J Nat Prod Plant Resour, 2021, 11 (1): 1-12 Legesse Available online at www.scholarsresearchlibrary.com Scholars Research Library Journal of Natural Product and Plant Resources, 2021, 11 (1): 1-12 (http://scholarsresearchlibrary.com/archive.html) Phytochemical Investigation of the Root Extract of Carduus Chevallieri Natnael Shamebo, Legesse Adane Bahiru, Tegene Tesfaye Tole Hawassa University, Department of Chemistry, College of Computational and Natural Sciences, Hawassa University Corresponding author: Legesse Adane Bahiru; Hawassa University, Department of Chemistry, College of Computational and Natural Sciences, P. O. Box 005; Hawassa, Ethiopia; E-mail: adanelegesse@gmail.com ABSTRACT Plant-derived substances have recently become of great interest owing to their versatile applications. The species Carduus chevallieri (C. chevallieri) is a traditional medicinal plant used to treat abdominal disorder, wounds, diabetes and hypertension among the peoples of Southern Nations Nationalities Peoples Regional State, Ethiopia. The chemical constituents of C. chevallieri, however, have not yet investigated although it has a wide traditional use for different ailments. The aim of this study was to carry out phytochemical screening, compound isolation, and structure elucidation of compounds isolated from the root of C. chevallieri.A standard phytochemical screening method was used to investigate the presence or absence of secondary metabolites, in the crude extracts. Compound isolation was performed using chromatographic separation techniques. Spectral data for structure elucidation of the isolated compounds were obtained by using IR, 1H-NMR and 13C-NMR spectroscopic techniques. The structure elucidation of the compounds was performed by interpretation and analyzing the IR, 1H-NMR and 13C NMR spectral data and comparison with literature reports. The root of C. chevallieri was collected from Angacha town, Kembata Tembaro Zone, South Nations and Nationalities Regional State, Ethiopia. The crude extracts were obtained by maceration technique by drying the root (500 g) under shed, finely grinding, and soaking the root powder in solvents n-hexane chloroform, acetone, chloroform/methanol and methanol, sequentially. The solvent was removed by rotary evaporator and the extracts were recovered. The phytochemical screening tests of the chloroform and methanolic extracts revealed the presence of cardiac glycosides, terpenoids, steroids, tannins, alkaloids, flavonoids, anthraquinones, saponins and phenols. Syringin and stigmasterol were isolated from the methanolic extract. Other than the two isolated compounds, its richest bio-resource of several bioactive secondary metabolites that can be used as candidates in drug discovery and development programs. This makes the species a valuable medicinal plant. Keywords: Carduus Chevallieri, Phytochemical Investigation, Stigmasterol, Syringin, Secondary Metabolite INTRODUCTION Plant-derived substances have recently become of great interest owing to their versatile applications. Medicinal plants are the richest bio-resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs [1]. Traditional medicine has been brought into focus for meeting the goals of a wider coverage of primary Page | 1 Scholar Research Library Legesse J Nat Prod Plant Resour, 2021, 11 (1): 1-12 healthcare delivery, not only in Africa but also, in all countries of the world. It is the first choice healthcare treatment for at least 80% of Africans who suffer from high fever and other common ailments [2]. Ethiopians used traditional medicines for many centuries, the use of which has become an integral part of the different cultures. The indigenous peoples of different localities in the country have developed their own specific knowledge of plant resource uses, management and conservation [3]. The genus Carduus belongs to the family Asteraceae, and consists of approximately 90 species worldwide [4-8]. In this genus, the different species are widely used for medicinal purposes by communities of various countries where the plants are available in abundant. The treatment of various human diseases such as cold, stomachache, and rheumatism are some examples of their medicinal uses [9]. They showed pharmacological activities such as antispasmodic, hypertensive [10], anti-inflammatory, antioxidant, anticancer, antiviral, and antibacterial activities [11,12], antimicrobial, antidiabetic [13], and anti-atherosclerotic effect [14]. The phytochemical screening of the genus Carduus [10,11,13] and isolation of compounds [15] have been reported by several research groups. So far the investigation of phytochemical and biological activities of three Carduus species of Ethiopian origin namely C. macracanthus [15] and C. schimperi [12,16-21] were the only studies reported. Similar to communities elsewhere in the world, these species are being used by the local people to treat different human illnesses. For instance, the root of C. schimperi is used for its anti-inflammatory, antinociceptive and antidiabetic activity [25]. C macracanthus is used for its anti-hypertension, anti-oxidant and antibacterial activities [15]. The roots of C. chevallieri (Figure 1) are used to treat abdominal disorder, wounds, diabetes and hypertension [personal observation]. Though C. chevallieri has wide applications in traditional medicine in Southern Ethiopia, no phytochemical investigation of the root of the plant has been performed so far. The aim of this study was to carry out phytochemical screening, compound isolation, and structure elucidation of compounds isolated from the root extract of C. chevallieri. The results could contribute to a better understanding of the chemical constituents of the investigated plant. Figure 1. The aerial par (a) and root part of C. chevallieri: (b) photo by Natnael S., February, 2019; Angacha, SNNPR, Ethiopia MATERIALS AND METODS Page | 2 Scholar Research Library J Nat Prod Plant Resour, 2021, 11 (1): 1-12 Legesse Plant material collection, preparation and extraction The roots of C. chevallieri were collected from Angacha town, Kembata Tembaro Zone, South Nations Nationalities Peoples Regional Sate, Ethiopia, in the month of February, 2019. The sample collection site was 7033ʹN latitude, 37085ʹ E longitude and 1831 m above sea level. The species was authenticated by a botanist, and its specimen with voucher number (CC/001) was deposited at Gullele Botanic Center, Addis Ababa, Ethiopia. The collected plant materials (roots) were washed, and dried under shade. The dried roots were ground using a mechanical grinder below 30 C. The powdered root (500 g) was soaked in n-hexane (3L) in order to remove fatty and oily substances. The mixture was continuously shaken by orbital shaker (Grant GIS400) at room te mperature with a speed of 200 rpm for 48 hrs. The solution was then filtrated Page | 3 Scholar Research Library J Nat Prod Plant Resour, 2021, 11 (1): 1-12 Legesse And the filtrate was concentrated under reduced pressure using rotary evaporator (LABOROTA 400) at 40⁰C to get crude extract. Similar procedure was repeated on the marc with chloroform, acetone, chloroform/methanol (50:50% by volume) and methanol, respectively. The resulting crude extracts were placed in refrigerator [26] until used for phytochemical screening and chromatographic separation of compounds. Phytochemical screening Qualitative phytochemical analysis of the crude extracts of the C. chevallieri roots was performed by standard methods [27-29]. Test for gardiac glycosides (Keller-Kiliani test) Glacial acetic acid (1 ml) was added in 2 ml of crude extract. Then 1 ml of FeCl 3 solution was added into the mixture followed by addition of few drops conc. H2SO4. Formation of the green blue color indicates the presence of cardiac glycosides [30,31]. Test for terpenoids (Salkowski test) Chloroform (10 ml) was added into 5 ml of solution of crude extract. The mixture was filtered, 2 ml of filtrate was added into a test tube holding 2 ml of acetic anhydride. Then 3 ml of concentrated H2SO4 acid was added carefully into the mixture. Formation of blue-green ring indicates the presence of terpenoids in the mixture [30]. Test for steroids Acetic anhydride (10 ml) was added into a test tube containing 2 ml of alcoholic crude extract. Then 1 ml of sulphuric acid was added carefully into the mixture. Formation of violet or blue-green color indicates the presence of steroids [32]. Test for tannins Small amount (200 mg) of crude extract was boiled with 10 ml of distilled water in a 200 ml beaker. Then the mixture was filtered, and 2 ml of 0.1M FeCl 3 solution in 0.1N HCl and 0.8 ml of potassium ferocyanide was added into the filtrate. The formation of blue-black color precipitate indicates the presence of tannins in the plant extracts [33]. Test for alkaloids (Mayer’s test) Alkaloids were tested by adding small amount HCl into the 3 ml of alcoholic solution crude extract in a test tube. The mixture was heated, cooled and filtered. Then the filtrate was tested with 1 ml of Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow color precipitate indicates the presence of alkaloids [34,35]. Page | 4 Scholar Research Library J Nat Prod Plant Resour, 2021, 11 (1): 1-12 Legesse Test for flavonoids (Shinoda test) Flavonoids were determined by magnesium-hydrochloric acid reduction test. A piece of 1 mg magnesium ribbon (powder) and 1 ml of concentrated hydrochloric acid was added into the 3 ml of alcoholic solution of crude extract. Formation of red color indicates presence of flavonoids [34]. Test for anthraquinones (Borntrager’s test) Detection of anthraquinones was carried out by mixing 200 mg of crude extract with 10 ml of benzene. The mixture was shaken for five minute and filtered. Finally, 10% ammonia solution was added into the filtrate. Formation of pink or red or violet color in the ammonical (lower) phase indicates the presence of free anthraquinones [36]. Test for saponins Small amount (200 mg) of crude extract was mixed with 10 ml of distilled water in a test tube and was shaken vigorously. The formation of stable foam indicates the presence of saponins [37]. Test for phenols To test phenols, 2 ml solution of crude extract was treated with 2 ml of 2% FeCl 3 solution. Formation of violet color indicates the presence of phenols [29]. Isolation and structure elucidation of compounds The methanol extract was selected for chromatographic separation of compounds for its good TLC profile (in chloroform:ethanol solvent systems) and its highest yield. The methanolic extract (13.9 g) was dissolved in small amount of methanol, adsorbed onto silica gel (0.063 mm) and then allowed to dry at room temperature. The column was first loaded with n-hexane slurry of 140 g silica gel (0.063 mm) which then was followed by loading the adsorbed methanolic extract on top. The column chromatographic separation was started with chloroform and then mixture of ethanol in chloroform, eluent, with gradual increase in proportion of ethanol. Each fraction (15 ml) was collected and monitored with TLC. The components on the TLC plates were visualized with UV chamber at 254 and 365 nm (LF-2006). Fractions with identical Rf values were combined. Further fractionation of fractions with more than one component was performed to get the pure components. After isolating pure compounds, the samples were kept in refrigerator, to protect further oxidation, until they were sent for spectral analysis. The structures of the isolated compounds were elucidated by interpreting spectroscopic data obtained from 1H NMR and 13C-NMR (Bruker avance 400 MHz spectrometer), Infrared (IR) (Perk-Elmer BX infrared spectrometer, 4000 - 400 cm-1) and by comparison of the spectroscopic data with literature. All the spectral analyses were carried out at The Department of Chemistry, Addis Ababa University, Ethiopia. Reagents and chemicals were laboratory grade and were purchased from Sigma and Aldrich (Addis Ababa). RESULTS AND DISCUSSION Percentage yield of crude extracts The powdered plant material was extracted in n-hexane, chloroform, acetone, chloroform:methanol (50:50 % by volume), and methanol. The percent yields (calculated using Eq. 1) are presented below (Table 1). The most polar solvent (methanol) extracted the highest yield (3.54%). This indicates that the amount of polar compounds is highest in the plant. According to Cowan [38] methanolic extracts contain the most of the secondary metabolites like Page | 5 Scholar Research Library J Nat Prod Plant Resour, 2021, 11 (1): 1-12 Legesse anthocyanins, terpenoids, saponins, tannins, xanthoxyllines, totarol, quasinoids, lactones, flavones, phenones and polyphenols. The relatively small yield of acetone extract reveals the presence of small amount of hydrophilic and lipophilic components [29], more specifically phenols and flavonols [38]. The result is therefore in agreement with literature reports. The most polar solvent (methanol) is therefore the solvent chosen to extract most of the secondary metabolites of this plant. Reports suggest that the extract yield is based on the extent of polarity of the solvent used for extraction which also indicates the plant’s pharmacological importance and proves that a particular medicinal plant to possess high potential as source phytochemicals [39,40]. Mass of plant material (g) 500 Extract Mass of extract (g) % Yield Chloroform extract 6 1.2 Acetone extract 2 0.4 7 1.4 17.7 3.54 Chloroform/methanol (50:50% by volume) extract Methanol extract Table 1. The percentage yield of crude extracts Phytochemical screening of the crude extracts Phytochemical screening test of C. chevallieri root extracts revealed the presence of secondary metabolites such as cardiac glycosides, terpenoids, steroids, tannins, alkaloids flavonoids, anthraquinones, saponins and phenols. The acetone extract showed positive test for cardiac glycosides, steroids, flavonoids, anthraquinones and phenols whereas the chloroform extract showed positive result for all tests. The chloroform/methanol extracts showed positive test for terpenoids, tannins, alkaloids, anthraquinones and phenols. The methanolic extract showed positive result for most of the secondary metabolites except alkaloids and flavonoids (Table 2). This finding is consistent with literature reports that state a single solvent may not necessarily extract all useful bioactive compounds from a plant suggesting that several solvents need to be used to obtain as many secondary metabolites as possible [41]. Several reports revealed that phytochemicals or secondary metabolites possess several pharmacological activities. Cardiac glycosides are known to lower blood pressure; tannins exhibit antioxidant, antimicrobial and antiviral effects and terpenoids exhibit a potent analgesic as well as anti-inflammatory effects [42]. Alkaloids exhibit antioxidant, antiinflammatory activities, and flavonoids are used to reduce risk of cancer, heart disease, asthma and stroke. Anthraquinones are known to have anticancer, antimalarial, antileukemic, mutagenicity, anti-inflammatory and antimicrobial activities [43]. Saponins have anti-inflammatory cytotoxicity, antitumor, antimutagenic, antiviral, antihelmintic and hemolytic activities [44]. Phenolic compounds have the ability to intervene at all stages of cancer development [45]. Steroids are used to reduce the risk of cardiovascular diseases [46]. These facts substantiate the use of C. chevallieri in Southern Ethiopia, and it’s richest bio-resource of several bioactive compounds that could be used as candidates in drug discovery and development program. Extract Phytochemical Chloroform Cardiac glycosides + Acetone Chloroform/methanol (50:50% by volume) + - Methanol + Page | 6 Scholar Research Library J Nat Prod Plant Resour, 2021, 11 (1): 1-12 Legesse Terpenoids + - + + Steroids + + - + Tannins + - + + Alkaloids + - + _ Flavonoids + + - _ Anthraquinones + + + + Saponins + - - + Phenols + + + + “+” denotes presence of phytochemical; “-” denotes absence of phytochemical. Table 2. The phytochemical screening test results of the crude extracts of root of C. chevallieri Structural elucidations of the isolated compounds Compound NLT-1 was isolated as a pale yellow solid (43 mg) by combining fractions 31-48 which were obtained by a solvent system of chloroform:ethanol (20:80% by volume). Its Rf was 0.43 (80:20% chloroform:ethanol by volume). Its melting point was 190-193 C. Analysis of IR spectrum of compound NLT-1 showed a broad absorption band at 3379 cm -1 indicating O-H stretching of alcohol functionality. The absorption bands at 2923 and 2854 cm-1 indicate C-H stretching of CH3 and CH2 groups, respectively. The medium band at 1461 cm-1 could be attributed to a vinyl group bonded to an aromatic ring. The 1H-NMR spectrum (DMSO, 400 MHz) (Appendix 2) showed peak at 6.73 pm that could be attributed to aromatic methine (CH). On the other hand, doublet peak 6.5 ppm and triplet peak at 6.3 pm that correspond to proton of a vinyl group bonded to an aromatic ring and methylene group, respectively. The doublet peak at 4.91 ppm could be to glucose moiety methine (CH) group that bonded connected with alpha and anomeric O atoms. On the same spectrum, the doublet peak at 4.15 ppm and an intense singlet peak at 3.74 ppm correspond to aliphatic methylene (CH2) bonded with –OH group and methoxy group bonded to an aromatic ring (Table 3). The 13C-NMR spectrum (DMSO, 100 MHz, and Appendix 3) showed a peak at 61.3 ppm that can be attributed to the presence of methylene (CH2) group that bears an OH group. The peaks at 128.9 and 130.6 ppm could indicate aliphatic C=C bond that is bonded to an Page | 7 Scholar Research Library aromatic ring. Moreover, the peaks at 104.8, 134.2, 133.08 and 153.1 ppm could be attributed to carbon atoms of benzene ring (Table 3). The peaks in the range of 61.3-103.0 ppm suggest carbon atoms of sugar moiety (Appendix 2). The strong peak at 56.7 ppm indicates a methoxy group bonded to an aromatic/benzene ring. The 13C-NMR spectral data was consistent with the DEPT-135 spectrum (Appendix 4)) of compound NLT-1. The patterns of the spectra of the compound with literature reports suggest that compound NLT-1 is identical to Syringin (Figure 2) [47-50]. The NMR spectral data of compound NLT-1 and that of Syringin are summarized in Table 3. Isolation of Syringing has been reported from the aqueous root extracts C. schimperi, and its presence has been attributed to in vivo anti-inflammatory and antinociceptive effects of the plant [16]. Figure 2. The proposed structure of compound NLT-1 (or Syringin) Carbon C-1 13 C-NMR data of compound NLT1 61.9 1 H-NMR data of Reported 13C-NMR data of syringing[47-50] Reported 1H-NMR data of syringin [47-50] compound NLT1 4.15 (dd, J=4.54, 1.2 64.0 4.21 (dd, J= 5.6, 1.2 Hz, 2H) Nature carbon of CH2 Hz, 2H) C-2 128.9 C-3 130.6 6.3 (dt, J= 4.8, 5.05 130.5 6.35(dt,J=15.8,5.6Hz, 1H) CH 131.7 6.59(d,J=10.9Hz, 1H) CH Hz, 1H) 6.5(d, J=16.42 Hz, 1H) C-4 134.2 C-5 104.8 136.3 6.73 (s, 105.9 C 6.76 (s, 1H) CH 1H) C-6 153.1 154.7 C C-7 133.0 135.7 C C-6’ 153.1 C-5’ 104.8 C-8 56.7 154.7 6.73 (s, C 105.9 6.76 (s, 1H) CH 57.4 3.87 (s, 3H) -OCH3 57.4 3.87 (s, 3H) -OCH3 1H) 3.73(s, 3H) C-8’ 56.7 3.73 (s, 3H) C-1” 103.0 4.91 (d, J=6.58 Hz, 105.8 4.85(d, J=7.5Hz, 1H) CH 1H) C-2” 74.6 3.12 (m, 1H) 76.0 3.33 (m, 1H) CH C-3” 76.9 3.16 (m, 78.1 3.43 (m, 1H) CH 71.6 3.50 (m, 1H) CH 78.6 3.23 (m, 1H) CH 1H) C-4” 70.3 3.25 (m, 1H) C-5” 77.6 3.05 (m, 1H) C-6” OH on 3”and 61.3 3.60(dd,J=11.16Hz, 2H) 63.0 3.69(dd,J=12.0Hz,2H) 3.49 (m, CH2 OH 2H) 4” OH on 4.35 (s, C- OH 1H) 6” OH on C1 5.03(s, OH 1H) OH on C- 3.22 (m, OH 1H) 2” Table 3. 1H-NMR and 13C-NMR spectral data of compound NLT-1 and Syringin from column chromatographic separation that was eluted by a solvent system of chloroform: ethanol (90:10 % by volume). Its Rf value was found to be 0.52 (in 90:10 % chloroform: ethanol by volume). The melting point of this compound was found to be between 139-143°C. The analysis of IR spectrum (Appendix 5) showed a strong absorption band at 3402.20 cm−1 indicating the presence of hydroxyl (O-H) group. On the other hand, a band at 1647.10 cm-1 could be attributed to unconjugated olefinic (C=C) stretching. The 1H-NMR spectrum (Appendix 6) showed the presence of peaks in the range of 0.69-1.00 ppm and 1.15 - 1.98 ppm revealed the presence of methyl and methylene protons, respectively. On the other hand, peaks at 5.03 ppm and 5.15 ppm revealed the existence of olefinic protons whereas the multiplet at 3.61 pm may reveal a proton bonded to the carbon that bears OH group. The 13C-NMR spectrum (DMSO, 100 MHz) indicate existence of methyl and methylene carbon atoms in the range 12-60 ppm. The signals at 141.1, 121.4, 139.1 and 129.2 ppm indicate the presence of olefinic carbon atoms. The signal at 71.1 ppm could be attributed to a carbon atom bearing –OH group. The aforementioned interpretation of the spectral data of NLT-2 and similarity of its spectral data with the spectral data of stigmasterol in the literature [51,52] confirmed that compound NLT-2 is Stigmasterol (Figure 3). The DEPT-135 spectrum is also consistent with the above interpretation. The positive test observed (Table 2) for steroid for methanol crude extract can also support this suggestion. The NMR spectral data of compound NLT-2 and that of Stigmasterol are summarized in Table 4. Figure 3. The proposed structure of compound NLT-2 (or Stigmasterol) Carbon The 13C-NMR data of compound NLT2 Reported 13C-NMR data of Stigmasterol [51,52] C-1 37.1 37.15 C-2 29.2 31.56 C-3 71.1 71.71 The 1H-NMR data of compound NLT2 The reported 1H- NMR data of Stigmasterol [51,52] Nature of Carbon CH2 CH2 3.61 (dtt, 1H) 3.51 (tdd, 1H) -CHOH C-4 43.5 42.19 C-5 141.1 140.81 CH2 C-6 121.4 121.62 C-7 31.9 31.56 CH2 C-8 31.4 31.79 CH C-9 51.4 50.02 CH C-10 37.2 36.16 C C-11 21.1 21.12 CH2 C-12 39.3 39.57 CH2 C-13 40.5 42.10 C C-14 56.4 56.76 CH C-15 21.6 24.27 CH2 C-16 31.5 28.83 CH2 C-17 55.2 55.84 C-18 12.2 12.15 1.00 (s, 3H) 1.03 (s, 3H) CH3 C-19 19.1 19.88 0.79 (s, 3H) 0.71 (s, 3H) CH3 C-20 43.1 40.51 C-21 21.2 20.99 0.93(d,3H,J=6.5Hz) 0.91 (d, 3H) CH3 C-22 139.1 138.23 5.03(m, 1H) 4.98 (m, 1H) C=CH C-23 129.2 129.16 5.07(m, 1H) C-24 51.4 51.30 CH C-25 32.1 31.94 CH C-26 18.9 19.01 5.17 (t, 1H) C=C 5.31 (t, 1H) C=CH CH CH 1.15 (d, 5.14(m, 1H) C=CH 0.80 (d, 3H;6.6Hz) CH3 0.82 (d, 3H;6.6Hz) CH3 3H;J=6.6Hz) C-27 20.9 21.23 1.15 (d, 3H;J=6.6Hz) C-28 25.5 25.50 C-29 12.1 12.25 CH2 0.83(t, 3H; 6.9Hz) 0.83 (t, 3H;7.1Hz) Table 4. The 1H-NMR and 13C-NMR spectral data of compound NLT-2 and Stigmasterol CONCLUSIONS To the best of our knowledge there is no prior report on the chemical constituents of the root of C. chevallieri contrary to its high traditional use among the peoples of South Nations Nationalities Regional State, Ethiopia. Preliminary phytochemical screening of the extracts of the root extract revealed the presence of cardiac glycosides, terpenoids, steroids, tannins, alkaloids, flavonoids, anthraquinones, saponins and phenols. Chromatographic separation of the methanolic extract of the root afforded the glycoside Syringin and the steroid Stigmasterol. These findings substantiate the use of C. chevallieri in peoples of Southern Ethiopia, and its potential as the richest bioresource of several bioactive compounds that can be used as candidates in drug discovery and development program. 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