Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata DOI: https://doi.org/10.31351/vol29iss2pp48-61 48 Isolation, Structural Characterization and Identification of Major Constituents in Ephedra foliata Naturally Growing in Iraq by TLC, GC- MS and UPLC-ESI-MS/MS Ahmed S. Khaleefa*,1 and Maha N. Hamad** * Ministry of Health and Environment, Baghdad,Iraq. **Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad. Iraq Abstract The aerial part of Ephedra foliata Family Ephedraceae have long been used in traditional medicine and now Ephedra species have medicinal, ecological, and commercial value. The variety of pharmacological actions of this plant is due to its chemical constituents. Ephedrine and related alkaloids; are the new potential medicinal value of Ephedra supplements for weight loss or performance improvement. Other pharmacological actions like antibacterial and antifungal effects of the phenolic acid compounds, the immunosuppressive action of the polysaccharides, and the antitumor action of flavonoids. The genus of this plant wildly distributed throughout Asia, America, Europe, and North Africa. The study is aimed at screening the phytochemical constituents due to the importance of pharmacological actions of this plant. That is done by maceration the aerial part of Ephedra foliata with 80% ethanol for 9 days and fractionated by n-hexane, chloroform, ethyl acetate, and n-butanol. The n-hexane, chloroform, n-butanol fractions, and isolated compounds were analyzed by gas chromatography-mass spectrometry, thin layer chromatography; ultra-performance liquid chromatography coupled with electrospray ionization mass/ mass spectroscopy. The various chromatographic and spectroscopic results indicate the presence of a different type of phytochemicals like ephedrine, 6-hydroxy kynurenic acid, vicenin 2 and quercetin 3-sophoroside-7-rhamnoside. These active constituents of Ephedra foliata have been identified play a crucial role in our life due to its pharmacological actions. Keywords: Ephedra, Gas chromatography, Mass spectrometry, Ultra-performance liquid chromatography electrospray ionization mass/ mass , 6-hydroxy kynurenic acid. نمو بشكل طبيعي في ي ذيال نبات العلندىالمكونات الرئيسية في و تشخيص التوصيف الهيكلي ,العزل كروماتوغرافيا الغازو و كروماتوغرافياو الرقيقهبواسطه كروماتوغرافيا الطبقه العراق من خالل عالية االداء السائله **و مها نوري حمد 1*،احمد سعدي خليفة العراق ،وزارة الصحة والبيئة، بغداد* ، العراق .فرع العقاقير والنباتات الطبية ، كلية الصيدلة ، جامعة بغداد** الخالصة مجموعة .الهوائية لنبات االفيدرا في الطب القديم واالن نبات االفيدرا لديها فوائد دوائية وبيئية و تجاريةمنذ فترة طويلة تستخدم االجزاء قلويدات ذات صلة بها؛ لديها القيمة الطبية االفيدرين وال .متنوعة من االستعماالت الدوائية لهذا النبات ويرجع ذلك إلى مكوناته الكيميائية المختلفة االستخدامات الدوائية األخرى مثل التأثيرات المضادة للبكتيريا والفطريات لمركبات .ا لمكمالت اإليفيدرا لفقدان الوزن أو تحسين األداءالمحتملة حديث في جميع أنحاء آسيا بكثرةجنس هذا النبات الموزع .حمض الفينول ، المثبط للمناعة من السكريات ، والعمل المضاد للورم من مركبات الفالفونويد يتم ذلك .الدوائية لهذا النبات ستعمالتالدراسة إلى فحص المكونات الكيميائية النباتية بسبب أهمية اإل هذة تهدف .وأمريكا وأوروبا وشمال إفريقيا الهكسان ، والكلوروفورم ، وخالت بواسطةتجزيئة من ثمأيام و 9٪ لمدة 80مع اإليثانول بنسبة نبات االفيدرامن هوائيةء الاجزاال تنقيععن طريق و كروماتوغرافيا الطبقه الرقيقه الهكسان ، الكلوروفورم ، بيوتانول ، والمركبات المعزولة من قبلاجزاءوقد تم تحليل وتانول.باإليثيل ، و من المواد ة نوع مختلفاتشير النتائج الكروماتوجرافية والطيفية المختلفة إلى وجود .كروماتوغرافيا عالية االداء السائلهالغازو و كروماتوغرافيا . تم التعرف على سيفروسايد -7رامنوسايد -3ستين ور، والكي 2هيدروكسي ، والفينسينين -6الكيميائية النباتية مثل اإلفيدرين ، وحمض الكينورينيك .الدوائية فعاليتهالعب دورا حاسما في حياتنا بسبب ت نبات االفيدرا التي هذه المكونات النشطة من هيدروكسي.-6حمض الكينورينيك ، كروماتوغرافيا عالية االداء السائله ،الغازو كروماتوغرافياالكلمات المفتاحية: اإلفيدرا ، 1Corresponding author E-mail: ameerzayona88@gmail.com Received: 14/10 /2019 Accepted: 10/3 /2020 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol29iss2pp48-61 Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 49 For at least five thousand years, ephedra plants have been used in traditional medicines in which dry stems are used for symptoms derived from the common cold, flu, asthma, bronchitis, nasal congestion and hay fever(1). The ephedra plant is also used for the treatment of arthritis, fever, hives, dyspnea, headache, joint and bone pain, wheezing and hypotension(2). Ephedra corresponds to a genus of gymnosperms including over 50 species of tropical and subtropical, small, much-branched shrubs founds in the dry regions of both hemispheres(3). It is related to the Gnetophyta division of gymnosperms plants and is related to the conifers(4). The plant species are short, evergreen and virtually leafless shrubs that grow about (60to90cm) tall. The stems are green in color, slender, erect, small ribbed and channeled, about (1.5 mm) in diameter and commonly terminating in a sharp point. Nodes are (4to6 cm) apart, and small triangular leaves appear at the stem nodes which are usually reddish brown(5). These species grow in dry weather over wide parts of the Northern hemisphere including North America, Europe, North Africa, and Southwest and Central Asia(6). The chemical constituents and pharmacological actions of Ephedra species The aerial parts of various plant species first of all, ehedrine-type alkaloids, usually have from (0.02% to 3.4%) of six optically active alkaloids as shown in Figure 1,(−)-Ephedrine (EPH) is the major one including 30–90% of the total alkaloids, (+)-Pseudoephedrine (PSE), the diastereomer of (−)-EPH, (−)-N-Methylephedrine, (−)-Norephedrine , (+)-N-Methylpseudoephedrine and (+)-Norpseudoephedrine (7) . Secondly, non- ephedrine alkaloids and amino compounds in Ephedra species. Ephedroxane(8), Ephedradine A(9), cyclopropyl-α-amino acid(10), maokonine(11), 6- methoxykynurenic acid(12), N- methylbenzylamine(13), Tertmethylpyrazine(14), and 6-hydroxykynurenic acid(10). Thirdly, Miscellaneous Non-alkaloidal Natural Constituents of Ephedra: trans-cinnamic iacid, icatechin, isyringin, iepicatechin, symplocoside, ikaempferol i3-O- rhamnoside i7-O-glucoside, iisovitexin i2-O- rhamnoside, iherbacetin i7-O-glucoside, iand ipollenitin iB iand iherbacetin 7-O- neohesperidoside (15). Ephedra species have numerous pharmacological actions for instance anti- inflammatory due to the inhibition of prostaglandin E2 biosynthesis(8), antibacterial and antifungal (16), anti-cancer activity(17)(18), CNS stimulant and perhaps thermogenic effects (19), antiviral activity(20) and finally antioxidant and hepatoprotective activity(21). This study was designed to investigate the phytochemicals and their proportions of the aerial part of Ephedra foliata naturally growing plants in Iraq. Figure 1. Ephedrine- type alkaloids Material and methods Collection of plant materials: Ephedra foliata was collected during March – June 2018 from Tikrit province, Iraq. This plant was authenticated by Dr. khansaa rasheed / Iraq Natural History Research Center and Museum Plant and Environment Department / University of Baghdad. The stems and aerial parts were, dried in a shed, rendered into a coarse powder. Extraction Extraction by maceration then fractionation according to active constituents. About 650 grams of aerial part the powdered plant material was soaked in 2500ml (1:7) 80% ethanol, with regular shaking, at room temperature. After 3 days, the ethanol extracts are filtered, repeat the process 3 times for 9 days. The filtrate was evaporated to dryness under vacuum using a rotary evaporator, to get dried extract. The dark greenish residue was suspended in 250ml H2O and partitioned successively with n-hexane, chloroform, ethyl acetate, and n-butanol until reaching a clear layer for each fraction. The first three fractions are dried over anhydrous sodium sulfates, filtered, and evaporated to dryness(22). Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 50 Phytochemical investigation of chemical constituents of Iraq Ephedra foliata: Preliminary identification by chemical test: 1-Test for alkaloids:  Mayer’s reagent.  Wagner’s reagent 2-Test for flavonoids About 5% alcoholic potassium hydroxide is added and then a few drops of 5% hydrochloric acid are added. 3-Test for phenols Few imilligrams of ethanol plant extract are treated with ifew drops of 1% ferric chloride(23). Purification of crude alkaloidal extract: The chloroform fraction was acidified by adding hydrochloric acid (5%). This solution was then placed in a separatory funnel and partitioned with equal volume of chloroform (four times). The upper aqueous acidic layer was separated and basified with ammonium hydroxide (25% NH4OH) to PH 10 using PH meter. After the basification process, the solution becomes warm and allowed to stand for 2 hours. Then partitioned with an equal volume of chloroform in a separatory funnel (three times). The chloroform ilayer iwas iseparated, dried with anhydrous sodium sulfate, filtered and evaporated under reduced pressure then tested with Dragendorff and Mayer’s reagents(24). Isolation of some phytochemicals by using preparative TLC Thin-layer chromatography was used to determine phytochemical compounds by using different solvent systems like chloroform; methanol (90: 1), Chloroform: acetone: formic acid (75: 16.5: 8.5) and Ethyl acetate: formic acid: acetic acid: water (80:5: 6: 10) for n-butanol fraction(25). While toluene-chloroform-ethanol-methanol (20:50:30:10), ethyl acetate-isopropanol-ammonia (100:2:1) and cyclohexane-ethanol-dietllyamine (80:10:10) for chloroform fraction that were tried for identification to get the best separation and the largest number of spots (26).  AS1 compound was isolated from n-butanol fraction using readymade preparative TLC silica gel GF254 plates and mobile phase Ethyl acetate: formic acid: acetic acid: water (80:5: 6: 10) (25). Detection of the AS1 compound was done by examination under UV light with wavelengths, 254 and 366 nm.  AS2 compound tertiary amine alkaloid was isolated from purified chloroform fraction after basification using readymade aluminum oxide on TLC-glass plates and mobile phase toluene- chloroform-ethanol-methanol (20:50:30:10 ) (26). Detection of the AS2 compound was done using Dragedorff,s spray reagent is detected as a brown zone.  The purity of each bands are verified by analytical TLC until a single point are obtained on the TLC plates for identification. Identification and structural characterization of isolated compounds and phytochemicals in fractions were done by I-GC-MS analysis The conditions used in the GC / MS analysis are compatible with the thermal desorption system (TD-20), GC / MSQP / 2010 Plus (Shimadzu, Japan) composed of an automatic sampler. The mass spectrometer instrument was connected. Column RTX-5MS (30 mm × 0.25 mm × 0.25 µm), operating in electronic printing mode at 70 eV. In this instrument, (99.99%) of helium gas is used as a carrier gas with a movement frequency of (1.2 ml / min). The initial temperature of the column oven is 80 ° C (isothermal for four minutes) with a constant increase from (5 ° C / min to 310 ° C), flow rate of (1.21 ml / min) and column pressure of 81, 7 kPa In the scanning interval of 0.50 s, the mass spectrum is prepared with a mass scan of (40to650) m /z(27). II-Ultra performance liquid chromatography- electrospray ionization mass/ mass spectrometry (UPLC-ESI-MS/MS) analysis Electrospray ionization mass spectrometry in negative and positive ions acquisition mode is performed in XEVO TQD triple quadruple instruments. Water Corporations, Milford, MA01757 USA UU. The sample solution (100 μg / ml) is prepared using high-performance liquid chromotography (HPLC) analytical grade methanol, the filtrate uses a membrane disk filter (0.2 μm), then subjected to LC / ESI / MS. The sample injection volume (10 μL) is injected into the UPLC instruments Equipped with C-18 reverse phase columns (ACQUITYS UPLC / BEH C18 Particle size of 1.7µm-2.1 ×50mm column). The mobile phase is prepared by filtration using a 0.2μm filter membrane disk and degassed by sonication before injections. The elution of the mobile phase is carried out with a flow rate of 0.2 ml per minute using a mobile gradient phase which includes two eluents: the eluent A is acidified in water with 0.1% of HCOOH and the eluent B is methanol acidified with 0.1% of HCOOH. The elution is performed using the gradient. The parameters for the analysis are performed using the negative ion mode as follow:150° C source temperature, 30eV cone voltage, 3kV capillary voltage, desolvation temperature about (440 ° C, 50L / h) cone gas flow and desolvation gas flow of (900L / h)(28). Mass spectra are detected in electrospray ionization between m/ z (100–1000). Peaks and spectra are processed using Maslynx (4.1) software and are tentatively identified by comparing their retention times and masses spectra with the reported data (27). https://www.sigmaaldrich.com/catalog/product/sial/90066?lang=en®ion=IQ https://www.sigmaaldrich.com/catalog/product/sial/90066?lang=en®ion=IQ Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 51 Results Phytochemical investigation of chemical constituents of Ephedra foliata: 1-Preliminary identification by chemical test: Various qualitative phytochemical screening tests were done to establish the chemical composition of each extract shown in Table1. Tabe1. Phytochemical screening tests for crude extract Phytochemical test Type of phytochemical Results Mayer’s Alkaloids + Wagner’s Alkaloids + KOH Flavonoids + Fecl3 Phenols + 2-Thin layer chromatography TLC (analytical and preparative): According to TLC results which are shown below A1 and A2 were found the best mobile phases for separation and isolation of AS1 and AS2 respectively as result shown below. Figure 2. TLC for chloroform fraction before basify (1), after basify (2) and pseudoephedrine standard (S) developed with A2 solvent system, at 254 nm and after Dragedorff,s spray reagent. Figure 3. preparative TLC for isolated AS2 from chloroform fraction after basify with developed the A2 solvent system with Dragendorff reagent. Figure 4. TLC of n-butanol fraction before hydrolysis with different titration under UV 253nm and 366nm. Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 52 Figure 5. Preparative TLC of n-butanol fraction before hydrolysis with different titration under UV366 nm to isolate AS1. Figure 6. Preparative TLC of n-butanol fraction before hydrolysis with different titration under UV 254 nm to isolate AS1. 3-Gas chromatography mass spectrometry GC/MS: A.GC/MS of n-hexane fraction: identification of phytochemical compounds in n-hexane fraction by gas chromatography mass spectrometry. Figure 7. GC/MS of n-hexane fraction. Table 2. Compounds identified in n-hexane fraction by GC/MS. NO. of Peaks Retention time (R.t) name base peak 1 31.725 1-Octadecyne 41.00 2 34.885 n-Heptadecanol-1 43.10 3 34.948 Hexadecanoic acid, ethyl ester 88.05 4 35.966 Hexadecanoic acid, trimethylsilyl ester 73.00 5 38.430 1-Methyl-1-(2-tridecyl)oxy-1- silacyclopentane 143.15 6 38.681 1-Octadecene 43.05 7 44.870 Di-n-octyl phthalate 149.00 8 52.711 17-Pentatriacontene 43.00 9 56.446 gamma.-Sitosterol 43.05 10 59.144 Stigmast-4-en-3-one 43.00 B. GC/MS of chloroform fraction: identification of phytochemical compounds in chloroform fraction by gas chromatography mass spectrometry. Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 53 Figure 8. GC/MS for chloroform fraction. Table 3. Compounds identified in chloroform fraction GC/MS. No. of peak Retention time Name M.WT Base peak 1 20.663 3,4-dimethyl-5-phenyl-2- oxazolidinone 191 57.05 2 20.958 1-Undecene 154 41.05 3 21.175 Ephedrine 165 58 4 24.257 Phenol, 3,5-bis(1,1-dimethylethyl)- 206 191.05 21 34.537 Aziridine, 1,2-dimethyl-3-phenyl-, trans 147 146 33 38.194 Linoleic acid ethyl ester 196 57.05 58 48.991 Squalene 410 69 C. GC/ MS for isolated AS2: identifiation of isolated AS2 compound form chloroform fraction by gas chromatography mass spectrometry. Figure 9. GC MS for isolated AS2 compound from chloroform fraction after basify. Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 54 Figure 10. Fragmentation and structure elucidation of isolated AS2 compound by GC/MS. Table 4. Isolated AS2 compound identified by GC MS(29-30) No. of peak Retention time Name Area % M.WT Base peak 2 20.818 Ephedrine 80.7 165 58.05 4- Ultra-performance liquid chromatography electrospray ionization mass/ mass ((UPLC-ESI- MS/MS): Identification of the results from UPLC-ESI-MS / MS depended on molecular weight, retention time and mass fragmentation through different sites specialized in the identification and confirms the result of a search with previous studies. A. UPLC for isolated AS2: identifiation of isolated AS2 compound form chloroform fraction by ultra- performance liqiud chromatography Figure 11. UPLC for isolated AS2 from chloroform fraction after basification. Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 55 Figure 12. First mass for isolated AS2 peak 1 at retention time 4.1min with major molecular ion [M+H]+ 166.093. Figure 13. Mass fragmentations for isolated AS2 compound. Table 5. UPLC ESI MS/MS for isolated AS2 compound peak no. of MS1 R.T [M+H] Peak no. of MS2 R.T Base peak Name Reference 1 4.1 166 14 3.93 148.0392 Ephedrine (31-32) Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 56 Figure 14. Structural elucidations of AS2 fragmentations (31)(32). B. UPLC for isolated AS1: identifiation of isolated AS1 compound form n-butanol fraction by ultra- performance liqiud chromatography Figure 15. UPLC for isolated AS1 compound from n-butanol fraction before hydrolysis at peak 3. Figure 16. First mass for isolated AS1 compound peak 3 at retention time 3.92 min with major molecular ion [M-H]- 204.0892 Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 57 Figure 17. Mass fragmentation for isolated AS1 compound. Table 6. UPLC ESI MS/MS for isolated AS1 compound Figure 18. Structural elucidations of AS2 fragmentations(33). C. UPLC n-butanol fraction: identification of phytochemical compounds in n-butanol fraction by ultra-performance liqiud chromatography Figure 19. UPLC for n-butanol fraction. peak no. of MS1 R.T1 [M-H] Peak no. of MS2 R.T2 Base peak Name Reference 3 3.92 204.0892 10 4.04 159.9218 6-hydroxykynurenic acid (33-10-34-35) Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 58 Figure 20. First mass for peak 6 at retention time 7.29 min with molecular ion [M-H] 593.1935 from n- butanol. Figure 21. First mass for peak 9 at retention time 7.99 min with molecular ion [M-H] 771.2651 from n- butanol fraction. Table 7. Identified compounds by UPLC-ESI-MS/MS fragmentation of n-butanol fraction: Peak no. Compound name Class Rt.1 M.W MS1 M-H Rt.2 MS/MS References 6 vicenin 2 Flavonoid glycosides 7.29 594 593.19 31 7.58 593,575, 565,533, 503,475,459,445,431,38 2,353, 311,105,87,73 (36-37-38) 9 Quercetin 3- sophoroside- 7- rhamnoside Flavonoid glycosides 7.99 772 771.26 51 7.3 771,505,461,447,341,30 1,299,271,253,179,161,1 47,133,103,73,59,43 (39-40-41) 3 6-hydroxy kynurenic acid quinoline-2- carboxylic acid 3.32 205 204.08 92 3.8 204,176,159.9, 158,132.9,117.9 (33-10-34-35) Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 59 Discussion Natural products have always been a preferred choice of all as it plays a great role in discovering new medicines. The Hexane fraction of the plant was investigated by GC-MS which revealed the presence of gamma.-Sitosterol and Stigmast-4-en-3-one, the chromatogram showed peaks with retention times (56.446 and 59.114) respectively and corresponding to the molecular ion peaks in comparison with NIST database as shown in (Figure7, Table 2). The chloroform fraction of the plant was investigated by TLC, GC MS and UPLS- ESI- MS/MS which showed the presence of a different type of secondary metabolites like alkaloids and triterpene. AS2 compound isolated from chloroform fraction after basify by alumina TLC Plates investigated as ephedrine due to its results. First of all, analytical TLC gives a brown zone with Dragedorff,s spray reagent as shown in (Figure 2-3). Furthermore, the GC MS result showed the presence of ephedrine in chloroform fraction at peak 3 (Figure 8 and Table 3) also, isolated AS2 investigate by GC MS as ephedrine according to its retention time, molecular weight [165] and base peak [58] depending on NIST database (Figure 9,10 ,Table 4). Finally, UPLS-ESI- MS/MS characterized AS2 compound as ephedrine according to its retention time, Molecular ion peak at m/z 166 [M+H]+ and mass fragmentation show loss of water [M+H-H2O] to give 148(base peak), then [M+H-CH3] yield m/z 132 and loss of methyl group from nitrogen atom yield m/z 117 (Figure 11- 12-13-14, Table 5)(31). The n-butanol fraction was investigated by TLC and UPLS-ESI- MS/MS which showed the presence of flavonoid glycosides which play important anticancer activity (Figure 19-20-21, Table7). AS1 compound was isolated from n- butanol fraction by preparative TLC recognized as 6-hydroxykynurenic acid since it is given under ultraviolet light at 254 nm reddish-white fluorescence and 366 nm strong fluorescence and a very small amount could be detected(42). Besides UPLS-ESI- MS/MS results of AS1 compound and n-butanol fraction predicted 6-hydroxykynurenic acid depending on its retention time, molecular ion peak at m/z 204.0892 [M-H]- and mass fragmentation suggesting the loss of 44 Da [M - H -44]- to give m/z 159.9(base peak) in comparison with mass bank database. Beside, 6-hydroxykynurenic acid was previously isolated from Ephedra foliata (Figure 15- 16- 17-18, Table 6) (33-10-34-35). Conclusion The results of the current study showed isolate ephedrine from chloroform fractions after purification. While 6-hydroxykynurenic acid presence in n-butanol fraction due to its acidity. The active components of E. foliata have been identified play a crucial role in our life due to its pharmacological actions. References 1. Morton JF. Major medicinal plants: Botany, culture, and uses. Charles C. Thomas, Springf. 1977;111:80. 2. Yakubu MT, Bilbis LS, Lawal M, Akanji MA. Evaluation of selected parameters of rat liver and kidney function following repeated administration of yohimbine. Biokemistri. 2003;15(2):50–6. 3. LaFeber W, Abbott B. America, Russia, and the Cold War, 1945-1975. Wiley; 1972. 4. Rydin C, Korall P. Evolutionary relationships in Ephedra (Gnetales), with implications for seed plant phylogeny. Int J Plant Sci. 2009;170(8):1031–43. 5. Blumenthal M, King P. Ma huang: ancient herb, modern medicine, regulatory dilemma. HerbalGram (USA). 1995; 6. Caveney S, Charlet DA, Freitag H, Maier-Stolte M, Starratt An. New Observations On The Secondary Chemistry Of World Ephedra (Ephedraceae ). Am J Bot. 2001;88(7):1199– 208. 7. Leung AY. Ephedrine, Ephedra, Mahuang, Mahuanggen-What are They. In: AHPA Ephedra international symposium, Arlington, VA. 1999. 8. Kasahara Y, Hikino H, Tsurufuji S, Watanabe M, Ohuchi K. Antiinflammatory actions of ephedrines in acute inflammations. Planta Med. 1985;51(04):325–31. 9. Hikino H, Kiso Y, Ogata M, Konno C, Aisaka K, Kubota H, et al. Pharmacological Actions of Analogues of Feruloylhistamine, an Imidazole Alkaloid of Ephedra Roots1. Planta Med. 1984;50(06):478–80. 10. Starratt AN, Caveney S. I .Quinoline -2- carboxylic acids from Ephedra species . Phytochemistry .1996;42(5):1477–1478. 11. Tamada M, Endo K, Hikino H. Maokinine, hypertensive principle of Ephedra roots [drug plants]. Planta Medica (Germany, FR). 1978; 12. Nawwar MAM, Barakat HH, Buddrust J, Linscheidt M. Alkaloidal, lignan and phenolic constituents of Ephedra alata. Phytochemistry. 1985;24(4):878–9. 13. Chen AL, Stuart EH, Chen KK. The occurrence of methylbenzylamine in the extract of Ma Huang. J Am Pharm Assoc. 1931;20(4):339– 45. 14. Khan IA, Abourashed EA. Leung’s encyclopedia of common natural ingredients: used in food, drugs and cosmetics. John Wiley & Sons; 2011. 15. Amakura Y, Yoshimura M, Yamakami S, Yoshida T, Wakana D, Hyuga M, et al. Characterization of phenolic constituents from Ephedra herb extract. Molecules. 2013;18(5):5326–34. 16. Khan A, Jan G, Khan A, Gul Jan F, Bahadur A, Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 60 Danish M. In vitro antioxidant and antimicrobial activities of Ephedra gerardiana (root and stem) crude extract and fractions. Evidence-Based Complement Altern Med. 2017;2017. 17. Oshima N. Efficient Preparation of Ephedrine Alkaloids-free Ephedra Herb Extract and Its Antitumor Effect and Putative Marker Compound. Yakugaku zasshi J Pharm Soc Japan. 2017;137(2):173–7. 18. Hyuga S, Hyuga M, Oshima N, Maruyama T, Kamakura H, Yamashita T, et al. Ephedrine alkaloids-free Ephedra Herb extract: a safer alternative to ephedra with comparable analgesic, anticancer, and anti-influenza activities. J Nat Med. 2016;70(3):571–83. 19. Murray MT. The Healing power of herbis: The enlightened person’s guide to the worders of medicinal plants. California, US: Prime Publishing; 1995. 20. Murakami T, Harada H, Suico MA, Shuto T, Suzu S, Kai H, et al. Ephedrae herba, a component of Japanese herbal medicine Mao- to, efficiently activates the replication of latent human immunodeficiency virus type 1 (HIV-1) in a monocytic cell line. Biol Pharm Bull. 2008;31(12):2334–7. 21. Al-Rimawi F, Abu-Lafi S, Abbadi J, Alamarneh AAA, Sawahreh RA, Odeh I. Analysis of phenolic and flavonoids of wild Ephedra alata plant extracts by LC/PDA and LC/MS and their antioxidant activity. African J Tradit Complement Altern Med. 2017;14(2):130–41. 22. Wu C, Wang F, Liu J, Zou Y, Chen X. A comparison of volatile fractions obtained from Lonicera macranthoides via different extraction processes: ultrasound, microwave, Soxhlet extraction, hydrodistillation, and cold maceration. Integr Med Res. 2015;4(3):171–7. 23. Gul R, Jan SU, Faridullah S, Sherani S, Jahan N. Preliminary Phytochemical Screening , Quantitative Analysis of Alkaloids , and Antioxidant Activity of Crude Plant Extracts from Ephedra intermedia Indigenous to Balochistan. 2017;2017(Figure 1). 24. Kumar S. Asian Journal of Pharmaceutical Science & Technology Alkaloidal Drugs - A Review. 2014;4(3):107–19. 25. Performance H. Chromatographic Fingerprint Analysis of Herbal Medicines. Vol. 5, Chromatographic Fingerprint Analysis of Herbal Medicines. 2011. 26. Edition S. " SIS, Characterization of phenolic constituents from Ephedra herb extract. 27. Yang FQ, Li SP, Zhao J, Lao SC, Wang YT. Optimization of GC–MS conditions based on resolution and stability of analytes for simultaneous determination of nine sesquiterpenoids in three species of Curcuma rhizomes. J Pharm Biomed Anal. 2007;43(1):73–82. 28. Hassan WHB. Uplc-Pda-Esi-Ms / Ms Analysis , Isolation Of Chemical Constituents , Cytotoxic , Antioxidant , Antiviral And Antimicrobial Activities Of The Aerial Parts Of Lycium Shawii. 2017;(October). 29. index @ webbook.nist.gov. Available from: https://webbook.nist.gov/30. 30. (+)-Ephedrine @ www.restek.com. Available from: https://www.restek.com/compound/view/321- 98-2/(+)-Ephedrine 31. Bijlsma L, Sancho J V., Hernández F, Niessen WMA. Fragmentation pathways of drugs of abuse and their metabolites based on QTOF MS/MS and MS E accurate-mass spectra. J Mass Spectrom. 2011;46(9):865–75. 32. Fernández M del MR, Samyn N. Ultra- performance liquid chromatography- tandem mass spectrometry method for the analysis of amphetamines in plasma. J Anal Toxicol. 2011;35(8):577–82. 33. Macnicol PK. Isolation for 6-hydroxykynurenic acid from the tobacco leaf. Biochem J. 1968;107(4):473–9. 34. Shen Z, He K, Xu M, Zeng K, Pan J, Ou F, et al. Development and validation of a sensitive LC-MS/MS method for the determination of 6- hydroxykynurenic acid in rat plasma and its application to pharmacokinetics study. J Chromatogr B Anal Technol Biomed Life Sci. 2019;1116(February):44–50. Available from: https://doi.org/10.1016/j.jchromb.2019.03.033 35. HMDB0033528 @ www.hmdb.ca .Available from: http:/ /www .hmdb .ca/ metabolites / HMDB0033528 36. index @ spectra.psc.riken.jp . Available from: http://spectra.psc.riken.jp/ 37. Hassan WHB, Abdelaziz S, Al Yousef HM. Chemical Composition and Biological Activities of the Aqueous Fraction of Parkinsonea aculeata L. Growing in Saudi Arabia. Arab J Chem [Internet]. 2019;12(3):377–87. Available from: https://doi.org/10.1016/j.arabjc.2018.08.003 38. Hussein SAM, Barakat HH, Nawar MAM, Willuhn G. Flavonoids from Ephedra aphylla. Phytochemistry. 1997;45(7):1529–32. 39. 536f34b50fc643530879518ec737d8d18ac4913 5 @ cfmid.wishartlab.com [Internet]. Available from: http://cfmid.wishartlab.com/queries/536f34b50 fc643530879518ec737d8d18ac49135 40. Trapero A, Ahrazem O, Rubio-Moraga A, Jimeno ML, Gómez MD, Gómez-Gómez L. Characterization of a glucosyltransferase enzyme involved in the formation of kaempferol and quercetin sophorosides in Crocus sativus. Plant Physiol. Iraqi J Pharm Sci, Vol.29(2) 2020 Identification of major constituents in Ephedra foliata 61 2012;159(4):1335–54. 41. index @ cfmid.wishartlab.com [Internet]. Available from: http://cfmid.wishartlab.com/ 42. Roy Jk, Price Jm. The identification of quinoline derivatives obtained from the urine of normal rabbits and swine. J Biol Chem. 1959;234:2759–63. Baghdad Iraqi Journal Pharmaceutical Sciences by bijps is licensed under a Creative Commons Attribution 4.0 International License. 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