Purification and Characterization of a Lectin from Leaf Pulp of Aloe Vera (L.) BURM

Manpreet Kaur et al. / Journal of Pharmacy Research 2011,4(7),2441-2446 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Purification and characterization of a lectin from leaf pulp of Aloe vera (L.) BURM. F. Manpreet Kaur1, Jatinder Singh 2*, Sukhdev Singh Kamboj2, A.K. Saxena3 1 Assistant Professor, Department of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab 143 005, India 2 Professor, Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, Punjab 143 005, India 3 Scientist, Department of Pharmacology, Indian Institute of Integrative Medicine, Jammu and Kashmir, 180 001, India Received on: 12-04-2011; Revised on: 18-05-2011; Accepted on:21-06-2011

ABSTRACT A mucin specific lectin was isolated from leaf pulp of Aloe vera (L.) Burm. f., family Aloaceae. The isolation procedure employed affinity chromatography on mucin-linked amino activated silica beads. The purity of the lectin preparation is showh by a single band in SDS-PAGE at pH 8.3 and in native PAGE at pH 4.5. However, multiple bands were obtained in isoelectric focusing and native PAGE at pH 8.3 showing the presence of isolectins. A. vera leaf lectin (AVLL) showed a single peak in gel permeation chromatography on Biogel P-200 and native molecular mass was calculated to be 53 kDa. The hemagglutinating activity of the lectin was not affected upto the temperature of 70°C and in the pH range of 1.5–12.5. The lectin was stable upto 7.0 M concentration of various denaturants tested i.e. urea, thiourea, guanidine hydrochloride and do not require metal ions for its activity. Out of 13 human cancer cell lines tested, AVLL inhibited proliferation of colon cancer cell lines i.e. HCT-15, HT-29 and SW-620 and a lung cancer cell line, HOP-62. This lectin also showed significant inhibitory effect on the development of melon fruit fly, Bactrocera cucurbitae (Coquillett), one of the most important pests of cucurbits. Furthermore, this lectin was found to be virtually non-mitogenic towards human peripheral blood monocytes. However, it was mitogenic towards murine splenocytes with relative mitogenic index approximately double i.e. 232.9, as compared to ConA, a well-known standard mitogen.

Key words: Aloe vera, anti-cancer, anti-insect, Bactrocera cucurbitae, lectin, mucin

INTRODUCTION Lectins are carbohydrate-binding proteins or glycoproteins of non-immune fly is an important pest of cucurbits, besides affecting a number of other plants origin possessing at least one non-catalytic domain [1]. These are usually con- from different families in tropical countries and till date no suitable active sidered a heterogenous group of proteins because of the apparent differences ingredient is available for controlling these tephritids. in molecular structure, sugar specificity and biological activity between individual lectins. Lectins are ubiquitous in distribution being present in all types MATERIALS AND METHODS of living organisms [2,3]. The abundance and the variety of carbohydrate speci- ficities of lectins raised the interest to use these molecules as one of the Materials required important tools in glycobiology. Lectins display a variety of biological activities including antitumor[4], antifungal[5], antiinsect[6], immunomodulatory[7] ac- Plant material: tivities. Most plant lectins are storage proteins with a potential function of Fresh leaves of A. vera were collected from Botanical gardens of Guru Nanak defense against insects and fungi suggesting not only their role in plant defense Dev University, Amritsar, Punjab, India. but also demonstrated their potential use in crop protection against phy- tophagous invertebrates[6,8]. Tumor-cell lines: The human cancer cell lines i.e. Colo-205 (colon), HT-29 (colon), HCT-15 Aloe vera (L.) Burm. f. is a well-known medicinal plant, which has been (colon), SW-620 (colon), HEP-2 (liver), HOP-62 (lung), A-549 (lung), SNB- endowed with many marvelous properties and over the years around the world, 78 (CNS), IMR-32 (neuroblastoma), SKOV-3 (ovary), MCF-7 (breast), PC-3 it has been given many wonderful names such as Burn Plant, Wand of Heaven (prostate) and SiHa (cervix) were procured from National Center for Cell and Plant of Life. It is a succulent member of family Asphodelaceae. Many Sciences, Pune, Maharashtra, India. The cells were cultured in RPMI-1640 health-promoting and therapeutic effects have been attributed to this plant medium supplemented with 10% FCS, glutamine (2 mM), penicillin (100 IU/ and it has been employed commercially such as in soothing burns, reducing ml) and streptomycin (100 mg/ml) at 37oC, in humidified atmosphere with 5% blood glucose level in diabetes, treating bowel diseases, reducing arthritic swell- CO2 in CO2 incubator (Heraeus, USA). ing[9]. Insect cultures: The present study describes the purification of a lectin from leaf pulp of A. The melon fruit flies were reared by the procedure described by Gupta and vera (L.) Burm. f. by affinity chromatography on mucin-linked amino acti- coworkers (1978) [14] in wire mesh cages (Rescholar equipment: 45´45´50 vated silica beads and its further physicochemical and biological characteriza- cm). The flies were provided with proteinex (Pfizer India) and 20% sugar tion. The lectin reported in present study have novel sugar specificity i.e. it solution as food and with pumpkin fruit Cucurbitae moschata Dusch, for ovi- differs from the sugar specificity of Aloe vera L. (=barbadensis Miller) [10] and position. Cultures of B. cucurbitae were maintained at 25°C±2°C, photoperiod Var. Chinensis (Haw.) Berger[11] reported earlier from Turkey and China re- (L10:D14) and 70-80% relative humidity. spectively. One study showed the tumour preventive effect of A. vera leaf pulp (=barbadensis Miller) lectin on Ehrlich ascites tumours in mice[12]. Carbohy- drates expressed on cell surface undergo certain changes during transforma- Chemicals: tion, for example aberent expression of mucin, is one of the most important All sugars/derivatives, glycoproteins, bovine serum albumin, sulphorhodamine process leading to malignant transformation of cells[13]. The lectin was evalu- B, MTT, 3H- thymidine, ConA, blue dextran, RPMI-1640 HEPES modifica- ated for in-vitro anti-cancer activity towards 13 human cancer cell lines and tion, histopaque-1077, 5-fluorouracil, mitomycin C and neuraminidase were mitogenic activity towards human peripheral blood mononuclear cells (PBMC)/ obtained from Sigma Chemical Co., St. Louis, USA. Standard molecular weight murine splenocytes. Further, AVLL was also tested for anti-insect activity markers, gel filtration markers and ampholines were from Amersham against Bactrocera cucurbitae (Coquillett), also known as melon fruit fly. This Pharmacia, New Jersey, USA. Amino activated silica beads used were from Clifmar, UK. Biogel P-200 was purchased from Bio-Rad, U.S.A. Fetal calf *Corresponding author. serum was procured from Sera Lab (GB). All other chemicals were of analytical Dr. Jatinder Singh grade. Professor, Crude Extract Preparation Department of Molecular Biology and Biochemistry, 100 g of leaf pulp was homogenized in 10 mM phosphate buffered saline (PBS), pH 7.2 (1:5) containing 0.02% sodium azide. The homogenate was Guru Nanak Dev University, kept overnight at 4ºC and then centrifuged at 20,000 ´ g for 20 min. The Amritsar, Punjab, pellet was discarded and the clear supernatant constituted the crude extract. Unless otherwise mentioned, 10 mM PBS, pH 7.2 was used in rest of the 143 005, India experiments. Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2441-2446 Manpreet Kaur et al. / Journal of Pharmacy Research 2011,4(7),2441-2446 Hemagglutination and Hapten-Inhibition assays (pH 9.0-12.5). Hemagglutination assay was performed for each sample using Lectin-mediated agglutination of red blood cells from rabbit was determined as 2% suspension of rabbit erythrocytes using lectin sample in PBS at pH 7.0 as described previously[15]. The hemagglutination titre (HU/ml) was recorded as positive control. For checking thermal stability, the lectin in 10 mM PBS was the reciprocal of the highest dilution giving visible agglutination. Specific heated in a water bath, for 15 min at a defined temperature in the range 35- activity was expressed as hemagglutination units/mg of soluble protein (HU/ 100ºC and cooled to room temperature. The inactivation kinetics of the mg). The carbohydrate-binding specificity of the purified lectin was accessed protein was performed using serially diluting 30 µl lectin with equal amount of by the ability of a series of sugars i.e. D-arabinose, L-arabinose, D-ribose, D- PBS and performing hemagglutination assay as explained above. Lectin sample xylose, D-fructose, D-galactose, D-glucose, D-mannose, L-sorbose, L-fucose, without heating served to estimate as control. L-rhamnose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, N-acetyl- D-mannosamine, methyl-a- and methyl-b-D-glucopyranosides, methyl-a-D- Circular Dichroism Spectroscopy mannopyranoside, sialic acid, methyl-a- and methyl-b-D-galactopyranosides, The circular dichroism measurements were performed using JASCO J-720 b-phenyl-D-glucopyranoside, adonitol, myo-inositol, ß-gentiobiose, D-lac- spectropolarimeter, calibrated with ammonium d-10 camphorsulphonate. The tose, D-maltose, D-melibiose, D-trehalose, T-disaccharide, N-acetyl-D- spectra were studied over a range of wavelength of 200-250 nm, under con- lactosamine, D-melizitose, D-raffinose, N,N’,N”–triacetylchitotriose, chitin, [25] stant N2 purging . The lectin was used at a concentration of 0.15 mg/mlin glycogen, inulin and glycoproteins (fetuin, asialofetuin, porcine stomach mu- deionized water, in quartz cuvettes of 0.1 mm path length. CD spectra were cin and thyroglobulin) to inhibit its haemagglutination activity against rabbit analyzed using K2D programme. erythrocytes. The hapten inhibition tests were carried out by 2-fold serial dilutions of sugar (100 mM) or glycoprotein (4 mg/ ml) solutions in PBS[15]. An Effect of denaturants and metal ions on lectin activity equal volume of lectin solution (30 ml) was added to each well and the mixture The effect of three denaturing agents i.e. urea, thiourea, and guanidine-hydro- was allowed to interact for 1h at room temperature. 60 ml of 2% suspension of chloride, at a concentration range of 0.5-8.0 M, was tested on lectin activity rabbit erythrocytes was added to each well and this mixture was incubated at by incubating 30 ml of each denaturant solution with equal volume of lectin at 37°C. The lowest concentration of a specific sugar or glycoprotein that inhib- 37ºC for 1 h. Then the hemagglutination activity was checked for untreated ited hemagglutination (Minimum Inhibitory Concentration, MIC) was re- and treated samples. To determine the metal ion requirement for lectin activ- corded and used to define inhibitory potency. ity, lectin was demetallized by the method of Paulova et al, 1971[26] followed by remetallization with Ca2+ and Mn2+ ions. The effect of these factors was Lectin Purification determined by comparing the hemagglutination activity after each step. The lectin was purified from the crude extract by single step affinity chromatography. The dialyzed crude extract was applied to a mucin-linked amino Atomic absorption spectrophotometry activated silica beads column (0.8´6.0 cm) equilibrated with PBS. The column The affinity-purified lectin was examined for the presence of various divalent was prepared as explained elsewhere[16]. After washing of unbound proteins and metal ions i.e. Ca2+, Co2+, Cr2+, Cu2+, Fe2+, Mn2+, Ni2+ and Zn2+ by Atomic absorp- pigments with PBS, adsorbed lectin was eluted from the column with 100 mM tion spectrophotometer (Shimadzu model AA6200) using an air-acetylene glycine-HCl buffer, pH 2.5. The eluted fractions were immediately neutralized flame. The lectin samples were digested in the presence of mixture of concen- with 2 M Tris-HCl-buffer, pH 8.3. Fractions showing agglutination activity trated HNO3 and HClO4 (1:1) for 30 min. Prior to analysis, lectin samples were were pooled and dialyzed against equilibration buffer in order to remove Tris extensively dialyzed against deionized water to remove any ions contributed ions, which may interfere in protein estimations. by the buffer. Standards were made by diluting commercial stock solutions of the respective metals. PHYSICOCHEMICAL CHARACTERIZATION BIOLOGICAL CHARACTERIZATION Determination of protein concentration and neutral sugar content The protein content of crude as well as affinity purified fractions was deter- Assay of anti-cancer activity on tumor cell lines mined by the Lowry assay, 1951[17] using bovine serum albumin (BSA) as a The inhibitory potential of A. vera leaf lectin against 13 human cancer cell standard. The amount of neutral sugars in the purified lectin was estimated by lines was checked using SRB assay described by Monks et al, 1991[27]. 100 ml of the anthrone method[18] using D-glucose as standard. cell suspension (105 cells/ml) was added to each well of 96 well plates and plates were incubated for 24 h in carbon dioxide incubator maintained at the Determination of molecular mass above-mentioned conditions. Subsequently 100 ml of lectin solution was added The purified lectin was subjected to sodium dodecyl sulfate–polyacrylamide in concentrations 10, 30 and 100 µg/ml and the culture was incubated for an gel electrophoresis (SDS– PAGE), pH 8.3 in accordance with the procedure of additional 48 h. The adherent cell cultures were fixed in situ by adding 50 ml of Laemmli (1970) [19] using 11% (w/v) separating and 5% stacking gel. The 50% (w/v) trichloroacetic acid (final concentration, 10% TCA) and incubated subunit molecular mass of the lectin was determined by running a mixture of for 1 h at 4°C. The supernatant was discarded and plates were washed five six standard proteins i.e. phosphorylase b (97 kDa); albumin (66 kDa); oval- times with deionized water and dried. 100 ml of sulforhodamine B (SRB, 0.4 w/ bumin (45 kDa); carbonic anhydrase (30 kDa); trypsin inhibitor (20.1 kDa) v in 1% acetic acid) was added to each well and the culture was incubated for 10 and a-lactalbumin (14.4 kDa), simultaneously along with the lectin. min at room temperature. The unbound SRB was removed by washing five times with 1% acetic acid and the plates were air dried. The dye bound to basic Gel permeation chromatography on Biogel P-200 column (1.6´67 cm), which amino acids of the cell membrane was solubilized with Tris buffer (10 mM, pH had been calibrated with molecular mass markers in the range of 12.4 kDa to 10.5) and the absorption was measured at 540 nm by ELISA reader (Multiscan 66 kDa, was conducted to determine the native molecular mass of the lectin[20]. EX, Labsystem) to determine the relative cell growth or viability in the treated as well as untreated cells. The standard anticancer drugs i.e. 5-flurouracil The void volume (Vo) was determined using blue dextran at a concentration of 2 mg/ml dissolved in PBS. The column was equilibrated and eluted with PBS. and mitomycin C at a concentration of 1´10-4 M were used as positive controls. Occurrence of isoforms and determination of isoelectric point PAGE at pH 4.5 was carried out using 7.5% tube gels[21] while PAGE at pH 8.3 Assay of lectin for anti-insect activity. was carried out using 10% tube gels[22]. Affinity purified leaf lectin was sub- Artificial diet bioassay jected to isoelectric focusing in 5% polyacrylamide tube gel using carrier The effect of this lectin was studied on the development of second instar of ampholines having pH range 3.5 to 9.5 according to[23]. A set of pI markers was melon fruit fly, Bactrocera cucurbitae by artificial diet bioassay. The artificial also loaded onto the separate tube gel. Before staining, ampholines were eluted diet was prepared according to the method described by Srivastava, 1975[28]. from the gels by incubation in 10% trichloroacetic acid (TCA) for 10 minutes Lectin was incorporated in diet at the concentrations of 10, 20, 40, 80, 160 followed by 1% TCA for 30 minutes at room temperature[24]. After electro- and 320 mg/ml. Seven replicas were used for each concentration as well as for phoresis, the gels were stained with 0.25% Coomassie Brilliant blue (R 250) control. Ten second instar larvae were released into each vial. The vials were prepared in destaining solution (1:3:6, acetic acid: methanol: water, v/v). maintained in culture room/B.O.D. incubator and daily observations were Isoelectric point was calculated by comparing the mobility of lectin with that made for the collection of data regarding pupation and adult emergence. The of pI markers. data were calculated for larval period, pupal period, total developmental period, percentage pupation, percentage emergence and lethal dose (LC50). Effect of pH and thermal treatment on lectin activity To determine the effect of pH on lectin-induced hemagglutination, 1 ml of Influence on enzyme activity affinity-purified lectin sample was extensively dialyzed against the buffers of The effect of lectin was also studied on activity of enzymes involved in various pH for 24h. Various buffers employed were: 0.1 M glycine-HCl (pH metamorphosis, hydrolysis and detoxification i.e. esterases and phosphatases 1.5-3.5), 0.1 M sodium acetate-acetic acid (pH 4.0-5.0), 0.1 M maleic acid- (acid and alkaline) in melon fruit fly. The larvae (64-72 h old) were harvested NaOH (pH 5.5-6.0), 0.1 M Tris-HCl (pH 6.5-8.5) and 0.1 M Glycine-NaOH from the charged pumpkin pieces and transferred to artificial diet containing

Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2441-2446 Manpreet Kaur et al. / Journal of Pharmacy Research 2011,4(7),2441-2446

LC50 concentration of lectin. The larvae were harvested from these vials after beads (Fig. 1). The PBS fractions were devoid of any lectin activity indicating three time intervals i.e. 24, 48 and 72 h at the age of 96, 120 and 144 h, the complete adsorption of lectin to the matrix. The data are summarized in respectively, and enzyme activity was assayed on fresh weight basis (mM/g Table 1. The low purification factor can be attributed to the fact that lectin fresh weight). Similar experiment was conducted on larvae reared on untreated fraction represents one of the major proteins in the storage parts of various medium (control). There were six replications for each experiment. The plant species as reported earlier[35] and may play a role in plant defence[7,8] enzyme activity of larvae reared on lectin-incorporated diet was compared with respective controls. Methodology given by Katzenellenboger and Kafatos, Table 1. Affinity purification of lectins from Aloe vera leaf 1971[29] was followed for the estimation of esterases while the method of Mc [30] Step Total Total Specific activity Purification Recovery MEAPC Intyre, 1971 was used for the measurement of phosphatases activities. protein (mg) Activity (HU)a,b (HU/ mg) fold (%) (µg/ml) The results obtained in artificial diet bioassay as well as enzyme activity, were Crude 780.0 19,200 24.6 1.0 100 40.6 expressed as the mean±SE. One-way analysis of variance (ANOVA) was ap- Affinity Purification *410.0 - - - - - plied where the multiple variants were to be accessed for their significance in PBS fractions the experiment for development period, percentage pupation and percentage Glycine-HCl fractions ¨268.0 10,880 40.2 1.6 57 24.8 emergence. The Student’s ‘t’ test was applied in order to compare two vari- Data are for 100 g of plant material; Volume of crude extract: 500 ml a b ables at a particular treatment interval in case of enzyme studies. LC50 values Total hemagglutination units; 2% rabbit erythrocytes were used for hemagglutination were calculated using probit analysis. All these tests were carried out with the *Unbound protein; ¨Eluted lectin help of SPSS for windows[31]. MEAPC: Minimal erythrocyte agglutinating protein concentration The lectin gave a single band in SDS-PAGE at pH 8.3 and native PAGE at pH Mitogenic potential 4.5 revealing the purity of the lectin preparation (Fig. 2A and 2B). As revealed Mitogenic potential of affinity-purified AVLL was checked towards inbred by gel permeation chromatography, AVLL has native molecular mass of 53 female BALB/c mice splenocytes and human peripheral blood mononuclear kDa (data not shown). In SDS-PAGE, the lectin appeared as a single band, both cells (PBMC). The test lectin and standard ConA were filtered though 0.22 µ under reducing as well as non-reducing conditions, with a molecular mass of membrane filters (13 mm diameter Schleicher and Schull, Germany) under 13.4 kDa, thus showing the absence of disulphide linkages in holding the aseptic conditions. subunits. This finding is consistent with the various earlier reported monocot lectins, which showed absence of cysteine residues and hence lack disulphide 3 [ H] thymidine uptake assay linkages[35]. Further, the results of gel permeation chromatography and SDS- To check the mitogenic potential of lectin on the proliferation of BALB/c PAGE showed that the lectin molecule was a homotetramer. Apparently, in 3 mice splenocytes, a radioactive assay, known as Methyl- H thymidine uptake terms of their subunit and molecular structure, this lectin differs from the assay, was carried out by the method of Kilpatrick, 1999[32]. BALB/c splenocytes lectins reported from genus Aloe[11] while it is similar to some other monocot at a concentration of 1.5´105/well in RPMI-1640 medium supplemented with lectins, from family Araceae[36,37] . 10% FCS were cultured with the various concentrations of AVLL ranging from 0.62 to 10.0 mg/ml. Con A, at the concentration of 2 mg/ml, was employed as a positive control. In the control wells, the cells were cultured only with medium without lectin. The cultures were set in an atmosphere of 5% CO2 for 72 h. Sixteen hours before the termination of cultures, 0.5 mCi of [3H] thymidine was added to each well. Emissions were monitored by liquid scintillation counter, Rackbeta (LKB). The CPM (counts per minute) is expressed as mean±SD.

MTT assay The assay of mitogenic activity was performed by MTT (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, as described by Mosmann, 1983[33]. Human peripheral blood mononuclear cells were isolated on Ficoll-Histopaque by the method of Bøyum, 1968 [34]. The cells were diluted with RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 mg/L streptomycin, and 100 IU/ml penicillin and then seeded (2 ´ 106cells/ ml/well)) in 96-well microplates (Nunc, Denmark). The lectin was then added Fig. 2A and 2B. please send the legend Fig. 3. please send the legend at various concentrations ranging from 2.5-40 mg/ml. Cells cultured in the absence of the lectin served as control and the same concentrations of Con A In native PAGE under alkaline conditions act as standard. The cells were incubated at 37°C in humidified atmosphere of three bands were obtained (Fig. 3B), while, 5% carbon dioxide for 24 h. in isoelectric focusing the lectin showed a series of bands ranging between isoelectric RESULTS AND DISCUSSION points 4.38-6.38 (Fig. 4). It seems that the The present investigation deals with the purification, physicochemical char- lectin carries a net negative charge at pH acterization and evaluation of anti-cancer, anti-insect and, mitogenic poten- 8.3 and the multiple bands may be due to tial of lectin from leaf pulp of A. vera. The lectin was purified by single step the difference in charge to mass ratio of affinity chromatography using porcine mucin-linked amino activated silica isolectins present in purified lectin as reported earlier for some monocot lectins[36- 38]. Fig. 4. AVLL was not inhibited by any of the simple sugars as well as complex glycoproteins like fetuin, asialofetuin and thyroglobulin, except porcine stomach mucin. However, individual components of mucin i.e. galactose, GalNAc, fucose, GlcNAc, N-acetyl–D-lactosamine, T- antigen were found to be non-inhibitory. Thus, it indicates that AVLL has complex binding specificity having above components as part of the complex glycoconjugate requirement for this lectin. In this respect, this lectin seems to be different from already reported varieties of A. vera[11], which have been reported to have specificity for mannose, N-acetyl-D—galactosamine. Fur- thermore, this finding is important in the view that mucins have been reported to show an aberrant expression profile in various malignancies[13,39]. In the light of these observations, this mucin-binding lectin, is studied for its in vitro anti-proliferative potential towards various human cancer cell lines.

AVLL was found to be glycoprotein having 4.2% carbohydrate. The lectin was found to be stable over a wide pH range i.e. 1.5-12.5 and thus may hold promise as drug delivery agents into the extreme physiological conditions Fig.1. please send the legend Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2441-2446 Manpreet Kaur et al. / Journal of Pharmacy Research 2011,4(7),2441-2446 such as gastrointestinal and urinary tracts[40]. In this aspect, this lectin re- semble most of the proteins like chitinases, glucanases, protease inhibitors and some anti-fungal proteins which are involved in the defense mechanisms of plants due to their stability over a broad pH range[41]. Further, the results of thermal denaturation suggested that this lectin remained stable upto 70ºC, thereafter the hemagglutination activity started declining and lost completely at 90ºC (Fig. 5). One probable reason for thermal stability of lectin is the presence of ß-pleated sheet structure as revealed by circular dichorism (Fig. 6). Lectin from A. vera leaf lost its activity completely above 7.0 M of all of these denaturants. These denaturants are known to disturb the three-dimen- sional conformation and binding sites of lectins by affecting the hydrophobic interactions that play crucial roles in carbohydrate binding activity[30]. Role of divalent cations in hemagglutination activity was determined by comparing the titre of lectin samples after demetallization and remetallization. No difference in titre value of these lectins showed that lectin activity was indepen- dent of the presence/absence of metal ions or metal ions have no role in hemagglutination activity of this monocot lectin. Atomic absorption spectroscopy was performed to examine the presence/absence of various divalent metal ions. This indicated the presence of Cr+2, Co+2, Zn+2, Ca+2 at a concentration ranging between 0.006-0.05 mole per mole of lectin subunits thus representing a very small fraction of divalent metal ions in lectin protein, which might suggest adventitious metal ion binding. The other ions tested i.e. Cu+2, Fe+2, Mn+2 and Ni+2 were found to be absent. Fig.7. cer cell lines while but failed to restrict the proliferation of others. Variations in glycosylation pattern of cell surface in various tumor cell lines can be one of the reasons for differential anti-cancer activity of AVLL. Studies have demonstrated that dramatic changes occur in glycosylation during malignant transformation including mucin expression. As every lectin has unique fine sugar specificity, there is a need to investigate a range of lectins against a number of cancer cell lines to generate a battery of anti-cancer agents.

Furthermore, AVLL was found to effect the development of B. cucurbitae. Second instar larvae reared on diet containing A. vera leaf lectin at the concentration of 320 µg/ml prolonged the larval period by 3.73 days while pupal period increased by 3.01 days. Thus the total development period got prolonged by 6.74 days (Table 2). At the highest concentration investigated i.e. 320µg/ml, there was 41% pupation compared to control while percentage Table 2. Influence of Aloe vera leaf lectin on the developmental parameters of B. cucurbitae larvae Lectin Larval period Pupal period Total development concentration in days in days period in days (mg ml-1) (Mean+SE) (Mean+SE) (Mean+SE)

0 8.36±0.25 7.47±0.36 15.83±0.14 Fig.5. 10 8.43±0.19 8.18±0.19 16.60±0.20 20 8.64±0.12 9.06±0.12 17.70±0.18 40 9.60±0.36 9.24±0.40 18.83±0.39 1 80 11.14±0.27 9.40±0.28 20.57±0.24 160 11.28±0.28 9.86±0.39 21.21±0.26 320 12.09±0.47 10.48±0.21 22.57±0.30 f-value 29.59* 12.24* 90.30* 0 *significant at 1%; values given in parenthesis are percentages of control 250 240 230 220 210 200

-1

CD (mdeg) -2

-3

-4 Wavelength (nm)

Fig.6.

In vitro anti-cancer activity of AVLL was evaluated against thirteen human cancer cell lines representing different organs. In SRB assay, AVLL was found to inhibit the proliferation of three colon cancer cell lines i.e. HT-29, SW-620 and HCT-15 by 79%, 71% and 42% respectively and HOP-62 (85%), a lung cancer cell line (Fig. 7). However, it showed no inhibitory effect towards other cancer cell lines tested. At present, it is difficult to reason out why AVLL showed significant inhibitory effect on the proliferation of some of the can- Fig.8.

Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2441-2446 Manpreet Kaur et al. / Journal of Pharmacy Research 2011,4(7),2441-2446 emergence was 36% compared to control. The dose-related influence of this Thus, in conclusion, AVLL manifests potent anti-proliferative activity to- affinity-purified lectin preparation on percentage pupation and percentage ward human colon and lung cancer cell lines. The anti-proliferative property emergence of B. cucurbitae compared to control is represented in Fig. 8. LC50 of AVLL suggests the binding of lectin to certain receptor on the cell surface was calculated to be 158 mg/ml. Ultrastructural studies have shown insecticidal which are responsible for cell growth. Therefore the lectin may also be de- lectins to be bound to midgut epithelial cells in a number of insect pests[42]. tected as histochemical marker in these type of cancers. The area of lectin Alternatively, evidence for the delivery of ingested lectins to the haemolymph research has great potential for cancer detection and treatment, therefore in Homopteran and Lepidopteran[43] species has highlighted the possibility for more study is required in this field. Further, the present study is significant in systemic mode of lectin action. An additional anti-nutritional complication view of the fact that A. vera leaf lectin have shown anti-insect potential for the insects fed on lectin-containing artificial diet is the possibility that against melon fruit fly, belonging to order Diptera, in particular and insect lectins may destabilize insect metabolism by interfering with gut enzymatic pests in general. Lectin genes from these plants may be employed to develop function either indirectly or by binding to glycosylated digestive enzymes in transgenics as a component of integrated pest management strategy but it the gut[44]. may require further studies.

Table 3. Influence of Aloe vera leaf lectin on enzyme activity of B. cucurbitae larvae ACKNOWLEDGEMENTS at various time intervals The authors acknowledge the financial assistance of CSIR in carrying out this Lectin Enzyme activity (mM g-1) work. 0h t-value 24 h t-value 48 h t-value 72 h REFERENCES Esterases 1. Van Damme EJM, Goossens K, Smeets K, Van Leuven F, Verhaert P, Peumans WJ. The Control 293.08±0.22 17.83* 251.22±2.24 3.86* 265.02±2.18 12.53* 309.38±2.14 Lectin - 3.09** 332.94+2.24 10.57* 480.88+0.16 14.11* 628.06+3.31 major tuber storage protein of araceae species is a lectin. Characterization and molecular (6.24*) (35.33*) (34.76*) cloning of the lectin from Arum maculatum L. Plant Physiol 1995; 107: 1147-1158. Acid Phosphatase 2. Oliveira MD, Andrade CA, Santos-Magalhães NS, Coelho LC, Teixeira JA, Carneiro- Control 9.48±0.22 27.21* 13.26±0.018 9.40* 15.41±0.14 15.01* 19.03±0.07 Lectin - 49.47* 13.58±0.12 7.24* 12.20±0.17 29.15* 10.76±0.15 da-Cunha MG, Correia MT. 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The lectin employed in the present study turned out to be potent mitogen Phytother Res 1999; 13: 489-493. towards BALB/c splenocytes in thymidine uptake assay. AVLL gave optimum 11. Suzuki, I., Saito, H., Inoue, S., Migita, S. and Takahashi, T. Purification and Charac- response at the concentration of 2.5µg/ml. The relative mitogenic index was terization of Two Lectins from Aloe arborescens Mill. J. Biochem. 1978; 8: 163-171 almost double i.e. 232.9, as compared to ConA, a well-known standard mito- 12. Akev N, Turkay G, Can A, Gurel A, Yildiz F, Yardibi H, Ekiz EE, Uzun H. Tumour gen. However, the lectin was virtually non-mitogenic towards human preventive effect of Aloe vera leaf pulp lectin (Aloctin I) on Ehrlich ascites tumours lymphocyes. This finding further poses an advantage to use this lectin in the in mice. Phytother Res. 2007;21:1070-5. development of transgenics plants for human use. 13. Simms MS, Hnges ODM, Limb M, Price MP, Bishop MC. 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Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2441-2446