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Extraction of β-Carboline alkaloids and preparation of extract nanoparticles from Peganum harmala L. capsules using supercritical fluid technique
Journal Pre-proof Extraction of β-Carboline alkaloids and preparation of extract nanoparticles from Peganum harmala L. capsules using supercritical fluid technique Hamze Salehi, Mehrnaz Karimi, Neda Rezaie, Farhad Raofie PII:
S1773-2247(19)31658-2
DOI:
https://doi.org/10.1016/j.jddst.2020.101515
Reference:
JDDST 101515
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 30 October 2019 Revised Date:
28 December 2019
Accepted Date: 9 January 2020
Please cite this article as: H. Salehi, M. Karimi, N. Rezaie, F. Raofie, Extraction of β-Carboline alkaloids and preparation of extract nanoparticles from Peganum harmala L. capsules using supercritical fluid technique, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/ j.jddst.2020.101515. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
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For submission to Journal of Drug Delivery Science and Technology, October, 2019
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Extraction of β-Carboline alkaloids and preparation of extract nanoparticles
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from Peganum harmala L. capsules using supercritical fluid technique
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Hamze Salehi, Mehrnaz Karimi, Neda Rezaie, Farhad Raofie*
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Department of Analytical Chemistry and pollutants, Shahid Beheshti University, Tehran, 1983969411, Iran * Corresponding author, email: [email protected] Fax: +98-21-22431661
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Abstract:
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Poor water solubility of pharmacologically active ingredients causes to incomplete absorption
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through the gastrointestinal tract. Increasing surface area by micronization has been performed to
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overcome these problems. In this study, the first step entailed an optimization of supercritical fluid
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extraction from the capsules of Peganum harmala L. As the second step, micronization process was
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accomplished using a new technique based on the expansion of CO2 supercritical solvent and
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collected precipitates were characterized by Field Emission Scanning Electron Microscopy
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(FESEM). The herbal extract was qualified using HPLC- mass spectrometry and Harmaline,
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Harmine and Harmalol were identified as the main β-carboline alkaloids. Effect of different
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supercritical parameters on the extraction yield and particle size were investigated. As a result,
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particle size decreased as the precipitation time decreased, whereas temperature and loaded volume
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did not significantly affect the morphology. Optimum micronization conditions were determined
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as: Volume of loaded extract (50 µL), oven temperature (40 °C), equilibrium and precipitation
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pressure (350 and 100 bar), equilibration and precipitation time (10 min). This supercritical process
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is proposed as a green technique for extraction and preparation of micronized β-Carbolines and
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nanoparticles with an average diameter ranging from 7 to 100 nm were successfully formed.
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Key words:
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Peganum harmala L.; β-Carbolines; Supercritical Fluid; Nanoparticle; Extraction.
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Abbreviation:
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SC-CO2, Supercritical Carbon dioxide fluid; CCD, Central composite design; LC-MS, High
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performance liquid chromatography- Mass spectrometry; FESEM, Field Emission Scanning
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Electron Microscopy; P-harmala, Peganum harmala L.
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1. Introduction 2
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Peganum harmala L. (Zygophyllaceae), commonly called Syrian rue, Harmel or Aspand is a plant
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of the family Nitrariaceae and grows in the Middle East and in part of South Asia mainly in India
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and Pakistan, South America and Southern USA [1]. The fruit of P. harmala contains
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pharmacologically active alkaloids such as Harmine, Harmaline and Harmalol (2 to 6%) which are
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mostly known as β-carbolines (Fig. 1) [2, 3]. Many therapeutic activities have been reported for
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these alkaloids such as anticancerous effects, antibacterial and antifungal activities, antinociceptive
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and analgesic effects, vasorelaxant, hypothermic properties and monoamine oxidase inhibition
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activities [4-12].
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Traditional methods for the extraction of chemical compounds from plant tissues such as liquid
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solvent extraction or distillation have some disadvantages such as long extraction times, use of
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large amounts of solvent, low efficiencies and instability of many natural products in thermally
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conditions [13]. Supercritical fluid extraction (SFE) is an environment-friendly technology that
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offers several advantages over conventional solvent extraction methods. In this technique,
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supercritical carbon dioxide is widely used as extraction solvent and has the following advantages:
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chemically inert, low toxicity, no pollution problem, non-flammable and explosive, low density,
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viscosity and surface tension. For the extraction of polar compounds, organic solvents have been
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added as modifiers [14-18].
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The solubility of β-carbolines in water is very low (0.0676 mg/l) and therefore this may cause to
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poor bioavailability [19]. Solubility of powders strongly depends on the morphology and particle
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size distribution. Therefore, one of the most effective methods to increase the solubility of active
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pharmaceutical ingredients in aqueous media is to reduce particle size [20-22].
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Grinding, spray drying and recrystallization using antisolvents are different techniques for
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reduction of particle size of organic and inorganic compounds [23]. These techniques have several
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disadvantages such as instability and decomposition of some organic compounds under milling
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conditions and contamination with solvent in recrystallization process [24, 25]. Recently, the use of
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supercritical fluids for particle formation and reduction of particle size have been considered and 3
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depending on the nature of the substance, several techniques have been proposed. Rapid expansion
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of supercritical solutions (RESS), anti-solvent processes [gas anti-solvent (GAS), supercritical anti-
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solvent (SAS)], aerosol solvent extraction system (ASES), solution enhanced dispersion by
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supercritical fluids (SEDS), controlled expansion of supercritical solution (CESS), and particles
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from gas-saturated solutions/suspensions (PGSS) are some of these techniques [26-35].
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In this study, a supercritical extraction process was carried out to extract β-carbolines from the
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capsule of P. harmala using SC-CO2 as a solvent and the experimental parameters such as pressure,
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temperature, modifier volume and dynamic extraction time were optimized using an experimental
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design methodology based on the central composite design (CCD). In the next, alkaloid compounds
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in the extract samples were identified by High performance liquid chromatography – mass
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spectrometry (LC–MS). Micronization process was based on a technique which described recently
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[36-38]. In this method, particle collection occurs in supercritical media as a solvent while
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precipitation in common methods such as RESS or SAS is based on CO2 evaporation or
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supercritical anti-solvent activity. Compared to mentioned methods, large number of collisions
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between pressurized SC-CO2 solvent molecules and primary nuclei decreases particle growth
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through coagulation and resulted particles are much smaller than those prepared from the other
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processes. On the other hand, expansion step in this method is much slower than RESS process and
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therefore the turbulent behavior of fluid at the time of particle collection are reduced.
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Precipitated nanoparticles were characterized by Field Emission Electron Microscopy (FESEM)
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and the effects of the different parameters such as concentration and volume of loaded extract,
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temperature, pressure of precipitation and time of expansion on the particles shape and size were
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investigated and optimum conditions were determined.
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2. Materials and methods
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2.1. Chemicals
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Acetonitrile, Methanol, Ethanol and Cyclohexane of LC grade were purchased from Merck Co.
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(Darmstadt, Germany). Formic acid and acetone were of analytical grade were supplied by Fluka
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Co. (distributed by Sigma–Aldrich, Allentown, PA, USA) and Analytical grade (99.99% purity)
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Carbon dioxide stored in a cylinder with an eductor tube was obtained from Roham Co. (Tehran,
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Iran) and used as received.
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2.2. Plant materials and sample preparation
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Capsules of P. harmala (pod and seeds) were collected from their natural habitats in the central
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regions of Iran (Esfahan, Iran). These plants were separated, cleaned, and stored at room
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temperature in dark for one week, so as to be dried. Next, the dried samples were ground to coarse
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powder by a laboratory mill (Myson, China) and used for extraction.
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2.3. Herbal extraction process
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2.3.1 Supercritical fluid extraction
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Herbal extraction process was carried out in the SFE mode of a Suprex MPS/225 system equipped
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with syringe pump unit (Pittsburg, Virginia, U.S.A.) using SC-CO2 as shown in Fig.2. In each
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extraction, 1g of the powder of P. harmala was mixed with glass beads (1 mm in diameter) and
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loaded into stainless steel extraction vessel (5ml) equipped with two sinter metal filters (5).
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Different volumes of modifier were added directly into the sample. The temperature of CO2 was
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cooled down to 0°C using the chiller (2) and a high-pressure syringe pump (3) was used to
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pumping the CO2 into extractor. The pressure was controlled by a back-pressure regulator and the
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electrically heated restrictor (6) (Dura flow manual variable; Suprex Co.) was used to prevent the
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sample plugging. For preventing solvent evaporation and improve the efficiency, the collection
5
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vessel (8) containing 3 mL of ethanol was placed in an ice bath (7) and the flow rate of SC-CO2
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during dynamic extraction time was about 0.30 ± 0.05 mL min-1.
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In order to prepare the sample for LC-MS analysis, the extracted solution was diluted to 5mL with
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ethanol and filtered through a 0.22µm syringe filter (Microlab, china). The extraction yield was
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calculated by weighting the collected extract after evaporation of solvent at room temperature at the
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end of each run.
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2.3.2. Conventional solvent (modifier) selection
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1 g of accurately weighted P. harmala powder was placed in a 20 ml volumetric flask and made up
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to volume with of acetonitrile, methanol, ethanol and cyclohexane separately. These samples were
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kept in an ultrasonic cleaning bath (SB-5200D type, 40 kHz, 250 W, Ningbo Scientz
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Biotechnology Co. Ltd., China) and sonicated for 40 min at 40°C. At the end of time, the
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supernatant and the sediment were separated by vacuum filtration and the solvent was evaporated to
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dryness at 60°C. Finally, the weight of the extracted compounds was determined gravimetrically
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and extraction yield (EY) was calculated using the following equation: %=
2 × 100 1
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Where W2 is the weight of the dried collected extract and the parameter W1 is the weight of the P.
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harmala powder sample.
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2.3.3. Optimization strategy
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In this study, a 2n fractional factorial design was applied to screening of the different variables to
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choose the main factors. This process was followed with central composite design (CCD) for
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optimization of the effective parameters. The experimental design matrix and data analysis were
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performed using the StatGraphics plus 5.1 package and the measured response was extraction yield
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(EY %). 6
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Preliminary experiments showed that extraction pressure, dynamic extraction time and modifier
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concentration are main parameters affecting the extraction yield as experimental response.
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Therefore, these factors at two levels with three center points were studied. The low and high
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values of these parameters included: extraction pressures of 100 and 350 atm, modifier volumes of
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0 and 100 µL and dynamic extraction times of 10 and 60 min. According to Previous similar
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experiments, the static extraction time and oven temperature were fixed at 10 min and 40°C
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respectively. The number of experiments is defined by the expression (2P + 2P + C), where P is the
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number of factors and C is the number of center points that was 3 in this study and hence, 17 runs
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to be carried out randomly.
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2.4. Identification of β-carbolines alkaloids in herbal extract
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β-carbolines were identified using High performance liquid chromatography–mass spectrometry
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(HPLC–MS) and an Agilent 1200 LC system via an ESI source in a post-column was connected to
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an Agilent 6410 triple quadrupole tandem mass spectrometer (Waldron, Germany). The mass
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spectrometer was run in the positive ion mode (ESI+) and set up in the multiple reaction monitoring
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mode and electrospray capillary potential was set to 4000V. The ultrahigh pure Nitrogen and Argon
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gas were used as a nebulizer and drying gas for solvent evaporation and collision gas respectively.
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The flow arte of the drying and nebulizer gas were set at 10 L min-1 and 40 psi and temperature of
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capillary head was 300◦C. Data acquisition and analyses were performed using Mass Hunter work-
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station.
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Chromatographic conditions were selected according to a validated method reported by M. Kartal
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et al as described in the following[39]. An isocratic elution with the flow rate 0.25 mL min−1 was
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performed. The mobile phase composition was a mixture of isopropyl alcohol, acetonitrile, water,
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formic acid (100:100:300:0.3) (v:v:v:v) and pH adjusted to 8.6 with trimethylamine. The column
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used for chromatographic system was an Agilent rapid resolution HT zorbax SB-C18 (2.1 mm × 50
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mm, 1.8µm) (Agilent Technologies, Santa Clara, CA). 1mL of extraction sample diluted to 10mL 7
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with ethanol and 5 µL of the resulting solution was injected into the LC-MS system. The full-scan
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mode over the mass range of m/z 100 – 400 spectra of the extract solution was obtained.
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2.5. Micronization process
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2.5.1. Supercritical fluid precipitation of extract nanoparticles
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The equipment used in the nanoparticles precipitation using supercritical fluid solvent is shown in
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Fig.3. This process is based on a method reported recently [37]. In this technique, a stainless-steel
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precipitation vessel (6) was coupled to an equilibration vessel (5) and both sides of each chamber
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were equipped with stainless steel filters (with the pore size of 0.1 µm). The capacity of the
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equilibration and precipitation vessel was 5 and 25 mL respectively.
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A 1 mL polyethylene vial (7) with loaded V µL solutions of the collected extract was placed in
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equilibrium chamber and two sheets of Mica (5×50 mm, Agar Scientific Ltd.) for collecting of the
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precipitate (8) were set in the vessel as shown in Fig.3. Equilibrating pressure and time (P1, t1),
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precipitation pressure and time (P2, t2), oven temperature (T) and loaded volume of extract (V)
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were variable parameters in precipitation process and equilibration time and pressure were
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constant.
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In first step, valve 1 was opened and CO2 was purged to equilibration vessel until the pressure was
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raised to P1. After the equilibrating time was elapsed, dissolved extract in SC-CO2 was introduced
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to precipitation vessel trough valve 2 while the valve 3 was also opened at a flow rate of 0.1 mL
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min-1. When the pressure was reached to P2, the valve 2 and 3 was closed. The expansion of the
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supercritical solvent during this pressure dropping program leads to decreasing in solvent density
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and hence decreasing of solubility of the dissolved substances in SC-CO2. Eventually, these
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conditions caused to precipitation of compounds in vessel and a part of the precipitate was
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collected on the surface of the mica sheets. At the end of experiment, the precipitation vessel was
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depressurized through valve 3 and then was disclosed. Collectors (mica sheets) were placed in the 8
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closed tube until the particle size determination was accomplished. Precipitation experiments were
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carried out in 6 different conditions which are shown in Table.2 and each experiment was repeated
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for second times.
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2.4.2. Characterization of precipitate
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Size analysis and morphology of collected precipitates was carried out using Field Emission
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Scanning Electron Microscopy (FESEM, MIRA3 TESCAN-XMU, and Czech Republic). In this
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analysis, the mica collector sheets were coated with gold using a sputter coater (Pelco, model Sc-7)
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and Microstructure Measurement Software was applied for image-analysis and particle size
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determination.
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Qualification analysis of β-carboline alkaloids in extract nanoparticles was done by LC-MS as
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described in 2.4. Sample solution preparation was done using ultrasonication of the mica sheet
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collector in 10 mL of ethanol for 20 min at 40°C. The solution was filtered through a 0.22 µm
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syringe filter before injection.
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3. Result and Discussion
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3.1. Optimization of supercritical fluid extraction conditions
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3.1.1. Modifier selection results
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In order to investigate effects of different solvents on the extraction yield, the extraction of β-
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carbolines from P. harmala fruit powder in four organic solvents (Acetonitrile, Methanol, Ethanol
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and Cyclohexane) using sonication as described in 2.3.2 was done. Results showed that, using
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methanol and ethanol as extraction solvent leads to the highest extraction yield (4.64% and 4.23%
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respectively). Based on these results and compare safety data sheet of mentioned solvents, ethanol
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was chosen as conventional solvent for supercritical fluid extraction and particle production
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process. 9
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3.1.2. Optimization design
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Analysis of the results was visualized at a 95% confidence level using standardized main effect
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Pareto charts (Fig.4a). According to this chart, modifier volume was the most significant variable,
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having a negative effect (corresponding to blue) on the extraction yield and dynamic extraction
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time and extraction pressure were the next most important parameters, respectively.
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In the next step, a CCD statistical model was applied to optimize the three selected factors at three
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levels. The examined levels of the factors and resulted extraction yield are shown in Table.1. The
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second-order polynomial model was acquired for the experimental results as the following
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equation:
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Y = 8.41816 - 0.0355801 X1 + 0.0576763 X2 - 0.0290452 X3 + 0.0000301381 X12 +
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0.000376667 X1 X2 + 0.000087 X1 X3 - 0.00209286 X22 - 0.00002 X2 X3 + 0.0000202389 X32
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Where X1, X2 and X3 are extraction pressure, dynamic extraction time, and volume of modifier
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respectively and Y is extraction yield.
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Fig.4b shows the response surface obtained by plotting pressure dynamic extraction time versus
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modifier volume whereas extraction pressure was fixed at 260 atm. This figure shows that
235
extraction yield increased when the dynamic extraction time was increased to 40 min and decreased
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at more times. This result could be due to the loss of volatile compounds during the collection of
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extract in long periods of time. On the other hand, the extraction efficiency has been increased by
238
decreasing the modifier volume. These observations indicate that most of the extracted compounds
239
are non-polar and the extraction yield decrease by increasing the polar solvents.
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Fig.4c shows the response surface developed for pressure and modifier volume, while keeping
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dynamic extraction time at 35 min. This figure shows that reducing the amount of the polar solvent
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at low pressures leads to increased extraction yield and at high pressure, increasing the amount of
243
modifier increases the extraction efficiency. This can be justified by this fact that at low pressures, 10
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non-polar compounds are extracted, and a decrease in modifier content leads to an increase in
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extraction yield. Also, at high pressures, the extraction of polar compounds is mainly accomplished
246
and solvent reduction leads to increased efficiency.
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Fig.4d indicates the response surface of pressure and time whereas modifier volume was 100 µL
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and confirms the results mentioned in the interpretation of Fig.4b. The effect of the mentioned
249
parameters on the extraction yield is well illustrated in Fig.4e.
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Based on the obtained results, the optimum extraction condition was obtained as: extraction
251
pressure of 176 atm, modifier volume of 16 µL (1.6%), oven temperature of 40°C and static and
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dynamic extraction times of 10 and 40 min respectively.
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3.1.3 Qualification of herbal extract
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Supercritical fluid extraction of the capsule of P. harmala in optimum conditions was carried out as
256
described in Section 2.2. Accordingly, an extraction yield of 3.9% was obtained (with reference to
257
the initial weight of seeds). As described, Identification of β- carbolines in the extract samples was
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done using LC-MS. The chromatogram and spectrogram of an extract sample are shown in Fig.5a
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and b respectively. According to the mass spectra, it was possible to identify Harmaline, Harmine
260
and Harmalol in P. harmala samples. The peak at retention time of 11.9 min in EIC at m/z ratio of
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213.1 can be related to the Harmine component (MW: 212.25 g mol-1), while the peak at retention
262
time of 11.8 min in the EIC at m/z ratio of 215.1 is attributed to Harmaline (MW: 214.26 g mol-1)
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and the peak at retention time of 10.9 min in the EIC at m/z ratio of 201.1 (Fig.6d) is related to
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Harmalol (MW: 200.24 g mol-1).
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3.2. Optimization of supercritical fluid micronization conditions
267
Optimization of operating conditions to obtain a minimum size of precipitant is critical step in the
268
micronization process. In this study, supercritical precipitation of herbal extract of P. harmala was
269
done as described and variable parameters such as equilibration time (t1), oven temperature (T), 11
270
volume of extract (V) and precipitation time (t2) and pressure (p2) were evaluated. Different
271
experiment conditions and observed results are shown in Table.2 and in order to evolution of each
272
parameter, other parameters were kept constant.
273
Figure 6 shows FESEM images and particle size distribution diagrams of collected extract particles
274
under different conditions. The optimum operating conditions for producing nanoparticles were
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determined as: volume of loaded extract sample (50 µL), oven temperature (40 °C), equilibrium
276
and precipitation pressure (350 and 100 bar), equilibration and precipitation time (10 min). In this
277
process, mean size of micronized particles was between 5 to 70 nm.
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3.2.1. The effect of equilibration time and pressure
280
As showed in Table.2 (run 1), The loaded extract solution did not dissolve completely in SC-CO2
281
phase and a lot of sample remained in the vial. Increasing of oven temperature from 40 to 60 ºC in
282
run 2 did not lead to significant change in the solubility of loaded compounds.
283
In next experiment (Table1, run 3), the equilibration pressure was increased to 350 atm. In these
284
conditions, no extract solution was remained in the vial and loaded sample were dissolved in SC-
285
CO2 completely. This result clearly shows that the solubility of β-carbolines and other components
286
of herbal extract increased with increasing of pressure and density of SC-CO2 solvent.
287 288
3.2.2. The effect of precipitation time, pressure and temperature
289
Low amounts of the collected precipitates on the mica sheets in run 3 shows that most of dissolved
290
compounds were remained in SC-CO2 phase and the pressure drop has not been sufficient.
291
Decreasing of precipitation pressure from 140 to 100 atm in run 4 caused to precipitation of big
292
crystalline compounds with a wide size distribution as shown in Fig.6a. These results illustrate that,
293
precipitation process and dissolution depends significantly to the density of solvent and also intense
294
agglomeration of particles has happened during precipitation step. 12
295
To investigate the impact of temperature on the shape of particles and preventing of accumulation,
296
oven temperature increased from 40 to 60 ºC (Table 2, run 5). Increasing of temperature can reduce
297
the density of the fluid and on the other hand it leads to increasing in the solutes vapor pressure.
298
Depending on which of these factors is dominant, solubility and hence supersaturation
299
concentration may enhance, decrease or constant with increasing of temperature. According to the
300
classical nucleation theory, increasing of supersaturation concentration cause to decreasing of
301
particle size of precipitants [36]. FESEM image of particles (Fig.6b) showed that increase in
302
temperature did not have impressive effect on the shape of particles in this experiment. Therefore,
303
supersaturation concentration of herbal extract components of P. harmala was reminded constant
304
during increase the temperature.
305
In next run, precipitation time reduced from 30 to 10 minutes (Table 2, Run 6). FESEM image of
306
collected particles (Fig.6c) and analysis of this image using imageJ software (Fig.6e) show that
307
these conditions caused to precipitation of spherical nanoparticles with particle size distribution
308
between 5 to 70 nm. These results illustrate that morphology and particle size of precipitants were
309
dependent to duration of pressure drop and rate of crystallization significantly. It proves that,
310
nucleation process was preferred to crystal growth and agglomeration reduced significantly in this
311
experiment.
312
In order to increase the number of collected nanoparticles, volume of loaded solution was increased
313
to 50 and 70 µL (Table 2, Run 7 and 8) and precipitation process was done under optimum
314
conditions as described in previous run. Accordingly, FESEM image of collected particles during
315
run 7 and analysis of this image as shown in Fig.6d and f respectively prove that the number of
316
collected particles on mica sheets has increased from 1329 to 2543 particles while the size and
317
morphology of the nanoparticles remained unchanged.
318
In experiment 8, extra amount of the loaded extracted solution (70 µL) was remained in the vial and
319
results were similar to those obtained in the previous experiment and optimum loaded volume was
320
considered about 50 µL. 13
321 322
3.3. Qualification analysis of extract nanoparticles
323
HPLC chromatogram of extract nanoparticles was similar to the chromatogram of extract sample
324
and related peaks of Harmine, Harmaline and Harmalol in the positive ion mode (ESI+) are shown
325
in Fig.7.
326 327
4. Conclusion
328
In this study, Supercritical fluid extraction of β-carboline alkaloids from the capsule of P. harmala
329
was described. The optimum extraction condition was obtained as: extraction pressure of 176 atm,
330
modifier concentration (ethanol %) of 1.6 %, oven temperature of 40°C and static and dynamic
331
extraction times of 10 and 40 min respectively. Micronization of herbal extracted using a
332
precipitation technique based on the expansion of supercritical solution was investigated and
333
nanoparticles with an average diameter ranging from 7 to 100 nm were collected. Optimum
334
micronization conditions were determined as: Volume of loaded extract sample (50 µL), oven
335
temperature (40°C), equilibrium and precipitation pressure (350 and 100 bar), equilibration and
336
precipitation time (10 min).
337
This technique can be an innovative method to improve the solubility and bioavailability of drugs
338
with low solubility in water, and it is possible to investigate other active pharmaceutical ingredients
339
which are extracted directly from the medicinal plants.
340
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Zargari, A., Medicinal plants. Vol 2. 1992, Tehran University Press, Tehran. Lamchouri, F., et al., In vitro cell-toxicity of Peganum harmala alkaloids on cancerous cell-lines. Fitoterapia, 2000. 71(1): p. 50-54. Wang, Z., et al., Analysis of alkaloids from Peganum harmala L. sequential extracts by liquid chromatography coupled to ion mobility spectrometry. Journal of Chromatography B, 2018. 1096: p. 73-79. Sobhani, A.M., S.-A. Ebrahimi, and M. Mahmoudian, An in vitro evaluation of human DNA topoisomerase I inhibition by Peganum harmala L. seeds extract and its beta-carboline alkaloids. J Pharm Pharm Sci, 2002. 5(1): p. 19-23. 14
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Arshad, N., et al., Effect of Peganum harmala or its β-carboline alkaloids on certain antibiotic resistant strains of bacteria and protozoa from poultry. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, 2008. 22(11): p. 1533-1538. Moloudizargari, M., et al., Pharmacological and therapeutic effects of Peganum harmala and its main alkaloids. Pharmacognosy reviews, 2013. 7(14): p. 199. Nenaah, G., Antibacterial and antifungal activities of (beta)-carboline alkaloids of Peganum harmala (L) seeds and their combination effects. Fitoterapia, 2010. 81(7): p. 779-782. Patel, K., et al., A review on medicinal importance, pharmacological activity and bioanalytical aspects of beta-carboline alkaloid “Harmine”. Asian Pacific journal of tropical biomedicine, 2012. 2(8): p. 660-664. Ouachrif, A., et al., Comparative study of the anti-inflammatory and antinociceptive effects of two varieties of Punica granatum. Pharmaceutical biology, 2012. 50(4): p. 429-438. Berrougui, H., et al., Vasorelaxant effects of harmine and harmaline extracted from Peganum harmala L. seed's in isolated rat aorta. Pharmacological research, 2006. 54(2): p. 150-157. Abdel-Fattah, A.-F.M., et al., Hypothermic effect of harmala alkaloid in rats: involvement of serotonergic mechanism. Pharmacology Biochemistry and Behavior, 1995. 52(2): p. 421-426. Herraiz, T., et al., β-Carboline alkaloids in Peganum harmala and inhibition of human monoamine oxidase (MAO). Food and Chemical Toxicology, 2010. 48(3): p. 839-845. Mendes, R.L., et al., Supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae. Inorganica Chimica Acta, 2003. 356: p. 328-334. Azwanida, N., A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med Aromat Plants, 2015. 4(196): p. 2167-0412.1000196. Li, S.-D. and L. Huang, Pharmacokinetics and biodistribution of nanoparticles. Molecular pharmaceutics, 2008. 5(4): p. 496-504. Lin, M.-C., M.-J. Tsai, and K.-C. Wen, Supercritical fluid extraction of flavonoids from Scutellariae Radix1Presented at the 22nd International Symposium on High-Performance Liquid Phase Separations and Related Techniques, St. Louis, MO, 3–8 May 1998.1. Journal of Chromatography A, 1999. 830(2): p. 387-395. Liu, J., et al., Supercritical fluid extraction of flavonoids from Maydis stigma and its nitritescavenging ability. Food and bioproducts processing, 2011. 89(4): p. 333-339. Lin, M.-C., M.-J. Tsai, and K.-C. Wen, Supercritical fluid extraction of flavonoids from Scutellariae Radix. Journal of Chromatography A, 1999. 830(2): p. 387-395. Saraf, S., Applications of novel drug delivery system for herbal formulations. Fitoterapia, 2010. 81(7): p. 680-689. Midoux, N., et al., Micronization of pharmaceutical substances in a spiral jet mill. Powder Technology, 1999. 104(2): p. 113-120. Sievers, R., et al., Micronization of water-soluble or alcohol-soluble pharmaceuticals and model compounds with a low-temperature Bubble Dryer®. The Journal of supercritical fluids, 2003. 26(1): p. 9-16. Li, Y., et al., Enhancement of dissolution rate and oral bioavailability in beagle dogs of oleanolic acid by adsorbing onto porous silica using supercritical carbon dioxide. Journal of Drug Delivery Science and Technology, 2014. 24(4): p. 380-385. Asghari, I. and F. Esmaeilzadeh, Formation of ultrafine deferasirox particles via rapid expansion of supercritical solution (RESS process) using Taguchi approach. International Journal of Pharmaceutics, 2012. 433(1): p. 149-156. Abuzar, S.M., et al., Enhancing the solubility and bioavailability of poorly water-soluble drugs using supercritical antisolvent (SAS) process. International journal of pharmaceutics, 2018. 538(1-2): p. 113. Girotra, P., S.K. Singh, and K. Nagpal, Supercritical fluid technology: a promising approach in pharmaceutical research. Pharmaceutical development and technology, 2013. 18(1): p. 22-38.
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29. 30.
31.
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33. 34. 35.
36. 37. 38. 39.
Thakur, R. and R.B. Gupta, Formation of phenytoin nanoparticles using rapid expansion of supercritical solution with solid cosolvent (RESS-SC) process. International Journal of Pharmaceutics, 2006. 308(1): p. 190-199. Kumar, B., et al., Recent advances in nanoparticle-mediated drug delivery. Journal of Drug Delivery Science and Technology, 2017. 41: p. 260-268. Türk, M., et al., Micronization of pharmaceutical substances by rapid expansion of supercritical solutions (RESS): experiments and modeling. Particle & Particle Systems Characterization: Measurement and Description of Particle Properties and Behavior in Powders and Other Disperse Systems, 2002. 19(5): p. 327-335. Chang, Y.-P., M. Tang, and Y.-P. Chen, Micronization of sulfamethoxazole using the supercritical anti-solvent process. Journal of Materials Science, 2008. 43(7): p. 2328-2335. Asghari, I. and F. Esmaeilzadeh, Formation of ultrafine deferasirox particles via rapid expansion of supercritical solution (RESS process) using Taguchi approach. International journal of pharmaceutics, 2012. 433(1-2): p. 149-156. Charoenchaitrakool, M., et al., Micronization by rapid expansion of supercritical solutions to enhance the dissolution rates of poorly water-soluble pharmaceuticals. Industrial & engineering chemistry research, 2000. 39(12): p. 4794-4802. Huang, J. and T. Moriyoshi, Preparation of stabilized lidocaine particles by a combination of supercritical CO 2 technique and particle surface control. Journal of Materials Science, 2008. 43(7): p. 2323-2327. Knez, Z. and E. Weidner, Particles formation and particle design using supercritical fluids. Current opinion in solid state and materials science, 2003. 7(4-5): p. 353-361. Warwick, B., et al., Synthesis, purification, and micronization of pharmaceuticals using the gas antisolvent technique. Industrial & engineering chemistry research, 2000. 39(12): p. 4571-4579. Jiao, Z., et al., Preparation of vitamin C liposomes by rapid expansion of supercritical solution process: Experiments and optimization. Journal of Drug Delivery Science and Technology, 2019. 51: p. 1-6. Karimi, M. and F. Raofie, Micronization of vincristine extracted from Catharanthus roseus by expansion of supercritical fluid solution. The Journal of Supercritical Fluids, 2019. 146: p. 172-179. Momenkiaei, F. and F. Raofie, Preparation of silybum marianum seeds extract nanoparticles by supercritical solution expansion. The Journal of Supercritical Fluids, 2018. 138: p. 46-55. Momenkiaei, F. and F. Raofie, Preparation of Curcuma Longa L. Extract Nanoparticles Using Supercritical Solution Expansion. Journal of Pharmaceutical Sciences, 2019. 108(4): p. 1581-1589. Kartal, M., M.L. Altun, and S. Kurucu, HPLC method for the analysis of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L. Journal of Pharmaceutical and Biomedical Analysis, 2003. 31(2): p. 263-269.
437 438 439 440 441 442 443 16
444
Figure caption
445
Fig.1. Chemical structure of (a) Harmine, (b) Harmaline and (c) Harmalol as main β-carboline
446
alkaloids in the capsule of Peganum harmala L.
447
Fig.2. Schematic diagram of supercritical fluid extractor including: (1) CO2 Cylinder, (2) Chiller,
448
(3) Syringe pump, (4) Heat exchanger, (5) Extraction vessel, (6) Back pressure valve, (7) Ice bath,
449
(8) collection vessel
450
Fig.3. Schematic representation of supercritical fluid precipitation apparatus: (1) CO2 Cylinder, (2)
451
Chiller, (3) Syringe pump, (4) Heat exchanger, (5) Equilibration vessel, (6) Precipitation vessel, (7)
452
Loaded extract vial, (8) Collector (mica sheet).
453
Fig.4. (a) Standardized Pareto chart in the central composite design for SFE, representing the
454
estimated effects of parameters and parameter interactions on extraction yield.
455
Response surfaces using central composite design obtained by (b) dynamic extraction time (min)
456
vs. modifier volume (µl) at 260 atm. (c) Pressure (atm.) vs. modifier volume (µL) at 35 min of
457
dynamic extraction time (d) pressure (atm.) vs. dynamic extraction time at 100 µL of modifier
458
volume. (e) main effect plots of pressure, modifier volume and dynamic extraction time.
459
Fig.5. HPLC chromatogram of extract sample (a) and Mass spectrum of Harmine, Harmaline and
460
Harmalol (b).
461
Fig.6. FESEM images of precipitated extract particles under different conditions (a) crystaline
462
particles produced during experiment 4 (size bar > 500nm), (b) crystaline particles produced during
463
experiment 5 (size bar > 500nm), (c) Nanoparticles produced during experiment 6, (d)
464
Nanoparticles produced during experiment 8, (e) The particle size distribution diagrams correspond
465
to experiment 6 (size bar < 50nm, Count: 1329), (f) The particle size distribution diagrams
466
correspond to experiment 7 (size bar < 50nm, Count: 2543).
467
Fig.7. HPLC chromatogram of extract nanoparticles in the positive ion mode (ESI+). 17
468 469
Table.1.
470 471
Orthogonal array design and extraction yield for supercritical fluid extraction from Peganum harmala L. capsules. Test set
Pressure
Extraction Time
Modifier volume
Extraction Yield
(atm)
(min)
(µL)
(%)
1
260
35
100
3.50
2
310
50
150
3.63
3
310
50
50
3.72
4
310
20
150
3.10
5
175.91
35
100
3.59
6
210
50
150
2.54
7
260
35
100
3.64
8
260
35
15.91
3.85
9
260
60.22
100
2.54
10
260
9.77
100
1.90
11
260
35
184.09
3.54
12
210
20
150
2.94
13
210
50
50
3.30
14
310
20
50
2.93
15
210
20
50
3.84
16
260
35
100
3.60
17
344.09
35
100
3.94
472 473 474 475 476 18
477
Table.2.
478
Supercritical micronization conditions. Run
T
V
t1
t2
P1
P2
observation
figure
1
40
30
10
30
176
140
Extra amount remained in the vial.
-
2
60
30
10
30
176
140
Extra amount remained in the vial.
-
3
40
30
10
30
350
140
A few particles were formed.
-
4
40
30
10
30
350
100
Big crystals were formed
6a
5
60
30
10
30
350
100
Big crystals were formed
6b
6
40
30
10
10
350
100
Nanoparticles were formed
6c
7
40
50
10
10
350
100
Nanoparticles were formed.
6d
8
40
70
10
10
350
100
Extra amount remained in the vial.
-
479 480 481 482 483 484 485 486
19
Author statement
Hamze Salehi: SFE Extraction, plant colection, LC-MS analysis,
Mehrnaz Karimi:
micronization process, Data interpretation, Writing- Reviewing and Editing , Neda Rezaie: software and characterization by Field Emission Scanning Electron Microscopy (FESEM), Farhad Raofie: Supervision
Conflict of interests
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
S1773-2247(19)31658-2
DOI:
https://doi.org/10.1016/j.jddst.2020.101515
Reference:
JDDST 101515
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 30 October 2019 Revised Date:
28 December 2019
Accepted Date: 9 January 2020
Please cite this article as: H. Salehi, M. Karimi, N. Rezaie, F. Raofie, Extraction of β-Carboline alkaloids and preparation of extract nanoparticles from Peganum harmala L. capsules using supercritical fluid technique, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/ j.jddst.2020.101515. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
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For submission to Journal of Drug Delivery Science and Technology, October, 2019
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Extraction of β-Carboline alkaloids and preparation of extract nanoparticles
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from Peganum harmala L. capsules using supercritical fluid technique
5
Hamze Salehi, Mehrnaz Karimi, Neda Rezaie, Farhad Raofie*
6 7
Department of Analytical Chemistry and pollutants, Shahid Beheshti University, Tehran, 1983969411, Iran * Corresponding author, email: [email protected] Fax: +98-21-22431661
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14 15 16 17 18 19 20 1
21
Abstract:
22
Poor water solubility of pharmacologically active ingredients causes to incomplete absorption
23
through the gastrointestinal tract. Increasing surface area by micronization has been performed to
24
overcome these problems. In this study, the first step entailed an optimization of supercritical fluid
25
extraction from the capsules of Peganum harmala L. As the second step, micronization process was
26
accomplished using a new technique based on the expansion of CO2 supercritical solvent and
27
collected precipitates were characterized by Field Emission Scanning Electron Microscopy
28
(FESEM). The herbal extract was qualified using HPLC- mass spectrometry and Harmaline,
29
Harmine and Harmalol were identified as the main β-carboline alkaloids. Effect of different
30
supercritical parameters on the extraction yield and particle size were investigated. As a result,
31
particle size decreased as the precipitation time decreased, whereas temperature and loaded volume
32
did not significantly affect the morphology. Optimum micronization conditions were determined
33
as: Volume of loaded extract (50 µL), oven temperature (40 °C), equilibrium and precipitation
34
pressure (350 and 100 bar), equilibration and precipitation time (10 min). This supercritical process
35
is proposed as a green technique for extraction and preparation of micronized β-Carbolines and
36
nanoparticles with an average diameter ranging from 7 to 100 nm were successfully formed.
37 38
Key words:
39
Peganum harmala L.; β-Carbolines; Supercritical Fluid; Nanoparticle; Extraction.
40
Abbreviation:
41
SC-CO2, Supercritical Carbon dioxide fluid; CCD, Central composite design; LC-MS, High
42
performance liquid chromatography- Mass spectrometry; FESEM, Field Emission Scanning
43
Electron Microscopy; P-harmala, Peganum harmala L.
44
1. Introduction 2
45
Peganum harmala L. (Zygophyllaceae), commonly called Syrian rue, Harmel or Aspand is a plant
46
of the family Nitrariaceae and grows in the Middle East and in part of South Asia mainly in India
47
and Pakistan, South America and Southern USA [1]. The fruit of P. harmala contains
48
pharmacologically active alkaloids such as Harmine, Harmaline and Harmalol (2 to 6%) which are
49
mostly known as β-carbolines (Fig. 1) [2, 3]. Many therapeutic activities have been reported for
50
these alkaloids such as anticancerous effects, antibacterial and antifungal activities, antinociceptive
51
and analgesic effects, vasorelaxant, hypothermic properties and monoamine oxidase inhibition
52
activities [4-12].
53
Traditional methods for the extraction of chemical compounds from plant tissues such as liquid
54
solvent extraction or distillation have some disadvantages such as long extraction times, use of
55
large amounts of solvent, low efficiencies and instability of many natural products in thermally
56
conditions [13]. Supercritical fluid extraction (SFE) is an environment-friendly technology that
57
offers several advantages over conventional solvent extraction methods. In this technique,
58
supercritical carbon dioxide is widely used as extraction solvent and has the following advantages:
59
chemically inert, low toxicity, no pollution problem, non-flammable and explosive, low density,
60
viscosity and surface tension. For the extraction of polar compounds, organic solvents have been
61
added as modifiers [14-18].
62
The solubility of β-carbolines in water is very low (0.0676 mg/l) and therefore this may cause to
63
poor bioavailability [19]. Solubility of powders strongly depends on the morphology and particle
64
size distribution. Therefore, one of the most effective methods to increase the solubility of active
65
pharmaceutical ingredients in aqueous media is to reduce particle size [20-22].
66
Grinding, spray drying and recrystallization using antisolvents are different techniques for
67
reduction of particle size of organic and inorganic compounds [23]. These techniques have several
68
disadvantages such as instability and decomposition of some organic compounds under milling
69
conditions and contamination with solvent in recrystallization process [24, 25]. Recently, the use of
70
supercritical fluids for particle formation and reduction of particle size have been considered and 3
71
depending on the nature of the substance, several techniques have been proposed. Rapid expansion
72
of supercritical solutions (RESS), anti-solvent processes [gas anti-solvent (GAS), supercritical anti-
73
solvent (SAS)], aerosol solvent extraction system (ASES), solution enhanced dispersion by
74
supercritical fluids (SEDS), controlled expansion of supercritical solution (CESS), and particles
75
from gas-saturated solutions/suspensions (PGSS) are some of these techniques [26-35].
76
In this study, a supercritical extraction process was carried out to extract β-carbolines from the
77
capsule of P. harmala using SC-CO2 as a solvent and the experimental parameters such as pressure,
78
temperature, modifier volume and dynamic extraction time were optimized using an experimental
79
design methodology based on the central composite design (CCD). In the next, alkaloid compounds
80
in the extract samples were identified by High performance liquid chromatography – mass
81
spectrometry (LC–MS). Micronization process was based on a technique which described recently
82
[36-38]. In this method, particle collection occurs in supercritical media as a solvent while
83
precipitation in common methods such as RESS or SAS is based on CO2 evaporation or
84
supercritical anti-solvent activity. Compared to mentioned methods, large number of collisions
85
between pressurized SC-CO2 solvent molecules and primary nuclei decreases particle growth
86
through coagulation and resulted particles are much smaller than those prepared from the other
87
processes. On the other hand, expansion step in this method is much slower than RESS process and
88
therefore the turbulent behavior of fluid at the time of particle collection are reduced.
89
Precipitated nanoparticles were characterized by Field Emission Electron Microscopy (FESEM)
90
and the effects of the different parameters such as concentration and volume of loaded extract,
91
temperature, pressure of precipitation and time of expansion on the particles shape and size were
92
investigated and optimum conditions were determined.
93 94
2. Materials and methods
95
2.1. Chemicals
4
96
Acetonitrile, Methanol, Ethanol and Cyclohexane of LC grade were purchased from Merck Co.
97
(Darmstadt, Germany). Formic acid and acetone were of analytical grade were supplied by Fluka
98
Co. (distributed by Sigma–Aldrich, Allentown, PA, USA) and Analytical grade (99.99% purity)
99
Carbon dioxide stored in a cylinder with an eductor tube was obtained from Roham Co. (Tehran,
100
Iran) and used as received.
101 102
2.2. Plant materials and sample preparation
103
Capsules of P. harmala (pod and seeds) were collected from their natural habitats in the central
104
regions of Iran (Esfahan, Iran). These plants were separated, cleaned, and stored at room
105
temperature in dark for one week, so as to be dried. Next, the dried samples were ground to coarse
106
powder by a laboratory mill (Myson, China) and used for extraction.
107 108
2.3. Herbal extraction process
109
2.3.1 Supercritical fluid extraction
110
Herbal extraction process was carried out in the SFE mode of a Suprex MPS/225 system equipped
111
with syringe pump unit (Pittsburg, Virginia, U.S.A.) using SC-CO2 as shown in Fig.2. In each
112
extraction, 1g of the powder of P. harmala was mixed with glass beads (1 mm in diameter) and
113
loaded into stainless steel extraction vessel (5ml) equipped with two sinter metal filters (5).
114
Different volumes of modifier were added directly into the sample. The temperature of CO2 was
115
cooled down to 0°C using the chiller (2) and a high-pressure syringe pump (3) was used to
116
pumping the CO2 into extractor. The pressure was controlled by a back-pressure regulator and the
117
electrically heated restrictor (6) (Dura flow manual variable; Suprex Co.) was used to prevent the
118
sample plugging. For preventing solvent evaporation and improve the efficiency, the collection
5
119
vessel (8) containing 3 mL of ethanol was placed in an ice bath (7) and the flow rate of SC-CO2
120
during dynamic extraction time was about 0.30 ± 0.05 mL min-1.
121
In order to prepare the sample for LC-MS analysis, the extracted solution was diluted to 5mL with
122
ethanol and filtered through a 0.22µm syringe filter (Microlab, china). The extraction yield was
123
calculated by weighting the collected extract after evaporation of solvent at room temperature at the
124
end of each run.
125 126
2.3.2. Conventional solvent (modifier) selection
127
1 g of accurately weighted P. harmala powder was placed in a 20 ml volumetric flask and made up
128
to volume with of acetonitrile, methanol, ethanol and cyclohexane separately. These samples were
129
kept in an ultrasonic cleaning bath (SB-5200D type, 40 kHz, 250 W, Ningbo Scientz
130
Biotechnology Co. Ltd., China) and sonicated for 40 min at 40°C. At the end of time, the
131
supernatant and the sediment were separated by vacuum filtration and the solvent was evaporated to
132
dryness at 60°C. Finally, the weight of the extracted compounds was determined gravimetrically
133
and extraction yield (EY) was calculated using the following equation: %=
2 × 100 1
134
Where W2 is the weight of the dried collected extract and the parameter W1 is the weight of the P.
135
harmala powder sample.
136 137
2.3.3. Optimization strategy
138
In this study, a 2n fractional factorial design was applied to screening of the different variables to
139
choose the main factors. This process was followed with central composite design (CCD) for
140
optimization of the effective parameters. The experimental design matrix and data analysis were
141
performed using the StatGraphics plus 5.1 package and the measured response was extraction yield
142
(EY %). 6
143
Preliminary experiments showed that extraction pressure, dynamic extraction time and modifier
144
concentration are main parameters affecting the extraction yield as experimental response.
145
Therefore, these factors at two levels with three center points were studied. The low and high
146
values of these parameters included: extraction pressures of 100 and 350 atm, modifier volumes of
147
0 and 100 µL and dynamic extraction times of 10 and 60 min. According to Previous similar
148
experiments, the static extraction time and oven temperature were fixed at 10 min and 40°C
149
respectively. The number of experiments is defined by the expression (2P + 2P + C), where P is the
150
number of factors and C is the number of center points that was 3 in this study and hence, 17 runs
151
to be carried out randomly.
152 153
2.4. Identification of β-carbolines alkaloids in herbal extract
154
β-carbolines were identified using High performance liquid chromatography–mass spectrometry
155
(HPLC–MS) and an Agilent 1200 LC system via an ESI source in a post-column was connected to
156
an Agilent 6410 triple quadrupole tandem mass spectrometer (Waldron, Germany). The mass
157
spectrometer was run in the positive ion mode (ESI+) and set up in the multiple reaction monitoring
158
mode and electrospray capillary potential was set to 4000V. The ultrahigh pure Nitrogen and Argon
159
gas were used as a nebulizer and drying gas for solvent evaporation and collision gas respectively.
160
The flow arte of the drying and nebulizer gas were set at 10 L min-1 and 40 psi and temperature of
161
capillary head was 300◦C. Data acquisition and analyses were performed using Mass Hunter work-
162
station.
163
Chromatographic conditions were selected according to a validated method reported by M. Kartal
164
et al as described in the following[39]. An isocratic elution with the flow rate 0.25 mL min−1 was
165
performed. The mobile phase composition was a mixture of isopropyl alcohol, acetonitrile, water,
166
formic acid (100:100:300:0.3) (v:v:v:v) and pH adjusted to 8.6 with trimethylamine. The column
167
used for chromatographic system was an Agilent rapid resolution HT zorbax SB-C18 (2.1 mm × 50
168
mm, 1.8µm) (Agilent Technologies, Santa Clara, CA). 1mL of extraction sample diluted to 10mL 7
169
with ethanol and 5 µL of the resulting solution was injected into the LC-MS system. The full-scan
170
mode over the mass range of m/z 100 – 400 spectra of the extract solution was obtained.
171 172
2.5. Micronization process
173
2.5.1. Supercritical fluid precipitation of extract nanoparticles
174
The equipment used in the nanoparticles precipitation using supercritical fluid solvent is shown in
175
Fig.3. This process is based on a method reported recently [37]. In this technique, a stainless-steel
176
precipitation vessel (6) was coupled to an equilibration vessel (5) and both sides of each chamber
177
were equipped with stainless steel filters (with the pore size of 0.1 µm). The capacity of the
178
equilibration and precipitation vessel was 5 and 25 mL respectively.
179
A 1 mL polyethylene vial (7) with loaded V µL solutions of the collected extract was placed in
180
equilibrium chamber and two sheets of Mica (5×50 mm, Agar Scientific Ltd.) for collecting of the
181
precipitate (8) were set in the vessel as shown in Fig.3. Equilibrating pressure and time (P1, t1),
182
precipitation pressure and time (P2, t2), oven temperature (T) and loaded volume of extract (V)
183
were variable parameters in precipitation process and equilibration time and pressure were
184
constant.
185
In first step, valve 1 was opened and CO2 was purged to equilibration vessel until the pressure was
186
raised to P1. After the equilibrating time was elapsed, dissolved extract in SC-CO2 was introduced
187
to precipitation vessel trough valve 2 while the valve 3 was also opened at a flow rate of 0.1 mL
188
min-1. When the pressure was reached to P2, the valve 2 and 3 was closed. The expansion of the
189
supercritical solvent during this pressure dropping program leads to decreasing in solvent density
190
and hence decreasing of solubility of the dissolved substances in SC-CO2. Eventually, these
191
conditions caused to precipitation of compounds in vessel and a part of the precipitate was
192
collected on the surface of the mica sheets. At the end of experiment, the precipitation vessel was
193
depressurized through valve 3 and then was disclosed. Collectors (mica sheets) were placed in the 8
194
closed tube until the particle size determination was accomplished. Precipitation experiments were
195
carried out in 6 different conditions which are shown in Table.2 and each experiment was repeated
196
for second times.
197 198
2.4.2. Characterization of precipitate
199
Size analysis and morphology of collected precipitates was carried out using Field Emission
200
Scanning Electron Microscopy (FESEM, MIRA3 TESCAN-XMU, and Czech Republic). In this
201
analysis, the mica collector sheets were coated with gold using a sputter coater (Pelco, model Sc-7)
202
and Microstructure Measurement Software was applied for image-analysis and particle size
203
determination.
204
Qualification analysis of β-carboline alkaloids in extract nanoparticles was done by LC-MS as
205
described in 2.4. Sample solution preparation was done using ultrasonication of the mica sheet
206
collector in 10 mL of ethanol for 20 min at 40°C. The solution was filtered through a 0.22 µm
207
syringe filter before injection.
208 209
3. Result and Discussion
210
3.1. Optimization of supercritical fluid extraction conditions
211
3.1.1. Modifier selection results
212
In order to investigate effects of different solvents on the extraction yield, the extraction of β-
213
carbolines from P. harmala fruit powder in four organic solvents (Acetonitrile, Methanol, Ethanol
214
and Cyclohexane) using sonication as described in 2.3.2 was done. Results showed that, using
215
methanol and ethanol as extraction solvent leads to the highest extraction yield (4.64% and 4.23%
216
respectively). Based on these results and compare safety data sheet of mentioned solvents, ethanol
217
was chosen as conventional solvent for supercritical fluid extraction and particle production
218
process. 9
219 220
3.1.2. Optimization design
221
Analysis of the results was visualized at a 95% confidence level using standardized main effect
222
Pareto charts (Fig.4a). According to this chart, modifier volume was the most significant variable,
223
having a negative effect (corresponding to blue) on the extraction yield and dynamic extraction
224
time and extraction pressure were the next most important parameters, respectively.
225
In the next step, a CCD statistical model was applied to optimize the three selected factors at three
226
levels. The examined levels of the factors and resulted extraction yield are shown in Table.1. The
227
second-order polynomial model was acquired for the experimental results as the following
228
equation:
229
Y = 8.41816 - 0.0355801 X1 + 0.0576763 X2 - 0.0290452 X3 + 0.0000301381 X12 +
230
0.000376667 X1 X2 + 0.000087 X1 X3 - 0.00209286 X22 - 0.00002 X2 X3 + 0.0000202389 X32
231
Where X1, X2 and X3 are extraction pressure, dynamic extraction time, and volume of modifier
232
respectively and Y is extraction yield.
233
Fig.4b shows the response surface obtained by plotting pressure dynamic extraction time versus
234
modifier volume whereas extraction pressure was fixed at 260 atm. This figure shows that
235
extraction yield increased when the dynamic extraction time was increased to 40 min and decreased
236
at more times. This result could be due to the loss of volatile compounds during the collection of
237
extract in long periods of time. On the other hand, the extraction efficiency has been increased by
238
decreasing the modifier volume. These observations indicate that most of the extracted compounds
239
are non-polar and the extraction yield decrease by increasing the polar solvents.
240
Fig.4c shows the response surface developed for pressure and modifier volume, while keeping
241
dynamic extraction time at 35 min. This figure shows that reducing the amount of the polar solvent
242
at low pressures leads to increased extraction yield and at high pressure, increasing the amount of
243
modifier increases the extraction efficiency. This can be justified by this fact that at low pressures, 10
244
non-polar compounds are extracted, and a decrease in modifier content leads to an increase in
245
extraction yield. Also, at high pressures, the extraction of polar compounds is mainly accomplished
246
and solvent reduction leads to increased efficiency.
247
Fig.4d indicates the response surface of pressure and time whereas modifier volume was 100 µL
248
and confirms the results mentioned in the interpretation of Fig.4b. The effect of the mentioned
249
parameters on the extraction yield is well illustrated in Fig.4e.
250
Based on the obtained results, the optimum extraction condition was obtained as: extraction
251
pressure of 176 atm, modifier volume of 16 µL (1.6%), oven temperature of 40°C and static and
252
dynamic extraction times of 10 and 40 min respectively.
253 254
3.1.3 Qualification of herbal extract
255
Supercritical fluid extraction of the capsule of P. harmala in optimum conditions was carried out as
256
described in Section 2.2. Accordingly, an extraction yield of 3.9% was obtained (with reference to
257
the initial weight of seeds). As described, Identification of β- carbolines in the extract samples was
258
done using LC-MS. The chromatogram and spectrogram of an extract sample are shown in Fig.5a
259
and b respectively. According to the mass spectra, it was possible to identify Harmaline, Harmine
260
and Harmalol in P. harmala samples. The peak at retention time of 11.9 min in EIC at m/z ratio of
261
213.1 can be related to the Harmine component (MW: 212.25 g mol-1), while the peak at retention
262
time of 11.8 min in the EIC at m/z ratio of 215.1 is attributed to Harmaline (MW: 214.26 g mol-1)
263
and the peak at retention time of 10.9 min in the EIC at m/z ratio of 201.1 (Fig.6d) is related to
264
Harmalol (MW: 200.24 g mol-1).
265 266
3.2. Optimization of supercritical fluid micronization conditions
267
Optimization of operating conditions to obtain a minimum size of precipitant is critical step in the
268
micronization process. In this study, supercritical precipitation of herbal extract of P. harmala was
269
done as described and variable parameters such as equilibration time (t1), oven temperature (T), 11
270
volume of extract (V) and precipitation time (t2) and pressure (p2) were evaluated. Different
271
experiment conditions and observed results are shown in Table.2 and in order to evolution of each
272
parameter, other parameters were kept constant.
273
Figure 6 shows FESEM images and particle size distribution diagrams of collected extract particles
274
under different conditions. The optimum operating conditions for producing nanoparticles were
275
determined as: volume of loaded extract sample (50 µL), oven temperature (40 °C), equilibrium
276
and precipitation pressure (350 and 100 bar), equilibration and precipitation time (10 min). In this
277
process, mean size of micronized particles was between 5 to 70 nm.
278 279
3.2.1. The effect of equilibration time and pressure
280
As showed in Table.2 (run 1), The loaded extract solution did not dissolve completely in SC-CO2
281
phase and a lot of sample remained in the vial. Increasing of oven temperature from 40 to 60 ºC in
282
run 2 did not lead to significant change in the solubility of loaded compounds.
283
In next experiment (Table1, run 3), the equilibration pressure was increased to 350 atm. In these
284
conditions, no extract solution was remained in the vial and loaded sample were dissolved in SC-
285
CO2 completely. This result clearly shows that the solubility of β-carbolines and other components
286
of herbal extract increased with increasing of pressure and density of SC-CO2 solvent.
287 288
3.2.2. The effect of precipitation time, pressure and temperature
289
Low amounts of the collected precipitates on the mica sheets in run 3 shows that most of dissolved
290
compounds were remained in SC-CO2 phase and the pressure drop has not been sufficient.
291
Decreasing of precipitation pressure from 140 to 100 atm in run 4 caused to precipitation of big
292
crystalline compounds with a wide size distribution as shown in Fig.6a. These results illustrate that,
293
precipitation process and dissolution depends significantly to the density of solvent and also intense
294
agglomeration of particles has happened during precipitation step. 12
295
To investigate the impact of temperature on the shape of particles and preventing of accumulation,
296
oven temperature increased from 40 to 60 ºC (Table 2, run 5). Increasing of temperature can reduce
297
the density of the fluid and on the other hand it leads to increasing in the solutes vapor pressure.
298
Depending on which of these factors is dominant, solubility and hence supersaturation
299
concentration may enhance, decrease or constant with increasing of temperature. According to the
300
classical nucleation theory, increasing of supersaturation concentration cause to decreasing of
301
particle size of precipitants [36]. FESEM image of particles (Fig.6b) showed that increase in
302
temperature did not have impressive effect on the shape of particles in this experiment. Therefore,
303
supersaturation concentration of herbal extract components of P. harmala was reminded constant
304
during increase the temperature.
305
In next run, precipitation time reduced from 30 to 10 minutes (Table 2, Run 6). FESEM image of
306
collected particles (Fig.6c) and analysis of this image using imageJ software (Fig.6e) show that
307
these conditions caused to precipitation of spherical nanoparticles with particle size distribution
308
between 5 to 70 nm. These results illustrate that morphology and particle size of precipitants were
309
dependent to duration of pressure drop and rate of crystallization significantly. It proves that,
310
nucleation process was preferred to crystal growth and agglomeration reduced significantly in this
311
experiment.
312
In order to increase the number of collected nanoparticles, volume of loaded solution was increased
313
to 50 and 70 µL (Table 2, Run 7 and 8) and precipitation process was done under optimum
314
conditions as described in previous run. Accordingly, FESEM image of collected particles during
315
run 7 and analysis of this image as shown in Fig.6d and f respectively prove that the number of
316
collected particles on mica sheets has increased from 1329 to 2543 particles while the size and
317
morphology of the nanoparticles remained unchanged.
318
In experiment 8, extra amount of the loaded extracted solution (70 µL) was remained in the vial and
319
results were similar to those obtained in the previous experiment and optimum loaded volume was
320
considered about 50 µL. 13
321 322
3.3. Qualification analysis of extract nanoparticles
323
HPLC chromatogram of extract nanoparticles was similar to the chromatogram of extract sample
324
and related peaks of Harmine, Harmaline and Harmalol in the positive ion mode (ESI+) are shown
325
in Fig.7.
326 327
4. Conclusion
328
In this study, Supercritical fluid extraction of β-carboline alkaloids from the capsule of P. harmala
329
was described. The optimum extraction condition was obtained as: extraction pressure of 176 atm,
330
modifier concentration (ethanol %) of 1.6 %, oven temperature of 40°C and static and dynamic
331
extraction times of 10 and 40 min respectively. Micronization of herbal extracted using a
332
precipitation technique based on the expansion of supercritical solution was investigated and
333
nanoparticles with an average diameter ranging from 7 to 100 nm were collected. Optimum
334
micronization conditions were determined as: Volume of loaded extract sample (50 µL), oven
335
temperature (40°C), equilibrium and precipitation pressure (350 and 100 bar), equilibration and
336
precipitation time (10 min).
337
This technique can be an innovative method to improve the solubility and bioavailability of drugs
338
with low solubility in water, and it is possible to investigate other active pharmaceutical ingredients
339
which are extracted directly from the medicinal plants.
340
References
341 342 343 344 345 346 347 348 349
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Zargari, A., Medicinal plants. Vol 2. 1992, Tehran University Press, Tehran. Lamchouri, F., et al., In vitro cell-toxicity of Peganum harmala alkaloids on cancerous cell-lines. Fitoterapia, 2000. 71(1): p. 50-54. Wang, Z., et al., Analysis of alkaloids from Peganum harmala L. sequential extracts by liquid chromatography coupled to ion mobility spectrometry. Journal of Chromatography B, 2018. 1096: p. 73-79. Sobhani, A.M., S.-A. Ebrahimi, and M. Mahmoudian, An in vitro evaluation of human DNA topoisomerase I inhibition by Peganum harmala L. seeds extract and its beta-carboline alkaloids. J Pharm Pharm Sci, 2002. 5(1): p. 19-23. 14
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437 438 439 440 441 442 443 16
444
Figure caption
445
Fig.1. Chemical structure of (a) Harmine, (b) Harmaline and (c) Harmalol as main β-carboline
446
alkaloids in the capsule of Peganum harmala L.
447
Fig.2. Schematic diagram of supercritical fluid extractor including: (1) CO2 Cylinder, (2) Chiller,
448
(3) Syringe pump, (4) Heat exchanger, (5) Extraction vessel, (6) Back pressure valve, (7) Ice bath,
449
(8) collection vessel
450
Fig.3. Schematic representation of supercritical fluid precipitation apparatus: (1) CO2 Cylinder, (2)
451
Chiller, (3) Syringe pump, (4) Heat exchanger, (5) Equilibration vessel, (6) Precipitation vessel, (7)
452
Loaded extract vial, (8) Collector (mica sheet).
453
Fig.4. (a) Standardized Pareto chart in the central composite design for SFE, representing the
454
estimated effects of parameters and parameter interactions on extraction yield.
455
Response surfaces using central composite design obtained by (b) dynamic extraction time (min)
456
vs. modifier volume (µl) at 260 atm. (c) Pressure (atm.) vs. modifier volume (µL) at 35 min of
457
dynamic extraction time (d) pressure (atm.) vs. dynamic extraction time at 100 µL of modifier
458
volume. (e) main effect plots of pressure, modifier volume and dynamic extraction time.
459
Fig.5. HPLC chromatogram of extract sample (a) and Mass spectrum of Harmine, Harmaline and
460
Harmalol (b).
461
Fig.6. FESEM images of precipitated extract particles under different conditions (a) crystaline
462
particles produced during experiment 4 (size bar > 500nm), (b) crystaline particles produced during
463
experiment 5 (size bar > 500nm), (c) Nanoparticles produced during experiment 6, (d)
464
Nanoparticles produced during experiment 8, (e) The particle size distribution diagrams correspond
465
to experiment 6 (size bar < 50nm, Count: 1329), (f) The particle size distribution diagrams
466
correspond to experiment 7 (size bar < 50nm, Count: 2543).
467
Fig.7. HPLC chromatogram of extract nanoparticles in the positive ion mode (ESI+). 17
468 469
Table.1.
470 471
Orthogonal array design and extraction yield for supercritical fluid extraction from Peganum harmala L. capsules. Test set
Pressure
Extraction Time
Modifier volume
Extraction Yield
(atm)
(min)
(µL)
(%)
1
260
35
100
3.50
2
310
50
150
3.63
3
310
50
50
3.72
4
310
20
150
3.10
5
175.91
35
100
3.59
6
210
50
150
2.54
7
260
35
100
3.64
8
260
35
15.91
3.85
9
260
60.22
100
2.54
10
260
9.77
100
1.90
11
260
35
184.09
3.54
12
210
20
150
2.94
13
210
50
50
3.30
14
310
20
50
2.93
15
210
20
50
3.84
16
260
35
100
3.60
17
344.09
35
100
3.94
472 473 474 475 476 18
477
Table.2.
478
Supercritical micronization conditions. Run
T
V
t1
t2
P1
P2
observation
figure
1
40
30
10
30
176
140
Extra amount remained in the vial.
-
2
60
30
10
30
176
140
Extra amount remained in the vial.
-
3
40
30
10
30
350
140
A few particles were formed.
-
4
40
30
10
30
350
100
Big crystals were formed
6a
5
60
30
10
30
350
100
Big crystals were formed
6b
6
40
30
10
10
350
100
Nanoparticles were formed
6c
7
40
50
10
10
350
100
Nanoparticles were formed.
6d
8
40
70
10
10
350
100
Extra amount remained in the vial.
-
479 480 481 482 483 484 485 486
19
Author statement
Hamze Salehi: SFE Extraction, plant colection, LC-MS analysis,
Mehrnaz Karimi:
micronization process, Data interpretation, Writing- Reviewing and Editing , Neda Rezaie: software and characterization by Field Emission Scanning Electron Microscopy (FESEM), Farhad Raofie: Supervision
Conflict of interests
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.