Iraqi J Pharm Sci, Vol.31(1) 2022 Campsis grandiflora DOI: https://doi.org/10.31351/vol31iss1pp176-183 176 Isolation of beta-sitosterol and evaluation of antioxidant Activity of Iraqi Campsis grandiflora flowers Sarah Saad Hasson *,1, Ibrahim Saleh Abbas ** and Bahir Abdul Razzaq Mshimesh*** *Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, Mustansiriyah University, Baghdad, Iraq. **Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, Mustansiriyah University, Baghdad, Iraq. ***Department of Pharmacology and Toxicology, College of pharmacy, Mustansiriyah University, Baghdad, Iraq. Abstract Campsis grandiflora (Bignoniaceae) is a fast growing deciduous climber, the dried flowers have been used as a carminative, blood tonic, and febrifuge in Chinese traditional medicine. This plant has an anti- inflammatory, anti-oxidant, anti-depressant, and anti-bacterial effect; with a beneficial role in stagnant blood and endometriosis conditions. In this study, the detection of beta-sitosterol in the hexane extract of Iraqi C.grandiflora flowers was performed using thin layer chromatography (TLC) and high performance liquid chromatography(HPLC); while the isolation done by preparative layer chromatography then structure elucidation of isolated compound was done by FTIR and 1HNMR. Furthermore, assessment of the anti-oxidant activity of the ethyl acetate extract of Iraqi C.grandiflora flowers using three different methods and total flavonoid content, then measuring the pearson’s correlation coefficient between these methods. The results showed that the hexane extract of Iraqi C.grandiflora flowers contain beta-sitosterol compound and the ethyl acetate extract of this plant possesses an excellent anti-oxidant effect using the single-electron transfer (SET) pathway in scavenging the free radicals, and this activity attributed to the potent antioxidant i.e. polyphenols. Keywords: Anti-oxidant activity, Betasitosterol, Campsis grandiflora, Total flavonoids content. ألزهار نبات البوق الزاحف العراقي لألكسدة ةالمضاد مركب البيتا سيتوستيرول وتقييم الفعاليةعزل *** مشيمش باهر عبد الرزاق و ** ابراهيم صالح عباس ،1*’ سارة سعد حسون العراق ، المستنصرية، بغداد الجامعة، الصيدلة كلية الطبية، فرع العقاقير والنباتات * العراق ، بغداد ، الجامعة المستنصرية ، كلية الصيدلة الطبية، فرع العقاقير والنباتات ** العراق ، بغداد ، المستنصريةالجامعة ، الصيدلةكلية والسموم، فرع االدوية *** الخالصة تم ايلتددام الزهو المجضضة لذاا النبات كماد اا د للري ، منطل هو متسلل نضيلس يلريل النمو ، )عائلة البنونية( نبات البوق الزاحف ا مل دو للدم ، وميلاد للمم فس الط التقليد الصلينسه هاا النبات لت تيرير ميلاد لهلتذابات ض ميلاد لةكسلد ض ميلاد لهكتضاد وميلاد للب تيري د ايلة تم ال طلف عم مرك البيتا يليتويلتيرو فس مسلتدلك الذ سلار نبها نبات مضيد فس حاالت كود الدم واالنتباذ البطانس الرحمسه فس هاه ال بينما تم العز بوايللطة ( HPLCوال روماتوارافيا السللائلة عالية اندا ) (TLC)البوق الزاحف العراقس بايللتددام كروماتوارافيا الطبقة الرقيقة عهو عل ذلك ، (HNMRو FTIRكجر ال يميائس للمرك المعزو بوايلطة) رم تم توضلي السلتر ((PLCكروماتوارافيا الطبقة التميليرية وقياس ممتوى بييلللتددام رهر ارق مدتلضة تم تقييم النطلللاا الميلللاد لةكسلللد لمسلللتدلك ليللليتات اتيايب نبها نبات البوق الزاحف العراقس لظذرت النتائج لر مسلللتدلك الذ سلللار مم لبها نبات البوق الزاحف الضهفونويد ال لسض رم حسلللاد معامب اال تباا )بيريلللور( بيم هاه الطرقه ا ميلادما لةكسلد بايلتددام ا ممتابم مسلا نقب العراقس يمتو عل مرك البيتا يليتويلتيرو ولر مسلتدلك ليليتات اتيايب لذاا النبات يمتلك تيريرم إل ميادات انكسد القوية ماب البوليضينو هالجاو المر ، و يُعزى هاا النطاا كس فس (SET) اتل ترور الضرد . محتوى الفالفونويد الكلي ، نبات البوق الزاحف، بيتاسيتوستيرول ، الكلمات المفتاحية: الفعالية المضادة لالكسدة Introduction Campsis grandiflora (Bignoniaceae) is a fast growing deciduous climber, native to central and southern China; its flowers are hermaphrodite, curled, and bright orange-red in color. This plant blooms for about eight months, between the last week of March and the second week of October(1).Flowering of C.grandiflora plant belongs to the cornucopia pattern, in which a large number of plant flowers are produced in each inflorescence for about several months(2). The dried flowers of this plant, also known as ''aborticide'' in chinese folk, have been used as a carminative, blood tonic, febrifuge, and depurative diuretic in Chinese traditional medicine. Rheumatoid pains and menstrual problems exacerbated by blood stagnation, swelling breast after child birth, rubella, bleeding rectum, and diabetes have been treated with a decoction of this flowers(3,4). C.grandiflora flowers have several pharmacologic activities e.g. cellular protection and anti -oxidant effect(3), anti- 1Corresponding author E-mail: sara.saad.hassoon@gmail.com Received: 27/8/2021 Accepted: 22/9 /2021 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31iss1pp176-183 Iraqi J Pharm Sci, Vol.31(1) 2022 MRSA/VRSA nasal carriage of school students 177 -depressant effect(3), with a beneficial role in stagnant blood, and endometriosis conditions(6).The objective of this study was detection and isolation of the beta-sitosterol present in the Iraqi C.grandiflora flowers with the evaluation of the anti-oxidant effect and total flavonoid content of the ethyl acetate extract of Iraqi C.grandiflora flowers and measuring the correlation between these methods. Materials and Methods Plant material collection Flowers of Campsis grandiflora were collected from Baghdad – hay Aljamea`a in July 2020. The plant material was authenticated at the University of Baghdad / College of sciences by a professional taxonomist. Flowers were separated from the rest of the plant and washed thoroughly. It was then dried under shade conditions at room temperature for seven days. After that, the dried flowers were ground in a mechanical grinder and finally stored in a glass container at 4◦C. Chemicals and reagents Beta-sitosterol and quercetin standards were purchased from Changdu Biopurify; hexane, ethanol, acetone, DPPH, TPTZ, ABTS, sodium nitrite were obtained from Sigma Aldirch. Ethyl acetate, methanol, FeCl3 hexahydrate, Potassium persulfate, chloroform and toluene were supplied from Alpha Chemika, Ascorbic acid was purchased from Merk, Sulphuric acid and aluminum chloride were obtained from BDH. Preparation of extract for the detection and isolation of compound 1 Powdered plant (100g) was extracted by soxhlet apparatus with n-hexane (1200ml) till exhaustion. The extract was dried using a rotary evaporator, weighted and labelled as hexane extract (HE)(7). TLC analysis An aliquot of hexane extract dissolved in about 2ml of hexane then applied to analytical TLC plate against standard beta-sitosterol, and developed in the following mobile phases(MP) (8–11): • MP1: aceton:hexane (1:3) • MP2: hexane :ethyl acetate (7:2) • MP3: toluene: chloroform :ethyl acetate (5:4:1) • MP4: chloroform :acetone (9:1) The plates were sprayed with anisaldehyde – sulphuric acid reagent followed by heating for the detection of distinct spots. HPLC analysis One milligram from hexane extract and standard beta-sitosterol was dissolved separately in HPLC grade methanol (1ml) using a mobile phase consisting of acetonitrile:methanol(70: 30, v/v). The flow rate was 1 ml/min, and the detector was monitored at 210 nm(12). Isolation of compound 1 Hexane extract (1.5 gram) was dissolved in hexane and conducted on Preparative Layer Chromatography plates (PLC) against standard beta- sitosterol(13) and developed in MP2 mobile phase(10). The detection was done by spraying the plates' side by anisaldehyde-sulphuric acid reagent followed by heating. The bands at Rf = 0.32 were scrapped off and the scrapped silica then eluted with warmed hexane, the purity of the C1 compound was confirmed by analytical TLC using the MP4 mobile phase. Spectral analysis of the isolated compound 1 The isolated compound was subjected to different spectral analysis e.g. FTIR and 1HNMR. Preparation of extract for the anti-oxidant assay Twenty-five gram of powdered plant material was defatted with 250 ml n-hexane by soxhlet apparatus. The marc was further extracted with ethanol 80% (250 ml) till exhaustion also by using the soxhlet apparatus. Firstly, the ethanolic extract was dried by rotary evaporator and weighted. Secondly, the dried extract was dissolved in 20 ml distilled water and partitioned with ethyl acetate (3×50 ml). The upper layer was collected, dried by rotary evaporator, weighted, labelled as ethyl acetate extract (EAE) and stored at 4◦C while the lower aqueous layer was discarded(14). The DPPH [ 1,1–Diphenyl – 2 picryl–hydrazyl] assay The DPPH assay was performed using a colourimetric method described by Anna Floegel and coworkers(15) in which a methanolic dilution of DPPH was used. (2.95) ml of DPPH solution (1 Mm DPPH in 80% methanol ) was mixed with (0.05) ml of EAE or ascorbic acid (concentrations ranging from 12.5 to 200 mcg per 1 ml methanol). The assay was performed in triplicate, and the developing mixture was incubated in the dark for 30 minutes with aluminium foil over it. At 517 nm, a decrease in the absorbance was observed. The control was prepared by replacing the amount of EAE or standard by 80% methanol. The findings were reported as a percentage of radical scavenging and estimated using the formula below(16): % 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴0 − 𝐴1) 𝐴0 × 100 𝐴0 is the absorbance of the control, 𝐴1 is the absorbance of plant extract (EAE) or standard ascorbic acid. The ABTS [2,2'–azino – bis (3– ethylbenzothiazoline–6–sulphonic acid) diammonium salt ] assay A colourimetric method(17) has been used to measure the antioxidant activity of EAE against ABTS•+ radical in which aqueous solutions (1:1) of ABTS (7 mM) and potassium persulphate (140 mM) were mixed. Then, this mixture was incubated in a Iraqi J Pharm Sci, Vol.31(1) 2022 MRSA/VRSA nasal carriage of school students 178 dark place for 12 hours at room temperature enabling the radical ABTS•+ to formed. After that, (1.96) ml from the previous solution was mixed with (0.04) ml of EAE or ascorbic acid (concentrations ranging from 12.5 to 200 mcg per 1 ml ethanol) and let to sit at room temperature. The assay was performed in triplicate, and the decrease in the absorbance was measured at 734 nm. The control was prepared by replacing the amount of EAE or standard by ethanol, and the findings were reported as a percentage of radical scavenging using the previously mentioned formula. The FRAP (ferric reducing antioxidant power) assay The FRAP assay was carried out using a colourimetric method(18) in which 3.8 ml of FRAP reagent was mixed with 0.2 ml of EAE or ascorbic acid (concentrations ranging from 12.5 to 200 mcg per 1 ml methanol). The FRAP reagent (10:1:1) was made by incorporating 10 parts of sodium acetate buffer solution (300 mM, pH=3.6) with 1 part from each of ferric chloride hexahydrate (20 mM) and TPTZ (10 mM). The assay was performed in triplicate, and the mixture was kept at 37◦C for 30 minutes. The absorbance was determined at 593 nm, and the control was made by replacing the diluted sample with the same volume of methanol. The findings were expressed in microgram of ascorbic acid equivalents (AAEs) per milligram of dried EAE(19) and calculated using the formula from the ascorbic acid standard curve: 𝑦 = 0.0025𝑥 + 0.1802 𝑦 is the sample's absorbance at 593nm, 𝑥 is the concentration of ascorbic acid equivalent measured in mcg/ml. The Total Flavonoids Content (TFC) The total flavonoids content of Iraqi C.grandiflora was estimated by using the aluminium chloride colourimetric method(20) in which 1ml of plant EAE extract (100 mcg/ml) or standard quercetin solution (5,10,20,40,60,80 and 100 mcg/ml ) was mixed with 4ml of distilled water and then adding 0.3 ml of sodium nitrite solution (5%) to all test tubes. After five minutes, 0.3 ml of aluminium chloride solution (10%) was applied, followed subsequently by 2 ml of sodium hydroxide solution (1%). The assay was performed in triplicate, and the mixture was mixed well, then, the absorbance was measured at 510 nm against blank. Total flavonoid content (TFC) has been determined using an equation derived from the quercetin standard curve(20) 𝑦 = 0.0009𝑥 + 0.0283 y is the sample's absorbance at 510 nm, x is the concentration of the flavonoids in the sample measured in mcg/ml. Statistical analysis The pearson’s correlation coefficient (r) was computed using the SPSS-16.0 (Statistical Packages for Social Sciences- version 16), the correlation is significant at (p≤0.05). Results and Discussion TLC results Phytosterol, such as beta-sitosterol, was reported in C.grandiflora flower extract(21). The results in (Table1) showed a matching in the Rf values of both beta-sitosterol and compound 1(C1) spots in four mobile phases, and this finding suggested the presence of beta-sitosterol in hexane extract of Iraqi C.grandiflora flower, as shown Figure.1. Table 1. The Rf values of compound 1(C1) as compared with the Rf values of standard beta- sitosterol. Figure1.Analytical TLC for the hexane extract and beta-sitosterol using MP1 solvent system:(A) C1 spot, (B) standard beta-sitosterol spot. HPLC results The peak at (6.973) minutes in the HPLC analysis of hexane extract of Iraqi C.grandiflora flowers was matched the peak of standard beta- sitosterol with a retention time (6.913) minutes, as shown in Figure 2. Mobile phase The Rf value of C1 The Rf value of standard beta- sitosterol MP1 0.51 0.50 MP2 0.32 0.33 MP3 0.42 0.43 MP4 0.69 0.67 Iraqi J Pharm Sci, Vol.31(1) 2022 MRSA/VRSA nasal carriage of school students 179 Figure2.The HPLC analysis: (I) hexane extract of Iraqi C.grandiflora flowers, (II) standard beta-sitosterol. Isolation of compound1 Hexane extract of C.grandiflora flowers was applied to PLC plates to give 52 mg (3.46 % w/w) of C1, and then the purity was confirmed using analytical TLC plate, as shown in Figure 3. Fourier Transform Infrared Spectrometry (FTIR) of compound1 The wavenumbers of the isolated compound (C1) showed a characteristic broad peak at 3242-3439 cm-1 that confirmed a hydroxyl group with peaks at 3007 and 1641 cm-1 that established a double bond. These results were confirmed by comparing the wavenumbers with the reported literature(22),as in Figure 4 Figure 3. Isolation and purity confirmation of C1 from hexane extract: (I) PLC plate for isolation using MP2, (II) TLC plate for purity confirmation using MP4, (A)C1 spot, (A') standard beta-sitosterol spot. Figure 4. The IR spectrum of the isolated compound 40060080010001200140016001800200024002800320036004000 1/cm 45 52.5 60 67.5 75 82.5 90 97.5 %T 34 39 .1 9 33 77 .4 7 33 50 .4 6 30 07 .1 2 29 26 .1 1 28 54 .7 4 17 45 .6 4 16 41 .4 8 14 54 .3 8 13 73 .3 6 12 42 .2 0 11 51 .5 4 1 08 0. 17 10 28 .0 9 93 9. 36 86 4. 14 75 8. 05 71 3. 69 57 8. 66 53 0. 44 48 4. 15 1 Iraqi J Pharm Sci, Vol.31(1) 2022 MRSA/VRSA nasal carriage of school students 180 Nuclear Magnatic Resonance (NMR) of compound1 The isolated compound 1 (C1) exhibited four distinct chemical shifts in the 1HNMR spectrum (Figure5), which reflected the active functional groups. The chemical shifts began at 0.65 parts per million (ppm), which correspond to the hydrogens of saturated hydrocarbons, followed by the allylic protons, which have a chemical shift of 2.1 ppm. At 4.62 ppm, a proton next to a carbon adjacent to heteroatom can be observed. Finally, at 5.32 ppm, the vinylic proton can be seen. Figure 5. The 1HNMR spectrum of C1 The results of the different chromatographic techniques and various spectral analysis proved that the compound 1 was beta-sitosterol. The DPPH assay results The DPPH radical is a popular substrate for quickly determining the antioxidant activity of biological samples because of the simplicity of the assay and the stability of radical. As the DPPH radical is quenched by the antioxidant using the single electron transfer (SET) and hydrogen atom transfer (HAT) reaction mechanisms, the color of the DPPH solution changes from purple to yellow and the absorbance was read at 517 nm(23). The results of DPPH assay were illustrated in Table (2) and more visually expressed in Figure. 6. Table 2.The DPPH-radical scavenging activity of C.grandiflora EAE DPPH :1,1-Diphenyl-2-picryl-hydrazyl, EAE: ethyl acetate extract of Iraqi C.grandiflora. Figure 6.The antioxidant activity of EAE measured by DPPH method and compared with ascorbic acid standard: (EAE) ethyl acetate extract of Iraqi C.grandiflora,(DPPHassay)1,1- Diphenyl-2-picryl-hydrazyl assay. The ABTS assay results The ABTS can react with potassium persulfate in an oxidation reaction in which the colored-ABTS radical is formed. The degree of decolorization indicates that the antioxidants had the ability to transfer electrons or hydrogen atoms to inactivate this radical(24). The results of ABTS assay were illustrated in Table (3) and more visually expressed in Figure 7. DPPH assay Conc. of vit C or extract (mcg/ml) % scavenging activity of vit C % scavenging activity of EAE extract 12.5 22.90 17.63 25 39.00 40.43 50 57.60 48.68 100 74.06 52.50 200 86.03 71.69 Iraqi J Pharm Sci, Vol.31(1) 2022 MRSA/VRSA nasal carriage of school students 181 Table 3.The ABTS-radical scavenging activity of C.grandiflora EAE. ABTS: [2,2'–azino – bis (3– ethylbenzothiazoline – 6– sulphonic acid) diammonium salt], EAE: ethyl acetate extract of Iraqi C.grandiflora. The FRAP assay results In FRAP assay, the antioxidant activity was assessed by measuring the capacity of the antioxidants in the EAE to convert ferric (Fe+3) to ferrous (Fe+2) in a redox-linked colorimetric assay using single electron transfer (SET) as a reaction mechanism(25). The results were expressed as ascorbic acid equivalents (AAEs), measured in microgram ascorbic acid per milligram dried EAE, depending on the equation from ascorbic acid standard curve (table 4). Figure 7. The antioxidant activity of EAE measured by ABTS method and compared with ascorbic acid standard: (EAE) ethyl acetate extract of Iraqi C.grandiflora, (ABTS assay ) 2,2'azino–bis(3–ethylbenzothiazoline–6– sulphonic acid) diammonium salt. Table 4. The antioxidant activity measured by the FRAP method. Each value represents the mean±SEM of 3 samples; FRAP: ferric reducing antioxidant power, EAE:ethyl acetate extract of Iraqi C.grandiflora, AAEs: ascorbic acid equivalents. The total flavonoids content result The main antioxidant compounds in plants are phenols, which have an aromatic ring that enables the unpaired electrons in their arrangement to be stabilized and relocated, allowing electrons and hydrogen atoms to be donated from their hydroxyl groups(18). Since phenolic compounds are one of the main classes of compounds known to serve as primary antioxidants, and the litreture have shown a good correlation between the total flavonoids content (TFC) and the antioxidant activity(23), it's essential to calculate the amount of these compounds that present in the EAE. The results of the total flavonoids content illustrated in table (5). Table 5. The total flavonoid content of EAE of Iraqi C.grandiflora. Each value represents the mean±SEM of 3 samples; EAE: ethyl acetate extract of Iraqi C.grandiflora. The pearson's correlation coefficient results The data of pearson's correlation coefficient between the three different antioxidant methods and between these methods and TFC were summerized in Table (6). ABTS assay Conc. of vit C or extract (mcg/ml) % scavenging activity of vit C % scavenging activity of EAE extract 12.5 23.43 15.97 25 37.93 15.35 50 52.43 28.89 100 64.7 37.92 200 71.96 45.10 EAE concentration (mcg/ml) EAE absorbance (nm) Mcg Ascorbic acid equivalent (mcg/ml) AAEs (mcg/mg dried extract) 12.5 0.190±0.005 3.92±2.309 313.6±184.7 25 0.216±0.017 14.58±7.055 583.46±282.2 50 0.265±0.011 34.18±4.42 683.73±88.4 100 0.362±0.025 72.72±10.283 727.2±102.8 200 0.590±0.005 163.92±2.309 819.6±11.5 EAE concentration (mcg/ml) EAE absorbance (nm) Mcg Quercetin equivalent (mcg/ml) Mcg Quercetin equivalent /mg EAE 100 0.0903±0.0008 68.925±0.979 689.25±9.799 Iraqi J Pharm Sci, Vol.31(1) 2022 MRSA/VRSA nasal carriage of school students 182 Table 6.The pearson's correlation coefficient. DPPH assay :1,1-Diphenyl-2-picryl-hydrazyl assay ; ABTS assay : 2,2'–azino – bis (3– ethylbenzothiazoline – 6– sulphonic acid) diammonium salt assay ; FRAP assay : ferric reducing antioxidant power assay ; TFC : total flavonoid content ; * : means correlation is significant at (p≤0.05). The results demonstrated that Iraqi C.grandiflora ethyl acetate extract possesses strong antioxidant capacity measured with DPPH, ABTS, and FRAP assays compared with ascorbic acid. These findings were consistent with another study in which C.grandiflora ethyl acetate extract exhibit the highest radical scavenging potency against DPPH and ABTS radicals with IC50=1.625mcg/ml; this result was excellent when compared to IC50 of ascorbic acid (8.6 mcg/ml), also the IC50 of other fractions such as ethanol, petroleum, butanol, and water were (8.75, 32.25, 10, 7.6 mcg/ml), respectively(3). There was a positive correlation between the DPPH, ABTS, and FRAP assays, and this may be explained by the fact that these assays were suitable and accurate for determining the plant extracts' overall antioxidant capacities; these results were agreed with another research(26) in which a high correlation between these methods have been showed. A significantly high correlation coefficient was obtained from the DPPH and FRAPS assays (0.976) with a p-value ≤0.05. These findings suggested that the antioxidants in EAE of Iraqi C.grandiflora were used the single-electron transfer (SET) pathway in scavenging the free radicals; since the DPPH and FRAP assays were based on the SET pathway as a reaction mechanism. There was a negative correlation between the antioxidant assays and the TFC, and these findings pointed that, although there are a relatively high amount of flavonoids in the extract but there are various factors affecting the anti-oxidant activity of a substance and results in negative correlation, these include substance solubility, pH of the medium, oxidation state, etc(23). These results suggested that flavonoids not only the major contributor to the antioxidant effect of this plant , but there are other phytochemicals i.e. the non-flavonoidal compounds such as acetoside, caffeic acid, gallic acid, etc. that reported in C.grandiflora flower extract may contribute to this excellent activity since these compounds also exhibited a pronounced antioxidant effects(27,28). Conclusion Beta-sitosterol was isolated form the hexane extract of Iraqi C.grandiflora flowers, and this compound was confirmed with different chromatographic and spectroscopic techniques. 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