DMD # 61473dmd.aspetjournals.org/content/dmd/early/2015/02/24/... · DMD # 61473 - 2 - Running Title: Systemic Exposure to Catechols from i.v. DanHong Injection Address correspondence

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Systemic Exposure to and Disposition of Catechols

Derived from Salvia miltiorrhiza Roots (Danshen)

after Intravenous Dosing DanHong Injection in

Human Subjects, Rats, and Dogs

Meijuan Li, Fengqing Wang, Yühong Huang, Feifei Du, Chenchun

Zhong, Olajide E. Olaleye, Weiwei Jia, Yanfen Li, Fang Xu, Jiajia

Dong, Jian Li, Justin B. R. Lim, Buchang Zhao, Lifu Jia, Li Li, and

Chuan Li

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,

China (M.L., F.W., F.D., C.Z., O.E.O., W.J., F.X., J.D., J.L., J.B.R.L., L.L., C.L.);

University of Chinese Academy of Sciences, Shanghai, China (M.L., L.L., C.L.);

Second Affiliated Hospital, Tianjin University of Traditional Chinese Medicines, Tianjin,

China (Y.H. Y.L.); Buchang Pharmacy Group, Xi’an, Shaanxi Province, China (B.Z.,

L.J.); Institute of Chinese Materia Medica, China Academy of Chinese Medical

Sciences, Beijing, China (C.L.)

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on February 10, 2015 as DOI: 10.1124/dmd.114.061473

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Running Title: Systemic Exposure to Catechols from i.v. DanHong Injection

Address correspondence to: Drs. Chuan Li and Li Li, Laboratory for DMPK

Research of Herbal Medicines, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai 201203,

China. E-mail: [email protected] (C. Li); [email protected] (L. Li)

Number of Text Pages: 26

Number of Tables: 2

Number of Figures: 6

Number of References: 40

Number of Words in Abstract Section: 250

Number of Words in Introduction Section: 738

Number of Words in Discussion Section: 1532

ABBREVIATIONS: ALDH, aldehyde dehydrogenase; AUC0-∞, area under plasma

concentration-time curve to infinity; COMT, catechol-O-methyltransferase; Cum.Ae-B,0-8h,

cumulative amount excreted into bile collected 0–24 h after dosing started; Cum.Ae-U,0-24h,

cumulative amount excreted into urine collected 0–24 h after dosing started; fe-B, fraction of

dose excreted into bile; fe-U, fraction of dose excreted into urine; PK, pharmacokinetic; SAH,

S-adenosylhomocysteine; SAM, S-adenosylmethionine; SULT, sulfotransferase; t1/2,

elimination half-life; TOF-MS, time-of-flight mass spectrometer; UGT, uridine

5’-diphospho-glucuronosyltransferase.


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ABSTRACT

DanHong injection is a Danshen (Salvia miltiorrhiza roots)-based injectable solution

for treatment of coronary artery disease and ischemic stroke. Danshen catechols are

believed to be responsible for the injection’s therapeutic effects. This study aimed to

characterize systemic exposure to and elimination of Danshen catechols in human

subjects, rats, and dogs receiving intravenous DanHong injection. A total of 28

catechols were detected, with content levels of 0.002–7.066 mM in the injection, and

the major compounds included tanshinol, protocatechuic aldehyde, salvianolic acid B,

rosmarinic acid, salvianolic acids A and D, and lithospermic acid with their daily doses

≥10 μmol/subject. After dosing, tanshinol, salvianolic acid D, and lithospermic acid

exhibited considerable exposure in human subjects and rats. However, only tanshinol

had considerable exposure in dogs. The considerable exposure to tanshinol was due

to its having the highest dose, while that to salvianolic acid D and lithospermic acid

was due to their relatively long elimination half-lives in the human subjects and rats.

Protocatechuic aldehyde and rosmarinic acid circulated in the bloodstream

predominantly as metabolites; salvianolic acids A and B exhibited low plasma levels

with their human plasma metabolites little or not detected. Tanshinol and salvianolic

acid D were eliminated mainly via renal excretion. Elimination of other catechols

involved hepatobiliary and/or renal excretion of their metabolites. Methylation was

found to be the primary metabolism for most Danshen catechols and showed

intercompound and interspecies differences in rate and degree in vitro. The

information gained here is relevant to pharmacological and toxicological research on

DanHong injection.


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Introduction

Traditionally, herbal medicines are orally administered. However, there are 134 types of

herbal injections that are approved by the China Food and Drug Administration (China FDA)

for clinical use. The herbal injections are prepared from a single herb or from a mixture of

several herbs and contain multiple bioactive compounds. The Chinese pharmaceutical

industry is required to manufacture herbal injection products in compliance with Good

Manufacturing Practice. Unlike orally administered herbal medicines, herbal injections are

injected directly into the bloodstream and potentially have immediate effects. Many approved

herbal injections are extensively used in Chinese hospitals for treating cardiovascular or

infectious diseases and for alleviating related symptoms; these herbal products appear to

exhibit comparable incidence of side effects with synthetic drug injections. However, active

compounds responsible for the therapeutic effects have remained poorly defined for most

herbal injections. This hampers investigating the mechanisms of action, optimizing the herbal

therapies, and assessing the safety and associated potential herb-drug interactions. Bioactive

herbal compounds that exhibit considerable levels of body exposure after administration are

most likely to account for the pharmacological effects of an herbal injection and form the

basis of therapeutic efficacy. Accordingly, pharmacokinetic (PK) studies should be designed

for herbal injections and implemented in human subjects and laboratory animals to reveal

body exposure to the herbal compounds after dosing. The body exposure to herbal compounds

depends on the doses of the compounds from the administered injection and their

pharmacokinetics and disposition after dosing. In the first step of PK studies of an herbal

injection, three questions should be answered: (i) what chemical compounds from the herbal

injection are injected into the bloodstream and how much are they dosed? (ii) Which herbal

compounds and herb-related metabolites exhibit considerable systemic exposure after dosing

and how long are their elimination half-lives? (iii) How are the herbal compounds that are


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considerably dosed from the injection principally eliminated? The first step of PK studies

provides information on which compounds derived from the herbal injection are worth further

investigation with respect to pharmacokinetics, pharmacology, and toxicology. The follow-up

PK studies should focus on these important herbal substances and should be carried out to

define their detailed pharmacokinetics and associated interindividual variations, to

characterize their principal routes of elimination, to quantify the contribution of enzymes and

transporters to their pharmacokinetics and disposition, and to assess associated potential

herb-drug and herb-herb interactions. Here, the aforementioned "first-step" PK studies were

designed for DanHong injection and implemented in human subjects, rats, and dogs.

DanHong injection is prepared from a 3:1 mixture of Salvia miltiorrhiza roots (Danshen

in Chinese) and Carthamus tinctorius flowers (Honghua). It is an injectable solution available

as a sterile, nonpyrogenic parenteral dosage form for intravenous injection. This herbal

injection is approved by the China FDA for the treatment of atherosclerotic coronary artery

disease and acute ischemic stroke. Several reports on meta-analyses of randomized trials

show that DanHong injection is effective in treating angina pectoris, congestive heart failure

and acute ischemic stroke (Peng et al., 2010; Peng et al., 2011; Yang et al., 2012). DanHong

injection contains multiple water-soluble polyphenols of Danshen-origin (Liu et al., 2013; Li

et al., 2013; Xie et al., 2014). The Danshen polyphenols contain one or more ortho-dihydroxy

benzene rings, known as catechols; have one or more carboxylic acid functional groups,

except for protocatechuic aldehyde. They are caffeic acid derivatives, occurring as monomers

(such as tanshinol and caffeic acid), dimers (such as rosmarinic acid and salvianolic acid D),

trimers (such as salvianolic acid A and lithospermic acid) and tetramers (such as salvianolic

acid B) (Jiang et al., 2005; Li et al., 2009). Cell- and isolated-tissue-based studies have shown

that Danshen catechols exhibited vasodilating, endothelial protective, cardioprotective,

antithrombotic, antioxidant, and antiinflammatory properties; tanshinol and other Danshen


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catechols also decreased blood pressure in rats and attenuated venular thrombosis, cardiac

hypertrophy, and methionine-induced hyperhomocysteinemia in rats (Jiang et al., 2005; Han

et al., 2008; Cao et al., 2009; Ho and Hong, 2011; Wang et al., 2013). Despite increasing

knowledge of the chemistry and pharmacology of DanHong injection, the systemic exposure

to and disposition of its bioactive compounds after dosing remain to be elucidated. This report

presents the Danshen-derived chemical profile of DanHong injection, the systemic exposure

to and elimination of Danshen catechols. It also presents comparative in vitro metabolic

stability of Danshen catechols towards various metabolic reactions and their in vitro

methylation profiles. The information gained here will be relevant to the optimization of

DanHong injection-based therapy.


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Materials and methods

DanHong injection. DanHong injection was manufactured by Heze Buchang

Pharmaceutical Co., Ltd. (Heze, Shandong Prov., China) with a China FDA ratification no. of

GuoYaoZhunZi-Z20026866. Each milliliter of DanHong injection was prepared from 0.75 g

of Salvia miltiorrhiza roots (Danshen) and 0.25 g of Carthamus tinctorius flowers (Honghua).

To prepare the herbal injection, Danshen and Honghua were mixed and extracted with water.

After filtration and concentration under reduced pressure, the concentrated extract was

precipitated with ethanol and centrifuged. The resulting supernatant was treated with active

carbon, evaporated under reduced pressure to remove the ethanol, adjusted to pH 6.5–7.5,

ultrafiltered, and sterilized. The finished product of DanHong injection was standardized to

contain not less than 0.35 mg/ml concentration of tanshinol and protocatechuic aldehyde

combined. Samples of six DanHong injection lots (lot numbers: 13011037, 13072016,

13082026, 13092035, 13102030, and 13122024) were obtained for analysis and all the

products were manufactured in 2013. DanHong injection that had the lot number 13011037

was used in the current human and animal studies.

Chemicals and reagents. Analytical reference standards of caffeic acid, lithospermic

acid, protocatechuic acid, protocatechuic aldehyde, rosmarinic acid, salvianolic acids A, B, C,

and D, tanshinol, chrysin, (−)-epicatechin, 7-hydroxyflavone, isovanillin, isovanillin acid and

vanillic acid were purchased from Tauto Biotech (Shanghai, China). The purity of the

preceding compounds was ≥98%. Pentobarbital was obtained from Shanghai Westang

Biotechnology (Shanghai, China).

S-adenosylmethionine (SAM), uridine 5′-diphospho-glucuronic acid (UDPGA),

3′-phosphoadenosine-5′-phosphosulfate (PAPS), tris-hydroxymethyl-aminomethane,

nicotinamide adenine dinucleotide (NAD+), taurocholic acid, formic acid, and methanol were

obtained from Sigma-Aldrich (St. Louis, MO, USA). Rat liver cytosol and microsomes and


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dog liver cytosol and microsomes were prepared in-house by differential centrifugation.

Human liver cytosol and microsomes were obtained from Corning Gentest (Woburn, MA,

USA). (−)-Epicatechin was used as the positive control to assess the

catechol-O-methyltransferase (COMT) activities of the human, rat, and dog liver cytosols

(Zhu et al., 2000; Li et al., 2012), and the in vitro t1/2 values were 0.46, 0.08, and 0.21 h,

respectively. 7-Hydroxyflavone was used as the positive control to assess the sulfotransferase

(SULT) activities of the human, rat, and dog liver cytosols (Li et al., 2012; Hu et al., 2013),

and the in vitro t1/2 values were 0.07, 0.29, and 0.10 h, respectively. Chrysin was used as the

positive control to assess the UDP-glycosyltransferase (UGT) activities of the human, rat, and

dog liver microsomes (Li et al., 2012; Hu et al., 2013), and the in vitro t1/2 values were 0.08,

0.05, 0.05 h, respectively. Isovanillin was used as the positive control to assess the aldehyde

dehydrogenase (ALDH) activities of the human, rat, and dog liver cytosols (Panoutssopoulos

et al., 2004), and the in vitro t1/2 values were 0.04, 0.05, and 0.05 h, respectively.

Human study. The protocol for human study was approved by the ethics committee of

clinical investigation at Second Affiliated Hospital of Tianjin University of Traditional

Chinese Medicine (Tianjin, China). The study was registered in Chinese Clinical Trials

Registry (www.chictr.org) with a registration number of ChiCTR-ONRC-13003571. Healthy

volunteers gave written informed consent to participate in the study. The human subjects were

considered to be in good health on the basis of medical history, physical examination, vital

signs measurement, electrocardiogram, and clinical laboratory tests. They were required to be

aged between 18 and 35 years, have a body mass index between 19 and 24 kg/m2, be a

nonsmoker, and not be allergic to products prepared from Danshen and/or Honghua. Synthetic

drugs and herbal products were prohibited starting from 2 weeks before the study began until

the end of the study period. In addition, alcoholic beverages were prohibited starting from 2

days before the study began until the end of the study period.


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A single-period, open-label human study was performed at a national clinical research

center of the Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine

(Tianjin, China). Six male subjects were asked to fast overnight before receiving a 120 min i.v.

infusion dose of DanHong injection (40 ml/person, a label daily dose) the next morning.

Before dosing, 40 ml of DanHong injection was diluted in 250 ml of 5% glucose injection

(GuoYaoZhunZi-H12020021; China Otsuka Pharmaceutical Co., Ltd., Tianjin, China). Serial

blood samples (~1 ml, collected in heparinized tubes) were taken from an antecubital vein

catheter at 0, 0.25, 0.5, 1, 2, 2.17, 2.5, 3.5, 5, 11, and 24 h after the i.v. infusion was started.

The blood samples were heparinized and centrifuged to obtain plasma fractions. 0.1 ml

aliquots of the resulting plasma samples of the same time point were pooled and stored at

−70°C until analysis. Urine samples were collected predose and at 0–2, 2–6, 6–10, and 10–24

h postdose and weighed. 1 ml aliquots of the urine samples within the same time interval were

pooled prior to storage at −70°C without use of any preservative. Serum alanine

aminotransferase, aspartate aminotransferase, total protein, albumin/globulin ratio, total

bilirubin, and direct bilirubin were monitored as liver function markers for the human subjects

before and 24 h after dosing of DanHong injection; serum creatinine and blood urea nitrogen

were also assessed to monitor human renal function.

Laboratory animals. All animal studies were conducted in compliance with the

Guidance for Ethical Treatment of Laboratory Animals (the Ministry of Science and

Technology of China, 2006). The experimental protocols were approved by the Institutional

Animal Care and Use Committee at Shanghai Institute of Materia Medica (Shanghai, China).

Male Sprague-Dawley rats (230–270 g) were obtained from Sino-British SIPPR/BK

Laboratory Animal (Shanghai, China). Rats were maintained in a unidirectional airflow room

at 20–24°C and relative humidity between 30% and 70% with a 12 h light/dark cycle. Rats

were given filtered tap water and commercial rat chow ad libitum and allowed to acclimate to


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the facilities and environment for three days before use. Rats received in-house

femoral-artery-cannulation for blood sampling or bile-duct-cannulation for bile sampling

(Chen et al., 2013; Cheng et al., 2013). After surgery, rats were housed individually and

allowed to regain their preoperative body weights before further use. During the bile

collection period, a sodium taurocholate solution (pH 7.4) was infused into the duodena of

bile-duct-cannulated rats at 1.5 ml/h. After use, rats were euthanized with CO2.

Male beagle dogs (7.8–9.0 kg) were obtained from the Teaching and Research Farm of

Agricultural College, Shanghai Jiao Tong University (Shanghai, China) and were individually

housed in stainless steel cages (1.7 × 1.0 × 0.9 m), which could be used to collect urine

samples. The dogs were maintained at a controlled temperature of 20–24°C, relative humidity

between 40% and 70%, and a 12-h cycle of light and dark. They were given commercial diets,

except for an overnight fasting period before dosing, and filtered tap water ad libitum. The

dogs were acclimated to the facilities for 2 weeks before use.

Rat studies. Conscious and freely moving rats received a single i.v. bolus dose of

DanHong injection at 4 ml/kg. The dose of the herbal injection for the rats was derived from

the clinical daily dose (40 ml/person) according to dose normalization by body surface area

(Reagan-Shaw et al., 2008). Serial blood samples (~150 µl; immediately before and 5, 15, and

30 min and 1, 2, 4, 6, 8, 11, and 24 h after dosing) were collected from three

femoral-artery-cannulated rats into heparinized tubes. The blood samples were centrifuged to

obtain the plasma fractions and the resulting plasma samples (0.05 ml) of the same time point

were pooled. Bile samples were collected from three bile-duct-cannulated rats before and 0–1,

1–2, 2–4, 4–6, and 6–8 h after dosing. Three rats that did not undergo any surgical treatment

were housed individually in metabolic cages. Urine samples were collected before and 0–4,

4–8, and 8–24 h after dosing; samples (0.5 ml) of the same collection period from different

rats were pooled and stored at −70°C until analysis.


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Dog study. Dogs received a single i.v. bolus dose of DanHong injection at 1.2 ml/kg.

The dog dose was derived from the clinical daily dose of the herbal injection (40 ml/person)

according to dose normalization by body surface area (Reagan-Shaw et al., 2008). Serial

blood samples (~500 µl; immediately before and 5, 15, 30 min and 1, 2, 4, 6, 8, 11, and 24 h

after dosing) were collected from three dogs into heparinized tubes. The blood samples were

centrifuged to obtain the plasma fractions and the resulting plasma samples (0.1 ml) of the

same time point were pooled. Urine samples were also collected from the dogs before and 0–4,

4–8, and 8–24 h after dosing. The urine samples (0.5 ml) from the same collection period

were pooled. The samples from dogs were stored at −70°C until analysis.

In vitro metabolism studies. Comparative metabolic stability of several Danshen

catechols toward COMT, SULT, UGT, and ALDH (ALDH for protocatechuic aldehyde only)

was assessed with human, rat, and dog liver cytosols and microsomes. The test Danshen

catechol compounds were tanshinol, protocatechuic aldehyde, protocatechuic acid, salvianolic

acid B, rosmarinic acid, salvianolic acids A and D, and lithospermic acid. Before use, the

COMT, SULT, and ALDH activities of the liver cytosols were assessed with the reference

compounds (−)-epicatechin, 7-hydroxyflavone, and isovanillin, respectively; the UGT

activities of liver microsomes was assessed with chrysin. The liver cytosol was fortified with

an appropriate cofactor (SAM for methylation, PAPS for sulfation, and NAD+ for oxidation)

and the liver microsomes were fortified with UDPGA for glucuronidation. The reaction was

initiated by adding the test compound (10 μM) at 37ºC and terminated at 0, 15 (for rat

enzyme-mediated reactions only), 30 (for dog enzyme-mediated reactions only), and 60 min

(for human enzyme-mediated reactions only) by adding a double volume of ice-cold methanol.

The time of incubation for tanshinol was double that for the other Danshen catechol

compounds. Negative control reactions were carried out by incubating mixtures that excluded

the cytosol or the microsomes. The quenched incubation was centrifuged at 1369g for 10 min


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and 5 μl of the supernatant were analyzed by liquid chromatography/mass spectrometry.

Metabolic stability was calculated as percentage of unchanged compound remaining after

incubation. When determining the metabolic stability of protocatechuic aldehyde toward

COMT and SULT present in human liver cytosol, isovanillin (100 μM) was used to inhibit the

catalytic activity of the coexisting ALDH.

In vitro methylation was further examined individually for the preceding Danshen

catechol compounds by incubating with human, rat, and dog liver cytosols fortified with

COMT. The incubation broth contained 0.5 mg protein/ml liver cytosol, 50 mM Tris-HCl

buffer (pH 7.4), 1 mM dithiothreitol, 2 mM MgCl2, and 1 mM SAM and was preincubated for

3 min at 37ºC before initiation of reaction by adding the test compound (10 μM). After

incubation for 0, 5, 15, 30, 60, 90, and 120 min at 37ºC, the reactions were terminated by

adding a double volume of ice-cold methanol. After centrifugation, the supernatant (5 μl) was

analyzed to profile unchanged substrate and methylated metabolites. In the determination of

in vitro t1/2, the concentrations of the test Danshen catechol in the incubation broth over time

were calculated as percentage of compound remaining, using the initial concentration (the

incubation time, 0 min) as 100%. The slope of the linear regression from log percentage

remaining versus incubation time relationship, i.e., −k, was used in the calculation of in vitro

t1/2, according to the following equation:

In vitro t1/2 = 0.693/k (1)

Analysis of Danshen catechol compounds and the metabolites. A Waters Synapt G2

high definition time-of-flight mass spectrometer (TOF-MS; Manchester, UK) was interfaced

via a Zspray/LockSpray ESI source with a Waters Acquity UPLC separation module (Milford,

MA, USA). The mass spectrometer was operated in resolution mode giving a resolving power

of around 20000. The ESI source worked in the negative ion mode under the following

conditions: capillary voltage at −2.5 kV, source temperature of 120°C, desolvation gas at 800


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L/h and 400°C, sampling cone at 25 V, and extraction cone at 4.0 V. The mass spectrometer

was externally calibrated over a range of m/z 50–1000 using a 5 mM sodium formate solution

at 20 μL/min and mass shifts during acquisition were corrected using leucine enkephalin (m/z

554.2615) as a lockmass. MSE data acquisition (in centroid mode, m/z 50–1000) was achieved

using a trap collision energy of 4 V, a trap collision energy ramp of 15–30 V simultaneously

and a scan time of 0.3 s. Chromatographic separation of Danshen catechol compounds in

DanHong injection was achieved on a Waters Acquity UPLC HSS T3 1.8-μm column (100 ×

2.1 mm i.d.; Dublin, Ireland; kept at 45°C) using a mobile phase that consisted of solvent A

(methanol/water, 1:99, v/v, containing 25 mM formic acid) and solvent B (methanol/water,

99:1, v/v, containing 25 mM formic acid). The mobile phase was delivered at 0.3 ml/min. A

gradient program was used as follows: 0–2 min, at 2% solvent B; 2–32 min, from 2% to 98%

solvent B; 32–37 min, at 98% solvent B; and 37–42 min, at 2% solvent B. For quantitative

analysis of Danshen compounds, a modified gradient elution program with a mobile phase

flow rate of 0.3 ml/min was used as follows: 0–10 min, from 2% to 70% solvent B; 10–12

min, from 70% to 98% solvent B; and 12–15 min, at 2% solvent B. Sample preparation for

human, rat, and dog samples was performed using methanol as precipitant with a volumetric

precipitant-to-sample ratio of 3:1. After precipitation and centrifugation, the supernatants

were reduced to dryness by centrifugal evaporation under reduced pressure. The residues were

reconstituted in 50% methanol for LC/TOF-MSE-based analysis.

Profiling of Danshen compounds in the samples of DanHong injection was conducted.

The detected compounds were identified by comparing with the reference standards, with

respect to accurate molecular mass, fragmentation, and chromatographic retention time. When

the reference standard was not available, the compound identification was achieved by

comparing the compound’s accurate molecular mass, fragmentation, and elution order with

the reported data of Danshen compounds. To support the metabolite profiling, an Accelrys


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metabolite database (version 2012.1; Accelrys, Inc., San Diego, CA, USA) was used to get

prior knowledge of likely metabolic pathways of Danshen compounds. Metabolite profiling

was conducted with the human, rat, and dog samples according to the molecular mass gains or

losses predicted for the possible metabolites compared with those of the parent compounds

and according to the generation of fragment ions from the precursor deprotonated molecules

by collision-induced dissociation. The MS fragmentation data were also used to assign the

metabolite structures. Methylated metabolites were characterized by the comparative

fragmentation profiles of metabolite and parent compound. Glucuronides and sulfates were

characterized by neutral losses of 176 and 80 Da, respectively. Quantification of Danshen

catechols and the metabolites in the biological samples was performed using the available

reference standards for calibration or using the calibration curve of an analog (also called

“brother compound”) that bore close structural similarity to the analyte (Li et al., 2012).

Using the reference standards, matrix-matched calibration curves (13.7–10000 nM) were

constructed using weighted linear regression of the peak area against the corresponding

nominal analyte concentration (nM), which showed good linearity (r2 >0.99). Assay

validation was carried out according to the US FDA guidance on bioanalytical validation

(2013) to prove that the bioanalytical assays were reliable for the intended applications.

PK data analysis. The area under plasma concentration-time curve up to the last

measured time point (AUC0-t) was calculated by the trapezoidal rule. The AUC0-∞ was

generated by extrapolating the AUC0-t to infinity using the elimination rate constant (k) and

the last measured concentration. The elimination half-life (t1/2) was calculated using the

relationship 0.693/k. The cumulative amounts excreted into urine (Cum.Ae-U) and bile

(Cum.Ae-B) were calculated by numeric integration of the amount excreted per collection

interval. The fractions of dose excreted into urine (fe-U) and bile (fe-B) were established by

Cum.Ae-U/dose and Cum.Ae-B/dose, respectively.


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Results

Danshen catechols present in DanHong injection. Three literature references by Liu et

al. (2007), Wang et al. (2007), and Li et al. (2009) give the most comprehensive information

about chemical constituents present in Danshen. Among the Danshen constituents,

hydrophilic catechols and lipophilic diterpene quinones are pharmacologically relevant.

However, only the Danshen catechols, but not the diterpene quinones, were detected in

DanHong injection. This is probably because of water extraction of the herb involved in

manufacturing the injection. As shown in Table 1 and Fig. 1, a total of 28 Danshen catechol

compounds were detected in six lots of DanHong injection with tanshinol (6) present in the

highest content level (6.139–7.066 mM). Other major Danshen compounds present were

protocatechuic aldehyde (1; 1.474–1.840 mM), salvianolic acid B (26; 1.014–1.245 mM),

rosmarinic acid (10; 0.684–0.795 mM), salvianolic acids A (16; 0.563–0.886 mM) and D (13;

0.467–0.704 mM), lithospermic acid (22; 0.235–0.322 mM), salvianolic acid B isomer (25;

0.159–0.227 mM), and salvianolic acid E (27; 0.136–0.154 mM). These were major Danshen

catechols, which have LogP values of −0.28–2.69 (calculated using Pallas software, Pallas

3.7.1.1; CompuDrug International, Sedona, AZ, USA). The remaining Danshen compounds,

which were also detected in the herbal injection samples, were salvianolic acid B isomer (24),

lithospermic acid isomers (18, 19, and 20), caffeic acid (3), protocatechuic acid (2),

salviaflaside (17), salvianolic acids H/I (21), F (7), J (23), G (8), and C (15), salvianic acid C

(11) and salvianic acid C isomer (12), isosalvianolic acid C (14), prolithospermic acid (9),

isoferulic acid (5), ferulic acid (4), and 4-methoxyl salvianolic acid B (28). These minor

Danshen compounds exhibited content levels of 0.002–0.076 mM with LogP values of

0.17–3.02. Lot-to-lot variability of DanHong injection was also investigated with the major

constituents (≥0.136 mM). The herbal injection showed consistency in the content levels of 6,

1, 26, 10, and 27 from lot to lot. This was indicated by relative standard deviations (RSD) of


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4.3–9.4%. Such RSD values for 16, 13, 22, and 25 were greater, i.e., 12.0%–16.3%.

(Insert Table 1 and Figure 1 here)

DanHong injection (lot number, 13011037) was used in the current study; the content

levels of tanshinol (6), protocatechuic aldehyde (1), salvianolic acid B (26), rosmarinic acid

(10), salvianolic acid A (16), salvianolic acid D (13), and lithospermic acid (22) were 7.066,

1.833, 1.013, 0.769, 0.886, 0.704, and 0.247 mM, respectively. Clinical daily dose levels of

the Danshen catechols from the herbal injection could be divided into three classes: >100,

10–100, and <10 μmol/subject (Fig. 1C). The dose level of 6 was 282.8 μmol/subject, which

was 52.7% of the total dose of Danshen catechols detected in the injection (Fig. 1D); 6 was a

Danshen catechol of class I. The dose levels of 1, 26, 10, 16, 13, and 22 were 10.0–73.2

μmol/subject, the sum of which accounted for 40.7% of the total dose of Danshen catechols

detected in the injection; these compounds were of class II. The remaining Danshen catechols

were of class III with dose levels of 0.1–7.2 μmol/subject, the sum of which accounted for

only 6.6% of the total dose of Danshen catechols detected in the injection.

Unchanged Danshen catechols detected in plasma and urine samples of human

subjects receiving an i.v. infusion dose of DanHong injection. In the current study, pooled

human plasma samples and pooled human urine samples were used for the detection of

unchanged Danshen catechols and related metabolites. This was because the detection of

Danshen compounds depended on factors, such as assay sensitivity and interindividual

variations in the elimination mediated by drug metabolizing enzymes and/or drug transporters.

The samples were pooled to minimize the possibility of failed detection of important herbal

compounds and their metabolites due to the potential influence of interindividual variation in

their elimination. A total of 11 Danshen catechols were detected in human plasma samples

during and after i.v. infusion of DanHong injection; they were not detected in the control

plasma samples before dosing. Tanshinol (6) was detected in considerable amount in the


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plasma samples after dosing started. Although salvianolic acid D (13) and lithospermic acid

(22) were Danshen catechols of class II with content levels being only 10.0% and 3.5% of that

of 6, respectively, their levels of systemic exposure were comparable with that of 6 (Fig. 2A).

The other Danshen catechols detected in plasma, including salvianolic acids B (26) and A (16),

rosmarinic acid (10), and protocatechuic acid (2), were at low AUC0-∞ levels (Fig. 2A). The

observed high levels of systemic exposure to 13 and 22 were probably associated with their

longer t1/2 values (2.5 and 5.7 h, respectively) compared with those of 6 (0.6 h) and the other

Danshen catechols (around 0.2 h). Only trace amounts of protocatechuic aldehyde (1),

lithospermic acid isomers (19 and 20) and salvianolic acid C (15) were detected in plasma.

Plasma concentrations of 6, 13, and 22 over time during and after an i.v. infusion dose of

DanHong injection in human subjects are shown in Supplemental Fig. 1.

A considerable amount of tanshinol (6) from i.v. dosed DanHong injection was recovered

in human urine with a fe-U of 63.3%. Although the fe-U value of salvianolic acid D (13; 62.7%)

was almost the same as that of 6, its Cum.Ae-U,0-24h was only 8.6% of that of 6 (Fig. 2B). The

difference in urinary Cum.Ae-U,0-24h between the two catechols was associated mainly with the

difference in their content levels in DanHong injection; the content level of 13 was 10.0% of

that of 6 (Fig. 1B). Only 3.8% of lithospermic acid (22) from i.v. dosed DanHong injection

was excreted unchanged into the urine and its Cum.Ae-U,0-24h was quite low (Fig. 2B). Other

plasma Danshen catechols [including salvianolic acid B (26) and rosmarinic acid (10)] were

also detected in urine with fe-U values of 0.2% and 18.2%, respectively; salvianolic acid A (16)

was not detected in urine.

Metabolites of Danshen catechols detected in plasma and urine samples of human

subjects receiving the i.v. infusion dose of DanHong injection. Metabolites detected in the

human samples after dosing were those of the major Danshen catechols. As shown in Fig. 3A,

the major Danshen catechols could be divided into three categories according to their


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circulating pattern; (i) tanshinol (6), salvianolic acid D (13), and lithospermic acid (22):

circulating mainly as parent compound with minor or major metabolites, (ii) protocatechuic

aldehyde (1) and rosmarinic acid (10): circulating in the bloodstream predominantly as

metabolites with a minor parent compound, and (iii) salvianolic acids B (26) and A (16): only

minor parent compound circulating in the bloodstream with metabolites very little or not

detected. The major metabolites of Danshen catechols were the methylated metabolites and

their further sulfated and glucuronized products. Sulfates and glucuronides of Danshen

catechols were also detected in plasma after dosing. Table 2 shows LC/TOF-MSE-based

detection of the individual metabolites of Danshen catechols in human plasma and/or urine

during and after the i.v. infusion dose of DanHong injection, as well as the metabolite ID

notation system. Fig. 3A and B exhibit plasma AUC0-∞ and Cum.Ae-U,0-24h of metabolites in

comparison with the respective data of their parent Danshen catechols. Fig. 4 depicts major

metabolic pathways proposed for Danshen catechols from i.v. dosed DanHong injection in

humans. The major metabolite of 6 was M6M; this metabolite was eliminated mainly via renal

excretion. M13M was a detected metabolite of 13 and its Cum.Ae-U,0-24h was 6.6% of the

amount of 13 dosed from DanHong injection. The major metabolite of 22 was M222M, which

was poorly eliminated via renal excretion. Protocatechuic aldehyde has been reported to

convert into protocatechuic acid by ALDH (Panoutsopoulos and Beedham, 2005; Xu et al.,

2007). Consistent with this, the total Cum.Ae-U,0-24h of protocatechuic acid (2) and its

metabolites (including M2M, M2M-G-1, M2M-G-2, M2S-1, and M2S-2) after dosing exceeded the

amount of 2 dosed from DanHong injection by 28 fold. This suggested that 1 was eliminated

mainly by oxidation into 2, which was further methylated, glucuronized, and sulfated before

renal excretion of 2 and the metabolites. The major metabolites of 10 included M10M-2,

M102M, M102M-S-1, M102M-S-2, M102M-G-1, and M102M-G-2; these metabolites were eliminated

considerably via renal excretion with a total Cum.Ae-U,0-24h being 44.3% of the amount of 10


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dosed from DanHong injection. Like in the plasma samples, no metabolite of 26 was detected

in the associated urine samples. Unchanged 16 from DanHong injection was not detected in

urine; only four minor urinary metabolites (M16M-G-1, M16M-G-2, M16M-G-3, M16G-2) were

detected for 16. The total Cum.Ae-U,0-24h of these metabolites was only 1.0% of the amount of

16 dosed from DanHong injection. These data suggested that 26, 16, and their metabolites in

humans were not eliminated mainly via renal excretion.

Danshen catechols and metabolites detected in plasma, urine, and bile samples of

rats and dogs after dosing DanHong injection. For rats and dogs, samples of the same kind

and from the same collection time points/intervals were pooled to minimize the possibility of

failed detection of important herbal compounds and their metabolites; this sample pooling

treatment was similar to that for human subjects. Like for human subjects, tanshinol (6),

salvianolic acid D (13), and lithospermic acid (22) were the most abundant Danshen

compounds detected in rat plasma samples after dosing DanHong injection (Fig. 2C). In rats,

the t1/2 values of 13 and 22 (1.8 and 1.9 h, respectively) were longer than that of 6 (0.3 h).

Protocatechuic aldehyde (1), Rosmarinic acid (10), salvianolic acids B (26) and A (16) were

also detected in the rat plasma samples, but at low AUC0-∞ levels (Fig. 2C). Their t1/2 values

ranged from 0.2 h to 0.8 h.

In rats, both tanshinol (6) and salvianolic acid D (13) were eliminated mainly via renal

excretion (fe-U, 54.3% and 53.6%, respectively), rather than hepatobiliary excretion (fe-B, 0.5%

and 1.0%, respectively), after dosing DanHong injection (Fig. 2D and E). Their major

metabolites M6M, M6S, and M13M exhibited lower levels of systemic exposure than their

respective parent compounds (Fig. 3C). These metabolites were also eliminated mainly via

renal excretion (Fig. 3D and E); the total Cum.Ae-U,0-24h of M6M and M6S and Cum.Ae-U,0-24h of

M13M were 29.1% and 20.0% of the amounts of 6 and 13, respectively, dosed from DanHong

injection. Lithospermic acid (22) was not eliminated mainly via renal excretion (fe-U, 2.7%) or


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hepatobiliary excretion (fe-B, 1.2%) (Fig. 2D and E). Its metabolites M222M and M22M were

substantially detected in rat plasma and bile after dosing (Fig. 3C and E). The hepatobiliary

excretion of these metabolites were significantly greater than their renal excretion (Fig. 3D

and E); their total Cum.Ae-B,0-8h and total Cum.Ae-U,0-24h were 76.9% and 4.7%, respectively, of

the amount of 22 dosed from DanHong injection. Protocatechuic aldehyde (1) was eliminated

mainly via oxidation into protocatechuic acid (2) followed by further methylation and

sulfation; sulfation also considerably occurred in rats for 1 (Fig. 3D and E). The urinary

recovery of 2 and the follow-up metabolites (M2M-S, M2S-1, and M2S-2) in rats exceeded the

amount of 2 dosed from DanHong injection by 109 fold, which was markedly greater than

that observed in human subjects (28 fold). This was, at least in part, because the rat diet

contained a considerable amount of p-hydroxybenzoic acid ethylester. It has been reported

that p-hydroxybenzoic acid esters can be hydrolyzed and hydroxylated into protocatechuic

acid (Wang et al. 2013). Rosmarinic acid (10) was eliminated more extensively via renal

excretion (fe-U, 25.2%) than hepatobiliary excretion (fe-B, 0.12%) (Fig. 2D and E). Unlike in

the human subjects, 10 exhibited a higher level of systemic exposure than its metabolites

(M102M, M102M-S-1, M102M-S-2, M102M-G-1, and M102M-G-2) in rats (Fig. 3C). These

metabolites were eliminated via both renal and hepatobiliary excretion (Fig. 3D and E); their

total Cum.Ae-U,0-24h and total Cum.Ae-B,0-8h were 29.0% and 49.4%, respectively, of the amount

of 10 dosed from DanHong injection. Salvianolic acid B (26) was eliminated more

extensively via hepatobiliary excretion (fe-B, 8.1%) than renal excretion (fe-U, 1.0%) (Fig. 2D

and E). Its major metabolites included M26M, M262M-1, M262M-2, M263M-1, and M263M-2;

neither sulfate nor glucuronide of 26 was detected. These metabolites were eliminated mainly

via hepatobiliary excretion, rather than renal excretion (Fig. 3D and E); their total

Cum.Ae-B,0-8h and total Cum.Ae-U,0-24h were 90.1% and 0.6%, respectively, of the amount of 26

dosed from DanHong injection. Salvianolic acid A (16) was eliminated more extensively via


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hepatobiliary excretion (fe-B, 9.3%) than renal excretion (fe-U, 0.5%) (Fig. 2D and E). Unlike in

the human subjects, the metabolites M16M-G-1, M162M-G-1, M162M-G-3, and M16G-1 appeared to

have higher levels of systemic exposure than unchanged 16 in rats (Fig. 3C). In addition to

these plasma metabolites, M16M-G-2, M16M-G-3, and M162M-G-2 were considerably detected in

bile. Like the parent compound, the detected metabolites were eliminated mainly via

hepatobiliary excretion (Fig. 3D and E); their total Cum.Ae-B,0-8h was 71.8% of the amount of

16 dosed from DanHong injection. Supplemental Table 1 shows LC/TOF-MSE-based

detection of the individual metabolites of Danshen catechols in rat plasma, urine, and/or bile

after dosing DanHong injection.

In dogs, only plasma tanshinol (6) was considerably detected after dosing DanHong

injection (Fig. 2F). Unlike in the human subjects and in the rats, plasma salvianolic acid D (13)

and lithospermic acid (22) were detected at low levels in dogs after dosing. Their t1/2 values

were 0.2 h and 0.5 h, which were shorter than that of 6 (1.0 h). Salvianolic acids B (26) and A

(16) and rosmarinic acid (10) were also detected in plasma but at low levels of systemic

exposure with t1/2 of 0.2–0.3 h (Fig. 2F). Only trace amounts of protocatechuic aldehyde (1)

was detected in canine plasma. The fe-U values of 6, 13, and 22 were 34.5%, 43.6%, and 5.8%,

respectively (Fig. 2G). M6M and M6S appeared to be substantially formed in dogs (Fig. 3G);

their Cum.Ae-U,0-24h were 14.1% and 48.4%, respectively, of the amount of 6 dosed from

DanHong injection. However, their plasma AUC0-∞ values were lower than that of the parent

compound 6 (Fig. 3F). The elimination of 1 mainly involved oxidation into protocatechuic

acid (2) followed by further methylation and sulfation (Fig. 3F and G). The urinary recovery

of 2 and the follow-up metabolites (M2M and M2M-S) in dogs exceeded the amount of 2 dosed

from DanHong injection by 12 fold. Multiple metabolites of 10 were detected in plasma but at

lower levels than the parent compound (Fig. 3F); they included M10M-1, M10M-2, M102M,

M102M-S-1, and M102M-S-2. The Cum.Ae-U,0-24h of unchanged 10 and the total Cum.Ae-U,0-24h of


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its metabolites were 5.1% and 21.9%, respectively, of the amount of 10 dosed from DanHong

injection. The metabolites of 13, 22, 16, and 26 were detected at low levels in canine plasma

and urine after dosing (Fig. 3F and G). Supplemental Table 2 shows LC/TOF-MSE-based

detection of the individual metabolites of Danshen catechols in canine plasma and/or urine

after dosing DanHong injection.

Comparative metabolism of Danshen catechols mediated by hepatic COMT, SULT,

UGT, and ALDH. In vitro methylation, sulfation, and glucuronidation of the Danshen

catechols (tanshinol, protocatechuic aldehyde, protocatechuic acid, salvianolic acid B,

rosmarinic acid, salvianolic acid A, salvianolic acid D, and lithospermic acid) were compared,

with respect to percentage of compound remaining after incubation with human, rat, and dog

hepatic enzymes for a certain length of time. Among the human hepatic enzyme-mediated

metabolic reactions, the rates of methylation of the test compounds were greater than those of

associated sulfation and glucuronidation, except for protocatechuic aldehyde and salvianolic

acid A (Fig. 5A). Sulfation and glucuronidation of salvianolic acid D, lithospermic acid, and

salvianolic acid B appeared to be negligible, because the sulfates and glucuronides were not

detected after incubation. Glucuronidation and oxidation of protocatechuic aldehyde were

significantly faster than its methylation. Glucuronidation of salvianolic acid A was

significantly faster than its methylation. Similar scenarios were observed for the rat and dog

hepatic enzyme-mediated metabolic reactions, except for rat COMT-mediated methylation of

protocatechuic aldehyde comparably as fast as the ALDH-mediated oxidation (Fig. 5B and C).

The in vitro catalytic activities of the rat and dog enzymes appeared to be higher than those of

the human enzymes. Accordingly, the incubation times used for the rat and dog enzymes to

mediate metabolism reactions were shorter than those of the human enzymes, i.e., 15 min (30

min for tanshinol only), 30 min (60 min for tanshinol only), 60 min (120 min for tanshinol

only), respectively.


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The rates of human hepatic COMT-mediated methylation of the different test Danshen

catechol compounds were considerably different (Fig. 6A). This is indicated by in vitro t1/2

values, which were ranked as follows: salvianolic acid D (0.31 h) ≈ lithospermic acid (0.41 h)

≈ protocatechuic acid (0.44 h) < salvianolic acid B (0.73 h) < rosmarinic acid (1.23 h) <

salvianolic acid A (3.25 h) ≈ protocatechuic aldehyde (3.71 h) ≈ tanshinol (3.73 h). The results

of in vitro metabolite profiling of the incubations indicated that the methylation degree was

catechol-moiety-number-dependent and incubation-time-dependent. Tanshinol, protocatechuic

aldehyde, and protocatechuic acid contain only one catechol moiety; only mono-methylated

metabolites (M6M, M1M, and M2M) were detected for these Danshen catechol compounds.

Rosmarinic acid and lithospermic acid contain two unsubstituted catechol moieties. Both

monomethylated (M10M-1, M10M-2, and M22M) and dimethylated metabolites (M102M and

M222M) of rosmarinic acid and lithospermic acid were found to be predominant and the

formation of dimethylated metabolites was significantly delayed as compared with that of

monomethylated metabolites. Salvianolic acid D contains one unsubstituted catechol moiety

and one substituted catechol moiety; it was converted predominantly into a monomethylated

metabolite (M13M-1). Its dimethylated metabolite was detected at very low levels. Salvianolic

acid B contains three unsubstituted catechol moieties and salvianolic acid A contains two

unsubstituted catechol moieties and one substituted catechol moiety. Human COMT-mediated

methylation of these catechol compounds resulted in the formation of monomethylated

(M16M-1, M16M-2, and M26M) and dimethylated metabolites (M162M-1, M162M-2, M262M-1,

and M262M-2), but only trace amounts of the trimethylated metabolites were detected. The

rates of methylation mediated by the rat and the dog COMTs were significantly faster than

those by the human enzyme (Fig. 6B and C). Due to this faster methylation by the rat COMT,

trimethylated metabolites (M163M-1, M163M-2, M263M-1, and M263M-2) were significantly

detected for salvianolic acids A and B (Fig. 6B). For the dog COMT, these trimethylated


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metabolites were also considerably detected only after 120-min incubation (Fig. 6C). For the

rat hepatic COMT-mediated methylation, the in vitro t1/2 values were ranked as follows:

lithospermic acid (0.04 h) = salvianolic acid B (0.04 h) = rosmarinic acid (0.04 h) ≈

salvianolic acid D (0.05 h) < salvianolic acid A (0.09 h) ≈ protocatechuic acid (0.12 h) <

protocatechuic aldehyde (0.24 h) < tanshinol (0.94 h). For the dog hepatic COMT-mediated

methylation, the in vitro t1/2 values were ranked as follows: lithospermic acid (0.10 h) ≈

salvianolic acid D (0.13 h) < protocatechuic acid (0.25 h) ≈ rosmarinic acid (0.28 h) ≈

salvianolic acid B (0.30 h) ≈ salvianolic acid A (0.39 h) < protocatechuic aldehyde (1.36 h) <

tanshinol (1.94 h).


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Discussion

Danshen catechols are pharmacologically important for DanHong injection; they have

been reported to have multiple antianginal properties. Because systemic exposure to

compounds after dosing an herbal injection depends on their dose levels and elimination

kinetics, defining the correlations between exposure and influencing factors are important in

PK research on the injection. The analysis of DanHong injection revealed a total of 28

Danshen catechols present. Because of their different content levels, these Danshen catechols

could be divided into three classes according to their daily dose levels from the i.v. injection.

Tanshinol (6; 282.8 μmol/subject) was the only Danshen catechol of class I (>100

μmol/subject). Protocatechuic aldehyde (1), salvianolic acids B (26) and A (16), rosmarinic

acid (10), salvianolic acid D (13), and lithospermic acid (22) had dose levels of 10.0–73.2

μmol/subject; they were Danshen catechols of class II (10–100 μmol/subject). The other 21

minor Danshen catechols (0.1–7.2 μmol/subject) were of class III (<10 μmol/subject). In the

human subjects receiving DanHong injection, Danshen catechols of classes I and II were all

detected in plasma. However, only few Danshen catechols of class III, including lithospermic

acid isomers (19 and 20; 5.2 and 3.9 μmol/subject, respectively) and protocatechuic acid (2;

1.9 μmol/subject), were detected in human plasma after dosing. Similar

compound-dose-dependent detection of plasma Danshen catechols took place in rats and

dogs.

Among the detected Danshen catechols, tanshinol (6), salvianolic acid D (13), and

lithospermic acid (22) exhibited the most significant systemic exposure in the human subjects

receiving DanHong injection, suggesting that these herbal compounds are worth special

consideration in pharmacological research on DanHong injection. Unlike 6 (the only Danshen

catechol of class I), 13 and 22 were Danshen catechols of class II and exhibited content levels

in the dosed injection lower than the other catechols of the same class. The considerable


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systemic exposure to 13 and 22 appeared to be correlated with their elimination half-lives (t1/2,

2.5 and 5.7 h, respectively) significantly longer than those of 6 and the other Danshen

catechols of class II (0.2–0.6 h). Rats and dogs are laboratory animals extensively used in

cardiovascular pharmacological research. Rats receiving DanHong injection exhibited an

exposure profile of unchanged Danshen catechols similar to the human subjects; 6, 13, and 22

exhibited levels of systemic exposure higher than the other Danshen catechols. In the rats, the

t1/2 values of 13 and 22 (around 2 h) were longer than those of 6 and the other Danshen

catechols of class II (<1 h). Unlike the human subjects and the rats, dogs exhibited

considerable systemic exposure to 6 only after dosing DanHong injection. In the dogs, 13 and

22 exhibited short t1/2 (<0.5 h). The observed PK interspecies difference should be considered

in pharmacological research of DanHong injection. In PK studies of other herbal medicines,

significant differences in elimination half-life have also been found to cause considerably

different systemic exposure to herbal compounds; long elimination half-life may considerably

counterbalance influence of poor oral bioavailability on systemic exposure to an herbal

compound (Liu et al., 2009; Chen et al., 2013; Jiang et al., 2015). With rapidly increasing

application of analytical techniques, such as liquid chromatography/mass spectrometry, in

research on herbal medicines, content levels of compounds in herbal formulations and their

elimination half-lives have become more and more available in literature; such data of herbal

compounds could be used together as potential PK marker to predict their systemic exposure

after dosing herbal injections.

Besides the systemic exposure to unchanged Danshen catechols, their elimination was

also investigated in the current study. After dosing DanHong injection, tanshinol (6),

salvianolic acid D (13), and lithospermic acid (22) circulated mainly as parent compound with

minor or major metabolites. Elimination of 6 and 13 mainly involved renal excretion of the

parent compounds, while 22 was eliminated mainly via hepatic uptake and methylation


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followed by hepatobiliary excretion of the metabolites. Unlike these three catechols,

protocatechuic aldehyde (1) circulated in the blood stream predominantly as metabolites,

rather than the parent compound, and was first transformed into protocatechuic acid (2),

which was then eliminated by conversion into sulfates and methylated glucuronides followed

by renal excretion. Rosmarinic acid (10) also circulated in the blood stream predominantly as

metabolites, rather than the parent compound. This catechol was eliminated mainly via

metabolism and only about 20% of dosed 10 was eliminated via renal excretion of the parent

compound. The metabolism of 10 involved formation of dimethylated products and their

sulfates and glucuronides; these metabolites were eliminated comparably via renal excretion

and hepatobiliary excretion. Salvianolic acids A (16) and B (26) were detected at low plasma

levels and their plasma metabolites exhibited little or no detection in the human subjects.

These two Danshen catechols appeared to be eliminated mainly via hepatobiliary excretion of

glucuronized and methylated metabolites of 16 and methylated metabolites of 26. Zhang et al.

(2004) reported several hepatobiliary metabolites of salvianolic acid B in rats, including

3-monomethyl-, 3,3"-dimethyl-, 3,3'"-dimethyl-, and 3,3",3'"-trimethyl-salvianolic acid B.

The Danshen catechols have poor membrane permeability, except for protocatechuic

aldehyde (Lu et al., 2008). During elimination, these compounds tend to necessitate

transporter-mediated basolateral uptake from blood as the first step in hepatobiliary and/or

renal tubular elimination. Hepatic OATP1B3 and renal OAT1 were found to mediate cellular

uptake of Danshen catechols (the details to be published elsewhere). Because of their catechol

moieties, most Danshen catechols underwent COMT-mediated methylation as the primary

metabolic pathway in humans, rats, and dogs. Significant intercompound and interspecies

differences in rate and degree of methylation were observed for Danshen catechols. Hepatic

methylation of tanshinol by human COMT was significantly slower than those of salvianolic

acid D and lithospermic acid, demonstrating their in vitro t1/2 3.73, 0.31, and 0.41 h,


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respectively. However, the elimination t1/2 of tanshinol (6; 0.6 h) after dosing DanHong

injection in human subjects was significantly shorter than those of salvianolic acid D (13; 2.5

h) and lithospermic acid (22; 5.7 h). This is probably because the transporter-mediated hepatic

and/or renal tubular uptake from blood, rather than the methylation, was the rate-limiting step

in the elimination of these Danshen catechols. Although methylation of drugs often hampers

their pharmacological activities (Goldstein et al., 2003), the antianginal properties of the

major methylated metabolites of Danshen catechols are unknown.

Methylation of xenobiotics with catechol moieties involves COMT-catalyzed transfer of

methyl groups from SAM to the catechol hydroxyl groups in the presence of Mg2+ (Zhu et al.,

2002). This produces S-adenosylhomocysteine (SAH), which can be further converted to

homocysteine by SAH hydrolase in the liver and kidneys (to less extent). An abnormally high

blood concentration of homocysteine is recognized as a risk factor for vascular diseases and

neurodegenerative conditions (McCully et al., 2007; Vogle et al., 2009; Smith et al., 2010).

This has raised concerns regarding untoward effects of hepatic methylation of xenobiotics on

the blood concentration of homocysteine. A typical example is hyperhomocysteinemia, which

is caused by chronic levodopa treatment of patients with Parkinson’s disease (Kuhn, et al.,

1998; Müller et al., 1999; Miller et al., 2003). The Danshen catechols from i.v. DanHong

injection undergo COMT-mediated methylation in the body. Further investigation of the

relationship between methylation of Danshen catechols and hyperhomocysteinemia risk may

facilitate hazard identification for this herbal injection. This may also improve understanding

of the endothelial protection properties of Danshen catechols (Chan et al., 2004; Yang et al.,

2010; Cao et al., 2009; Zhang et al., 2013).

In summary, Danshen catechols present in DanHong injection could be divided,

according to their daily dose levels, into three classes (>100, 10–100, and <10 μmol/subject).

Tanshinol (6) was the only Danshen catechol of class I; protocatechuic aldehyde (1),


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salvianolic acids B (26) and A (16), rosmarinic acid (10), salvianolic acid D (13), and

lithospermic acid (22) were of class II; the other 21 minor Danshen catechols were of class III.

Unchanged Danshen catechols of classes I and II were all detected in plasma after i.v. dosing

DanHong injection; only three catechols of class III were detected in plasma, but at very low

levels. Only 6, 13, and 22 exhibited considerable systemic exposure in human subjects

receiving the injection. These three catechols exhibited considerable systemic exposure also

in rats after dosing, but only 6 had considerable exposure in dogs. The considerable exposure

to 6 was due to its highest daily dose, while that to 13 and 22 was due to their relatively long

elimination half-lives. The catechols 1 and 10 circulated in the blood stream predominantly as

metabolites, rather than the parent compound; 16 and 26 were detected at low plasma levels

with their plasma metabolites little or not detected in the human subjects. Both 6 and 13 were

eliminated mainly via renal excretion of the parent compounds; 22 was eliminated mainly via

hepatobiliary excretion of its methylated metabolites. The other Danshen catechols were

eliminated mainly via hepatobiliary and/or renal excretion of their metabolites.

COMT-mediated methylation was found to be the primary metabolism for most Danshen

catechols and showed intercompound and interspecies differences in rate and degree. Because

the only known mechanism of homocysteine production involves SAM-dependent

methylation, further studies are warranted to fully characterize the methylation of Danshen

catechols and to address safety concern associated with possible alteration of blood

concentrations of homocysteine related to using DanHong injection.


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Authorship Contributions

Participated in research design: C. Li, L. Li, Huang.

Conducted experiments: L. Li, M. Li, Wang, Huang, Y. Li, Zhong, Du, W. Jia, Dong, J. Li, Xu.

Contributed new reagents: Zhao, L. Jia.

Performed data analysis: C. Li, M. Li, L. Li.

Wrote or contributed to the writing of the manuscript: C. Li, M. Li, L. Li, Olaleye, Lim.


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Footnotes

This work was supported by the National Science Fund of China for Distinguished Young Scholars

[Grant 30925044]; the National Science and Technology Major Project of China “Key New Drug Creation

and Manufacturing Program” [Grants 2009ZX09304-002 and 2013ZX09201020001003]; and the National

Basic Research Program of China [Grant 2012CB518403].

This work was present in part as a poster presentation: Li L et al (2014) In vivo chemical profiles and

metabolic profiles of phenolic acids after intravenous administration of DanHong injection in different

species. 5th Asian Pacific Regional Meeting of ISSX; 2014 May 9-12; Tianjin, China. Justin B. R. Lim was a

visiting student from Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, USA.


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Legends for Figures

FIG. 1. LC/TOF-MSE-based detection and measurement of Danshen catechols present in

DanHong injection. A, chromatogram of DanHong injection; B, average content levels of

Danshen catechols in different lots of DanHong injection in mM; C, clinical daily doses of

Danshen catechols from i.v. DanHong injection (lot number, 13011037) in μmol/subject; D,

percentages of individual catechols in total content level of Danshen catechols in DanHong

injection. Danshen catechols (shown as compound ID) in panels B and C are ranked

according to their average content levels in different lots of the herbal injection; their names

and detection information are shown in Table 1.

FIG. 2. Systemic exposure to and excretion of Danshen catechols in human subjects (A and B),

rats (C–E), and dogs (F and G) receiving i.v. DanHong injection (lot number, 13011037).

Danshen catechols (shown as compound ID) in panels A–G are ranked according to their

average content levels (Fig. 1B); their names are shown in Table 1. Red, black, and green bars

are used to show the plasma, urinary, and biliary levels of the Danshen catechols after dosing,

respectively. The symbol “×” (in red) denotes the Danshen compound that was not detected.

The human subjects received a 120 min i.v. infusion dose of the herbal injection at 40

ml/subject. The rats and the dogs received an i.v. bolus dose of the herbal injection at 4.0 and

1.2 ml/kg, respectively. Because of the sample pooling, no standard deviation is shown.

FIG. 3. Systemic exposure to and excretion of metabolites of major Danshen catechols

[tanshinol (6), protocatechuic aldehyde (1), rosmarinic acid (10), salvianolic acid A (16),

salvianolic acid D (13), lithospermic acid (22), and salvianolic acid B (26)] in human subjects

(A and B), rats (C–E), and dogs (F and G) receiving i.v. DanHong injection (lot number,

13011037). The parent Danshen catechols are ranked according to their average content levels

(Fig. 1B) and their respective metabolites are shown on the right (in a left-to-right ranking


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order of methylated metabolites, sulfates and glucuronides of methylated metabolites, sulfates

and glucuronides of parent compounds, and oxidized metabolites). Only metabolites (shown

as metabolite ID) exhibiting plasma AUC0-∞ values and/or excretory amounts, which were

10% of those of their respective parent compounds at least in one species, are shown in this

figure. The metabolite ID is used to indicate the compound as a metabolite, its parent

compound, the type of metabolism, and metabolite isomer. For instance, “M10” in M102M-S-1

denotes “metabolite of Danshen catechol 10 (rosmarinic acid)”; the subscript number and

letter “2M” denotes “dimethylation”; the subscript letter “S” denotes “sulfation”, and the last

subscript number “1” denotes “the first eluted metabolite isomer”. The subscript letter “G” for

other metabolite IDs denotes “glucuronidation”. Red/pink, black/gray, and green/light green

bars represent the plasma, urinary, and biliary levels of the parent Danshen catechols/their

metabolites, respectively. The symbol “×” (in red) denotes the Danshen compounds that was

not detected. The names of metabolites and associated detection information are shown in

Table 2 (for human metabolites) and Supplemental Tables 1 (for rat metabolites) and 2 (for

dog metabolites). The human subjects received a 120 min i.v. infusion dose of the herbal

injection at 40 ml/subject. The rats and the dogs received an i.v. bolus dose of the herbal

injection at 4 and 1.2 ml/kg, respectively. Because of the sample pooling, no standard

deviation is shown.

FIG. 4. Proposed major metabolic pathways of tanshinol (6), salvianolic acid D (13),

lithospermic acid (22), protocatechuic aldehyde (1), and rosmarinic acid (10) in human

subjects receiving i.v. DanHong injection. The metabolites of salvianolic acids B (26) and A

(16) exhibited little or no detection in human plasma and urine after dosing. The names and

detection information of metabolites are shown in Table 2; their systemic exposure levels and

excretory amounts are depicted in Fig. 3A and B. Sulfation of vanillic acid (M2M) and

tanshinol (6) mainly took place in rats and dogs after dosing DanHong injection. The


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chemical structures of M6M and M6S were inferred to be 3-O-methyltanshinol and

tanshinol-3-O-sulfate, respectively, according to the data by Tian et al. (2015). The chemical

structures of M22M and M222M were inferred to be 3'-O-methyllithospermic acid and

3',3"-O-dimethyllithospermic acid, respectively, according to the data by Wang et al. (2008).

FIG. 5. Comparative in vitro metabolic stability of Danshen catechols (including tanshinol,

protocatechuic aldehyde, protocatechuic acid, salvianolic acid B, rosmarinic acid, salvianolic

acid A, salvianolic acid D, and lithospermic acid) towards metabolism reactions mediated by

COMT, SULT, UGT, and ALDH (ALDH, for protocatechuic aldehyde only). Human, rat, and

dog liver cytosols containing COMT, SULT, and ALDH were used to induce methylation,

sulfation, and oxidation of Danshen catechols, while human, rat, and dog liver microsomes

containing UGT were used to induce glucuronidation of the compounds. Green, brown, black,

and purplish-red semi-open bars denote the data of Danshen catechols undergoing

methylation, sulfation, glucuronidation, and aldehyde dehydrogenation, respectively. Green,

brown, black, and purplish-red open bars denote the data of positive controls, i.e.,

(−)-epicatechin for COMT-mediated methylation, 7-hydroxyflavone for SULT-mediated

sulfation, chrysin for UGT-mediated glucuronidation, and isovanillin for ALDH-mediated

aldehyde dehydrogenation, respectively. Values represent the means ± standard deviations (n

= 3).

FIG. 6. Methylation of Danshen catechols (including tanshinol, protocatechuic aldehyde,

protocatechuic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, salvianolic acid D,

and lithospermic acid) mediated by COMT in SAM-fortified human (A), rat (B), and dog

liver cytosols (C) over time. Blue lines/blue open circles denote the substrate compounds.

Green lines/green open circles, green lines/green semi-open circles, and green lines/green

solid circles denote monomethylated, dimethylated, and trimethylated metabolites,


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respectively. For the methylation of protocatechuic aldehyde, isovanillin was used to inhibit

the catalytic activity of ALDH in human liver cytosol. Values represent the means ± standard

deviations (n = 3).


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TABLE 1

Detection of Danshen catechol compounds in DanHong injection.

tR, chromatographic retention time; Number in bold, product ion of base peak.

ID Compound

LC/TOF-MSE data Molecular mass (Da)

Molecular formula

tR

(min)a [M−H]–

(m/z) Fragmentation profileb

(m/z) 1 Protocatechuic aldehyde 8.38 137.0241 109.0311, 108.0233 138.0317 C7H6O3 2 Protocatechuic acid 7.11 153.0191 109.0295, 108.0221 154.0266 C7H6O4 3 Caffeic acid 11.30 179.0342 135.0451, 134.0370 180.0423 C9H8O4 4 Ferulic acid 14.12 193.0505 178.0258, 134.0370, 133.0305 194.0579 C10H10O4 5 Isoferulic acid 14.75 193.0506 178.0257, 134.0373, 133.0292 194.0579 C10H10O4 6 Tanshinol 6.63 197.0455 179.0346, 135.0454, 123.0453 198.0528 C9H10O5 7 Salvianolic acid F 11.01 313.0718 295.0621, 269.0818, 203.0376,159.0446 314.0790 C17H14O6 8 Salvianolic acid G 15.92 339.0502 311.0565, 295.0614, 267.0659 340.0583 C18H12O7 9 Prolithospermic acid 11.67 357.0606 313.0706, 203.0349, 159.0458 358.0689 C18H14O8 10 Rosmarinic acid 16.85 359.0765 197.0453, 179.0345, 161.0242 360.0845 C18H16O8 11 Salvianic acid C 12.07 377.0869 359.0774, 197.0459, 179.0352, 161.0237 378.0951 C18H18O9 12 Salvianic acid C isomer 12.52 377.0862 359.0772, 197.0448, 179.0355, 161.0244 378.0951 C18H18O9 13 Salvianolic acid D 16.32 417.0827 373.0917, 197.0447, 179.0345, 175.0393 418.0900 C20H18O10 14 Isosalvianolic acid C 19.50 491.0975 311.0563, 293.0455, 249.0556 492.1056 C26H20O10 15 Salvianolic acid C 21.27 491.0978 311.0559, 293.0455, 267.0663 492.1056 C26H20O10 16 Salvianolic acid A 18.51 493.1131 313.0716, 295.0611, 185.0246 494.1213 C26H22O10 17 Salviaflaside 15.79 521.1292 359.0773, 197.0453, 161.0241 522.1373 C24H26O13 18 Lithospermic acid isomer 13.48 537.1038 493.1134,313.0721, 295.0607 538.1111 C27H22O12 19 Lithospermic acid isomer 13.80 537.1030 493.1133, 313.0726, 295.0603, 185.0249 538.1111 C27H22O12 20 Lithospermic acid isomer 14.32 537.1039 493.1134, 313.0717, 295.0604, 185.0244 538.1111 C27H22O12 21 Salvianolic acid H/I 15.38 537.1031 339.0502, 295.0617, 185.0243 538.1111 C27H22O12 22 Lithospermic acid 16.78 537.1036 493.1141, 295.0607, 185.0241 538.1111 C27H22O12 23 Salvianolic acid J 17.97 537.1037 493.1142, 295.0609, 185.0244 538.1111 C27H22O12 24 Salvianolic acid B isomer 16.03 717.1461 519.0933, 339.0511, 321.0408 718.1534 C36H30O16 25 Salvianolic acid B isomer 16.36 717.1448 519.0932, 339.0502, 321.0407 718.1534 C36H30O16

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26 Salvianolic acid B 17.41 717.1455 519.0935, 339.0504, 321.0399 718.1534 C36H30O16 27 Salvianolic acid E 18.18 717.1454 519.0938, 339.0503, 321.0403 718.1534 C36H30O16 28 4-Methoxyl salvianolic acid B 18.38 731.1599 533.1101, 353.0676, 335.0564 732.1690 C37H32O16 T

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TABLE 2

Detection of major metabolites of tanshinol (6), protocatechuic aldehyde (1), rosmarinic acid (10), salvianolic acid A (16), salvianolic acid D (13),

and lithospermic acid (22) in human plasma and/or urine samples collected during and after an i.v. infusion dose of DanHong injection.

tR, chromatographic retention time; Number in bold, product ion of base peak. The metabolite ID is used to indicate the compound as a metabolite, its parent compound, the

type of metabolism, and metabolite isomer. For instance, “M10” in M102M-S-1 denotes “metabolite of Danshen catechol 10 (rosmarinic acid)”; the subscript number and letter

“2M” denotes “dimethylation”; the subscript letter “S” denotes “sulfation”, and the last subscript number “1” denotes “the first eluted metabolite isomer”. The subscript letter

“G” for other metabolite IDs denotes “glucuronidation”.

ID Parent compound/metabolite

LC/TOF-MSE data Molecular mass

(Da) Molecular formula

tR (min)

[M−H]− (m/z)

Fragmentation profile (m/z)

Metabolites of tanshinol (6) 6 Tanshinol 6.61 197.0455 179.0346, 135.0454, 123.0453 198.0528 C9H10O5 M6M Methyltanshinol 9.90 211.0611 193.0507, 150.0323, 134.0371 212.0684 C10H12O5

Metabolites of protocatechuic aldehyde (1)1 Protocatechuic aldehyde 8.36 137.0240 109.0311, 108.0233 138.0317 C7H6O3 2 Protocatechuic acid 7.07 153.0194 109.0295 154.0266 C7H6O4 M2M Methylprotocatechuic acid 10.8 167.0348 123.0451 168.0422 C8H8O4 M2M-G-1 Methylprotocatechuic acid glucuronide 8.04 343.0660 167.0345, 123.0443 344.0743 C14H16O10 M2M-G-2 Methylprotocatechuic acid glucuronide 8.96 343.0671 167.0351, 123.0441 344.0743 C14H16O10 M2S-1 Protocatechuic acid sulfate 10.3 232.9767 153.0193, 109.0291 233.9838 C7H6SO7 M2S-2 Protocatechuic acid sulfate 10.7 232.9759 153.0195, 109.0295 233.9838 C7H6SO7

Metabolites of rosmarinic acid (10) 10 Rosmarinic acid 16.70 359.0775 197.0453, 179.0345, 161.0242 360.0845 C18H16O8 M10M-1 Methylrosmarinic acid 16.26 373.0918 197.0446,175.0392, 179.0346 374.1001 C19H18O8 M10M-2 Methylrosmarinic acid 18.18 373.0919 197.0452,175.0389, 135.0447 374.1001 C19H18O8 M102M Dimethylrosmarinic acid 19.47 387.1078 211.0600, 193.0492, 175.0388 388.1158 C20H20O8 M102M-S-1 Dimethylrosmarinic acid sulfate 21.33 467.0649 387.1076, 211.0605, 175.0392 468.0726 C20H20SO11 M102M-S-2 Dimethylrosmarinic acid sulfate 21.44 467.0650 387.1085, 211.0601, 175.0388 468.0726 C20H20SO11




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M102M-G-1 Dimethylrosmarinic acid glucuronide 17.17 563.1411 387.1085, 211.0606, 193.0484 564.1479 C26H28O14 M102M-G-2 Dimethylrosmarinic acid glucuronide 17.59 563.1405 387.1093, 211.0607, 193.0501 564.1479 C26H28O14

Metabolites of salvianolic acid A (16) 16 Salvianolic acid A 18.41 493.1127 313.0716, 295.0611, 185.0246 494.1208 C26H22O10 M16M-G-1 Methylsalvianolic acid A glucuronide 18.62 683.1606 507.1295,335.0576,295.0599 684.1690 C33H32O16 M16M-G-2 Methylsalvianolic acid A glucuronide 19.03 683.1611 507.1285,335.0553,295.0608 684.1690 C33H32O16 M16M-G-3 Methylsalvianolic acid A glucuronide 19.46 683.1616 507.1293,335.0557,295.0608 684.1690 C33H32O16 M162M-G-2 Dimethylsalvianolic acid A glucuronide 19.76 697.1771 521.1448, 327.0865, 309.0767 698.1847 C34H34O16

Metabolites of salvianolic acid D (13) 13 Salvianolic acid D 16.30 417.0833 373.0917, 197.0447, 175.0393 418.0900 C20H18O10 M13M Methylsalvianolic acid D 17.79 431.0981 387.1073, 211.0608, 175.0392 432.1056 C21H20O10

Metabolites of lithospermic acid (22) 22 Lithospermic acid 16.75 537.1037 493.1141, 313.0725, 295.0625 538.1111 C27H22O12 M22M Methyllithospermic acid 18.00 551.1198 507.1316, 327.0869, 309.0774 552.1268 C28H24O12 M222M Dimethyllithospermic acid 19.22 565.1342 521.1436, 327.0879, 309.0753 566.1424 C29H26O12




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Documents

DMD # 61473dmd.aspetjournals.org/content/dmd/early/2015/02/24/... · DMD # 61473 - 2 - Running Title: Systemic Exposure to Catechols from i.v. DanHong Injection Address correspondence