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A comparative study of phytohaemagglutinin and extract of Phaseolus vulgaris seeds by characterization and cytogenetics
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 143–147
Contents lists available at ScienceDirect
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 Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India c University of Madras, Chennai, Tamil Nadu, India b
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
Article history: Received 12 March 2014 Received in revised form 13 May 2014 Accepted 25 May 2014 Available online 20 June 2014 Keywords: Phytohaemagglutinin Phaseolus vulgaris Mitogen Cytogenetics Human leucocyte cultures
a b s t r a c t Phytohaemagglutinin (PHA) is a lectin obtained from Phaseolus vulgaris (red kidney beans), that acts as a mitogen in human leucocyte culture and is commercially available from GibcoÒ. This PHA (GibcoÒ) was found to be very expensive, hence other inexpensive sources that can be used in all kinds of cytogenetics labs (rich and poor), were attempted. One such successful attempt was PHA extract from seeds of 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 the extract, analyzed by various spectroscopic and analytical techniques. The analysis clearly indicates that the product from Phaseolus seeds extract was found to be similar to the commercially available PHA (GibcoÒ) in the cytogenetic study of human leucocyte cultures. The present study enforces the possible utility of the plant extract directly for human leucocyte cultures. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Phytohaemagglutinin (PHA) is a lectin (mucoprotein) from Phaseolus vulgaris comprises of two 30-kDa subunits along with N-terminal sequence [1]. This un-degraded mitogen binds to cultured cells in the 3-day incubation period [2] and increases DNA synthesis (mitogenicity) making little or no target-cell
⇑ Corresponding author. Tel.: +91 9443328424. E-mail address: [email protected] (R. Saraswathy). http://dx.doi.org/10.1016/j.saa.2014.05.086 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
damage [3]. The mature lymphocytes which normally are unable to undergo the cell division, with the help of the lectin binding to carbohydrates moieties on cell surface helps mitogenic stimulation and cell agglutination [4]. The kidney beans extract on human peripheral lymphocytes promotes the synthesis of c-globulin, ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and induces 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 an attempt was made to utilize the crude seed extract of P. vulgaris in the leucocyte cultures, which is less expensive. Also there has
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 PHA in human leucocyte cultures [4]. Therefore a comparative study of commercial PHA and crude seed extract of P. vulgaris is the need of the hour. This paper discusses the methodology of preparation of PHA from seeds of P. vulgaris and its comparison with commercially available PHA (GibcoÒ) by characterization and cytogenetic analysis.
Materials and methods Materials With informed consent and ethical clearance, 5 ml of peripheral blood samples of clinically confirmed type II diabetes mellitus (T2DM, n = 20), type II diabetic neuropathy (T2DN, n = 20), type II diabetic nephropathy (T2DNP, n = 20) and Cataract (n = 20) were collected in heparinized vacutainer from Medzon Hospital, Vellore, India. P. vulgaris seeds (red kidney bean seeds) were purchased from super market and commercial PHA obtained from GibcoÒ.
Methods Preparation of PHA from seeds of P. vulgaris 25 g of red kidney bean seeds were surface sterilized with 70% ethanol and soaked overnight in 100 mL of sterile Ringer’s solution (NaCl-0.9 g, KCl-0.042 g, CaCl2-0.025 g, Distilled water-100 mL). The next day, the soaked seeds were ground along with the Ringer’s solution till a paste-like consistency was obtained. This was transferred to 50 mL sterile centrifuge tubes and centrifuged at 3000 rpm for 20 min, which resulted in the separation of four layers. The top two layers were carefully collected and centrifuged again at 3000 rpm for 20 min. The supernatant was separated and transferred to a sterile container. 0.5 mL of mycostatin (HiMediaÒ) was added to the supernatant. This constituted the stock solution, which was stored at 20 °C. 9 mL of sterile water was added to 1 mL of this stock solution to obtain the working PHA solution and stored at 20 °C.
Characterization of the PHA solution by chemical studies Both the PHA solutions were subjected to various chemical studies as follows.
Analysis by thin layer chromatography (TLC) TLC was done for both the samples in different ratios with methanol: chloroform (20:80, 30:70, 40:60, and 50:50) ratio.
UV visible spectroscopy The absorption spectra of both samples in water were recorded in the range of 200–800 nm.
FT-IR spectroscopy The lyophilised samples were mixed with potassium bromide (KBr) and grinded to form a very fine powder and was then compressed into a thin pellet and analyzed for infrared spectra.
Instrumentation 1
H and 13C NMR spectra were determined in D2O, CDCl3 and DMSO-d6 solutions at 400 MHz respectively using Bruker Ascend Model. FT-IR Spectra were recorded on a Schimadzu IR Affinity-1 Spectrophotometer. UV/Visible spectra were recorded on a SYSTRONIC 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 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: 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, Transfer Temp = 230 °C, Source Temp = 230 °C, Scan: 50–600 Da. Activity of PHA solutions on human cytogenetic studies Leucocyte culture method Working P. vulgaris crude extract (42 lg/ml) and the commercial PHA GibcoÒ (0.2 mL) was added to the human leucocyte cultures 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 from the vials at intervals of 24 h till harvest. Several test cultures were made using both commercial PHA and the extracted sample by modified method of Hungerford [6]. Chromosomal analysis The Giemsa stained metaphase slides were scored blind fold to estimate for mitotic index and harvesting time. A minimum of 100 well spread metaphases were analyzed for each sample and photographed using Olympus BX51 microscope wherever needed. Patient-control studies The cytogenetic studies were carried out for both control samples (n = 20) and Human Genetic Disorders, n = 80 [T2DM (n = 20), T2DN (n = 20), T2DNP (n = 20), Cataract (n = 20)]. The mitotic index and harvesting time was found to be similar. 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 human genetic disorders. A minimum of 1000 binucleated cells were analyzed for each sample to check the presence of micronuclei and/or other abnormalities using the scoring criteria given by Fenech et al. [8]. Microphotographs of selected binucleated cells were taken using Olympus BX51 microscope. Results and discussion Thin layer chromatography (TLC) Initially the samples were analysed on TLC plates in 20:80, 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 the ninhydrin spray in UV chamber, the spots were clearly appeared after eluting with methanol and chloroform mixture (1:1). The spots in both the commercial as well as the extract are exactly similar to each other. UV spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy Both the samples were subjected to lyophilisation using freeze drier, active fractions were collected and were dissolved in D2O and used for proton NMR, 13C NMR and DEPT analysis.
The following Fig. 2 shows the UV visible spectra absorption of commercial and extracted sample at 267 nm and 266 nm 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.
at 3400 cm 1, which is characteristic feature of hydroxyl group present in PHA Fig. 3. The aliphatic CAH stretching was clearly seen in range of 2877–2920 cm 1 in both the samples. Similarly to commercial PHA band 1641.42 cm 1 for C@O was appeared for the extract. The Appearance of moderate intense bands at 1062 cm 1 and 1053 cm 1 due to alcoholic CAO stretching was observed for both the samples.
4 204 nm 3 203 nm
Abs 2 267 nm
The results of nuclear magnetic resonance (NMR)
1 266 nm
400
600
800
Wavelength [nm]
Fig. 2. Comparison of UV absorption spectrum for commercial PHA and Phaseolus vulgaris crude extract.
Infrared analysis The infrared spectroscopy (IR) spectra of commercial PHA and extract were observed at the range of 4000–200 cm 1. IR bands for commercial PHA as well as extract sample showed a broad band
4000
3000
623.01
594.08
524.64 536.21
696.30
Fig. 3. FT-IR analysis commercial PHA and Phaseolus vulgaris crude extract.
605.65
1139.93
997.20
1000
621.08
1062.78
1236.37
1153.43
1402.25
921.97
1500
1053.13
1641.42
2000
1402.25
1641.42
2877.79 2916.37
3396.64
2939.52
3379.29
3400.50
2968.45
%T
2837.29
RSY 1 RSY 2
1535.34
200
1629.85
0
In 1H1 NMR (Figs. 4 and 5), both the commercial and the extract showed the complex spectra by giving majority of the signals in the range of d 1.0–5.0 See Fig. 1. It is predicted that the intense 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 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. It is significant to note that the extract is a combination of several unknown constituents. Attempts were made to compare the signals of both the extract and the commercial. On comparison, 1 H and 13C NMR spectra of crude extract showed signals pertaining to aromatic groups (d 7.0–12.5 in 1H NMR and d 122–160 in 13C
500
4
2
2.745
2.654
2.702
3.695
3.656
3
1
0
-1
-2
-3
-4
-5
ppm
75.24
3.841
3.881
4.398
4.351
5
107.78
6
5.007
7.166
7.778
8.818
8.859
8.498
7 5.42
8.880
8 11.70
9
5.74
8.983
9.077
9.183
9.201
10
48.69
11
5.73
10.098
9.221
10.118
12 5.43
13
5.90
12.177
A.R.S. Badari Nath et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 143–147 10.903
146
Fig. 4. Showing proton NMR for Phaseolus vulgaris crude extract.
Fig. 5. Showing proton NMR for commercial PHA.
(C) With Commercial PHA
(A) With Commercial PHA
(B) With crude extract of PHA
Fig. 6. (A and B) Metaphase spreads.
NMR respectively) apart from the other signals matching with the NMR data of commercial sample. These results were reproducible and they were further compared and confirmed with the commercially available compound. In this current study, results obtained from TLC, UV spectrophotometer, FT-IR, NMR (1H, 13C, DEPT analysis) confirmed the presence of the compound in the extract (see Table 1).
(D) With crude extract of PHA
Fig. 7. (C and D) Binucleate cells (CBMN cyt assay).
Cytogenetics studies In order to assess the biological activity, extracted PHA from seeds and Commercial PHA were utilized in cytogenetic studies (see Figs. 6 and 7). The blindfold scoring was carried out in chromosomal studies and CBMN cyt assay (Table 2). As the extracted PHA showed functional similarity to that of the commercial PHA, both the PHA were used in the cytogenetic studies of the following patients groups T2DM (n = 20), T2DN (n = 20), T2DNP (n = 20), Cataract (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 (kmax) (nm)
FT-IR vibrational frequency (cm
Commercial PHA
266
Phaseolus vulgaris crude extract
267
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) 3400.50 abroad band (OAH), 1402.25, 1062.78 (CAC), 1153.13 (CAO), 1641.42 (NAC@O)
Table 2 Comparison of cytogenetic activities of the commercial and Phaseolus vulgaris PHA in human leucocyte cultures.
Chromosomal aberration frequency per cell (Mean ± SE) Frequency of nuclear aberrations per cell (Mean ± SE)
Commercial PHA
Extracted PHA
0.6 ± 0.1
0.9 ± 0.2
1.3 ± 0.1
1.3 ± 0.15
Table 3 The mean frequencies of chromosomal and nuclear aberrations observed in the patient groups using Phaseolus vulgaris crude extract.
Chromosomal aberration frequency per cell, (Mean ± SE) Frequency of nuclear aberrations per cell (Mean ± SE)
T2DM (n = 20)
T2DN (n = 20)
T2DPN (n = 20)
1.5 ± 0.2
0.75 ± 0.1
1.45 ± 0.2
1.1 ± 0.25
1.0 ± 0.2
1.75 ± 0.31
1.5 ± 0.2
1.3 ± 0.28
Cataract (n = 20)
The results show that the extracted PHA can be used in the leucocyte cultures for the cytogenetic studies. In the developing countries like India where cytogenetic tests are becoming a routine in the hospitals and in research, the extracted PHA may be utilized, which is less expensive and has shelf-life for more than one year when stored at 20 °C. Conclusions It is important to note that the commercial PHA is expensive. Since the seed extract exhibits the bioactivity which is similar to
) (nm)
the activity of commercial PHA, the present work enforces the possible utility of the plant extract directly without further processing. The similarities in spectroscopic data and cytogenetic studies in the commercial and the crude extract guided the authors to investigate further. Acknowledgements Badari Nath A.R.S. is grateful to VIT University for the research associateship. The authors and the group members thank DSTVIT-FIST for NMR and SIF-VIT University for instrumentation facilities. References [1] Arishya Sharma, Tzi Bun Ng, Jack Ho Wong, Peng Lin, Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi Beans), J. Biomed. Biotechnol. (2009). Article ID 929568. [2] Daniel P. Stites, Martin C. Carr, Hugh. Fudenberg, Development of cellular immunity in the human fetus: dichotomy of proliferative and cytotoxic responses of lymphoid cells to phytohemagglutinin (T and B cells/DNA synthesis/target cells), Proc. Nat. Acad. Sci. USA 69 (6) (1972) 1440–1444. [3] W.J. Penhale, Anne Farmer, A.C. Maccuish, W.J. Irvine, A rapid micro-method for the phytohaemagglutinin-induced human lymphocyte transformation test, Clin. Exp. Immunol. 18 (1) (1974) 155–167. [4] G. Gandhi, V. Bhel, Phytohaemagglutinin laboratory preparation used as a mitogen in peripheral blood lymphocyte cultures of infertile couples, South Asian Anthropol. 9 (1) (2009) 37–44. [5] K. Hirschhorn, R.L. Kolodny, N. Hashem, F. Bach, Mitogenic-action of phytohemagglutinin, Lancet 2 (1983) 305. [6] David A. Hungerford, Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCL, Biotechnic Histochem. 40 (6) (1965) 333–338. [7] Michael Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81–95. [8] M. Fenech, WP. Chang, M. Kirsch-Volders, N. Holland, S. Bonassi, E. Zeiger, Human MicronNucleus project, HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures, Mutat. Res. 534 (1–2) (2003) 65–75.
Contents lists available at ScienceDirect
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 Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India c University of Madras, Chennai, Tamil Nadu, India b
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
Article history: Received 12 March 2014 Received in revised form 13 May 2014 Accepted 25 May 2014 Available online 20 June 2014 Keywords: Phytohaemagglutinin Phaseolus vulgaris Mitogen Cytogenetics Human leucocyte cultures
a b s t r a c t Phytohaemagglutinin (PHA) is a lectin obtained from Phaseolus vulgaris (red kidney beans), that acts as a mitogen in human leucocyte culture and is commercially available from GibcoÒ. This PHA (GibcoÒ) was found to be very expensive, hence other inexpensive sources that can be used in all kinds of cytogenetics labs (rich and poor), were attempted. One such successful attempt was PHA extract from seeds of 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 the extract, analyzed by various spectroscopic and analytical techniques. The analysis clearly indicates that the product from Phaseolus seeds extract was found to be similar to the commercially available PHA (GibcoÒ) in the cytogenetic study of human leucocyte cultures. The present study enforces the possible utility of the plant extract directly for human leucocyte cultures. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Phytohaemagglutinin (PHA) is a lectin (mucoprotein) from Phaseolus vulgaris comprises of two 30-kDa subunits along with N-terminal sequence [1]. This un-degraded mitogen binds to cultured cells in the 3-day incubation period [2] and increases DNA synthesis (mitogenicity) making little or no target-cell
⇑ Corresponding author. Tel.: +91 9443328424. E-mail address: [email protected] (R. Saraswathy). http://dx.doi.org/10.1016/j.saa.2014.05.086 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
damage [3]. The mature lymphocytes which normally are unable to undergo the cell division, with the help of the lectin binding to carbohydrates moieties on cell surface helps mitogenic stimulation and cell agglutination [4]. The kidney beans extract on human peripheral lymphocytes promotes the synthesis of c-globulin, ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and induces 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 an attempt was made to utilize the crude seed extract of P. vulgaris in the leucocyte cultures, which is less expensive. Also there has
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 PHA in human leucocyte cultures [4]. Therefore a comparative study of commercial PHA and crude seed extract of P. vulgaris is the need of the hour. This paper discusses the methodology of preparation of PHA from seeds of P. vulgaris and its comparison with commercially available PHA (GibcoÒ) by characterization and cytogenetic analysis.
Materials and methods Materials With informed consent and ethical clearance, 5 ml of peripheral blood samples of clinically confirmed type II diabetes mellitus (T2DM, n = 20), type II diabetic neuropathy (T2DN, n = 20), type II diabetic nephropathy (T2DNP, n = 20) and Cataract (n = 20) were collected in heparinized vacutainer from Medzon Hospital, Vellore, India. P. vulgaris seeds (red kidney bean seeds) were purchased from super market and commercial PHA obtained from GibcoÒ.
Methods Preparation of PHA from seeds of P. vulgaris 25 g of red kidney bean seeds were surface sterilized with 70% ethanol and soaked overnight in 100 mL of sterile Ringer’s solution (NaCl-0.9 g, KCl-0.042 g, CaCl2-0.025 g, Distilled water-100 mL). The next day, the soaked seeds were ground along with the Ringer’s solution till a paste-like consistency was obtained. This was transferred to 50 mL sterile centrifuge tubes and centrifuged at 3000 rpm for 20 min, which resulted in the separation of four layers. The top two layers were carefully collected and centrifuged again at 3000 rpm for 20 min. The supernatant was separated and transferred to a sterile container. 0.5 mL of mycostatin (HiMediaÒ) was added to the supernatant. This constituted the stock solution, which was stored at 20 °C. 9 mL of sterile water was added to 1 mL of this stock solution to obtain the working PHA solution and stored at 20 °C.
Characterization of the PHA solution by chemical studies Both the PHA solutions were subjected to various chemical studies as follows.
Analysis by thin layer chromatography (TLC) TLC was done for both the samples in different ratios with methanol: chloroform (20:80, 30:70, 40:60, and 50:50) ratio.
UV visible spectroscopy The absorption spectra of both samples in water were recorded in the range of 200–800 nm.
FT-IR spectroscopy The lyophilised samples were mixed with potassium bromide (KBr) and grinded to form a very fine powder and was then compressed into a thin pellet and analyzed for infrared spectra.
Instrumentation 1
H and 13C NMR spectra were determined in D2O, CDCl3 and DMSO-d6 solutions at 400 MHz respectively using Bruker Ascend Model. FT-IR Spectra were recorded on a Schimadzu IR Affinity-1 Spectrophotometer. UV/Visible spectra were recorded on a SYSTRONIC 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 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: 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, Transfer Temp = 230 °C, Source Temp = 230 °C, Scan: 50–600 Da. Activity of PHA solutions on human cytogenetic studies Leucocyte culture method Working P. vulgaris crude extract (42 lg/ml) and the commercial PHA GibcoÒ (0.2 mL) was added to the human leucocyte cultures 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 from the vials at intervals of 24 h till harvest. Several test cultures were made using both commercial PHA and the extracted sample by modified method of Hungerford [6]. Chromosomal analysis The Giemsa stained metaphase slides were scored blind fold to estimate for mitotic index and harvesting time. A minimum of 100 well spread metaphases were analyzed for each sample and photographed using Olympus BX51 microscope wherever needed. Patient-control studies The cytogenetic studies were carried out for both control samples (n = 20) and Human Genetic Disorders, n = 80 [T2DM (n = 20), T2DN (n = 20), T2DNP (n = 20), Cataract (n = 20)]. The mitotic index and harvesting time was found to be similar. 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 human genetic disorders. A minimum of 1000 binucleated cells were analyzed for each sample to check the presence of micronuclei and/or other abnormalities using the scoring criteria given by Fenech et al. [8]. Microphotographs of selected binucleated cells were taken using Olympus BX51 microscope. Results and discussion Thin layer chromatography (TLC) Initially the samples were analysed on TLC plates in 20:80, 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 the ninhydrin spray in UV chamber, the spots were clearly appeared after eluting with methanol and chloroform mixture (1:1). The spots in both the commercial as well as the extract are exactly similar to each other. UV spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy Both the samples were subjected to lyophilisation using freeze drier, active fractions were collected and were dissolved in D2O and used for proton NMR, 13C NMR and DEPT analysis.
The following Fig. 2 shows the UV visible spectra absorption of commercial and extracted sample at 267 nm and 266 nm 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.
at 3400 cm 1, which is characteristic feature of hydroxyl group present in PHA Fig. 3. The aliphatic CAH stretching was clearly seen in range of 2877–2920 cm 1 in both the samples. Similarly to commercial PHA band 1641.42 cm 1 for C@O was appeared for the extract. The Appearance of moderate intense bands at 1062 cm 1 and 1053 cm 1 due to alcoholic CAO stretching was observed for both the samples.
4 204 nm 3 203 nm
Abs 2 267 nm
The results of nuclear magnetic resonance (NMR)
1 266 nm
400
600
800
Wavelength [nm]
Fig. 2. Comparison of UV absorption spectrum for commercial PHA and Phaseolus vulgaris crude extract.
Infrared analysis The infrared spectroscopy (IR) spectra of commercial PHA and extract were observed at the range of 4000–200 cm 1. IR bands for commercial PHA as well as extract sample showed a broad band
4000
3000
623.01
594.08
524.64 536.21
696.30
Fig. 3. FT-IR analysis commercial PHA and Phaseolus vulgaris crude extract.
605.65
1139.93
997.20
1000
621.08
1062.78
1236.37
1153.43
1402.25
921.97
1500
1053.13
1641.42
2000
1402.25
1641.42
2877.79 2916.37
3396.64
2939.52
3379.29
3400.50
2968.45
%T
2837.29
RSY 1 RSY 2
1535.34
200
1629.85
0
In 1H1 NMR (Figs. 4 and 5), both the commercial and the extract showed the complex spectra by giving majority of the signals in the range of d 1.0–5.0 See Fig. 1. It is predicted that the intense 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 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. It is significant to note that the extract is a combination of several unknown constituents. Attempts were made to compare the signals of both the extract and the commercial. On comparison, 1 H and 13C NMR spectra of crude extract showed signals pertaining to aromatic groups (d 7.0–12.5 in 1H NMR and d 122–160 in 13C
500
4
2
2.745
2.654
2.702
3.695
3.656
3
1
0
-1
-2
-3
-4
-5
ppm
75.24
3.841
3.881
4.398
4.351
5
107.78
6
5.007
7.166
7.778
8.818
8.859
8.498
7 5.42
8.880
8 11.70
9
5.74
8.983
9.077
9.183
9.201
10
48.69
11
5.73
10.098
9.221
10.118
12 5.43
13
5.90
12.177
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146
Fig. 4. Showing proton NMR for Phaseolus vulgaris crude extract.
Fig. 5. Showing proton NMR for commercial PHA.
(C) With Commercial PHA
(A) With Commercial PHA
(B) With crude extract of PHA
Fig. 6. (A and B) Metaphase spreads.
NMR respectively) apart from the other signals matching with the NMR data of commercial sample. These results were reproducible and they were further compared and confirmed with the commercially available compound. In this current study, results obtained from TLC, UV spectrophotometer, FT-IR, NMR (1H, 13C, DEPT analysis) confirmed the presence of the compound in the extract (see Table 1).
(D) With crude extract of PHA
Fig. 7. (C and D) Binucleate cells (CBMN cyt assay).
Cytogenetics studies In order to assess the biological activity, extracted PHA from seeds and Commercial PHA were utilized in cytogenetic studies (see Figs. 6 and 7). The blindfold scoring was carried out in chromosomal studies and CBMN cyt assay (Table 2). As the extracted PHA showed functional similarity to that of the commercial PHA, both the PHA were used in the cytogenetic studies of the following patients groups T2DM (n = 20), T2DN (n = 20), T2DNP (n = 20), Cataract (n = 20) (Table 3).
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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 (kmax) (nm)
FT-IR vibrational frequency (cm
Commercial PHA
266
Phaseolus vulgaris crude extract
267
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) 3400.50 abroad band (OAH), 1402.25, 1062.78 (CAC), 1153.13 (CAO), 1641.42 (NAC@O)
Table 2 Comparison of cytogenetic activities of the commercial and Phaseolus vulgaris PHA in human leucocyte cultures.
Chromosomal aberration frequency per cell (Mean ± SE) Frequency of nuclear aberrations per cell (Mean ± SE)
Commercial PHA
Extracted PHA
0.6 ± 0.1
0.9 ± 0.2
1.3 ± 0.1
1.3 ± 0.15
Table 3 The mean frequencies of chromosomal and nuclear aberrations observed in the patient groups using Phaseolus vulgaris crude extract.
Chromosomal aberration frequency per cell, (Mean ± SE) Frequency of nuclear aberrations per cell (Mean ± SE)
T2DM (n = 20)
T2DN (n = 20)
T2DPN (n = 20)
1.5 ± 0.2
0.75 ± 0.1
1.45 ± 0.2
1.1 ± 0.25
1.0 ± 0.2
1.75 ± 0.31
1.5 ± 0.2
1.3 ± 0.28
Cataract (n = 20)
The results show that the extracted PHA can be used in the leucocyte cultures for the cytogenetic studies. In the developing countries like India where cytogenetic tests are becoming a routine in the hospitals and in research, the extracted PHA may be utilized, which is less expensive and has shelf-life for more than one year when stored at 20 °C. Conclusions It is important to note that the commercial PHA is expensive. Since the seed extract exhibits the bioactivity which is similar to
) (nm)
the activity of commercial PHA, the present work enforces the possible utility of the plant extract directly without further processing. The similarities in spectroscopic data and cytogenetic studies in the commercial and the crude extract guided the authors to investigate further. Acknowledgements Badari Nath A.R.S. is grateful to VIT University for the research associateship. The authors and the group members thank DSTVIT-FIST for NMR and SIF-VIT University for instrumentation facilities. References [1] Arishya Sharma, Tzi Bun Ng, Jack Ho Wong, Peng Lin, Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi Beans), J. Biomed. Biotechnol. (2009). Article ID 929568. [2] Daniel P. Stites, Martin C. Carr, Hugh. Fudenberg, Development of cellular immunity in the human fetus: dichotomy of proliferative and cytotoxic responses of lymphoid cells to phytohemagglutinin (T and B cells/DNA synthesis/target cells), Proc. Nat. Acad. Sci. USA 69 (6) (1972) 1440–1444. [3] W.J. Penhale, Anne Farmer, A.C. Maccuish, W.J. Irvine, A rapid micro-method for the phytohaemagglutinin-induced human lymphocyte transformation test, Clin. Exp. Immunol. 18 (1) (1974) 155–167. [4] G. Gandhi, V. Bhel, Phytohaemagglutinin laboratory preparation used as a mitogen in peripheral blood lymphocyte cultures of infertile couples, South Asian Anthropol. 9 (1) (2009) 37–44. [5] K. Hirschhorn, R.L. Kolodny, N. Hashem, F. Bach, Mitogenic-action of phytohemagglutinin, Lancet 2 (1983) 305. [6] David A. Hungerford, Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCL, Biotechnic Histochem. 40 (6) (1965) 333–338. [7] Michael Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81–95. [8] M. Fenech, WP. Chang, M. Kirsch-Volders, N. Holland, S. Bonassi, E. Zeiger, Human MicronNucleus project, HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures, Mutat. Res. 534 (1–2) (2003) 65–75.