Effects of Viola odorata syrup on sleep quality in menopausal women: a randomized, triple-blind, controlled trial.

PURPOSE: The present study aimed to assess the effects of Viola odorata syrup on the sleep quality of postmenopausal women. METHODS: This triple-blinded randomized clinical trial was conducted on menopausal women presenting to the healthcare centers in Mashhad, Iran, in 2019. The participants were selected using simple random sampling. Participants received 5 ml syrup V. odorata or placebo twice a day for 1 month. Data were collected using the Pittsburgh Sleep Quality Index. The data were analyzed using SPSS version 25. RESULTS: The 118 eligible women enrolled in the study were divided into two groups of V. odorata syrup and placebo (n = 59 each). The analysis was conducted on only 84 menopausal women (42 in each group) due to exclusions. Exclusions consisted of 12 participants who withdrew from the study due to unwillingness to cooperate, 8 who had irregular consumption of the therapeutic syrup, 6 with inaccurate completion of the questionnaire, and 8 due to lack of accessibility. The two study groups were homogenous in terms of demographic characteristics. Before the intervention, no significant difference was observed in the mean PSQI score between the two groups (9.2 ± 2.9 vs. 8.4 ± 2.5) (P = 0.18). However, a significant difference was seen in the mean PSQI score between the two groups (4.9 ± 1.9 vs. 8.1 ± 2.1, P < 0.001) after the intervention. CONCLUSIONS: The findings of this study suggest that V. odorata syrup may be a useful therapeutic agent to improve the sleep quality of menopausal women. REGISTRATION CODE: IRCT20180514039660N1.

Development and Characterization of Antioxidant and Antibacterial Films Based on Potato Starch Incorporating Viola odorata Extract to Improve the Oxidative and Microbiological Quality of Chicken Fillets during Refrigerated Storage.

In this research, the antioxidant and antibacterial activities of active films based on potato starch containing Viola odorata extract (VOE) were investigated both in vitro and in chicken fillets. The VOE was added to the starch film formulation at 0, 1, 2, and 3% (w/v). The results showed that by increasing the extract level, the total phenol content and antioxidant and antibacterial activity of the films against Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium improved remarkably. The results of the meat tests indicated the significant antioxidant and antimicrobial activity of active films containing different levels of VOE in chicken fillets, and a direct relationship was observed between the concentration of the extract and the functional activity of the films, so with the increase in the concentration of the extract in the films, the rate of lipid oxidation and growth of microorganisms in the chicken fillets decreased significantly during the storage period, and less volatile nitrogen bases, metmyoglobin, and oxidation products were produced in the fillets. In general, the results of this research demonstrated that an active film based on potato starch containing VOE (especially 2 and 3% levels) has the ability to extend the oxidative and microbiological shelf life of chicken fillets during cold storage for at least eight days.

Antibacterial Activity of Economically Important Medicinal Plants in Pakistan Against Different Bacterial Strains.

The emergence of medication resistance and unfavorable side effects from existing antibiotics has prompted the quest for novel antimicrobial agents over the last 2 decades. Plant extracts have been shown to have antibacterial effects in numerous studies. The objective of this study was the evaluation of the antibacterial effect of economically important medicinal plants found in Pakistan. Onosma bracteatum (flowers and leaves), Viola odorata (flowers and leaves), Cuscuta reflexa (whole plant), Swertia chirata (whole plant), and Fagonia arabica (whole plant) were used against Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. Water and ethanol extracts were obtained from different parts of the plants. To evaluate the antibacterial effect of these plants, qualitative assay agar well diffusion method was performed. The minimum inhibitory concentration (MIC) was determined by the broth micro dilution method. Results revealed that the highest inhibition zone (18 mm) was shown by ethanol extract of V odorata flower against P aeruginosa. Ethanol extract of C reflexa plants is best for all 3 tested microbes (P aeruginosa, B subtilis, and E coli). The results concluded that all these plants have abilities to fight against these tested bacteria. Ethanol extract of V odorata flower has the highest activity against P aeruginosa.

Commercially Available Viola odorata Oil, Chemical Variability and Antimicrobial Activity.

Viola odorata L. oil is frequently recommended in the aromatherapeutic literature for treating respiratory, urinary, and skin infections; however, antimicrobial evidence is lacking. In addition, in aromatherapy, combinations of essential oils are predominantly utilized with the goal of achieving therapeutic synergy, yet no studies investigating the interaction of essential oil combinations with V. odorata oil exists. This study thus aimed to address these gaps by investigating the antimicrobial activity of three Viola odorata oil samples, sourced from different suppliers, independently and in combination with 20 different commercial essential oils, against micro-organisms involved in respiratory, skin, and urinary tract infections associated with global resistance trends. These pathogens include several of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) The chemical profile of the oils was determined using gas chromatography coupled with mass spectrometry. The minimum inhibitory concentrations (MIC) were determined using the broth micro-dilution method. The interactive profiles for the combinations were assessed by calculating the fractional inhibitory concentration index (ΣFIC). The main compounds varied across the three samples, and included phenethyl alcohol, isopropyl myristate, 2-nonynoic acid, methyl ester, α-terpineol, α-cetone, and benzyl acetate. The V. odorata oil samples displayed overall poor antimicrobial activity when tested alone; however, the antimicrobial activity of the combinations resulted in 55 synergistic interactions where the combination with Santalum austrocaledonicum resulted in the lowest MIC values as low as 0.13 mg/mL. The frequency of the synergistic interactions predominantly occurred against Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterococcus faecium with noteworthy MIC values ranging from 0.25-1.00 mg/mL. This study also reports on the variability of V. odorata oils sold commercially. While this warrants caution, the antimicrobial benefit in combination provides an impetus for further studies to investigate the therapeutic potential.

Efficacy and safety of add-on Viola odorata L. in the treatment of COVID-19: A randomized double-blind controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Severe Acute Respiratory Syndrome (SARS) due to the novel coronavirus has become the highest priority that threatens human health. This situation demands widespread vaccination and the innovation of new therapeutic methods. Despite drug discoveries, the need for approving new medicaments is felt because of adverse effects and lack of efficacy. Several medicinal plants including Viola odorata L. are recommended in traditional Persian medicine for alleviating respiratory infection symptoms. Recent studies showed anti-inflammatory, antioxidant, anti-asthmatic, antitussive, analgesic, and antibacterial activities of sweet violet. These enhance respiratory functions, reduce pulmonary inflammation, and decline mucous membrane edema. This study aimed to evaluate the efficacy of sweet violet syrup in alleviating the manifestations of COVID-19 infection. MATERIAL AND METHODS: A randomized parallel-group double-blind controlled trial was conducted at Al-Zahra general hospital, Isfahan, Iran. A total of 108 outpatients were enrolled in the study. The patients were randomly allocated to intervention and placebo groups, with 54 patients in each group. The allocation was concealed using sealed opaque envelopes. The intervention group received violet syrup and the control group received placebo syrup, an add-on to the conventional treatment. The outcomes were COVID-19 manifestations, such as dyspnea, cough, myalgia, headache, and diarrhea, considered as outcomes of the study and were evaluated twice using a visual analog scale before the intervention and after 7 days, at the end of the study. Patients were followed daily by phone calls to monitor proper drug consumption and possible side effects. RESULTS: No significant difference was between groups regarding demographic characteristics and vital signs before and after the treatment. Although all symptoms have improved significantly in both groups, patients who received violet syrup recovered faster and the mean severity scores of cough (P = 0.025), myalgia (P = 0.036), headache (P = 0.037), and diarrhea (P = 0.044) decreased greater in comparison to control group. CONCLUSION: This study, the first clinical trial on the effectiveness of Viola odorata on SARS-CoV-2 patients, showed that Viola odorata L. effectively controls prevalent manifestations of COVID-19 including cough, myalgia, headache, and diarrhea. Regarding this survey, the violet syrup can be mentioned as a complementary treatment for viral influenza-like infections in which cough, myalgia, headache, and diarrhea are prominent.

Attenuating NF-κB/VCAM-1 Expression in the Middle Cerebral Artery Occlusion Model by Viola Odorata: Protection Against Ischemia-Reperfusion Injury in Rats.

Introduction: The death of neurons and cerebral edema are the main consequences of stroke. However, inflammatory processes play key roles in aggravating cerebral damage following a stroke. This study aimed to investigate the effects of Viola odorata extract (VOE) on the infarct volume (IV), neurologic deficits (ND), and the expression of NF-κB and VCAM-1 in the Middle Cerebral Artery Occlusion (MCAO) model. Methods: The animals were randomly separated into 6 groups: (1) control group, (2) vehicle-treated group, (3) MCAO group, (4) VOE25 group, (5) VOE50 group, and (6) VOE75 group (n= 12). VOE (25, 50, and 75 mg/kg) and distilled water were administered daily for 30 days. Two hours after the last gavage, the rats were exposed to MCAO for 60 minutes. Twenty-four hours later, IV, ND, and NF-κB/VCAM-1 expressions were evaluated. Results: Viola odorata extract exhibited excellent neuroprotective effects by reducing IV (mainly in the core and subcortex areas), and induced downregulation of NF-κB and VCAM-1 expression. Conclusion: Viola odorata could also activate intracellular pathways, reducing the expression of NF-κB and VCAM-1. It is useful for developing a novel medical herb for treating cerebral ischemia. Highlights: A stroke occurs when the blood supply to a part of the brain is interrupted or reduced.Viola odorata extract (VOE) reduced the infarct volumes (IV) in rats' brains 24 h after middle cerebral artery occlusion (MCAO).VOE may decrease IV in the MCAO model by downregulating the NF-κB/VCAM-1 expression. Plain Language Summary: A stroke is a medical condition in which poor blood flow to the brain results in cell death. After an interruption of blood flow, energy stores are rapidly depleted, and complex cellular cascades induce excitotoxic cell death. So far, no effective and specific treatment has been suggested for the stroke. In this study, the neuroprotective effects of the viola odorata extract (VOE) were screened. The V. odorata extract exhibited excellent neuroprotective effects by reducing infarct volume and inducing downregulation of NF-κB/VCAM-1 expression. This finding suggests that V. odorata may be useful for developing a novel medical herb for treating cerebral ischemia.

Immunomodulatory effects of cyclotides isolated from Viola odorata in an experimental autoimmune encephalomyelitis animal model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that causes chronic inflammation. Cyclotides are small plant proteins with a wide range of biological activity, making them a target for researchers to investigate. This study was conducted to investigate the possible effects of cyclotide-rich fractions from Viola odorata as an immunomodulatory agent in an experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: At room temperature, the plant materials were subjected to maceration in methanol: dichloromethane (1:1; v/v) for 3 days. The extraction was repeated 3 times, and the final concentrated extract was partitioned 3 times by 1/2 volume of double-distilled water. The aqueous phases were separated and freeze-dried. Finally, the crude extract was fractionated by C18 silicagel using vacuum liquid chromatography, with mobile phases of 30%, 50% and 80% of ethanol: water, respectively. The 50%, and 80% fractions were analyzed by HPLC and MALDI-TOF analysis and administrated intraperitoneally to forty-five female C57BL/6 EAE-induced mice, at 5, 25, and 50 mg/kg doses. After 28 days, the animals were evaluated using EAE clinical scoring which was done every 3 days, cytokine levels, and myelination level. RESULTS: The results confirmed the presence of cyclotides in V. odorata based on their retention time and the composition of mobile phase in HPLC and the molecular weight of the peaks in MALDI-TOF analysis. It was observed that cyclotides, especially in the 80% fraction group at the dose of 50 mg/kg significantly reduced the clinical scores, inflammation, and demyelination in EAE mice compared with the normal saline group (P<0.05), and the results of this group were comparable with fingolimod (P>0.05). CONCLUSION: It could be concluded that V. odorata is a rich source of cyclotides which they could be extracted by an easily available process and also, they could be used as immunomodulatory agents in MS, with similar effects to fingolimod.

The Efficacy of an Oral Formulation of Glycyrrhiza glabra, Viola odorata, and Operculina turpethum as an Add-on Therapy for Mild-to-moderate Childhood Asthma: A Randomized Placebo-Controlled Clinical Trial.

Objective: We aimed to evaluate the efficacy of an oral combined tablet of Glycyrrhiza glabra, Viola odorata, and Operculina turpethum (Anti-Asthma®) as an add-on therapy for the relief of the severity of symptoms in mild-to-moderate childhood asthma. Methods: This randomized placebo-controlled clinical trial was performed on 60 children and adolescents with chronic mild-to-moderate childhood asthma. Patients were randomly divided into cases who received Anti-Asthma® oral combined tablets 2 tablets twice dailt for 1 month and controls, received placebo tablets identically the same to Anti-Asthma® (2 tablets, twice daily, for 1 month) as add-ons to their standard therapy according to the guideline. The severity and frequency of cough attacks and shortness of breath, respiratory test indices (based on spirometry), and the extent of disease control and treatment adherence were measured clinically by validated questionnaires at the beginning and after the study. Findings: Respiratory test indices improved and the severity of activity restriction decreased significantly in the cases compared to the controls However, the mean difference before and after the study was significantly different between the cases and controls only for the number and severity of coughs and the severity of activity restriction. In the scores of the Asthma Control Questionnaire, the cases group had a significant improvement compared to the controls. Conclusion: Anti-Asthma® oral formulation may be effective as an adjunct add-on treatment in the maintenance therapy of mild-to-moderate childhood asthma.

Pharmacological Screening of Viola odorata L. for Memory-Enhancing Effect via Modulation of Oxidative Stress and Inflammatory Biomarkers.

Purpose: Alzheimer disease (AD) is a progressive neurodegenerative disorder that is caused by neuroinflammation and oxidative stress. The present study aimed to characterize and then investigate the memory-enhancing potential of Viola odorata methanolic extract in lipopolysaccharide (LPS)-treated mice. Methods: V. odorata characterization was done by using the GCMS technique. Neuroinflammation was induced by the intracerebroventricular administration of LPS at a dose of 12 µg. Animals were divided randomly into six groups (n = 10). Group I was normal control, which was given vehicle. Group II was disease control, which received LPS (12 µg) via the intracerebroventricular route. Group III was standard, which was administered with donepezil (3 µg) orally for 21 days. Groups IV-VI were the treatment groups, which were administered with the extract at 100, 200, and 400 mg/kg dose levels orally respectively for 21 days. Groups III-VI received LPS (12 µg) on the first day along with their treatments. During the treatment, the animals were assessed for memory retention by employing different behavioral paradigms namely elevated plus maze, passive avoidance, foot shock and open field. Various mediators [endogenous antioxidants, neurotransmitters, and acetylcholinesterase (AChE)] involved in the pathogenesis of AD were quantified by using the UV spectrophotometric method. Results: Extract-treated groups showed a remarkable improvement in cognitive impairment in all behavioral paradigms. Oxidative stress biomarkers, that is, superoxide dismutase, catalase, and glutathione were raised dose-dependently in the treatment groups with a dose-dependent decrease in the malonaldehyde and AChE levels in the brains of the treated animals. The treatment groups showed decreased levels of inflammatory biomarkers, that is, tumor necrosis factor-alpha, nuclear factor kappa light-chain enhancer of activated ß-cells, and cyclo-oxygenase, which supports the therapeutic effectiveness of the treatment. Conclusion: Based on behavioral, oxidative stress biomarker, and neuroinflammatory data, it is concluded that V. odorata possesses memory-enhancing activity and may prove a beneficial role in the management of AD.

Isolation and characterization of cyclotides from the leaves of Viola odorata L. using peptidomic and bioinformatic approach.

Cyclotides are true gene products characterized by the presence of six conserved cysteine residues and knotted arrangement of three disulfide bonds. These macrocyclic peptides show exceptional resistance to thermal, chemical and enzymatic degradation which is defined due to their three-dimensional folding. The current study describes an efficient strategy involving reduction, enzymatic digestion and mass spectroscopy sequencing for the identification of the precursor sequences and the cyclotide domains present in the leaf tissue of Viola odorata. We observed 122 partial peptide sequences containing 31 cyclotide domains along with 19 unique sequences consisting of putative novel cyclotides and acyclotides. Four precursor sequences consisting of putative new and already reported domains were further characterized for cyclotide domains, their structure and subfamilies. The sequences revealed the presence of classic knotted cyclotide folds with similar six characteristic loops but different amino acid residues. Molecular modeling indicated that the secondary structures present in the cyclotides are mainly α-helix and random coils. Variation in the sequences and conservation in cysteine residues in the cyclotides was revealed by protein diversity wheel. The significant information observed in the current study expands our knowledge about the structure and type of cyclic peptides in V. odorata leaves. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02763-2.

Development, characterization and in vitro antioxidant activity of chitosan-coated alginate microcapsules entrapping Viola odorata Linn. extract.

Viola odorata Linn or sweet violet has several biological activities due to the presence of flavonoids, tannins, alaloid, glycoside, and saponins. However, susceptibility of these compounds to harsh conditions and low solubility is a great challenge for their incorporation into food products. Therefore, encapsulation can be an effective approach in this respect. In the present study, chitosan-coated microcapsules loaded with Viola extract were prepared for the first time and the effects of independent variables (sodium alginate: 1-1.5%, calcium chloride: 0.6-1.5% and extract concentrations: 5-10%) on encapsulation efficiency (EE%) were investigated. After evaluation of the model, the optimum condition for preparation of microcapsules was selected as 1.47% sodium alginate, 5.02% extract and 1.42% CaCl2 with EE% of 83.21%. The microcapsules developed at this condition had an acceptable spherical shape and the results obtained in Fourier transform-infrared spectroscopy (FTIR) indicated the presence of the extract within the microcapsules. The mean diameters of the uncoated and chitosan-coated microcapsules were 73 and 141 µm, respectively. The in vitro release in acidic medium (pH 1.5) and phosphate buffer saline (pH 7) were 43.21% and 95.39%, respectively. The prepared extract-loaded microcapsules have potential to be used in food products providing acceptable antioxidant activity.

Effects of Viola odorata as an Add-On Therapy on Insomnia in Patients with Obsession or Depression: A Pilot Randomized Double-Blind Placebo-Controlled Trial.

Objective: The present study was conducted to investigate the effect of Viola odorata extracted syrup on the quality and patterns of sleep in patients with depression or obsessive-compulsive disorder (OCD) as add-on therapy. Design: A pilot double-blind randomized placebo-controlled trial. Settings/Location: Psychiatric Clinic of Imam Hossein Hospital, Tehran, Iran. Subjects: Participants were 16-15 years of age with mild and moderate depression or OCD having insomnia. Interventions: This pilot study was conducted on patients with insomnia divided into two groups with depression (40 patients) or OCD (43 patients). Each group randomly assigned into two arms with the same conditions at baseline. The intervention arm daily received 5 mL V. odorata syrup every 12 h for 4 weeks, and the control arm received 5 mL placebo syrup every 12 h for 4 weeks. None of the participants was deprived of their routine treatment for depression or OCD. Outcome measures: The scores of insomnia symptoms were evaluated using total score of the Pittsburgh Sleep Quality Index (PSQI) and the scores of its components, the depression score using the final Beck depression inventory-II (BDI-II) score, and OCD score using the Yale-Brown Obsessive-Compulsive Scale (YBOCS). Results: The total PSQI score was found to be improved significantly in the intervention arms with depression or OCD (p < 0.001) compared with the corresponding control arms. Significant improvements were also observed in the final mean difference of BDI-II (p = 0.009) and YBOCS (p = 0.001) scores in the intervention arms. Conclusions: V. odorata syrup significantly improved insomnia symptoms and the scores of depression and OCD.

Suppressive role of Viola odorata extract on malignant characters of mammosphere-derived breast cancer stem cells.

BACKGROUND: Mammospheres are breast cancer stem cells (BCSCs) that could be yielded through culturing cells in non-adherent and non-differentiating condition. With regard to therapy resistance of cancer stem cells (CSCs), it is essential to discover efficient approaches targeting CSCs. Viola odorata extract has been considered as a traditional herbal anti-metastatic drug in several cancer cells. Effect of this drug on BCSCs has not been clearly identified. Current study tries to detect and to compare effect of Viola odorata extract on malignant characterization of breast cancer cell lines and BCSCs. MATERIALS AND METHODS: MCF7 and SKBR3 and their derived mammospheres as BCSCs were used and the effect of alcoholic extraction of Viola odorata on apoptosis and malignant characters of MCF7, SKBR3 and their derived BCSCs were analyzed and compared. RESULTS: Viola odorata extract induced cell death in MCF7, SKBR3 and their derived mammospheres through apoptosis without any effects on MCF10A. Also, this extract showed anti-migratory, anti-invasion and anti-colony formation activity in MCF7, SKBR3 and their derived mammospheres which was significantly more in MCF7- and SKBR3-derived mammospheres. Also, this extract decreased size and volume of tumors generated by MCF7, SKBR3 and their derived mammospheres in chicken embryo model. CONCLUSION: Viola odorata extract exerted anti-cancerous activity on both breast cancer cell lines and their derived BCSCs. Anti-cancerous activity of this extract was significantly more in MCF7-, SKBR3-derived mammospheres in comparison with dedicated cell lines. Data suggest that Viola odorata extract mostly targets cancerous cells, not normal cells with exception in high concentration. It acts in a cell-dependent manner.

The Effect of Viola Odorata L. Oil for Fever in Children: A Randomized Triple-blinded Placebo-controlled Clinical Trial.

INTRODUCTION: Finding non-systemic antipyretic option in cancer patients who simultaneously receive several other drugs seems be logical. This study was designed to evaluate complementary therapy with Viola odorata L. oil for fever control in febrile neutropenic children. METHODS AND MATERIALS: In a randomized placebo controlled clinical trial, 41 febrile children were divided into two groups. Children in the active drug group received viola oil (20 drops) to be rubbed on the peripheral margin of the patient umbilicus. Primary outcome measure of the study was the mean axillary temperature in the 30, 60, and 240 minutes after the intervention. RESULTS: The mean temperature reduced significantly in the viola group after 30 minutes of administration (p =0.005), while there was no significant change in the placebo group (p =1.00). The number of patients who received paracetamol as the rescue treatment was significantly lower in the viola group than that in the placebo group (5 vs. 17, p =0.001). CONCLUSION: The results of our study showed the safety and efficacy of complementary therapy with Viola odorata L. oil for fever control in febrile neutropenic children during hospital course.

The mechanism of neuroprotective effect of Viola odorata against serum/glucose deprivation-induced PC12 cell death.

OBJECTIVE: Oxidative stress is associated with the pathogenesis of brain ischemia and other neurodegenerative disorders. Previous researches have shown the antioxidant activity of Viola odorata L. In this project, we studied neuro-protective and reactive oxygen species (ROS) scavenging activities of methanol (MeOH) extract and other fractions isolated from V. odorata in PC12 cell line in serum/glucose deprivation (SGD) condition. MATERIALS AND METHODS: The PC12 neuronal cells were pretreated for 6 hr with MeOH extract and fractions of V. odorata (1 to 25 µg/ml) followed by 24 hr incubation under SGD condition. Cell viability was measured by Alamar Blue® assay. The level of ROS was calculated using DCFH-DA. Also, Bax/Bcl-2 protein ratio was analyzed by western blot assay. RESULTS: SGD condition significantly decreased cells viability (p<0.001). Pretreatment with EtOAc (12.5 and 25 µg/ml), BuOH (12, 25, 50 µg/ml) and CH2Cl2 (1.5 µg/ml) fractions of V. odorata reduced SGD-induced cytotoxicity. MeOH extract could not increase the viability significantly. All four semi polar fractions (EtOAc, BuOH, CH2Cl2 and MeOH) decreased SGD-induced ROS production and changed Bax/Bcl-2 ratio. CONCLUSION: V. odorata showed promising effects against SGD condition; further mechanistic and clinical studies are warranted before application of V. odorata as a neuro-protective agent.

The synergistic cytotoxic effects of doxorubicin and Viola odorata extract on human breast cancer cell line T47-D.

BACKGROUND: Breast cancer accounts for one-third of cancer cases in women. Doxorubicin (Dox) is one of the chemotherapeutical compounds widely used to treat breast cancer. Chemical drugs have several side effects and their continuous administration leads to drug resistance in patients. To decrease such side effects in cancer treatment, combination therapy as well as application of natural and herbal compounds has been taken into consideration. The aim of this study was to investigate the cytotoxic effect of Viola odorata (Vo) extract on T47-D human breast cancer cells, alone and in combination with Dox. MATERIALS AND METHODS: The cytotoxic effects of V. odorata and Dox were studied by morphological examination and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flowcytometric analysis was performed to determine the type of cell death. Moreover, scratch healing assay was conducted to investigate antimigration effect of V. odorata. RESULTS: The results of MTT assay showed that V. odorata and Dox-induced cell death in T47-D cells in a dose- and time-dependent manner. Morphological analysis revealed that V. odorata and Dox-induced features of apoptotic cell death in T47-D cells. These results were confirmed by flow cytometry analysis. Scratch healing assay revealed that migration rate was reduced in the V. odorata- treated cells. CONCLUSIONS: Our findings suggest that components of V. odorata exert antitumor effects on human breast cancer and could be administered with lower doses of antitumor agent Dox, in combination therapy, to decrease its side effects.

Antihypertensive Indigenous Lebanese Plants: Ethnopharmacology and a Clinical Trial.

Hypertension is highly prevalent among the Lebanese adult population and is indeed the major cause of mortality in Lebanon. Traditional use of antihypertensive medicinal plants has long been practiced. The aim of this study is to document this traditional knowledge and clinically test the antihypertensive capacity of three of the most commonly used wild plant species Mentha longifolia, Viola odorata and Urtica dioica. Ethno-pharmacological data was collected by personal interviews with herbalists and traditional healers using a semi structured survey questionnaire and assessing relative frequency of citation (RFC). The clinical study was conducted by a randomized, blind, placebo-controlled trial in 29 subjects with mild hypertension distributed in four groups, three plant extract treatments and one placebo. Systolic (SBP) and diastolic blood pressures (DBP) as well as mean arterial blood pressures (MAP) were monitored at weeks 4, 8, 12 and 16 during the treatment with 300 mL/day of plant extract. Results showed that M. longifolia, U. dioica and V. odorata exhibited the highest values of RCF (0.95) followed by Allium ampeloprasum (0.94), Apium graveolens (0.92) and Crataegus azarolus (0.90). The clinical trial revealed dose- and duration-dependent significant reductions in SBP, DBP and MAP of subjects treated with M. longifolia, U. dioica or V. odorata. Our findings indicate that extracts of these plants present an effective, safe and promising potential as a phyto-therapuetical approach for the treatment of mild hypertension. More research on the phytochemistry, pharmacological effects and the underlying mechanisms is necessary.

Effects of intranasal administration of violet oil in dry eye disease.

BACKGROUND: Dry eye disease is a disorder of the tear film. In this study, the effect of Viola odorata L. oily extract was examined for the treatment of patients suffering from dry eye disease. METHODS: A randomised, double-blind, placebo-controlled study was designed. During the trial, Schirmer's test, tear breakup time, Oxford staining and the Ocular Surface Disease Index were assessed. Overall, 105 patients with dry eye symptoms between the ages of 18 and 60 years were allocated to the violet-almond oil, almond oil and placebo (1% w/v hydroxypropyl methylcellulose solution) groups. The treatment and placebo were administered intranasally, two drops three times a day for one month. The patients were followed up for four weeks. A total of 91 patients (32, 29 and 30 in the violet-almond oil, almond oil and placebo groups, respectively) completed the study. RESULTS: At baseline, there was no difference between the three groups in terms of demographic data and the measurement parameters. After the intervention, the results revealed that the Schirmer's score without local anaesthesia and the tear breakup time results significantly improved in the violet-almond oil group. One-way ANOVA indicated a significant improvement in the Schirmer's score, tear breakup time and Ocular Surface Disease Index of the treatment group, as compared with the other groups (p < 0.05). However, the obtained results did not present any significant mean difference between and within the groups of the Oxford staining grade (p > 0.05). CONCLUSIONS: This trial showed that the intranasally administered V. odorata L. oily extract enhances tear production and improves tear film stability.

Design, Formulation, and Physicochemical Evaluation of Vaginal Cream Containing Eucalyptus camaldulensis, Viola odorata, and Mentha piperita extracts for Prevention and Treatment of Trichomoniasis.

BACKGROUND: Trichomoniasis is a common sexually transmitted disease that is caused by infection with protozoan parasite called Trichomonas vaginalis. Metronidazole is the drug of choice for the treatment of this infection. In this study, design, formulation, and physicochemical evaluation of vaginal cream containing Eucalyptus camaldulensis, Viola odorata, and Mentha piperita extracts for the prevention and treatment of trichomoniasis has been investigated. METHODS: Ethyl acetate extract of E. camaldulensis leaves, water fraction of V. odorata root, and hydroalcoholic extract of M. piperita leaves was prepared and used for anti-trichomonas experiments. Then, based on results, different formulations of vaginal cream containing mixed extracts were prepared and physicochemical evaluation was conducted. In the next step, anti-trichomonas effect of selective formulation was tested in vitro. RESULTS: The mixed concentrates containing 2.5 mg/ml E. camaldulensis, 0.06 mg/ml V. odorata, and 1 mg/ml M. piperita showed 100% growth inhibition (GI) during 24 h. Furthermore, the mixture containing 1.25 mg/ml E. camaldulensis, 0.03 mg/ml V. odorata, and 0.5 mg/ml M. piperita showed 92% GI in the first 24 h. The selective formulation passed all of physicochemical test and also showed 100% GI for in vitro anti-trichomonas experiments in the first 24 h. CONCLUSIONS: The mixed concentrates containing 2.5 mg/ml E. camaldulensis, 0.06 mg/ml of V. odorata, and 1 mg/ml of M. piperita are the mixture which showed the highest percentage of GI (100%) after 24 h. The selective formulation of vaginal cream containing this mixture of extracts was detected 100% GI in the first 24 h.

Production of bioactive cyclotides in somatic embryos of Viola odorata.

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.