Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Cupressus sempervirens DOI: https://doi.org/10.31351/vol31issSuppl.pp121-130 121 Isolation and Structural Characterization of Quercetin 3-O-Rhamnoside and Essential oil Estimation from Leaves of Iraqi Cupressus sempervirens L (Conference Paper )# Amani Amer Tawfeeq*,1 and Shatha H Ali ** # 10th scientific conference sponsored by College of Pharmacy, University of Baghdad 2-3 June 2022 *Department of Pharmacognosy and Medicinal Plants, Baghdad College of Medical Sciences, Baghdad, Iraq ** Department of Clinical Laboratory Science, College of Pharmacy, University of Baghdad, Baghdad, Iraq Abstract Cupressus sempervirens L., Cupressaceae, which is known as evergreen cypress, Mediterranean cypress, and in Arabic called “al -Sarw. It is evergreen, has a medium-sized, and longevity, and is widely distributed over the world. The plant represents an important member of conifer plants which are characterized by aromatic leaves and cones. Cupressus sempervirens have been ethnobotanicals as an antiseptic, relief of cough, astringent, antispasmodic, wound healing, and anti-inflammatory. The aims of this work are phytochemical analysis, isolation, and structural identification of quercetin 3-O-rhamnoside (quercitrin) and essential oil in Iraqi C. sempervirens. Isolation of quercitrin was performed using a semi preparative HPLC from n.butanol fraction and extracted from Cupressus sempervirens leaves using ultrasound probe extraction, the structural identification of isolated quercitrin done by TLC, HPLC, UV spectrophotometry, FT-IR characterization according to a variety of frequency ranges, and LC-MS/MS revealed a molecular ion at 448 m/z and base peak m/z 301. Furthermore, isolation of essential oil using hydro-distillation and estimated by GC-MS shows a good essential oil yield of 0.9% with an interesting concentration of alpha-pinene 44%, Carene10%, Cedrol 4.86%, and β- myrcene 3.67%. Hence, the isolation of a new Quercetin-glycoside and 0.9% essential oil yield from leaves of Cupressus sempervirens species is considered an important valuable source of quercetin 3-O-rhamnoside and essential oil in the Iraqi cypress plant and might prefer the agriculture of this plant and widespread it in different regions. Keywords: Cupressus, Conifers, Al Sarw, Mass, Pinene, Quercitrin, Spectroscopy. الزيت العطريتقدير وquercetin 3-O-rhamnoside سيترينالكويرمركب وتشخيصعزل # ) بحث مؤتمر ( العراقينبات السرو من أوراق المعزولة **شذى حسين علي و 1*،اماني عامر توفيق 2022حزيران 3 – 2# المؤتمر العلمي العاشر لكلية الصيدلة، جامعة بغداد بغداد، بغداد، العراق الصيدلة، جامعهكلية الطبية، رع العقاقير والنباتات ف* بغداد، بغداد، العراق الصيدلة، جامعه ، كليةالسريرية المختبرية**فرع العلوم الخالصة منتشر ر ، ومعم دائم الخضرة ، متوسط الحجم. النبات السرو المتوسطي والسرو دائم الخضرة نبات السرو من عائلة السرويات وايضا معروف ب العطرية. والمخاريط باألوراق تتميز التي الصنوبرية النباتات في مهما عضوا النبات يمثل العالم. أنحاء جميع في واسع نطاق لديهعلى السرو نبات ي التحليل الكيميائي استخدامات نباتية عرقية كمطهر ، وتخفيف السعال ، وقابض ، ومضاد للتشنج ، والتئام الجروح ومضاد لاللتهابات. أهداف هذا العمل ه تم إجراء .المستزرع في العراق الوراق السروالزيت العطري وتحليل هوالكشف عن quercitrinاالكيميائي وتشخيص المركب المعزول النباتي والعزل تشخيص فوق الصوتية ، الموجات باستخدام جهاز نبات السرومن أوراق استخالصه الذي تم n-butanolمن جزء HPLCبواسطة quercitrin عزل األيون الجزيئي عند الذي اظهر LC-MS/MSتشخيص بواسطه، UV،FT-IR قياس الطيف الضوئي ، TLC, ،HPLC بواسطهالمعزول المركب 448 m/z واعلى قمه m/z 301 . عالوة على ذلك ، عزل الزيت العطري باستخدام التقطير المائي وتقديره بواسطةGC-MS . تظهر النتائج عائدا جيدا عزل مركب جديد من ،لذا٪3.67 .٪ ، وميرسين 4.86٪ ، سيدرول 10٪ ، كارين 44 لماده ال باينينتركيز اعلى ٪ مع0.9من الزيت العطري quercetin-glycosideنبات السرو العراقي،وقد تفضل زراعة في مصدر مهم يعتبر من اوراق صنف نبات السرو,إنتاج الزيت العطري ٪ من0.9 و . شاره في مناطق مختلفةهذا النبات وانت التحليل الطيفي ،سيترينكوير، باينين، كتله ، نبات السرو، الصنوبريات ، يات: السروالكلمات المفتاحية Introduction Medicinal plants are considered an indigenous source of a wide variety of compounds possessing different therapeutic applications (1). Sumerian and Babylonian civilizations employed clay tablets to document the usage of medical plants in Iraq for thousands of years, and they used a variety of treatment methods for medicinal plants (2). The Conifers are woody plants formed from varieties of genera and species. The plants are generally characterized by needle-shaped leaves, single-veined, and consist of both female and male unisexual cones categorized with bract scales (3). The conifers consist of eight families, the most common families were Cupressaceae, Pinaceae, Podocarpaceae, Cephalotaxaceae, Taxaceae, and Phyllocladanecea (4). Cupressus sempervirens L. (C.S) is an evergreen, aromatic, longevity, and widest extant tree among the Cupressaceae family (5). It is usually known as evergreen cypress in different regions, while in Arabic called "al -Sarw" (6,7). 1Corresponding author E-mail: amani.tawfiq1103@copharm.uobaghdad.edu.iq Received:2 /7/2022 Accepted: 1/11 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31issSuppl.pp121-130 Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Cupressus sempervirens 122 The origin of this plant is back to several ancient Greek mythology legends according to a man named Cypresses, the Provence cypress was used as a coating for the wells and the cypress wood was considered as "dowry of the daughter"(8). The previous literature review indicates that various parts of Cupressus around the world including wood, cones, leaves, stem and bark different ethnomedicinal applications and have been used as a topical to relieve muscle pain and rheumatoid arthritis, relief of gout, have an expectorant effect, relief whooping cough, asthma (9,10), bronchitis, diabetes and have a diuretic effect. Furthermore, the essential oil of coniferous plants is used as an antiseptic for wound healing, as an anti-scarring, and astringent effect (11,12). The major chemotaxonomic secondary metabolites are polyphenols; flavonoids, bi-flavonoids, phenolic acids and terpenes, fatty acids, carbohydrates, and resins (13). Amongst the polyphenols reported in the Cupressus genus are flavonoid glycosides such as 3,5,7-trihydroxy-4-O-methoxy flavone, quercetin- 3-D-xyloside, and kaempferol-7 neo-hesperidin - side (14,15). Chemically, Quercitrin is also known as Quercitroside or quercetin O-glycoside; Figure 1 has a potential role in biological activity such as antioxidant, control of cancer, relief of rheumatoid disorders antiangiogenic, hepatoprotective, and antiproliferative activities (16-18). In addition, these constituents were revealed to have acted as protection from skin ageing through the cytoprotective effect of this constituent on UVB- induced cell injury in humans (19). Furthermore, essential oil (EO) is considered one of the major components in C.sempervirens and almost conifer plants. The chemical unit of monoterpenes containing ten carbon atoms with unsaturated carbon atoms (the building unit isoprene) joined head-to-tail and is considered important volatile oil, which is widely spread in medicinal plants with potential biological activity (20,21). Moreover, pharmacologic activities of C.sempervirens includes; anti-inflammatory(22), antimicrobial, antiplatelet, and anticoagulant activity (23), and hepatoprotective activity(24). Our work aimed to phytochemically investigate Iraqi Cupressus sempervirens leaves, isolation of quercitrin (quercetin 3-O-rhamnoside ), and structural identification by UV, FT-IR, and LC-MS/MS analysis moreover, GC-MS analysis of estimation of essential oil. Figure 1. Quercetin 3-O-rhamnoside (16) Materials and Methods Plant material collection and authentication Cupressus sempervirens L leaves were collected from Baghdad in September 2020. Prof. Dr Khansaa Ghazi Rasheed identified and authenticated this plant at the Iraqi Natural History Research Center and Museum -Plant and Environment Department- Baghdad. Leaves were cleaned, air-dried in shade, ground in an electrical grinder to a fine powder, and subsequently used for extraction and phytochemical analysis. Equipment and chemicals A rotary evaporator was used in this work from BȔCHI Rotavapor R-205, Swiss). All chemicals used in this study were of high purity the solvents and chemicals were used in analytical grade from BDH, Ltd. Poole, England, and the reference standard was purchased from Chengdu Biopurify Phytochemicals, China (purity >97). Silica gel GF254 TLC plates belonging to Merck trademark Ltd., India. Extraction and isolation of essential oil (EO) The extraction and isolation of (EO) from the leaves of C. sempervirens were performed with the hydro distillation method using the Clevenger apparatus. The procedure for the extraction of (E.O) was applied as follows: for every 200 grams of air- dried leaves extracted with 1500L water in Clevenger-apparatus for 4 -5 hours. A pale-yellow oil was isolated and then it was dried using anhydrous sodium sulfate. The storage was at about 4 °C in an air-tight and dark glass container until analysis by Gas chromatography/Mass spectrometry (GC/MS) (25). Preparation of plant extract and standard Ultrasound-assisted extraction (UAE) is an efficient, one of the new non-conventional extraction methods, and a favoured technique to isolate phytochemicals from botanical sources. Sonication realizes a complete extraction and enhanced extraction yields are obtained quickly (26). Ultrasonic extraction was performed using a probe Sonicator. 300 gm of powdered leaves of C. sempervirens L was defatted with hexane, then the Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Cupressus sempervirens 123 defatted leaves powder was extracted with 85 % methanol using Ultrasound-assisted extraction (probe) with the following parameters;20 min, solid - solvent ratio 1:8mg/ml, temperature 25 0C, and frequency 20 kHz;), then concentrated under reduced pressure and suspended with water for fractionation method three times with 150 ml of different solvents polarity in ascending manner (chloroform, ethyl acetate, and n. butanol fraction). The n.butanol fraction was dried and subjected to chemical screening tests for the detection of flavonoids and flavonoid glycosides. On other hand, the target component is determined and isolated by semi-preparative HPLC compared with standard. the sample to be analyzed by HPLC was dissolved in methanol -HPLC grade and filtered through a 0.45μm Millipore membrane filter. Furthermore, the standard solution; quercitrin was prepared by dissolving 1 mg /1 ml of methanol (analytical HPLC grade) to compare with the sample to determine of isolated compound. Preliminary phytochemical screening Chemical screening tests were used for the detection of flavonoids and to reduce sugar in n. butanol fraction of leaves extract (27-29). Alkaline reagent test The alkaline reagent was used for detection of flavonoids, 3ml of the extract was mixed with approximately 1.5 ml of alkaline reagent (5% KOH), and the yellow color was considered an appositive indication of flavonoids, and when the addition of a few drops of dilute acid the solution became colourless. Ferric chloride test Phenolic groups were screened using 1% alcoholic ferric chloride added to 2ml of extract (1:1) the color change to deep green or deep blue color indicates the presence of the phenolic compound. Reducing sugar test Benedict's reagent was used to screen the reducing sugar in n. butanol fraction. Approximately 2 ml of sample and 3 ml of reagent were added in the test tube and then the mixture was heated in a water bath for 10 minutes. The greenish brown precipitate according to the concentration of sugar was an indication of the presence of the reducing sugar. Salkowski test Salkowski test was used to detection of terpenoids in Cupressus Sempervirens leaves. 5ml of extracts were dissolved in 1.5 ml of chloroform then added 2 ml of concentrated H2SO4 carefully. Reddish brown color indicates a positive result. Libermann-burchard test Sterols and steroids were detected using acetic anhydride (2 ml) and 2ml of H2SO4 was added in a test tube with 0.5 gm of extract. The appearance of green or blue color confirmed steroids in the sample. Gas chromatography-mass spectrometer (GCMS) conditions GC/MS -SHIMADZU -QP-2010 ULTRA with Scan mode -ACQ was used at the University of Basrah/ College of Agriculture/ Department Food Science, for this analysis and adjusted at these conditions: The capillary column that was used to separate constituents was (30 m× 0.25mm, with a thickness of 0.25µm) at a flow rate of 1.0 mL/min. in addition, the carrier gas that was used is helium, the split ratio was 2.0, the Injector port was 250˚C, oven temperature: was from 80 º C for I min, then rise to 240 ºC at a rate of 10 ºC /min and the detector was 280º C. electronic impact mode (SEI) was used as an ionization mode at 70e (30). Structural identification was based on the comparison of component mass spectra with components in the NIST mass spectral library. Chromatographic analysis for an n. butanol fraction Thin-Layer Chromatography (TLC) In this work, a qualitative analysis of the n. butanol fraction was performed using silica gel 60 F254 plates with 0.25mm thickness and activated at 110 °C (Merck), using the mobile phase system EtOAc/ H2O/ HOAc (glacial) (10:10:40) v/v/v (31). The target compound compared with the quercitrin standard was detected under UV light at 254 nm. Semi preparative high-performance liquid chromatography In this work, n. butanol fraction of leaves extract was subjected to an analytical HPLC system equipped with a fraction collector unit. HPLC -UV system, Shimadzu, 10AV-LC, Japan was carried out for separation and isolation of target compound (quercitrin) in n.butanol fraction compared with quercitrin standard the chromatographic separation done by using specific chromatographic conditions (32,33): HPLC Column: C18- reverse phase, 3 nuclear µm particle size (50 x 2.0 mm). The mobile phase consisted of two solvents mixture in gradient Mode; (A) 0.1% formic acid in water and (B) methanol with gradient elution program from 10% A to:95% A(v/v). The flow rate was 1 ml /min Injection volume was 20 µL and the detection was at ƛ 280 nm -310 nm. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Cupressus sempervirens 124 Chromatographic analysis for Isolated compound (quercitrin) Analytical HPLC and TLC for identification of isolated compound Regardless of HPLC, the purity and identification of the isolated compound were directly checked compared with the standard by using the same analytical HPLC (without a fraction collector unit) with the observant of the same chromatogram conditions used in isolation of the quercitrin as mentioned above. TLC was considered a simple and also the rapid method used for further screening of purity and simple identification of isolated compound compared with quercitrin standard using the same mobile phase system that was used above; EtOAc/ H2O/ HOAc (glacial) (10:10:40 v/v/v). Characterizations for isolated compound (quercetin-3-O-rhamnoside) Ultraviolet spectroscopy (UV) The Ultra Violet spectra analysis was done using a double beam Shimadzu-spectrophotometer (UV-1700) at Al-Mustansirya University - College of pharmacy. The isolated compound and quercetin 3-O- rhamnoside standard were dissolved in methanol and then analyzed between a range of 200 nm to 600 nm. Fourier transforms infrared spectroscopy FT-IR spectroscopy was used for structural identification of isolated quercitroside (quercetin 3- O- rhamnoside) and carried out at Baghdad University-College of Pharmacy. FTIR spectra Shimadzu were performed using the KBr disk method and the results were recorded in the range of 400–4000 cm-1. Liquid Chromatography /Mass /Mass Spectrometry (LC/MS/MS) The mass spectrometric analysis was done using API (ion trap mass spectrometer) /3200/ LC- MS/MS system with Ion Spray Ionization (ESI) source Ion spray, ESI is more frequently used in flavonoid and flavonoid glycosides analysis (34). The liquid chromatographic separation of the target compound was carried out on a Phenomenex Gemini C18 (250 mm x 4.6 mm i.d.; 5 μm particle diameter), column temperature was kept at 250 C0, and 20µl was injected and a flow rate (FR) was 1 ml/min. Full scan spectra were acquired in ESI-MS spectra and were carried out in the positive /negative ionization mode. The mass range was measured, m/z 50-900m/z; drying nitrogen gas nebulizing N2 30 psi and the flow rate was 45 ml/min. MS/MS fragmentation experiments were performed on the selected precursor ion and the conditions were; Ion spray voltage, 4500 V; Data Analysis (Analyst 1,6.3 version –Germany-Darmstadt) was used to analyze the mass spectra. Results and Discussion In this work, the results of phytochemical screening of the C.sempervirens plant indicate the presence of flavonoids, phenolic groups, steroids, reducing sugar, and terpenoids (35). GC-MS analysis of essential oil (EO) of C.sempervirens The chemical components of the essential oil of C.sempervirens leave estimated in this study and identified by GC-MS analysis as in Figure 2. In this work, the extraction yield of the EO from the leaves of Iraqi Cupressus sempervirens L. was 0.9%. Table 1 shows the chemical components of the essential oil obtained by GC-MS analysis and revealed 50 compounds;46 compounds identified and 4 unidentified., the results show the essential oil (EO) contains monoterpenes(C10) and sesquiterpenes(C15) as the major constituents in the essential oil at different retention times (Rt) and concentrations. Hence, the reported previous studies, GC- MS analysis revealed the essential oil components of C.sempervirens leaves in different geographical regions such as in Turkey;α-pinene was; 35.60 %, Δ-2-carene concentration was 22.7, 14.9 %, trans- pino-carveol (5.22 %), α-phellandrene-8-ol percentage was 4.56 %, β-pinene (3.06 %), D- limonene(36); in Iran different concentration in this plant that is identified in both parts leaves and fruits such as α-pinene (46.2, 59.2 %) highest one, while its isomer β-pinene was (3.3, 2.8 %), germacrene-D (6.3-2.1 %), in addition, myrcene percentage value was (4.6, 3.4 %), isoterpinolene (3.7, 3.2 %), sabinene (2.2, 2.0 %), limonene (2.8, 2.4 %) as cyclic hydrocarbon, and as ester was α- terpenylacetate (2.6, 3.2 %) (37), and in North of Tunisia were 𝛼-pinene 47.51%, 𝛿-3-carene7.40,% 𝛼-terpinyl acetate4.11%, 𝛽-caryophyllene, 4.53%, and 𝛼-cedrol4.99% (38). However, these different qualitative and quantitative of volatile oil composition belongs to many factors such as environment, soil composition, and plant organ (39). Iraqi J Pharm Sci, Vol.31(2) 2022 Cupressus sempervirens 125 Figure 2. GC-MS chromatogram of essential oil from Iraqi C. sempervirens leaves Table 1. Essential oil composition from leaves of Cupressus sempervirens L. Peak NO. R. Time Area% *SI Name 2 4.488 44.09 95 alpha.-Pinene 3 4.752 1.44 93 Camphene 5 5.459 7.49 96 Thujene , Sabinene 6 5.737 3.67 90 beta.-Myrcene 7 5.939 10.01 95 3-Carene 9 6.146 6.41 90 m-Mentha-6,8-diene, (R)-(+)- (+)-m-Mentha-1(6),8-diene 10 6.437 0.89 95 gamma.-Terpinene 11 6.757 2.63 94 (+)-4-Carene 12 6.817 0.23 91 alpha.-Terpinene 13 6.967 0.18 85 beta.-Terpineol 14 7.283 0.41 86 alpha.-Campholenal 16 7.519 0.48 90 L-trans-Pinocarveol,L-pinocarveol 21 8.163 2.07 94 p-Menth-1-en-4-ol, Terpinen-4-ol 25 9.820 1.16 95 Bornyl acetate 28 10.840 2.64 90 alpha.-Terpineol acetate 29 11.464 0.16 91 beta.-Elemene, Iraqi J Pharm Sci, Vol.31(2) 2022 Cupressus sempervirens 126 Continued table 1. Peak NO. R. Time Area% *SI Name 30 11.842 0.48 82 beta.-Cedrene 31 11.911 1.03 93 Caryophyllene 32 11.967 0.23 81 beta.-Cedrene 33 12.126 0.40 93 Thujopsene, Sesquichamene 34 12.434 0.76 95 alpha.-Caryophyllene 36 12.804 0.39 87 alpha.-Cubebene 37 13.202 0.16 92 alpha.-Curcumene 39 13.801 0.25 93 Hedycaryol 43 14.646 4.86 92 Cedrol 44 14.915 0.19 86 Cubenol 46 15.21 0.24 87 beta.-Selinenol SI: similarity index Separation and detection of quercitrin in an n. butanol fraction by TLC Thin-Layer Chromatography was applied for the separation of components in n. butanol fraction and screening of quercitrin in the extract. The result under UV light at 254 nm shows the separation of different components of n. butanol Extract (E), and one component shows identical Rf values with quercitrin standard(Q) at 0.8, as seen in Figure 3. Figure 3. TLC analysis of n. butanol Extract (E)compared with quercitrin standard(Q) Isolation and identification of (target compound) quercitrin The results of The Semi-preparative HPLC for separation and isolation of chemical components in n. butanol fraction of C.sempervirens leaves was indicated at three peaks with different retention times and the concentration was at Rt 6.44 for the target compound to be isolated compared with quercitrin standard as in figures 4 (A) and (C). After that, the analytical HPLC was also performed for identification, and checking the purity of the isolated target compound (quercitrin) was achieved at Rt 6.55 min as a single peak as in Figures 4 :(B) and (C). Figure 4. The HPLC chromatogram (A): n. butanol fraction of C. sempervirens leaves; (B): Isolated target compound (quercitrin ); (C): quercitrin standard. Iraqi J Pharm Sci, Vol.31(2) 2022 Cupressus sempervirens 127 Furthermore, A thin-layer chromatographic (TLC) method is considered simple and fast screening for separation and simple identification of organic compounds (40). The results revealed the purity of the isolated compound (one spot) and optimized compatibility with the standard at Rf value 0.52 and detected under UV light at 245nm as shown in Figure 5. Figure 5. Analytical TLC for identification of the isolated compound and checking the purity: (A)quercitrin standard; (B) the isolated compound. Structural elucidations of quercetin-3-O- rhamnoside For structural characterization, Figure 6 shows the UV spectra characteristic of the isolated compound with quercetin-3-O-rhamnoside (quercitrin) at the following spectra of UV max: λ 210, 250, and 350 nm. Taken together, these data indicated the binding of quercetin to sugar molecules. The characteristics of FT-IR spectra were in the range of 400–4000 cm −1as seen in Figure 7. The results show, that the band at 3160 cm−1and broadband range at 3414 -3340 cm - 1 represented the OH stretching vibration of the phenolic O-H and alcoholic O-H due to the intramolecular hydrogen bonding. The strong band observed at 1651 cm - 1 assigned to conjugated carbonyl C=O stretching vibration, A prominent peak at 1500 –1454cm - 1 represents C=C of an aromatic ring; stretching vibration, the prominent peak at 1056 and 1288 cm -1 represent stretching v. of conjugated ether linkage. The bands near 802 cm -1 indicate the bending vibration of the Ar– H group. However, the spectral patterns are in agreement with those data reported previously (41,42). Figure 6. UV- Spectra of quercetin-3-O-rhamnoside compared with standard. Figure 7. FTIR spectra of the isolated quercetin-3-O-rhamnoside Iraqi J Pharm Sci, Vol.31(2) 2022 Cupressus sempervirens 128 Furthermore, for further characterization, full scan product ion liquid chromatography coupled with negative ES ionization spectra was carried out of the isolated quercetin-3-O- rhamnoside. The scan mode for the identification of isolated compounds was MRM (multiple reaction monitoring). The retention time at 19.11 min and MS-MS of the isolated compound was compared with the standard in library Data. Figure 8 shows MS/MS spectra of the target compound showing different fragment ions at m/z 448 indicating a precursor ion [M+1] + and base peak at m/z 301 because of loss of C6H11O4 . that represents quercetin. In addition, the characteristic product ions at m/z 177 represent C6H11O4 .-CH2O, and at m/z 194 represent C6H11O4 . – CO +H2O and at m/z 181 represent C6H11O5 . -OH (L-rhamnose - H2O). However, all the fragmentation patterns are in agreement with those data reported previously (43). Figure 8. LC/MS-MS Spectra of isolated quercetin 3-O-rhamnoside As a final consideration, the above findings approved that the isolated compound from n. butanol fraction of Iraqi C.sempervirens was quercetin 3- O- rhamnoside . This compound was reported in previous studies in conifer plants and in different species of the genus Cupressus such as; C. funebris and C. lusitanica, Cupressus junebris L, Cupressus glabra L., Cupressus goveniana, and C. macrocarpa, while it was not detected previously in species C.sempervirens (44,45). However, an important flavonoid glycosyl; quercetin 3- O- rhamnoside was determined in this species (C.sempervirens )which is cultivated in Iraq. Conclusion The findings of this work evidenced a new Quercetin-glycoside from the species Cupressus sempervirens L. cultivated in Iraq. quercetin 3-O- rhamnoside was isolated as a major compound in an n-butanol fraction extracted by ultrasonic extraction. Moreover, the study also provides a good concentration of 0.9% essential oil yield. Hence, the Iraqi cypress plant might be considered a valuable source of quercetin 3-O-rhamnoside and essential oil. Conflict of interest The authors declare no conflict of interest. Acknowledgements We like to extend our thanks and sincere gratitude to the University of Baghdad /College of Pharmacy/Department of Pharmacognosy and Medicinal plant for providing a facility to complete this work study. References 1. Mujeeb F., P. Bajpai, and N. Pathak. Phytoche mical evaluation, antimicrobial activity , and determination of bioactive components from leaves of Aegle marmelos. BioMed Res. Intl. 2 014; 6 (1): 1–11. 2. Nedhal A. 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