Extraction, Isolation and Characterization of Bioactive Compounds from Artemisia and Their Biological Significance: A Review.

Diverse medicinal plants such as those from the genus Artemisia have been employed globally for centuries by individuals belonging to different cultures. Universally, Artemisia species have been used to remedy various maladies that range from simple fevers to malaria. A survey conducted by the World Health Organization (WHO) demonstrated that 80% of the global population is highly reliant on herbal medicine for their primary healthcare. WHO recommends artemisinin-based combination therapies (ACT) for the treatment of global diseases such as malaria. Artemisinin is a bioactive compound derived from Artemisia annua leaves. It is a sesquiterpene endoperoxide with potent antimalarial properties. This review strives to instill natural products to chemists and others in diverse fields with a heterogeneous set of knowledge compiled from multifaceted researchers and organizations in literature. In particular, the various Artemisia species and effective extraction, isolation, and characterization methodologies are discussed in detail. An in-depth investigation into the literature reveals that divergent species of Artemisia exhibit a vast array of biological activities such as antimalarial, antitumor, and anti-inflammatory activities. There is substantial potential for bioactive compounds from Artemisia to provide significant relief from differing human ailments, but more meticulous research in this field is needed.

In vitro reduction of Plasmodium falciparum gametocytes: Artemisia spp. tea infusions vs. artemisinin.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua has a long history of use in Southeast Asia where it was used to treat "fever", and A. afra has a similar history in southern Africa. Since their discovery, A. annua use, in particular, has expanded globally with millions of people using the plant in therapeutic tea infusions, mainly to treat malaria. AIM OF THE STUDY: In this study, we used in vitro studies to query if and how A. annua and A. afra tea infusions being used across the globe affect asexual Plasmodium falciparum parasites, and their sexual gametocytes. MATERIALS AND METHODS: P. falciparumstrain NF54 was grown in vitro, synchronized, and induced to form gametocytes using N-acetylglucosamine. Cultures during asexual, early, and late stage gametocytogenesis were treated with artemisinin, methylene blue, and A. annua and A. afra tea infusions (5 g DW/L) using cultivars that contained 0-283 µM artemisinin. Asexual parasitemia and gametocytemia were analyzed microscopically. Gametocyte morphology also was scored. Markers of early (PfGEXP5) and late stage (Pfs25) gametocyte gene expression also were measured using RT-qPCR. RESULTS: Both A. annua and A. afra tea infusions reduced gametocytemia in vitro, and the effect was mainly artemisinin dependent. Expression levels of both marker genes were reduced and also occurred with the effect mainly attributed to artemisinin content of four tested Artemisia cultivars. Tea infusions of both species also inhibited asexual parasitemia and although mainly artemisinin dependent, there was a weak antiparasitic effect from artemisinin-deficient A. afra. CONCLUSIONS: These results showed that A. annua and to a lesser extent, A. afra, inhibited parasitemia and gametocytemia in vitro.

The birth of artemisinin.

As the first-line antimalarial drugs, artemisinins gained wide acceptance after the emergence of resistance to chloroquine in the 1950s. Artemisinin-based drugs have saved lives, especially in developing countries. The discovery of artemisinin was unique, timely, and fascinating, and the benefits of artemisinin were with far-reaching implications. Herein, we will give a brief description of various aspects of the development of artemisinin and discuss the position and perspectives of artemisinin-based drugs.

Biodegradable functional chitosan membrane for enhancement of artemisinin purification.

Artemisinin is mainly derived from Artemisia annua L. Since the leaves composition is complex, artemisinin purification faces great challenges. In this work, functional chitosan membranes were fabricated by a one-step hydrolysis method through grafting long-chain alkyl group on the surface of chitosan to increase its hydrophobicity. The as-prepared membranes were used to adsorb wax oil (i.e., the impurity components) in Artemisia annua L. and to avoid co-precipitation of wax oil along with artemisinin using the crystallization technique for purification. Octyl-trimethoxysilane modified chitosan membrane (FCM-C8) showed excellent capability to intensify this purification process. The product purity could reach more than 98 % using one crystallization step under the optimal conditions, and in this case, adsorption capacity of FCM-C8 for wax oil was 478.9 mg/g. In addition, the adsorption kinetics and mechanism of wax oil on FCM-C8 were studied. The membrane can simultaneously adsorb multiple components in wax oil through interactions like electrostatic forces, hydrogen bondings.

Artemisinin and its derivatives; ancient tradition inspiring the latest therapeutic approaches against malaria.

Artemisinin (ART) is an endoperoxide sesquiterpene lactone, commonly used in the treatment of malaria. Although it was isolated from Artemisia annuaL., a plant widely applied in Chinese Traditional Medicine, its mechanism of action remains uncertain and its clinical use is still limited due to its low solubility, its poor bioavailability and short in vivo half-life. Over time, several studies have been aimed towards the discovery of potent ART derivatives that could overcome clinical drawbacks. In this review, we focus on the multifaced aspects of ART and on the efforts spent to improve its pharmacological profile that so far culminated in the discovery of more effective drugs. Lastly, we outline the new perspectives in the ART-derivatives scenario.

Optimisation of artemisinin and scopoletin extraction from Artemisia annua with a new modern pressurised cyclic solid-liquid (PCSL) extraction technique.

INTRODUCTION: Artemisia annua is a small herbaceous plant belonging to the Asteraceae family declared therapeutic by the World Health Organisation, in particular for its artemisinin content, an active ingredient at the base of most antimalarial treatments, used every year by over 300 million people. In the last years, owing to low artemisinin content, research of new ways to increase the yield of the plant matrix has led to the use of the total extract taking advantage from the synergic and stabilising effects of the other components. OBJECTIVE: In this work we evaluated and compared the content of artemisinin and scopoletin in extracts of A. annua collected in the Campania Region (southern Italy), by two different extraction processes. METHODOLOGY: Artemisia annua plants were extracted by traditional maceration (TM) in hydroalcoholic solution as a mother tincture prepared according to the European Pharmacopeia and by pressurised cyclic solid-liquid (PCSL) extraction, a new generation method using the Naviglio extractor. RESULTS: The results showed that the PCSL extraction technique is more effective than traditional methods in extracting both phytochemicals, up to 15 times more, reducing the extraction times, without using solvents or having risks for the operators, the environment and the users of the extracts. CONCLUSION: The Naviglio extractor provides extracts with an artemisinin and scopoletin content eight times higher than the daily therapeutic dose, which should be evaluated for its stability over time and biological properties for possible direct use for therapeutic purposes.

Investigation of the component in Artemisia annua L. leading to enhanced antiplasmodial potency of artemisinin via regulation of its metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: The chemical matrix of the herb Artemisia annua L. (A. annua), from which artemisinin (QHS) is isolated, can enhance both the bioavailability and efficacy of QHS. However, the exact mechanism of this synergism remains unknown. The biotransformation of QHS and potential "enzyme inhibitors" in plant matrix could be of great importance in understanding the improved efficacy of QHS in A. annua, which has been limited to the synergism with flavonoid components. AIM OF THE STUDY: To investigate the component in A. annua extracts (MAE) leading to enhanced antiplasmodial potency of QHS via regulation of its metabolism. The efficacy of QHS in combination with the synergistic component was also evaluated. MATERIALS AND METHODS: The total MAE extract and its three MAE fractions (MAE-I eluted using 3% methanol, MAE-II eluted using 50% methanol and MAE-III eluted using 85% methanol) were obtained from dry plant materials and prepared after lyophilization. The pharmacokinetic profiles of QHS and its major phase I metabolite monohydroxylated artemisinin (QHS-M) were investigated in healthy rats after a single oral administration of QHS in each MAE extract. Major components isolated from the target MAE fraction were evaluated for their enzyme inhibition. The antimalarial activity of QHS in combination with the potential synergistic component against Plasmodium falciparum was studied in vivo (murine Plasmodium yoelii). The recrudescence and survival time of infected mice were also recorded after drug treatment. RESULTS: Compared to pure QHS, a 2-fold increase in QHS exposure (AUC and Cmax) was found in healthy rats after a single oral dose of QHS in the total MAE extract or its fraction MAE-III. In addition, metabolic biotransformation of QHS to the metabolite QHS-M (mediated by CYP3A) was inhibited by MAE or MAE-III. Among nine major components isolated from MAE-III (five sesquiterpenenes, three flavonoids and one phenolic acid), only arteannuin B (AB) showed an inhibition of CYP3A4 (IC50 1.2µM). The synergism between QHS and AB was supported using in vivo antiplasmodial assay and a pharmacokinetic study in mice. Unfortunately, the synergism cannot reduce the rate of recrudescence. CONCLUSIONS: AB was one of main contributors in A. annua leading to enhanced antiplasmodial potency of QHS via regulation of its metabolism. The final recrudescence indicated the careful use of A. annua for malaria treatment unless additional contributing components or antiplasmodial mechanism were found.

Antiprotozoal Effect of Artemisia indica Extracts and Essential Oil.

Diverse solvent extracts of Artemisia indica leaves originating from the West Bengal region (India) were assessed for the content of artemisinin and characteristic Artemisia polymethoxyflavonoids, namely eupatin (1), casticin (2), chrysoplenetin (3), cirsilineol (4), chrysophenol-D (5), and artemetin (6). HPLC-DAD and HPLC-MS were used to investigate the extracts macerated by solvents of increasing polarity, i.e., petroleum ether, n-hexane, dichloromethane, acetone, MeOH, or EtOH (either 96, 80, or 60 % v/v), and hot water. Artemisinin was absent in all extracts. The acetone and EtOH extracts comprised the highest levels of polymethoxyflavonoids, whereas no flavonoid could be detected in the infusion. None of the remaining extracts contained chryosphenol-D (5) or artemetin (6), while chrysoplenetin (3) was found in all extracts. The essential oil of the plant was also obtained by hydrodistillation and analysed by gas chromatography and gas chromatography-mass spectrometry simultaneously. Of the 92 compounds detected in the oil, camphor (13.0 %) and caryophyllene oxide (10.87 %) were the major components. All solvent extracts and the volatile oil showed in vitro antimalarial activity, plus a potential malaria prophylactic effect by inhibiting at least two recombinant plasmodial fatty acid biosynthesis (PfFAS-II) enzymes. Except for the infusion, all extracts were also active against other parasitic protozoa and displayed low cytotoxicity against mammalian cells. This is the first detailed study investigating both artemisinin and polymethoxyflavonoid content as well as in vitro malaria prophylactic and detailed antiprotozoal potential of A. indica extracts against a panel of protozoan parasites. This is also the first report of antiparasitic activity of the essential oil of the plant.

Influence of Artemisia annua extract derivatives on proliferation, apoptosis and metastasis of osteosarcoma cells.

Regarding the Artemisia annua extract derivatives called dihydroarteminin (DHA) as the object, we studied about its influence to the proliferation, apoptosis and metastasis of human osteosarcoma cells. First, we cultured in vitro the osteosarcoma cell strain and divided them into groups, then detected the cell proliferation, apoptosis and cell metastasis, etc by multiple measurement technique. Finally, we observed the influence of DHA to human osteosarcoma cells. Osteosarcoma cells were all sensitive to DHA, and the appropriate concentration range was 10~40µM. DHA could effectively restrain its protein expression, and there was a significant difference between experimental group and control group. These finding suggest that, the Artemisia annua extract derivatives (DHA) has a biological effect of observably restraining the proliferation and metastasis of human osteosarcoma cells and promoting the tumour cell apoptosis.

The biological characteristics of a novel camptothecin-artesunate conjugate.

A novel conjugate of camptothecin and artesunate (C-Q) was prepared and its cytotoxicity was evaluated using the MTT assay. In addition, the antitumour activity and toxicity of C-Q were investigated in mice, and interaction between transferrin (TF) and C-Q was investigated to evaluate its interaction with biological macromolecules. In the MTT assay, C-Q showed better inhibitory activity against MCF7 breast cancer cells and SMMC-7721 liver cancer cells than camptothecin or artesunate. In vivo, C-Q showed lower toxicity and better antitumour activity compared with camptothecin. Fluorescence spectroscopy showed static quenching of TF in the presence of C-Q, and thermodynamic parameters (ΔH>0 and ΔG<0) indicated that the reaction was spontaneous and endothermic. The main binding force between C-Q and TF was hydrophobic, as indicated by thermodynamic parameters (ΔH>0 and ΔS>0). Thus, synchronous fluorescence spectra showed that C-Q had no influence on the conformation of TF. Our results indicated that C-Q represents a novel potential anticancer therapeutic vector with advantages over current methods of CPT and ART administration. This novel drug delivery system allows the use of these drugs in a manner associated with few side effects for normal tissue, and which facilitates synergistic effects of anti-tumour drugs.

[Relationship between artesunate influence on the process of TGF-beta1 induced alveolar epithelial cells transform into mesenchymal cells and on idiopathic pulmonary fibrosis].

This study is to investigate the effect of artesunate on transforming growth factor-beta1 (TGF-beta1) induced epithelial-mesenchymal transition (EMT) and its possible mechanism. After the in vitro cultured RLE-6TN cells were treated with TGF-beta1 then artesunate intervened on it, after 24 h, expression of the markers of mesenchymal cell was assayed using Western blotting and real-time PCR analysis. Western blotting was also used to detect the effect of TGF-beta1 on the Smad3 and Smad7 expressions of RLE-6TN cells. Morphological alterations were examined by phase-contrast microscope, and ultrastructure changes by electron microscope. Incubation of RLE-6TN cells with TGF-beta1 resulted in the up-regulation of the expression of the mesenchymal cell markers, after artesunate intervened on it, resulted in the down-regulation of the expression. Meanwhile, incubation with artesunate intervened on RLE-6TN cells could lead to the apparent down-regulation of the expression of Smad3 and up-regulation of Samd7 and the transition of RLE-6TN cells to mesenchymal-like by TGF-beta1 induction, after artesunate intervened on it, RLE-6TN cells to epithelial-like. TGF-beta1 induced epithelial-mesenchymal transition process; artesunate can inhibit TGF-beta1-induced epithelial-mesenchymal transition process, the possible mechanism is up-regulation of the expression of Smad7 and down-regulation of the expression of Smad3, meanwhile inhibits phosphorylation of Smad3.

Antimicrobial activity of artemisinin and precursor derived from in vitro plantlets of Artemisia annua L.

Artemisia annua L., a medicinal herb, produces secondary metabolites with antimicrobial property. In Malaysia due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. In this study, the leaves of three in vitro A. annua L. clones were, extracted and two bioactive compounds, artemisinin and a precursor, were isolated by thin layer chromatography. These compounds were found to be effective in inhibiting the growth of Gram-positive and Gram-negative bacteria but not Candida albicans. Their antimicrobial activity was similar to that of antibactericidal antibiotic streptomycin. They were found to inhibit the growth of the tested microbes at the minimum inhibition concentration of 0.09 mg/mL, and toxicity test using brine shrimp showed that even the low concentration of 0.09 mg/mL was very lethal towards the brine shrimps with 100% mortality rate. This study hence indicated that in vitro cultured plantlets of A. annua can be used as the alternative method for production of artemisinin and its precursor with antimicrobial activities.

Role of traditional antimalarial plants in the battle against the global malaria burden.

Malaria continues to be a major global public health problem with 3.3 billion people at risk in 106 endemic countries. Globally, over 1000 plants have been used as potential antimalarials in resource-poor settings due to fragile health-care systems and lack of accessibility and affordability of artemisinin-based combination therapies (ACTs). Although many believe that the use of medicinal plants that have folklore reputations for antimalarial properties is relatively safe, many herbs may be potentially toxic due to their intrinsic adverse side effects. Therefore, herbal-derived remedies require powerful and deep assessment of their pharmacological qualities to establish their mode of action, safety, quality, and efficacy. In addition, the evolution of drug resistance also demands new antimalarial agents. This can be achieved by forming a vibrant antimalarial discovery pipeline among all stakeholders, including traditional healers, ethnobotanists, scientists, entomologists, pharmacists, and research institutions, for the isolation and characterization of the bioactive compounds with the ultimate objective of finding novel modes of action antimalarial compounds that can be used to fight against drug-resistant malarial parasites.

Is artemisinin the only antiplasmodial compound in the Artemisia annua tea infusion? An in vitro study.

In our ongoing investigation into Artemisia annua for the treatment of malaria, we decided to study the possibility that synergism might enhance the efficacy of artemisinin. Our main objective was to test tea infusions and nonpolar extracts prepared from different A. annua varieties against Plasmodium falciparum in vitro in order to determine if synergism will increase the effectiveness of artemisinin in the samples as compared to pure artemisinin. We found that the IC50 of artemisinin in the tea and nonpolar extracts was not significantly different to the IC50 of pure artemisinin. We could show that the year and country of harvest or storage conditions did not have any influence on the activity and that it narrowly followed the concentration of artemisinin in all the extracts. In conclusion, based on these in vitro results, artemisinin seems to be the only active antiplasmodial compound in A. annua.

NAD(P)H : quinone oxidoreductase 1 inducer activity of some Saudi Arabian medicinal plants.

Medicinal plants are a rich source of biologically-active phytochemicals and have been used in traditional medicine for centuries. Specific phytochemicals and extracts of their plant sources have the ability to reduce the risk for chronic degenerative diseases by induction of enzymes involved in xenobiotic metabolism, many of which also have antioxidant and anti-inflammatory functions. One such multifunctional cytoprotective enzyme is NAD(P)H : quinone oxidoreductase. In this study, we prepared extracts of 27 Saudi Arabian medicinal plants which belong to 18 different plant families and tested their ability to induce NAD(P)H : quinone oxidoreductase in murine hepatoma cells grown in microtiter plate wells. In addition to the Brassicaceae, a known source of NAD(P)H : quinone oxidoreductase inducer activity, we found substantial inducer activity in extracts from the Apiaceae, Apocynaceae, and the Asteraceae families. Five out of a total of eight active extracts are from plants which belong to the Asteraceae family. We further show that artemisinin, an agent which is used clinically for the treatment of malaria, contributes but does not fully account for the inducer activity of the extract of Artemisia monosperma. In contrast to artemisinin, deoxyartemisinin is inactive in this assay, demonstrating the critical role of the endoperoxide moiety of artemisinin for inducer activity. Thus, the NAD(P)H : quinone oxidoreductase inducer activity of extracts of some Saudi Arabian medicinal plants indicates the presence of specific phytochemicals which have the potential to protect against chronic degenerative diseases.

Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents.

A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g(-1) ), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC-MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 µg mL(-1) . Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step-purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high-purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process.

[Determination of artemisinic acid in Artemisia annua at different growth stages based on spot area].

OBJECTIVE: Based on the important medicinal applications of artemisinic acid and the superiority of Thin Layer Chromagraphy (TLC), the spot area method of TLC was presented to determine the content changes of artemisinic acid of Artemisia annua at different growing stages. METHODS: The separation conditions including chromatographic solutions and chromogenic agent were optimized. The detection limit and the linear concentration range were analyzed. And the content changes of artemisinic acid of Artemisia annua at different growing stages were detected. RESULTS: The results showed that artemisinic acid extracted from Artemisia annua could be separated completely by the chromatographic solutions composed by petroleum ether,acetone and ethyl acetate (80: 19: 1). The artemisinic acid was clearly colored using the chromogenic agent consisting by ethanol, bromophenol blue and sulfuric acid. The detection limit of TLC was 0.05 mg/mL. The spot area of TLC had a good linear relationship within the range of 0.05-0.6 mg/mL, accorded with regression equation of y = 11.162 x + 0.0823. The results showed that the content of artemisinic acid at 0.041 mg/g in April which below the detection limit of TLC had no color spot. Contrarily, the spots of artemisinic acid were obvious in materials growing from May to September, and content was about 0.7, 1.2, 2.1, 2.4 and 2.7 mg/g, respectively corresponding to results by HPLC. CONCLUSION: The method can be applied to the quantitative analysis of artemisinic acid in Artemisia annua.

The impact of impurities in various crude A. annua extracts on the analysis of artemisinin by liquid chromatographic methods.

Analysis of Artemisia annua extracts by liquid chromatographic methods has traditionally been complicated by the presence of significant quantities of impurities. It has been observed that these impurities often remain as a solid residue after sample reconstitution, but the possibility of artemisinin remaining entrained within this waxy layer has not been detailed in the literature. This investigation found that A. annua extract impurities have a critical impact on the quantification of artemisinin by liquid chromatographic methods. Extended sample reconstitution times of up to 24h are required in order for the mobile phase (acetonitrile) to penetrate the residue and solubilise the artemisinin contained within. Extracts produced using ethyl acetate, hexane-ethyl acetate (95:5, v/v), hexane and ethanol were examined in the study. Extended residue reconstitution times resulted in a significant increase in the number and concentration of impurities in the mobile phase, requiring the development of a new HPLC-UV analytical method to exact adequate separation of artemisinin for quantification. The solvent selectivity and capacity for each of the solvent extraction approaches was then determined using the new reconstitution and HPLC-UV methods.

[Variation of content of artemisic acid in different growth stages of Artemisia annua].

OBJECTIVE: To study the variation of content of Artemisic acid of Artemisia annua from eight areas of four provinces around Wuling Mountain. METHODS: Artemisic acid of plants were extracted by organic solvent method. Qualitative and quantitative analysis of Artemisic acid were measured by thin layer chromatography (TLC) and high performance thin layer chromatography (HPLC), respectively. RESULTS: The results showed the average levels of Artemisic acid in May and August changed from 0.964 to 2.288 mg/g and from 1.837 to 3.737 mg/g, respectively. The average level in August was 1.5 times as that in May. The Artemisic acid in cultured plants was higher than the levels in wild plants, and Artemisic acid in plant collected below 300 m altitude was higher than that of the plant collected above 300 m altitude. CONCLUSION: The biosynthesis of Artemisic acid depends on the plant growth stage,which is mainly accumulated in plant at the mature stage.

Simultaneous isolation of artemisinin and its precursors from Artemisia annua L. by preparative RP-HPLC.

It is still a major challenge to simultaneously isolate artemisinin and its precursors, especially dihydroartemisinic acid and artemisinic acid, from herbal Artemisia annua. A rapid, economical and automatical chromatographic separation process to isolate and purify artemisinin, dihydroartemisinic acid and artemisinic acid at the same time on a preparative scale was developed. The procedure included solvent extraction of ground Artemisia annua leaves by refluxing and purification of crude extract by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). Fractions containing artemisinin and its precursors were collected and identified by gas chromatography and mass spectrometry. High purity of artemisinin, dihydroartemisinic acid and artemisinic acid was obtained by preparative HPLC with a C(18) column and 60% acetonitrile in water as the mobile phase. The techniques described here are useful tools for the preparative-scale isolation of artemisinin and its precursors in a fast, cost-effective and environmental friendly manner.