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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 4  |  Page : 239-247

Preparation of Vetas Ghana (a semisolid form of Salix alba L. stem bark aqueous extract) and its analysis by using high-performance thin-layer chromatography and liquid chromatography–mass spectrometry


1 Department of Ayurveda, Central Ayurveda Research Institute, Jhansi, Uttar Pradesh, India
2 Department of Chemistry, Central Ayurveda Research Institute, Jhansi, Uttar Pradesh, India
3 Department of Chemistry, Central Council for Research in Ayurvedic Sciences, New Delhi, India
4 Department of Ayurveda, Central Council for Research in Ayurvedic Sciences, New Delhi, India

Date of Submission09-Jan-2022
Date of Acceptance24-Jan-2022
Date of Web Publication17-May-2022

Correspondence Address:
Vijay Kumar
Department of Chemistry, Central Ayurveda Research Institute, Jhansi 284003, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdras.jdras_1_22

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  Abstract 

BACKGROUND: Vetas Ghana is a semi-solid prepared from the aqueous extract of stem bark of Salix alba L. (Vetas). Vetas Ghana is an important Ayurvedic intermediate used in various herbal and cosmetic formulations. There is no advance analysis of Vetas Ghana preparation reported yet. OBJECTIVE: The aim of this work was to prepare Vetas Ghana and to study phytochemicals by using advanced analytical instruments such as high-performance thin-layer chromatography (HPTLC) and tandem liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS/MS). MATERIALS AND METHODS: Vetas Ghana was analyzed by physicochemical parameters such as loss on drying, pH, total ash, acid insoluble ash, total soluble solids, and its major metabolites were checked by using advanced chromatographic tools such as HPTLC and LC-QTOF-MS/MS. RESULTS: In comparison of Vetas with Vetas Ghana, drastic changes in the parameters such as pH, and ash (total and acid insoluble) were observed. In the HPTLC study, after derivatization marker compound epicatechin is detectable in Vetas and Vetas Ghana. LC-QTOF-MS/MS analysis of Vetas and Vetas Ghana reveals the presence of eight major metabolites including the presence of marker compound epicatechin. CONCLUSIONS: Vetas Ghana was prepared in in-house pharmacy and phytochemicals were detected by using HPTLC and LC-QTOF-MS/MS analysis, where biomarkers like epicatechin are identified including other biomarkers such as salicin and isosalicin.

Keywords: API, epicatechin, HPTLC, LC-MS, Salix alba, Vetas Ghana


How to cite this article:
Jagtap C, Charde V, Kushwaha V, Garewal J, Shakya SK, Kumar V, Babu G, Singh A, Singh R, Sharma BS, Khanduri S, Srikanth N. Preparation of Vetas Ghana (a semisolid form of Salix alba L. stem bark aqueous extract) and its analysis by using high-performance thin-layer chromatography and liquid chromatography–mass spectrometry. J Drug Res Ayurvedic Sci 2021;6:239-47

How to cite this URL:
Jagtap C, Charde V, Kushwaha V, Garewal J, Shakya SK, Kumar V, Babu G, Singh A, Singh R, Sharma BS, Khanduri S, Srikanth N. Preparation of Vetas Ghana (a semisolid form of Salix alba L. stem bark aqueous extract) and its analysis by using high-performance thin-layer chromatography and liquid chromatography–mass spectrometry. J Drug Res Ayurvedic Sci [serial online] 2021 [cited 2022 Jun 29];6:239-47. Available from: http://www.jdrasccras.com/text.asp?2021/6/4/239/345392

Chandrashekhar Jagtap, Vaibhav Charde, Vikram Kushwaha, Jyotika Garewal, Santosh K. Shakya, Vijay Kumar2; Equal contribution





  Introduction Top


It is a well-known fact that higher plants are the basis of modern medicine. Well-known drug molecules such as taxol, aspirin, and morpine are originated from the plants or natural system.[1],[2],[3],[4],[5],[6],[7],[8],[9] As per World Health Organization (WHO) reports, for primary healthcare, more than 70% population of the world is relying on the natural resources.[10] More importantly, traditional medicines have played an important role in COVID-19.[1] All the drugs, either herbal or modern, need standardization for the safety of consumers and to gain their faith.[8],[10] Recently, for the global acceptance of herbal drugs, WHO and European Medicines Agency (EMEA) have released guidelines for the standardization of herbal drugs.[10],[11],[12],[13]

Salix alba L. (genus Salix) is a member of Salicaceae family.[14] The distribution of genus Salix is widespread.[15]Salix alba L. is known for salicylate-based phytochemicals including salicin and isosalicin, where salicylic acid is the basic precursor.[15],[16],[17] The versatile medicinal applications of genus Salix and its utility cosmetic industry make it more significant.[14],[15],[16] Various parts of Salix plant are used to treat a variety of disorders or aliments such as obesity, hyperlipidemia, inflammation, cardiovascular diseases, rheumatoid arthritis, and cancer.[14],[15],[16],[17],[18],[19] Stem bark of Salix alba plant is antioxidant, anti-acetylcholinesterase, neuroprotective, anti-inflammatory, and anti-migraine in nature.[17],[18],[19] The major biological active compounds of Salix are salicin, catechins, and procyanidins.[3] In Indian Medicine System, Salix alba L. is known as Vetas. Aqueous extract of stem bark of Vetas is known as Vetas Ghana, which is an intermediate of various Ayurvedic formulations.

There are very few selected phytochemicals studies on Salix genus where high-performance liquid chromatography (HPLC),[19],[20] high-performance thin-layer chromatography (HPTLC),[21],[22] liquid chromatography mass spectrometry (LC-MS),[16],[18] and gas-liquid chromatography mass spectrometry (GC-MS)[22],[23] have been reported by various research groups. Moreover, there is no report on preparation of Vetas Ghana and its chromatographic analysis has been published yet. The aim of this study was to prepare Vetas Ghana and characterization of its metabolites by using HPTLC and LC-MS analytical tools. Moreover, quality control parameters such as loss on drying, pH, total ash, acid insoluble ash, water extractive value, and ethanol extractive value and total soluble solids as appropriate were performed as per the Ayurvedic Pharmacopeia of India (API).[24],[25],[26]


  Experimental Top


Materials

Marker compounds epicatechin (Make: Cayman R; Purity: 98%) was used. Solvents including toluene, ethyl acetate, and methanol were of AR grade. TLC plates 60F-254 (Make: M/s Merck Ltd India, Bangalore; Purity: 98% or AR grade) were used under this study. The authentic material of Vetas (Salix alba L. stem bark) was collected from Kashmir, India by the Botanist of Kashmir University (Herbarium Number: 3160-KASH).

Preparation of Vetas Ghana from Vetas stem bark

Vetas stem bark was cleaned, washed, and crushed in pieces of 1–3 cm, and subjected to drying in a tray drier maintaining temperature around 40°C. Vetas stem bark was converted into Kvatha Churna (coarse powder: 40 mesh) and boiled with 16 times potable water, further reduced to 1/8th of original volume. The heating was carried out maintaining the temperature at 90–100°C along with intermittent stirring. The Kvatha was filtered through muslin cloth. It took around 25 hours to complete Kvatha preparation from the raw drug (Vetas stem bark). This Kvatha was again subjected to mild heating (with peak temperature of 70–75°C) with continuous stirring till the entire mass converted into semi solid state (Ghana). It took 16.5 hours (average) for preparation of Ghana from Kvatha. The semisolid mass was shifted into an open tray and kept in tray dryer at 40–50°C for the proper drying of Vetas Ghana.

Physicochemical parameter analysis

The quality control parameters of Vetas and Vetas Ghana were performed as per the API in triplicate.[24],[25],[26] Quality control parameters such as loss on drying, pH, total ash content, acid insoluble ash content, water extractive value, ethanol extractive value and total soluble solids were analyzed as appropriate. The ultraviolet–visible (UV–visible) spectrum of methanolic extracts of Vetas and Vetas Ghana were recorded by using methanolic as blank solvent.

High-performance thin-layer chromatography Instrumentation and chromatographic conditions

CAMAG HPTLC instrument, syringe (100 µL), and glass twin trough developing chamber were used for the HPTLC profiling of Vetas, Vetas Ghana, and marker compound epicatechin. Methanolic extracts (10 µL) of Vetas and Vetas Ghana, and methanolic standard solutions (5 µL) of epicatechin were applied on aluminum plate pre coated with silica gel 60 F254 of 0.2 mm thickness (Merck, India) using applicator CAMAG Linomat-5 and software VisionCATS 2.5. The TLC plate track number details are as follows: Track 1: Vetas (V), Track 2: Vetas Ghana (VG), and Track 3: epicatechin (EC).

The plate was developed in glass twin through chamber presaturated (for 15 min) with the mobile phase. The developed plate was visualized at 254 and 366 nm by using a CAMAG visualizer. The plate was derivatized with anisaldehyde-sulphuric acid reagent and heated at 105°C till the development of visible spots. The visible spots were captured by using CAMAG TLC Visualizer 2. The developed visible spots on the derivatized plate were scanned using CAMAG Scanner-4 at 514 nm. The Rf values and densitograms were recorded by using the CAMAG VisionCATS 2.5 software and the final report was generated. Images were captured by keeping plates in photo-documentation chamber and Rf values were recorded by VisionCATS 2.5 software.

Stationary Phase: TLC Silica gel 60 F254 (Merck)

Developing Solvent: Toluene: Ethyl acetate: Acetone: Formic acid: (8.5: 0.5: 1: 1 v/v/v/v)

Saturation Time: 15 min at 25 ± 2°C

Test Solutions: A 100 mg/mL methanolic solutions of Vetas and Vetas Ghana was prepared. To prepare the methanolic solutions, 100 mg sample was dissolved in 100 mL methanol, sonicated for 10 min at 25°C, centrifuged at 10,000 rpm for 5 min and supernatant keep in glass vials for HPTLC analysis.

Standard solution: The standard solution of epicatechin with concentration 1 mg/mL was prepared in methanol.

Tandem liquid chromatography-quadrupole time of flight-mass spectrometry instrumentation and chromatographic conditions

Metabolite profiling was done by using tandem liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS/MS) in positive mode (ESI+). The 5 µL of test solution (as mentioned under the HPTLC section) was injected by diluting the test solution to 1 mg/mL. The flow rate was 0.300 mL/min. Column Hypersil column Gold C18 100 mm × 2.1 mm – 3 Micron was used. The solvent composition was as follows: A = 100% water with 0.1% formic acid and B = 100% acetonitrile with 90% acetonitrile + 10% water with 0.1% formic acid. The run time of experiment was 20 min. with following steps: 1 to 20 min, solvent A = 95% and B = 5%; 21 to 25 min, solvent B = 100%; 26 to 30 min, solvent A = 95% and B = 5%. The compound databases, namely PubChem, KEGG Compound, and ChemSpider, were used to search the metabolites.


  Results and Discussion Top


Preparation of Vetas Ghana from Vetas

During the preparation of Vetas Ghana from Vetas, the following facts and results were observed.

Vetas Kvatha extract results

General observations during preparation

  • ✓ Initially, the liquid was light brown in color and subsequently turned into dark brown.


  • ✓ Evaporation started at 70ºC, which was aggravated on stirring.


  • ✓ Maximum temperature was recorded up to 90°C–100°C.


Precautions

  • ✓ It was ensured that all vessels were nonreactive and cleaned properly before using.


  • ✓ During boiling, liquid should be stirred continuously.


  • ✓ Care was taken to avoid direct contact with product.


Result

  • ✓ Amount of Kvatha Churna taken for Vetas Kvatha prepration:13 kg


  • ✓ Amount of water taken for Vetas Kvatha preparation: 208 L


  • ✓ Total time taken for preparation of Vetas Kvatha = 25 h


  • ✓ Final quantity of Kvatha obtained = 25 L


Results for Vetas Ghana obtained from Vetas Kvatha

General observations during preparation of Ghana

  • ✓ With gradual heating, liquid becomes more intense in color and the liquid turned stickier in nature.


  • ✓ Stickiness of the liquid and adhesiveness to the vessel increased with time.


Precaution

  • ✓ Continuous stirring was carried out to avoid burning of Kvatha.


  • ✓ Temperature was maintained between 70°C and 75°C.


  • ✓ The semisolid mass was shifted into a tray dryer for the proper drying at 40°C–50°C.


Result

  • ✓ Total time taken for preparation of Ghana from Kvatha = 16.5 h (avg.)


  • ✓ Final quantity of semisolid Ghana obtained = 7657 g


  • ✓ Weight of dried Ghana = 530 g


  • ✓ Percentage of Ghana obtained w. r. t. semisolid = 6.92%


  • ✓ Percentage of Ghana obtained w. r. t. raw drug = 4.07%


Physicochemical parameter analysis

The observed results of various physicochemical parameter analyses for Vetas and Vetas Ghana are mentioned in [Table 1]. Slight amount of foreign matter was observed in Vetas since it was collected from the natural resource. The variation in the moisture content is due to the interaction of Vetas with water molecules during the preparation of Vetas Ghana. Decrease in pH of the Vetas Ghana indicates the probable reduction of the basic metabolites of Vetas. The water extractive value was not applicable for Vetas Ghana since it is an aqeous extract of Vetas. Thus the total soluble solid was analysed. The observed value of total soluble solid for Vetas Ghana was calculated (93.62% w/w) which is quite acceptable, as experimental value is more than 90%. The increased ethanol extractive value of Vetas Ghana indicates that the polar solvents are favourable for its extraction. The extractive values were found increased in Vetas Ghana due to the extraction of hydrophilic or polar compounds during its preparation. The increase in total ash after preparation of the Vetas Ghana confirms the facts and trends observed in water and ethanol extractive values. No change in the observed values of λmax of Vetas and Vetas Ghana in methanol means major molecules of Vetas are not depleted after the preparation of Vetas Ghana. Direct heating or higher temperature can affect the compounds like Syringol, 4-Methylsyringol, 4-Ethylsyringol, and 4-(I-Propenyl)-syringol.[22],[23]
Table 1: Physicochemical parameters and HPTLC analysis of Vetas and Vetas Ghana

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High-performance thin-layer chromatography analysis of Vetas and Vetas Ghana

In the HPTLC study, mobile phase optimization is the main phase. Better mobile phase means good resolution and better separation of compounds. In this study, the mobile phase consisting of Toluene: Ethyl acetate: Acetone: Formic acid: (8.5: 0.5: 1: 1 v/v/v/v) at room temperature (25°C ± 2°C) was optimized for the HPTLC analysis. The observed results of HPTLC analysis for Vetas, Vetas Ghana, and marker compound (epicatechin) are mentioned under [Table 1]. In HPTLC analysis, at 254 nm, three bands were observed at Rf 0.013, 0.073, and 0.948, respectively [Figure 1]. At 366 nm, one band was observed at Rf 0.929. Marker compounds were not detected at 254 and 366 nm. After derivatization of TLC plate, three bands were observed in Vetas and Vetas Ghana samples at Rf 0.0714 (orange), 0.867 (light purple), 0.930 (pink) including the marker compound epicatechin band at Rf 0.0714 (orange) [Table 1] and [Figure 1].
Figure 1: Comparative HPTLC analysis of Vetas (V), and Vetas Ghana (VG) at 254 (A), 366 (B), and 514 nm (C) w.r.t. their marker compounds (Track 1: Vetas [V], Track 2: Vetas Ghana [VG], and Track 3: epicatichin [EC])

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There are selected studies on the quantification of Salix alba stem bark. In the first study, mobile phase 1,2-dichloroethaneethanol-methanol-water (50:20:20:6 v/v/v) was used and HPTLC plates were used instead of TLC plates.[27] In another study, mobile phase chloroform–ethanol–formic acid (50:40:6 v/v/v) was used and HPTLC plates were used instead of TLC plates. Moreover, before the HPTLC analysis, C18 column elution with mobile phase methanol–water 7:93 v/v was performed.[21] The basic advantage of this study was the high resolution and usage of TLC plates HPTLC plates are costly and every laboratory cannot afford it. In terms of resolution, [Figure 1] shows the bands of high resolution with blade-like sharpness, which is very rare and need more experimental skills are required to develop such plates.

Liquid chromatography mass spectrometry analysis of Vetas and Vetas Ghana

To verify and validate the HPTLC analysis results, LC-QTOF-MS/MS analysis of Vetas and Vetas Ghana was performed. To attain the significant results, same concentration of Vetas and Vetas Ghana and same method conditions were used. The, LC-QTOF-MS/MS analysis of methanolic extract of Vetas and Vetas Ghana revealed the presence of 38 compounds (in ESI+) including the presence of marker compound epicatechin [Figure 2]. A detailed literature and library search has confirmed the presence of eight major phytochemicals as indicated by yellow-green lines in the chromatographs of Vetas and Vetas Ghana under [Figure 2]. [Figure 2] and [Figure 3] show the presence and fragmentation pattern of Caffeoylhexose I-III (Rt 1.10), Salicin/Isosalicin (Rt 3.10), Quercetin methyl-pentoside (Rt 3.71), Procyanidin dimer I-III (Rt 4.95), Epigallocatechin I-III (Rt 9.10), (Epi)catechin methyl-hexoside I-II (Rt 16.25), Catechin (Rt 22.98)/epicatechin (Rt 22.98), and Naringenin (Rt 26.66). There are few peaks highlighted with # sign where significant changes in intensity and resolution are observed. Few new peaks were noticed (mentioned by * symbol) and intensity of few peaks (mentioned by $ symbol) were also reduced [Figure 2].
Figure 2: Comparative LC-MS chromatograph of Vetas and Vetas Ghana ESI (+ve) mode [caffeoylhexose I-III (Rt 1.10), salicin/isosalicin (Rt 3.10), salicortin (Rt 3.71), procyanidin dimer I-III (Rt 4.95), epigallocatechin I-III (Rt 9.10), (Epi)catechin methyl-hexoside I-II (Rt 16.25), catechin (Rt 22.98)/epicatechin (Rt 22.98), and naringenin (Rt 26.66)]
*New peaks
#Intensity increased
$Intensity decreased


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Figure 3: Major phytochemical fragmentations pattern of Vetas and Vetas Ghana observed in LC-MS ESI (+ve) mode study (salicin/isosalicin, procyanidin dimer I-III, and catechin/epicatechin)]

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The compound databases, namely PubChem, KEGG Compound, and ChemSpider, were used to search the metabolites. Moreover, a detailed literature search was executed to confirm the phytochemical of Vetas and Vetas Ghana as shown under [Table 2]. The pattern of elution of phytochemicals was confirmed as almost the same conditions were used by Piatczak et al.[16] and Lavola et al.[18] In the HPLC study they confirmed the elution pattern as follows: Caffeoylhexose I-III (Rt 1.10), Salicin/Isosalicin (Rt 3.10), Salicortin (Rt 3.71), Procyanidin dimer I-III (Rt 4.95), Epigallocatechin I-III (Rt 9.10), (Epi)catechin methyl-hexoside I-II (Rt 16.25), Catechin (Rt 22.98)/epicatechin (Rt 22.98), and Naringenin (Rt 26.66). LC-MS study has confirmed that phytochemical compound Caffeoylhexose I-III with m/z 343 [M + H]+ has the fragmentations at m/z 338, 229, 171, and 118. Phytochemical compounds salicin/isosalicin with m/z 313 [M+Na]+ have the fragmentations at m/z 301, 296, 279, and 127. Phytochemical compound Salicortin with m/z 447 [M + H]+ having the fragmentations at m/z 433, 301, 283, 265, 227, 137 167, and 111.[16],[18] Phytochemical compounds Procyanidin and its dimer I-III with m/z 600 [M+Na]+ having the fragmentations at m/z 432, 415, 411, 401, 249, 207, and 111. Phytochemical compounds Epigallocatechin I-III with m/z 353 [M+2Na]+ having the fragmentations at m/z 295, 277, 242, and 195. Phytochemical compounds (Epi)catechin methyl-hexoside I-II with m/z 489 [M+Na]+ having the fragmentations at m/z 359, 338, 312, and 242. Phytochemical compounds catechin/epicatechin with m/z 663 [2M+Na]+ having the fragmentations at m/z 607, 312, and 242. Phytochemical compound Naringenin with m/z 274 [2M + H]+ having the fragmentations at m/z 247, 214, and 124 [Table 2] and [Figure 2].[16],[18],[20],[21],[27]
Table 2: LC-MS data of phytochemicals detected in Vetas and Vetas Ghana

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  Conclusions Top


The following are the main conclusions drawn from this study:

  • In comparison of Vetas with Vetas Ghana, drastic changes in the parameters such as pH, ethanol extractive value, and ash (total and acid-insoluble) were observed.


  • Comparative HPTLC analysis revealed that marker compound epicatechin is detectable in Vetas and Vetas Ghana with mobile phase Toluene: Ethyl acetate: Acetone: Formic acid: (8.5: 0.5: 1: 1 v/v/v/v) after derivatization.


  • The comparative LC-QTOF-MS/MS analysis of Vetas and Vetas Ghana reveals the presence of eight major metabolites including the presence of marker compound epicatechin.


  • Acknowledgement

    SAIF, IIT, Bombay is highly acknowledged for LC-MS analysis facility. DG-CCRAS is highly acknowledged for funding and facilities at CARI, Jhansi (Uttar Pradesh).

    Financial support and sponsorship

    This work was supported by the Central Council for Research in Ayurvedic Sciences (CCRAS), Ministry of Ayush under IMR Project (F.No.3–87/2020-CCRAS/Admn/IMR/2868 dated September 18, 2020).

    Conflicts of interest

    There are no conflicts of interest.

    Ethical policy and institutional review board statement

    Not applicable.

    Graphical Abstract







     
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        Figures

      [Figure 1], [Figure 2], [Figure 3]
     
     
        Tables

      [Table 1], [Table 2]



     

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    Abstract
    Introduction
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