Storage and time course effects on the quality of oil extracted from Phyllanthus amarus Schumach and Annona muricata Linn and their antidiabetic potentials.

With the advent of modern technology, advancements in processing and storage techniques, and increasing medical knowledge, people are becoming aware of deterioration in the quality of medicinal products due to storage methods and time. In most cases, herbal products are not consumed immediately after production; as such, improper storage can result in physical, chemical, and microbiological changes. The study evaluated the effect of storage methods and time on the quality of oil extracted from Phyllanthus amarus Schumach and Annona muricata Linn and assessed their antidiabetic and antioxidative effects. Plants were air-dried, pulverized, and then subjected to Soxhlet extraction in petroleum ether. The oil was evaluated for phytochemical constituents and the effects of time and storage methods on its physicochemical properties. Characterization of the oil was done by spectroscopic techniques. Oils from both plants contained tannins, flavonoids, alkaloids, steroids, glycosides, terpenoids, phlobotannins, resins, reducing sugar, phenols, and saponins in different proportions. The oil from A. muricata had higher phenolic (3.11 ± 0.31 mg GAE/g), flavonoid (11.82 ± 0.08 mg QUE/g), alkaloid (16.37 ± 0.56 mg APE/g), and tannin (7.13 ± 0.47 mg CE/g) contents than the oil from P. amarus, which had 0.54 ± 0.08 mg GAE/g, 7.83 ± 0.13 mg QUE/g, 9.87 ± 0.15 mg APE, and 3.16 ± 0.12 mg CE/g for total phenolic, flavonoids, alkaloids, and tannins, respectively. Initial acid, iodine, peroxide, and saponification values recorded for P. amarus were 5.63 ± 0.82 mg KOH/g, 97.17 ±0.53 Wijis, 9.31 ± 0.15 mEq/kg, and 116.11 ± 0.74 mg KOH/g, respectively, significantly different from those of A. muricata , which had values of 1.17 ± 0.08 mg KOH, 76.23 ± 0.03 Wijis, 6.75 ± 0.47 mEq/kg, and 193.31 ± 0.52 mg KOH/g, respectively. FT-IR characterization of the oils revealed the presence of carboxylic acid, alkyl, alkene, alkane, haloalkane, aldehyde, aromatic amine, α-unsaturated and ß-unsaturated esters, and phenol functional groups. P. amarus oil inhibited α-amylase (IC50 0.17 ± 0.03 mg/ml), α-glucosidase (IC50 0.64 ± 0.03 mg/ml), and xanthine oxidase (0.70 ± 0.01 mg/ml) to a greater extent than A. muricata oil, with IC50 values of 0.43 ± 0.05 mg/ml (α-amylase), 2.25 ± 0.31 mg/ml (α-glucosidase), and 0.78 ± 0.07 mg/ml (xanthine oxidase). This study showed that oils from the tested plants have low rancidity with a moderate shelf life. The extracts contained essential phytoconstituents that significantly inhibited α-glucosidase and xanthine oxidase. These effects of the oil indicate their potential to prevent diabetes, gout, and oxidative stress. Consequently, the supply of P. amarus and A. muricata in homemade diets is strongly encouraged for healthy living.

Identification of authentic Phyllanthus amarus species in India.

The Phyllanthus is a plant used in the traditional Ayurvedic Medicine system and has more than 800 species. These species grow in the same area and there are chances of adulteration of other species and, incorrect identification may also lead to wrong reports. This study was attempted by Enovate Biolife Pvt. Ltd. to identify authentic Phyllanthus amarus. The nine raw material samples were collected from different populations/suppliers from various zones of India for the study. All the samples were analysed using microscopic and macroscopic ID, and by using the High Performance Thin Layer Chromatography (HPTLC) fingerprint method. The samples collected from the Central zone (Lucknow PA-08, Uttar Pradesh) and the Southern zone (Coimbatore PA-05, and Chennai PA-09, Tamil Nadu) of India were found to be authentic P. amarus by the mentioned identification methods.

Aqueous leaf extract of Phyllanthus amarus protects against oxidative stress and misfiring of dopaminergic neurons in Paraquat-induced Parkinson's disease-like model of adult Wistar rats.

BACKGROUND OF THE STUDY: Phyllanthus amarus has high nutritional value and is beneficial in managing and treating diverse ailments. This study assessed the role of aqueous leaf extract of Phyllanthus amarus on Paraquat (PQ) induced neurotoxicity in the substantia nigra of Wistar rats. MATERIALS AND METHODS: The role of aqueous leaves extract of Phyllanthus amarus was assessed using an open field test (OFT) for motor activity, oxidative stress biomarkers [Catalase (CAT), and Superoxide Dismutase (SOD)], histological examination (H and E stained) for cytoarchitectural changes and immunohistochemical studies using tyrosine hydroxylase (TH) as a marker for dopaminergic neurons. Forty-two (42) rats were categorized into six groups (n = 7); group 1: control was administered 0.5 ml/kg distilled water, group 2: received 10 mg/kg PQ + 10 mg/kg L-dopa as reference drug, group 3; received 10 mg/kg PQ, while group 4: received 10 mg/kg PQ + 200 mg/kg P. amarus, group 5: received 10 mg/kg PQ + 300 mg/kg P. amarus, and group 6: received 10 mg/kg PQ + 400 mg/kg P. amarus respectively, for 14 days. All administrations were done orally; a significant difference was set at p < 0.05. RESULTS AND DISCUSSION: The study's open field test (OFT) revealed no motor activity deficit with Paraquat (PQ) exposure. Also, cytoarchitectural distortions were not observed with Paraquat (PQ) only treatment group compared to the control and other groups pretreated with P. amarus and L-dopa. Moreover, the Paraquat (PQ) only treatment group showed oxidative stress by significantly decreasing the antioxidant enzyme (SOD) compared to the control and L-dopa pretreated group. A significant decrease in tyrosine hydroxylase (TH) expressing dopaminergic neurons was also observed in Paraquat (PQ) only treatment. However, P. amarus treatment showed therapeutic properties by significantly increasing tyrosine hydroxylase (TH) expressing dopaminergic neuron levels relative to control. CONCLUSION: Aqueous leaf extract of Phyllanthus amarus possesses therapeutic properties against Paraquat (PQ) induced changes in the substantia nigra of Wistar rats.

Safety and antimalarial therapeutic index of alkaloid-rich extract of Phyllanthus amarus Schumach. & Thonn. in mice.

Background: Malaria fever is known to cause around one million passings per annum. This life-threatening infection is predominant in most part of Africa. Malaria vaccinations are challenging in Nigeria, particularly in rural areas. Drugs derived from plants have been utilized customarily to treat malaria. In this manner, assurance of the harmfulness and antimalarial capacity of plant derived drugs can demonstrate to be the source of novel lead compound to control malaria. The aim of this study was to evaluate the safety and antimalarial therapeutic index of alkaloid-rich extract of Phyllanthus amarus in mice. Methods: Thirty rats (n = 5/group) were used for the oral acute toxicity study and administered with varying doses (0, 100, 500, 1000, 2000 and 5000 mg/kg b.wt) of alkaloid-rich extract of P. amarus. The oral acute toxicity was carried out according to OECD guidelines.After 21 days of monitoring, serum liver function tests and liver histology were performed using documented methods. The antimalarial index was determined using median effective dose (ED50) of thirty five mice divided into 7 groups (n = 5). Results: showed that up to the highest dose (5000 mg/kg), there were no biochemical derangements in liver function. Physical signs of toxicity were also not observed. Antimalarial activity indices showed high potency with therapeutic index of 30.13. Conclusion: Alkaloid-rich extract of Phyllanthus amarus is therefore, non-toxic with reputable antimalarial activity. The active alkaloid(s) deserve further study as source for possible development of new and more potent antimalarial agent.

Anti-inflammatory, anticholinesterase, antioxidant, and memory enhancement potential of Phyllanthus amarus in potassium-dichromate induced neurotoxicity of male Wistar rats.

This study investigated the protective effect of aqueous Phyllanthus amarus leaf extract (APALE) in Potassium dichromate (PDc)-induced neurotoxicity. Seventy young adult male, Wistar rats with a weight of 130-150 g, were randomised into seven groups (n = 10): Group 1; distilled water; Group 2: 300 mg/kg APALE; Group 3: 17 mg/kg PDc; Group 4: 5 mg/kg Donepezil (DPZ); Group 5: 17 mg/kg PDc + 400 mg/kg APALE; Group 6:17 mg/kg PDc + 200 mg/kg APALE; Group 7: 17 mg/kg PDc + 5 mg/kg DPZ. All administrations were given once daily via an orogastric cannula for 28 consecutive days. Cognitive assessment tests were employed to ascertain the treatments' effects on the rats' cognitive function. At the end of the experiment, the rats were sacrificed, morphometric analysis was done, and the brains were dissected for histology, enzyme, and other biochemical analysis. Findings from this study showed that APALE significantly improved locomotive activity, recognition memory sensitivity, protection against fear and anxiety, enhanced decision-making, and improved memory function in a dose-dependent manner comparably to DPZ. In addition, APALE significantly increased antioxidants level, reducing oxidative stress in PDc-induced neurotoxic rats and significantly reducing brain acetylcholinesterase (AchE) activity by regulating gamma amino butyric acid (GABA) levels in PDc-induced neurotoxic rats compared to DPZ. Furthermore, APALE alleviated neuroinflammatory responses via maintaining histoarchitecture and down-regulation of IBA1 and Tau in PDc-induced rats. In conclusion, APALE protected against PDc-induced neurotoxicity via a combination of anti-inflammatory, anticholinergic, and antioxidant effects on the prefrontal cortex of rats.

Polyphenolic compounds of Phyllanthus amarus Schum & Thonn. (1827) and diabetes-related activity of an aqueous extract as affected by in vitro gastrointestinal digestion.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of the aerial part of Phyllanthus amarus have been extensively used in several countries to cure diabetes. No data is available on the impact of gastrointestinal digestion of such crude extracts on their antidiabetic activity. AIM OF THE STUDY: The aim of this study was to identify active fractions and compounds of fresh aerial parts of P. amarus extracted by an infusion method that are responsible for antidiabetic effects occurring at the level of glucose homeostasis. MATERIALS AND METHODS: An aqueous extract was obtained by an infusion method and its polyphenolic composition was analysed by reverse phase UPLC-DAD-MS. The influence of in vitro gastrointestinal digestion was evaluated both on the chemical composition and on the antidiabetic effect of P. amarus infusion extract using glucose-6-phosphatase enzyme inhibition and stimulation of glucose uptake. RESULTS: Analysis of the chemical composition of the crude extract revealed the presence of polysaccharides and various families of polyphenols such as phenolic acids, tannins, flavonoids and lignans. After simulated digestion, the total content of polyphenols decreased by about 95%. Caffeoylglucaric acid derivates and lignans exhibited strong stimulation of glucose uptake similar to metformin with an increase of 35.62 ± 6.14% and 34.74 ± 5.33% respectively. Moreover, corilagin, geraniin, the enriched polysaccharides fraction and the bioaccessible fraction showed strong anti-hyperglycemic activity with about 39-62% of glucose-6-phosphatase inhibition. CONCLUSION: Caffeoylglucaric acid isomers, tannin acalyphidin M1 and lignan demethyleneniranthin were reported for the first time in the species. After in vitro gastroinstestinal digestion, the composition of the extract changed. The dialyzed fraction showed strong glucose-6-phosphatase inhibition.

Revealing the Acetylcholinesterase Inhibitory Potential of Phyllanthus amarus and Its Phytoconstituents: In Vitro and in Silico Approach.

The inhibition of acetylcholinesterase plays a vital role in the treatment of Alzheimer disease. This study aimed to explore the acetylcholinesterase inhibition potential of Phyllanthus amarus and its phytoconstituents through an in vitro and in silico approach. The in vitro acetylcholinesterase inhibitory activity of P amarus was carried out, followed by the molecular docking studies of its phytoconstituents. The top-ranked molecules identified through molecular docking were subjected to molecular dynamics simulation (MDS) and density functional theory (DFT) studies. The results obtained revealed the methanolic extract of P amarus as a potent acetylcholinesterase inhibitor, while amarosterol A, hinokinin, ß-sitosterol, stigmasterol and ellagic acid were identified as potential acetylcholinesterase inhibitors. The MDS and DFT results are in agreement with those obtained from the docking studies. Our findings suggest further studies on the hit molecules.

Restoration of Hepatic and Intestinal Integrity by Phyllanthus amarus Is Dependent on Bax/Caspase 3 Modulation in Intestinal Ischemia-/Reperfusion-Induced Injury.

Ethnopharmacological relevance: Oxidative stress is a key player in intestinal ischemia/reperfusion (I/R) injury (IIRI) with a tendency to trigger systemic inflammatory response, resulting in progressive distal organ injury. To date, the role of Bax/caspase 3 signaling in IIRI has not been reported. Furthermore, the discovery of a safe and effective drug remains pertinent in improving the outcome of IIRI. Therefore, this study investigated the role of Bax/caspase 3 signaling in intestinal I/R-induced intestinal and hepatic injury. In addition, the protective effect and possible associated mechanism of action of methanolic Phyllanthus amarus leaf extract (PA) against intestinal I/R-induced intestinal and hepatic injury were evaluated. Materials and methods: Fifty male Wistar rats were randomized into five groups (n = 10). The sham-operated group was received 0.5 mL of distilled water for seven days prior to the sham surgery, while the IIRI, febuxostat (FEB) + IIRI, low-dose PA (LDPA) + IIRI, and high-dose PA (HDPA) + IIRI groups underwent the I/R procedure. In addition to the procedure, IIRI, FEB + IIRI, LDPA + IIRI, and HDPA + IIRI received 0.5 mL of distilled water, 10 mg/kg of febuxostat, 200 mg/kg of PA, and 400 mg/kg of PA, respectively, for seven days prior to the I/R procedure. Results: Administration of methanolic Phyllanthus amarus leaf extracts attenuated the intestinal I/R-induced rise in intestinal and hepatic injury markers, malondialdehyde, nitric oxide, TNF-α, IL-6, and myeloperoxidase activities. In addition, Phyllanthus amarus ameliorated I/R-induced suppression of reduced glutathione, thiol and non-thiol proteins, and superoxide dismutase, catalase, and glutathione peroxidase activities in intestinal and hepatic tissues. These were coupled with the suppression of I/R-induced bacterial translocation, downregulation of I/R-induced activation of Bax/caspase 3 signaling, and improvement of I/R-induced distortion of intestinal and hepatic histoarchitecture by Phyllanthus amarus. Conclusion: Methanolic Phyllanthus amarus leaf extract protects against intestinal and hepatic injuries associated with intestinal I/R by suppressing oxidative-stress-mediated activation of Bax/caspase 3 signaling. The beneficial effects of Phyllanthus amarus may be ascribed to its constituent bioactive molecules, especially tannins, anthocyanin, alkaloids, and phenolics.

Phyllanthus amarus attenuated derangement in renal-cardiac function, redox status, lipid profile and reduced TNF-α, interleukins-2, 6 and 8 in high salt diet fed rats.

High salt diet (HSD) has been implicated in the etiopathogenesis of immune derangement, cardiovascular disorders and, metabolic syndromes. This study investigated the protective effect of ethanol extract of Phyllanthus amarus (EEPA) against high salt diet (HSD) induced biochemical and metabolic derangement in male Wistar rats. The rats were divided into 5 groups of 6 animals each as follows; control group fed with normal rat chow, negative control group, fed HSD only, animals on HSD treated orally with 75 mg/kg, 100 mg/kg, and, 150 mg/kg EEPA once daily. At the end of 8 weeks treatment, lipid profile (TG, TC, LDL, and VLDL), oxidative stress (catalase, reduced glutathione, and malondialdehyde), inflammatory (TNF-a, interleukins 2, 6, and 8), cardiac (lactate dehydrogenase, creatine kinase) and kidney function markers (urea, uric acid, creatinine) were assessed. Serum TG, TC, LDL, and VLDL content were significantly (p < 0.05) elevated in HSD-only fed rats, while HDL was significantly elevated in a concentration-dependent manner in EEPA treated animals. The extract produced a significant (p < 0.05) and dose-dependent increase in the antioxidant enzymes activities and a significant reduction in the malondialdehyde level. A significant (p < 0.05) dose-dependent reduction in serum TNF-alpha, IL-2, 6, and 8 of EEPA treated rats compared with HSD-fed rats was observed. More so, reduction in serum LDH, creatine kinase, creatinine, urea, and uric acid activity of extract-treated animals were noted. EEPA attenuated high salt diet-induced oxidative stress, inflammation, and dyslipidemia in rats.

In vitro antiplasmodial activity of Phyllanthus amarus against Plasmodium falciparum and evaluation of its acute toxicity effect in mouse model.

BACKGROUND: The emergence of widespread resistance of Plasmodium species to most antimalarial drugs has led to a more vigorous and concerted research on traditional medicinal plants for the treatment of malaria. OBJECTIVE OF STUDY: The study was aimed to investigate the in vitro antiplasmodial activity of crude ethanolic and aqueous extracts of Phyllanthus amarus against clinical isolates of Plasmodium falciparum in Northwestern Nigeria. MATERIALS AND METHODS: The plant was extracted using two solvents, water and ethanol, where a high yield was obtained from the aqueous extracts (11.9%) as compared to the ethanolic extract (9.64%). The extracts were evaluated in vitro at concentrations of 6.25, 12.5, 25, 50, and 100 µg/ml, and the level of potency in each case was expressed as the concentration of the extract that exhibited a 50% reduction of the parasites relative to control (100%) parasitemia. Artemether-lumefantrine was used as a positive standard in the assay. RESULTS: All extracts showed a significant reduction in parasite growth relative to control (P ≤ 0.05). Ethanolic extract exhibited a higher antiplasmodial activity of 76.8%, half-maximal inhibitory concentration (IC50) of 5.80 µg/ml, and aqueous extract had an activity of 75.3%, IC50 of 7.94 µg/ml. Both extracts exhibited very active antiplasmodial activity. Oral acute toxicity test in the doses of 500, 1000, and 1500 mg/kg showed no sign of toxicity on albino mice after 48 h. CONCLUSION: Although there was an increase in appetite after 24 and 48 h, the findings from this study show that P. amarus possesses a promising antimalarial activity which can be exploited for malaria therapy and justifies the traditional use of the plant in malaria treatment.

In Vitro Anti-Leptospiral Activity of Phyllanthus amarus Extracts and Their Combinations with Antibiotics.

Despite modern medicine, there is an increasing trend for cases of the bacterial infection leptospirosis, and this has led to the exploration of alternative medicines from various sources including plants. The aim of this study was to investigate the in vitro anti-leptospiral activity of Phyllanthus amarus extracts alone and combined with penicillin G, ceftriaxone, and doxycycline. Antimicrobial susceptibility testing was performed using the microdilution broth technique upon methanol extract (ME), aqueous extract (AE), and antibiotics against the Leptospira interrogans serovars Australis, Bataviae, Canicola, and Javanica, to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The results were analyzed using an ELISA microplate reader combined with microscopic analysis. Synergy testing using a checkerboard assay was performed to determine the fractional inhibitory concentration index values of extracts combined with antibiotics against leptospires. Scanning electron microscopy (SEM) was used to investigate morphological changes of leptospires caused by potential anti-leptospiral agents alone and combined with antibiotics. The MICs and MBCs for P. amarus extracts ranged from 100 to 400 µg/mL for AEs and from 400 to 800 µg/mL for MEs. Penicillin G was the most effective anti-leptospiral drug, with MICs and MBCs ranging from <0.01 to 0.78 and <0.01 to 3.13 µg/mL, respectively, followed by ceftriaxone, with both MICs and MBCs ranging from 0.05 to 0.78 µg/mL, and doxycycline, with MICs and MBCs ranging from 0.39 to 3.13 µg/mL and 12.5 to 25 µg/mL, respectively. Combinations of P. amarus extracts and antibiotics did not show synergistic effects on all tested Leptospira serovars, with some combinations demonstrating antagonistic effects. SEM analysis, however, showed distorted Leptospira surfaces. P. amarus AE performed better anti-leptospiral activity than P. amarus ME. The morphological effects of P. amarus extract alone and its combination with antibiotic on Leptospira cells revealed promising anti-leptospiral properties.

In silico docking analysis revealed the potential of phytochemicals present in Phyllanthus amarus and Andrographis paniculata, used in Ayurveda medicine in inhibiting SARS-CoV-2.

The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in outbreak of global pandemic, fatal pneumonia in human referred as Coronavirus Disease-2019 (Covid-19). Ayurveda, the age old practice of treating human ailments in India, can be considered against SARS-CoV-2. Attempt was made to provide preliminary evidences for interaction of 35 phytochemicals from two plants (Phyllanthus amarus and Andrographis paniculata used in Ayurveda) with SARS-CoV-2 proteins (open & closed state S protein, 3CLpro, PLpro and RdRp) through in silico docking analysis. The nucleotide analogue remdesivir, being used in treatment of SARS-CoV-2, was used as a positive control. The results revealed that 18 phytochemicals from P. amarus and 14 phytochemicals from A. paniculata shown binding energy affinity/dock score < - 6.0 kcal/mol, which is considered as minimum threshold for any compound to be used for drug development. Phytochemicals used for docking studies in the current study from P. amarus and A. paniculata showed binding affinity up to - 9.10 kcal/mol and - 10.60 kcal/mol, respectively. There was no significant difference in the binding affinities of these compounds with closed and open state S protein. Further, flavonoids (astragalin, kaempferol, quercetin, quercetin-3-O-glucoside and quercetin) and tannins (corilagin, furosin and geraniin) present in P. amarus have shown more binding affinity (up to - 10.60 kcal/mol) than remdesivir (up to - 9.50 kcal/mol). The pharmacokinetic predictions suggest that compounds from the two plants species studied in the current study are found to be non-carcinogenic, water soluble and biologically safe. The phytochemicals present in the extracts of P. amarus and A. paniculata might have synergistic effect with action on multiple target sites of SARS-CoV-2. The information generated here might serve as preliminary evidence for anti SARS-CoV-2 activity of phytochemicals present from P. amarus and A. paniculata and the potential of Ayurveda medicine in combating the virus. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02578-7.

An Insight Into the Anxiolytic Effects of Lignans (Phyllanthin and Hypophyllanthin) and Tannin (Corilagin) Rich Extracts of Phyllanthus amarus : An In-Silico and In-vivo approaches.

The extracts and the compounds isolated from Phyllanthus amarus Schumm and Thonn (Family: Euphorbiaceae) have shown a wide spectrum of pharmacological activities including antiviral, antibacterial, antiplasmodial, antimalarial, antimicrobial, anticancer, antidiabetic, hypolipidemic, antioxidant, hepatoprotective, nephroprotective and diurectic properties. BACKGROUND: This investigation was aimed at exploring the anxiolytic potential of Phyllanthus amarus standardized extracts and predict probable role of marker phyto constitutents. OBJECTIVE AND METHODS: Three standardized extracts of Phyllanthus amarus plant viz. standardized aqueous extract of Phyllanthus amarus whole plant (PAAE), standardized methanolic extract of P. amarus leaf (PAME) and the standardized hydro-methanolic extract of P. amarus leaf (PAHME) were tested in the classical animal models of anxiety: Elevated plus-maze model and Light & Dark Exploration test. RESULTS: The lower doses of the tannin rich extract (PAHME) of the P. amarus possess significant anxiolytic activity compared to lignin rich (PAME) and aqueous extracts (PAAE), while at a higher dose (400mg/kg) the results of all three extracts appears to be potentially sedative. While the molecular docking studies support these probable anxiolytic, the sedative effects of the Phyllanthus amarus extracts could be due to the interaction of tannins and lignans with the GABAbenzodiazepine receptor complex. CONCLUSION: The results of the present study indicate that the tannin-rich extract of the P. amarus may have potential clinical applications in the management of anxiety. It can be further studied for optimum dosage to be used as a future of anti-anxiety drug development or as a standardized Phytomedicine.

Lignans and Polyphenols of Phyllanthus amarus Schumach and Thonn Induce Apoptosis in HCT116 Human Colon Cancer Cells through Caspases-Dependent Pathway.

BACKGROUND: The anticancer effects of Phyllanthus amarus extract on various cancer cells have been investigated, however, the effects of its major constituents on HCT116 human colorectal cancer cells have not been reported. OBJECTIVE: In the present study, we investigated the cytotoxic effect of 80% ethanol extract of P. amarus and its marker constituents (phyllanthin, hypophyllanthin, gallic acid, niranthin, greraniin, phyltetralin, isolintetralin, corilagin and ellagic acid) on HCT116 and their underlying mechanisms of action. METHODS: Their antiproliferative and apoptotic effects on HCT 116 were performed using MTT assay and flow cytometric analysis, respectively, while caspases 3/7, 8 and 9 activities were examined using the colorimetric method. The expression of cleaved poly ADP ribose polymerase enzyme (PARP) and cytochrome c proteins was investigated by the immune-blot technique. RESULTS AND DISCUSSION: HPLC and LC-MS/MS analyses demonstrated that the extract contained mainly lignans and polyphenols. The plant samples markedly suppressed the growth and expansion of HCT116 cells in a concentration- and time-dependent manner with no toxicity against normal human fibroblast CCD18 Co. P. amarus extract, phyllanthin and gallic acid induced mode of cell death primarily through apoptosis as confirmed by the exteriorization of phosphatidylserine. Caspases 3/7, 8, and 9 activities increased in a concentration-dependent manner following 24h treatment. The expressions of cleaved PARP (Asp 214) and cytochrome c were markedly upregulated. CONCLUSION: P. amarus extract, phyllanthin and gallic acid exhibited an apoptotic effect on HCT116 cells through the caspases-dependent pathway.

In Vitro Calli Production Resulted in Different Profiles of Plant-Derived Medicinal Compounds in Phyllanthus amarus.

The efficient production of plant-derived medicinal compounds (PDMCs) from in vitro plants requires improvements in knowledge about control of plant or organ development and factors affecting the biosynthesis pathway of specific PDMCs under in vitro conditions, leading to a realistic large-scale tool for in vitro secondary metabolite production. Thus, this study aimed to develop an in vitro technique, through the induction and proliferation of calli, for production of plant fresh weight, and to compare the PDMC profile obtained from the plants versus in vitro calli of Phyllanthus amarus. It was successfully possible to obtain and proliferate two types of calli, one with a beige color and a friable appearance, obtained in the dark using Murashige and Skoog (MS) medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and a second type with a green color, rigid consistency, and nonfriable appearance obtained under light conditions and MS medium plus 6-benzyladenine (6-BA). In vitro micropropagated plants that gave rise to calli were also acclimatized in a greenhouse and cultivated until obtaining the mass for PDMC analysis and used as a control. While the micropropagated-derived plants concentrated the lignans niranthin, nirtetralin, and phyllanthin, the Phyllanthus amarus calli proliferated in vitro concentrated a completely different biochemical profile and synthesis of compounds, such as betulone, squalene, stigmasterol, and ß-sitosterol, in addition to others not identified by GC-MS database. These results demonstrate the possibility of applying the calli in vitro from Phyllanthus amarus for production of important PDMCs unlike those obtained in cultures of differentiated tissues from field plants.

Effects of Phyllanthus amarus extract on nonspecific immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp Litopenaeus vannamei.

This study investigates the effects of Phyllanthus amarus extract (PAE) on immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp (Litopenaeus vannamei). In vitro PAE treatment did not alter the cell viability of haemocytes and significantly enhanced immune parameters such as phenoloxidase (PO) activity, phagocytic activity, and superoxide anion (O2-) production. We conducted two feeding trials to examine the effects of PAE on the growth, disease resistance, and innate immune parameters of white shrimp. In the first in vivo trial, shrimps (4.01 ± 0.03 g) were fed a diet containing 0 g (control), 10 g (PAE10), 20 g (PAE20), or 40 g (PAE40) of PAE per kilogram of feed for 56 days. After the feeding period, the PAE20 group showed a significantly higher weight gain and specific growth rate than shrimp fed the control diet. Furthermore, after challenge with V. alginolyticus, shrimp fed a diet containing PAE showed significantly higher survival than those fed the control diet. The second in vivo trial (28 days) was performed to identify the mechanisms of enhanced immunity in PAE-fed shrimp. Shrimp fed the PAE20 diet generally had the highest total haemocyte count, PO activity, phagocytic activity, and O2- production, followed by the PAE40 and PAE10 groups. Thus, our results suggest that administration of 20 g of PAE per kilogram of feed can enhance immunity, growth, and resistance to V. alginolyticus in white shrimp.

Phyllathin From Phyllanthus Amarus Ameliorates Epileptic Convulsion and Kindling Associated Post-Ictal Depression in Mice via Inhibition of NF-κB/TLR-4 Pathway.

BACKGROUND: Epilepsy is a chronic, complex, unprovoked, and recurrent disorder of the nervous system that affected several people worldwide. Phyllanthus amarus (PA) has been documented to have neuroprotective potential. AIM: To evaluate the potential of standardized extract of PA and its possible mechanism of action against the Pentylenetetrazol (PTZ)-induced convulsion and kindling associated post-ictal depression in experimental mice. MATERIALS AND METHODS: Phyllathin was isolated from methanolic extract of PA and well-characterized using HPTLC, ESI-MS/MS, and LC/MS. Phyllathin containing a standardized extract of PA (50, 100, and 200 mg/kg) was administered in convulsed and kindled mice, followed by an assessment of various parameters. RESULTS: The spectral analysis confirmed the molecular formula and weight of phyllanthin as C24H34O6 and 418.2342 Da. PA (100 and 200 mg/kg) significantly ameliorated PTZ-induced (p < 0.05) duration, onset of tonic-clonic convulsion, and mortality in mice. It also significantly attenuated (p < 0.05) PTZ-induced kindling in mice. Alteration in brain GABA, dopamine, and glutamate, Na+K+ATPase, Ca+2-ATPase activities, and oxido-nitrosative stress in kindled mice was significantly restored (p < 0.05) by PA treatment. It also significantly (p < 0.05) down-regulated brain mRNA expressions of NF-κB, TNF-α, IL-1ß, COX-2, and TLR-4. Histological aberrations induced by PTZ in the brain of a kindled rat was significantly (p < 0.05) ameliorated by PA. CONCLUSION: Phyllanthin containing a standardized extract of PA exerts its antiepileptic potential via balancing excitatory (glutamate) and inhibitory (GABA) brain monoamines, voltage-gated ion channels (Na+K+/Ca+2-ATPase) and inhibition of NF-κB/TLR-4 pathway to ameliorate neuroinflammation (TNF-α, IL-1ß, and COX-2) in experimental mice.

Phyllanthus amarus prevents LPS-mediated BV2 microglial activation via MyD88 and NF-κB signaling pathways.

BACKGROUND: Phyllanthus amarus has been shown to attenuate lipopolysaccharide (LPS)-induced peripheral inflammation but similar studies in the central nervous system are scarce. The aim of the present study was to investigate the neuroprotective effects of 80% ethanol extract of P. amarus (EPA) in LPS-activated BV2 microglial cells. METHODS: BV2 microglial cells c for 24 h, pre-treated with EPA for 24 h prior to LPS induction for another 24 h. Surface expression of CD11b and CD40 on BV2 cells was analyzed by flow cytometry. ELISA was employed to measure the production of pro-inflammatory mediators i.e. nitric oxide (NO) and tumor necrosis factor (TNF)-α. Western blotting technique was used to determine the expression of inducible nitric oxide synthase (iNOS), myeloid differentiation protein 88 (MYD88), nuclear factor kappa B (NF-κB), caspase-1, and mitogen activated protein kinase (MAPK). RESULTS: Qualitative and quantitative analyses of the EPA using a validated ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method indicated the presence of phyllanthin, hypophyllanthin, niranthin, ellagic acid, corilagin, gallic acid, phyltetralin, isolintetralin and geraniin. EPA suppressed the production of NO and TNFα in LPS-activated BV2 microglial cells. Moreover, EPA attenuated the expression of MyD88, NF-κB and MAPK (p-P38, p-JNK and p-ERK1/2). It also inhibited the expression of CD11b and CD40. EPA protected against LPS-induced microglial activation via MyD88 and NF-κB signaling in BV2 microglial cells. CONCLUSIONS: EPA demonstrated neuroprotective effects against LPS-induced microglial cells activation through the inhibition of TNFα secretion, iNOS protein expression and subsequent NO production, inhibition of NF-κB and MAPKs mediated by adapter protein MyD88 and inhibition of microglial activation markers CD11b and CD40.

Immunomodulatory potential of extracts, fractions and pure compounds from Phyllanthus amarus and Psidium guajava on striped catfish (Pangasianodon hypophthalmus) head kidney leukocytes.

This study aimed to identify major phytochemical constituents, as well as compare the immunomodulatory effects of Psidium guajava L. and Phyllanthus amarus Schun and Thonn crude ethanol extracts and their fractions on striped catfish (Pangasianodon hypophthalmus) head kidney leukocytes (HKLs). Moreover, pure constituents were also investigated for their effects on those cells: hypophyllanthin, identified as a major constituent of P. amarus crude extracts and its hexane fraction; corosolic acid, ursolic acid, and oleanolic acid, identified in P. guajava crude extract, ethyl acetate and dichloromethane fractions; with other terpenic derivatives, as well as guajaverin and avicularin, identified with other flavonoids by LC-UV-MS in the crude P. guajava extract and its ethyl acetate fraction. Cell viability, respiratory burst assay (RBA), nitric oxide synthase (NOS) and lysozyme activity in HKLs were analyzed after 24 h stimulation with each extract (10, 20 and 40 µg/mL) or pure compound (7.5, 15 and 30 µM). Our results show that the hexane fraction of both plant extracts inhibited the viability of HKLs, while several other fractions enhanced the cell viability. All P. guajava fractions at all or some concentration considerably enhanced the RBA production in HKLs. Similarly, NOS production was also significantly increased by some or all concentrations of P. guajava dichloromethane and ethyl acetate fractions. However, the NOS production was dose-dependently inhibited in HKLs treated with Pa ethyl acetate and both plants aqueous fractions at 10 or 10 and 40 µg/mL respectively. The lysozyme activity in cells treated with P. guajava crude extracts and all its organic solvent fractions were stronger than those in P. amarus treatments. Pure compounds including corosolic acid, guajaverin, ursolic acid, hypophyllanthin inhibited the HKLs viability according to concentration and type of compound. All pure compounds except avicularin significantly stimulated, at certain or all concentrations, the RBA production and/or the lysozyme activity in HKLs. The NOS production was significantly reduced in HKLs treated with oleanolic acid (30 µM) and hypophyllanthin (7.5 µM) while its level was increased by hypophyllanthin at 30 µM. These results highlighted that the crude ethanol extracts of P. guajava and P. amarus, their fractions and some of their pure components at certain concentrations can potentially act as immunomodulators, and could be considered as valuable candidates in fishery sciences.

Single or Combined Dietary Supply of Psidium guajava and Phyllanthus amarus Extracts Differentially Modulate Immune Responses and Liver Proteome in Striped Catfish (Pangasianodon hyphophthalmus).

Guava Psidium guajava L (Pg) and bhumi amla Phyllanthus amarus Schum. et Thonn (Pa) are well-known plants in traditional medicine. However, the capacity of these plants for improving the immune system of aquatic species has received less attention so far. This study aimed to investigate the effects of single supply or mixture of Pg and Pa extracts on immune responses, disease resistance and liver proteome profiles in striped catfish Pangasianodon hypophthalmus. Fish were fed diets including basal diet 0% or one of three doses of each plant extract, either alone or in mixture, 0.08, 0.2, or 0.5% Pg, Pa or mixture (Pg:Pa, v/v) for 6 weeks. The immune parameters (respiratory burst activity (RBA); nitric oxide synthase (NOS), total immunoglobulin, lysozyme, and complement activities) were examined at W3, W6 post-feeding, and after challenge test. The growth parameters and the challenge test with Edwardsiella ictaluri were done at W6. The liver proteome profiles were analyzed in W6 at 0.08 and 0.5% of each extract. The results showed that extract-based diets significantly improved growth parameters in the Pg0.2 group compared to control. The cellular immune responses in spleen and the humoral immune responses in plasma were significantly improved in a dose and time-dependent manner. Diets supplemented with single Pg and Pa extracts, and to lesser extent to combined extracts, could significantly decrease the mortality of striped catfish following bacterial infection compared to control. The proteomic results indicated that some pathways related to immune responses, antioxidant and lipid metabolism were enriched in liver at W6. Several proteins (i.e., CD8B, HSP90AA1, HSP90AB1, PDIA3, CASP8, TUBA1C, CCKAR, GNAS, GRIN2D, PLCG1, PRKCA, SLC25A5, VDAC2, ACTN4, GNAI2, LCK, CARD9, NLRP12, and NLRP3) were synergistically upregulated in mixture of Pg and Pa-based diets compared to control and single dietary treatments. Taken together, the results revealed that single Pg and Pa extracts at 0.2 and 0.5% and their mixture at 0.08 and 0.5% have the potential to modulate the immune mechanisms and disease resistance of striped catfish. Moreover, the combination of Pg and Pa in diets suggested positive synergistic effects liver proteome profile related to immune system processes.