Untargeted metabolomics using UHPLC-Q-Orbitrap HRMS for identifying cytotoxic compounds on MCF-7 breast cancer cells from Annona muricata Linn leaf extracts as potential anticancer agents.

INTRODUCTION: The leaves of Annona muricata L., known as "soursop" or "sirsak" in Indonesia, are used traditionally for cancer treatment. However, the bioactive components remain largely unidentified. OBJECTIVE: This study used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics to identify potential cytotoxic compounds in A. muricata leaf extracts on MCF-7 breast cancer cells in vitro. METHODS: A. muricata leaves were macerated with water, 99% ethanol, and aqueous mixtures containing 30%, 50%, and 80% ethanol. Cytotoxic activity of the extracts against MCF-7 breast cancer cells was determined using the MTT assay. Ultra-high-performance liquid chromatography-Q-Orbitrap high-resolution mass spectroscopy (UHPLC-Q-Orbitrap-HRMS) was used to characterize the metabolite composition of each extract. The correlations between metabolite profile and cytotoxic activities were evaluated using orthogonal partial least square discriminant analysis (OPLS-DA). The binding of these bioactive compounds to the tumorigenic alpha-estrogen receptor (3ERT) was then evaluated by in silico docking simulations. RESULTS: Ninety-nine percent ethanol extracts demonstrated the greatest potency for reducing MCF-7 cell viability (IC50 = 22 µg/ml). We detected 35 metabolites in ethanol extracts, including alkaloids, flavonoids, and acetogenins. OPLS-DA predicted that annoreticuin, squadiolin C, and xylopine, and six unknown acetogenin metabolites, might reduce MCF-7 cell viability. In silico analysis predicted that annoreticuin, squadiolin C, and xylopine bind to 3ERT with an affinity comparable to doxorubicin. CONCLUSION: Untargeted metabolomics and in silico modeling identified cytotoxic compounds on MCF-7 cells and binding affinity to 3ERT in A. muricata leaf extracts. The findings need to be further verified to prove the screening results.

Identification of volatile compounds contained in the therapeutic essential oils from Pogostemon cablin, Melaleuca leucadendra, and Mentha piperita and their purified fractions.

Pogostemon cablin, Melaleuca leucadendra, and Mentha piperita are three aromatic plants that have been reported to produce a high yield of volatile components with medicinal and therapeutic properties. This present study aimed to perform qualitative and semi-quantitative analysis on the volatile components present in the aforementioned aromatic plants. Essential oils from P. cablin and M. leucadendra were obtained from community-based enterprises in Aceh Province, Indonesia. The essential oils were further purified using vacuum rotary evaporator. In addition, we also investigated the essential oils from M. piperita based on the priorly optimized parameters. The volatile components contained in the essential oils were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The qualitative data were derived from the MS data based on the fragmented components separated by the GC and compared with the database. The abundance of each volatile component was determined based on the area percentage of the chromatographic peak. In P. cablin oil, the relative abundance of α-guaiene and seychellene was higher in heavy fraction (17.11 and 10.29, respectively), while patchouli alcohol in light fraction (69.92%). Eucalyptol was found higher in the light fraction of M. leucadendra oil (MO) than that in the heavy fraction (78.87% vs. 17.34%, respectively). As for the M. piperita oil, menthone was found as the predominant component with relative abundance of 21.6%. Essential oils extracted from P. cablin, M. leucadendra, and M. piperita consist of volatile components with medicinal and therapeutic potentials, in which their compositions are affected by the purification process.

Atherosclerotic Lesion of the Carotid Artery in Indonesian Cynomolgus Monkeys Receiving a Locally Sourced Atherogenic Diet.

The atherosclerotic lesion is a principal hallmark of atherosclerotic animal models. This study aimed to assess lesions of the carotid artery in Indonesian cynomolgus monkeys exposed to an IPB-1 atherogenic diet. A total of 20 adult male cynomolgus monkeys received the local IPB-1 diet for two years. Blood lipid profiles, morphology, and carotid ultrasound of monkeys were measured. Nine of them were euthanized to confirm atherosclerotic lesions. Common carotid arteries (CCA) and carotid bifurcation (BIF) samples were collected and stained using Verhoef-van Giessen and CD68 immunohistochemistry. The results reveal the presence of severe atherosclerosis plaques in six out of nine animals (66.7%) corresponding to intermediately and hyper-responsive groups. The hyper-responsive group displayed the highest response in the developing intimal area (IA) at the CCA (0.821 mm2), whereas the hyporesponsive group had the smallest IA (0.045 mm2) (p = 0.0001). At the BIF, the hyporesponsive group showed the smallest IA (p = 0.001), but there was no difference between the intermediately and hyper-responsive groups (p = 0.312). The macrophage marker CD68 was also expressed on the cartotid of the intermediately and hyper-responsive groups. These results indicate that severe atherosclerotic lesions with high infiltration of macrophages were formed in the carotid arteries of intermediately and hyper-responsive Indonesian cynomolgus monkeys fed with the local atherogenic diet IPB-1 over two years, thus confirming atherosclerosis in a nonhuman primate model.

Chemokine Therapy in Cats With Experimental Renal Fibrosis and in a Kidney Disease Pilot Study.

Background: Chronic tubulointerstitial fibrosis is a common final pathway leading to end stage kidney disease in cats and has no effective treatment. The use of cell-based molecules to treat kidney fibrosis may be a promising approach. The objectives were to test the effects of intra-renal chemokine CXCL12 injection in a pre-clinical cat model of unilateral ischemia/reperfusion (I/R)-induced kidney fibrosis and then, within a clinical pilot study, test the safety/feasibility of CXCL12 injection in cats that might have early chronic kidney disease (CKD). Methods: Pre-clinical: Thirty cats received intra-renal injection of 100, 200, or 400 ng of recombinant human CXCL12, or sterile saline, into the I/R kidney 70 days post-injury, or were non-injured, non-injected controls (n = 6/group). Kidney collagen content was quantified 4 months post-treatment using Masson's Trichrome and Picrosirius Red (PSR) stained tissues. In a separate study (n = 2) exploring short-term effects of CXCL12, 200 ng CXCL12 was injected into I/R kidneys and then harvested either 30 min (n = 1) or 1 month (n = 1) post-injection. Kidney concentrations of CXCL12, matrix metalloproteinase 1 (MMP-1), and lysyl oxidase-like enzyme 2 (LOXL-2) were quantified via ELISA. Clinical Pilot: 14 client-owned cats with potential early kidney disease received a single-treatment, bilateral intra-renal injection of 200 ng CXCL12 (n = 7), or received no injection (n = 7). Blood/urine samples were collected monthly for 9 months to assess renal function and CKD staging. Results: Pre-clinical: I/R increased the affected kidney collagen content, which both mid and high doses of CXCL12 restored to normal (ps < 0.05 vs. untreated). I/R increased collagen fiber width, which both mid and high doses of CXCL12 restored to normal (p < 0.001 vs. untreated). Early changes in kidney MMP-1, associated with collagen breakdown, and subsequent decreases in LOXL-2, associated with collagen cross-linking, in response to CXCL12 treatment may contribute to these findings. Clinical Pilot: Bilateral intra-renal injection of CXCL12 using ultrasound guidance in cats with CKD was feasible and safe in a general practice clinical setting with no obvious side effects noted during the 9-month follow-up period. Conclusions: Intra-renal injection of CXCL12 may prove to be an effective treatment for kidney fibrosis in cats with CKD. Additional mechanistic and clinical evaluations are needed.

Mammosphere Culture of Mammary Cells from Cynomolgus Macaques (Macaca fascicularis).

The mammary gland contains adult stem cells that are capable of self-renewal. Although these cells hold an important role in the biology and pathology of the breast, the studies of mammary stem cells are few due to the difficulty of acquiring and expanding undifferentiated adult stem cell populations. In this study, we developed mammosphere cultures from frozen mammary cells of nulliparous cynomolgus macaques (Macaca fascicularis) as a culture system to enrich mammary stem cells. Small samples of mammary tissues were collected by surgical biopsy; cells were cultured in epithelial cell growth medium and cryopreserved. Cryopreserved cells were cultured into mammospheres, and the expression of markers for stemness was evaluated by using quantitative PCR analysis. Cells were further differentiated by using 2D and 3D approaches to evaluate morphology and organoid budding, respectively. The study showed that mammosphere culture resulted in an increase in the expression of mammary stem cell markers with each passage. In contrast, markers for epithelial cells and pluripotency decreased across multiple passages. The 2D differentiation of the cells showed heterogeneous morphology, whereas 3D differentiation allowed for organoid formation. The results indicate that mammospheres can be successfully developed from frozen mammary cells derived from breast tissue collected from nulliparous cynomolgus macaques through surgical biopsy. Because mammosphere cultures allow for the enrichment of a mammary stem cell population, this refined method provides a model for the in vitro or ex vivo study of mammary stem cells.

Metabolic and morphometric changes in Indonesian cynomolgus monkeys (Macaca fascicularis) fed an atherogenic diet composed of locally sourced ingredients.

BACKGROUND AND AIM: This study was designed to determine the effects of a new atherogenic diet formulated at Institut Pertanian Bogor (IPB) (Bogor, Indonesia) on metabolic, morphometric, and carotid artery imaging of cynomolgus monkeys. MATERIALS AND METHODS: A total of 20 adult male cynomolgus monkeys fed IPB-1 atherogenic diet for 1 year. Total plasma cholesterol (TPC), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, and morphometric measurements were evaluated at baseline and monthly during the study. Carotid plaques and intima-media thickness (IMT) were measured using ultrasonography at baseline and after 8 months of treatment. RESULTS: This diet increased TPC, LDL, and TPC/HDL ratio and induced carotid atherosclerosis in this model. The TPC, LDL, and TPC/HDL ratio were positively associated; however, HDL was negatively associated with carotid plaques and IMT. CONCLUSION: The IPB-1 atherogenic diet formulated with locally and readily available ingredients provides an economically and scientifically feasible monkey model to study atherosclerosis in Indonesia and Southeast Asia.

Cell Versus Chemokine Therapy Effects on Cell Mobilization to Chronically Dysfunctional Urinary Sphincters of Nonhuman Primates.

PURPOSE: A major question remaining in approaches to tissue engineering and organ replacement is the role of native mobilized native cells in the regeneration process of damaged tissues and organs. The goal of this study was to compare the cell mobilizing effects of the chemokine CXCL12 and cell therapy on the urinary sphincter of nonhuman primates (NHP) with chronic intrinsic urinary sphincter dysfunction. METHODS: Either autologous lenti-M-cherry labeled skeletal muscle precursor cells (skMPCs) or CXCL12 were injected directly into the sphincter complex of female NHPs with or without surgery-induced chronic urinary sphincter dysfunction (n=4/treatment condition). All monkeys had partial bone marrow transplantation with autologous lenti-green fluorescent protein (GFP) bone marrow cells prior to treatment. Labeled cells were identified, characterized and quantified using computer-assisted immunohistochemistry 6 months posttreatment. RESULTS: GFP-labeled bone marrow cells (BMCs) were identified in the bone marrow and both BMCs and skMPCs were found in the urinary sphincter at 6-month postinjection. BMCs and skMPCs were present in the striated muscle, smooth muscle, and lamina propria/urothelium of the sphincter tissue. Sphincter injury increased the sphincter content of BMCs when analyzed 6-month postinjection. CXCL12 treatment, but not skMPCs, increased the number of BMCs in all layers of the sphincter complex (P<0.05). CXCL12 only modestly (P=0.15) increased the number of skMPCs in the sphincter complex. CONCLUSION: This dual labeling methodology now provides us with the tools to measure the relative number of locally injected cells versus bone marrow transplanted cells. The results of this study suggest that CXCL12 promotes mobilization of cells to the sphincter, which may contribute more to sphincter regeneration than injected cells.

Effects of inhaled citronella oil and related compounds on rat body weight and brown adipose tissue sympathetic nerve.

Citronella oil is one of the most famous Indonesian essential oils, having a distinctive aroma. As with other essential oils, it is crucial to explore the effects of inhalation of this oil. Therefore, the aim of this research was to elucidate the effects of inhalation of citronella oil and its components isolated from Cymbopogon nardus L. (Poaceae), Indonesian local name: "Sereh Wangi" on the body weight, blood lipid profile, and liver function of rats, as well as on the sympathetic nerve activity and temperature of brown adipose tissue. Sprague-Dawley male adult rats fed with high fat diet (HFD) were made to inhale citronella oil, R-(+)-citronellal, and ß-citronellol for five weeks, and the observations were compared to those of HFD rats that were not subjected to inhalation treatment. The results showed that inhalation of ß-citronellol decreased feed consumption. As a consequence, the percentage of weight gain decreased compared with that in control group and the blood cholesterol level in the ß-citronellol group was significantly lowered. Concentration of liver function enzymes were not significantly different among the groups. In conclusion, inhalation of citronella oil, specifically ß-citronellol, decreased body weight by decreasing appetite, without any marked changes in liver enzyme concentrations.

Effects of hormone therapy and dietary soy on myocardial ischemia/reperfusion injury in ovariectomized atherosclerotic monkeys.

OBJECTIVE: Hormone therapy (HT) and dietary soy (Soy) inhibit myocardial ischemia/reperfusion (I/R) injury in nonatherosclerotic animals. The aim of this study was to determine their independent and interactive effects on I/R in monkeys previously fed an atherogenic diet for 15 months. DESIGN: Ovariectomized atherosclerotic monkeys (n = 40) were divided into one of four dietary treatment groups: (1) casein as the protein source, (2) casein and added HT (the equivalent of 5 mug ethinyl estradiol + 1 mg norethindrone acetate daily), (3) Soy protein providing 141 mg total isoflavones daily, or (4) Soy + HT. After 12 months monkeys were anesthetized, and their left anterior descending coronary artery was occluded for 1 hour and reperfused for 4 hours. Infarct size was the percentage of the area at risk not staining with triphenyltetrazolium chloride. Additional measures were myocardial blood flow, stroke volume, coronary output, myeloperoxidase, and malondialdehyde. RESULTS: There was an interactive negative effect of HT + Soy to increase infarct size from approximately 30% (in other groups) to 55% (P = 0.0004). Additionally, there were negative main effects of Soy on blood flow, coronary output, and stroke volume during I/R (all P values <0.05). There were no effects of treatment on either myeloperoxidase or malondialdehyde. CONCLUSIONS: Neither HT nor Soy had beneficial effects, whereas their combination had harmful effects, on myocardial I/R injury in monkeys with preexisting atherosclerosis. The mechanism of this negative interaction remains unclear but may relate to Soy's negative effects on hemodynamics.

A comparison of two progestins on myocardial ischemia-reperfusion injury in ovariectomized monkeys receiving estrogen therapy.

OBJECTIVE: It has been reported that the progestin medroxyprogesterone acetate (MPA), but not norethindrone acetate (NETA), inhibits the beneficial vascular effects of post-menopausal estrogen therapy, but their effects on the myocardium are unclear. The goal of this study is to compare the effects of these two progestins on post-ischemic myocardial damage. METHODS: Ovariectomized monkeys were fed an atherogenic diet for 18 months while receiving, or not receiving (control, n=15), the monkey equivalent to a woman's dose of 5 mug ethinyl estradiol with either 1 mg NETA daily (n=15) or 2.5 mg MPA daily (n=15). The left anterior descending coronary artery was occluded for 1 h and then released to allow myocardial reperfusion for 4 h. Infarct size was quantified using the histochemical stain triphenyl-tetrazolium chloride. Regional myocardial blood flow was measured by 15 mum neutron-activated microspheres, blood pressure and heart rates with a pneumatic cuff, stroke volume by echocardiography, coronary output by thermodilution and neutrophil accumulation in the myocardium using myeloperoxidase (MPO) activity. RESULTS: The infarct size (area of necrosis/area at risk) was similar between the control group (21+/-3%) and the MPA group (29+/-3%) (P<0.05) but significantly less in the NETA group (3+/-2%) than other groups (P<0.05). The hemodynamic myocardial function and regional myocardial blood values were similar among groups before, during and 4 h after reperfusion (all P-values >0.05). Similarly, there were no treatment effects on MPO activity (P>0.05). CONCLUSIONS: NETA, but not MPA, diminished ischemia-reperfusion injury in estrogen-treated post-menopausal females. The mechanism(s) of this difference remains unclear.

Hormone replacement therapy and cardiovascular disease: lessons from a monkey model of postmenopausal women.

Concerns exist about the cardiovascular effects of hormone replacement therapy (HRT) in postmenopausal women because results from the Women's Health Initiative (WHI) and the Heart and Estrogen/Progestin Replacement Study (HERS) are contradictory. In both of these studies, postmenopausal conjugated equine estrogens + medroxyprogesterone acetate did not reduce risk, and somewhat increased the risk of myocardial infarction in both primary (WHI) and secondary (HERS) prevention. These results appear to contradict numerous observational clinical trials and animal studies, which reported profound beneficial effects of HRT on cardiovascular disease risk. Results of both human and monkey studies indicate that estrogen replacement therapy (ERT)/HRT is effective in inhibiting progression of early stage (fatty streak) atherosclerosis but that ERT/HRT is much less effective in inhibiting progression of more advanced (established plaque) atherosclerosis. Results of these monkey studies are consistent with those of studies in women wherein ERT/HRT was initiated in postmenopausal women with different initial amounts of atherosclerosis. Based on these findings, it is speculated that ERT/HRT may be more cardioprotective in younger postmenopausal women with less coronary artery disease, and less effective in women with established coronary artery disease. Researchers are challenged to define the relative cardiovascular risk/benefit in different populations of postmenopausal women based on differences in age, amounts of pre-existing atherosclerosis, and risk factors.

Contrasting effects of two hormone replacement therapies on the cardiovascular and mammary gland outcomes in surgically postmenopausal monkeys.

OBJECTIVE: The purpose of this study was to compare the effects of two hormone replacement therapies on the intermediate end points of coronary heart disease and mammary gland hyperplasia in postmenopausal monkeys. STUDY DESIGN: Surgically postmenopausal cynomolgus monkeys were fed an atherogenic diet for 12 months while receiving no treatment (control, n = 19), conjugated equine estrogens plus continuous medroxyprogesterone acetate (n = 19), or ethinyl estradiol plus norethindrone acetate (n = 21) at doses that were scaled from those doses taken by women. RESULTS: Quantitative coronary angiography revealed that the arteries of the control group and the conjugated equine estrogens plus continuous medroxyprogesterone acetate-treated animals constricted in response to acetylcholine (-5.4% +/- 1.4% and -6.2% +/- 1.5%, respectively), whereas those arteries in the animals in the ethinyl estradiol plus norethindrone acetate group did not (P =.002). The incidence of dobutamine-induced ST-segment depression in the ethinyl estradiol plus norethindrone acetate group (10.5%) was significantly less than in the control group (68.8%, P =.001) or the conjugated equine estrogens plus continuous medroxyprogesterone acetate group (50%, P =.01). Conjugated equine estrogens plus continuous medroxyprogesterone acetate, but not ethinyl estradiol plus norethindrone acetate, induced diffuse epithelial tissue proliferation in the mammary glands (P =.0006). CONCLUSION: Ethinyl estradiol plus norethindrone acetate protected against atherosclerosis-induced endothelium-mediated vasoconstriction of coronary arteries and heart rate-induced myocardial ischemia and did not induce epithelial tissue proliferation (tissue density) in the mammary gland.

Effects of exercise on cardiovascular outcomes in monkeys with risk factors for coronary heart disease.

OBJECTIVE: Exercise reduces the risk for coronary heart disease. However, the mechanisms mediating the beneficial effects of exercise remain ambiguous. In particular, it is uncertain whether exercise inhibits the development of atherosclerosis, a major pathobiologic process underlying heart disease. METHODS AND RESULTS: To address this question, adult male monkeys were fed an atherogenic diet while assigned to one of four experimental conditions for 34 months: 1) runner/no group disruption, ie, "stable" (n=19); 2) runner plus frequent social group disruption, ie, "unstable" (n=19); 3) sedentary/stable (n=15); or 4) sedentary/unstable (n=18). Neither exposure to exercise nor social group disruption significantly affected the resulting coronary artery atherosclerosis extent or lumen areas (all ANOVA values, P>0.05). When compared with sedentary individuals, exercise animals had lower resting heart rates (119.0+/-3 vs 132.0+/-3 bpm, P=0.002), greater echocardiographically measured left ventricular ejection fractions (77.2+/-0.01% vs 73.8+/-0.01%, P=0.02), greater quantitative angiographically measured dilation of coronary arteries to phenylephrine (2.6+/-1% vs -3.7+/-1% change from baseline diameter, P=0.003), and a reduced cortisol response to an adrenocorticotropin challenge. These measures were not significantly affected by social condition. CONCLUSIONS: Thus, exercise improved some measures of cardiovascular health and reduced stress responsivity but did not inhibit progression of coronary artery atherosclerosis or promote positive artery remodeling. It is concluded that exercise may exert cardioprotective effects without influencing atherosclerosis extent.