Major biochemical constituents of Withania somnifera (ashwagandha) extract: A review of chemical analysis

Mahmoud Tareq Abdelwahed; Maha A. Hegazy; Ekram H. Mohamed

doi:10.1515/revac-2022-0055

Article Open Access

Major biochemical constituents of Withania somnifera (ashwagandha) extract: A review of chemical analysis

Mahmoud Tareq Abdelwahed , Maha A. Hegazy and Ekram H. Mohamed

Published/Copyright: March 29, 2023

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From the journal Reviews in Analytical Chemistry Volume 42 Issue 1

Abstract

Ashwagandha (Withania somnifera) is a very popular medicinal herb originated mainly in India and found in the Middle East and parts of Africa. Ashwagandha has gained recognition as the most revered plant in Ayurvedic medicine. Studies indicated that it is used as anxiolytic, anti-inflammatory, antioxidant, adaptogen, memory enhancer, and possess an effect on neurodegenerative diseases. Additionally, it was revealed that the plant exerts antibacterial, antiviral, antitumor, aphrodisiac, and cardiovascular protection activities. Due to the biological and nutritional importance of nutraceuticals, particularly ashwagandha, and as part of the ongoing research of the natural products and its bioactive phytochemicals, this review aims to summarize the recent analytical techniques that have been reported for the determination of different chemical constituents of Withania somnifera quantitatively and qualitatively, and to highlight new challenges.

Keywords: Withania somnifera ; ashwagandha; chemical analysis; liquid chromatography; spectroscopy

1 Introduction

The important use of herbal medicines in preventing and treating different diseases has expanded throughout the world because of its distinctive therapeutic effect and fewer side effects compared to traditional pharmaceuticals [1].

Withania somnifera (WS), in the Solanaceae family, commonly known as ashwagandha, Indian ginseng or winter cherry, is an herb natively originated in Asia, particularly India, and found in the Middle East and parts of Africa [2]. Ashwagandha, is used in Ayurvedic medicine as it gained recognition in the treatment of anxiety, stress, and various health conditions [3]. The plant is also categorized as an anti-inflammatory agent [4] and one of the most prominent herbs for treating certain neurodegenerative diseases including Alzheimer disease [5]. Moreover, the plant is also prescribed for the use as energy booster and adaptogen as it has been demonstrated that the plant possesses free radical scavenging, antioxidant activities [6], and it increases the state of nonspecifically increased resistance of the organisms, protecting these organisms from the stresses [7]. Regarding the radio sensitizing and antitumor effects of ashwagandha, it has been documented that the plant is used in the treatment of various cancer types [8] as in urethane induced lung adenomas and Chinese hamster ovary cells carcinoma. The plant is also considered an aphrodisiac and it is reported that the plant root extract is used to treat sexual weakness, erectile dysfunction, and performance anxiety in men [9]. Additionally, some compounds in the plant have revealed antiviral activity with recognizable effects on the viral receptors which might be valid against coronavirus [10,11]. In folk medicine, the plant roots powder and the extract of roots and leaves serve as a major ingredient in hundreds of formulations [12].

The plant contains different classes of chemical compounds and a huge assortment of nutrients and phytochemicals that have gained active research interest because they possess a wide array of health benefits and due to their multidimensional importance [13]. The primary active constituents of the plant that have been identified as bioactive are withanolides A-Y, withaferin A, withasomniferin A, withasomnidienone, withasomnierose A–C, withanone, etc. [14]. Along with these lactones, the plant extract also contains alkaloids as isopelletierine, anaferine, cuseohygrine, and anahygrine [15]. Moreover, it has been demonstrated that the extract of both roots and leaves of the plant also contain sitoindosides, withanamides, reducing sugars, peroxidases, glycosides, starch, withanicil, benzyl alcohol, dilcitol, 2-phenyl ethanol, 3, 4, 5-trihydroxy cinnamic acid, benzoic acid, and phenyl acetic acid [16–18].

Withanolides are a group of naturally occurring polyoxygenated steroidal lactones arranged on a C₂₈ ergostane skeleton and the structural variation of withanolides is due to the number and nature of oxygenated substituents and the rings’ degree of unsaturation [19,20]. Withanolides possess the major biological activities of the plant and plant’s extract and different mechanisms have been reported for these pharmacological activities. For insistence, the antioxidant and free-radical scavenging activities of the plant is reported to be due to the presence of certain withanolides such as withanoside V, withaferin A, withanolide B, withanone, and 1,2-deoxywithastramonolide [21].

The anti-inflammatory activities of withanolides result from targeting various signaling pathways simultaneously, including, the nuclear factor kappa B, signal transducer and activator of transcription, and ubiquitin proteasome pathways [22–25].

Regarding the antitumor activities of the plant, withanolides such as withaferin A and withalongolide A, block the heat shock proteins which ultimately leads to cancer cell apoptosis [26–28]. Withanolides can also target the cancer stem cells (CSCs) by blocking different developmental pathways which are important in CSC epithelial-to-mesenchymal transition, inflammation, and self-renewal [29–34].

Several withanolides and their derivatives including, withanolide A, withanosides, withaferin A, and withanolidedenosomin, have been reported in treating neurodegenerative disorders through neuroinflammatory modulation and stimulating neurite outgrowth and regeneration [35–39].

The potent aphrodisiac effect of ashwagandha and its extract is attributable to the presence of number of active compounds, particularly withanolides. Human and animal studies confirmed the aphrodisiac and testosterone-enhancing properties of ashwagandha [40–43]. The anti-stress effect of the plant has the capability in managing psychogenic erectile dysfunction and this activity is imposed due to some active compounds in the plant extract including serotonin agonists and other neurotransmitters [40,42,44,45]. Moreover, investigators have reported that ashwagandha is beneficial in treating male infertility through inducing testicular development and spermatogenesis [46]. The plant extract, especially root extract, improves sperms quality and sperm count in men and reduce the effect of chemical toxins on both men’s and women’s gonads and enhance hormonal balance of LH, FSH, and testosterone as well [47–49].

Because of the growing interest of using natural plants and nutraceuticals due to their valuable pharmacological effects, this brings a special focus on screening the biologically active compounds and on the problems associated with using these natural compounds. One of the problems is the prevalent use of nutraceuticals without medical supervision owing to the wrong assumption that they are safer, and more affordable than synthetic drugs. Illicit nutraceuticals, from scrupulous manufacturers, are often adulterated endangering the lives of the consumers. Accordingly, this review aims to summarize the recent advances and techniques used in the chemical analysis of the bioactive compounds of ashwagandha’s extracts and proposing some analytical tools for the detection of the possible adulterants.

2 Chemical compounds

2.1 Chemical compounds presented in the plant

The phytochemical profile of the plant revealed that the bioactive compounds are distributed in leaves, roots, fruits, and stem bark [12,50]. The plant leaves are reported to contain 12 withanolides, condensed tannins, flavonoids, glycosides, and free amino acids; whereas, the roots contain alkaloids, steroids, volatile oils, and reducing sugars [51]. These compounds sparked intense study attention around the world because of their multidimensional significance as mentioned above. Bioactive steroidal lactones namely withanolides have been discovered as the key chemical ingredients of WS. Withanolides are a group of C-28 steroidal lactones formed on ergostane-type skeleton with C-22 and C-26 oxidized to form a six-membered lactone ring [52–54]. Different structural variations of withanolides, that have biological activities, have been reported and described, including withanone and withaferin A and withanolide (A–D) [50,53,55–58] (Table 1). The plant contains wothanolide glycosides, or glycowithanolides identified as withanosides [59–62]. In addition to the withanolide alkaloids and the glycowithanolides, the plant methanolic extract also contains tropine, choline, pseudotropine, withasomnine, mesoanaferine, withanine, somniferine, hentriacontane, withananine, visamine, ashwagandhine, pseudowithanine, withaniol, reducing sugars, iron, and amino acids such as aspartic acid, proline, tyrosine, alanine, glycine, glutamic acid, cysteine, and tryptophan [63–65].

Table 1

Summary of the major bioactive compounds in the plant extract

Bioactive compound	Chemical properties	Reference
Withaferin A	Molecular formula: C₂₈H₃₈O₆	[54,55,66–69]
Withaferin A	Molecular weight: 470.6 g·mol⁻¹	[54,55,66–69]
Withanolide A	Molecular formula: C₂₈H₃₈O₆	[54,70,71]
Withanolide A	Molecular weight: 470.6 g·mol⁻¹	[54,70,71]
Withanolide B	Molecular formula: C₂₈H₃₈O₅	[54,70]
Withanolide B	Molecular weight: 454.6 g·mol⁻¹	[54,70]
Withanolide C	Molecular formula: C₂₈H₃₉ClO₇	[72]
Withanolide C	Molecular weight: 523.1 g·mol⁻¹	[72]
Withanolide D	Molecular formula: C₂₈H₃₈O₆	[73,74]
Withanolide D	Molecular weight: 470.6 g·mol⁻¹	[73,74]
Withanone	Molecular formula: C₂₈H₃₈O₆	[11,54]
Withanone	Molecular weight: 470.6 g·mol⁻¹	[11,54]
Withanoside IV	Molecular formula: C₄₀H₆₂O₁₅	[54]
Withanoside IV	Molecular weight: 782.9 g·mol⁻¹	[54]
Withanoside V	Molecular formula: C₄₀H₆₂O₁₄	[54]
Withanoside V	Molecular weight: 766.9 g·mol⁻¹	[54]

2.2 Extraction of plant chemical compounds

Since the phytochemical screening of raw plant materials is necessary for the optimization of the known constituents’ concentrations, to maintain their activity [75], different techniques of extraction, using mainly water and alcohol as solvents for extraction, were reported for the extraction of WS bioactive molecules. These techniques included conventional heat reflux [76], Soxhlet extraction, ultrasonic-assisted extraction, microwave-assisted extraction [77], maceration, and subcritical water extraction [78]. Prior to the extraction process, the plant is collected, sorted then sieved, to remove any contaminants or dust, and then pulverized to obtain the powder ready for extraction. The indicator used for assessment of efficiency and potency of the extraction method is the percentage of the extraction yield (EY%), which is calculated as mass ratio between quantity of the recovered extract and the initial quantity of the plant material [79] as summarized in Table 2.

Table 2

Yield values of the extracts of WS prepared using different extraction techniques

Extraction method	Extraction yield (EY%)	Reference
Conventional heat reflux	45	[76,80]
Soxhlet	9.08:9.51	[77]
Maceration	20.8	[79]
Subcritical water extraction	30.5:65.6	[79]
Ultrasonic-assisted extraction	2.85:9.74	[77]
Microwave-assisted extraction	10.01:11.39	[77,80]

3 Chemical analysis

3.1 Analysis using chromatographic methods

3.1.1 Reported high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UPLC) methods with UV detection

Several methods have been implemented for the determination of WS chemical compounds using HPLC and UPLC with UV detection as illustrated in Table 3.

Table 3

Summary of the reported methods for analysis of WS active chemical compounds using HPLC and UPLC with UV detection

Stationary phase	Mobile phase	Flow rate (mL·min⁻¹)	UV detection	Application	Reference
Reverse-phase (RP) C18 Phenomenex Luna column (250 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) potassium dihydrogen orthophosphate and 0.05% o-phosphoric acid in HPLC grade water and (B) acetonitrile. From 0–12 min A:B (90:10; v/v), from 12–18 min (80:20; v/v), from 18:25 min (55:45; v/v), from 25–28 min (20:80; v/v), from 28–35 min (55:45; v/v) and from 35 min to end of run (90:10; v/v)	1.5	350 nm	Simultaneous estimation and determination of flavonoid glycosides in WS aerial parts extract	[81]
RP C18 Hibar LiChrocart Purospher Star, end capped column (250 mm × 4.6 mm and 5 µm particle size)	Acetonitrile:water (40:60; v/v)	1.5	227 nm (for the initial 10 min) and 278 nm	Analysis of withaferin-A and 6-gingerol in their polyherbal formulations	[82]
RP C18 column (150 mm × 3.9 mm and 4 µm particle size)	Gradient elution using (A) 0.1% acetic acid in methanol and (B) 0.1% acetic acid in water. A:B from (45:55; v/v) to (65:35; v/v) within 45 min	0.6	227 nm	Analysis of withanolides (withanolide A) in cultured roots of WS	[83]
C18 column (125 mm × 4.6 mm and 5 µm particle size)	Acetonitrile:(0.2%; v/v) aqueous formic acid (70:30; v/v)	1.0	360 nm	Analyses of phytochemical compounds in WS leaf extract	[84]
C18 column (250 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) orthophosphoric acid in HPLC grade water and (B) acetonitrile	1.5	227 nm	Analytical standardization for studying WS roots and leaf extract effect on improving sleep	[85]
Spherisorb ODS2 column (250 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) water and (B) acetonitrile. From 0–10 min A: B (70:30; v/v) and from 10–20 min (60:40; v/v)	(0–10 min) from 1.0 to 1.5 (10 min-end of run) 1.5 to 1.0	225 nm	Determination of withaferin-A in human plasma	[86]
Phenomenex Luna, C₁₈, end capped column (250 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) potassium dihydrogen phosphate and 0.05% phosphoric acid in HPLC grade water and (B) acetonitrile. A:B from (95:5; v/v) to (55:45; v/v) to (20:80; v/v) to (95:5; v/v)	1.5	227 nm	Quantitative analysis of withanolides in WS root extract	[87]
MAX-RP column (150 mm × 4.6 mm and 4 µm particle size)	Gradient elution using (A) water and (B) methanol:alcohol (1:1; v/v). A:B from (65:35; v/v) in 25 min to (55:45; v/v) and 5 min wash with (0:100; v/v)	1.0	UV 6000LP detector	Quantitative analysis of withanolides in WS extract	[88]
Waters RP column (150 mm × 3.9 mm and 4 µm particle size)	Gradient elution using (A) 0.1% (v/v) acetic acid in water and (B) 0.1% (v/v) acetic acid in methanol. In 60 min, A:B from (60:40; v/v) to (40:60; v/v) to (25:75; v/v) to (5:95; v/v) to (0:100; v/v)	0.6 then adjusted to 1.0 during the run	227 nm	Analysis of withanolides in roots and leaf extract of WS	[89]
Merck Purospher Star, RP-18e (125 mm × 4 mm and 5 µm particle size)	Gradient elution using (A) ultra-pure water with 0.1% (v/v) of phosphoric acid and (B) methanol with 0.1% (v/v) of phosphoric acid. In 10 min, A:B from (65:35; v/v) to (45:55; v/v). from 10–25 min (38:62; v/v) and from 25–30 min (15:85; v/v)	0.5	Photodiode array detector (PDA)	Detection of high catechin concentrations in WS roots, fruits, and leaf extract	[90]
Lichrocart Purospher STAR RP-18e column (250 mm × 4.5 mm and 5 µm particle size)	Methanol:0.01 M ammonium acetate buffer (60:40; v/v)	1.0	228 nm	Separation of isomeric withanolides in rat intestine	[91]
Phenomenex Luna C18 column (250 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) 0.05% (v/v) orthophosphoric acid in water and (B) acetonitrile. From 0–18 min A:B (55:45; v/v), from 18–28 min (20:80; v/v), from 28:35 min (55:45; v/v), and from 35–45 min (95:5; v/v)	1.5	227 nm	Determination of withanolides in WS root extract	[92]

The isocratic elution is rarely applied for the separation of withanolides due to the complexity of the plant’s extract but rather the gradient elution is mostly used as it resulted in sufficient separation and adequate peak purity. Separation of withanolide A, withaferin A, and withanone in rat intestine matrix was carried out using the isocratic elution of a mobile phase consisting of methanol and 0.01 M ammonium acetate buffer (60:40; v/v) in the range of 1.56–50.00 µg·mL⁻¹. The accuracy and precision of this method were in the range of 96.37–104.21% and 0.63–6.12%, respectively.

Based on literature, the optimum conditions for analyzing the major withanolides in the plant extract were found to be using RP column as a stationary phase and a gradient solvent system with a mobile phase consisting of 0.1% (v/v) acetic acid in water and 0.1% (v/v) acetic acid in methanol under UV detection range between 225 and 230 nm. These conditions enabled reliably efficient qualitative and quantitative analyses for the major active constituents in the plant extract and can also be applied for the analysis in their marketed dosage forms. The method was validated, and its accuracy was in the acceptable range and the relative standard deviation% of intra- and inter-day precision were in the ranges 1.1–2.1% and 0.8–8.3%, respectively.

For the estimation of flavonoid glycosides in the aerial parts of the plant, a validated UPLC method was developed using RP C₁₈ column as a stationary phase and a mobile phase consisting of potassium dihydrogen orthophosphate and 0.05% o-phosphoric acid in HPLC grade water and acetonitrile under gradient elution program. This method was validated according to ICH guidelines and showed accuracy range from 90% to 108% and the inter-day and intra-day precision was in the acceptable range. In terms of sensitivity and baseline separation of the compounds without the interference from sample matrix, the method exhibited adequate performance.

3.1.2 HPLC and UPLC with mass spectroscopy detectors

Analytical techniques using LC-MS and LC-MS/MS that have been implemented for the analysis of WS, extracts are summarized in Table 4.

Table 4

Summary of methods for analysis of WS active chemical compounds using HPLC and UPLC with UV-mass detectors

Stationary phase	Mobile phase	Flow rate (mL·min⁻¹)	Detection	Application	Reference
RP-18, Merck column (4.6 mm × 250 mm and 5 µm particle size)	Methanol:water (60:40; v/v)	0.5	DAD coupled with mass detector	Separation and quantification of withanolides in WS root extract	[93]
Waters ACQUITY BEH C18 column (50 mm × 2.1 mm and 1.7 µm particle size)	Gradient elution using (A) 0.1% (v/v) formic acid in water and (B) acetonitrile. From 0–1.8 min, A:B (70:30; v/v) from 1.8–2.5 min, (10:90; v/v) and from 2.5–4 min, (70:30; v/v)	0.3	PDA coupled with mass detector	Quantitative determination of markers in leaf, stem, and root of WS extracts and in a pharmaceutical dosage form	[94]
Phenomenex Luna C8 column (250 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) water and (B) acetonitrile. At 0–7 min, A:B, from, (95:5; v/v) to (75:25; v/v) at 7–22 min, (55:45; v/v), at 22–32 min, (20:80; v/v), at 32–37 min, (0:100; v/v) and at 37–40 min (95:5; v/v)	1.5	DAD coupled with mass detector	Investigation of 11 withanosides and withanolides in WS root extract	[54]
Zorbax Bonus RP C18 Column (250 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) acetonitrile: water (2:98, v/v) and (B) 0.1% (v/v) formic acid in water. From 0–5 min, A:B, (98:2; v/v), from 5–10 min (90:10; v/v), from 10–70 min (20:80; v/v) and from 70–72 min (5:95; v/v)	1.0	PDA coupled with mass detector	Identification of metabolites WS fruit extract	[95]
ZORBAX XDB, RP, C18 capillary column (150 mm × 4.6 mm and 5 µm particle size)	Gradient elution using (A) 0.5% (v/v) formic acid in water and (B) 0.5% (v/v) formic acid in acetonitrile. At 0–6 min, A:B, from, (90:10; v/v) to (65:35; v/v) at 6–10 min, (55:45; v/v), at 10–20 min, (55:45; v/v), and at 20–25 min, (10:90; v/v)	0.02	PDA coupled with mass detector	Simultaneous analysis of withanolides in WS leaf extract	[96]
Venusil MP C18 column (50 mm × 2.1 mm and 5 µm particle size)	Gradient elution using (A) acetonitrile:water (5:95; v/v) and (B) acetonitrile:water, (5:95; v/v); both solvents contained 10 mM ammonium acetate. From 0–0.3 min, A:B, (70:30; v/v), from 0.3–2.8 min (0:100; v/v), from 2.8–4 min (70:30; v/v)	0.25	MS/MS	Analysis and bioavailability studies of withaferin-A in rats’ plasma	[97]
RP ACQUITY BEH, C18 column (100 mm × 2.1 mm and 1.7 µm particle size)	Gradient elution using (A) 2 mM ammonium acetate in water and (B) acetonitrile. From 0–15 min, A:B, (95:5; v/v), from 15–21 min (3:97; v/v), from 21–25 min (95:5; v/v)	0.3	PDA coupled with mass detector	Analysis and metabolite profiling of WS hydroalcoholic root extract	[98,99]

Conventionally, analysis using LC-MS or LC-MS/MS offers several advantages over the use of UPLC such as improved precision and accuracy, less time of analysis and higher sensitivity and selectivity. The use of C18 columns as stationary phase and binary mixtures, applied on gradient flow, as mobile phase was optimally considered as suitable conditions for the analysis of the plant extract using LC-MS and LC-MS/MS.

A MS/MS coupled with UHPLC–PDA method was developed for the quantitative and qualitative determination of the major withanolides and withanosides. The compounds were separated on Phenomenex Luna C8 column (250 mm × 4.6 mm and 5 µm particle size) using water and acetonitrile as mobile phase applied in a gradient elution system. For the optimum mass ion fragmentation, electrospray ionization (ESI) at a temperature of 300°C resulted in better selectivity and high efficiency. The method was validated following the ICH guidelines and showed excellent linearity in the range of 1–150 µg·mL⁻¹ for the studied compounds. The limit of detection and limit of quantification values were in the range of 0.213–0.362 and 0.646–1.098 µg·mL⁻¹, respectively. The developed method exhibited overall recovery percentages ranged between 84.77 ± 0.547% and 100.11 ± 0.631% for the studied analytes. The inter-day and intra-day precision of the method was evaluated, and the results were in the acceptable range.

For the metabolite profiling of WS extract, a UPLC with positive ion ESI tandem mass spectrometry method, was found to be an advanced analytical technique for the identification of known and unknown metabolites in the plant extract. The method was carried out using RP C₁₈ column (100 mm × 2.1 mm and 1.7 µm particle size) as a stationary phase and a mobile phase consisting of 2 mM ammonium acetate in water and acetonitrile applied in a gradient elution program with a flow rate of 0.3 mL·min⁻¹. The Triple Quad mass spectrometer was equipped with ESI source with a solving temperature of 300°C and electrospray capillary voltage of 3.5 kV. The method was able to identify a total of 43 possible metabolites and their structures were proposed based on the mass of molecular and fragment ions.

3.1.3 Gas chromatography (GC)

As part of the advanced separation technology to identify the compounds of the crude herbal extracts, GC has been used in the identification of WS metabolites. GC analytical methods that have been reported in literature for the analysis of WS are summarized in Table 5.

Table 5

Summary of methods for analysis of WS active chemical compounds using GC with different detectors

Stationary phase	Carrier gas	Flow rate (mL·min⁻¹)	Detection	Temperature	Application	Reference
DB-624 silica capillary column (30 m × 0.32 mm i.d. × 1.8 µm film thickness)	Helium	0.9	Mass selective detector (MSD) equipped with quadrupole detector	Initial oven temperature of 70°C then increased to 200°C and ended up at 280°C within 44 min	Analysis and metabolite profiling of WS hydroalcoholic root extract	[98]
DB-624 silica capillary column (30 m × 0.32 mm i.d. × 1.8 µm film thickness)	Helium	0.9	MSD equipped with quadrupole detector and flame ionization detector (FID)	Initial oven temperature of 70°C then increased to 200°C and ended up at 280°C within 44 min	Investigation of the effect of energy of consciousness healing treatment in WS root extract	[99]
Dimethylpolysiloxane capillary column (50 m × 0.32 mm i.d. × 1 µm film thickness)	Nitrogen	1.0	FID	Initial temperature of 65°C then increased to 210°C and ended up at 250°C	Quantification of tropinone and tropine alkaloids in different parts of WS	[100]
Fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness)	Helium	1.0	MSD	Initial temperature of 70°C then increased to 150°C to 250°C and ended up at 320°C	Metabolomic profiling of WS extract	[101]
SE-30 capillary column (30 m × 0.32 mm i.d. × 0.25 µm film thickness)	Helium	1.2	MSD	Initial temperature of 100°C and ended up 300°C within 44 min	Identification and quantification of alkaloids in WS extracts	[102]
Rtx-5MS capillary column	Helium	4.0	MSD equipped with quadrupole detector	Inlet temperature of 280°C	Analysis and bioactivity studies of WS extract	[103]

Based on literature, the GC technique is not commonly used as it is limited to detect only the volatile compounds present in the plant.

A GC-MS method was able to identify 39 metabolites in the methanolic extract of the plant. The method was performed using fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) as a stationary phase and helium as the carrier gas and initial temperature of 70°C then increased to 150–250°C and ended up at 320°C.

For screening the alkaloids found in WS, a GC-MS method was established and resulted in the identification of 17 alkaloids and confirmed that withasomine was main alkaloid in roots. The column used was SE-30 capillary column (30 m × 0.32 mm i.d. × 0.25 µm film thickness) and the carrier gas was helium pumped at a flow rate of 1.2 mL·min⁻¹. The initial temperature was recorded to be 100°C and ended up at 300°C with a total run time of 44 min.

3.1.4 Thin layer chromatography (TLC) and high-performance thin layer chromatography (HPTLC)

Different analysis methods using TLC and HPTLC were reported in literature and summarized in Table 6.

Table 6

Summary TLC and HPTLC methods for the analysis of WS active constituents

Stationary phase	Mobile phase	Application	Reference
Si 60F₂₅₄ (10 cm × 10 cm) HPTLC plates	Dichloromethane:toluene:methanol:acetone:diethyl ether (7.5:7.5:3.0:1.0:1.0, by volume)	Simultaneous quantification of chemical compounds in WS root extract and in dosage forms containing plant material	[104]
Si 60F₂₅₄ (10 cm × 10 cm) HPTLC plates	Dichloromethane:toluene:methanol:acetone:diethyl ether (6.5:7.0:4.0:1.5:1.0, by volume)	Estimation of antioxidant potential of certain compounds in WS extract	[105]
(10 cm × 10 cm) HPTLC plates	Toluene:ethyl acetate:formic acid (5.0:5.0:1.0, by volume)	Quantification of bioactive compounds in WS extract and quality control validation of marketed formulations	[80]
	Dichloromethane:methanol:acetone:diethyl ether (15:1.0:1.0:1, by volume)
	Ethyl acetate:toluene:formic acid:2-propanol (7.0:2.0:0.5:0.5, v/v/v/v)
	Chloroform:methanol:toluene: formic acid (6.5:0.5:3:0.25, v/v v/v)
	Acetone:methanol:dichloromethane:diethyl ether (1.0:1.0:15:1.0, v/v v/v)
60F₂₅₄ HPTLC plates coated with 200 µm layers of silica gel (20 cm × 10 cm)	Toluene:ethyl acetate:formic acid (5.0:5.0:1.0, v/v/v)	Quantification and identification of withaferin-A in WS	[106]
TLC silica plates	Methanol:water (6.0:4.0, v/v)	Quantitative and qualitative evaluation of an herbal formulation containing WS extract	[107]
TLC silica plates	Toluene:ethyl acetate (7.0:3.0, v/v)	Qualitative analysis of pharmaceutical formulation containing ashwagandha	[108]

The major bioactive withanolides, particularly withaferine A, 1,2 deoxy-withastramonolide, withanolide A, and withanolide B, were quantified using a HPTLC method using Si 60F₂₅₄ (10 cm × 10 cm) HPTLC plates and a mobile phase consisting of dichloromethane:toluene:methanol:acetone:diethyl ether (7.5:7.5:3.0:1.0:1.0, by volume). The method was validated and showed proper linearity in the range of 200–1,200 ng for all the standards. The accuracy of the applied method was more 98.00% and the precision was less than 2.00%.

3.2 Analysis using spectroscopic methods

3.2.1 UV-Vis spectroscopy

Withanolide A in marketed polyherbal formulation was estimated using a validated UV-Vis spectroscopic method using UV-Vis spectrophotometer single beam (model UV mini-1240) and double beam (model UV-1650PC) and quartz cuvette in the range of 200–400 nm. The solvent used was methanol:water 50:50 (v/v) and the λmax was found to be 223 nm [109].

A UV-Vis spectrophotometric method for WS extract determination was carried out using UV-Visible spectrophotometer (Shimadzu UV-2450) and a 1.0 cm quartz cell with a slit width of 1.0 nm. The solvent used was methanol and the detection range was 190–800 nm for recording the spectra and the maximum absorbance was observed at 206.4 nm [110].

3.2.2 Fourier-transform infrared spectroscopy (FT-IR)

The FT-IR spectra of ashwagandha root extract was recorded using Fourier transform infrared spectrometer (Perkin Elmer) with the frequency array range between 400 and 4,000 cm⁻¹ using pressed KBr disk technique [110].

FT-IR analysis of WS samples was performed and the spectrum was recorded in the frequency range between 500 and 3,300 cm⁻¹ [111]

FT-IR analysis for the methanol extract of WS using (SHIMADZU IR Preastage-21) FT-IR spectrometer with a frequency scan range between 400 and 5,000 cm⁻¹ and the spectrum was recorded [103].

3.2.3 Nuclear magnetic resonance spectroscopy (NMR)

¹H-NMR spectrum of the hydro-alcoholic extract of WS was recorded in a 400 MHz Varian FT-NMR spectrometer and ¹³C-NMR spectrum was measured at 100 MHz on a VarianFT-NMR spectrometer [98].

The ¹H NMR spectra was recorded using HR-MAS NMR (Bruker Biospin Avance-III 800 MHz) spectrometer which operates at a proton frequency of 800.21 [112].

4 Discussion

Based on literature, in general, chromatographic analysis offers several advantages over spectroscopic analytical methods in the analysis of botanical samples. These advantages include the accurate and precise determination and quantification of the studied samples and the full characterization of the phytochemical profile of the samples of interest [113]. Furthermore, the chromatographic methods can also separate the complex mixtures and the components with very similar chemical and physical properties and collect these components individually. Whereas, although the spectroscopic methods are conventional and inexpensive, they have several limitations including low sensitivity and selectivity compared to the chromatographic methods.

In the phytochemical analysis, it is found that the use of columnar chromatography is very useful in the separation of the complicated compound mixtures, purification process, the isolation of the bioactive constituents and the separation of diastereomers [114]. Considering that there are few limitations for the use of columnar chromatography including the high time consumption for the separation of the compounds, the high consumption of the solvents, and the complication of the process automation. In comparison of UPLC to HPLC, UPLC has superior chromatographic resolution, increased selectivity and sensitivity, increased sample throughout, and separation in a shorter timeframe. Regarding detectors in columnar chromatography, the diode array detectors (DAD) have several advantages over the conventional UV detectors as the spectral profile in DADs has the ability to determine the unknown peaks in the chromatograms, can be used in peak purity analysis, and can measure the peaks at all wavelengths in short time. Whereas the mass detectors can be used as a powerful tool in determining and identifying the unknown components in the sample and can calculate the purity of the sample with higher sensitivity than other techniques. Overall, the columnar chromatography techniques have been used in the identification, quantification, and separation of the major bioactive phytochemical compounds in ashwagandha and have been used in the metabolite profiling of the plant as well.

As most of the plants contain different assortments of volatile oils, the GC acts as the premier analytical technique for identifying and separating the volatile compounds and has been used in the metabolite profiling of different plants including ashwagandha. The advantages of GC include the high efficiency and the separation of the components in reasonable time and improved resolution, while the major drawback is that it is only limited to the volatile compounds and most of the GC detectors are destructive. Regarding the use of GC in the analysis of ashwagandha, this technique has been used in the identification of quantification of the alkaloid content of the plant and in the analysis and bioactivity studies of the plant’s extract.

Regarding planar chromatography techniques including TLC and HPTLC, both techniques have several advantages such as simplicity, separation of non-volatile compounds, and small sample size is required [115]. On the other hand, planar chromatography has some disadvantages including the automation is difficult, sensitivity and accuracy in quantitative analysis is lower than other techniques, and the results obtained are difficult to reproduce. Comparing TLC to HPTLC in phytochemical analysis, TLC is considered the preliminary step to identify the phytochemical constituents of the studied sample, whereas the HPTLC can detect the presence of marker compounds in the sample as it can produce an electronic image of the fingerprint chromatogram and can provide a densitogram of the studied analyte. TLC and HPTLC have been used in the simultaneous determination and identification of certain biologically active compounds in the extract of ashwagandha.

The use of spectroscopic techniques, including UV-Vis spectroscopy, NMR spectroscopy, and FT-IR spectroscopy, in the botanical analysis is limited and needs further investigations due to the complexity of the plants’ phytochemical compositions and the sophistication of their extracts. The spectroscopic techniques are mostly coupled to the conventional chromatographic technique to further confirm the presence of certain compounds in plants and in their extracts.

5 Conclusion

The analysis of phytochemicals in medicinal plants gained recognition in scientific research due to the therapeutic effect of these plants and its minimal side effects compared to traditional medicines. WS (ashwagandha) contains various phytochemical compounds that possess therapeutic activity and used in the treatment of multiple diseases including different types of cancer, neurodegenerative diseases, autoimmune diseases, and sexual dysfunction diseases. Additionally, the plant works as immunomodulator and is used against stress, anxiety, and inflammation.

The chemical analysis of the plant constituents and understanding its bioactivity are major concerns and different analytical techniques have been used for these studies. Chromatographic methods, including HPLC, UPLC, LC/MS, TLC, HPTLC, and GC, have been illustrated in this review with the conditions used and the application criteria.

Spectrophotometric methods, for analysis of plant bioactive compounds, including, UV-Vis spectroscopy, FT-IR spectroscopy, and NMR spectroscopy, have been also summarized in this review.

Acknowledgements

The authors are also grateful to Cairo University and The British University in Egypt for providing the necessary facilities to perform this study.

Funding information: Authors state no funding is involved.
Author contributions: Maha Hegazy: conceived the proposed idea, collected data, revised the final version of the manuscript (reviewing and editing), final approval of the manuscript to be published; Ekram Hany: designed the framework of the presented review, collected the data, revised the final version of the manuscript (reviewing and editing), data analysis and interpretation; Mahmoud Tareq: collected the data, designed the framework of the manuscript, wrote the first draft of the manuscript, data analysis and interpretation, finalization of the manuscript.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: The files used to support the data findings of this study are available from the corresponding author on request.

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Received: 2022-11-04

Revised: 2023-01-31

Accepted: 2023-02-22

Published Online: 2023-03-29

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/revac-2022-0055

Keywords for this article

Withania somnifera; ashwagandha; chemical analysis; liquid chromatography; spectroscopy

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