Artemisinin, an endoperoxide sesquiterpene lactone has proven effective in treating drug resistant cases of malaria and cancer. Artemisia annua [sweet wormwood] is the sole source for artemisinin production in many countries. To counter the low content in leaves and costly chemical synthesis process in India, alternative ways to produce artemisinin have been sought. In current study, we collected A. pallens, A. japonica and A. nilagirica from Western Ghats of Maharashtra, India and analyzed artemisinin content. Samples were extracted from leaves and florets in various extraction conditions and analyzed using different chromatographic techniques. Thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) analysis showed the presence of compound with endoperoxide linkage in A. nilagirica. High performance liquid chromatography (HPLC) analysis showed the detection of artemisinin in methylene dichloride florets extract of A. japonica, but the concentration was too low [1.3 mg/g dry wt.] for further analyses. Gas chromatography/mass spectrometry (GC/MS) analysis identified structurally important components in the A. nilagirica ethyl acetate extract which explores the biosynthetic pathway of artemisinin from its most important precursor amorpha-4,11-diene. This is the first report of chromatographic screening of these Indian varieties of Artemisia spp. for artemisinin content.
Artemisia annua (family Asteraceae) commonly called Quinghao, sweet wormwood, is an annual herb native to China [
Several studies in tissue culture have shown potential and convenience of using roots transformed by Agrobacterium rhizogenes for the biosynthesis of secondary metabolites in numerous species [
A. annua plant still remains sole source for artemisinin and relatively low yield in available germplasms became serious limitation in large-scale production of artemisinin [
Two different extract were prepared to assess the artemisinin detection. In the Hexane extract of plants, no spots were found to be related to standard spot (Rf 0.63) of artemisinin. However, TLC analysis of dichloromethane leaves extract of A. pallens and A. japonica showed Rf of 0.80 and 0.78 respectively with a very faint pink spot and leaves extract of A. nilagirica showed intense pink spot with Rf value of 0.38 (
Sample | Kolhapur | Panchgani | Lonavala | ||
---|---|---|---|---|---|
A. pallens | A. nilagirica | A. japonica | A. nilagirica | A. nilagirica | |
Leaves | 0.80 | 0.41 | 0.78 | 0.38 | 0.39 |
Florets | 0.78 | 0.53 | 0.80 | 0.47 | 0.50 |
For HPTLC analysis, Toluene extract was prepared for the solvent extraction. The Rf value of standard obtained was 0.60 units. The intensity of fluorescent pink band in spiked samples was found to be more intense in comparison to normal plant extracts (
Artemisinin detection in collected Artemisia species was found to be difficult and results are shown in
GC-MS analysis of methanol extract and ethyl acetate extract revealed the presence of structures mainly components of essential oil in Artemisia species.
The pathway reported for amorpha-4,11-diene by Geoffery (2010) and Edward et al. (2000) was reinvestigated [
Sample | Kolhapur | Panchgani | Lonavala | ||
---|---|---|---|---|---|
A. pallens | A. nilagirica | A. japonica | A. nilagirica | A. nilagirica | |
Leaves | 19.0 | 18.7 | 18.4 | 18.0 | 18.7 |
Florets | 19.1 | 18.7 | 18.3 | 18.9 | 18.7 |
A. Japonica (Panchagani) | Retention Time | Area | Concentration (mg/g) |
---|---|---|---|
Leaves | 18.4 | 660720 | 1.3 |
Florets | 18.3 | 192926 | 0.4205 |
Sr. No. | Name of the compound | Mol. wt | Structure | Hit | ||||
---|---|---|---|---|---|---|---|---|
1 | Cyclohexanecarboxylic acid, methyl ester | 142 | ||||||
2 | Ethyl 2-cyclopentanone | 152 | ||||||
3 | 3,4-Dihydro-2H-pyran- 2-carboxylic acid | 128 | ||||||
4 | 2-Hydroxy-5-imidazolic acid, ethyl ester | 156 | ||||||
5 | Isoaromadendrene epoxide | 220 | ||||||
6 | Epiglobulol | 222 | ||||||
7 | Globulol | 222 | ||||||
8 | Thunbergol | 290 | ||
---|---|---|---|---|
9 | Indan, 3a,4,5,6,7,7a-beta- hexahydro-2alpha-isopropyl | 208 | ||
10 | 1-Buten-3-one, 1-(1-acetyl-5, 5-dimethylcyclopentyl)- | 208 | ||
11 | 2,4,4-Trimethyl-3- (3-methylbutyl) cyclohex-2-enone | 208 | ||
12 | Deoxyartemisinin | 266 |
formed cation (C-7 bisabolyl) is deprotonated to an unknown intermediate which further results into C-1 bisabo- lylcation after a 1,3-hydride shift. This cation with positive charge at C1 promotes a nucleophillic attack by the double bond C10-C11, thus second ring closes to form a C-11 amorphanecation. Finally, deprotonation on C12 or C13 gives γ-muurolene.
The work on Germacrene D and γ-Muurolene by Setzer (2008) and Yoshihara et al. (1969) is reinvestigated [
Besides, highly conserved DDxxD sequence found in all terpene synthases, additional DDxxD motif in γ- humulene synthase (residues 487 - 491) employs multiple product formation resulting from diphosphate binding at these conserved sites [
Sr. No. | Name | Mol. Wt. | Structure | Hit |
---|---|---|---|---|
1. | Cis-verbenol | 152 | ||
2 | d-verbenol | 152 | ||
3 | 2,6-Dimethyl- 3,5,7-octatriene-2-ol | 152 | ||
4 | Caryophyllene | 204 | ||
5 | Beta-sesquiphelandrene | 204 | ||
6 | (Z)-beta-farnesene | 204 | ||
7 | Cedrene | 204 | ||
8 | Germacrene D | 204 | ||
9. | Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-7- methyl-4-methylene-1- (1-methylethyl)-, (1.alpha.,4a.alpha.,8a.alpha.) | 204 | ||
10. | Caryophyllene oxide | 220 | ||
11 | Lanceol | 220 | ||
12 | Spathulenol | 220 | ||
13 | (-)-spathulenol | 220 | ||
14. | Eudesmol | 222 | ||
15. | n-hexadeconic acid | 256 | ||
16. | n-ecosanoic acid | 312 | ||
17. | 5,6,6-Trimethyl-5- (3-oxobut-1-enyl)-1-oxaspiro [2.5]octan-4-one | 236 |
18. | Beta-carotene | 536 | ||
---|---|---|---|---|
19. | Provitam-in D4 | 398 |
charged C11 in Germacrylcation forces a 1,3-hydride shift to give another Germacrylcation leaving a positive charge at C1 atom. Through a nucleophillic attack on C1 atom by C5-C6 double bond promotes second ring closure to form Muurolenylcation. Further, formed cation forces a proton at C7 position out of the structure to give γ-muurolene.
This study reported screening of new plant sources of artemisinin from different regions of India. The content of artemisinin in A. japonica was significantly lower when compared to A. annua. Further, elicitors can be used to increase the concentration of artemisinin in tissue cultures of A. japonica. Structures present in ethyl acetate extract of A. nilagirica explore the biosynthetic mechanism involved in artemisinin formation from its precursor amorpha, 4,11-diene. The pathway can be further analyzed to identify any possible intermediate or derivative of artemisinin in A. nilagirica.
The species were collected from different regions in Western Ghats of Maharashtra. A. pallens was collected from Shikharshinganapur, Satara and Jyotiba hill, Kolhapur. A. nilagirica was collected from Panhala fort (Kolhapur), Panchgani (Mahabaleshwar) and Pawana hill (Lonavala). Furthermore, Artemisia japonica was collected from Panchgani (Mahabaleshwar). The species were authenticated by taxonomist at Department of Botany, Shivaji University, Kolhapur. The samples were air dried at room temperature in well ventilated room.
Artemisinin (98% pure) was obtained from Sigma-Aldrich and dissolved in acetonitrile at 1 mg/ml concentration.
Plant material (1 gm) of respective species was weighed and crushed to fine powdered using liquid nitrogen. Two different extracts were prepared in 20 ml of hexane and dichloromethane separately. The mixture of powdered plant material and respective solvents was incubated for 6 - 7 h in shaker at 150 RPM at room temperature. After incubation, 2 ml of filtrate was lyophilized in water bath at 80˚C - 90˚C. Acetonitrile was added and ultra-sonicated for 2 - 3 seconds (80 watt, 220 V AC). Finally, filtrate was centrifuged at 10,000 rpm and supernatant was used as test solution.
The samples were applied to the TLC Si 60 F254 , 10 × 10 cm (Merck) plate as a 5 mm spot or 10 mm band, 1 cm apart at 1 cm height from the lower edge of the plate. After complete air drying, plate was placed in a TLC chamber saturated with mobile phase as Hexane:Diethylether [1:1], and allowed to run till the front of mobile phase reached to the upper edge of the plate. After running plate was air dried and dipped into anisaldehyde developing agent for 10 - 15 seconds (anisaldehyde developing agent was prepared by slowly adding 9 ml of 98% sulphuric acid to an ice cooled mixture of 85 ml of methanol and 10 ml of glacial acetic acid). Plate was air dried and incubated at 100˚C for 5 - 7 min for spot development and examined visually.
Dried plant material (200 mg) was mixed with 10 ml of toluene and sonicated for 10 min. Mixture was centrifuged at 10,000 rpm, and supernatant was used as test solution for screening.
The samples (20 µl) were applied by automated injection system on HPTLC Si 60 F254, 10 × 10 cm (Merck) plate and air dried. Plate was placed in a chromatographic chamber saturated with Cyclohexane, Ethyl acetate, Acetic acid [20:10:1] and allowed to run till the front of mobile phase reaches at 3/4th height of the TLC plate. After air drying, plate was developed using anisaldehyde reagent by immersing for 10 - 15 s. Then plate was air dried and heated to 100˚C for 5 - 7 min and examined under Camag Scanner 4.
Different HPLC systems were used to analyze artemisinin in different plant extracts. Waters HPLC (model 2487), using a hypersil C18 reversed phase column 15 cm with 5 µ particle size and Agilent 1200 series, using zorbax SB-C18, 250 mm × 4.6 mm, 5 µm, prepacked column. A constant rate of 1 ml/min was used with two mobile phases: [A] acetonitrile: water [50:50] and solvent [B] acetonitrile. The elution was performed employing a program from [A]-100% [t = 0 min], 2% [t = 30 - 40 min], to [B]-0% in a period of 60 minute and detected at 211 nm [UV-VIS]. The retention time of sample peak obtained was compared with that of standard.
Prepared standard (1 mg/ml) was completely evaporated and dissolved in 1 ml of methanol.
Air dried leaves (1 gm) were powdered using liquid nitrogen. Two extracts were prepared in 20 ml of methanol and ethyl acetate and incubated overnight at room temperature. This mixture was centrifuged at 5000 rpm for 10 min and supernatant was collected. The supernatant was treated with activated charcoal and centrifuged at 12,000 rpm for 10 min. The supernatant obtained was filtered through 0.22 µ filter and used for analysis.
Analysis was performed on Agilent 5973 Mass Selective Detector coupled to anagilent 6890 N Gas Chromatograph using G1701 MSD Chemstation software and equipped with a 30 M × 0.25 mm DB-5MS column with 0.25 µm film thickness. The chromatograph conditions were a split injection [20:1] onto the column using a helium flow of 0.4 ml/min and temperature programmed with either initial temperature of 70˚C for 1 min or temperature ramped at 10˚C/min to 300˚C and held for 10 min. The mass selective detector was run under standard EI+ conditions scanning on effective mass ranging from 40 to 1000 at 2.26 scan/second.
We thank Dr. Sandeep Kale for providing HPLC facility at DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai, India. HPTLC and GC-MS facility was provided by Prof. S.R.Yadave at Department of Botany, Shivaji University, Kolhapur, India.