Evaluation of natural gum-based cryogels for soft tissue engineering.

In this study, three natural biomaterials, Locust bean gum (LBG), Xanthan gum (XG), and Mastic gum (MG), were combined to form cryogel scaffolds. Thermal and chemical characterizations revealed the successful blend formation from LBG-XG (LX) and LBG-XG-MG (LXM) polymers. All blends resulted in macro-porous scaffolds with interconnected pore structures under the size of 400 µm. The swollen cryogels had similar mechanical properties compared with other polysaccharide-based cryogels. The mean tensile and compressive modulus values of the wet cryogels were in the range of 3.5-11.6 kPa and 82-398 kPa, respectively. The sustained release of the small molecule Kartogenin from varying concentrations and ratios of cryogels was in between 32 and 66% through 21 days of incubation. Physical, mechanical, and chemical properties make LX and LXM polysaccharide-based cryogels promising candidates for cartilage and other soft tissue engineering, and drug delivery applications.

Nutrigenetic Interactions Might Modulate the Antioxidant and Anti-Inflammatory Status in Mastiha-Supplemented Patients With NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease with no therapeutic consensus. Oxidation and inflammation are hallmarks in the progression of this complex disease, which also involves interactions between the genetic background and the environment. Mastiha is a natural nutritional supplement known to possess antioxidant and anti-inflammatory properties. This study investigated how a 6-month Mastiha supplementation (2.1 g/day) could impact the antioxidant and inflammatory status of patients with NAFLD, and whether genetic variants significantly mediate these effects. We recruited 98 patients with obesity (BMI ≥ 30 kg/m2) and NAFLD and randomly allocated them to either the Mastiha or the placebo group for 6 months. The anti-oxidative and inflammatory status was assessed at baseline and post-treatment. Genome-wide genetic data was also obtained from all participants, to investigate gene-by-Mastiha interactions. NAFLD patients with severe obesity (BMI > 35kg/m2) taking the Mastiha had significantly higher total antioxidant status (TAS) compared to the corresponding placebo group (P value=0.008). We did not observe any other significant change in the investigated biomarkers as a result of Mastiha supplementation alone. We identified several novel gene-by-Mastiha interaction associations with levels of cytokines and antioxidant biomarkers. Some of the identified genetic loci are implicated in the pathological pathways of NAFLD, including the lanosterol synthase gene (LSS) associated with glutathione peroxidase activity (Gpx) levels, the mitochondrial pyruvate carrier-1 gene (MPC1) and the sphingolipid transporter-1 gene (SPNS1) associated with hemoglobin levels, the transforming growth factor-beta-induced gene (TGFBI) and the micro-RNA 129-1 (MIR129-1) associated with IL-6 and the granzyme B gene (GZMB) associated with IL-10 levels. Within the MAST4HEALTH randomized clinical trial (NCT03135873, www.clinicaltrials.gov) Mastiha supplementation improved the TAS levels among NAFLD patients with severe obesity. We identified several novel genome-wide significant nutrigenetic interactions, influencing the antioxidant and inflammatory status in NAFLD. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03135873.

Development, Validation and Application of a UHPLC-MS Method for the Quantification of Chios Mastic Gum Triterpenoids in Human Plasma.

Chios mastic gum is the resinous secretion obtained from the barks of the shrub Pistacia lentiscus var. Chia, which is endemic to the Greek island of Chios. Since antiquity, Chios mastic gum has found several uses as a phytotherapeutic remedy, primarily for the treatment of gastrointestinal disorders while recently, Chios mastic gum was also recognized by EMA as an herbal medicinal product with specific indications. Chios mastic gum's biological properties are attributed to triterpenes which comprise the major chemical group (approx. 70%) and notably isomasticadienonic acid and masticadienonic acid. However, due to their structural characteristics, the isolation thereof in high yield and purity is challenging and since they are not commercially available, pharmacological studies aiming to assess their biological properties are limited. In the present work, mastic's phytochemical investigation by UPLC-HRMS is followed by the isolation and characterization of isomasticadienonic acid and masticadienonic acid to be used as analytical standards for their accurate and reliable quantification in human plasma. A UHPLC-tQ-MS method that was developed and validated (in terms of specificity, linearity, limit of quantification, accuracy and precision), for the direct quantification of the targeted compounds in the low ng/mL range of concentration, was subsequently implemented on plasma samples of healthy volunteers thus demonstrating its fitness for purpose. The results presented herein might provide insight to the understanding of this traditional natural product consumed notably for its anti-inflammatory, antioxidant and lipid lowering properties. Moreover, this method might serve as a starting point for any study aiming to monitor bioactive triterpenes in biological fluids.

Chemical Characterization and Biological Activity of the Mastic Gum Essential Oils of Pistacia lentiscus Var. Chia from Turkey.

The essential oils (EOs) were isolated by hydrodistillation from wild and cultivated Pistacia lentiscus L. var. chia-mastic gum tree (Anacardiaceae) from two natural habitats, namely from Cesme-Uzunkoy (1) and Mordogan (2), and one cultivated source, Cesme-Germiyan (3), in Izmir, Turkey. This comparative study evaluated the chemical composition and biological activity of mastic gum essential oils (MGEOs). For this purpose, MGEOs 1-3 were analyzed by gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and chiral GC for α-pinene. Laboratory assays were conducted to assess for potential in vitro cytotoxicity (multiple in vitro cancer cell lines), antimicrobial properties (five bacterial species and yeast), anti-inflammatory activity (inhibition of inducible nitric oxide synthase, iNOS), and the attraction of Ceratitis capitata (Mediterranean fruit fly, medfly), respectively. Chemical analysis indicated that MGEOs 1 and 2 were rich in α-pinene (56.2% and 51.9%), myrcene (20.1% and 18.6%), and ß-pinene (2.7% and 3.1%), respectively; whereas MGEO-3 was characterized by a high level of α-pinene (70.8%), followed by ß-pinene (5.7%) and myrcene (2.5%). Chiral GC analyses showed that concentration ratios between (-)/(+)-α-pinene and (-)-α-pinene/myrcene allowed for differentiation between wild and cultivated MGEO sources. In biological assays, MGEOs 1-3 did not exhibit significant antimicrobial effects against the pathogens evaluated and were not strong attractants of male medflies; however, all three MGEOs displayed a dose-dependent inhibition of iNOS, and MGEOs 1 and 2 exhibited selective in vitro cytotoxicity against human cancer cells. These results suggest that wild-type mastic gum oils from Cesme and Mordogan (MGEOs 1 and 2) are potential sources of beneficial products and warrant further investigation.

Aroma Investigation of Chios Mastic Gum (Pistacia lentiscus Variety Chia) Using Headspace Gas Chromatography Combined with Olfactory Detection and Chiral Analysis.

Chios mastic gum (Pistacia lentiscus var. Chia) exhibits intensely sourish, green, resinous, and woody odor notes with hints of citrus and pine. Despite its attractive flavor, no description of its aroma properties by molecular sensory techniques has been published thus far. A total of 25 odor-active compounds with flavor dilution (FD) factors of 1-512 were identified by gas chromatography-mass spectrometry-olfactometry (GC-MS-O) combined with headspace solid-phase microextraction (HS-SPME) and stir bar sorptive extraction (HS-SBSE). Quantitative analyses performed by multiple HS-SPME and calculation of odor activity values of 10 odorants with high FD factors revealed an essential role of several minor components (e.g., ß-myrcene, limonene, ß-linalool, and perillene) for the overall aroma of mastic gum, besides the dominating compound α-pinene. The indispensable contribution of the minor odorants to mastic gum was further confirmed by aroma recombination and omission tests. Varying enantiomeric excess values of the key odorants were observed by multidimensional GC-MS.

Toxicity and Toxicokinetic Study of Subcutaneously Administered RPh201 in Minipigs.

Mastic gum extracts are widely used as herbal remedies and are being tested for several clinical indications. Nevertheless, information on their safety is limited. RPh201 is an extract of the mastic gum, formulated and stabilized in a proprietary method, which is being developed as a novel drug candidate for neurological indications. The aim of this study was to assess the systemic toxic potential of RPh201, administered twice weekly by subcutaneous injections to minipigs, after 39 weeks of administration followed by a recovery period of 6 weeks. No clinical or dose-related signs were observed, but treatment-related findings were seen at the injection sites of the high-dose animals, composed of abscesses, chronic inflammation, and subcutaneous fibrosis. Abscesses >30 mm in size, graded as marked severity, were confined to the high-dose group and were considered as adverse. Minimal-slight subcutaneous and lymph nodes abscesses seen in control, low, and intermediate doses, related to the vehicle (cottonseed oil), were not considered as adverse. Additionally, minimal-to-slight cystic spaces or vacuolation related to the vehicle were observed in the skin, lymph nodes, kidney, and lungs. These findings were considered not to be adverse. The no-observed-adverse-effect level was considered to be 12.5 mg/kg/occasion.

Bioavailability of Terpenes and Postprandial Effect on Human Antioxidant Potential. An Open-Label Study in Healthy Subjects.

SCOPE: To assess bioavailability of terpenes in human plasma and their effect on oxidative stress biomarkers. METHODS AND RESULTS: In this open-label and single arm postprandial trial, seventeen healthy male volunteers (20-40 years old) follow a low-phytochemical diet for 5 days. Next, after overnight fasting, volunteers consume Mastiha powder (a natural resin rich in terpenes) dispersed in water. Blood samples are collected on time points 0 h (before ingestion) and 0.5, 1, 2, 4, 6, and 24 h (post-ingestion). Ultra-high-pressure liquid chromatography high-resolution MS (UHPLC-HRMS/MS) is applied for high throughput analysis of plasma. Serum resistance to oxidation and oxidized LDL (oxLDL) levels are measured. UHPLC-HRMS/MS analysis shows that major terpenes are bioavailable since 0.5 h after administration, reaching a peak between 2 h and 4 h. Serum resistance to oxidation, expressed as difference of tLAG (time point-0 h), starts to increase from 0.5 h. This increase reaches statistical significance at 4 h (402.3 ± 65.0 s), peaks at 6 h (524.6 ± 62.9 s), and remains statistically significant until 24 h (424.2 ± 48.0 s). oxLDL levels, expressed as %change from 0 h, are reduced significantly from time point-1 h until time point-6 h. CONCLUSION: Results demonstrate the terpene bioavailability pattern after oral administration of Mastiha. Terpenes are potential mediators of antioxidant defense in vivo.

Human Adenocarcinoma Cell Line Sensitivity to Essential Oil Phytocomplexes from Pistacia Species: a Multivariate Approach.

Principal component analysis (PCA) multivariate analysis was applied to study the cytotoxic activity of essential oils from various species of the Pistacia genus on human tumor cell lines. In particular, the cytotoxic activity of essential oils obtained from P. lentiscus, P. lentiscus var. chia (mastic gum), P. terebinthus, P. vera, and P. integerrima, was screened on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian), and LoVo (colon). The results indicate that all the Pistacia phytocomplexes, with the exception of mastic gum oil, induce cytotoxic effects on one or more of the three cell lines. PCA highlighted the presence of different cooperating clusters of bioactive molecules. Cluster variability among species, and even within the same species, could explain some of the differences seen among samples suggesting the presence of both common and species-specific mechanisms. Single molecules from one of the most significant clusters were tested, but only bornyl-acetate presented cytotoxic activity, although at much higher concentrations (IC50 = 138.5 µg/mL) than those present in the essential oils, indicating that understanding of the full biological effect requires a holistic vision of the phytocomplexes with all its constituents.

Dietary mastic oil extracted from Pistacia lentiscus var. chia suppresses tumor growth in experimental colon cancer models.

Plant-derived bioactive compounds attract considerable interest as potential chemopreventive anticancer agents. We analyzed the volatile dietary phytochemicals (terpenes) present in mastic oil extracted from the resin of Pistacia lentiscus var. chia and comparatively investigated their effects on colon carcinoma proliferation, a) in vitro against colon cancer cell lines and b) in vivo on tumor growth in mice following oral administration. Mastic oil inhibited - more effectively than its major constituents- proliferation of colon cancer cells in vitro, attenuated migration and downregulated transcriptional expression of survivin (BIRC5a). When administered orally, mastic oil inhibited the growth of colon carcinoma tumors in mice. A reduced expression of Ki-67 and survivin in tumor tissues accompanied the observed effects. Notably, only mastic oil -which is comprised of 67.7% α-pinene and 18.8% myrcene- induced a statistically significant anti-tumor effect in mice but not α-pinene, myrcene or a combination thereof. Thus, mastic oil, as a combination of terpenes, exerts growth inhibitory effects against colon carcinoma, suggesting a nutraceutical potential in the fight against colon cancer. To our knowledge, this is the first report showing that orally administered mastic oil induces tumor-suppressing effects against experimental colon cancer.

Evaluation of Biological Activity of Mastic Extracts Based on Chemotherapeutic Indices.

BACKGROUND: Most previous mastic investigators have not considered its potent cytotoxicity that may significantly affect the interpretation of obtained data. In the present study, we re-evaluated several biological activities of mastic extracts, based on chemotherapeutic indexes. MATERIALS AND METHODS: Pulverized mastic gum was extracted with n-hexane and then with ethyl acetate or independently with methanol or n-butanol. Tumor specificity (TS) of the extracts was determined by their cytotoxicity against human malignant and non-malignant cells. Antibacterial activity was determined by their cytotoxicity against bacteria and normal oral cells. Antiviral activity was determined by their protection of viral infection and cytotoxic activity. Cytochrome P-450 (CYP) 3A4 activity was measured by ß-hydroxylation of testosterone. RESULTS: Ethyl acetate extract showed slightly higher tumor specificity (TS=2.6) and one order higher antibacterial activity (selectivity index (SI)=0.813) than other extracts (TS=1.4-2.5; SI=0.030-0.063). All extracts showed no anti-human immunodeficiency virus (HIV) activity, but some anti-herpes simplex virus (HSV) activity, which was masked by potent cytotoxicity. They showed strong inhibitory activity against CYP3A4. CONCLUSION: Ethyl acetate extraction following the removal of cytotoxic and CYP3A4 inhibitory substances by n-hexane can enhance antitumor and antibacterial activity of mastic.

A simple and reliable analytical method based on HPLC-UV to determine oleanonic acid content in Chios gum mastic for quality control.

A reliable analytical method based on high-performance liquid chromatography-ultraviolet detection was established for the determination of oleanonic acid (OA) content in Chios gum mastic (CGM). A simple method involving methanol extraction of CGM powder followed by basification and ether extraction was developed to isolate the triterpenic fraction including OA. The triterpenic fraction was chromatographed on a Phenomenex Gemini C18 column (150 × 4.6 mm, 5 µm) under a simple gradient elution of a mobile phase containing acetonitrile and water at a flow rate of 1.0 mL min-1. The detection wavelength was set at 210 nm. Good linearity was achieved in the range of 100.0-1000.0 µg mL-1 with r2 > 0.9993, and the limit of quantification was 32.22 µg mL-1. Accuracy measured at three concentration levels was in the range of 93.72-99.56%, while intra-day and inter-day precisions estimated using both OA standard and CGM samples were no more than 2.83 and 4.57% RSD, respectively. Finally, this method was applied to real CGM samples from various batches, revealing that the OA contents were between 88.13 and 100.83 µg mg-1. These results suggest that the current method can be applied as an efficient analytical method for quality control of CGM.