Phytochemicals in the treatment of inflammation-associated diseases: the journey from preclinical trials to clinical practice.

Advances in biomedical research have demonstrated that inflammation and its related diseases are the greatest threat to public health. Inflammatory action is the pathological response of the body towards the external stimuli such as infections, environmental factors, and autoimmune conditions to reduce tissue damage and improve patient comfort. However, when detrimental signal-transduction pathways are activated and inflammatory mediators are released over an extended period of time, the inflammatory process continues and a mild but persistent pro-inflammatory state may develop. Numerous degenerative disorders and chronic health issues including arthritis, diabetes, obesity, cancer, and cardiovascular diseases, among others, are associated with the emergence of a low-grade inflammatory state. Though, anti-inflammatory steroidal, as well as non-steroidal drugs, are extensively used against different inflammatory conditions, they show undesirable side effects upon long-term exposure, at times, leading to life-threatening consequences. Thus, drugs targeting chronic inflammation need to be developed to achieve better therapeutic management without or with a fewer side effects. Plants have been well known for their medicinal use for thousands of years due to their pharmacologically active phytochemicals belonging to diverse chemical classes with a number of these demonstrating potent anti-inflammatory activity. Some typical examples include colchicine (alkaloid), escin (triterpenoid saponin), capsaicin (methoxy phenol), bicyclol (lignan), borneol (monoterpene), and quercetin (flavonoid). These phytochemicals often act via regulating molecular mechanisms that synergize the anti-inflammatory pathways such as increased production of anti-inflammatory cytokines or interfere with the inflammatory pathways such as to reduce the production of pro-inflammatory cytokines and other modulators to improve the underlying pathological condition. This review describes the anti-inflammatory properties of a number of biologically active compounds derived from medicinal plants, and their mechanisms of pharmacological intervention to alleviate inflammation-associated diseases. The emphasis is given to information on anti-inflammatory phytochemicals that have been evaluated at the preclinical and clinical levels. Recent trends and gaps in the development of phytochemical-based anti-inflammatory drugs have also been included.

Corrigendum: Phytochemicals in Cancer Treatment: From Preclinical Studies to Clinical Practice.

[This corrects the article DOI: 10.3389/fphar.2019.01614.].

Phytochemicals in Cancer Treatment: From Preclinical Studies to Clinical Practice.

Cancer is a severe health problem that continues to be a leading cause of death worldwide. Increasing knowledge of the molecular mechanisms underlying cancer progression has led to the development of a vast number of anticancer drugs. However, the use of chemically synthesized drugs has not significantly improved the overall survival rate over the past few decades. As a result, new strategies and novel chemoprevention agents are needed to complement current cancer therapies to improve efficiency. Naturally occurring compounds from plants known as phytochemicals, serve as vital resources for novel drugs and are also sources for cancer therapy. Some typical examples include taxol analogs, vinca alkaloids such as vincristine, vinblastine, and podophyllotoxin analogs. These phytochemicals often act via regulating molecular pathways which are implicated in growth and progression of cancer. The specific mechanisms include increasing antioxidant status, carcinogen inactivation, inhibiting proliferation, induction of cell cycle arrest and apoptosis; and regulation of the immune system. The primary objective of this review is to describe what we know to date of the active compounds in the natural products, along with their pharmacologic action and molecular or specific targets. Recent trends and gaps in phytochemical based anticancer drug discovery are also explored. The authors wish to expand the phytochemical research area not only for their scientific soundness but also for their potential druggability. Hence, the emphasis is given to information about anticancer phytochemicals which are evaluated at preclinical and clinical level.

Hepatoprotective Activity of Satwa, an Ayurvedic Formulation, Against Alcohol-induced Liver Injury in Rats.

Context • Guduchi Satwa is an Ayurvedic formulation prepared from Tinospora species. It has been used since ancient times to treat liver disorders. Objectives • The study intended to assess the hepatoprotective potential of Satwa prepared from 3 forms of Tinospora against alcohol-induced hepatotoxicity. Design • Male, albino Wistar rats were divided into 6 groups, with 6 rats each: 3 control groups-healthy controls, negative controls, and positive controls-and 3 intervention groups-Tinospora cordifolia, Tinospora sinensis, and Neem-Guduchi. Setting • The study was carried out at the Animal House facility of Bharati Vidyapeeth Deemed University's Medical College (Maharashtra, India). Intervention • Hepatotoxicity was induced by repeated dosing with alcohol for 15 d for all groups except for the healthy controls. To induce hepatotoxicity, the 5 groups received 1 mL of 30% alcohol PO per 100 g of body weight per day. The healthy controls and the negative controls received no hepatoprotective treatments. The other 4 groups received the dosing with alcohol 30 min after the hepatoprotective treatment, which they also received for 15 d: (1) positive controls-100 mg of silymarin per kg of body weight per day PO; (2) intervention group 1 (T cordifolia group)-200 mg of T cordifolia per kg of body weight per day PO; (3) intervention group 2 (T sinensis group)-200 mg of T sinensis per kg of body weight per day PO; and (4) intervention group 3 (Neem-Guduchi group)-200 mg of Neem-Guduchi per kg of body weight per day PO. Outcome Measures • Serum and liver tissue were used for biochemical analysis. Results • For the negative and positive control groups and the 3 intervention groups, the repeated dosing with alcohol produced elevations in the levels of liver-marker enzymes and changes in the lipid-profile status of the animals. Satwa from T cordifolia had a specific action in maintaining the lipid profile: total cholesterol, high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein. Improvement in the hepatic function, normalization of the lipid profile in the serum and liver, and improvements in the levels of antioxidant enzymes and oxidative-stress markers were observed in the animals treated with T sinensis Satwa. Neem-Guduchi Satwa was found to have a specific action in maintaining the lipid profile. The differential hepatoprotective effect of that Satwa was also evident from the liver histology. Conclusions • The data suggest that the 3 Satwa might be used in combination as a liver tonic that can help restore and strengthen the liver functions. The current study shows that the combination has the potential to be an effective liver tonic in animals. Scientific data from clinical trials of the 3 Satwa are not available. Systematic clinical trials are required that can yield information on their effects in humans.

Antidiabetic, Lipid Normalizing, and Nephroprotective Actions of the Strawberry: A Potent Supplementary Fruit.

The study was designed to assess the effect of different strawberry extracts on glucose levels, lipid profiles, and oxidative stress in nicotinamide-streptozotocin (NIC-STZ) induced diabetic rats. The associated changes were evaluated through biochemical, molecular, and histological assays. Diabetes was induced by intraperitoneal injection of STZ to albino Wistar rats after treatment with nicotinamide. Aqueous, hydroalcoholic, and alcoholic strawberry extracts were administrated orally to diabetic rats. Treatment of strawberry extracts improved lipid profile, liver function, and serum creatinine and led to a significant increase in antioxidant status in diabetic rats. Real-time PCR expression analysis of genes from the liver of animals treated with strawberry extracts exhibited downregulation of several fatty acid synthesis genes, transcription factors, such as Sterol regulatory Element Binding Transcription factor (SREBP) and Nuclear Factor-κß (NF-κß), and inflammatory markers, like Interleukin 6 (IL6) and Tumor Necrosis Factor-α (TNF-α). Strawberry extracts also upregulated liver Peroxisome Proliferator Activated Receptor-γ (PPAR-γ). Histological examination confirmed the nephroprotective and ß-cell regeneration/protection effects of strawberry extracts. The present study demonstrates several beneficial effects of strawberry extracts along with its probable mechanism of action.

Evaluating the anticancer activity and nanoparticulate nature of homeopathic preparations of Terminalia chebula.

BACKGROUND: Breast cancer is the most common cancer diagnosed among women and is the second leading cause of cancer death. Homeopathic medicines are part of the alternative medicines that are given as a supportive therapy in breast cancer. The objective of this study was to investigate the anticancer activity of commercially available homeopathic preparations of Terminalia chebula (TC) and evaluate their nanoparticulate nature. METHODS: Mother tincture (MT) and other homeopathic preparations (3X, 6C and 30C) of TC were tested for their effect on the viability of breast cancer (MDAMB231 and MCF7) and non-cancerous (HEK 293) cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell growth assay was performed to analyze the effect of the different potencies on the growth kinetics of breast cancer cells. MT and 6C were evaluated for the presence of nanoparticles by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: MT decreased the viability of breast cancer (MDAMB231 and MCF7) and non-cancerous (HEK 293) cells. However, the other potencies (3X, 6C and 30C) decreased the viability of only breast cancer cells without affecting the viability of the non-cancerous cells. All the potencies, MT, 3X, 6C and 30C, reduced growth kinetics of breast cancer cells, more specifically at 1:10 dilution at 24, 48 and 72 h. Under SEM, MT appeared as a mesh-like structure whereas under TEM, it showed presence of nanoclusters. On the other hand, 6C potency contained 20 nm sized nanoparticles. CONCLUSION: The current study reports the anticancer activity of homeopathic preparations of TC against breast cancer and reveals their nanoparticulate nature. These preliminary results warrant further mechanistic studies at both in vitro and in vivo levels to evaluate the potential of TC as nanomedicine in breast cancer.

Inflammatory response of cultured rat synoviocytes challenged with synovial fluid from osteoarthritis patients correlates with their radiographic grading: a pilot study.

The inflammatory nature of synovial fluid (SF) of varying grade osteoarthritis (OA) patients was estimated by measuring pro-inflammatory factors and through a unique cell-challenge experiment. SF samples were collected from six OA and one non-OA patient; spanning Kellgren-Lawrence (KL) grades were analyzed for interlukin-1-beta (IL-1ß), nitric oxide (NO) and its derivatives, and glycosaminoglycan (GAG). Levels of IL-1ß, NO, and GAG in SF did not correlate with KL grades of the patients studied. In the cell-challenge experiment, cultured rat synoviocyte fibroblasts (RSFs) were challenged by the patient's SFs with and without pre-treatment of IL-1ß and lipopolysaccharide (LPS). NO released by the cells was taken as an indicator of inflammation. SFs from KL grades 2 and 3 induced maximum inflammation in cultured RSFs (grade 2 64.61 ± 4.8 and 89.51 ± 5.6 µM/ml after 48 and 72 h, grade 3 58.27 ± 2.7 and 64.22 ± 2.8 µM/ml after 48 and 72 h, respectively). Similar trend was observed in RSF pretreated with either recombinant IL-1ß or LPS suggesting that SF from patients KL grades 2 and 3 accumulates more pro-inflammatory factors. IL-1ß-pre-treated RSFs challenged by SF for 72 h showed 234.41 ± 17.6 µM/ml increase (patient 3, grade 3), whereas higher NO after LPS pre-treatment was recorded (118.92 ± 6.2 µM/ml; patient 3, grade 3). Interestingly, SFs from grade 1 and non-OA patient could reduce released NO to 27.10 ± 2.2 µM/ml showing potency to alleviate inflammation. These interesting findings, however, need to be confirmed on a wider number of patients, which may offer significant therapeutic application in treatment of OA.

Circulatory adipocytokines and lipid profile variations in type-2 diabetic subjects: desirable side-effects of antidiabetic drugs.

AIM: Inspite of availability of a variety of drugs to treat type 2 diabetes, little is known about their effects on other systems. Normalization of glucose metabolism by these drugs may consequently affect the secretory function in adipocytes. Secretory adipocytokines like adiponectin and leptin are emerging as novel therapeutic targets for type 2 diabetes mellitus (T2DM). The present study was undertaken to analyze the effects of commonly used Oral Hypoglycemic Agents (OHAs) alone, or in combination with other drugs and/or insulin on circulatory adiponectin and leptin levels, lipid profile, and blood pressure in diabetic subjects. METHODS: The study was undertaken at IRSHA and Bharati Vidyapeeth Medical College and Hospital, MS, India. Clinically diagnosed T2DM subjects and age, gender matched healthy controls were recruited. Fasting blood was collected from each subject and the blood samples were analyzed for circulatory adipocytokines and lipid parameters using commercial kits. RESULTS: Serum adiponectin levels were significantly increased while leptin significantly decreased in diabetic men (p<0.05) and women (p<0.001) on OHA, as compared to healthy controls. Triglyceride levels significantly decreased (p<0.05) in diabetic men, however, they remained unchanged in women despite same drug treatment. Serum HDL and LDL levels (p<0.001) were significantly lower in diabetic women as compared to healthy women. Systolic (p<0.05) and diastolic (p<0.001) blood pressure was significantly high in diabetic men but remained unchanged in women. CONCLUSIONS: Frequently used OHAs significantly improve circulatory levels of adipocytokines. Selecting best treatment option for each patient is a key, and 2012 European Association for the Study of Diabetes (EASD) and ADA guidelines recommend diabetes treatment to be individualized depending on various socioeconomic and lifestyle factors. We recommend regular analysis of circulatory adipocytokines in T2DM patients to help clinicians select the best treatment option to normalize levels of these important therapeutic targets.

Therapeutic implications of targeting energy metabolism in breast cancer.

PPARs are ligand activated transcription factors. PPARγ agonists have been reported as a new and potentially efficacious treatment of inflammation, diabetes, obesity, cancer, AD, and schizophrenia. Since cancer cells show dysregulation of glycolysis they are potentially manageable through changes in metabolic environment. Interestingly, several of the genes involved in maintaining the metabolic environment and the central energy generation pathway are regulated or predicted to be regulated by PPARγ. The use of synthetic PPARγ ligands as drugs and their recent withdrawal/restricted usage highlight the lack of understanding of the molecular basis of these drugs, their off-target effects, and their network. These data further underscores the complexity of nuclear receptor signalling mechanisms. This paper will discuss the function and role of PPARγ in energy metabolism and cancer biology in general and its emergence as a promising therapeutic target in breast cancer.

Functional and bioinformatic characterisation of sequence variants of Fad3 gene from flax.

BACKGROUND: Desaturases are enzymes that drive the multi-step fatty acid biosynthetic pathway. As evident from directed mutagenesis, single base changes in their polypeptide can potentially alter their structure and may result in altered substrate specificity, regioselectivity and even loss of function. The authors have previously isolated several sequence variants of Δ15 desaturase from flax while attempting to clone that gene. The aim of the present study was to analyse these gene variants for their functionality and to predict the tertiary structure of the protein in order to correlate the functional differences with the protein structure. RESULTS: The variants differed in the rate at which they could convert linoleic acid to α-linolenic acid. The highest conversion rate was 7.03%, while the lowest was 2.39%. The overall shape of the predicted 3D model of the protein is a compact cylinder containing α-helices and ß-sheets. The Ramchandran plot of this model revealed that 98.5% of the residues are located in allowed region, which denotes a stable structure. CONCLUSION: Although the structures of the variants are apparently similar, subtle changes account for variation in their activity. Besides, these substitutions may alter their cross-talk with other proteins and thus differentially influence their specificity, localisation and stability, which in turn may explain the diversity in their function.

Rectal gel application of Withania somnifera root extract expounds anti-inflammatory and muco-restorative activity in TNBS-induced inflammatory bowel disease.

BACKGROUND: Inflammatory Bowel Disease (IBD) is marked with chronic inflammation of intestinal epithelium driven by oxidative stress. Traditional treatments with plant extracts gained renewed interest due to their ability to ameliorate the multi factorial conditions like inflammation. We investigated the beneficial effects of Withania somnifera in Trinitro Benzyl Sulfonic Acid (TNBS) induced experimental IBD through a rectally applicable formulation. METHODS: The study included (i) preparation of gel formulation from aqueous Withania somnifera root extract (WSRE), (ii) biochemical assays to determine its performance potential, (iii) testing of formulation efficacy in TNBS-induced IBD rat model, and (iv) histo-patholgical studies to assess its healing and muco-regenerative effect in IBD-induced rats. For this purpose, concentration dependant antioxidant activity of the extracts were evaluated using biochemical assays like (a) inhibition of lipid peroxidation, (b) NO scavenging, (c) H2O2 scavenging, and (d) ferric reducing power assay. RESULTS: The extract, at 500 µg/ml, the highest concentration tested, showed 95.6% inhibition of lipid peroxidation, 14.8% NO scavenging, 81.79% H2O2 scavenging and a reducing capacity of 0.80. The results were comparable with standard antioxidants, ascorbic acid and curcumin. WSRE treatment positively scored on histopathological parameters like necrosis, edema, neutrophil infiltration. The post treatment intestinal features showed restoration at par with the healthy intestine. In view of these results, gel formulation containing an aqueous extract of W. somnifera, prepared for rectal application was tested for its anti-inflammatory activity in TNBS-induced rat models for IBD. Commercially available anti-inflammatory drug Mesalamine was used as the standard in this assay. CONCLUSIONS: Dose of the rectal gel applied at 1000 mg of WSRE per kg rat weight showed significant muco-restorative efficacy in the IBD-induced rats, validated by histo-pathological studies.

Scientific basis for the use of Indian ayurvedic medicinal plants in the treatment of neurodegenerative disorders: ashwagandha.

Ayurveda is a Sanskrit word, which means "the scripture for longevity". It represents an ancient system of traditional medicine prevalent in India and in several other south Asian countries. It is based on a holistic view of treatment which is believed to cure human diseases through establishment of equilibrium in the different elements of human life, the body, the mind, the intellect and the soul [1]. Ayurveda dates back to the period of the Indus Valley civilization (about 3000 B.C) and has been passed on through generations of oral tradition, like the other four sacred texts (Rigveda, Yajurveda, Samaveda and Atharvanaveda) which were composed between 12(th) and 7(th) century B.C [2, 3]. References to the herbal medicines of Ayurveda are found in all of the other four Vedas, suggesting that Ayurveda predates the other Vedas by at least several centuries. It was already in full practice at the time of Buddha (6(th) century B.C) and had produced two of the greatest physicians of ancient India, Charaka and Shushrutha who composed the basic texts of their trade, the Samhitas. By this time, ayurveda had already developed eight different subspecialties of medical treatment, named Ashtanga, which included surgery, internal medicine, ENT, pediatrics, toxicology, health and longevity, and spiritual healing [4]. Ayurvedic medicine was mainly composed of herbal preparations which were occasionally combined with different levels of other compounds, as supplements [5]. In the Ayurvedic system, the herbs used for medicinal purposes are classed as brain tonics or rejuvenators. Among the plants most often used in Ayurveda are, in the descending order of importance: (a) Ashwagandha, (b) Brahmi, (c) Jatamansi, (d) Jyotishmati, (e) Mandukparni, (f) Shankhapushpi, and (g) Vacha. The general appearance of these seven plants is shown in Fig.1. Their corresponding Latin names, as employed in current scientific literature, the botanical families that each of them belongs to, their normal habitats in different areas of the world, as well as the common synonyms by which they are known, are shown in the Table 1. The scientific investigations concerning the best known and most scientifically investigated of these herbs, Ashwagandha will be discussed in detail in this review. Ashwagandha (Withania somnifera, WS), also commonly known, in different parts of the world, as Indian ginseng, Winter cherry, Ajagandha, Kanaje Hindi and Samm Al Ferakh, is a plant belonging to the Solanaceae family. It is also known in different linguistic areas in India by its local vernacular names [6]. It grows prolifically in dry regions of South Asia, Central Asia and Africa, particularly in India, Pakistan, Bangladesh, Sri Lanka, Afghanistan, South Africa, Egypt, Morocco, Congo and Jordon [7]. In India, it is cultivated, on a commercial scale, in the states of Madhya Pradesh, Uttar Pradesh, Punjab, Gujarat and Rajasthan [6]. In Sanskrit, ashwagandha, the Indian name for WS, means "odor of the horse", probably originating from the odor of its root which resembles that of a sweaty horse. The name"somnifera" in Latin means "sleep-inducer" which probably refers to its extensive use as a remedy against stress from a variety of daily chores. Some herbalists refer to ashwagandha as Indian ginseng, since it is used in India, in a way similar to how ginseng is used in traditional Chinese medicine to treat a large variety of human diseases [8]. Ashwagandha is a shrub whose various parts (berries, leaves and roots) have been used by Ayurvedic practitioners as folk remedies, or as aphrodisiacs and diuretics. The fresh roots are sometimes boiled in milk, in order to leach out undesirable constituents. The berries are sometimes used as a substitute to coagulate milk in cheese making. In Ayurveda, the herbal preparation is referred to as a "rasayana", an elixir that works, in a nonspecific, global fashion, to increase human health and longevity. It is also considered an adaptogen, a nontoxic medication that normalizes physiological functions, disturbed by chronic stress, through correction of imbalances in the neuroendocrine and immune systems [9, 10]. The scientific research that has been carried out on Ashwagandha and other ayurvedic herbal medicines may be classified into three major categories, taking into consideration the endogenous or exogenous phenomena that are known to cause physiological disequilibrium leading to the pathological state; (A) pharmacological and therapeutic effects of extracts, purified compounds or multi-herbal mixtures on specific non-neurological diseases; (B) pharmacological and therapeutic effects of extracts, purified compounds or multi-herbal mixtures on neurodegenerative disorders; and (C) biochemical, physiological and genetic studies on the herbal plants themselves, in order to distinguish between those originating from different habitats, or to improve the known medicinal quality of the indigenous plant. Some of the major points on its use in the treatment of neurodegenerative disorders are described below.

Maternal caloric restriction spares fetal brain polyunsaturated fatty acids in Wistar rats.

There is increasing interest in the role of developmental programming; however, the impact on fetal oxidative stress and brain fatty acid levels has been relatively unexplored. Recent reports have shown that caloric restriction regimens in adult animals reduce the occurrence of chronic diseases by reducing the oxidative stress and altering the long chain polyunsaturated fatty acids (LCPUFA). The present study examined whether caloric restriction during pregnancy alters oxidative stress and essential fatty acid metabolism in mother and offspring at birth. Pregnant female rats were fed either a standard chow (C, n=7) or were calorie restricted (CR, n=7) by feeding 60% of the intake of the control. Oxidative stress marker (malondialdehyde) and polyunsaturated fatty acid profiles in brain and liver were analyzed in both dams and offspring. Total weight gain during pregnancy was lower (p<0.01) in the CR group as compared to the control group but did not affect the litter size and weight. Brain malondialdehyde levels were lower (p<0.05) in dams from the CR group. There was no change in brain and liver LCPUFA levels in both male and female offspring in the CR group. Most of the polyunsaturated fatty acids were reduced (p<0.05) in plasma and brain in the CR dams. Caloric restriction during pregnancy did not alter LCPUFA metabolism in the offspring suggesting that during maternal caloric restriction mothers own stores are mobilized to provide docosahexaenoic acid and arachidonic acid for fetal brain development.

Cloning of a novel omega-6 desaturase from flax (Linum usitatissimum L.) and its functional analysis in Saccharomyces cerevisiae.

The Delta12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Delta12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids-putative membrane-bound Delta12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Delta12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence 'YNNKL' was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.

Assessment of genetic diversity in Trigonella foenum-graecum and Trigonella caerulea using ISSR and RAPD markers.

BACKGROUND: Various species of genus Trigonella are important from medical and culinary aspect. Among these, Trigonella foenum-graecum is commonly grown as a vegetable. This anti-diabetic herb can lower blood glucose and cholesterol levels. Another species, Trigonella caerulea is used as food in the form of young seedlings. This herb is also used in cheese making. However, little is known about the genetic variation present in these species. In this report we describe the use of ISSR and RAPD markers to study genetic diversity in both, Trigonella foenum-graecum and Trigonella caerulea. RESULTS: Seventeen accessions of Trigonella foenum-graecum and nine accessions of Trigonella caerulea representing various countries were analyzed using ISSR and RAPD markers. Genetic diversity parameters (average number of alleles per polymorphic locus, percent polymorphism, average heterozygosity and marker index) were calculated for ISSR, RAPD and ISSR+RAPD approaches in both the species. Dendrograms were constructed using UPGMA algorithm based on the similarity index values for both Trigonella foenum-graecum and Trigonella caerulea. The UPGMA analysis showed that plants from different geographical regions were distributed in different groups in both the species. In Trigonella foenum-graecum accessions from Pakistan and Afghanistan were grouped together in one cluster but accessions from India and Nepal were grouped together in another cluster. However, in both the species accessions from Turkey did not group together and fell in different clusters. CONCLUSIONS: Based on genetic similarity indices, higher diversity was observed in Trigonella caerulea as compared to Trigonella foenum-graecum. The genetic similarity matrices generated by ISSR and RAPD markers in both species were highly correlated (r = 0.78 at p = 0.001 for Trigonella foenum-graecum and r = 0.98 at p = 0.001 for Trigonella caerulea) indicating congruence between these two systems. Implications of these observations in the analysis of genetic diversity and in supporting the possible Center of Origin and/or Diversity for Trigonella are discussed.

Decreased antioxidant enzymes and membrane essential polyunsaturated fatty acids in schizophrenic and bipolar mood disorder patients.

Oxidative stress-mediated cell damage has been considered in the pathophysiology of schizophrenia. Abnormal findings have often been considered related to differences in ethnicity, life style, dietary patterns and medications, all of which influence indices of oxidative stress and oxidative cell damage. To minimize these confounds, schizophrenic patients were compared with age-matched control subjects with the same ethnic background and similar lifestyle, as well as with bipolar mood disorder (BMD) patients. Levels of antioxidant defense enzymes (i.e. superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GPx) were lower in schizophrenic patients than in controls, indicating conditions for increased oxidative stress. The contents of plasma thiobarbituric acid reactive substances (TBARS) were only marginally higher in schizophrenic patients, who had normal levels of arachidonic acid (AA), a major source of TBARS, indicating no significant oxidative membrane lipid peroxidation. Levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), however, were significantly lower in schizophrenic patients. When the same indices in BMD patients were compared with findings in matched controls, levels of only SOD and CAT were lower in the patients, whereas GPx was not. Again, as in schizophrenia, the contents of TBARS were marginally higher in BMD patients with no change in levels of AA. Levels of alpha-linolenic acid and EPA were significantly lower and levels of DHA were slightly lower in BMD patients. These data indicate that certain biochemical characteristics may be common to a spectrum of psychiatric disorders, and suggest supplementation of antioxidants and essential fatty acids might affect clinical outcome.

Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura.

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.

Supplementation with a combination of omega-3 fatty acids and antioxidants (vitamins E and C) improves the outcome of schizophrenia.

Reduced levels of membrane essential polyunsaturated fatty acids (EPUFAs), namely, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acids (DHAs), and their association with psychopathology have been consistently reported in both chronic-medicated schizophrenic patients as well as in never-medicated patients soon after the first episode of psychosis. Past supplementation studies with either omega-6 or omega-3 or both EPUFAs generally in chronic-medicated-older patients have reported varying degrees of therapeutic effects, and have suggested that supplementation with primarily omega-3 EPUFAs (EPA>DHA) may be preferable. We report the supplementation with a mixture of EPA/DHA (180:120 mg) and antioxidants (vitamin E/C, 400 IU:500 mg) orally morning and evening to schizophrenic patients (N=33) for 4 months. The red blood cell (RBC) membrane fatty acid levels, plasma lipid peroxides and clinical measures were carried out by established procedures at pretreatment, posttreatment and after 4 months of postsupplementation period to determine the stability of treatment effects within patients. Levels of fatty acids and lipid peroxides were compared with their levels in normal controls (NC) (N=45).Posttreatment levels of RBC EPUFAs were significantly higher than pretreatment levels as well as levels in normal controls without any significant increase in plasma peroxides. Concomitantly, there was significant reduction in psychopathology based on reduction in individual total scores for brief psychiatric rating scale (BPRS) and positive and negative syndrome scale (PANSS), general psychopathology-PANSS and increase in Henrich's Quality of Life (QOL) Scale. The EPUFA levels returned to pretreatment levels after 4 months of supplementation washout. However, the clinical improvement was significantly retained. Future studies need be done in placebo-controlled trials and also with a comparison group supplemented with fatty acids alone in a larger number of patients, both chronic as well as never medicated, and for a longer duration of treatment while the dietary intake is monitored. This may establish the EPUFA supplementation a very effective treatment to improve the outcome for an extended period of time.

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds.

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.

Essential polyunsaturated fatty acid and lipid peroxide levels in never-medicated and medicated schizophrenia patients.

BACKGROUND: Reduced levels of membrane essential polyunsaturated fatty acids (EPUFAs) and increased levels of lipid peroxidation products (thiobarbituric acid reactive substances; TBARS) have been observed in chronic medicated schizophrenics. The relationship of EPUFA and TBARS to psychopathology is unclear, since their levels may be altered differentially by duration of illness and antipsychotic treatment. To minimize these confounds, their levels were compared among never-medicated patients in early illness, medicated patients and control subjects with similar lifestyle and common ethnic background. METHODS: RBC membrane EPUFAs, plasma TBARS, and various dimensions of psychopathology were measured using established procedures in never-medicated (n = 20) and medicated (n= 32) schizophrenia patients and in control subjects (n= 45). RESULTS: Reduced levels of EPUFAs, particularly arachidonic acid (AA) and docosahexaenoic acid (DHA), were found in never-medicated compared with control subjects; however, the reductions in levels of both AA and DHA were much smaller in medicated versus never-medicated patients; AA levels were similar to levels in control subjects. Only DHA levels were significantly reduced in medicated patients. Lower membrane AA levels were associated with increased levels of plasma TBARS in never-medicated patients. Lower levels of membrane EPUFAs and higher levels of plasma TBARS were associated with the severe symptoms in never-medicated versus medicated patients. CONCLUSIONS: Data indicate that reduced EPUFAs and increased TBARS exist in never-medicated patients, and these measures correlate with the severity of psychopathology indicating that the membrane EPUFA status may reflect the outcome of schizophrenia.