The Antiprotozoal Activity of Papua New Guinea Propolis and Its Triterpenes

Profiling a propolis sample from Papua New Guinea (PNG) using high-resolution mass spectrometry indicated that it contained several triterpenoids. Further fractionation by column chromatography and medium-pressure liquid chromatography (MPLC) followed by nuclear magnetic resonance spectroscopy (NMR) identified 12 triterpenoids. Five of these were obtained pure and the others as mixtures of two or three compounds. The compounds identified were: mangiferonic acid, ambonic acid, isomangiferolic acid, ambolic acid, 27-hydroxyisomangiferolic acid, cycloartenol, cycloeucalenol, 24-methylenecycloartenol, 20-hydroxybetulin, betulin, betulinic acid and madecassic acid. The fractions from the propolis and the purified compounds were tested in vitro against Crithidia fasciculata, Trypanosoma congolense, drug-resistant Trypanosoma congolense, Trypanosoma b. brucei and multidrug-resistant Trypanosoma b. brucei (B48). They were also assayed for their toxicity against U947 cells. The compounds and fractions displayed moderate to high activity against parasitic protozoa but only low cytotoxicity against the mammalian cells. The most active isolated compound, 20-hydroxybetulin, was found to be trypanostatic when different concentrations were tested against T. b. brucei growth.     

Physico-chemical comparison of betulinic acid, betulin and birch bark extract and in vitro investigation of their cytotoxic effects towards skin epidermoid carcinoma (A431), breast carcinoma (MCF7) and cervix adenocarcinoma (HeLa) cell lines

Betulin and betulinic acid are pentacyclic triterpenes present in the bark of the birch tree and other vegetal sources. Quantitatively, in birch bark betulin is more significant than betulinic acid; therefore, birch can be a large and feasible source of raw material for betulin extraction. Betulin can be used as extracted or, after chemical modification, as a starting compound for its acid, betulinic acid, with both substances possessing various interesting pharmacological properties. The purpose of this study is to analyse the betulin and betulinic acid content of a birch tree bark extract, as well as its cytotoxic activity. The extraction was done using a Soxhlet extractor and chloroform/dichlormethane/methanol (1?:?1?:?1) as solvent. The betulin and betulinic acid content of the extract was estimated using standards of pure betulin and betulinic acid, by thermal analysis as opposed to pure substance (thermogravimetric and differential thermal analysis). The extract and the main compounds were also analysed by NMR. The results indicated a high amount of betulin in the final extract (up to 50%), and an important quantity of betulinic acid: over 3%. The cytotoxic activity indicated a high proliferation inhibition for the birch tree extract but was still comparable with betulinic acid and betulin.     

Determination of Pentacyclic Triterpenes from Betula utilis by High-Performance Liquid Chromatography and High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

A rapid and reliable method was developed and validated for determining betulin and betulinic acid in bark in Betula utilis by high-resolution magic angle spinning ¹H nuclear magnetic resonance (HR-MAS NMR) spectroscopy. HR-MAS NMR spectroscopy clearly distinguished the resonances of betulin and betulinic acid in the bark of all accessions of B. utilis. The concentrations of betulin and betulinic acid were calculated and added to the spectra. The determination of the targeted metabolites in chloroform extract of bark of each accession of B. utilis was performed by high-performance liquid chromatography (HPLC). Quantitatively, betulin was present at higher concentrations than betulinic acid in all accessions. The HR-MAS NMR and HPLC results showed that betulin and betulinic acid varied significantly among accessions of B. utilis. Principal component analysis of the NMR and HPLC results provided classification into three metabolic groups in which the betulin concentration was high, moderate, or low. The results show that HR-MAS NMR is rapid for fingerprinting of betulin and betulinic acid in the bark of B. utilis, while minimizing the drawbacks associated with solvent extraction.     

Total Triterpenoid Extraction from Inonotus Obliquus Using Ionic Liquids and Separation of Potential Lactate Dehydrogenase Inhibitors via Ultrafiltration High-Speed Countercurrent Chromatography

Extracts of the fungus Inonotus obliquus exhibit cytotoxic properties against different cancers; hence, this fungal species has been extensively studied. This study aimed to extract total triterpenoids from Inonotus obliquus using ionic liquids (ILs) and separate potential lactate dehydrogenase (LDH) inhibitors via ultrafiltration (UF)-high-speed countercurrent chromatography (HSCCC). Total triterpenoids from Inonotus obliquus were extracted by performing a single-factor experiment and employing a central composite design via ultrasonic-assisted extraction (UAE) and heat-assisted extraction (HAE). The extract was composed of 1-butyl-3-methylimidazolium bromide as the IL and methanol as the dispersant. Ultrafiltration-liquid chromatography (UF-LC) was used to rapidly scan the LDH inhibitors and betulin and lanosterol were identified as potential inhibitors. To obtain these target compounds, betulin and lanosterol with the purities of 95.9% and 97.8% were isolated from HSCCC within 120 min. Their structures were identified using several techniques, among which IL-HAE was fast and effective. This study reports the extraction of triterpenoids from Inonotus obliquus by IL for the first time. Collectively, the findings demonstrate that UF-LC is an effective tool for screening potential LDH inhibitors from crude extracts of I. obliquus and may help to identify bioactive substances against myocardial infarction, whereas high-purity compounds can be separated via UF-HSCCC.     

Crassiflorone, a new naphthoquinone from Diospyros crassiflora (Hien)

A new naphthoquinone, 11-hydroxy-1,9-dimethyl-6H-naphtho[2′,3′:4,5]furo[3,2-c]chromene-6,7,12-trione, named crassiflorone, was isolated from the stem bark of Diospyros crassiflora together with the known compounds plumbagin, cyclocanaliculatin, gerberinol, lupeol, lupenone and betulinic acid. The structures of the compounds were established on the basis of 1D and 2D NMR spectroscopic data, as well as co-TLC with authentic samples. Some of the above compounds exhibited significant antimicrobial activity against bacteria and yeasts.     

Chemical profiles of birch and alder bark by ambient mass spectrometry

Desorption atmospheric pressure photoionization (DAPPI) is an ambient mass spectrometry (MS) technique that allows the analysis of both polar and nonpolar compounds directly from the surfaces of various sample types. Here, DAPPI was used to study the chemical profiles in different parts of birch and alder tree barks. Four distinct fractions of Betula pendula (silver birch) bark were collected from three different developmental stages of the stem, after which the chemical profiles of the different tissue types were measured. Of special interest were triterpenoids, a class of important defensive substances, which are found in the bark of the silver birch. Additionally, the chemical profiles of lenticels and the surrounding surfaces in the phellem of B. pendula (silver birch), Alnus glutinosa (black alder), and Alnus incana (gray alder) were screened with DAPPI. Another ambient MS technique, laser ablation atmospheric pressure photoionization (LAAPPI), was further used for the mass spectrometry imaging of lenticels on the B. pendula phellem. All the studied birch bark fractions showed individual chemical profiles in DAPPI. The mass spectra from the young apical stem and the transition zone resembled each other more than the mature stem. Instead, the phellem was found to contain a high amount of triterpenoids in all the developmental stages of the stem. The most intense peaks in the DAPPI mass spectra of the birch bark fractions were those of betulin and lupeol. Betulinic and betulonic acid peaks were intense as well, and these compounds were detected especially in the lenticels of the tree samples.

Graphical abstract

     

Chemical Profile of Lipophilic Fractions of Different Parts of Zizyphus lotus L. by GC-MS and Evaluation of Their Antiproliferative and Antibacterial Activities

Zizyphus lotus L. is a perennial shrub particularly used in Algerian folk medicine, but little is known concerning the lipophilic compounds in the most frequently used parts, namely, root bark, pulp, leaves and seeds, which are associated with health benefits. In this vein, the lipophilic fractions of these morphological parts of Z. lotus from Morocco were studied by gas chromatography–mass spectrometry (GC–MS), and their antiproliferative and antimicrobial activities were evaluated. GC–MS analysis allowed the identification and quantification of 99 lipophilic compounds, including fatty acids, long-chain aliphatic alcohols, pentacyclic triterpenic compounds, sterols, monoglycerides, aromatic compounds and other minor components. Lipophilic extracts of pulp, leaves and seeds were revealed to be mainly composed of fatty acids, representing 54.3–88.6% of the total compounds detected. The leaves and seeds were particularly rich in unsaturated fatty acids, namely, (9Z,12Z)-octadeca-9,12-dienoic acid (2431 mg kg⁻¹ of dry weight) and (9Z)-octadec-9-enoic acid (6255 mg kg⁻¹ of dry weight). In contrast, root bark contained a high content of pentacyclic triterpenic compounds, particularly betulinic acid, accounting for 9838 mg kg⁻¹ of dry weight. Root bark extract showed promising antiproliferative activity against a triple-negative breast cancer cell line, MDA-MB-231, with a half-maximal inhibitory concentration (IC₅₀) = 4.23 ± 0.18 µg mL⁻¹ of extract. Leaf extract displayed interesting antimicrobial activity against Escherichia coli, methicillin-sensitive Staphylococcus aureus and Staphylococcus epidermis, presenting minimum inhibitory concentration (MIC) values from 1024 to 2048 µg mL⁻¹ of extract. Our results demonstrate that Zizyphus lotus L. is a source of promising bioactive components, which can be exploited as natural ingredients in pharmaceutical formulations.     

Constituents of the Heartwood of Diospyros Eriantha

The isolation and identification of fifteen compounds from the heartwood of Diospyros eriantha are described. Their structures were determined on the basis of spectral evidence and/or comparison with authentic samples. These compounds include three steroids (stigmast-4-en-3-one, stigmast-4-ene-3,6-dione, and 3β-hydroxystigmast-5-en-7-one), seven triterpenoids (friedelin, lupeol, betulinaldehyde, 3β-acetoxyurs-11-en-13,28-olide, 3β-acetoxyoleanolic acid, betulin, and betulinic acid), three quinones (2-ethoxy-7-methyljuglone, 3-ethoxy-7-methyljuglone, and 2,6-dimethoxy-1,4-benzoquinone), and two phenols (3,4,5-trimethoxyphenol, and syringic acid).     

Synthesis of conjugates of lupane triterpenoids with chromane antioxidants and <Emphasis Type="Italic">in vitro</Emphasis> study of their influence on the production of nitrogen monoxide and on the arginase activity in activated macrophages

Conjugates of lupane triterpenoids (betulin and betulonic and betulinic acids) with synthetic analogs of α-tocopherol were obtained via ester bond formation and tested in vitro. Showing low cytotoxicity, some of them suppress nitrogen monoxide production without affecting the activity of arginase, which suggests their antiinflammatory and immunomodulating properties.     

A Concise Semi-Synthetic Approach to Betulinic Acid from Betulin

Betulin was convert to betulinic acid using two different synthetic routes. The first approach involved an oxidation of betulin using Jones' reagent to betulonic acid and subsequent NaBH₄ reduction to betulinic acid. The second approach involved steps utilizing different protecting groups on the alcohol functional groups of betulin and Jones' oxidation to circumvent the isomerization of the secondary alcohol of betulinic acid.     

Extraction and characterization of total phenolic and flavonoid contents from bark of Swietenia macrophylla and their antimicrobial and antioxidant properties

In the current study, the bioactive compounds such as total phenols, flavonoids, hydrolyzable tannins, and gallic acid were extracted from the bark of Swietenia macrophylla using four different solvents such as ethanol, methanol, acetone, and water. Among them, acetone exhibited the highest contents of bioactive compounds. To optimize the extraction process, a statistical approach was adopted using the central composite design (CCD) of response surface methodology (RSM). The five parameters at five different levels were chosen in the design of the experiments. A total of 32 experimental runs given by the design were fitted into the second-order regression model equation. The analysis of the model shows the best fit of the experimental data with an R² of 0.9971 and a model F-value of 191.73. The optimal conditions of acetone concentration (56 %), the volume of acetone (22 mL), agitation speed (173 rpm), extraction temperature (31 °C), and extraction time (28 h) were noted from desirability function and showed a 2.0-fold increase in the contents of bioactive compounds when compared to unoptimized conditions. Further, the antimicrobial activity of 5 % (w/v) extract was tested against two-gram positive strains Bacillus sp, and Staphylococcus aureus, and two-gram negative strains Escherichia coli, and S. marcescens. The extract exhibited the 21 mm and 18 mm clear zone of diameter with 5 mm standard disc against the gram-positive strains tested whereas no clear zone was found against gram-negative strains. Finally, the antioxidant property was electrochemically analyzed using cyclic voltammetry and Differential pulse voltammetry, which confirmed the presence of multiple antioxidants in the extract.     

Metabolomic Profiling and Antioxidant Activities of Breonadia salicina Using 1H-NMR and UPLC-QTOF-MS Analysis

Breonadia salicina (Vahl) Hepper and J.R.I. Wood is widely used in South Africa and some other African countries for treatment of various infectious diseases such as diarrhea, fevers, cancer, diabetes and malaria. However, little is known about the active constituents associated with the biological activities. This study is aimed at exploring the metabolomics profile and antioxidant constituents of B. salicina. The chemical profiles of the leaf, stem bark and root of B. salicina were comprehensively characterized using proton nuclear magnetic resonance (¹H-NMR) spectroscopy and ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The antioxidant activities of the crude extracts, fractions and pure compounds were determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging and reducing power assays. A total of 25 compounds were tentatively identified using the UPLC-QTOF-MS. Furthermore, the ¹H-NMR fingerprint revealed that the different parts of plant had differences and similarities among the different crude extracts and fractions. The crude extracts and fractions of the root, stem bark and leaf showed the presence of α-glucose, β-glucose, glucose and fructose. However, catechin was not found in the stem bark crude extracts but was found in the fractions of the stem bark. Lupeol was present only in the root crude extract and fractions of the stem bark. Furthermore, 5-O-caffeoylquinic acid was identified in the methanol leaf extract and its respective fractions, while the crude extracts and fractions from the root and dichloromethane leaf revealed the presence of hexadecane. Column chromatography and preparative thin-layer chromatography were used to isolate kaempferol 3-O-(2″-O-galloyl)-glucuronide, lupeol, d-galactopyranose, bodinioside Q, 5-O-caffeoylquinic acid, sucrose, hexadecane and palmitic acid. The crude methanol stem bark showed the highest antioxidant activity in the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity with an IC₅₀ value of 41.7263 ± 7.6401 μg/mL, whereas the root crude extract had the highest reducing power activity with an IC_0.5 value of 0.1481 ± 0.1441 μg/mL. Furthermore, the ¹H-NMR and UPLC-QTOF-MS profiles showed the presence of hydroxycinnamic acids, polyphenols and flavonoids. According to a literature survey, these phytochemicals have been reported to display antioxidant activities. Therefore, the identified hydroxycinnamic acid (caffeic acid), polyphenol (ellagic acid) and flavonoids (catechin and (epi) gallocatechin) significantly contribute to the antioxidant activity of the different parts of plant of B. salicina. The results obtained in this study provides information about the phytochemistry and phytochemical compositions of Breonadia salicina, confirming that the species is promising in obtaining constituents with medicinal potential primarily antioxidant potential.     

Two new xanthones from Calophyllum nodusum (Guttiferae)

The air-dried powdered stem bark of Calophyllum nodusum (Guttiferea) collected from Sandakan (Sabah, Malaysia), was extracted sequentially with hexane, chloroform and methanol. The solvents were removed by rotary evaporator to give dark viscous extracts. Detailed and repeated chromatographic separation of the extracts lead to isolation of two new xanthones, identified as nodusuxanthone and trapezifolixanthone A. Other common terpenoids such as betulinic acid, lupeol, stigmasterol and friedelin were also isolated from the extracts and identified. The structures of the compounds were established by detailed spectral analysis and comparison with previously reported data.     

Antimicrobial and cytotoxic activities of leaves, twigs and stem bark of Scutia buxifolia Reissek

The antimicrobial and cytotoxic activities of the dichloromethane, ethyl acetate and butanolic fractions from the leaves, twigs and stem bark of Scutia buxifolia were evaluated using the broth microdilution method and the brine shrimp lethality method, respectively. Phytochemical analysis was performed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The antimicrobial results demonstrated that the strongest effect occurred with the butanol fraction from the twigs and the ethyl acetate fraction from the stem bark against Saccharomyces cerevisiae (minimal inhibitory concentration; MIC?=?62.5?μg?mL(-1)), whereas the ethyl acetate and butanolic fractions from the twigs and stem bark were effective against Pseudomonas aeruginosa and Staphylococcus aureus, with MIC values ranging from 125 to 500?μg?mL(-1). LD(50) values varied from 50.00?±?0.22 to 82.23?±?0.34?μg?mL(-1). Quercetin, quercitrin, isoquercitrin and rutin were identified by HPLC and may be partially responsible for the antimicrobial activities observed. This study reports for the first time the antimicrobial and cytotoxic activities of S. buxifolia leaves, twigs and stem bark.     

Lupane-triterpenes from Bupleurum flavum

Two new triterpenoids, lup-20(29)-en-3 alpha, 28-diol (1) and lup-20(29)-en-3 alpha, 28,30-triol (2), have been isolated from aerial parts of Bupleurum flavum, a native plant of the Eastern Mediterranean area (Bulgaria, Greece, and Turkey). Their structures were elucidated by spectral analysis. The known compounds betulin, betulinic acid, jasminol, together with the lignan nemerosin and eight flavonoids (kaempferol, isokaempferide, gossipetin, quercetin, luteolin, isorhamnetin 3-O-beta-D-glucopyranoside, isorhamnetin 3-rutinoside, and rutin) were also characterized.     

Self‐assembly of Renewable Nano‐sized Triterpenoids

Studies on plant metabolites have gained renewed interest in recent years because these can serve as renewable chemicals for the development of a sustainable society. Among various plant secondary metabolites, terpenoids constitute the major component and triterpenoids are the 30C subset of it. In recent years, triterpenoids have drawn the attention of scientific community due to many of its potential and realized applications in medicine, drug delivery, thermochromic materials, pollutant capture, catalysis, liquid crystals, etc. In this personal review, we have discussed our computational results carried out on sixty representative naturally occurring triterpenoids demonstrating that all the triterpenoids are renewable functional nano‐entities. Study of the self‐assembly of several triterpenoids such as betulin, betulinic acid, oleanolic acid, glycyrrhetinic acid and arjunolic acid and their derivatives in different liquids have also been discussed. Moreover, the utilization of the resulting supramolecular architectures such as vesicles, spheres, flowers and fibrillar networks of nano‐ to micrometer dimensions and gels have also been discussed in the perspective of green, renewable and nanos.     

Chemical Fingerprint and Determination of Triterpenoids in Cultured Cyclocarya paliurus Cells by High-Performance Liquid Chromatography

Cyclocarya paliurus is a medicinal plant containing various bioactive components with significant health benefits. Cell cultures of C. paliurus have been used to produce these bioactive metabolites. A chemical fingerprint was obtained by high-performance liquid chromatography (HPLC) to monitor the synthesis of major triterpenoids in cultured C. paliurus cells and provide a reliable quality assessment for the cell strain screening. The determination of five triterpenoids, namely, maslinic acid, corosolic acid, betulinic acid, oleanic acid, and ursolic acid, was also performed. The HPLC method for the determination of triterpenoids in the cultured cells was accurate, stable, and reliable, and therefore suitable for chemical fingerprint analysis. Sixteen C. paliurus cell strains varied dramatically in their triterpenoid accumulations. The concentrations of the triterpenoids were 0.45–2.19 (maslinic acid), 0.92–5.34 (corosolic acid), 2.58–4.70 (betulinic acid), 4.07–12.47 (oleanic acid), and 12.64–40.98 (ursolic acid) mg/g. A high yield cell strain had a total triterpenoid concentration of 66.34?mg/g. Ten peaks in the HPLC chromatogram with reasonable height and high resolution were assigned as characteristic for fingerprint and analysis. A reference fingerprint was also obtained for cell strain assessment.     

Anticancer potential of Annona genus: A detailed review

Genus Annona is widely distributed in tropical and subtropical regions around the world. From root to shoot, plant parts of different species of Annona were used traditionally in many countries for treatment of different types of diseases and as a general health supplement. It is naturally enriched with a large number of bioactive molecules which attributed to the several biological activities including antioxidant, anti-inflammatory, antimicrobial and anticancer effects. These bioactive constituents isolated from the leaf, bark, fruit and stem of this plant genus have found terpenoids, steroids, flavonoids, cardiac glycosides, tannins, phenols and alkaloids. Studies have reported that annonaceous acetogenins and alkaloids from this genus were very effective against different types of tumour cell lines. This review highlights anticancer effects of Annona species in cancer therapy, the efficacy of its bioactive components on diverse cancer types and their mechanism of action and also summarizes the use of these phytochemicals for the purpose of developing a promising anticancer drug candidate in future.     

In vitro anthelmintic and cytotoxic activities of extracts from the stem barks of Berlinia confusa (C. Hoyle) and identification of its active constituents

Phytochemical investigation of EtOAc extract from the stem bark of Berlinia confusa yielded a new and two known polysaturated monoacylglycerides characterised as 1-O-docosanoyl-sn-glyceride (3), 1-O-(13-methyltetradecanoyl)-sn-glycerol (4) and 1-O-pentadecacanoyl-sn-glycerol (5); along with the known compounds betulinic acid (1) and sitosteryl-β-d-glucoside (2). The structures of these compounds were elucidated using analytical methods, including 1D and 2D-NMR together with MS spectroscopy. The extracts and isolated compounds demonstrated concentration-dependent anthelmintic activities against Fusciola gigantica (liver flukes) and Taenia solium (tapeworm) at 10–100 mg/ml. The extracts and isolated compounds were evaluated for cytotoxicity against a small panel of three human tumour cell lines.     

Vibrational spectroscopy of betulinic acid HIV inhibitor and of its birch bark natural source

Raman and IR spectra of betulinic acid (BA) (3beta-hydroxy-20(19) lupaen-28 oic acid) and of the natural birch bark have been successfully recorded and discussed. A high selectivity of the Raman technique was remarked for evidencing the betulin in the birch bark. Surface enhanced Raman spectrum (SERS) of BA on colloidal silver has been obtained. The presence and shifting of the symmetric stretching mode of the COO group in the SERS spectrum suggests a chemisorption of the steroid structure on the metallic particles.