WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Jadhao et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 10, 747-763. Research Article ISSN 2278 4357

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
Article Received on
21 July 2014, In the study, the purification of Andrographolide from the
Revised on 14 August 2014,
Accepted on 05 September Andrographis Paniculata was carried out using different physical
2014
separation techniques such as extraction and crystallization followed
by drying. The extraction of the andrographolide from the A. Paniculta
*Correspondence for was carried out using different solvents. The effect of andrographolide
Author
to solvent ratio on extraction efficiency was studied. It was found that
Dr. Dilip Jadhao
the andrographolide to solvent ratio of 1:3.5 w/v gives higher
Advanced Drying
Laboratory, Department of percentage purity of andrographolide. The solubility study of
Chemical Engineering, andrographolide was studied in order to find out the best solvent for
Institute of Chemical crytsallization. Subsequently, the extract which was obtained after
Technology (Formerly
extraction was treated with activated charcoal to get rid of the
UDCT), N. P. Road,
undesired impurity which may hinder the process of crystallization.
Matunga (E), Mumbai 400
019, India 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 30m- 40 m.

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

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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,
[7, 8]
neo-andrographolide, and deoxyandrographolide . 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
HO

CH3 CH2

HO
H3C CH2OH

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 max 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.

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It was obvious that crystallization should be an effective method applied to separate natural
[13-14]
product from herbal extract . 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
[15]
dichloromethane . 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.

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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 30C 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 50C. 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.

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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 65-70C
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

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stainless steel silica based Zorbax Eclipse XDBC18 column (4.6 mm150 mm, 5 m). The
column temperature was maintained at 30C. 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 m filter to remove any
suspended particles. The amount of sample injected in the column was kept constant at 10 l.
All the solvents used in the HPLC analysis were first filtered through 0.22 m 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.

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1.2

0.8

0.6

0.4

0.2

0
Acetone Ethanol Methanol Dicloromethane Hexane

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
[9, 10]
extraction of andrographolide . 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.

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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.

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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,

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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 650C, almost by a factor of two. Before
450C and post 650C, the change in the solubility is small as compared to 15-650C 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

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0.0003 mass fraction of solute was the degree of supersaturation and the corresponding
supersaturation () 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, T c 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 T s, where Ts > T. Point C
refers to temperature Tc and the concentration equal to that of supersaturated solution.

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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 20C which was
slightly smaller than the actual difference in temperature, T of the solution and the
corresponding saturation temperature T s.

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

Crystals procured Recovery or

Melting Andrographolide
from different Overall
point C (%)
operations Yeild (%)
Andrographolide
Crystallization by
evaporation followed 223-230 93.67 94.1
by cooling
Recrystallization 228-230 96 92.67

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
30m- 40 m 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

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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.

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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

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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, 20C 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.

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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. (Solidliquid extraction of
andrographolide from plantsexperimental study, kinetic reaction and model), Sep.
Purif. Technol, 2004; 40; 147154.
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: 8589.

www.wjpps.com Vol 3, Issue 10, 2014. 762

Jadhao et al. World Journal of Pharmacy and Pharmaceutical Sciences

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: 342346.
14. Xu WL, Huang YB, Qian JH, Sha O, Wang YQ. (Separation and purification of
stigmasterol and -sitosterol from phytosterol mixtures by solvent crystallization method..
Sep. Purif. Technol, 2005, 41: 173178.
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: 52975298.
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.

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