Estimation and Quantification of Andrographolide in Herbal Powder and Polyherbal Asava by HPTLC

 

Monika Jadhao* and Vikrant Wankhade

Dept. of Pharmaceutics, Vidya Bharti College of Pharmacy-Amravati, District- Amravati, (M. S) India - 444602.

*Corresponding Author E-mail: monikajadhao2006@yahoo.co.in

ABSTRACT:

Herbal powder and polyherbal formulation containing Andrographis peniculata were standardized and validated by high performance thin layer chromatographic method. Andrographolide in sample was identified and amount was estimated densitometrically. This method involves separation of compounds by TLC on pre-coated silica gel 60F 254 plates with the solvent system of benzene: ethyl acetate (5:5) and scanned using densitometric scanner in UV reflectance photomode at 220 nm. The linearity was observed in the range of 360ng-660 ng /spot. The andrographolide content of 237.2 µg/ 100 mg in powder and 41.80 mg/ 5ml. was observed in the test sample. The average percentage recovery value of 97.68 was obtained. The proposed method being precise and sensitive can be used for the detection, monitoring and quantification of andrographolide in Andrographis peniculata.

 

 

 

INTRODUCTION:

Standardization of ayurvedic drug and plant materials is the need of the day. Several pharmacopoeias containing monographs on plant material describes only the physico-chemical parameter¹. Andrographis peniculata Nees (Acanthaceae), commenly known as kalmegh, is widely used in the traditional system of Indian medicine in the treatment of hepatitis. The drug is mainly for its diterpenoid andrographolide and related compound^1,2. The plant is reported to possess protective activity against various liver disorders. Andrographolide and related compound were investigated for their pharmacological properties and all shows varying degrees of anti pyretic, anti-malarial, anthelmintic, stomachic, antityphoid, antibiotic activity³. Among the complex mixture of biologically active compound in the plant, andrographolide can be used as an analytical marker compound to determine the quality of plant material of different sources^4,5.

 

During crop improvement and drug analysis, a sensitive and accurate analytical method is required for the quantitation of important compound like andrographolide, which is present in the plant. Although few methods such as gravimetric, colorimetric, spectrophotometric methods have been reported for the quantitative estimation of andrographolide⁶.

 

Many of these methods are time consuming, not very precise and required multiple step extraction and purification. The present HPTLC method offers a sensitive, accurate and reliable method for routine detection and quantification of andrographolide present in andrographis peniculata^7,8.

 

MATERIALS AND METHOD:

Standard solution:

The andrographolide was isolated by using following procedure. An accurately weighed quantity of of std. andrographolide (1 mg) was dissolved in methanol (5-7 ml) and sonicated for 5 min. The volume was made up with methanol (10 ml) to give conc. (100 ng/µl). From this solution, 3 ml was further diluted to 10 ml to get final conc. (30 ng/ul).

 

Sample solution

The kalmegh powder (0.5 g) was weighed accurately and dissolved in methanol (7 ml). The solution was sonicated for 5 min and filtered though Whatman filter paper (No.01). The volume was made up to with methanol (10 ml)⁹. The std. solution (30 ng/µl) and the sample solution (50 µg/µl) were applied as 10 µl and 5 µl on the TLC plate, respectively. The plate was then developed with the mobile phase in the twin trough chamber and scanned. The sample solution (5 ml) was accurately pipette out and diluted with water (10 ml). The diluted sample was extracted with diethyl ether (3 X 20 ml).The separated ether layer was combined and filter though the bed of sodium sulphate. The filtrate then evaporated and reconstitute with methanol (5 ml). From this, 20 µl (565 µg/µl) of sample solution and 5 µl (30 ng/µl) of std. solution were applied on the TLC plate, respectively. The sample solution (5 ml) was accurately pipette out and diluted with water (10 ml). The diluted sample was extracted with diethyl ether (3 X 20 ml).The separated ether layer was combined and filter though the bed of sodium sulphate. The filtrate then evaporated and reconstitute with methanol (5 ml). From this, 20 µl (565 µg/µl) of sample solution and 5 µl (30 ng/µl) of std. solution were applied on the TLC plate, respectively^10,11. The chromatogram was developed and evaluated in the scanner.

 

RESULT AND DISCUSSION:

Different compositions of the mobile phase were tested and a good resolution was achieved by using benzene: ethyl acetate (5:5) as mobile phase. Andrographolide showed Rf value of 0.10 in plant extract, which was verified by comparing with Rf of the standard samples [Figure - 1].

 

Figure 1. Chromatogram of kalmegh powder containing andrographolide

 

µl = microliter ,  Rf = Retention factor

 

Thus they do not interfere with the quantification of andrographolide. A spectrum of andrographolide exhibited maxima at 222 nm UV. Comparing the spectra of standard and sample tracks did peak purity test [Figure - 2]. The calibration graph of andrographolide was linear in the range of 360 to 600 ng. The calibration plots were y= a ₀ +a ₁ x type, where y is the response and x is the amount of andrographolide, a ₀ is the intercept of the plot on the y-axis, a ₁ is its slope. The regression equation obtained was Y= 138.411+4221.617×X, r=0.99983, sdv=1.8%. For the examination of recovery of andrographolide, known amount of stock solution of pure andrographolide was added to preparation and quantitative analysis was repeated three times. The average recovery of andrographolide was 97.68% [Table - 1]. Results showed mean andrographolide content of 237.2 (µg) per 100mg in dry powder whereas andrographolide content ranged from 41.80 (µg /5 ml) in the preparation.

 

Figure 2.  Chromatogram of asava containing andrographolide

 

µl = microliter, Rf = Retention factor

 

This HPTLC method offers high degree of selectivity, sensitivity and rapidity combined with single-step sample preparation. Simultaneously a large number of samples along with the standard can be analyzed in one TLC plate and solvent requirement is also very negligible, thus making it inexpensive compared to HPLC. In addition it requires very small amount of sample and can detect active principle concentration in nanograms level. Thus this method can be conveniently adopted for routine quality control analysis.

 

CONCLUSION:

It can be concluded that the proposed HPTLC method is rapid, simple and accurate for quantitative monitoring of andrographolide from andrographis peniculata.

 

ACKNOWLEDGEMENTS:

Authors express their sincere gratitude to Mr. B. K. Shrikhande, Director, Baidyanath Research Foundation Ltd., Nagpur, for valuable guidance and providing valuable support to carry out research work.

 

Table 1. Statistical data for recovery study of andrographolide

Track No.	Sample applied (µg) [A]	Andrographolide present in[ A] (ng) [B]	Std added to [A] (ng) [C]	Total andrographolide applied (ng) [D]	Total  andrographolide recovered (ng) [E]	% Recovery E/D x 100 [F]
1	100	379.55	100	479.55	255.11	94.70
2	100	379.55	150	529.55	512.87	96.87
3	100	379.55	----	379.55	379.55	100
4	----	----	600	600	----	----
Mean						97.68
S.D.						1.801
R.S.D.						1.080

(n= 3), µg = microgram, ng = nanogram

 

 

REFERENCES:

1.       Chaudhari RD. Herbal Drug Industry, 1st ed. New Delhi: Eastern publishers, 2004:513.

2.       Negi A,Kumar JK, Shankar K, Khanuja SP. Recent advances in plant hepatoprotectives. J Wiley Intersci 2007; 23-35.

3.       Jayaram S, Gopalkrishnan V, Hari R, Shripathi M. Herbal medicines for liver diseases in India. J Gastroenterol Hepatol. 2002; 17:24-25.

4.       Bhopale GM, Nanda RK. Hepatitis B vaccine, present status. Indian Drugs 2003; 40-379.

5.       Bolton S. Pharmaceutical Statistics, 3rd ed. New York: Marcel Dekker, 2000.444.

6.       Raina AP, Kumar A, Pareek SK. HPTLC analysis of diterpenoid andrographolide from Andrographis peniculata. Indian J Pharm Sci 2007; 75-473.

7.       Saxena S, Jain DC, Gupta MM, Bhakuni RS, Hari O. Analysis of diterpenoid andrographolide from Andrographolide peniculata. J liq chromatogr1998; 27-123.

8.       Bhutani KK. Fingerprintings of Ayurvedic drugs. Eastern Pharmacist 2000; 5:21-24.

9.       Sheridon J. An overview of different types of viral hepatitis. Eastern Pharmacist 1999; 12:37-40.

10.     Chowdary KP, Rao GD, Kumar KK, Kanth RB. Validation of ayurvedic products and process. Eastern Pharmacist 1997; 21-33.

11.     Merck E. HPLC/TLC/HPTLC Application Manual; Darmastadt. FRG:1986

 

 

Received on 09.07.2010        Modified on 22.07.2010

Accepted on 01.08.2010        © AJRC All right reserved