Leaf spot disease adversely affects human health-promoting constituents and withanolide biosynthesis in Withania somnifera (L.) Dunal.

AIMS: The present work investigates the implication of leaf spot disease on the antioxidant potential and commercial value of pharmaceutically important constituents of Withania somnifera, a high-valued medicinal plant. METHODS AND RESULTS: Leaf spot disease was induced in W. somnifera by inoculating Alternaria alternata (Fr.) Keiss. pathogen. Total polyphenolic content and antioxidant potential showed a significant decrease during leaf spot disease. Evaluation of pharmaceutically active constituents withaferin A, withanone and withanolide A utilizing high-performance liquid chromatography showed a significant decrease in diseased samples as compared to healthy ones. Quantitative expression of major genes involved in withanolide biosynthesis also showed down-regulation in diseased samples. Alterations in the ultra-structure of chloroplasts were also analysed under transmission electron microscopy to get a better insight into the changes of withanolide biosynthesis in leaf during disease infestation. CONCLUSIONS: The present work suggests that when the pathogenic fungus invades the host plants, it evokes multiple responses, which could be studied at various levels. The knowledge gained from this work will provide appropriate rationale for controlling the bio-deterioration of the pharmaceutically active metabolites in W. somnifera and development of suitable strategies against leaf spot disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate the effect of leaf spot disease on the human health-promoting constituents and withanolide biosynthesis in this high-valued medicinal plant.

Biodiversity of endosymbiont fungi associated with a marine sponge Lamellodysidea herbacea and their potential as antioxidant producers.

This study aims to isolate endosymbiontic fungi from the marine sponge Lamellodysidea herbacea and to explore their antioxidant potential. Marine-derived fungi, with their vast biodiversity, are considered a promising source of novel antioxidants which can replace synthetic ones. Marine sponges have previously reported bioactive properties that could ameliorate oxidative stress, particularly their associated fungi, producing high-frequency bioactive molecules (adaptogenic molecules) in response to stressors. 19 endosymbiont fungi associated with marine sponges were isolated, and their extracts were evaluated for their antioxidant capacities. Extract of an endosymbiont fungus, isolate SPG6, identified as Alternaria destruens, through surface electron microscopy (SEM) and ITS gene sequencing, showed broad range antioxidant activities (EC50 values) (free radical scavenging 32.54 mg L-1, Hydroxyl radical scavenging activity < 0.078 g L-1, total reducing power 0.114 g L-1, Chelating power 0.262 g L-1, H2O2 scavenging activity < 0.078 g L-1, and Superoxide radical scavenging activity > 5.0 g L-1). The extract of isolate SPG6 was fractioned and analyzed through GC-MS. Marine sponge-associated endosymbiont fungi are a rich source of antioxidant molecules. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03972-1.

Validated high-performance thin-layer chromatography method for steviol glycosides in Stevia rebaudiana.

A high-performance thin-layer chromatographic (HPTLC) method was developed and validated as per ICH (International Conferences on Harmonization) guidelines for simultaneous quantification of three steviol glycosides, i.e. steviolbioside, stevioside and rebaudioside-A in Stevia rebaudiana leaves. For achieving good separation, mobile phase of ethyl acetate-ethanol-water (80:20:12, v/v/v) on pre-coated silica gel 60 F254 HPTLC plates were used. The densitometric quantification of steviol glycosides was carried out at lambda=510 nm in reflection-absorption mode after spraying with acetic anhydride:sulphuric acid:ethanol reagent. The calibration curves were linear in the range of 160-960 ng/spot for steviolbioside, 1-6 microg/spot for stevioside and 0.5-3 microg/spot for rebaudioside-A with good correlation coefficients (0.998-0.999). The method was found to be reproducible for quantitative analysis of steviol glycosides in S. rebaudiana leaves collected from ten different locations and will serve as a quality control indicator to monitor the commercial production of stevioside and its allied molecules during different stages of its processing.

Studies on leaf spot disease of Withania somnifera and its impact on secondary metabolites.

During an investigation of the disease profile of Withania somnifera, it was observed that leaf spot is the most prevalent disease. Repeated isolations from infected leaf tissues and pathogenicity tests showed the association of fungal pathogen identified as Alternaria alternata (Fr.) Keissler. Scanning electron microscopy showed various histological changes in the leaf tissues of infected plants. A decrease in total content of reducing sugars (20%) and chlorophyll (26.5%) was observed in diseased leaves whereas an increase was noticed in proline (25%), free amino acids (3%) and proteins (74.3%). High performance thin layer chromatography (HPTLC) analysis of secondary metabolites viz. withanolides, withaferin-A and total alkaloids of the diseased leaves vis-à-vis control revealed reduction in withaferin-A and withanolides contents by 15.4% and 76.3% respectively, in contrast to an increase in total alkaloids by 49.3%, information hitherto unreported in W. somnifera.

A new alkylated benzoquinone from rhizomes of Iris kumaonensis.

A novel alkylated unsaturated p-benzoquinone designated as 3-[(z)-12'-heptadecenyl]-2-hydroxy-5-methoxy-1,4-benzoquinone was isolated from hexane extract of the rhizomes of Iris kumaonensis and it's structure was confirmed by extensive spectroscopic analysis, IR, MS, HREIMS, 1D, 2D NMR and comparison with the literature data of known compounds.

Nitrogen use scenario in India.

Nitrogen is one of the major plant nutrients without which the agricultural production is not possible. Nitrogen use in Indian agriculture was nearly 55000 tons in 1950-1951 that increased to 11.31 million tons in 2001-2002. The total food production of the country has also experienced the similar increase from 50.83 to 222 million tons in the respective years. Interestingly the N fertilizer consumption of India remained almost constant during the last six years indicating the possibility of reducing N consumption. The highest N consumption is in North zone owing to the introduction of rice-wheat cropping system followed by West, South and East. The N use efficiency has been reported to be varying between 30% to 50% depending on the crops and the management. But in most of the cases, N use efficiency has been calculated based on the total N removed by the crops (above ground part only) ignoring the N content left in the roots. It has been observed in controlled experiments that the total N uptake by roots varied from 18% to 44% of the total N removed by the above ground parts, i.e. grain and straw. If the root N is also accounted, the N use efficiency will be higher than reported. The management of other organic sources has to be improved so as to increase the fertilizer use efficiency as well as to check the direct release of N in the atmosphere. In this review all these issues will be dealt.

Localization of a putative inositol 1,4,5-triphosphate receptor in the Limulus granulocyte.

The horseshoe crab (Limulus polyphemus) granulocyte (GR) degranulates upon contact with bacteria and release factors that mediate an immune response. Stimulated cells produce IP3, which binds to receptors (IP3R, M.W.240-300 kD) that function to release stored Ca2+ into the cytoplasm that mediates degranulation. This mechanism is believed to mediate exocytosis in the Limulus GR but IP3R in the GR has not been shown. The present study utilized monoclonal antibody 4C11 and a commercially available anti-IP3R antibody, both of which label amino acids of the N-terminal of all known isoforms. Electron microscopy, immunohistochemistry, SDS-PAGE, and Western blot analysis, which employed the use of the two antibodies, demonstrates that a putative IP3R exists in the: plasma membrane, smooth surfaced vesicles, nucleus and nuclear membrane. We hypothesize that this putative IP3R is involved in mediating the immune response of the Limulus GR.

Signal transduction during exocytosis in Limulus polyphemus granulocytes.

Bacterial lipopolysaccharide (LPS)-induced exocytosis is one of the primary immune responses of the Limulus granulocyte (GR). Exocytosis can be mediated by guanine nucleotide-binding protein (G-protein)-linked surface receptors that activate phospholipase C (PLC) to produce inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 mobilizes intracellular Ca2+ ([Ca2+]i), which can lead to exocytosis. We used activators and inhibitors of known signal transduction pathways to investigate the signaling pathway responsible for LPS-induced exocytosis in the GR. These compounds have been shown to similarly effect pathways in vertebrate and invertebrate systems and this assumption is made here. Pretreatment of GRs with cholera and pertussis toxins, which modulate G-proteins, and U73122, which inhibits PLC, inhibited LPS-induced exocytosis, but pretreatment with the tyrosine kinase inhibitor herbimycin did not. In contrast, exocytosis was induced with fluoride (a G-protein activator) and thapsigargin with Mg2+ (an inhibitor of endomembranous Ca(2+)-ATPase). Exocytosis was not induced by phorbol ester, which mimics DAG to activate protein kinase C (PKC) and it was not effected by ethanol or chelerythrine, which inhibit phospholipase D and PKC, respectively. Microinjection of GRs with different concentrations of IP3, an IP3 analog (DL-2,3,6,trideoxy-myo-inositol 1,4,5-triphosphate), Mg2+, or Ca2+ induced different percentages of exocytosis in individual cells, while HEPES buffer did not. Microfluorometric analysis of intracellular Mg2+ ([Mg2+]i) and [Ca2+]i, using the dyes Mag Fura-2AM and Calcium Green 5N, respectively, revealed [Mg2+]i and [Ca2+]i fluxes during LPS-induced exocytosis. This study suggests that LPS induces exocytosis in the Limulus GR through activation of G-protein-coupled receptors, which stimulate the IP3 signaling pathway to induce both [Ca2+]i and [Mg2+]i fluxes to facilitate vesicular and plasma membrane fusion. This is the first demonstration of the signal transduction pathway responsible for the primary immune response of the GR.

Plasma membrane specializations in resting, stimulated and phagocytosing arthropod (Limulus polyphemus, Gromphadorhina portentosa and Blattella germanica) immunocytes: structural and functional analogies with those of vertebrate macrophages and neutrophils.

Using indirect immunofluorescence and scanning electron microscope techniques, we have shown that the plasma membrane specializations, such as pseudopods, filopods, lamellipods, and zeiotic blebs occur in activated and/or phagocytosing arthropod (Limulus polyphemus, Gromphadorhina portentosa, and Blattella germanica) immunocytes (hemocytes), as they do in challenged vertebrate neutrophils and macrophages. All four specializations may also be caused by some of the chemicals in the preparative media, which suggests that arthropod immunocytes, like the vertebrate neutrophils and macrophages, have evolved as highly reactive cells that are sensitive to chemical and/or biological foreign agonists/antigens. These attributes are necessary for the effectiveness of a cellular defense mechanism. The plasma membrane specializations perform the same functions in arthropod immunocytes as reported for those of vertebrates. For example, pseudopods and lamellipods are needed for spreading and locomotion and filopodia for cell adhesion and crawling. Because they are formed as a result of similar reactions to foreign antigens, as in vertebrate cells, it is suggested that similar terminologies be consistently used for them in arthropods.

Arthropod immune system. III. Septate junctions in the hemocytic capsule of the german cockroach, Blattella germanica.

Granulocytes (GRs) and/or plasmatocytes (PLs), the two major immunocytes in arthropods, participate in cellular encapsulation of foreign tissue. Although gap and desmosome junctions have been reported in insect capsules, smooth septate junctions are being reported for the first time by both thin section and freeze-fracture techniques in Blattella germanica. In 7-day-old capsules, the septa are 23 nm thick, faintly 'scalloped' and slightly curved in appearance; the interseptal space has a periodicity of about 5 nm. In freeze-fractured capsules, the septa are associated on both sides with the corresponding intramembranous structures, belonging to the plasma membranes of the two junction-forming GRs. The intercellular space is 27 nm wide. There are 36-40 septa/1 microm junctional length. The junctions show furrows on the extracellular fracture face (E) and the complementary regular rows of intramembranous particles on the cytoplasmic face (P). The septate junctions often occur in the region of the capsule that also shows the presence of gap junctions, but only rarely that of desmosomes. The septate junctions are in close proximity with mitochondria. It is suggested that the function of these junctions is to produce compact capsules.

Presence of Dirofilaria repens and an insect immunocyte (plasmatocyte) in a human subcutaneous nodule, induced by a mosquito bite.

It is well known that the nematode Dirofilaria repens is transmitted to humans by vector mosquito bite. Examination of a fine needle aspiration biopsy drawn from a month-old nodule on the chest of a woman, residing in Garlasco, province of Pavia, Northern Italy, revealed the presence of not only one immature female of D. repens, but also some scattered cells that we believe to be mosquito's blood cells, plasmatocytes (immunocytes). We presume that plasmatocytes were carried into the bite wound with the mosquito's hemolymph that had oozed from a rupture in its mouthparts during feeding. Because Aedes albopictus recently colonized certain areas in the above region, we suspect that the nodule resulted from the bite of this mosquito.

Metoclopramide as a lactogogue.

Thirty-two mothers with no or inadequate breast milk were treated with metoclopramide. Improved lactation occurred in 66.67 percent of mothers with no breast milk and 100 percent of those with inadequate milk output. Improvement persisted after discontinuing the treatment. No untoward effects were noticed in any of the mothers or their infants.

Hepatoprotective activity of LC-ESI-MS standardized Iris spuria rhizome extract on its main bioactive constituents.

The study was designed to evaluate the hepatoprotective activity of Iris spuria against paracetamol induced toxicity at two different doses 100 and 200 mg/kg. The extract showed significant protective activity (p>0.01) at both the doses in dose dependent manner. Administration of the plant extract restored the paracetamol induced elevated levels of serum marker and distorted hepatic tissue architecture. The lipid peroxides (LPO) and glutathione (GSH) levels were also restored towards normal in liver tissue significantly. The main chemical constituents of the extract identified by the liquid chromatography-tandem mass spectrometry (LC-ESI-MSMS) were found to be flavones and isoflavonoids. Tectoridin and iristectorigenin A were the principal compounds present in the methanolic extract of Iris spuria.

Volatile (As and Hg) and non-volatile (Pb and Cd) toxic heavy metals analysis in rhizome of Zingiber officinale collected from different locations of North Western Himalayas by Atomic Absorption Spectroscopy.

Ginger is an important ingredient of spice and herbals. The monitoring of toxic heavy metals in the rhizome of ginger is important for protecting public health against the hazards of metal toxicity. The concentration of volatile and non-volatile metals (As, Hg, Pb and Cd), in the soil and rhizome of Zingiber officinale were analyzed using AAS. Soil analysis profile showed uniformity in the metal contents, in active root zone and subsoil, except mercury, which was present in higher quantity in one, out of the four sectors, of the field. The infield metal content in the soil in increasing order was, cadmium < arsenic < lead < mercury. In ginger rhizome the volatile toxic heavy metals arsenic (As) and mercury (Hg) varied from not detected to 0.13 µg/g and 0.01 to 0.42 µg/g, respectively. The non-volatile metals lead (Pb) and cadmium (Cd) ranged from 0.06 to 0.64 µg/g and 0.002 to 0.03 µg/g, respectively(.) The results illustrated the findings that soil is the major but not the only source of metal accumulation in the plants. In our study, the volatile metal content (As, Hg) was found more in rhizomes collected from Himachal Pradesh while the non-volatile metals were predominant in samples from Uttarakhand.