Natural product

At an IC50 of about 42 μM, rosmarinol demonstrated the greatest inhibitory effect on cell viability. Rosmarinol may prevent cancer cells from proliferating or from undergoing apoptosis, which would stop the cells from growing. Following administration of 50 μM rosmarinol, the proportion of COLO 205 cells undergoing apoptosis had a significant rise, rising from 29% at 9 hours to 51% at 24 hours. After being treated with 50 μM rosmarinol for 24 hours, COLO 205 cells showed notable morphological alterations and chromosomal condensation, which are indicative of apoptosis [1]. Lipid protectant known as carbosol fends against O2 assault [2].

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Rosemary (Rosmarinus officinalis), a culinary spice and medicinal herb, has been widely used in European folk medicine to treat numerous ailments. Many studies have shown that rosemary extracts play important roles in anti-inflammation, anti-tumor, and anti-proliferation in various in vitro and in vivo settings. The roles of tumor suppression of rosemary have been attributed to the major components, including carnosic acid, carnosol, and rosmarinic acid, Rosmanol, and ursolic acid. This study was to explore the effect of Rosmanol on the growth of COLO 205 human colorectal adenocarcinoma cells and to delineate the underlying mechanisms. When treated with 50 μM of rosmanol for 24h, COLO 205 cells displayed a strong apoptosis-inducing response with a 51% apoptotic ratio (IC(50) ∼42 μM). Rosmanol increased the expression of Fas and FasL, led to the cleavage and activation of pro-caspase-8 and Bid, and mobilized Bax from cytosol into mitochondria. The mutual activation between tBid and Bad decreased the mitochondrial membrane potential and released cytochrome c and apoptosis-inducing factor (AIF) to cytosol. In turn, cytochrome c induced the processing of pro-caspase-9 and pro-caspase-3, followed by the cleavage of poly-(ADP-ribose) polymerase (PARP) and DNA fragmentation factor (DFF-45). These results demonstrate that the rosmanol-induced apoptosis in COLO 205 cells is involvement of caspase activation and involving complicated regulation of both the mitochondrial apoptotic pathway and death receptor pathway.[1]

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In previous studies, other carnosic acid-derived metabolites, such as Rosmanol, epirosmanol, or rosmaridiphenol, also were found to possess some antioxidative capacities. For instance, carnosol, Rosmanol, and epirosmanol were able to inhibit the oxidation of lipoproteins in vitro (Zeng et al., 2001). Methyl carnosate was reported to be even more active than carnosic acid in the protection of triglyceride emulsions at 60°C (Huang et al., 1996). Rosmanol and epirosmanol were reported to inhibit mitochondrial and microsomal lipid peroxidation (Haraguchi et al., 1995), and the antioxidative activity of rosmanol and 20-deoxocarnosol was observed using the 2,2-diphenyl-1-picrylhydrazyl antioxidant assay (Escuder et al., 2002). The in vitro antioxidant activity of rosmanol, epirosmanol, and isorosmanol was found to be higher than that of α-tocopherol (Nakatani and Inatani, 1984). Thus, when scavenging ROS, carnosic acid can generate a variety of secondary antioxidants. This cascade-type process is likely to amplify the antioxidative power of carnosic acid and to constitute an effective defense mechanism. Moreover, ROS scavenging by carnosic acid can be fueled by the very large pools of this compound (representing several percentages of leaf dry weight) that rosemary plants are able to accumulate in their leaves [2].

Cell survival assay [1]
Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Briefly, COLO 205 cells were plated at a density of 1 × 105 cells/mL into 24-well plates. After overnight growth, cells were pretreated with a series of concentrations of Rosmanol for 24 h. The final concentrations of dimethyl sulfoxide in the culture medium were <0.1%. At the end of treatment, 30 μL of MTT was added, and cells were incubated for a further 4 h. Cell viability was determined by scanning with an ELISA reader with a 570-nm filter.

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DNA extraction and electrophoresis analysis [1]
After Rosmanol treatment, COLO 205 cancer cells were harvested, washed with phosphate-buffered saline (PBS), and then lysed with digestion buffer containing 0.5% sarkosyl, 0.5 mg/mL proteinase K, 50 mM tris(hydroxy methyl)aminomethan (pH 8.0), and 10 mM EDTA at 56 °C overnight and treated with RNase A (0.5 μg/mL) for 3 h at 56 °C. The DNA was extracted by phenol/chloroform/isoamyl (25:24:1) before loading and was analyzed by 2% agarose gel electrophoresis. The agarose gels were run at 50 V for 120 min in Tris–borate/EDTA electrophoresis buffer (TBE). Approximately 20 μg of DNA was loaded in each well and visualized under UV light, and photographed (Pan et al., 2001).

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Analysis of mitochondrial transmembrane potential [1]
The change of the mitochondrial transmembrane potential was monitored by flow cytometry. Briefly, COLO 205 cells were exposed to Rosmanol (50 μM) for different time periods and the mitochondrial transmembrane potential was measured directly using 40 nM 3,3′-dihexyloxacarbocyanine [DiOC6(3)]. Fluorescence was measured after staining of the cells for 30 min at 37 °C. Histograms were analyzed using Cell Quest software and were compared with histograms of control untreated cells.

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Western blotting [1]
The cellular proteins were isolated from COLO 205 cells after treatment with 50 μM Rosmanol for 0, 3, 6, 9, 12, and 24 h. The total proteins were extracted via the addition of 200 μL of gold lysis buffer (50 mM Tris–HCl, pH 7.4; 1 mM NaF; 150 mM NaCl; 1 mM EGTA; 1 mM phenylmethanesulfonyl fluoride; 1% NP-40; and 10 μg/mL leupeptin) to the cell pellets on ice for 30 min, followed by centrifugation at 10,000g for 30 min at 4 °C. The cytosolic fraction (supernatant) proteins were measured by Bio-Rad protein assay. The samples (50 μg of protein) were mixed with 5 × sample buffer containing 0.3 M Tris–HCl (pH 6.8), 25% 2-mercaptoethanol, 12% sodium dodecyl sulfate (SDS), 25 mM EDTA, 20% glycerol, and 0.1% bromophenol blue. The mixtures were boiled at 100 °C for 5 min and were subjected to 12% SDS–polyacrylamide minigels at a constant current of 20 mA. Subsequently, electrophoresis was ordinarily carried out on SDS–polyacrylamide gels. For electrophoresis, proteins on the gel were electrotransferred onto an immobile membrane with transfer buffer composed of 25 mM Tris–HCl (pH 8.9), 192 mM glycine, and 20% methanol.

[1]. Rosmanol potently induces apoptosis through both the mitochondrial apoptotic pathway and death receptor pathway in human colon adenocarcinoma COLO 205 cells. Food Chem Toxicol. 2011 Feb;49(2):485-93.

[2]. Carnosic Acid and Carnosol, Two Major Antioxidants of Rosemary, Act through Different Mechanisms. Plant Physiol. 2017 Nov;175(3):1381-1394.

Rosmanol has been reported in Salvia officinalis, Isodon grandifolius, and other organisms with data available.

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To sum up, our study suggested that the Rosmanol-induced apoptosis in COLO 205 cells was via both a mitochondrial pathway and a receptor-mediated pathway. Rosmanol could trigger the engagement of Fas and FasL, followed by the cleavage of caspase-8 and Bid. Rosmanol could also initiate the translocation of Bax to the mitochondrial membrane and cause the formation of membrane pores. Furthermore, the mutual effect between the death-receptor pathway and the mitochondrial pathway led to the decrease in the membrane potential of the mitochondria and the release of cytochrome c and AIF from the mitochondria. Cytochrome c is linked to the caspase-dependent apoptotic signaling, which causes the activation of the downstream caspase cascade, followed by the cleavage of PARP and DFF-45. AIF is involved in the caspase-independent responses and provokes chromatin condensation and DNA fragmentation. In conclusion, the rosmanol extracted from rosemary is capable of inducing apoptosis through both a mitochondria-mediated pathway and a receptor-mediated pathway in COLO 205 cells. These findings suggest that rosmanol may have clinical application in the prevention and treatment of cancer. [1]

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Carnosic acid, a phenolic diterpene specific to the Lamiaceae family, is highly abundant in rosemary (Rosmarinus officinalis). Despite numerous industrial and medicinal/pharmaceutical applications of its antioxidative features, this compound in planta and its antioxidant mechanism have received little attention, except a few studies of rosemary plants under natural conditions. In vitro analyses, using high-performance liquid chromatography-ultraviolet and luminescence imaging, revealed that carnosic acid and its major oxidized derivative, carnosol, protect lipids from oxidation. Both compounds preserved linolenic acid and monogalactosyldiacylglycerol from singlet oxygen and from hydroxyl radical. When applied exogenously, they were both able to protect thylakoid membranes prepared from Arabidopsis (Arabidopsis thaliana) leaves against lipid peroxidation. Different levels of carnosic acid and carnosol in two contrasting rosemary varieties correlated with tolerance to lipid peroxidation. Upon reactive oxygen species (ROS) oxidation of lipids, carnosic acid was consumed and oxidized into various derivatives, including into carnosol, while carnosol resisted, suggesting that carnosic acid is a chemical quencher of ROS. The antioxidative function of carnosol relies on another mechanism, occurring directly in the lipid oxidation process. Under oxidative conditions that did not involve ROS generation, carnosol inhibited lipid peroxidation, contrary to carnosic acid. Using spin probes and electron paramagnetic resonance detection, we confirmed that carnosic acid, rather than carnosol, is a ROS quencher. Various oxidized derivatives of carnosic acid were detected in rosemary leaves in low light, indicating chronic oxidation of this compound, and accumulated in plants exposed to stress conditions, in parallel with a loss of carnosic acid, confirming that chemical quenching of ROS by carnosic acid takes place in planta.[2]

C20H26O5

346.4174

346.178

C, 69.34; H, 7.57; O, 23.09

80225-53-2

13966122

White to off-white solid powder

1.33

574.186ºC at 760 mmHg

205.663ºC

1.625

3.257

3

5

1

25

572

4

O=C1O[C@H]2[C@@H]3[C@]1(CCCC3(C)C)C1C(O)=C(O)C(C(C)C)=CC=1[C@@H]2O

LCAZOMIGFDQMNC-FORWCCJISA-N

InChI=1S/C20H26O5/c1-9(2)10-8-11-12(15(23)13(10)21)20-7-5-6-19(3,4)17(20)16(14(11)22)25-18(20)24/h8-9,14,16-17,21-23H,5-7H2,1-4H3/t14-,16+,17-,20-/m0/s1

(1R,8S,9S,10S)-3,4,8-trihydroxy-11,11-dimethyl-5-propan-2-yl-16-oxatetracyclo[7.5.2.01,10.02,7]hexadeca-2,4,6-trien-15-one

Rosmanol; 80225-53-2; UNII-F25TV383OC; F25TV383OC; (1R,8S,9S,10S)-3,4,8-trihydroxy-11,11-dimethyl-5-propan-2-yl-16-oxatetracyclo[7.5.2.01,10.02,7]hexadeca-2,4,6-trien-15-one; 2H-10,4a-(Epoxymethano)phenanthren-12-one, 1,3,4,9,10,10a-hexahydro-5,6,9-trihydroxy-1,1-dimethyl-7-(1-methylethyl)-, (4aR,9S,10S,10aS)-; 2H-10,4a-(Epoxymethano)phenanthren-12-one, 1,3,4,9,10,10a-hexahydro-5,6,9-trihydroxy-1,1-dimethyl-7-(1-methylethyl)-, (4ar-(4aalpha,9beta,10alpha,10abeta))-; (6beta,7alpha)-7,11,12-Trihydroxy-6,20-epoxyabieta-8(14),9(11),12-trien-20-one;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (~288.67 mM)

Solubility in Formulation 1: ≥ 7.5 mg/mL (21.65 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 75.0 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: ≥ 7.5 mg/mL (21.65 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 75.0 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.)