Betulinic Acid

Due to the disadvantages of biological experiments as being time-consuming and of high cost, identification of ADME (absorption, distribution, metabolism, and excretion) properties by in silico tools has now become an inevitable paradigm in pharmaceutical research. In this study, three ADME-related models, namely, the evaluation of oral bioavailability (OB), Caco-2 permeability, and drug-likeness (DL), are employed to identify the potential bioactive compounds of DXP [33 (link)].
Oral Bioavailability. OB prescreening is used to determine the fraction of the oral dose of bioactive compound which reaches systemic circulation in the TCM remedy. Here, a reliable in silico model OBioavail 1.1 [34 (link)] which integrates the metabolism (P450 3A4) and transport (P-glycoprotein) information was employed to calculate the OB values of herbal ingredients.
Caco-2 Permeability. The Caco-2 cell monolayers are widely applied as standard permeability-screening assay for prediction of the compound's intestinal absorption and fraction of the oral dose absorbed in humans [35 (link)]. The Caco-2 cell permeation values of all molecules are calculated by in silico model using the VolSurf approach [36 ].
Drug-Likeness Evaluation. Drug-likeness is a qualitative profile used in drug design to evaluate whether a compound is chemically suitable for the drug, and how drug-like a molecule is with respect to parameters affecting its pharmacodynamic and pharmacokinetic profiles which ultimately impact its ADME properties [37 (link)]. In order to identify drug-like compounds, we apply a database-dependent model using the Tanimoto coefficient to calculate the DL (see (1)) of each compound in DXP. $\begin{array}{c}f\left(\mathcal{x},\mathcal{y}\right)=\frac{\mathcal{x}\mathcal{y}}{{\left|\mathcal{x}\right|}^{\mathrm{2}}+{\left|\mathcal{y}\right|}^{\mathrm{2}}-\mathcal{x}\mathcal{y}}.\end{array}$ 𝓍 represents the molecular parameters of herbal ingredients and 𝓎 represents the average molecular properties in DrugBank database (available online: http://www.drugbank.ca).
In this work, the compounds of OB ≥ 30%, Caco-2 > −0.4, and DL ≥ 0.18 are selected for subsequent research, and others are excluded.
According to these indexes, several compounds are included: ergosterol peroxide, ethyl oleate (NF), glabridin, glycyrrhetinic acid, linoleyl acetate, longikaurin A, mairin, mandenol, MOL000273, MOL001910, 508-02-1, 64997-52-0, 8β-ethoxy atractylenolide III, pachymic acid, paeonidanin, palbinone, saikosaponin C, beta-sitosterol, supraene, trametenolic acid, troxerutin, α-amyrin, MOL000285, 4-O-methylpaeoniflorin, glabrene, poricoic acid A, glycyrrhizin, sudan III, ZINC02816192, kaempferol, 7,9(11)-dehydropachymic acid, licochalcone G, paeoniflorgenone, areapillin, quercetin, stigmasterol, isoliquiritigenin, (+)-anomalin, isorhamnetin, vestitol, crocetin, 113269-36-6, α-spinasterol, licochalcone A, 113269-37-7, 3β-acetoxyatractylone, licoricone, 113269-39-9, petunidin, hederagenin, dehydroeburicoic acid, licochalcone B, ergosta-7,22E-dien-3beta-ol, MOL000280, MOL000287, mudanpioside H, NSC684433, octalupine, 18103-41-8, formononetin, 1-methoxyphaseollidin, paeoniflorin, glycyrin, ammidin, poricoic acid B, poricoic acid C, sainfuran, sitosterol, isoimperatorin, isolicoflavonol, cerevisterol, 3-methylkempferol, licoisoflavone B, cubebin, and (+)-catechin, 3′-hydroxy-4′-O-methylglabridin.

Viscum album L. extracts were kindly supplied by Birken AG (Niefern-Oeschelbronn, Germany). Preparation of Viscum album L. extracts were performed as described before [47 , 51 ].
Sprouts from Viscum album L. were harvested from apple trees (Malus domestica Borkh.) and identified by the co-author S. Jaeger. Two different extracts were prepared to obtain the oleanolic acid-containing TT- extract and the mistletoe lectin containing viscum- extract. The combination of both extracts is further described as viscumTT.
Preparation of the oleanolic acid containing TT- extract: Oleanolic acid (OA) was extracted from dried plant material resulting in a dry extract containing 69.4% OA and 6.9% betulinic acid [52 (link)]. 100 mg of these triterpene acids were mixed with 2-HP-β-cyclodextrins (2-hydroxypropyl-β-cyclodextrin) and suspended in water. The dried (105°C) mixture was pestled, the resulting powder suspended in sodium dihydrogen phosphate buffer (30 mM, pH 8.0) and the mixture sonicated for 30 min. After adjusting the pH to 7.5 (100 mM phosphoric acid) the volume was made up to 25 mL (30 mM sodium phosphate buffer pH 7.5). After filtration OA was quantified in triplicate in each solution using GC-FID and external calibration with OA as reference substance (>97%, Extrasynthese, Genay Cedex, France) [30 (link)].
Preparation of the mistletoe lectin-containing viscum- extract: For the aqueous extract (lectin and viscotoxin-containing viscum) plant material was milled under liquid nitrogen using a cryo mill (Retsch, Germany) and extracted using ascorbate phosphate buffer (30 mM sodium phosphate, 3.4 g/L ascorbic acid, pH 9.1) resulting in a filtered (0.22 μm) extract pH 7.5. Preparation of viscumTT: Viscum album L. extracts (VAE) are combined to viscumTT by mixing of both extracts. The concentrations of the different extracts are described in Table 1.
Table 1 gives a schematic presentation of the concentrations of the applied Viscum album L. extracts TT, viscum and viscumTT. OA = oleanolic acid; ML = mistletoe lectin-I, BA = betulinic acid; CD = 2-hydroxypropyl-β-cyclodextrin.

All commercial reagents were purchased from Sigma-Aldrich and Acros organics (Verona, Italy). Betulinic acid was prepared from commercially available betulin by a reported procedure [16 (link)]. All commercially available solvents and reagents used were of analytical grade and without further purification. Reactions were monitored by TLC on Sorbfil plates. Column chromatography was carried out on Acrus silica gel (0.060–0.200 mm). Optical rotations were measured on a Perkin–Elmer 341 polarimeter (Waltham, MA, USA). Melting points were recorded on Stuart SMP3. IR spectra were recorded on Bruker VERTEX 70 V using KBr discs over the range of 400–4000 cm⁻¹ (Billerica, MA, USA). ¹H and ¹³C NMR spectra were obtained using a Bruker Ascend 500 spectrometer in CDCl₃ operating at 500 MHz for ¹H and 125 MHz for ¹³C and a Bruker AVANCE 400 spectrometer in CDCl₃ operating at 400 MHz for ¹H and 100 MHz for ¹³C. Mass spectra of MALDI TOF/TOF positive ions (matrix of sinapic acid) are recorded on a mass spectrometer Bruker AutoflexTM III Smartbeam. Elemental analyses were measured on 1106 Carlo Erba apparatus (Val de Reuil, France). Figures S1–S99 show the ¹H and ¹³C NMR spectra of the synthesized compounds.

Citrate-capped gold nanoparticles (GNP) were produced by employing trisodium citrate to reduce chloroaurate. Specifically, 68 mg (0.2 mmoles) of chloroauric acid (HauCl4, Merck KGaA, Darmstadt, Germany) is dissolved in 180 mL of deionized water with constant stirring. After bringing the solution to a boil, a 20-mL solution of sodium citrate dihydrate (Merck KGaA, Darmstadt, Germany) containing 176.5 mg (0.6 mmoles) is added. When the solution’s color turns ruby red, the heat is turned off and it is allowed to cool to room temperature for 24 h. UV-VIS spectroscopy confirmed the production of GNP. GNP was purified using successive centrifugations at 13,000× g, water removal and resuspension in water until a neutral pH value was reached.
Triterpene derivative-loaded GNP was obtained using the following method: 0.01 mmoles of benzotriazolyl esters of betulinic acid (BA-HOBt), oleanolic acid (OA-HOBt) and ursolic acid (UA-HOBt), respectively, were each added to 10 mL of previously obtained citrate-capped GNP nanosuspension. The suspension was sonicated for 1 h (0.8 cycles, 80% amplitude, 200 W) with a UP200S ultrasonic homogenizer (Hielscher Ultrasonics GmbH, Germany, Teltow) outfitted with a volume-appropriate sonotrode. After 10 min, all three samples began to turn purple, and by the end of the sonication cycle, all three samples were completely purple, indicating that GNP surface functionalization has occurred. The samples were kept at room temperature in the dark for 24 h. All three samples were later extracted with ethyl acetate (4 × 5 mL) (Merck KgaA, Darmstadt, Germany) in order to remove potentially unattached triterpene derivatives, after which each sample was purified using successive centrifugations at 13,000× g, water removal and resuspension in water (three cycles) until all three samples reached a neutral pH.