Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 75 Extraction and Isolation of -sitosterol from Iraqi Wild Lycium barbarum by Different Techniques (Probe and Bath Ultrasound, HPTLC and PHPLC) Thukaa Z. Abdul-Jalil*,1, Ahmed A.Hussein** and Kawkab Y. Saour*** *Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad, Iraq. **Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq. *** Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract Lycium barbarum (Awsaj) is a plant belong to family Solanaceae serves as a good source of bioactive compounds like phytosterols which have many important biological activity. Literature survey available so far revealed that there was no studies about Iraqi wild Awsaj phytosterols especially ß-sitosterol, there for the objective of this study was to examine the efficiency of ultrasound assisted extraction (probe and bath) as compared to the conventional (Soxhlet) extraction method for extraction of phytosterols especially -sitosterol from fruits, leaves, stems and roots of Iraqi wild Awsaj plant. This goal was achieved by comparing the extraction mass yield, also by a quick and easy approach for identification and quantification of bioactive -sitosterol in four parts of Awsaj using high performance thin layer chromatography (HPTLC) which confirm the presence of -sitosterol in four parts of Iraqi Awsaj plant and the results clarify that the root part of Iraqi Awsaj plant extracted by probe UAE give the highest concentration (0.610 mg/ml) of B-sitosterol followed by fruits (0.465mg/ml), leaves(0.405mg/ml) and the least was stems(0.275mg/ml); according to these; isolation and purification of ß-sitosterol from petroleum ether fraction of Awsaj roots was done by preparative HPLC. The isolated compound (-sitosterol) was identified by measuring melting point, FT-IR, TLC and preparative HPLC. Keywords: Awsaj, Ultrasound assisted extraction (UAE), -sitosterol, high performance thin layer chromatography (HPTLC), Preparative high performance liquid chromatography (PHPLC). قنيات استخالص وفصل مركب بيتاستوستيرول من نبات العوسج البري العراقي بواسطة ت االنجاز مختلفة ) مسبارد حوض االمواج الفوق الصوتية ، الفصل اللوني للطبقة الرقيقة ذات العالي والفصل اللوني السائل ذو االنجاز العالي ( ***و كوكب يعقوب ساعور **احمد عباس حسين ، 1*,ذكاء زهير عبد الجليل فرع العقاقير والنباتات الطبية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق .* فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . ** فرع الكيمياء الصيدالنية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . *** الخالصه مصدر جيد للكثير من المركبات مثل الستيرويدات ويعتبر من العائله الباذنجانيه هو نبات ( Lycium barbarumالعوسج ) -حول الستيرويدات النباتيه و خصوصا مركب بيتاحتى الوقت الحالي النباتيه ذات الفعاليه االحيائيه. و نظرا لعدم وجود دراسات بمساعدة ستلاصلفحص كفائة االري العوسج, لذلك اصبح هدف العمل في هذه الدراسه هو سيتوستيرول في النبات العراقي الب المسبار و الحوض( و مقارنته بطريقة االستلاص التقليدي بوساطة جهاز السوكسليت ) االمواج فوق الصوتيه )بوساطة (Soxhletمار, االوراق, السيقان و الجذور لنبات العوسج الجل استلاص الستيرويدات النباتيه و خصوصا البيتاسيتوستيرول من الث العراقي البري. هذا الهدف تم انجازه بمقارنة حصيلة االستلاص الكتلي و كذلك عمل مقارنة سريعه و بسيطه لغرض الكشف و الرقيقه للطبقه )كروماتوغرافيا( اللوني الفصل باستعمال نبات العوسجلفي االجزاء االربعه التحديد الكمي لمركب البيتاسيتوستيرول المستللصه ن جذور النبات في االجزاء االربعه لنبات العوسج. اوضحت النتائج اسيتوستيرول -بيتاالذي اكد وجود ال العالي االنجاز ذات نسبة مل( يتبعها الثمار ب\مغ016.0بوساطة مسبار االمواج فوق الصوتيه تحتوي على اعلى نسبة من البيتاسيتوستيرول ) مركب و تنقية فصل مل(. وفقا لتلك النتائج تم \مغ012.0مل( و اخرا السيقان بنسبة )\مغ01400مل( و االوراق بنسبة )\مغ01460) باستعمال تقانة الفصل اللوني السائل ذات االنجاز العالي. كذلك من المستللص اثير البترول لجذور العوسج سيتوستيرول-بيتاال درجة قياس سيتوستيرول( و درجة نقاوته و التي شملت -تقانات للتحقق من نوعية المركب المفصول )بيتااستلدمت مجموعه من ال .للطبقة الرقيقه و الفصل اللوني السائل ذات االنجاز العاليالفصل اللوني االشعه تحت الحمراء, االنصهار , ،الفصل اللوني للطبقه الرقيقه ذات االنجاز العالي ،سيتوستيرول -تاالب،بمساعدة االمواج فوق الصوتيه ستخالصاالالكلمات المفاتيحيه: العوسج, الفصل اللوني السائل ذات االنجاز العالي. 1Corresponding author E-mail: aljas_dadid@yahoo.com Received: 3/10/ 2017 Accepted:15/11/2017 mailto:aljas_dadid@yahoo.com Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 76 Introduction Nature has blessed mankind with a treasure of bioactive ingredients which are an emerging field in the context of health and nutrition. The American dietetic association (ADA) define the bioactive ingredients of food as a physiologically active constituent in food or dietary supplement derived from both animal and plant sources including those needed to meet basic human nutritional needs that have been demonstrated to have a role in health and safe of human consumption (1, 2) . Phytosterols especially -sitosterol is a predominant sterol in human herbal nutrition forming 65% diet contents can be considered as constituent of bioactive foods ,(figure 1) (3) . Awsaj (Lycium barbarum), also known as Goji, is a shrub belong to Solanaceae family and is native to Asia, (figure 2) (4). Awsaj has been used in Asian countries as a traditional herbal medicine and functional food which possess a large variety of beneficial effects, including antiaging activity (5), immune modulation (6), antioxidant(7), neuroprotective effect(8), anticancer(9), male fertility facilitatory action (10), lower blood pressure and blood cholesterol level(11) as well as hypoglycemic properties (12). Figure (1): structure of -sitosterol Figure (2): Iraqi Lycium barbarum (Awsaj) plant Literature survey available so far revealed that there are no study concerned with Awsaj ß-sitosterol in Iraq, therefore the objective of the present study is to extract ß- sitosterol from four parts of Iraqi wild Awsaj plant (fruits, leaves, stems and roots) then identification and quantification by high performance thin layer chromatography (HPTLC) further more isolation of ß-sitosterol from Awsaj by preparative high performance liquid chromatography (PHPLC). Materials and Methods Plant material Four parts (fruits, leaves, stems and roots) of Awsaj (Lycium barbarum L.), which grows as a wild plant in Iraq, were collected in October and November 2016 from the University of Baghdad in Al-Jadiryia District, Baghdad, then authenticated by the herbarium of the Department of Biology, College of Science/University of Baghdad registered at BUH No. 50777. These parts were left to dry in shade then grinded using an electric blender, weighted and subsequently subjected to extraction procedure. Extraction methods Two different extraction methods were used; the first representing a classical conventional (Soxhlet) method (A) and the second is a simple, fast and nonconventional extraction (ultrasound assisted extraction UAE) method (B). Method A: Approximately 250 gm of grounded fruits, leaves, stems and roots were subjected to extraction using a Soxhlet apparatus in which each part of Awsaj were extracted separately with 85% methanol, then the crude methanol extract of each part was filtered, concentrated under reduced pressure then suspended in distal water and partitioned with petroleum ether (B.P, 60-80 C) three times. Later on the fractions of petroleum ether were concentrated to dryness over anhydrous sodium sulfate, weighted and subjected for identification of ß-sitosterol by HPTLC (13, 14) . Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 77 Method B: the UAE was done using probe ultrasonicator and bath ultrasonicator with 85% methanol was used as a solvent. The UAE was carried out under the following experimental conditions in case of probe ultrasonicator the plant material is in direct contact with ultrasonic waves, temperature 25 C ,time 20 minutes, solid to solvent ratio 1:8 gm/ml and sonication frequency 20KHz and power of 60 watt. While in bath ultrasonicator the ultrasonic waves have to pass through the glass material in order to come in contact with plant material, temperature 50 C ,time 60 minutes, solid to solvent ratio 1:8 gm/ml and sonication frequency 60KHz and power of 60 watt. In the current work, both instruments were used in order to study the effect of direct and indirect contact of ultrasonic waves on extraction of ß-sitosterol from four parts of Iraqi Awsaj plant. The same schematic procedure was used in conventional method (Soxhlet) was applied, in which the crude methanolic extract filtered, concentrated, suspended in distal water, partitioned by petroleum ether. Later on the fractions of petroleum ether were concentrated to dryness over anhydrous sodium sulfate, weighted and subjected for identification of -sitosterol by HPTLC (15-18) . Identification and quantification of ß- sitosterol by HPTLC The sample solutions were spotted in the form of bands of width 8.0 mm with a Camag microliter syringe on precoated silica gel glass plate 60F254 (20 cm × 10 cm with 250 μm thickness; E. Merck KJaA) using a Camag Linomat 5 (Switzerland). A constant application volume of 4 μl was employed and space between two bands was 3.3 mm. The slit dimension was kept at 4.0mm × 0.3 mm and 20 mm/s scanning speed was employed and each track was scanned thrice and baseline correction was used. The mobile phase consisted of toluene – ethyl acetate ‐ chloroform, in the volume ratio of 5:1:4 (v/v) and 10 ml of mobile phase was used per chromatography. Linear ascending development was carried out in 20 cm x 10 cm automatic developing chamber (Camag, ADC 2) saturated with filter paper Whatmann no.1 in the mobile phase. The optimized chamber saturation time for mobile phase was 5 min at room temperature (25 C ± 2) at relative humidity of 50% ± 5. The length of chromatogram run was 7.5 cm. Later on to the scanning, TLC plates were dried for 5 min with the help of an air dryer. Densitometry scanning was performed with Camag TLC scanner in the reflectance absorbance mode at 196 nm and operated by Win CATS software using tungsten lamp. Subsequent to the development, TLC plate was sprayed with 5% sulphuric acid reagent followed by drying in oven at 110 C for few min, all these steps was done at Baghdad collage of pharmacy (2, 19) . Quantitative estimation of B-sitosterol was done by using calibration curve in which series of diluted solutions (0.5, 0.25, 0.125, 0.0625 mg/ml) were prepared from ß-sitosterol standard stock solution (0.5mg/ml) (20) . Isolation and purification of ß-sitosterol by preparative HPLC ß-sitosterol was isolated from petroleum ether fraction of Awsaj roots by preparative high performance liquid chromatography (PHPLC) using Mediterranean Sea 18s µm (15x2.12, ambient) column and methanol (HPLC grade) as mobile phase. The isocratic elution was carried out for 10 min at flow rate 25ml/min, injection volume was 2 ml/min and detection was recorded with UV detector at 196 nm. All these steps was done at college of pharmacy/Baghdad University. The isolated compound was recrystallized using hot methanol and compared with standard ß- sitosterol by different identification method including: (21, 22) 1. Thin layer chromatography (TLC) using the best mobile phase toluene – ethyl acetate ‐ chloroform 5:1:4 and detection by spraying with 5% H2SO4 + heating at 110 Co 2. Melting point. 3. Fourier transforms infrared (FT-IR) spectroscopy (FTIR) in KBr disk using FT/IR 4200 Type A. 4. Preparative high performance liquid chromatography (PHPLC) using the previously mentioned mobile phase. Results and Discussion Plant extract yield comparison The results showed that probe UAE produced the higher yield in the extraction fraction followed by Soxhlet extraction fraction and finally bath UAE give the lower yield (table 1). The advantage of probe UAE is increased as it is considered the reduction of the extraction time and in the solvent consumption. Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 78 Table (1): Difference in extract content yield (gram of fraction / 250 gm) in different parts of Awsaj plant using different methods. Parts of Awsaj Soxhlet petroleum ether fraction UAE petroleum ether fraction Probe Bath Fruits 8.5 9.2 7.5 Leaves 10.5 11.3 9.4 Stems 3.2 3.8 2.9 Roots 5.3 6.9 4.8 Identification and quantification of ß- sitosterol by HPTLC The results indicated that the HPTLC method was developed for the first time for qualitative identification and quantitative estimation of -sitosterol from four parts of Iraqi Awsaj plant in which qualitative identification was made by comparison of maximum retardation factor (Rf) and UV spectrum of petroleum ether fraction of each part with that of stander -sitosterol as shown in figures 3-6. Figure (3): HPTLC chromatogram of ß- sitosterol standard Figure (4): HPTLC chromatogram of petroleum ether fraction extracted from four parts of Awsaj plant by conventional (Soxhlet) method. A= Roots, B= Fruits, C= Leaves, D= stems. Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 79 Figure (5): HPTLC chromatogram of petroleum ether fraction extracted from four parts of Awsaj plant by UAE probe method. A= Roots, B= Fruits, C= Leaves, D= stems. Figure ( 6 ): HPTLC chromatogram of petroleum ether fraction extracted from four parts of Awsaj plant by UAE bath method. A= Roots, B= Fruits, C= Leaves, D= stems. Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 80 For quantification measurements, the calibration curve were plotted using area under the curve (AUC) versus four concentration levels of ß-sitosterol standard. A straight line equation were obtained from which the concentration of the analyte was calculated in each part of Awsaj plant as shown in figure 7. Figure(7): Calibration curve of - sitosterol standard on HPTLC Quantitative concentration of -sitosterol in petroleum ether fraction revealed that roots contain the higher concentration of ß-sitosterol followed by fruits, leaves and the least was stems. The high concentration of -sitosterol in roots of Iraqi wild grown Awsaj may be attributed to the availability of biochemical factors which are considered essential elements for the formation of building back of this secondary metabolite. The probe UAE gives the higher concentration of ß-sitosterol followed by Soxhlet and last by bath UAE, as shown in (table 2) and (figure 8). Table(2): Quantitative analysis of - sitosterol in each part of Awsaj by HPTLC Part of plant Concent ration mg/ml by Soxhlet Concentra tion mg/ml by probe UAE Concentr ation mg/ml by bath UAE Roots 0.465 0.610 0.412 Fruits 0.295 0.485 0.122 Leaves 0.135 0.405 0.113 Stems 0.120 0.275 0.0534 Figure (8): HPTLC, track 1-4= -sitosterol standard, track 5-16= petroleum ether fraction obtained by probe UAE, bath UAE and Soxhlet methods respectively for each part of Awsaj. Isolation of -sitosterol by preparative HPLC According to HPTLC results; isolation and purification of ß-sitosterol from the root parts of Iraqi Awsaj plant extracted by probe UAE will be done by preparative HPLC which is considered now the most powerful and versatile method for purification tasks in pharmaceutical industries. PHPLC chromatogram showed twelve peaks which represent twelve different compounds according to their retention time, one of them (-sitosterol) at retention time of 1.142, is a major peak which was identified by comparison with standard -sitosterol at retention time of 1.123. The major peak was collected by fractions collector after monitoring it according to the time ( time from Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 81 beginning of peak appearance until start to disappearing of the peak) figure 9 and 10, then the sample obtained from PHPLC was dried over anhydrous sodium sulfate, weighted and subjected to different identification methods. Figure (9): PHPLC chromatogram of standard -sitosterol Figure( 10): PHPLC chromatogram of roots petroleum ether fraction Characterization of the isolated ß-sitosterol 1- Thin layer chromatography (TLC): The isolated compound appeared as a single spot having the same color and Rf value as that of standard -sitosterol as shown in (figure 11). 2- Measuring melting point: The isolated compound had a sharp melting point 138-140 Co, compared to melting point 139-140 Co for the ß-sitosterol standard (23) . 3- Fourier transforms infrared (FT-IR) spectra: FT-IR spectroscopy is most commonly used in phytochemical studies as a finger printing device, for comparing a natural with the synthetic reference standard, so IR spectra of isolated compound gave identical results which was compared with standard -sitosterol as shown in figure 12 and 13, and the characteristic IR absorption bands of isolated compound are listed in (table 3) (24) . Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 82 Figure (11): TLC Chromatogram of isolated compound (ISO) with -sitosterol standard (ST) on silica gel GF 254, developing in toluene – ethyl acetate ‐ chloroform 5:1:4 and detection by spraying with 5% H2SO4 + heating at 110 Co. Figure (12): IR Spectrum of standard -sitosterol Figure (13): IR Spectrum of isolated -sitosterol Iraqi J Pharm Sci, Vol.26(2) 2017 -sitosterol in Iraqi Awsaj plant 83 Table( 3): Characteristic infra-red frequencies of isolated ß-sitosterol compound Phytosterol compound Approximated position of characteristic bands (cm-1) -sitosterol 3361.32 (free O-H stretching band), 2931.27 and 2854.13 (aliphatic C-H stretching), 1654.62 (C=C absorption peak), 1458.89 (C-H bending /-CH2), 1053.91 and 874.56 (cycloalkane) 4- Preparative HPLC PHPLC was used again for characterization and identification by comparing the retention time of the isolated (1.117) with that of standard -sitosterol (1.123) as shown in (figure 14). Figure (14): PHPLC chromatogram of isolated ß-sitosterol Conclusion From the above findings, the utilization of probe UAE has proved to be much simpler and more effective than the conventional Soxhlet and bath UAE extraction methods for obtaining ß-sitosterol from fruits, leaves, stems and roots of Iraqi Awsaj plant. HPTLC and comparison of standard facilitated the identification and quantification of B- sitosterol present in petroleum ether fraction of the four parts of Awsaj plant. ß-sitosterol was isolated from petroleum ether fraction of root parts of Awsaj and characterization was carried out by measuring of melting point follow by running two chromatographic procedures (TLC and PHPLC) and finally spectral characteristic (FTIR). The current research also recommends using the other bioactive Phytosterols in order to confirm the presence of them in Iraqi Awsaj plant. Acknowledgment The authors wish to thank Mr. Zaid M. 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