Suitability evaluation on material specifications and edible methods of Dendrobii Officinalis Caulis based on holistic polysaccharide marker.

BACKGROUND: Dendrobii Officinalis Caulis (DC) is a well-known tonic herbal medicine worldwide and has favorable immunomodulatory activity. Various material specifications of DC are available in herbal markets, and DC is ingested by different edible methods. However, whether these specifications and edible methods are suitable or not remains unknown. METHODS: In this study, we evaluated the suitability of four material specifications (fresh stem, dried stem, fengdou and powder) and three edible methods (making tea, soup and medicinal liquor) based on holistic polysaccharide marker (HPM), the major polysaccharide components in DC. First, the HPMs were extracted from the four specifications of DC by the three edible methods in different conditions. Second, qualitative and quantitative characterization of the extracted HPMs was performed using high performance gel permeation chromatography (HPGPC). Third, immunomodulatory activities of the extracted HPMs were evaluated in vivo. RESULTS: The results showed that the HPMs were found to be quantitatively different from various specification of DC and edible methods. In vivo analysis indicated that the HPMs exerted positive effects on innate immune responses by increment in proliferation of splenocytes, secretion of IL-2 and cytotoxicity activity of NK cells. Moreover, the dosage amount of HPM should be defined as a certain range, but not the larger the better, for exerting strong immunological activities. CONCLUSION: According to the both chemical and biological results, fengdou by boiling with water for 4 h is the most recommended specification and edible method for DC.

A novel ultra-performance liquid chromatography hyphenated with quadrupole time of flight mass spectrometry method for rapid estimation of total toxic retronecine-type of pyrrolizidine alkaloids in herbs without requiring corresponding standards.

Nearly 50% of naturally-occurring pyrrolizidine alkaloids (PAs) are hepatotoxic, and the majority of hepatotoxic PAs are retronecine-type PAs (RET-PAs). However, quantitative measurement of PAs in herbs/foodstuffs is often difficult because most of reference PAs are unavailable. In this study, a rapid, selective, and sensitive UHPLC-QTOF-MS method was developed for the estimation of RET-PAs in herbs without requiring corresponding standards. This method is based on our previously established characteristic and diagnostic mass fragmentation patterns and the use of retrorsine for calibration. The use of a single RET-PA (i.e. retrorsine) for construction of calibration was based on high similarities with no significant differences demonstrated by the calibration curves constructed by peak areas of extract ion chromatograms of fragment ion at m/z 120.0813 or 138.0919 versus concentrations of five representative RET-PAs. The developed method was successfully applied to measure a total content of toxic RET-PAs of diversified structures in fifteen potential PA-containing herbs.

Proteomic study of pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome in rats.

Pyrrolizidine alkaloids (PAs) are a group of phytotoxins that can induce human liver injury, particularly hepatic sinusoidal obstruction syndrome (HSOS). To date, the molecular targets of PA-induced HSOS are largely unknown. In this study, retrorsine (RTS), a known hepatotoxic PA, was used as a representative PA for proteomic studies. Toxicological assessment demonstrated that 35 mg/kg RTS (designated as RTS-L) caused early lesions of HSOS at 24 h after dosing. A proteomic approach revealed 17 up-regulated and 31 down-regulated proteins in RTS-L-treated rats. Subsequently, bioinformatic analysis suggested that two proteins, carbamoyl-phosphate synthase (CPS1) (p < 0.05) and ATP synthase subunit beta (ATP5B) (p < 0.01) were associated with RTS-L intoxication. Using immunohistochemical staining, we further verified the down-regulation of CPS1 and ATP5B in RTS-L-treated rats. These findings indicated that CPS1 and ATP5B were altered in the RTS-induced early lesions of HSOS in rats, and therefore, these two proteins and their involved pathways might play important roles in the initiation of HSOS. To the best of our knowledge, our study using a proteomic approach combined with conventional toxicological assessment is the first systems toxicology study on PA-induced HSOS. The results of this study provide novel findings on protein profiles in response to PA exposure, which can serve as a starting point to further investigate potential protein targets and their interactions with PAs to induce HSOS.

The Presystemic Interplay between Gut Microbiota and Orally Administered Calycosin-7-O-ß-D-Glucoside.

Presystemic interactions with gut microbiota might play important roles in the holistic action of herbal medicines in their traditional oral applications. However, research interests usually focus on biologic activities of the in vivo available herb-derived components and their exposure in circulation. In this study, we illustrated the importance of studying the presystemic interplay with gut microbiota for understanding the holistic actions of medicinal herbs by using calycosin-7-O-ß-D-glucoside (C7G), the most abundant flavonoid and chemical marker in Astragali Radix, as a model compound. When C7G was orally administrated to rats, calycosin-3'-O-glucuronide (G2) was the major circulating component in the blood together with a minor calycosin but not C7G. Rat gut microbiota hydrolyzed C7G in vitro rapidly and produced its aglycone calycosin. Calycosin exhibited higher permeability than C7G and further underwent extensive glucuronidation to yield 3'-glucuronide as the dominant metabolite. Bioactivity assays revealed that G2 exhibited similar or more potent proangiogenic effects than calycosin in human umbilical vein endothelial cells in vitro and in the vascular endothelial growth factor receptor tyrosine kinase inhibitor II-induced blood vessel loss model in zebrafish. More interestingly, the incubation of C7G with gut microbiota from both normal and colitic rats showed a probiotics-like effect through stimulating the growth of the beneficial bacteria Lactobacillus and Bifidobacterium. In conclusion, C7G interacts reciprocally with gut microbiota after oral dosing, which makes it not only an angiogenic prodrug but also a modulator of gut microbiota.

Regioselective glucuronidation of the isoflavone calycosin by human liver microsomes and recombinant human UDP-glucuronosyltransferases.

Hepatic conjugation plays important roles in systemic exposure and drug interactions of flavonoids. In the present study, the hepatic metabolism of calycosin, a major isoflavone from Astragali Radix, was characterized and the regioselectivity in the predominant glucuronidation pathway was first delineated in human liver microsomes (HLMs) and a panel of recombinant human UDP-glucuronosyltransferases (UGTs). Calycosin underwent major glucuronidation and minor oxidation and sulfation in human liver subcellular fractions. The major glucuronide (G2) of calycosin was isolated and identified as calycosin 3'-glucuronide by NMR analysis, and thus, the minor glucuronide (G1) was tentatively assigned as calycosin 7-glucuronide. The formations of both glucuronides in HLMs fit typical Michaelis-Menten kinetics. HLMs exhibited higher affinity (Km, G2 12.37±1.20 µM vs G1 40.90±5.51 µM) and velocity (Vmax, G2 5.39±0.13 nmol/min/mg protein vs G1 2.80±0.13 nmol/min/mg protein) on G2 formation, leading to the intrinsic clearance of calycosin via 3'-glucuronidation 6 times that through 7-glucuronidation. UGT1A1, 1A3 and 1A10 showed activities on both 3'-OH and 7-OH, whereas UGT1A7, 1A8, 1A9, and 2B7 were only capable of catalyzing 3'-OH glucuronidation of calycosin. Among them, UGT1A9 exhibited the highest activity (Clint, 2169.50 µL/min/mg protein) for 3'-glucuronide formation followed by UGT1A7 (Clint, 396.38 µL/min/mg protein). UGT1A1 showed the highest activity towards 7-OH glucuronidation (Clint, 224.34 µL/min/mg protein), which was comparable to its activity on 3'-OH glucuronidation (Clint, 203.82 µL/min/mg protein). Propofol (UGT1A9 inhibitor) produced a complete inhibition of 3'-glucuronide formation accompanied by an increase of 7-glucuronide in HLMs, while bilirubin (UGT1A1 inhibitor) only partially (∼60%) inhibited the 7-OH glucuronidation. These findings demonstrated the regioselective glucuronidation at the 3'-OH of the isoflavone calycosin in HLMs and shed light on potential drug interactions of calycosin with other UGT1A9 substrates.

Biotransformation of ginsenoside Rb1 via the gypenoside pathway by human gut bacteria.

BACKGROUND: Bacterial conversion of ginsenosides is crucial for the health-promoting effects of ginsenosides. Previous studies on the biotransformation of ginsenoside Rb1 (Rb1) by gut bacteria have focused on the ginsenoside Rd (Rd) pathway (Rb1 → Rd → ginsenoside F2 (F2) → compound K (Cpd K)). This study aims to examine the gypenoside pathway in human gut bacteria in vitro. METHODS: The metabolic pathways of ginsenoside Rb1 and its metabolites ginsenoside Rd and gypenoside XVII in human gut bacteria were investigated by incubating the compounds anaerobically with pooled or individual gut bacteria samples from healthy volunteers. Ginsenoside Rb1, the metabolites generated by human gut bacteria, and degraded products in simulated gastric fluid (SGF) were qualitatively analyzed using an LC/MSD Trap system in the negative ion mode and quantitatively determined by HPLC-UV analysis. RESULTS: When incubated anaerobically with pooled gut bacteria, Rb1 generated five metabolites, namely Rd, F2, Cpd K, and the rare gypenosides XVII (G-XVII) and LXXV (G-LXXV). The gypenoside pathway (Rb1 → G-XVII → G-LXXV → Cpd K) was rapid, intermediate, and minor, and finally converted Rb1 to Cpd K via G-XVII → F2 (major)/G-LXXV (minor). Both the Rd and gypenoside pathways exhibited great inter-individual variations in age-and sex-independent manners (P > 0.05). Rb1 was highly acid-labile and degraded rapidly to form F2, ginsenoside Rg3, ginsenoside Rh2, and Cpd K, but did not generate the gypenosides in SGF. The formation of the gypenosides might be explained by the involvement of a gut bacteria-mediated enzymatic process. CONCLUSIONS: Rb1 was metabolized to G-XVII, F2 (major) or G-LXXL (minor), and finally Cpd K by human gut bacteria in vitro.

Pharmacokinetic evidence on the contribution of intestinal bacterial conversion to beneficial effects of astragaloside IV, a marker compound of astragali radix, in traditional oral use of the herb.

Astragaloside IV (AIV) is the most abundant saponin and a marker compound in Astragali Radix, a Chinese herb notable for its anti-aging and immune-enhancing effects. The present study investigated the role of intestinal bacterial conversion in the in vivo fate of AIV administered through a traditional oral route for the first time. When incubated anaerobically with rat intestinal bacteria, AIV generated five metabolites with three [monoglycosides brachyoside B and cyclogaleginoside B, the aglycone cycloastragenol (CA)] via stepwise deglycosylation and two from further epimerization (CA-iso) and dehydrogenation (CA-2H). Hydrolytic removal of C-6 glucose was a rate-limiting step for formations of CA and its derivatives. When AIV was orally administered to the rat, CA and CA-iso presented as the main components in plasma following AIV, and the AUC(0-∞) were 88.60 ± 9.66 (CA), 179.06 ± 28.53 (CA-iso) and 452.28 ± 43.33 nM·h (AIV). CA-2H was the predominant form in feces but was not detected in urine or plasma. This agreed well with in vitro data including rapid hepatic metabolism of CA-2H to form CA and CA-iso and reversible conversions between CA-2H and CA/CA-iso by intestinal bacteria. These findings support a crucial role of gut bacterial conversion of AIV in the traditional application of Astragali herb and warrant further investigational emphasis on CA and CA-iso.

In vitro pharmacokinetic characterization of mulberroside A, the main polyhydroxylated stilbene in mulberry (Morus alba L.), and its bacterial metabolite oxyresveratrol in traditional oral use.

Mulberroside A (MulA) is one of the main bioactive constituents in mulberry (Morus alba L.). This study examined the determining factors for previously reported oral pharmacokinetic profiles of MulA and its bacterial metabolite oxyresveratrol (OXY) on in vitro models. When incubated anaerobically with intestinal bacteria, MulA underwent rapid deglycosylation and generated two monoglucosides and its aglycone OXY sequentially. MulA exhibited a poor permeability and predominantly traversed Caco-2 cells via passive diffusion; yet, the permeation of OXY across Caco-2 cells was much more rapid and involved efflux (both p-glycoprotein and MRPs)-mediated mechanisms. Moreover, OXY underwent extensive hepatic glucuronidation; yet, the parent MulA was kept intact in liver subcellular preparations. There was insignificant species difference in intestinal bacterial conversion of MulA and the extent of OXY hepatic glucuronidation between humans and rats, while OXY exhibited a distinct positional preference of glucuronidation in the two species. Overall, these findings revealed a key role of intestinal bacterial conversion in absorption and systemic exposure of MulA and its resultant bacterial metabolite OXY in oral route in humans and rats and warranted further investigational emphasis on OXY and its hepatic metabolites for understanding the benefits of mulberry.

Characterization of metabolism and in vitro permeability study of notoginsenoside R1 from Radix notoginseng.

As a main and characteristic constituent in Radix notoginseng, the fate of notoginsenoside R1 (NGR1) in human is largely unknown. The present study investigated, for the first time, NGR1 metabolism by human intestinal bacteria and liver subcellular fractions, and permeability properties of NGR1 and resultant metabolites on a Caco-2 model. Samples were qualitatively analyzed using HPLC-MS/MS and quantitatively determined using HPLC-UV. When incubated with pooled human intestinal bacteria anaerobically, NGR1 showed biphasic elimination: an insignificant decrease in the first 8 h followed by a rapid elimination during 8-48 h. Four metabolites, three unambiguously identified as ginsenosides Rg1, F1 and 20(S)-protopanaxatriol formed via stepwise deglycosylation, and one tentatively assigned as a dehydrogenated protopanaxatriol with transformation occurring at the tetracyclic triterpenoid skeleton, were produced sequentially. Rg1 and F1 were formed transiently at low apparent velocities, while 20(S)-protopanaxatriol was the major metabolite with a formation rate close to the rate of NGR1 elimination and a low elimination rate. NGR1 remained intact in human liver S9 or microsomes over 1 h. Transport study of NGR1 and its metabolites revealed an ascending permeability order with stepwise deglycosylation. Taken together, the results revealed a determinant role of intestinal bacteria in the overall disposition and potential bioactivity of NGR1 in human.