Research Article

InPharm Communique (Suppl.) Vol 2, No 2 | 51

An improved HPTLC – UV method for rapid estimation of

andrographolide in Andrographis paniculata (Burm. f) Nees

Himanshu Misra1, 2, Manish Soni3, Darshana Mehta1, B. K. Mehta1 and D. C. Jain2, 3*

1 School of Studies in Chemistry and Biochemistry, Vikram University, Ujjain – 456 010, Madhya Pradesh, India.

2 Green Technology Department, Ipca Laboratories Limited, Ratlam –457 001, Madhya Pradesh, India.

3 Mandsaur Institute of Pharmacy, Mandsaur- 458 001, Madhya Pradesh, India.

[e-mail: dc_52@rediffmail.com ; himanshumisra1@rediffmail.com (for correspondence)]

ABSTRACT

A simple, rapid and precise (%RSD<2.5) high-performance thin-layer chromatographic method was

developed for quantitative estimation of a hepatoprotective diterpenoid andrographolide.

Separation was performed on 60 F254 HPTLC plates using chloroform : methanol :: 27 : 3 v/v as

mobile phase for elution of extract components. The determination was carried out in ultra-violet

mode using densitometric absorbance-reflection mode at 232 nm. The concentration of

andrographolide in the whole plant powder was 0.178 (±0.003) %, dry weight basis.

Andrographolide response was found to be linear over a broad range 100 – 2500 ng spot-1

. Limit of

detection and quantification were 40ng and 100ng spot-1

. The developed method is capable of

quantifying smaller as well as higher amounts of andrographolide in its plant raw-material. The

HPTLC method was evaluated in terms of precision, accuracy, sensitivity and robustness.

Keywords: Andrographis paniculata, Acanthaceae, whole plant, HPTLC method, ultra-violet

detection.

INTRODUCTION

Andrographis paniculata (Burm. f) Nees of family Acanthaceae is traditionally known as kalmegh. The plant is widely used in ayurvedic and homeopathic systems of medicine. The plant is also known as ‘king of bitters’ due to its bitter taste and weak odor. The whole plant is used in medicine and is official in the Indian Pharmacopoeia (Farooqi & Sreeramu, 2001). In ayurveda, the drug has been described as antipyretic and hepatopratective. Cold infusion of the drug is mentioned in sushruta samhita for fever and liver disorders. Plant drug contains flavones and lactones. Among lactones andrographolide (Figure 1) is the main constituent and is active constituent of the plant. Andrographolide has been isolated in pure form and it has shown various pharmacological activities (Caceres et al., 1997). In vitro and in vivo studies suggest that andrographis has antiinfective, antiviral, antidiarrhoeal, antipyretic and analgesic activities (Farnsworth & Bunyapraphatsara; Caceres et al., 1997).

Although some methods was previously developed on colorimetric (Maiti et al., 1959) and spectrophotometric (Shah et al., 2007) but they suffers with being multisteps and are not very precise. Later on few HPTLC / TLC methods (Raina et al., 2007; Saxena et al., 2000 & Srivastava et al., 2004) developed but they were utilizing unsafe solvents like, benzene and toluene and also detection methods were either involves post-chromatographic derivatization with strong acid or ultraviolet light with narrower detection range for andrographolide in crude plant extract. Recently, Akowuah et al. (2006) established a nice comparison of quantitative analysis of andrographolide and 14-deoxy-11, 12-didehydroandrographolide (DIAP) by HPLC and HPTLC together with LPI assay and free radical scavenging activity of extracts but HPTLC method suffers with narrow linear range. Here, we developed an HPTLC – UV method for broad range detection of andrographolide, which will be very useful for newer plant variety development to quantify very small as well

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as very large quantities of andrographolide present in Andrographis paniculata at once. EXPERIMENTAL

Plant material

Plant material was grown in our experimental field at Mandsaur institute of pharmacy, Mandsaur (M.P.). Whole plant was taken, air-dried and pulverized to fine powder of mesh 22. Sowing was performed in the month of July and sample taken after 30-35 days before application of first dose of fertilizer. Chemicals

Chloroform (99.0 to 99.4% purity) and methanol (99.5% purity) were purchased from s. d. fine-chem. Limited, Mumbai. Ethanol was of commercial grade and was utilized after distillation. Standard andrographolide was previously isolated and characterized (Saxena et al., 2000). Apparatus HPTLC analysis was performed on a Camag’s TLC Scanner 3 controlled by winCATS planar chromatography manager version 1.4.2 (CAMAG, Switzerland). Drying and concentration stepts were performed using rotatory evaporator. Ultrasonicator was used for homogenizing of test and standard samples. Preparation of standard solution and calibration curve

50 mg of standard andrographolide was dissolved in methanol, maintained to mark in a 50 mL volumetric flask and sonicated for 5 minutes. 1-25 mL of this solution pipetted out and transferred to seven different 50 mL volumetric flasks, made up to mark with methanol and sonicated for 5 min. for homogenizing reference standard of andrographolide. A calibration curve was plotted between increasing amounts of andrographolide per spot and their peak area response. A straight line was obtained between 100 - 2500 ng spot-1. Extraction and test sample preparation

Three samples each of 5 g fine powder of mesh 22 was extracted with chloroform, methanol and ethanol (180mL each) through hot soxhlet extraction for 8 hrs. The extract was reduced in vacuo and volume made up to 50 mL with methanol in a volumetric flask. Chloroform extract was made up to the mark with methanol : chloroform (1:1). These test solutions are used for quantification purpose of test andrographolide. Thin-layer chromatographic analysis

Thin-layer chromatography was performed on aluminum backed HPTLC plates (60 F254, E. Merck, Germany, 200 x 100

mm). Test and standard sample (2µL each) spots were applied via camag’s Linaomat 5 as 6.0 mm wide bands at the height of 10 mm from base; spots were simultaneously dried with N2 gas supply on to HPTLC plates. Plates were developed in a camag twin trough chamber of size more than 200 x 200 mm at 95 mm height from base using chloroform – methanol (27 : 3, v/v) as mobile phase. Plates were air-dried for complete evaporation of mobile phase and scanned using camag’s TLC scanner 3 equipped with winCATS software in absorption – reflection detection mode at 232 nm (Deuterium lamp). Chromatogram of andrographolide separation was obtained after completion of scanning (Figure 2).

Method validation

Linearity

The linearity of the andrographolide calibration plot (Figure 4) was evaluated on seven point-scale by spotting increasing amounts of the andrographolide working standard solution, starting from 100 - 2500 ng spot-1 (100, 500, 900, 1300, 1700, 2100 and 2500 ng spot-1). For this, a stock solution of 100mg mL-1 was prepared and then pipette out 1, 5, 9, 13, 17, 21 and 25 mL and transferred to seven different volumetric flask of volume 50mL, respectively. Volume made to the mark with methanol and homogenized properly under sonication followed

by manual shaking. 5 µL of each solution spayed over HPTLC plate and chromatographic separation performed. The method showed good linearity in the given range with a correlation coefficient of 0.99727 and the linear regression equation was Y = 4.032 X + 687.3 (sdv = 4.35). Precision

Precision of the method determined by three replications of each sample. The precision (%RSD) of the replications was found to be less than 2, which is indicative of a precise method (table 1). Limit of detection and quantitation (LOD and LOQ)

Limit of detection and quantitation was determined by spotting increasing amounts (20 – 140 ng; n = 2) of standard

andrographolide solution of concentration 10 µg mL-1 (1mg of andrographolide per 100 mL) until the average responses were 3 and 10 times of noise for LOD and LOQ respectively. LOD and LOQ were found to be 40 and 100 ng spot-1 respectively. Specificity

The developed HPTLC – UV method was found to be specific as no interfering peak found during detection of andrographolide. Peaks of andrographolide eluted on to HPTLC plate were found to be pure, which was also evidenced by peak purity data as shown in figure 3. Robustness

Robustness of the method was determined by performing small variations in mobile phase ratio, height of plate development and TLC tank saturation time. The results indicated insignificant differences in assay and thus indicative of a robust method. RESULTS AND DISCUSSION

Optimization of extraction

Extraction solvent was standardized for better extraction of andrographolide using three different solvents (chloroform, methanol and ethanol) under hot soxhlet extractor and associated screening of andrographolide content using presently developed method (table 1). Methanol was found to be good for better recovery of andrographolide, which was also supported by previous literature [Srivastava et al., 2004]. Optimization of mobile phase

Mobile phase for the HPTLC separation of andrographolide in plant extract was optimized using different binary mixtures of few solvents but chloroform : methanol (27 :3, v/v) was finalized for better extraction and associated resolution with the neighboring spots.

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Table 1: Screening of solvents for better extraction

Extraction Wt. of sample (g) Extraction time

(h)

Extraction

Solvent

% Mean content of

andrographolide

(n = 3)

(±SD) %RSD

Soxhlet 5.0 8.0 Chloroform 0.045 0.001 2.22

Soxhlet 5.0 8.0 Methanol 0.178 0.003 1.69

Soxhlet 5.0 8.0 Ethanol 0.166 0.002 1.20

CH3

CH2

OH

CH3

O

OOH

H

OH

Figure 1: Structure of Andrographolide [C20H30O5 ; Molecular weight = 350.45]

Figure 2: HPTLC Chromatogram of Andrographolide separation

Figure 3: Overlapping UV spectrum of standard andrographolide spot (spot start, middle and end) eluted on to HPTLC plate showing λmax. at

232 nm

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InPharm Communique (Suppl.) Vol 2, No 2 | 54

Figure 4: Calibration curve of andrographolide

CONCLUSION

The developed HPTLC method is not only rapid but also reliable for plants containing low andrographolide contents at early stages. The method can be utilized for the development of newer plant varieties as well as for the selection of high-yielding plant varieties of A. paniculata. Validation parameters were found to be in good agreement with ideal ones. ACKNOWLEDGEMENTS

Authors are thankful to Ipca Laboratories Limited for valuable support and facilities during the course of work. REFERENCES

Akowuah, G.A., Zhari, I., Norhayati, I., Mariam, A., (2006). HPLC and HPTLC densitometric determination of andrographolides and antioxidant potential of Andrographis paniculata. J. Food Composition Anal. 19: 118.

Caceres D.D. et al. (1997) Prevention of common colds with Andrographis paniculata dried extract – a pilot double blind trial, Phytomed. 4: 101.

Farnsworth, N. R., Bunyapraphatsara, N., (Eds.) Thai Medicinal Plants, Bangkok: Medicinal Plant Information Center, Mahidol University, Thialand.

Farooqi, A. A., Sreeramu, B. S., (2001). Cultivation of medicinal plants, University Press (India) Limited, Hyderabad, pp. 152.

Maiti, P.C., Kanji, S.K., Chatterjee, R., (1959). Studies in Kalmegh extract Indian J. Pharm., 21: 169.

Raina,A.P., Kumar, A., Pareek, S. K., (2007). HPTLC analysis of hepatoprotective diterpenoid andrographolide from

Andrographis paniculata nees (kalmegh). Indian J. Pharm. Sci. 69: 473.

Saxena, S., Jain, D.C., Gupta, M.M., Bhakuni, R.S., Misra, H.O., Sharma, R.P., (2000). High-performance thin-layer chromatographic analysis of hepatoprotective diterpenoids from Andrographis paniculata. Phytochem.

Anal. 11: 34. Shah, K., Trivedi, P., Praksh Shiv and Pundarikakshudu, .K.,

(2007). Spectrophotometric determination of Andrographoloides in Andrographis paniculata Nees and its formulations. Indian J. Pharm. Sci. 69: 457.

Srivastava, A., Misra, H., Verma, R.K., Gupta, M.M., (2004). Chemical fingerprinting of Andrographis paniculata using HPLC, HPTLC and densitometry. Phytochem. Anal., 15: 282.

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