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Chapter Title Techniques for Extraction, Isolation, and Standardization of Bioactive

Compounds from Medicinal Plants
Copyright Year 2019
Copyright Holder Springer Nature Singapore Pte Ltd.
Author Family Name Kamil Hussain
Particle
Given Name Mohammad
Suffix
Division Department of Chemistry
Organization/University Government Raza Post Graduate
College
Address Rampur, Uttar Pradesh, India
Corresponding Author Family Name Saquib
Particle
Given Name Mohammad
Suffix
Division Department of Chemistry
Organization/University S.S. Khanna Girls Degree College
Address Allahahbad, Uttar Pradesh, India
Corresponding Author Family Name Faheem Khan
Particle
Given Name Mohammad
Suffix
Division Department of Biotechnology, Era’s
Lucknow Medical College and
Hospital
Organization/University Era University
Address Lucknow, Uttar Pradesh, India
Abstract Naturally occurring plant-based compounds have been used by generations of
practicing physicians of indigenous systems of medicine, since hundreds years.
Currently, these are in much demand due to their efficacy, safety, and minimal
side effects. Extraction of plant materials can be done by various conventional
and nonconventional extraction procedures including maceration, infusion,
percolation, digestion, decoction, Soxhlet extraction, ultrasound-assisted
extraction, turbo-extraction, countercurrent extraction, microwave-assisted
extraction, ultrasound extraction, supercritical fluid extraction, solid-phase
extraction, and column chromatography. Recent techniques of extraction
include ionic liquid extraction, HPLC, gas-phase chromatography, chiral
phase chromatography, etc. The plant extracts obtained are ready for use as a
medicinal agent in the form of dried powder or fluid extracts, and these may be
further processed to be incorporated in dosage form such as tablets or capsules,
or it may be fractionated to isolate individual chemical entities which are then
marketed in the form of modern drugs. Thus, the aim of this chapter is to
provide an overview on the different extraction techniques used for isolation
of bioactive constituents from medicinal plants and also to provide a detailed
account on the various phytodrugs.
Keywords (separated Bioactive compounds - Chromatography - Extraction techniques - HPLC -
by “ - ”) Phytomedicine
Techniques for Extraction, Isolation,
and Standardization of Bioactive
8
1

Compounds from Medicinal Plants 3

Mohammad Kamil Hussain, Mohammad Saquib, 4

and Mohammad Faheem Khan 5

Contents 6

8.1 Introduction 000 7

8.2 Pre-extraction Operations for Crude Drugs 000 8
8.2.1 Selection of Crude Drug 000 9
8.2.2 Collection and Identification 000 10
8.3 Drying and Grinding 000 11
8.4 Procedure for Extraction of Herbal Drugs 000 12
8.4.1 Conventional Extraction Techniques 000 13
8.4.2 Nonconventional Extraction Techniques 000 14
8.5 Conclusions and Future Prospects 000 15
References 000 16

Abstract 17

Naturally occurring plant-based compounds have been used by generations of 18

practicing physicians of indigenous systems of medicine, since hundreds years. 19
Currently, these are in much demand due to their efficacy, safety, and minimal 20
side effects. Extraction of plant materials can be done by various conventional 21
and nonconventional extraction procedures including maceration, infusion, per- 22
colation, digestion, decoction, Soxhlet extraction, ultrasound-assisted extraction, 23

M. Kamil Hussain
Department of Chemistry, Government Raza Post Graduate College, AU1
Rampur, Uttar Pradesh, India
M. Saquib (*)
Department of Chemistry, S.S. Khanna Girls Degree College,
Allahahbad, Uttar Pradesh, India
M. Faheem Khan (*)
Department of Biotechnology, Era’s Lucknow Medical College and Hospital, Era University,
Lucknow, Uttar Pradesh, India

© Springer Nature Singapore Pte Ltd. 2019

M. K. Swamy, M. S. Akhtar (eds.), Natural Bio-active Compounds,
[Link]
 M. Kamil Hussain et al.

24 turbo-extraction, countercurrent extraction, microwave-assisted extraction, ultra-

25 sound extraction, supercritical fluid extraction, solid-phase extraction, and col-
26 umn chromatography. Recent techniques of extraction include ionic liquid
27 extraction, HPLC, gas-phase chromatography, chiral phase chromatography, etc.
28 The plant extracts obtained are ready for use as a medicinal agent in the form of
29 dried powder or fluid extracts, and these may be further processed to be incorpo-
30 rated in dosage form such as tablets or capsules, or it may be fractionated to
31 isolate individual chemical entities which are then marketed in the form of mod-
32 ern drugs. Thus, the aim of this chapter is to provide an overview on the different
33 extraction techniques used for isolation of bioactive constituents from medicinal
34 plants and also to provide a detailed account on the various phytodrugs.

35 Keywords
36 Bioactive compounds · Chromatography · Extraction techniques · HPLC ·
37 Phytomedicine

38 8.1 Introduction

39 Medicinal plants belong to the oldest known health-care products that have been
40 used by mankind all over the word in the form of folklore medicine, traditional
41 medicine, or ethnic medicine (Newman and Cragg 2012). These traditional systems
42 are mainly based on the usage of crude plant drugs against debility and disease.
43 Despite tremendous advances in modern medicine through chemotherapy, vaccines,
44 and diagnostics, indigenous drugs continue to be of interest not only to scientists but
45 also to health providers and layman alike because plant-based drugs are not only
46 effective but have better compatibility with human systems (Heinrich 2000).
47 Compounds present in the plant already have biological functions and may exhibit
48 biologically relevant chemistry even in human system, hence lesser side effects
49 (Kumar et al. 2014).
50 It was with the gradual advancement in organic chemistry in the early part of the
51 nineteenth century that the refinement in the methods of purification, isolation, and
52 characterization of the active principles from medicinal plants becomes a reality
53 (McRae et al. 2007). Since, then a large number of active principles have been iso-
54 lated from plants (Clark 1996). They have not only enriched modern medicine but
55 have also provided valuable leads for drug design and have been extensively used to
56 elucidate complex cellular mechanisms, including signal transduction and cell cycle
57 regulation leading to the identification of the important targets for therapeutic inter-
58 vention (Fabricant and Farnsworth 2001). Early humans treated illness by using AU2
59 plants, animal parts, and minerals as medicaments. The first records of indigenous
60 drugs are reported from Mesopotamia and date back to about 2600 B.C. Among the
61 substances used were oils of Cedrus species (cedar) and Cupressus sempervirens
62 (cypress), Glycyrrhiza glabra (licorice), Commiphora species (myrrh), and Papaver
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

somniferum (opium poppy), all of which are still in use today for the treatment of 63
various ailments (Atanasov et al. 2015). Cardiotonic digitalis extracts from fox- 64
glove have beneficial effects for treating some manifestations of heart disease in the 65
eighteenth century (Becker 2007). The Catharanthus roseus was used in Cuba, 66
Philippines, and South Africa for the treatment of inflammation, rheumatism, and 67
diabetes (Tolambiya and Mathur 2016). The unique bisindole alkaloid, vinblastine 68
(1), and vincristine (2) were reported to possess significant clinical antitumor activ- 69
ity against Hodgkin’s and non-Hodgkin’s lymphomas, acute lymphoblastic leuke- 70
mia, breast carcinoma, Wilms’ tumor, Ewing’s sarcoma, neuroblastoma, 71
hepatoblastoma, and small cell lung cancer (Verma et al. 2007). The most signifi- 72
cant anticancer drug discovered and developed is Taxol (3), isolated from the bark 73
of the Pacific yew tree (Taxus brevifolia) (Wani et al. 1971) (Fig.8.1). 74
Camptothecin (3), an anticancer drug, was isolated from the Chinese ornamental 75
tree, Camptotheca acuminata Decne (Nyssaceae) (Fig. 8.1). It was clinically 76
approved by the National Cancer Institute (NCI) in the 1970s but was dropped 77
because of severe bladder toxicity (Wall et al. 1966). Other plant-derived agents in 78
clinical use are homoharringtonine (4) (Fig. 8.1), isolated from the Chinese tree 79
Cephalotaxus harringtonia (Zhou et al. 1995), and elliptinium (5), a derivative of 80
ellipticine (6) (Fig. 8.2), isolated from Bleekeria vitensis (Ochrosia vitiensis), a 81
medicinal plant with potent anticancer properties (Colegate and Molyneux 2007). 82
Homoharringtonine has shown efficacy against various leukemias, including some 83
resistant to standard treatment, and has been reported to produce complete hemato- 84
logic remission in patients with late chronic phase chronic myelogenous leukemia. 85
The isolation of the antimalarial drug, quinine (7) (Fig. 8.2), from the bark of 86
Cinchona species (Cinchona officinalis), was reported in 1820 by Caventou and 87

Fig. 8.1 Chemical structures of important anticancer drugs isolated from medicinal plants
 M. Kamil Hussain et al.

Fig. 8.2 Chemical structures of anticancer agents (5 and 6) and antimalarial drugs (7 and 8) from
traditionally used medicinal plants

88 Pelletier. The bark had long been used by indigenous people of the Amazon region
89 for the treatment of fevers and was introduced in Europe to treat malaria in the
90 medieval period 117. Another plant used in the treatment of fevers for more than AU3
91 2000 years in traditional Chinese medicine is Artemisia annua (Quinhaosu) from
92 which the antimalarial agent artemisinin (8) (Fig. 8.2) was isolated in 1985 (Youyou
93 2011). The use of medicinal plants for the cure of pain possibly began with the use
94 of the crude extract of the poppy (Papaver somniferum) for this purpose. Although
95 traditionally opium has been reported for various uses like as astringent, antispas-
96 modic, aphrodisiac, diaphoretic, expectorant, hypnotic, narcotic, and sedative, the
97 ability of opium as an analgesic is by far the most well known. Opium and its
98 derivatives are used in the pharmaceutical industry as narcotic analgesics, hypnot-
99 ics, and sedatives (Mani and Dhawan 2014).
100 A number of additional plants served as sources of natural product-derived
101 agents that are still used currently in routine medical practice. Presently, more than
102 50% of all prescribed drugs worldwide are derived or synthesized from natural
103 sources, i.e., from animals, marine, plants, and microorganisms (Jacob 2009). Due
104 to structural and biological diversity, natural resources offer a unique and renewable
105 resource for the discovery of new drugs and biologically active chemical entities
106 (Fig. 8.3). About half of the marketed drugs are derived from natural products or
107 derivatives thereof with the large majority being based on terrestrial natural product
108 scaffolds. The World Health Organization (WHO) has estimate that about 88% of
109 the world’s population rely mainly on traditional medicine for their primary health
110 care (Ekor 2003). It was reported that at least 29.5% of the 132 FDA-approved NP
111 and NP-derived drugs like NP semisynthetic derivatives, mimetics, and
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

Fig. 8.3 A flow chart showing sources of natural product

pharmacophore-­guided synthetic molecules have been launched into the market up 112
to 2012. Currently, approximately 100 NP and NP-derived compounds are in clini- 113
cal trials (Butler et al. 2014). 114
This chapter provides an overview about the extraction procedures of herbal 115
drugs from medicinal plants which are mainly classified into conventional and non- 116
conventional operations. Conventional procedures include maceration, infusion, 117
digestion, decoction, percolation, and Soxhlet extraction, whereas nonconventional 118
procedures include ultrasound-assisted extraction (UAE), supercritical fluid extrac- 119
tion (SFE), pressurized liquid extraction (PLE), and microwave-assisted extraction 120
(MAE). It also describes the effect of solvents on extraction procedures and charac- 121
teristics of chemical constituents isolated from different plants. Since millions of 122
structures of natural products derived from plants are known, only selected groups 123
and compounds are presented. Post-extraction techniques to get pure drugs from 124
crude herbal drugs are also described. 125

8.2 Pre-extraction Operations for Crude Drugs 126

Before going on to the extraction procedures, it is beneficial to dry, powder, and 127
grind the plant material because the presence of bioactive constituents responsible 128
for particular diseases may be localized in any part of plant such as bark, leaves, 129
flowers, roots, fruits, and seeds, as well as whole plant (Vongsak et al. 2013). Hence, 130
carefully pre-extraction operations enhance or maximize the yield of active con- 131
stituents in crude drug. These operations include the following criteria: 132
 M. Kamil Hussain et al.

133 8.2.1 Selection of Crude Drug

134 Plant material to be investigated can be selected randomly or on the basis of some
135 specific traditional ethnomedical uses. The use of literature databases early in the
136 selection process can provide some preliminary information on the type of chemical
137 constituent, already isolated from the plant and the extraction methods employed to
138 isolate them. However, a more targeted approach is often preferred to a random
139 selection. Some plants can be selected on the basis of taxonomical view; if the
140 genus contains specific compounds, then there is a probability that the plants
141 belonging to the same genus may contain similar compounds (Bucar et al. 2013)
142 (Fig. 8.4).

143 8.2.2 Collection and Identification

144 Plant part (s) or the whole plant may be collected qualitatively or quantitatively on
145 the basis of suitable time or specific traditional uses. The selection criteria might be
146 based on ethnomedicinal data, chemosystematics or chemotaxonomic relationships,
147 or ecological observations. Legal and ethical issues like the convention on biodiver-
148 sity have to be implemented with respect to guidelines. The material is best col-
149 lected when the part to be extracted has reached its optimal state of development.
150 Roots and rhizomes are collected at the end of the vegetation period, i.e., usually in
151 the autumn. Bark is collected in the spring; leaves and herbs are collected at the
152 flowering stage. Flowers are usually gathered when fully developed. Fruits and
153 seeds are collected when fully ripe. Medicinal plants must be largely collected by
154 hand. This is especially true in the case of wild plants. With cultivation on a large
155 scale, it may be possible to use modern agricultural harvesters. The plant material

Fig. 8.4 A flow chart showing steps before extraction procedure

8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

must first be preserved so that the active compounds will remain unchanged during 156
transport and storage. After preservation, identification of plant species must be 157
done by a specialized taxonomist who should provide an authentication of sample 158
through a detail classification into its species, genus, family, order, class, etc. A 159
thorough collection report, such as the name of the plant, the identity of the part(s), 160
the place, and date of collection, must be recorded as part of a voucher specimen 161
deposited in a herbarium for future reference (Bucar et al. 2013). 162

8.3 Drying and Grinding 163

Drying is the most common method to preserve the plant material from enzymatic 164
degradation such as hydrolysis of glycoside, etc. They must be dried in the open 165
under primitive conditions at ambient or room temperature. However, they should 166
be spread out on shallow trays with good atmospheric air up to dryness. The choice 167
of sunshine or shade is determined by the sensitivity to light of the chemical con- 168
stituents (if they are known). Protection from direct sunlight is advisable to mini- 169
mize the chemical reactions which are responsible for the formation of artifact. 170
Alternatively, plant material should be dried under optimum conditions at 40–50 °C 171
temperature, or they can be dried in an oven if needed. In addition, plants having 172
volatile or thermolabile compounds may be freeze-dried (lyophilized). Freeze-­ 173
drying is a very mild method. Frozen material is placed in an evacuated apparatus 174
which has a cold surface maintained at −60 °C to −80 °C. Water vapor from the 175
frozen material then passes rapidly to the cold surface to yield the dry material 176
(Azwanida 2015). 177
Grinding is the fragmentation of the plant material into smaller particles to 178
improve the subsequent extraction by rendering the sample more homogenous, 179
increasing the surface area and facilitating the penetration of solvent into the cells. 180
It is important that the particles are of as uniform a size as possible because large 181
particles take a longer time to complete extraction process. Several types of 182
machines are available for grinding crude drugs which include mechanical grinders 183
(e.g., hammer and cutting mills which are employed conveniently to shred the plant 184
tissues to various particle sizes). Knife and tooth mills are used for the production 185
of very fine powders of leaves, barks, and roots for the subsequent extraction pro- 186
cess. For thermolabile compounds in crude drugs, mills cooled with liquid nitrogen 187
or cold grinding are preferable methods. After grinding, the powdered material is 188
passed through a sieve of suitable mesh size to separate the smaller particle from the 189
larger ones. The larger particles are returned to the mill for continued grinding. 190
Blast shifting is a significant grinding process in which crude material is blown with 191
compressed air into an apparatus which allows the particles to sediment according 192
to their weight. The coarse or heavy particles settle faster, whereas smaller, lighter 193
particles stay for a longer time in the air stream (Azwanida 2015). 194
 M. Kamil Hussain et al.

195 8.4 Procedure for Extraction of Herbal Drugs

196 Isolation of herbal drugs from plant materials mostly relies on the selection of
197 proper extraction procedure (Sasidharan et al. 2011), which is the first step in crude
198 herbal drug preparation and plays a significant and crucial role in the final outcome
199 as a pure drug. Extraction may be defined as the treatment of the plant material with
200 solvent whereby the medicinally active constituents are dissolved and most of the
201 inert matter remains undissolved. The solvent used for extraction is known as men-
202 struum, and the inert insoluble material that remains after extraction is called marc.
203 In order to obtain biologically active compounds, herbal material can be extracted
204 by various conventional and nonconventional techniques. Most of the conventional
205 techniques are based on the extracting power of the solvents with different polarities
206 and effect of heat and/or mixing power of particles present in the material. In order
207 to obtain biologically active compounds, the techniques used include maceration,
208 infusion, decoctions, digestion, percolation, serial exhaustive extraction, and
209 Soxhlet extraction (Fig. 8.5).

210 8.4.1 Conventional Extraction Techniques

211 [Link] Maceration

212 Maceration is the simplest and still widely used procedure which involves the soak-
213 ing of coarsely powdered plant material in a suitable solvent in a closed container at
214 room temperature for a definite period of time. This method is suitable for both

Fig. 8.5 Various methods for extraction of natural compounds from medicinal plants
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

initial and bulk extraction for the production of thermolabile herbal drugs. 215
Occasional or constant stirring with mechanical shakers or mixers can be used to 216
increase the speed of the extraction. The liquid is then strained off, the solid residue, 217
known as marc, is pressed to remove as much solution as possible, and the com- 218
bined liquids are clarified by filtration or decantation after standing. 219

[Link] Infusion 220

Infusion is a simple chemical process used for extraction of plant materials that are 221
volatile and dissolve readily or release their active ingredients easily in organic 222
solvents. It is prepared by macerating the crude drug for a short period of time with 223
cold or boiling water which is then allowed to steep in the liquid for a period of 224
time. The liquid then may be separated and concentrated under vacuum by using 225
rotatory evaporator. Quantities of the herb and solvent used will vary according to 226
the herb or how strong an infusion is required. 227

[Link] Digestion 228

Digestion is a modified form of maceration with gentle heating during the process 229
of extraction, provided that the temperature does not alter the active ingredients of 230
plant material, so there is greater efficiency in the use of menstruum. This process 231
is used for the herbal material or plant parts which contain poorly soluble sub- 232
stances or polyphenolic compounds. 233

[Link] Decoction 234

If the herbal material is boiled for short period of time or if boiling water is poured 235
over it and it is allowed to stand for some time, the process is called decoction. This 236
procedure is suitable for extracting water-soluble and heat-stable constituents from 237
crude drug. This process is typically used in preparation of ayurvedic extracts called 238
“quath” or “kwath.” The starting ratio of crude drug to water is fixed, e.g., 1:4 or 239
1:16; the volume is then brought down to one-fourth its original volume by boiling. 240
The concentrated extract is then filtered and used as such or processed further. 241

[Link] Percolation 242

This is the procedure most frequently used to extract powdered herbal material in a 243
solvent in a percolator (a narrow, cylindrical, or cone-shaped apparatus with a tap at 244
the bottom). Additional solvent is then poured on top of the herbal material, and the 245
sample is allowed to stand for a given time. It is then allowed to percolate slowly or 246
dropwise out of the bottom of the percolator. To obtain a significant amount of 247
extract, successive percolations can be performed by refilling the percolator with 248
fresh solvent and pooling all extracts together. Percolation steps may be increased 249
to insure the completion of extraction after the analysis of the herbal material for the 250
presence of metabolites with specific reagents. In addition if the solvent is not dis- 251
tributed homogenously in the percolator between herbal materials, heating may also 252
be applied because it increases the extraction yield by providing better contact time 253
between solvent and herbal material. The major disadvantage of percolation is that 254
large volumes of solvents are required and the process can be time-consuming. 255
 M. Kamil Hussain et al.

256 [Link] Serial Exhaustive Extraction

257 Serial exhaustive extraction (SEE) is a mild technique of extraction which involves
258 the fractionation of a crude extract with solvents of increasing polarity, from a non-
259 polar (hexane) to a more polar (butanol) solvent, to ensure that a wide range of
260 compound is extracted. It was chosen as the first fractionation step in the isolation
261 of the major compounds for biological importance. This method cannot be used for
262 thermolabile compounds as prolonged heating may lead to degradation of the
263 compounds.

264 [Link] Soxhlet Extraction

265 Soxhlet extractor was first designed by Ritter Von Soxhlet in 1879 (Soxhlet 1879)
266 mainly for the extraction of lipid. But now it is widely used for extracting the bioac-
267 tive constituents from various herbal materials. Soxhlet extractor consists of three
268 parts:

269 1. Flask containing boiling solvent.

270 2. Soxhlet extractor in which the drug to be extracted is packed. It has a side tube
271 which carries the vapors of the solvent from the flask to the condenser and a
272 siphon tube which siphons off the extract from Soxhlet extractor to the flask.
273 3. A condenser in which the vapors of the solvent condense back into the solvent
274 (Fig. 8.6).

275 In this method, a finely divided herbal sample is placed in a porous bag or “thimble”
276 made of strong filter paper in an extraction chamber, which is placed on top of a
277 collecting flask, beneath a reflux condenser. A suitable solvent is added to the flask,
278 and the setup is heated under reflux. When a certain level of condensed solvent has

Fig. 8.6 A flow chart of

assembly of Soxhlet
extractor water outlet

Condensor
water inlet

Extraction
chamber

Boiling flask
Plant material

heating plate
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

accumulated in the thimble, it is siphoned off into the flask beneath. The main 279
advantage of this method is that it is a continuous process and is carried out until the 280
last drop of solvent from the siphon tube leaves the residue. In addition, a large 281
amount of crude drug is also extracted with a much smaller amount of solvent. In 282
terms of time, economy, energy, and financial inputs, it becomes much more viable 283
when converted into a continuous extraction procedure in small to medium batches 284
or on a larger scale. This method cannot be used for thermolabile compounds as 285
prolonged heating may lead to degradation of the compounds. 286

8.4.2 Nonconventional Extraction Techniques 287

The majority of conventional extraction techniques still utilize simple extraction 288
procedures with organic solvents of different polarity, water or their mixtures. They 289
have disadvantages such as long extraction time, high solvent consumption, and low 290
extraction yield. This leads to the need for more promising extraction techniques 291
which are referred to as nonconventional extraction techniques. The promising non- 292
conventional techniques are ultrasound-assisted extraction, enzyme-assisted extrac- 293
tion, microwave-assisted extraction, pulsed electric field-assisted extraction, 294
supercritical fluid extraction, and pressurized liquid extraction. The major advan- 295
tages of these techniques are the use of less hazardous chemical, design for energy 296
efficiency, design to prevent degradation, atom economy, and time analysis for pol- 297
lution prevention. 298

[Link] Ultrasound-Assisted Extraction (UAE) 299

It is a sonication process of extraction carried out at ultrasound frequencies ranging 300
from 20 KHz to 2000 KHz in order to increase the permeability of cell walls and 301
produce cavitation. In this method, plant material is put into an ultrasonic bath, usu- 302
ally covered by the extraction solvent in a temperature-controlled glass container 303
(Esclapez et al. 2011) (Fig. 8.7). It decreases extraction time and improves extrac- 304
tion yields. UAE showed superior extraction efficiency compared to steam distilla- 305
tion or superheated water extraction. A disadvantage of this method is the deleterious 306
effect of ultrasound energy on the active constituents of extract via free radical for- 307
mation, and it may also lead to undesirable changes in the drug molecules (Vilkhu 308
et al. 2008). Examples of natural product extracted by UAE include anthocyanidins 309
(9) (Fig. 8.8), flavonols (10) (Fig. 8.8), and phenolic acids from Delonix regia, cap- 310
saicinoids (11) (Fig. 8.7) from Capsicum frutescens, cyanidin-3-rutinoside (12) 311
(Fig. 8.8) from Litchi chinensis, or essential oils from laurel, rosemary, thyme, oreg- 312
ano, and tuberose. UAE method was used to optimize the extraction of phenolic 313
compounds from pumpkins and peaches through response surface methodology 314
(RSM) study on three independent variables each with three treatments. Authors 315
were able to find optimal conditions to extract the total phenolics from pumpkins 316
and peaches many times better than simple solvent extraction procedures (Altemimi 317
et al. 2016). Jin Wang et al. applied an optimized sonication condition to extract 318
phenolic compounds from Inula helenium. Under the optimal conditions, the yield 319
 M. Kamil Hussain et al.

Fig.8.7 A flow chart showing assembly of ultrasound-assisted extraction (UAE)

320 of total phenolic compounds and chlorogenic acid (13) (Fig. 8.8) was 6.13 ± 0.58
321 and 1.32 ± 0.17 mg/g, respectively (Wang et al. 2013). Recovery yield of fatty acid
322 composition and triacylglycerol profile of papaya seed oil were evaluated using
323 UAE method which proved that this method significantly influenced the triacylglyc-
324 erol profile of papaya seed oil, but no significant differences were observed in the
325 fatty acid composition of papaya seed oil extracted by different extraction methods
326 (SXE, SE, and UAE) and conditions (Samaram et al. 2013). Ionic liquid (IL)-based
327 UAE extraction methods have also been developed for the effective extraction of
328 alkaloids and phenolic compounds from plant material with optimal extraction effi-
329 ciency within 30–40 min. The extraction efficiency of the optimized IL-UAE
330 approach increases by 30–45% when compared with UAE (Dai et al. 2013).

Fig. 8.8 Chemical structures of different phenolic and acids isolated from food and medicinal
plants
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

[Link] Supercritical Fluid Extraction (SFE) 331

Supercritical fluid extraction (SFE) is a technique of separating one component 332
(extractant) from another (matrix) by using supercritical fluids as the extracting 333
solvent. This technique resembles Soxhlet extraction except that the solvent used is 334
a supercritical fluid above its critical temperature and pressure. Supercritical fluid 335
(sCO2) provides a wide range of useful properties such as non-toxicity, non-­ 336
flammability, odorlessness, tastelessness, inertness, and less expensive. In addition, 337
SFE technique is superior over the others with less use of harmful organic solvents, 338
shorter extraction times, and high diffusion rates of lipids. Since the critical tem- 339 AU4
perature and pressure of CO2 are only 31 °C and 74 bar, respectively, extraction 340
done at these conditions will not damage heat-labile molecules, and the absence of 341
oxygen minimizes oxidative degradation of lipid as compared to other conventional 342
liquid solvents. Addition of modifiers such as ethanol or methanol may slightly alter 343
this. Supercritical fluids can produce a product with no solvent residues. 344
The system of SFE comprises a pump for the sCO2, a pressure cell to contain the 345
sample, a means of maintaining pressure in the system, and a collecting vessel 346
(Fig. 8.9). The liquid is pumped to a heating zone, where it is heated to supercritical 347
conditions. It then passes into the extraction vessel, where it rapidly diffuses into the 348
solid matrix and dissolves the material to be extracted. The dissolved material is 349
swept from the extraction cell into a separator at lower pressure, and the extracted 350
material settles out. The sCO2 can then be cooled, recompressed and recycled, or 351
discharged to atmosphere (Sapkale et al. 2010). Applications of SFE include (1) 352
decaffeinating of coffee and tea, (2) extraction of essential oils (vegetable and fish 353
oils), (3) extraction of flavors from natural resources (nutraceuticals), (4) extraction 354

Fig. 8.9 A flow chart showing assembly of supercritical fluid extraction (SFE)
 M. Kamil Hussain et al.

355 of ingredients from spices and red peppers, (5) extraction of fat from food products,
356 (6) fractionation of polymeric materials, and (7) extraction from natural products.
357 Extraction of active ingredients includes various flavors and medicinal constitu-
358 ents from natural product, e.g., docosahexaenoic acid (DHA) (14) (Fig. 8.8),
359 advanced unsaturated fatty acids and fatty esters such as eicosapentaenoic acid
360 (EPA) (15) (Fig. 8.8), fat-soluble vitamins, and pharmaceuticals. The extraction
361 efficiencies of SFE and conventional solvent methods like Soxhlet or liquid-liquid
362 extractions were frequently compared and shown to be in good agreement (Eller
363 and King 1998). It is well known that the extraction yield and composition of tri-
364 glycerides depend on the extracting conditions. This was verified for the extraction
365 of oil from seaweed (Cheung et al. 1998), tomato seeds (Roy et al. 1996), or soy-
366 bean oil (Snyder et al. 1986).
367 SFE has been used for producing fat-free or fat-reduced potato chips since the
368 early 1970s. Therefore, the amount of remaining fat in the potato chips can easily be
369 controlled (Abbas et al. 2008). Tocochromanols (tocopherols 16, tocotrienols 17,
370 enrichment of vitamin E) (Fig. 8.10) exhibit a much higher solubility in sCO2 than
371 the triglycerides; that is why SFE offers advantages for the production of enriched
372 fractions of tocochromanols (Fang et al. 2007). When compared with three other
373 extraction procedures, supercritical carbon dioxide extraction enables an optimal
374 recovery of unsaturated fatty acids (mainly oleic acid, linoleic acid, and linolenic
375 acid) in Nitraria tangutorum (Zygophyllaceae) seed lipids (up to 79%) (Suo and
376 Wang 2010).

377 [Link] Pressurized Liquid Extraction (PLE)

378 Pressurized liquid extraction (PLE) (Fig. 8.11) is a common procedure employed
379 for the extraction of nonpolar and semipolar nutraceuticals such as phenolic com-
380 pounds, lignans, carotenoids, oils and lipids, and essential oils from foods and
381 herbal plants. Pressurized liquid extraction is similar to Soxhlet extraction, except
382 that during the extraction process the solvent condition inside the PLE cell
383 approaches the supercritical region which results in more efficient extractions. The
384 elevated temperature allows the sample to become more soluble and achieve a

Fig. 8.10 General

structures of
tocochromanols
(tocopherols 16,
tocotrienols 17)
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

Fig. 8.11 A flow chart showing assembly of pressurized liquid extraction (PLE)

higher diffusion rate, while the elevated pressure keeps the solvent below its boiling 385
point. At elevated pressures and temperatures, solvents can penetrate solid samples 386
more efficiently which reduces solvent usage. This procedure is carried out with 387
nonpolar and/or semipolar solvents (pentane, hexane, dichloromethane, acetone, 388
and diethyl ether) or their mixtures advantageously in azeotropic ratio. PLE uses 389
conventional liquid solvents at elevated pressures (10–15 MPa) and temperatures 390
(50–200 °C) for short time periods (5–10 min) and with much less solvent than 391
conventional techniques for completing total extraction of all analytes (Saim et al. 392
1998 and Richter et al. 1996) (Fig. 8.12). PLE technique was used to extract the 393
carotenoids in food (Denery et al. 2004). Moreaua et al. extracted the samples of 394
freshly ground corn kernels and freshly ground rolled oats via PLE technique using 395
four different organic solvents (hexane, dichloromethane, isopropanol, and ethanol) 396
at two temperatures (40 °C and 100 °C) with significant yield variation from 2.9 wt 397
% to 5.9 wt % for ground corn and from 5.5 wt % to 6.7 wt % for ground oats 398
(Moreaua et al. 2003). 399
This method was also used to isolate tocopherols from several seeds and nuts and 400
was shown to yield very clean extracts and recoveries similar to conventional tech- 401
niques (Delgado-Zamarreño et al. 2004). Optimized extraction parameters were 402
described for the maximal extraction efficiency of cereal tocopherols (16) and toco- 403
trienols (17) (Bustamante-Rangel et al. 2007) or carotenoids from a microalga (Kim 404
et al. 2012). PLE has also been found to show good efficiency in the determination 405
of carotene, tocopherols, and tocotrienols in residue oil from palm (Sanagi et al. 406
2005). A comparison with Soxhlet and ultrasound-assisted extractions showed that 407
pressurized liquid extraction was more effective for terpenes, fatty acids, and vita- 408
min E contained in leaves of Piper species (Péres et al. 2006). 409

[Link] Microwave-Assisted Extraction (MAE) 410

Microwave-assisted extraction (MAE) is the rapid extraction technique that enables 411
the heating of solvent and plant tissues with the combined effort of microwave and 412
traditional solvent extraction (Delazar et al. 2012). After the first report on the use 413
of microwave heating for extraction process of plant material, numerous reports 414
 M. Kamil Hussain et al.

Fig. 8.12 A flow chart showing assembly of microwave-assisted extraction (MAE)

415 have investigated the analytical possibilities of this new extraction technique
416 (Sinquin et al. 1993; Waksmundzka-Hajnos et al. 2004). It utilizes microwave
417 energy to heat the solvent and the sample to increase the mass transfer rate of the
418 solutes from the sample matrix into the solvent through dual mechanism of ionic
419 conduction and dipole rotation of solute particles. Microwaves are generated by
420 nonionizing electromagnetic waves positioned between the X-ray and infrared rays
421 in the electromagnetic spectrum with frequency between 300 MHz and 300 GHz.
422 Microwaves interact with solvent particles and caused heating effect. Consequently,
423 it causes electrophoretic migration of ions. The process of heating takes place at a
424 frequency of 2450 MHz; above this range, no heating occurs. Only dielectric mate-
425 rial or solvents with permanent dipoles get heated up under microwave. The value
426 of dissipation factor (tan δ) is a measure of the efficiency with which different sol-
427 vents heat up under microwave (Tatke and Jaiswal 2011). Microwave systems for
428 extraction are available in two forms: (i) closed extraction vessels/multimode micro-
429 wave ovens and (ii) focused microwave ovens. The extraction in a closed extraction AU5
430 vessel is brought about by controlled pressure and temperature, whereas in focused
431 microwave-assisted Soxhlet or solvent extraction (FMASE), as the name indicates,
432 only the part of the extraction vessel containing the sample is focused for irradiation
433 with microwaves (Moen et al. 2012). The MAE assembly comprises of four major
434 components. A microwave generator also known as the magnetron is responsible for
435 generation of microwaves; a wave guide is used to direct the propagation of micro-
436 waves from the source to the microwave cavity. The third component is the
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

applicator, where the sample holder along with the sample is placed. The next com- 437
ponent is the circulator which regulates the movement of microwaves only in the 438
forward direction (Kristenson 2006) (Fig. 8.12). MAE has a number of advantages 439
over conventional techniques, e.g., shorter extraction time, less solvent, higher 440
extraction rate, and lower cost. Therefore, it has now become one of the most popu- 441
lar extraction methods, and several advanced MAE setup and methodologies have 442
become available, e.g., pressurized microwave-assisted extraction (PMAE) and 443
solvent-free microwave-assisted extraction (SFMAE). MAE seems particularly 444
promising for the extraction of compounds with medium to high polarity from solid 445
matrix. 446
Its application includes extraction of active constituents from natural sources, 447
nutraceutical and functional food ingredients, and also pharmaceutical from bio- 448
mass. Kothari and Seshadri used MAE for the extraction of flavonoids from finely 449
ground seeds of Annona squamosa and Carica papaya (Kothari and Seshadri 2010). 450
Total phenolics were extracted from Rosmarinus officinalis using microwave-­ 451
assisted extraction in terms of reduced time, less consumption of solvent, and high 452
yields in comparison with traditional extraction methods (Proestos and Komaitis 453
2008). Microwave-assisted extraction was also applied to extract the coumarin (18) 454
and coumaric (19) and melilotic acids (20) (Fig.8.13) in the flowering tops of 455
Melilotus officinalis (Martino et al. 2006). Extraction of curcumin (21) from 456
Curcuma longa was optimized using Taguchi L9 orthogonal test from microwave-­ 457
assisted method and has been found to have high yield with reduced time period 458
(Mandal et al. 2008). MAE of anthraquinones from the roots of M. citrifolia was 459
found to have high antioxidant activity than those extracted by Soxhlet, maceration, 460
and ultrasonic-assisted extraction (Hemwimon et al. 2007). Optimization of 461
microwave-­assisted extraction of saikosaponins (22) from the roots of Bupleurum 462
falcatum was also studied (Kwon et al. 2006). It was extracted from peel of 463
Dimocarpus Longan (Pan et al. 2008). 464
Several bioactive compounds have been extracted with the help of microwave-­ 465
assisted extraction, such as extraction of taxanes (Taxol (3), 10-deacetyltaxol (23), 466AU7
and cephalomannine (24) (Fig. 8.13) from Taxus brevifolia needles (Mattina et al. 467
1997); azadirachtin-related limonoids (25) from Azadirachta indica seed kernels 468
(Dai et al. 1999); glycyrrhizic acid (26) from Glycyrrhiza glabra roots (Pan et al. 469
2000); tanshinones such as tanshinone I (27), tanshinone II A (28), and cryptotan- 470
shinone I (29) from Salvia miltiorrhiza Bunge (Pan et al. 2002); artemisinin (23) 471
from Artemisia annua (Hao et al. 2002); and ginsenosides (30) from Panax ginseng 472
root (Shu et al. 2003) (Fig. 8.13). In addition, there are few reports on the use of 473
microwave-assisted extraction in the large-scale industrial processing of plant sec- 474
ondary metabolites. Microwave-assisted extraction is currently regarded as a robust 475
alternative to traditional extraction techniques, especially in the case of the sample 476
preparation for analytical purpose (Zhang et al. 2011). 477
 M. Kamil Hussain et al.

Fig. 8.13 Chemical structures of various therapeutically important molecules form different AU6
medicinal plants

478 8.5 Conclusions and Future Prospects

479 A large proportion of the human population relies on traditional medicine, which is
480 based predominantly on materials derived from plants, for primary health care. This
481 is especially true for the Indian subcontinent which has a well-established system of
482 traditional medicine in the form of Ayurveda and Unani system of medicines. In the
483 past few decades, there has been a renewed interest in the investigation of medicinal
484 plant due to the growing realization of the important role that phytomedicine can
485 play in the development and improvement of modern medicine. In fact, there has
486 been talk of phytomedicine ushering in a new era of health-care system for the man-
487 agement of human diseases in the next few decades.
8 Techniques for Extraction, Isolation, and Standardization of Bioactive Compounds…

However, the full potential of phytodrugs in modern drug development cannot be 488
realized until and unless the virtually inexhaustible treasure troves of phytocom- 489
pounds are systematically explored and exploited with a curious and objective mind, 490
without prejudice toward the concept of phytomedicine. In this context, there is a 491
need for the development of better screening and extraction and purification meth- 492
ods of plants and other natural sources to identify potential plant-based therapeu- 493
tics. In this context, the present chapter with its in-depth discussion on recent 494
advances in modern extraction and purification techniques as well as its review of 495
various phytodrugs that have been studied to scientifically validate their claims as 496
biologically active agents would be of utmost interest not only to natural product 497
chemists and pharmaceutical chemists but also to practitioners of modern medicine 498
and traditional medicine. 499

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 Author Queries
Chapter No.: 8 470705_1_En_8_Chapter

Queries Details Required Author’s Response

AU1 Please confirm the identified given name and family name for
the authors “Mohammad Kamil Hussain” and “Mohammad
Faheem Khan” are fine.
AU2 Reference citations Eller et al. (1991), Youyou et al. (2011),
Tolambiya and Mathur (2015), Ekor (2015), Fabricant et al.
(2001), Becker et al. (2007) Mani et al. (2014) Jacob et al.
(2009), Tatke et al. (2011), Proestos et al. (2008) Colegate et
al. (2007) have been changed to Eller and King (1998), Youyou
(2011) Tolambiya and Mathur (2016), Ekor (2003), Fabricant
and Farnsworth (2001), Becker (2007), Mani and Dhawan
(2007), Jacob (2009), Tatke and Jaiswal (2011) Proestos and
Komaitis (2008) and Colegate and Molyneux (2007) as per
reference list. Please check if okay.
AU3 Please check “117” for significance.
AU4 Please check if edit to sentence starting “Since the critical…”
is okay.
AU5 Please check if edit to sentence starting “The extraction in...” is
okay.
AU6 Please provide better quality image for Fig. 8.13.
AU7 Please provide missing closing parenthesis in the sentence
starting “Several bioactive compound.....”.