L-Dopa Extraction and Analytical Determination in Plant Matrices

L-Dopa Extraction and Analytical Determination in Plant Matrices: Comparison

Please note this is a comparison between Version 2 by Peter Tang and Version 1 by Rosanna Ciriello.

L-dopa is a precursor of dopamine used as the most effective symptomatic drug treatment for Parkinson’s disease. Most of the L-dopa isolated is either synthesized chemically or from natural sources, but only some plants belonging to the Fabaceae family contain significant amounts of L-dopa. Due to its low stability, the unambiguous determination of L-dopa in plant matrices requires appropriate technologies. Several analytical methods have been developed for the determination of L-dopa in different plants. The most used for quantification of L-dopa are mainly based on capillary electrophoresis or chromatographic methods, i.e., high-performance liquid chromatography (HPLC), coupled to ultraviolet-visible or mass spectrometric detection. HPLC is most often used.

levodopa
plant matrices
extraction
chromatographic methods

1. Introduction

L-Dopa or levodopa (LD) is an amino acid analogue belonging to the class of catecholamine compounds. It is a precursor of dopamine (DP) and norepinephrine that act as neurotransmitters in brain areas related to psychomotor and emotional functions. LD is currently considered the most effective oral dopaminergic treatment for the main motor symptoms of Parkinson’s disease (PD). This latter is the most widespread neurodegenerative movement disorder in the world: only Europe has a prevalence rate of around 108–257/100,000 and an incidence rate of 11–19/100,000 per year [1,2]^[1][2]. PD arises when the substantia nigra neuronal cells die and cannot biosynthesize dopamine (DA), a fundamental neurotransmitter, as it plays an essential role in physiological motor control. The symptoms of PD can be kept under control with strategies to replace or improve dopamine ^[1]. The LD pharmacological treatment is based on its replacement for DP to increase its bioavailability at the peripheral synaptic level, where the LD is decarboxylated to DP because of the amino acid aromatic decarboxylase (AADC) enzyme ^[3]. The pharmacological efficacy decreases after a certain period of intake; serious side effects such as motor fluctuations (commonly called on-off phenomenon), orthostatic hypotension, hallucinations and dyskinesias occur after a half-life time t1/2 of 50 to 90 min. These reasons led to the development of extended-release LD formulations, combined with other drugs, to extend the half-life and bioavailability and reduce side effects [2,4,5,6,7,8]^{[2][4][5][6][7][8]}.

The LD drug is chemically synthesized through a process that requires a costly metal catalyst and advanced technologies ^[9]. There are also natural sources, and the production of LD from different plants has advantages compared to chemical methods, such as a pure enantiomerically compound and low-cost approach. LD from natural sources also reduces the secondary effects and helps slow the disease’s progression. Some plants belonging to the Fabaceae family naturally contain significant amounts of LD ^[10]. Among these, the genus Mucuna includes the highest concentration of LD, which explains its widespread use in the management of Parkinson disease. The Mucuna pruriens is the most considered, containing up to 10% of LD in its seeds [11,12]^[11][12]. However, the seeds are covered by stinging hairs, and the beans contain elevated levels of tryptamines which may cause hallucinations in humans, so other plant matrices as a natural source of LD are also investigated. The control of crucial human body functions can be affected by a lack or excess of LD and its metabolites. Consequently, it is necessary to monitor the concentration of LD in all plant matrices destined for human consumption.

LD’s low molecular weight and polar nature generally make its determination by reversed phase liquid chromatography challenging. A possible solution is to use an ion pair reagent to increase retention time. In general, it is necessary to work below the pK_a of the compound, where it will be protonated and not charged, and to decrease the organic content of the mobile phase ^[13]. In addition, LD aqueous solutions are unstable and degrade naturally over time, so the extraction procedure also requires special attention ^[13].

1.1. Chemical and Physical Properties

LD structure is characterized by the catechol moiety bonded to the amino acid functionality (-CH₂NH₂COOH) in -meta and -para positions to the hydroxyl groups in positions 3 and 4, respectively (Figure 1). The main chemical and physical properties are summarized in Figure 1.

Figure 1.

L-Dopa, (3,4-dihydroxyphenyl)-L-alanine, structure and its chemical and physical properties.

Furthermore, LD has three ionizable groups (Figure 2). When the pH value is on average between pK_a1 = 2.3 and pK_a2 = 8.11, LD is present as zwitterion that forms a network of intermolecular bonds where the protonated amine groups and the deprotonated carboxylic acid groups are linked. For this reason, LD is not very soluble in this pH range (LD solubility in water is 3.3 g/L), and acids are required to prepare aqueous solutions. This point is especially crucial regarding LD pharmacological bioavailability along the gastrointestinal lumen as well [14,15]^[14][15].

Figure 2.

Ionization of L-Dopa at various pH values.

1.2. Biosynthesis and Conversion Routes of Levodopa in Plants

Plants produce hundreds of non-protein amino acids, among which LD, a secondary metabolite belonging to the class of catecholamines. Metabolism refers to the whole regulatory aspects implied in the biosynthesis of functional compounds, generally called metabolites. Metabolism in plants can be primary or secondary: primary metabolism generates all the essential compounds for the organism’s growth (primary metabolites); secondary metabolism produces all compounds that are considered not essential for the organism’s growth (secondary metabolites) but are equally important since directly involved in the interaction with the external environment [16,17]^[16][17]. Among catecholamines, norepinephrine (NE), epinephrine (EP), dopamine (DP) and normetanephrine (NMP) are other secondary metabolites whose structures are shown in Figure 3.

Figure 3.

Norepinephrine (NE), epinephrine (EP), dopamine (DP) and normetanephrine (NMP) structures.

Shikimic acid pathway is the most important metabolic pathway of secondary metabolites in plants, and it represents the plant’s biosynthetic key for L-tyrosine [16,18]^[16][18]. Like in mammals, L-tyrosine is involved in plants as a precursor for the catecholamines’ biosynthetic pathway.

L-tyrosine can be converted into LD by tyrosine hydroxylase, or in tyramine upon decarboxylation of the same substrate. DP can derive both from tyramine hydroxylation and L-dopa decarboxylation (Figure 4) [16,19]^[16][19]. This last synthetic route has been reported in plants such as Cytisus scoparius Scottish broom, Monostroma fuscum marine alga, Lophophora williamsii peyote cactus and Portulaca callus [19,20]^[19][20].

Figure 4.

Plant catecholamine synthesis pathway.

In plant organisms, LD plays an important role as a precursor for other classes of compounds. LD can be converted by 4,5-DOPA dioxygenase into betalamic acid, which is a key compound in the biosynthesis of betalaines, red-purple and yellow pigments found in plants of the order Caryophyllales and two genera of fungi: Amanita and Hygrocybe [21,22,23]^[21][22][23]. Furthermore, it can be oxidized toward melanin: at first, LD is oxidized to dopaquinone by the enzyme polyphenol oxidase (PPO), and then it is metabolized to melanin by the plant lipoxygenase [18,19,24]^[18][19][24]. LD also represents a key precursor in the biosynthesis of benzylisoquinoline alkaloids, involved in specific plants (like basal eudicots in the order Ranunculales) for defense against herbivores and pathogens ^[21]. As far as the defense is concerned, in some legumes (e.g., Mucuna pruriens), LD plays an important role as an allelopathic compound that is exuded from the roots in order to inhibit the growth of surrounding plants [18,21,24,25]^{[18][21][24][25]}. The catecholamines catabolism in some plant species also involves their methylation: this is the case of the peyote cactus, Lophophora williamsii, in which LD is decarboxylated to dopamine and subsequently leads to the biosynthesis of mescaline, a hallucinogenic alkaloid, through the key compound 4-hydroxy-3-methoxyphenethylamine ^[26].

2. Levodopa Extraction Techniques

As it was previously outlined, extraction from natural products is nowadays considered the method of choice for providing LD over chemical synthesis which is time consuming, requires expensive and harmful chemicals and generates a racemic mixture of LD. This justifies the growing interest in developing an extraction protocol to ensure LD recoveries are as high as possible, to remove interfering endogenous compounds and to be quick, easy and cheap.

In a general workflow, analytes are extracted directly from the plant matrices after undergoing simple pre-treatment steps consisting of homogenization and freeze drying. Homogenization, in particular, was found to be effective in increasing LD concentration in extracts from Mucuna pruriens seeds with respect to the extract obtained without any pre-conditioning (151.5 ± 5.1 µg/g dw vs. 146.0 ± 4.5 µg/g dw) ^[15].

Typical steps within food sample preparation after pre-treatment and extraction generally include clean-up and concentration. To this regard, literature data show that in the case of plant matrices, LD pre-concentration and clean-up steps are rarely provided. In contrast, for biological samples (e.g., plasma, blood, animal tissues or urine), a sample pre-concentration step or solid phase extraction (SPE) is always required. Such a distinction in sample preparation may be ascribed to the different content of LD in biological and plant samples. For samples containing low levels of LD, like biological ones, pre-concentration and SPE are essential in order to guarantee the minimum levels for analyte detection and quantification [27,28,29,30,31,32,33,34]^{[27][28][29][30][31][32][33][34]}. On the other hand, plant samples mainly involved in LD extraction studies, e.g., different varieties of Mucuna pruriens seeds and Vicia faba broad beans, are rich in this analyte. To get an idea of the LD content in plants, an average concentration of 4.96 and 4.39 g/100 g were estimated, respectively, in white and black variety of Mucuna pruriens seeds ^[35], whereas an average concentration of 7.68 mg/g dw was found in Vicia faba seeds ^[36]).

The extraction techniques used for LD, whose specifications are reported in Table 1, range from the traditional liquid–solid extraction (LSE), Soxhlet extraction, maceration extraction and reflux extraction to the latest and less used microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). The last two techniques involve substantial improvements in terms of automation and solvent consumption so to be referred as green techniques.

Regardless of the particular extraction technique used, in all cases the transferring of LD from the solid plant matrix into the extracting liquid phase revealed to be highly dependent on the pH: the extracting solutions used are all acidic in order to inhibit the LD oxidation process and avoid the formation of its zwitterionic form, which is poorly soluble. Controversies arise about the appropriateness of using solutions slightly acidified with acid acetic/formic acid or strongly acidified with hydrochloric/perchloric acid. The use of mineral or concentrated organic acids for L-dopa extraction is surely efficient but it is limited by the requirement of costly and energy-demanding downstream processes. The potential degradation of L-dopa in a strongly acidic environment is also to be considered. Acids of moderate strength meet the need to find more sustainable solvents even if they could be less efficient in preserving LD stability towards oxidation or formation of aggregated structures.

While the pH strongly influences the extraction yield, on the other hand, the sample ionic strength does not seem to affect the extraction process. IUPAC defines the salting-out effect as “the addition of particular electrolytes to an aqueous phase in order to increase the distribution ratio of a particular solute” ^[37]. It is usually exploited to improve the extraction efficiency (as it generally occurs for solid-phase microextraction SPME), but in the case of LD extraction from plant matrices it seemed to play no role.

Table 1.

Overview of the methods used for LD extraction occurring in plant samples.

Extraction Technique	Matrix	Variety

3. Levodopa Detection Methods

In the last 20 years, many attractive papers have been published showing the optimized techniques used to identify and quantify the LD in plant matrices (Table 2). Most of these require a preliminary separation step, and the HPLC is most often used; others allow a direct detection of the analyte.

Table 2.

Analytical methods and operation conditions employed in the last twenty years for the LD detection with their main strengths and drawbacks.

Methods	Sample Source	LOD Range	Solvents and Optimized Conditions	Stationary Phase Recovery Percent (Mean Value Percent)	References
	Mobile Phase			Detection Mode		Strengths	Drawbacks	References
Solid–liquid extraction (LSE)	Mucuna pruriens dehulled and whole seed	White and black var. utilis	HCl 0.1 M; solvent: sample ratio 100:1 (v/w
	HPLC-UV	Broad bean, cocoa and beans	),	10 ng/mL–15 µg/mL	extraction time 2 × (30 sec under homogenization and 1 h under stirring); extraction temperature 22 °C	101.8%		RP-C18 (mean particle diameter 5 µm, 125 × 3 mm I.D.)	Solvent (A): acetate buffer, pH = 4.66; solvent (B): methanol ^[35]
			Photodiode array detector (DAD)				It is highly reproducible, rapid and efficient				Sensitivity is rather limited so it is suitable for plant matrices with medium and high concentrations of LD. Selectivity is also limited since it does not allow the unambiguous identification of structurally similar molecules.	^[38]	Broad bean, cocoa and beans
												Mucuna pruriens dehulled and whole seed	//		Solvent (A): water/methanol/phosphoric acid 975.5:19.5:1 (v/v/v), pH = 2.0; solvent (B): 70% methanol. HClO₄ 0.2 M, solvent: sample ratio 5:1 (v/w), extraction time 24 h under shaking time for time; extraction temperature 25 °C	^[35] Within-day 84.4–96.0% Between-day 84.0–83.1%	^[38]
												Vicia faba seeds (cotyledons and embryo axis)
												Vicia faba seeds (cotyledons and embryo axis)	var. Alameda var. Brocal	HClO₄ 0.83 mol/kg;	Ammonium phosphate buffer (0.05 mol/kg, pH = 2.0) solvent: sample ratio 100:1 (v/w); extraction time 1 min under homogenization; extraction temperature 4 °C	^[25 //	^] ^[25]
												Vicia faba broad beans
												Vicia faba broad beans	Iambola, San Francesco, FV5, Cegliese, Extra-early purple and Aguadulce supersimonia		Water (H₂O) and acetonitrile (ACN) both containing 0.1% (v/v%) acid formic 5% w/v HClO₄ solution; solvent: sample ratio 10:1 (v/w); extraction time 5 min under homogenization; extraction temperature 4 °C	^[39] //	^[39]
												Vicia Faba roots, sprouts and seeds
												Vicia faba	//	roots, sprouts, seedling, leaf, flower, pod, stem Formic acid:ethanol (1:1 v/v); solvent: sample ratio (10–40):1 (v/w); extraction time 5 × 120 min at 120 rpm; extraction temperature 4 °C	94.1–116.6%		Solvent (A): 0.1% acetic (98%); ^[40]
												Solvent (B): methanol (2%).		^[43]	Mucuna pruriens seed cooked and raw
												Vicia faba sprouts	//	Solvent (A): 82% buffer solution (32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM Na₂EDTA, 0.215 mM octyl sulphate pH = 4); Solvent (B): 18% methanol Water; solvent: sample ratio 400:1 (v/w); extraction time 20 min under stirring; extraction temperature 25 °C	^[44 //	^] ^[41]
												Avena sativa seeds	GK Iringo, GK Kormorán and GK Zalán
Mucuna and													Aqueous solution of 0.1% (m/v) ascorbic acid and 1% (v/v) MeOH; solvent: sample ratio 6:1 (v/w); extraction time 5 h under shaking; extraction temperature 25 °C	95.2–99.6%	^[42]
Vicia Faba roots, sprouts, leaf, seediling, pod, flower, stem								//				Ethanol solution 95% (v/v); solvent: sample ratio //; extraction time 72 h in freezer; extraction temperature −18 °C	//	^[43]
Stizolobium pruriens seed								Solvent (A): 0.1 N acetic acid (90%); Solvent (B): methanol (10%)				^[45]	Vicia Faba
Mucuna pruriens seed								Solvent (A): 0.1% formic acid (98%); Solvent (B): methanol (2%)				^[48]	sprouts	//	Ethanol solution 95% (v/
Vicia faba flowers, fruits and leaves	Solution of 50 mM potassium dihydrogen phosphate (pH = 2.3)			v); solvent: sample ratio //;				^[36] extraction time 48–72 h; extraction temperature −18 °C				//	^[44]
Mucuna and Stizolobium pruriens seed	M. sempervirens, M. birdwoodiana, M. macrocarpa, M. interrupta, M. paohwashanica, Stizolobium pruriens var. pruriens, S. pruriens var. utilis
Vicia faba sprouts, leaves, flowers, pods, roots	Solvent (A): water with 0.3% formic acid; Solvent (B): acetonitrile with 0.3% formic acid	HCl 0.1 M; solvent: sample ratio 20:1 (v/w); extraction time 2 × 5–10 min; extraction temperature 100 °C with a steam bath.	//	^[51] ^[45]
Mucuna pruriens seed	//	0.1 M phosphate-buffered solution (pH = 7.0);
Vicia faba seeds	Solvent (A): 97% v/v of an aqueous solution of 0.2% v/v acetic acid; Solvent (B): 3% v/v methanol	solvent: sample ratio 5000:1( v/w); extraction time 5 h;	^[ extraction temperature 25 °C under stirring.	^15] 99.35%	^[46]
	Mucuna pruriens leaves	//			^[46]
M. pruriens seeds	Water, methanol and acetonitrile (5:3:2) containing 0.2% triethylamine, pH = 3.3	0.1 M phosphate-buffered solution (pH = 7.0);	^[54 solvent: sample ratio 500:1 (v/w); extraction time 5 h; extraction temperature 25 °C under stirring	^] 98.30%
Mucuna pruriens seed
Mucuna pruriens powder formulation	//	Solvent (A): water 80% v/v; Solvent (B): methanol 20% v/v Citric acid 58% (wt%); solvent: sample ratio 7:1; extraction time 90 min; extraction temperature 60 °C	80–84%	^[	^[47]
⁵⁷	^]		Ultrasound-assisted solvent extraction (UASE)	Mucuna pruriens seed	Arka Dhanwantri	Water acidified with 0.1 M HCl (pH: 2.6);
Mucuna pruriens powder and extracts		solvent: sample ratio 10:1 (			v /		RP-C18 (mean particle diameter 5 µm)	w	Water: Methanol: Acetonitrile (100:60:40) containing 0.2% Triethylamine, pH = 3.3	); frequency 35 kHz; extraction time 5, 10, 15 min; extraction temperature 25 °C.	(5 min) 30.7%	^[55] (10 min) 25.6% (15 min) 31.5%	^[48]
Arka Ashwini
Mucuna sanjappae Seed							(5 min) 29.0% (10 min) 27.7% (15 min) 26.8%
				RP-C18 (250 × 4.6 mm I.D.)		Methanol	^[53]		White		(5 min) 29.3% (10 min) 31.4% (15 min) 30.8%
Brown	(5 min) 23.9% (10 min) 28.7% (15 min) 30.6%
Vicia faba sprouts and seeds	//	MeOH and water mixture (80:20); solvent: sample ratio 1:5 (v/w); frequency //; extraction time 30 min; extraction temperature 25 °C.		//	^[49]

Mucuna utilis seed	RP-C18 (250 × 4.0 mm I.D.)	Solvent (A):0.5% v/v of acetic acid 30%; Solvent (B): methanol 70%		^[59]
LC-MS	Vicia faba roots, sprouts and seeds	18 µg/Kg		RP-C18 (mean particle diameter 2.6 µm, 100 × 4.6 mm I.D.)	Solvent (A): ultrapure water with 0.5% (v/v) formic acid 50%; Solvent (B): methanol 50%	Photo diode array detector (DAD) and triple quadrupole (TQ) mass spectrometer	Robust analytical technique that provides higher sensitivity and selectivity than LC-UV methods. It allows to unambiguously identify the compounds under analysis, through the possibility of fragmentation.	It is a technique susceptible to matrix effects: co-eluting compounds could interfere with the ionization of the analyte under examination. Detection in MRM mode is to be preferred.	^[40]
	Vicia faba roots, sprouts and seeds	0.01 µg/mL		Not reported	Not reported	Photo diode array detector (DAD) and quadrupole-time-of-flight (QTOF)- mass spectrometer			^[49]	Vicia faba flowers, fruits and leaves
	Avena sativa seeds	0.01 µg/mL		//	RP-C18 (mean particle diameter 4 µm, 250 × 2 mm I.D.) Water boiling deionized; solvent: sample ratio 50:1 (v/w); frequency //; extraction time 15 min; extraction temperature 100 °C.	Solvent (A): solution 0.1% ( 100.32%			v/v) of formic acid (97%); Solvent (B): ACN/MeOH 75/25 containing 0.1% (v/v) formic acid (3%)	Ion Trap mass spectrometer ^[36]
	^[	⁴²		^]		Vicia Faba seeds	//	HCl 10 mM 5 mL;
HPTLC	M. pruriens seeds	solvent: sample ratio // ( v/w); frequency //; extraction time 2 × 60 min; extraction temperature 25 °C.	Not reported 99.8%		Silica-coated aluminum sheet (10 cm × 10 cm with 0.2 mm thickness)	^[50]	//	HCl 10 mM 5 mL;
	n-butanol,			acetic acid and water were used as mobile phase at 4:1:1		^[50]	UV-Vis thin layer scanner	It makes it possible to obtain a preliminary separation of the analytes in a fast, efficient, easy and low cost analysis	It is generally employed only for qualitative analysis. It is poorly reproducible, as it works in an open system, whose environmental conditions could alter the results.	^[54]	Lens culinaris seeds	105.0%

CE-UV	Vicia faba seeds	LOD value 0.7 µg/mL.	47 cm (40 cm from inlet to the detector) × 75 µm i.d. fused-silica capillary	35 mM NaH₂PO₄, pH = 4.55, 17.5 kV and 30 °C.	Photo diode array detector (DAD)	It allows faster analysis and higher efficiency than LC-UV	It is less sensitive than HPLC-UV	^[50]	Vicia Faba sprouts, leaves, flowers, pods, roots	//
CE-UV	Lens culinaris seeds	LOD value 0.7 µg/mL.		35 mM NaH₂PO₄, pH = 4.55, 17.5 kV and 30 °C.	Photo diode array detector (DAD)	It allows faster analysis and higher efficiency than LC-UV	It is less sensitive than HPLC-UV	^[50]		Aqueous MeOH 50% (v/v); solvent: sample ratio 200:1 (v/w); frequency //; extraction time 30 min; extraction temperature below 40 °C.	//	^[51]
Vicia faba seeds	Bachus, Bolero White, Windsor Bonus, Rambo Amigo, Olga Granit, Albus Fernando, Amulet	Aqueous CH₃COOH 0.2% (v/v); solvent: sample ratio 25:1 (v/w); frequency 40 kHz; extraction time 2 × 20 min; extraction temperature 25 °C.
Electrochemical methods	Mucuna pruriens seed, leaves	LOD value 1.54 µM	//		Working electrode: ^[15]
	gold modified pencil graphite			Supporting electrolyte:	0.1 M phosphate-buffered solution (pH = 7.0)	Differential pulse voltammetry (DPV)	It makes it possible to identify and quantify the analyte in a fast and economical way, through the use of conventional or modified nanostructured electrodes, which permits a better selectivity and sensitivity of analysis.	The technique still shows limitations especially related to the problem of electrode poisoning and oxidizable interfering compounds in the same range of anode potential.	^[46]	Wild type legume grain	Acacia nilotica, Bauhinia purpurea, Canavalia ensiformis, Cassia hirsuta, Caesalpinia bonducella, Erythrina indica, Mucuna gigantea, Pongamia pinnata, Sebania sesban, Xylia xylocarpa	HCl 0.1 M; solvent: sample ratio 10:1 (
	Mucuna pruriens seed cooked and raw	LOD value 5.12 ng/mL	v /w); frequency // kHz; extraction time 30 min and stirring for 1 h. extraction temperature 25 °C.	RP-C18 (mean particle diameter 3.5 µm, 150 × 2.1 mm I.D.) Working electrode: Glassy carbon //	Eluent/supporting electrolyte: 103 mM sodium acetate, 0.88 mM citric acid, 2.14 mM 1-octanesulphonic acid sodium salt with pH adjusted to 2.38 by orthophosphoric acid ^[52]
			Amperometric detection at a potential of +0.7 V after micro-high performance		liquid chromatography separation	^[41]			Mucuna sanjappae seed	//	HCl 0.1 M; solvent: sample ratio 300:1 (v
Sunflower seed, sesame seed, pumpkin seed and fava bean seed	LOD value 14.3 nmol/L	/ w); frequency // kHz;	Working electrode: glassy carbon modified by graphene quantum dots decorated with Fe₃O extraction time 20 min; extraction temperature 25 °C.	₄ //	nanoparticles/functionalized multiwalled carbon ^[53]
nanotubes			Supporting electrolyte: 0.1 mol/L PBS at pH = 5.5	Differential Pulse Voltammetry (DPV)	[64]^[60]	M. pruriens seeds	Macrocarpa	HCl 0.1 M; solvent: sample ratio 300:1 (v/w); frequency // kHz;
Sweet potato	17 nM	extraction time 20 min; extraction temperature 25 °C.	Working electrode: nitrogen-doped graphene supported with nickel oxide nanocomposite //	^[54]
	Supporting electrolyte: 0.05 M PBS at pH = 7			Differential pulse voltammetry (DPV)	[65]^[61]	Microwave-assisted solvent extraction (MASE)
Spectrophotometry UV-Vis	Mucuna pruriens seed	Wild type legume grain Arka Dhanwantri	LOD 1.12 µg/mL	Water acidified with 0.1 M HCl (pH = 2.6); solvent: sample ratio 10:1 (v/w); MW power 400 W; irradiation time 5, 10, 15 min; extraction temperature 60 °C.	//		(5 min) 53.5% (10 min) 58.7% (15 min) 58.4%	//	^[48]
Spectrophotometry UV-Vis			LOD 1.12 µg/mL		//		//	//				It is an easy to use and low-cost technique that allows both qualitative and quantitative evaluation			It is generally not preceded by a separation step. This implies that the sample can contain interfering compounds causing potential false positives.	^[52]	Arka Ashwini
Phaseolus vulgaris dried seed, seeding and callus			(5 min) 50.6% (10 min) 59.6% (15 min) 54.0%				//
		^[58]	White
NMR	(5 min) 50.5% (10 min) 49.6% (15 min) 58.5%
	Mucuna pruriens seed	LOD value 0.0175 mg/g	//	//	//	It is a highly reproducible technique. It makes it possible to get structural details of the compounds under examination.	Requires expensive equipment and provides low sensitivity compared to LC-MS. It is hardly used for quantification, due to the chemical noise and signal overlapping.	^[56]	Brown	(5 min) 56.1% (10 min) 54.9% (15 min) 54.8%
Reflux extraction	Mucuna pruriens seed	Arka Dhanwantri	Water acidified with 0.1 M HCl (pH = 2.6); solvent: sample ratio 10:1 (v/w); extraction time 300 min, extraction temperature 100 °C	60.2%	^[48]
		Arka Ashwini		65.7%
		White		57.2%
		Brown		59.8%
	Mucuna pruriens powder and extracts	//	MeOH and 0.1 M HCl mixture (70:30); solvent: sample ratio 100:1 (v/w); extraction time 30 min; extraction temperature 25 °C	98.83%	^[55]
	Mucuna pruriens seed	Preta Kaunch	HCl 0.1 M; solvent: sample ratio 2:1 (v/w); extraction time 180 min; extraction temperature 25 °C	98.1–106.7%	^[56]
Maceration	Mucuna pruriens powder formulation	//	Water and EtOH mixture (30:70); solvent: sample ratio //; extraction time 7 days; extraction temperature cold	94.5%	^[57]
Maceration	Phaseolus vulgaris dried seed, seeding and callus	//	HCl 0.1 M and EtOH mixture (1:1); solvent: sample ratio 1:10; extraction time 5 days; extraction temperature 25 °C	99.55–100.27%	^[58]
Soxhlet extraction	Mucuna utilis seed	//	MeOH; solvent: sample ratio //; extraction Soxhlet time //; extract obtained sonication for 60 min with 100 mL HCl 0.1 M; extraction temperature 25 °C	98.67–100.4%	^[59]

// This indicates that values were not reported.

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