|Year : 2018 | Volume
| Issue : 2 | Page : 130-136
Chemical profile and biological activities of the aerial parts of Senecio acaulis (L.f.) Sch.Bip
Rehab M S. Ashour1, Abeer M El Sayed1, Mohamed F Elyamany2, Dina R Abou-Hussein3
1 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
2 Department of Pharmacology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
3 Department of Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt; Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
|Date of Web Publication||20-Apr-2018|
Dr. Rehab M S. Ashour
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El Aini, Cairo 11562
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Genus Senecio is known by its phenolic constituents, terpenoids, essential oil (EO), and pyrrolizidine alkaloids. No previous reports could be traced about the phytochemical study of Senecio acaulis. Objectives: To investigate the chemical composition and biological potentiality of EO of S. acaulis aerial parts and to study the phytoconstituents of the plant extract and its spasmolytic activity. Materials and Methods: The EO was obtained by hydrodistillation and its chemical composition was analyzed by gas chromatography coupled to mass spectrometry. In-vitro screen of antimicrobial, antimalarial, and antileishmanial activities was determined against positive controls. Column chromatography was used to isolate the phytoconstituents from chloroform and ethyl acetate fractions; their structures were elucidated using physical and spectral methods. Spasmolytic activity was measured before and after K+-induced contractions on isolated rabbit jejunum. DNA-fingerprint was established by RAPD-PCR technique using 12 primers. Results: The study of EO revealed the detection of 22 compounds representing 81.08% of the oil composition. The major constituents were D-limonene (13.32%), β-pinene (11.54%), and sabinene (10.79%). Eight compounds were isolated from the plant extract and identified as β-amyrin, β-sitosterol, lupeol, oleanolic acid, β-amyrin-3-O-β-glucopyranoside, isorhamnetin 3-O-β-glucopyranoside, isoquercitrin, and quercitrin. The oil exhibited moderate antimalarial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. The oil showed a significant antimicrobial activity against methicillin-resistant Staphylococcus aureus and Cryptococcus neoformans and weak antileishmanial activity. In isolated rabbit jejunum, the ethanol extract produced a relaxation of spontaneous and moderate effect against high K+ (80 mM)-induced contractions. Amplification of DNA yielded 87 RAPD fragments. Conclusion: Senecio acaulis (L.f.) Sch.Bip essential oil can be used as antimicrobial agent against Staphylococcus aureus and Cryptococcus neoformans. In addition, the spasmolytic activity of its ethanolic extract suggests its incorporation in antidiarrheal preparations. Further clinical trials are required to evaluate these effects on humans. Identification of twenty two compounds in its essential oil, isolation of eight compounds for the first time as well as authentication of the plant via DNA finger print may play an important role in its chemotaxonomic classification.
Abbreviations Used: EO: Essential oil, GC: Gas Chromatography, GC-MS: Gas Chromatography-Mass Spectrophotometry, ETOAc: Ethyl acetate, SEM: Standard error mean, RRI: Relative retention indices, SI: Selectivity Index, D6: chloroquine sensitive Plasmodium falciparum, W2: chloroquine resistant Plasmodium falciparum. SI: selectivity index, IC50, IC90: concentration that affords 50 and 90% inhibition, respectively, 1H-NMR: Proton Nuclear magnetic resonance, 13C-NMR: Carbon-13 Nuclear magnetic resonance.
Keywords: DNA-fingerprinting, essential oil, phytoconstituents, Senecio acaulis, spasmolytic activity
|How to cite this article:|
S. Ashour RM, El Sayed AM, Elyamany MF, Abou-Hussein DR. Chemical profile and biological activities of the aerial parts of Senecio acaulis (L.f.) Sch.Bip. Phcog Res 2018;10:130-6
|How to cite this URL:|
S. Ashour RM, El Sayed AM, Elyamany MF, Abou-Hussein DR. Chemical profile and biological activities of the aerial parts of Senecio acaulis (L.f.) Sch.Bip. Phcog Res [serial online] 2018 [cited 2019 May 26];10:130-6. Available from: http://www.phcogres.com/text.asp?2018/10/2/130/230755
Investigation of the essential oil of Senecio acaulis aerial parts revealed the identification of twenty-two compounds representing 81.08% of the oil composition. It demonstrated significant antimicrobial activity against Staphylococcus aureus and Cryptococcus neoformans and moderate antimalarial activity. Eight compounds belonging to sterols, triterpenes and flavonoids were isolated for the first time from the plant extract. The ethanolic extract showed the most potent spasmolytic activity compared to its fractions in reference to atropine. Furthermore, DNA-fingerprint was performed to distinguish S. acaulis from other species.
| Introduction|| |
Genus Senecio (Family Asteraceae) is huge among the flowering plants, comprising several chemical constituents particularly terpenoids,, phenolic and flavonoid compounds,, essential oils (EOs),, and pyrrolizidine alkaloids., In folk medicine, Senecio species were used as antiemetic, anti-inflammatory, and vasodilator and in the treatment of wounds. Many Senecio species in the previous studies had demonstrated an antispasmodic activity.,,, To the best of our knowledge, there is no report on the investigation of Senecio acaulis (L.f.) Sch. Bip. plant constituents or biological activities. This study aimed to explore the chemical composition of its EO and evaluate certain of its biological potentialities. Furthermore, isolation of the phytoconstituents and studying the spasmolytic activity of the ethanol extract and its fractions to verify the reported antidiahrreal activity of Senecio species. DNA fingerprint was also studied as contribution in the plant characterization.
| Materials and methods|| |
Plant material and extracts
The aerial parts (leaves and stems) of S. acaulis (L.f.) Sch. Bip. were collected from the Experimental Plant Station of Faculty of Pharmacy, Cairo University, Giza, Egypt, in May 2014. Mrs. Therese Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture, and Dr. Mohamed El-Gibaly, Ex-curator of National Research Centre Herbarium, Dokki, Giza, kindly verified the identification of the plant. A voucher specimen (numbered 8.5.2016) has been located at the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Egypt.
Hydrodistillation for 3 h using a Clevenger-type apparatus was used for separation of the EO, from fresh aerial parts (1 kg); the oil obtained was dried over anhydrous sodium sulfate, saved in a closed vial, and stored in a refrigerator for further investigation.
One kilogram of air-dried powdered aerial parts of S. acaulis was extracted by cold percolation (5 × 3 L) with ethanol (95%) till exhaustion. A greenish-brown residue, collected after evaporation of the solvent, was perched in distilled water (500 mL) and sequentially partitioned among n-hexane, chloroform, ethyl acetate, and n-butanol saturated with water till exhaustion. Solvent in each case was totally evaporated under reduced pressure, and residues obtained were kept for the study.
For alkaloid extraction, air-dried powdered aerial parts (10 g) were extracted with 0.5 N HCl (500 ml). The resulting extract was defatted with chloroform. Half of the aqueous phase was made alkaline with 25% NH4 OH solution (pH 10) and extracted with chloroform. The resulting extract was evaporated to dryness to give the free tertiary bases. The second half of the aqueous phase was brought to 2 N HCl and reduced with Zn dust overnight under continuous stirring, filtered, and treated as above to obtain the total alkaloids.
Before the isolation of DNA, a sample of fresh leaves was kept at −70°C, freeze-dried, and then pulverized under liquid nitrogen.
Chemicals used in spasmolytic activity on smooth muscle
Acetylcholine, sulfate ester of atropine, and potassium chloride were obtained from Sigma Chemicals Co, St Louis, MO, USA. All chemicals in the study were of the analytical grade and solubilized in distilled water/saline.
Electrothermal 9100 (U.K.) was used for melting point (m.p.) determination.1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) were assigned on Bruker NMR-spectrometer at 25°C using tetramethylsilane as an internal standard. Mass spectral data were performed using Ion Trap LC/Mass Spectrometry (MS) Agilent. UV-visible spectrophotometer, Shimadzu UV (P/N 204-58000), was used for footage UV spectra and measuring the absorbance in UV range. Reagents for UV spectroscopic analysis of flavonoids, viz., NaOMe, AlCl3, HCl, NaOAc, and H3 BO3, were prepared according to a method previously described. Vacuum liquid chromatography was performed using silica gel H-type 60 (Merck); silica gel 60 (70–230 Mesh, Merck) and Sephadex LH-20 (Pharmacia Fine Chemicals) were used for column chromatography (CC). Thin-layer chromatographic (TLC) analyses were conducted on precoated silica gel 60 F254 (0.2-mm thickness, Merck); chromatographic spray reagents were P-anisaldehyde-sulfuric acid for sterols and triterpenes  and aluminum chloride for flavonoids. Paper chromatographic (PC) analyses of sugars were conducted on Whatman no. 1 mm sheets (Whatman, Ltd., Madison, Kent, England) using aniline phthalate spray reagent. Solvent systems for TLC were 95% chloroform/methanol (S1), ethyl acetate/formic acid/acetic acid/water (100:11:11:20) (S2), and 90% chloroform/methanol with the addition of two drops of formic acid (S3), while solvent system (S4) for PC was composed of the upper phase of butanol/acetic acid/water (4:1:5).
Detection of pyrrolizidine alkaloids (Ehrlich's reagent test)
For the preparation of Ehrlich's reagent, 5 g of 4-dimethylamino benzaldehyde was separately dissolved in 3 ml distilled water, 10 ml of 60% perchloric acid, and 60 ml of acetic acid. Powdered plant, fraction of free tertiary bases, and total alkaloid fraction (1.5 g) along with 5% ascorbic acid (40 mg) were mixed with small amount of sand, separately. Each of them was shaken, and after 5 min, they were filtered and divided into sample and blank. Twelve milliliters of alkaline sodium nitroprusside 5% reagent was added to the sample tube. Ehrlich's reagent was added to both tubes, and they were heated on the water bath for few minutes. The red color determines the presence of pyrrolizidine alkaloid.
Gas chromatography and gas chromatography-mass spectrometry analysis for essential oil
The EO analysis of S. acaulis was performed by gas chromatography (GC) with a flame ionization detector and GC-MS using an Agilent GC − a mass selective detector system. Mass spectral libraries (Wiley GC/MS Library, MassFinder 3 Library) and the in-house “Baser Library of Essential Oil Constituents,” in addition to MS literature data, served in the identification of compounds.,,
Isolation of phytoconstituents
Column chromatographic fractionation of chloroform fraction
Seven grams of the chloroform fraction was subjected to fractionation by VLC on 150 g silica gel H column (7 cm length × 12.5 cm diameter) using n-hexane-CHCl3-EtOAc mixtures for gradient elution where the polarity was gradually increased by 5%. Fractions (100 mL, each) were collected and monitored by TLC. Five collective fractions were obtained upon elution with 15% CHCl3/n-hexane (Fr.a, 200 mg), 20%–25% CHCl3/n-hexane (Fr.b, 120 mg), 50% CHCl3/n-hexane (Fr.c, 220 mg), 80% CHCl3/n-hexane (Fr.d, 235 mg), and 10%–15% EtOAc/CHCl3(Fr.e, 300 mg). Fr.a–e were, separately, subjected to chromatographic purification on silica gel columns. Fr.a was purified on silica gel CC using isocratic elution by 10% EtOAc/n-hexane to afford compound 1 (16 mg). Fr.b gave compound 2 (12 mg) upon rechromatography on silica gel CC eluted with 20% EtOAc/n-hexane. Similarly, Fr.c and Fr.d were separately subjected to purification on silica gel CC using isocratic elution by 0.5% MeOH/CHCl3 for Fr.c and 1% MeOH/CHCl3 for Fr.d and yielded compound 3 (22 mg) and compound 4 (18 mg), respectively, while Fr.e afforded the isolation of compound 5 (25 mg) upon isocratic elution with 2% MeOH/CHCl3 on silica gel column.
Column chromatographic fractionation of ethyl acetate fraction
Five hundred milligrams of ethyl acetate fraction was purified on Sephadex LH-20 column eluted with methanol. The purified fraction (400 mg) was further subjected to silica gel CC eluted with CHCl3/MeOH gradient. Fractions (20 mL, each) were collected and monitored by TLC. Three major fractions were eluted with 7.5%–10% MeOH/CHCl3(Fr.f, 80 mg), 12.5% MeOH/CHCl3(Fr.g, 40 mg), and 15%–17% MeOH/CHCl3(Fr.h, 35 mg). Fr.g and Fr.h were then separately rechromatographed on silica gel CC using CHCl3/MeOH gradient and produced compound 6 (21 mg) from Fr.f, compound 7 (6.8 mg) and compound 8 (6.8 mg) from Fr.g and Fr.h, respectively.
Hydrolysis of glycosides
Acid hydrolysis of the glycosidic compound 5 was accomplished by refluxing with 15% H2 SO4 in MeOH for 6 h, while compounds 6–8 were separately hydrolyzed by 6% aqueous HCl on a steam bath for 45 min. Resulting aglycone and sugar in each case were separated, purified, and identified by TLC and/or PC.
Toxicity study: Determination of the median lethal dose
Median lethal dose (LD50) of the ethanolic extract of the aerial parts was estimated after oral administration of single doses ranging from 1 to 5 g/kg, which was the maximum soluble dose, using adult male albino mice (20–30 g) obtained from the National Research Centre, Giza, Egypt.
EO was tested for its antimicrobial activity on ten microorganisms, including five fungi and five bacteria. Microorganisms were obtained from the American Type Culture Collection (Manassas, VA, USA). Assay was carried out according to a reported process; amphotericin B and ciprofloxacin served as control for bacteria and fungi, respectively. Detailed experimental procedure was attached in the [Supplementary File (S-1) [Additional file 1]].
In-vitro antimalarial activity of the EO was investigated against D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) strains of Plasmodium falciparum. Evaluation was based on the determination of parasite lactate dehydrogenase activity using Malstat reagent. Chloroquine and artemisinin were the control drugs in the study. Cell viability was determined by means of the supravital dye neutral red. Detailed experimental procedure was attached in the [Supplementary File (S-2) [Additional file 2]].
The antileishmanial activity of the EO was initially assessed against Leishmania donovani according to a previous procedure. Pentamidine and amphotericin B served as positive controls. Oil sample was firstly tested in a primary screen at 80 μg/mL in duplicate, and percentage inhibition was considered relative to negative and positive controls. Detailed experimental procedure was attached in the [Supplementary File (S-3) [Additional file 3]].
Spasmolytic activity on smooth muscle
In control experiments, the medium used for solubilization on isolated tissue preparations was found inert. Stockpile solutions of chemicals were completed fresh in normal saline on the day of the experiment.
Rabbits (weighing 1.5–1.8 kg) of both sexes were obtained from the National Research Centre, Laboratory Animal Facility, Dokki, Giza. They were housed in stainless steel cages (34 cm × 47 cm × 18 cm) with softwood shavings as bedcovers, fed with standard pellets diet, and given water ad libitum. They were allowed to acclimate to standard laboratory conditions (24°C–28°C temperature, 60%–70% relative humidity, and 12-h light/dark cycle) for 1 week before the experiments. They were underprivileged of food for at least 18 h previous to experiments but allowable free access to drinking water. The gear usage, management, and sacrificing of the animals were performed in accordance with the Ethics committee (No. 9-031) in agreement with the recommendations for the appropriate care and use of laboratory animals (NIH Publication No. 80-23; revised 1978).
In-vitro study on isolated rabbit jejunum preparation
The spasmolytic activity of (0.01 mg/mL) of S. acaulis 95% ethanol extract in addition to the successive fractions (n-hexane, chloroform, ethyl acetate, and n-butanol-soluble fractions) was deliberated using isolated rabbit jejunum., Spontaneous rhythmic contractions of the rabbit jejunum were achieved, where the spasmolytic (relaxant) activity can be tested directly without using an agonist; then, the inhibition of duodenum contraction was expressed as percentage of mean ± standard error of the mean from six experiments. In addition, relaxation of rabbit jejunum was studied after the use of a spasmogenic; in this case, the preparations were depolarized by potassium ion (K+) as reported. To the tissue bath, high K + (80 mM) was added which fashioned a sustained contraction. The intestinal preparations relaxation, which was previously contracted with K + (80 mM), was expressed as the percentage of the control response mediated by K +. Isolation of rabbit jejunum was described in [Supplementary File (S-4) [Additional file 4]].
All IC50s in antimicrobial, antimalarial, and antileishmanial activities were calculated using the XLfit curve fitting software. Data for spasmolytic activity were expressed as mean ± standard error of mean (SEM, n = experiments number). Student's t-test was used as a statistical parameter; P < 0.05 was considered as significant difference.
DNA extraction and quantification
The sample was prepared adopting the procedure previously described. Detailed experimental procedure was attached in the [Supplementary File (S-5) [Additional file 5]].
| Results and Discussion|| |
Extraction yield, gas chromatography-mass spectrometry analysis, and biological activities of essential oil
The EO of the aerial parts of S. acaulis was investigated for the first time in this study; it was faint yellow in color, with aromatic odor and obtained by hydrodistillation in a yield of 0.11%. GC/MS analysis allowed the identification of 22 compounds, representing 81.08% of the total oil composition as presented in [Table 1]; identified constituents demonstrated the predominance of monoterpenes (77.68%) where hydrocarbons constituted 45.5% and the oxygenated compounds were 32.18%. Sesquiterpenes hydrocarbons represented 1.19% with complete absence of oxygenated sesquiterpenes. The main identified compound was D-limonene (13.32%) followed by β-pinene (11.54%) and then sabinene (10.79%). Cryptone (10.13%) was the major identified oxygenated compound. The results of analysis were in accordance with those of certain prestudied Senecio species regarding the predominance of monoterpene hydrocarbons,, while the prevalence of limonene was only reported for the EO obtained from Senecio polyanthemoides.
|Table 1: Chemical composition of the essential oil of Senecio acaulis aerial parts|
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Percentage inhibitions in the primary screen against ten strains of microorganisms revealed that the EO exhibited only good antifungal activity against Cryptococcus neoformans and moderate antibacterial activity against methicillin-resistant strain of Staphylococcus aureus relative to negative and positive controls. IC50 calculated in the secondary screen was 36.02 and 197.98 μg/mL, respectively. Moderate antimalarial activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of P. falciparum was displayed by the EO. IC50s was 8020.3 ng/mL (D6) and 5785.4 ng/mL (W2) and the selectivity indices were calculated in both cases, >5.9 (D6) and >8.2 (W2) with no cytotoxicity against the tested VERO mammalian cell line. Antileishmanial evaluation revealed weak activity with IC50 and IC90 values of 24.3 and 34.37 μg/mL, respectively. The results of biological activity of the EO, as recorded in [Table 2], may be attributed to the total composition of the oil as the single constituents were not tested separately and need further investigation. However, it was reported that D-limonene, the main oil constituent, inhibited isoprenylation of proteins in P. falciparum, arrested its development  and also possessed antimicrobial activity. In addition, it was worthy to mention that antileishmanial activity was proved by limonene β-amino alcohol derivatives.
|Table 2: Results of in vitro biological screening of essential oil of Senecio acaulis aerial parts|
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Yield of extractives and preliminary phytochemical screening of the aerial parts
The percentage yield of ethanolic extractive was 12% w/w. Successive fractionation yielded 0.78, 0.96, 0.17, and 0.74% of n-hexane, chloroform, ethyl acetate, and n-butanol fractions, respectively. Preliminary phytochemical screening revealed the presence of carbohydrates and/or glycosides, tannins, flavonoids, alkaloids and/or nitrogenous bases, sterols, and/or triterpenes in the aerial parts of the plant under investigation. Powdered plant, total ethanolic extract, and total alkaloid fraction gave negative results (no red color) when tested with Ehrlich's reagent.
Structure elucidation of isolated compounds
Chromatographic fractionation of chloroform fraction of the aerial parts led to the isolation of five known compounds: β-amyrin 1 (white powder, m.p. 188°C–190°C), β-sitosterol 2 ( white powder, m.p. 137°C–139°C), lupeol 3 (white powder, m.p. 120°C–122°C), oleanolic acid 4 (white powder, m.p. 270°C–273°C), and β-amyrin-3-O-β-glucopyranoside 5 (white powder, m.p. 220°C–222°C); their data were presented in [Table 3] and [Table 4]. However, chromatographic fraction of the ethyl acetate fraction afforded three compounds identified by their spectral data recorded in [Table 5] as isorhamnetin 3-O-β-glucopyranoside 6 (yellow powder, m.p. 157°C–160°C), isoquercitrin 7 (yellow powder, m.p. 225°C–228°C), and quercitrin 8 (yellow powder, m.p. 180°C–182°C). Compounds 1–8 illustrated in [Figure 1] were isolated for the first time from the plant extract, but certain of them were previously isolated from other species of Senecio.
Spasmolytic activity of the crude ethanolic extract and its fractions
No signs of acute toxicity were observed in animals after the oral administration of ethanolic extract. The results of toxicity study, together with the absence of pyrrolizidine alkaloids (Erhlich's test), encourage the authors to carry out biological studies designated for acute diseases, viz., spasmolytic activity on the ethanolic extract of the plant. Effects of the crude ethanolic extract and its fractions on the isolated rabbit duodenum were examined. Results showed a reduction of the spontaneous contraction by the ethanolic extract and the n-hexane fraction at 0.01 mg/mL concentration, the intestinal contraction was reduced by 62.0% ± 1.2% and 50% ± 3.1%, respectively, compared to standard antimuscarinic drug (atropine). The chloroform and the ethyl acetate fractions exerted a nearly matching reduction effect to the intestinal contractions 46% ± 2.1% and 45% ±2.0%, respectively. The butanol fraction (0.01 mg/mL) induced the lowest reduction of the spontaneous contraction of the duodenum, reaching 20% ± 3.1% as compared to atropine. In tissues pretreated with high K + (80 mM), the relaxant effect of ethanol extract, n-hexane, chloroform, and ethyl acetate fractions was markedly observed by 50%, 40%, 37%, and 36%, respectively, while n-butanol fraction showed weak relaxation. The ethanol extract of S. acaulis and its fractions possess antidiarrheal and antispasmodic activities that could be mediated by a mechanism involving the decrease of gastrointestinal mobility, resulting in inhibition of spontaneous contraction and/or mediated through K +-channel activation. The descending order of potency, ethanolic extract > n-hexane > chloroform > ethyl acetate > n-butanol as demonstrated in [Table 6], may suggest that the crude ethanol extract had the highest concentration of the phytochemical principle(s) with synergistic action responsible for the activity observed. Phytochemical study revealed the presence of triterpenes and phenolic compounds as plant constituents. Our results are in agreement with many previous studies as it was reported that triterpenes, flavonoids, and lipidosterolic constituents ,, were responsible for spasmolytic properties of certain medicinal plants.
|Table 6: Results of spasmolytic activity of the ethanol extract and its fractions of Senecio acaulis aerial parts on isolated rabbit jejunum|
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Results of DNA ampliflication
The DNA of S. acaulis was amplified using 12 decamer primers; a total of 87 different RAPD fragments were recorded showing 10 bands by each of OPA-12 and OPG-O5 ranging from 1.15 to 0.17 and 1.04 to 0.31 Kbp, respectively, 9 bands by OPH-15 primer (1.53–0.24 Kbp), 8 bands by OPA-02 primer (0.87–0.23 Kbp), 7 bands by each of OPA-07, OPB-14, OPC-12, and OPM-13 primers (1.87–0.22, 1.41–0.22, 1.20–0.33, and 0.79–0.25, Kbp, respectively), 6 bands by each of OPB-13, OPG-13, and OPE-01 primers (1.45–0.33, 1.05–0.20, and 0.69–0.24 Kbp, respectively), and 4 bands by OPB-02 primer (0.74–0.33 Kbp). The analysis of RAPD data, under the experimental conditions, can thus be used to distinguish the plant from other species.
| Conclusion|| |
The EO of the aerial parts of S. acaulis showed the predominance of monoterpenes in its composition and demonstrated significant antimicrobial activity against S. aureus and C. neoformans and moderate antimalarial activity. Eight compounds were isolated for the first time from the plant extract. The spasmolytic activity revealed the ethanolic extract is more potent than its fractions in reference to atropine. The descending order of potency, ethanolic extract > n-hexane > chloroform > ethyl acetate > n-butanol may suggest that the crude ethanol extract had the highest concentration of the phytochemical principle(s) with synergistic action responsible for the activity observed.
We were grateful to Professors Ahmed Osman, Shabana Khan, and Mei Wang from National Center for Natural Products Research, School of Pharmacy University of Mississippi, for carrying the analysis of EO and its biological activities.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]