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April-June 2019
Volume 11 | Issue 2
Page Nos. 103-191

Online since Tuesday, April 16, 2019

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Secondary metabolites from lichen Usnea longissima and its pharmacological relevance Highly accessed article p. 103
Moumita Dandapat, Santanu Paul
DOI:10.4103/pr.pr_111_18  
The objective of the study was to isolate chemical compounds from lichen Usnea longissima (UL) and to use lichen metabolites as antibiotics, antifungal, anti-HIV, anticancer, antiprotozoan, and antibacterial agents. Preparation of the lichen extracts: Fine powder of UL lichen was extracted using acetone, methanol, and water in a Soxhlet extractor. In a rotary evaporator, the extracts were filtered and then concentrated under reduced pressure. The dry extracts were stored at 18°C until they were used in the tests. The extracts were used for various assays. Sixteen compounds were isolated from lichen UL using various chromatographic techniques, including silica gel, Sephadex LH-20, operational data store, and semipreparative high-performance liquid chromatography. By spectroscopic data analyses, their structures were identified as (1) useanol, (2) lecanorin, (3) 3-hydroxy-5-methylphenyl 2-hydroxy-4-methoxy-6-methylbenzoate, (4) lecanorin E, (5) 3'-methylevernic acid, (6) evernic acid, (7) barbatinic acid, (8) 3,7-dihydroxy-1,9-dimethyldibenzofuran, (9) orcinol, (10) O-methyl orcinol, (11) methyl orsellinate, (12) methyl everninate, (13) 2,5-dimethyl-1,3-benzenediol, (14) 2-hydroxy-4-methoxy-3,6-dimethyl benzoic acid, (15) ethyl everninate, and (16) ethyl 2,4-dihydroxy-6-methylbenzoate. UL lichen is a great source of secondary metabolites, which can be used as antimicrobial, antiproliferative, and anticancer agents. This lichen can be used as a natural antioxidant or antitumor agent in clinical practice; commercially, it can be used as an anticancer drug.
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ORIGINAL ARTICLES Top

Biological screening of tri-jannarose as a recipe from Thai traditional medicine p. 110
Ladachart Taepongsorat, Ampa Konsue
DOI:10.4103/pr.pr_178_18  
Context: Tri-Jannarose (TJ) is a Thai traditional medicine recipe, the ingredients of which are betel palm seed (Areca catechu L.), Siamese neem tree root (Azadirachta indica A. Juss.), and heart-leaved moonseed vines (Tinospora cordifolia [Thunb.] Miers). The equal mixture of three plants indicated to treatment of antipyretic, diuretic, expectorate, nourishment and appetizing. Aims: Phytochemical screening, antioxidant, and α-glucosidase inhibitory activities of TJ using different extractions were evaluated. Materials and Methods: The three plants of recipe were extracted using different solvents such as aqueous extract (ATJ), 50% ethanolic extract (HETJ), and 95% ethanolic extract (ETJ). The phytochemical screening was determined by total phenolic compounds and total flavonoid contents. The anti-oxidation were tested using by 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging and 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) assay. The α-glucosidase inhibitory activity was determined for glucose transferase mechanism. Results: Phytochemical screening found that this recipe had both phenolic and flavonoid substances. The ETJ (IC50 = 0.0463 ± 0.002) was exerted on antioxidation higher than HETJ and ATJ (IC50 = 0.0511 ± 0.000 and 0.1485 ± 0.005 mg/mL). Surprisingly, ABTS+ assay, ETJ (IC50 = 0.015 ± 0.000 mg/mL), and HETJ (IC50 = 0.022 ± 0.000 mg/mL) showed high effect on free radical scavenging activity than the standard controls, ascorbic acid (IC50 = 0.025 ± 0.001 mg/mL), and Trolox (IC50 = 0.032 ± 0.001 mg/mL). The α-glucosidase inhibitory activity found that all of the extract including ATJ (IC50= 0.0127±0.02 mg/mL), ETJ (IC50= 0.0154±0.01 mg/mL) and ETJ (IC50= 0.0202±0.01 mg/mL) were more potent to inhibit α-glucosidase emzyme than acarbose (IC50=0.745±0.026 mg/mL) as a positive control. Conclusion: The pharmaceutical preliminary scarring was confirmed to treatment on Thai traditional medicine. The recipe composed with phenolic compounds and flavonoids contents which chemical substance were more potent anti-oxidation, and bittersweet flavor was stronger to α-glucosidase inhibitory activity.
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Antidiabetic activity of hydroalcoholic extract of Myrtus communis (myrtle) fruits in streptozotocin-induced and dexamethasone-induced diabetic rats p. 115
Mohammad Sharif Talebianpoor, Mohammad Shafee Talebianpoor, Mahboubeh Mansourian, Tahereh Vafaiee-Nejad
DOI:10.4103/pr.pr_160_18  
Background: Diabetes mellitus is characterized by an increase in blood glucose levels, resulting in insufficiency of insulin secretion, insulin resistance, or both. Objective: The effects of hydroalcoholic extract of the Myrtus communis (myrtle) fruits on Wistar rats were investigated in two types of diabetes mellitus. Materials and Methods: Intraperitoneal administration of streptozotocin (60 mg/kg) was used to induce Type I diabetes. Type II diabetes was induced by subcutaneous injection of 1 mg/kg/day dexamethasone for 10 days. Two groups of the diabetic animals received the hydroalcoholic extract of the fruit by gavage (250 mg/kg and 500 mg/kg for 45 and 10 days) and the diabetic control groups receiving distilled water. Results: The hydroalcoholic extract of M. communis fruits reduced the serum levels of the glucose, triglyceride, urine volume, urine protein, and malondialdehyde at the end of the 45 day. In Type II diabetic rats, there was a significant effect on plasma glucose levels. On this day, blood glucose-lowering effect was significantly observed after insulin administration as C2>D2M500>D2M250>D2 (P ≤ 0.05). The plasma level of insulin was completely reversed. There were no differences in the other biochemical parameters. Conclusions: The hydroalcoholic extract of M. communis fruits has a significant effect on the improvement of diabetes mellitus complications especially Type II diabetic animals, which begins with insulin resistance.
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Identification of bioactive diterpenoid lactones using nuclear magnetic resonance from Andrographis paniculata p. 121
Raghava N Sriramaneni, Amirin Sadikun, Naveen Kumar Hawala Shivashekaregowda, Annegowda Venkatappa, Mohamad Zaini Asmawi
DOI:10.4103/pr.pr_117_18  
Background: Andrographis paniculata (AP) (Burm. F) Nees, belongs to the family Acanthaceae, it is also known as a highly bitter plant. In South East Asia this plant is commonly used for cold, cough and respiratory tract infections. Andrographolide (ANG), 14-deoxy-11, 12-didehydroandrographolide (DDA) are the major bioactive diterpenoid lactone compounds of the plant shown vasorelaxant effect, anticancer, anti-inflammatory, and to treat variety of diseases which is well-documented. Materials and Methods: The high-performance liquid chromatography (HPLC) and 1H-NMR analysis of AP chloroform extract (APCE) revealed the presence of ANG, 14-deoxyandrographolide (DA), and DDA. The main advantage of 1H-NMR is simple, rapid and successfully applied to quantify the active diterpenoids and an alternative to HPLC method to check the presence of various bioactive compounds and its presence in the active raw materials of AP. Results: The results revealed that most active bioactive compounds such as ANG, DA, and DDA are present in the APCE portion of the AP. The chemical shifts of various groups confirmed through 1H-NMR that all the three ANG, DA, and DDA has potent vasorelaxant effect and majority of these compounds are present in the APCE portion and not in the methanol or aqueous fractions. The 1H-NMR study confirmed that diterpenoids such as ANG, DA, and DDA from APCE has vasorelaxant effect on the animal study.
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In vitro screening for antioxidant and antimicrobial properties of three lebanese medicinal plants crude extracts p. 127
Josiane Alhage, Hoda Elbitar
DOI:10.4103/pr.pr_171_18  
Background: Despite its small geographic area, Lebanon is characterized by a natural wealth in medicinal plants. Lebanese flora has about 2600 species, where >100 are endemic and are used in traditional medicine for their therapeutic effects. Objective: To evaluate the antioxidant and antimicrobial activities of dichloromethane, methanol, and aqueous crude extracts of three Lebanese medicinal plants: Geranium robertianum, Asphodelus microcarpus, and Alcea setosa. Materials and Methods: The antioxidant activity of different crude extracts was determined using the free radical, 1,1-diphenyl-2-picrylhydrazyl. Antimicrobial activity was evaluated in a plate-hole diffusion assay against two Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacterial strains, one Gram-positive (Staphylococcus aureus) bacterial strain, and a fungal strain (Candida albicans). Results: Methanol and aqueous extracts of G. robertianum possessed high scavenging activity (Radical scavenging activity (RSA) >85%) at 0.3 mg/mL followed by the extracts of A. microcarpus which showed a moderate activity. G. robertianum extracts exhibited good inhibition diameters against S. aureus growth. Conclusion: Obtained results give an overall view on the bioactivities of three Lebanese medicinal plants crude extracts.
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Phytochemical and antimicrobial evaluation of Lauridia tetragona (L.F) R.H. Archer: A medicinal plant used for the management of dysentery in the Eastern Cape Province of South Africa p. 134
Olubunmi Abosede Wintola, Anthony Jide Afolayan
DOI:10.4103/pr.pr_41_18  
Objective: The present investigation evaluated the in vitro phytochemical and antimicrobial potential of the methanol and acetone leaf extracts of Lauridia tetragona (L.F), a plant consumed as a herb in South Africa. Materials and Methods: Spectrophotometry assays using Folin–Ciocalteu, aluminum chloride, and vanillin–hydrochloric acid were used for the determination of phenols, flavonoids, and proanthocyanidin contents of the extracts, respectively. The agar dilution technique was used for the antimicrobial evaluation. The inhibitory activity of the extracts was tested on five Gram-negative and five Gram-positive bacteria and four fungi. Results: The methanol extract had a higher phenol (54.87 ± 4.01 mg gallic acid equivalent [GAE]/g) and proanthocyanidin (78.55 ± 0.83 mg catechin equivalent [CE]/g) content than the acetone extracts with 45.27 ± 3.93 mg GAE/g and 63.54 ± 1.67 mg CE/g, respectively. The acetone extract, however, had higher flavonoid content (462.45 ± 1.93 mg quercetin equivalent [QE]/g) than the methanol extract (412.20 ± 3.85 mg QE/g). The minimum inhibitory concentration (MIC) of the antibacterial assay ranged from 2.5 to 5 mg/mL. All the bacteria except Staphylococcus aureus were susceptible to the acetone extract. Five bacteria, three Gram positive and two Gram negative, were resistant while the remaining five were susceptible to the methanol extract. Conversely, all the fungi tested were susceptible to both extracts with a MIC which ranged from 0.63 to 10 mg/mL. Conclusion: The results obtained revealed that Gram-positive and Gram-negative bacteria and fungi showed some degree of susceptibility to the plant extracts. This gives an indication of broad-spectrum activity exhibited by the crude extracts of L. tetragona and supports its ethnomedicinal usage.
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Biochemical screening and determination of bioactive components of commercially cultured pacific white shrimp Penaeus vannamei p. 140
Jayalakshmi Muniyappan, Vanitha Varadharajan, Pushpabharathi Namadevan
DOI:10.4103/pr.pr_60_18  
Background: Marine waste is an extraordinarily renewable aid for the restoration of several valued metabolites with potential biological applications. Objectives: The investigation is planned to detect the biochemical components present in the prawn shell waste by qualitative, quantitative methods, to assess the antioxidant potential, and also to find the bioactive compounds existing in the prawn shell waste by gas chromatography-mass spectrometry (GC-MS) analysis. Materials and Methods: Penaeus vannamei shell wastes are collected, cleaned, dried, and powdered well. The bioactive compounds present in crude ethyl acetate extract of P. vannamei shell was determined by qualitative, quantitative, and GC-MS analysis. The free radical scavenging activity of the extract was studied by different in vitro antioxidant assays. Results: The bio-compounds such as carbohydrates, saponins, flavonoids, tannins, and quinones show the positive result by qualitative analysis. The higher tannin content 49.2 ± 0.084 mg/g was observed in the ethyl acetate extract of P. vannamei shell and the flavonoid was found to be 5.994 ± 0.044 mg/g. The GC-MS analysis of the P. vannamei shell shows the various numbers of bio-compounds. Some of the identified compounds are Timonacic which has the powerful antioxidant property, Octadecane, 3ethyl5(2ethylbutyl) is a good antifungal agent, Acetamide possesses antioxidant and anti-inflammatory property. The results of the in vitro assays revealed that P. vannamei shell extract possess significant antioxidant activity. Conclusion: The present study suggests that the effective utilization of prawn shell waste enhances biomedical research field for the development of the natural drug for many chronic diseases with no side effects and at the same time can reduce environmental pollution.
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Characterization of the gain and loss of resistance to antibiotics versus tolerance to honey as an antimutagenic and antimicrobial medium in extended-time serial transfer experiments p. 147
Salma G Abdel-Azim, Ahmad G Abdel-Azim, Brian P Piasecki, Gamal A Abdel-Azim
DOI:10.4103/pr.pr_175_18  
Background: Honey contains several substances with antimicrobial properties that appear more recalcitrant to generating bacterial-evolved resistance than traditional antibiotics. Objectives: This study seeks to characterize the evolution of bacteria grown for successive generations in honey as an antimutagenic medium versus ampicillin. Materials and Methods: A naive strain of Staphylococcus aureus was serially cultured for 17 days in Lysogeny broth (LB) containing sublethal concentrations of medical-grade Manuka honey, polyfloral honey, and ampicillin. Glucose as an osmotic control and pure LB were included for comparison. A portion of each culture was removed every 24 h to (a) determine the amount of growth that occurred during the previous 24 h and (b) use as a genetic stock in serially transferred tubes containing the same inhibitory compound as used previously. Results: As indicated by an increase in growth over sequential 24-h period, bacteria rapidly gained resistance to ampicillin in a step-wise pattern. However, bacteria grown in Manuka and polyfloral honey never exceeded their initial growth levels. Bacteria grown in relatively high concentrations of honey for a single 24-h period consistently lost viability after one transfer, a phenomenon that has not been reported in the literature before and which indicates the inability of bacteria to adapt to honey as an antimutagenic medium. While bacteria grown in honey did not evolve the ability to grow at higher concentrations, a single isolate grown in Manuka honey gained tolerance to Manuka by successfully surviving and proliferating beyond second transfers. Bacteria that developed antibiotic resistance were found to remain sensitive to honey. Moreover, bacteria lost their resistance to ampicillin upon a single exposure to Manuka honey.
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Evaluation of acute toxicity of plants' mixture used in traditional treatment of kidney diseases in Morocco p. 155
Mohamed Chebaibi, Dalila Bousta, Laila Chbani, Imane Iken, Sanae Achour
DOI:10.4103/pr.pr_191_18  
Background: The use of plants' mixture in the traditional treatment of kidney diseases in Morocco is widespread. Objective: To evaluate the toxic effects of plants' mixture used in the traditional treatment of kidney diseases in Morocco. Materials and Methods: The phytochemical screening was performed. For acute toxicity, single doses of low dose (300 mg/kg), medium dose (500 mg/kg), high dose (2000 mg/kg), lethal dose (6000 mg/kg), and traditional dose (10 ml/kg) body weight of aqueous extracts of plants' mixture were administered orally in Wistar rats. Animals were monitored daily for at least 15 days after an oral administration of aqueous extract of the mixture to detect any changes in body weight, behavior, autonomic profiles, or mortality. Calculation of relative organ weight (ROW), hematological, biochemical analysis, and histopathology evaluation were carried out. Results: The acute oral toxicity study showed diarrhea, somnolence, and agitation of different groups of rats, while no mortality and no statistically significant decrease in body weight was observed. Statistically, the kidneys, liver, and spleen showed significant decrease in the ROW of the treated groups of rats when compared to the control group. In biochemical analysis, there was a significant increase in aspartate aminotransferase, creatinine, urea, and uric acid. Hematological parameters showed a significant decrease in leukocytes, eosinophil, basophil, lymphocytes, monocytes, and hematocrit. Histopathological evaluation which revealed major histology changes of liver sections of rats treated with low and medium doses had lymphocyte and plasma cell inflammatory infiltrates; whereas, the liver sections of rats treated with high, lethal, and traditional doses exhibited lymphocyte, plasma cell, and eosinophilic inflammatory infiltrates. Conclusion: The study finds that the plants' mixture marketed by herbalists for the treatment of kidney diseases is toxic to body organs such as the liver and the hematopoietic system.
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Bioactive secondary metabolites from the locally isolated terrestrial fungus, Penicillium sp. SAM16-EGY p. 162
Mosad A Ghareeb, Manal M Hamed, Amal M Saad, Mohamed S Abdel-Aziz, Ahmed A Hamed, Laila A Refahy
DOI:10.4103/pr.pr_102_18  
Background: Penicillium is a diverse genus occurring worldwide; its species are of major importance in the natural environment as decomposer of organic materials as well as food and drug production. Objective: Chromatographic isolation and identification of its bioactive secondary metabolites and their evaluation as antimicrobial agents. Materials and Methods: Disc agar plate method has been recognized to assess the antimicrobial activities. The antioxidant activity was determined using phosphomolybdenum method. The fungus strain SAM16-EGY was isolated from soil and was molecularly identified as Penicillium sp. SAM16-EGY using 18S ribosomal ribonucleic acid technique (acc. no., KP125952). Results: Seven compounds namely 3-O-docosyl-4-benzoyloxy methyl-3-oxobicyclo (4.1.0) heptane-1,5,6,7 tetrol (3-O-docosyl-3-debenzol rotepoxide) (1), (4bE, 6Z, 8E, 9aS, 10S)-1,4-dihydroxy-9a, 10-dihydro-10,12-epoxy-5-methylbenzo[a] azulen-12-one (2), 7α,9β,15β-triacetoxy-3-β-hydroxy jatropha-5E, 11E-diene (3), sesquiterpene I diol dihexoside malonate ester (4), piperogalone (5), (5R, 8Z, 11Z)-5 β-(6'-O-malonyl-β-glucopyranozyloxy-6-hydroxy tetradeca-8, 11-dienoic acid (6), and n-trcosanyl-n-octaced-9-enoate (7) were isolated and identified from this fungus. Their structures were determined on the basis of proton nuclear magnetic resonance and carbon-13 nuclear magnetic resonance spectroscopy. Compounds 1, 2, 4, and 5 exhibited antimicrobial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans only, whereas compound 3 exerted higher antimicrobial activity against S. aureus (9 mm), P. aeruginosa (9 mm), C. albicans (11 mm), and Aspergillus niger (13 mm) as compared to the other compounds. In the phosphomolybdenum assay, compound 5 showed high total antioxidant capacity value of 608.59 mg ascorbic acid equivalent/g compound, followed by compound 2 (443.66 mg) and compound 1 (332.16 mg). Conclusion: The isolated compounds showed promising antimicrobial and antioxidant activities.
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Anatomical and phytochemical characterization of Physalis angulata L.: A plant with therapeutic potential p. 171
Léa Maria dos Santos Lopes Ferreira, Ademir Evangelista do Vale, Amancio José de Souza, Kelly Batista Leite, Cássia Sacramento, Maria Lucia Vieira Moreno, Thessika Hialla Araújo, Milena Botelho Pereira Soares, Maria Fernanda Rios Grassi
DOI:10.4103/pr.pr_97_18  
Background: Physalis angulata L. is widely used in folk medicine. Secondary metabolites with pharmacological potential, including physalins that exhibit anti-inflammatory/immunomodulatory and antiparasitic activities, have been identified in this specie. To date, few studies have investigated storage sites for secondary metabolites in P. angulata. Objective: The objective of the study is to characterize the anatomical structures and determine the phytochemical composition of the vegetative organs of P. angulata. Materials and Methods: Electron and conventional optical microscopy was used for the anatomical characterization of P angulata organs (leaves, roots, stems, and fruits). Methanolic extracts from leaves, roots, stems, and fruits were chemically characterized for the presence of steroids, terpenoids, tannins, alkaloids, saponins, flavonoids, anthraquinones, coumarins, and phenolic compounds. Phenolic compounds, flavonoid contents, and antioxidant capacity of these extracts were determined by 2,2-diphenyl-1-picrylhydrazyl-free radical scavenging activity. Results: Abaxial leaf stomata were more abundant than the adaxial stomata. Trichomes were more abundant along veins in the petioles and stems, beyond the margin in the sepals and petals, and dispersed in the ovary. Steroids and terpenoids were present in leaves, stems, and fruits of P. angulata. Saponins were exclusive to fruits. Phytochemical screening did not detect flavonoids, anthraquinones, and alkaloids in all tested plant parts. The highest antioxidant capacities were identified in leaf and fruit extracts, possibly due to the presence of phenolic compounds in these organs. Conclusion: This study describes anatomical and biochemical features from P angulata that will assist in future phytochemistry and pharmacological studies, particularly pointing toward organs abundant in antioxidants (leaves and fruits) and steroids (possibly physalins; leaves).
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Pretreatment and optimization of processing conditions for extraction of oleuropein from olive leaves using central composite design p. 178
P Sucharitha, SV Satyanarayana, K Bhaskar Reddy
DOI:10.4103/pr.pr_179_18  
Background: The extraction methods used for isolation of biomolecule from Olive leaves show risk of residual solvent and less extraction efficiency. Hence, there is a need to develop novel techniques to encapsulate the risks headed with extraction process. Objective: The goal was to unravel the effect of novel extraction techniques on the extraction efficiency of Oleuropein from Olea europaea, a major secoiridoid resided in Olive leaf. Materials and Methods: Olive leaves were collected, authenticated, and subjected to proximate, phytochemical analysis to contemplate the source of active moiety. The précised solvent, i.e., water: glycerol (3:1%v/v) was functionalized to depict the influence of independent variable on response using central composite design. For hot blanching, the independent factors selected were treatment temperature (50°C–70°C) and duration of blanching (10–30 min) whereas the observed response is percentage extraction efficiency of Oleuropein. The hot blanched leaves were subjected to extraction by cold maceration, microwave-assisted extraction (MAE), and ultrasound-assisted extraction (UAE). The content of Oleuropein was analyzed by high-performance thin-layer liquid chromatography. Results: From the design space, the model is stable at a range of 0.002–0.80 which indicates lack of fit is very less and more curvature effects are clearly visualized with P = 5% level of significance. Maximum response was attained at a temperature of 60°C–65°C and duration of 15–20 min. Microstructural changes in leaf were observed through scanning electron microscopy studies. From the study, pretreated leaves followed by UAE result in higher yield of Oleuropein compared to MAE and maceration. Conclusion: Hot blanching technique shows a significant linear upswing in the concentration of Oleuropein when compared to direct extraction techniques. Blanching of Olive leaves causes deactivation of enzymes, and further exposure to ultrasonic waves enhances mass transfer of solvent and promotes the release of Oleuropein.
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SHORT COMMUNICATION Top

Efficacy of topical herbal remedies for insomnia in Iranian traditional medicine p. 188
Elham Haghjoo, Asie Shojaii, Mohammad Mahdi Parvizi
DOI:10.4103/pr.pr_163_18  
Background: Insomnia is one of the most prevalent sleep disorders which affect the quality of life. Due to high prevalence of this disease and the side effects of sedative drugs, people tend to use herbal remedies. There are some oral or topical prescriptions in Persian medicine texts for the treatment of insomnia. The aim of this study was to investigate topical treatments for insomnia in Iranian traditional medicine (ITM) and comparing them with current therapies in modern medicine. Methods: In this study, ITM textbooks including the Canon of Medicine, Teb-e-Akbari, Kholasat al-Hekmah, Makhzan al-Advieh, Exir-e-azam, Al-Aghraz al-Tibbia, Val Mabahess al-Alaiia, Sharh-al-Asbab-val-Alamat, Tohfa-al-Momen, and Qarabadeen-e-Kabir were searched to investigate effective topical therapies for treating insomnia. Further, relevant studies on these topical remedies were searched at databases such as PubMed, Google Scholar, Scopus, and ScienceDirect from the beginning till June 2018 and the results were presented as tables. Results: There are some herbs which have been recommended in ITM for the treatment of insomnia in topical dosage form such as ointment, lotion, nasal drop, and oil. The most important herbs used in these topical dosage forms were lettuce, violet, almond, pumpkin, and water lily. Some of these herbal therapies including lettuce and violet have been studied in clinical trial for insomnia. Conclusion: Findings of the present study showed that according to ITM texts, there are some effective topical herbal remedies which can be used in treatment of insomnia with less adverse effects.
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