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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 8
| Issue : 2 | Page : 181-189 |
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Determination of total alkaloid content in unpurified Dhattura beeja (Datura metel L. seeds) and purified Dhattura beeja (D. metel L. seeds) by UV-spectroscopic method
Poornachandra Tejaswini1, Hassan Rangaswamy Pradeep2, Divya Khare3, Avinash Math4
1 ALN Rao Memorial Ayurvedic Medical College, Koppa, Chikmagalur District, Karnataka, India
2 Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa, Chikmagalur District, Karnataka, India
3 Department of Dravyaguna, KAHER’s Shri B. M. Kankanawadi Ayurveda Mahavidyalaya, Belagavi, Karnataka, India
4 Department of Biochemistry, USM KLE International Medical Programme, Nehru Nagar, Belagavi, Karnataka, India
| Date of Submission | 09-Jun-2022 |
| Date of Acceptance | 26-Nov-2022 |
| Date of Web Publication | 31-Mar-2023 |
Correspondence Address:
Dr. Divya Khare
Department of Dravyaguna, DTL, Central Research Facility, KAHER’s Shri B. M. Kankanawadi Ayurveda Mahavidyalaya, Belagavi, Karnataka
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/jdras.jdras_92_22
BACKGROUND: Dhattura herbal tea has been reported for toxicity, which is attributed to use without purification. Purification of Dhattura as per classics is essential as it contains toxic alkaloids. The present study highlights the role of classical Shodhana procedures in rendering the drug safe for therapeutics. METHODS: Dhattura seeds were collected and purified by Swedana (sudation) using Goksheera (cow milk) and Gomutra (cow urine). The unpurified and purified Dhattura seeds were analyzed for alkaloid content by UV spectroscopy. RESULTS: Unpurified Dhattura seeds contained 40.83 mg atropine equivalent (AE)/100 g of drug. Purification using Goksheera reduced alkaloid content to 20.46 mg AE/100 g of drug, and purification using Gomutra resulted in 26.45 mg AE/100 g of drug. CONCLUSION: UV spectroscopy is an effective method to determine the total alkaloid content in the raw and purified Dhattura seeds. This study supports that the purificatory methods stated in Ayurveda are effective in reducing atropine levels in Dhattura seeds. Keywords: Alkaloid, Ashodhita, Dhattura beeja, Shodhita, UV-spectroscopic method
How to cite this article:
Tejaswini P, Pradeep HR, Khare D, Math A. Determination of total alkaloid content in unpurified Dhattura beeja (Datura metel L. seeds) and purified Dhattura beeja (D. metel L. seeds) by UV-spectroscopic method. J Drug Res Ayurvedic Sci 2023;8:181-9 |
How to cite this URL:
Tejaswini P, Pradeep HR, Khare D, Math A. Determination of total alkaloid content in unpurified Dhattura beeja (Datura metel L. seeds) and purified Dhattura beeja (D. metel L. seeds) by UV-spectroscopic method. J Drug Res Ayurvedic Sci [serial online] 2023 [cited 2023 Jun 10];8:181-9. Available from: http://www.jdrasccras.com/text.asp?2023/8/2/181/373022 |
| Introduction | |  |
The herbal drug industry is flourishing today because of increasing awareness among the public about Ayurveda and other traditional systems of medicine. Though the herbal medicine is considered safe, it is a fact that many visha dravyas (drugs that can exert toxic effects) are used in Ayurveda for treating certain diseases.[1] Drug safety is a major challenge for the Ayurvedic drug industries, as there are reports mentioning toxicity in Ayurvedic formulations. The classical texts of Ayurveda mention certain purification procedures (Shodhana) for such drugs before using them in therapeutics.[2] These procedures are aimed to nullify the harmful properties of drugs and make them biocompatible. But we need to ensure whether by the application of these purificatory techniques, the herbals are rendered safe or not. It is need of the hour to develop evaluation methods by applying advanced techniques of drug standardization. These will also help us to understand the pharmacokinetics and safety profile of herbal drugs/formulations.
Alkaloids are nitrogen-containing organic compounds. They are naturally present in plant kingdom and are pharmacologically active. They have a vast application and hence responsible for a major impact on plant medicine. Alkaloids derived from plants are one of the largest groups of natural products. They represent a very diverse class of chemical compounds. Alkaloids comprise of an enormous class of around 12,000 natural products.[3]Rasa being an important criterion for explaining drug action in Ayurveda, the drugs having Tikta (bitter) and Katu (pungent) taste are found to contain alkaloids. Dhattura is one such Tikta-Katu rasa pradhana dravya (contains bitter and pungent as predominant tastes), which is also mentioned under Visha dravyas (drugs with toxic properties). Its therapeutic actions are Vishaghna (anti-poisonous), Jwaraghna (anti-pyretic), Kushtaghna (cures skin diseases), Madakari (intoxicating effect), Deepana (carminative), Vrana-shothahara (anti-inflammatory), and krimighna (anthelmintic and anti-microbial).[4] The alkaloids present in Dhattura are responsible for many of its therapeutic actions and even the toxic effects. The present study was an attempt to see whether the classical Shodhana procedure by Goksheera and Gomutra is able to reduce the alkaloid content of Dhattura seeds. The total quantity of alkaloids in the Dhattura seeds was determined by the UV-spectrophotometric method. Many methods are reported for the determination of alkaloids, which include high-performance liquid chromatography (HPLC), fluorometry, ion chromatography, colorimetry, gas chromatography, and electrochromatography. Compared with other methods, UV spectrophotometry assay is less sensitive but is used widely for its simplicity and rapid determination. This method is based on the reaction of alkaloid with Bromocresol green (BCG), forming a yellow-colored product.
| Materials and Methods | | |
Procurement of raw material
Dhattura seeds were collected in and around Koppa. Authentication was done by a botanist, Department of Botany, Jagadguru Chandrashekhara Bharathi Mahaswamiji College, Sringeri (authentication number ALNC/05/2020).
UV spectrophotometer was procured from SHIMADZU CORPORATION, Kyoto, Japan, with model number UV-1800 240V, CAT number 206-25400-38, Serial number A11454804654 CD (220–240 V, 50–60 Hz, 140 VA).
Chemicals and reagents
The details of the chemicals used are depicted in [Table 1].
Shodhana (purification)
Shodhana (purification) of Dhattura seeds was done at the pharmacy attached with Aroor Lakshmi Narayan Rao Memorial Ayurvedic Medical College, Koppa. Dhattura seeds were purified by two methods as stated in the classics.
Method 1
The Shodhana of Dhattura seeds was carried out as per the method mentioned in Rasatarangini (24/346–47).[5],[6]
Materials required
Cotton cloth for Pottali (seeds tied in a cotton cloth to make a ball-like structure), Dolayantra set up (seeds tied in a cotton cloth and suspended in a vessel with a rod), Dhattura seeds—48 g, cow’s milk—5 L, warm water—quantity sufficient and gas stove with a proper regulator.
Forty-eight grams of raw Dhattura seeds was measured and tied in a cotton cloth to prepare a Vastra pottali (seeds tied in a cloth to make a ball). This pottali was tied to a rod with a thread and the rod was kept on the vessel so that the pottali was suspended in the vessel. This arrangement is called Dolayantra. Cow’s milk was poured inside the dolayantra until the pottali (cloth ball containing raw Dhattura seeds) was immersed in the milk. The length of the cord was adjusted in such a way that the bottom of the vessel was undisturbed. Then this arrangement was placed over a gas stove with medium flame and was subjected to Swedana with Godugdha for 3 h in this Dolayantra. After 25 min, the milk started to boil and the time was noted. For every 30 min, time was noted and the temperature of the apparatus was also recorded (a temperature of 144 ± 10 °C recorded during the procedure). As the quantity of milk reduced due to boiling, fresh milk was added frequently and stirred with a spatula. After 3 h (1 yama), the pottali was taken out of dolayantra. It was opened and the seeds were washed with warm water. The seeds were then dried [Figure 1][Figure 2][Figure 3][Figure 4][Figure 5][Figure 6]. Thus the purified Dhattura seeds were obtained. After proper drying, the seeds were weighed and the weight was 46 g.[6]
The second method was the same as method 1, but instead of cow’s milk, cow’s urine was used for purification in Dola yantra and the duration of Shodhana in this case was 12 h (3 yama) as per the reference [Figure 7][Figure 8][Figure 9][Figure 10][Figure 11][Figure 12].[6],[7] At the end of purification, the seeds were weighed and the weight was 38 g.
Determination of alkaloids
Determination of Alkaloids was carried out by the UV-spectrophotometric method.[8],[9]
Preparation of the sample
20 g each of raw Dhattura (RD) seeds, milk-processed Dhattura seeds (MDS), and cow-urine-processed Dhattura seeds (UDS) were grinded and then extracted with methanol for 24 h in a continuous extraction (Soxhlet) apparatus maintaining a temperature of 65 ± 1 °C throughout the extraction procedure. The extract was filtered and methanol was evaporated in a water bath to dryness.
Qualitative test for alkaloids
The presence of alkaloids was confirmed by Dragendroff’s method.[10] A part of the extract was dissolved in dilute HCl and two drops of Dragendroff’s reagent was added. A crystalline precipitate was formed, which indicated the presence of an alkaloid. The sample which showed a positive test for alkaloid was then subjected to further quantitative evaluation.
Preparation of reagents
Bromocresol green solution was prepared by heating 69.8 mg bromocresol green with 3 mL of 2N NaOH and 5 mL distilled water until it completely dissolved and the solution was diluted to 1000 mL with distilled water. Phosphate buffer solution (pH 4.7) was prepared by adjusting the pH of 2 M sodium phosphate (71.6 g Na2 HPO4 in 1 L distilled water) to 4.7 with 0.2 M citric acid (42.02 g citric acid in 1 L distilled water). Atropine standard solution was made by dissolving 1 mg of pure atropine (AR-grade procured from Sigma Company) in 10 mL distilled water.
Separation of alkaloid
About 10 mg of extract residue was dissolved in 10 mL 2N HCl and then filtered. An aliquot of 1 mL of this solution (with concentration 1 mg/mL) was transferred to a separating funnel and washed with 10 mL chloroform (10 mL × 3 times washes). The pH of this solution was adjusted to neutral with 0.1N NaOH. Then 5 mL of BCG solution and 5 mL of phosphate buffer were added to this solution. The mixture was shaken and the complex was extracted with 1, 2, 3, and 4 mL of chloroform by vigorous shaking. The extracts were then collected in a 10-mL volumetric flask and diluted with chloroform to adjust the volume to 10 mL.
Preparation of standard curve
Atropine standard solution of 100 µg/mL (1 mg atropine in 10 mL distilled water) concentration was used to prepare accurately measured aliquots of six different concentrations (20, 40, 60, 80, 100 and 120 µg/mL). The aliquots of atropine standard solution was transferred to different separating funnels. Then 5 mL of pH 4.7 phosphate buffer and 5 mL of BCG solution were taken and the mixture was shaken with 1, 2, 3, and 4 mL of chloroform. The extracts were then collected in a 10-ml volumetric flask and then diluted to adjust the solution volume to 10 mL with chloroform. The absorbance of the complex in chloroform was measured at a wavelength of 470 nm in a UV spectrophotometer (SHIMADZU UV-1800) against the blank (prepared by adding 5 mL of pH 4.7 phosphate buffer and 5 mL of BCG solution to 1 mL distilled water but without atropine).[8]
| Observations and Results | | |
Observations during the Shodhana procedure
In the first method of Shodhana, it took 5 L of milk to complete the procedure. At the end of the procedure, the milk turned yellowish brown in color and bitter in taste. At the end of Shodhana, a slightly yellowish thick cream was formed over milk and also around the pottali. After thorough washing with warm water and drying, the wings over the seeds were removed and the seeds became glossier.
In the second method, by the end of the procedure, Gomutra turned dark in color. After opening pottali, it was observed that the seeds were bulged and had acquired a strong odor of Gomutra.
All three samples of Dhattura seeds showed positive results for the qualitative test for alkaloids. Methanol extractive value showed that Dhattura beeja purified by Gomutra has a significantly higher extractive value [Table 2].
During spectrophotometric determination of alkaloids, the absorbance of standard at 470 nm ranged from 0.04 to 0.18 for different concentrations of atropine with an R2 value of 0.9635. A calibration curve was plotted for various concentrations of atropine [Figure 13].
The unpurified/raw Dhattura beeja showed a significantly higher content of alkaloids (40.83 mg AE per 100 g) than the purified Dhattura beeja, as shown in [Table 3]. The Dhattura seeds purified by Gomutra showed a lower concentration of atropine when compared with unpurified one, i.e., 26.45 mg AE per 100 g and the one processed with Goksheera showed the lowest concentration of 20.46 mg AE per 100 g of the drug. | Table 3: Mean values of the alkaloid content in three samples of Dhattura seeds
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| Discussion | | |
Ayurveda mentions about drugs with toxic effects under the heading of Mahavisha (poison) and Upavisha (moderate poison).[11] These drugs have to be used only after specific Shodhana (purification) procedures, which do not only detoxify the drugs but also help in potentiating their actions. Shodhana also helps in reducing the adverse reactions caused by consuming those drugs. Acharya Charaka opines that even the deadly poison can become a very good medicine if it is administered properly.[12] There are very few studies available so far which discuss the scientific validity and rationale behind adopting specific Shodhana procedures for herbal drugs. The present study explores the effect of classical Shodhana procedure by determining the total alkaloid content of raw and purified Dhattura seeds by the UV-spectrophotometric method.
Dhattura (Datura metel Linn.) has been mentioned under upavisha varga (moderate poison). It has also been mentioned under Schedule E (1) of the Drugs and Cosmetics Act, 1940, which is the list of poisonous substances under Ayurveda, Siddha, and Unani systems of medicine.[13]Datura stramonium L. seeds are highly toxic due to the presence of 28 various belladonna alkaloids in them. There are interspecies variations within Datura genus with respect to the concentration of alkaloids. Most of the side effects are due to blocking of peripheral muscarinic receptors of smooth muscle, cardiac muscle, and exocrine glands. Dryness of the mouth, excessive thirst, tachycardia, fever, erythema, dilatation of pupil, confusion, and disorientation are due to anti-cholinergic property of the alkaloids present in the plant. Though fatalities are rare, adverse reactions are very common due to Dhattura poisoning.[14] A comparative acute toxicity study of D. stramonium L. and D. metel L. has shown that D. metel L. produces less anatomical abnormalities.[15]
Alkaloids, which are the nitrogen-containing secondary metabolites, are found in many herbal drugs. They form one of the major groups of pharmacologically active phytochemicals and represent a diverse group of chemicals. Man has been using plant-based alkaloids such as herbal teas, potions, and so on, since many centuries.[3] Plants of Datura genus contain mostly tropane alkaloids such as atropine, hyoscyamine, and scopolamine. Also other alkaloids such as indole, beta-carboline, and pyrrolidine have been identified. Many Dhattura alkaloids are usually organic soluble in uncharged free base forms. Polar aqueous extracts of these can be basified (e.g., sodium hydroxide) and extracted with a semipolar solvent such as chloroform.[16]
Few studies so far have tried to analyze the alkaloid content in different species of Datura genus. Different quantitative determination techniques are being used in validating the classical Shodhana procedures. An analytical study of Datura innoxia Mill. and D. metel Linn. seeds before and after Shodhana revealed that there is a 70–90% reduction in hyosciamine content, whereas scopolamine reduced to almost zero. This study included thin layer chromatography and gas chromatography–mass spectrometry, apart from regular physicochemical and phytochemical analyses.[17] Another study by Gupta et al.[18] performed quantification of atropine and hyoscine in the seeds of D. metel L. before and after Shodhana using markers. The study concluded that there was a remarkable decrease in the quantity of marker compounds after purification and the results were depicted in terms of percentage.
Many methods such as HPLC, fluorometry, ion chromatography, colorimetry, gas chromatography, and electrochromatography are applied for the determination of alkaloids. In the present study, the total quantity of alkaloids in the Dhattura seeds was determined by the UV-spectrophotometric method. Compared with other methods, UV spectrophotometry assay is less sensitive but is used widely for its simplicity and rapid determination. This method is based on the reaction of alkaloid with BCG, forming a yellow-colored product, which can be extracted with chloroform. Through this study, it was found that unpurified/raw Dhattura beeja contained a significantly higher content of alkaloids (40.83 mg AE per 100 g) than the purified Dhattura beeja. A yellow-colored complex with maximum absorption was developed in the case of unpurified/raw Dhattura beeja. This complex was completely extractable by chloroform at pH 4.7. The Dhattura seeds purified by Gomutra (cow’s urine) showed an alkaloid content of 26.45 mg AE per 100 g, and the one processed with Goksheera (cow’s milk) showed 20.46 mg AE per 100 g of the drug. The sample purified using cow’s milk showed the lowest alkaloid content proving Goksheera (cow’s milk) to be more efficient media for detoxification.
Goksheera is mild in its properties in comparison with Gomutra. It has madhura rasa, madhura vipaka (sweet in taste and post-digestion), guru (heavy for digestion), sheeta (cold in potency), and snigdha (induces unctuousness), whereas Gomutra has contradictory properties such as katu-tikta-kashaya rasa (pungent, bitter, astringent tastes), tikshna (corrosive), laghu (light for digestion), ushna (hot in potency), and pittakara (increases pitta dosha) properties. Goksheera might have been able to reduce the alkaloid content more effectively due to its mild properties. Dhattura being one of the upavisha (moderate poison), the poisonous nature of the drug is nullified by Snigdha Sheeta and Madhura properties of cow milk. On the other hand, cow urine might have acted because of its Ruksha-Tikshna-Ushna properties which might have helped in reducing the poisonous nature of Dhattura seeds but not able to nullify it to the extent as seen in cow milk purified seeds.[19]
Apart from atropine, hyoscyamine and scopolamine, Dhattura contains other alkaloids such as withanolide type steroidal alkaloids. These withanolides isolated from leaves of Dhattura were evaluated against human lung cell carcinoma cells (A549) and human colorectal adenovarcinoma cells (DLD-1). They were found to block the cell cycle and induced apoptosis.[20]
Purification of drugs mentioned in the Ayurvedic classical literature must be resulting in reduction of such cytotoxic chemicals, thus making the drug more compatible. As observed in the present study, the Shodhana procedure was able to decrease the atropine alkaloids content, thus validating the classical methods of purification of drugs. At the same time, Shodhana procedure might have resulted in decrease of other pharmacologically active phytochemicals and minerals/heavy metals. But this was not assessed in the present study.
Further BCG can only react with a certain class of alkaloids (alkaloids that have nitrogen inside their structure). Amine or amide alkaloids do not react with this reagent. Hence, the obtained data may provide a gross understanding of total quantity of alkaloids in the drug. Furthermore, our study lacked quantification of different alkaloids in Dhattura seeds using reference standard phytochemicals. Also the exploration and observation of difference in the biological activity before and after purification of Dhattura seeds was not in the scope of our study.
Future aspect
The study will be repeated with multiple samples collected at different time points. Inclusion of quantitative estimation of standard alkaloid as a reference will be done.
| Conclusion | | |
Results presented in this study reveal that UV spectroscopy is an effective method to determine the total alkaloid content in the raw and purified Dhattura seeds. Furthermore, it proved that unpurified/raw Dhattura beeja contains higher amount of total alkaloids. The Dhattura beeja which was purified by Gomutra showed lower concentration of alkaloids, whereas the one processed with Goksheera contained alkaloids in lowest concentrations. The classical purification procedures were able to reduce the content of alkaloids present in the drug. This shows the relevance and importance of Shodhana (purification) procedures of herbal drugs to nullify the toxic effects. Further in-vivo studies could be conducted to support this by assessing the acute toxicity and organ toxicity of raw and purified Dhattura seeds in animals.
Acknowledgements
The authors are grateful to the Central Research Facility, KAHER’s Shri B. M. Kankanawadi Ayurved Mahavidhyalaya, Belagavi, for conducting the analysis. Also, we extend our sincere thanks to Dr. K. S. Sanjay, Principal, ALNRMAMC, Koppa, and the Principal, KAHER’s Shri B. M. Kankanawadi Ayurveda Mahavidyalaya, Belagavi, for their valuable support and encouragement.
Financial support and sponsorship
The project was funded by the Rajiv Gandhi University of Health Sciences, Bangalore, under research fund for UG students.
Conflicts of interest
The authors declare that there is no conflict of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13]
[Table 1], [Table 2], [Table 3]
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