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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 7  |  Issue : 4  |  Page : 229-242

Detoxification of Datura metel L. seeds using Shodhana (purifying process) and estimation of scopolamine content


1 Department of Chemistry, Regional Ayurveda Research Institute, Gwalior, Madhya Pradesh, India
2 Department of Chemistry, Captain Srinivasa Murthy Central Ayurveda Research Institute, Chennai, Tamil Nadu, India
3 Central Council for Research in Ayurvedic Sciences, Ministry of Ayush, New Delhi, India
4 Department of Biotechnology, ITM University, Gwalior, Madhya Pradesh, India
5 Department of Biochemistry, Central Ayurveda Research Institute, Kolkata, West Bengal, India

Date of Submission05-Aug-2021
Date of Acceptance13-Jun-2022
Date of Web Publication21-Nov-2022

Correspondence Address:
Dr. Ajay Kumar Meena
Department of Chemistry, Regional Ayurveda Research Institute, Amkhoh, Gwalior 474001, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdras.jdras_45_21

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  Abstract 

BACKGROUND: The seeds of Datura metel L. are classified as toxic in the classical texts of Ayurveda and hence need to be purified by the traditional method as mentioned before using for medications. METHODS: In this study, the seeds of Datura metel L. were detoxified by Shodhana, an Ayurvedic purifying process. The process of detoxification (Shodhana) was performed by soaking the seeds in gomutra (cow’s urine) and boiling them in godugdha (cow’s milk). The preliminary phytochemical and physicochemical analyses were performed simultaneously for the processed and unprocessed Datura metel L. seeds. The total ethanol and chloroform extracts of the processed and unprocessed Datura metel L. seed samples were used for chemical profiling using high-performance thin-layer chromatography, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometer, and gas chromatography-mass spectrometer. Scopolamine content was quantified by HPLC in both processed and unprocessed seeds. RESULTS: Scopolamine was not detected in the chloroform extract. The ethanol extract found that the detoxification process has removed scopolamine content in the processed Datura metel L. seed. An attempt has been made to identify the chemical constituents responsible for the toxicity by measuring the level of major chemical constituents before and after the detoxification of seeds. CONCLUSION: This study reveals that the process of Shodhana was effective by reducing the content of scopolamine, and hence it was mandatory and supports its use in medications.

Keywords: Datura metel, detoxification, HPLC, scopolamine, Shodhana


How to cite this article:
Meena AK, Venktaraman P, Singh R, Ganji K, Srikanth N, Dhiman KS, Motiwale M, Chaturvedi S, Dixit AK. Detoxification of Datura metel L. seeds using Shodhana (purifying process) and estimation of scopolamine content. J Drug Res Ayurvedic Sci 2022;7:229-42

How to cite this URL:
Meena AK, Venktaraman P, Singh R, Ganji K, Srikanth N, Dhiman KS, Motiwale M, Chaturvedi S, Dixit AK. Detoxification of Datura metel L. seeds using Shodhana (purifying process) and estimation of scopolamine content. J Drug Res Ayurvedic Sci [serial online] 2022 [cited 2022 Nov 29];7:229-42. Available from: http://www.jdrasccras.com/text.asp?2022/7/4/229/361577




  Introduction Top


Ayurveda, an ancient Indian traditional medicinal system, is gaining popularity worldwide. Medicinal plants play a vital role in drug discovery in traditional medicine systems. In recent days, herbal drugs usage has reached global markets. Hence it is required to provide a scientific study and analytical data on the toxic content of the plant material used in medications. In Ayurvedic texts, these poisonous drugs are classified as Visa (poison) and Upavisa (poison but not lethal) under Schedule E-1 of Drugs and Cosmetics Act, 1940 and Rules 1945.[1]Shodhana (detoxification) is the process used to remove physical or chemical impurities, to minimize the side effects and increase the therapeutic activity of poisonous materials used for medicinal purposes. In the classical Ayurvedic literature, a specific detoxification method (Shodhana process) has been mentioned for each plant, which must be followed before using it for formulations.[2] It says that, by executing the correct procedure of Shodhana, a toxic material can be converted into amrita (nectar).[3] The Shodhana process helps reduce the toxic content of the drug, which improves the potency/therapeutic activity.

The seeds of the Datura metel L. plant are considered to be highly toxic.[4] The toxicity is mainly due to the tropane alkaloids atropine (dl-hyoscyamine) and scopolamine (l-hyoscine); a competitive antagonist of muscarinic cholinergic receptors is central nervous system depressants. Most of the side effects (the dryness of the mouth, excessive thirst, cramps, unconsciousness, and giddiness) are due to the anticholinergic property of the alkaloids present in this plant.[2],[5],[6],[7],[8] Various species of Datura are now cultivated for the production of secondary metabolites. In Ayurvedic medicine, the seeds of Datura metel are used to treat skin rashes, ulcers, bronchitis, jaundice, diabetes, etc.[9]

The Datura metel L. plant contains different functional groups such as saponins, tannins, steroids, alkaloids, flavonoids, phenols, and glycosides. The seeds of Datura metel contain scopolamine [Figure 1],[10] atropine, fastunine, fastudine, fastusidine, daturanolone, and fastusic acid. The most important natural tropane alkaloids are hyoscyamine and scopolamine (also known as hyoscine). Scopolamine represented the primary tropane alkaloid in Datura metel.[11] The plant has alkaloids hyoscyamine, hyoscine, and atropine.[10] The seeds held a total alkaloid content of 0.426%, which was mainly atropine. All plant parts are toxic, but the principal amount of alkaloids is contained in the ripe seeds.[12],[13],[14]
Figure 1: Chemical structure of scopolamine

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Medicinal plants can cause toxic effects, being used as an individual drug or as an ingredient of many compound formulations; if not processed properly. Many poisonous drugs are used as a single drug or a compound formulation in Ayurveda. Datura metel is used as an ingredient in Kanakasava, Sutasekhara Rasa, Jwarankusa Rasa, Dugdha vati, Kanakasundara Rasa, Lakshmivilasa Rasa, Mahajwarankusha Rasa, Bruhat kanakasundara Rasa, Pralapantaka Rasa and Kanakaprabha vati. The poisonous plants should be added to the compound formulations only after proper Shodhana (purification/detoxification) processing.[15],[16]

In the present study, the seeds of the Datura metel L. were subjected to the Shodhana process as mentioned in the classical texts.[17] An analysis for the presence of secondary metabolites was performed and tabulated. The analysis of physicochemical parameters was carried out for the seeds of Datura metel before and after the detoxification process. The ethanol and chloroform extracts prepared by the Soxhlet extraction method were used to analyze and quantify the compounds. Analytical techniques such as high-performance thin-layer chromatography (HPTLC), liquid chromatography-mass spectrometer (LC-MS), and gas chromatography-mass spectrometer (GC-MS) techniques were used for the qualitative identification of chemical constituents of the Datura metel seeds. High-performance liquid chromatography (HPLC) method was used to quantify the scopolamine content in Datura metel seeds before and after the detoxification process.


  Materials and Methods Top


Collection of plant material

The Datura metel L. seed was procured from the local crude drug market Chennai, Tamil Nadu, and authenticated at the Botany Department, CCRAS, Ministry of Ayush, Chennai. A specimen voucher was deposited in the Botany Department of the Institute.

Chemicals, reagents, and reference standards

All the chemicals and solvents utilized in the present study were of analytical grade and HPLC grade only. The solvents and chemicals were procured from E-Merck, Sigma Aldrich, and other renowned companies from Chennai, India. The scopolamine hydrobromide reference standard was procured from Sigma Aldrich (≥98% purity), India.

Sample preparation of Datura metel L. seeds

The procured raw drug was cleaned physically to remove dust and foreign matters, authenticated by botanist with accession no. F/Sd 33 and used for the study. Air-dried cleaned raw drug was powdered and stored in polythene containers for further study.

The processed and unprocessed powdered Datura metel L. seeds were taken separately. Extraction was carried out using ethanol (for more effective extraction of alkaloids)[18] and chloroform (to study chemical profiling of the seed) as solvents. The hot Soxhlet extraction method was used to collect the extracts, which was used in all instrumental analyses.

Phytochemical and physicochemical analysis

The powdered samples were weighed (5 g) separately, and 10 mL of ethanol was added to them. The samples were sonicated, filtered, and subjected to phytochemical analysis. The secondary metabolites such as alkaloids, tannins, flavonoids, steroids, saponins, phenols, coumarins, glycosides, acids, proteins, and sugar were qualitatively analyzed and tabulated. Powdered samples are taken as such for the analysis. Physicochemical parameters such as alcohol and water-soluble extractive values, pH, total ash, acid-insoluble ash, and loss on drying were quantitatively analyzed based on the standard procedures in Ayurvedic Pharmacopeia of India (API) methods.

Instrumentation

High-performance thin-layer chromatography

This technique was used to identify the presence of the marker compound in the plant extracts and for the chemical profiling of the extracts. The study was performed using CAMAG automatic thin-layer chromatography (TLC) sampler-4, 10 × 10 cm twin trough glass chambers, TLC visualizer, and scanner 3 for developing, imaging, and scanning the TLC plates. The process was supported by Win CATS software 1.4.4. Precoated silica gel aluminum plates 60F254 (E. Merck, Darmstadt, Germany) with thickness 0.2 mm were used for all determinations.

High-performance liquid chromatography

The marker compound in the plant extract was quantified using Agilent (1200 series) HPLC system with a manual sample injector. It was equipped with a quaternary pump, degasser unit with diode array detector (DAD), and variable wavelength detector (VWD). Chemstation32 Software was used for data generation. A calibration graph was prepared with reference standards to quantify the percentage of the marker compound present in the seed of the plant used.

Gas chromatography-mass spectrometer

The chemical profiling of Datura metel L. seeds was carried out at VIT-SIF Lab, SAS, Chemistry Division for GC-MS Analysis, Vellore, Tamil Nadu. Perkin Elmer Clarus 680 model gas chromatograph coupled with Clarus 600 (E.I.) mass spectroscopy detector (GC-MS) was used.

Liquid chromatography-mass spectrometer

The analysis of Datura metel seeds was carried out at the Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM University, Kattankulathur, Chennai. Shimadzu 2020 L.C. system coupled with single quadruple mass spectrometer (LC-MS) was used.

Shodhana of Datura metal seeds

The Shodhana process of Datura metal seeds was performed using cow’s urine and cow’s milk as per the classical Ayurvedic texts.[19] The raw seeds of Datura metel were physically cleaned from dust and other foreign matter. Seeds were weighed and taken for the process. It was then fully soaked in gomutra (cow’s urine) for 12 h. After which, the seeds were removed from gomutra, cleaned in running water, and then tied in a piece of muslin cloth as a bundle. It was hung inside the mud pot without touching the bottom (Dola yantra), and a sufficient amount of godugdha (cow’s milk) was added to immerse it completely. The process of Swedana (boiling under a liquid bath) was performed for 3 h. After cooling, it was taken out, washed, and air-dried utterly. The testa (outer cover) of the dried seeds was then removed with the help of a blender. After separation, the purified seeds were powdered and used for further studies.[20],[21] The images are shown in [Figure 2].
Figure 2: Images of Shodhana process

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  Results and Discussion Top


Preliminary phytochemical analysis

Preliminary phytochemical screening results showed the presence or absence of certain phytochemicals in the Datura metel L. seeds.[18],[22],[23] The comparative qualitative test results of the secondary metabolite analysis in the processed and unprocessed seeds of Datura metel L. are tabulated in [Table 1].
Table 1: Preliminary phytochemicals screening of the processed and unprocessed Datura metel L. seeds

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Determination of physicochemical analysis

Physicochemical analysis was done to ascertain the quality of the raw material used in the study. The comparative results of the processed and unprocessed seeds of Datura seeds are given in [Table 2]. The results of all the unprocessed Datura seeds’ parameters comply with the API standards.[9],[24],[25],[26]
Table 2: Physicochemical parameters of the unprocessed and processed Datura metel L. seeds

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The percentage of water-soluble extractive, ash content, acid-insoluble ash, and loss on drying at 105°C was decreased in the processed sample compared with the unprocessed sample of Datura metel L. seeds. The percentage of alcohol-soluble extractive and pH value was increased in the processed sample compared with the unprocessed sample.

Sample preparation by extraction method

The extraction of processed and unprocessed Datura metel L. seed samples was performed simultaneously. Each sample was weighed in thimble around 8 g and subjected to hot Soxhlet extraction using 200 mL of ethanol as a solvent. The extract was then concentrated, dried, and stored in a refrigerator for future analysis. The same procedure was followed using chloroform as the solvent, and the extract was collected and stored. The weight of the obtained extracts was tabulated in [Table 3].
Table 3: Extractive values of the processed and unprocessed Datura metel L. seeds

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Chemical profiling of the processed and unprocessed Datura metel L. seeds

High-performance thin-layer chromatography chromatographic profiling

The ethanol and chloroform extracts of processed and unprocessed seed samples were taken and dissolved in 1 mL of methanol, filtered through 0.22 μ membrane filters, and used for HPTLC analysis. 30 μL of each test solution of ethanol extracts, and chloroform extracts in different tracks on a precoated silica gel 60 F254 TLC plate (E. Merck) of 0.2 mm thickness was given. The plate was developed in the suitable solvent system of toluene:chloroform:methanol (2:6:2) till the solvent rises to a distance of 8 cm.[27],[28],[29],[30]

The plate was observed through CAMAG TLC visualizer under ultraviolet (UV) at 254 nm and 366 nm, and images were documented. Finally, the plate was dipped in anisaldehyde-sulphuric acid reagent and heated in a hot air oven at 105°C until the color of the spots had appeared. The image was documented under white light, and the calculated Rf value details are given in [Figure 3] and [Table 4][Table 5][Table 6], respectively. Marker compound scopolamine spot is not visible in the TLC plats.
Figure 3: HPTLC fingerprint profiling of the processed and unprocessed Datura metel seeds’ chloroform and ethanol extracts

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Table 4: Rf values of the processed and unprocessed Datura metel L. seeds’ chloroform and ethanol extracts at UV 254 nm

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Table 5: Rf values of the processed and unprocessed Datura metel L. seeds’ chloroform and ethanol extracts at UV 366 nm

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Table 6: Rf values of the processed and unprocessed Datura metel L. seeds’ chloroform and ethanol extracts at 540 nm

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Before derivatization, the plate was scanned under UV at 254 and 366 nm using deuterium and mercury lamps, respectively. The plate has been scanned at 540 nm using a tungsten lamp after derivatization. The Rf values and fingerprint data were recorded by WIN CATS software. Details of the HPTLC fingerprint profiling at UV at 254 nm are given in [Figure 4].
Figure 4: HPTLC fingerprint profiling of the processed and unprocessed Datura metel L. seeds’ chloroform and ethanol extracts at UV 254 nm

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High-performance liquid chromatography profiling of Datura metel seeds

Chloroform and ethanol extracts of the Datura metel seeds of the processed and unprocessed samples are evaluated under similar chromatographic conditions.[31],[32] A known quantity of each extract is taken separately and was made up to the volume of 5 mL using HPLC grade methanol. The solution was then filtered through a 0.22 µ filter and used for further analysis. The chromatographic conditions are given below:

  • ✓ Column type: ZORBAX Eclipse XBD-C18 (4.6 mm × 150 mm), 5 µm particle size


  • ✓ Mobile phase: Phosphate buffer:methanol (85:15)


  • ✓ Detection: DAD detector @ 230 nm


  • ✓ Flow rate: 0.8 mL/min


  • ✓ Injection volume: 10 μL


Comparing the chloroform extracts from the above data, 13 peaks in the unprocessed and 10 peaks in the processed samples were detected. The 13 peaks were detected in the processed and unprocessed samples of each ethanol extract. It is observed that the peak area of all peaks of the processed samples was reduced compared with the unprocessed samples. The detailed peak identification and peak area results are shown in [Figure 5] and [Figure 6] and [Table 7] and [Table 8]. The remarkable changes in the area of both processed and unprocessed samples have been observed in the chloroform and ethanol extracts of Datura metel seeds.
Figure 5: HPLC profiling chromatogram of the chloroform extract of the processed and unprocessed Datura metel L. seeds

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Figure 6: HPLC profiling chromatogram of the ethanol extract of the processed and unprocessed Datura metel L. seeds

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Table 7: HPLC peak details of the chloroform extract of the processed and unprocessed Datura metel L. seeds

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Table 8: HPLC peak details of the ethanol extract of the processed and unprocessed Datura metel L. seeds

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Gas chromatography-mass spectrometer profiling of Datura metel L. seeds

To prepare the test solution, the dried chloroform extracts of Datura metel seeds were dissolved in chloroform and methanol solvents of the desired volume.[33] It is filtered and sent for GC-MS analysis.

The chloroform extracts of the Datura metel seeds in the processed and unprocessed samples are compared under the same chromatographic conditions. GC-MS analysis of the chloroform extracts of Datura metel seeds shows eight peaks in the unprocessed sample and 13 peaks in the processed sample. The detailed peak identification is shown in [Figure 7], and the retention time, peak area, area percentage, compound name, and molecular weight are given in [Table 9]. The remarkable changes have been observed in the chloroform extracts of GC-MS profiling chromatograms of the Datura metel seeds.
Figure 7: GC-MS chromatogram of the chloroform extracts of the processed and unprocessed Datura metel seeds

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Table 9: GC-MS peak details of the chloroform extract of the processed and unprocessed Datura metel L. seeds

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Liquid chromatography-mass spectrometer chromatographic profiling of Datura metel L. seeds

Before and after the Shodhana process, 20 mg of each ethanol extract was dissolved in 1.0 mL methanol solvents to prepare the test solution.[34] It is filtered and sent for LC-MS analysis.

The ethanol extracts of the Datura metel seeds before and after the Shodhana process, the processed and unprocessed samples, are compared under the same chromatographic conditions. LC-MS analysis of the ethanol extracts of Datura metel seeds shows 20 peaks in the unprocessed sample and 17 peaks in the processed sample. The detailed peak identification is shown in [Figure 8], and the retention time, peak area, and area percentage are given in [Table 10]. The remarkable changes have been observed in the ethanol extracts of LC-MS profiling chromatograms of the Datura metel seeds before and after the Shodhana process.
Figure 8: LC-MS chromatogram of Datura metel L. seeds’ ethanol extracts before and after the Shodhana process

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Table 10: LC-MS peak details of Datura metel L. seeds’ ethanol extracts before and after the Shodhana process

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Quantitative estimation of scopolamine in the processed and unprocessed Datura metel L. seeds

The ethanol extracts of the processed and unprocessed seed samples were weighed in triplicate and dissolved in 1 mL of methanol, filtered through 0.22 μ membrane filters, and used for HPLC analysis; 2.4 mg of scopolamine (≥98%) was accurately weighed and added to a 10 mL volumetric flask, dissolved in HPLC grade methanol, and the volume was made up to 10 mL to obtain 0.24 mg/mL of scopolamine stock solution. Further dilutions are made to get a concentration of 0.120, 0.060, 0.030, and 0.0150 mg/mL of scopolamine.[35],[36] A calibration graph was established for peak area versus the concentration of scopolamine applied is shown in [Figure 9].
Figure 9: HPLC chromatogram of the scopolamine standard and calibration curve

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The chromatographic conditions for the analysis were given below:

  • ✓ Column: ZORBAX Eclipse XBD-C18 (4.6 mm × 150 mm), 5 µm particle size


  • ✓ Detection: DAD detector at 205 nm


  • ✓ Mobile phase: Phosphate buffer:methanol (85:15)


  • ✓ Flow rate: 2.0 mL/min


  • ✓ Injection volume: 10 μL


  • ✓ Mode of operation: Isocratic elution


  • ✓ Retention time: 3.237.


The calibration was repeated five times at different conditions to confirm the method. 29.2 mg of the unprocessed and 40.0 mg of the processed ethanol extract of Datura seeds were taken and dissolved in 1 mL of methanol, filtered through 0.22 μ membrane filters, and used for HPLC analysis. Each 10 μL of the prepared ethanol extract solution was injected, and the corresponding chromatogram was recorded. The amount of scopolamine present in the extract was calculated, and the values are given in [Table 11].
Table 11: Estimation of scopolamine in the ethanol extracts of the processed and unprocessed Datura metel L. seeds

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From the above data, it is shown that the scopolamine content was completely removed in the process of detoxification (Shodhana). Hence, scopolamine is quantified only in the unprocessed ethanol extract sample. The results given above are the means of the triplicate analysis. Also, scopolamine was not detected in both processed and unprocessed samples of the chloroform extracts. Hence, the data were not given. [Figure 10] shows the chromatogram of ethanol extracts of the processed and unprocessed samples along with the standard. Purification of Datura metel seeds was performed by the traditional shodhana process. The study showed significant and remarkable changes in different physicochemical parameters and chromatographic profiling of Datura metel seeds before and after the shodhana. There was a significant reduction in the peak area of all peaks for processed (shodhit) samples as compared to unprocessed (Ashodhit) samples in HPTLC, HPLC, GC-MS, and LC-MS profiling.
Figure 10: Estimation of scopolamine in the ethanol extracts of the processed and unprocessed Datura metel L. seeds

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In case of HPLC studies revealed that, the main poisonous constituent of Datura metel seeds, that is scopolamine was not found detectable after shodhana process, it indicates that the percentage of scopolamine in ethanol extract reduced nearby 100% after shodhana. While studies on sophisticated techniques viz. LC-MS and GC-MS showed remarkable reduction of scopolamine content in processed sample of Datura metel seeds. The present study reflects the importance of Shodhana of poisonous plant drugs by means of which one can lessen the toxic constituents.


  Conclusion Top


In the current study, an attempt has been made to purify the Datura metel seeds by the classical Ayurvedic Shodhana process. The percentage of physicochemical parameters such as water-soluble extractive, ash content, acid-insoluble ash, and loss on drying at 105°C were decreased in the processed sample compared with the unprocessed sample of Datura metel seeds. The percentage of alcohol-soluble extractive and pH value was increased in the processed sample.

The remarkable changes have been observed in different physicochemical parameters, HPTLC, HPLC, GC-MS, and LC-MS chromatographic profiling of before and after Shodhana process in the Datura metel seeds. In HPLC chromatographic profiling, it is observed that the peak area of all peaks of the processed samples was reduced compared with the unprocessed samples.

The quantification results obtained from HPLC analysis showed the complete depletion of toxic alkaloid scopolamine in the processed sample compared with the unprocessed Datura metel seeds. In this study, the complete removal of scopolamine reflects the importance of the Shodhana process in the seeds of Datura metel L., using which the toxic effects are removed.

Acknowledgment

The authors are very grateful to the Director-General, CCRAS, Ministry of Ayush, New Delhi, for providing encouragement and facilities for carrying out this work.

Financial support and sponsorship

IMR project funded by CCRAS, Ministry of Ayush, Government of India.

Conflicts of interest

There are no conflicts of interest.





 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11]



 

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