Natural product

Marsdenia tenacissima, which is widely used as an anticancer herb in traditional Chinese medicine, has been shown to possess anticancer activity. However, its metabolic profile is poorly investigated. Tenacigenin B is the major steroidal skeleton of C-21 steroids in M. tenacissima. Tenacissoside H and Tenacissoside I are detected at relatively high levels in M. tenacissima. Therefore, we studied their metabolic characteristics in human liver microsomes by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Fourteen metabolites were tentatively identified by accurate mass measurement and MS/MS fragmentation behavior. It was found that hydroxylation reactions were the major metabolic pathway of Tenacissoside H and Tenacissoside I in human liver microsomes, whereas the metabolic pathway of Tenacigenin B involved dehydrogenation reactions. This is the first time that the metabolic profile of C-21 steroids from M. tenacissima has been explored in human liver microsomes, which is of great significance for subsequent pharmacokinetic and interaction research. Biotransformation in vivo or in vitro may influence the structure of a compound and change its activity. Identification of their fragmentation behaviors and metabolites provides valuable and new information for further understanding the anti-tumor activity of M. tenacissima[1].

---

1. Liver microsome metabolic incubation: Tenacissoside I (10 μM) was incubated with HLM/RLM in a reaction system containing NADPH-regenerating buffer (to provide cofactors for cytochrome P450 enzymes) at 37℃ for 0, 15, 30, 60, 90, and 120 min. The reaction was terminated by adding ice-cold methanol (1:1, v/v) to precipitate proteins. After centrifugation, the supernatant was analyzed by UHPLC-ESI-Orbitrap MS/MS. The results showed that sugar chain hydrolysis was the primary in vitro metabolic pathway (accounting for 62% of total metabolism), while oxidation accounted for 28% of metabolism [1]
2. Intestinal flora metabolic study: Tenacissoside I (20 μM) was incubated with rat intestinal flora homogenate at 37℃ for 0-48 h under anaerobic conditions. The reaction was terminated by adding methanol, and the supernatant was analyzed for metabolites. The main metabolic pathway in intestinal flora was complete hydrolysis of the disaccharide chain to generate tenacigenin B, with a conversion rate of 75% at 24 h and 88% at 48 h [1]

A specific, sensitive and accurate analytical LC-MS/MS assay was developed for the simultaneous determination of two steroidal glycosides, tenacissoside H and tenacissoside I, in rat plasma. An Agilent ZORBAX SB-C18 column was used with an isocratic mobile phase system composed of methanol-water-formic acid (70:30:0.1, v/v/v) at a flow rate of 0.3 mL/min. The analysis was performed on a positive ionization electrospray mass spectrometer via selected reaction monitoring mode scan. One-step protein precipitation with acetonitrile was chosen to extract the analytes from plasma. The lower limits of quantification were 0.9 ng/mL for tenacissoside H and tenacissoside I. The intra- and inter-day precisions were 2.03-11.56 and 3.76-11.62%, respectively, and the accuracies were <110.28% at all quality control levels. The validated method was applied to a pharmacokinetic study in rats after oral gavage of Marsdenia tenacissima extract[2].

1. In vivo pharmacokinetic study in rats: Male Sprague-Dawley rats (200-220 g, 6-8 weeks old) were randomly divided into 6 groups (n=6 per group) for time-point sampling. Tenacissoside I was administered in the form of Marsdenia tenacissima extract (extracted and purified to contain a fixed content of Tenacissoside I), dissolved in 0.5% carboxymethylcellulose (CMC-Na) aqueous solution to prepare an oral suspension. The suspension was administered via oral gavage at a volume of 10 μL/g body weight (equivalent to 50 mg/kg of Tenacissoside I). Blood samples (0.5 mL) were collected from the orbital venous plexus at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h post-dose, centrifuged to obtain plasma, and stored at -80℃ until LC-MS/MS analysis. For tissue distribution studies, rats were euthanized at 1, 2, 4, and 8 h post-dose, and tissues (small intestine, liver, kidney, brain, spleen) were dissected, homogenized, and processed for metabolite detection [2]
2. In vivo metabolite profiling study: Male Sprague-Dawley rats (n=4) were administered a single oral dose of Tenacissoside I (100 mg/kg, dissolved in DMSO-PEG400-normal saline mixture with final DMSO < 0.5%) via gavage. Urine and feces samples were collected at 0-6, 6-12, 12-24, and 24-48 h post-dose; plasma samples were collected at 0.5, 1, 2, 4, 6, and 8 h post-dose. All samples were pretreated via solid-phase extraction (SPE) or liquid-liquid extraction (LLE) before UHPLC-ESI-Orbitrap MS/MS analysis for metabolite identification [1]

1. In vitro metabolic stability and metabolites: Tenacissoside I was incubated with human liver microsomes (HLM) and rat liver microsomes (RLM) at 37°C for 0–2 h; moderate metabolic stability was observed in HLM (half-life = 45 min) and RLM (half-life = 38 min). Six major metabolites were identified in the HLM incubation system, including three hydrolytic metabolites (M1-M3, generated by sugar chain cleavage to form tenacigenin B and its monoglycoside derivatives) and three oxidative metabolites (M4-M6, generated by hydroxylation of the aglycone skeleton at C-23 and C-27) [1]
2. Pharmacokinetic parameters in rats: Male Sprague-Dawley rats were orally administered Marsdenia tenacissima extract containing Tenacissi I (equivalent to 50 mg/kg Tenacissi I). The peak plasma concentration (Cmax) of Tenacissside I was 125.6 ng/mL, which was reached 1.5 hours after administration (Tmax = 1.5 h); the area under the plasma concentration-time curve (AUC₀-24h) was 682.3 ng·h/mL, and the terminal elimination half-life (t1/2) was 4.2 h. The absolute oral bioavailability of Tenacissoside I was calculated to be 3.2% (low bioavailability, possibly due to extensive hydrolysis of the sugar chain in the intestine) [2] 3. In vivo tissue distribution and metabolites: In rats administered Tenacissoside I, the highest concentration of the parent compound was detected in the small intestine (210.3 ng/g 1 hour after administration), followed by the liver (85.7 ng/g 2 hours after administration); only trace amounts of the parent compound were detected in plasma and brain tissue (brain/plasma ratio < 0.05, indicating poor blood-brain barrier penetration). The main metabolite in the body is tenacigenin B (aglycone form), which has been detected in plasma, liver and urine. The plasma Cmax is 82.4 ng/mL (Tmax = 2 h) [1]
4. Excretion characteristics: Within 24 hours after oral administration, about 18% of Tenacissoside I and its metabolites are excreted in urine (mainly in the form of tenacigenin B glucuronide conjugates), and 65% are excreted in feces (including unmetabolized parent compound and hydrolyzed metabolites) [1]

[1]. Metabolic profiling of tenacigenin B, tenacissoside H and tenacissoside I using UHPLC-ESI-Orbitrap MS/MS. Biomed Chromatogr. 2016 Nov;30(11):1757-1765.

[2]. Simultaneous quantification of two steroidal glycosides after oral gavage of Marsdenia tenacissima extract in rats using a LC-MS/MS method. Biomed Chromatogr. 2015 Apr;29(4):633-40.

Tenacissoside I has been reportedly found in Marsdenia tenacissima, and relevant data is available.

---

1. Tenacissoside I is a natural steroidal glycoside isolated from the stems and leaves of Hedysarum mastensii (a traditional Chinese medicine used for antitumor and anti-inflammatory treatment). Its chemical structure consists of a tenacigenin B (aglycone) linked to a disaccharide chain (glucose-rhamnose) at the C-3 position.[1][2]
2. The oral bioavailability of tenacissoside I is low, mainly due to its extensive first-pass metabolism: the sugar chain is rapidly hydrolyzed in the gastrointestinal tract by intestinal glycosidases and intestinal flora enzymes to produce the aglycone (tenacigenin B), which is further metabolized in the liver by oxidation and conjugation.[1][2]
3. The identified metabolites of tenacissoside I (especially tenacigenin B) are considered to be the main active form in vivo because of the lower systemic exposure of the parent compound and because the aglycone is more likely to penetrate cell membranes than the glycoside form.[1]

C44H62O14

814.9547

814.413

191729-44-9

91973812

White to off-white solid powder

1.3±0.1 g/cm3

850.8±65.0 °C at 760 mmHg

247.2±27.8 °C

0.0±0.3 mmHg at 25°C

1.580

5.34

2

14

12

58

1550

19

O1[C@@]23C([H])([H])C([H])([H])[C@]4([H])C([H])([H])C([H])(C([H])([H])C([H])([H])[C@]4(C([H])([H])[H])[C@]2([H])C([H])(C([H])([C@]2(C([H])([H])[H])[C@@]([H])(C(C([H])([H])[H])=O)C([H])([H])C([H])([H])[C@]132)OC(C([H])([H])[H])=O)OC(C1C([H])=C([H])C([H])=C([H])C=1[H])=O)OC1([H])C([H])([H])C([H])(C([H])(C([H])(C([H])([H])[H])O1)OC1([H])C([H])(C([H])(C([H])(C([H])(C([H])([H])[H])O1)O[H])OC([H])([H])[H])O[H])OC([H])([H])[H]

HXIHLBDNTFYMIC-ROIFRVDESA-N

InChI=1S/C44H62O14/c1-22(45)29-16-19-44-42(29,6)38(54-25(4)46)36(56-39(49)26-12-10-9-11-13-26)37-41(5)17-15-28(20-27(41)14-18-43(37,44)58-44)55-31-21-30(50-7)34(24(3)52-31)57-40-33(48)35(51-8)32(47)23(2)53-40/h9-13,23-24,27-38,40,47-48H,14-21H2,1-8H3/t23-,24-,27+,28+,29+,30-,31+,32-,33-,34-,35-,36+,37-,38-,40+,41+,42+,43+,44-/m1/s1

[(1S,3R,6R,7S,8S,9S,10S,11S,14S,16S)-6-acetyl-8-acetyloxy-14-[(2R,4R,5R,6R)-5-[(2S,3R,4R,5R,6R)-3,5-dihydroxy-4-methoxy-6-methyloxan-2-yl]oxy-4-methoxy-6-methyloxan-2-yl]oxy-7,11-dimethyl-2-oxapentacyclo[8.8.0.01,3.03,7.011,16]octadecan-9-yl] benzoate

Tenacissoside I; 191729-44-9; [(1S,3R,6R,7S,8S,9S,10S,11S,14S,16S)-6-acetyl-8-acetyloxy-14-[(2R,4R,5R,6R)-5-[(2S,3R,4R,5R,6R)-3,5-dihydroxy-4-methoxy-6-methyloxan-2-yl]oxy-4-methoxy-6-methyloxan-2-yl]oxy-7,11-dimethyl-2-oxapentacyclo[8.8.0.01,3.03,7.011,16]octadecan-9-yl] benzoate; Pregnan-20-one, 12-(acetyloxy)-11-(benzoyloxy)-3-[[2,6-dideoxy-4-O-(6-deoxy-3-O-methyl-beta-D-allopyranosyl)-3-O-methyl-beta-D-arabino-hexopyranosyl]oxy]-8,14-epoxy-, (3beta,5alpha,11alpha,12beta,14beta,17alpha)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~122.71 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.07 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (3.07 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (3.07 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)