Natural product from Aloe vera; Emodin metabolite

Natural anthraquinone compounds have emerged as potent anticancer chemotherapeutic agents because of their promising DNA-binding properties. Aloe vera is among one of the very well-known medicinal plants, and the anthraquinone derivatives like aloe emodin (ALM), aloins (ALN), and Aloe emodin-8-glucoside (ALMG) are known to have immense biological activities. Here, we have used biophysical methods to elucidate the comparative DNA-binding abilities of these three molecules. Steady-state fluorescence study indicated complexation between calf thymus DNA (ctDNA) and both the molecules ALM and ALMG whereas ALN showed very weak interaction with DNA. Displacement assays with ctDNA-bound intercalator (ethidium bromide) and a groove binder (Hoechst 33258) indicated preferential binding of both ALM and ALMG to minor groove of DNA. Isothermal titration calorimetric (ITC) data suggested spontaneous exothermic single binding mode of both the molecules: ALM and ALMG. Entropy is the most important factor which contributed to the standard molar Gibbs energy associated with relatively small favorable enthalpic contribution. The equilibrium constants of binding to ctDNA were (6.02 ± 0.10) × 104 M-1 and (4.90 ± 0.11) × 104 M-1 at 298.15 K, for ALM and ALMG, respectively. The enthalpy vs temperature plot yielded negative standard molar heat capacity value, and a strong negative correlation between enthalpy and entropy terms was observed which indicates the enthalpy entropy compensation behavior in both systems. All these thermodynamic phenomena indicate that hydrophobic force is the key factor which is involved in the binding process. Moreover, the enhancement of thermal stability of DNA helix by ALM and ALMG fully agreed to the complexation of these molecules with DNA [1].

Fluorescence spectroscopy studies [1]
Fluorescence titration study of the aloe active compounds in the presence of ctDNA was performed by recording the emission spectra on a Hitchi-F4010 fluorimeter using quartz cuvette (fluorescence free) of 1-cm path length. ALM and Aloe emodin-8-glucoside (ALMG) both have characteristic fluorescence emission maxima at 535 nm (Figure 2A) and 576 nm (Figure 2C) when excited at their absorption maxima at 430 and 410 nm, respectively, although ALN does not show any fluorescent property (Figure 2B). The fluorescence emission spectrum of free molecule of known concentration was monitored, and then, the changes in the intensity with increasing concentration of ctDNA were recorded. All the titrations were performed at 298.15 K. The data obtained from spectrofluorimetric titrations of ALM and Aloe emodin-8-glucoside (ALMG) were fitted with Scatchard plot of (r/Cf) versus r, where r denotes the moles of the bound ligand per mole of DNA and Cf is the concentration of the free ligand in solution. The fits were analyzed by the Origin 7.0 using the cooperative McGhee-von Hippel equation,39 r/Cf = Ki(1 − nr)[(1 − nr) / {1 − (n − 1)r}](n − 1), where Ki is the intrinsic binding constant to an isolated DNA-binding site, whereas n is the number of DNA base pairs excluded because of binding of single ligand molecule.

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Competitive dye displacement assay [1]
Dye displacement experiments were carried out in Shimadzu RF-5301PC spectrofluorometer using two well-established DNA-binding dyes, ethidium bromode (EtBr), an intercalator, and Hoechst 33258, a minor groove binder. In a typical experiment, dye-DNA complex was formed which gave a significant fluorescence signal at respective emission maxima. Aloe active compound was then added to the complex gradually with increasing concentration, and the changes in emission maxima were studied at 298.15 K.40 Drug that binds to DNA would be able to compete and displace the dyes at their respective binding site leading to fluorescence quenching of the DNA-dye complexes.

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Isothermal titration calorimetry [1]
Isothermal titration calorimetry studies were carried out in a MicroCal VP-ITC unit at 298.15 K. The solutions were prepared in degassed buffer as reported earlier.22 Ligand solution (1.4235 mL) kept in the isothermal sample chamber was titrated by the DNA solution, injected from a rotating syringe (290 rpm). After injection of DNA solution (10 μL aliquots) into the isothermal chamber containing the ligand solution, a heat burst curve (microcalories per second) was generated, which, on integration with respect to time, gave the measure of total heat associated per injection. The heat of dilution of DNA was measured by injecting identical volume of same DNA solution into the buffer alone by keeping all other parameters same. The actual heat of reaction per injection was calculated by subtracting the corresponding heat of dilution and was plotted against the molar ratio (ligand)/(DNA). The data obtained here were fitted and analyzed with a one site binding model using Origin 7.0 software, which provided the binding affinity (K), binding stoichiometry (N), and enthalpy of binding (∆H°). The free energies (∆G°) and TΔS° were calculated using the standard thermodynamic relationship.

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Differential scanning calorimetry [1]
The influence of aloe active molecules on thermal denaturation of DNA was evaluated by differential scanning calorimeter (DSC). The system (both the sample and the reference cells) was equilibrated with the degassed buffer solution at 30°C for 15 minutes. After equilibration, the sample solution was scanned from 35°C to 100°C with a rate of 60°C/h, and the excess heat capacities were measured with the change in temperature and repeated for several times until a stable overlapping baseline was achieved. Then, on the cooling cycle, the buffer solution was removed from the sample cell only, and it was again loaded with ctDNA solution and its complex and scanned to get the DSC thermogram of DNA helix melting. The plot of excess heat capacity versus temperature was analyzed (Origin 7.0 software) to determine the sharp peak of transition temperature (Tm) at which excess heat capacity is at the maximum. The model-independent calorimetric transition enthalpy (ΔHcal) and the model-dependent van't Hoff enthalpy (ΔHv) were also obtained from the analysis.

[1]. Interaction of aloe active compounds with calf thymus DNA. J Mol Recognit. 2019 Oct;32(10):e2786.

Emodin-8-glucoside is a dihydroxyanthraquinone. It has been reported to be found in Rumex nepalensis, Rheum palmatum, and several other organisms with relevant data. See also: Rhinotria multiflora root (partial). Naturally occurring anthraquinones are important therapeutic molecules due to their known anticancer properties. This paper investigates the interactions between three anthraquinone derivatives from aloe plants—ALM, ALN, and ALMG—and circulating tumor DNA (ctDNA) using fluorescence spectroscopy, differential scanning calorimetry (DSC), melting curve analysis, and isothermal titration calorimetry. Steady-state fluorescence titration data showed that both ALM and ALMG could form complexes with ctDNA, with binding affinities of (6.76 ± 0.12) × 10⁴ M⁻¹ and (4.31 ± 0.14) × 10⁴ M⁻¹, respectively, while ALN showed extremely low affinity for ctDNA. Competitive dye substitution experiments indicated that ALM and ALMG molecules bind to ctDNA in a non-intercalation manner. Significant fluorescence quenching in the Hoescht substitution experiment suggested that these two molecules may bind to ctDNA via minor groove binding rather than intercalation. DSC thermogram data also perfectly matched the results indicating that complex formation led to the stabilization of double-stranded DNA. The binding affinities of ALM and ALMG obtained from ITC data were (6.02 ± 0.10) × 10⁴ M⁻¹ and (4.90 ± 0.11) × 10⁴ M⁻¹, respectively, very close to the fluorescence spectral data. The binding process in both cases showed negative enthalpy change and positive entropy change, as well as negative Gibbs free energy, indicating that there is a favorable exothermic spontaneous binding within a certain temperature range. The binding of ALM and ALMG is mainly driven by entropy, which can be seen from the thermodynamic parameters. The contribution of enthalpy change is small, which again shows that the molecules have small groove binding characteristics, which is due to the release of water molecules during the formation of the complex. The negative standard molar heat capacity change and enthalpy-entropy compensation phenomenon indicate that significant non-covalent hydrophobic components contribute to the binding process. In summary, this study demonstrates the effectiveness of structurally homologous aloe active compounds ALM, ALN and ALMG as DNA targeting molecules, and may help design molecules with important therapeutic value derived from natural products. [1]

C21H20O10

432.381

432.105

C, 58.34; H, 4.66; O, 37.00

23313-21-5

99649

Light yellow to yellow solid powder

1.7±0.1 g/cm3

798.7±60.0 °C at 760 mmHg

284.2±26.4 °C

0.0±3.0 mmHg at 25°C

1.725

1.71

6

10

3

31

700

5

O1[C@]([H])(C([H])(C([H])([C@@]([H])(C1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1=C([H])C(=C([H])C2C(C3C([H])=C(C([H])([H])[H])C([H])=C(C=3C(C1=2)=O)O[H])=O)O[H]

HSWIRQIYASIOBE-JNHRPPPUSA-N

InChI=1S/C21H20O10/c1-7-2-9-14(11(24)3-7)18(27)15-10(16(9)25)4-8(23)5-12(15)30-21-20(29)19(28)17(26)13(6-22)31-21/h2-5,13,17,19-24,26,28-29H,6H2,1H3/t13-,17-,19+,20-,21-/m1/s1

1,6-dihydroxy-3-methyl-8-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyanthracene-9,10-dione

Emodin 8-O-β-D-glucopyranoside; Emodin 8-glucoside; Emodin 8-β-D-glucoside; Emodin 8glucoside; NSC257449; Emodin-8-glucoside; 23313-21-5; Emodin 8-glucoside; Emodin glucoside B; Anthraglycoside B; emodin 8-O-glucoside; Emodin-8-beta-D-glucoside; Emodin-1 (8)-monoglucoside; NSC 257449; NSC-257449; Emodin 8 glucoside

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 (~115.64 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.81 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: 1.67 mg/mL (3.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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.)