Topclass Journal of Herbal Medicine Vol. 2(4) pp. 65-74, 26 April, 2013 Available online at http://www.topclassglobaljournals.

org ISSN 2315-8840 2013 Topclass Global Journals

Submitted 29/01/13 Accepted 04/03/13

Full Length Research Article

Extraction and Identification of alkaloids from wild and In Vitro germinated plants of Datura metel L.
Sabeh D. Al-Utbi1, Niran J. Al-Salihi2*, Suhad A. Al-Kanany1
2

Department of Biology, college of Science, university of Basrah Department of Chemistry, college of Science, university of Basrah

******************************************************************************************************************************* Abstract Plant alkaloids which are one of the largest groups of natural products provide many pharmacologically active compounds. Naked seeds of Datura metel L. were cultured on solid (MS) Murashinge and skoog 0 medium at 27 c for 16 h of light and 8 h of darkness. The seeds then germinated into plants. The extraction of alkaloids was carried out for leaves of In vitro seed germinated plant and wild plants using 95% ethanol ,three alkaloidal compounds (A, B, C) were found and identified from both wild and In vitro germinated plants leaves. Thin layer chromatographic technique(TLC) using BAW(Butanol, acetic acid, water ) shows one spot Rfc= 0.7 for In Vitro leaves and two spots Rfa= 0.9 and Rfb=.98 for wild leaves. Some primary qualitative tests of In vitro and wild Datura leaves were carried out as well. Separation using silica gel G60 emulsion as a stationary phase in glass column and BAW were used as mobile phases, chemical identification of isolated compounds using UV-visible spectrophotometric technique shows maximum absorption at max.=326nm and max.=400nm for compound A and max.=406 nm and max.=660nm for compound B from wild plants ; and at max.=404nm and max.=666nm for compound C from In vitro germinated plants. IR spectrophotometer was used to identify the functional groups in In vitro germinated plants and the wild plants, the results show the full scan of IR spectrum of isolated compounds A, B and C. Finally the results of C.H.N showed there was a good agreement between theoretical and practical values of compounds A and C. Key words: Extraction, In vitro, alkaloids, Datura metel L, Idetification ******************************************************************************************************************************* INTRODUCTION Datura is an annual wild plant belonging to the family solanaceae and it is considered as one of the most important medicinal plants. It is a major source of a variety of alkaloids required for pharmaceutical industries. (Al-Humaid 2003). Datura was regenerated from In vitro cultured embryo (Overbeek et al., 1942); and anther (Gupta and Babbar, 1980). Zayed et al. (2006) found that alkaloid formation is nearly absent or very low in cultured callus and is induced upon differentiation of callus to root and shoots. Robins et al. (1991) stated that obtaining these alkaloids through In vitro culture techniques remains the focus of considerable research. Iranbakhsh et al. (2006) explained that alkaloids production begins at the end of the second week after seed germination, this increases up to the tenth week and then the decreasing occurs later. Al-samaree (1983) found by using (TLC) and (GLC) that hyosine is the main compound in Datura innoxia whereas hyoscyamine was the main compound in both organs of Datura by TLC tests. The aim of this study is to recognize if there is a difference between the extracts of wild plants and in vitro germinated of plants of Datura metel L. and the identification of these extracts.

*Corresponding Author's Email : niranjassim@yahoo.com

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MATERIAL AND METHODS Tissue culture Media preparation 10 ml from each of five groups of Murashige and Skoog (MS) basal salt stock solutions (Murashige and Skoog 1962) were put in a container and supplemented with the following (mg/L): NaH2PO4 [170], Meso-Inositol [100], Sucrose [30000], Thiamine-HCl [0.5], Pyridoxine-HCl [0.5], Glycine [0.5], Glutamic acid [0.5], Nahthalene acetic acid [NAA] [2], Kinetin [1]. The pH was adjusted to 5.7 and then activated charcoal [3000] and agar [7000] were added. After heating, the cooling medium was dispensed o in containers and autoclaved at 121 C for 15 min.
Figure 1. Datura metel plantlets after six-week culture of naked seeds on MS medium

In vitro Culture Establishment Naked seeds of Datura metel were sterilized with 20% clorax bleech (6% sodium hypochlorite) and tween 20 (2 drops) for (15-20) min., successive washings with distilled water were performed for seed before culturing one seed in each tube. The leaves of In Vitro germinated plants of Datura metel L. were air dried at a dark room temperature, grinded by morter and preserved in polyethylene bags. The leaves of Datura metel plants were air dried at a dark room temperature, grinded by electrical mill and preserved in polyethylene bags until use.

Alkaloid extraction Alkaloids were extracted from leaves of each of in vitro seed germinated plant and wild plants according to AlSamaree (1983). 1.050 gm powder of in vitro plants leaves were put in thumble and the soxhlet; extraction was carried out for 24 h by adding 200 ml of 95% ethanol. The extract was dried by rotary evaporator; 30 ml of 2% H2SO4 was added to the ethanol extract and the ethanol was removed by rotary evaporator, 10% ammonium hydroxide was added until the pH reached 9, basic solution was extracted four times by separating funnel using chloroform, 10 ml of chloroform were used at each time. Chloroform extract was collected and dried by adding 10 gm anhydrous sodium sulfate using rotary evaporator. Alkaloid extract was collected and preserved in petridish. Alkaloids from wild plant leaves were extracted by similar method for in vitro germinated plants leaves except that 10 gm of dry weight of wild plants leaves were used. 300 mg of alkaloid extract of In vitro plant leaves were dissolved in 2 ml of BAW (butanol, acetic acid, water). Separation was carried out using column chromatographic technique. Silica gel G60 emulsion was

used as a stationary phase in glass column. (1.550cm) and BAW (4:1:5) were used as mobile phases. Separated compounds were examined by TLC (20202mm) and manifested by Dragendroff`s and Mayer`s reagents, then the compounds of similar Rf were collected in one group (David et al., 2001). Column chromatography was used for wild plants leaves for alkaloid extraction in the same manner. Chemical identification of isolated compounds: UV-VIS spectra were recorded on UV-VIS spectrophotometer SP8-100. IR spectra were recorded on ETIR 8400S spectrophotometer and the C.H.N analysis was determined by using EURO EA Elemental Analyzer.

RESULTS In Vitro Culture Datura metel plantlets were obtained from naked seeds after six weeks culture on solid MS media; plantlets were initiated with tap roots with secondary roots, violet to green-violet stem with branches and simple alternate leaves (Figure1)

Chemical extraction Thin layer chromatographic (TLC), study of alkaloidal extract for both in vitro and wild Datura leaves has been studied; the results in (Table 1, Figures 2 and 3) showed the retardation factor (Rf) values. The Rf showed positive results using Drangendr and Mayer reagent. Two compounds were isolated from wild plant leaves (A and B) whereas one compound was isolated from in vitro germinated plants leaves (c). (Figures 4, 5 and 6) show UV-visible spectrum of the isolated compounds A, B and

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Table 1. TLC of alkaloidal extract of Datura metel L. in vitro germinated plants and wild plants leaves

Extracts Reagent

Alkaloidal extract of in vitro germinated plant leaves 1-spot

Alkaloidal extract of wild plant leaves 2-spots Rf value 0.98 0.94 0.98 0.94

Color

Observation

Dragedroff reagent

Rf value 0.71

Orange

Indicate on alkaloids presence

Mayer reagent

0.71

Marble White

Indicate on alkaloids presence

C. The UV-visible spectrum shows maximum absorption at max.=326nm and max.= 400nm for compound A (n* transition) due to non bonding. The maximum absorption band at max.=406nm and max.=660nm for compound B whereas it was at max.=404nm and max.=666nm for compound C due to non-bonding transition as well. (Figures 7, 8 and 9) show full scan of IR spectrum of the isolated compounds A, B and C. The results of C.H.N. (elementary analysis of isolated compounds A and C show that there is a good agreement between theoretical and practical values as shown in Table 2.

DISCUSSION Tissue culture

Figure 2. Thin layer chromatography (by using Dragendroffs reagent) for alkaloidal extract of Datura metel plant leaves from In Vitro germinated naked seeds on MS medium.

The results showed morphological similarities between in vitro germinated plants and wild plants of Datura metel except some differences in plant size and color intensity of leaves of in vitro germinated plants, these differences are common in tissue culture. The results also showed enhancement of seed germination in vitro by the removal of their coats, this may be owing to hard natural barriers which prevent germination, however Hartman et al (2002) stated that the removal of seed coats involves the removal of natural barriers preventing the germination; Juan et. al. (2006) stated that the difficulties in the propagation of juniper (Juniper oxycedrus) by seeds are due to the hardness of seed coat and their contents of inhibitors. Hajar (1991) pointed out that the hardness of seed coats of hawthorn (Crateagus azarolus) makes them impermeable to water, gasses and other essential requirements for germination. Alkaloids extraction and identification The appearance of one spot Rf = 0.7 for In vitro leaves and two spots Rf a =0.9, Rf b =0.98 for wild leaves by thin

Figure 3. Thin layer chromatography (by using Mayer`s reagent) for alkaloidal extract of Datura metel wild plant leaves

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Figure 4. Ultra violet-visible spectroscopy for isolated compound A

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Figure 5. Ultra violet-visible spectroscopy for isolated compound B

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Figure 6. Ultra violet-visible spectroscopy for isolated compound C

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Figure 7. Infra red spectroscopy for isolated compound A

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Figure 8. Infra red spectroscopy for isolated compound B

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1/cm Figure 9. Infra red spectroscopy for isolated compound C

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Table 2. Elementary analysis of isolated compounds: A (from alkaloidal extract of wild plant leaves and C (from alkaloidal extract of In vitro germinated plants leaves of Datura metel L

Found value Compound a C N% 7.410 8.145 H% 9.245 9.099 C% 58.492 58.304 N% 7.56 7.56

Calculated value H% 8.16 8.16 C% 58.36 58.36

layer chromatography (Table 1), Figures 2 and 3 may reflect the developmental differences between wild and In vitro leaves. (Figures 4, 5 and 6) showed two bands in UV-Visible spectrum of compounds A, B and C which are due to n transition which indicates the presence of double non covalent bonds (Silverstein et. al. 1981). The IR spectra of the isolated compounds A and B indicate -1 that the compounds are aliphatic (freq. at 2923.88 cm belongs to CH3 of aliphatic compounds). The compounds contain NH and OH group and C-N group that belong to cyclic compounds. The elemental analysis showed good agreement between theoretical and practical values of C and N. The molecular weight of A and C was 185.22 gm/mol. The result nearly agreed with De Simone et al. (2008).
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