Metatranscriptome analysis of symptomatic bitter apple plants revealed mixed viral infections with a putative novel polerovirus.

BACKGROUND: Next-generation Sequencing (NGS) combined with bioinformatic analyses constitutes a powerful approach for identifying and characterizing previously unknown viral genomes. In this study, leaf samples from bitter apple plants (Citrullus colocynthis (L.) Schrad) exhibiting symptoms such as dwarfing, leaf crinkling, and chlorosis were collected from the southern part of Kerman province, Iran. RESULTS: Putative infecting viruses were identified through de novo assembly of sequencing reads using various tools, followed by BLAST analysis. Complete genomes for Squash vein yellowing virus (SqVYV), Citrus-associated rhabdovirus (CiaRV), and a novel polerovirus-related strain termed Bitter apple aphid-borne yellows virus (BaABYV) were assembled and characterized. Additionally, a partial genome for Watermelon mosaic virus (WMV) was assembled. The genomic organization of the BaABYV was determined to be 5'-ORF0-ORF1-ORF1,2-ORF3a-ORF3-ORF3,5-ORF4-3'. Amino acid sequence identities for inferred proteins (P0 and P1, P1,2) with known poleroviruses were found to be the 90% species delineation limit, implying that BaABYV should be considered a new member of the genus Polerovirus. Recombination events were observed in the BaABYV and WMV strains; such events were not found in the CiaRV strain. CONCLUSIONS: Molecular evidence from this study suggests that C. colocynthis is a reservoir host of several plant viruses. Among them, BaABYV is proposed as a new member of the genus Polerovirus. Furthermore, the CiaRV strain has been reported for the first time from Iran.

Two new cucurbitane-type triterpenoid saponins from the fruit of Citrullus colocynthis.

Two new cucurbitane-type triterpenoid saponins, 2,20ß,22ß-trihydroxy-16α,23(R)-epoxycucurbita-1,5,24-triene-3,11-dione 2-O-ß-D-glucopyranoside (1), 2,20ß,22α-trihydroxy-16α,23(S)-epoxycucurbita-1,5,11,24-tetraene-3-one 2-O-ß-D-glucopyranoside (2) were isolated from the fruit of Citrullus colocynthis (L.) Schrad. Their structures were elucidated by mass spectrometry, IR, 1D, and 2D NMR spectroscopy, etc. Besides, both of the compounds showed significant hepatoprotective activities at 10 µM against paracetamol-induced HepG2 cell damage.

Investigating the effects of Citrullus colocynthis on cognitive performance and anxiety-like behaviors in STZ-induced diabetic rats.

Background: Diabetes can impair cognitive performance and lead to dementia. Patients with type 1 diabetes mellitus (T1DM) are reported with different levels of cognitive dysfunctions in various cognitive domains ranging from general intellectual testing to specific deficits with visuospatial abilities, motor speed, writing, attention, reading, and psychomotor efficiency. The present study aimed to investigate the effect of Citrullus colocynthis on cognitive functions.Methods: A total of 42 male Wistar rats (3-4 months old and weighing 200-250 g) were tested in the current study. Rats were randomly allocated into 3 groups of control, Diabetes, and Diabetes + Drug. The diabetic rats received Citrullus colocynthis extraction orally. The behavioral tests included the open field, elevated plus maze (EPM), novel object recognition (NOR), passive avoidance tests, and Morris Water Maze (MWM) tests. Data were analyzed using student and paired t-tests via SPSS software version 16.Results: Our results showed the protective effects of Citrullus colocynthis administration against cognitive impairments. This is followed by STZ-induced diabetes in the MWM, novel object recognition, and passive avoidance tasks. Also, it was found that Citrullus colocynthis improved anxiety in diabetic rats.Conclusion According to the findings of this study, the administration of 200 mg/kg C. colocynthis once per day for 40 days can lead to ameliorated cognitive impairments and antidiabetic effects such as increasing body weight and decreasing FBS.

Citrullus colocynthis (L.) Schrad.: A Promising Pharmaceutical Resource for Multiple Diseases.

Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) is widely distributed in the desert areas of the world. The fruit bodies of C. colocynthis are recognized for their wide range of nutraceutical potential, as well as medicinal and pharmaceutical uses. The plant has been reported for various uses, such as asthma, bronchitis, cancer, colic, common cold, cough, diabetes, dysentery, and jaundice. The fruit has been extensively studied for its biological activities, which include insecticide, antitumor, and antidiabetic effects. Numerous bioactive compounds have been reported in its fruit bodies, such as essential oils, fatty acids, glycosides, alkaloids, and flavonoids. Of these, flavonoids or caffeic acid derivatives are the constituents associated with the inhibition of fungal or bacterial growth, whereas eudesmane sesquiterpenes or sesquiterpene lactones are most active against insects, mites, and nematodes. In this review, the scientific evidence for the biological activity of C. colocynthis against insecticide, cytotoxic, and antidiabetic effects is summarized.

Characterization of Bioactive Compounds and Novel Proteins Derived from Promising Source Citrullus colocynthis along with In-Vitro and In-Vivo Activities.

Herbal products are preferable to synthetic medicines, and the use of traditional medicines is increasing day-by-day. The current study was designed to evaluate the potentials of bioactive compounds from Citrullus colocynthis by performing FTIR, HPLC, and GC-MS analyses, which explore the good concentration of the secondary metabolites, such as gallic acid (74.854 ppm), vanillic acid (122.616 ppm), and ferulic acid (101.045 ppm) with considerable bioactivities. Antimicrobial protein was estimated by performing SDS-PAGE, ranging from 15 to 70 kDa in all protein fractions. The current study also checked the cytotoxicity of the bioactive compounds in the active fraction of C. colocynthis, and to perform this activity, the groups of rats were arranged with 16 rats randomly divided into four groups (three experimental and one control) by administering various dosage of methanolic fractions in dose-dependent manner. Histopathology was conducted on the livers of the rats after 15 days of sacrifice under deep anesthesia. In liver cell slides examined at the maximum dose of 600 mg/kg, minimal morphological changes, such as slight ballooning, nuclear variation, vacuolar degeneration, and hydropic degeneration, were observed. Furthermore, the in silico analysis identified bioactive compounds as potential drug candidates.

[Three new cucurbitane-type triterpenoid glycosides from Citrullus colocynthis and their anti-inflammatory activity].

Three new cucurbitane-type triterpenoid glycosides were separated from the ethyl acetate extract of Citrullus colocynthis by a variety of chromatographic techniques. According to the data of NMR, HR-ESI-MS, and/or comparison with the reported data, the three novel cucurbitane-type triterpenoid glycosides were identified as colocynthenin E(1), colocynthenin G(2), and colocynthenin H(3). The cell inflammation model was established with RAW264.7 macrophages exposed to lipopolysaccharide and then used to determine the anti-inflammatory activities of the three compounds. Compounds 2 and 3 showed mild anti-inflammatory activities with the IC_(50) of 48.21 and 40.11 µmol·L~(-1), respectively, compared with that(IC_(50)=7.57 µmol·L~(-1)) of the positive control dexamethasone.

Ruta graveolens, Peganum harmala, and Citrullus colocynthis methanolic extracts have in vitro protoscolocidal effects and act against bacteria isolated from echinococcal hydatid cyst fluid.

Echinococcosis is a common and endemic disease that affects both humans and animals. In this study, the in vitro activities of methanolic extracts of Ruta graveolens, Peganum harmala aerial parts, and Citrullus colocynthis seeds against protoscolosis and isolated bacterial strains from hydatid cysts were assessed using disc diffusion methods and Minimum Inhibitory Concentration (MIC). The chemical composition of three methanolic extracts was studied using LC-MS. After 3 h of exposure to 40 mg/mL R. graveolens extract, a tenfold protoscolocidal effect was seen when compared to the convintional medication (ABZ) for the same duration (P < 0.05). The bacteria listed below were isolated from hydatid cyst fluid collected from a variety of sick locations, including the lung and liver. Micrococcus spp., E. coli, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter amnigenus, Pseudomonas aeruginosa, Staphylococcus xylosus, and Achromobacter xylosoxidans are among the bacteria that have been identified. The most effective extract was R. graveolens, followed by P. harmala and C. colocynthis, according to the results of antibacterial activity using the disc diffusion method. R. graveolens extract had the lowest MIC values (less than 2 mg/mL) against all microorganisms tested. This shows that the R. graveolens extract has additional properties, such as the ability to be both scolocidal and bactericidal. Because these bacteria are among the most prevalent pathogenic bacteria that increase the risk of secondary infection during hydatid cysts, the results of inhibitory zones and MICs of the R. graveolens methanol extract are considered highly promising.

Isolation and identification of endophytic actinobacteria from Citrullus colocynthis (L.) Schrad and their antibacterial properties.

BACKGROUND: Antibiotic resistance poses a major threat to human health globally. Consequently, new antibiotics are desperately required to discover and develop from unexplored habitats to treat life-threatening infections. Microbial natural products (NP) are still remained as primary sources for the discovery of new antibiotics. Endophytic actinobacteria (EA) which are well-known producers of bioactive compounds could provide novel antibiotic against pathogenic bacteria. This research aimed to isolate EA from the Citrullus colocynthis plant and explore the antibacterial properties of their metabolites against pathogenic bacteria. RESULTS: The healthy samples were collected, dissected and surface-sterilized before cultured on four different selection media at 28 °C. Six endophytic actinobacteria were isolated from Citrullus colocynthis plant. They were taxonomically classified into two family namely Streptomycetaceae and Nocardiopsaceae, based on colony morphological features, scanning electron microscope analysis and molecular identification of isolates. This is the first report on the identification of EA form Citrullus colocynthis and their antibacterial activity. The strains generated a chain of vibrio-comma, cubed or cylindrical shaped spores with indenting or smooth surfaces. Three of those were reported as endophytes for the first time. The strain KUMS-C1 showed 98.55% sequence similarity to its closely related strains which constitutes as a novel species/ strain for which the name Nocardiopsis colocynthis sp. was proposed for the isolated strain. Five isolated strains had antagonist activity against S. aureus, P. aeruginosa, and E. coli. Among those, stain KUMS-C6 showed the broadest spectrum of antibacterial activity against all test bacteria, whereas the strain KUMS-C4 had no antibacterial activity. CONCLUSIONS: NPs have a long history of safe and efficient use for development of pharmaceutical products. Our study highlights that Citrullus colocynthis is an untapped source for the isolation of EA, generating novel and bioactive metabolites by which might lead to discovery of new antibiotic(s). This study reveals the future of new antibiotic developments looks bright against multi-drug resistance diseases by mining under- or unexplored habitats.

A Bitter Experience-A Bitter Apple (Citrullus colocynthis) Ingestion in a Child.

Here we present the case of a 4-year-old child who presented to the emergency department (ED) with bloody diarrhea. The causative agent was confirmed to be Citrullus colocynthis (bitter apple), which had been given to the patient by his grandmother for constipation. Hemorrhagic colitis can be induced by Citrullus colocynthis. Treatment is essentially support. Hypoglycemia and transaminitis have been associated with Citrullus colocynthis ingestion. The case highlights that pediatric patients can be inadvertently exposed to herbal medications that can cause harm. It also highlights that certain herbal medications with toxic potential, such as Citrullus, can be seen in emergency departments that are geographically quite distant from the most common origins of the plant. To our knowledge, this is the first American pediatric case report of bitter apple induced hemorrhagic colitis.

Phytochemicals Mediated Synthesis of AuNPs from Citrullus colocynthis and Their Characterization.

Engineered nanoparticles that have distinctive targeted characteristics with high potency are modernistic technological innovations. In the modern era of research, nanotechnology has assumed critical importance due to its vast applications in all fields of science. Biologically synthesized nanoparticles using plants are an alternative to conventional methods. In the present study, Citrullus colocynthis (bitter apple) was used for the synthesis of gold nanoparticles (AuNPs). UV-Vis's spectroscopy, XRD, SEM and FTIR were performed to confirm the formation of AuNPs. UV-Vis's spectra showed a characteristic peak at the range of 531.5-541.5 nm. XRD peaks at 2 θ = 38°, 44°, 64° and 77°, corresponding to 111, 200, 220 and 311 planes, confirmed the crystalline nature of AuNPs. Spherical AuNPs ranged mostly between 7 and 33 nm, and were measured using SEM. The FTIR analysis confirmed the presence of phytochemicals on the surface of AuNPs. Successful synthesis of AuNPs by seed extract of Citrullus colocynthis (bitter apple) as a capping and reducing agent represents the novelty of the present study.

Norcolocynthenins A and B, two cucurbitane 3-nor-Triterpenoids from Citrullus colocynthis and their cytotoxicity.

Two novel cucurbitane 3-nor-triterpenoids, named norcolocynthenins A (1) and B (2), were isolated from the fruits of Citrullus colocynthis. The structures including their absolute configurations were determined by extensive spectroscopic analyses and theoretical calculations. Compound 1 features an unprecedented 5/6/6/5-fused ring system while compound 2 possesses a rare lactone moiety at modified ring A. Compounds 1 and 2 showed significant cytotoxic activity against human cancer cell lines of HL-60 (IC50 = 8.32, 6.49 µM) and PC-3 (IC50 = 31.26, 13.42 µM). The plausible biosynthetic pathway of compounds 1 and 2 via a key enzymatic Baeyer-Villiger reaction is proposed.

Spinasterol, 22,23-Dihydrospinasterol and Fernenol from Citrullus Colocynthis L. with Aphicidal Activity against Cabbage Aphid Brevicoryne Brassicae L.

Brevicoryne brassicae is a problematic pest in cabbage and other field crops. Synthetic pesticides are used to control this pest, but they are injurious for human health and the environment. The present study aimed to purify and identify the active compounds from Citrullus colocynthis leaves with an appraisal of their efficacy against B. brassicae. Separation and purification were performed via different chromatographic techniques. Molecular analysis and chemical structures were recognized by mass spectrum (MS) and nuclear magnetic resonance (NMR), respectively. Moreover, in vitro and in vivo aphicidal activity was assessed using various concentrations, i.e., 6.25, 12.5, 25 and 50 µg/mL at 12, 24, 48 and 72 h exposure. The outcome shows that mass spectrum analyses of the purified compounds suggested the molecular formulae are C30H50O and C29H50O, C29H48O. The compounds were characterized as fernenol and a mixture of spinasterol, 22,23-dihydrospinasterol by 1H-NMR and 13C-NMR spectrum analysis. The toxicity results showed that the mixture of spinasterol and 22,23-dihydrospinasterol showed LC50 values of 32.36, 44.49 and 37.50 µg/mL by contact, residual and greenhouse assay at 72 h exposure, respectively. In contrast, fernenol recorded LC50 values as 47.99, 57.46 and 58.67 µg/mL, respectively. On the other hand, spinasterol, 22,23-dihydrospinasterol showed the highest mortality, i.e., 66.67%, 53.33% and 60% while, 30%, 23.33% and 25% mortality was recorded by fernenol after 72 h at 50 µg/mL by contact, residual and greenhouse assay, respectively. This study suggests that spinasterol, 22,23-dihydrospinasterol are more effective against B. brassicae which may be introduced as an effective and suitable substitute of synthetic chemical pesticides.

[Research progress on chemical constituents of Citrullus colocynthis and their pharmacological effects].

Citrullus colocynthis is widely distributed in the desert regions of the world. C. colocynthis has shown to improve constipation, liver diseases, jaundice, typhoid fever, diabetes and asthma in traditional use. As a kind of exterritorialy medicinal material, C. colocynthis has been used in China and introduced successfully. The main active ingredients of C. colocynthis are cucurbitacin, flavonoids, alkaloids and phenolic acids, which have been proven to have antioxidant, anti-diabetic, anti-pathogenic microorganisms and anti-cancer activities in modern pharmacological research. This paper reviews the traditional application, chemical composition and pharmacological effects of C. colocynthis, and provides reference for the in-depth study for the efficacy and mechanism of different components of C. colocynthis.

Effects of temperature on the cuticular transpiration barrier of two desert plants with water-spender and water-saver strategies.

The efficacy of the cuticular transpiration barrier and its resistance to elevated temperatures are significantly higher in a typical water-saver than in a water-spender plant growing in hot desert.

Application of NIR Spectroscopy Coupled with PLS Regression for Quantification of Total Polyphenol Contents from the Fruit and Aerial Parts of Citrullus colocynthis.

INTRODUCTION: Citrullus colocynthis (L.) Schrad is extensively used to treat diabetes, obesity, fever, cancer, amenorrhea, jaundice, leukemia, rheumatism, and respiratory diseases. Chemical studies have indicated the presence of several cucurbitacins, flavones, and other polyphenols in this plant. These phytochemical constituents are responsible for the interesting antioxidant and other biological activities of C. colocynthis. OBJECTIVE: In the present study, for the first time, near infrared (NIR) spectroscopy coupled with partial least square (PLS) regression analysis was used to quantify the polyphenolic phytochemicals of C. colocynthis. METHODOLOGY: The fruit and aerial parts of the C. colocynthis were extracted individually in methanol followed by fractionation in n-hexane, chloroform, ethyl acetate, n-butanol, and water. Near infrared (NIR) spectra were obtained in absorption mode in the wavelength range 700-2500 nm. The PLS regression model was then built from the obtained spectral data to quantify the total polyphenol contents in the selected plant samples. RESULTS: The PLS regression model obtained had a R2 value of 99% with a 0.98 correlationship value and a good prediction with a root mean square error of prediction (RMSEP) value of 1.89% and correlation of 0.98. These results were further confirmed through UV-vis spectroscopy and it is found that the ethyl acetate fraction has the maximum value for polyphenol contents (101.7 mg/100 g; NIR, 100.4 mg/100 g; UV-vis). CONCLUSIONS: The polyphenolic phytochemicals of the fruit and aerial parts of C. colocynthis have been quantified successfully by using multivariate analysis in a non-destructive, economical, precise, and highly sensitive method, which uses very simple sample preparation. Copyright © 2017 John Wiley & Sons, Ltd.

Topical Anti-Inflammatory and Analgesic Activities of Citrullus colocynthis Extract Cream in Rats.

Background and objectives:Citrullus colocynthis (CC), known as bitter apple, is used to treat diabetes in Iranian traditional medicine. The aim of this study is to evaluate the anti-inflammatory and analgesic activities of CC cream in rats. Materials and Methods: The carrageenan-induced edema in a rat hind paw was carried out to evaluate the topical anti-inflammatory effect of the CC fruit extract cream (2⁻8%) and the tissue levels of IL-6 and TNF-α were estimated by using a commercial ELISA kit. The topical antinociceptive activity of CC cream (2⁻8%) was evaluated in the rat formalin test. To determine the role of opioid receptors in the local antinociceptive effect of the CC cream, naloxone (20 µg/paw, i.pl.), a non-selective opioid antagonist, was used. Results: The results showed that the CC cream (2⁻8%) dose-dependently reduced the carrageenan-induced paw edema and reversed the changes in the level of TNF-α and IL-6 due to carrageenan-induced edema (p < 0.01). The anti-inflammatory effect of CC cream 8% was comparable to that of hydrocortisone ointment 1%. Furthermore, the application of CC cream (2⁻8%) dose-dependently inhibited both first and second phases of the formalin test (p < 0.05). The antinociceptive effect of the CC cream (8%) was comparable to that of methyl salicylate cream 30%. Moreover, the administration of naloxone significantly reversed the topical antinociceptive effect of the CC cream (p < 0.05). Conclusions: For the first time, this study indicated that the topical application of CC cream possesses significant anti-inflammatory and antinociceptive activities in animal models, which were probably mediated by opioid receptors and the suppression of pro-inflammatory cytokines (TNF-α and IL-6). Thus, the CC cream can be used to treat inflammatory pain and inflammatory diseases.

Green Microwave-Assisted Combustion Synthesis of Zinc Oxide Nanoparticles with Citrullus colocynthis (L.) Schrad: Characterization and Biomedical Applications.

In this paper, a green microwave-assisted combustion approach to synthesize ZnO-NPs using zinc nitrate and Citrullus colocynthis (L.) Schrad (fruit, seed and pulp) extracts as bio-fuels is reported. The structure, optical, and colloidal properties of the synthesized ZnO-NP samples were studied. Results illustrate that the morphology and particle size of the ZnO samples are different and depend on the bio-fuel. The XRD results revealed that hexagonal wurtzite ZnO-NPs with mean particle size of 27-85 nm were produced by different bio-fuels. The optical band gap was increased from 3.25 to 3.40 eV with the decreasing of particle size. FTIR results showed some differences in the surface structures of the as-synthesized ZnO-NP samples. This led to differences in the zeta potential, hydrodynamic size, and more significantly, antioxidant activity through scavenging of 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals. In in vitro cytotoxicity studies on 3T3 cells, a dose dependent toxicity with non-toxic effect of concentration below 0.26 mg/mL was shown for ZnO-NP samples. Furthermore, the as-synthesized ZnO-NPs inhibited the growth of medically significant pathogenic gram-positive (Bacillus subtilis and Methicillin-resistant Staphylococcus aurous) and gram-negative (Peseudomonas aeruginosa and Escherichia coli) bacteria. This study provides a simple, green and efficient approach to produce ZnO nanoparticles for various applications.

Effect of Citrullus colocynthis aqueous seed extract on beta cell regeneration and intra-islet vasculature in alloxan induced diabetic male albino rats.

OBJECTIVE: To observe the effect of Citrullus colocynthis on beta cell regeneration and intra-islet vasculature. METHODS: This experimental study was conducted at the University of Health Sciences, Lahore, Pakistan, from February 2013 to January 2014. It comprised male wistar rats weighing 100-150gand aged 6-8 weeks. The animals were divided into 6 groups. Group A1 served as control. Diabetes was induced in groups A2, B2 and C2 using single intravenous injection of 50mg/kg of alloxan. Animals having fasting blood glucose>250mg/dl were considered diabetic. Diabetic rats in groups B2 and C2 and their controls B1 and C1 were given 1ml/kg and 2ml/kg of Citrullus colocynthis aqueous seed extract orally per day for 14 days. Animals were sacrifised on day 15. RESULTS: Of the 48 rats, there were 8(16.7%) in each group. Citrullus colocynthis has stabilized the body weight of rats and difference was statistically significant on days 7(p<0.013) and 14(p<0.001). Citrullus colocynthis significantly reduced (p<0.001) the fasting blood sugar levels in a dose- and time-dependent manner. It increased the islet diameter (p<0.001) and beta cell count (p<0.001). The number of intra-islet capillaries was increased in group C2, but the difference was not statistically significant (p>0.05). CONCLUSIONS: Citrullus colocynthis aqueous seed extract stabilised animal body weight and ameliorated hyperglycaemia in a dose- and time-dependent manner which was attributable to regenerative effect on beta cells and intra-islet vasculature.

High efficient somatic embryogenesis development from leaf cultures of Citrullus colocynthis (L.) Schrad for generating true type clones.

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L(-1) BAP + 1.0 mg L(-1) 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L(-1) BAP + 0.5 mg L(-1) NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.

Two new cucurbitane-type triterpenoid saponins isolated from ethyl acetate extract of Citrullus colocynthis fruit.

Two new cucurbitacins I (1 and 2), together with eight known compounds (3-10), were isolated from the ethyl acetate extract of the fruit of Citrullus colocynthis. Compounds 3, 5-9 were isolated from C. colocynthis for the first time. The structures of new compounds were determined primarily from IR, HR-MS, 1D-, and 2D-NMR analysis.