High cytotoxic activity is demonstrated by manool against HeLa (IC50=6.7 µg/mL) and U343 (IC50=6.7 µg/mL) cells [1]. In HepG2 cells, manool has a chromosomal damage-inducing effect that prevents damage [3].

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Manool exhibited cytotoxic activity against several cancer cell lines. The IC50 values (μg/mL) were: B16F10 (murine melanoma) 15.6 ± 0.1; MCF-7 (human breast adenocarcinoma) 17.1 ± 0.8; HeLa (human cervical adenocarcinoma) 6.7 ± 1.1; HepG2 (human hepatocellular carcinoma) 28.5 ± 0.3; MO59J (human glioblastoma) 9.6 ± 0.8; U343 (human glioblastoma) 6.7 ± 1.2; U251 (human glioblastoma) 13.1 ± 1.6. The IC50 for normal V79 cells (Chinese hamster lung fibroblasts) was 49.3 ± 3.3 μg/mL, indicating selectivity. The selectivity index (SI) for HeLa and U343 was 7.4. In comparison, positive controls doxorubicin (DXR), (S)-(+)-camptothecin (CPT), and etoposide (VP16) showed varying IC50 values across cell lines. [1]
Manool (isolated from Greek propolis) showed antiproliferative activity against HT-29 human colorectal adenocarcinoma cells (IC50 = 6.5 ± 0.3 μg/mL; 22.3 ± 0.9 μM), HT-1080 fibrosarcoma cells (IC50 = 19.3 ± 2.2 μg/mL; 66.5 ± 7.7 μM), and HL-60 human promyelocytic leukemia cells (IC50 = 15.6 ± 1.2 μg/mL; 51.2 ± 3.8 μM). Against normal human skin fibroblasts (AG01523c, DSF9, DSF60), IC50 values were >29 μg/mL (>100 μM). [2]
Manool (at 50 μM; 14.5 μg/mL) incubated with exponentially growing HT-29 cells for 24-48 hours exerted cytostatic activity, reduced the percentage of malignant cells in the S phase, and blocked cells in the G2/M phase of the cell cycle, increasing the percentage of cells at G2/M by approximately 2.5-fold compared to control. [2]
Manool demonstrated genotoxic effects at higher concentrations. In V79 cells, a concentration of 6.0 μg/mL caused a significant increase in micronuclei frequency (12.00 ± 1.00) compared to negative control (3.33 ± 0.58). In HepG2 cells, a concentration of 8.0 μg/mL caused a significant increase in micronuclei frequency (29.39 ± 3.79) compared to negative control (9.33 ± 0.58). [3]
Manool exhibited antigenotoxic effects against methyl methanesulfonate (MMS)-induced DNA damage in HepG2 cells but not in V79 cells. In HepG2 cells, co-treatment with 0.5 μg/mL manool + MMS (44 μg/mL) reduced micronuclei frequency to 37.33 ± 0.58 (33.33% reduction), and 2.0 μg/mL manool + MMS reduced micronuclei frequency to 30.67 ± 3.51 (49.19% reduction), both significantly different from MMS alone (51.33 ± 4.93). No significant reduction was observed in V79 cells. [3]

Cytotoxicity testing (XTT colorimetric assay): Cells (10⁴ per well) were plated in 96-well microplates. Each well received 100 μL of medium containing different concentrations of Manool (0.49 - 1000 μg/mL, diluted in DMSO with final concentration ≤0.1%). After 24 hours incubation at 37°C in 5% CO₂, culture medium was removed, cells were washed with phosphate-buffered saline, and exposed to 100 μL phenol red-free medium. Then 25 μL XTT was added to each well and plates were incubated for 17 hours at 37°C. Absorbance was read at 450 nm with reference at 620 nm. Doxorubicin, (S)-(+)-camptothecin, and etoposide were used as positive controls. [1]
Cytotoxicity assay (MTT method): Cells (approximately 5,000 per well) were plated in 96-well flat-bottomed microplates. After 24 hours, test compounds (including Manool) were added, appropriately diluted with DMSO. After 72 hours incubation, medium was replaced with MTT (1 mg/mL) in serum-free, phenol red-free medium for 4 hours. The MTT formazan was solubilized in 2-propanol, and optical density was measured at 550 nm (reference 690 nm). Daunorubicin hydrochloride was used as positive control. [2]
Cell cycle analysis: Exponentially growing HT-29 cells were incubated with Manool (50 μM) for 24 or 48 hours. Treated cultures were trypsinized, washed in phosphate-buffered saline, fixed in 50% ethanol, and stained with RNase-containing propidium iodide solution. DNA content was analyzed by flow cytometry. [2]
Colony-forming assay (cytotoxicity): V79 cells were treated with Manool concentrations ranging from 1.0 to 256 μg/mL for 3 hours. Negative control, solvent control (DMSO, 14.5 μg/mL), and positive control (MMS, 110 μg/mL) were included. Then 300 cells were seeded per culture flask (three flasks per concentration). Experiments were carried out for 10 days. Cells were fixed in methanol/acetic acid/distilled water (1:1:8) for 30 minutes and stained with 3% Giemsa for 30 minutes. Survival fraction (FS%) was calculated as (number of colonies in treatment / number of colonies in negative control) × 100. [3]
Cytokinesis-block micronucleus assay (genotoxicity and antigenotoxicity): V79 or HepG2 cells were seeded into culture flasks and incubated for 25 hours. Cells were treated with Manool alone (V79: 0.5, 1.0, 2.0, 4.0, 6.0 μg/mL for 3 hours; HepG2: 0.5, 1.0, 2.0, 4.0, 8.0 μg/mL for 24 hours) or combined with MMS (44 μg/mL). After treatment, cells were washed with PBS and fresh medium containing cytochalasin B (3 μg/mL) was added (V79 for 17 hours, HepG2 for 24 hours). Cells were then trypsinized, centrifuged, hypotonized in 1% sodium citrate at 37°C, fixed in methanol:acetic acid (3:1), and stained (V79 with 3% Giemsa for light microscopy; HepG2 with acridine orange and ethidium bromide for fluorescence microscopy). A total of 3000 binucleated cells were scored per treatment. The nuclear division index (NDI) and cytotoxicity index (CI) were calculated. [3]

Manool showed selective cytotoxicity: it was significantly more cytotoxic against tumor cell lines than against normal V79 cells (IC50 49.3 ± 3.3 μg/mL). The selectivity index (SI) for HeLa and U343 cells was 7.4. [1]
Manool showed low toxicity to normal human skin fibroblasts (AG01523c, DSF9, DSF60) with IC50 values >29 μg/mL (>100 μM). [2]
Manool exhibited cytotoxic effects in V79 cells starting at 8.0 μg/mL as assessed by colony-forming assay. Concentrations of 8.0, 16.0, 32.0, 64.0, 128.0, and 256.0 μg/mL significantly reduced viability compared to negative control. [3]
Manool was genotoxic at the highest concentration tested in V79 cells (6.0 μg/mL) with a significant increase in micronuclei frequency (12.00 ± 1.00 vs. control 3.33 ± 0.58). No significant increase was observed at lower concentrations (0.5, 1.0, 2.0, 4.0 μg/mL). In HepG2 cells, genotoxicity was observed at 8.0 μg/mL (micronuclei frequency 29.39 ± 3.79 vs. control 9.33 ± 0.58). The nuclear division index (NDI) for HepG2 at 8.0 μg/mL was 1.58 ± 0.06 (significantly lower than control 1.74 ± 0.05), with a cytotoxicity index (CI) of 21.62%. No significant cytotoxicity was observed for other concentrations in either cell line as measured by NDI and CI. [3]

[1]. Manool, a Salvia officinalis diterpene, induces selective cytotoxicity in cancer cells. Cytotechnology. 2016;68(5):2139-2143.

[2]. Antiproliferative activity of Greek propolis. J Med Food. 2010;13(2):286-290.

[3]. Differential effect of manool--a diterpene from Salvia officinalis, on genotoxicity induced by methyl methanesulfonate in V79 and HepG2 cells. Food Chem Toxicol. 2014;72:8-12.

Manolochic acid is a latanane diterpenoid compound with double bonds at positions 8(17) and 14 of its latanane skeleton and an R-hydroxyl group at position 13. It possesses antitumor activity and is a plant metabolite and antibacterial agent. It is a latanane diterpenoid compound and also a tertiary alcohol. Manolochic acid has been reported in Glycosmis pentaphylla, Salvia officinalis, and other organisms with relevant data.

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According to the US National Cancer Institute, substances presenting IC50 values < 30 μg/mL in tumor cell lines are considered promising candidates for anticancer drug development. Manool meets this criterion for several cancer cell lines (HeLa, U343, MO59J, B16F10, MCF-7, U251). [1]
Manool has been previously identified by GC/MS in Brazilian propolis and in propolis from various regions of Greece. The present study isolated it from Greek propolis for the first time, confirming its chemical structure by NMR, MS, and optical rotation data compared to literature values. [2]
Manool is a labdane-type diterpene. Labdane-type diterpenoids have been suggested to possess potential for anticancer drug development. Other labdane-type diterpenoids (yunnanconorarin A and B, villosin, ellipticine) have shown interesting IC50 values against various cancer cell lines. [1]
Manool exhibits characteristics of a "Janus compound" (a term for substances that behave as genotoxic or antigenotoxic agents depending on experimental conditions). It was genotoxic at the highest concentration tested but exerted chemopreventive effects against MMS-induced chromosome damage at lower concentrations in HepG2 cells. [3]
The differential effect of Manool on MMS-induced genotoxicity in V79 vs. HepG2 cells may be due to biotransformation occurring in HepG2 cells, which possess phase I and II metabolism, active Nrf2 electrophile responsive system, functionally active p53 protein, active DNA repair, and protective enzymes such as superoxide dismutases, catalase, glutathione peroxidase, glutathione reductase, and thioredoxin reductase. [3]

C20H34O

290.48336

290.261

596-85-0

3034394

White to off-white solid

0.93g/cm3

368.2ºC at 760mmHg

49-52ºC(lit.)

118.2ºC

1.5

5.502

1

1

4

21

422

4

C=C[C@@](CC[C@@H]1C(=C)CC[C@@H]2C(CCC[C@@]12C)(C)C)(C)O

CECREIRZLPLYDM-QGZVKYPTSA-N

InChI=1S/C20H34O/c1-7-19(5,21)14-11-16-15(2)9-10-17-18(3,4)12-8-13-20(16,17)6/h7,16-17,21H,1-2,8-14H2,3-6H3/t16-,17-,19-,20+/m0/s1

(3R)-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylidene-3,4,4a,6,7,8-hexahydro-1H-naphthalen-1-yl]-3-methylpent-1-en-3-ol

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.

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 (~344.26 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.61 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.61 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.61 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.

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