CHAPTER 4

CHARACTERIZATION OF ACTIVE COMPOUNDS FROM

GARCINIA GUMMI-GUTTA FRUITS

4.1 Introduction

Plants often exhibit a wide range of biological and pharmacological activities.

Due to the need for development of new drugs with better pharmacological activities,

depends on plants grew increasingly as scientists continuously exploited them for

isolation of bioactive compounds. The premier steps to utilize the biologically active

compound from plant resources are extraction, pharmacological screening, isolation and

characterization of bioactive compound, toxicological evaluation and clinical evaluation

(Sasidharan et al., 2011).

Extraction is essential for the analysis of medicinal plants, because it is necessary

to extract the desired chemical components from plant materials for further separation

and characterization. The selection of solvent system largely depends on the specific

nature of the bioactive compound being targeted. Different solvent systems are available

to extract the bioactive compound from natural products. The extraction of hydrophilic

compounds uses polar solvents such as methanol, ethanol or ethyl acetate. As the target

compounds may be non-polar to polar and thermaly labile, the suitability of the methods

of extraction must be considered (Cos et al., 2006).

Plant extracts usually occur as a combination of various types of bioactive

compounds or phytochemicals with different polarities, their separation still remains a

big challenge for the process of identification and characterization of bioactive

compounds. It is a common practice in isolation of these compounds that a number of

different separation techniques such as TLC, column chromatography and HPLC should

153
be used to obtain pure compounds. The pure compounds are then used for the

determination of structure and biological activity (Sasidharan et al., 2011). The

evaluation of bioactive compounds can be accomplished by appropriate method,

including HPLC with UV (DAD), ELSD, MS detection or GC-MS, FT-IR, NIR, NMR

or a combination of these techniques (Hashimoto and Kameoka, 2008; Gong et al., 2009;

Giri et al., 2010).

TLC is a simple, quick and inexpensive procedure that gives the researchers a

quick answer as to how many components are in a mixture. TLC is also used to support

the identify of a compound in a mixture when the Rf of a compound is compared with the

Rf of a known compound. Additional tests involve the spraying of phytochemical

screening reagents, which cause color changes according to the phytochemicals existing

in plant extract or by viewing the plate under the UV light. This has also been used for

confirmation of purity and identify of isolated compounds (Sasidharan et al., 2011).

UV detectors are polar because they offer high sensitivity (Li et al., 2004) and

also because majority of naturally occurring compounds encountered have some UV

absorbance at low wavelengths (Cannell, 1998). The high sensitivity of UV detection is

bonus if a compound of interest is only present in small amounts within the sample. UV-

Vis spectra are still valuable for a preliminary identification and for quantification using

characteristic absorption maxima, whereas mass spectra contribute greatly to structural

characterization (Gunasekaran, 2003). Besides UV, other detection methods are also

being employed to detect phytochemicals among which is the diode array detector

(DAD) coupled with mass spectrometer (MS) (Tsao and Deng, 2004). Liquid

chromatography coupled with mass spectrometry (LC/MS) is also a powerful technique

for the analysis of complex botanical extracts (Cai et al., 2002).

154
 IR spectroscopy is more sensitive and selective than colorimetric methods. The

Fourier Transform Infrared (FTIR) spectroscopy allows the analysis of a relevant amount

of compositional and structural information in plants (Kogel-Knaber, 2000). The analysis

can be performed both on pure compounds and complex mixtures, without separation

into individual components. Moreover, FT-IR spectroscopy is an established time-saving

method to characterize and analyze microorganisms and monitor biotechnological

processes (Grube et al., 2008). Fourier Transform Infrared spectroscopy (FTIR) offers a

rapid and non-destructive investigation, easy to use to fingerprint herbal extracts or

powders, is relatively uncommon compared with chromatographic and classical methods

(Hussain et al., 2009).

FTIR along with the statistical method principal component analysis (PCA) was

applied to identify and discriminate herbal medicines for quality control in the

fingerprint region 400 - 2000 cm1. The ratio of the areas of any two marked

characteristic peaks was found to be nearly consistent for the same plant from different

regions, thereby, an additional discrimination method for herbal medicines. PCA clusters

herbal medicines into different groups, clearly showing that IR method can adequately

discriminate different herbal medicines using FTIR data (Singh, et al., 2010).

Mass spectroscopy provides information about the molecular mass and

fragmentation pattern of the analyte. The ionization may be performed in the positive

and / or negative ion mode and anthocyanins are analyzed in the positive mode, whereas

other groups are usually analyzed in the negative mode (Maatta et al., 2003). In the

negative ionization mode, acids deprotonate easily, and in the positive mode, they form

adducts with the cations in the sample or mobile phase, for example, sodium ions

(Swatsitang et al., 2000). The recent applications of electrospray ionization (ESI) for

155
analysis of the polyphenolic compounds involve detection of the pseudo-molecular ion in

order to investigate the degree of polymerization.

NMR spectroscopy is a powerful technique for structure elucidation of organic

molecules. Therefore, the coupling of LC and NMR could lead to the complete

assignment and structure determination of analytes. LC-NMR has become an important

technique for the biomedical, pharmaceutical, environmental, food and natural products

analysis, as well as for the identification of drug metabolites (Tatsis et al., 2007). LC-

NMR improves speed and sensitivity of detection and found useful in the areas of

pharmacokinetics, toxicity studies, drug metabolism and drug discovery process

(Dachtler et al., 2003; Pasch et al., 2008; Patil and Rajani, 2010).

Nuclear Magnetic Resonance (NMR) spectroscopy uses radiofrequency waves to

reveal information about magnetic nuclei. Since the word ‘spectroscopy’ describes any

technique in which electromagnetic (EM) radiation is used to probe atoms. The field

where multi-dimensional NMR spectroscopy has most impressively been used is its use

in the determination of protein structures as first suggested by among others Wuthrich

(1990).

Proteins may have thousands of NMR resonances associated with them, and thus

simple one-dimensional 1H NMR spectra are highly congested. To circumvent this

dispersion problem, spectra from proteins are usually collected across multiple

dimensions often associated with different nuclei. In particular, because the backbone of

peptides consists of amide linkages, the use of 1H, 13C and 15N multi-dimensional NMR

spectra is common in protein NMR spectroscopy. These experiments can consist of two-

dimensional NMR spectra such as 1H - 13

C HSQC and 1H - 15
N HSQC experiments

which take hours to 2–3 days to acquire depending on the protein, whether it has been

156
 13
artificially labelled with amino acids containing C and 15N and the field strength of the

magnet used. Alternatively, more time-consuming three-dimensional experiments can be

13 15
performed that simultaneously measure the interactions between C, N and 1H or

experiments (Wuthrich, 1990).

Plants of the genus Garcinia are known to be a rich source of bioactive

compounds of oxygenated and prenylated phenol derivatives such as prenylated

xanthones, polyisoprenylated benzophenones, bioflavonoids and triterpenoids (Babu et

al., 1988; Oliveira et al., 1999; Nyemba et al., 1990). Many bioactive chemicals such as

xanthones, benzophenone, flavonoids have been isolated from the fruits, leaves and stem

of Garcinia mangostana. The medicinal properties of plants can be attributed to a larger

extent to xanthones, the most bioactive constituents (Pedraza-Chaverri et al., 2008).

Garcinia parvifolia is recognized as rich source of xanthone and xanthonoid natural

products with high pharmaceutical and medicinal potential (Xu et al., 2001).

Tetraoxygenated xanthones, cowaxanthones A-E, together with 10 previously

reported tetraoxygenated xanthones, were isolated from the crude hexane extract of

Garcinia cowa fruits (Panthong et al., 2006). Polyisoprenylated benzophenones such as

garcinol, guttiferone I, J, M, N and oxidized derivative of guttiferone K were found in

the fruits of Garcinia gummi-gutta (Masullo et al., 2008). Hence the present study of the

isolation and characterization of active compounds were evaluated by TLC, Mass

spectroscopy, UV, IR and NMR is of importance.

4.2 Materials and Methods

4.2.1 Preparation of crude extracts

Fresh fruits of Garcinia gummi-gutta growing at site S5 (Kodayar basin) was

collected, sun dried and ground into powered. 5 kg of powder sequentially soaked in
157
methanol in 1:3 (W/V) ratio for 72 hours respectively. After 72 hours filtered using

whatman filter paper no.1 and the filtrate was concentrated under reduced pressure using

rotary vaccuam evaporator. The filtrate was air dried to yield 32.48g of methanol extract.

Only methanol extract was subjected to column chromatography techniques for the

isolation of active principle.

4.2.2 Thin Layer Chromatography

Thin Layer Chromatography (TLC) is the simplest and cheapest method of

detecting plant constituents since the method is easy to run, reproducible and requires

little equipment (Sasidharan et al., 2011). TLC is an important method for the isolation,

purification and confirmation of natural products. Compared with other chromatographic

methods, TLC is often considered to be deficient in reproducibility and accuracy, but

some distinctive attributes of this tool should be considered: low cost analysis, high-

throughput screening of samples, minimal sample preparation, whole sample integrity,

disposable stationary phase. Thin Layer Chromatography (TLC) is a solid-liquid type in

which the two phases are a solid (stationary phase) and a liquid (moving phase). Solids

most commonly used in chromatography are silica gel (SiO2 x H2O) and alumina

(AL2O3 x H2O). In our experiments Thin Layer Chromatography (was done using 5l

of a 100 mg extract/ml solution) is loaded on Merck TLC F254 or manual silica gel glass

plates using hexane: ethyl acetate mixture as eluents.

4.2.3 Column chromatography

The active methanolic crude extract of fruit of Garcinia gummi-gutta (28.8 g)

was mixed (adsorbed) with silica gel (60-120 mesh) and chromatographed on a silica gel

(100-200 mesh) initially eluting with continuous suitable solvent system and gradually

increasing the polarity mixture of solvent (Hexane : ethyl acetate and ethyl acetate:

158
methanol). The fractions were eluted using TLC and similar TLC pattern were pulled in

to major fraction. The major fractions were concentrated and air dried. Finally the active

compounds were subjected to UV, IR, NMR and MASS spectroscopic studies.

4.2.4 Spectroscopic studies of active compounds (Chen et al., 2004)

4.2.4.1 Ultraviolet Spectroscopy (UV)

The active fraction (sample) was dissolved in methanol and subjected to UV

spectroscopic study. The UV spectrum was recorded in Shidmazu UV

spectrophotometer. The UV absorption was recorded from 100 to 700 nm.

4.2.4.2 Infrared Spectroscopy (IR)

Sample was ground along with Potassium bromide (KBr) and pellet was

prepared. IR spectrum was recorded in Shidmazu IR spectrophotometer by KBr pellet

method. IR gives a strong absorption pattern at a particular frequency (400 - 4000) for a

particular functional group.

4.2.4.3 Nuclear Magnetic Resonance (NMR)

1
H NMR and 13C NMR were taken on Bruker AV- 400 JOEL instrument at 400

and 100 MHz respectively in CDCL3. The chemical shift values were given in scale

with tetra-methylsillane (TMS) as the internal standard.

4.2.4.4 Mass Spectroscopy

By mass spectroscopy the molecular mass of a compound and its elements

composition can be easily determined. Further this method involves very little amount of

the test sample, which will give molecular weight accurately using ESI MS

chromatography. Mass spectroscopic studies were carried out by using Shimazu.

159
4.3 Results

Column chromatography of methanol extract of Garcinia gummi-gutta fruit gave

223 major fractions which were eluted (Fig 4.1). TLC was carried out for each fraction

with suitable mobile phase (n-hexane / EtOAc 90:10, v/v). The eluted fractions were

evaluated by TLC and were combined to give 27 major fractions. The spots were

visualized by exposing to iodine vapours and UV light. In column chromatography,

fractions 9-13 was eluted with hexane: ethyl acetate (90:10 V/V) which yielded 63 mg

and gave yellowish oil.

Fig 4.1: Isolation of active principle from crude methanol extract of

Garcinia gummi-gutta fruit

Garcinia gummi-gutta fruit

160
The single spot on TLC plate with Rf value 4.5 were shown in Figure 4.2.

Fig 4.2: TLC profile of compound 1 isolated from methanol extract of

Garcinia gummi-gutta fruit

Fractions 24-25 was eluted with hexane: ethyl acetate (60:40 V/V) and 48 mg

was obtained which showed single spot on TLC plate with Rf value 3.9 (Fig 4.3). The

purified compounds were subjected to spectroscopic analysis.

Fig 4.3: TLC Profile of compound 2 isolated from methanol extract of

Garcinia gummi-gutta fruit

161
4.3.1 Compound 1 (2–hydroxyl-1-(hydroxymethoxy) propane-1,2,3 tricarboxylic acid)

The compound 1 was eluted and identified from methanolic crude extract of

Garcinia gummi-gutta fruit. The eluted major fractions 9-13 were considered as

compound 1(Fig 4.4).

Fig 4.4: Structure of compound 1 (2 – hydroxyl-1-(hydroxymethoxy) propane-

1,2,3-tricarboxylic acid) isolated from methanol extract of
Garcinia gummi-gutta fruit

162
4.3.1.1 Mass Spectroscopy

Metabolite of the active compound was isolated from Garcinia gummi-gutta fruit

and identified using ESI-MS (Electrospray Ionization Mass Spectroscopy). The mass

spectrum of compound 1 showed a strong molecular ion peak at m/z 238.0 in its ESI-MS

which corresponds to the molecular formula C7H10O9 (Fig 4.5).

Fig 4.5: ESI-MS compound 1 (2 – hydroxyl-1-(hydroxymethoxy) propane-1,2,3-

tricarboxylic acid) isolated from methanol extract of Garcinia gummi-gutta fruit

163
4.3.1.2 UV Spectroscopy

Methanol extract of fruit showed a wavelength of !max at 210, 250 and 275 nm.

The results of the UV spectral study was shown in Fig 4.6.

Fig 4.6: UV Spectrum of compound 1 (2 – hydroxyl-1-(hydroxymethoxy)

propane-1,2,3-tricarboxylic acid) isolated from methanol extract of
Garcinia gummi-gutta fruit

164
4.3.1.3 IR Spectroscopy

IR "max (KBr) cm-1: 3287 (–OH str.), 2943 (methyl –CH str.), 1650 cm-1 (C=O)

(Fig 4.7).

Fig 4.7: IR Spectrum of compound 1 (2 – hydroxyl-1-(hydroxymethoxy)

propane-1,2,3-tricarboxylic acid) isolated from methanol extract of
Garcinia gummi-gutta fruit

165
4.3.1.4 1H NMR

1
H NMR (CDCl3, 300 MHz) , ppm: 2.501 (1H, s), 2.506 (1H, s), 2.510 (1H, s),

3.375 (2H, s), 5.613 (2H, s), 6.916 (1H, s), 8.832 (1H, s), 9.192 (1H, s) (Fig 4.8).

Fig 4.8: 1H NMR Spectrum of compound 1 (2 – hydroxyl-1-(hydroxymethoxy)

propane-1,2,3-tricarboxylic acid) isolated from methanol extract of
Garcinia gummi-gutta fruit

166
4.3.1.5 13C NMR

13
C NMR (CDCl3, 100 MHz), ppm: 39.247 (C3), 90.024 (CH2), 98.324 (C1 &

C2), 167.407 (C3) ppm) (Fig 4.9).

13
Fig 4.9: C NMR Spectrum of compound 1 (2 – hydroxyl-1-(hydroxymethoxy)
propane-1,2,3-tricarboxylic acid) isolated from methanol extract of
Garcinia gummi-gutta fruit

167
4.3.2 Compound 2 ( amyrin)

The compound 2 was eluted and identified from methanolic crude extract of

Garcinia gummi-gutta fruit. The eluted major fractions 24-25 were considered as

compound 2 (Fig 4.10).

Fig 4.10: Structure of compound 2 ( amyrin) isolated from methanol

extract of Garcinia gummi-gutta fruit

168
4.3.2.1 Mass spectroscopy

Metabolite of the active compound was isolated from methanolic crude extract of

Garcinia gummi-gutta fruit and identified using ESI-MS (Electrospray Ionization The

mass spectrum of compound 2 showed a strong molecular ion peak at m/z 426.0 in its

ESI-MS which corresponds to the molecular formula C30H50O (Fig 4.11).

Fig 4.11: ESI-MS of compound 2 ( amyrin) isolated from methanol

extract of Garcinia gummi-gutta fruit

169
4.3.2.2 UV spectroscopy

Methanol extract of fruit showed a wavelength of !max at 220, 280 and 330 nm.

The results of the UV spectral study were shown in Fig 4.12.

Fig 4.12: UV Spectrum of compound 2 ( amyrin) isolated from methanol extract

of Garcinia gummi-gutta fruit

170
 4.3.2.3 IR Spectroscopy

IR "max (KBr) cm-1: 3380, 2947 (methyl –CH str.), 1032, 686 cm-1 (olefinic

moiety) (Fig 4.13).

Fig 4.13: IR Spectrum of compound 2 ( amyrin) isolated from methanol

extract of Garcinia gummi-gutta fruit

100.0

95

90 2051
2521
85
79
80

75

70

65
1653
60
1113
55

50
%T 1453 686
1414
45

40

35

30

25
2834
20

15
2947
10 3380 1032

0.0
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
cm-1

171
4.3.2.4 1H NMR

1
H NMR (CDCl3, 300 MHz,), ppm: 0.52 – 2.10 (14H, m), 0.987 (1H, s), 1.045

– 1.104 (1H, s), 1.177 – 1.234 (1H, s), 1.301 (1H, s), 3.361 (1H, s) (Fig 4.14).

Fig 4.14: 1H NMR Spectrum of compound 2 ( amyrin) isolated from methanol

extract of Garcinia gummi-gutta fruit

172
4.3.2.5 13C NMR

13
C NMR (CDCl3, 100 MHz), ppm: 29.698 (C6, C25), 31.082 (C24, C11), 32.493

(C2, C15, C16, C28, C29, C30), 32.667 (C7, C17, C20, C21), 33.337 (C1, C4, C10, C8, C22, C23),

34.743 (C14), 36.958 (C9, C19), 37.149 (C5). 121.733 (C12), 145.195 (C13) (Fig 4.15).

13
Fig 4.15: C NMR Spectrum of compound 2 ( amyrin) isolated from methanol

extract of Garcinia gummi-gutta fruit

173
4.4 Discussion

The methanol extract of Garcinia gummi-gutta fruit was better activity than other

extracts. So methanol extract of Garcinia gummi-gutta fruit was used for the spectral

analysis. Many bioactive compounds were isolated from the genus of Garcinia.

Methanol extract of Garcinia xanthochymus fruit was identified to have two new

benzophenones, guttiferone H and gambogenome (Lim, 2012). Two polyisoprenylated

benzophenones, isoxanthochymol and camboginol were identified and quantified in the

fruit rinds of Garcinia gummi-gutta by Chattopadhyay and Kumar (2007).

Spectroscopic technique has become a powerful and analytical tool for the

isolation and identification of bioactive compounds. A brief summary of the general

approaches in extraction, isolation and characterization of bioactive compound from

methanol extract of Garcinia gummi-gutta fruit. In the present investigation, two

compounds were eluted from the methanolic extract of Garcinia gummi-gutta fruit.

These two purified compounds were subjected to spectroscopic analysis.

4.4.1 Mass Spectroscopy

The mass spectrum of compound 1 showed a strong molecular ion peak at m/z

238 in its Electrospray Ionization Mass Spectroscopy (ESI-MS) which corresponding to

the molecular formula C7H10O9. The mass spectrum of compound 2 showed a strong

molecular ion peak at m/z 426 in its ESI-MS which corresponding to the molecular

formula C30H50O. Similar result was observed by Dias et al. (2011). Klaiklay et al.

(2011); Mawa and Said (2012) who isolated compounds such as flavanone glucuronides,

niflavonoids, amenthoflavone and friedelin from methanol extract of leaves of Garcinia

prainiana. The structures of these compounds were evaluated based on the spectroscopic

174
analysis. Han et al. (2005) evaluated the isolated compounds from Garcinia kola,

molecular weight was detected by Electrospray Ionization Mass Spectroscopy (ESI-MS).

4.4.2 UV-Vis Spectroscopy

The UV-Vis profile of two compounds isolated from methanolic extract of

Garcinia gummi-gutta fruit showed peaks between wavelength of 100 nm to 700 nm.

The profile showed the characteristic absorption bands of compound 1 at 210, 250 and

275 nm respectively. The UV-Vis spectrum profile of compound 2 showed the

characteristic absorption peaks at 220, 280 and 330 nm respectively. Klaiklay et al.

(2011) has studied the UV-Vis spectrum of Garcinia prainiana, and absorption bands of

a flavanone chromophore at 283 and 337nm.

4.4.3 IR Spectroscopy

IR has proven to be a valuable tool for the characterization and identification of

compounds or functional groups (chemical bonds) present in an unknown mixture of

plant extracts (Eberhardt et al., 2007; Hazra et al., 2007). In addition, FT-IR spactra of

pure compounds are usually so unique that they are like a molecular “fingerprint”. For

most common plant compounds, the spectrum of an unknown compound can be

identified by comparison to a library of known compounds available in the internet. In

the present study, the functional groups identification was based on the IR peaks

attributed to stretching and bending vibrations. The IR spectrum was used to identify the

functional group of the two active compounds based on the peak value in the region of

infrared radiation.

The two isolated compounds from methanolic crude extract of Garcinia gummi-

gutta fruit was passed into the IR and the functional group of the components were

separated based on its peak ratio. The IR (KBr) spectrum of the compound 1 displayed
175
absorption for hydroxyl ("O-H) band at 3287 cm-1 due to OH group of compound 1.

Vibration of "C-H stretching of alkane (methyl group) was observed at 2943 cm-1. The

stretching vibration of ("COOH) carboxyl group was attributed at 1650 cm-1.

In compound 2, IR (KBr) spectrum displayed absorption for ("O-H) hydroxyl

group, band at 3380 cm-1. Vibration of "O-H stretching of alkane (methyl group) was

observed bands at 2947 cm-1 and 2834 cm-1. The results of the present study, confirms

the presence of alkane (methyl, ethyl and propyl), alkene, hydroxyl, carboxylic acid and

ether in purified two compounds of methanolic extract of Garcinia gummi-gutta fruit.

Characteristic functional groups like aliphatic amines, alkyl halides, aromatics, alkanes,

alcohols and phenols are responsible for the various medicinal properties of Commiphora

mukul, Garcinia gummi-gutta and Plantago ovata as identified by Pravinkumar et al.

(2015). The present results agree with the above authors in correlating the presence of

alkanes, alkenes and carboxylic acid etc. with the medicinal principle of the plant.

Baker (1982) have found out that alkanes in the plant cuticle and epicuticular wax

of many species, protect the plant against water loss, prevent the leaching of important

minerals by the rain, and against bacteria, fungi and harmful insects. Carboxylic acids

are biologically very important in the body, while aldehydes are mainly used in the

production of resins when combined with urea, melamine and phenol (Reuss et al.,

2005). The present study showed that Garcinia gummi-gutta fruit contain rich sources of

phytoconstituents which can be isolated and screened for different kinds of bioactive

compounds, depending on their therapeutic uses. The potential of IR spectroscopy for

easy and identification of various functional group responsible for medicinal properties.

4.4.4 NMR Spectrum

The 1H spectrum of compound 1 revealed the presence of three carboxyl

176
hydrogen appearing as singlet at  9.192. The singlet at 3.375 is due to hydroxyl

hydrogen present in compound 1. One methylene group corresponds to two hydrogens,

the singlet at 2.506 attributed to two hydrogen attached to C3 carbon. The 1H spectrum of

compound 2 showed the peak at 0.819 – 0.903 eight methyl groups (24 hydrogen) and

two olefinic hydrogen. The singlet at 3.361 is due to hydroxyl hydrogen present in

compound 2.

13
The C NMR spectrum of compound 1 showed three carboxylic hydrogen (

167.407 ppm),  98.324 ppm was typical for C1, C2 and methylene carbon,  90.024 ppm
13
corresponds to methylene carbon and  39.456 ppm attributed to C3 carbon. The C

spectrum of compound 2 revealed 30 carbon signals including eights methyls, ten

methylenes, ten quaternary and two methane carbons. The results are supported by

Mawa and Said (2012). Several other workers have done the NMR studies in Garcinia

species like Garcinia mangostana (Inas et al., 2014), Garcinia linii (Chen et al., 2004),

Garcinia griffithii (Alkadi et al., 2013), Garcinia cowa (Na et al., 2013), Garcinia

chapelieri (Rambeloson et al., 2014) and Garcinia cf cymosa (Ernawati et al., 2014).

The present work is one of its kind as isolation and characterization of active compounds

from Garcinia gummi-gutta plants growing at site S5 (Kodayar) in Kanyakumari district

of Tamil Nadu has not yet been reported.

177