Diosgenin biosynthesis investigation in medicinal herb (Tribulus terrestris) by transcriptome analysis.

Next-generation sequencing (NGS) has revolutionized the analysis of specific genes, pathways, and their regulation in various species. Tribulus terrestris L., an annual medicinal herb of Zygophyllaceae family, has gained significant attention due to its diverse medicinal properties, including anti-inflammatory, antimicrobial, and anti-cancer effects. Diosgenin, a steroidal saponin, is the major bioactive compound responsible for the medicinal importance of T. terrestris. However, there is a paucity of information regarding the genes involved in the diosgenin biosynthetic pathway in T. terrestris. To address this gap, this study aimed to identify candidate genes associated with diosgenin biosynthesis through whole transcriptome profiling. A total of ∼7.9 GB of data, comprising 482 million reads, was obtained and assembled into 148,871 unigenes. Subsequently, functional annotations were assigned to 50 % of the unigenes using sequence similarity searches against the NCBI non-redundant (NR), Uniprot, KEGG, Pfam, GO, and COG databases, primarily based on Gene Ontology and KEGG-KAAS pathways. The majority of unigenes associated with the biosynthesis of the steroidal diosgenin backbone exhibited up-regulation in the fruit, leaf, and root tissues, except the SQE gene in root. The differential expression of selected genes was further validated through quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the study identified 21,026 unigenes related to transcription factors and 15,551 unigenes containing simple sequence repeats (SSR). Notably, di-nucleotide SSR motifs exhibited a high repeat frequency. These findings greatly enhance our understanding of the diosgenin biosynthesis pathway and provide a basis for future research in molecular investigation and metabolic engineering, specifically for boosting diosgenin content.

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.

Can flaxseed, chia or puncture vine affect mare ovarian cell functions and prevent the toxic effect of the environmental contaminant toluene?

The aim of this in vitro study was to examine the potential effect of functional food plant extracts, namely, extracts of flaxseed (Linum usitatissimum L.), chia (Salvia hispanica) and puncture vine (Tribulus terrestris L.), on basic mare ovarian cell functions and their response to the environmental contaminant toluene. Mare granulosa cells were incubated with and without toluene (0, 0.02, 0.2 or 2.0 µg/mL) in the presence or absence of flaxseed, chia and puncture vine extracts (10 µg/mL). Markers of cell proliferation (accumulation of proliferating cell nuclear antigen, PCNA) and apoptosis (accumulation of bax), viability (Trypan blue extrusion) and the release of progesterone (P), oxytocin (OT) and prostaglandin F 2 alpha (PGF) were measured. Toluene reduced all other measured parameters except OT release. All the tested plants were able to reduce cell viability and the release of P and PGF, but they did not influence other indexes. Moreover, flaxseed mitigated toluene action on ovarian cell proliferation, apoptosis, OT and PGF, whilst puncture vine prevented and inverted toluene action on P and PGF ourput. Chia extract did not modify toluene action on any parameter. On the other hand, toluene was able to promote the inhibitory action of flaxseed on cell viability and P release and to prevent the inhibitory action of all the plant extracts on PGF release. The present study (1) is the first demonstration, that flaxseed, chia and puncture vine can directly suppress mare ovarian cell functions, (2) shows that toluene can suppress basic ovarian cell functions and modify the reproductive effect of food plants and (3) demonstrates the ability of flaxseed and puncture vine, but not of chia, to prevent some toxic effect of toluene on mare ovarian cell functions.

Herbal Formulation Comprised of Methanol Extracts of Tribulus terristris L. and Zingiber officinale Roscoe Has Antihypertensive Effects.

People prefer to use medicinal plants rather than chemical compounds because they are low cost and have fewer adverse events. Zingiber officinale Roscoe is a natural dietary rhizome with anti-oxidative, anti-inflammatory and anti-carcinogenic properties. Tribulus terrestris L. has been used for the treatment of impotence, to enhance sexual drive and performance and for its diuretic and uricosuric effects. The aim of this study was to evaluate the combined effect of 2 extracts, Tribulus terristris and Zingiber officinale (TZ) for antioxidant, enzyme modulation, liver function, kidney function, blood profile and anti-hypertensive effects, which may pave the way for possible therapeutic applications. Antioxidant potential was measured with the 2,2-diphenyl-1-picryl-hydrazyl-hydrate free radical method antioxidant assay (DPPH) and kojic acid was used as the standard drug for tyrosine inhibition assay. The effect of TZ on biochemical parameters of the liver (alanine transferase [ALT], alkaline phosphatase [ALP], aspartate aminotransferase [AST], total serum protein, total serum albumin, serum bilirubin), kidney (blood urea and creatinine) and hematology (hemoglobin, red blood cells [RBC], platelets, thin-layer chromatography, neutrophils, eosinophils, lymphocytes, monocytes, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration) of Wister rats were studied by administering 100, 250 and 500 mg/kg-1 body weight TZ dose orally for 28 days. Antihypertensive effects were measured by the invasive method. The results showed that the scavenging percentage of TZ was 78.5 to 80.4, with an IC50 value of 1166.7 µg/ ml and tyrosinase inhibition was 72% compared with 93% for kojic acid. Different doses (100, 250 and 500 mg/kg) did not show an increase in serum biomarkers of liver and renal parameters. A significant increase in hemoglobin, erythrocytes, hematocrit, white blood cells (WBC) and lymphocytes with no significant increase/decrease in platelet count was observed but blood pressure was significantly decreased. Therefore, we concluded that TZ is safe and can be used in the treatment of hypertension.

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.

In vitro evaluation of immunomodulatory, anti-diabetic, and anti-cancer molecular mechanisms of Tribulus terrestris extracts.

Dampened immunity and impaired wound healing in diabetic patients may lead to diabetic foot ulcer disease, which is the leading cause of limb amputations and hospitalization. On the other hand, cancer is the most significant cause of mortality globally, accounting for over 10 million fatalities in 2020, or nearly one in every six deaths. Plants and herbs have been used to treat chronic diseases due to their essential pharmaceutical attributes, such as mitigating drug resistance, ameliorating systemic toxicities, reducing the need for synthetic chemotherapeutic agents,and strengthening the immune system. The present study has been designed to evaluate the effects of Tribulus terrestris on wound healing, cytotoxic and anti-inflammatory responses against HepG-2 liver cancer cell line. Two solvents (methanol and ethanol) were used for root extraction of T. terrestris. The wound healing potential of the extracts was studied on diabetic cell culture line by scratch assay. The anti-oxidant and cytotoxic potentials were evaluated by in vitro assays against HepG2 cell line. The methanolic root extract resulted in the coverage of robust radical scavenging or maximum inhibition of 66.72%,potent cytotoxic activity or reduced cell viability of 40.98%, and anti-diabetic activity having mighty α-glucosidase inhibition of 50.16% at a concentration of 80 µg/ml. Significant reduction in the levels of LDH leakage (56.38%), substantial ROS (48.45%) and SOD (72.13%) activities were recorededMoreover, gene expression analysis demonstrated the down-regulation of inflammatory markers (TNF-α, MMP-9, Bcl-2, and AFP) in HepG-2 cells when treated with T. terresteris methanolic extract as compared to stress. Furthermore, the down-regulation of inflammatory markers was validated through ELISA-mediated protein estimation of IL-1ß and TNF-α. It is expected that this study will lay a foundation and lead to the development of efficient but low-cost, natural herbs extract-based dressing/ointment for diabetic patients and identify potential drug metabolites to treat out-of-whack inflammatory responses involved in cancer onset, progression, and metastasis.

Fructose milieu undermines the therapeutic effect of Tribulus terrestris extract on neuroblastoma cell line via maintaining mitochondrial function.

Fructose overconsumption promotes tumor progression. Neuroblastoma is a common extracranial tumor with about 50% 5-year survival rate in high-risk children. The anti-tumor effect of Tribulus terrestris might bring new hope to neuroblastoma therapy. However, whether fructose disturbs the therapeutic effect of T. terrestris is currently unknown. In this study, the mouse neuroblastoma cell line, Neuro 2a (N2a) cells, was used to investigate the therapeutic effects of T. terrestris extract at various dosages (0.01, 1, 100 ng/ml) in regular EMEM medium or extra added fructose (20 mM) for 24 h. 100 ng/ml T. terrestris treatment significantly reduced the cell viability, whereas the cell viabilities were enhanced at the dosages of 0.01 or 1 ng/ml T. terrestris in the fructose milieu instead. The inhibition effect of T. terrestris on N2a migration was blunted in the fructose milieu. Moreover, T. terrestris effectively suppressed mitochondrial functions, including oxygen consumption rates, the activities of electron transport enzymes, the expressions of mitochondrial respiratory enzymes, and mitochondrial membrane potential. These suppressions were reversed in the fructose group. In addition, the T. terrestris-suppressed mitofusin and the T. terrestris-enhance mitochondrial fission 1 protein were maintained at basal levels in the fructose milieu. Together, these results demonstrated that T. terrestris extract effectively suppressed the survival and migration of neuroblastoma via inhibiting mitochondrial oxidative phosphorylation and disturbing mitochondrial dynamics. Whereas, the fructose milieu blunted the therapeutic effect of T. terrestris, particularly, when the dosage is reduced.

The effect of Tribulus terrestris supplementation on inflammation, oxidative stress, and performance of recreational runners: study protocol for a randomized placebo-controlled trial.

BACKGROUND: High intensity and endurance exercises lead to exercise-induced oxidative stress (EIOS), exercise-induced muscle damage (EIMD), and inflammation, which are the influencing factors on muscle soreness, localized swelling, and sports performance decrease. Therefore, the purpose of this study is to determine the effectiveness of Tribulus terrestris (TT) as an herbal supplement with antioxidant and anti-inflammatory properties on the nutritional, oxidative, inflammatory, and anti-inflammatory status, as well as the sports performance of recreational runners. METHODS/DESIGN: This study is a double-blind, randomized, placebo-controlled trial, which will be conducted among recreational runners of Tabriz stadiums, Iran. Thirty-four recreational runners will be selected, and participants will be assigned randomly to two groups: to receive 500 mg TT supplement or placebo capsules twice daily for 2 weeks. Both groups will do high-intensity interval training (HIIT) workouts during the study. Baseline and post-intervention body composition, muscle pain, and aerobic and anaerobic performance will be assessed. In addition, assessment of malondialdehyde (MDA), total antioxidant capacity (TAC), total oxidant status (TOS), superoxide dismutase (SOD), glutathione peroxidase (GPx), uric acid (UA), 8-iso-prostaglandin F2α (8-iso-PGF2α), protein carbonyl (PC), catalase (CAT), glutathione (GSH), nitric oxide (NO), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), creatine kinase (CK), myoglobin (MYO), lactate dehydrogenase (LDH), insulin-like growth factor-1 (IGF-1) irisin, cortisol, and brain-derived neurotrophic factor (BDNF) will be done during three blood samplings. Changes in oxidative stress, anti/inflammatory biomarkers, and sports performance will be assessed as primary outcomes. DISCUSSION: This study will be the first to assess the potential effects of TT on recreational runners. Our results will contribute to the growing body of knowledge regarding TT supplementation on the nutritional, oxidative, inflammatory, and anti-inflammatory status and sports performance in recreational runners. TRIAL REGISTRATION: Iranian Registry of Clinical Trials ( www.irct.ir ) (ID: IRCT20150205020965N8 ). Registration date: 13 February 2021.

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.

Tribulus terrestris L. extract ameliorates atherosclerosis by inhibition of vascular smooth muscle cell proliferation in ApoE-/- mice and A7r5 cells via suppression of Akt/MEK/ERK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis (AS) is one of major threatens of death worldwide, and vascular smooth muscle cell (VSMC) proliferation is an important characteristic in the progression of AS. Tribulus terrestris L. is a well-known Chinese Materia Medica for treating skin pruritus, vertigo and cardiovascular diseases in traditional Chinese medicine. However, its anti-AS activity and inhibition effect on VSMC proliferation are not fully elucidated. AIMS: We hypothesize that the furostanol saponins enriched extract (FSEE) of T. terrestris L. presents anti-AS effect by inhibition of VSMC proliferation. The molecular action mechanism underlying the anti-VSMC proliferation effect of FSEE is also investigated. MATERIALS AND METHODS: Apolipoprotein-E deficient (ApoE-/-) mice and rat thoracic smooth muscle cell A7r5 were employed as the in vivo and in vitro models respectively to evaluate the anti- AS and VSMC proliferation effects of FSEE. In ApoE-/- mice, the amounts of total cholesterol, triglyceride, low density lipoprotein and high density lipoprotein in serum were measured by commercially available kits. The size of atherosclerotic plaque was observed by hematoxylin & eosin staining. The protein expressions of α-smooth muscle actin (α-SMA) and osteopontin (OPN) in the plaque were examined by immunohistochemistry. In A7r5 cells, the cell viability and proliferation were tested by MTT and Real Time Cell Analysis assays. The cell migration was evaluated by wound healing assay. Propidium iodide staining followed by flow cytometry was used to analyze the cell cycle progression. The expression of intracellular total and phosphorylated proteins including protein kinase B (Akt) and mitogen-activated protein kinases (MAPKs), such as mitogen-activated extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), were detected by western blotting analysis. RESULTS: FSEE significantly reduced the area of atherosclerotic plaque in high-fat diet-fed ApoE-/- mice. And FSEE increased the protein expression level of α-SMA and decreased the level of OPN in atherosclerotic plaque, which revealed the inhibition of VSMC phenotype switching and proliferation. In A7r5 cells, FSEE suppressed fetal bovine serum (FBS) or oxidized low density lipoprotein (oxLDL)-triggered VSMC proliferation and migration in a concentration dependent manner. FSEE protected against the elevation of cell numbers in S phase induced by FBS or oxLDL and the reduction of cell numbers in G0/G1 phase induced by oxLDL. Moreover, the phosphorylation of Akt and MAPKs including MEK, ERK and JNK could be facilitated by FBS or oxLDL, while co-treatment of FSEE attenuated the phosphorylation of Akt induced by oxLDL as well as the phosphorylation of MEK and ERK induced by FBS. In addition, (25R)-terrestrinin B (JL-6), which was the main ingredient of FSEE, and its potential active pharmaceutical ingredients tigogenin (Tigo) and hecogenin (Heco) also significantly attenuated FBS or oxLDL-induced VSMC proliferation in A7r5 cells. CONCLUSION: FSEE presents potent anti- AS and VSMC proliferation activities and the underlying mechanism is likely to the suppression of Akt/MEK/ERK signaling. The active components of FSEE are JL-6 and its potential active pharmaceutical ingredients Tigo and Heco. So, FSEE and its active compounds may be potential therapeutic drug candidates for AS.

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.

Potential protective effect of puncturevine (Tribulus terrestris, L.) against xylene toxicity on bovine ovarian cell functions.

The action of the medicinal plant Tribulus terrestris (TT) on bovine ovarian cell functions, as well as the protective potential of TT against xylene (X) action, remain unknown. The aim of the present in vitro study was to elucidate the influence of TT, X and their combination on basic bovine ovarian cell functions. For this purpose, we examined the effect of TT (at doses of 0, 1, 10, and 100 ng/mL), X (at 20 ?g/mL) and the combination of TT + X (at these doses) on proliferation, apoptosis and hormone release by cultured bovine ovarian granulosa cells. Markers of proliferation (accumulation of PCNA), apoptosis (accumulation of Bax) and the release of hormones (progesterone, testosterone and insulin-like growth factor I, IGF-I) were analyzed by quantitative immunocytochemistry and RIA, respectively. TT addition was able to stimulate proliferation and testosterone release and inhibit apoptosis and progesterone output. The addition of X alone stimulated proliferation, apoptosis and IGF-I release and inhibited progesterone and testosterone release by ovarian cells. TT was able to modify X effects: it prevented the antiproliferative effect of X, induced the proapoptotic action of X, and promoted X action on progesterone but not testosterone or IGF-I release. Taken together, our observations represent the first demonstration that TT can be a promoter of ovarian cell functions (a stimulator of proliferation and a suppressor of apoptosis) and a regulator of ovarian steroidogenesis. X can increase ovarian cell proliferation and IGF-I release and inhibit ovarian steroidogenesis. These effects could explain its anti-reproductive and cancer actions. The ability of TT to modify X action on proliferation and apoptosis indicates that TT might be a natural protector against some ovarian cell disorders associated with X action on proliferation and apoptosis, but it can also promote its adverse effects on progesterone release.

Synchronized resistance training and bioactive herbal compounds of Tribulus Terrestris reverse the disruptive influence of Stanozolol.

Androgenic-Anabolic Steroids (AAS) consumption may have irreversible effects on athletes' hearts. The beneficial effects of Tribulus Terrestris (TT) have been shown to reduce cardiovascular risks through disruption in apoptosome complex construction. Therefore, this study aimed to investigate the effect of eight weeks of resistance training (RT) with TT consumption in the heart tissue of rats exposed to Stanozolol. Thirty-five male rats were divided into seven groups, Control group, Stanozolol (ST), ST + 100 mg/kg TT, ST + 50 mg/kg TT, RT + ST, RT + ST + 100 mg/kg TT, and RT + ST + 50 mg/kg TT. Differential genes expression was measured by q-RT-PCR. Artificial intelligence highlighted apoptosis pathways as a vital process in cardiovascular risks. Hence, we estimated the binding affinity of chemical and bioactive molecules on the cut point hub gene by pharmacophore modeling and molecular docking. Moreover, ST increased IL-6, Cat, Aif-1, and Caspase-9. 100 mg/kg TT has a more favorable effect than 50 mg/kg T. Also, RT with TT had interactive effects on reducing IL-6, Cat, Aif-1, and Caspase-9. RT and TT consumption seemed to synergistically reduce the apoptotic pathway markers in the heart tissue of rats exposed to the supra-physiologic dose of ST. Moreover, TT could be added to supplements and sports drink to increase an athlete's performance.

Antiglycation and antioxidant activities of the crude extract and saponin fraction of Tribulus terrestris before and after microcapsule release.

OBJECTIVE: The present study investigated antiglycation and antioxidant activities of crude dry extract and saponin fraction of Tribulus terrestris. It also developed a method of microencapsulation and evaluated antiglycation and antioxidant activities of crude dry extract and saponin fraction before and after microcapsule release. METHODS: Antiglycation activity was determined by relative electrophoretic mobility (REM), free amino groups and inhibition of advanced glycation end-product (AGE) formation. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric ion-reducing antioxidant power (FRAP), nitric oxide (NO) and thiobarbituric acid reactive species (TBARS) tests. Microcapsules were prepared using maltodextrin as wall material and freeze-drying as encapsulation technique. Morphological characterization of microcapsules was evaluated by scanning electron microscopy, and encapsulation efficiency and microcapsule release were determined by total saponins released. Antiglycation and antioxidant assays were performed using crude dry extract and saponin fraction of T. terrestris before and after release. RESULTS: Saponin fraction showed an increase of 32.8% total saponins. High-performance liquid chromatography-mass spectrometry analysis showed the presence of saponins in the obtained fraction. Antiglycation evaluation by REM demonstrated that samples before and after release presented antiglycation activity similar to bovine serum albumin treated with aminoguanidine. Additionally, samples inhibited AGE formation, highlighting treatment with saponin fraction after release (89.89%). Antioxidant tests demonstrated antioxidant activity of the samples. Crude dry extract before encapsulation presented the highest activities in DPPH (92.00%) and TBARS (32.49%) assays. Saponin fraction before encapsulation in FRAP test (499 µmol Trolox equivalent per gram of dry sample) and NO test (15.13 µmol nitrite formed per gram of extract) presented the highest activities. CONCLUSION: This study presented antiglycation activity of crude dry extract and saponin fraction of T. terrestris, besides it demonstrated promising antioxidant properties. It also showed that the encapsulation method was efficient and maintained biological activity of bioactive compounds after microcapsule release. These results provide information for further studies on antidiabetic and antiaging potential, and data for new herbal medicine and food supplement formulations containing microcapsules with crude extract and/or saponin fraction of T. terrestris.

Identification of steroidal saponins from Tribulus terrestris and their in silico docking studies.

Tribulus terrestris is known to possess many pharmacological properties, most notably its anticancer activities, owing to its rich steroidal saponin contents. Even though many reports are available elucidating the anticancer potential of the herb, we, for the very first time have attempted to isolate and identified the active compound present in seed crude saponin extract and confers its in silico docking ability with various cellular targets proteins. High performance thin layer chromatography confirms the presence of active saponins in leaf and seed saponin extracts which were further fractionated by silica gel column chromatography. Fractions collected were assessed for cytotoxicity on human breast cancer cells. High resolution liquid chromatography and mass spectroscopy was employ to identify the active components present in fraction with highest cytotoxicity. Intriguingly, Nautigenin type of steriodal saponin was identified to present in the active fraction of seed extract (SF11) and the identified compound was further analyzed for its in silico docking interaction using PyRx AutodockVina. Docking studies revealed the high binding affinity of Nuatigenin at significant sites with apoptotic proteins Bcl-2, Bax, caspase-3, caspase-8, p53 and apoptosis inducing factor along with cell surface receptors estrogen receptor, projesterone receptor, epidermal growth factor receptor, and human epidermal growth factor receptor-2. Thus, the conclusions were drawn that saponin fraction of Tribulus terrestis possesses active compounds having anticancer property and specifically, Nuatigenin saponin can be considered as an important therapeutic drug for the breast cancer treatment.

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.

Antilithiatic activity of a non-pharmacopoeial Unani formulation in chemically induced urolithiasis in rats.

OBJECTIVES: Parshioshan (Adiantum capillus-veneris L.), Duqu (Peucedanum grande C.B. Clarke), Kaknaj (Physalis alkekengi L.) and Kharekhasak (Tribulus terresteris L.) have been selected for this study as they have been associated with medicinal actions for litholytic activity. METHODS: The experiment was carried out in Sprague Dawley rats divided into seven groups, serving as plain control, disease control, standard control, curative A and B and preventive A and B groups. Animals of plain control received distilled water. Remaining six groups received Ethylene glycol 0.75% and Ammonium chloride 1% by adding in the drinking water for the first three days followed by 0.75% Ethylene glycol for 18 days. From 8th day till 21st day, standard control received Cystone in the dose of 750 mg/kg. Preventive and curative test groups were treated with hydroalcoholic extract of the test drug in the dose of 132 mg/kg and 264 mg/kg from 1st to 21st day and 8th to 21st day of calculi induction. RESULTS: Test drug reduced the number of calcium oxalate crystals in the urine; the level of urinary calcium, creatinine, magnesium, phosphorus, sodium and chloride decreased significantly in standard and test groups. The urine volume increased significantly in all the test groups. The level of serum calcium, urea, phosphorus and creatinine were significantly reduced in all the test groups. CONCLUSIONS: These results indicated that the test drug reduced and prevented the growth of urinary stones. Moreover, the test drug also possessed significant antiurolithiatic activity. However, the protective effect was found more than its curative effect.

Tribulus terrestris fruit extract inhibits autophagic flux to diminish cell proliferation and metastatic characteristics of oral cancer cells.

Elevated autophagy is highly associated with cancer development and progression. Fruit extracts of several plants inhibit activity of autophagy-related protease ATG4B and autophagy activity in colorectal cancer cells. However, the effects of these plant extracts in oral cancer cells remain unclear. In this study, we found that the extracted Tribulus terrestris fruit (TT-(fr)) and Xanthium strumarium fruit had inhibitory effects on autophagy inhibition in both SAS and TW2.6 oral cancer cells. Moreover, the fruit extracts had differential effects on cell proliferation of oral cancer cells. In addition, the fruit extracts hampered cell migration and invasion of oral cancer cells, particularly in TT-(fr) extracts. Our results indicated that TT-(fr) extracts consistently inhibited autophagic flux, cell growth and metastatic characteristics of oral cancer cells, suggesting TT-(fr) might contain function ingredient to suppress oral cancer cells.