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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
Qualifier:
no guideline followed
Principles of method if other than guideline:
Neohesperidin was determined by UHPLC-MS/MS in rat plasma after a single administration of Poncirus trifoliata extract to 6 rats.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The powdered P. trifoliata fruits (100 g) were extracted by refluxing with water (1 : 15, w/v) for 2 h.2 The aqueous extract was filtered, concentrated under reduced pressure by using a rotary evaporation apparatus, and lyophilized to give a powder (15.6 g). The powder was suspended in 0.5% CMC–Na solution to get a concentration equivalent to 0.156 g/ml of P. trifoliata extract.
- Purity: Neohesperidin, NE: 0.604 mg/mL.
- Hesperidin, HE: 1.61 mg/mL
- Hesperidin Methyl chalcone, HMC: 0.378 mg/mL.
- Poncirin, PO: 2.06 mg/mL.
- Phellopterin, PH: 0.196 mg/mL.
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Laboratory Animal Center of China Medical University (Shenyang, China).
- Weight at study initiation: 230 ± 20 g

ENVIRONMENTAL CONDITIONS
- All animals were kept in conformity with the European Community guidelines for the use of experimental animals (86/609/EEC).
Route of administration:
oral: unspecified
Vehicle:
other: 0.5% CMC–Na solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: lyophylized p. trifoliata extract was suspended in 0.5% CMC–Na solution to get a concentration equivalent to 0.156 g/mL of extract (0.604 mg/mL neohesperidin).
Duration and frequency of treatment / exposure:
single dose.
Dose / conc.:
6.04 mg/kg bw/day (nominal)
Remarks:
Neohesperidin in plant extract. Dose of P. trifoliata extract of 1.56 g/kg (equivalent to 16.1, 6.04, 3.78, 20.6, and 1.96 mg/kg of HE, NE, HMC, PO, and PH, respectively).
No. of animals per sex per dose:
6 males
Control animals:
no
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, plasma.
- Time and frequency of sampling: Serial blood samples (approximately 0.3 mL) were collected through the oculi chorioideae vein in heparinized tubes at 0, 5, 15, and 30 min, and then at 1, 1.5, 2, 3, 5, 8, 12, and 24 h.
- Other: Plasma was separated by centrifugation (2500 x g, 10 min), and stored at -20 ºC until analysis.

ANALYTICAL METHOD:
- Separation method: UHPLC. UHPLC system (Dionex UltiMate 3000) with a cooling autosampler and column oven and a TSQ Quantum Ultra mass spectrometer (Thermo Scientific, San Jose, USA) with an electrospray ionization (ESI) interface.
- Conditions: Agilent XDB C18 column (100 x 2.1 mm and 1.8 mm). The mobile phase consisted of water containing 0.1% formic acid (solvent A) and acetonitrile (solvent B). The gradient elution program
was set as follows: 20 / 45% B (v/v) at 0–5.0 min; 45 / 90% B at 5.0–5.5 min, 90% B at 5.5–6.5 min; 90 / 20% B at 6.5–7.0 min; 20% B at 7.0–10.0 min. The flow rate was set at 0.35 mL/min and injection volume was 2 mL using a partial loop mode for sample injection. The temperatures of the column and autosampler were maintained at 30 and 10ºC, respectively.
- Detection method: MS/MS. Mass spectrometric detection was performed using an ESI source via polarity switching between positive and negative (for NE) ionization mode; m/z 609.2 / 301.0 was used as quantifier for NE.
- Limits of detection and quantification: LOQ = 2.0 ng/ml for NE.
- Extraction recovery (indicate if results are corrected or not for recoveries): 88.4 - 93.9%. Results not corrected.
- Validation: Method validation was performed according to the guidelines set by the United States Food and Drug Administration (FDA) for bioanalytical method validation. Linear range for NE was 2.0 - 250 ng/ml, r = 0.9962, accuracy 99.5%, RSD = 2.5%.
Type:
absorption
Results:
quick absorption, detection in plasma 5 min after oral administration.
Details on absorption:
These compounds were quickly absorbed into the body and were detected 5 min after oral administration.
Key result
Test no.:
#1
Toxicokinetic parameters:
Tmax: 1.18 ± 0.57 h
Key result
Test no.:
#1
Toxicokinetic parameters:
Cmax: 19.1 ± 3.6 ng/mL
Key result
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 4.17 ± 1.04 h
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 79.2 μg/(L·h)
Remarks:
to the last measurable concentration
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 94.8 μg/(L·h)
Remarks:
to infinity
Metabolites identified:
not measured
Conclusions:
The substance was quickly absorbed and found in plasma 5 minutes after oral administration, reaching a maximum plasma concentration of 19.1 ± 3.6 ng/mL, the half-life of elimination (T1/2) was 4.17 ± 1.04 h.
Executive summary:

A pharmacokinetic study was performed after a single oral administration of Poncirus trifoliata extract to 6 male Sprague-Dawley rats, containing 0.604 mg/mL of neohesperidin, which resulted in a dose of 6.04 mg/kg bw of neohesperidin. Neohesperidin content was determined in rat plasma by means of a validated UHPLC-MS/MS method. The substance was quickly absorbed and found in plasma 5 minutes after oral administration. The relevant toxicokinetic parameters obtained were the following: The time of maximum plasma concentration (Tmax) and maximum plasma concentration (Cmax) were 1.18 ± 0.57 h and 19.1 ± 3.6 ng/mL, the half-life of elimination (T1/2) was 4.17 ± 1.04 h, and the area under the curve to the last measurable concentration (AUC0t) and to infinity (AUC) were 79.2 and 94.8 mg/Lh.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The metabolism of the test item by human intestinal microflora was studied by incubating the test item for 48h at 37ºC with 5 bacterial strains isolated from human fecal microflora and analysing the resulting metabolites by UPLC-Q-TOF analytical method, and verifying the metabolites identity by MS/MS.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Jiangsu Institute for Food and Drug Control (Nanjing, China).
- Purity: > 98%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid: The standard solution of neohesperidin was prepared by dissolving neohesperidin that was weighed accurately in 80 % methanol to give a final concentration of 5.0 mg/mL. The solution was stored in refrigeration at 4ºC before analysis.


Radiolabelling:
no
Species:
other: human fecal microflora.
Strain:
other: Clostridium sp.8, Bacteroides sp.15, Bacillus sp.46, and Enterobacter sp.41-1 and sp.73.
Details on test animals and environmental conditions:
COLLECTION AND ISOLATION OF HUMAN INTESTINAL BACTERIA
- Source: Fresh human fecal samples were obtained from a healthy female volunteer who was without a history of intestinal diseases or metabolic diseases and had not taken any medicine and avoided alcohol in 3 months before fecal collection.
- Sample preparation: 4.0 g fecal sample was weighted and thoroughly suspended in a centrifuge tube covered with 20 mL aseptic physiological saline, then was homogenized adequately by a vortex-mixer for 2 min. The mixture was centrifuged at 1,000xg for 5 min and the suspension was used as human intestinal bacterial mixture. The bacterial mixture was diluted tenfold serially in sterile water and inoculated on non-selective and selective agars. Aerobic agar plates were incubated for 24 h at 37ºC and anaerobic plates were incubated for 48 h at 37ºC in anaerobic jars.
- Isolation of bacteria: From the highest dilution containing equolforming bacteria, serial dilutions were repeatedly prepared until a pure bacterial culture was obtained. Following incubation, different colony types were counted, isolated in pure culture and identified to genus level. All isolates were analyzed according to Gram-stain appearance and colony morphology followed by biochemical tests.
Details on exposure:
INCUBATION
Intestinal bacterial mixture and different pure bacterial colonies were inoculated into 1.0 mL GAM broth which contained neohesperidin with a final concentration of 0.1 mmol/L, respectively. The media were anaerobically incubated at 37 ºC for 48 h. Both the preparation and the incubation of microbial cultures were carried out under strict anoxic conditions. Two control samples were run in parallel.
Duration and frequency of treatment / exposure:
48 h
Dose / conc.:
0.1 other: mmol/L
Details on study design:
After termination of the incubation, the incubated solution was extracted with 3 mL ethyl acetate by mixing for 2 min on a vortex-mixer for three times. The supernatant were removed and dried at 50ºC. After that, the residues were dissolved in 0.3 mL chromatographic pure MeOH, centrifuged at 13,0009g for 10 min at 4ºC, the obtained supernatant were used for being analyzed by UPLC-Q-TOF/MS.
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: human fecal microflora
- Time and frequency of sampling: 48h
- From how many animals: 1 person
- Method type(s) for identification: UPLC-Q-TOF/MS.

ANALYTICAL METHOD
- Apparatus: Acquity UPLC system (Waters Corp., Milford, MA, USA), consisting of a quaternary solvent delivery system, an on-line degasser, an autosampler, a column temperature controller, and a Waters PDA detector.
- Conditions: Syncronis C18 column (100x2.1 mm i.d., 1.7 μm; Thermo, USA). The column was eluted with a gradient mobile phase which consisted of acetonitrile (solvent system A) and 0.1 % aqueous formic acid (V/V) (solvent system B). Gradients were programmed as follows: 10–35 % A at 0–5.5 min; 35–90 % A at 5.5–9 min; 9–10 min, held at 90 % A for 1 min; 11–12 min, 10 % A for equilibration of the column. The column temperature was maintained at 35ºC, the autosampler temperature was set at 4ºC. The flow rate was 0.3 mL/min and the injection volume was 5 μL.
- Detector: Synapt mass spectrometer (Waters Corp., Milford, MA, USA). Full scan mass spectra were acquired in the range of m/z 100–1,000.
Statistics:
Post-acquisition analyses were performed using a MetaboLynxTM (v4.1) program (Waters Corp., Milford, MA, USA), which employs an extensive list of potential biotransformation reactions (e.g., hydroxylation, methylation), in combination with the elemental compositions of the substrate molecules, to generate a series of extracted ion chromatograms (XICs). These XICs are compared between the control and sample to eliminate those chromatographic peaks in the sample that also appear in the control. Finally, post-acquisition data analysis employing MetabolynxTM was finished and the results file contained information of metabolites that existed only in the test samples.
Metabolites identified:
yes
Details on metabolites:
5 metabolites were detected, the results indicated that hydrolysis, dehydroxylation, demethylation and acetylation were the major metabolism of neohesperidin: M2 = naringin, M3 = dehydroxylated neohesperidin, M4 = acetylated neohesperidin, M5 = dehydroxylated and hydrolysed neohesperidin, M6 = hesperetin.

Table 1. Metabolites (M1-M7) of neohesperidin identified in the samples of human intestinal bacteria.

Metabolite

Identification

Bacteria

Mixed

Sp. 8

Sp. 15

Sp. 41-1

Sp. 46

Sp. 73

M1 (Parent)

Neohesperidin

X

 

 

 

 

 

M2 (M1-CH2-O)

Naringin

 

 

 

 

X

 

M3 (M1-O)

dehydroxylated

neohesperidin

 

X

 

 

 

 

M4 (M1 + C2H2O)

acetylated

neohesperidin

X

 

 

X

 

 

M5 (M1-rha)

dehydroxylated

+ hydrolyzed

neohesperidin

 

 

 

 

 

 

M6 (M1-rha-glu)

hesperetin

X

 

X

 

 

X

 

Conclusions:
Five metabolites were detected: naringin, dehydroxylated neohesperidin, acetylated neohesperidin, deglycosylated + dehydroxylated neohesperidin, and hesperetin. The results indicated that hydrolysis, dehydroxylation, demethylation and acetylation were the major steps in the metabolism of the test item.
Executive summary:

The metabolism of the test item by human intestinal microflora was studied by incubating the test item with 5 bacterial strains isolated from human fecal microflora of a healthy donor and analysing the resulting metabolites by UPLC-Q-TOF, and verifying the metabolites identity by MS/MS. Clostridium sp.8, Bacteroides sp.15, Bacillus sp.46, and Enterobacter sp.41-1 and sp.73 were incubated with the test item for 48h at 37ºC, under strict anoxic conditions. A rapid and simple liquid–liquid extraction method was used for sample pretreatment. A highly sensitive and specific ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry was developed for the analysis. The components in the extract were identified and confirmed according to the mass spectrometric fragmention mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of five metabolites were detected: naringin, dehydroxylated neohesperidin, acetylated neohesperidin, deglycosylated + dehydroxylated neohesperidin, and hesperetin. The results indicated that hydrolysis, dehydroxylation, demethylation and acetylation were the major steps in the metabolism of the test item.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
Qualifier:
no guideline followed
Principles of method if other than guideline:
Neohesperidin was determined by UFLC-MS/MS in rat plasma after a single administration of Zhi-Zi-Da-Huang extract to normal and cholestatic liver injury rats.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Zhi-Zi-Da-Huang decoction (ZZDHD) is composed of four crude drugs: Gardenia jasminoides Ellis, Rheum palmatum L., Citrus aurantium L. and Sojae Semen Praeparatum. Gardenia jasminoides Ellis, Rheum palmatum L., Citrus aurantium L. and Sojae Semen Praeparatum were purchased from the Tianyitang TCM shop (Shenyang, China) and authenticated by Associate Professor Jiuzhi Yuan (Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China).
- Purity: ZZDHD at a dose of 8 g/kg (crude drug/body weight) was equivalent to 35.9 mg/kg geniposide, 11.4 mg/kg genipin gentiobioside, 2.50 mg/kg rhein, 0.23 mg/kg emodin, 11.2 mg/kg isonaringin, 83.5 mg/kg naringin, 12.4 mg/kg hesperidin and 79.6 mg/kg neohesperidin.
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China).
- Weight at study initiation: 220–250 g
- Diet: standard laboratory food.
- Water: tap water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 40-60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):
- The experiment was carried out in accordance with the Guidelines for Animal Experimentation of Shenyang Pharmaceutical University, and the protocol was approved by the Animal Ethics Committee of the institution (no further data).
Route of administration:
oral: unspecified
Vehicle:
other: 0.5% CMC–Na solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Duration and frequency of treatment / exposure:
single dose.
Dose / conc.:
79.6 mg/kg bw/day (nominal)
Remarks:
of neohesperidin (in 8 g/kg ZZDHD extract).
No. of animals per sex per dose:
6 male rats
Control animals:
no
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma.
- Time and frequency of sampling: Blood samples (approximately 0.3 mL) were collected from the fosse orbital vein using heparinized 1.5 mL polythene tubes before the dose and at 0.08,
0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36 and 48 h after dosing, and then centrifuged immediately at 13,000 rpm for 5 min. Plasma was transferred into clean tubes and stored at −80 ◦C until analysis.
- Other:

ANALYTICAL METHOD:
- Separation method: UFLC. The UFLC-MS/MS system consisted of a XR LC-20AD ProminenceTM UFLC system equipped with a binary pump, a degasser, an autosampler, a thermostatted column compartment (Shimadzu, Japan) and a 4000 QTRAPTM MS/MS system equipped with a turbo ion spray source (AB Sciex, Foster city, California, USA).
- Conditions: Shim-pack XRODS C18 column (75 mm × 3.0 mm, 2.2 μm) (Shimadzu, Japan), preceded by a high pressure column pre-filter (2 μm). The mobile phase consisted of water (containing 0.1% formic acid) (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. The gradient elution program was as follows: 0–10 min, 10%B–30%B; 10–12 min, 30%B–60%B; 12–16 min, 60%–80%B, and the re-equilibration time of gradient elution was 2 min. The temperature of column and autosampler were maintained at 30 ◦C and 4 ◦C, respectively. The injection volume was 5 μL.
(Shimadzu, Japan).
- Detection method: MS/MS. Quantification was performed using multiple reaction monitoring (MRM) in the negative ionization mode.
- Limits of detection and quantification: LOQ = 2.0 ng/mL.
- Extraction recovery (indicate if results are corrected or not for recoveries): . Results not corrected.
- Validation: For neohesperidin, the linear range was 2.0 - 1000 ng/mL, r = 0.9975, RSD = 4.5%, RE = -2.1%
Statistics:
Non-compartmental pharmacokinetic parameters were calculated by DAS 2.1 pharmacokinetic program (Chinese Pharmacological Society). Significant difference between normal and CLI groups was determined using SPSS 16.0 (Statistical Package for the Social Science) by independent sample t-tests after natural logarithmic transformation for AUC0–t, AUC0–∞, Cmax and by the nonparametric Mann-Whitney test for Tmax, t1/2, MRT0–t and CL. A p value less than 0.05 was considered statistically significant.
Details on absorption:
The plasma concentration–time curves of the four flavonoid glycosides (isonaringin, naringin, hesperidin and neohesperidin) showed multiple peaks, which demonstrated that an
enterohepatic circulation may exist.
Details on excretion:
excretion of most drugs in vivo was mainly by bile and urine.
Key result
Test no.:
#1
Toxicokinetic parameters:
Tmax: 1.10 ± 0.42 h
Key result
Test no.:
#1
Toxicokinetic parameters:
Cmax: 91.8 ± 35.6 ng/mL
Key result
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 2.74 ± 0.99 h
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 201.2 ± 21.4 ng·h/mL
Remarks:
AUC(0–t)
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 222.8 ± 30.6 ng·h/mL
Remarks:
AUC(0–∞)
Test no.:
#1
Toxicokinetic parameters:
other: MRT0–t (ng h/mL)
Remarks:
3.55 ± 0.69
Metabolites identified:
not measured
Conclusions:
The relevant toxicokinetic parameters obtained were the following: The time of maximum plasma concentration (Tmax) was 1.10h, the maximum plasma concentration (Cmax) was 91.8 ng/mL, the half-life of elimination (T1/2) was 2.74 h, and the area under the curve to the last measurable concentration (AUC,0–t) and to infinity (AUC,0–∞) were 201.2 and 222.8 ng·h/mL.
Executive summary:

A pharmacokinetic study was performed after a single oral administration of Zi-Da-Huang decoction to 6 male Sprague-Dawley rats, at a dose of 8 g/kg, which resulted in a dose of 79.6 mg/kg bw of neohesperidin. Neohesperidin content was determined in rat plasma by means of a validated UFLC-MS/MS method. The relevant toxicokinetic parameters obtained were the following: The time of maximum plasma concentration (Tmax) was 1.10h, the maximum plasma concentration (Cmax) was 91.8 ng/mL, the half-life of elimination (T1/2) was 2.74 h, and the area under the curve to the last measurable concentration (AUC0t) and to infinity (AUC0–∞) were 201.2 and 222.8 ng·h/mL.

Description of key information

Weight of evidence:

Adsorption: A study on the absorption of the test item in Poncirus trifoliata extract was performed by Wang et al. (2016) in male Wistar rats. The test item was quickly absorbed into the body, being found in plasma 5 min after administration. The plasma concentration also declined quickly, with a half-life of 4.17h. Another study on the pharmacokinetics of the test item in Zi-Da-Huang decoction, performed in Sprague-Dawley rats by Zhu et al. (2014), also shows rapid adsorption (Tmax = 1.1h) and rapid elimination from plasma (T1/2 = 2.7 h).

Metabolism: An in vitro study on the metabolism of the test item by multiple bacteria isolated from human intestinal microflora was performed by Zhang et al. (2014). Five metabolites were detected by UPLC-Q-TOF and confirmed by MS/MS: naringin, dehydroxylated neohesperidin, acetylated neohesperidin, deglycosylated + dehydroxylated neohesperidin, and hesperetin. The results indicated that hydrolysis, dehydroxylation, demethylation and acetylation were the major steps taking place in the intestine.

Neohesperidin is rapidly absorbed in the bacterially colonized segments of the gastrointestinal tract after bacterial hydrolysis, yielding the aglycone hesperetin, which can be then further degraded by the microflora or conjugate with glucuronic acid or sulphate, to be excreted. Based on the available information, it can be concluded that there is no bioaccumulation potential for neohesperidin.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information