Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 143–147

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

A comparative study of phytohaemagglutinin and extract of Phaseolus

vulgaris seeds by characterization and cytogenetics
A.R.S. Badari Nath a, A. Sivaramakrishna b, K.M. Marimuthu c, Radha Saraswathy a,⇑
a
Biomedical Genetics Research Laboratory (BMGRL), TT120, School of Bio Sciences and Technology, VIT University, Vellore 632 014, Tamil Nadu, India
b
Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India
c
University of Madras, Chennai, Tamil Nadu, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 P. vulgaris seed extract exhibits the

bioactivity similar to commercial
PHA.
 Our study enforces the use of seed
extract directly in human leucocyte
cultures.
 This less expensive extract may be
utilized for cytogenetics test in
hospitals.
 Extracted PHA has shelf-life for more
than one year when stored at 20 °C.

a r t i c l e i n f o a b s t r a c t

Article history: Phytohaemagglutinin (PHA) is a lectin obtained from Phaseolus vulgaris (red kidney beans), that acts as a
Received 12 March 2014 mitogen in human leucocyte culture and is commercially available from GibcoÒ. This PHA (GibcoÒ) was
Received in revised form 13 May 2014 found to be very expensive, hence other inexpensive sources that can be used in all kinds of cytogenetics
Accepted 25 May 2014
labs (rich and poor), were attempted. One such successful attempt was PHA extract from seeds of
Available online 20 June 2014
P. vulgaris. This paper details the methodology of extraction and application of PHA from seeds of
P. vulgaris. Attempts has been made to identify the chemical and physical properties of the products in
Keywords:
the extract, analyzed by various spectroscopic and analytical techniques. The analysis clearly indicates
Phytohaemagglutinin
Phaseolus vulgaris
that the product from Phaseolus seeds extract was found to be similar to the commercially available
Mitogen PHA (GibcoÒ) in the cytogenetic study of human leucocyte cultures. The present study enforces the
Cytogenetics possible utility of the plant extract directly for human leucocyte cultures.
Human leucocyte cultures Ó 2014 Elsevier B.V. All rights reserved.

Introduction damage [3]. The mature lymphocytes which normally are unable
to undergo the cell division, with the help of the lectin binding
Phytohaemagglutinin (PHA) is a lectin (mucoprotein) from to carbohydrates moieties on cell surface helps mitogenic stimula-
Phaseolus vulgaris comprises of two 30-kDa subunits along with tion and cell agglutination [4]. The kidney beans extract on human
N-terminal sequence [1]. This un-degraded mitogen binds to peripheral lymphocytes promotes the synthesis of c-globulin,
cultured cells in the 3-day incubation period [2] and increases ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and induces
DNA synthesis (mitogenicity) making little or no target-cell mitosis in a large fraction of lymphocytes [5]. The commercially
available PHA has been used for many years in human leucocyte
cultures for cytogenetic studies, but it is very expensive, therefore
⇑ Corresponding author. Tel.: +91 9443328424. an attempt was made to utilize the crude seed extract of P. vulgaris
E-mail address: r_saraswathy@yahoo.com (R. Saraswathy). in the leucocyte cultures, which is less expensive. Also there has

http://dx.doi.org/10.1016/j.saa.2014.05.086
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
144 A.R.S. Badari Nath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 143–147

been very few and inconclusive studies using the crude extract of Instrumentation
PHA in human leucocyte cultures [4]. Therefore a comparative
1
study of commercial PHA and crude seed extract of P. vulgaris is H and 13C NMR spectra were determined in D2O, CDCl3 and
the need of the hour. This paper discusses the methodology of DMSO-d6 solutions at 400 MHz respectively using Bruker Ascend
preparation of PHA from seeds of P. vulgaris and its comparison Model. FT-IR Spectra were recorded on a Schimadzu IR Affinity-1
with commercially available PHA (GibcoÒ) by characterization Spectrophotometer. UV/Visible spectra were recorded on a SYS-
and cytogenetic analysis. TRONIC AU-2701 UV/Visible spectrophotometer. Mass spectra were
recorded on a Perkin–Elmer clarus 680 (GC) and clarus 600 (EI,
Mass). ACQUISITION PARAMETERS: Oven: Initial temp. 60 °C for
Materials and methods 2 min, ramp 10 °C/min to 300 °C, hold 6 min, Total Run Time:
32.00 mint; InjAuto = 250 °C, Volume = 1 lL, Split = 10:1, Flow Rate:
Materials 1 mL/min, Carrier Gas = He, Column = Elite-5MS (30.0 m, 0.25 mm
ID, 250 lm df). MASS CONDITION (EI): Solvent Delay = 2.00 min,
With informed consent and ethical clearance, 5 ml of peripheral Transfer Temp = 230 °C, Source Temp = 230 °C, Scan: 50–600 Da.
blood samples of clinically confirmed type II diabetes mellitus
(T2DM, n = 20), type II diabetic neuropathy (T2DN, n = 20), type II Activity of PHA solutions on human cytogenetic studies
diabetic nephropathy (T2DNP, n = 20) and Cataract (n = 20) were
collected in heparinized vacutainer from Medzon Hospital, Vellore, Leucocyte culture method
India. Working P. vulgaris crude extract (42 lg/ml) and the commer-
P. vulgaris seeds (red kidney bean seeds) were purchased from cial PHA GibcoÒ (0.2 mL) was added to the human leucocyte cul-
super market and commercial PHA obtained from GibcoÒ. tures containing 5 mL of RPMI medium, 1.2 mL of Serum
(HiMediaÒ) and 0.5 mL of blood and incubated for 72 h before
treating with (1 mg/ml) colchicine for 20 min. CO2 was released
Methods from the vials at intervals of 24 h till harvest. Several test cultures
were made using both commercial PHA and the extracted sample
Preparation of PHA from seeds of P. vulgaris by modified method of Hungerford [6].
25 g of red kidney bean seeds were surface sterilized with 70%
ethanol and soaked overnight in 100 mL of sterile Ringer’s solution Chromosomal analysis
(NaCl-0.9 g, KCl-0.042 g, CaCl2-0.025 g, Distilled water-100 mL). The Giemsa stained metaphase slides were scored blind fold to
The next day, the soaked seeds were ground along with the Ring- estimate for mitotic index and harvesting time. A minimum of 100
er’s solution till a paste-like consistency was obtained. This was well spread metaphases were analyzed for each sample and photo-
transferred to 50 mL sterile centrifuge tubes and centrifuged at graphed using Olympus BX51 microscope wherever needed.
3000 rpm for 20 min, which resulted in the separation of four lay-
ers. The top two layers were carefully collected and centrifuged Patient-control studies
again at 3000 rpm for 20 min. The supernatant was separated The cytogenetic studies were carried out for both control sam-
and transferred to a sterile container. 0.5 mL of mycostatin (HiMe- ples (n = 20) and Human Genetic Disorders, n = 80 [T2DM (n = 20),
diaÒ) was added to the supernatant. This constituted the stock T2DN (n = 20), T2DNP (n = 20), Cataract (n = 20)]. The mitotic index
solution, which was stored at 20 °C. 9 mL of sterile water was and harvesting time was found to be similar.
added to 1 mL of this stock solution to obtain the working PHA
solution and stored at 20 °C. Cytokinesis-block micronucleus (CBMN) assay
Both PHA solutions were further analysed for CBMN Cyt assay
by modified method of Fenech 2000 [7] in the controls and various
Characterization of the PHA solution by chemical studies
human genetic disorders.
A minimum of 1000 binucleated cells were analyzed for each
Both the PHA solutions were subjected to various chemical
sample to check the presence of micronuclei and/or other abnor-
studies as follows.
malities using the scoring criteria given by Fenech et al. [8]. Micro-
photographs of selected binucleated cells were taken using
Analysis by thin layer chromatography (TLC) Olympus BX51 microscope.
TLC was done for both the samples in different ratios with
methanol: chloroform (20:80, 30:70, 40:60, and 50:50) ratio. Results and discussion

Thin layer chromatography (TLC)

UV visible spectroscopy
The absorption spectra of both samples in water were recorded Initially the samples were analysed on TLC plates in 20:80,
in the range of 200–800 nm. 30:70, 40:60, and 50:50 ratios of methanol and chloroform. As
the spots were identified not only by iodine alone but by applying
FT-IR spectroscopy the ninhydrin spray in UV chamber, the spots were clearly
The lyophilised samples were mixed with potassium bromide appeared after eluting with methanol and chloroform mixture
(KBr) and grinded to form a very fine powder and was then com- (1:1). The spots in both the commercial as well as the extract are
pressed into a thin pellet and analyzed for infrared spectra. exactly similar to each other.

UV spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy
Both the samples were subjected to lyophilisation using freeze The following Fig. 2 shows the UV visible spectra absorption of
drier, active fractions were collected and were dissolved in D2O commercial and extracted sample at 267 nm and 266 nm
and used for proton NMR, 13C NMR and DEPT analysis. respectively.
 A.R.S. Badari Nath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 143–147 145

(Source: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=45480564#itabs-2d)

Fig. 1. Structure of PHA containing polyhydroxy units.

4 at 3400 cm 1, which is characteristic feature of hydroxyl group

present in PHA Fig. 3. The aliphatic CAH stretching was clearly
204 nm
seen in range of 2877–2920 cm 1 in both the samples. Similarly
3 to commercial PHA band 1641.42 cm 1 for C@O was appeared
for the extract. The Appearance of moderate intense bands at
203 nm
1062 cm 1 and 1053 cm 1 due to alcoholic CAO stretching was
Abs 2 observed for both the samples.
267 nm
The results of nuclear magnetic resonance (NMR)
1

266 nm
In 1H1 NMR (Figs. 4 and 5), both the commercial and the extract
showed the complex spectra by giving majority of the signals in
0
200 400 600 800 the range of d 1.0–5.0 See Fig. 1. It is predicted that the intense
Wavelength [nm] and broad signals between d 3.0 and 5.0 are could be due to the
presence of cyclic CHAOH protons. Since D2O is the solvent for
Fig. 2. Comparison of UV absorption spectrum for commercial PHA and Phaseolus
vulgaris crude extract. recording the spectra, there is possibility for deuterium exchange
with hydrogens.
In addition to this, 13C NMR data exhibited the peaks in the
range of d 20.0–105.0.
Infrared analysis It is significant to note that the extract is a combination of sev-
eral unknown constituents. Attempts were made to compare the
The infrared spectroscopy (IR) spectra of commercial PHA and signals of both the extract and the commercial. On comparison,
extract were observed at the range of 4000–200 cm 1. IR bands 1
H and 13C NMR spectra of crude extract showed signals pertaining
for commercial PHA as well as extract sample showed a broad band to aromatic groups (d 7.0–12.5 in 1H NMR and d 122–160 in 13C

RSY 1
RSY 2
1236.37

%T
1153.43

524.64
696.30

594.08
623.01
2837.29

1402.25

1062.78
2877.79
2968.45

1535.34
1641.42
3379.29
3400.50

921.97

536.21
605.65
621.08
1139.93

997.20
1402.25
2916.37
2939.52

1053.13
1629.85
1641.42
3396.64

4000 3000 2000 1500 1000 500

Fig. 3. FT-IR analysis commercial PHA and Phaseolus vulgaris crude extract.
146 A.R.S. Badari Nath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 143–147

12.177
10.903
10.118
10.098
9.221
9.201
9.183
9.077
8.983
8.880
8.859
8.818
8.498
7.778
7.166
5.007
4.398
4.351
3.881
3.841
3.695
3.656
2.745
2.702
2.654
13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 -4 -5 ppm
11.70

107.78
5.43

5.90

5.73

48.69

5.74

5.42

75.24
Fig. 4. Showing proton NMR for Phaseolus vulgaris crude extract.

Fig. 5. Showing proton NMR for commercial PHA.

(C) With Commercial PHA (D) With crude extract of PHA

(A) With Commercial PHA (B) With crude extract of PHA Fig. 7. (C and D) Binucleate cells (CBMN cyt assay).

Fig. 6. (A and B) Metaphase spreads. Cytogenetics studies

In order to assess the biological activity, extracted PHA from

NMR respectively) apart from the other signals matching with the seeds and Commercial PHA were utilized in cytogenetic studies
NMR data of commercial sample. (see Figs. 6 and 7). The blindfold scoring was carried out in chro-
These results were reproducible and they were further com- mosomal studies and CBMN cyt assay (Table 2). As the extracted
pared and confirmed with the commercially available compound. PHA showed functional similarity to that of the commercial PHA,
In this current study, results obtained from TLC, UV spectropho- both the PHA were used in the cytogenetic studies of the following
tometer, FT-IR, NMR (1H, 13C, DEPT analysis) confirmed the pres- patients groups T2DM (n = 20), T2DN (n = 20), T2DNP (n = 20), Cat-
ence of the compound in the extract (see Table 1). aract (n = 20) (Table 3).
 A.R.S. Badari Nath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 143–147 147

Table 1
Comparison of the properties of commercial PHA and Phaseolus vulgaris crude extract compounds based on UV visible spectrum, FT-IR vibrational frequency.
1
Type of sample UV visible spectrum FT-IR vibrational frequency (cm ) (nm)
(kmax) (nm)
Commercial PHA 266 3400 cm 1 (due to OAH stretching), 2916.37 and 2877 aliphatic CAH stretching, 1641.42 (C@O), 1053.13,
1402.25 (CAC), 1139.93 (CAO)
Phaseolus vulgaris crude 267 3400.50 abroad band (OAH), 1402.25, 1062.78 (CAC), 1153.13 (CAO), 1641.42 (NAC@O)
extract

Table 2
the activity of commercial PHA, the present work enforces the pos-
Comparison of cytogenetic activities of the commercial and Phaseolus vulgaris PHA in
human leucocyte cultures. sible utility of the plant extract directly without further processing.
The similarities in spectroscopic data and cytogenetic studies in
Commercial Extracted
the commercial and the crude extract guided the authors to inves-
PHA PHA
tigate further.
Chromosomal aberration frequency per cell 0.6 ± 0.1 0.9 ± 0.2
(Mean ± SE)
Frequency of nuclear aberrations per cell 1.3 ± 0.1 1.3 ± 0.15
Acknowledgements
(Mean ± SE)
Badari Nath A.R.S. is grateful to VIT University for the research
associateship. The authors and the group members thank DST-
Table 3 VIT-FIST for NMR and SIF-VIT University for instrumentation
The mean frequencies of chromosomal and nuclear aberrations observed in the
facilities.
patient groups using Phaseolus vulgaris crude extract.

T2DM T2DN T2DPN Cataract References

(n = 20) (n = 20) (n = 20) (n = 20)
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