Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 µg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

Comprehensive Metabolomic Fingerprinting Combined with Chemometrics Identifies Species- and Variety-Specific Variation of Medicinal Herbs: An Ocimum Study.

Identification of plant species is a crucial process in natural products. Ocimum, often referred to as the queen of herbs, is one of the most versatile and globally used medicinal herbs for various health benefits due to it having a wide variety of pharmacological activities. Despite there being significant global demand for this medicinal herb, rapid and comprehensive metabolomic fingerprinting approaches for species- and variety-specific classification are limited. In this study, metabolomic fingerprinting of five Ocimum species (Ocimum basilicum L., Ocimum sanctum L., Ocimum africanum Lour., Ocimum kilimandscharicum Gurke., and Hybrid Tulsi) and their varieties was performed using LC-MS, GC-MS, and the rapid fingerprinting approach FT-NIR combined with chemometrics. The aim was to distinguish the species- and variety-specific variation with a view toward developing a quality assessment of Ocimum species. Discrimination of species and varieties was achieved using principal component analysis (PCA), partial least squares discriminate analysis (PLS-DA), data-driven soft independent modelling of class analogy (DD-SIMCA), random forest, and K-nearest neighbours with specificity of 98% and sensitivity of 99%. Phenolics and flavonoids were found to be major contributing markers for species-specific variation. The present study established comprehensive metabolomic fingerprinting consisting of rapid screening and confirmatory approaches as a highly efficient means to identify the species and variety of Ocimum, being able to be applied for the quality assessment of other natural medicinal herbs.

In vivo therapeutic efficacy of Curcuma longa extract loaded ethosomes on wound healing.

Since ancient times, medicinal plants are widely accepted to promote the health and wellness of animals and mankind. The medicinal plant-based therapies have limitations of delayed onset of action, inconsistent absorption, low bioavailability, oxidation, and poor solubility. The encapsulation studies suggested improved efficacy. Therefore, the present study attempts to evaluate the efficacy of Curcuma longa extracts encapsulated in Ethosome on wound healing model compared to crude extract. The Curcuma longa extract swere prepared by cold percolation method and total curcuminoid content was determined by Reverse phase-HPLC. Three Ethosomal suspensions (ETS1, ETS2, and ETS3) were prepared and characterized for particle distribution, morphology, and absorption spectrum by Zetasizer, Scanning Electron Microscopy, and FTIR respectively. The Ethosomal suspension with the highest entrapment efficiency was applied topically at a varying concentrations (0.25, 0.5, and 1 g/cm2) on the surgically created wounds in rats. The efficacy of wound healing was evaluated by clinical observation, macroscopic evaluation of granulation tissue, colour digital image processing, and histology. The methanolic extract of Curcuma longa showed better antibacterial potential than ethanolic and aqueous. The total Curcuminoid content in the Curcuma longa rhizome was 4.03%. The size, PDI, zeta potential, and viscosity of Ethosomal suspension ranged from 34.8 to 371 nm, 0.236-1.178, 15.6-36.8mV, and 0.8460-0.8510, respectively. The ETS3 was found the most optimum combination with the highest entrapment efficiency and the topical application at a dose rate of 0.5 g/cm2 and 1.0 g/cm2 resulted in comparable wound contracture, pain score, histopathological score as compared to control groups.It was concluded that the Curcuma longa encapsulation in Ethosome resulted in improved wound appearance, granulation tissue score, and appearance with a shortened period of wound resolution at the cellular level as compared to crude extract.

Assessment of antidiabetic effect of 4-HIL in type 2 diabetic and healthy Sprague Dawley rats.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia and insulin resistance. 4-hydroxyisoleucine (4-HIL) is a non-proteinogenic amino acid isolated from the fenugreek seeds and has enormous pharmacological activities. The present study was undertaken to investigate the antihyperglycemic effect of 4-HIL in streptozotocin (STZ)-induced diabetic rats. Moreover, its toxicity was evaluated in vitro and in vivo employing human embryonic kidney cells (HEK-293) and healthy rats, respectively. In experiment 1, STZ-induced diabetic male rats were subjected to an oral treatment of 4-HIL (100 mg/kg), while experiment 2 deals with the effects of 4-HIL on healthy male and female rats following oral administration. The treatment (experiment 1) declined the elevated blood glucose level, feed intake, and increased body weight(s). Additionally, blood glucose impairment was improved as observed by OGTT and IPGT tests. Pancreatic histopathology revealed mild changes in the 4-HIL group. Moreover, experiment 2 showed increased body weight, normal blood glucose levels (male-106.06 ± 7.49 mg/dl and female-100.06 ± 14.69 mg/dL), hematological parameters, and histopathological profiles in the treatment group. 4-HIL did not affect the viability of HEK-293 cells, and no signs of toxicity were observed in healthy rats. Therefore, the study concludes that 4-HIL has potential antihyperglycemic activity without any toxic effects.

Effects of Spirulina (Arthrospira platensis) as a drinking water supplement during cyclical chronic heat stress on broiler chickens: Assessing algal composition, production, stress, health and immune-biochemical indices.

Spirulina, the blue green algae is considered to exhibit multifaceted benefits on both human health and animal production. Three hundred sixty day old unsexed broiler chicks of CARIBROVISHAL strain were assigned to five treatment groups each comprising nine replicates of 8 chicks. The experiment was carried out during the hot humid summer season (Mid-April to May) under deep litter rearing system with uniform managemental conditions. Birds were administered orally with Spirulina through drinking water in the morning (06:00-12:00 PM) on daily basis throughout the experimental period at 5, 10, 15 and 20 gL-1 concentration. Spirulina supplementation neither improved nor compromised production performance of broilers reared during hot climatic condition. Results based on one way analysis of variance indicated a significant effect on haemoglobin and total red blood cell count. Serum lipid content and transaminases were reduced, while serum protein concentration was higher (P < 0.01) in the groups administered with 15 and 20 gL-1 of Spirulina. The extent of imparting shank pigmentation was improved in all the supplemented groups. Cell mediated and humoral immunity against Phytoheamagglutunin-P and Newcastle disease vaccination respectively were maximized (P < 0.05) at 20 gL-1. These findings provide direct evidence of dose-related modulation of production, physiological and immunological attributes by Spirulina engendering its further investigation as a potential source of drinking water supplement for stress alleviation in broilers. From the results, it may concluded that Spirulina can be incorporated at 15 or 20 gL-1 for achieving optimal improvement of health and welfare attributes in broilers reared during hot summer without compromising production.

Regulatory safety pharmacology and toxicity assessments of a standardized stem extract of Cassia occidentalis Linn. in rodents.

Cassia occidentalis Linn (CO) is an annual/perennial plant having traditional uses in the treatments of ringworm, gastrointestinal ailments and piles, bone fracture, and wound healing. Previously, we confirmed the medicinal use of the stem extract (ethanolic) of CO (henceforth CSE) in fracture healing at 250 mg/kg dose in rats and described an osteogenic mode of action of four phytochemicals present in CSE. Here we studied CSE's preclinical safety and toxicity. CSE prepared as per regulations of Current Good Manufacturing Practice for human pharmaceuticals/phytopharmaceuticals and all studies were performed in rodents in a GLP-accredited facility. In acute dose toxicity as per New Drug and Clinical Trial Rules, 2019 (prior name schedule Y), in rats and mice and ten-day dose range-finding study in rats, CSE showed no mortality and no gross abnormality at 2500 mg/kg dose. Safety Pharmacology showed no adverse effect on central nervous system, cardiovascular system, and respiratory system at 2500 mg/kg dose. CSE was not mutagenic in the Ames test and did not cause clastogenicity assessed by in vivo bone marrow genotoxicity assay. By a sub chronic (90 days) repeated dose (as per OECD, 408 guideline) study in rats, the no-observed-adverse-effect-level was found to be 2500 mg/kg assessed by clinico-biochemistry and all organs histopathology. We conclude that CSE is safe up to 10X the dose required for its osteogenic effect.

Hesperidin-rich ethanol extract from waste peels of Citrus limetta mitigates rheumatoid arthritis and related complications.

The aim of this study is to explore the possible pharmacological effects of fruit waste that may have a key role in converting the fruit waste into pharmaceutical agents. Citrus limetta (Rutaceae) is an important commercial citrus fruit crops used by juice processing industries. C. limetta peels are perishable waste material, which creates a big challenge in juice processing industries. Initial pharmaco-chemical profile of peels' extracts revealed that the ethanol extract (ClPs) has promising anti-inflammatory activity and rich in hesperidin content. In vivo experimental pharmacology profile of ClPs against arthritis and related complications revealed that oral administration of ClPs significantly reduced the arthritis score and arthritis index in elbow and knee joints against collagen-induced arthritis (CIA) in rats. Biochemical parameters include pro-inflammatory cytokines (TNF-α, IL-6, and IL-17A), and C-RP level in blood serum of CIA rats further confirmed the anti-arthritic profile of ClPs. Further individual experiments related to arthritis-related complications in experimental animals demonstrated the analgesic, anti-inflammatory, and antipyretic potential of ClPs in dose-dependent manner. The result of this study suggests the suitability of ClPs as a drug-like candidate for further investigation toward the management of arthritis and related complications.

Evaluation of antimicrobial potential of successive extracts of Ulmus wallichiana Planch.

BACKGROUND: The plant Ulmus wallichiana Planch. is found in hills of Uttarakhand, India. Bark of U. wallichiana is commonly used as traditional healer for bone fracture of animals as well as human beings and also used as wound healer remedy. OBJECTIVE: The present study was designed to evaluate antimicrobial potential of various extracts of U. wallichiana bark. MATERIALS AND METHODS: Soxhlet extraction method was used for preparation of different extracts viz. petroleum ether, chloroform, ethyl acetate, ethanol and aqueous. Antioxidant activity was determined by DPPH and FRAP assay method. In vitro antimicrobial activity was evaluated using agar well diffusion method. RESULTS: Ethyl acetate extract exhibited the highest significant antioxidant activity. Antibacterial activity was performed against Escherichia coli, Bacillus subtilis, Staphylococcusaureus and Pseudomonas aeruginosa. Amongst the various extracts tested, only ethyl acetate exhibited highest zone of inhibition as compared to other extracts and greater than standard drug. Chloroform extract also showed moderate zone of inhibition. Antifungal activity was evaluated against Aspergillus fumigates and Aspergillus flavus. The ethyl acetate extract showed maximum zone of inhibition as compared to other extracts. Chloroform extract showed mild antifungal activity. Chloramphenicol and nystatin were used as a positive control as antibacterial and antifungal agent respectively. Furthermore, the highest percentage of phenolic and flavonoid compounds was estimated in ethyl acetate extract. CONCLUSION: The ethyl acetate extract of U. wallichiana showed the highest antimicrobial activity, and should be further investigated for isolating active compound(s) responsible for antimicrobial activity.

Dose- and Ion-Dependent Effects in the Oxidative Stress Response to Space-Like Radiation Exposure in the Skeletal System.

Space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized that ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-weeks old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 MeV/n) or high-LET 56Fe ions (600 MeV/n) using either low (5 or 10 cGy) or high (50 or 200 cGy) doses at NASA's Space Radiation Lab. Five weeks or one year after irradiation, tissues were harvested and analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed by RT-PCR during the proliferative or mineralizing phase of growth, and differentiation was analyzed by imaging mineralized nodules. As expected, a high dose (200 cGy), but not lower doses, of either 56Fe or protons caused a loss of cancellous bone volume/total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; 56Fe (200 cGy) inhibited osteoblastogenesis by more than 90% (5 weeks and 1 year post-IR). After 5 weeks, irradiation (protons or 56Fe) caused few changes in gene expression levels during osteoblastogenesis, although a high dose 56Fe (200 cGy) increased Catalase and Gadd45. The addition of exogenous superoxide dismutase (SOD) protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET (137Cs γ) when irradiated in vitro, but had limited protective effects on high-LET 56Fe-exposed cells. In sum, either protons or 56Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET 56Fe increased redox-related gene expression, albeit to a limited extent, and inhibited osteoblastogenesis. Doses below 50 cGy did not elicit widespread responses in any parameter measured. We conclude that high-LET irradiation at 200 cGy impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss.

Genotoxicity evaluation of zinc oxide nanoparticles in Swiss mice after oral administration using chromosomal aberration, micronuclei, semen analysis, and RAPD profile.

The expanded uses of zinc oxide nanoparticles (ZnO NPs) have grown rapidly in the field of nanotechnology. Thus, rising production of nanoparticles (NPs) increases the possible risks to the environment and occupationally exposed humans. Hence, it is indispensable to appraise the safety toxicity including genotoxicity for these NPs. In the present study, we have evaluated the genotoxic effect of ZnO NPs after oral administration to Swiss mice at dose levels of 300 and 2000 mg/kg body weight. These doses were administered for 2 days at 24 h apart. Chromosomal aberration (CA) and micronucleus tests were conducted following Organization for Economic Co-operation and Development guidelines. DNA damage was evaluated at 0, 24, 48, and 72 h posttreatment using a randomly amplified polymorphic DNA (RAPD) assay; additionally, semen analyses were also performed at 34.5 days post oral exposure. The reactive oxygen species (ROS), 8-oxo-2'-deoxyguanosine and CAs were increased ( p < 0.05) at the highest dosage (2000 mg/kg) of ZnO NPs compared to controls. Aberrant sperm morphology with reduced sperm count and motility were also present ( p < 0.05) in the high-dose group. Based on the RAPD assay, the genomic template stability within the high-dose group (<90%) was less than the controls (100%). The results suggested that ZnO NPs are mildly genotoxic in a dose-related manner and this toxicity were induced by generation of ROS.

Lanosterol synthase-like is involved with differential accumulation of steroidal glycoalkaloids in potato.

MAIN CONCLUSION: Phytosterol homeostasis may be maintained in leaves through diversion of intermediates into glycoalkaloid biosynthesis, whereas in tuber flesh, excess intermediates are catalyzed by tuber-specific StLAS - like , resulting in low tuber glycoalkaloids. Lanosterol synthase (LAS) and cycloartenol synthase (CAS) are phylogenetically related enzymes. Cycloartenol is the accepted precursor leading to cholesterol and phytosterols, and in potato, to steroidal glycoalkaloid (SGA) biosynthesis. LAS was also shown to synthesize some plant sterols, albeit at trace amounts, questioning its role in sterol homeostasis. Presently, a potato LAS-related gene (StLAS-like) was identified and its activity verified in a yeast complementation assay. A transgenic approach with targeted gene expression and metabolic profiling of sterols and SGAs was used. Analyses of StLAS-like transcript levels and StLAS-like-promoter::GUS reporter assays indicated specific expression in tuber flesh tissue. Overexpression of Arabidopsis AtLAS in leaves where the endogenic StLAS-like is not expressed, resulted with increased SGA level and reduced phytosterol level, while in the tuber flesh SGA level was reduced. StLAS-like expression only in tuber flesh may explain the differential accumulation of SGAs in commercial cultivars-low in tubers, high in leaves. In leaves, to maintain phytosterol homeostasis, an excess of intermediates may be diverted into SGA biosynthesis, whereas in tuber flesh these intermediates are catalyzed by tuber-specific StLAS-like instead, resulting in low levels of SGA.

Silicon fertilization of potato: expression of putative transporters and tuber skin quality.

MAIN CONCLUSION: A silicon transporter homolog was upregulated by Si fertilization and drought in potato roots and leaves. High Si in tuber skin resulted in anatomical and compositional changes suggesting delayed skin maturation. Silicon (Si) fertilization has beneficial effects on plant resistance to biotic and abiotic stresses. Potatoes, low Si accumulators, are susceptible to yield loss due to suboptimal growth conditions; thus Si fertilization may contribute to crop improvement. The effect of Si fertilization on transcript levels of putative transporters, Si uptake and tuber quality was studied in potatoes grown in a glasshouse and fertilized with sodium silicate, under normal and drought-stress conditions. Anatomical studies and Raman spectroscopic analyses of tuber skin were conducted. A putative transporter, StLsi1, with conserved amino acid domains for Si transport, was isolated. The StLsi1 transcript was detected in roots and leaves and its level increased twofold following Si fertilization, and about fivefold in leaves upon Si × drought interaction. Nevertheless, increased Si accumulation was detected only in tuber peel of Si-fertilized plants--probably due to passive movement of Si from the soil solution--where it modified skin cell morphology and cell-wall composition. Compared to controls, skin cell area was greater, suberin biosynthetic genes were upregulated and skin cell walls were enriched with oxidized aromatic moieties suggesting enhanced lignification and suberization. The accumulating data suggest delayed tuber skin maturation following Si fertilization. Despite StLsi1 upregulation, low accumulation of Si in roots and leaves may result from low transport activity. Study of Si metabolism in potato, a major staple food, would contribute to the improvement of other low Si crops to ensure food security under changing climate.

An ethnobotanical survey of medicinal plants used in Terai forest of western Nepal.

BACKGROUND: Nepal Himalayas have been known as a rich source for valuable medicinal plants since Vedic periods. Present work is the documentation of indigenous knowledge on plant utilization as natural remedy by the inhabitants of terai forest in Western Nepal. METHODS: Study was conducted during 2010-2011 following standard ethnobotanical methods. Data about medicinal uses of plants were collected by questionnaire, personal interview and group discussion with pre identified informants. Voucher specimens were collected with the help of informants, processed into herbarium following standard methods, identified with the help of pertinent floras and taxonomic experts, and submitted in Department of Botany, Butwal Multiple Campus, Tribhuvan University, Nepal for future references. RESULTS: During the present study 66 medicinal plant species belonging to 37 families and 60 genera has been documented. These plants were used to treat various diseases and ailments grouped under 11 disease categories, with the highest number of species (41) being used for gastro-intestinal disorders, followed by dermatological disorders (34). In the study area the informants' consensus about usages of medicinal plants ranges from 0.93 to 0.97 with an average value of 0.94. Herbs (53%) were the primary source of medicine, followed by trees (23%). Curcuma longa (84%) and Azadirachta indica (76%) are the most frequently and popularly used medicinal plant species in the study area. Acacia catechu, Bacopa monnieri, Bombax ceiba, Drymaria diandra, Rauvolfia serpentina, and Tribulus terrestris are threatened species which needs to be conserved for future use. CONCLUSIONS: The high degree of consensus among the informants suggests that current use and knowledge are still strong, and thus the preservation of today's knowledge shows good foresight in acting before much has been lost. The connections between plant use and conservation are also important ones, especially as the authors note that neither the local inhabitants nor the government is addressing the potential loss of valuable species in this region.

Documentation and determination of consensus about phytotherapeutic veterinary practices among the Tharu tribal community of Uttar Pradesh, India.

PURPOSE: The aim of the present investigation was to document the phytotherapeutic knowledge and veterinary healthcare management practices among the Tharu tribal community of Uttar Pradesh, India and to determine the consensus of such practices, in order to evaluate the potential for new veterinary drugs of herbal origin. METHODS: This study was conducted in 2000-2004 using semistructured, open-ended questionnaires, informal interviews and group discussions with farmers engaged in animal husbandry. RESULTS: In the present study, 59 phytotherapeutic practices using 48 plant species were documented for management of 18 types of healthcare problems of domesticated animals. Crude drug formulations keep the animal healthy, increase lactation, and reduce estrus interval and puberty period to make them economically more important. There was great agreement among informants regarding phytotherapeutic uses of medicinal plants with factor of informants' consensus (F(IC)) value ranging from 0.84 to 1, with an average value of 0.94. CONCLUSION: Study reveals that there is great agreement among informants for the usages of Azadirachta indica A Juss, Bombax ceiba L, Bambusa arundinacea (Retz) Willd, Corianderum sativum L, Cuscuta reflexa Roxb, Datura metal L, Nelumbo nucifera Gaertn, and Parthenium hysterophorus L. These species may be used for the development of new, cheep, effective, and eco-friendly herbal formulations for veterinary healthcare management. Further investigation of these herbal formulations for veterinary healthcare management will require safety and efficacy testing. There is an urgent need to formulate suitable conservation strategies for wildly growing phytotherapeutics to overcome their depletion from natural resources and to make these practices more eco-friendly.

Antiangiogenic and proapoptotic activities of allyl isothiocyanate inhibit ascites tumor growth in vivo.

The authors investigate the antiangiogenic and proapoptotic effects of mustard essential oil containing allyl isothiocyanate (AITC) and explore its mechanism of action on Ehrlich ascites tumor (EAT) cells. Swiss albino mice transplanted with EAT cells were used to study the effect of AITC. AITC was effective at a concentration of 10 mum as demonstrated by the inhibition of proliferation of EAT cells when compared with the normal HEK293 cells. It significantly reduced ascites secretion and tumor cell proliferation by about 80% and inhibited vascular endothelial growth factor expression in tumor-bearing mice in vivo. It also reduced vessel sprouting and exhibited potent antiangiogenic activity in the chorioallantoic membrane and cornea of the rat. AITC arrested the growth of EAT cells by inducing apoptosis and effectively arrested cell cycle progression at the G1 phase. The results clearly suggest that AITC inhibits tumor growth by both antiangiogenic and proapoptotic mechanisms.