects on l-arginine absorption in human intestinal cells, in vitro and in vivo experiments were performed using a standardized mixture of Astragalus membranaceus and Panax notoginseng saponins (APS). Our studies indicated that APS increased the expression of cationic amino acid transporter 1 (CAT1) and L- arginine transport in human Caco-2 cells. In addition, APS benefits TNBS-induced-colitis rats by increasing food intake, body weight, intestinal epithelial integrity, CAT1 gene expression, and blood l-arginine level. APS also reduces intestinal inflammation associated with increased myeloperoxidase activity in colitis rats. Further studies indicated that the plasma l-arginine level significantly increases in human subjects administered APS.

Introduction

l-Arginine plays a key role in cellular metabolism, as it is involved in many important physiological functions, including protein synthesis, creatine biosynthesis, polyamine formation, and NO production (Morris, 2016; Agarwal et al., 2017). Studies have indicated that l-arginine is required during times of development and pathophysiological stress. Thus, l-arginine has been increasingly considered as a potential therapeutic target in certain disease states, such as cardiovascular disorders, wound healing, and cancer (Pernow and Jung, 2013; Albaugh et al., 2017). Previous studies also indicate that l-arginine promotes intestinal and colonic epithelial wound repair and enhances cell proliferation and restoration of intestinal epithelial cell (IEC) integrity (Lee et al., 2017). l-Arginine is transported across the plasma membrane via several transporter systems. Among the transporters, the y⁺ transport system, which includes the cationic amino acid transporters (CATs) is considered to be the primary transporters for l-arginine uptake in a variety of tissues (White et al., 1982; Closs et al., 2004). l-Arginine uptake into IECs displays significant first-pass metabolism in the intestinal tract, as recent studies have indicated that the small intestine is a major site for extensive catabolism of amino acids (Castillo et al., 1993). Modulation of l-arginine in IECs and blood circulation represents an important mechanism responsible for regulating plasma l-arginine levels under different conditions (Lee etal., 2017; White etal., 1982; Closs et al., 2004).

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is a chronic gastrointestinal disorder. The pathogenesis of IBD is a complex process and is thought to be associated with genetic factors, environmental factors, gut immune function, and the enteric microbiome (Ng et al., 2018). It has been reported that the overall prevalence of IBD surpasses 0.3 % in Western countries. However, IBD is an emerging health issue, as the incidence of IBD is rising worldwide as the typical human lifestyle changes (Ng et al., 2018). Current medications, such as non-steroidal anti-inflammatory drugs, steroids, and immune-modulators, are limited in their applications because of their poor efficacy and adverse effects (Kozuch and Hanauer, 2008). Thus, it is crucial to develop novel therapeutic strategies for IBD. Previous studies have indicated that increased intestinal l-arginine uptake may facilitate intestinal barrier protection, ameliorating gut disorders in IBD; this represents a promising strategy for managing IBD (Coburn et al., 2012; Maloy and Powrie, 2011; Lee et al., 2017).

Radix Astragali and Panax notoginseng are among the most popular medicinal herbs for reinforcing vital energy in traditional Chinese medicine (Shahrajabian et al., 2019; Park et al., 2012). They are included as key components in many traditional therapeutic formulas and as dietary supplements, despite the lack of a well-defined mechanism of action. The major active component of notoginseng is a group of dammarane triterpenoid saponins, which includes protopanaxtriol and protopanaxdiol type ginsenosides. Similar to ginseng, research indicates that notoginsenosides exhibit anti-inflammatory, energy metabolism-modulating, anti-aging, and anti-cancer activities (Park et al., 2012; Lee et al., 2019; Shang et al., 2019; Zhao et al., 2017). The main constituents of Astragalus extract include flavonoids, polysaccharides, polyphenols, and cycloartane-type saponins (Guo et al., 2019). Astragalus and astraglosides have been used as anti-inflammatory, immune-modulatory, and wound-healing agents (Guo et al., 2019; Di Cesare Mannelli et al., 2017). A number of clinical and physiological effects of astragalus and notoginseng have been investigated. Overall, astragalus and notoginseng are generally considered safe, non-toxic, and effective herbal supplements (Dong et al., 2005; Lee et al., 2012; Attele et al., 1999).

Given the potential beneficial applications of ginseng and astragalus for health, the effects of a formulation consisting of enriched fractions of Astragalus membranaceus and Panax notoginseng saponins (APS) on l-arginine uptake and intestinal inflammation were investigated. The study was performed in vitro using Caco-2 cells, in vivo on normal and TNBS-induced-colitis rats and healthy human subjects. The results indicate that APS exerts protective effects against intestinal inflammation in rats with colitis and significantly promotes blood plasma l-arginine levels. The knowledge gained from this study will provide valuable information for the development of novel and effective natural nutrient supplements.

Materials and Methods

Materials  APS is a mixture of dried extracts of Astragalus membranaceus (10:1 hydroethanolic extract) and Panax notoginseng (50:1 aqueous extract) roots blended with maltodextrin as an excipient; its production is compliant with current Good Manufacturing Practice. The final blend is beige to light yellow powder and is standardized to contain ≥1.5% saponins. The approximate ratio of the two saponins in APS of ginsenosides and astragalosides is 5:1 (wt/wt). The APS used in this study was provided by NuLiv Science USA, Inc. (Brea, CA, USA) (Murbach et al., 2019). The APS capsules consisted of 50 mg of the proprietary extract mixtures, and the placebo capsules contain maltodextrin. l-Arginine and sodium 2, 4, 6-trinitrobenzene sulfonate (TNBS) (Sigma-Aldrich, St. Louis, MO, USA), l-[³H] -arginine (American Radiolabeled Chemicals, St. Louis, MO, USA), and Ultima Gold liquid scintillation cocktails (PerkinElmer, Waltham, MA, USA) were used in this study.

Cell culture The human intestinal epithelial cell line Caco-2, a widely used model for studying the intestinal barrier, permeability, and wound healing (Sambuy et al., 2005), was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 0.584 g/L glutamine, 10 % fetal bovine serum, 3.7 % sodium bicarbonate, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 1 % nonessential amino acids. In this study, APS was dissolved in DMSO to get stock solutions. The APS working solutions were prepared by 1:1 000 dilution of the stock solutions into culture medium to make final APS concentrations to 1.0, 0.1, and 0.01μg/mL with 0.1 % DMSO. The APS-containing medium is clear, which were filter sterilized before applying to the cells. For western blot and Q-PCR analysis, Caco-2 cells were plated on 6-cm culture dishes at a density of 1 x 10⁶ cells/dish and the cells were left to undergo differentiation for 8 days prior to treatment with APS for 24 h before harvested for analysis. Control cells were treated with the 0.1 % DMSO-containing medium, which did not show any significant cytotoxicity compared to cells not exposed to DMSO.

l-Arginine uptake assay For l-arginine uptake analysis, 0.3 mL (10⁵ cell/mL) of Caco-2 cells were seeded into each transwell inserts (the apical chamber, 6.5 mm diameter, 0.4 μm pore size, No. 3470, Costar, Corning Inc., NY, USA). The cells were left to differentiate for 14–17 days and the culture medium was regularly changed three times a week in both of the apical and basolateral chambers. The integrity and the full development of the tight junctions of the Caco-2 monolayers were monitored by determining the transepithelial electrical resistance (TEER) by use of a commercial apparatus (Millicell ERS; Millipore, Bedford, MA). Only cell monolayers with TEER values of 500–600 Ω-cm² were used for uptake analysis. The TEER values were taken before and after each experiment to justify consistency of the data collected. In measuring l-arginine transport across the Caco-2 cell monolayer, both of the apical and basolateral chambers of the transwells were washed three times with l-arginine transport buffer (137 mM NaCl, 10 mM Hepes, 0.3 mM NH2PO4, 0.3 mM K2HPO4, 5.4 mM KC1, 2.8 mM CaC1₂, 1 mM MgSO₄, 10 mM glucose, adjusted to pH 7.4). The Caco-2 cell monolayers were then incubated in fresh transport buffer for 5 min, 0.2 mL and 0.9 mL in the apical and basolateral chambers, respectively. The l-arginine uptake assay was initiated by replacing the transport buffer on the apical side with transport buffer containing 0.1 mM of l-arginine and 1 μCi/mL of l-[³H]-arginine. A series of five 10-μL samples were taken from the basolateral chamber at every 1 min intervals. In order to maintain the constant buffer volume, same amount of transport buffer were added back to the basolateral chamber after withdrawn of each sample. The 10-μL sample was then mixed with 2 mL of liquid scintillation cocktail and radioactivity was measured using a scintillation counter (Tri-Carb 2900TR, Packard BioScience, Meriden, CT, USA). Results are expressed as the steady-state rate of l-arginine transport across the Caco-2 monolayers (nmoles/min). The uptake of [³H]-mannitol (0.1 mM) was involved to correct for the nonspecific transport of molecules across the Caco-2 monolayers. To study the involvement of cationic amino acid transporters (CATs) in l-arginine transport in the cells, the water-soluble CAT1 specific inhibitor N-ethylmaleimide (NEM) is employed. The differentiated cells were pre-treated with 0.5 mM NEM in culture medium for 10 min and the NEM-containing medium was then washed out with transport buffer prior to the l-arginine uptake assay.

Animal study Adult Sprague-Dawley rats (6–8 weeks) were purchased from LASC Inc. (AAALAC accreditation, Taipei, Taiwan). Animal feeding and experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center and performed in accordance with the relevant guidelines and regulations. The rats were randomly divided into three groups with eight rats in each group. Before the experiment, the rats were acclimated to the environment for one week. TNBS-induced intestinal colitis is commonly used to simulate the IBD model. As shown in Figure 2A, the rats in the control group were gavaged with normal saline once daily for 13 consecutive days. The TNBS group was administered normal saline for 13 days and treated with TNBS on day 9. The TNBS+APS groups were administered 5.14 mg/kg APS for 13 days and treated with TNBS on day 9. To induce colitis in the TNBS and TNBS+APS groups, the rats were anesthetized with sodium pentobarbital injected intraperitoneally (100 mg/kg), and TNBS 10 mg of in 0.25 mL ethanol (50 % v/v) was instilled into the colon through the cannula to induce acute colitis on the 9th day (Scheiffele and Fuss 2002). The body weight and amount of food intake of the rats were taken daily until termination. Blood samples were collected from the tail vein of each rat before the experiment (Day 1), before TNBS induction (Day 8), and at the end of the experiment (Day 13). The animals were euthanized using CO₂ to allow for harvesting of tissues.

Myeloperoxidase activity assay Myeloperoxidase (MPO) activity was assayed as previously described (Lee et al., 2017). Briefly, the tissue was lysed and freeze-thawed for three cycles in extraction buffer (1:20, w/v). Homogenates were then centrifuged at 15 000 g for 20 min. Ten microliters of supernatant was collected and mixed with 190 μL assay buffer (1.68 mM 3,3',5,5'-tetramethylbenzidine and 0.00015 % hydrogen peroxide). MPO activity was determined at 650 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Western blot analysis Rat tissue fragments were excised and homogenized in solubilizing buffer at 4 °C. Insoluble material was removed by centrifugation for 20 min at 9 000 g at 4 °C. The protein concentrations of the supernatants were determined using a BCA (bicinchoninic acid) kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein samples of cell lysate were separated by SDS-PAGE and transblotted onto a PVDF membrane (Millipore, Billerica, MA, USA). Immunoblotting was performed with primary antibodies against CAT1 (14195-1-AP, Proteintech, Rosemont, IL, USA), CAT2 (GTX66268, GeneTex, Irvine, CA, USA) and GAPDH (GTX100118, GeneTex). These antibodies possess reactivity toward human, mouse and rat serum as indicated by commercial suppliers. After incubation with horseradish peroxidase-conjugated secondary antibodies for goat-anti-rabbit (111-035-003, Jackson ImmunoResearch, West Grove, PA, USA) or goat-anti-mouse (ab205719, Abcam, Cambridge, UK), signals were visualized with an enhanced chemiluminescence kit (ECL, Amersham Biosciences, Little Chalfont, UK) followed by exposure to X-ray films.

RNA isolation and real-time quantitative PCR The relative level of CAT1 transcript expressed in human Caco-2 cells was determined using real-time quantitative PCR (qPCR). Briefly, total RNA was isolated from Caco-2 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA samples were reverse-transcribed into cDNA using high-capacity cDNA reverse transcription kits (Applied Biosystems, Foster City, CA, USA). Real-time quantitative PCR was performed on an Applied Biosystems 7 500 system using SYBR Green PCR master mix (Applied Biosystems). The sequences of the primers used for this study are as follows: CAT1 forward, 5 ’-GCCATCGTCATCTCCTTCCTG-3'; CAT1 reverse, 5'- CCCTCCCTCACCGTATTTCAC-3'; GAPDH forward, 5'-TGGTATCGTGGAAGGACTCA-3'; and GAPDH reverse, 5'-AGTGGGTGTCGCTGTTGAAG-3'. Three independent triplicate experiments were performed, and the obtained threshold cycle values were averaged using the comparative Ct method according to the manufacturer's instructions. The data are presented as the fold differences in gene expression normalized to the endogenous reference GAPDH mRNA.

Histological examination Histological examination was performed as previously described (Lee et al., 2017). Intestinal tissues were soaked in a 10 % formaldehyde solution for 24 h and then embedded in paraffin. Two 4 μm-thick sections were cut and stained with hematoxylin–eosin (H&E) for histological evaluation.

Human study The protocol for this study was approved by the ethical committee at the Chung Shan Medical University Hospital IRB (Protocol No. CSMUH No: CS2-20203). This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. All participants signed the informed consent form before the trial. In a randomized, double-blind, placebo-controlled crossover study, seven (five males and two females) healthy, non-obese volunteers [33–74 years old, with a body mass index (BMI) less than 27 kg/m²] were randomly allocated into placebo and APS-treatment groups. All participants had a normal clinical history and physical examination. Diabetes, obesity, hypertension, cardiovascular disease, liver or kidney disease, current infections, and smoking were exclusion criteria. None of the volunteers received any drugs that might alter amino acid or vitamin status, and dietary habits were kept constant during the study. Written informed consent was obtained from all volunteers. All procedures performed in this study were approved and in accordance with the relevant guidelines and regulations. Subjects in this study took a 50 mg placebo (maltodextrin) capsule at 9 p.m. and began fasting for 12 h, except for a small amount of water the night before the experiment. All subjects had their first blood sample (5 mL) collected at 9 a.m. the next morning with the heparinized tube from the median cubital vein using the indwelling catheter method. The blood samples were centrifuged at 3 000 rpm for 10 min to separate the plasma from blood. The plasma sample was kept at -20 °C until analysis. Immediately after the first blood collection, all subjects took 50 mg placebo and 5 g of l-arginine with 250 mL of water. Additional blood samples were collected at 15, 30, 45, 60, 90, 120, 150, and 180 min for analysis of the plasma concentration of l-arginine. Vital signs, adverse events, concomitant medications, and intercurrent illnesses were assessed and recorded. Continuation criteria were reviewed. Seven days later, the same procedures were repeated as in the first round, except that the placebo was replaced with an APS capsule.

Determination of plasma l-arginine concentration The HPLC system (Waters Inc., Milford, MA, USA) was employed for the measurement of plasma l-arginine levels. The system is equipped with two Waters 515 pumps, a high-pressure mixer, a 717-plus autosampler, a 474 scanning fluorescence detector. Sample separation was achieved using a 4 μm; AccQ.Taq C18 column (3.9 x 150 mm I.D.) with a Nova-Pak C₁₈ Sentry Guard column (3.9 × 20 mm, 4 μm). The plasma l-arginine assay was carried out as described previously (Bosch et al., 2006). Briefly, plasma samples (100 μL) were precipitated by vortexing with ice-cold methanol (395 μL) and incubated for 15 min at 4 °C. The samples were then centrifuged, and the supernatants were collected. The protein-free supernatants were processed immediately for HPLC analysis or stored at −70 °C for further analysis. The derivatization reaction was then initiated by adding the AccQ Fluor reagent, and the mixture was immediately vortexed for several seconds. After 1 min of incubation at room temperature, the mixture was transferred to an autosampler vial. The vial was placed in a heating block for 10 min at 55 °C before automatic injection of 5-μL sample into the HPLC. The column was maintained at 38 °C with a flow rate of 0.8 mL/min. Mobile phase A consisted of AccQ. Tag eluent A [AccQ. Tag A concentrate-Milli-Q water, (1:10, v/v), pH5.2]. Mobile phase B was acetonitrile-Milli-Q water (6:4, v/v). The linear gradient system consisted of mobile phase A and mobile phase B: 0–0.5 min, 100 % A; 0.5–15 min, 98–93 % A; 15–19 min, 93–88 %A; 19–26 min, 88–68 %A; 26–35 min, 68–60 %A; 35–50 min, 60 %A; and 50–52 min, 0 %A. Detection was carried out by fluorescence (λ excitation 250 nm and λ emission 395 nm).

Calculations and statistical analysis In Caco-2 cells and animal studies, the values for each group were expressed as mean ± SD (n ≥ 3). The experimental groups are compared by one-way ANOVA and followed by Scheffe's post-hoc test using SPSS software (IBM, USA). For clinical trial, all pharmacokinetic parameter (Cmax, Tmax, and AUC) calculations for human studies were performed using the WinNonlin program (v. 5.2, Pharsight Corp., Cary, NC). The difference between the placebo and APS groups was determined by paired t-test for paired data at the same time using SPSS software. A p-value of less than 0.05 is considered statistically significant.

Results

APS enhances l-arginine uptake and CAT1 expression in Caco-2 cells To assess the effects of APS on l-arginine uptake, differentiated human intestinal Caco-2 cells were treated with APS. Results shown in Fig. 1A indicate that APS increases l-arginine absorption in a dose-dependent manner. Twenty-four hours after incubation with 0.01, 0.1 and 1 μg/mL APS, l-arginine uptake increased by 1.41, 1.67 and 1.92 fold, respectively in the cells (p < 0.05 for APS at 0.1 and 1 μg/mL, relative to untreated control cells) (Fig. 1A). A background mannitol (0.1 mM) uptake is observed, which was performed to correct for non-specific uptake (Fig. 1A). As l-arginine uptake is primarily through the y⁺ transporter CAT system in intestinal cells, the CAT specific inhibitor NEM was included in the l-arginine uptake analysis. Results shown in Fig. 1B indicates that l-arginine uptake is significantly inhibited in the presence of NEM, which demonstrates that CAT transporters play a crucial role in APS-stimulated l-arginine uptake in human intestinal cells.

Fig. 1.

Effect of APS on arginine uptake and CAT expression in human intestinal cells. Differentiated human Caco-2 cells were incubated with or without the indicated concentrations of APS for 24 h, prior to further analysis. (A) The relative rate of arginine (Arg) transported across the monolayer cells and accumulated in the basolateral chamber were measured. Mannitol (Mtl, 0.1 mM) was included as the background control of non-specific uptake level. (B) The arginine uptake activity were determined in absence or presence of CAT inhibitor N-ethylmaleimide (NEM, 0.5 mM). The effect of APS on (C) CAT mRNA and (D) CAT protein levels in the cells were examined. The results are presented as the mean ± SD (n = 3). Statistical analysis was performed by one-way ANOVA and followed by Scheffe's post-hoc test using SPSS software. *p > 0.05 and ***p < 0.001 versus the untreated control.

The effect of APS on the expression of CAT1 and CAT2 l-arginine transporters were then investigated. As shown in Fig. 1C, in the presence of 0.01, 0.1, and 1 μg/mL APS, the abundance of CAT1 mRNA considerably increased by 1.49, 2.15 and 2.14 fold, respectively, in the differentiated Caco-2 cells (p < 0.05 for APS at 0.1 and 1 μg/mL, relative to untreated control cells). APS also enhanced the expression levels of CAT1 protein. As shown in Fig. 1D, with increased concentrations from 0.01, 0.1, to 1 μg/mL, APS significantly induced CAT1 protein levels by 1.65, 1.82, and 2.16 fold, respectively (p < 0.05 for APS at 0.01, 0.1 and 1 μg/mL, relative to untreated control cells). Our results also demonstrated that the CAT2 mRNA and protein expression levels are weakly induced by APS treatment, but not significantly (Fig. 1C and 1D) in human intestinal cells.

APS ameliorates TNBS-induced colitis in rats The TNBS-induced rat colitis model was employed to assess the in vivo effects of APS. The flow chart in Fig. 2A depicts the design and performance of animal study. As shown in Fig. 2B, before TNBS treatment, there was no significant difference in food intake between the three groups. After TNBS induction, food intake was drastically decreased, but it was significantly recovered in the TNBS+APS group (from 28.3 ± 9.5 % to 54.8 ± 7.7 %, relative to the untreated control group,p < 0.05). APS also slightly decreases TNBS-induced body weight loss in colitis rats, although not significantly (Fig. 2C).

Fig. 2.

Effect of APS on the TNBS-induced rat colitis model. (A) Scheme of the animal study design. The effect of APS (0.1 μg/mL) on food intake (B) and body weight (C) of TNBS-induced-colitis rats. (D) MPO activity in rat colon tissues was measured. (E) Histological examination was performed by photomicrography (original magnification at 100x magnification). Results are expressed as the mean ± SD (n = 8). Statistical analysis was performed by one-way ANOVA and followed by Scheffe's post-hoc test using SPSS software. *p < 0.05, **p < 0.01 and ***p < 0.001 versus the untreated control group; #p < 0.05 and ###p < 0.001 versus the TNBS-treated group.

MPO activity is used as an index of neutrophil infiltration, which is frequently used as a biomarker for inflammation of the intestinal mucosa. As shown in Fig. 2D, administration of TNBS resulted in increased MPO activity in rat intestinal tissues 5.92-fold relative to the untreated control group. APS significantly suppressed TNBS-induced MPO activity (from 592.2 ± 69.7 % to 158.7 ±32.3 % relative to the untreated control group, p < 0.001). Intestinal epithelial integrity was examined by H&E staining. The results shown in Fig. 2E indicate that intestinal mucosal tissue was damaged in the TNBS group of rats, and the presence of APS significantly improved the integrity of the intestinal mucosa.

APS increases blood l-arginine level and CAT1 expression in TNBS-colitis rats In addition to its effects on colitis in rats with intestinal inflammatory disorders, the effect of APS on blood l-arginine levels was also investigated. The results shown in Fig. 3 indicate a significant increase of 16.1 % in blood l-arginine levels in rats treated with APS for a week before TNBS induction (Day 8 of Fig. 3A, p < 0.05). Between the day of TNBS induction and day 13, the blood l-arginine level was significantly decreased to 70.0 % of that in the normal control rat group (p < 0.001). However, pretreatment with APS significantly prevented the TNBS-induced reduction in blood l-arginine levels on day 13 (91.5 % in the TNBS+APS group relative to the normal control group), which represents a 30.7 % increase relative to the TNBS-treated group (from 70.0 % to 91.5 %,p < 0.01).

Fig. 3.

APS significantly increased blood arginine levels and jejunum CAT1 expression in TNBS-treated rats. (A) APS (0.1 μg/mL) significantly increased blood arginine levels in the TNBS-induced group of rats. APS also increased CAT1 (B) mRNA and (C) protein levels in the rat jejunum. The results are presented as the mean ± SD (n = 3). Statistical analysis was performed by one-way ANOVA and followed by Scheffe's post-hoc test using SPSS software. *p < 0.05 and ***p < 0.001 relatives to untreated normal control group, and #p < 0.05 and ##p < 0.01 relative to TNBS-treated group.

In addition to l-arginine uptake, the effect of APS on CAT1 gene expression level was examined. As shown in Fig. 3, our results indicate that TNBS induction markedly suppressed CAT1 expression in the rat jejunum (74 % in mRNA (Fig. 3B) and 46.6 % in protein (Fig. 3C) levels relative to the normal control group, p < 0.05). Pretreatment with APS significantly restored jejunum CAT1 mRNA and protein levels in TNBS-induced-colitis rats (91 % in mRNA (Fig. 3B) and 129.2 % in protein (Fig. 3C) levels relative to the normal control group, p < 0.05). These results suggest that the blood l-arginine level is directly related to the CAT1 expression level in intestinal epithelial cells.

APS increases blood l-arginine levels in healthy human subjects The effect of APS on human blood l-arginine levels was examined by orally administered 5 g l-arginine in seven healthy human subjects, and the results are shown in Fig. 4A. The plasma l-arginine concentration was significantly increased 30–90 min after administration of 50 mg APS compared with the placebo (50 mg maltodextrin) (p < 0.05). Based on this study, APS was found to increase the pharmacokinetic parameters of blood l-arginine AUC and C_max by 10.5 % and 15.4 %, respectively, in human subjects (p < 0.05) compared with the placebo. T_max was not significantly different between the placebo and APS (53.6 ± 19.1 min and 55.7 ± 18.8 min, respectively) (Fig. 4B and 4C).

Fig. 4.

APS stimulates blood arginine level in human subjects. (A) Effect of 50 mg APS on plasma concentration of arginine in healthy human subjects. The kinetic parameters (B) AUC and (C) C_max of arginine in human plasma after oral supplementation with either APS or placebo (maltodextrin). The results are presented as the mean ± SD (n = 7). The difference between the placebo and APS groups was determined by p Figure 4. APS stimulates blood arginine level in human subjects. (A) Effect of 50 mg APS on plasma concentration of arginine in healthy human subjects. The kinetic parameters (B) AUC and (C) C_max of arginine in human plasma after oral supplementation with either APS or placebo (maltodextrin). The results are presented as the mean ± SD (n = 7). The difference between the placebo and APS groups was determined by paired t-test for paired data at the same time using SPSS software. *p < 0.05 relatives to untreated placebo control aired t-test for paired data at the same time using SPSS software. *p < 0.05 relatives to untreated placebo control.

Discussion

In the present study, the in vitro and in vivo effects of APS on l-arginine absorption and intestinal protection were investigated. APS was found to significantly increase l-arginine absorption level and enhance the expression of the l-arginine transporter CAT1 in human intestinal Caco-2 cells as well as the in vivo rat model. Using a TNBS-induced-colitis rat model, APS was found to ameliorate the severity of colitis symptoms, including food intake, weight loss, and intestinal inflammation. The increased l-arginine uptake in rat intestines may help explain the protective and anti-inflammatory responses of APS in the colitis rats. Moreover, APS also enhanced human blood l-arginine levels in human subjects, suggests the therapeutic potential of APS in inflammatory disorders. Fig. 5 summarizes the results observed for APS in stimulating l-arginine uptake and intestinal protection in vitro and in vivo.

Fig. 5.

The potential efficacy of APS in stimulating arginine uptake and intestinal protection.

Astragalus and astragalosides have been shown to display efficacy in the healing of intestinal inflammatory disorders via increased l-arginine uptake and improved intestinal epithelial barrier integrity in in vitro and in vivo colitis animal models (Lee et al., 2017; Guo et al., 2019; Di Cesare Mannelli et al., 2017). Notoginseng displays anti-inflammation, energy metabolism-modulating, anti-aging, and anti-cancer activities (Xu et al., 2018; Zhao et al., 2017; Wen et al., 2014). Documents indicate that notoginseng extract or ginsenosides effectively ameliorate inflammatory disorders in TNBS-induced colitis in rats (Wen et al., 2014; Xu et al., 2018; Lee et al., 2015). Fermented wild ginseng ameliorates DSS-induced acute colitis by inhibiting NF-kB signaling and protecting the intestinal epithelial barrier (Park et al., 2018). Many traditional Chinese medicine formulations contain both ginseng and astragalus as the main ingredients. The formulas were commonly used to augment the vital energy (“qi”) as well as to suppress inflammatory disorders of the body. A previous study demonstrated the effects of a formula containing both ginseng and astragalus in protecting intestinal mucositis and strengthening immune functions in mice (Gou et al., 2016; Wang et al., 2018). In this study, the effects of APS, a formula consisting of astragalus and notoginseng extracts, were shown to markedly stimulate CAT1 expression, l-arginine absorption and protective effects against intestinal colitis in in vivo and in vitro model systems. It should be noted that most studies focused on astragalus or ginseng saponins alone for their effect on increased l-arginine uptake and intestinal protection, but not on the combination of these two saponins (Lee et al., 2017; Guo et al, 2019; Di Cesare Mannelli et al, 2017; Wen et al, 2014; Xu et al., 2018). However, previous study reported that the combined use of Astragaloside IV and ginsenoside Rg1 can synergistically inhibit the ROS-induced autophagic injury in human cells (Huang et al, 2017). Similarly, the combination of ginseng and astragalus extracts was shown to synergistically promote the activation of mouse splenic lymphocytes (Wang et al., 2018). Thus, the combination of these two saponins in APS is very likely can provide a synergistic or accumulative effect on improving l-arginine absorption and gut healthy, though further investigation is needed.

l-Arginine, a conditionally essential amino acid, is involved in protein synthesis and many other cellular functions, including immune response, hormone secretion, wound healing, and cardiovascular maintenance (Albaugh et al., 2017; Morris, 2009; Morris, 2016; Pernow and Jung, 2013). Given its vital roles in whole-body homeostasis, l-arginine has drawn considerable research interest and practical applications. In addition to maintaining homeostasis, l-arginine has been very popular among athletes and bodybuilders because it is required for muscular metabolism and maintaining the nitrogen balance (Helms et al., 2014; Campbell et al., 2004). Moreover, l-arginine has been shown to facilitate maintenance of weight control, as it helps to increase muscle mass while reducing body fat (Paddon-Jones et al., 2004). Thus, dietary l-arginine supplementation or facilitation of intestinal l-arginine absorption may be an effective method for increasing blood l-arginine levels. Although l-arginine can be transported by a variety of amino acid transport systems, it is transported primarily by CAT1 and CAT2 (Closs et al., 2004). CAT1 is the sole precursor for NO biosynthesis and is constitutively expressed in most tissues, whereas CAT2 is expressed mostly in the liver, muscle, and macrophages (Closs et al., 1993). In addition, documents indicates that CAT1 plays a crucial role on l-arginine import in erythrocytes and renal tubular epithelial cells (Shima et al., 2006; Schwartz et al., 2006). These studies indicated that the blood l-arginine level is associated with the expression level of the l-arginine transporter CAT1 in intestinal epithelial cells (Yuan et al., 2015).

IBD is a chronic gastrointestinal inflammatory disorder with a complex pathogenesis process. As current therapeutic strategies for IBD still carry issues including potential side effects, costs, and immunogenicity, novel and effective therapies remain to be explored (Kozuch and Hanauer, 2008). Previous studies have indicated that the integrity of the intestinal epithelial barrier plays a role in IBD progression (Porras et al., 2006). Recent studies have indicated that increased l-arginine absorption is an important factor for the restoration of colonic epithelial cells (Liu et al., 2017; Yuan et al., 2015). This is also confirmed by evidence that dietary l-arginine supplementation can suppress intestinal permeability and improve the gut response to inflammatory injury in IBD (Iacucci and Ghosh, 2011). The intestinal protective effects of l-arginine were found to be associated with multiple mechanisms. l-arginine is known to play important physiological roles in increasing NO production, polyamine, creatine biosynthesis, and these pathways are critical of intestinal function and protection (Morris, 2016). In addition, l-arginine is shown to enhance antioxidant capacity through improving mitochondria function and ATP production (Zhang et al., 2021). Furthermore, l-arginine is involved in regulating the activation of various signaling pathways, including focal adhesion kinase (FAK) and AMP-activated protein kinase (AMPK) (Rhoads et al, 2004; Xia et al, 2019). Our results demonstrate that APS effectively reduces intestinal inflammation and restores intestinal epithelial integrity in TNBS-induced colitis. Thus, APS may serve as a therapeutic option that promises better management of IBD.

The small intestine is the primary site for amino acid absorption, and the amount of l-arginine absorbed in the gut is dependent on the amount of cationic amino acid transporter protein. A previous study indicated that once l-arginine is absorbed in human subjects, 38% of dietary l-arginine is removed in the first pass within the splanchnic bed for its own metabolism (Castillo et al., 1993). In the present study, APS was shown to significantly increase l-arginine absorption in human intestinal Caco-2 cells compared with the untreated control cells (Fig. 1). APS was also shown to increase the blood level of l-arginine by 16.1 % and 30.7 % in normal and TNBS-induced-colitis rats, respectively (Fig. 3). Moreover, in our human study, APS was found to significantly increase the blood plasma l-arginine level (AUC) by 10.5 % when the subjects took 5 g of dietary l-arginine combined with 50 mg of APS (Fig. 4). Considering the complexity of intestinal absorption and first-pass metabolism in l-arginine absorption (Wu, 1998; Morris, 2016), the above-mentioned increase in l-arginine AUC level of 10.5 % in the APS group may not reflect the real value in the human body. To optimize the amount of l-arginine absorbed in the human body system, 38 % of the absorbed l-arginine consumed during first-pass metabolism should be counted. Thus, the collective amount of the APS-mediated increase in l-arginine absorption in healthy human subjects was calculated to be 16.9 %, including the 10.5 % in the bloodstream and 6.4 % used by the splanchnic bed. Previous reports indicated that l-arginine absorption and first-pass metabolism are similar in rats and human (Wu, 1998; Windmueller and Spaeth, 1976). The effect of APS on l-arginine absorption is similarly calculated, if the 38 % first-pass fraction was applied to the rat model, the total amount of l-arginine absorbed would be 26.0 % and 49.4 % for normal and colitis rats, respectively (supplementary information). These results demonstrate that APS displays promising efficacy in enhancing l-arginine absorption, and the effect is even more pronounced in weakened subjects, such as those in the colitis rat group. In addition to be the building blocks of protein, growing evidence indicates that amino acids display multiple physiological functions. Along with l-arginine, many studies showed that glutamine plays a critical role in maintenance of intestinal integrity, promote enterocyte growth, as well as protect against intestinal injury or inflammatory disorders (Wang, 2015). Furthermore, recent studies revealed that some other amino acids, including glycine, cysteine, proline, and branched-chained amino acids (BCAAs) also exhibits prominent metabolic functions in supporting intestinal healthy (Liu, 2017; Zhou, 2018). These findings provide evidence that dietary supply of these functional amino acids are crucial to animal intestinal and whole body health. Thus, the natural nutraceutical product such as APS deserves further investigated on its efficacy in facilitating gastrointestinal uptake of these amino acids in animals and human.

In conclusion, the beneficial effects of APS on dietary l-arginine absorption and intestinal protection were investigated. The pharmacokinetics of dietary l-arginine flow from intestinal absorption to blood circulation was evaluated. The results demonstrate that APS increases l-arginine absorption, reduces intestinal inflammation, and protects intestinal damage in colitis rats. As overly processed foods, stress, and medication all contribute to degrees of compromised gut function, even in healthy people (Abraham and Cho, 2009), APS could be used as a potential dietary supplement not only for increasing the absorption of l-arginine, but also for optimizing intestinal function. Although the detailed mechanism of APS still warrants further investigation, our findings illustrate the promising therapeutic potential of APS in IBD.

Acknowledgements  This work was supported by grants from the Ministry of National Defense (MAB-109-055, MAB-110-118, and MND-MAB-111092 to T-C.C.) and Cheng-Hsin General Hospital (CH-NDMC-106-15 and CH-NDMC-107-2 to T-C. C.), Taipei, Taiwan, ROC. We are also grateful to Nuliv Science, Inc., for generous assistance in this work.

Conflict of interest  There are no conflicts of interest to declare.

Author Contributions  C-H. O., I-C T., S-F. H., Y-C. S., W-L. C. and T-C. C. participated in the concept of the study and the experimental design. Y-T. K. and W-L. C. isolated and characterized the APS. C-H. O., I-C T., S-F. H., and Y-C. S. were involved in laboratory experiments and data analysis. T-C. C. wrote the manuscript.

Supplementary Materials  The online version of this article contains Supplementary Materials.

Astragalus membranaceus and Panax notoginseng saponins improves intestinal arginine absorption and protects against intestinal disorder in vivo

Supplementary information

The small intestine is the primary site for amino acids absorption. Previously, Ten Half, et al. indicated a 90% absorption efficiency for amino acids in human intestine ¹⁾. They also show that virtually all dietary glutamine, glutamate and aspartate and arterial blood glutamine are used as major fuels for small intestinal mucosa and are responsible for providing energy required for intestinal ATP-dependent metabolic processes such as active nutrient transport and high rates of intracellular protein turnover ¹⁾. According to Wu, et al., as much as 50% of amino acids are used by the gut for protein synthesis ²⁾. The amount of arginine absorbed in the gut is dependent on the amount of cationic amino acid transporter 1 (CAT1) protein. Once arginine is absorbed, a portion of that is used by the splanchnic bed for its own metabolism. Castillo, et al. found that in adult humans, 38% of dietary arginine is removed in the first pass within the splanchnic bed ³⁾.

In the present study, we showed that APS significantly increase the arginine absorption in human intestinal Caco-2 cell (Figure 1A). APS has also been shown to increase the blood level of arginine by 16.1% and 30.7% in normal and TNBS-induced-colitis rats, respectively (Figure 3). In human subjects, APS was found to significantly increase the blood plasma arginine level (AUC) by 10.5% when the subjects took 50 mg of APS (Figure 4). Considering 38% of the absorbed arginine was consumed in first-pass metabolism ³⁾, the collective amount of the APS-mediated increased arginine absorption in healthy adults would be 16.9%. Similarly, if the 38% first pass fraction be applied to rat model, the total arginine absorbed should be 26.0% and 49.4% for normal and colitis rats, respectively. These results demonstrated that APS displays promising efficacy in enhancing arginine absorption and the effect is even more pronounced in weakened subjects such as in the colitis rat group.

Based on the published documents ^1–3) and results shown in this study, we calculate the absorption efficiency of arginine in human intestine, with assumption that the data obtained from human intestinal Caco-2 cells can be applied to the human system. The absorption efficiency in human subjects was deduced from the following processes: based on the increased 10.5% of blood arginine AUC of APS group as shown in Figure 4, which represent the fraction of absorbed arginine entering into blood circulation; that is, 100%–38% = 62%. Accordingly, the APS-mediated increase in total systemic bioavailability of arginine (first pass use of arginine by the splanchnic bed plus arginine in blood plasma) would be (110.5% - 100%)/62% = 16.9%. Thus, it is 116.9% for total systemic bioavailability in APS group administered with 5 g dietary arginine, which also means that a maximum of 5 g arginine is absorbed. The arginine absorbed in subjects of placebo group with 100% of systemic bioavailability administered with 5 g arginine is 5/116.9% = 4.28 g. Consequently, the arginine absorption in normal subjects in placebo group is 4.28 g/5 g x 100% = 85.6%, which is very close to the previous published value of 90% ¹⁾.

From abovementioned calculations, we find the arginine absorption efficiency to be 85.6% in human intestine. Thus, in placebo group, 4.28 g of arginine will be absorbed by the gut after a 5 g oral intake of arginine. As 38% of dietary arginine was extracted in first-pass metabolism in adult men with arginine-rich diet ³⁾, 1.63 g of this 4.28 g should be used up by the gut for its own metabolism, and 2.65 g will appear in the blood plasma. In APS group, we showed APS-mediated 66.7% increase in arginine absorption in the in vitro study; thus, the absorption efficiency is well over 100% when 5 g of arginine is taken together with 50 mg of APS. Out of this 5 g, 1.9 g is used up by the gut for its own metabolism, and 3.1 g will appear in blood plasma. The results are summarized in table 1.

Table 1. Absorption efficiency, gut first pass utilization, blood level, and systemic bio-availability of arginine in human after oral administration of 5 g arginine without (placebo) and with APS.

	Placebo	APS
Arginine Oral intake	5 g	5 g
Arginine absorption (85.6%)	4.28 g	5 g (4.28 g × 1.667 = 7.1 g)
Gut first pass utilization (38%)	1.63 g	1.9 g
Blood level (62%)	2.65 g	3.1 g
Total systemic bio-availability	100%	116.9% (5 g/4.28 g)

In conclusion, APS showed in this human study to increase the absorption rate of arginine in otherwise healthy human gut by 16.9% comparing to the same tested subjects when they did not take APS. The 16.9% consists of the 10.5% in blood level and 6.4% consumed in first pass metabolism. The results presented here is an attempt to piece together the distribution of arginine from ingestion to blood plasma. It presented the scenario on how the absorption efficiency of arginine may be affected by physiological condition of individuals and the concept of total systemic bioavailability and not blood plasma only as customary used. Total systemic bioavailability is the total benefit a person would receive from taking arginine supplementation. Further studies need be performed to -verify the validity of the results presented in this work.

References for Supplementary information

1. Ten Have, G. A., Engelen, M. P., Luiking, Y. C., and Deutz, N. E. (2007) Absorption kinetics of amino acids, peptides, and intact proteins. International journal of sport nutrition and exercise metabolism 17 Suppl, S23–36

2. Wu, G. (1998) Intestinal mucosal amino acid catabolism. The Journal of nutrition 128, 1249–1252

3. Castillo, L., Chapman, T. E., Sanchez, M., Yu, Y. M., Burke, J. F., Ajami, A. M., Vogt, J., and Young, V. R. (1993) Plasma arginine and citrulline kinetics in adults given adequate and arginine-free diets. Proceedings of the National Academy of Sciences of the United States of America 90, 7749–7753

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