Extraction, Purification and Drying of Andrographolide from A

www.wjpps.com Vol 3, Issue 10, 2014.

747

Jadhao et al. World Journal of Pharmacy and Pharmaceutical Sciences

PURIFICATION (CRYSTALLIZATION) OF BIOACTIVE

INGREDIENT ANDGROGRAPHOLIDE FROM ANDROGRAPHIS

PANICULATA

Dilip Jadhao* and Bhaskar Thorat

Advanced Drying Laboratory, Department of Chemical Engineering, Institute of Chemical

Technology (Formerly UDCT), N. P. Road, Matunga (E), Mumbai 400 019, India.

ABSTRACT

In the study, the purification of Andrographolide from the

Andrographis Paniculata was carried out using different physical

separation techniques such as extraction and crystallization followed

by drying. The extraction of the andrographolide from the A. Paniculta

was carried out using different solvents. The effect of andrographolide

to solvent ratio on extraction efficiency was studied. It was found that

the andrographolide to solvent ratio of 1:3.5 w/v gives higher

percentage purity of andrographolide. The solubility study of

andrographolide was studied in order to find out the best solvent for

crytsallization. Subsequently, the extract which was obtained after

extraction was treated with activated charcoal to get rid of the

undesired impurity which may hinder the process of crystallization.

Subsequent to extract clarification, extract was concentrated by

evaporation. The process of cooling crystallization was effectively

employed for further purification of andrographolide with the recovery of 95%

andrographolide with high purity. The process of crystallization was studied in terms of

supersaturation (more refined product). The andrographolide was confirmed by LCMS and

Melting point. Scanning electron and inverted microscopy were applied to find out the

morphology and the size of purified andrographolide. It was observed that andrographolide

gives different size of cube shaped whitish crystals in the range to 30µm- 40 µm.

Keywords: Crystallization, herbal products, medicinal plant, anti-pyretic.

WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS

SSJJIIFF IImmppaacctt FFaaccttoorr 22..778866

VVoolluummee 33,, IIssssuuee 1100,, 774477--776633.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357

Article Received on 21 July 2014,

Revised on 14 August 2014,

Accepted on 05 September

2014

*Correspondence for

Author

Dr. Dilip Jadhao

Advanced Drying

Laboratory, Department of

Chemical Engineering,

Institute of Chemical

Technology (Formerly

UDCT), N. P. Road,

Matunga (E), Mumbai 400

019, India


748


INTRODUCTION

Andrographis paniculata (Burm. f.) Nees (Acanthaceae), native to Taiwan, Mainland China

and India, is a medicinal herb with an extremely bitter taste used to treat liver disorders,

bowel complaints of children, colic pain, common cold and upper respiratory tract infection

[1-3]. The aerial part of A. paniculata is commonly used in Chinese medicine. According to

Indian ayurveda, A. paniculata 'cools' and relieves internal heat, inflammation and pain and is

used for detoxication [4-6]

.

The three main diterpenoid lactones identified in A. paniculata leaves were andrographolide,

neo-andrographolide, and deoxyandrographolide [7, 8]

. Andrographolide, which is grouped as

an unsaturated trihydroxy lactone has the molecular formula of C20H30O5. The molecular

structure of andrographolide is shown in Fig. 1.

O

CH2

H3C CH2OHHO

CH3

HOO

Figure 1: Molecular structure of Andrographolide

Andrographolide, the main component in the leaves of A. paniculata can be easily dissolved

in methanol, ethanol, pyridine, acetic acid, and acetone, but has limited solubility in ether and

water. Its physical properties are: melting point at 228-230oC, and the ultraviolet spectrum in

ethanol λmax 223nm [8]

.

Various methods of extraction of andrographolide have been reported, such as hydrotropic,

microwave assisted and Soxhlet extraction etc

[9-12]. Followed by extraction, to achieve better

enrichment of andrographolide, various columns chromatographic techniques for

andrographolide purification from the crude drug of Andrographis Paniculata have been

reported. However, these techniques were found to be time consuming, expensive and

tedious, leaving impure andrographolide. By keeping these things in mind, the objective of

this research work was to develop a solvent based extraction of andrographolide from

Andrographis Paniculata.


749


It was obvious that crystallization should be an effective method applied to separate natural

product from herbal extract [13-14]

. Crystallization is surely rank as the oldest units operation

in the biochemical engineering sense. Apart from being one of the best and cheapest methods

available for the production of pure solids from impure solutions, crystallization has the

additional advantage of giving an end product that had many desirable properties. There are a

large number of solvents and solvent mixtures suitable for the crystallization purification

process. However, when aiming at simple purification process it is beneficial to use only one

solvent instead of a solvent mixture. The solubility characteristics of a solute in a given

solvent have a considerable influence on the choice of a method of crystallization. Both the

solubility power and the solubility power with temperature should be considered when

choosing a solvent for a crystallization process; the former quantity influenced the volume of

the crystallizer, and the latter determined the crystal yield. Thus, the choice of the suitable

solvent and proper operation condition became especially important during the process of

separation. The selection of the ‘best’ solvent for a given crystallization operation was not

always an easy matter.

There are some reports on the solubility studies of andrographolide in ethanol, methanol and

dichloromethane [15]

. Hence, the similar available solubility data was used to carry out

crystallization and to study the effects of solvents on the polymorphs formation.

Moreover, the development of drugs from natural plants usually requires the isolation and

purification of the target compound from complex multi-component mixture to produce high

purity product. Therefore, the objective of present work is to investigate the possibility of

combining the advantages of crystallization to generate a hybrid process for the isolation and

purification of andrographolide from the crude extract of Andrographis Paniculata.

MATERIALS AND METHODS

Chemicals

The finely grounded to 80 mesh size leaves powdered of A. paniculata was collected from

local herbal supplier, Mumbai, India. The pure (approx. 98%) andrographolide was obtained

from Sigma Aldrich, Mumbai. Thin layer chromatographic (TLC) plates and silica gels were

obtained from S.D. Fine Chemicals, India. Solvents such as methanol, ethanol, ethyl acetate,

acetone, petroleum ether, dichloromethane, chloroform was obtained from S.D. Fine

Chemicals, India.


750


Solid-Liquid Extraction

In the first set of experiments, the extraction was performed using different solvents. The

suitable solvent which gives higher andrographolide enrichment in the extracted phase was

selected for further experiments. The leaves were ground to a powder (80 mesh size) and

extracted at reflux temperature for 3 h. The extraction of andrographolide from the ground

powder was carried by mixing powder and solvent using Sox-let extraction technique. The

powder to the different solvent ratio (w/v) used for all the studies. The solvent was removed

and the process was repeated for one more time to remove the final traces of andrographolide

from the ground powder of leaves. The extracts were then combined and concentrated by

recovering the solvent using Buchi rotavapour. The obtained brownish enriched extract of

andrographolide was used for further studies. The effect of andrographolide to solvent ratio

on the extraction efficiency was also studied by varying the andrographolide to solvent ratio.

Enrichment of Andrographolide Extract

The most important operation in phytochemical separation process is the extract clarification

because it results in better visual quality of the final product. Since, the leaf of A. Paniculata

contains the coloring matters such as chlorophyll which gets sticky in the extraction phase

after extraction and makes andrographolide purification difficult. To get rid of this difficulty,

the crude green and dark brown extract was treated with different percentages in the range of

5 to 25% of activated charcoal and reflux for 20 min. The extract was filtered and the residual

charcoal was again mixed with methanol and reflux one more time for 10 min. The filtrates

were then combined and concentrated. The total content of chlorophyll was determined to

check the level of andrographolide in the extract and it was done by taking the absorbance at

646 and 662 nm [16-17]

. The obtained yellow colored extract used for further study.

Determination of Solubility

The solubility of andrographolide in methanol was measured at different temperatures. To the

10 mL of solvent, excess quantity of andrographolide was added. Subsequently, the liquid-

solid suspension was constantly agitated at 120 rpm at 30°C for 2h in REMI Shaker to

achieve uniform mixing. The clear solution was then removed using syringe filter and dried

in the vacuum oven at 50ºC. The obtained solids were weighed and the solubility was

reported as mg of andrographolide per ml of solvent. The same procedure was repeated at

different temperatures in order to get solubility curve.


751


Purification of Andrographolide Using crystallization

The isolation of andrographolide from diterpene lactones mixture of A. Paniculata was

carried out by using evaporation followed by slow cooling crystallization technique. The

supersaturation of the solution is the driving force for both crystal growth and nucleation. To

achieve supersaturation, the methanol was recovered using evaporation process from final

extract which obtained after clarification step. This leads to the increase in the solid

concentration of andrographolide. Andrographolide extract which was obtained after

clarification process was concentrated by recovering the methanol by evaporation at 65-70ºC

till the volume of extract reduced to the initial extract. Yellowish clear solution was then

filtered and the filtrate was allowed to cool slowly at room temperature to attain the

supersaturation level. During cooling, subsequent to attaining the supersaturation level after

over period of time expels the yellowish solids from the solution. The formation of yellowish

solid can be referred to the appearance of supersaturation and thereafter, crystal formation.

The mother liquid was decanted and crystals were collected and dried in a vacuum dryer at

50oC for 3-4 hrs. By carring out crystallization repeatedly for couple of times, more refined,

whitish, high purity andrographolide could be obtained.

Furthermore, in order for crystallization to take place a solution must be "supersaturated".

The supersaturation is the concentration difference between that of the supersaturated

solution in which the crystals are growing and that of a solution in equilibrium with the

crystal. The supersaturation can be defined by equations (1) and (2).

(1)

The supersaturation ratio is defined by,

(2)

Where,

y = Supersaturation, mass fraction of solution

y = mass fraction of solute in solution

ys = mass fraction of solute in saturated solution.

HPLC Analysis of Andrographolide

The Agilent (Germany) HPLC system, consisting of a model G1329A standard auto-sampler,

model G1316A thermostat column, model G1322 A vacuum degasser, quaternary pump,

model G1314B variable wavelength detector, was used. The separation was achieved on a


752


stainless steel silica based Zorbax Eclipse XDB–C18 column (ф4.6 mm×150 mm, 5 µm). The

column temperature was maintained at 30ºC. Andrographolide was eluted using mobile phase

consisting of methanol and 0.1% v/v H3PO4 (70:30) at the flow rate of 1 ml/min. The eluent

was monitored at 223 nm. The standard curve was obtained by analyzing known

concentration of Andrographolide. The standard curve was plotted between the concentration

of andrographolide and the area under the curve. This plot was used for the determination of

concentration of the andrographolide in the unknown solution. All the samples were prepared

in the methanol of 10 mg/l concentration and filtered through 0.22 µm filter to remove any

suspended particles. The amount of sample injected in the column was kept constant at 10 µl.

All the solvents used in the HPLC analysis were first filtered through 0.22 µm filter and then

sonicated for 10 min to remove any dissolved gases.

Crystal Morphology and Characterization Study

To know more about morphology and size of andrographolide crystals, electron micrographs

of crystals were obtained using a scanning electron microscope (Leica Cambridge S360, UK)

operating at 5 kV. The specimens were mounted on plasma coated with JEOL-JFC-1600

AUTO FINE COATER.

Extracted pure andorgrapholide was characterized by melting point apparatus from Acumen

Labware and LCMS/MS at department of chemical engineering, Institute of chemical

technology, Mumbai.

RESULT AND DISCUSSION

Extraction

The effect of different solvents such as ethanol, methanol, DCM etc was studied. Fig.2 shows

the % yield of andrographolide obtained with different solvents. It shows that methanol

extraction which gives higher yield compared to other solvents.


753


Figure 2: Effect of solvents on the extraction of Andrographolide

Different solvent extraction using methanol, ethanol and dichloromethane were all

performed. Fig.2. displayed HPLC chromatograms of the extracts, where a represent main

andrograopholide. In evidence, Fig. 2 and Fig.3 implied that there were less undesired

components in the methanol extract than those obtained by them methanol or

dichloromethane extraction. As for the extracts, the purity of the product is the prime

characteristic and quality factor. It would be easy to judge extraction methods if the target

compound to be enriched was single and well-defined. In comparison to non – polar solvents,

polar solvents could extract andrographolide at higher yield except water, where hydrolysis

and thermal degradation might occur. Methanol was found to be the best solvent for the

extraction of andrographolide [9, 10]

. Ethanol and aqueous acetone extracted andrographolide

at lower yield although their solubility parameters are closer to that of andrographolide.

Solvents having moderate polarity extracted andrographolide much lower than methanol did.

Non - polar solvents were almost not able to extract andrographolide.

Isolation of andrographolide

The crude leaf extract was deep green in color due to the presence of pigment such as

chlorophyll and flavonoides. Figure 4 is the chromatogram of the crude extract showing

several peaks along with one major peak of andrographolide at the retention time 4.1 min.

which was exactly matching with standard andrographolide sample peak which is show in the

Figure 9.

0

0.2

0.4

0.6

0.8

1

1.2

Acetone Ethanol Methanol Dicloromethane Hexane


754


Figure 3: Effect of dry feed to solvent ratio on the percent yield of andrographolide

Figure 4: Chromatogram of initial extract of Andrographis Paniculata leaves

The chromatogram shows that the pigments such as chlorophyll present in the crude extract

affects to the great extent in the purification process and therefore the purity of the

andrographolide. To isolate the andrographolide in the pure form, we thought of simple

crystallization process other than column chromatography, membrane separation technology

or filtration technology, since these technologies are very costly, tedious and not physible for

transfer for large scale.


755


To get rid from the chlorophyll and other impurities, charcoal was selected as adsorbent since

it is very economical and therefore suitable for large scale also. Apart from charcoal, there is

other the most common industrial adsorbents are activated silica gel, and alumina, because

they present enormous surface areas per unit weight. In Case of activated charcoal, present

more surface area than other adsorbent, since it is available commercially in the different

granular sizes. Previous to use in the purification or isolation experiment, it is necessary to

wash it from water because it may contains high iron, acid, ash, and water soluble impurities

which may hinder in the isolation process. After washing the activated carbon, the crude

green extract was treated with different percentages to optimize the percentage of activated

charcoal, i.e., 5 to 20% of activated charcoal to remove the chlorophyll and other impurities.

This resulted into substantial removal of chlorophyll from the crude mixture as visually

observed as fairly higher color of solution. The pigment content reduces by about 90% in

terms of chlorophyll as shown in this figure.

Figure 5: Total chlorophyll content after charcoal treatment

Crystallization

To select the appropriate solvent for a crystallization process, solubility and the ICH

guidelines are the key aspects of the process. Furthermore, the starting point for most

crystallization processes is a saturated solution. Crystallization is generally achieved by

reducing the solubility of the product in this solution by cooling, antisolvent addition,


756


evaporation or some combination of these methods. The fundamental of solubility which

often depends on temperature and also it was noticed that the solubility of many substances

increases with increasing temperature. In general, at a given temperature, there is a maximum

quantity of solute that can be dissolved in a given solvent. At this point, the solution is

saturated. The quantity of solute dissolved per unit of solvent at this point is the solubility. In

case of androgrpaholide crystallization and solubility, andrographolide are highly soluble in

methanol, consequently the crystallization was carried out in the same medium. Figure (6)

shows the solubility profile of crystallization in methanol as a function of temperature.

Figure 6: Equilibrium relationship for bulk andrographolide crystals

The solubility remarkably increases from 150C to 65

0C, almost by a factor of two. Before

450C and post 65

0C, the change in the solubility is small as compared to 15-65

0C temperature

range. Moreover, the solubility profile represents an equilibrium relationship in

crystallization processes. Equilibrium is reached when the solution is saturated and the

equilibrium relationship indicates the significant crystallization point where, maximum

recovery of crystallized product was obtained which was shown in Table 1. Such type of

curve is an ideal one for cooling crystallization, where supersaturation by means of cooling

brings about the separation of two phases rather easily. Figure (7) shows the plot of

supersaturation as a function of temperature difference and it can seen that approximately


757


0.0003 mass fraction of solute was the degree of supersaturation and the corresponding

supersaturation (α) given by equation (2) was found to be in the range of 1.18.

Figure 7: Supersaturation and Temperature potential

When the solubility of andrographolide increases appreciably with temperature, the

supersaturation can be expressed as an equivalent temperature difference instead of mass

fraction difference. The relation between these driving potential is shown in Figure (7) which

contains a small section of the solubility curve of andrographolide in mass fraction solute.

The field above the line at 650C temperature represents the unsaturated solution and that

below the line, supersaturated solutions. Point A refers to a saturated solution at temperature

Tc, which is the temperature of the growing crystal, and point D to the supersaturated solution

at temperature T. Since, the heat is evolved by the crystal as it grows, Tc is slightly larger

than T, providing the driving force of Th for heat transfer from crystal to the liquid. The

supersaturation ∆y is normally based on the bulk temperature and, as shown by the difference

in point E and D.

Point B refers to a saturated solution of the same composition as the supersaturated solution

in which the crystals are growing. It would be at a temperature Ts, where Ts > T. Point C

refers to temperature Tc and the concentration equal to that of supersaturated solution.


758


Using Equation (1), the supersaturation potential can be represented by the line segment A .

The equivalent temperature driving potential can be shown by line segment BC. Segment AB

of the solubility curve can be considered linear over the small concentration spanned by the

line AC and the temperature potential defined by

(3)

Ts = Supersaturation Temperature, Tc = Saturation Temperature

From the above equation, the temperature potential (Tc) was found to be 20°C which was

slightly smaller than the actual difference in temperature, T of the solution and the

corresponding saturation temperature Ts.

Table 1. Isolation of Andrographolide after Crystallization and Re-crystallization

Andrographolides crystals as viewed via SEM images at 1000x magnification and it was

observed that andrographolide gives different size of rod shaped whitish crystals and many

rods reunited together in the absence of magnetic field and it was found in the range of

30µm- 40 µm which was having very good solubility in 20 % ethanol. Since having very

good solubility, this type of crystals can be used in the preparation of different type of herbal

formulations or medicines such as antipyretic, cough, antiviral under approval of GRAS

(Generally Recognized as Safe).

Characterization of Andrographolide by LCMS/MS

Extracted pure Andrographolide and standard andrographolide were characterised by

LCMS/MS and it was found that the molecular weight of andrographolide was found exactly

corresponding with standard andrographolide. To achieve this, Pure compound was

solubilized in DMSO and a (500ng/ml) solution of analyte in Acetonitrile:water::50:50 was

prepared for characterization. First a Q1 (Parent ion) scan was done, mass range from 300 to

500 m/z in both positive and negative mode. An intense peak at m/z 349.20 in negative mode

was obtained and MS2 scan of 349.20(Q1) was done in the mass range of m/z(mass/ion) 80

Crystals procured

from different

operations

Melting

point ºC

Andrographolide

(%)

Recovery or

Overall

Yeild (%)

Andrographolide

Crystallization by

evaporation followed

by cooling

223-230 93.67 94.1

Recrystallization 228-230 96 92.67


759


to 400. Two intense daughter ions 287.1 and 331.10 were obtained. 331.1 daughter ion may

be due to water loss from the parent ion.


760


Figure 8: SEM images of Andrographolide

HPLC Analysis

The chromatograms of andrographolide from the crude drug of A. Paniculata were compared

with standard andrographolide and the percent purity of andrographolide crystals was found

to be 96%. Figure 8 and 9 shows the chromatograms of standard andrographolide and crystals

obtained in this study, respectively. The presence of andrographolide at 4.18 min retention

time, clearly shows the intrinsic advantage of crystallization in attaining more bitter

component as a substantial fraction in the extracted purified product.

Figure 9: Chromatogram of Standard andrographolide


761


Figure 10: Chromatogram obtained crystals of andrographolide

CONCLUSION

The extraction of andrographolide from the powder of A. Paniculata using methanol as a

solvent was carried out. The optimized ratio of dried A. Paniculata powder to methanol was

found to be 1:3.5. Followed by conventional extraction, the extract clarification was

successfully carried out using charcoal treatment. Evaporation followed by cooling

crystallization was effectively employed for the recovery of andrographolide and it was found

to be in the range of 90-96% of recovery of andrographolide with 96% purity. Solubility

study at the different temperature of Andrographolide was carried out in methanol. Purified

andrographolide was effectively characterized by LCMS and Melting Point. The process

parameters of crystallization were studied in terms of such as supersaturation (y),

supersaturation ratio () and temperature potential (TC). To obtain substantial yield of

andrographolide, 20°C super cooling were found to be sufficient practically. The simple and

novel approach based on extraction followed by clarification of extract and crystallization

suggested in the present work might be one of the most promising techniques for this kind of

natural bitter separation and purification.

ACKNOWLEGEMENT

The authors would like to thanks profusely to Rajiv Gandhi commission for Science and

Technology, Government of Maharashtra, India for providing the funding for this research

work.


762


REFERENCES

1. Negi AS, Kumar, JK, Luqman SS, Banker K, Gupta MM, Khanuja SP. (Recent advances

in plant hepatoprotectives: a chemical and biological profile of some important leads).

Med. Res. Rev, 2008; 28(5): 821.

2. Roxas M, Jurenka J. (Colds and influenza: A review of diagnosis and conventional,

botanical and nutritional considerations). Altern. Med. Rev, 2007; 12: 25-48.

3. Kligler B, Ulbricht C, Basch E, Kirkwood CD, Abrams TR, Miranda M, Singh Khalsa KP,

Giles M, Boon H, Woods J. (Andrographis paniculata for the treatment of upper

respiratory infection: a systematic review by the natural standard research collaboration).

Explore, 2006; 2(1): 25-29.

4. Huang C J, Wu M C. (Differential effects of foods traditionally regarded as 'heating' and

'cooling' on prostaglandin E2 production by a macrophage cell line). J. Biomed. Science,

2008; 9: 596-606.

5. Chao WW, Koon YH, Li WC, Lin BF. (The production of nitric oxide and prostaglandin

E2 in peritoneal macrophages is inhibited by Andrographis paniculata, Angelica sinensis

and Morus alba ethyl acetate fractions). J. Ethnopharmacol, 2009; 122: 68-75.

6. Mandela SC, Dhara, AK, Maiti BC. (Studies on Psychopharmacological Activity of

Andrographis paniculata Extract). Phytother. Res, 2001; 15: 253-256

7. Choudhury BR, Haque SJ, Poddar M.K. (In vivo and in vitro effects of kalmegh

(Andrographis paniculata) extract and andrographolide on hepatic microsomal drug

metabolising enzymes), Planta. Med, 1987; 53: 135-140.

8. Rajani M, Shrivastava N, Ravishankara MN. (A rapid method for isolation of

andrographolide from Andrographis paniculata Nees (Kalmegh)”, Pharmaceuts. Biol,

2000; 38: 204-209.

9. Mishra SP, Gaikar VG. (Aqueous hydroropic solutions as an efficient solubilizing agent

for andographolide from andrographis paniculata leaves. Sep. Sci. Technol, 2006; 41 (6):

1115-1134.

10. Wongkittipong R, Prat L, Damronglerd, S, Gourdon C. (Solid–liquid extraction of

andrographolide from plants—experimental study, kinetic reaction and model), Sep.

Purif. Technol, 2004; 40; 147–154.

11. Xu HN, He CH. (Extraction of isoflavones from stem of Pueraria lobata (Willd.) Ohwi

using n-butanol/water two-phase solvent system and separation of daidzein. Sep. Purif.

Technol, 2007; 56: 85–89.


763


12. Patil VV, Galge RV, Thorat BN. (Extraction and Purification of phosphatidyl choline

from soyabean lecithin). Sep. Purif. Technol, 2010, 75 (2): 138-144.

13. Nie Q, Wang, J, Qiuxiang, Y. (Separation and Purification of two isomorphic steroids by

a novel extractive drowning out crystallization). Sep. Purif. Technol, 2006, 50: 342–346.

14. Xu WL, Huang YB, Qian JH, Sha O, Wang YQ. (Separation and purification of

stigmasterol and β-sitosterol from phytosterol mixtures by solvent crystallization method..

Sep. Purif. Technol, 2005, 41: 173–178.

15. Chen M, Xie C, Liu L:(Solubility of Andrographolide in Various Solvents from 288.2 to

323.2 K). J. Chem. Eng. Data, 2010; 55: 5297–5298.

16. Iain L, Suryana, N, Lukulay P,Marcus I. (Determination of chlorophyll a and b by

simultaneous multi-component spectrophotometry). Fresenius' J. of Anal. Chem, 1995;

352: 456-460.

17. Sukran D, Tohit G, Ridvan S. (Spectrophotometric Determination of Chlorophyll - A, B

and Total Carotenoid Contents of Some Algae Species Using Different solvent). Tr. J. of

Botany, 1998; 22: 13-17.

18. Siripong, P, Kongkathip, K, Preechanukool, P, Picha, P, Tunsuwan, K, Talor, WC.

(Cytotoxic Diterpenoid Constituents from Andrographis Paniculata Ness Leaves). J. Sci.

Soc. Thailand, 1992; 18: 187-194.

19. Chan WR, Taylor DR, Willis CR, Bodden RI, Fehlhaber HW. (The structure and

stereochemistry of neoandrographolide, a diterpene glucoside lactone from Andrographis

Paniculata Nees). Tetrahydron Letters, 1997; 46: 4803-4806.

Documents

Extraction, Purification and Drying of Andrographolide from A