Simultaneous determination of vasicine and vasicinone by High-performance liquid chromatography in roots of eight Sida species.

INTRODUCTION: Sida L. is a medicinally important genus widely used in conventional systems of medicine in India. AIM: The present study aims toward simultaneous determination of two bioactive compounds vasicine and vasicinone in root extracts of eight Sida spp. from Western Ghats, India.
MATERIALS AND METHODS: Determination of vasicine and vasicinone was undertaken in methanolic root extracts (10% w/v) of Sida acuta, Sida cordata, Sida cordifolia, Sida rhombifolia, Sida spinosa, Sida indica, Sida retusa and Sida mysorensis by high performance liquid chromatography (HPLC) method. The standards were prepared with the concentration of mg/mL. Data were expressed as mean values of three reading and relative standard deviations. The separation was achieved on a Waters, Nova-Pack, C18 (250 mm × 4.6 mm, 5 μ) column, with acetonitrile - 0.1 M phosphate buffer-glacial acetic acid (15: 85: 1, v/v/v) as solvent system at a flow-rate of 1.0 mL/min. The effluent was monitored using ultraviolet detection at a wavelength of 300 nm.
RESULTS: Both calibration curves of standard showed good linear regression (R2 > 0.994). The limit of detection and the limit of quantification for vasicine was 0.110 and 0.333 μg/mL and for vasicinone was 0.059 and 0.179 μg/mL respectively. The vasicine content was highest in S. cordifolia (9.891 ± 0.495 μg/100 mg) and vasicinone content was rich in S. cordata (33.013 ± 1.651 μg/100 mg.) The content of vasicinone was higher than vasicine.
CONCLUSION: HPLC method provides simple, accurate, and reproducible quantitative analysis for simultaneous determination of vasicine and vasicinone. Among the selected Sida species, S. cordifolia and S. cordata were found to be rich in the vasicine and vasicinone contents, respectively.

Introduction

Genus Sida L. (Malvaceae) comprises of 200 species distributed throughout the world, out of which 17 species are confined to India.[1] The plant is well documented in ancient Indian system of medicine “Ayurveda.” Bala is an important plant belonging to Karpasakula, [2] used as Rasayana[3] to treat various ailments such as Vatavyadhi[4] and Pradara.[5] Even though species belonging to genus Sida are well known for their medicinal properties, especially in Ayurveda, the issue in their correct botanical identity persists. Bhavaprakasha Nighantu mentions four varieties of Bala (Balachatushtaya)[6] while Dhanvantari Nighantu mentions five varieties (Panchabala).[7] In general, as per the classics, Bala is Sidacordifolia L., Mahabala is correlated to two plants, namely, Kshetrabala (Sida rhombifolia L.) and Sahadevi (Vernonia cinerea L.). Similarly, the name Atibala is attributed to Abutilon indicum L. and Nagabala indicate three plants, i.e. Bhumibala (Sida veronicaefolia L.), Kantakinibala (Sida spinosa L.) and Gudasharkara (Grewia hirsuta Vanb.).[8] Other classical references mentioned Rajabala or Bruhat Nagabala as Sida acuta Burm. F.[9] There are different of opinion regarding botanical identities of Bala, which are still controversial.[10]

It is known for various pharmacological activities such as hepatoprotective, anti-arthritic, gonorrheal, end also as an immune enhancer.[11] Several Sida species have been investigated for their phytochemical constituents and have reported many compounds using various analytical techniques.[1213141516] The previous study suggested, that Sida species contain polyphenols and possess antioxidant activity.[17] Reports suggested high performance thin layer chromatography (HPTLC) estimation of ephedrine, a major active principle from Sida spp., [18] optimization and validation of high performance liquid chromatography (HPLC) and HPTLC method for quantification of vasicine (IUPAC name: [3R]-1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-ol; Mol. Formula: C11H12N2O; Mol. Wt.: 188.226) and vasicinone (IUPAC name: 3S-hydroxy-2,3-dihydro-1H-pyrrolo [2,1-b] quinazolin-9-one; Mol. Formula: C11H10N2O2; Mol. Wt.: 202.209) two other major compounds reported in roots of two Sida species (viz. S. cordifolia and S. acuta).[12] Vasicine is a light sensitive compound and when exposed to light, it is auto-oxidized to vasicinone.[1920] Both vasicine and vasicinone are chief alkaloids known to possess interesting biological activities.[212223] Vasicine and vasicinone are quinazoline alkaloids known to possess bronchodilatory activity; the first was reported to be more potent and also showed anti asthmatic activity.[2425]

Hence, the present study aims at quantifying vasicine and vasicinone and also to identify the highest yielding species from roots of 8 Sida species (viz. S. acuta Burm. F., S. cordata Boiss., S. cordifolia L., S. rhombifolia L., S. spinosa L., S. indica L., S. retusa L., and S. mysorensis Wt. and Arn.) collected from Western Ghats, India.

Materials and Methods

Eight selected Sida species were collected from Belagavi region of Western Ghats, India during September, 2013. Plants were identified, authenticated, and voucher specimens were deposited at ICMR - NITM, Belagavi (KA), India for future reference (S. cordata Boiss., S. spinosa L., S. rhombifolia L., S. acuta Burm. F., S. cordifolia L., S. indica L., S. mysorensis Wt. and Arn., S. retusa L.; Voch. Nos.: RMRC 475, 477, 479, 484, 938, 939, 970, and 971, respectively). All the solvents viz. methanol, water, acetonitrile, and glacial acetic acid were of HPLC grade (Fischer Scientific, Mumbai, India). Vasicine (>95% pure) and vasicinone (>95% pure) were procured from, Natural Remedies, Bengaluru, India.

Roots of the eight species were washed, dried, ground to fine powder, and was stored in air tight containers until further use. Continuous shaking extraction was performed on an orbital shaker (Orbitek, India) by subjecting 10 g of dried plant materials in 100 mL of methanol in 250 mL Erlenmeyer flask at 150 rpm, overnight at ambient temperature. Samples were filtered through Whatman filter paper no. 1, to obtain a residue. The filtrate was passed through 0.45 μ nylon filters before analysis to remove impurities from the extract and to avoid damaging the column. The extracts were diluted to 10% (w/v) for HPLC analysis.

A previously described solvent system[12] was employed during the present study. The separation was achieved on a Waters, Nova-Pack, C18 (250 mm × 4.6 mm, 5 μ) column, with acetonitrile - 0.1 M phosphate buffer-glacial acetic acid (15:85: 1, v/v/v) (pH 4.0 by phosphoric acid) as solvent system at a flow-rate of 1.0 mL/min. The effluent was monitored using ultraviolet detection at a wavelength of 300 nm. The mobile phase was filtered through 0.45 μm nylon filter before use.

Standard stock solution of vasicine and vasicinone (5 mL) were prepared with the concentration of mg/mL. The concentrations of both the standards were made from 0.1 to 40 μg/mL with 7 point calibration. The system suitability test was assessed by triplicate injections of the standard solutions at a particular concentration. The peak areas were used to evaluate repeatability of the proposed method, and their peaks were analyzed for resolution.

Data were expressed as mean values of three reading and relative standard deviations. The statistical analyses were performed using Microsoft Excel 2007 software.

Results

Vasicine and vasicinone were determined quantitatively using HPLC analysis in roots of eight, Sida species from Western Ghats, India. The identification of these compounds in extracts was achieved by comparing retention time of authentic standards. Chromatograms of 2 standards and comparative report of representative samples are depicted in Figures 1–4. The method of extraction and HPLC conditions were unchanged throughout the study.

Figure 1

Simultaneous detection of Vasicine and Vasicinone standards (100 μg/mL)

Figure 4

Comparative chromatograms of 8 Sida species

Seven point calibration of vasicine

Seven point calibration of vasicinone

Different concentrations of vasicine and vasicinone were detected at 300 nm using HPLC system. Profiles with retention times of 3.722 ± 0.318 (vasicine) min and 6.272 ± 0.265 min (vasicinone) were obtained as the output of standard injection by HPLC analysis. Seven point calibration curves of both the standards within the concentration range of 0.1– 40 μg/mL were constructed with the coefficient of determination (R2) above 0.994. The regression equation showed a significant relationship between peak areas and concentrations and this equation was used to estimate content from samples. The results of the regression equations were y = 17449x + 24238 (vasicine) and y = 19946x + 11986 (vasicinone), a linear relationship between peak area and concentration was established. Limit of detection and limit of quantification as determined by signal to noise ratio for vasicine was 0.110 and 0.333 μg/mL and for vasicinone was 0.059 and 0.179 μg/mL, respectively.

The contents (μg/100 mg) were calculated using standard calibration curves [Table 1]. Simultaneous determination of both the standards from roots of eight Sida spp. varied from 3.194 ± 0.160–9.891 ± 0.495 μg/100 mg (vasicine), and 02.328 ± 0.116–33.013 ± 1.651 μg/100 mg (vasicinone).

Table 1

Content of vasicine and vasicione as determined by high performance liquid chromatography in roots of eight species of Sida

Discussion

The highest vasicine content was determined in S. cordifolia and vasicinone in S. cordata. It was also interesting to note that the retention times for detection of both the compounds were less than the earlier reports.[12] The results were in accordance with the earlier reports[12] of S. acuta and S. cordifolia using HPLC analysis, wherein it has been reported the similar content of vasicine (10.00 μg/100 mg) in S. cordifolia and vasicinone (2.30 μg/100 mg) in S. acuta. However, the content of vasicinone in S. cordifolia (6.00 μg/100 mg) was lower and vasicine in S. acuta (8.00 μg/100 mg) was higher than the present study [Table 1]. It is well evident from the earlier studies on medicinal plants that the variation in chemical makeup may be attributed to change in the area of collection and climatic factor.[262728] Furthermore, it is evident that vasicine and vasicinone are the chief marker compounds of Adhatoda vasica (Syn.Adhatoda zeylanica).[29] Vasicine and vasicinone content as determined in methanolic extracts of A. vasica leaves were 0.1200% and 0.0340%, respectively. However the content of vasicine in Sida spp. during the present study was lower than that reported earlier in A. vasica and vasicinone content in S. cordata [Table 1] was almost equal to that mentioned by Srivastava et al. in A. vasica.[30]

Conclusion

Conclusively, HPLC method provides simple and reproducible quantitative analysis for simultaneous determination of vasicine and vasicinone. Vasicinone was available in higher amount as compared to vasicine in Sida species. S. cordifolia and S. cordata were the species with a higher amount of vasicine and vasicinone, respectively. The study helps to select the plant species with higher content of these compounds required for its maximum therapeutic potential.

Financial support and sponsorship

Drugs have been collected from wild source, the lab facility was utilized from ICMR-NITM, Belagavi.

Conflicts of interest

There are no conflicts of interest.