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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 3  |  Page : 162-176

Assessing the effect of Shodhana (detoxification) process using chromatographic profiling (HPTLC, HPLC, LC-MS, and GC-MS) and estimation of toxic content Abrine in Abrus precatorius L. (Gunja) seeds


1 Regional Ayurveda Research Institute (RARI), CCRAS, Gwalior, Madhya Pradesh, India
2 Captain Srinivasa Murthy Central Ayurveda Research Institute, CCRAS, Chennai, Tamil Nadu, India
3 Central Council for Research in Ayurvedic Sciences, New Delhi, India
4 Central Ayurveda Research Institute, CCRAS, Kolkata, West Bengal, India
5 Central Council for Research in Ayurvedic Sciences, Janakpuri, New Delhi, India
6 Department of Shalakya Tantra, Faculty of Ayurveda, IMS, BHU, Varanasi, Uttra Pradesh, India

Date of Submission21-Jul-2021
Date of Acceptance22-Dec-2021
Date of Web Publication25-Mar-2022

Correspondence Address:
Dr. Ajay Kumar Meena
Regional Ayurveda Research Institute, CCRAS, Ministry of AYUSH, Government of India, Gwalior, Madhya Pradesh.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdras.jdras_10_21

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  Abstract 

BACKGROUND AND OBJECTIVE: Abrus precatorius L. (Gunja) is popular in Ayurveda system of medicine for its seeds, which are toxic because of the presence of Abrine–an alkaloid. It is used for the management of skin disease (Kustha), itching (kandu), cough (kasa), wound (vrana), and alopecia (indralupta). The shodhana process in Ayurveda is used for the purification and detoxification of toxic plant materials. This study aimed to observe the effect of the shodhana (purification) on phytochemicals and the Abrine content of A. precatorius L. (Gunja) seeds. MATERIALS AND METHODS: The ethanolic and chloroform extracts of A. precatorius L. (Gunja) seeds are used to determine the Rf value through high-performance thin-layer liquid chromatography (HPTLC) technique. Chemical profiling of seeds was carried out using sophisticated modern chromatographic different such as HPTLC, high-performance liquid chromatography (HPLC), gas chromatography with mass spectrometry (GC-MS), and liquid chromatography with mass spectrometry (LC-MS). RESULTS: The Rf value 0.45 corresponds to Abrine and confirms its presence in Gunja seeds. GC-MS and LC-MS results show the presence of 19–22 compounds. Results of HPLC analysis showed that Abrine content in chloroform extract and ethanol extract reduced to 30.36% and 10.20%, respectively, after shodhana process. CONCLUSIONS: These results showed the significance and effectiveness of shodhana process in reducing the toxins present in A. precatorius.

Keywords: Abrine, Abrus precatorius, detoxification, GC-MS, HPLC, LC-MS, seeds, shodhana


How to cite this article:
Meena AK, Venktaraman P, Ganji K, Kumar N, Singh R, Dixit AK, Ilavarasan R, Srikanth N, Dhiman KS. Assessing the effect of Shodhana (detoxification) process using chromatographic profiling (HPTLC, HPLC, LC-MS, and GC-MS) and estimation of toxic content Abrine in Abrus precatorius L. (Gunja) seeds. J Drug Res Ayurvedic Sci 2021;6:162-76

How to cite this URL:
Meena AK, Venktaraman P, Ganji K, Kumar N, Singh R, Dixit AK, Ilavarasan R, Srikanth N, Dhiman KS. Assessing the effect of Shodhana (detoxification) process using chromatographic profiling (HPTLC, HPLC, LC-MS, and GC-MS) and estimation of toxic content Abrine in Abrus precatorius L. (Gunja) seeds. J Drug Res Ayurvedic Sci [serial online] 2021 [cited 2022 Sep 27];6:162-76. Available from: http://www.jdrasccras.com/text.asp?2021/6/3/162/340868




  Introduction Top


Abrus precatorius L. (Gunja; Rosary pea or Jequirity bean) is an herbaceous woody twining flowering plant of Fabaceae family.[1] Its seeds are highly toxic and red in color with black spots at the base and leaves resemble with that of tamarind leaves with 20–30 leaflets, as shown in [Figure 1]A and B. Abrus precatorius L. is the poisonous plant reported in ancient scriptures of Ayurveda but presence of a toxic alkaloid Abrine limits its use.[2],[3] This plant is mainly found in South Africa, Brazil, and Indian continent.[4],[5],[6] In terms of toxicity, intake of well-chewed single seed may be fatal for mammals. It is being used as a fish poison, arrow poison, and poisoning both humans and cattle since ancient times.[7] In addition to this, its seeds are rich source of essential amino acids, that is, tryptophan, serine, and hederagenin, kaikasaponin III, sophoradiol, Abrusin,[8] trimethyl,[9] and alanine.[10] The chief chemical constituents of gunja seeds are Abrine, abraline, and hypaphorine.[11] Seeds are the official part of A. precatorius L. In Ayurveda, it is used for the management of skin disease (Kustha), itching (kandu), cough (kasa), wound (vrana), and alopecia (indralupta). The purified seed is also known for its neurostimulant properties and is widely used in joint pains and paralysis.
Figure 1: Abrus precatorius L. seeds (A) attached with plants and (B) collected seeds

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A few studies have reported the effect of shodhana process of Gunja seeds, and its acute toxicity before and after shodhana process.[12],[13] Very few reports are available on liquid chromatography with mass spectrometry (LC-MS) and gas chromatography with mass spectrometry (GC-MS) data for Gunja seeds. Therefore, an attempt was made regarding detailed chemical profiling of compounds present in A. precatorius L. seeds via high-performance thin-layer liquid chromatography (HPTLC), high-performance liquid chromatography (HPLC), GC-MS, and LC-MS techniques. This study also evaluated the impact of the shodhana process (purification) on Gunja seeds by estimating the abrine content using HPLC. This study may lead to some valuable research for the future of the Indian system of medicines.


  Materials and Methods Top


Plant collection, reagents, and chemicals

The seeds of A. precatorius L. were procured from local herbal drugs market of Chennai, India. The authentication of the seeds has been done by the botanist of CSM- Regional Ayurveda Drug Development Institute, CCRAS, Ministry of Ayush, Chennai, with the help of flora. All chemicals and solvents were of Analytical and HPLC grade. L-Abrine standard was procured from sigma Aldrich.

Shodhana procedure

In this study, we carried out shodhana of A. precatorius L. seeds by the classically approved methods. Approximately 100 g of raw A. precatorius seeds were cleaned and weighed. The seeds were tied within a muslin cloth piece and it has been suspended in a mud pot in such a way that it would not touch the bottom of the pot, and a sufficient amount of freshly prepared Kanji (fermented liquid) was added to immerse it completely. The pot was heated continuously for 3 h in Dolayantra and allowed to cool down steadily. It was then taken out, washed, and air-dried completely. Upon washing, the embryo of the seed was removed. The testa (outer cover) of the seeds were separated with iron mortar and pestle. The seeds were separated, weighed, powdered, and stored for further studies.[14],[15] The images of the process are shown in [Figure 2].
Figure 2: Shodhana (purification) process of Abrus precatorius seeds

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Physicochemical and phytochemical analysis

Phytochemical constituents, that is, alkaloids, coumarins, flavonoids, glycosides, phenols, proteins, saponins, steroids, sugar, tannins, and physicochemical parameters were determined as per standard procedures. The standardization and quality measures are followed as mentioned in Indian Pharmacopoeia methods and WHO guidelines.[16],[17],[18],[19]

Test solution and standard solution preparation

10 g of powdered raw and purified seeds were taken in round bottom flask; 250 mL of ethanol was added. Soxhlet apparatus was used for extraction on a water bath for 6 h. The extracted material was collected, dried, and used for analytical studies. The same procedure was performed with chloroform. A known quantity of each extract was dissolved in known volume of methanol and filtered through 0.22 micron membrane filter before using for HPTLC and HPLC analysis. For the preparation of the standard solution, 0.29 mg/mL Abrine stock solution was prepared in methanol.

High-performance thin-layer liquid chromatography instrumentation, development, and scanning

HPTLC profiling was done using CAMAG made HPTLC system with inbuilt autosampler, TLC Scanner-3 linked with WINCATS software version 1.4.4 (Muttenz, Switzerland).

The 10 μL of samples, that is, test and standard, were applied on different tracks in a E. Merck Aluminium plate precoated with silica gel 60F254 of size 10 cm × 10 cm, and 0.2 mm thick. Samples were applied on the plates by using a Linomat-IV applicator fitted with a 100 μL syringe as bands 10 mm wide. Linear ascending development to a distance of 80 mm with n-Butanol: Acetic Acid: Water (7:3:1 v/v/v) as mobile phase for both chloroform and ethanol extract was performed in a twin-trough CAMAG TLC glass chamber (20 cm × 10 cm) previously saturated with vapours of mobile phase for exact 20 min.[20] The developed plate was dried with a dryer and visualized through CAMAG-TLC visualizer under UV at 254 and 366 nm. Finally, the plate was dipped in anisaldehyde sulphuric acid reagent (derivatizing agent) and heated in a hot air oven at 105°C until the colour of the spots appeared (max. 23 min.), and the photo was obtained under white light. The plate was scanned at deuterium lamps UV wavelengths of 254 nm and 366 nm (CAMAG TLC Scanner with WINCATS software) before derivatization and after derivatization, the plate was again scanned using tungsten lamp at 540 nm.

High-performance liquid chromatography profiling of Abrus precatorius seeds

HPLC profiling was done using Agilent 1200 series HPLC system. Filtration of all samples and standards was done with 0.22 μm filters before manual injection. Separation was carried onC18Eclipse, XDB (4.6 mm × 150 mm) 5 μm particle size Agilent Column. Phosphate buffer: methanol (80:20 v/v) were used as a mobile phase in anisocratic elution mode with a flow rate of 0.8 mL/min. The test solution 10.0 μL injected in to the HPLC system. The temperature of the column was 40°C. Diode array detector (DAD) was used for the detection of analytes.

Liquid chromatography–mass spectrometry profiling of Abrus precatorius seeds

The chemical constituents of the ethanol extract of A. precatorius seeds were determined by using the LC-MS system. The analysis was done using LC-MS 2020 equipped with LC10 A D V P binary pump (Shimadzu, Japan) and DAD with an ESI source. Full-scan mode from m/z 100 to 1000 was performed with a source temperature of 150°C. Ethanol extracts of A. precatorius seeds before (6.1 mg) and after (6.0 mg) shodhana process in methanol upto 1.0 mL volume were used as test solution. It is filtered with a 0.22 µm nylon filter. The flow rate of solvents were maintained at 0.8 mL/min with isocratic elution. The mass spectrophotometer spectra were obtained in the positive ion mode. The temperature was set for the curved desolvation line (CDL) with heat block at 280–320°C, and nitrogen was used for nebulisation with a 1.5 mL/min flow rate.

Gas chromatography-mass spectrometry profiling of Abrus precatorius seeds

GC-MS studies of the chloroform extract of A. precatorius seeds was performed through G.C. (Perkin Elmer Model Clarus 680) coupled with Clarus 680 (E.I.) mass analyzer. The interpretation of GC-MS data was conducted by comparison with National Standard and Technology (NIST) library 2008 database.

Quantitative estimation of Abrine in Abrus precatorius seeds by high-performance liquid chromatography

Excellent separations and suitable retention time of A. precatorius seeds were obtained in isocratic elution. Separation was carried on ZORBAX Eclipse XBD- C18 (4.6 x 150 mm), 5 μm particle size column. Phosphate buffer:acetonitrile (80:20 v/v) was used as a mobile phase at a flow rate of 0.9 mL/min. The detection was carried out by using DAD at 220 nm. 10 μL of the test solution was injected in to the HPLC system. The column temperature was kept at 40°C.

Calibration curve and estimation of Abrine

L-Abrine stock solution was diluted in different proportions to get a concentration of 0.232, 0.188, 0.145, and 0.101 mg/mL of L-Abrine and the same run on HPLC and respective peak areas were recorded. A calibration curve (in situ was prepared for peak area. 10 μL of the test solution was injected to the HPLC system. Chromatograms were recorded and concentration of Abrine was determined using the peak area of the test solution corresponding to standard calibration curve of Abrine.


  Results and Discussion Top


Preliminary phytochemicals analysis

The results of qualitative phytochemicals screening revealed that all phytochemicals are present before and after the shodhana process in A. precatorius L. seeds except sugar. The list of phytochemicals analyzed is tabulated in [Table 1].
Table 1: Preliminary phytochemicals of purified and unpurified Abrus precatorius seeds

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Physicochemical analysis

The physicochemical analysis was performed for quality assurance of the raw materials [Table 2]. The pH value, water-soluble extractive, and alcohol-soluble extractive values were reduced in the processed (purified) samples compared to unprocessed (unpurified) A. precatorius seeds samples. The percentage of acid insoluble ash, ash content, and loss on drying (at 105°C) were increased in the purified samples as compared to the unpurified samples.
Table 2: Physicochemical parameters of purified and unpurified Abrus precatorius seed

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High-performance thin-layer liquid chromatography analysis

The comparative phytochemical analysis using HPTLC ultraviolet detection method for A. precatorius seeds was not studied before. Phytochemical fingerprints for chloroform and ethanol extract of gunja in purification process (before and after) are shown in [Figure 3]. Different bands and color observed at 254 nm, 366 nm, and 540 nm for each extracts are tabulated in [Table 3]A,[Table 3]B–[Table 3]C. A suitable solvent system n-butanol:acetic acid:water (7:3:1) for Abrine standard and extract was optimized and eluted along with test samples. A dark band at Rf 0.45 corresponds to Abrine standard, which was clearly visible at 254 nm and at 540 nm derivatised in anisaldehyde sulphuric acid solution. Ethanol extract confirms the presence of Abrine in A. precatorius seeds. The fingerprint profile of the extracts is given in [Figure 4]A–C.
Figure 3: HPTLC profiling of Abrus precatorius seeds in chloroform and ethanol extracts and Abrine reference standard Rf

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Table 3A: HPTLC Rf values of Abrus precatorius seed ethanol and chloroform extracts in shodhana process and Abrine standard at 254 nm

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Table 3B: HPTLC Rf values of Abrus precatorius seed ethanol and chloroform extracts in shodhana at 366 nm

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Table 3C: HPTLC Rf values of Abrus precatorius seed ethanol and chloroform extracts in shodhana and Abrine standard at 540 nm

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Figure 4: (A) HPTLC chromatograms of Abrus precatorius seeds chloroform and ethanol extracts and Abrine standard at UV 254 nm. (B) HPTLC chromatograms of Abrus precatorius seeds chloroform and ethanol extracts and Abrine standard at UV 366 nm. (C) HPTLC chromatograms of Abrus precatorius seeds chloroform and ethanol extracts and Abrine standard at UV 540 nm

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High-performance liquid chromatography analysis

In HPLC profiling of chloroform extracts, 17 peaks in unprocessed and 11 peaks in processed samples were detected. 13 and 15 peaks in processed and unprocessed samples of each ethanol extract, respectively, were obtained. It has been shown that the corresponding peak areas of all the peaks of processed samples were reduced as compared to unprocessed samples. Significant changes were obtained in the chloroform and ethanol extracts HPLC chromatograms of the A. precatorius seeds in shodhana [Figure 5] and [Figure 6] and [Table 4] and [Table 5].
Figure 5: HPLC chromatogram of Abrus precatorius seeds chloroform extracts (A) before (unprocessed) and (B) after (processed) shodhana

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Figure 6: HPLC profiling of Abrus precatorius seeds ethanol extracts (A) before (unprocessed) and (B) after (processed) shodhana

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Table 4: HPLC peak details of Abrus precatorius seed chloroform extracts in shodhana

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Table 5: HPLC peak of Abrus precatorius seed ethanol extracts in shodhana

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Gas chromatography-mass spectrometry profiling of Abrus precatorius seeds

GC-MS studies of the chloroform extract of A. precatorius seeds lead to the identification of several compounds through mass spectrum. The lists of various components found in the seeds of A. precatorius analyzed by GC-MS are shown in [Table 6]A and [Table 6]B. The chloroform extracts of A. precatorius seeds before and after shodhana were compared under the same chromatographic conditions. GC-MS analysis of chloroform extracts of A. precatorius showed 19 peaks in both unprocessed processed sample. There were remarkable and significant changes observed in the chloroform extracts chromatograms of A. precatorius seeds during shodhana. The composition determined for this chloroform extract corresponds to 89.53% of the entire GC-MS chromatogram. The GC-MS chromatogram is illustrated in [Figure 7]. The mass spectrometer generally analyzes and identifies the nature and structure of the compounds eluted at different times. In this MS analysis larger compounds fragments into very small compounds according to different m/z ratios. Results suggest that this study helps to predict the compounds with molecular weight of 19 biomolecules.
Table 6A: GC-MS peak details of Abrus precatorius seed chloroform extracts before (unprocessed) shodhana

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Table 6B: GC-MS peak details of Abrus precatorius seed chloroform extracts after (processed) shodhana

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Figure 7: GC-MS profiling of Abrus precatorius seeds chloroform extracts (A) before (unprocessed) and (B) after (processed) shodhana

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Liquid chromatography–mass spectrometry chromatographic profiling of Abrus precatorius seeds

LC-MS analysis with full-scan mode from m/z 100 to 1000 was performed with a source temperature of 150°C. The test solution was made by dissolving ethanolic extract of A. precatorius seeds before (6.1 mg) and after (6.0 mg) shodhana process in methanol up to 1.0 mL volume. It is filtered with a 0.22 µm nylon filter and used for LC-MS analysis. Solvents were delivered at 0.8 mL/min flow rate with isocratic elution and the mass spectra were obtained in the +ve ion mode. The temperature of nitrogen (N2) gas (drying) was 320°C at 5 mL/min flow rate. This study on ethanol extracts of unprocessed and processed sample of A. precatorius seeds showed the presence of 17 peaks and 22 peaks, respectively [Table 7] and [Figure 8].
Table 7: LC-MS peak details of Abrus precatorius seed ethanol extracts in shodhana

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Figure 8: LC-MS profiling of Abrus precatorius seeds ethanol extracts (A) before (unprocessed) and (B) after (processed) shodhana

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Quantitative estimation of Abrine biomarker in Abrus precatorius seeds by high-performance liquid chromatography

In isocratic elution mode, excellent separations with proper retention time of A. precatorius seeds were obtained. HPLC chromatogram of the extract showed a smooth baseline with a good resolution and marker peak was properly identified. The calibration curve was made by measuring the peak area, which was linear (R2 > 0.99624) in the concentration range of 0.101–0.232 mg/ml. The marker compound Abrine showed at retention time 2.222 for standard and 2.242 [Figure 9] for formulation in both extract’s chromatograms before and after shodhana. For the rest of the other compound identification, the LC-MS technique was used. [Figure 10] and [Table 8] show the amount of Abrine in extracts, that is, ethanol, chloroform, for each test sample obtained during shodhana process. The results of the HPLC analysis showed remarkable and significant decrease Abrine content after the shodhana process of A. precatorius seeds. HPLC analysis revealed that the percentage of Abrine in chloroform and ethanol extract reduced by 30.36% and 10.20%, respectively, after shodhana.
Figure 9: HPLC chromatogram of standard Abrine and calibration curve (in situ)

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Figure 10: Analysis of Abrine in the Abrus precatorius seeds chloroform and ethanol extracts during shodhana process (EB = ethanol before shodhana extract, EA = ethanol after shodhana extract, STD = Abrine standard, CB = chloroform before shodhana extract, CA = chloroform after shodhana extract)

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Table 8: Estimation of Abrine in the Abrus precatorius seed during shodhana

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Conclusion

Purification of A. precatorius seeds was performed by the classical Ayurvedic shodhana process. The study showed significant and remarkable changes in different physicochemical parameters and chromatographic profiling of A. precatorius seeds before and after the shodhana. There was a significant reduction in the peak area of all peaks for processed (purified) samples as compared to unprocessed samples in HPLC profiling. In the HPLC chromatogram of A. precatorius seeds, the peak of standard Abrine was observed at 227 nm wavelength at a retention time of 2.223 min. HPLC studies also revealed that the chief poisonous constituent of A. precatorius seeds, that is, Abrine has been reduced after shodhana. The HPLC analysis revealed that the percentage of Abrine in chloroform and ethanol extract reduced by 30.36% and 10.20%, respectively, after shodhana. Further studies related on isolation of nutraceuticals compounds of A. precatorius seeds along with their structural elucidation will be helpful for future drug development.

Acknowledgement

The authors are very thankful to the Director General, CCRAS, for providing encouragement and facilities. The authors are also thankful to VIT-SIF Lab, Chemistry Division, Vellore, Tamil Nadu and Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM University, Chennai for GC-MS and LC-MS analysis.

Financial support and sponsorship

Nil.

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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