Optimal Enzyme-Assisted Extraction of Phenolics from Leaves of Pongamia pinnata via Response Surface Methodology and Artificial Neural Networking.

This research work seeks to evaluate the impact of selected enzyme complexes on the optimised release of phenolics from leaves of Pongamia pinnata. After preliminary solvent extraction, the P. pinnata leaf extract was subjected to enzymatic treatment, using enzyme cocktails such as kemzyme dry-plus, natuzyme, and zympex-014. It was noticed that zympex-014 had a greater extract yield (28.0%) than kemzyme dry-plus (17.0%) and natuzyme (18.0%). Based on the better outcomes, zympex-014-based extract values were subsequently applied to several RSM parameters. The selected model is suggested to be significant by the F value (12.50) and R2 value (0.9669). The applicability of the ANN model was shown by how closely the projected values from the ANN were to the experimental values. In terms of total phenolic contents (18.61 mg GAE/g), total flavonoid contents (12.56 mg CE/g), and DPPH test (IC50) (6.5 g/mL), antioxidant activities also shown significant findings. SEM analysis also revealed that the cell walls were damaged during enzymatic hydrolysis, as opposed to non-hydrolysed material. Using GC-MS, five potent phenolic compounds were identified in P. pinnata extract. According to the findings of this study, the recovery of phenolic bioactives and subsequent increase in the antioxidant capacity of P. pinnata leaf extract were both positively impacted by the optimisation approaches suggested, including the use of zympex-014.

Harnessing Nature's Gifts: Salix nigra and Its Potential for Combating Hepatitis C Virus (HCV).

Hepatitis C virus (HCV) causes various liver complications, including fibrosis, cirrhosis, and steatosis, and finally progresses toward hepatocellular carcinoma (HCC). The current study aimed to explore the antiviral activity of the traditional Pakistani medicinal plant Salix nigra (S. nigra) known as black willow against the hepatitis C virus (HCV). The anti-HCV activity of S. nigra was established against stable Hep G2 cell lines expressing the HCV NS3 gene. Various plant-derived compounds with anti-HCV activity were identified, making phytotherapy a promising alternative to conventional treatments due to their cost-effectiveness and milder side effects. The two extraction methods (Maceration and Soxhlet) and four solvents (n-hexane, methanol, ethyl acetate, and water) were used to obtain crude extracts from S. nigra. Cytotoxicity testing showed that methanol (CC50 25 µg/mL) and water (CC50 30 µg/mL) extracts were highly toxic, while ethyl acetate and n-hexane (CC50 > 200 µg/mL) extracts were nontoxic at low concentrations (10-50 µg/mL), making them suitable for further anti-HCV investigations. Stable transfection of the NS3 gene was successfully performed in Hep G2 cells, creating a cellular expression system for studying virus-host interaction. The ethyl acetate extract of S. nigra exhibited significant inhibition of NS3 gene expression (mRNA and protein levels). The phytochemical analysis of S. nigra was also performed using the high-performance liquid chromatography (HPLC) technique. The phytochemical analysis identified several polyphenolic substances in the extracts of S. nigra. Our results concluded that the extracts of S. nigra have significantly reduced the expression of the NS3 gene at mRNA and protein levels. These findings contribute to the global efforts to combat hepatitis C by offering plant-based treatment options for HCV management.

Hepatoprotective and nephroprotective effects of Sedum adenotrichum in paracetamol-induced hepatotoxicity in rabbits.

Sedum adenotrichum (SA) has been traditionally used against various ailments due to the presence of several important phenolic compounds. This study provides a scientific basis for the hepatoprotective and nephroprotective potentials of Sedum adenotrichum in paracetamol-induced toxicity in rabbits. Methanolic extract of Sedum adenotrichum along with paracetamol was administered orally to rabbits alone or in combination with paracetamol for 2 weeks. Results showed that paracetamol-induced toxicity was correlated with a significant change in biochemical, hematological parameters, and loss in body weight of rabbits, and the curative effect of SA methanolic extract was found. Liver and kidney histological studies showed significant induced toxicity of paracetamol and correspondence restoration power of SA methanolic extract. The levels of reduced glutathione, radical scavenging activity, and lipid peroxidation in the liver were restored to normal by the methanolic extract of SA. This study confirmed the protective effect of SA methanolic extract against hepatotoxicity and nephrotoxicity caused by paracetamol. Biochemical analysis of the methanolic extract of SA confirmed the presence of bioactive phenolic compounds which have hepatic and nephroprotective potentials.

Evaluation of the whole proteome to design a novel mRNA-based vaccine against multidrug-resistant Serratia marcescens.

Serratia marcescens, a Gram-negative bacterium, is one of the known disease-causing pathogens. It is resistant to ampicillin, macrolides, cephalosporins, cefotaxime, and ceftazidime. The only antibiotic that has been proven to be effective against S. marcescens is gentamicin. By causing epigenetic alterations, bacteria can also become resistant to all antibiotics. Many epigenetically related proteins were studied, and four proteins were selected in this regard for epitope evaluation and their subsequent use in the development of a messenger ribonucleic acid (mRNA) vaccine. A series of immune-informatics tools used to build this mRNA vaccine elicited cellular and humoral immunity. Molecular docking between epitopes and alleles of the major histocompatibility complex (MHC) was performed. The vaccine was developed using 37 epitopes, an adjuvant that is a TLR-4 agonist known as resuscitation-promoting factor E (RpfE), subcellular trafficking structures, secretion boosters, and linkers. This proposed architecture was found to cover 99.6% of the population during testing. During testing, it was proven that it was both effective and safe. To confirm our idea, we performed an in silico immunological simulation of vaccination. The codon was also optimized to ensure that the mRNA reached the cytoplasm of a human host and underwent efficient translation. TLR-4 and TLR-3 were also docked against the secondary and tertiary structures of the vaccine peptide. Furthermore, the vaccine's stability was confirmed by molecular dynamics simulation. In summary, this vaccine construct can be a potential candidate against S. marcescens and is suitable for in vitro analyses to validate its effectiveness.

The hematological and histological studies for the hepatoprotective-like effect of the hydromethanolic extract and the fractions of Viola serpens Wall.

Traditionally, Viola serpens has been used in the treatment of several human disorders including liver diseases without any scientific evidence. As the current therapies are not very effective and face challenges of unwanted effects and patient compliance, therefore more effective and safe agents are highly needed. The current study aimed to evaluate the hepatoprotective potential of the crude extract and subsequent fractions of the whole plant in the in-vivo model using various hematological and histopathological parameters followed by an HPLC study for the identification of phenolic compounds. Rabbits (1000-1200 g) were used in the study. Paracetamol (2g) was used to induce hepatotoxicity in experimental rabbits. The plant extract was used in two doses (150 and 300 mg/kg body weights) for eight days. The hematological parameters AST, ALT and ALP values were determined along with the histopathology of the liver. Phenolic compounds were identified by high-performance liquid chromatography (HPLC) Agilent-1260 infinity from their retention time, UV spectra and available standards while quantification was done taking the percent peak area. The doses 150 and 300 mg/kg body weight seemed to be more effective. The hematological values and the histopathological slides show the hepatoprotective effect of the plant. Regeneration indicated the presence of nuclei, nuclear cleaning, prominent nucleoli, RBC's, central veins and plates of hepatocytes. The HPLC studies revealed the presence of a number of phenicol compounds. The crude extract and the subsequent fractions of the plant possess strong hepatoprotective activity, providing a scientific rationale for its uses in the treatment of liver toxicities.

Anabasis articulata (Forssk.) Moq: A Good Source of Phytochemicals with Antibacterial, Antioxidant, and Antidiabetic Potential.

Anabasis articulata is medicinally used to treat various diseases. In this study, A. articulata was initially subjected to extraction, and the resultant extracts were then evaluated for their antimicrobial, antioxidant, and antidiabetic potentials. After obtaining the methanolic extract, it was subjected to a silica gel column for separation, and fractions were collected at equal intervals. Out of the obtained fractions (most rich in bioactive compounds confirmed through HPLC), designated as A, B, C, and D as well hexane fraction, were subjected to GC-MS analysis, and a number of valuable bioactive compounds were identified from the chromatograms. The preliminary phytochemical tests were positive for the extracts where fraction A exhibited the highest total phenolic and flavonoid contents. The hexane fraction as antimicrobial agent was the most potent, followed by the crude extract, fraction A, and fraction D. DPPH and ABTS assays were used to estimate the free radical scavenging potential of the extracts. Fraction C was found to contain potent inhibitors of both the tested radicals, followed by fraction D. The potential antidiabetic extracts were determined using α-glucosidase and amylase as probe enzymes. The former was inhibited by crude extract, hexane, and A, B, C and D fractions to the extent of 85.32 ± 0.20, 61.14 ± 0.49, 62.15 ± 0.84, 78.51 ± 0.45, 72.57 ± 0.92 and 70.61 ± 0.91%, respectively, at the highest tested concentration of 1000 µg/mL with their IC50 values 32, 180, 200, 60, 120 and 140 µg/mL correspondingly, whereas α-amylase was inhibited to the extent of 83.98 ± 0.21, 58.14 ± 0.75, 59.34 ± 0.89, 81.32 ± 0.09, 74.52 ± 0.13 and 72.51 ± 0.02% (IC50 values; 34, 220, 240, 58, 180, and 200 µg/mL, respectively). The observed biological potentials might be due to high phenolic and flavonoid content as detected in the extracts. The A. articulata might thus be considered an efficient therapeutic candidate and could further be investigated for other biological potentials along with the isolation of pure responsible ingredients.

Anatomical Characterization, HPLC Analysis, and Biological Activities of Ilex dipyrena.

Ilex dipyrena Wall (Aquifoliaceae), is a traditional medicinal plant abundantly found in India and Pakistan. In the current research work, initially, the anatomical characteristics were recorded through microscopic examination of selected plant parts, such as leaf, petiole, and midrib. Then, the quantitative phytochemical screening was performed using standard tests reported in literature. The whole-plant powdered sample was then soaked in methanol to obtain crude extract, which was then fractionated into solvents of different polarities to obtain ethyl acetate, chloroform, butanol, hexane, and aqueous extracts. The phytochemical composition of the crude ethyl acetate and chloroform extracts (being the most active fractions) was then confirmed through HPLC analyses, where the possible phytochemical present were predicted through comparison of retention time of a given compound peak with the available standards. The extracts were also evaluated for their in vitro antioxidant and ani-lipoxygenase potentials using standard methods. The microscopic examination revealed the presence of anomocytic type stomata on the abaxial side of the leaf as well as unicellular trichrome and calcium oxalate druses crystals in the midrib and petiole, with a single, centered U-shaped collateral arterial bundle, which was directed toward the adaxial and the phloem toward the abaxial sides of the selected plant parts, respectively. Almost all tested representative groups of phytochemicals and essential minerals were detected in the selected plant, whereas five possible phytochemicals were confirmed in crude and chloroform extract and seven in ethyl acetate fraction. As antioxidant, chloroform fraction was more potent, which exhibited an IC50 value of 64.99, 69.15, and 268.52 µg/mL, determined through DPPH, ABTS, and FRAP assays. Ethyl acetate extract was also equally potent against the tested free radicals. Chloroform and ethyl acetate extracts were also potent against lipoxygenase, with IC50 value of 75.99 and 106.11 µg/mL, respectively. Based on the results of biological studies, Ilex dipyrena was found to good inhibitor of free radicals and lipoxygenase that could be further investigated to isolate compounds of medicinal importance.

Genetic diversity in nutritional composition of oat (Avena sativa L.) germplasm reported from Pakistan.

In the present study, 30 potential germplasm of oat (Avena sativa L.) were subjected to proximate, elemental, and HPLC analysis to provide a scientific basis to genetic diversity present among them. The extracts of the selected germplasms were also evaluated for their antioxidant potentials through DPPH and ABTS assays. Proximate analysis showed protein contents to be in the range 8.35-17.72% with the highest protein contents in the accession line 22,365 (17.72 ± 0.38%). The genotype-725 showed the highest carbohydrate, and dry matter (53.35 ± 0.01 and 93.50 ± 0.07% respectively) contents whereas, the germplasm-830 contained the highest fat (7.88 ± 0.12%) contents while the highest moisture contents were there in germplasm-22348 (11.95 ± 0.06%). The crude fiber contents (19.67 ± 0.19%) were found high in germplasm-832. The mentioned contents were also correlated to each other where a negative (-0.431*) correlation was noted for crude protein and carbohydrate while ash content to crude protein has a positive (0.38*) correlation. A positive and a negative correlation were there in Crude fats/crude protein (0.30*) and crude fats/moisture contents (-0.39*) respectively. Principal component analysis showed an Eigenvalue of 0.76 with a total variation of 85.01% when applied to proximate components. Based on cluster analysis to proximate composition all the oat germplasms were divided into 5 sub-clusters, where accession numbers 769 and 817 were found to be the most diverse genotypes. The elemental analysis confirmed the presence of magnesium (2.89-7.62 mg/L), sodium (3.71-8.03 mg/L), manganese (0.93-3.71 mg/L), copper (0.35-3.36 mg/L), iron (2.15-6.82 mg/L), zinc (1.30-3.37 mg/L), chromium (0.37-3.34 mg/L), and potassium (50.70-59.60 mg/L) in the selected germplasms. Principal component analysis for elemental composition showed the total variation of 73.75% with the Eigenvalue of 0.97. Cluster analysis on an elemental basis divided all the oat germplasms into 7 sub-clusters where accession numbers 769 and 22,350 were found to be the most diverse germplasm. Phytochemical analysis performed through HPLC resulted in the identification of nine possible compounds (malic acid, epigallocatechin gallate, quercetin, morin, ellagic acid, catechin hydrate, rutin, pyrogallol, and mandelic acid) in various germplasm of oat. A concentration-dependent antioxidant response was recorded when extracts were tested as an inhibitor of DPPH and ABTS free radicals. The results revealed that oat grains are a good source of nutrients, minerals, and phytochemicals that can be used as nutraceuticals and as food. The genetic differences revealed that this plant can be grown under varied environmental conditions.

Phytochemical profiling and antioxidant potential of Daphne mucronata Royle and action against paracetamol-induced hepatotoxicity and nephrotoxicity in rabbits.

The paracetamol-induced injuries of liver and kidneys in animals are mostly used to screen out the hepato and nephroprotective effect of extract or other therapeutic agents. In the present study total phenolic and flavonoid contents, in vitro antioxidant, and in vivo hepato/nephroprotective (on paracetamol-induced intoxication in experimental rabbits) potentials of the Daphne mucronata leaves methanolic extract were determined. For the identification of possible phytochemicals, HPLC (high performance liquid chromatography) analysis was carried out and a total of eight phenolic compounds; malic acid, gallic acid, chlorogenic acid, epigallocatechin gallate, quercetin, morin, ellagic acid, and rutin were identified. D. mucronata extract at doses of 250 and 500 mg/kg body weight were given for eight days to paracetamol intoxicated rabbits and the observed results were compared with standard Silymarin. The level of liver enzymes like aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, serum triglyceride, serum cholesterol, serum bilirubin, and kidneys biomarkers like serum urea, uric acid, and creatinine, as well as lipid peroxidation malondialdehyde contents were increased while the antioxidant enzymes like reduced glutathione and total antioxidant capacity were decreased. Furthermore, histopathological analysis of the liver and kidney tissues of control and treated groups also confirmed the hepatoprotective and nephroprotective effect of the D. mucronata which was most probably due to its high antioxidant phenolic and flavonoid phytoconstituents.

Antioxidants Isolated from Elaeagnus umbellata (Thunb.) Protect against Bacterial Infections and Diabetes in Streptozotocin-Induced Diabetic Rat Model.

The increase in resistance of microbes against conventional drugs is currently a hot issue, whereas diabetes is another main cause of mortalities encountered throughout the world after cancer and heart attacks. New drug sources in the form of plants are investigated to get effective drugs for the mentioned diseases with minimum side effects. Elaeagnus umbellata Thunb. is a medicinal plant used for the management of stress related disorders like diabetes and other health complications. The active constituents of the chloroform extract derived from E. umbellata berries was isolated by silica gel column chromatography which were identified as morin, phloroglucinol, and 1-hexyl benzene through various spectroscopic techniques (electron ionization mass spectrometry, 1H-NMR, and 13C-NMR spectroscopy). The possible protective effects (antioxidant, antibacterial, and antidiabetic activity) of isolated compounds were evaluated using reported methods. Morin exhibited strong in vitro antiradical potential against DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) radicals along with prominent antibacterial activities against selected bacterial strains (Escherichia coli, Bacillus cereus, Salmonella typhi, Klebsiella pneumonia, Pseudomonas aeruginosa and Proteus mirabilis). Among the isolated compounds the more potent one (morin) was assessed for its in vivo antidiabetic potential in streptozotocin-induced diabetic rat model. The in vivo effects observed were further confirmed in ex vivo experiments where the effect of isolated compound on antioxidant enzyme like glutathione peroxidase (GPx), total content of reduced glutathione (GSH), % DPPH inhibition, and the lipid peroxidation MDA (Malondialdehyde) level in pancreatic tissues homogenates were evaluated. In vivo morin at tested doses (2, 10, 15, 30 and 50 mg/kg body weight) significantly restored the alterations in the levels of fasting blood glucose level and body weight loss along with significant decrease in levels of cholesterol, triglycerides, low density lipoprotein, HbA1c level, and significantly increased the high-density lipoprotein in diabetic rats. Morin also effectively ameliorated the hepatic enzymes, and renal functions like serum creatinine. Morin significantly increased the antioxidant enzyme like GPx activity, GSH content, and % DPPH inhibition activity, while reduced the lipid peroxidation MDA (malondialdehyde) level in pancreatic tissues homogenates, and modification of histopathological changes in diabetic rats. Morin exhibited high antioxidant, antibacterial, and antidiabetic potentials as compared to phloroglucinol and 1-hexyl benzene, that could, therefore, be considered as a promising therapeutic agent to treat diabetes mellitus and bacterial infections.

Phytochemical and Biological Screening of Leaf, Bark and Fruit Extracts from Ilex dipyrena Wall.

The Aquifoliaceae is an important family and has been used traditionally for some time. One of the members of this family is the Ilex dipyrena wall, which itself possesses a potential medicinal importance. This plant is traditionally used for the treatment of various ailments including pain, swelling, burns, and fever. The current study was designed to screen out the antioxidant and analgesic potential of this plant and to verify its traditional uses, along with its phytochemical profile. Extracts were subjected to antioxidant, analgesic, and phytochemical analysis using DPPH, chemical-induced (acetic acid and formalin) nociception models and GC-MS analysis, respectively. The leaf, bark, and fruit extracts showed significant antioxidant activity compared to that of standard. Likewise, all the extracts demonstrated significant (p < 0.01) analgesic activity in a mice model. In acetic acid induced analgesia, the leaf, bark, and fruit extracts caused 51.64, 56.13 and 59.52% inhibition, respectively at a dose of 100 mg/kg while at 200 mg/kg it showed 83.01, 71.69 and 75.47% inhibition, respectively. In Formalin-induced paw-licking assay, fruit extract showed 59.42 and 64.19% inhibition at 200 mg/kg dose in the first and second phase, respectively. The GC-MS analysis revealed the presence of cathinone, phenylpropanolamine, dl-phenylephrine, amphetamine, myristic acid, and palmitic acid. Results of the study suggest that crude extracts from different parts of this plant may be a useful source for the development of novel analgesics. However, further investigation in terms of isolation of bioactive compounds and their toxicological evaluations are needed to validate the observed results.

A Comprehensive Review on the Medicinal Importance; Biological and Therapeutic Efficacy of Lagenaria siceraria (Mol.) (Bottle Gourd) Standley Fruit.

Herbal remedies have been employed for the treatment and management of different diseases for ages. Herbal medicines are a promising choice over modern synthetic drugs because of their low side effects and are thus considered to be safe and effective in treating human diseases. Lagenaria siceraria (Mol.) Standley fruit (Bottle gourd) belongs to the Cucurbitaceae family that has been used in a different system of traditional medication to treat various diseases. This is a domestic plant that provides food as well as medication. This vegetable have low caloric values and high water contents. The edible portion of it contains phytochemicals like vitamins, proteins, choline, minerals, terpenoids, flavonoids, etc. Several bioactive compounds have been isolated from L. siceraria, including triterpenoids, sterols, cucurbitacins, flavones, C-glycosides and ß-glycosides. Researchers have evaluated various parts of this plant viz. fruit, root, flowers, and leaves for pharmacological activities like antianxiety, antidepressant, diuretic, antimicrobial, cytotoxic, antihyperlipidemic, cardio protective, analgesic, anti-inflammatory, anthelmintic, anti-hyperglycemic, antihepatotoxic, anti-urolithiatic, antistress, antiulcer, anticancer, hepatoprotective, anthelmintic, immunomodulatory, and antioxidant. In this review, an attempt has been made to explore its phytochemical constituents, traditional, medicinal, and pharmacological uses to highlight the therapeutic importance of this well-known plant. This would be helpful in reviving its importance and will highlight its several promising aspects to encourage researchers for further research on L. siceraria.

Evaluation of antinociceptive activity of Ilex dipyrena Wall. in mice.

BACKGROUND: In order to find a new natural resource for pain-relief, the analgesic effects of Ilex dipyrena crude extract, fractions, and subfractions were evaluated in in-vivo mouse models with possible mechanism of action. METHODS: Analgesic effects of crude extract (100 and 200 mg/kg body weight), fractions and subfractions (75 mg/kg body weight) were screened using heat-induced (tail-immersion and hot plate test) and chemical-induced (formalin and acetic acid) nociception models in mice. The samples were also tested for the elucidation of a possible mechanism through opioidergic and GABAergic systems. RESULTS: The administration of crude extract, fractions and subfractions produced analgesic responses in acetic acid, formalin, tail immersion, and hot plate model for pain similar to those obtained with the standard. Naloxone antagonized the antinociceptive effects of the tested samples, whereas bicuculline showed partial inhibition. Considering the analgesic response, crude extract, fractions, and subfractions demonstrated promising inhibitory activity against all test models for pain, which was further supported by the possible involvement of opioidergic and GABAergic systems. CONCLUSION: The results suggest that this plant may be useful in the development of new analgesic drugs. Further research with regard to the isolation of bioactive compounds is required to verify these findings.

Iron-doped zinc oxide nanoparticles-triggered elicitation of important phenolic compounds in cell cultures of Fagonia indica.

The callus cultures of Fagonia indica could prove as factories for the production of important phytochemicals when triggered through different types of stress. In this study, we initiated callus cultures from healthy stem explants in the presence of iron-doped zinc oxide nanoparticles (Fe-ZnO-NPs). We performed experiments with the callus cultures of F. indica to determine the impact of Fe-ZnO-NPs in concentrations (15.62-250 µg/mL) on biomass accumulation, production of important phenolic and flavonoids, and antioxidative potential. Our results showed that maximum callus biomass [Fresh weight (FW) = 13.6 g and Dry weight (DW) = 0.58 ± 0.01] was produced on day 40 when the media was supplemented with 250 µg/mL Fe-ZnO-NPs. Similarly, maximum total phenolic content (268.36 µg GAE/g of DW) was observed in 40 days old callus added with 125 µg/mL Fe-ZnO-NPs. Maximum total flavonoid content (78.56 µg QE/g of DW) was recorded in 20 days old callus grown in 62.5 µg/mL Fe-ZnO-NPs containing media. Maximum total antioxidant capacity (390.74 µg AAE/g of DW) was recorded in 40 days old callus with 125 µg/mL Fe-ZnO-NPs treated cultures, respectively. Similarly, the highest free radical scavenging activity (93.02%) was observed in callus derived from media having 15.62 µg/mL Fe-ZnO-NPs. The antioxidant potential was observed to have positive correlation with TPC (r = 0.44). HPLC analysis showed that Fe-ZnO-NPs produced compounds (e.g., Epigallocatechin gallate) that were either absent or in lesser quantities in the control group. These results showed that Fe-ZnO-NPs elicitors could increase the biomass and activate secondary metabolism in F. indica cells. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02123-1.

Phytochemical Analysis, In Vitro Anticholinesterase, Antioxidant Activity and In Vivo Nootropic Effect of Ferula ammoniacum (Dorema ammoniacum) D. Don. in Scopolamine-Induced Memory Impairment in Mice.

BACKGROUND: Ferula ammoniacum (D. Don) is one of the endemic medicinal plants that is traditionally used to treat a number of diseases. Although the plant has been used to enhance memory, the investigational evidence supporting the nootropic effect was unsubstantial. Hence, the rationale for this study was to assess the potential beneficial effect of F. ammoniacum seed extracts on learning and memory in mice. METHODS: The powdered plant samples (aerial parts) were subjected to extraction ad fractionation. Among the extracts, crude and ethyl acetate extracts were screened for major phytochemicals through HPLC analysis. All the extracts were evaluated for the in vitro anticholinesterase (AChE and BChE) and antioxidant potentials. Among the extracts the active fraction was further assessed for improving learning and memory in mice using behavioural tests like Y-maze and novel object recognition test (NORT) using standard protocols. After behavioural tests, all the animals were sacrificed and brains tissues were assessed for the ex vivo anticholinesterase and antioxidant potentials. RESULTS: Phytochemicals like chlorogenic acid, quercetin, mandelic acid, phloroglucinol, hydroxy benzoic acid, malic acid, epigallocatechin gallate, ellagic acid, rutin, and pyrogallol were identified in crude methanolic extract (Fa.Met) and ethyl acetate fraction (Fa.EtAc) through HPLC. Fa.EtAc and Fa.Chf extracts more potently inhibited AChE and BChE with IC50 values of 40 and 43 µg/mL, and 41 and 42 µg/mL, respectively. Similarly highest free radical scavenging potential was exhibited by Fa.EtAc fraction against DPPH (IC50 = 100 µg/mL) and ABTS (IC50 = 120 µg/mL). The extract doses, 100 and 200 mg/kg body weight significantly (p < 0.01) improved the short-term memory by increasing the percent spontaneous alternation in the Y-maze test along with increasing discrimination index in the NORT that clearly indicated the enhancement in the recognition memory of mice. CONCLUSION: The extracts more potently scavenged the tested free radicals, exhibited anticholinesterase activities, improved the learning abilities and reduced the memory impairment induced by scopolamine in mice model thus suggesting that these extracts could be effectively used for the management of oxidative stress, neurodegenerative diseases and memory loss.

Chemical composition, in vitro antioxidant, anticholinesterase, and antidiabetic potential of essential oil of Elaeagnus umbellata Thunb.

BACKGROUND: Elaeagnus umbellata Thunb. (autumn olive) is a high valued medicinal plant. It belongs to Elaeagnaceae family and is widely distributed in Himalayan regions of Pakistan. In the present study essential oil were extracted from the fruit of this plant and their antioxidant, anticholinesterase and antidiabetic potentials were also evaluated. METHODS: Essential oils were extracted from the fruit of E. umbellata using hydro-distillation method and were characterized by GC-MS. The extracted oil were tested for its antioxidant, anticholinesterase, and antidiabetic potentials using standard protocols. RESULTS: About 68 compounds were identified by GC-MS. The extracted oil exhibited a fairly high free radical scavenging activities against DPPH and ABTS radicals with IC50 values of 70 and 105 µg/mL respectively (for ascorbic acid, used as standard, the IC50 values were 32 and 29 µg/mL, respectively against the mentioned radicals). The essential oil also exhibited anticholinesterase activities with IC50 values of 48 and 90 µg/mL respectively against AChE and BChE (for galantamine used as standard, the IC50 values were 25 and 30 µg/mL respectively). The essential oil also exhibited antidiabetic potential with IC50 values of 120 and 110 µg/mL respectively against α-glucosidase and α-amylase (IC50 values for standard acarbose = 28 and 30 µg/mL respectively). CONCLUSION: Essential oil extracted from the fruits of E. umbellata exhibited reasonable antioxidant, anticholinesterase, and antidiabetic potentials that could be used as alternative medicine in treating diabetes and neurodegenerative disorders. However, further studies are needed to isolate responsible compounds and evaluate the observed potential in animal models.

Evaluation of Cholinesterase Inhibitory Potential of Different Genotypes of Ziziphus nummularia, Their HPLC-UV, and Molecular Docking Analysis.

Ziziphus nummularia is an important source of valuable phytoconstituents, which are widely used in traditional medicine system of Indo-Pak sub-continent. In this study we investigated the distribution of phenolic compounds in the fruit pericarps of six different genotypes (ZNP01-06) of Z. nummularia growing in the unexplored hilly areas of Pakistan. The methanolic extracts of these genotypes were screened for total phenolic content (TPC), total flavonoid content (TFC), antioxidant, and cholinesterase inhibitory potentials. The observed biological potentials were explained in terms of the outcome of molecular docking and HPLC analyses. Among them, genotype ZNP02 displayed high TPC (88.50 ± 1.23 µg/mL) and showed potent scavenging activity against DPPH (67.03 ± 1.04 µg/mL) and ABTS (65.3 ± 1.74 µg/mL) in comparison to ascorbic acid (68.7 ± 0.47 µg/mL). Moreover, genotypes ZNP01, ZNP02, and ZNP04 displayed potent inhibition against acetyl and butyryl cholinesterases (AChE and BChE) with IC50 values of 21.2, 20.5, and 23.7 µg/mL (AChE) and 22.7, 24.4, and 33.1 µg/mL (BChE), respectively. Furthermore, the individual compounds in the most potent species ZNP01 responsible for potent enzyme inhibition (identified through HPLC-UV analysis), were computed via docking simulation software to the enzyme structures. Among these compounds rutin exhibited significant binding affinity with value of -9.20 kcal/mol. The differences amongst the phytochemical compositions of the selected genotypes highlighted the genotypic variations in them. Based on our results it was concluded that the selected plant can be used as remedy of oxidative stress and neurodegenerative diseases. However, further studies are needed to isolate responsible compounds and test the observed potential in vivo, along with toxicological evaluations in animal models.

Evaluation of neuroprotective and anti-amnesic effects of Elaeagnus umbellata Thunb. On scopolamine-induced memory impairment in mice.

BACKGROUND: Elaeagnus umbellata is abundantly found in Himalayan regions of Pakistan which is traditionally used to treat various health disorders. However, the experimental evidence supporting the anti-amnesic effect is limited. Therefore the study was aimed to evaluate the prospective beneficial effect of E. umbellata on learning and memory in mice. OBJECTIVES: To assess neuroprotective and anti-amnesic effects of E. umbellata fruit extracts and isolated compounds on the central nervous system. METHODS: Major phytochemical groups present in methanolic extract of E. umbellata were qualitatively determined. The total phenolic and flavonoid contents were also determined in extract/fractions of E. umbellata. On the basis of in vitro promising anticholinesterases (AChE & BChE) and antioxidant activities observed for CHF. Ext and isolated compound-I (Chlorogenic acid = CGA), they were further evaluated for learning and memory in normal and scopolamine-induced cognitive impairment in mice using memory behavioral tests such as the Y maze and Novel object recognition using standard procedures. The test sample were further assessed for in vivo anticholinesterases (AChE & BChE) and DPPH free radical scavenging activities in mice brain sample and finally validated by molecular docking study using GOLD software. RESULTS: The extract/fractions and isolated compounds were tested for their anticholinesterase and antioxidant potentials. The CHF. Ext and CGA showed maximum % inhibition of tested cholinesterases and free radicals. The CHF. Ext and CGA reversed the effects of scopolamine in mice. The CHF. Ext and CGA significantly increased the alternate arm returns and % spontaneous alteration performance while escape latency times (second) significantly decreased in Y maze test. The CHF. Ext and CGA significantly increased the time spent with novel object and also increased the discrimination index in the Novel object recognition test. Furthermore, molecular docking was used to validate the mechanism of cholinesterases inhibition of isolated compounds. CONCLUSION: The data obtained from behavioral and biochemical studies (AChE/BChE and DPPH/ABTS inhibition) have shown that E. umbellata possessed significant memory enhancing potency. These results suggest that E. umbellata extract possess potential antiamnesic effects and amongst the isolated compounds, compound I could be more effective anti-amnesic therapeutics. However, further studies are needed to identify the exact mechanism of action.

Characterization of phenolic compounds in two novel lines of Pisum sativum L. along with their in vitro antioxidant potential.

Like other vegetables, Pisum sativum L. also faces storage and degradation problems. To enhance their resistance and make them enable to cope with the deterioration problems during storage, the current study was designed to develop two resistant lines of P. sativum in terms of phenolic contents and genotypes. The phenolic compounds generally have antioxidant properties and deterioration during storage which are usually due to oxidation caused by free radicals. Thus, if a variety has high phenolic contents these problems will be coped in a better way. The genotype of a plant is also important in this regard, and the best adopted species would survive in unfavorable conditions. First, the phenolic and flavonoid contents were determined in the crude extract using the Folin-Ciocalteu method. Then, the identification and quantification of phenolic compounds were carried out in the developed lines of selected plants PL-04 and PL-05, as well as in the parental varieties [Climax (female) and Falan (male)] via HPLC. DPPH assay was used to determine the free radical scavenging capabilities of the extracts of the developed verities. The genotypic differences were confirmed by DNA fingerprinting using advanced simple sequence repeat (SSR) markers. The HPLC analysis of PL-04 confirmed the presence of three phenolic compounds in an appreciable amount which exhibited a higher antioxidant activity against DPPH radicals, while in the parental varieties, two phenolic compounds were identified and exhibited lower antioxidant activities. PL-04 was found rich in phenolic compounds and affectively scavenge-free radicals which would therefore be resistant to oxidation and degradation caused by free radicals. Comparing the present findings with our previous one, P-04 was found to be resistant to powdery mildew; it was concluded that the most probable reason of the resistance was the high phenolic contents and thus long shelf life.

Curative Effect of Catechin Isolated from Elaeagnus Umbellata Thunb. Berries for Diabetes and Related Complications in Streptozotocin-Induced Diabetic Rats Model.

In this study, catechin (CTN) isolated from Elaeagnus umbellata was evaluated for in vitro antioxidant potential and inhibition of carbohydrate digestive enzymes (α-amylase and α-glucosidase). The compound was also tested for its in vivo antidiabetic potential using Sprague-Dawley rats as experimental animals. The effects of various doses of catechin in STZ (Streptozotocin) induced diabetic rats on fasting blood glucose level, body weight, lipid parameters, hepatic enzymes, and renal functions were evaluated using the reported protocols. The CTN exhibited the highest percent antioxidant for free radical scavenging activity against DPPH and ABTS free radicals, and inhibited the activity of carbohydrate digestive enzymes (with percent inhibition values: 79 ± 1.5% α-amylase and 80 ± 1.1% α-glucosidase). Administration CTN and standard glibenclamide significantly decreased the fasting blood glucose level and increased the body weight in STZ-induced diabetic rats. CTN significantly decreased the different lipid parameters, hepatic, and renal function enzyme levels along with Hb1c level in diabetic rats, while significantly increasing the high-density lipoprotein (HDL) level with values comparable to the standard glibenclamide. Further, the altered levels of glutathione and lipid peroxides of liver and kidney tissues were restored (by CTN) to levels similar to the control group. CTN significantly increased the antioxidant enzyme activities, total content of reduced glutathione, and reduced the malondialdehyde (MDA) level in rat liver and kidney tissues homogenates, and also corrected the histopathological abnormalities, suggesting its antioxidant potential.