In-depth investigation of the therapeutic effect of Tribulus terrestris L. on type 2 diabetes based on intestinal microbiota and feces metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Tribulus terrestris L. (TT) is extensively documented in the Tibetan medical literature 'Si Bu Yi Dian', has been used to treat diabetes mellitus for more than a thousand years. However, the underlying mechanisms and comprehensive effects of TT on diabetes have yet to be investigated. AIM OF THE STUDY: The aim of the study was to systemically elucidate the potential mechanisms of TT in treating diabetes mellitus, and further investigate the therapeutic effects of the water extract, small molecular components and saccharides from TT. MATERIALS AND METHODS: Fecal metabolomics was employed to draw the metabolic profile based on UHPLC-Q-TOF-MS/MS. The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified to explore the structural changes of the intestinal microbiome after TT intervention and to analyze the differential microbiota. The microbial metabolites SCFAs were determined by GC-MS, and the BAs and tryptophan metabolites were quantified by UPLC-TQ-MS. Spearman correlation analysis was carried out to comprehensively investigate the relationship among the endogenous metabolites profile, intestinal microbiota and their metabolites. RESULTS: TT exhibited remarkably therapeutic effect on T2DM rats, as evidenced by improved glucolipid metabolism and intestinal barrier integrity, ameliorated inflammation and remission in insulin resistance. A total of 24 endogenous biomarkers were screened through fecal metabolomics studies, which were mainly related to tryptophan metabolism, fatty acid metabolism, bile acid metabolism, steroid hormone biosynthesis and arachidonic acid metabolism. Investigations on microbiomics revealed that TT significantly modulated 18 differential bacterial genera and reversed the disordered gut microbial in diabetes rats. Moreover, TT notably altered the content of gut microbiota metabolites, both in serum and fecal samples. Significant correlation among microbial community, metabolites and T2DM-related indicators was revealed. CONCLUSIONS: The multiple components of TT regulate the metabolic homeostasis of the organism and the balance of intestinal microbiota and its metabolites, which might mediate the anti-diabetic capacity of TT.

Exploring the in vitro anti-inflammatory activity of gross saponins of Tribulus terrestris L. fruit by using liquid chromatography-mass spectrometry-based cell metabolomics approach.

Our previous studies confirmed the efficacy of gross saponins of Tribulus terrestris L. fruit in treating cerebral ischemia. This study aimed to investigate the related mechanisms in vitro. The lipopolysaccharide-induced BV2 cells model was constructed and treated with gross saponins at different concentrations to explore its anti-inflammatory activity. The cell metabolite changes were tracked by liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, and the metabolic biomarkers and related metabolic pathways were analyzed. Molecular biochemistry analysis was further used to verify the relevant inflammatory pathways. The results showed that the saponins reduced nitric oxide release and the secretion of tumor necrosis factor-alpha, interleukin-1ß, and interleukin-6 from lipopolysaccharide-induced BV2 cells. Metabolic perturbations occurred in lipopolysaccharide-treated BV2 cells, which could be reversed by drug treatment via mainly regulating glycerophospholipid metabolism, tryptophan metabolism, purine metabolism pathways, etc. The western blot analysis demonstrated that saponin could suppress the activation of the inflammatory-related signaling pathway. The present study explored the in vitro anti-inflammatory mechanism of gross saponins of Tribulus terrestris L. fruit using an LC-MS-based cell metabolomics approach, which confirms the great potential of LC-MS for drug efficacy evaluation and can be applied in other herbal medicine-related analyses.

Tribulus terrestris L. induces cell apoptosis of breast cancer by regulating sphingolipid metabolism signaling pathways.

BACKGROUND: Tribulus terrestris L. (TT) was initially documented in Shen-Nong-Ben-Cao-Jing and has been used for thousands of years in China as a herb to calm liver, dispel melancholy and wind, promote blood circulation, improve eyesight, and relieve itching. Moreover, it was also used to treat breast cancer in ancient China. However, the pharmacological activities of TT extract on breast cancer have received little attention. PURPOSE: In this study, we investigated the anti-breast cancer effects and possible mechanisms of action of this herbal drug. METHODS: Network pharmacology analysis the study of network pharmacology was done to analyze the possibility of TT's anti-breast cancer effect. And then, molecular docking between TT7/TT8 and vascular endothelial growth factor receptor 2 (VEGFR2) were performed by Autodock software as well as the related protein expressions were analyzed by western blot to verify this effect. In vivo experiment: The mouse model of breast cancer was established by injection of 4T1 cells. Then drugs were intragastrically administered to the mice once daily for fourteen days. Body weight, tumor size, and tumor weight were recorded at the end of the experiment. Moreover, tumor inhibitory rate was calculated. Finally, pathological changes and apoptosis of breast cancer tissues were respectively evaluated by HE and Hoechst staining. Proteomics and metabonomics analyses: The tumor tissues were chosen to perform conjoint analysis. Firstly, differential proteins and metabolites were found. Furthermore, the functional analyses of them were analyzed by software. At the last, immunofluorescent staining of SGPP1, SPHK1 and p-SPHK1 in tumor tissue were done. RESULTS: 12 active ingredients of TT, 127 targets of active ingredients, 15,253 targets of breast cancer, 1,225 targets of Ru yan, and 123 overlapping genes were obtained in the network pharmacology study. There was firm conjunction between TT7/TT8 and VEGFR2. Besides, tumor size and weight were markedly reduced in TT groups compared to the model group. The tumor inhibitory rate was more than 26% in TTM group. After drug treatment, many adipocytes and cracks between tumor and apoptosis were discovered. The western blot results showed that TT aqueous extract lowered the levels of VEGFR2, ERK1/2, p-ERK1/2 (Thr202, Tyr204) and Bcl2, while increasing the levels of Bax and the ratio of Bax/Bcl2. Furthermore, 495 differential proteins and 76 differential metabolites were found between TTM and model groups with the sphingolipid metabolism pathway being enriched. At last, TT treatment significantly reduced the levels of SGPP1, SPHK1 and p-SPHK1 in tumor tissue. CONCLUSIONS: In conclusion, TT demonstrates therapeutic effects in a mouse model of breast cancer, and its mechanism of action involves the regulations of sphingolipid metabolism signaling pathways. This study lends credence to the pharmacological potential of TT extract as a breast cancer therapy.

Promoting liver cancer cell apoptosis effect of Tribulus terrestris L. via reducing sphingosine level was confirmed by network pharmacology with metabolomics.

Background: Tribulus terrestris L. (TT) is one of the most common Chinese herbs and distributes in various regions in China. TT was first documented to treat breast cancer in Shen-Nong-Ben-Cao-Jing. However, the pharmacological activities of TT extract on liver cancer have not been reported. In this study, we investigated its anti-liver cancer activity and underlying mechanism. Methods: Traditional Chinese Medicine Systems Pharmacology (TCMSP) and PharmMapper databases were used to obtain the active ingredients and the targets of TT. Genecards database was employed to acquire TT targets in liver cancer. Furthermore, Venny 2.1, Cytoscape 3.8.2, DAVID 6.8 software were utilized to analyze the relationship between TT and liver cancer. In vivo experiment: The animal model of liver cancer was established by injection of H22 cells into Balb/c mice. After five days, drugs were intragastrically administered to the mice daily for 10 days. Body weight, tumor size and tumor weight were recorded. Tumor inhibitory rate was calculated. Protein levels were examined by Western blotting. Pathological changes of liver cancer tissues were evaluated by HE and Tunel staining. Metabolomics study: LC-MS was used to analyze different metabolites between model and TTM groups. Results: 12 active ingredients of TT, 127 targets of active ingredients, 17,378 targets of liver cancer, and 125 overlapping genes were obtained. And then, 118 items of GO biological processes (BP), 54 items of GO molecular function (MF), 35 items of GO cellular component (CC) and 128 pathways of KEGG were gotten (P < 0.05). Moreover, 47 differential metabolites were affirmed and 66 pathways of KEGG (P < 0.05) were obtained. In addition, after TT and sorafenib treatment, tumor size was markedly reduced, respectively, compared with model group. Tumor weight was significantly decreased and tumor inhibitory rate was more than 44% in TTM group. After TT treatment, many adipocytes, cracks between tumor cells and apoptosis were found. The levels of pro-Cathepsin B, Cathepsin B, Bax, Bax/Bcl2, Caspase3 and Caspase7 were markedly increased, but the level of Bcl2 was significantly reduced after TT treatment. Conclusion: TT has a broad range of effects on various signaling pathways and biological processes, including the regulation of apoptosis. It exhibits antitumor activity in an animal model of liver cancer and activates the apoptotic pathway by decreasing Sph level. This study provides valuable information regarding the potential use of TT extract in the treatment of liver cancer and highlights the importance of investigating the underlying molecular mechanisms of traditional medicines for the development of new therapeutic drugs in liver cancer.

Integrated LC-MS/MS and network pharmacology approach for predictingactive ingredients and pharmacological mechanisms of Tribulus terrestris L. against cardiac diseases.

Tribulus terrestris L. (Gokshura) is a medicinal herb used for treating cardiac diseases and several other diseases. However, the active ingredients and the possible mechanism of action for treating cardiac diseases remain unclear. Hence, the study was designed to identify the active ingredients and to explore the potential mechanism of action of Tribulus terrestris L. for treating cardiac diseases by an integrated approach of metabolomics and network pharmacology. We performed HPLC-QTOF-MS/MS analysis to identify putative compounds and network pharmacology approach for predictive key targets and pathways. Using molecular docking and molecular dynamics simulation, we identified the active ingredients in Tribulus terrestris L. that can act as putative lead compounds to treat cardiac diseases. A total of 55 putative compounds were identified using methanolic extract of Tribulus terrestris L. using HPLC-QTOF-MS/MS analysis. Network pharmacology analysis predicted 32 human protein targets from 25 secondary metabolites, which have shown direct interaction with cardiac diseases. Based on the degrees of interaction, the hub targets such as TACR1, F2, F2R, ADRA1B, CHRM5, ADRA1A, ADRA1D, HTR2B, and AVPR1A were identified. In silico molecular docking and simulation resulted in the identification of active ingredients such as Kaempferol 3-rutinoside 7-glucuronide, Keioside, rutin, moupinamide, aurantiamide, quercetin-3-o-α-rhamnoside, tribuloside, and 3'',6''- Di-O-p-coumaroyltrifolin against hub protein targets. Hence, these compounds could be potential lead compounds for treating cardiac diseases. A further assessment of its efficacy can be made based on in vivo and in vitro studies for better understanding and strong assertion.Communicated by Ramaswamy H. Sarma.

Effect of Tribulus terrestris L. supplementation on Exercise-Induced Oxidative Stress and Delayed Onset Muscle Soreness Markers: A Pilot Study.

Tribulus terrestris L. contains compounds with antioxidant and anti-inflammatory properties, but its effects on exercise-induced oxidative stress and inflammatory responses are unclear. The aim of this study was to examine whether Tribulus terrestris L. supplementation can attenuate oxidative stress and inflammatory responses to acute aerobic exercise and improve DOMS. In a randomized, double-blind, crossover design study, thirteen healthy men received either a daily supplement of Tribulus terrestris L. or a placebo for 4 weeks (2-week wash-out period between trials). Before and after the supplementation periods, participants performed an exercise test to exhaustion (75% VO2max). DOMS, thigh girth, and knee joint range of motion (KJRM) were assessed before and after the exercise (2, 24, and 48 h). Blood samples were analyzed for reduced (GSH) and oxidized (GSSG) glutathione, GSH/GSSG ratio, protein carbonyls, total antioxidant capacity, creatine kinase activity, white blood cell count, and TBARS. Acute exercise to exhaustion induced inflammatory responses and changed the blood redox status in both Tribulus and Placebo groups (p < 0.050). Tribulus terrestris L. improved GSH fall (p = 0.005), GSSG rise (p = 0.001) and maintained a higher level of GSH/GSSG ratio at the 2 h point (p = 0.034). TBARS were lowered, protein carbonyls, creatine kinase activity, and white blood cell count elevation diminished significantly (p < 0.050). Tribulus terrestris L. administration did not affect DOMS, thigh girth, or KJRM (p > 0.050). 4-weeks of Tribulus terrestris L. supplementation effectively attenuates oxidative stress responses but cannot improve DOMS.

6-Week Supplementation with Tribulus terrestris L. to Trained Male CrossFit® Athletes on Muscle, Inflammation, and Antioxidant Biomarkers: A Randomized, Single-Blind, Placebo-Controlled Trial.

Tribulus terrestris L. (TT) ingredients have anti-inflammatory and antioxidant activities, but their effects on exercise-induced muscle damage (EIMD) in trained athletes are uncertain. The purpose of this single-blind placebo-controlled trial, in accordance with CONSORT guidelines, was to examine the effect of 6 weeks of TT supplementation on muscle metabolism, inflammation biomarkers, and oxidant status. Thirty trained male CrossFit® athletes were randomly assigned to be supplemented with 770 mg/day of TT (intervention group (IG)) or receive a placebo daily (control group (CG)) for 6 weeks. Muscle damage enzymes, inflammation biomarkers, and Total Antioxidant Status (TAS) were assessed at baseline (T1), 21 days after baseline (T2), and after 42 days (T3). Grace, a Workout of the Day, was measured in T1 and T3. Statistical significance (p < 0.05) was found between IG and CG in Lactate Dehydrogenase (LDH), C-reactive protein (CRP), and TAS levels at the end of the follow-up. Furthermore, TAS levels were significantly (p < 0.05) lower at T2 and T3 relative to baseline in the IG, also LDH and CRP increased significantly (p < 0.05) at T2 and T3 relative to baseline in the CG. No significant (p > 0.05) decreases in muscle damage or inflammation biomarkers were observed, although a slight downward trend was observed after 6 weeks for supplemented athletes. TT supplementation could attenuate the CrossFit® training program-induced oxidative stress, muscle damage, and inflammation which could be due to the natural antioxidant and anti-inflammatory properties of TT.

Tribulus terrestris can suppress the adverse effect of toluene on bovine and equine ovarian granulosa cells.

Influence of oil-related product toluene and herbal remedy puncturevine Tribulus terrestris L. (TT) on female reproduction is known. Yet, mechanisms of their action on ovaries in different species and potential protective effect of TT against adverse toluene action remain to be established. We studied the effect of toluene, TT, and their combination on ovarian granulosa cells from two mammalian species (cows and horses). Viability, markers of proliferation (PCNA) and apoptosis (bax), steroid hormones, IGF-I, oxytocin, and prostaglandin F (PGF) release were analyzed by trypan blue exclusion test, quantitative immunocytochemistry, and EIA/ELISA. Toluene suppressed all analyzed parameters. In both species, TT stimulated proliferation and reduced progesterone, oxytocin, and PGF. In horses, TT inhibited testosterone and IGF-I. In both species, TT supported toluene effect on viability, steroids, IGF-I, and PGF, and inverted its action on apoptosis. In cows, TT promoted toluene effect on proliferation. In horses, TT supported toluene effect on oxytocin but suppressed its influence on proliferation. In both species, toluene induced inhibitory action of TT on viability, steroids, IGF-I, and PGF, and prevented its stimulatory action on proliferation. In cows, toluene supported inhibitory action of TT on oxytocin and prevented its stimulatory action on apoptosis. In horses, toluene induced stimulatory effect of TT on apoptosis. Our results indicate potential toxic toluene effect on farm animal ovaries, applicability of TT as a biostimulator of farm animal reproduction and as a protector against the adverse influence of toluene on female reproduction.

Ameliorative effect of methanolic extract of Tribulus terrestris L. on nicotine and lead-induced degeneration of sperm quality in male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of herbal and medicinal plants to treat male infertility is well known in history. Tribulus terrestris L. (TT) belongs to the Zygophyllaceae family and it is used in folk medicine to vitalize and also improve both physical performance and sexual function in men in addition to the protective effect of the gross saponins of TT against ischemic stroke and its clinical anti-inflammatory property. AIM OF THE STUDY: This study aimed to investigate the effects of methanol extract of T. terrestris on nicotine hydrogen tartrate and lead-induced degeneration of sperm quality in male rats and to identify the volatile bioactive non-polar compounds thought to be responsible for its activity using gas chromatography-mass spectrometry (GC-MS). MATERIALS AND METHODS: The effect of T. terrestris on nicotine hydrogen tartrate and lead-induced infertility was evaluated in male rats. Fifty-four mature male albino rats weighing 220-250 g body weight were used. The rats were randomly divided into 9 equal groups (n = 6). Infertility was induced by administering nicotine hydrogen tartrate (0.50 mg/kg) through peritoneal injection (i.p.) or lead acetate (1.5 g/L) orally with drinking water for sixty days. Two doses (50 and 100 mg/kg body weight of the animal) of T. terrestris were also used. At the end of the experimental period, the rats were anesthetized and sacrificed. Blood samples were collected. Hormonal analyses were carried out on the serum. The testicle, epididymis, and accessory sex organs (seminal vesical and prostates) were removed for histopathological analysis. Gas chromatography-mass spectrometry (GC-MS) analysis of the methanol extract was also carried out to identify major volatile compounds in T. terrestris methanol extract. RESULTS: Nicotine and lead toxicity caused a significant (p < 0.05) decrease in the number of sperm, motility, and an increase in the sperm abnormalities such as the reduction in weight and size of sexual organs (testis, epididymis, and accessory sex glands), reduction of diameter and length of seminiferous tubules. The administration of T. terrestris methanol extract, however, improved the semen quantity and quality, sexual organ weights, and fertility of male rats and, thus, ameliorated the adverse effects of nicotine and lead. Ten major compounds were found from the GC-MS analysis of the extract of T. terrestris methanol extract. CONCLUSION: Findings showed that T. terrestris plant methanolic extracts ameliorated nicotine hydrogen tartrate and lead-induced degeneration of sperm quality in male rats. The GC-MS analysis of the T. terrestris plant methanolic extracts revealed the presence of several important bioactive compounds which were thought to be responsible for the ameliorative effect. Further isolation and evaluation of the individual components would provide relevant lead to finding new drugs.

Eucommia ulmoides Oliver-Tribulus terrestris L. Drug Pair Regulates Ferroptosis by Mediating the Neurovascular-Related Ligand-Receptor Interaction Pathway- A Potential Drug Pair for Treatment Hypertension and Prevention Ischemic Stroke.

Background: In this study, we used the network pharmacology approach to explore the potential disease targets of the Eucommia ulmoides Oliver (EUO)-Tribulus terrestris L. (TT) drug pair in the treatment of hypertension-associated neurovascular lesions and IS via the ferroptosis pathway. Methods: We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform to search for the key active compounds and targets of the drug pair. Based on the GeneCards database, the relevant targets for the drug pair were obtained. Then, we performed the molecular docking of the screened core active ingredients and proteins using the DAVID database and the R AutoDock Vina software. Based on the GSE22255 dataset, these screened target proteins were used to build random forest (RF) and support vector machine (SVM) models. Finally, a new IS nomogram prediction model was constructed and evaluated. Results: There were 36 active compounds in the EUO-TT drug pair. CHRM1, NR3C1, ADRB2, and OPRD1 proteins of the neuroactive ligand-receptor interaction pathway interacted with the proteins related to the ferroptosis pathway. Molecular docking experiments identified 12 active ingredients of the drug pair that may tightly bind to those target proteins. We constructed a visual IS nomogram prediction model using four genes (CHRM1, NR3C1, ADRB2, and OPRD1). The calibration curve, DCA, and clinical impact curves all indicated that the nomogram model is clinically applicable and diagnostically capable. CHRM1, NR3C1, ADRB2, and OPRD1, the target genes of the four effective components of the EUO-TT drug pair, were considered as risk markers for IS. Conclusions: The active ingredients of EUO-TT drug pair may act on proteins associated with the neuroactive ligand-receptor interaction pathway to regulate ferroptosis in vascular neurons cells, ultimately affecting the onset and progression of hypertension.

Multi-omics analysis of brain tissue metabolome and proteome reveals the protective effect of gross saponins of Tribulus terrestris L. fruit against ischemic stroke in rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Gross Saponins of Tribulus terrestris L. Fruit (GSTTF) has been reported to have a protective effect against ischemic stroke, but the related mechanism is complex and still not fully investigated. AIM OF THE STUDY: The combination of metabolomics and proteomics approach was applied to reveal the mechanisms of GSTTF in treating ischemic stroke. MATERIALS AND METHODS: The metabolite and protein changes in brain tissue were analyzed by the LC-MS-based untargeted metabolomics method and tandem mass tags (TMT)-based quantitative proteomics technology. The multivariate statistical analysis and protein-protein interaction (PPI) analysis were conducted to screen out the biomarkers, and their related pathway was further investigated by the joint pathway analysis. RESULTS: A total of 110 metabolites and 359 differential proteins, which were mainly associated with complement and coagulation cascades, sphingolipid metabolism, glycerophospholipid metabolism, glutathione metabolism, and platelet activation, etc. were screened out from the rat brain tissue. The PPI network exhibited that the protein F2, Fga, Fgb, Fgg, Plg, and C3, which are greatly involved in the complement and coagulation cascades, have a relatively high connectivity degree, indicating their importance in the process of middle cerebral artery occlusion (MCAO). The GSTTF exerted a protective effect against MCAO via modulating multiple proteins on this pathway. Moreover, F2 played a key role during the protective process and worth to be further investigated due to it has been reported as one of the therapeutic targets of ischemic stroke. CONCLUSION: The present study could improve the understanding of the potential therapeutic mechanism of GSTTF against ischemic stroke.

Optimization of ultrasound extraction of Tribulus terrestris L. leaves saponins and their HPLC-DAD-ESI-MSn profiling, anti-inflammatory activity and mechanism in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tribulus terrestris L., as an annual herb plant from Zygophyllaceae, exhibits many biological activities, and its main chemical constituents are saponins. However, the extraction process, chemical compositions, anti-inflammatory effect and mechanism of total saponins from Tribulus terrestris L. leaves are still unclear. AIM OF THE STUDY: The present study extensively evaluated the extraction process, major components, anti-inflammatory action and mechanism of Tribulus terrestris L. leaves saponins. MATERIALS AND METHODS: The ultrasonic extraction and response surface methods were adopted for optimization of extraction technology of total saponins from Tribulus terrestris L. leaves, and its compositions were detected with LC-MSn method. The anti-inflammatory activity of total saponins was studied by lipopolysaccharide induced RAW 264.7 cells and acute lung injury mice models. RESULTS: The ultrasonic extraction parameters of saponins fraction, including ethanol concentration 30%, extraction time 55 min, ratio of solvent to material 35:1 ml/g and extraction temperature 46 °C, were screened by response surface method with the extracting rate 5.49%, and thirty compositions were detected with LC-MSn method. Moreover, saponins fraction can play a stronger anti-inflammatory effect by reducing the phagocytic activity and pulmonary edema, and protection of morphology of RAW 264.7 cells and lung tissues, and decreasing the content of NO and TNF-α. Moreover, it was revealed that total saponins extract can exert the anti-inflammatory action by the inhibition of the activation of the TLR4-TRAF6-NF-κB signalling pathway. CONCLUSION: These studies imply that Tribulus terrestris L. leaves saponins may be an important anti-inflammatory drug in clinic.

Phytochemical and Biological Evaluation of a Newly Designed Nutraceutical Self-Nanoemulsifying Self-Nanosuspension for Protection and Treatment of Cisplatin Induced Testicular Toxicity in Male Rats.

The incorporation of cisplatin (CP) as a cytotoxic antineoplastic agent in most chemotherapeutic protocols is a challenge due to its toxic effect on testicular tissues. Natural compounds present a promising trend in research, so a new nutraceutical formulation (NCF) was designed to diminish CP spermatotoxicity. A combination of three nutraceutical materials, 250 mg Spirulina platensis powder (SP), 25 mg Tribulus terrestris L. extract (TT), and 100 mg fish oil (FO) were formulated in self-nanoemulsifying self-nanosuspension (SNESNS). SP was loaded into the optimized self-nanoemulsifying system (30% FO, 50% span 80/cremophor EL and 20% isopropanol) and mixed with TT aqueous solution to form SNESNS. For the SP, phytochemical profiling revealed the presence of valuable amounts of fatty acids (FAs), amino acids, flavonoids, polyphenols, vitamins, and minerals. Transmission electron microscopy (TEM) and particle size analysis confirmed the formation of nanoemulsion-based nanosuspension upon dilution. Method validation of the phytochemical constituents in NCF has been developed. Furthermore, NCF was biologically evaluated on male Wistar rats and revealed the improvement of spermatozoa, histopathological features, and biochemical markers over the CP and each ingredient group. Our findings suggest the potential of NCF with SNESNS as a delivery system against CP-induced testicular toxicity in male rats.

Integrating metabolomics and network pharmacology to explore the protective effect of gross saponins of Tribulus terrestris L. fruit against ischemic stroke in rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Tribulus terrestris L. belongs to the family Zygophyllaceae and has been widely used as a folk medicine for a long history in Asian countries. Gross saponins of Tribulus terrestris L. fruit (GSTTF) has an obvious neuroprotective effect on the treatment of ischemic stroke, but its potential therapeutic mechanisms have not been thoroughly studied. AIM OF THE STUDY: To investigate the protective effect of GSTTF against ischemic stroke in rat. MATERIALS AND METHODS: The combination of metabolomics and network pharmacology analysis was applied to investigate the protective effects of GSTTF on ischemic stroke and its putative mechanism. The related pathway of the biomarkers highlighted from metabolomics analysis was explored, then the possible targets of GSTTF were further revealed by network pharmacology analysis. Molecular docking was conducted to investigate the interaction between the active compound and target protein. RESULTS: Metabolomics analysis showed that metabolic disturbances were observed in serum for the rats in middle cerebral artery occlusion (MCAO). These MCAO-induced deviations in serum metabolism can be reversely changed by GSTTF via metabolic pathways regulation. Twenty-four proteins with the connectivity degree larger than 15 were selected by the network pharmacology analysis, which are considered as the possible therapeutic targets of the GSTTF against ischemic stroke. The results of molecular docking showed that the active compounds were capable of binding to the representative potential targets HSD11B1 and AR, respectively. And the docking mode of two compounds with the lowest binding energy to their target protein was illustrated by the ribbon binding map. CONCLUSION: The present study combines metabolomics and network pharmacology analysis to investigate the mechanism of MCAO-induced ischemic stroke and reveal the efficiency and possible mechanisms of GSTTF for ischemic stroke. Further studies on the bioactive saponin as well as their synergistic action on ischemic stroke will be conducted to better reveal the underlying mechanisms.

Saponins from Tribulus terrestris L. Extract Down-regulate the Expression of ICAM-1, VCAM-1 and E-selectin in Human Endothelial Cell Lines.

Atherosclerosis is an inflammatory disease in which intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (SELE) are consistently expressed in the vascular endothelium. Several evidence support the crucial role of adhesion molecules in the development of atherosclerosis and plaque instability. Due to the anti-inflammatory activity of Tribulus terrestris (TT), the present study investigated the effect of aqueous extract and saponin fraction of TT on the expression of ICAM-1, VCAM-1, and SELE genes in endothelial cells during normal and lipopolysaccharide (LPS) induced conditions. Human umbilical vein endothelial cells (HUVEC) and human bone marrow endothelial cells (HBMEC) were cultured, stimulated by LPS, and treated with aqueous extract and saponin fraction of TT. Finally, the expression of ICAM-1, VCAM-1, and SELE genes were measured using quantitative real-time polymerase chain reaction. LPS-induced HUVECs and HBMECs significantly increased the expression of ICAM-1, VCAM-1, and SELE in comparison with control groups (P<0.001). Treatment of LPS-induced HUVECs and HBMECs by aqueous extract and saponin fraction of TT decreased the expression of all three mentioned genes significantly (P<0.001) in comparison with LPS-induced cells. Taken together, our data suggest that TT has an anti-inflammatory effect. In vivo study about anti-inflammatory effect of this herb may provide new insights into the development of a herbal drug for the prevention/therapy of atherosclerosis.

Extraction technology, component analysis, antioxidant, antibacterial, analgesic and anti-inflammatory activities of flavonoids fraction from Tribulus terrestris L. leaves.

Research on the extraction technology, component analysis, and antioxidant, antibacterial, analgesic and anti-inflammatory activities of flavonoids fraction from Tribulus terrestris L. leaves was carried out. The extraction process was optimized by response surface method, and the maximum yield 0.27% for flavonoids fraction was achieved with concentration of ethanol solution 25.87%, ratio of solvent to material 30:1 ml/g, and ultrasonic extraction time 27.93 min. Moreover, 14 compositions were identified separately from flavonoids fraction by HPLC-DAD-ESI-MS2. In addition, flavonoids fraction exhibited a better antioxidant activity demonstrated by assays of ABTS, DPPH radical scavenging activity and ferric reducing antioxidant power activity. Furthermore, the antibacterial, analgesic and anti-inflammatory activities of flavonoids fraction were also proved to be stronger. Therefore, the present findings suggest that flavonoids fraction from Tribulus terrestris L. leaves can be a very interesting candidate for the research and development of natural and healthy herbal medicine for the pharmaceutical and food industries.

Pretreatment of Tribulus terrestris L. causes anti-ischemic cardioprotection through MAPK mediated anti-apoptotic pathway in rat.

The aim of the present investigation is the evaluation and elucidation of the mechanisms by which Tribulus terrestris L. methanol extract (TTM) devoid of fruit exhibits protection against cardiac ischemia in in vitro (H9c2 cell line) and in vivo (Wistar rat) model. Tribulus terrestris L. (TT) was used in this study to evaluate the efficacy against cardiac ischemia employing in vitro and in vivo models of myocardial ischemia. H9c2 cells were used for the in vitro induction of ischemia. Male Wistar rats (10 weeks old) weighing 180-220 g were used for the in vivo experiments. ECG and clinically relevant cardiac biomarkers like serum lactate dehydrogenase, serum creatinine kinase, serum creatinine kinase myocardial B fraction, serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase were analysed to evaluate efficacy in the rat. For elucidation of molecular mechanisms of its beneficial activity in vitro, expression of apoptotic markers like Bax, Bad, Bcl-2 and signalling pathways involving mitogen-activated protein kinases like p38α, JNK, and Akt were studied. Tribulus terrestris L. was found effective against cardiac ischemia in the rat which was evident from ECG and various cardiac biomarkers analysis. Tribulus terrestris L. was found to act through the mitogen-activated signalling pathway leading to prevention of apoptosis during ischemic insult. The beneficial effect of Tribulus terrestris L. against cardiac ischemia was seen both in in vitro and in vivo models via its anti-apoptotic potential.

Effect of Tribulus terrestris L. on sperm parameters in men with idiopathic infertility: A systematic review.

BACKGROUND AND OBJECTIVE(S): Tribulus terrestris L. is an herbal plant that has long been used as sex stimulant and to treat male infertility. This systematic review collected the clinical trials and/or quasi-experimental studies on the effect of T. terrestris on sperm parameters in idiopathic male infertility. MATERIALS AND METHODS: To search the related articles, Cochrane Library, EMBASE, ProQuest, Clinicaltrial.gov, WHO, Google Scholar, MEDLINE via Pubmed, Web of Science, SID, Magiran, Irandoc, and Iranmedex databases were used without any time limitation. Words used to search were T. terrestris L., Tribestan, male infertility and sperm parameters based on the MeSH glossary. To assess the eligibility of the articles, the views of the two authors and in cases where there was no agreement, the third person was used. RESULTS: Through searching of the databases, 5775 articles were identified, of which 3509 were entered after removing the duplicates. Afterward, 102 articles were screened for inclusion. Finally, 7 articles were included in this systematic review. Only one quasi-experimental without control arm article reported that the use of T. terrestris L. in the treatment of idiopathic male infertility was not effective, In the others, T. terrestris L. was reported to be effective in improving some or all parameters of the sperm, namely number, motility and morphology. CONCLUSION: The results of the present systematic review showed that the consumption of T terrestris L., in general, resulted in the improvement of sperm parameters. This result was obtained from 6 out of the 7 articles.

Extraction technology, component analysis, and in vitro antioxidant and antibacterial activities of total flavonoids and fatty acids from Tribulus terrestris L. fruits.

The current study focused on the extraction technology, components analysis, in vitro antioxidant and antibacterial activities of total flavonoids and fatty acids from Tribulus terrestris L. fruits. The extraction process of total flavonoids and fatty acids was optimized by the response surface method, and the compositions were identified from the two extracts by HPLC-DAD-ESI-MS- and GC-MS, respectively. In addition, the antioxidant and antibacterial activities were evaluated by assay of ABTS, DPPH radical scavenging activity, ferric reducing antioxidant power and minimal inhibitory concentration. The yields of total flavonoids and fatty acids were 0.46 and 9.76% under the optimized conditions. Moreover, nine and eight compositions were identified from the two extracts based on the related references, respectively. In addition, total flavonoids and fatty acids extracts both exhibited certain antioxidant and antibacterial activities. The present findings suggest that total flavonoids extracted from T. terrestris L. fruits comprised a more interesting candidate than fatty acids for the research and development of natural and healthy antioxidants and antibacterial agents for the pharmaceutical and food industries.

Análise histológica do fêmur de camundongos suíços fêmeas ovariectomizadas e não ovariectomizadas suplementadas com extrato de Tribulus terrestris L / Histological analysis of the fem of female swiss mice ovariectomized and not ovariectomized supplements with extract of Tribulus terrestris L

Introdução: a incapacidade de gerar filhos é chamada de infertilidade, e acomete grande parte da população, principalmente mulheres entrando no período da menopausa. Tal condição afeta a qualidade de vida, pois aumenta o desgaste ósseo, fazendo com que a busca por tratamentos de reposição hormonal aumente exponencialmente. Entretanto, tais tratamentos possuem efeitos colaterais, o que apresenta plantas medicinais como tratamentos alternativos. Objetivo: avaliar a histologia do fêmur de camundongos suíços fêmeas ovariectomizadas e não ovariectomizadas tratadas ou não com Tribulus terrestris L. (TT) Métodos: Foram utilizados 28 camundongos suíços fêmeas divididos em 4 grupos, sendo dois controles com e sem suplementação e dois que sofreram o processo de ovariectomia bilateral, com e sem suplementação. O extrato da planta foi administrado por 21 dias por gavagem. Ao final do tratamento, os animais foram eutanasiados e o fêmur coletado para análise histológica. Resultados: o TT não foi capaz de alterar significativamente o diâmetro do osso. A castração, como esperado, mostrou possível osteopenia. A planta pode ter efeito protetor aos ossos devido a efeito antiartrítico. Conclusão: na análise histológica descritiva do fêmur, foram evidenciadas alterações que se associam à osteopenia nos animais castrados e efeito ósseo-protetor nos animais suplementados com o extrato dos frutos do Tribulus terrestris L.

Introduction: inability to generate children is called infertility and affects a large part of the population, especially women entering the menopausal period. Such a condition affects the quality of life, including the use of the bone system, causing the search for hormone replacement services exponentially. However, such treatments shave light collectors, such as medicinal plants as alternative treatments. Objective: to evaluate the histology of the treatment of pairs of ovariectomized and non-ovariectomized women treated with or without Tribulus terrestris L. (TT). Methods: twenty-eight Swiss mice were divided into four groups, two controls with and without supplementation, and two with bilateral ovariectomy, with and without supplementation. The plant extract was administered for 21 days by way of gavage. After the evaluation, the animals were eutanasiated and their femur was collected for histological analysis. Results: the TT was not able to change the diameter of the bone. Castration, as expected, allowed for osteopenia. The plant may have a protective effect on bones because of an anti-arthritic effect. Conclusion: in the descriptive histological analysis of phosphorus, there were evidences alterations that associate the osteopenia in the animals and the bony-protective effects in the animals supplemented with the extract of the fruits of Tribulus terrestris L.