A reverse phased high-pressure liquid chromatographic method for the estimation of a poisonous matter in Strychnos nux-vomica.

Seeds of Strychnos nux-vomica were subjected to preliminary phytochemical tests and its presence was confirmed by thin layer chromatography (TLC) method. The TLC profile of the methanolic extract of seeds of S. nux-vomica was developed using the solvent system toluene:chloroform:methanol in the ratio 8:2:1. The plate was observed in visible light after spraying with Dragendorff's reagent (specific method). High-performance liquid chromatography (HPLC) profile of the methanolic extracts of S. nux-vomica was developed, and the amount of strychnine seems to be 0.36% (w/w) in the seeds. The TLC and HPLC profiles developed are very valuable for the identification of the original drug from their adulterants. The TLC profile identifies the presence of strychnine in the plant material. The quantification method for the strychnine in the seeds can be used for the quality standardization of the raw drug because the strychnine is reported to have some toxicity.

INTRODUCTION

Some plants contain toxic substances in their roots, leaves, fruits or seeds. These toxic substances are the secondary metabolites or alkaloids and are prepared by the plants to protect them from herbivorous animals and insects. Poisonous plants are also dangerous to man, as they can be eaten by children or mistaken for edible plants by adults. Particularly dangerous to children that are plants with attractive fruits or seeds, such as the deadly nightshade (Atropa belladonna) and Abrus precatorius seeds while hemlock is easily mistaken for parsley or other edible umbelliferous herbs. In the past, epidemics due to plant toxins have occurred, including St. Anthony's fire caused by ergot (fungus) infection of rye (used for making bread) and milk sickness due to Eupatorium rugosum – White Snakeroot (Ageratina altissima).[1] Strychnos nux-vomica L. (Loganiacea) seen in almost all parts of India is classified as a spinal poison [Figure 1].[2] The main active principles of S. nux-vomica L. are known to be alkaloids, such as strychnine, brucine and loganin. The active principles are in the highest concentration in seeds. Nux-vomica seeds usually contain about 3% total alkaloids of which about one half is strychnine.[34] Pharmacologically brucine is much less active than strychnine being only 1/10th as toxic; the pharmacopeial assay of nux-vomica is a determination, not of total alkaloids but of strychnine only.[5]

Figure 1

Chemical structure of strychnine (R=H)[4]

In the present study, preliminary phytochemical tests were done to find out various chemical constituents in methanolic extract of S. nux-vomica and presence of strychnine was confirmed by thin layer chromatography (TLC) method. High-performance liquid chromatography (HPLC) profile of S. nux-vomica was developed and standardized. The amount of strychnine in the sample was calculated by considering the area under the peak for the standard strychnine and the strychnine peak in the sample profile. The study was conducted on 2007.

Aim and objectives

To perform the preliminary phytochemical screening to identify the major chemical groups present in the seeds of S. nux-vomica by carrying out specific chemical tests

To detect the presence of strychnine in the methanolic extracts of S. nux-vomica by carrying out TLC analysis

To develop HPLC profile for methanolic extract of S. nux-vomica and to quantify the strychnine in the same.

MATERIALS AND METHODS

Collection of seeds

Fresh seeds of S. nux-vomica were collected from Herb garden, Arya Vaidya Sala, Kottakkal and authenticated by the Botany Division of Center for Medicinal Plants Research, Kottakkal, India. The collected raw drug was then dried in the shade and powdered.

Preparation of extracts

Five gram of dried powdered seeds were extracted by refluxing in methanol for 8 h. The extracts were filtered and concentrated, and these were used for the phytochemical screening, TLC profiling and HPLC profiling.

The methanolic extract of the seeds of S. nux-vomica was subjected to preliminary phytochemical screening for the detection of major class of compounds. The details of the tests are shown in Table 1.

Table 1

Preliminary phytochemical screening of plant extract

Thin layer chromatography

Materials

Precoated silica gel plates 60 F254 with layer thickness 0.25 mm (E. Merck), micro syringe (25 μl, Hamilton = 802) and a UV chamber.

Chemicals

Toluene, ethyl acetate, chloroform, methanol, ethanol, anisaldehyde, glacial acetic acid, H2SO4, antimony trichloride (GR grade, E. Merck, India).

Method

Sample preparations: One milliliter of each concentrated extract was diluted to 10 ml in respective solvents (pet, ether, chloroform and methanol) and was used for the analysis.

Chromatographic condition

Stationary phase – TLC precoated silica Gel F254 aluminum sheet

Solvent system – Tolune:chloroform:methanol (8:2:1) (methanol extract)

Detection – By visualization under visible light

Spray reagent: Dragendorff reagent

Development distance: 8 cm.

The chromatogram of plant extracts was eluted under the chromatographic conditions mentioned above, and the analytic detection was done under daylight. A corresponding band in the sample solution is observed to the standard strychnine.[6]

Assay/analytical method

High-performance liquid chromatographic estimation of strychnine in S. nux-vomica.

Chromatographic system

Shimadzu® High Performance Liquid Chromatographic system equipped with LC-10ATVP pump, a rheodyne 7725i injector with 20 μl loop volume, A Shim-Pack CLC, ODS (C- 18), (250 × 4.6 mm i.d., 5 μ) was used for the separation. SPD M10AVP photodiode array detector in combination with CLASS-VP 6.12 SP5 integration software. The mobile phase was composed of mixture of methanol and water (55:45 v/v). It was filtered through a 0.45 μ membrane filter and degassed for 10 mins. Detection was carried out at 254 nm.

Chromatographic conditions

Mobile phase: Methanol: Water (55:45)

Column: C18 - ODS (Octadecylsilane), Lichrospher RP 18e (5 μm) (Merck)

Detector: SPD M10AVP PDA

Wavelength: 254 nm

Flow rate: 1 ml/min

Runtime: 30 min

Injection volume: 20 μl.

Five gram of each powdered drug were weighed accurately and extracted in a reflux apparatus with (150 × 3) ml of methanol for about 8 h. The filtrate was filtered and evaporated to remove methanol. The residue was dissolved in 25 ml methanol. One drop of concentrated HCl was added to liberate the alkaloids, and the same was filtered through 0.45 μm membrane filter and used for HPLC analysis.

Standard solution

Ten milligram of strychnine was dissolved in 10 ml methanol.

Procedure

Twenty microliter of suitably diluted sample solution/standard solution was injected, and the peak responses recorded. Retention time of strychnine was 3.7 min. The UV spectrums of the corresponding times were also compared to confirm the identity of the peaks. The percentages of the marker compound in the test solution were calculated by comparing the peak areas of the standard compounds with those of the corresponding peaks present in the chromatogram of the test solution.[6]

RESULT

Dried and powdered seeds of S. nux-vomica were extracted by refluxing with methanol and subjected to preliminary phytochemical screening for the presence of different chemical groups [Table 2]. Tannins, alkaloids, and glycosides were found to be the major groups present, along with phenol, steroids and flavonoids in smaller quantities [Table 3].

Table 2

Preliminary phytochemical screening

Table 3

Preliminary phytochemical screening of methanolic extract of Strychnos nux-vomica

The TLC profile of the methanolic extract of seeds of S. nux-vomica was developed using the solvent system toluene:chloroform:methanol in the ratio 8:2:1. The plate was observed in visible light [Figure 3] after spraying with Dragendorff's reagent (specific method). A corresponding band in the sample solution was observed in the standard strychnine [Figure 2].

Figure 3

HPLC chromatogram of the methanolic extract of Strychnos nux-vomica. HPLC: High-performance liquid chromatography

Figure 2

Thin layer chromatography identity test

High-performance liquid chromatography profile of the methanolic extracts of S. nux-vomica is given in Table 4. In our study, the amount of strychnine in the sample was calculated by considering the area under the peak for the standard strychnine (0.2–14.0 μg/ml) and the strychnine peak in the sample profile [Figure 3].

Table 4

HPLC profile of the methanolic extract of Strychnos nux-vomica

DISCUSSION

Now-a-day HPLC is being used a major tool for fingerprinting and routine quality control analysis. HPLC chromatogram of standard strychnine was also developed under the same chromatographic conditions. By comparing the chromatogram of standard and sample, the purity of the compound can be known. If additional peaks are obtained, impurities are present. Patel et al. studied the phytochemical constituents of S. nux-vomica and found that alkaloid, carbohydrate, tannin, steroid, triterpenoid, and glycoside are the major contents when compared to flavonoid and phenol.[7] The poisonous matters presented in S. nux-vomica are strychonos alkaloids, mainly monomeric tertiary indole alkaloids; chief constituent being strychnine and brucine.[2] The TLC identity was developed to confirm the presence of strychnine in the plant. For selection of TLC plate we choose precoated plate over handmade plate as precoated plates with different support material and with different sorbent layers are available in different format and thickness by various manufactures. Usually, plates with sorbent thickness of 100–250 mm are used for qualitative and quantitative analysis.[6] Proper sample preparation is an important process for the success of the TLC separation. For sample, preparation 1 ml of each concentrated extract of S. nux-vomica was diluted to 10 ml in respective solvents (pet, ether, chloroform and methanol). Sharma suggested that for detecting colorless spots, techniques like nonspecific and specific method can be used.[6] Gaitonde and Joshi studied the TLC-spectrophotometric analysis of strychnine and brucine from the ayurvedic pills of S. nux-vomica and developed a chromatogram for the same.[8]

High-performance liquid chromatography fingerprint profile of S. nux-vomica was developed and standardized. In this study, the amount of strychnine seems to be 0.36% (w/w) in the seeds. Choi et al. compared the content of strychnine in unprocessed and detoxified S. nux-vomica seeds and found that unprocessed seed contains 0.411 ± 0.028 mg/g by HPLC-ESI/MS method.[9]

CONCLUSION

From the present investigation, the methanol extract of the seeds S. nux-vomica contains tannins, alkaloids and glycosides in higher amounts along with phenols, steroids and flavonoids in smaller quantities.

The TLC and HPLC profiles developed are very valuable for the identification of the original drug from their adulterants. The TLC profile identifies the presence of strychnine in the plant material.

The quantification method for the strychnine in the seeds can be used for the quality standardization of the raw drug because the strychnine is reported to have some toxicity. So the variation in the content of this compound may affect the biological efficacy of S. nux-vomica.

This HPLC method applied for the quantification of S. nux-vomica was accurate, and sensitivity was also good. From the calculated value for standard solution of strychnine was 0.2–14.0% (w/w), and the value for the test was 0.36% (w/w), so that accurate quantitation down to low levels is possible, making it easy to test the quality of different Ayruvedic products, as well as crude drugs.