world journal of pharmaceutical research subash et al

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Subash et al. World Journal of Pharmaceutical Research

RAPID METHOD FOR ISOLATION OF ANDROGRAPHOLIDE FROM

AERIAL PARTS OF ANDROGRAPHIS PANICULATA USING

MICROWAVE-ASSISTED EXTRACTION AND ITS

QUANTIFICATION BY NORMAL PHASE

HIGH-PERFORMANCE TLC

Subash Chandra Verma1*

, Amita Kumari1, Ektaa Vashishth

1, Kuntal Basu

2, Madan

Mohan Padhi 1

1Central Council for Research in Ayurvedic Sciences, 61-65, Institutional Area, Opp.-D-

Block, Janakpuri, New delhi-110058, India

2Arbro Analytical Division, Kirti Nagar, New Delhi, India

ABSTRACT

Andrographolide (AD) is the most important bioactive

phytoconstituent of Andrographis paniculata (Burm.f.) Ness and

exhibits antitumor metastasis, anti-HIV, antibacterial, anti-

inflammatory, antimalarial, antidiabetic activities etc. In this study,

microwave-assisted extraction (MAE) technique was employed for

rapid isolation of andrographolide from the aerial part of Andrographis

paniculata and simultaneously HPTLC was carried out for the

quantification of andrographolide in various solvent polarity based

extracts. Effects of several experimental parameters on the extraction

efficiencies of andrographolide, such as type and volume of extraction

solvents, microwave power and extraction times were evaluated. The

optimal extraction conditions were found to be 25 mL of a mixture of

chloroform/methanol, 50:50; preleaching time, 10 min; microwave power, 450 W;

temperature, 50οC; and microwave irradiation time, 8 min. Under the optimum conditions,

the percentage of andrographolide was quantified to be 0.05 ± 0.021 to 2.24 ± 0.016% in the

MAE extracts of aerial parts. Rapidly andrographolide was isolated from the

chloroform/methanol MAE extracts with 1.4% yield and 62.5% recovery by precipitation

process. The purity of AD was found ≥97.0%. MAE is a faster, convenient, and appropriate

World Journal of Pharmaceutical Research SJIF Impact Factor 5.045

Volume 3, Issue 4, 2069-2084. Research Article ISSN 2277 – 7105

Article Received on

30 April 2014,

Revised on 25 May

2014,

Accepted on 19 Jun 2014

*Correspondence for

Author

Dr. S. C. Verma

Central Council for Research in

Ayurvedic Sciences, 61-65,

Institutional Area, Opp.-D-

Block, Janakpuri, New delhi-

110058, India


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method for rapid isolation of andrographolide from A. paniculata than the conventional

methods with higher yield and lower solvent consumptions.

Key Words: Andrographis paniculata (Burm.f.) Ness; HPTLC methods; Microwave assisted

extraction (MAE); Andrographiloide

INTRODUCTION

Andrographolide is a labdane diterpenoid (Fig. 1) that is the main bioactive component of

Andrographis paniculata (Burm.f.) Ness. Andrographolide is an extremely bitter substance

extracted from the aerial part, stem and leaves of A. paniculata. The extracts and constituents

exhibit a wide spectrum of biological activities including antimicrobial [1], antihypertensives

and antidiabetics [2, 3], antifilarial [4], anti-inflammatory [5], antidiarrhoeal [6], antiviral [7],

antimalarial [8-10] activities etc. Being effective in reducing the growth of a variety of cancer

cell lines in vitro due to in vitro anticancer activity in many tumor cell lines i.e. leukemia,

myeloma, HeLa, colon (HT-29), human peripheral blood lymphocytes (HPBLs), and human

breast cancer MCF-7 [11], human breast cancer TD-47 cell line [12], it has been the subject

of interest for anticancer and antidiabetic research. Due to enormous applications of

andrographolide in treatment of cancer and diabetes many methods are reported for the

separation and determination of andrographolide by micellar electrokinetic capillary

chromatography, high-pressure liquid-chromatography [13- 14]. Isolation and purification of

andrographolide from plants by classical methods, such as maceration, soxhlet extraction, and

cold percolation, are tedious, time consuming, and expensive [15]. Consequently,

microwave-assisted extraction (MAE) is used to accelerate the extraction process of target

compounds from a variety of sources. The MAE extraction method is now widely used

because it is simple and rapid, involves use of lesser amount of solvent for extraction, and

with better yield [16–18], which utilizes microwaves power to enhance extraction efficiency

and provides a new dimension in the area of isolation of target molecules from the plant

drugs. Several applications of MAE for biologically active compounds have been described

in the literature, such as extraction of oleanolic acid from Lantana camara roots [19],

extraction of vanillin from Vanilla planifolia pods [20], rapid extraction and isolation of

arjunic acid and ursolic acid from Terminalia arjuna and Eucalyptus hybrida leaves

respectively [21-22], extraction of oleanolic acid and ursolic from Ligustrum lucidum Ait

[23], Actinidia deliciosa root [24] and Eriobotrya japonica residues [25] etc. and so on.

http://en.wikipedia.org/wiki/Labdane

http://en.wikipedia.org/wiki/Diterpenoid


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Isolation and purification of bioactive compounds from natural sources have become very

important for the use of phytochemicals due to least toxicity.

Figure 1. Structure of andrographolide

The present study describes solvent polarity based microwave-assisted extraction and rapid

isolation of andrographolide from the aerial part of Andrographis paniculata including the

effects of extraction solvent, volume, microwave power, and extraction time. Moreover,

quantification of andrographolide in extracts of aerial part of Andrographis paniculata was

assessed using the HPTLC method.

MATERIALS AND METHODS

Materials, standard and reagents

Fresh aerial parts of Andrographis paniculata (Burm.f.) Ness were collected from M/s

Azafran Innovacion Ltd., Ahmedabad, Gujarat, India, in the month of April 2010 and washed

thoroughly with water. Botanical identification of the plant was carried out at the National

Institute of Sciences Communication and Information Resources (NISCAIR), New Delhi and

a voucher specimen (NISCAIR/RHMD/Consult/2010–2011/1421/19) dated 31 May 2010 has

been deposited to NISCAIR, New Delhi. Aerial parts of plant were cleaned and dried under a

gentle stream of air in the laboratory for 4–5 days till no loss in weight was observed

(temperature, 28 2C; relative humidity, 45 5%) and powdered in an electric grinder.

Andrographolide was isolated from Andrographis paniculata (Burm.f.) Ness aerial parts and

used as a standard for HPTLC analysis. The purity of the andrographolide was found to be

≥97.0% based on the percentage of total peak area obtained by HPTLC.


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METHODS

Conventional extraction

Conventional extraction of aerial parts of A. paniculata was performed at room temperature

(28 3C) with solvents of increasing polarity i.e. n-hexane, dichloromethane (DCM),

chloroform, ethyl acetate: chloroform (50:50 v/v), ethyl acetate, chloroform: methanol (50:50

v/v), methanol, methanol: water (50:50 v/v). Dried and coarsely powdered aerial parts of A.

paniculata (5 g each) were extracted three times (25 mL) for 24 h of each extraction by

maceration with each of the above-mentioned solvents separately. Each extract was filtered

by using Whatman filter paper No. 1 and the solvents were removed under vacuum at 50°C,

separately and lypholized till each extract was free from solvents. The concentrated extracts

were re-dissolved separately in corresponding solvents and 100 mg/mL solutions of each

extract were prepared for the analysis of andrographolide.

Microwave-assisted extraction (MAE)

Kenstar domestic microwave (900 W; frequency, 2450 MHz) was used for the MAE. The

conditions of extraction such as amount of aerial parts of A. paniculata, nature and volume of

solvents were kept as mentioned in Section 2.2.1. Before microwave irradiation, each

suspension was allowed a preleaching time of 10 min. Each suspension was extracted at

temperature below the boiling point of the solvent and microwave power of 450W was

applied for 8 min. The sample was treated under microwave irradiation in an intermittent

way, i.e. irradiation cooling–irradiation. Other processes such as filtration, concentration of

MAE extracts, and sample preparing for HPTLC analysis were same as in Section 2.2.1. For

precision study, repeatability of the optimized method was measured as %RSD. Thus, 5.0 g

of sample was extracted under conventional method and optimized MAE conditions (n = 3)

on the same day (intraday precision) and on three consecutive days (interdays precision, n =

6) and then analyzed by proposed HPTLC method. Analyte was expressed as percentage of

the total peak area of the identified AD. Finally, the extracts obtained by the conventional and

MAE methods were analyzed by using HPTLC method. Peak identification was performed

on the basis of their relative retention time and comparison of UV spectra of standard AD.


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Figure-2 HPTLC method development and validation

Figure 2: HPTLC fingerprint of andrographolide and MAE extracts of A. paniculata arial

parts; chromatographic conditions are reported in section 2.2.1; AD, andrographolide;

extracts of T1, n-hexane; T2,dichloromethane; T3, ethylacetate; T4, EtOAc: CHCl3 (50:50);

T5, Chloroform; T6, CHCl3/MeOH (50:50); T7, methanol and T8, MeOH/water (50:50,v/v).

Chromatographic conditions

Separation of andrographolide in the extracts of aerial parts of A. paniculata was performed

on a CAMAG HPTLC system equipped with TLC applicator (Linomat-IV), TLC scanner-3,

photo documentation (Reprostar-3), winCATS version 1.3.3 software with and twin trough

glass tank (24.5 × 8 × 22.5 cm). The samples were applied on 20 × 10 cm aluminum packed

TLC plate coated with 0.2 mm layer of silica gel 60F254 (E. Merck Ltd, Darmstadt, Germany)

in 10 mm bands at 8 mm from the bottom, both sides and 8 mm space between the two bands.

TLC plates were developed in a Camag twin-trough glass tank which was pre-saturated with

developing solvent. The composition of the developing solvent was optimized by using

varying polarity of solvents. The plates were developed to a height of about 10 cm from the

base using Toluene: Acetone: Formic acid (06:04:0.4, v/v/v) as a solvent system. After

development, the TLC plate was removed, dried and derivatized with anisaldehyde-sulfuric

acid reagent. The TLC plate was kept at 105oC in oven for 5 minutes or till appeared the

spots and visualized under white light at 520 nm (Fig.2). Quantitative evaluation of the TLC

plate was performed in the reflectance/absorbance mode at 520 nm, with the following

conditions: slit width 6 × 0.45 mm, scanning speed 20 mm s-1

, data resolution 100 μm step-1

and HPTLC chromatograms are represented in Fig.3-4.


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HPTLC method validation

In order to make method useful in laboratories as a routine analysis procedure, performance

criteria were examined for parameters including analytical limits [26], linearity [27],

repeatability [27, 28], accuracy and recovery [26, 29-30] using standard laid-down protocols.

Figure 3. HPTLC chromatogram of andrographolide at 520 nm wavelength

Linearity curve, limit of detection (LOD) and limit of quantification (LOQ)

Standard stock solution of andrographolide (1 mg/mL) was prepared by dissolving 10 mg in

10 mL of methanol. Working standard solutions were prepared in a range of concentrations

(200–1000 ng/mL) by serial dilution of the standard stock solution. For preparing a

calibration curve of AD, each working standard solution was applied to the HPTLC system in

triplicate. Six-point calibration curve of AD was obtained by plotting the concentration of AD

versus peak area to check the linearity of response, LOD, and LOQ. LOD and LOQ were

calculated based on the SD of the y-intercept and the slope (S) as 3.3 and 10 SD/S,

respectively [26].

Repeatability and recovery studies

System repeatability was determined by running six replicate applications of the sample and

evaluated by calculating coefficient of variation (%CV) of the analysis. Peak areas of

andrographolide in six replicates of 400 ng/mL sample (5 µL) of A. paniculata were

measured.

The accuracy of the method was determined by analyzing the percentage recovery of

andrographolide in MAE extract, as it showed the presence of compounds of interest. The


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samples were spiked with different concentrations (200,400 and 800 ng/mL) of

andrographolide before extraction. The spiked samples were extracted in triplicate and then

analyzed by proposed HPTLC method to calculate the percentage recovery.

Figure 4. HPTLC chromatogram of microwave-assisted extractions (MAE) extract of A.

paniculata arial parts obtained from a mixture of chloroform/MeOH (50:50, v/v) at 520

nm.

RESULTS AND DISCUSSION

Rapid isolation of andrographolide from stem bark of A. paniculata

The coarsely powdered arial part (25 g) of A. paniculata was accurately weighed and placed

in a capped conical flask (1 L) and then mixed with 125 mL of a mixture of chloroform/

methanol (50:50). After soaking for 10 min that permitted solvent to wet plant material, the

flask with sample was kept in the microwave device, and irradiated for 8 min with preset

extraction temperature 50°C and microwave power 450 W. The irradiation time was kept for

1 min and 1 min was taken to cool the sample solution between two irradiations. Extract was

filtered and this process was repeated twice with marc. Each filtrate of same polarity was

pooled together and removed solvent by vacuum rotary evaporator at 50°C. The crude was

dissolved in a mixture of CHCl3/MeOH (90:10, v/v) and left overnight for precipitation. The

precipitate so obtained was crystallized with methanol and afforded compound AD, 0.35 g,

white amorphous powder with 1.4% yield and 62.5% recovery. The purity of AD was found

≥97.0% by HPTLC (Fig. 2-3). Compound AD showed melting point 229–230°C. It gave

emerald green color with copper acetate solution indicated diterpenoid nature of the

molecule. It did not respond Molisch’s test showing non-glycosidic nature of the molecule.


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The UV spectrum of the AD sample showed λmax 230 nm (CAMAG TLC Scanner 3). The

13C-NMR data of compound AD showed signals with deuterated pyridine at d (ppm), 37.3

(C1), 28.8 (C2), 79.8 (C3), 43.1 (C4), 55.2 (C5), 24.2 (C6), 38.0 (C7), 147.8 (C8), 56.2 (C9),

39.0 (C10), 24.9 (C11), 147.7 (C12), 130.8 (C13), 65.8 (C14), 75.0 (C15), 170.4 (C16), 108.4

(C17), 23.4 (C18), 64.0 (C19) and 15.0 (C20) which confirmed the molecular formula

C20H30O5. It forms triacetate with acetic anhydride-pyridine suggesting the presence of three

hydroxyl group in the molecule. On the basis of above spectral and chemical evidences

compound AD was identified as andrographolide (Fig. 1). The identity of the compound was

finally confirmed by comparison of melting point, UV and 13

CNMR-chemical shifts with the

reported data [31-32].

Optimization of MAE

Orthogonal array design (L9) procedure was followed for optimization of MAE of

andrographolide. All the samples were subjected to MAE. Four main factors i.e. nature of

solvents, solvent volume, microwave power and irradiation time were the optimized variables

with the constant sample amount (5 g). According to L9 design experiments were needed to

complete the optimization process. MAE was carried out for different time of irradiation at

different volumes with the microwave oven operating at different power levels as shown in

Table 1.

Effect of solvent and volume on extraction yield of andrographolide

Selection of suitable solvent is of most important significance in MAE [33]. It is common

practice to use a mixture of organic solvent and water at varying ratios to improve recovery

of phytoconstituents with MAE. For this reason, different solvents such as n-hexane,

dichloromethane, chloroform, ethyl acetate, methanol, and mixture (50:50, v/v) of ethyl

acetate/chloroform, chloroform/methanol and methanol/water were investigated to determine

the effective extraction of AD. Quantity of coarsely powdered aerial parts (5 g) was extracted

with 25 mL of different solvents mentioned in Table 2 under microwave power of 450 W and

a temperature of 50C for 8 min. The results of percentage AD in the extracts are showed in

Table 2. The yields of AD in both methods i.e. in MAE (2.24 ± 0.016 %) and conventional

(2.00 ± 0.005 %) reached the maximum when the solvent was a mixture of

chloroform/methanol (50:50, v/v). The polarity of solvent is an important factor which affects

the extraction yield. However, the dielectric constant and the dissipation factor of solvent

significantly influence the extraction yield in MAE [30]. Different solvent volumes (10, 25,


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50 mL) of a mixture of chloroform/methanol (50:50, v/v) were taken for the extraction of

AD, while other conditions such as microwave power, irradiation time, and temperature were

kept as Table 1. The yields of AD (2.24 ± 0.016%) reached the maximum when the solvent

volume was 25 mL as evident in Table 1. Therefore, the ratio of solvent to material (5:1) was

chosen for optimum extraction of AD from aerial parts of A. paniculata.

Effect of microwave power on extraction yield of andrographolide

Yield obtained with a mixture of CHCl3/MeOH (50:50, v/v) was maximum at microwave

power 450 W (60%) followed by 750W (100%) and 150W (20%). These results indicate that

microwave power is factor which has variable effect on the yield of the extracts. Hence trials

1-9 have to be carried out to choose appropriate power level for extraction. It is apparent

from the results that a microwave power of 450 W showed high yield of AD (2.24 %). Thus,

450 W microwave powers were chosen for MAE of AD from the whole plant of A.

paniculata.

Effect of irradiation time on extraction yield of AD

The extraction time must be optimized to ensure maximum recovery in the minimum analysis

time. The yield obtained with a mixture of CHCl3/MeOH (50:50, v/v) was maximum at 8

minute followed by 4 minute and 2 minute. Results showed that in order to get maximum

yield of the extracts appropriate irradiation times is necessary and it must be selected by

carrying out extraction trials with different irradiation time. Highest extraction of AD

occurred at 8 min according to results shown in Table 1. Therefore, extraction time of 8 min

was chosen for microwave irradiation to get the optimum yield of AD.

Precision study of MAE

The RSD% values for the yield of AD in the extract are represented in Table 3. In MAE

experiments, intraday repeatability and interday repeatability of the developed method found

was < 0.1. Intraday precision was lower than the interday precision in both methods, and

methods showed a good repeatability overall.


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Table 1: Optimization of MAE of andrographolide using a mixture of CHCl3/MeOH

(50:50, v/v) at 50°C

TR.

NO.

POWER

(W)

SOLVENT

VOLUME (ML)

TIME

(MIN.)

ANDROGRAPHOLIDE (%),

W/W

1. 750 50 2 1.65 ± 0.014

2. 750 25 4 1.72 ± 0.012

3. 750 10 8 1.93 ± 0.008

4. 450 50 4 1.86 ± 0.011

5. 450 25 8 2.24 ± 0.016

6. 420 10 2 1.28 ± 0.018

7. 150 50 8 1.22 ± 0.012

8. 150 25 2 1.06 ± 0.016

9. 150 10 4 1.15 ± 0.017

HPTLC method development and validation of AD

The selectivity of the method was determined by comparison of chromatographic profile of

AD with samples, considering the following parameters such as retention time, maximum

wavelength of absorption, and UV spectrum overlay [28]. The spots of AD in samples were

identified by comparing their retention times and UV spectra obtained from the spots with

standard andrographolide. Calibration curves were constructed by plotting the peak area of

AD versus nominal concentration. A weighed (1/x) linear regression was used to perform

standard calibration, giving a mean linear regression equation for the calibration curve of y =

3.727x + 94.89 (n = 6, r 2 = 0.981), where y represents the peak area and x represents the

concentration. A good linearity was achieved in the range of 200–1000 ng/spot, r2 0.981 for

AD. LOD and LOQ for AD were found 100 ng/spot and 200 ng/spot, respectively. Six

replicate injections of same solution of AD (200 ng/mL) and six injections of extract samples

(chloroform/methanol extract, 1000 µg/mL) of same concentration were analyzed by the

proposed method to determine the system and method precision, respectively. In system

precision, %RSD value of Rt and peak area were 0.01% and 0.23%, respectively, whereas in

method precision %RSD value of Rt and peak area were 0.01 % and 0.16%, respectively; it

indicated that the %RSDs of Rt and peak area of AD were within 0.3%, which indicated very

less variation of the measured values. Recoveries of the experiment were performed in order

to determine the accuracy of the method. Recoveries at three level – i.e., level 1 (50%), level

2 (75%), and level 3 (100%) were carried out, which showed percentage recoveries of 94.82

± 0.24, 95.35 ± 0.12 and 96.24 ± 0.21 respectively. All the percentage recoveries of AD were

found to be within the range of 98–102% represented the good accuracy of the method. The


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results of linearity, LOD, LOQ, precision, and recovery have been summarized in Table 3

and indicated that the proposed method was found in accord with the required criteria.

Application of the MAE method

To improve the efficiency of the optimized MAE method, it was compared with the

conventional extraction method. With respect to the extraction time, MAE method was the

fastest one that require only 8 min for providing the highest yield of AD 2.24 ± 0.016 of dry

weight, whereas the yield in conventional extraction method did not exceed 2.000.005% in a

mixture of chloroform/MeOH (50:50, v/v), which could thus be safely assumed as optimum

solvent for extraction of AD (Table 2).

Table 2: Comparison of andrographolide % yield in extract of A. paniculata arial parts

obtained by MAE and conventional methods

SOLVENT

MAE EXTRACTION

(AT 50°C, 450 W, 8 MIN) CONVENTIONAL EXTRACTION

(AT 70°C, 4 HRS)

EXTRACT

YIELD(%±

SD)

ANDROGRAPHOLI

DE

(%± SD)

EXTRACT

YIELD(%± SD)

ANDROGRAPHOLID

E (%± SD)

n-Hexane 2.12 ± 0.02 Not detected 2.85 ± 0.07 Not detected

Dichloromethane 5.68 ± 0.05 1.69 ± 0.012 6.27 ± 0.28 1.66 ± 0.006

Chloroform 9.58 ± 0.14 1.95 ± 0.018 10.72 ± 0.08 1.82 ± 0.002

EtOAc:CHCl3

(50:50,v/v)

8.78± 0.09 1.64 ± 0.025 9.60 ± 0.18 1.58 ± 0.003

Ethyl acetate 8.21 ± 0.24 1.98 ± 0.008 9.47 ± 0.12 1.69 ± 0.003

CHCl3:MeOH

(50:50,v/v)

11.75 ± 0.21 2.24 ± 0.016 12.78 ± 0.11 2.00 ± 0.005

Methanol 13.46 ± 0.05 2.01 ±0.015 14.40 ± 0.22 1.96 ±0.005

MeOH: water

(50:50,v/v)

17.34 ± 0.16 0.05 ± 0.021 18.10 ± 0.05 Not detected

Although, maximum yields of extracts of aerial parts were obtained by the conventional

method (18.10±0.05%) against MAE (17.34±0.16) in methanol/water (50:50, v/v). From the

above-mentioned results, it is evident that MAE method is more efficient than conventional

extraction methods for extraction of AD (Table 2).


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Table 3: Summary of HPTLC method validation for andrographolide

PARAMETERS ANDROGRAPHOLIDE

Linearity

Range (ng/ mL)

Linear equation

Slope (m)

Intercept (C)

Correlation coefficient (r2)

200 – 1000

Y= 94.89+3.727x

3.209

989.6

0.981

Precision (%RSD)

System precision ( n=6)

Repeatability of peak area

Repeatability of Rt (0.51)

method precision ( n=6)

Repeatability of peak area

Repeatability of Rt (0.51)

0.23%

0.01%

0.16%

0.01%

Limit of Detection(LOD) 100 ng/spot

Limit of Quantitation

(LOQ)

200 ng/spot

Specificity Specific

Recovery (%)

Level 1- 50%

Level 2- 75%

Level 3- 100%

94.82 ± 0.24

95.35 ± 0.12

96.24 ± 0.21

However, AD was not detected from the extract obtained from n-hexane (Fig 2). The

percentage yield of AD in the MAE extracts was found in the range from 1.69 ± 0.012 % to

2.24 ± 0.016 % while percentage yield of AD in convention extracts was reported from 1.66

± 0.006 % to 2.00 ± 0.005 % as revealed in Table 2. Further, quantification and identification

of remaining secondary metabolites present in aerial extracts of by HPTLC is under progress.

CONCLUSION

In this study, a new precise, economical, and time-saving extraction method based on the use

of microwave energy has been modified for the analysis of AD from A. paniculata, aerial

parts by using a HPTLC technique. Further, a rapid method for the isolation of AD was

developed. The proposed MAE method allows the extraction of AD in a shorter time (8 min)

with higher efficiency when compared to the conventional solvent method. Therefore, MAE

proved to be an attractive alternative to conventional extraction methods for the extraction of

AD compounds from A. paniculata. The optimal MAE conditions were as follows: extraction

solvent, mixture of chloroform/MeOH (50:50, v/v); microwave power, 450 W; extraction

solvent, 25 mL; extraction time, 8 min. Under the optimal conditions, the yield of AD was

2.24 ± 0.016 %. Developed and validated HPTLC method could be helpful for the


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determination of the individual AD in extracts with excellent precision, accuracy, and

linearity. The MAE extract could be used as either components of some complex herbal

medicines or for further isolation and purification of andrographolides (anticancer and

antidiabetic molecule) with better yield.

ACKNOWLEDGMENT

This research was supported by Director General, Central Council for Research in Ayurvedic

Sciences, New Delhi. M/s Arbro Analytical Division, Kirti Nagar, New Delhi is highly

appreciated for providing the desired facilities.

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