Antimycobacterial

The polyacetylene (3R,8S)-Falcarindiol and the furanocoumarins bergapten, isobergapten, angelicin, sphondin, pimpinellin, isopimpinellin and 6-isopentenyloxyisobergapten were isolated from the Heracleum maximum root extract. (3R,8S)-Falcarindiol and 6-isopentenyloxyisobergapten exhibited MICs of 24 μM and 167 μM and IC50s of 6 μM and 27 μM against Mycobacterium tuberculosis H37Ra respectively. The remaining furanocoumarins bergapten, isobergapten, angelicin, sphondin, pimpinellin, and isopimpinellin were less active, with MICs of 925, 1850, 2149, 1859, 812 and 1625 μM and IC50s of 125, 344, 350, 351, 389 and 406 μM. Conclusions: (3R,8S)-Falcarindiol, bergapten, isobergapten, angelicin, sphondin, pimpinellin, isopimpinellin and 6-isopentenyloxyisobergapten were identified as the principal constituents responsible for the antimycobacterial activity of the roots of Heracleum maximum. This work supports the ethnopharmacological use of Heracleum maximum by Canadian First Nations and Native American communities as a treatment for infectious diseases, specifically tuberculosis. [1]

Objectives: (3R)-Falcarinol (FaOH) and (3R,8S)-Falcarindiol (FaDOH) have previously been shown to reduce the number of neoplastic lesions and the growth rate of polyps in the colon of azoxymethane (AOM) treated rats. Based on previous investigations, it appears that different mechanisms of actions are involved in the antineoplastic effect of FaOH and FaDOH. One mechanism of action may be related to the antibacterial effect of FaOH and FaDOH and thus their effect on the gut microbiota. This study aimed to determine the effect of FaOH and FaDOH on gut microbiota composition of AOM treated rats. Results: Azoxymethane treated rats were fed either a standard rat diet or a rat diet supplemented with FaOH and FaDOH. The gut microbiota of AOM-induced rats was determined by 16S rRNA gene-amplicon sequencing. Analysis of fecal cecum samples demonstrated a significant gut microbiota change in rats receiving standard rat diet supplemented with FaOH and FaDOH compared with the control group that only received the rat diet. Comparison of the gut microbiota of rats who developed large neoplasms in the colon with rats without large neoplasms showed that the gut microbiota was significantly different in rats who developed large colon neoplasms compared to rats with no macroscopic colon neoplasms.[2]

Determination of minimum inhibitory concentrations (MIC) and median inhibitory concentrations (IC50) [1]
MIC and IC50 values were determined against Mycobacterium tuberculosis H37Ra using the MRA as described in Section 2.4. Stock solutions (800 µg/mL) in sterile 4% DMSO in MGIT growth medium and used immediately. Serial twofold dilutions of test compound solutions (100 µL) were performed in 96-well microtitre assay plates with MGIT growth medium (100 µL) to give a series of 12 concentrations (400–0.20 µg/mL) in triplicate. However, when 4, 5 and 6 were tested at concentrations greater than 100 µg/mL, inconsistent results were obtained due to problems associated with the solubility of these compounds. Compounds 4, 5 and 6 were therefore tested at a more dilute series of 12 concentrations (100, 75, 50, 37.5, 25, 18.75, 12.5, 9.38, 6.25, 4.69, 3.13, 2.34 µg/mL; obtained by twofold serial dilutions of 200 and 150 µg/mL solutions in 4% DMSO). The MIC of a compound was considered to be the lowest concentration at which it inhibited mycobacterial growth by more than a mean value of 90% (Collins and Franzblau, 1997) and the corresponding IC50 was estimated by fitting a four parameter logistic (4PL) curve (Sebaugh, 2011) to the mycobacterial growth data using Masterplex 2010 Readerfit (Hitachi). In cases when the data were insufficient to obtain reliable estimates by four parameter logistic regression (Sebaugh, 2011), absolute IC50s were obtained by probit analysis (Finney, 1971, Morgan, 1992) performed by fitting percentage inhibition values calculated from the growth data to the probit model by the maximum likelihood method (Mantel et al., 1985) using SPSS Statistics 20 (IBM).

Powder/meal maintenance rat diet was used as standard diet for feeding the rats. Diet group 1 received standard rat diet supplemented with 7 µg FaOH/g feed and 7 µg (3R,8S)-Falcarindiol/FaDOH/g feed. The polyacetylenes FaOH and FaDOH were isolated from carrots by flash chromatography and preparative HPLC and identified by liquid chromatography tandem mass spectrometry (LC–MS/MS), NMR spectroscopy and optical rotation as described previously. Diet group 2 only received standard rat diet. Because the purified FaOH and FaDOH (purity > 99%) was added to the diet in the form of an ethanol solution, the diet of the control group (group 2) was added the same amount of ethanol. Portions of 3.5 kg diet were prepared weekly for each of the two groups. The concentrations of FaOH and (3R,8S)-Falcarindiol/FaDOH in the rat diets were determined by LC–MS/MS before use. Diet group 2 was used as a negative control. No sign of degradation, oxidation or isomerization of FaOH and FaDOH was observed during the animal study as well as no significant differences in the content of FaOH or FaDOH in the weekly prepared diet [2].

[1]. The Canadian medicinal plant Heracleum maximum contains antimycobacterial diynes and furanocoumarins. J Ethnopharmacol. 2013 May 2;147(1):232-7.

[2]. Effect of the dietary polyacetylenes falcarinol and falcarindiol on the gut microbiota composition in a rat model of colorectal cancer. BMC Res Notes. 2018 Jun 27;11(1):411.

Falcarindiol has been reported in Anthriscus nitida, Eleutherococcus koreanus, and other organisms with data available.

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The weak antimycobacterial activity observed for 2–8 in conjunction with the significant levels of general cytotoxicity typically displayed by furanocoumarins (Santana et al., 2004) indicates that the furanocoumarins isolated from Heracleum maximum are unlikely to provide promising therapeutic leads. However, reports that falcarindiol displays very low levels of toxicity towards mammalian cells both in vitro (Deng et al., 2008, Inui et al., 2010) and in vivo (Matsuda et al., 1998, Ohnuma et al., 2011) suggest that it would have a favorable therapeutic index as an antimycobacterial agent. It is therefore surprising to note that, despite the antimycobacterial activity of falcarindiol stereoisomers being reported previously (Deng et al., 2008, Inui et al., 2010, Kobaisy et al., 1997, Lechner et al., 2004, Schinkovitz et al., 2008, Stavri and Gibbons, 2005), little is known about the mechanism by which this and related C-17 diynes inhibit the growth of mycobacteria (Li et al., 2012, Schinkovitz et al., 2008). The isolation of antimycobacterial compounds from extracts of the root of Heracleum maximum supports the traditional medicinal uses of this plant by the indigenous peoples of North America as a treatment for respiratory ailments that include tuberculosis. Our research has provided further evidence that medicinal plants used by the First Nations communities of the Canadian Maritime provinces represent an important source of biologically active compounds and has identified (3R,8S)-Falcarindiol and related antimycobacterial C-17 diynes as natural products that may possesses significant potential to contribute to the development of antimycobacterial agents in the future.[1]

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In conclusion, this study revealed that FaOH and (3R,8S)-Falcarindiol/FaDOH, which have previously been shown to inhibit the formation of neoplastic tumors in the colon in a rat model of colon cancer, affect the composition of low abundant GM members, which in turn is associated with a reduced formation of macroscopic neoplasms. Thus, the present investigation has shown that changes in the GM may play an important role in the preventive effect of FaOH and FaDOH towards neoplastic transformation in the colon. Limitations Chronic infection and inflammation contributes to CRC, although there is growing evidence that the GM play an important role in the progression of this disease. Even though microbiota-based cancer prevention, diagnosis, and therapy in humans are beginning to emerge, we still need more information about the microbiota composition to identify, which changes in the GM that may result in a preventive effect towards CRC in humans as well as in animals. Consequently, we are not able to conclude, whether the significant changes of the low abundant GM members in the microbiota of rats receiving FaOH and FaDOH in the diet compared with the control group, are essential, and thus contribute to an explanation to the preventive effects of FaOH and FaDOH towards CRC in the AOM treated rats. [2]

C17H24O2

260.37126

260.177

C, 78.42; H, 9.29; O, 12.29

225110-25-8

Falcarindiol;55297-87-5

5281148

Colorless to light yellow liquid

1.0±0.1 g/cm3

408.2±45.0 °C at 760 mmHg

184.7±23.3 °C

0.0±2.2 mmHg at 25°C

1.524

6.32

2

2

9

19

394

2

CCCCCCC/C=C\[C@@H](C#CC#C[C@@H](C=C)O)O

QWCNQXNAFCBLLV-YWALDVPYSA-N

InChI=1S/C17H24O2/c1-3-5-6-7-8-9-10-14-17(19)15-12-11-13-16(18)4-2/h4,10,14,16-19H,2-3,5-9H2,1H3/b14-10-/t16-,17+/m1/s1

(3R,8S,9Z)-heptadeca-1,9-dien-4,6-diyne-3,8-diol

Falcarindiol; (3S,8S)-Falcarindiol; (3R,8S,9Z)-heptadeca-1,9-dien-4,6-diyne-3,8-diol; AC1NQY3Z; Falcalindiol; 55297-87-5; Heptadeca-1,9-diene-4,6-diyne-3,8-diol; ...; 225110-25-8;

2934.99.9001

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 : ~150 mg/mL (~576.10 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.60 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 (9.60 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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 (Please use freshly prepared in vivo formulations for optimal results.)