Iraqi J Pharm Sci, Vol.31( Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala DOI: https://doi.org/10.31351/vol31issSuppl.pp62-74 62 Phytochemical Screening of Petroleum Ether Fractions by GC/MS and Iso- lation of Lupeol from Two Different Parts of Iraqi Leucaena leucocephala. (Conference Paper )# Azal Satar Al-Baaj *,1 and Thukaa Z. Abdul-Jalil1* # 10th scientific conference sponsored by College of Pharmacy, University of Baghdad 2-3 June 2022 *Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract This work is considered the first study for the components of the Iraqi Leucaena leucocephala plant, where the different phytochemical compounds that present in the aerial parts were identified by using the gas chromatography/mass spectrometry technique (GC/MS). The type of the components and their concentration will differ according to the part of the plant used and the method of extraction (hot and cold). This study made a comparison in lupeol concentration that was identified and isolated from petroleum ether fractions of Leucaena leucocephala by using Gas Chromatography/Mass Spectrometry (GC/MS), High-performance thin-layer chroma- tography (HPTLC), and Preparative High-Performance Liquid Chromatography (P-HPLC). The plant leaves and stems were collected in September, dried under shade, and powdered (separately), then extracted by two extraction methods: hot Soxhlet and cold maceration method using 85% ethanol, then the result crude extract was fraction- ation with petroleum ether by using a separator funnel. The results of GC/MS, HPTLC, and PHPLC indicated that the leaves contain a higher concentration of lupeol than the stems and the cold maceration method is more efficient than the hot Soxhlet extraction method. Lupeol has many pharmacological activities applied in alternative medi- cine such as anti-inflammatory, antimicrobial, anti-arthritic, anticancer, antidiabetic, and antioxidant activities with wide future applications. Keywords: Leucaena leucocephala, Lupeol, Gas Chromatography/Mass Spectrometry (GC/MS), High-performance thin-layer chromatography (HPTLC), and Preparative High-Performance Liquid Chromatography (P-HPLC). الفحص الكيميائي النباتي الجزاء االثير البترولي بواسطة تقنيات كروماتوغرافيا الغاز / قياس الطيف ) بحث مؤتمر (# وعزل مادة اللوبيول من جزأين مختلفين من نبات الليوسينا العراقي GC/MSالكتلي *زهير عبد الجليل و ذكاء 1*،أزل ستار ألبعاج 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي جامعة بغداد ، بغداد ، العراق . الصيدلة، كلية الطبية، فرع العقاقير والنباتات * الخالصة ) شجرة الرصاص أالبيض ( المزروع في ألعراق ) ألتابع هذا العمل ، وألول مرة ، دليل على وجود مادة اللوبيول في نبات الليوسينا اجراء مقارنة في الى عائلة البقوليات( من خالل تحديد المركبات الكيمونباتية في االجزاء العليا من النبات ) االوراق و السيقان (. في هذه الدراسة تم كروماتوغرافيا الغاز /قياس الطيف ستخلصة من نبات الليوسينا باستخدام تقنياتتركيز مادة اللوبيول المشخص والمعزول من أجزاء البتروليم ايثر الم تم جمع أوراق و سيقان . PHPLC ، و الفصل اللوني السائل العالي االداء HPTLC ، الفصل اللوني للطبقة الرقيقة عالية االداءGC/MS الكتلي ، بعدها تم االستخالص بإستخدام طريقتين: طريقة االستخالص منفصل عن االخر( النبات في شهر سبتمبر ، و جففت تحت الظل، ثم طحنه )كل جزء ٪ من االيثانول، بعدها تم تجزئة ٨٥الساخن باستخدام جهاز السوكسلت و طريقة االستخالص البارد باستخدام طريقة النقع ، و كال الطريقتين باستخدام .ام قمع الفصلالمستخلص النباتي الخام مع البتروليم أيثر بأستخد الى ان االوراق تحتوي على تركيز اعلى من اللوبيول من السيقان و أن طريقة (PHPLC ,HPTLC ,GC/MS) أشارت نتائج التقنيات المستخدمة البدي الطب في المطبقة الدوائية أالنشطة من العديد اللوبيول يمتلك الساخن. االستخالص طريقة من كفاءة أكثر البارد فاعليته االستخالص مثل ل .كمضاد لاللتهابات، مضاد للميكروبات، أللتهاب المفاصل، مضاد للسرطان، مضاد للسكر، ومضاد لالكسدة مع تطبيقات مستقبلية واسعة الكروماتوجرافيا السائلة عالية ، كروماتوغرافيا الطبقة الرقيقة عالية األداء ، ، كروماتوجرافيا الغاز / قياس الطيف الكتلي وبيولل ،: ليوسينا مفتاحية كلمات . األداء التحضيري Introduction From the beginning of life on earth, there was an association between humans, animals, and plants in which it supplied some of the needs that are important for life continence such as oxygen, food, and medicine for treating their diseases. Over time and with many tries , humans learned how to use herbal materials in their life in a beneficial way. Aft er thousands of years, the traditional medicine systems were used all over the world, the Ayurvedic and Unani of the Indian subcontinent, the Chinese and Tibetan of other parts of Asia, the Native Amer- icans of North America, the Amazonian of South America, and several local systems within Africa. (1) azal.satar1200m@copharm.uobaghdad.edu.iq :mail-Corresponding author E1 Received:18 /5 / 2022 Accepted:5 /9 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31issSuppl.pp62-74 Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 63 Leucaena belongs to the Fabaceae family and Mimosoideae’s subfamily which contain ap- proximately 50 species of shrubs and trees. L. leuco- cephala has high nutritious value and a lot of uses around the world such as human food, firewood, timber, green manure, and shade; so, it is known as “a miracle tree’’. (2) L. leucocephala is used in many countries by a hu- man who eats the young leaves, flowers, and young pods in soups such as in Indonesia, Central America, and Thailand. (3), (4) L. leucocephala tree proved to have a high medicinal value as studies revealed that it contains various active chemical compounds such as flavonoids, car- diac glycosides, tannins, phylobatanins, alkaloid, saponins, ester, and ketone. So, it has a lot of phar- macological activities such as antimicrobial, anthel- mintic, antibacterial, anti-proliferative, antidiabetic, anticancer, diuretic, anti-inflammatory, antioxidant, antitumor, antihistaminic, anti-androgenic, and hepatoprotective properties. (2) This study articulates the frame to view the first report with regarded phy- tochemical screening by GC/MS, determination and isolation of Lupeol from petroleum ether fractions of two different parts of Iraqi Leucaena leucoceph- ala by HPTLC and PHPLC respectively. Figure1. Photos of flowers, leaves, fruits, seeds, and stems of the Iraqi Leucaena leucocephala plant (27) . Materials and Methods Plant material collection and authentication In September 2021, the plant was collected from one of the farms located in Al_ Diwaniyah city. The plant parts (leaves and stems) were isolated, cleaned, and dried for a week under the shade until they dried completely. The leaves and stems were separately ground to be ready for extraction. The plant has been authenticated by Dr. Zainab Abed Aoun Ali, Ph.D. in Plant Taxonomy / Department of Life Sciences / College of Science / the University of Baghdad. Preparation of petroleum ether extracts. The dry powdered leaves and stems were extracted by two extraction methods: 1. Soxhlet (hot method): 100 grams of each pow- dered part was extracted with 1000 ml of 85% etha- nol for 21 hours, and the result fractions were con- centrated by a rotary evaporator and then fractiona- tion two times with 200 ml of petroleum ether in a separatory funnel, the results P.E fractions were an- alyzed by using GC/MS, HPTLC, and PHPLC. 2. Maceration (cold method): 100 grams of each powdered part was macerated with 1000 ml of 85% ethanol (two times, each time for two days), and the result fractions were concentrated by a rotary evap- orator and then fractionation two times with 250 ml of petroleum ether in a separatory funnel, the results P.E fractions were analyzed by using GC/MS, HPTLC, and PHPLC. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 64 Figure 2. Extraction, fractionation & identification scheme of lupeol from Iraqi Leucaena leucocephala. leaves stems Evaporate by using a rotary evaporator Extraction with 85% ethanol Hot method by Soxhlet Cold method by maceration Crude ethanolic extracts of leaves and stems (separately) that result from the hot and cold extraction methods Result of four fractions of petroleum ether crude extracts: 1st: from leaves by hot extraction method 2nd: from leaves by cold extraction method 3rd: from stems by hot extraction method 4th: from stems by cold extraction method All the resultant organic layers were concentrated by using a rotary evaporator to get crude extracts GC/MASS HPLC HPLC HPLC P-TLC HPLC TLC HPLC Drying under shade, Pulverized into powder In separatory funnel, all ethanolic extracts are fractionation with water and petroleum ether (100-250ml) *2 Evaporate Petroleum ether layers Aqueous layer P_HPLC HPLC GC/MS HPLC HPTLC HPLC Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 65 Preliminary identification and isolation of lupeol in petroleum ether extracts of Iraqi Leucaena leu- cocephala Gas chromatography/mass spectrometry (GC/MS) analysis for petroleum ether fractions. Four fractions of petroleum ether were an- alyzed by using GC/MS chromatography which is found in Ibn Al-Bittar Center / Ministry of Science and Technology / Baghdad / Iraq. Lupeol in P.E fractions of Leucaena leucocephala was identified by matching the mass spectra with libraries spectra. The quantity of lupeol is calculated as a percentage of the relative peak area. Table (1) shows the GC/MS conditions that were used in the analysis of the P.E fractions. Table 1. GC/MS conditions. (4) Instrument Agilent (7820A) USA GC Mass Spectrometer Analytical Column Agilent HP-5ms Ultra (30 m length x 250 µm diameter x 0.25 µm inside di- ameter) Injection volume 1µl Pressure 11.933 psi Temperature GC Inlet Line Temperature: 250 ˚C Aux heaters Temperature: 310 ˚C Carrier Gas He 99.99% Injector Temperature 250 ˚C Scan Range: m/z 25-1000 Injection Type Split less Oven Program Tempera- ture Ramp 1 60˚C hold to 3 min. Ramp 2 60˚C to180 ˚C 7 ˚C/min. Ramp 3 180˚C to 300˚C 8 ˚C/min Ramp 4 300˚C hold to 3 min. Qualitative estimation of lupeol by using High- Performance Thin Layer Chromatography (HPTLC(. All P.E extract fractions of leaves and stems that were prepared by different extraction methods were qualitatively identified by using the HPTLC which is found in Baghdad College of Med- ical Sciences/ Baghdad/ Iraq. Table (2) shows the HPTLC conditions that were used in the analysis of the P.E fractions. Table 2. HPTLC conditions. (5) Instrument Eike-Reich/CAMAG-laborator/ Switzerland and operated by Win CATS soft- ware using a tungsten lamp Stationary phase TLC plates silica gel 60 F254 pre-coated layer (20 cm X 10 cm), thickness 0.2mm Band length 8 mm Standards Lupeol Samples Four P.E fractions of leaves and stems from two extraction methods for Iraqi Leucaena leucocephala Solubility Methanol Application volume 3 μl Development chamber CAMAG, ADC 2- chamber (20X 10) Chamber saturation time 5 minutes Development mode Ascending mode Distance run 75 mm Slit dimensions 4.00 x 0.30 mm Scanning speed 20 mm/s Measurement mode Absorbance Mobile phase and detection Lupeol Toluene: ethyl acetate: chloroform (5:1:4). (26) UV 225 nm and 5% H2SO4 spray Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 66 Qualitative and quantitative Identification of pro- posed Lupeol by Preparative High-Performance Liquid Chromatography (PHPLC). Lupeol was identified and quantified from petroleum ether fractions of Leucaena leucocephala by using preparative high-performance liquid chromatography (PHPLC) which is found in the Ministry of Science and Technology/ Department of Water and Environment/ Baghdad/ Iraq. Table (3) shows the PHPLC conditions that were used in the analysis of the P.E fractions. Table 3. PHPLC conditions. (6) Instrument CYKNM high-performance liquid chromatography Column MEDITERRANEA C18 (5 µm 15 X 2.12 cm) Mobile phase Acetonitrile (A) and water (B) Gradients 0-1 min 3% A; 10-45 min 3-21% A; 45-60 min 21-40% A. (25) Samples Petroleum ether fractions (leaves and stems) Standard Lupeol standard Column temperature room temperature Application volume 100 μl for identification, 1 ml for isolation Injection concentration 1mg /1ml for each sample Detection wavelength UV detector at λ 210 nm Results and Discussion This work is considered the first attempt to recognize the biologically active constituents of Leucaena leucocephala in Iraq. The weights of the result crude ethanolic extracts were (cold 16.78, hot 14,53) grams, after fractionation with P.E, the weight of the P.E fractions were (leaves, cold 2.89 / leaves, hot 1.85 / stems, cold 1.9 / stems, hot 0.4) grams. The results of GC/MS, HPTLC, and PHPLC indicate that there are the same compounds that are found in the leaves and stem but in different concentrations. These compounds are belonging to various chemical classes, these are; fatty acids, vol- atile oils, phytosterols, triterpene, diterpene, fatty al- cohols, vitamin E isomers, alkanes, and alkenes. Gas chromatography/mass spectrometry (GC/MS) analysis for petroleum ether fractions. After analyzing the petroleum ether ex- tracts of Leucaena leucocephala by GC/MS, it was found that they contain many active compounds, which are summarized in the following Figures and tables. Figure 3. GC-MS analysis of petroleum ether leaves extract of Leucaena leucocephala by hot extraction method. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 67 Table 4. GC-MS analysis of petroleum ether leaves extract of Leucaena leucocephala by hot extraction method. NO. Chemical class Compound Rt (min.) Molecular formula Similarity index Refer- ences 1 lactams caprolactam 12.039 C6H11NO 98 (7) 2 Natural monoterpenoid phenol Thymol 12.776 C10H14O 94 (8) 3 macrocyclic organosilox- ane cyclohexasiloxane Do- decamethyl 13.018 C12H36O6Si6 94 (9) 4 macrocyclic organosilox- ane Cycloheptasiloxane, tetradecamethyl- 16.187 C14H42O7Si7 93 (10) 5 fatty acid methyl ester Methyl Tetradecanoate 21.255 C15H30O2 99 (11) 6 fatty acid methyl ester. Hexadecanoic acid, me- thyl ester 25.240 C17H34O2 99 (11) 7 long-chain fatty acid ethyl ester Hexadecanoic acid, ethyl ester 27.099 C18H36O2 98 (12 8 fatty acid methyl ester 9,12-Octadecadienoic acid (Z, Z)-, methyl ester (Linoleic acid) 28.344 C19H34O2 98 (13) 9 essential fatty acid 9,12,15-Octadecatrienoic acid, (Z, Z, Z)- 28.488 C18H30O2 99 (9) 10 fatty acid methyl ester 9,12,15-Octadecatrienoic acid, methyl ester, (Z, Z, Z)- 28.488 C19H32O2 99 (14) 11 diterpenoid Phytol 28.657 C20H40O 99 (2) 12 fatty acid methyl ester Methyl Stearate 28.964 C19H38O2 99 (15) 13 long-chain fatty acid ethyl ester Linoleic acid ethyl ester 29.531 C20H36O2 91 (12) 14 Fatty acid ethyl ester 9,12,15-Octadecatrienoic acid ethyl ester, (Z,Z,Z)- 29.649 C20H34O2 95 (15) 15 diester Hexanedioic acid, bis(2- ethylhexyl) ester 33.399 C22H42O4 95 (16) 16 fatty acid methyl ester Docosanoic acid, methyl ester 35.636 C23H46O2 97 (17) 17 triterpene Squalene 39.790 C30H50 97 (14) 18 steroid Stigmast-4-en-3-one 40.671 C29H48O 94 (11) 19 lipids gamma-Tocopherol 43.214 C28H48O2 99 (18) Figure 4. GC-MS analysis of petroleum ether leaves extract of Leucaena leucocephala by cold extraction method. https://pubchem.ncbi.nlm.nih.gov/#query=C6H11NO https://pubchem.ncbi.nlm.nih.gov/#query=C6H11NO https://pubchem.ncbi.nlm.nih.gov/#query=C6H11NO https://pubchem.ncbi.nlm.nih.gov/#query=C6H11NO https://pubchem.ncbi.nlm.nih.gov/#query=C6H11NO https://en.wikipedia.org/wiki/Monoterpene https://en.wikipedia.org/wiki/Phenols https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C12H36O6Si6 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/#query=C14H42O7Si7 https://pubchem.ncbi.nlm.nih.gov/compound/31284 https://pubchem.ncbi.nlm.nih.gov/#query=C15H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C15H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C15H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C15H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C15H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C15H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12-Octadecadienoic%20acid%20(Z%2CZ)-%2C%20methyl%20ester%22%5bCompleteSynonym%5d%20AND%205284421%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12-Octadecadienoic%20acid%20(Z%2CZ)-%2C%20methyl%20ester%22%5bCompleteSynonym%5d%20AND%205284421%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C19H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H34O2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12%2C15-Octadecatrienoic%20acid%2C%20(Z%2CZ%2CZ)-%22%5bCompleteSynonym%5d%20AND%205280934%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12%2C15-Octadecatrienoic%20acid%2C%20(Z%2CZ%2CZ)-%22%5bCompleteSynonym%5d%20AND%205280934%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C18H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H30O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H30O2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12%2C15-Octadecatrienoic%20acid%2C%20methyl%20ester%2C%20(Z%2CZ%2CZ)-%22%5bCompleteSynonym%5d%20AND%205319706%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12%2C15-Octadecatrienoic%20acid%2C%20methyl%20ester%2C%20(Z%2CZ%2CZ)-%22%5bCompleteSynonym%5d%20AND%205319706%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12%2C15-Octadecatrienoic%20acid%2C%20methyl%20ester%2C%20(Z%2CZ%2CZ)-%22%5bCompleteSynonym%5d%20AND%205319706%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C19H32O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H32O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H32O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H32O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H32O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H32O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C19H38O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H38O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H38O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H38O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H38O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H38O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H36O2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12%2C15-Octadecatrienoic%20acid%20ethyl%20ester%22%5bCompleteSynonym%5d%20AND%206371716%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2C12%2C15-Octadecatrienoic%20acid%20ethyl%20ester%22%5bCompleteSynonym%5d%20AND%206371716%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22Hexanedioic%20acid%2C%20bis(2-ethylhexyl)%20ester%22%5bCompleteSynonym%5d%20AND%207641%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22Hexanedioic%20acid%2C%20bis(2-ethylhexyl)%20ester%22%5bCompleteSynonym%5d%20AND%207641%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C30H50 https://pubchem.ncbi.nlm.nih.gov/#query=C30H50 https://pubchem.ncbi.nlm.nih.gov/#query=C30H50 https://pubchem.ncbi.nlm.nih.gov/#query=C30H50 https://pubchem.ncbi.nlm.nih.gov/#query=C29H48O https://pubchem.ncbi.nlm.nih.gov/#query=C29H48O https://pubchem.ncbi.nlm.nih.gov/#query=C29H48O https://pubchem.ncbi.nlm.nih.gov/#query=C29H48O https://pubchem.ncbi.nlm.nih.gov/#query=C29H48O https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22(%2B%2F-)-gamma-Tocopherol%22%5bCompleteSynonym%5d%20AND%2014986%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C28H48O2 https://pubchem.ncbi.nlm.nih.gov/#query=C28H48O2 https://pubchem.ncbi.nlm.nih.gov/#query=C28H48O2 https://pubchem.ncbi.nlm.nih.gov/#query=C28H48O2 https://pubchem.ncbi.nlm.nih.gov/#query=C28H48O2 https://pubchem.ncbi.nlm.nih.gov/#query=C28H48O2 Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 68 Table 5. GC-MS analysis of petroleum ether leaves extract of Leucaena leucocephala by cold extraction method. NO . Chemical class Compound Rt (min.) Molecular formula Similarity in- dex Refer- ences 1 caprolactams Caprolactam 12.013 C6H11NO 98 (8) 2 Natural monoterpe- noid phenol Thymol 12.763 C10H14O 94 (9) 3 macrocyclic orga- nosiloxane Cyclohexasiloxane, dodecamethyl 13.018 C12H36O6Si6 93 (10) 4 macrocyclic orga- nosiloxane Cycloheptasiloxane, tetradecamethyl 16.174 C14H42O7Si7 93 (11) 5 fatty acid methyl ester Methyl tetradecanoate 21.255 C15H30O2 99 (12) 6 fatty acid methyl ester Hexadecanoic acid, methyl ester 25.220 C17H34O2 89 (12) 7 long-chain fatty acid ethyl ester Hexadecanoic acid, ethyl ester 26.492 C18H36O2 98 (13) 8 fatty acid methyl ester 9,12-Octadecadienoic acid (Z, Z)-, methyl es- ter 28.318 C19H34O2 98 (2) 9 fatty acid 9,12,15-Octadeca- trienoic acid, (Z, Z, Z)- 28.612 C18H30O2 91 (2) 10 diterpenoid Phytol 28.612 C20H40O 99 (2) 11 fatty acid methyl ester Methyl Stearate 28.944 C19H38O2 99 (16) 12 triterpene squalene 39.803 C30H50 99 (15) 13 phytosterols Beta-sitosterol 39.953 C29H50O 96 (14) 14 pentacyclic triterpenoid Lupeol 42.327 C30H50O 96 (14) 15 very long-chain pri- mary fatty alcohol 1-Heptacosanol 44.414 C27H56O 98 (20) 16 fat soluble tocoph- erols VITAMIN E 44.669 C29H50O2 95 (14) Figure 5. GC-MS analysis of petroleum ether stems extract of Leucaena leucocephala by hot extraction method. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 69 Table 6. GC-MS analysis of petroleum ether stems extract of Leucaena leucocephala by hot extraction method. Figure 6. GC-MS analysis of petroleum ether stems extract of Leucaena leucocephala by cold extraction method. Table 7. GC-MS analysis of petroleum ether stems extract of Leucaena leucocephala by cold extraction method. NO . Chemical class Compound Rt (min.) Molecular formula Similarity index Refer- ences 1 fatty acid methyl ester Hexadecanoic acid, methyl ester 23.604 C17H34O2 99 (11) 2 fatty acid ethyl ester Hexadecanoic acid, ethyl ester 24.445 C18H36O2 98 (12) 3 fatty acid methyl ester 9-Octadecenoic acid (Z)-, methyl ester 25.864 C19H36O2 99 (21) 4 diterpenoid Phytol 26.100 C20H40O 93 (2) 5 diester Hexanedioic acid, bis(2-ethylhexyl) ester 29.211 C22H42O4 89 (16) 6 Diterpenoid al- cohol trans-Geranylgera- niol 33.276 C20H34O 90 (22) GC-MS chromatogram of the petroleum extracts of Leucaena leucocephala shows many peaks indicating the presence of different com- pounds. Some of these compounds are considered as a major and others are minor. Lupeol is one of the detected compounds in P.E extracts, was identified by matching its mass spectra with libraries spectra The quantity of lupeol is calculated as a percentage of the relative peak area.. Qualitative identification by HPTLC HPTLC is consider an advanced forms of TLC, it is very efficient for qualitative and quantita- tive analysis. Automated application of sample is more precise than manual application and that will prevent the difference in volume of application, so NO . Chemical class Compound Rt (min.) Molecular for- mula Similarity in- dex Refer- ences 1 fatty acid methyl ester Hexadecanoic acid, methyl ester 23.594 C17H34O2 96 (11) 2 phthalate ester that (diester) Dibutyl phthalate 24.464 C16H22O4 93 (19) 3 Fatty acid 10-Octadecenoic acid methyl ester 25.854 C19H36O2 99 (14) 4 Enol 2-Methyl-Z,Z-3,13- octadecadienol 28.161 C19H36O 93 (20) 5 phytosterols Beta -sitosterol 30.308 C29H50O 99 (13) https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C18H36O2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229-Octadecenoic%20acid%20(Z)-%2C%20methyl%20ester%22%5bCompleteSynonym%5d%20AND%205364509%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229-Octadecenoic%20acid%20(Z)-%2C%20methyl%20ester%22%5bCompleteSynonym%5d%20AND%205364509%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229-Octadecenoic%20acid%20(Z)-%2C%20methyl%20ester%22%5bCompleteSynonym%5d%20AND%205364509%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://pubchem.ncbi.nlm.nih.gov/#query=C20H40O https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22Hexanedioic%20acid%2C%20bis(2-ethylhexyl)%20ester%22%5bCompleteSynonym%5d%20AND%207641%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22Hexanedioic%20acid%2C%20bis(2-ethylhexyl)%20ester%22%5bCompleteSynonym%5d%20AND%207641%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22Hexanedioic%20acid%2C%20bis(2-ethylhexyl)%20ester%22%5bCompleteSynonym%5d%20AND%207641%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://pubchem.ncbi.nlm.nih.gov/#query=C22H42O4 https://en.wikipedia.org/wiki/Alcohol_(chemistry) https://en.wikipedia.org/wiki/Alcohol_(chemistry) https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22All-trans-Geranylgeraniol%22%5bCompleteSynonym%5d%20AND%205281365%5bStandardizedCID%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22All-trans-Geranylgeraniol%22%5bCompleteSynonym%5d%20AND%205281365%5bStandardizedCID%5d https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O https://pubchem.ncbi.nlm.nih.gov/#query=C20H34O https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C17H34O2 https://pubchem.ncbi.nlm.nih.gov/#query=C16H22O4 https://pubchem.ncbi.nlm.nih.gov/#query=C16H22O4 https://pubchem.ncbi.nlm.nih.gov/#query=C16H22O4 https://pubchem.ncbi.nlm.nih.gov/#query=C16H22O4 https://pubchem.ncbi.nlm.nih.gov/#query=C16H22O4 https://pubchem.ncbi.nlm.nih.gov/#query=C16H22O4 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O2 https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O https://pubchem.ncbi.nlm.nih.gov/#query=C19H36O https://pubchem.ncbi.nlm.nih.gov/#query=C29H50O https://pubchem.ncbi.nlm.nih.gov/#query=C29H50O https://pubchem.ncbi.nlm.nih.gov/#query=C29H50O https://pubchem.ncbi.nlm.nih.gov/#query=C29H50O https://pubchem.ncbi.nlm.nih.gov/#query=C29H50O Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 70 that will decrease the differences in development of spots through the plate that may occur. It’s flexible enough for one HPTLC System to analyze different samples. Use of stationary and mobile phase is de- pending on the number of samples being analyzed (23) . Two parts of Leucaena leucocephala and two extraction methods were selected to make a comparison between them based on the percentage yield obtained from each method. The presence of lupeol in petroleum ether fractions was obtained. Qualitative identification was made by comparison of the maximum retardation factor (max Rf) and UV spectrum of lupeol in petroleum ether fractions with its corresponding lupeol standard. Figure 7. HPTLC chromatogram of Lupeol standard. Figure 8. HPTLC chromatogram of Lupeol in petroleum ether fractions of leaves and stems of Leucaena leucocephala by hot and cold extraction methods. (A)Leaves, hot extraction method (B)Leaves, cold extrac- tion method (C)Stems, hot extraction method (D)Stems, cold extraction method. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 71 Identification and isolation of proposed Lupeol by PHPLC. Identification and isolation of lupeol from petroleum ether fractions of the plant was done by using PHPLC which is considered as the most com- mon method for purification in pharmaceutical in- dustries. (24) PHPLC chromatogram showed many peaks which represent many different compounds according to their retention time, one of them is lupeol at retention time of (30.08, 30.00, 30.07, 29.94), are the major peaks which were identified by compari- son with standard lupeol at retention time of (30.11). The major peaks were collected by fractions collec- tor after monitoring it according to the time (time from beginning of the appearance to disappearance of the peak), then the sample obtained from PHPLC was dried over anhydrous sodium sulfate, weighted and subjected to different identification methods. Figure 9. PHPLC chromatogram of petroleum ether fraction of leaves that extracted by cold method. Figure 10. PHPLC chromatogram of petroleum ether fraction of leaves that extracted by hot method. Figure 11. PHPLC chromatogram of petroleum ether fraction of stems that extracted by cold method. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 72 Figure 12. PHPLC chromatogram of petroleum ether fraction of stems that extracted by hot method. Figure 13. PHPLC chromatogram of standard lupeol. According to linear least square regression equation, quantitative determination was carried out by using a calibration curve for the isolated lupeol with a correlation factor equals’ 0.9998278 and peak areas of the new detected compound in fraction of petroleum ether was used to determine the concentration as shown in table 8 and Figure 14. Figure 14. Calibration curve of lupeol standard. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Phytochemical screening of two different parts of Iraqi Leucaena leucocephala 73 Table 8. Amount of lupeol in P.E extracted frac- tions. Fraction AUC (mV.s) Amount (ppm) P.E, Leaves, Cold extraction method 8963.08 589.787 P.E, Leaves, Hot extraction method 6589.11 277.548 P.E, Stems, Cold extraction method 5789.25 370.918 P.E, Stems, Hot extraction method 3658.99 296.946 PHPLC revealed that leaves contain higher amount of lupeol than stems, and the cold extraction method give best results than hot one, so it will be preferred in the extraction of Leucaena leucocephala. Conclusions The identification methods that applied on these fractions elucidate that leaves and stems of Leucaena leucocephala have many phytochemicals that belongs to different chemical classes (fatty ac- ids, volatile oils, phytosterols, triterpene, diterpene, fatty alcohols, vitamin E isomers, alkanes and al- kenes). These phytochemicals differ in their quantity and types from leaves to stems, and also differ ac- cording to the method of extraction (hot or cold method). Phytochemicals were detected in higher amount in the leaves that extracted by cold macera- tion method. Also, the findings of the study show that HPLC method can be adopted for the determi- nation of concentration of lupeol in various extract petroleum ether fractions from various plant parts and isolation with shorter run time and good effi- ciency. So, it is advisable to consume the leaves of Leucaena leucocephala for extraction and isolation of these compounds. Acknowledgement Thanks to the University of Baghdad, Col- lege of Pharmacy, members of the Pharmacognosy and Medicinal Plants branch for providing necessary facilities and assistance. References 1. Mamedov N. Medicinal Plants Studies: History, Challenges and Prospective. Med Aromat Plants. 2012;01(08). 2. Zayed MZ, Samling B. 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