Extraction of Andrographolide From Andrographis Paniculata Dried

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Extraction of Andrographolide From Andrographis Paniculata Dried Leaves using Supercritical CO2 And Ethanol Mixture Andri C. Kumoro, Masitah Hasan, and Harcharan Singh Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b02243 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 28, 2018

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Industrial & Engineering Chemistry Research

EXTRACTION OF ANDROGRAPHOLIDE FROM ANDROGRAPHIS PANICULATA DRIED LEAVES USING SUPERCRITICAL CO2 AND ETHANOL MIXTURE Andri C. Kumoro1,2*, Masitah Hasan1, Harcharan Singh1 1Department

of Chemical Engineering, Faculty of Engineering, Universitas Diponegoro,

Jl. Prof. Soedarto, SH, Tembalang - Semarang 50275, Indonesia. 2Department

of Chemical Engineering, Faculty of Engineering, University of Malaya, Kuala Lumpur 50603, Malaysia. *Corresponding author: [email protected] Abstract

The increase in the number of world’s elderly population and changes in their lifestyle has triggered to the formation of diseases susceptible communities. Inherent with the aforementioned conditions, the need in andrographolide as one of natural remedies isolated from the leaves of Andrographis paniculata also increases. In this work, supercritical fluid extraction employing CO2–ethanol mixture as solvent has shown its capability in extracting andrographolide from dried leaves of A. paniculata. The andrographolide yield largely increased when the ethanol concentration in the solvent mixtures was increased from 0 to 12.5 mol %, but no significant increase in andrographolide yield as ethanol concentration was further increased. Although an enhancement in both temperature and pressure gave favorable effects on the yield of andrographolide, a crossover effect existed in this supercritical system causing no significant increase of andrographolide yield. The down flow mode performed the highest extraction efficiency compared to both up flow and horizontal flow modes. Supercritical CO2–ethanol mixture containing 12.5 mol % ethanol flowed at 2 mL/min according to down flow mode at 323 K and 15 MPa was found to be the most effective in extracting andrographolide. The integral desorption model satisfactory described the extraction profile as shown by its low average error of about 5%. Keywords: andrographolide; carbon dioxide; ethanol; supercritical; extraction; modeling. Introduction 1 ACS Paragon Plus Environment

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Andrographolide is an unsaturated bicyclic diterpene -lactone fractionated from Andrographis paniculata dried leaf, a shrub vegetation from Acanthaceae tribes, which is broadly used as a natural remedy in China, India, and some other nations in South East Asia.1 Andrographolide is reported to exhibit various medicinal properties, such as antioxidative, antiinflammatory,

antibacterial,

antiretroviral,

antitumor,

antidiabetic,

antimalarial,

antihypertension, antirheumatism, hepatoprotective, anti-venom etc.2 With regard to those medicinal activities, it is highly essential to find the efficient ways to extract andrographolide from A. paniculata leaves to provide safe medicines for the people in needs. Although andrographolide is soluble in methanol and ethanol, it does not dissolve easily. It is slightly soluble in other less polar organic solvents such as acetic acid, pyridine, dichloromethane, acetone or ethyl acetate.3 The melting point of this light yellow compound is 228-230oC, while the maximum spectra of ultraviolet in ethanol (max) exists at 223 nm.4 Extraction method is one of the key factors to preserve the bioactive compounds and their pharmacological activity. The extraction method selection largely be determined by the nature of the metabolites to be extracted and isolated.6 This separation process should be simple, fast, cheap, efficient, high-throughput and should protect the natural condition of the targeted compound as well as the environment.7 Conventional extraction methods, which include cold and hot maceration, boiling, Soxhlet method and leaching have been used for the separation of various lactones from A. paniculata dried leaf.8-10 However, these conventional extraction methods are thermally unsafe, suffering from lower value of extraction yields, and requiring a substantial volume of solvent and lenghty extraction period.11 Recently, more advance extraction techniques, such as enzymatic-assisted extraction, ultrasound-assisted extraction (UAE), three phase partitioning, microwave-assisted extraction (MAE), solid phase extraction and supercritical fluid extraction of andrographolide have been applied.12-15 In current decennium, the supercritical fluid (SCF) extraction has gained remarkable attentions from researchers due to the conscientious environmental regulations, product safety and drawbakcs of the conventional extraction technique.6 This is particularly true for the extraction of functional food, phytochemical and nutraceutical products as the consumers are insisting natural products without the use of organic solvents.16 Principally, the importance in using SCFs as solvents is contributed by the excellent characteristics of this category of fluid, such as the ability to change density and to cause a shift in solubilization power by altering either 2 ACS Paragon Plus Environment

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the pressure or temperature.17 These adjustable characteristics are very crucial for the development of highly efficient extraction process of secondary metabolites from herbaceous matrices.15 The viscosity of an SCF is far below than that of a liquid, while its diffusivity is higher than for any other fluids by one to two orders of magnitude. This property allows speedy mass transfer rate, leading to a quicker extraction rate than the most of traditional liquid extractions.18 The use of SCF’s in extraction processes also facilitates a relatively easy separation between the solvent and the desired products.17 Carbon dioxide (CO2) is one of the extremely popular gases utilized as a SCF primarily because it is cheap, non-reactive, non-toxic, less explosive, less flammable, and abundantly obtainable in high purity levels with moderate critical temperature (304.26 K) and pressure (7.38 MPa).6 The low critical temperature of CO2 is interesting for the extraction of thermolabile or easily oxidized compounds.15 Similar to the most of other less-volatile high molecular weight organic compounds, andrographolide is almost insoluble in supercritical CO2.19 This low solubility value is probably due to low polarity and low capacity of CO2 for specific interactions with solute, which would lead to lacks of selectivity to polar organic compounds.17 The incorporation of a little portion of polar cosolvent to supercritical CO2 will essentially increase the SCF mixture polarity and density, which will subsequently enhance the solubility of polar organic compounds as a result of special chemical interactions mainly through hydrogen bonding or charge transfer for the formation of complex compounds.6 Therefore, the characteristics and portion of the cosolvent are also becoming the important factors responsible for the solubilization of the target compounds in the SCF.16 The selection of a suitable cosolvent must be made not only on a thermodynamics basis, but should also consider its food safety status, which is, it must be “Generally Recognized as Safe” (GRAS).20 In fact, the most common organic cosolvent employed to extract bioactive compounds is ethanol in a range of 5 to 10 mol % of CO2 flow.16 Although a number of researches on supercritical fluid extraction (SFE) had been conducted in the recent years, very limited number of literatures outlined the SFE of andrographolide from A. paniculata leaves. Supercritical CO2 and CO2–ethanol mixture had been utilized to extract andrographolide from A. paniculata dried leaves in a custom-made extractor.19, 21 Ge et al. studied the optimization of andrographolide extraction from A. paniculata dried leaves using supercritical CO2–ethanol mixture through an orthogonal experiment. They reported that the utiliztion of ethanol as cosolvent resulted in a larger extraction yield and andrographolide content than using pure 3 ACS Paragon Plus Environment


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supercritical CO2.21 Later, Kumoro and Hasan reported that highest yield of andrographolide was achieved when the extraction was operated by delivering supercritical CO2 at a volumetric rate of 2 mL/min under pressure and temperature of 10 MPa and 313 K, respectively. The yield of extraction was found to be 0.0174 gram andrographolide per gram of initial andrographolide contained in the A. paniculata leaf, which is corresponding to 2.01 × 10−3 gram andrographolide per gram of dried leaf.19 Charoenchaitrakool et al. extracted andrographolide from andrographolide crude extracts of various polarity organic solvents (methanol, ethanol, acetone and N, N-dimethylformamide) using gas anti-solvent (GAS) technique employing CO2 as antisolvent.22 Chen et al. optimized the SFE of andrographolide from A. paniculata dried leaf using pure supercritical CO2 employing response surface methodology (RSM) with three factor levels experimental design.15 Unfortunately, both Ge et al. and Charoenchaitrakool et al. did not provide detail information on the influence of each parameter studied on the andrographolide extraction performance with the presence of ethanol as cosolvent.21,22 In this work, a semibatch dynamic extraction technique combined with gravimetric and chromatographic analyses was used to study the kinetics of supercritical extraction of andrographolide from A. paniculata dried leaf powder using CO2–ethanol mixture. This research aimed to explore the effect of ethanol concentration, temperature, pressure and flow direction on the supercritical extraction of andrographolide from A. paniculata. Further, the integral desorption model was tested its adequacy to represent the experimental data. Materials The A. paniculata dried leaves were purchased from Malaysian Agricultural Research and Development Institute (MARDI) and were deposited in a closed air tight bottles at room temperature. Prior to extraction process, the dried leaves were directly milled into fine powder and were screened to get an average particle size of about 0.375 mm with 0.841 g/cm3 true density. The liquid carbon dioxide with a purity of 99.7% was procured from Air Product (M) Berhad. The absolute ethanol (99.5% purity), methanol (99.9% purity), andrographolide (98% purity) and deionized water were the products of Sigma-Aldrich and were obtained from an authorized chemicals distributor in Malaysia. All of the chemicals were directly utilized without any further treatment.

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Equipment and Experimental Procedures The supercritical fluid extraction experimental set-up employed in this work was similar to that previously used by Kumoro and Hasan with some modification to deliver and mix ethanol as co-solvent as shown in Figure 1.19 In a typical run, 15g of dried-powder of A. paniculata leaves was firstly introduced into a 50 mL extraction vessel that was immersed in an automatically controlled water bath heater. The dried leaf powder was distributed in the column as a mixture with tiny stainless steel balls (2 mm diameter with 8.027 g/cm3 true density) forming 0.32 bed porosity with 1.224 g/cm3 apparent bed density to enhance the leaf particles – supercritical solvent physical contact and to prevent the occurrence of channeling when the solvent flew through. Liquid solvents (CO2 and ethanol) were each delivered at pre-determined flow rate to reach the desired pressure and were mixed in a static mixer to obtain the desired ethanol concentration in the CO2–ethanol mixture with 2 mL/min flow rate. The CO2–ethanol mixture was then preheated by a preheating coil submerged in a water bath so that it could attain the exraction temperature before going into the extraction chamber. The CO2–ethanol mixture is close to the supercritical point, in which the critical temperature is around 333 K. A back pressure regulator (BPR) was operated to regulate the extraction pressure. The extraction was performed out at 10 – 20 MPa pressures, 303 – 333 K extraction temperatures, 0 to 15 mol % ethanol concentrations, (upflow, downflow and horizontal) flow directions and 2 mL/min flow rate of CO2–ethanol mixture. The concentrations of ethanol (mol %) were calculated based on the actual volume of ethanol mixed into a known volume of liquid carbon dioxide. The extract was obtained by depressurizing the CO2–ethanol mixture at the back pressure regulator and was directly accumulated into a vial glass cold trap at 4°C. The vial was firmly sealed with a septum equipped with an outlet to vent the decompressed CO2. The samples were withdrawn at one-hour intervenes period and the extractions were stopped after seven hours. The samples were dark green solutions with specific ethanol smell containing andrographolide. The extracts were then dried under a modest flow of nitrogen and were quantified gravimetrically. For each trial, at least two experiments were performed from which the relative error obtained between these two runs was lower than 5%. Prior to analysis, the carefully pre-weighed dried samples were dissolved in 25 mL of methanol. Each sample was filtered through a Whatman nylon syringe, and followed by transferring the filtrate into a 1.5 mL HPLC vial. The analyses of the content of andrographolide 5 ACS Paragon Plus Environment


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in the samples were carried out by high-performance liquid chromatography (HPLC) with a system from Shimadzu using UV detector according to the protocol previously developed by Kumoro and Hasan.19 The chromatographic column employed for andrographolide determination was reverse phase C18-Thermo Hypersil ODS (250mm × 4.6mm, containing particles with 5μm diameters). The mobile phase used in this analysis was methanol/water mixture (54/46, v/v) delivered constantly at 1 mL/min flow rate. The volume injection of the sample was 20 μL. The wavelength of the UV detector was set at 250 nm for 10 minutes. The peaks of the chromatogram were interpreted by comparing them with the retention time of the standard. Prior to analysis, linear calibration of standards at an accuracy of higher than 99% was performed using pure andrographolide standard compound. The calibration curve was linear from 1.1 – 24 μg/mL. In addition, an injection of solvent without extract sample (blank) was also performed to obtain the retention time of the solvent. The yield of andrographolide was defined as the ratio of the mass of andrographolide in the extract and initial mass of andrographolide in the leaf powder. Mathematical Modeling The simple integral desorption model was utilized to represent the physical event of the extraction process.23 The extraction vessel is loaded with leaf powder with an initial andrographolide content of S0 (g/g leaf particles) to form a packed bed system. Fresh supercritical solvent is introduced into the extraction chamber, the pore and void volumes () are initially solute free, and the system is isothermal. The total material balance in the supercritical fluid phase of this system can be described by the expression below:

(

)

∂𝐶 ∂𝐶 ∂2𝐶 ∂2𝐶 ∂𝑆 𝛼 + 𝑢 ― 𝛼 𝐷𝑧 2 + 𝐷𝑟 2 = ― (1 ― 𝛼) ∂𝑡 ∂𝑧 ∂𝑡 ∂𝑧 ∂𝑟

(1)

where C, S, Dz , Dr, u, r, z and t are respectively the concentration of andrographolide in the supercritical solvent (g/cm3), concentration of andrographolide in the leaf particles (g/g leaf particles), axial dispersion coefficient (cm2/hour), radial dispersion coefficient (cm2/hour), axial supercritical fluid velocity (cm/hour), radial position of the extraction cell (cm), axial position of the extraction cell (cm), and time (hour). In spite of extraction vessel dimension with length to diameter ratio (L/D) = 10

Extraction of Andrographolide From Andrographis Paniculata Dried

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