RESUMO
The aim of this study was to investigate the potential of Ipomoea carnea flower methanolic extract (ICME) as a natural gastroprotective therapy against ethanol-induced gastric ulcers, particularly in individuals exposed to ionizing radiation (IR). The study focused on the Nrf2/HO-1 signaling pathway, which plays a crucial role in protecting the gastrointestinal mucosa from oxidative stress and inflammation. Male Wistar rats were divided into nine groups, the control group received distilled water orally for one week, while other groups were treated with ethanol to induce stomach ulcers, IR exposure, omeprazole, and different doses of ICME in combination with ethanol and/or IR. The study conducted comprehensive analyses, including LC-HRESI-MS/MS, to characterize the phenolic contents of ICME. Additionally, the Nrf2/HO-1 pathway, oxidative stress parameters, gastric pH, and histopathological changes were examined. The results showed that rats treated with IR and/or ethanol exhibited histopathological alterations, increased lipid peroxidation, decreased antioxidant enzyme activity, and reduced expression levels of Nrf2 and HO-1. However, pretreatment with ICME significantly improved these parameters. Phytochemical analysis identified 39 compounds in ICME, with flavonoids, hydroxybenzoic acids, and fatty acids as the predominant compounds. Virtual screening and molecular dynamics simulations suggested that ICME may protect against gastric ulceration by inhibiting oxidative stress and inflammatory mediators. In conclusion, this study demonstrates the potential of ICME as a natural gastroprotective therapy for preventing gastric ulcers. These findings contribute to the development of novel interventions for gastrointestinal disorders using natural plant extracts particularly in individuals with a history of radiation exposure.
Assuntos
Extratos Vegetais , Úlcera Gástrica , Animais , Ratos , Antioxidantes/farmacologia , Etanol/química , Mucosa Gástrica/metabolismo , Metanol/química , Fator 2 Relacionado a NF-E2/metabolismo , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Ratos Wistar , Úlcera Gástrica/tratamento farmacológico , Úlcera Gástrica/etiologia , Úlcera Gástrica/prevenção & controle , Espectrometria de Massas em Tandem , Úlcera/patologiaRESUMO
The growing spread of infectious diseases has become a potential global health threat to human beings. According to WHO reports, in this study, we investigated the impact of co-cultivating the isolated endophytic fungus Aspergillus sp. CO2 and Bacillus sp. COBZ21 as a method to stimulate the production of natural bioactive substances. (GC/MS)-based metabolomics profiling of two sponge-associated microbes, Aspergillus sp. CO2 and Bacillus sp. COBZ21, revealed that the co-culture of these two isolates induced the accumulation of metabolites that were not traced in their axenic cultures. By detection of different activities of extracts of Bacillus sp. COBZ21 and Aspergillus sp. CO2 and coculture between Bacillus sp. COBZ21 and Aspergillus sp. CO2. It was noted that the coculture strategy was the reason for a notable increase in some different activities, such as the antimicrobial activity, which showed potent activity against Escherichia coli ATCC 25,922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC 10,231. The antibiofilm activity showed significant biofilm inhibitory activity toward Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10,145, and Staph aureus NRRLB-767, with activity up to 53.66, 71.17, and 47.89%, while it showed low activity against E. coli ATCC 25,922, while the antioxidant activity based on the DPPH assay showed maximum activity (75.25%). GC-MS investigations revealed the presence of variable chemical constituents belonging to different chemical categories, which reflected their chemical diversity. The main components are (+-) cis-Deethylburnamine (2.66%), Bis(3,6,9,12-tetraoxapentaethylene) crowno-N,N,N',N'-tetra methylpphanediamine (2.48%), and 11-phenyl-2,4,6,8-tetra(2-thienyl)-11-aza-5,13-dithiaeteracyclo[7.3.0.1(2,8)0.0(3,7)] trideca-3,6-diene-10,12,13-trione (3.13%), respectively, for Bacillus sp. axenic culture, Aspergillus sp. CO2, Aspergillus sp. CO2, and Bacillus sp. COBZ21 coculture. By studying the ADME-related physicochemical properties of coculture extract, the compound showed log Po/w values above 5 (8.82). The solubility of the substance was moderate. In order to provide a comprehensive definition of medicinal chemistry and leadlikness, it is important to note that the latter did not meet the criteria outlined in the rule of three (RO3). The toxicity prediction of the coculture extract was performed using the ProTox II web server, which showed that the selected compound has no pronounced toxicity.
Assuntos
Anti-Infecciosos , Bacillus , Humanos , Bacillus/metabolismo , Antioxidantes/farmacologia , Dióxido de Carbono/metabolismo , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Anti-Infecciosos/química , Aspergillus/metabolismo , Staphylococcus aureus , Extratos Vegetais/farmacologia , Antibacterianos/farmacologiaRESUMO
BACKGROUND: Trichinellosis is a public health threat infected both animals and humans as a result of eating undercooked meat. It caused by Trichinella spiralis that has widespread drug resistance and even developed many sophisticated strategies for their survival, this increases the demand in searching for new anthelmintic drugs from natural source. METHODS: Our objectives were to test the in vitro and in vivo anthelmintic activity of Bassia indica BuOH frac., and to characterize its chemical composition using UPLC-ESI-MS/MS. Besides an in silico molecular docking study with the prediction of the PreADMET properties. RESULTS: In vitro investigation of B. indica BuOH frac., showed severe destruction of the adult worm and larvae, marked cuticle swelling, areas with vesicles, blebs and loss of annulations. This was assured via in vivo study, which revealed a significant reduction (P < 0.05) in the mean adult worm count with efficacy of 47.8% along with a significant decrease (P < 0.001) in the mean larval count per gram muscle with efficacy 80.7%. Histopathological examinations of the small intestine and muscular sections showed marked improvement. In addition, immunohistochemical findings demonstrated that B. indica BuOH frac. depressed the proinflammatory cytokines expressions of TNF-α, which was obviously upregulated by T. spiralis. Precise chemical investigation of the BuOH frac. using UPLC-ESI-MS/MS resulted in the identification of 13 oleanolic type triterpenoid saponins; oleanolic acid 3-O-6´-O-methyl-ß-D-glucurono-pyranoside (1), chikusetsusaponin-IVa (2) and its methyl ester (3), chikusetsusaponin IV (4) and its methyl ester (5), momordin-Ic (6) and its methyl ester (7), betavulgaroside-I (8), -II (9) -IV (10), -X (11), licorice-saponin-C2 (12) and -J2 (13). In addition, 6 more phenolics were identified as syringaresinol (14), 3,4-di-O-caffeoylquinic acid (15), 3-O-caffeoyl-4-O-dihydrocaffeoylquinic acid (16), 3,4-di-O-caffeoylquinic acid butyl ester (17), 3,5-di-O-galloyl-4-O-digalloylquinic acid (18) and quercetin 3-O-(6´´-feruloyl)-sophoroside (19). The auspicious anthelmintic activity was further ascertained using in silico molecular docking approach that targeted certain protein receptors (ß-tubulin monomer, tumor necrosis factor alpha (TNF-α), cysteine protease (Ts-CF1), calreticulin protein (Ts-CRT)), all the docked compounds (1-19) fit into the binding site of the active pocket with binding affinities noteworthy than albendazole. In addition, ADMET properties, drug score and drug likeness were predicted for all compounds.
Assuntos
Anti-Helmínticos , Trichinella spiralis , Humanos , Camundongos , Animais , Antiparasitários , Espectrometria de Massas em Tandem , Simulação de Acoplamento Molecular , Fator de Necrose Tumoral alfa , Anti-Helmínticos/farmacologia , Extratos Vegetais/farmacologia , Componentes Aéreos da PlantaRESUMO
Herniaria hemistemon J.Gay is widely used in folk medicine to treat hernia. The present study aimed to annotate the phytoconstituents of H. hemistemon aerial-part extract and investigate its in vivo anticryptosporidial activity. The chemical characterization was achieved via the LC-ESI-MS/MS technique resulting in the annotation of 37 phytocompounds comprising flavonoids and phenolic acids. Regarding the anticryptosporidial activity, fifty dexamethasone-immunosuppressed mice were separated into five groups: GI, un-infected (normal control); GII, infected but not treated (model); GIII, infected and received NTZ, the reference drug; GIV, infected and received H. hemistemon extract (100 mg/kg); and GV, infected and received H. hemistemon extract (200 mg/kg). When GIII, GIV, and GV were compared to GII, parasitological analyses displayed highly significant differences in the mean numbers of Cryptosporidium parvum oocysts in the stool between the different groups. GV demonstrated the highest efficacy of 79%. Histopathological analyses displayed improvement in the small intestine and liver pathology in the treated groups (GIII, IV, and V) related to the model (GII), with GV showing the highest efficacy. Moreover, the docking-based study tentatively highlighted the potential of benzoic acid derivatives as lactate dehydrogenase inhibitors. The docked compounds showed the same binding interactions as oxamic acid, where they established H-bond interactions with ARG-109, ASN-140, ASP-168, ARG-171, and HIS-195. To sum up, H. hemistemon is a promising natural therapeutic agent for cryptosporidiosis.
RESUMO
Hepatocellular carcinoma (HCC) is a poor-prognosis type of cancer with high resistance to chemotherapy, making the search for safe drugs a mandatory issue. Plant-derived products have potential to reduce negative side effects of cancer treatments. In this work, ability of a defatted methanolic extract of Alocasia gigantea leaves to fight HCC was evaluated in an animal model. Overall, treatment of HCC-induced mice with the methanolic extract at 150 mg/kg body weight for four consecutive weeks caused induction of autophagy through silencing of the relative expression of autophagy suppressor (mTOR) and inducement of autophagy markers (AMPK, Beclin-1, and LC-3). Moreover, it improved preservation of the hepatic histological architecture of the animals, with minor hepatocytic changes but scattered foci of hepatocytic apoptosis. Chemical profiling of the methanolic extract via ultra-high-performance liquid chromatography coupled to a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI-MS/MS) allowed identification of di-C-glycosyl flavones, mostly represented by 6-C-hexosyl-8-C-pentosyl apigenin isomers, which may possibly be associated with inducement of the autophagy pathway in HCC. Overall, these outcomes gave an initial visualization of the operative effect of some compounds in A. gigantea leaves that are potential treatment for HCC.
Assuntos
Alocasia , Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Animais , Espectrometria de Massas em Tandem , Carcinoma Hepatocelular/tratamento farmacológico , Espectrometria de Massas por Ionização por Electrospray/métodos , Neoplasias Hepáticas/tratamento farmacológico , Cromatografia Líquida de Alta Pressão/métodos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Metanol/química , AutofagiaRESUMO
CONTEXT: Thais savignyi Deshayes (Muricidae) is widely distributed in the Red Sea. Its abundance and the history of Muricidae in traditional medicine make it a tempting target for investigation. OBJECTIVE: To investigate the chemical profile and biological activities of T. savignyi tissue extracts. MATERIALS AND METHODS: Methanol, ethanol, acetone, and ethyl acetate extracts from T. savignyi tissue were compared in their antioxidant by total antioxidant capacity, DPPH free radical scavenging, and total phenolic content. In addition, the antimicrobial, and antibiofilm properties (at 250 µg/mL) of the extracts were tested against Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans. The antioxidant extract with greatest activity was assessed for cytotoxicity (range 0.4-100 µg/mL) against 3 human cancer cell lines (UO-31, A549 and A431), and its chemical composition was investigated using GC-MS. Moreover, docking simulation was performed to predict its constituents' binding modes/scores to the active sites of thymidylate kinase. RESULTS: The ethyl acetate extract (Ts-EtOAc) showed the highest total antioxidant capacity (551.33 mg AAE/g dry weight), total phenolics (254.46 mg GAE/g dry weight), and DPPH scavenging (IC50= 24.0 µg/mL). Ts-EtOAc exhibited strong antibacterial (MIC: 3.9 µg/mL against K. pneumoniae), antibiofilm (MIC: 7.81 µg/mL against S. aureus), and antifungal (MIC: 3.9 µg/mL against C. albicans) activities and considerable cytotoxicity against cancer cells (UO-31: IC50= 19.96 ± 0.93, A549: IC50= 25.04 ± 1.15 µg/mL). GC-MS identified multiple bioactive metabolites in Ts-EtOAc extract belonging to miscellaneous chemical classes. Molecular docking studies revealed that the constituents of Ts-EtOAc have antibacterial potential. DISCUSSION AND CONCLUSIONS: T. savignyi extract has considerable antimicrobial and cytotoxic activities. Further studies are needed to isolate the active constituents of this snail for comprehensive drug discovery tests.
Assuntos
Anti-Infecciosos , Antioxidantes , Acetatos , Acetona , Antibacterianos , Anti-Infecciosos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Etanol , Radicais Livres , Humanos , Metanol , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Fenóis/farmacologia , Extratos Vegetais , Staphylococcus aureus , Tailândia , Extratos de TecidosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The genus Warburgia (family Canellaceae) is widely distributed over Afrotropical and Neotropical realms. Traditionally, W. salutaris (G. Bertol.) Chiov., and other Warburgia species are used as anti-inflammatory, antimalarial, antimicrobial, and for wound healing, and treating several skin complaints as well. Specifically, different extracts from W. salutaris were reported to possess diuretic, anti-inflammatory, and cytotoxic effects. AIM OF THE STUDY: This work aimed to investigate the phytochemical composition of an aqueous extract from W. salutaris bark, and evaluate its antioxidant and anti-aging activities in silico, in vitro, and in vivo. MATERIALS AND METHODS: HPLC-PDA-MS/MS was used to investigate the phytochemical components of the extract. The antioxidant potential of the extract was evaluated in vitro using DPPH and FRAP assays. The Caenorhabditis elegans nematodes model was adopted to investigate the antioxidant and the anti-aging effects in vivo by determining the worms' survival rate, level of ROS, HSP16 expression, and nuclear translocation of the transcription factor DAF16. Molecular operating environment (MOE) software was utilized for in silico molecular docking of the extract's components into different enzymes involved in the aging process. Anti-collagenase, anti-elastase, anti-tyrosinase, and anti-hyaluronidase assays were used to evaluate the anti-aging effects in vitro. RESULTS: HPLC-MS analysis furnished 30 compounds, among them catechin, 11α-hydroxy muzigadiolide, mukaadial, pereniporin B, and 11α-hydroxycinnamosmolide. The major components of the extract showed appropriate fitting in the binding site of the target enzymes adopted in the study with considerable minimum free binding energy relative to the standard inhibitors. The extract showed substantial in vitro antioxidant activity in DPPH and FRAP assays and in vitro anti-aging assays against collagenase, elastase, tyrosinase, and hyaluronidase with comparable IC50 values to the reference standards. Moreover, it attenuated oxidative stress in vivo as it significantly increased the survival rate of ROS stressed C. elegans worms, decreased intracellular ROS, decreased the juglone-induced HSP16 expression and enhanced the nuclear localization of DAF16 in a dose-dependent manner. CONCLUSION: Our results support the traditional use of W. salutaris to counteract inflammation and oxidative stress associated with several pathological conditions. In addition, W. salutaris bark extract can be considered as a substantial source for bioactive metabolites with strong potential as anti-aging and antioxidant agents.
Assuntos
Antioxidantes , Magnoliopsida , Envelhecimento , Animais , Antioxidantes/uso terapêutico , Caenorhabditis elegans , Magnoliopsida/química , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/farmacologia , Casca de Planta/química , Extratos Vegetais/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Diabetes mellitus is the most common disease in Egypt. In this context, Beta vulgaris subspecies cicla L. var. flavescens is an edible plant that has been used in traditional medicine as a therapy for treating some diseases. OBJECTIVES: The current study was performed to evaluate the antibacterial and potential anti-diabetic activities of different extracts and isolated flavone C-glycoside compounds isolated from Beta vulgaris subspecies cicla L. var. flavescens leaves. METHODS: Phytochemical investigation of n-butanol extract led to the isolation of five phytoconstituents. Their structures were determined by spectroscopic tools, including 1D-NMR (1H- & 13C-NMR) and 2D-NMR (HMQC & HMBC) besides the comparison of the data with the literature. The extracts and phytoconstituents were evaluated in vitro for their activity against some bacterial pathogens, which represent prominent human pathogens, particularly in hospital settings. The antibacterial activity was examined against three Gram-positive bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis & Enterococcus faecalis) and five Gram-negative ones (Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Proteus mirabilis & Salmonella typhimurium) relative to Ciprofloxacin as a reference drug. Furthermore, the in vitro antidiabetic activity (Type II) was evaluated using the alpha-glucosidase inhibitory assay. RESULTS: Five flavone C-glycosides namely; Apigenin 8-C-ß-D-glucopyranoside (vitexin) (1), 2''-Oxylopyranosylvitexin (2), acacetin 8-C-ß-D-glucopyranoside (3), acacetin 8-C-α-L-rhamnoside (4), and 6,8-di-C-ß-D-glucopyranosylapigenin (vecinin-II) (5) were isolated from n-butanol extract of B. vulgaris subspecies cicla L. var. flavescens. Compound 1 showed a promising antibacterial activity against most of the test bacterial strains with respect to the minimum inhibitory concentration values (MIC) ranged from 1.95 to 15.63 µg ml-1. On the other hand, compounds 1 and 3 demonstrated superior antidiabetic activities with IC50 values of 35.7 and 42.64 µg ml-1, respectively, while an inferior potential antidiabetic activity was recorded for compound 4 (IC50 = 145.5 µg ml-1) in comparison with Acarbose as a reference drug. CONCLUSION: B. vulgaris L. is an edible plant, which could be used as a natural source of antibiotic and hypoglycemic drugs.
Assuntos
Amaranthaceae/química , Antibacterianos/farmacologia , Beta vulgaris/química , Flavonas/farmacologia , Glicosídeos/farmacologia , Hipoglicemiantes/farmacologia , Amaranthaceae/crescimento & desenvolvimento , Antibacterianos/isolamento & purificação , Beta vulgaris/crescimento & desenvolvimento , Egito , Flavonas/isolamento & purificação , Glicosídeos/isolamento & purificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Hipoglicemiantes/isolamento & purificação , Testes de Sensibilidade Microbiana , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Folhas de Planta/químicaRESUMO
Reactive oxygen species (ROS) have been linked to several health conditions, among them inflammation. Natural antioxidants may attenuate this damage. Our study aimed to investigate the chemical composition of a methanol leaf extract from Alpinia zerumbet and its possible antioxidant, anti-inflammatory, anti-nociceptive, and antipyretic effects. Altogether, 37 compounds, representing benzoic and cinnamic acid derivatives and flavonoids (aglycones and glycosides), were characterized. The extract showed substantial in vitro antioxidant effects, and inhibited both cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) in vitro, with a higher selectivity towards COX-2. It also inhibited 5-lipoxygenase (LOX) activity in vitro with nearly double the potency of zileuton, a reference 5-lipoxygenase (LOX) inhibitor. The extract exhibited anti-inflammatory effects against carrageenan-induced rat hind paw edema, and suppressed leukocyte infiltration into the peritoneal cavity in carrageenan-treated mice. Furthermore, it possessed antipyretic effects against fever induced by subcutaneous injection of Brewer's yeast in mice. Additionally, the extract demonstrated both central and peripheral anti-nociceptive effects in mice, as manifested by a decrease in the count of writhing, induced with acetic acid and an increase in the latency time in the hot plate test. These findings suggest that the leaf extract from Alpinia zerumbet could be a candidate for the development of a drug to treat inflammation and ROS related disorders.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Antipiréticos/farmacologia , Extratos Vegetais/química , Polifenóis/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Zingiberaceae/química , Analgésicos/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Antioxidantes/farmacologia , Antipiréticos/química , Permeabilidade Capilar/efeitos dos fármacos , Carragenina , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Inibidores de Ciclo-Oxigenase/farmacologia , Edema/tratamento farmacológico , Edema/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Inibidores de Lipoxigenase/farmacologia , Metanol , Camundongos , Cavidade Peritoneal/patologia , Polifenóis/químicaRESUMO
The potential hepatoprotective activities of two Lannea species were explored in vivo. Furthermore, the binding activities of their main polyphenols to the antiapoptotic protein Bcl-2 were investigated. Based on HPLC-MS/MS results, 22 secondary metabolites were characterized in L. stuhlmannii (mainly tannins), while 20 secondary metabolites (mainly sulphated tannins) were identified in L. humilis. Both extracts exhibited substantial antioxidant activities in vitro and counteracted D-galactosamine induced intoxication in rats in vivo and increased the total antioxidant capacity (TAC) of liver tissues. In addition to reducing the elevated levels of AST and total bilirubin, both extracts significantly attenuated the deleterious histopathologic changes in liver after D-galactosamine-intoxication. Also, both extracts protected hepatocytes from apoptotic cell death and increased the expression of the anti-apoptotic protein Bcl-2. The identified compounds from both extracts can bind to the Bcl-2: Bim (BH3) interface with an appreciable binding free energy. Hydrogen and ionic bonds and hydrophobic interactions with amino acid residues in the hydrophobic face of Bim (BH3) domain were discovered. To sum up, L. humilis and L. stuhlmanni exhibited promising hepatoprotective activities in vivo against D-GalN-induced liver injury and their hepatoprotection is due to the antioxidant and anti-apoptotic effects of tannins and proanthocyanidins.
Assuntos
Anacardiaceae/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Taninos/química , Animais , Antioxidantes/metabolismo , Cromatografia Líquida de Alta Pressão , Galactosamina/farmacologia , Ligação de Hidrogênio , Imuno-Histoquímica , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espectrometria de Massas em TandemRESUMO
The potential role of hepatoprotective and antipathological effect of Ficus sycomorus and Azadirachta indica extracts was evaluated for scavenging the reactive oxygen species (ROS) and reduced the oxidative damage and pathological changes in the liver of S. mansoni infected mice. The levels of alanine aminotransferase (ALT), asparate aminotransferase (AST) and gamma glutamyl transferase (GGT) were evaluated in the infected mice and treated orally with each plant extract 12 weeks post infection (P.I.) in a dose of 500 mg/kg of each plant extract for five consecutive days and sacrificed two weeks P.I. The infection of mice showed an elevation of ALT, AST & GGT. Treatment of mice with 70% methanol extract of each plant extract reduced significantly ALT, AST & GGT elevation. The highest reduction was with the methanolic extract of F. sycomorus (42%, 35% &44% for ALT, AST & GGT respectively). Fractionation of the methanolic extract of each plant was carried out. The effect of ethyl acetate and butanolic fractions of each plant was also evaluated. The result showed that the two fractions lowered the levels of the tested enzymes and decreased the number and size of granuloma diameters with an increased in the percentage of degenerated ova.