Iranian Journal of Pharmaceutical Research (2019), 18 (2): 922-937 DOI: 10.22037/ijpr.2019.2390 Received: February 2017 Accepted: April 2018

Original Article

Aquaretic Activity of Solidago canadensis L. Cultivated in Egypt and Determination of the Most Bioactive Fraction

Passent Mahmoud Abdel Bakia*, Moshera Mohamed El-Shereia, Amal Elsayed Khaleela, Amira Ahmed Abdel Motaalb and Heba Mohammed Ibrahim Abdallahc

aDepartment of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt. bDepartment of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Kingdom of Saudi Arabia. cDepartment of Pharmacology, Medical Research Division, National Research Center, Giza 12622, Egypt.

Abstract

Despite the traditional use of Solidago canadensis L. (Asteraceae) as a diuretic drug, there is a scarcity in scientific data concerning the activity of its different extracts and fractions as well as the class of constituents responsible for this diuretic action. A comparative study was carried out for the diuretic activities of the different standardized extracts and fractions of the flowering aerial parts of S. canadensis, as well as isolation of compounds from the most biologically active fraction. The ethanol extract and its ethyl acetate fraction (EA) showed the highest aquaretic activities (91 and 58% at a dose of 400 mg/Kg b.wt., respectively) compared to 100% of furosemide at 20 mg/Kg b.wt.. Their activities were higher than Cystinol® and spironolactone reference standards (74% and 59% of furosemide, respectively). EA showed the highest total phenolic and flavonoid contents among the tested fractions of the ethanol and aqueous extracts (9.38 ± 0.004 g GAE and 39.75 ± 0.005 g RE/100 g dried extract, respectively). Eight flavonoids, 2 phenolic acids and 1 nucleoside were isolated from EA. This is the first report of a comparative study between the aquaretic activities of the different extracts, fractions and essential oil of S. canadensis, as well as isolation of thyimidine (1), isorhamnetin-3-O-β- ᴅ-glucopyranoside (2), kaempferol-3-O-(6”-O-acetyl)-β-ᴅ-glucopyranoside (4), quercetin-3- O-(6”-O-acetyl)-β-ᴅ-glucopyranoside (5), and kaempferol-3-O-β-ᴅ-apiofuranoside (7) from genus Solidago.

Keywords: Aquaretic activity; Flavonoids; Kaempferol-3-O-β-ᴅ-apiofuranoside; Phenolics; Saponins; Thymidine.

Introduction the rate of urine flow by the kidneys (2). The commonly used synthetic diuretics (viz thiazides Phytopharmaceuticals were successfully and furosemide) have been associated with many used in the therapy of the urinary tract with side effects such as disturbances of electrolytes, parallel administration of synthetic drugs acid-base and water balance, changes in uric- especially those used as diuretics (1). Diuretics acid, carbohydrate and lipid metabolism, and are commonly defined as drugs that promote drug interactions (3). Therefore, herbal diuretics could be considered as a better therapeutic * Corresponding author: option, having relatively safer and milder E-mail: [email protected] actions, compared to synthetic diuretics which Aquaretic activity of Solidago canadensis L. cause several adverse effects due to their strong mg. It is used for the treatment of urolithiasis saluretic actions (4). Numerous herbs were by promoting the excretion of water more than traditionally considered as diuretics. Among the electrolytes and increasing renal blood flow. those herbs are members of genus Solidago This facilitates the washout of bacteria from the belonging to family Asteraceae (5). urinary tract, prevents crystal formation, and Numerous interesting secondary metabolites hence kidney stones (46). such as flavonoids, triterpenoids, saponins, Having Cystinol® in the Egyptian market, it phenolic acids, glucosides, polysaccharides, was found interesting to investigate the diuretic diterpenes, and essential oils were reported in activity of the essential oil and crude extracts genus Solidago (6). Most commonly used in of the flowering aerial parts of S. canadensis phytotherapy are the apical shoots of goldenrod, cultivated in Egypt using different solvents known as Herba Solidaginis (7). Herba for extraction (70% ethanol and water). A Solidaginis were applied in the middle ages biologically guided fractionation of the crude in nephrolithiasis, urinary tract, and prostate extracts was carried out to determine the diseases, while the flowers and leaves were most active fraction and its content of active used as natural yellow dyes (6). Goldenrods constituents. were forgotten for a while, but found its place again in modern phytotherapy and their demand Experimental has been rising over the past few years (6, 8). Several species of Solidago were reported to Plant material exhibit diuretic, spasmolytic, cytotoxic, anti- Samples of Solidago canadensis L. were microbial, anti-mutagenic, anti-inflammatory, collected during the years 2010-2013 from immunobiological, gastroprotective, ulcer- El-Mansoureya, Giza, Egypt. The plant was healing, amoebicidal and amoebistatic activities kindly authenticated by the temperate regional (9-21). The essential oils of different goldenrods team of the Royal Botanical Gardens, Kew., were reported to possess anti-microbial activity London, U.K. Voucher specimen of the plant (9-14, 16-22). (24.02.2013) was deposited at the herbarium Solidago canadensis L., also known as of the Pharmacognosy Department, Faculty of Canadian goldenrod, is native to North America Pharmacy, Cairo University. (23, 24). It is an erect perennial herb that is widely used as a landscape flowering plant, and in cut Plant extraction flower arrangements and bouquets (8). Canadian A- Ethanolic extract goldenrod has been used in folk medicine for The air-dried powdered flowering aerial centuries as urological and antiphlogistical parts of S. canadensis (1 kg) was exhaustively therapy, febrifuge, analgesic, gastro-intestinal extracted with 70% ethanol by cold maceration tract and liver aids, and in burns and ulcer to give 230 g extract. An aliquot of the dry treatment (25-30). It was reported to exhibit residue (200 g) was subjected to liquid-liquid several biological activities including diuretic, fractionation with n-hexane, methylene chloride, anti-microbial, cytotoxic, antioxidant, inhibiting ethyl acetate and n-butanol saturated with water activity to the lyase of DNA polymerase, and concentrated to give 10.4, 6.8, 17.2 and 22 antimutagenic activities (9, 16, 24, 31-33). Its g of dried fractions, respectively. The remaining essential oil was reported to possess cytotoxic water of the ethanol extract was lyophilized and and anti-microbial activities (23, 34-36). Earlier weighed (122 g). investigations on the plant led to the isolation of flavonoids, phenolic acids, saponins, alkaloids, B- Aqueous extract polyacetylenes, terpenes and sterols (31, 33-45). The air-dried powdered flowering aerial parts In the past few years, the extract of the (1 kg) were macerated in boiling distilled water flowering aerial parts of Solidago virgaurea for 20 min. The aqueous extract was lyophilized L. was launched in the Egyptian market under to give 206 g residue. An aliquot of the the trade name of Cystinol® at a dose of 400 lyophilized residue (200 g) was extracted with

923 Abdel Baki PM et al. / IJPR (2019), 18 (2): 922-937

methylene chloride, ethyl acetate and n-butanol (85:15:0.2 v/v/v); S4, ethyl acetate-methanol- saturated with water to give 12, 18, and 26 g of water (100:16:13 v/v/v); S5, ethyl acetate-formic the dried fractions, respectively. The remaining acid-glacial acetic acid-water (100:11:11:10 water left after fractionation was lyophilized and v/v/v/v). The chromatograms were visualized weighed (138 g). The extracts and fractions were under UV light (at 254 and 366 nm) before stored at 5 °C for both the phytochemical and and after exposure to ammonia vapour and biological investigations. spraying with AlCl3, FeCl3 as well as after spraying with natural product-polyethylene C- Preparation of essential oil glycol (NP/PEG) and p-anisaldehyde spray The fresh flowering aerial parts (200 g) were reagents. Paper chromatography was conducted subjected to hydrodistillation using a Clavenger′s on Whatmann No. 1 filter paper (Whatmann, apparatus according to the procedures described Ltd., Maidstone, Kent, England) using solvent in the Egyptian Pharmacopœia (47). The system S6, n-butanol-acetic acid-water (4:1:2 obtained essential oil sample wasdried over v/v/v, upper phase) and visualized by spraying anhydrous sodium sulphate. The hydrodistilled with aniline phthalate spray reagent. Shift oil was saved in a refrigerator (4 ºC) in a tightly reagents for UV spectroscopy according to sealed container. the published procedures and chemicals used were obtained from E-Merck, Darmstadt, Chemicals and equipment Germany (51). Melting points (uncorrected) Cystinol® was purchased from Atos were determined on anelectrothermal 9100 Pharma (Cairo, Egypt). Furosemide and (UK). UV spectra were recorded in a Jenway spironolactonewere obtained from Sigma- model 6800 spectrophotometer. 1H-NMR (300, Aldrich (Darmstadt, Germany) and used as 400, 600 MHz) and 13C-NMR (75, 100, 150 reference diuretic drugs. MHz) were measured on a Varian Mercury The total phenolic content was estimated NMR-spectrometer (Japan), Bruker Ascend TM using Folin-Ciocalteu′s colourimetric assay, 400/R NMR spectrometer and Bruker Ascend while the total flavonoids were determined TM 600/R NMR spectrometer, respectively. The using the AlCl3colourimetric assay (48, 49). NMR spectra were recorded in CD3OD and The total saponin contentwas carried out using DMSO-d6 and chemical shifts were given in δ the vanillin colourimetric assay (50). Authentics (ppm) relative to TMS as an internal standard. as gallic acid, rutin and ursolic acid were EI-MS was performed on Varian Mat 711, obtained from E-Merck, Darmstadt, Germany. Finnigan SS Q 7000. Phenolics and sugars used as reference standards in co-chromatography (PC and TLC) were Evaluation of the pharmacological activity purchased from Sigma Chemicals Co. (St. Animals Louis, MO, USA). Diaion HP-20 AG (75-150 µ, Adult male albino rats of Wistar strain (120- Sigma-Aldrich Chemicals, Germany), silica gel 150 g), obtained from the animal house colony 60, and silica gel RP-18 (70-230 mesh, Fluka, at the National Research Center (Dokki, Giza, Sigma-Aldrich Chemicals, Germany), silica gel Egypt), were utilized for determination of the

60 (35-70 mesh, ASTM Germany) and sephadex LD50 and assessment of the diuretic activity. LH-20 (Pharmacia Fine Chemicals AB, Uppsala, They were housed in steel cages at standardized Sweden) were used for column chromatography conditions of temperature and humidity and (CC). Thin layer chromatography (TLC) was fed with standard pellets and water ad libitum. performed on silica gel 60 F254 and silica gel All experimental procedures were conducted RP-18 (Fluka, Sigma-Aldrich Chemicals, in accordance with the internally accepted Germany) using the following solvent systems: principles for laboratory animal use and care,

S1, methylene chloride-methanol-formic and were approved by the Ethics Committee No. acid (95:5:0.2 v/v/v); S2, methylene chloride- MP (4) in accordance with recommendations for methanol-formic acid (90:10:0.2 v/v/v); S3, the proper care and use of laboratory animals methylene chloride-methanol-formic acid (NIH Publication No. 85-23; revised 1985).

924 doses ranging from 1 to 4 g/kg b.wt. The LD50 of the tested extracts was calculated according to the following formula: doses ranging from 1 to 4 g/kg b.wt. The LD50 of the tested extracts was calculated according to the following formula: LD50 = �� ���� � �� Where: LD50 = � doses ranging from 1 to 4 g/kg b.wt. The LD50 of the tested extracts was calculated according Dm = The largest dose that killed all animals. �� ���� � �� Where:doses rangingto the fromfollowing 1 to 4 formula: g/kg b.wt. The LD50 of the tested� extracts was calculated according z = Mean of deaddoses animals ranging frombetween 1 to 4 2g/kg successive b.wt. The groups.LD50 of the tested extracts was calculated according Dm to= theThe following largest formula: dose that killed all animals. LD50 = �� ���� � �� d = The constantdoses rangingto the factor following from between 1 to formula: 4 g/kg 2 successive b.wt. The LD doses.50 of the tested extracts was calculated according z = Mean of Where: dead animals between LD2 successive50 = groups. � to the following formula: �� LD ����50 = � �� n = Number ofDm animals = The largest in each dose group. that killed all animals. d = TheWhere: constant factor between 2 successive �doses.�� ���� � �� LD50 = � Σ = TheDm sum = The ofzWhere: = largest(z Mean d). doseof dead that animals killed all between animals. 2 successive groups. n = Number of animals in each group. �� ���� � �� Dm = The largest dose that killed all animals. z =Where: Meand of= Thedead constant animals factorbetween between 2 successive 2 successive groups.� doses. Σ = The sumz = of Mean x(z of d). dead animals between 2 successive groups. d =Dm The = constantnThe = Number largest factor dose of betweenanimals that killed in2 successiveeach all animals.group. doses. d = The constant factor between 2 successive doses. n =z Number= MeanΣ = ofofThe deadanimals sumx animals of in (z each betweend). group. 2 successive groups. Evaluation of nthe = Number diuretic of activityanimals in each group. Σ =d The= The sum constant of (z factor d). betweenx 2 successive doses.

n = NumberΣ = The of sumanimals of (z in eachd). group. The 70% ethanol x and aqueous extracts, their fractions, and the essential oil of the Evaluation of the diuretic activity floweringΣ = aerial TheEvaluation sum parts of (z wereof thed). diuretic testedx foractivity their diuretic activities as well as their effect on the The 70% ethanol and aqueous extracts, their fractions, and the essential oil of the excretionEvaluation of potassium ofThe the 70% diuretic andx ethanol sodiumactivity and in aqueous urine according extracts, their to the fractions, method and of the Lipschitz essential et oil al. of 1943 the flowering aerial parts were tested for their diuretic activities as well as their effect on the The 70%floweringEvaluation ethanol aerial of the and partsdiuretic aqueous were activity extracts,tested for their their fractions, diuretic activitie and thes asessential well as oil their of effect the on the (53). Three diureticThe 70% drugs ethanol namely and furosemide aqueous extracts, (20 mg/kg their fractions, b.wt.), andspironolactone the essential (25oil of mg/kg the excretionfloweringEvaluation ofexcretion potassium aerial of the parts of diuretic potassium wereand activitysodium tested and for sodium in their urine in diuretic urine according according activitie tos astothe the well met met ashod hod their ofof effectLipschitz Lipschitz on theet al.et 1943al. 1943 b.wt.) and Cystinol®flowering (400 aerial mg/kg/b.wt.) parts were tested were for used their diureticas reference activitie sstandards. as well as Oral their doses effect onof the200 (53).excretion ThreeThe(53). of diuretic 70% potassium Three ethanol drugs diuretic and and sodium namely drugs aqueous in namely furosemideurine extracts, furosemideaccording their (20 to fractions, (20 the mg/kg mg/kgmet hod and b.wt.), b.wt.), of the Lipschitz essentialspironolactone et al. oil 1943 of (25 the (25 mg/kg mg/kg and 400 mg/kgexcretion b.wt.of of the potassium tested andSolidago sodium extractsin urine according and fractions to the metwerehod selectedof Lipschitz for et the al. study,1943 b.wt.)(53). floweringand Three Cystinol®b.wt.) diuretic aerial and partsCystinol® drugs(400 were namelymg/kg/b.wt.) (400 tested mg/kg/b.wt.) furosemide for their were diuretic (20 were used mg/kg used activitie as b.wt.),asreference references as spironolactone well s standards. astandards. their effect(25 Oral Oral mg/kg doses on doses the of 200 of 200 (53). Three diuretic drugs namely furosemide (20 mg/kg b.wt.), spironolactone (25 mg/kg based onb.wt.)excretion the and marketedand Cystinol® of400 potassium mg/kg dose (400 b.wt.of ofand mg/kg/b.wt.) Cystinol®. sodium the tested in wereurine SolidagoThe usedaccording rats asextracts were reference to fastedtheand met sfractionstandards. hodand ofdeprived were LipschitzOral selecteddoses of et ofwateral. for 200 1943 the for study, 18 h and 400 mg/kgb.wt.) b.wt.of and Cystinol® the tested (400 Solidagomg/kg/b.wt.) extracts were used and as fractionsreference sweretandards. selected Oral doses for ofthe 200 study, before and the(53). 400 experiment. Threebasedmg/kg on diureticb.wt.of the Theymarketed drugsthe tested were namely dose Solidago divided of furosemide Cystinol®. extracts into (20The28 and mg/kg groupsrats fractions were b.wt.), of fastedwere six spironolactone selected aanimalsnd deprived for each.the (25of study,water mg/kg The for rats 18 h of based on theand marketed 400 mg/kg dose b.wt.of of Cystinol®.the tested Solidago The ratsextracts were and fasted fractions and were deprived selected of for water the study, for 18 h basedb.wt.) on before andthe marketedCystinol® the experiment. dose (400 of mg/kg/b.wt.) Cystinol®. They were The were divided rats used were into as fasted reference 28 groups and sdeprivedtandards. of six animals of Oral water doses each. for 18of The 200h rats of each group werebased subjected on the marketed to the dose specified of Cystinol®. treatment The rats and were the fasted co ntroland deprived group of received water for 118 mLh beforebeforeand the 400 the each experiment. mg/kg experiment. group b.wt.of were They the subjected tested were were dividedSolidago to divided the specified into extracts into 28 groups treatment 28and groupsfractions of six and animals ofwere the sixco selectedntrol animals each. group Thefor the each. rats received study, of The 1 mL rats of 0.9% NaCl/100 g b.wt. The following parameters were estimated: eacheach groupbased group 0.9%before on were the were NaCl/100 marketed the subjected subjected experiment. gdose b.wt. to to of the The theTheyCystinol®. specified following specified were dividedThe treatment parameters treatmentrats intowere and 28 were fasted the groups and coestimated: a ntrolnd the ofdeprived co group six ntrol animals receivedof groupwater each. 1for received mL The 18 h rats of 1 mL Urine output each group were subjected to the specified treatment and the control group received 1 mL 0.9%0.9% NaCl/100before NaCl/100Urine the experiment. goutput gb.wt. b.wt. The They followingfollowing were parameters divided parameters into were 28 were groupsestimated: estimated: of six animals each. The rats of 0.9% NaCl/100 g b.wt. The following parameters were estimated: ImmediatelyUrineeach output group afterImmediately were the treatment, subjected after the to treatment, the specified animals the animalstreatment were wereindividually and individually the control placed groupplaced inreceived in metabolic metabolic 1 mL cages cages Urine output 0.9% NaCl/100speciallyUrine output designedg b.wt. The to followingseparate urineparameters and faeces. were estimated:During this period, neither food nor water speciallyImmediately designed after to separate the treatment,Aquaretic urineactivity the of Solidagoand animals faeces.canadensis were L. During individually this placed period, in neither metabolic food cages nor water Immediately after the treatment, the animals were individually placed in metabolic cages speciallyUrine wasImmediatelyoutput designed made available to afterseparate the to urinethe treatment, animals. and faeces. the The animals roomDuring temperature were this individually period, was neither mai placedntained food in nor at metabolic 27-29water °C. cages The was made availablespecially to designed the animals. to separate The urineroom and temperature faeces. During was this mai period,ntained neither at 27-29 food nor °C. water The speciallywasImmediately made designedurine available was after to collected to theseparate the treatment, animals. in urinemeasuring The the androom animals cylinders faeces. temperature were upDuring individually to was 24h maithis after ntainedplaced pe treariod, tmentat in 27-29 metabolicneither for °C. all food The controlcages nor and water Determination of median lethal dose (LD50) groups. Urine volume was expressed as mL/kg The LD of both the ethanol and urineaqueous was (54). collectedwas made in available measuring to the cylindersanimals. The up room to 24htemperature after treawas tmentmaintained for at all 27-29 control °C. The and 50 was urinemadespecially was availabletreated collecteddesigned groups. to in to the Urine measuringseparate animals. volume urine cylinders wasThe and expressed room faeces. up to temperature During 24has mL/kg after this (54). trea pe wasriod,tment maineither for ntained all food control norat 27-29 andwater °C. The extracts was determined following Karber′s urine was collectedUrinary in excretion measuring of test group cylinders up to 24h after treatment for all control and treatedtreated groups.was madegroups.Diuretic Urine available Urine action:volume volume to the was animals.was expressed expressed The room as as mL/kg mL/kgtemperature (55, (54). 56). (54). was maintained at 27-29 °C. The procedure (1931) (52). Five groups, each ofurine six was collected in measuring Urinary excretion cylindersof control group up to 24h after treatment for all control and rats, received both plant extracts separately in urine treated was Urinary collected groups.Urinary excretion excretion inUrine measuring of volume oftest test group group was cylinders expressed up to as 24hmL/kg after (54). trea tment for all control and DiureticDiuretic action: action: Diuretic action of extract (55, (55, 56). 56). doses ranging from 1 to 4 g/kg b.wt. The treatedLD groups.Diuretic Urine Urinaryactivity: volume excretion Urinary ofwas controlexcretion expressed group of test groupx 100 as mL/kg(55). (54). 50 Urinary excretionDiuretic of control action ofgroup standard dosesdoses rangingranging fromfrom 11 toto 44 g/kgg/kg b.wt.b.wt.of the TheThe tested LDLD 5050extracts ofof thethe wastestedtested calculated extractsextracts according waswas calculatedcalculated to treated accordingaccordingDiuretic groups. action:Urine volume was expressed as mL/kg(55, 56). (54). UrinaryDiuretic excretion action Urinary of extract excretionof test group of control group the following formula: Diuretic activity:Diuretic Urinary action excretion of extract of test xgroup 100 (55). toto thethe followingfollowing formula:formula: DiureticDiuretic Diureticactivity: action: action: Diuretic actionDiuretic of standard actionx of 100extract (55). (55,(55, 56). 56). DiureticDiuretic Urinary activity: Urinary action excretion excretionof standard of of control control groupgroup x 100 (55). × Diuretic action of standard LDLD5050 == DiureticDiuretic action action of of extract extract DiureticDiuretic activity: activity: ×xx 100100 (55). (55). ���� �������� �� ���� DiureticDiuretic action action of of standard standard Where:Where: Where: �� 6

DmDm == TheThe largestlargest dosedose thatthat killedkilled allallDm animals.animals. = The largest dose that killed all animals. z = Mean of dead animals between 2 EstimationEstimation ofof electrolytes electrolytes 6 zz == MeanMean ofof deaddead animalsanimals betweenbetween 22 successivesuccessive groups.groups. + + 6 successive groups. TheEstimation electrolyte of electrolytes (Na , K ) content+ + 6was d = The constant factor between 2 successive estimated Thein the electrolyte urine using the (Na commercially, K ) content was estimated in the urine using the commercially dd == TheThe constantconstant factorfactor betweenbetween 22 successivesuccessive doses.doses. + + 6 doses. available kitThe (Biodiagnostic electrolyte Co.,(Na Giza,, K ) Egypt).content was estimated in the urine using the commercially nn == NumberNumber ofof animalsanimals inin eacheach group.group.n = Number of animals in each group. availableavailable kit kit (Biodiagnostic (Biodiagnostic Co., Giza, 6Co., Egypt). Giza, Egypt). Σ = The sum of (z × d). + + ΣΣ == TheThe sumsum ofof (z(z d).d). + + + + Concentration of Na ofin Naurinein urine Na Na/ K/ Kratio: ratio: + . . Concentration of K ofin urineK+ in urine xx Evaluation of the diuretic activity The 70% ethanol and aqueous extracts, their Statistical analysis fractions, and the essential oil of the flowering StatisticalStatistical analysis EvaluationEvaluation ofof thethe diureticdiuretic activityactivityaerial parts were tested for their diuretic The data obtained were presented as mean activities as well as their effect on the excretion ± standardThe data error obtained (SE) and were statistically presented analyzed as mean ± standard error (SE) and statistically analyzed TheThe 70% 70% ethanol ethanol and and aqueous aqueous extracts, extracts, their their fractions, fractions, and and the the essentialessential oil oil of of the the of potassium and sodium in urine according Theusingusing ANOVA data ANOVA obtained followed followed by were LSD by LSD presentedpost-hoc post-hoc test. astest. mean The values ± standard were dete rmined error to (SE) be significant and statistically analyzed floweringflowering aerial aerial parts parts were were tested testedto for forthe their theirmethod diuretic diuretic of Lipschitz activitie activitie ss et as as al. well well 1943 as as their their(53). effect effectThe on on values the the were determined to be significant Three diuretic drugs namely furosemide (20 whenwhen p-value p-value was lesswas thanless than0.05 0.05(p < (0.05).p < 0.05). excretionexcretion ofof potassiumpotassium andand sodiumsodiummg/kg inin urineurine b.wt.), accordingaccording spironolactone toto thethe met met(25hodhod mg/kg ofof LipschitzLipschitz b.wt.) etetusing al.al. 19431943 ANOVA followed by LSD post-hoc test. The values were determined to be significant (53).(53). Three Three diuretic diuretic drugs drugs namely namelyand furosemide furosemide Cystinol® (20 (20(400 mg/kg mg/kgmg/kg/b.wt.) b.wt.), b.wt.), werespironolactonespironolactone used as (25 (25 mg/kgIsolation mg/kgIsolation of of the the components components of theof theethylacetate ethylacetate fraction of the 70% ethanolextract (EA) reference standards. Oral doses of 200 and 400 whenfraction p- of valuethe 70% was ethanolextract less than (EA) 0.05 (p < 0.05). b.wt.)b.wt.) andand Cystinol®Cystinol® (400(400 mg/kg/b.wt.)mg/kg/b.wt.)mg/kg b.wt.of werewere usedused the astestedas referencereference Solidago sstandards.tandards. extracts Oral Oraland dosesdoses ofofEAEA 200200 (16 (16 g) g) was was chromatographed chromatographed on on a Diaiona Diaion column (35 cm L × 3.5 cm D). Gradient elution andand 400400 mg/kgmg/kg b.wt.ofb.wt.of thethe testedtestedfractions SolidagoSolidago were extractsextracts selected andand fractionsfractionsfor the study, werewere basedselectedselected on forfor thethecolumn study,study,with (35 water/methanol cm L × 3.5 cm mixturesD). Gradient was elution adopted. Fractions, 200 mL each, were collected and the marketed dose of Cystinol®. The rats were Isolationwith water/methanol of the components mixtures was ofadopted. the ethylacetate fraction of the 70% ethanolextract (EA) basedbased onon thethe marketedmarketed dosedose ofof Cystinol®.Cystinol®. TheThe ratsrats werewere fastedfasted aandnd depriveddeprived ofof waterwater forformonitored 1818 hh by TLC using solvent system S2. Similar fractions were pooled together and the fasted and deprived of water for 18 h before the EAFractions, (16 g)200 was mL chromatographedeach, were collected andon a Diaion column (35 cm L × 3.5 cm D). Gradient elution beforebefore the the experiment. experiment. They They were wereexperiment. divided divided into intoThey 28 28 were groups groups divided of of six six into animalsanimals 28 groups each. each. The Themonitored rats ratssolvents of of by were TLC separately using solvent evaporated system under S2. reduced pressure yielding three major fractions (I- of six animals each. The rats of each group were Similar fractions were pooled together and eacheach group group were were subjected subjected to to the thesubjected specified specified to treatment treatment the specified and and the thetreatment cocontrolntrol group groupand the received received withthe 1 1solventsIII). mL mL water/methanol Fraction were Iseparately (0.8 g, eluted mixturesevaporated with 25%under was methanol adopted. in water) Fractions, was rechr omatographed 200 mL eac overh, a were collected and 0.9%0.9% NaCl/100NaCl/100 gg b.wt.b.wt. TheThe followingfollowingcontrol parametersparameters group received werewere estimated:estimated: 1 mL 0.9% NaCl/100 g reducedsilica pressure gel 60 yielding column three (30 major × 1 cm) fractions eluted with methylene chloride: methanol (95:5 v/v) b.wt. The following parameters were estimated: monitored(I-III). Fraction by I TLC(0.8 g,using eluted solvent with 25% system S2. Similar fractions were pooled together and the UrineUrine outputoutput yielding compound 1 (30 mg). Fraction II (6.5 g, eluted with 50% methanol in water) was solventsmethanol inwere water) separately was rechromatographed evaporated under reduced pressure yielding three major fractions (I- ImmediatelyImmediately after after the the treatment, treatment, the theUrine animals animals output were were individually individually placedplaced in in metabolic metabolicover cages cagesrechromatographed a silica gel 60 column over (30 a sephadex× 1 cm) eluted LH 20 column (30 × 3 cm) using methylene chloride: Immediately after the treatment, the animals with methylene chloride: methanol (95:5 v/v) speciallyspecially designeddesigned to to separateseparate urine urinewere and andindividually faeces. faeces. DuringDuring placed thisthis in pe pemetabolicriod,riod, neitherneither cages food food nor norIII).yielding waterwatermethanol Fraction compound (1:1 I v/v) 1 (0.8 (30 as mg). eluent g, elutedFraction to give withII three(6.5 25% fractions methanol (IIa, IIb and in IIc). water) Upon was evaporation rechromatographed of over a waswas mademade availableavailable toto thethe animals.animals.specially TheThe roomroom designed temperaturetemperature to separate waswas urine maimaintained ntainedand faeces. atat 27-2927-29 g, °C.°C. elutedfractions TheThe with IIa 50% and methanol IIb, two in pure water) compounds was were obtained: compound 2 (25 mg) and During this period, neither food nor water silicarechromatographed gel 60 columnover a sephadex (30 × LH 1 cm)20 eluted with methylene chloride: methanol (95:5 v/v) urineurine was was collected collected in in measuring measuring cylinders cylinders up up to to 24h 24h after after trea treatmenttment for for all all control controlcompound and and 3 (20 mg). Fraction IIc (5 g) was rechromatographed over a silica gel 60 column was made available to the animals. The room yieldingcolumn (30 compound× 3 cm) using 1methylene (30 mg). chloride: Fraction II (6.5 g, eluted with 50% methanol in water) was treatedtreated groups.groups. UrineUrine volumevolume waswastemperature expressedexpressed as aswas mL/kgmL/kg maintained (54).(54). at 27-29 °C. The methanol(25 × 3(1:1 cm). v/v) Elution as eluentwith methylene to give chloride:three methanol (95:5 v/v) was adopted yielding four UrinaryUrinary excretionexcretion ofof testtesturine groupgroup was collected in measuring cylinders up fractions (IIa, IIb and IIc). Upon evaporation DiureticDiuretic action:action: (55,(55, 56).56). rechromatographedfractions IIc1-4. Upon over evaporation, a sephadex IIc1 yielded LH compound20 column 4 (45 (30 mg) × while,3 cm) IIc 2usin yieldedg methylene chloride: UrinaryUrinary excretionexcretion ofof controlcontrolto 24 groupgroup h after treatment for all control and treated of fractions IIa and IIb, two pure compounds DiureticDiuretic actionaction ofof extractextract methanolcompound (1:1 5 (40 v/v) mg). asFractionIIc eluent3 (200to give mg) was three rechromatographed fractions (IIa, on RP-18IIb and column IIc). (10 Upon × evaporation of DiureticDiuretic activity:activity: xx 100100 (55).(55). DiureticDiuretic actionaction ofof standardstandard 1 cm), eluted with H2O: MeOH (80:20 v/v) to yield of compound 6 (45 mg). FractionIIc4 (2.8 925fractions IIa and IIb, two pure compounds were obtained: compound 2 (25 mg) and g) was rechromatographed over sephadex LH20 column (20 × 1.5 cm) using methanol: water

compound(1:1 v/v) as3 eluent(20 mg). yielding Fraction two main IIc fractions (5 g) (IIc was4i and rechromatographed IIc4ii). IIc4i gave compound over 7 a(20 silic a gel 60 column (25 mg).× 3 IIc cm).4ii (1g) Elution was rechromatographed with methylene over chloride:a silica gel 60methanol column (13 (95:5 × 1 cm) v/ andv) elutedwas adoptedby yielding four 66 methylene chloride: methanol (95: 5 v/v), yielding two compounds, compound 8 (45 mg) and fractions IIc1-4. Upon evaporation, IIc1 yielded compound 4 (45 mg) while, IIc2 yielded compound 9 (20 mg). Fraction III (1.8 g, eluted with 75 and 100% methanol in water) was 3 compoundrechromatographed 5 (40 mg). over aFractionIIc sephadex LH 20 (200 column mg) (30 was × 3 cm).rechromatographed Elution with methanol: on H 2ORP-18 column (10 ×

1 cm),(90:10 eluted v/v) was with adopted H2O: giving MeOH two fractions (80:20 IIIa v/v) and toIIIb. yield Fraction of IIIacompound yielded compound 6 (45 mg). 10 FractionIIc4 (2.8 g) was rechromatographed over sephadex LH20 column (20 × 1.5 cm) using methanol: water 7 (1:1 v/v) as eluent yielding two main fractions (IIc4i and IIc4ii). IIc4i gave compound 7 (20

mg). IIc4ii (1g) was rechromatographed over a silica gel 60 column (13 × 1 cm) and eluted by methylene chloride: methanol (95: 5 v/v), yielding two compounds, compound 8 (45 mg) and compound 9 (20 mg). Fraction III (1.8 g, eluted with 75 and 100% methanol in water) was

rechromatographed over a sephadex LH 20 column (30 × 3 cm). Elution with methanol: H2O (90:10 v/v) was adopted giving two fractions IIIa and IIIb. Fraction IIIa yielded compound 10

7

Abdel Baki PM et al. / IJPR (2019), 18 (2): 922-937 were obtained: compound 2 (25 mg) and compound 3 (20 mg). Fraction IIc (5 g) was rechromatographed over a silica gel 60 column acid

(25 × 3 cm). Elution with methylene chloride: 255, 346 253, 347 258, 350 259, 386 272, 303, 354 267, 291sh, 390 262, 308sh, 378 267, 295sh, 368 methanol (95:5 v/v) was adopted yielding four Na acetate/boric fractions IIc1-4. Upon evaporation, IIc1 yielded compound 4 (45 mg) while, IIc2 yielded compound 5 (40 mg). FractionIIc3 (200 mg)

was rechromatographed on RP-18 column (10 274, 382 Na acetate 274, 304, 382 × 1 cm), eluted with H2O: MeOH (80:20 v/v) 267, 318sh, 389 278, 325sh, 382 278, 328sh, 398 264, 300sh, 382 268, 329sh, 390 274, 303sh, 390 to yield of compound 6 (45 mg). FractionIIc4 (2.8 g) was rechromatographed over sephadex LH20 column (20 × 1.5 cm) using methanol: /HCl water (1:1 v/v) as eluent yielding two main 3

fractions (IIc4i and IIc4ii). IIc4i gave compound AlCl No Change No Change 268, 363sh, 398 279, 345sh, 400 276, 365sh, 408 274, 300sh, 394 7 (20 mg). IIc4ii (1g) was rechromatographed 268, 298sh, 366, 400 269, 303sh, 350, 424 over a silica gel 60 column (13 × 1 cm) and 274, 300sh, 348sh, 403 267, 303sh, 352sh, 429 eluted by methylene chloride: methanol (95: 5 v/v), yielding two compounds, compound 8

(45 mg) and compound 9 (20 mg). Fraction III 3

(1.8 g, eluted with 75 and 100% methanol in AlCl water) was rechromatographed over a sephadex 268, 402 269, 457 279, 355sh, 407 279, 303sh, 435 270, 306sh, 426 274, 302sh, 396

LH 20 column (30 × 3 cm). Elution with 268, 302sh, 350, 400 269, 304sh, 347, 425 methanol: H2O (90:10 v/v) was adopted giving two fractions IIIa and IIIb. Fraction IIIa yielded compound 10 (40 mg). Similarly, IIIb (83 mg)

was rechromatographed over a sephadex LH 277, 405 279, 329, 410 Na methoxide 20 column (13 × 1 cm). Elution with methanol: 271, 326sh, 410 276, 328sh, 400 268, 328sh, 410 274, 324sh, 400 247sh, 330, 406 280, 322sh, 418 water (1:1 v/v) yielded compound 11 (40 mg).

Spectroscopic data of the isolated compounds Compound 1 (thymidine) MeOH EI/MS (70 eV rel. int.), m/z at 126 [M– 254, 348 252, 347 268, 350 290, 326 deoxyribose]+, (32.22%); 83 [M-deoxyribose 268, 300sh, 350 257, 273sh, 365 258, 300sh, 358 245, 290sh, 330 256, 301sh, 372 – HNCO]+, (25.88%); 70 [M+ – deoxyribose– 265, 292sh, 329sh, 366 + C3H4O] , (31.53%); 55 [M –deoxyribose– HNCO –CO]+, (100%, base peak); 54 [M– deoxyribose–HNCO – CO –H]+, (76.10%); 28 + [M –deoxyribose– HNCO – CO – H – C2H2] , (89%); 27 [M –deoxyribose– HNCO – CO – H + – C H – H] , (34.43%). -D-glucopyranoside

2 2 β -D-glucopyranoside Structures of the isolated compounds are β shown in Figure 1 and their spectral data were recorded in Tables 1-4. -D-glucopyranoside β -D-apiofuranoside - β -(6"-O-acetyly)- - O O -(6"-O-acetyly)- Results O O

Determination of median lethal dose acids. flavonoids and phenolic of theisolated UV-shifts Both the ethanol and aqueous extracts kaempferol

isorhamnetin-3-

were safe and non-toxic under the present 1. Table 2 3 Narcissi 3- 4 kaempferol- 3- 5 quercetin- 6 Rutin 7 kaempferol-3- acid 8 caffeic acid 9 neochlorogenic 10 Quercetin 11

926 Aquaretic activity of Solidago canadensis L.

Table 2. 1H-NMR of the isolated phenolic acids and nucleoside.

Position 1 (400 MHz, DMSO) thymidine 8 (400 MHz, CD3OD) caffeic acid 9 (300 MHz, DMSO) neochlorogenic acid 2 6.94 (d, J = 2 Hz)

3 5.01 (d, J = 3.6 Hz)

4 3.82 (br.s)

5 6.67 (d, J = 8 Hz) 3.87 (d, J = 9 Hz)

6 7.70 (s) 6.82 (dd, J = 8, 2Hz)

7 7.41 (d, J = 16 Hz)

8 6.10 (d, J = 16Hz)

1' 6.15 (t)

2' 2.03 (2H, m) 7.02 (d, J = 1.8 Hz)

3' 4.22 (q)

4' 3.75 (dd, J = 10.4, 3.7 Hz)

5' 3.51 (o) 6.75 (d, J = 8.4)

6' 6.95 (dd, J = 8.1, 2 Hz)

7' 7.35 (d, J = 15.9 Hz)

8' 6.07 (d, J = 16.2 Hz)

2ax, eq 2.08 (2H, m)

6ax 1.73 (d, J = 12.9 Hz)

6eq 1.90 (dd, J = 13.8, 10.8 Hz)

CH3-5 1.78 (3H, s) N-H 11.27 (s)

experimental conditions up to 4 g/kg b.wt. The kg/b.wt.) exhibited the highest diuretic activity extracts are considered safe in the range of the of all tested samples comparable to that of administered doses (57). furosemide (91% of furosemide activity), and higher than spironolactone and Cystinol® which Effect on urine volume exhibited 59% and 74% of furosemide activity, The reference diuretic drugs furosemide, respectively (Table 5). The aqueous extract (400 spironolactone, and Cystinol® significantly mg/kg/b.wt.) exhibited lower diuretic activity increased the urine output when compared than that of the 70% ethanol extract and all the to the control group (Table 5). The diuretic used reference diuretics (46% of furosemide). activities of the tested samples were calculated Whereas, the ethyl acetate fraction of the 70% relative to furosemide, as it proved to be the ethanol extract (EA) (400 mg/kg/b.wt.) showed most potent diuretic reference drug. All the the highest diuretic activity amongst all fractions tested samples showed a significant increase representing 58% of furosemide activity and in the volume of urine output except the compared to spironolactone (potassium sparing n-butanol and the remaining water fractions diuretic). However, it was less potent than the of the aqueous extract (Table 5). The 70% parent 70% ethanol extract at the same dose level. ethanolextract of the aerial parts (400 mg/ The essential oil (400 mg/kg/b.wt.) showed poor

927 Abdel Baki PM et al. / IJPR (2019), 18 (2): 922-937 11 6.19 (d, J = 2Hz) 6.44 (d, J = 2Hz) (400 MHz, DMSO) 8.03 (d, J = 8.9Hz) 6.92 (d, J = 8.9Hz) 6.92 (d, J = 8.9Hz) 8.03 (d, J = 8.9 Hz) OD) 3 10 6.18 (br.s) 7.74 (br.s) 7.64 (d, J = 6 Hz) 6.89 (d, J = 8.1Hz) 6.40 (d, J = 1.5Hz) (300 MHz, CD OD) 3 7 4.11 5.01 4.00 3.55 6.09 (br.s) 3.31 (s, H-5") (d, J = 4 Hz,H-2" ) 6.81 (d, J = 8.9 Hz) 6.81 (d, J = 8.9 Hz) 7.99 (d, J = 8.8 Hz) 6.31 (d, J = 1.7 Hz) (400 MHz,CD (d, J = 3.7 Hz,H-1'') (d, J = 8 Hz, H-4a") (d, J = 8.9 Hz,H4b") 7.99 (d, J = 8.84 Hz) Rh) 6 3 7.52 5.28 4.39 0.99 CH 6.11 (br.s) 6.11 7.51 (br.s) 3.17- 3.72 6.30 (br.s) (br.s, H-1''') (br.s, (3H, d, J = 6.3Hz, (300 MHz, DMSO) (10 sugar protons) 6.79 (d, J = 8.7 Hz) (d, J = 7.2Hz, H-1'') (dd, J = 2.7, 8.4 Hz) ) 3 5 7.50 5.34 1.71 H-1'') 3.2- 3.4 6.15 (br.s) 6.36 (br.s) (d, J = 7.2Hz, (3H, s, COCH (6 sugar protons) (dd, J =1.8, 8.4Hz) (600 MHz, DMSO) 7.49 (d, J = 2.4Hz) 6.80 (d, J = 8.4Hz) ) 3 4 7.98 6.85 6.85 7.98 5.34 COCH 3.28 - 3.95 1.73 (3H, s, (dd, J = 9, 1.8Hz) (dd, J = 1.8, 9Hz) (6 sugar protons) (600 MHz, DMSO) 6.19 (d, J = 1.8Hz) 6.42 (d, J = 1.8Hz) (dd, J = 8.4, 1.8Hz) (dd, J = 8.4, 1.8Hz) (d, J = 7.8 Hz,H-1'') Rh) 3 3 7.58 5.36 1.29 3.30-3.60 3.65 (3H, s) 4.52 (br.s, H-1''') 4.52 (br.s, (10 sugar protons) (3H, br.s, CH (3H, br.s, 6.20 (d, J = 2.1Hz) 6.39 (d, J = 1.8Hz) 7.92 (d, J = 2.1Hz) 6.90 (d, J = 8.4Hz) (300 MHz, CD3OD) (d, J = 7.8Hz, H-1'') (dd, J = 8.4, 1.8 Hz) OD) 3

2 7.58 5.22 3.32-3.44 3.60 (3H, s) 6.88 (d, J = 9) (6 sugar protons) (d, J = 7.2Hz,H1'') 6.21 (d, J = 1.8Hz) 6.41 (d, J = 1.5Hz) (dd, J = 8.4,1.8Hz) 7.92 (d, J = 1.8Hz) (300 MHz, CD H-NMR of the isolated flavonoids. H-NMR of the isolated 1 3 Table 3. Table Position 6 8 2' 3' 5' 6' OCH Sugar protons

928 Aquaretic activity of Solidago canadensis L.

Table 4. 13C-NMR of the isolated compounds.

C 1 (100 MHz, DMSO) 2 (150 MHz, DMSO) 5 (150 MHz, DMSO) 7 (100 MHz, CD3OD) 8 (100MHz, CD3OD)

1 - - - 126.40

2 150.92 156.53 156.3 157.23 114.12

3 133.06 133.12 133.10 145.65

4 164.20 177.34 177.21 176.98 148.05

5 109.81 161.19 161.21 160.74 115.10

6 136.58 98.78 98.6 99.69 121.46

7 164.47 164.5 163.20 145.39

8 93.74 93.74 95.00 113.70

9 156.42 156.41 156.10 169.63

10 103.79 103.55 105.35

1' 84.19 120.76 121.56 122.11

2' 40.61 130.83 115.15 131.23

3' 70.90 115.05 148.65 115.92

4' 87.71 160.03 144.91 160.23

5' 61.80 115.05 116.08 115.92

6' 130.83 121.04 131.23

CO acetyl 169.84 169.97 -

CH3 of acetyl 20.17 20.12 - 1'' 101.11 101.12 107.17

2'' 74.09 74.03 77.20

3'' 76.12 76.31 78.24

4'' 69.77 69.98 73.54

5'' 73.88 74 62.31

6'' 62.74 62.83 -

CH3-5 12.72

diuretic activity (31% of furosemide activity). (200 and 400 mg/kg b.wt.) did not affect the urinary electrolyte excretion (Table 5). The Na+/ Effect on urinary electrolyte excretion K+ excretion ratio was uniform (1.27 to 1.36) in Furosemide significantly increased the all the tested plant samples. excretion of urinary electrolytes. Spironolactone (potassium sparing diuretic) increased the Spectrophotometric estimation of the total excretion of sodium iononly, while Cystinol® phenolic, flavonoid and saponin contents did not affect the excretion of sodium and The 70% ethanol extract of the total aerial potassium in urine (aquaretic). Administration parts showed higher total phenolic and flavonoid of all the tested extracts, fractions and the contents than their aqueous extract while the essential oil of the aerial parts at both dose levels saponins were more concentrated in the aqueous

929 Abdel Baki PM et al. / IJPR (2019), 18 (2): 922-937 1.31 1.49 1.78 1.26 1.30 1.31 1.32 1.31 1.31 1.31 1.31 1.31 1.31 1.31 1.32 1.31 1.33 1.34 Na+/K+Ratio * + K 1.94 ± 0.01 1.95 ± 0.02 1.98 ± 0.03 1.92 ± 0.01 1.91 ± 0.01 1.90 ± 0.02 1.96 ± 0.01 1.94 ± 0.03 1.91 ± 0.01 1.91 ± 0.01 1.91 ± 0.05 1.93 ± 0.02 1.93 ± 0.01 1.90 ± 0.01 1.91 ± 0.05 1.90 ± 0.01 1.89 ± 0.02 2.86 ± 0.04 L. + Electrolytes mEq/L Electrolytes Na 2.54± 0.03 2.54 ± 0.01 4.25 ± 0.04 3.48 ± 0.01 2.50 ± 0.05 2.50 ± 0.01 2.50 ± 0.02 2.50 ± 0.02 2.56 ± 0.01 2.51 ± 0.01 2.51 ± 0.05 2.51 ± 0.01 2.53 ± 0.02 2.52 ± 0.01 2.50 ± 0.07 2.50 ± 0.01 2.52 ± 0.03 2.53 ± 0.02 Solidago canadensis 14 59 74 25 31 28 91 22 24 23 24 41 58 31 54 22 30 100 activity Percent of Diuretic of Diuretic Percent 1.00 7.23 4.30 5.36 1.78 2.26 2.05 6.61 1.59 1.74 1.69 1.76 2.99 4.16 2.26 3.94 1.59 2.18 Diuretic action Diuretic ratios of the essential oil, extracts and fractions of the aerial parts of of the aerial and fractions oil, extracts ratios of the essential + /K + ± 0.70 ± 0.41 ± 0.19 ± 0.11 ± 0.40 ± 0.40 ± 0.16 ± 0.29 ± 0.30 ± 0.28 ± 0.19 ± 0.18 ± 0.16 ± 0.29 ± 0.37 ± 0.30 ± 0.31 * * * ± 0.12 * * * * * * * * * * * * * * * 1.6 3.62 6.88 8.57 2.85 3.62 3.28 2.54 2.78 2.70 2.82 4.78 6.30 2.54 3.49 6.66 11.56 10.58 Urine volume (mL/24 h) levels and Na levels + , K + O (200 mg/kg b.wt.) O (400 mg/kg b.wt.) fr. (200 mg/kg b.wt.) fr. (400 mg/kg b.wt.) fr. 2 2 2 2 Cl Cl 2 2 -But. fr. (200 mg/kg b.wt.) -But. fr. (400 mg/kg b.wt.) -But. fr. -Hex. fr. (200 mg/kg b.wt.) -Hex. fr. (400 mg/kg b.wt.) -Hex. fr. EtOAcfr. (200 mg/kg b.wt.) EtOAcfr. (400 mg/kg b.wt.) EtOAcfr. n n n n CH CH Rem. H Rem. H The diuretic action, diuretic activity, Na activity, diuretic action, The diuretic Treatment g b.wt.) saline/100 Control (1 mL Furosemide (20 mg/kg b.wt.) (25 mg/kg b.wt.) Spironolactone (400 mg/kg b.wt.) Cystinol® E.O. (200 mg/kg b.wt.) E.O. (400 mg/kg b.wt.) EtOH ext. (200 mg/kg b.wt.) EtOH ext. (400 mg/kg b.wt.) Fractions of the 70% ethanol extract Table 5. Table

930 Aquaretic activity of Solidago canadensis L. 1.30 1.31 1.31 1.35 1.27 1.36 1.31 1.32 1.34 1.33 Na+/K+Ratio + K 1.98 ± 0.03 1.92 ± 0.02 1.90 ± 0.08 1.89 ± 0.02 1.96 ± 0.02 1.90 ± 0.01 1.94 ± 0.01 1.90 ± 0.04 1.91 ± 0.01 1.91 ± 0.03 + Electrolytes mEq/L Electrolytes fr.: methylene chloride fraction; E.O.: essential oil; essential E.O.: fraction; chloride methylene fr.: Na 2 Cl 2 2.57 ± 0.03 2.52 ± 0.02 2.49 ± 0.02 2.56 ± 0.01 2.48 ± 0.01 2.58 ± 0.01 2.55 ± 0.04 2.50 ± 0.01 2.55 ± 0.01 2.54 ± 0.01 6 7 6 8 28 46 32 41 28 52 activity Percent of Diuretic of Diuretic Percent O, remaining water; SE: standard error. water; O, remaining 2 -butanol fraction; b.wt.: body weight; CH body weight; b.wt.: fraction; -butanol n 2.03 3.29 2.34 3.00 2.03 3.73 0.41 0.53 0.44 0.56 -But. fr.: fr.: -But. n Diuretic action Diuretic -hexane fraction; Rem.: H Rem.: -hexane fraction; n -Hex. fr.: -Hex. fr.: ± 0.31 ± 0.47 ± 0.29 ± 0.10 ± 0.21 ± 0.27 * * * * * * n 0.65 ± 0.05 0.85 ± 0.05 0.70 ± 0.08 0.90 ± 0.08 3.24 5.26 3.75 4.80 3.25 5.97 < 0.05. Aq.ext.: aqueous extract; Aq.ext.: < 0.05. Urine volume (mL/24 h) p O (200 mg/kg b.wt.) O (400 mg/kg b.wt.) fr. (200 mg/kg b.wt.) fr. (400 mg/kg b.wt.) fr. 2 2 2 2 Cl Cl 2 2 -But. fr. (200 mg/kg b.wt.) -But. fr. (400 mg/kg b.wt.) -But. fr. EtOAcfr. (200 mg/kg b.wt.) EtOAcfr. (400 mg/kg b.wt.) EtOAcfr. n n CH CH Rem. H Rem. H Continued. Statistically significant difference from zero time at time from zero difference significant Statistically Treatment Aq. ext. (200 mg/kg b.wt.) Aq. ext. (400 mg/kg b.wt.) Fractions of the aqueous extract Table 5. Table are expressed as Mean ± SE. Values * EtOAcfr.: ethyl acetate fraction; EtOH ext.: 70EtOH ext.: fraction; ethyl acetate EtOAcfr.: extract; % ethanol

931 Abdel Baki PM et al. / IJPR (2019), 18 (2): 922-937

Table 6. Total phenolic, flavonoid and saponin contents in the different extracts and fractions. Extract TPC* ± SE (GAE/100 g) TFC* ± SE (RE/100 g) TSC* ± SE (UAE/100 g)

70% Ethanol ext. a.p. 5.97±0.001 15.85 ± 0.001 16.83 ± 0.001

n-Hexane fr. 0.69 ± 0.001 2.25 ± 0.001 3.67 ± 0.001

Methylene chloride fr. 0.78 ± 0.001 2.75 ± 0.005 4.83 ± 0.002 Fractions of the 70% Ethyl acetate fr. (EA) 9.38 ± 0.004 39.75 ± 0.005 9.00 ± 0.005 ethanol extract of a.p. n-Butanolfr. 6.86 ± 0.001 13.30 ± 0.007 30.00 ± 0.001

Remaining water 1.38 ± 0.001 4.65 ± 0.001 27.00 ± 0.002

Aqueous ext. a.p. 5.63 ± 0.005 12.05 ± 0.004 31.50 ± 0.004

Methylene chloride fr. 2.68 ± 0.001 9.50 ± 0.006 5.33 ± 0.001

Fractions of the Ethyl acetate fr. 6.86 ± 0.001 11.15 ± 0.001 15.17 ± 0.001 aqueous extract of a.p. n-Butanolfr. 0.71 ± 0.001 2.50 ± 0.001 36.17 ± 0.003

Remaining water 0.69 ± 0.001 2.35 ± 0.001 31.67 ± 0.001 *average of three determinations; a.p.: aerial parts; ext.: extract; fr.: fraction; GAE:gallic acid equivalent; SE: standard error; TFC: total flavonoid content; TPC: total phenolic content; TSC: total saponin content.

extract. The most active EA showed the highest comparison with the available authentic samples total phenolic and flavonoid contents amongst and with the published data (51, 58-67). all the tested fractions of both extracts, whereas the n-butanol fraction of the aqueous extract was Discussion the highest in the saponin content (Table 6). Preliminary phytochemical screening of the Purification of the phenolic and flavonoid air-dried flowering aerial parts of S. canadensis rich fraction (EA) deduced that the main constituents of the plant The quantitative determination of the major were essential oils, free and combined flavonoids, constituents in the different fractions and and saponins. Guided by the available literature correlating their relative concentrations to their and the results of the phytochemical screening, diuretic activities, revealed that the total phenolics it was found interesting to study the main and flavonoids may be responsible for this active constituents of the plant and the solvent activity. EA was subjected to the purification and of choice for the extraction of each class of isolationof its major constituents as it possessed constituents knowing that the essential oil of the the highest diuretic activity and was standardized plant was previously investigated by our group to contain the highest amount of total phenolics (35). Higher yield of total phenolic compounds and flavonoidsamong other fractions. Thymidine and flavonoids was achieved by extraction with (1), isorhamnetin-3-O-β-ᴅ-glucopyranoside (2), 70% ethanol rather than water. This was in narcissin (3), kaempferol-3-O-(6”-O-acetyl)- accordance with the previous reports of Apati β-ᴅ-glucopyranoside (4), quercetin-3-O-(6”- et al. 2002 (39). EA showed the highest diuretic O-acetyl)-β-ᴅ-glucopyranoside (5), rutin (6), activity as well as the highest total phenolic kaempferol-3-O-β-ᴅ-apiofuranoside (7), caffeic and flavonoid contents relative to the other acid (8), neochlorogenic acid (9), quercetin (10), fractions, this made it the most proper candidate and kaempferol (11) were isolated from the for further phytochemical investigations. ethyl acetate fraction (Figure 1). Identification Optimum extraction of saponins was achieved of the isolated compounds was achieved by by water rather than 70% ethanol. The saponins their physicochemical and spectral data, and by were concentrated in the n-butanolfraction of the

932 Aquaretic activity of Solidago canadensis L.

Compound 1

Compound 3 Compound 2 Compound 1

CompoundOH 3 Compound 2 Compound 3 Compound 1 3` OH 2` 4` 1 8 Compound1` OH 3 Compound 2 HO O OH 5` 7 2 6`3` OH 3` OH 2` 2` 4` 6 3 4` 1 85 1 4 1`O OH HO 8 O 1` 5` HO O 5` 7 OH O 2 1`` 6`3` 7 2 6`H OH 2` O 4` 6 OH 6 3 1 H 3 85 4 1`O HO H 5 O 4 2``O 5` 5`` 7 OH O 2 H 1`` 6` 1```OH O 1`` H O 6`` H O H O 3``OH O 6 OH H 3 OH H 5 4 O H H H HO 2`` 2```H 2`` 5``OH OH O 5`` H 1`` Compound 5 5``` H 1``` H H Compound 4 1``` 6`` O 6`` O O OHH 6``` O H 3`` H 3```OH H 3`` H O OH H H H HO 2`` 4```H HO 2```H HO 2``` 5``OH OH H Compound 5 5```OH H 1``` Compound 4 Compound 5 5``` H O 6`` Compound 4 H 6``` 3`` 6``` O 3```OHH H H 3``` H H H HO 4``` HO 2``` 4``` HO OH Compound 5 Compound5```OH 6 Compound 4 OH H 6``` 3``` H H 4``` CompoundHO 6 CompoundOH 6

Compound 6

Compound 9 Compound 8

Compound 9 Compound 8

Compound 7 Compound 9 Compound 8

Compound 7 Compound 7

Compound 7

16

Compound 10 Compound 11 16

Figure 1. Structures of the isolated compounds.16 Figure 1. Structures of the isolated compounds.

933

Discussion

Preliminary phytochemical screening of the air-dried flowering aerial parts of S. canadensis deduced that the main constituents of the plant were essential oils, free and combined flavonoids, and saponins. Guided by the available literature and the results of the phytochemical screening, it was found interesting to study the main active constituents of the plant and the solvent of choice for the extraction of each class of constituents knowing that the essential oil of the plant was previously investigated by our group (35). Higher yield of total phenolic compounds and flavonoids was achieved by extraction with 70% ethanol rather than water. This was in accordance with the previous reports of Apati et al. 2002 (39). EA showed the highest diuretic activity as well as the highest total phenolic and flavonoid contents relative to the other fractions, this made it the most proper candidate for further phytochemical investigations. Optimum extraction of saponins was achieved by water rather than 70% ethanol. The saponins were concentrated in the n-butanolfraction of the aqueous extract.

Previous reports claimed that the flavonoids and saponins of the different Solidago species were responsible for the diuretic activity of the genus (7, 43 and 44). Others attributed the activity to their content of flavonoids and phenolics (6, 9 and 68). In our study, the highest diuretic activity of all tested samples was exhibited by the 70% ethanol extract. This high potency was presumably related to its high content of phenolics and flavonoids. This was further confirmed by the high diuretic activity of EA which possessed the highest

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Abdel Baki PM et al. / IJPR (2019), 18 (2): 922-937 aqueous extract. Purification of the active ethyl acetate fraction Previous reports claimed that the flavonoids (EA) yielded eleven compounds; thymidine (1), and saponins of the different Solidago species isorhamnetin-3-O-β-ᴅ-glucopyranoside (2), were responsible for the diuretic activity of narcissin (3), kaempferol-3-O-(6”-O-acetyl)- the genus (7, 43 and 44). Others attributed β-ᴅ-glucopyranoside (4), quercetin-3-O-(6”- the activity to their content of flavonoids O-acetyl)-β-ᴅ-glucopyranoside (5), rutin (6), and phenolics (6, 9 and 68). In our study, the kaempferol-3-O-β-ᴅ-apiofuranoside (7), caffeic highest diuretic activity of all tested samples acid (8), neochlorogenic acid (9), quercetin (10), was exhibited by the 70% ethanol extract. This and kaempferol (11). Compounds 1, 2, 4, 5 and high potency was presumably related to its high 7 were isolated for the first time from genus content of phenolics and flavonoids. This was Solidago. further confirmed by the high diuretic activity The spectral data of compound 1 showed of EA which possessed the highest phenolic and that the protons were assigned to the pyrimidine flavonoid contents among other fractions, the base thymine and deoxyribose sugar. The imino inactivity of the saponin rich n-butanol fraction proton of the thymine base appeared at δ 11.27 of the aqueous extract, as well as the poor activity ppm. The olefinic proton H-6 appeared at δ 7.70 of the essential oil. This was in accordance with ppm. The 3 protons of the methyl group at C-5 Apati et al. 2003 who stated that the flavonoids, appeared as a singlet integrated as 3H at δ 1.78 especially quercetin and its derivatives showed ppm. The H-2′, H-3′, H-4′, and H-5′ protons of a potential to inhibit the neutral endopeptidase the deoxyribose sugar appeared at the region enzyme, which is responsible for the interaction between 2.03 - 4.22 ppm. The anomeric proton of the atrial natriuretic peptide through the of the deoxyribose sugar appeared as a triplet at δ excretion of the sodium ions (68). Moreover, the 6.15 ppm. The 13C-NMR spectrum of compound flavonoid fractions of some previously studied 1 revealed the presence of ten carbon atoms in the Solidago species showed diuretic activities, and molecule. The 13C chemical shifts of a carbon at the diuretic actions of several plant species δ 12.72 indicated the presence of amethyl group were related to their flavonoid content (9, attached to C-5. The assignment of the carbons 69-71). The fractions of the 70% ethanol and of the pyrimidine base was determined using aqueous extracts proved to be less active than HMBC spectrum. Compound1 was identified the parent extracts, except for the ethyl acetate as thymidine. Compound 7 showed the signals fraction of the aqueous extract, suggesting the characteristic for a kaempferol nucleus and existence of additive and/or synergistic effects additional signals for a sugar moiety. The in the parent extracts. The 70% ethanol extract spectrum showed anomeric proton at 5.01 of the flowering aerial parts of S. canadensis ppm as a doublet with J= 3.7 Hz characteristic showed more powerful diuretic activity than for O-β-ᴅ-apiofuranose structure (63, 64, 72 the aquaretic drug Cystinol® (91% and 74% of and 73). 13C-NMR spectrum of compound furosemide activity, respectively) at the same 7 showed 18 carbon signals assigned to 20 dose level (400 mg/kg b.wt.) with a similar carbons, 13 of which assigned to kaempferol aquaretic property. It also showed a much (15 carbons) and 5 for apiose sugar. Signal of higher diuretic activity when compared to the C-3 at 133.10 δ ppm was shifted upfield by 2 potassium sparing diuretic spironolactone (91% ppm relative to kaempferol aglycone (74). This and 59% of furosemide activity, respectively) confirmed the 3-glycosylation of kaempferol. but without promoting the loss of sodium in Compound 7 was identified as kaempferol-3- urine. The tested 70% ethanol extract showed a O-β-ᴅ-apiofuranoside. 1H-NMR spectrum of comparable diuretic activity to the loop diuretic compound 9 showed the characteristic signals furosemide but without enhancing the loss of the for a caffeic acid molecule. Also the protons of electrolytes in urine (Na+ and K+). The increase a quinic acid moiety could be observed with a in urine volume without loss of electrolytes doublet at δ 1.73 (J = 12.9 Hz) assigned to H-6 showed that the tested samples were aquaretics, ax, a doublet of doublet at δ 1.90 ppm (J = 13.8, similar to the reference drug Cystinol®. 10.8 Hz) assigned to H-6 eq and a multiplet at

934 Aquaretic activity of Solidago canadensis L.

2.08 ppm integrated as two protons and assigned diuretics. Clin. Exp. Hypertens. A (1983) 5: 309-20. to H-2 and H-2 . A broad singlet at δ 3.82 and (4) Khan A, Bashir S and Gilani A. An in-vivo study on ax eq the diuretic activity of Holarrhena antidysenterica. a doublet at δ 3.87 ppm (J = 9 Hz) assigned to Afr. J. Pharm. Pharmacol. (2012) 6: 454-8. H-4 and H-5. The downfield shift of H-3 which (5) Yarnell E. Botanical medicines for the urinary tract. appeared at δ 5.01 ppm indicated the acylation World J. Urol. (2002) 20: 285-93. of the quinic acid by the caffeic acid at the OH (6) Thiem B, Wesolowska M, Skrzypczak L and on C-3 (75). The assignement of the protons of Budzianowski J. Phenolic compounds in two Solidago L. species from in-vitro culture. Acta Pol. Pharm. the quinic acid moiety was determined using (2001) 58: 277-81. 1 1 H- H COSY. Compound 9 was identified as (7) Skrzypczak L, Wesołowska M, Thiem B and neochlorogenic acid. Budzianowski J. Solidago L. Species (Goldenrod). In: Bajaj YPS. (ed.) In-vitro Regeneration and Conclusion Biologically Active Secondary Metabolites, Medicinal and Aromatic Plants XI, Biotechnology in Agriculture and Forestry, Vol 43. Springer, Berlin, Heidelberg Ethanol (70%) was the best solvent for (1999) 384. extracting phenolic compounds from Solidago (8) Osman AR and Sewedan E. Effect of planting density canadensis L., while water was the best solvent and gibberellic acid on quantitative and qualitative for the extraction of its saponins. The ethyl characteristics of Solidago canadensis “Tara” in acetate fraction of the 70% ethanol extract (EA) Egypt. Asian J. Crop Sci. (2014) 6: 89-100. (9) Chodera A, Dabrowska K, Sloderbach A, Skrzypczak of the flowering aerial parts possessed the highest L and Budzianowski J. Effect of flavonoid fractions total phenolic and flavonoid contents, as well as of Solidago virgaurea L. on diuresis and levels of the highest diuretic activity, amongst all tested electrolytes. Acta Pol. Pharm. (1991) 48: 35-7. fractions. A strong correlation existed between (10) Leuschner J. Anti-inflammatory, spasmolytic the total phenolic and flavonoid contents and the and diuretic effects of a commercially available investigated aquaretic activity of the different Solidagogigantea herb extract. Arzneimittel-Forsch. (1995) 45: 165-8. extracts and fractions. Thus, S. canadensis L. (11) Sung JH, Lee JO, Son JK, Park NS, Kim MR, Kim JG showed a pronounced aquaretic activity owing and Moon DC. Cytotoxic constituents from Solidago to its phenolic and flavonoid contents which virga-aurea var. gigantean Miq. Arch. Pharm. Res. was in accordance with Chodera et al., 1991 (1999) 22: 633-7. (9). Eight flavonoids, 2 phenolic acids and (12) Choi SZ, Choi SU and Lee KR. Pytochemical constituents of the aerial parts from Solidagovirga- 1 nucleoside were isolated and identified in aurea var. gigantea. Arch. Pharm. Res. (2004) 27: the most active ethyl acetate fraction (EA). 164-8. Four of them namely, isorhamnetin-3-O-β-ᴅ- (13) Thiem B and Goślińska O. Antimicrobial activity glucopyranoside (2), kaempferol-3-O-(6”-O- of Solidago virgaurea L. from in-vitro cultures. acetyl)-β-ᴅ-glucopyranoside (4), quercetin- Fitoterapia (2002) 73: 514-6. 3-O-(6”-O-acetyl)-β-ᴅ-glucopyranoside (5) (14) Morel AF, Dias GO, Potro C, Simionatto E, Stuker CZ and Dalcol II. Antimicrobial activity of extractives of andkaempferol-3-O-β-ᴅ-apiofuranoside (7) Solidago microglossa. Fitoterapia (2006) 77: 453-5. were isolated for the first time from genus (15) Starks CM, Williams RB, Goering MG, Johnson MO, Solidago, while the other isolated compounds Norman VL, Hu JF, Garo E, Hough GW, Rice SM were previously reported (31, 37-40). and Eldridge GR. Antibacterial clerodane diterpenes from Goldenrod (Solidago virgaurea). Phytochemistry (2010) 71: 104-9. References (16) Kolodziej B, Kowalski R and Kędzia B. Antibacterial and antimutagenic activity of extracts aboveground (1) Apati P. Antioxidant constituents in Solidago canadensis parts of three Solidago Speies: Solidago virgaurea L., L. and its traditional phytopharmaceuticals. Doctoral Solidago canadensis L. and Solidago gigantean Air. J. School of Pharmaceutical and Pharmacological Med. Plants Res. (2011) 5: 6770-9. Science, Ph. D. thesis. Budapest, Hungarian Academy (17) Goulart S, Moritz MIG, Lang KL, Liz R, Schenkel of Sciences (2003) 1. EP and Fröde TS. Anti-inflammatory evaluation (2) Dutta KN, Chetia P, Lahkar S and Das S. Herbal plants of Solidago chilensis Meyen in a murine model of used as diuretics: A comprehensive Review. Res. J. pleurisy. J. Ethnopharmacol. (2007) 113: 346-53. Pharm. Biol. Chem. Sci. (2014) 2: 27-32. (18) Tamura EK, Jimenez RS, Waismam K, Gobbo-Neto (3) Losse H, Zumkley H and Quante T. Side effects of L, Lopes NP, Malpezzi-Marinho EA, Marinho EA and

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