Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
|Iranian Journal of Pharmaceutical Research (2009), 8 (1): 65-70
Received: February 2008
Accepted: June 2008
Copyright ? 2009 by School of Pharmacy
Determination of Hyoscyamine and Scopolamine in
Four Hyoscyamus Species from Iran
Atefeh Bahmanzadegana, Fatemeh Sefidkona* and Ali Sonbolib
aDepartment of Phytochemistry, Research Institute of Forests and Rangelands, Tehran, Iran. bDepartment of Biology, Medicinal Plants and Drugs Research Institute, Shaheed Beheshti University, Tehran, Iran.
The purpose of the present study was to determine the tropane alkaloid content of four Hyoscyamus species from Iran, i.e. H. pusillus L., H. niger L., H. reticulatus L. and H. kurdicus Bornm. Determination of alkaloids was performed by the HPLC method. Samples were extracted with chloroform-methanol-25% ammonium hydroxide 15:5:1 (V/V/V). HPLC separation was performed on Eurospher C18 reversed phase column. An isocratic mixture of triethylammonium phosphate buffer (30 Mm, pH 6.2) and acetonitrile (75:25), was used as the eluent. Hyoscyamine and scopolamine were determined by the external standard method at 210 nm. All the four mentioned Hyoscyamus species contained hyoscyamine and scopolamine, but in different amounts. Scopolamine was the predominant tropane alkaloid in H. pusillus, H. niger and H. kurdicus, while, H. reticulatus contained a higher amount of hyoscyamine.
Hyoscyamine and scopolamine are medicinally important tropane alkaloids. Because of possessing anticholinergic and central nervous system activities, they have well-established therapeutic uses (in ophthalmology, cardiology, gastroenterology, etc.). They are synthesized in several species from Solanaceous family like the genus Atropa, Datura, Duboisia, Hyoscyamus and Scopolia. Industrially, these natural substances are exclusively produced by plants and the demand for them is continuous (1). Hyoscyamus species are rich sources of tropane alkaloids, mainly hyoscyamine and scopolamine, which are widely used for their mydriatic, antispasmodic, anticholinergic, analgesic and sedative properties (2).
Due to the pharmacological and toxicological importance of tropane alkaloids, determination of these compounds, in Hyoscyamus species were studied. There are several articles published in the past few decades,concerning the identification and determination of these alkaloids in medicinal plants by different analytical techniques (3-6). The total alkaloid content of the leaves of H. reticulatus from Anatolia was determined by a colorimetric method,and found to be in the range of 0.011-0.027% (7). Parallel with the improvement of biotechnological methods (8), HPLC techniques are also widely applied for the determination of tropane alkaloids content in different plant tissues (9-13). In vivo, hyoscyamine 6β-hydroxylase (H6H) catalyzes the epoxidation of hyoscyamine to scopolamine (14). The pharmacological uses of tropane alkaloids, especially scopolamine, are very extensive. For example scopolamine, an antimuscarinic agent, acts as a competitive inhibitor, mimicking acetylcholine at the neural synapses and depresses the central nervous system. Its most potent activity is manifested on the iris, ciliary body, and secretory (salivary, bronchial and sweat) glands (15).
In this study the extraction and determination of hyoscyamine and scopolamine from H. pusillus L., H. niger L., H. reticulatus L. and H. kurdicus Bornm. as four medicinal plants of Iran were investigated.
Different parts of four Hyoscyamus species, i.e. H. pusillus L., H. niger L., H. reticulatus L. and H. kurdicus Bornm. were collected from their natural habitats in Iran. The collecting date, locality information and voucher numbers of the studied species have been listed in Table 1.
Voucher specimens have been deposited at the herbarium of Medicinal Plants and Drugs Research Institute of Shahid Beheshti University (Tehran, Iran).
Extraction of total alkaloids
In order to ensure that the collected plants have tropane alkaloids, the Vitali-Morin test was used (16). Four different methods of alkaloid extraction were tested on each plant material and then the extracts analyzed using with HPLC (17-21). The best results are based on the reported procedure by Kamada at al (21), In this method higher amounts of tropane alkaloids were obtained compared to the other methods reported in the literature. However, this method detected a limited amount of additional and interference HPLC. The test procedure has been summarized in Figure 1. The resulting extract was directly subjected to HPLC analysis.
The standard methanolic solution of hyoscyamine and scopolamine (50 ppm) was prepared. Tropane alkaloids were recovered with 5ml aliquots of chloroform-25% and ammonium hydroxide (15:1 V/V), and then entered in to the previously described procedure shown in Figure 1.
HPLC analysis was carried out on a Knauer HPLC system (Berlin, Germany) equipped with a Eurospher C18 column (25 cm ? 4 mm i.d., RP) and a UV detector. Elution was monitored at 210 nm.
Isocratic elution with a mixture of triethylammonium phosphate buffer (30 Mm, pH 6.2) and acetonitrile (75:25) at a flow rate of 1.0 ml/min was selected to achieve maximum separation and sensitivity. Hyoscyamine and scopolamine hydrobromide were obtained from Sigma-Aldrich. The calibration graphs for standard samples were constructed by plotting the peak area of the alkaloids against their concentrations. Linear calibration graphs were obtained with good correlation for standard solutions (Table 2).
Results and Discussion
Quantitative analysis of hyoscyamine and scopolamine were determined on various parts of Hyoscyamus pusillus, H. niger, H. reticulatus (in two regions) and H. kurdicus. The results have been summarized in Table 3, where the amount of tropane alkaloids is reported in ppm (g/g) based on the weight of the ground dry plants. As shown, all four hyoscyamus species contained both the above mentioned tropane alkaloids. Our findings indicated that the distribution of tropane alkaloids was different in various parts of the hyoscyamus species investingated.
Scopolamine was the predominant tropane alkaloid in H. pusillus, H. niger and H. kurdicus. This findig is in agreement with data reported studies by Supria on H. niger and H. muticus (22). These data also indicated the presence of a higher hyoscyamine content in H. reticulates in both regions studied. That finding is in accordance with the results reported by Jonkova (23). Higher amounts of scopolamine have been observed in the leaves of H. pusillus and H. kurdicus, as well as the seeds of H. niger (Figures 2-4). The seeds of H. reticulates, in both the studied regions, contained hyoscyamine in higher amounts that the other plant parts (Figures 5 and 6).
It can be concluded that, among the four studied hyoscyamus species, H. pusillus, H. niger and H. kurdicus due to the higher amount of scopolamine and H. reticulatus with a higher content of hyoscyamine, could be used as a rich source of these tropane alkaloids for medicinal purposes.
In terms of the mean amount of hyoscyamine to scopolamine ratio, H. pusillus was the predominant species. This the due to high amount of conversion hyoscyamine to scopolamine, making it a suitable species for transportation of the H6H enzyme.
The authors would like to thank Dr. Jaimand from the Research Institute of Forests and Rangelands and also the Department of Phytochemistry, Shahid Beheshti University for their technical and financial assistances.
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