Response surface methodology to supercritical fluids extraction of artemisinin and the effects on rat hepatic stellate cell in vitro

doi:10.1016/j.supflu.2006.02.012

The Journal of Supercritical Fluids

Volume 39, Issue 1, November 2006, Pages 48-53

https://doi.org/10.1016/j.supflu.2006.02.012 Get rights and content

Abstract

This study examined supercritical carbon dioxide and near critical fluid extractions of artemisinin from Artemisia annua L. by varying pressure from 1000 psig (7.00 MPa) to 4500 psig (31.13 MPa), temperature from 30 to 60 °C, and co-solvent addition ratio ranged from 0 to 22.56 wt.%. The investigations included the total yield of the extracts, the recovery and the purity of artemisinin, and then the growth inhibition of rat hepatic stellate cells. Experimental results showed that dry supercritical carbon dioxide extraction was favorable for obtaining high purity but low recovery of artemisinin. A pre-loaded and continuing addition of co-solvent was proved to be effective in enhancing the recovery of artemisinin for the extractions near critical region. Moreover, a two-factor and three-level response surface methodology (RSM) disclosed that carbon dioxide added with 16.25 wt.% of N-hexane, extracted at 2700 psig (18.72 MPa), 33 °C, and for a period of 1.5 h could obtain an optimal value of quick recovery and high purity of artemisinin. Finally, treatments with the samples extracted using supercritical carbon dioxide and near critical fluids led to a strong inhibition of cell growth in primary cultured rat hepatic stellate cells.

Introduction

Both Artemisia annua L. (Abbr. as AA) and Artemisia capillaris Thumb. (Abbr. as AC) are used as traditional Chinese herb medicines. Although their physical outlooks are alike, it is still possible to distinguish AA from AC by the former unique floral trichome's morphology. Artemisinia annua L. has multi-seriate glandular trichomes and filamentous T-trichomes on flowers and leaf surfaces, but not for Artemisia capillaris Thumb. [1], [2], [3], [4], [5]. A. annua L. has long been useful for treating malaria and dysentery since it contains an active compound, i.e. artemisinin, which generally constitutes ranging from 0.01% to 0.947% depending on the growth conditions and species. Other than artemisinin, a few active constituents such as 0.24% artemisinic acid [6], 0.17% dihydroartemisinic acid [7], arteannuin-B [8], scopoletin [8], artemisitene [9], artemetin [9], deoxyartemisinin [10], quercetagetin 6,7,3′,4′-tetramethyl ether [11], artesunate [12], flavonoids, and 0.25% volatile oils, have been studied for their pharmaceutical effects. Artemisinin molecular formula is C₁₅H₂₂O₅ that belongs to a sesquiterpene trioxane lactone, which contains a peroxide bridge ring easily to be oxidized, has a melting point of 156–157 °C, a TGA point of 183.73 °C, and insoluble in water.

The purification of artemisinin has been carried out by traditional organic solvent extraction, chemical synthesis, histiocyte culture, and supercritical fluid extraction with carbon dioxide [13], [14], [15], [16], [17]. The traditional organic solvent extraction has residual solvent and low purity problems; chemical synthesis including 11 steps is complicated and the 37% conversion yield is quite low [18], [19]; histiocyte culture can possibly give 0.4% high yield but with an inconsistent yield [20]. Supercritical carbon dioxide extraction presents high extraction yield (1.49%) and high purity advantages [13], [14], [21].

A number of analytical methods have been used for qualifying and quantifying artemisinin, such as thin layer chromatography (TLC); high-performance liquid chromatography (HPLC) coupled with ultraviolet detection; electrochemical detection; polarographic detection; mass spectrometric detection; evaporative light scattering detection [9], [22], [23], [24]. Gas chromatography (GC) coupled with flame ionization detection, and mass spectrometric detection can be also used [25], [26].

The activation of the form cell of hepatic stellate cell (HSC) plays a major key role in a progressive course of the liver fibrotic formation. When a liver is damaged, the molecules such as liver cell, kuffer cell, endothelial cell, and immune cell, etc. try to secrete out. A various kinds of cytokines and their growth factors like PDGF, TGF-β1 can activate HSC. This is known as hyperplasia. These cells increase gradually become the fibrotic of hepatic stellate cells. By this fibrotic formation, a large number of extra-cellular matrices are further secreted and accumulated on the outside of normal liver cells. Hence more fiber tissues are formed, and finally cause the liver fiber. Current researches show that during a medical treatment, the activated HSC is observed to be depressed and the extra-cellular matrices are totally collapsed in the recovery of a fibrotic liver. However, in vivo curing of the fibrotic liver is still being under development.

Recent published literatures reported that artemisinin and its derivatives have anti-tumor [27], anti-fibrotic, and the depression of cancer cells growth [28]. Artemisinin exhibits potent cytotoxicity against P-388 (murine lymphocytic leukemia), A-549 (human lung carcinoma) and HT-29 (human colon adenocarcinoma) tumor cells with EC₅₀ values of 9.62 × 10⁻², 4.16, and 4.41 μg/ml, respectively.

In this study, supercritical carbon dioxide and near critical extractions of artemisinin from A. annua L. have been investigated. A response surface methodology is applied to optimize the recovery and the purity of artemisinin. Finally, treatments with the samples extracted using supercritical and near critical carbon dioxide on the cell growth in primary cultured rat hepatic stellate cells are examined.

Section snippets

Materials

Naturally air dried whole plant of A. annua L. and Artemisia capillaris T. were supplied by Kaiser pharmaceutical (Tainai, Taiwan). The moisture content of two raw materials is 9.7 and 7.1 wt.%, respectively measured by an Infrared moisture meter (AD4714, A&D corp., Japan). Artemisia capillaris T. was used for comparison purpose. Carbon dioxide (99.5% bone-dry) was purchased from a local supplier of the Air Products (Taichung, Taiwan). N-hexane (Merck, 99%, Germany); Ethanol (Merck, 99.8%,

Results and discussion

Table 1, Table 2 list all experimental conditions of response surface methodology for supercritical CO₂ (SC-CO₂) and near critical fluid extractions, respectively. The accuracies of pressure and temperature measurements are within ±0.40 MPa and ±1 K. For the comparison, the artemisinin yield of 11.01 mg/g of feed and the purity of 19.8 wt.% is used as the reference state, which is obtained from a 12-h Soxhlet N-hexane extraction. Table 1 presents total yield of SC-CO₂ extract (TY) ranged from 0.32

Conclusions

Supercritical and near critical carbon dioxide added with N-hexane extractions from A. annua L. show high recovery and high purity of artemisinin. This process proves to be feasible. The maximum purity of artemisinin attained by 2500 psig (17.34 MPa) and 60 °C extraction is more than three-fold of that by the Soxhlet N-hexane extraction. The two-factor and three-level RSM at near critical extractions of carbon dioxide added with 16.25% N-hexane indeed enhances the artemisinin recovery but with a

Acknowledgments

The authors thank the National Science Council of the Republic of China for financially supporting this research under Contract NSC92-2214-E005-003 and the cell culture work was supported by grants from the Taichung Veterans General Hospital and National Chung Hsing University (TCVGH-NCHU-937606) Taichung, Taiwan, ROC.

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It was shown that SCCO2 at 100 bar and 40 °C, artemisinin could effectively be extracted and yielded a 35% artemisinin containing extract (Baldino et al., 2017). Addition of a co-solvent such as n-hexane to SCCO2 results in consistently low recovery of artemisinin, but the extracts are of high purity (Lin et al., 2006). It is noted that SCCO2 with addition of ethanol and SCCO2 can be used as substitutes for flammable hexane and organic solvents (Ciftci et al., 2018).
Fifty years ago, in 1972, artemisinin was first isolated as a pure compound from Artemisia annua L. (Asteraceae) and was shown to possess potent antimalarial properties. This review aims to present a concise account of the history of the discovery of artemisinin and the development of artemisinin combination therapies. This is followed by the current production techniques (isolation and purification) to supply the growing global demand and finally the possibility to produce artemisinin in South Africa is discussed. Artemisinin and its derivatives are now used worldwide for treatment of malaria, largely in combinations with other antimalarial drugs known as artemisinin combination therapies (ACTs). The main source of artemisinin still relies on extraction from A. annua, with Vietnam and China presently providing over 80% of the global supply. However, the production of artemisinin is not sufficient to meet requirements for global treatment of malaria, and demand continues to outstrip supply. This supply problem will be exacerbated once artemisinin and its derivatives are adopted for treatment of other diseases including cancer, other parasitic diseases, and viral infections. Thus, higher yielding A. annua cultivars coupled with development of novel extraction and purification techniques are required for meeting the enhanced demands. One significant development involves the use of a pressurised hot water system to efficiently extract artemisinin. From a South African perspective, little work has been conducted on the production of A. annua and optimization of supply of artemisinin. A recent pilot study simulating an agrophotovoltaic (APV) system where the land area utilised is associated with both production of solar energy as well as the cultivation and growing of A. annua indicated that the plant produced roughly 20% more artemisinin when grown under these conditions as compared to the control group. Current activities focussing on production of artemisinin in South Africa to serve the Sub-Saharan African region are also discussed.
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SFE produced extracts with ART in the range of 1.0–23.4 g/100 g extract, resulting AY of 0.08–0.8 g/100 g plant, where the highest ART and AY values were obtained without the addition of ethanol to the scCO2. These results corroborate the literature that reports AY in the range of 0.2 – 0.9 g/100 g plant when pure scCO2 is used (Ciftzi et al., 2018; Kohler et al., 1997; Lin et al., 2006; Martinez-Correa et al., 2017; Quispe-Condori et al., 2005; Tzeng et al., 2007). It is possible to observe in Table 3 that the increase in the amount of co-solvent decreased the ART due to the coextraction of other compounds, similarly to the findings reported by Ciftzi et al. (2018).
Artemisia annua L. is the main artemisinin source, a sesquiterpene lactone used in the treatment against cancer and malaria, whose demand is high and the costs involving its production must be feasible. The main objective of the present work was to define a industrially and economically viable technology to obtain artemisinin. The experimental conditions of temperature, pressure and co-solvent use for obtaining extracts enriched in artemisinin by supercritical fluid extraction (SFE) were investigated, and an economic evaluation of the SFE was performed in comparison with the conventional extraction using ethanol. The techno-economic analysis showed that among the conditions evaluated in the present study, SFE at 60 °C and 250 bar, without co-solvent, was the most promising production condition. Nevertheless, for the commercial viability of the arteminisin extraction using SFE it is indicated to use A. annua rigorously selected, and to reduce costs of raw material.
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Quality by design (QbD) has lately been widening its domain at a winged pace among the diverse terrains. The current research findings suggest a spurt in the utilization of principles of QbD in modern herbal drug industries. QbD approach, verily, banks upon the combined use of risk assessment and design of experiments (DoEs) for efficient and cost-effective extraction of bioactives from diverse plant materials. Such approaches not only provide benefits of efficient extraction process but also help in understanding the scientific minutiae for controlling the variability encountered during the extraction process. This chapter endeavors to highlight the applications of QbD in improving extraction process of potent bioactives from medicinal plants using modern extraction techniques. In a nutshell, the utility of systematic QbD tools for extraction processes of varied kinds with minimal expenditure of time, cost, and other resources has been described in the present work.
Desorption of artemisinin extracts of CIM-Arogya by supercritical carbon dioxide
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Lin et al. [7], at 323.1 K and 173.4 MPa, achieve higher purity values of 73 wt%, with a total yield of 0.5%. Some papers report supercritical carbon dioxide artemisinin extractions, from Artemisia annua L., by adding co-solvents: 3% of methanol at 150 bar and 40 °C by Kohler et al. [8,9], 16.25% of n-hexane at 70–208 bar and 40–60 °C by Lin et al. [7] and 16.25% of ethanol at 173–311 bar and 40–60 °C by Tzeng et al. [10]. The co-solvent modified SFCO2 extraction produces more pure artemisinin than classical Soxhlet solvent extraction.
Artemisinin is a drug for chloroquine resistant malaria and cerebral malaria treatments. In the recent past, there was an acute shortage of this drug and hence World Health Organization made a strategy to fulfil the Artemisinin demand.
In this study, artemisinin was extracted by supercritical Carbon Dioxide (SFCO₂) from CIM-Arogya, a variety of Artemisia annua, in temperature and pressure ranges of 313.1-333.1 K and 15–25 MPa. Artemisinin global yield isotherms were determined obtaining a maximum yield of 3.65 wt%. Artemisinin extracts were also obtained by hexane Soxhlet extraction: then, the crude extracts were purified using SFCO₂, after adsorption on silica gel. Different desorption runs were performed with a 6 ml/min CO₂ flow rate, in temperature and pressure ranges of 313.1–333.1 K and 15–25 MPa. At different time intervals, extracts were collected and analysed: their yields varied from 2.75% to 4.34% function of the experimental conditions. Desorption trials were also correlated with different models.
An optimized process for SC-CO<inf>2</inf> extraction of antimalarial compounds from Artemisia annua L.
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Moreover, it has been demonstrated that extracts of A. annua have antiulcerogenic [2], antifibroric and antitumoral activity [3]. Potent cytotoxic activity against P-388 (murine lymphocytic leukemia), A-549 (human lung carcinoma) and HT-29 (human colon adenocarcinoma) tumor cells was also ascertained [1]. The chemical synthesis of artemisinin has been achieved in the 1983; but, it is not competitive with respect to the natural source, since it is complex and expensive.
Supercritical fractional extraction and separation scheme, was used to process Artemisia annua L., producing extracts enriched in active antimalarial principles. The best results were obtained when extraction was performed at 100 bar, 40 °C and the first separator was operated at the same pressure and at −7 °C. Paraffinic co-extracted compounds were selectively recovered in the first separator, confirming the efficiency of the fractional cooling separation and a concentration of 35% w/w of active compounds was obtained in the second separator. Artemisinin was the major active compound in the extract; but, other two active compounds, artemisin and dehydroartemisinin, were also largely found. Other co-extracted compounds belonged to Artemisia essential oil. Different SC-CO₂ flow rates were tested: an increase from 0.8 to 1.2 kg/h did not determine appreciable variations of the extraction rate of the various compounds, indicating that internal mass transfer resistance mainly controlled the extraction process.
Integrated extraction process to obtain bioactive extracts of Artemisia annua L. leaves using supercritical CO<inf>2</inf>, ethanol and water
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Artemisia annua L. is an herb used in traditional Chinese medicine due to presence of biocompounds with biomedical and pharmaceutical applications. In this work, an integrated process for extraction of bioactive compounds present in A. annua leaves was performed. The process comprised two-step extractions: in the first step supercritical carbon dioxide (scCO₂) was used as a solvent, and from the solid residue of the supercritical extraction other extracts were obtained (second stage) using ethanol or water as solvents. Single-step extractions using ethanol or water as solvents were also performed for comparison. In all extracts the variables analyzed were overall extraction yield, the content and yield of total phenolic, total flavonoids and artemisinin, as well as antimalarial activity. The supercritical (SC) and ethanolic (E) extracts obtained in a single step showed the highest yields of artemisinin and were very active against Plasmodium falciparum, with IC₅₀ values less than 0.1 μg mL⁻¹. On the other hand, the aqueous (SCA) and ethanolic (SCE) extracts from the second extraction step were free of artemisinin, but these extracts contained roughly 90 mg of phenolic compounds per gram of extract, including a high overall yield in the aqueous extract. The volatile fraction (SC-V) obtained from the supercritical extraction consisted mainly of camphor. Therefore, the two-step extraction in two steps proved to be advantageous because the residue of supercritical extraction could be used for obtaining aqueous or ethanolic extracts containing phenolic compounds.

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Response surface methodology to supercritical fluids extraction of artemisinin and the effects on rat hepatic stellate cell in vitro

Abstract

Introduction

Section snippets

Materials

Results and discussion

Conclusions

Acknowledgments

J. Chromatogr. A

Tetrahedr. Lett.

J. Chromatogr.

J. Chromatogr. B

J. Supercrit. Fluids

Development and fine structure of glandular trichomes of Artemisia annua L

Int. J. Plant Sci.

Glandular trichomes: a focal point of chemical and structural interactions

Int. J. Plant Sci.

Localization of artemisinin and artemisitene in foliar tissues of glanded and glandless biotypes of Artemisia annua L

Int. J. Plant Sci.

Floral morphology of Artemisia annua with special reference to trichomes

Int. J. Plant Sci.

Distribution of Artemisinin in Artemisia annua

Methoxylated flavones and coumarins from Artemisia annua

Phytochemistry

An improved method for the isolation of qinghao (artemisinic) acid from Artemisia annua

Planta Med.

Terpenoids and flavonoids from artemisia species

Planta Med.

Isolation and identification of dihydroartemisinic acid from Artemisinia annua and its possible role in the biosynthesis of artemisinin

J. Nat. Prod.

New trends in extraction, identification and quantification of artemisinin and its derivatives

Curr. Med. Chem.