Extraction of Alkaloids from Powdered Plants

Abstract

The dried and powdered plant material is extracted with pet ether (or hexane) to isolate alkaloids. Many alkaloids may be isolated straight from the alcoholic extract using chromatographic techniques, which works well for tropane alkaloids (atropine, cocaine, and scopolamine) comes first. Fats, oils, terpenes, waxes, and other substances are removed. This extract is thrown away. The substance is now extracted using alcohol, such as methanol or ethanol. To get to the crude alkaloids combination, the extract is evaporated. Using chromatographic techniques, several alkaloids may be extracted directly from the alcoholic extract. Column chromatography on SiO2 separates the alcohol extract into three fractions: solvent chloroform, chloroform with increasing methanol content, and chloroform with decreasing methanol concentration. Lipids and terpenes are separated from the crude alkaloid fraction in this way. To separate the pure alkaloids, the alkaloid fraction is chromatographed once more (SiO2; CHCl3: MeOH = 10:1).

Introduction

Alkaloids are found in plants as combinations of similar chemicals with inert components including tannins, proteins, lipids, resins, and pigments, which make it difficult to isolate them. Preparation of the plant sample, liberation and extraction of the free alkaloidal bases, purification and fractionation of the crude extract, and separation of specific alkaloids are the primary stages used in the isolation of alkaloids (7). Although most alkaloids are insoluble in petroleum ether, the extracted material is carefully dried, reduced to a sufficient size, and defatted if required using petroleum ether (1). Water or aqueous alcohol with dilute acid is used to remove the powdered particles. The salts of alkaloids, as well as any soluble contaminants, are removed (4). To eliminate pigments and other unwanted contaminants, the acidic extract is shaken with chloroform or another appropriate organic solvent (weak bases may be present). To liberate the free bases, the acid extracts (alkaloidal salts) from the procedure are treated with a dilute alkali, such as sodium bicarbonate or ammonia (2). Filtration or extraction with organic solvents separates the free bases.

When it comes to solvent extraction, the choice of solvent is critical. When choosing a solvent, take into account selectivity, solubility, cost, and safety (3). Solvents with polarity values close to the polarity of the solute are likely to perform better, and vice versa, according to the rule of resemblance and intermiscibility (like dissolves like). In solvent extraction for phytochemical research, alcohols (EtOH and MeOH) are ubiquitous solvents. In general, the smaller the particle size, the better the extraction outcome (10). The extraction efficiency will be improved by the tiny particle size due to increased solvent penetration and solute diffusion. However, too small a particle size will result in increased solute absorption in the solid and difficulties in subsequent filtering. Solubility and diffusion are increased at high temperatures.(6) Temperatures that are excessively high, on the other hand, might cause solvents to evaporate, resulting in unwanted impurity extracts and the breakdown of thermolabile components.

A natural product extraction technique has been developed. The novel approach is faster, more efficient, and uses less solvent than previous procedures. Alkaloids are extracted from natural products such as Hyoscyamus muticus, Datura stramonium, and Ruta graveolens using a sonicated solution with a surfactant as an extracting agent (10). Mayer reagent is used to precipitate the alkaloids, which are subsequently dissolved in an alkaline solution and extracted using chloroform. This article compares the findings obtained with those obtained using previous approaches, demonstrating the new method's benefits.

Many alternative techniques for detecting alkaloids in thin-layer chromatography (TLC) have been published, ranging from non-selective to very selective (5). Non-selective techniques for detecting various kinds of organic compounds in TLC, such as UV light quenching on fluorescent plates, iodine vapour or iodine spray reagents, and concentrated sulphuric acid, are generally quite sensitive, with detection limits of less than 1 g. However, other techniques for detecting alkaloids are typically chosen due to the lack of specificity of these reagents (7). In the organic lab, thin-layer chromatography (TLC) is a very useful analytical method. It allows for the fast separation of compounds, giving an indication of the number and type of the mixture's components. TLC can also be used to identify compounds by comparing them to known samples, to evaluate a compound's purity, or to keep track of the progress of a reaction, extraction, or purification operation (9). This experiment will teach you about TLC's mechanics as well as the chemical concepts that behind it.

Method

To 0.6 grams of each powdered plants from the samples of the leaves of datura stramonuim, atropa belladonna and hyoscyamus niger were used in the experiment. 15 millilitres of 0.05 sulphuric acid were added to the powdered plant extracts. The mixture was shaken for 15 minutes. The residue was then filtered and washed with more sulphuric acid until 20 millilitres of the filtrate were obtained. 1ml of concentrated ammonia solution was added to the filtrate which was then transferred to a separating funnel and shaken gently with 10ml of dichloromethane (DCM). The lower layers remaining were run off into a conical flask. The top layer was retained at the separating funnel as another 10ml of DCM was added and shaken. The lower layer was also run into a conical flask while top layer was run down the sink. A sufficient amount of anhydrous sodium sulphate was added to the extract to remove water. The extract was stirred and left to settle for 5 minutes. The supernatant liquid was decanted into an evaporating basin leaving the solid materials in the conical flask and evaporated to dryness a fume cupboard. The material was allowed to cool before 0.5ml of methanol was added to the residue.

Where DS – Datura Stramonium

AB- Atropa Belladonna

HN-hyoscyamus Niger

Results and Discussion

TLC plates pre-coated with aluminium oxide F-254 (Type E) with a layer thickness of 0.25 mm, produced in Germany, were used for all thin-layer chromatographic analyses (4). The active chemicals in the extracts were separated using a TLC plate (F-254, type E. The presence of atropine and hyoscine was found in the crude extracts of each powdered plant from samples of the leaves of Datura Stramonuim, Atropa Belladonna, and Hyoscyamus Niger, according to the results of phytochemical screening tests done on the crude extracts of each powdered plant.

Distance travelled by spots

A-Atropine

H- Hyoscine

Plant extracts were analysed by Vitali Morin test TLC, which were eluted with a chloroform/ethanol (A) solvent combination and stained with Dragendorff reagent, revealing orange and purple spots. Atropine was found in the leave extracts of DS (1.9) and AB(1.9) while hyoscine was found in HN(3.9) and DS (3.9). The presence of alkaloids in the leaves of datura stramonuim, atropa belladonna, and hyoscyamus niger was confirmed by these observations. As a result, more research is required to isolate these potent chemicals, clarify their chemical structures, and demonstrate their bio efficacy.

Colour after addition of dragendorff’s reagent

Colour of the spots after vitali-Morin test

Ratio of the distance moved by the spots

DS : RF= 1.9/5.2 = 0.37

DS : RF= 3.9/5.2= 0.75

AB: RF= 1.9/5.2 = 0. 37

HN: RF= 3.9/5.2 = 0.75

The findings of this investigation demonstrate that alkaloids are present in all three extracts. The results of the study show that this approach is more efficient, with the alkaloids recovered having qualitative features that are similar to those obtained using traditional extraction methods (8). The approach is a viable option that can most likely be developed to include other secondary metabolites (essential oils and polyphenols are in the course of study). Optimization of parameters such as temperature, type of surfactants, volume and concentration of surfactant solution will be required to make this approach more general.

Conclusion

The extracts were put through a qualitative chemical examination to see whether there were any secondary metabolites or active principles present. Alkaloids were discovered in the extracts. The plant matter is then dried and pulverized. The solvent distillation method is used to extract the ethanol. The extract is then purified using a variety of techniques. For chemical testing, however, crude extract can be utilized for qualitative detection. High performance liquid chromatography, or planar chromatography, is commonly used to separate and determine chemicals in multicomponent mixtures, such as plant extracts. In both approaches, sample complicity frequently entails the use of a two-dimensional system. The primary to compound identification in 2D-HPLC is the comparison of solute and standard retentions obtained during two separate analyses. Similarly, two chromatographic plates are used to separate the spots of two mixtures (plant extract and standards) in 2D-TLC. The sample components are identified by comparing their retardation factor (RF) values to the separated standards' values.

Looking for further insights on Immune System: Innate vs. Adaptive Defense Mechanisms? Click here.

References

1. Belwal T, Ezzat SM, Rastrelli L, Bhatt ID, Daglia M, Baldi A, Devkota HP, Orhan IE, Patra JK, Das G, Anandharamakrishnan C. A critical analysis of extraction techniques used for botanicals: Trends, priorities, industrial uses and optimization strategies. TrAC Trends in Analytical Chemistry. 2018 Mar 1;100:82-102.

2. Bribi N. Pharmacological activity of alkaloids: a review. Asian Journal of Botany. 2018;1(1):6.

3. Ciechomska M, Woźniakiewicz M, Nowak J, Świadek K, Bazylewicz B, Kościelniak P. Development of a microwave-assisted extraction of atropine and scopolamine from Solanaceae family plants followed by a QuEChERS cleanup procedure. Journal of Liquid Chromatography & Related Technologies. 2016 Jul 2;39(11):538-48.

4. Dong B, Tang J, Yonannes A, Yao S. Hexafluorophosphate salts with tropine-type cations in the extraction of alkaloids with the same nucleus from radix physochlainae. RSC advances. 2018;8(1):262-77.

5. Jiang ZM, Wang LJ, Gao Z, Zhuang B, Yin Q, Liu EH. Green and efficient extraction of different types of bioactive alkaloids using deep eutectic solvents. Microchemical Journal. 2019 Mar 1;145:345-53.

6. Rosales PF, Bordin GS, Gower AE, Moura S. Indole alkaloids: 2012 until now, highlighting the new chemical structures and biological activities. Fitoterapia. 2020 Jun 1;143:104558.

7. Roy A. A review on the alkaloids an important therapeutic compound from plants. IJPB. 2017;3(2):1-9.

8. Sharma A, Kamble SH, León F, Chear NJ, King TI, Berthold EC, Ramanathan S, McCurdy CR, Avery BA. Simultaneous quantification of ten key Kratom alkaloids in Mitragyna speciosa leaf extracts and commercial products by ultra‐performance liquid chromatography− tandem mass spectrometry. Drug testing and analysis. 2019 Aug;11(8):1162-71.

9. Takla SS, Shawky E, Hammoda HM, Darwish FA. Green techniques in comparison to conventional ones in the extraction of Amaryllidaceae alkaloids: Best solvents selection and parameters optimization. Journal of Chromatography A. 2018 Sep 14;1567:99-110.

10. Wang Z, Kang D, Jia X, Zhang H, Guo J, Liu C, Meng Q, Liu W. Analysis of alkaloids from Peganum harmala L. sequential extracts by liquid chromatography coupled to ion mobility spectrometry. Journal of Chromatography B. 2018 Oct 1;1096:73-9.