|Year : 2021 | Volume
| Issue : 1 | Page : 1-4
Phytochemical and biological studies on Muscari inconstrictum seeds distributed in Iran
Mahsa Kazemnezhad1, Mohammadali Torbati2, Solmaz Asnaashari3, Fariba Heshmati Afshar4
1 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Food Science and Technology, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran
3 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
4 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
|Date of Submission||25-May-2019|
|Date of Acceptance||20-Jun-2019|
|Date of Web Publication||31-May-2021|
Dr. Solmaz Asnaashari
Biotechnology Research Center, Tabriz University of Medical Sciences, Across from Shahid Madani Hospital, University Street, Tabriz
Source of Support: None, Conflict of Interest: None
Background and Purpose: Muscari Miller. (Asparagaceae family) contains about 50 species worldwide, which are distributed in the Central and Southeastern Europe, Southern Russia, Africa, and some area of Asia such as Iran, Iraq, Afghanistan, Anatolia, and Syria. This study was designed to assess the antioxidant and antimalarial activities of Muscari inconstrictum Rech. f. seeds as one of the Iranian species of Muscari genus. In addition, preliminary phytochemical analysis of the extracts with different polarities was performed. Materials and Methods: The essential oil of M. inconstrictum seeds was prepared using Clevenger and extracted with n-hexane, chloroform, and methanol (MeOH) by Soxhlet apparatus. Gas chromatography-mass spectrometry (GC-MS) was used for the characterization of essential oil. Total phenol and flavonoid contents were measured using Folin–Ciocalteu and aluminum chloride reagents. Free radical scavenging and antimalarial activities were investigated via 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and cell-free ß-hematin formation methods. Results: GC-MS analysis of the volatile oil of seeds demonstrated the presence of sesquiterpenoid, alkanes, fatty acid, and linear alcohol structures as the main constituents. Among different extracts of M. inconstrictum seeds, the methanolic extract showed significant antioxidant activity, which can be related to the presence of flavonoid and other phenolic structures. Furthermore, chloroform extract is introduced as the most potent antimalarial part. Conclusion: It seems that further studies on the M. inconstrictum seeds are necessary to focus on pure compounds and their biological activities.
Keywords: Antimalarial, antioxidant, gas chromatography-mass spectrometry, Muscari
|How to cite this article:|
Kazemnezhad M, Torbati M, Asnaashari S, Heshmati Afshar F. Phytochemical and biological studies on Muscari inconstrictum seeds distributed in Iran. J Rep Pharma Sci 2021;10:1-4
|How to cite this URL:|
Kazemnezhad M, Torbati M, Asnaashari S, Heshmati Afshar F. Phytochemical and biological studies on Muscari inconstrictum seeds distributed in Iran. J Rep Pharma Sci [serial online] 2021 [cited 2021 Jul 31];10:1-4. Available from: https://www.jrpsjournal.com/text.asp?2021/10/1/1/317249
| Introduction|| |
Muscari Miller. (Asparagaceae family) is introduced by approximately 50 species worldwide. These ornamental and garden plants are distributed in the Central and Southeastern Europe, Southern Russia, Africa, and some areas of Asia such as Iran, Iraq, Afghanistan, Anatolia, and Syria.,, These plants have pharmaceutical and economic values. According to previous literature, plants of Muscari genus have numerous medicinal and biological activities such as antioxidant, anti-inflammatory, emetic, diuretic, hypoglycemic, and stimulant effects.,,
A review on the chemical structures of the genus Muscari indicated the existence of alkaloids, flavonoids, steroids, and triterpenoids. Homoisoflavonoids are an uncommon subclass of flavonoid structures with an extra carbon atom that are obtained from the plants of this genus, which are found rarely in nature. These bioactive compounds have shown a broad range of activities such as antioxidant, antiplasmodial, antimutagenic, anticlastogenic, antimicrobial, antidiabetic, anti-inflammatory, immunomodulatory, antiangiogenic, vasorelaxant, and cytotoxic.,,,
This study was conducted to evaluate phytoconstituents and some biological activities of Muscari inconstrictum Rech. f. seeds as one of the bulbous species reported from Iranian flora. As per our recent studies, the chloroform and n-hexane extracts of the bulbs of this species showed significant antioxidant and antimalarial activities, respectively. The flavonoids and coumarins were reported as the responsible components of chloroform extract and the saponin structures were reported as the most potent antimalarial parts of n-hexane extract
| Materials and Methods|| |
Muscari inconstrictum Rech. f. was collected from the gardens of East Azerbaijan province of Iran (37° 53′ 51″ N, 45° 57′ 15″ E) in April 2016. It was authenticated and stored as No.8897 at the Herbarium of the East-Azarbaijan Agricultural and Natural Resources Research and Education Center, Tabriz, Iran.
The seeds of M. inconstrictum were dried after the harvest in a shady for a week, then they were packed in paper bags and stored in a dark and dry place at the room temperature.
For gathering of the essential oil, approximately 100g of powdered seeds were subjected to hydro-distillation for 4 h using a Clevenger apparatus. The obtained volatile oil was measured (V/W) and dried via anhydrous sodium sulfate and then stored in a sealed vial for further analysis.
In addition, 100g of the seeds were powdered and were Soxhlet-extracted with approximately 1 liter n-hexane, chloroform, and methanol (MeOH). Subsequent extracts were evaporated at 50 ℃ using a rotary evaporator instrument.
Identification of components
Gas chromatography/mass spectrometry analysis
Gas chromatography–mass spectrometry (GC–MS) and gas chromatography with flame ionization detector (GC/FID) analysis were performed, respectively, on a Shimadzu QP5050A GC/MS system (Kyoto, Japan) and GC17A equipped with a DB-1 fused silica column (60 m × 0.25mm i.d.; 0.25 µm film thickness). Helium was used as the carrier gas at a flow rate of 1.3 mL/min. The oil was diluted with a ratio of 1:10 in n-hexane and 1 µL of which was injected into the column. Split ratio, ionization energy, scan time, and acquisition mass range were 1:33, 70eV, 1s, and 30–600 amu, respectively.
The temperature program was an oven temperature of 50 ℃ rising to 260 ℃ at a rate of 3 ℃/min for a total run time of 75 min. Injector temperature was set at 220 ℃ and the detector temperature was 260 ℃.
Identification of the volatile constituents was completed based on the direct comparison of the retention times (Rt) and mass spectral data with standard alkanes (C8–C20) from Sigma Aldrich (Allentown, PA, USA), computer matching with the WILEY229, NIST21 and NIST 107 libraries, and comparing the fragmentation patterns of the mass spectra with those described in the literature,.
Total phenolic content
Determination of total phenolic content of extracts was performed using modified Folin–Ciocalteu method as gallic-acid equivalents (GAE). Approximately 1 mL of sample (5 mg/100 mL acetone in water 60:40) was mixed with 0.2 mL Folin–Ciocalteu and 0.5 mL Na2CO3 (2%) and centrifuged at 12000 rpm for 5 min. After a 30-min incubation at room temperature, the absorbance of samples was measured at 750 nm with UV/Visible Spectrophotometer (Spectronic Genesys 5 Spectrophotometer, San francisco, CA, USA). The same procedure was repeated in triplicate and average absorption was noted. Total phenolic content (TPC) was reported as GAE in mg/g of sample.
Total flavonoid content
The samples were assessed to determine their total flavonoid content (TFC) with aluminum chloride reagent and rutin as a positive control. Approximately 2 mL of each sample (1 mg/1 mL methanol in water 80:20) was mixed with 1 mL reagent (AlCl3 crystals and sodium acetate crystals in 100 mL of 80% of methanol in water) and 400 μL distilled water. Tubes were permitted to stay at room temperature for 30 min. Subsequently, the absorbance of samples was measured at 430 nm with UV/Visible Spectrophotometer (Spectronic Genesys Spectrophotometer). TFC was reported as rutin equivalents/g of sample.
Free radical scavenging activity
The concentrated n-hexane, chloroform, and methanol extracts were objected to antioxidant assay using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma, Germany) as a reagent. Approximately 4 mg of DPPH was dissolved in 50 mL methanol for methanolic extract and chloroform (for n-hexane and chloroform extracts) to obtain the stock solution. The methanolic extract was dissolved in methanol (1 mg/mL). Next, n-hexane and chloroform extracts were dissolved in chloroform (1 mg/mL). Concentrations of 0.25, 0.125, 0.0625, 0.0031, and 0.0015 mg/mL were made by dilution. In the next step, 2 mL of each dilution was mixed with DPPH (2 mL). Tubes were kept at room temperature for 30 min to allow any reaction to occur. The absorbance of samples was measured using UV–Visible Spectrophotometer (Spectronic Genesys Spectrophotometer) at 517 nm. The same procedure was repeated in triplicate and the average absorbance was recorded. Reduction percentage of DPPH (R %) was calculated as below and the result was reported as sample concentration providing 50% reduction of DPPH (RC50):
where Abs stands for absorbance of samples.
Positive control: quercetin (0.001–0.01 mg/mL) was used as the same procedure.
Cell-free beta hematin formation assay
The antimalarial activities of three extracts (n-hexane, chloroform, and methanol extracts) were assessed by the heme bio-crystallization method. Different concentrations of extracts (0.4–2 mg/mL in DMSO) were mixed with hematin (100 µL, dissolved in 0.1 M NaOH), oleic acid (10mM), and HCl (10 µM). The reaction volume was adjusted to 1000 µL of sodium acetate buffer with a pH of 5. The samples were incubated at 37 ℃ for 24 h with consistent shaking. Then, the samples were centrifuged for 10 min in 12,000 rpm and the hemozoin sediments were continually washed by 2.5% (w/v) sodium dodecyl sulfate in phosphate-buffered saline. In the next step, they were washed in sodium bicarbonate (0.1 M and pH 9.0) until the supernatant was clear (after 4–6 washes). Afterward, the supernatant was removed and the sediments were re-suspended using 1 mL of NaOH (0.1 M). The absorbance of the samples was measured at 400 nm with UV/Visible Spectrophotometer (Spectronic Genesys Spectrophotometer). The results were reported as inhibition percentage (I %) of heme crystallization calculated as below:
where Abs stands for absorbance of samples.
Positive control: chloroquine (0.01–0.5 mg/mL) was used as the same procedure.
| Results|| |
The GC–MS analysis results of the volatile oil of M. inconstrictum seeds (the yield of oil: 0.2%v/w) are shown in [Table 1].
|Table 1: Chemical composition of the essential oil of Muscari inconstrictum seeds|
Click here to view
In addition, total phenol and flavonoid contents of three main extracts were assessed by two colorimetric methods correspondingly. [Table 2] presents the TPC and TFC assay results.
|Table 2: TPC and TFC results of n-hexane, chloroform, and MeOH extracts of Muscari inconstrictum seeds|
Click here to view
In addition, antioxidant and antimalarial tests of three extracts were evaluated using DPPH and cell-free beta-hematin formation methods [Table 3].
|Table 3: Antioxidant and antimalarial results of n-hexane, chloroform, and MeOH extracts of Muscari inconstrictum seeds|
Click here to view
| Discussion|| |
Based on the GC–MS analysis obtained for the essential oil of M. inconstrictum seeds, a total of 17 volatile constituents were recognized [Table 1]. According to the relative content of identified volatile constituents, sesquiterpenes and derivatives (49.12%) constituted the main functional groups in the seeds oil of M. inconstrictum. The main sesquiterpene structure of the oil is spathulenol with an abundance of 34.38%, which is categorized as a member of tricyclic sesquiterpene alcohol compounds. Spathulenol could be responsible for some biological activities such as anti-inflammatory, antioxidant, antiproliferative, antimicrobial, and as a mosquito repellant agent against Aedes aegypti and Anopheles stephensi in the essential oil of various plant species.
The presence of n-hexadecanoic acid (7.52%) as the second most abundant component of M. inconstrictum seeds was also prominent. In addition, n-hexadecanoic acid or palmitic acid is one of the common saturated fatty acids in plants., Previous studies have reported the anti-inflammatory effect of n-hexadecanoic acid via inhibition of phospholipase A2 and the results validated the use of the n-hexadecanoic acid-rich medicinal oils for management of rheumatic symptoms in traditional medicine. In addition to the mentioned chief constituents, alkanes (19.99%) and linear alcohol (5.76%) were identified as the other functional groups.
Moreover, antioxidant and antimalarial activities of three extract of M. inconstrictum seeds were evaluated [Table 3]. The methanol extract showed the highest antioxidant activity, which was confirmed by total phenol and flavonoid contents [Table 2]. Earlier investigations on Muscari genus showed potent antioxidant activities of Muscari racemosum, which are related to the presence of homoisoflavonoid structures. Furthermore, the antimalarial assay showed a moderate effect of chloroform extract of the seeds of M. inconstrictum in comparison with chloroquine as the positive control of the test. Researchers have shown strong antimalarial effects of purified flavonoid and the potential synergistic effects between artemisinin and flavonoid structures., In this regard, Midiwo et al. reported antiplasmodial activities of a homoisoflavonoid structure against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum.
However, according to previous literature and our findings, it is necessary to conduct further studies on M. inconstrictum pure compounds and their biological activities.
| Conclusion|| |
Overall, according to this introductory biological and phytochemical investigation, among different extracts of M. inconstrictum seeds, the methanolic extract showed significant antioxidant activity, which can be related to the existence of flavonoid and other phenolic structures. Moreover, the chloroform extract with the moderate antimalarial effect is introduced as the most potent antimalarial part of M. inconstrictum seeds.
The GC–MS analysis of the volatile oil of M. inconstrictum seeds showed the presence of sesquiterpenes as the main constituents.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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