Tumor cells are dose-dependently inhibited by protosappanin B (12.5, 25, 50, 100, and 200 µg/mL, 48 hours); the IC50 values for SW-480 and HCT are 21.32 µg/mL, 26.73 µg/mL, and 76.53 µg/mL, correspondingly. -116 and BTT cells, in that order[1].

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- Protosappanin B (2 mg/mL) time‑dependently reduced the viability of human bladder cancer T24 cells and mouse bladder cancer BTT cells as assessed by trypan blue exclusion assay. After 100 minutes, death rates were 79.58% ± 4.13% for T24 and 81.12% ± 6.11% for BTT (P < 0.05). [1]
- Protosappanin B significantly inhibited the growth of human colon cancer HCT‑116, SW‑480, and mouse bladder cancer BTT cells in a concentration‑dependent manner as measured by MTT assay after 48 h treatment. IC₅₀ values: SW‑480 = 21.32 μg/mL; HCT‑116 = 26.73 μg/mL; BTT = 76.53 μg/mL. For comparison, brazilin IC₅₀ values were: SW‑480 = 7.79 μg/mL; HCT‑116 = 12.35 μg/mL; BTT = 8.76 μg/mL. [1]
- Protosappanin B (12.5–200 μg/mL, 48 h) caused concentration‑dependent inhibition of T24, 5637, and SV‑HUC‑1 cell growth. Growth inhibition rates at 200 μg/mL: T24 = 92.13%, 5637 = 75.86%, SV‑HUC‑1 = 56.54% (P < 0.05). IC₅₀ values: T24 = 82.78 μg/mL; 5637 = 113.79 μg/mL. [2]
- Morphological changes (shrinkage, rounding, membrane abnormalities, reduced adhesion, and cell debris) were observed in T24 and 5637 cells after treatment with Protosappanin B (200 μg/mL, 24 h) under light microscopy. [2]
- Protosappanin B (100–300 μg/mL, 24 h) induced concentration‑dependent apoptosis in T24 and 5637 cells as measured by annexin‑V/PI flow cytometry. Total apoptotic cells in T24: increased from control (approx. 5%) to about 55% at 300 μg/mL. In 5637 cells: from control (approx. 6%) to about 50% at 300 μg/mL (P < 0.05). [2]
- Western blot analysis showed that Protosappanin B (100–300 μg/mL, 48 h) decreased Bcl‑2 and increased Bax protein expression in a concentration‑dependent manner in both T24 and 5637 cells. [2]
- Cell cycle analysis by flow cytometry revealed that Protosappanin B (100–300 μg/mL, 48 h) caused G₁ phase arrest in T24 and 5637 cells. In T24 cells, G₁ phase increased from 62.96% (control) to 82.74% at 300 μg/mL, while proliferative index decreased from 37.04% to 17.26% (P < 0.05). In 5637 cells, G₁ phase increased from 37.43% to 64.59%, and proliferative index decreased from 62.57% to 35.41% (P < 0.05). [2]
- Proteomics analysis of T24 cells treated with Protosappanin B (48 h) identified 4914 proteins, with 3494 quantified. Differentially expressed proteins were clustered, and KEGG pathway analysis indicated that cell cycle‑related proteins were significantly affected, supporting that cell cycle arrest is involved in Protosappanin B‑induced apoptosis. [2]

- H22 mouse liver cancer cells were pretreated with Protosappanin B (final concentrations 6.25, 3.12, or 1.56 mg/mL) for 10, 20, or 40 minutes, then inoculated into KM mice (n=5 per group). Control groups received normal saline or mitomycin (0.66 mg/mL). Tumor formation and survival were observed for 60 days. High‑dose Protosappanin B (6.25 mg/mL) completely inhibited tumor formation (0% tumor formation rate) in all treatment time groups, with mean survival time of 60.00 ± 0.00 days, representing survival extension of 297.35% (10 min), 226.09% (20 min), and 166.67% (40 min) compared to control (P < 0.05). Moderate dose (3.12 mg/mL) gave tumor formation rates of 100% (10 min), 80% (20 min), and 60% (40 min), with mean survival times 20.40 ± 8.27, 32.10 ± 16.34, and 41.30 ± 19.73 days respectively. Low dose (1.56 mg/mL) gave 100% tumor formation rates in all groups, with mean survival times 21.60 ± 2.97 (10 min), 24.20 ± 12.6 (20 min), and 35.50 ± 14.11 (40 min) days. Mitomycin (0.66 mg/mL) resulted in 0‑20% tumor formation and 60 days survival in most groups. [1]
- BTT tumor‑bearing T739 mice (n=10 per group) received intraperitoneal injections of Protosappanin B (100, 200, or 300 mg/kg/day) or mitomycin (1 mg/kg/day) or normal saline (control) for 6 consecutive days, starting 24 h after intraperitoneal inoculation of BTT cells (5×10⁶ cells/mouse). Survival was observed for 60 days. Mean survival times: control = 19.28 ± 4.94 days; low‑dose Protosappanin B (100 mg/kg) = 22.64 ± 5.49 days (extension 17.43%, P < 0.05); medium dose (200 mg/kg) = 43.81 ± 10.21 days (extension 127.23%, P < 0.05); high dose (300 mg/kg) = 51.38 ± 11.31 days (extension 166.49%, P < 0.05); mitomycin (1 mg/kg) = 52.44 ± 8.83 days (extension 177.99%, P < 0.05). [1]

Cell Viability Assay[1]
Cell Types: CT-116, SW-480 and BTT Cell
Tested Concentrations: 12.5, 25, 50, 100, 200 µg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: Dose-dependent inhibition of tumor cells after 48 hrs (hours) Treatment.

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- Trypan blue exclusion assay: T24 and BTT cells were adjusted to 2×10⁶/mL in normal saline. 500 μL of cell suspension was mixed with 500 μL of Protosappanin B (final concentration 2 mg/mL) or normal saline (control). After incubation at 37°C with 5% CO₂, samples were taken at 0, 20, 40, 60, and 100 minutes. 100 μL of cell suspension was mixed with an equal volume of trypan blue. Dead (blue‑stained) and viable (unstained) cells were counted under an optical microscope (100× magnification) in 4 large grids of a hemocytometer. Death rate = (dead cell count / (dead cell count + viable cell count)) × 100%. [1]
- MTT assay for HCT‑116, SW‑480, and BTT cells: Cells were seeded into 96‑well plates (4×10³ cells/well) in 100 μL culture medium and incubated for 24 h. Medium was replaced with 200 μL fresh medium containing Protosappanin B or brazilin at final concentrations of 12.5, 25, 50, 100, or 200 μg/mL. Control wells received equal volume of medium. After 48 h, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h. Medium was removed, 150 μL DMSO added, and plates shaken for 10 min. Optical density was measured at 570 nm. Growth inhibition ratio (%) = (1 – mean OD₅₇₀ treatment / mean OD₅₇₀ control) × 100%. IC₅₀ was calculated using the formula: lgIC₅₀ = Xm – I(P – (3 – Pm – Pn)/4). [1]
- MTT assay for T24, 5637, and SV‑HUC‑1 cells: Cells were seeded in 96‑well plates (T24 and SV‑HUC‑1: 4×10³ cells/well; 5637: 9×10³ cells/well) in 100 μL medium and incubated for 24 h. Medium was replaced with 200 μL medium containing Protosappanin B (12.5, 25, 50, 100, or 200 μg/mL). After 48 h, 20 μL MTT (5 mg/mL) was added, incubated for 4 h, then medium removed, 150 μL DMSO added, shaken for 10 min. OD was measured at 570 nm. Growth inhibition rate and IC₅₀ were calculated as above. [2]
- Cell morphology observation: T24 (4×10⁵ cells/well) or 5637 (5×10⁵ cells/well) were seeded in 6‑well plates. Protosappanin B (200 μg/mL) or equal volume of medium (control) was added. After 24 h incubation, cells were observed under an inverted light microscope (100× magnification) and photographed. [2]
- Apoptosis assay (annexin‑V/PI): T24 (4×10⁵ cells/well) or 5637 (5×10⁵ cells/well) were cultured in 6‑well plates for 24 h. Medium was replaced with medium containing Protosappanin B (100, 150, 200, or 300 μg/mL) or control. After 24 h, cells were digested, collected, centrifuged (1000 rpm, 5 min), resuspended in PBS, filtered, and centrifuged again. Cells were resuspended in binding buffer, and 5 μL each of annexin‑V‑FITC and PI were added, incubated in dark for 15 min at room temperature. Apoptosis was detected by flow cytometry. Total apoptosis = early + late apoptosis rates. [2]
- Western blot: T24 and 5637 cells were treated with Protosappanin B (0, 100, 200, or 300 μg/mL) for 48 h. Cells were lysed with RIPA buffer. Protein concentration was determined by Bradford assay. 20 μg protein per lane were separated by polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were incubated with primary antibodies against Bax (1:200) and Bcl‑2 (1:200) overnight at 4°C, then with HRP‑labeled secondary antibody for 2 h. Bands were visualized using ECL reagent and analyzed with Image J software. [2]
- Cell cycle analysis: T24 (8×10⁵ cells/well) or 5637 (1×10⁶ cells/well) were cultured in 6‑well plates for 24 h. Medium was replaced with medium containing Protosappanin B (100, 150, 200, 250, or 300 μg/mL) or control. After 48 h, cells were digested, collected, centrifuged (1000 rpm, 5 min), washed with PBS, and fixed in 70% cold ethanol overnight at 4°C. Cells were then washed, filtered, centrifuged, and DNA content was examined using a cell cycle detection kit by flow cytometry. Each sample was analyzed in triplicate. Proliferative index = (S + G₂) / (G₁ + S + G₂) × 100%. [2]
- Proteomics analysis: T24 cells treated with Protosappanin B or vehicle for 48 h were collected. Proteins were extracted by sonication in lysis buffer (8M urea, 1% protease inhibitor cocktail), centrifuged (12,000×g, 10 min, 4°C), and quantified by BCA method. Protein was reduced with 5 mM dithiothreitol (56°C, 30 min), alkylated with 11 mM iodoacetamide (dark, 15 min, room temperature), diluted with 100 mM NH₄HCO₃ to 2M urea, and digested with trypsin (1:50 mass ratio overnight, then 1:100 for 4 h). Tryptic peptides were fractionated by high pH reverse‑phase HPLC using a C18 column with a gradient of 8‑32% acetonitrile (pH 9.0) over 60 min into 60 fractions, then combined into 4 fractions. LC‑MS/MS analysis was performed on a Q Exactive Plus instrument coupled to an EASY‑nLC 1000 UPLC. Peptides were loaded onto a reversed‑phase analytical column, eluted with a gradient of 6‑35% solvent B (0.1% formic acid in 98% acetonitrile) over 34 min at 400 nL/min. MS data were acquired in data‑dependent mode. For hierarchical clustering, pathway enrichment analysis was performed, and P values were transformed and z‑scored for clustering using Genesis. [2]

- H22 tumor cell invasion assay in KM mice: Frozen H22 cells were thawed and inoculated (2.5×10⁶/mL, 0.2 mL) into the abdominal cavity of KM mice. Ascites were collected aseptically on day 7. Cell density was adjusted to 2.5×10⁷/mL with normal saline. Protosappanin B (final concentrations 6.25, 3.12, or 1.56 mg/mL), mitomycin (final concentration 0.66 mg/mL), or normal saline (control) were added (1:9 ratio). Cells were incubated at 37°C and washed at 10, 20, or 40 minutes to remove drugs, then resuspended in the same volume of normal saline. 0.2 mL of the cell suspension was inoculated into the abdominal cavity of KM mice (n=5 per group). Mice were observed twice daily for 60 days; body weights measured every 3 days. Tumor formation rate, mean survival time, and rate of extended survival were calculated. For animals found dead in the morning, survival time was recorded as 1 day; in the afternoon, as 1.5 days. Postmortem examinations were performed. [1]
- Survival of BTT tumor‑bearing T739 mice: BTT cells in logarithmic phase (10⁷/mL) were injected subcutaneously (0.1 mL) into the right axilla of T739 mice. When tumor diameter reached 1.5‑2 cm, mice were euthanized, immersed in 75% ethanol for 2 min, and tumor tissue collected aseptically. Tumor tissues were homogenized to obtain single‑cell suspensions, washed twice with normal saline, and resuspended at 5×10⁶/mL. 0.2 mL of this suspension was inoculated into the abdominal cavity of T739 mice. After 24 h, mice were randomly divided into 5 groups (n=10): control, mitomycin (1 mg/kg), and Protosappanin B (100, 200, or 300 mg/kg). Drugs or normal saline (0.2 mL) were injected intraperitoneally daily for 6 consecutive days. Survival time was measured over a 60‑day observation period (60 days recorded as maximum). Rates of extended survival were calculated. [1]

[1]. Antitumor Effects of Purified Protosappanin B Extracted From Lignum Sappan. Integr Cancer Ther. 2016 Mar;15(1):87-95.

[2]. Protosappanin B promotes apoptosis and causes G1 cell cycle arrest in human bladder cancer cells. Sci Rep. 2019 Jan 31;9(1):1048.

- Protosappanin B is a polyphenolic compound with molecular formula C₁₆H₁₆O₆ and molecular weight 304.09. It is one of the two major components of Lignum Sappan, along with brazilin, and is listed in the Chinese Pharmacopoeia as an indicator of quality for Lignum Sappan preparations. [1][2]
- The antitumor mechanism of Protosappanin B in bladder cancer cells involves induction of apoptosis (via decreased Bcl‑2 and increased Bax expression) and G₁ phase cell cycle arrest, preventing transition from G₁ to S phase. Proteomics analysis confirmed that cell cycle‑related proteins are significantly affected. [2]
- Protosappanin B has been shown to possess anti‑inflammatory, antibacterial, antioxidant, and neuroprotective (protection of PC12 cells against oxygen‑glucose deprivation‑induced apoptosis) properties. [2]
- The compound is used clinically at 0.5 mg/kg. [2]
- In vivo studies suggest that Protosappanin B treatment is safe with no apparent toxicity at the tested doses (100‑300 mg/kg/day for 6 days). [1]

C16H16O6

304.29

304.094

102036-29-3

13846689

Light yellow to orange solid powder

1.5±0.1 g/cm3

655.5±55.0 °C at 760 mmHg

350.2±31.5 °C

0.0±2.1 mmHg at 25°C

1.700

1.13

5

6

1

22

391

0

QRTYTQTVJQUCEP-UHFFFAOYSA-N

InChI=1S/C16H16O6/c17-7-16(21)6-9-3-13(19)14(20)5-12(9)11-2-1-10(18)4-15(11)22-8-16/h1-5,17-21H,6-8H2

10-(hydroxymethyl)-8-oxatricyclo[10.4.0.02,7]hexadeca-1(16),2(7),3,5,12,14-hexaene-5,10,14,15-tetrol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~50 mg/mL (~164.32 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.84 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.84 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.84 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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