Immunoadjuvant and Humoral Immune Responses of Garlic (Allium sativum L.) Lectins upon Systemic and Mucosal Administration in BALB/c Mice.

Dietary food components have the ability to affect immune function; following absorption, specifically orally ingested dietary food containing lectins can systemically modulate the immune cells and affect the response to self- and co-administered food antigens. The mannose-binding lectins from garlic (Allium sativum agglutinins; ASAs) were identified as immunodulatory proteins in vitro. The objective of the present study was to assess the immunogenicity and adjuvanticity of garlic agglutinins and to evaluate whether they have adjuvant properties in vivo for a weak antigen ovalbumin (OVA). Garlic lectins (ASA I and ASA II) were administered by intranasal (50 days duration) and intradermal (14 days duration) routes, and the anti-lectin and anti-OVA immune (IgG) responses in the control and test groups of the BALB/c mice were assessed for humoral immunogenicity. Lectins, co-administered with OVA, were examined for lectin-induced anti-OVA IgG response to assess their adjuvant properties. The splenic and thymic indices were evaluated as a measure of immunomodulatory functions. Intradermal administration of ASA I and ASA II had showed a four-fold and two-fold increase in anti-lectin IgG response, respectively, vs. the control on day 14. In the intranasal route, the increases were 3-fold and 2.4-fold for ASA I and ASA II, respectively, on day 50. No decrease in the body weights of animals was noticed; the increases in the spleen and thymus weights, as well as their indices, were significant in the lectin groups. In the adjuvanticity study by intranasal administration, ASA I co-administered with ovalbumin (OVA) induced a remarkable increase in anti-OVA IgG response (~six-fold; p < 0.001) compared to the control, and ASA II induced a four-fold increase vs. the control on day 50. The results indicated that ASA was a potent immunogen which induced mucosal immunogenicity to the antigens that were administered intranasally in BALB/c mice. The observations made of the in vivo study indicate that ASA I has the potential use as an oral and mucosal adjuvant to deliver candidate weak antigens. Further clinical studies in humans are required to confirm its applicability.

Humoral immune and adjuvant responses of mucosally-administered Tinospora cordifolia immunomodulatory protein in BALB/c mice.

BACKGROUND: In traditional medicine, guduchi (Tinospora cordifolia) is considered as an adaptogen with immunomodulatory prowess. A 25 kDa protein from guduchi stem has been characterized as an immunomodulatory protein (ImP). OBJECTIVES: The aim of this study was to evaluate the intrinsic immunogenicity of guduchi ImP and adjuvant activity using ovalbumin (OVA) as antigen in BALB/c mice. MATERIALS AND METHODS: Mice were given guduchi ImP (30 and 60 µg) by intranasal administration to respective groups (n = 6) on days 1, 14 and thereafter weekly till day 42. Immunogenic response was monitored by serum IgG/IgA levels (days 14, 35 and 50). The adjuvant activity was measured by serum anti-OVA IgG/IgA responses to administration of 30 µg OVA with guduchi ImP. The effect of guduchi ImP on the spleen status was examined by splenic weight (day 50). RESULTS: Guduchi ImP administration displayed a significant increase in anti-guduchi ImP IgG (5-7 fold) and anti-guduchi ImP IgA (3-4 fold) on day 50 vs. control. Guduchi ImP showed a significant increase in anti-OVA IgG (6-7 fold) and anti-OVA IgA (4-5 fold) on day 50 vs. control. The splenic index of guduchi ImP group increased significantly in both the immune and adjuvant response groups; however, the splenic index in the adjuvant response group was markedly higher. CONCLUSION: The results indicate that guduchi ImP is a strong immunogen by itself and enhances the immunogenicity of mucosally-administered antigen in BALB/c mice. Based on the results of this animal study, it appears that guduchi ImP shows a potential for future studies in humans.

Immunoassays for riboflavin and flavin mononucleotide using antibodies specific to d-ribitol and d-ribitol-5-phosphate.

Riboflavin (vitamin B2), a water-soluble vitamin, plays a key role in maintaining human health. Though, numerous methods have been reported for the determination of total riboflavin (TRF) content in foods and biological samples, very few methods are reported for quantifying riboflavin and its coenzymes [flavin mononucleotide (FMN); flavin adenine dinucleotide (FAD)] individually. Recently, we have demonstrated that antibodies specific to d-ribitol and d-ribitol-5-phosphate also recognize riboflavin and FMN, respectively, and not vice-versa. In this study, we have evaluated these two antibodies for the analysis of riboflavin and FMN by indirect competitive ELISA (icELISA) in selected foods and pharmaceuticals. Under the optimal assay conditions, 50% inhibition concentration (IC50) and limit of detection (LOD, IC10) were 3.41ng/mL and 0.02ng/mL for riboflavin, and 7.84ng/mL and 0.24ng/mL for FMN, respectively, with detectable concentration range between 0.1 and 100ng of analytes and <0.1% cross-reactivity with other water-soluble vitamins. The amounts of TRF in food samples, as analyzed by icELISA using ribitol antibody, were 90-95% of the reported values in the literature or label values. Quantification of individual flavins (riboflavin and FMN) from the same food samples showed variation in their values compared to TRF, and were in good agreement with values obtained from HPLC and AOAC methods. Further, spiking and recovery analysis of food samples and pharmaceuticals showed no significant matrix effects. The immunoassays were validated in terms of accuracy and precision using inter- and intra-assays. The immunoassays developed in this study are sensitive and appears feasible for screening a large number of samples in the quantification of riboflavin and FMN in various biological samples, pharmaceuticals and natural/processed foods.

Emerging food allergens: Identification of polyphenol oxidase as an important allergen in eggplant (Solanum melongena L.).

Although many allergens have been detected in eggplant (Solanum melongena L.), their identity have not been elucidated. The aim of this study was to investigate whether polyphenol oxidase (PPO), an important eggplant enzyme, acts as an allergen. The proteins of eggplant peel extract were separated on phenyl-Sepharose (PS), and analyzed by skin prick test (SPT), ELISA and IgE-immunoblotting; the components were analyzed for PPO activity, presence of protein-bound copper, and recognition by rabbit polyclonal anti-sweet potato PPO antiserum. LC-MS/MS and in silico analysis were employed to identify the separated allergens and prediction of IgE epitopes. Eggplant allergens were separated into 5 components (PS1-PS5), of which component PS2 exhibited high specific PPO activity. SPT and ELISA with PPO-rich pool (PS2) were positive in all 6 eggplant-allergic subjects; the 43, 64 and 71kDa proteins displayed strong IgE-binding ability. The 64 and 71kDa IgE-binding proteins show PPO activity, presence of copper, and recognition by anti-sweet potato PPO antiserum, clearly identifying them as PPOs; the 43kDa protein appears to be a degradation product of the 64 or 71kDa proteins based on enzymic activity and recognition by PPO antiserum. The 64kDa protein upon further resolution by SDS-PAGE displayed two components (identified as eggplant PPO1 and PPO4 by LC-MS/MS). Based on bioinformatics approaches, PPO4 has been identified as an allergen since it harbors an IgE epitope. This study clearly demonstrates that the 64 and 71kDa allergens in eggplant peel are PPOs based on enzymic activity and recognition by PPO antiserum; the 64kDa copper-containing protein is identified as one of the several eggplant allergens (Sola m PPO4). This is the first instance of polyphenol oxidase being identified as a new food allergen.

Alleviation of cyclophosphamide-induced immunosuppression in Wistar rats by onion lectin (Allium cepa agglutinin).

ETHNOPHARMACOLOGICAL RELEVANCE: In various traditional medicines, onion has been classified as an immune-boosting food. Recent studies have claimed this property due to the presence of bioactive organosulfur compounds, prebiotic fructo-oligosaccharides and an immunomodulatory protein, lectin (Allium cepa agglutinin; ACA) (Prasanna and Venkatesh, 2015. Characterization of onion lectin (Allium cepa agglutinin) as an immunomodulatory protein inducing Th1-type immune response in vitro. Int. Immunopharmacol. vol. 26, pp. 304-313). AIM OF THE STUDY: The aim of this study was to evaluate the immunoprotective properties of ACA in normal and cyclophosphamide (CP; 100µg/kg)-induced immunosuppressed Wistar rats. MATERIALS AND METHODS: Wistar rats were administrated different doses of ACA (1, 10, and 100µg) to respective groups in normal as well as immunosuppressed animals. The effect of ACA on the status of immune organs was assessed by examining the splenic and thymic indices, and histopathological changes. The biomarkers for humoral immunity (serum IgG and IgA levels) and serum pro-inflammatory markers (COX-2, TNF-α and IL-10) were measured by ELISA. RESULTS: ACA showed immunoprotective properties by significantly promoting the restoration of lymphoid cell count by ~6 fold vs. model control (immunosuppressed animals) and promotes the immune response significantly (~1.5-fold) in CP-induced immunosuppressed animals compared to model control; production of pro-inflammatory molecules (COX-2 and nitric oxide) and expression levels of immune regulatory molecule (TNF-α) were elevated in a dose-dependent manner. CONCLUSIONS: The observed in vivo results suggest that ACA has the potential to be used as a nutritional therapeutic to boost the immune status of immunosuppressed subjects brought about by CP administration.

Occurrence, Functions and Biological Significance of Arginine-Rich Proteins.

Arginine, the most basic among the 20 amino acids, occurs less frequently than lysine in proteins despite being coded by six codons. Only a few important proteins of biological significance have been found to be abundant in arginine. It has been established that these arginine-rich proteins have been assigned important roles in the biological systems. Arginine-rich cationic proteins are known to stabilize macromolecular structures by establishing appropriate interactions (salt bridges, hydrogen bonds and cation-π interactions). These proteins are also known to be the key members of many regulatory pathways such as gene expression, chromatin stability, expurgation of introns from naïve mRNA, mRNA splicing, membrane-penetrating activity and pathogenesis-related defense, to name a few. Further, arginine occurs in various combinations with other amino acids (serine, lysine, proline, tryptophan, valine, glycine and glutamic acid) which diversify the potential functions of arginine-rich proteins. Arginine-rich proteins known till date from dietary sources have been described in terms of their structure and functional properties. A variety of activities such as bactericidal, membrane-penetrating, antimicrobial, anti-hypertensive, pro-angiogenic and others have been reported for arginine-rich proteins. This review attempts to collate the occurrence, functions and the biological significance of this unique class of proteins rich in arginine.

Characterization of onion lectin (Allium cepa agglutinin) as an immunomodulatory protein inducing Th1-type immune response in vitro.

Onion (Allium cepa), a bulb crop of economic importance, is known to have many health benefits. The major objective of the present study is to address the immunomodulatory properties of onion lectin (A. cepa agglutinin; ACA). ACA was purified from onion extract by D-mannose-agarose chromatography (yield: ~1 mg/kg). ACA is non-glycosylated and showed a molecular mass of ~12 kDa under reducing/non-reducing SDS-PAGE; glutaraldehyde cross-linking indicated that ACA is a non-covalent tetramer of ~12 kDa subunits. Its N-terminal sequence (RNVLLNNEGL; UniProt KB Accn. C0HJM8) showed 70-90% homology to mannose-specific Allium agglutinins. ACA showed specific hemagglutination activity of 8200 units/mg and is stable in the pH range 6-10 and up to 45° C. The immunomodulatory activity of ACA was assessed using the macrophage cell line, RAW264.7 and rat peritoneal macrophages; at 0.1 µg/well, it showed a significant increase (6-8-fold vs. control) in the production of nitric oxide at 24h, and significantly stimulated (2-4-fold vs. control) the production of pro-inflammatory cytokines (TNF-α and IL-12) at 24h. ACA (0.1 µg/well) enhanced the proliferation of murine thymocytes by ~4 fold (vs. control) at 24h; however, ACA does not proliferate B cell-enriched rat splenocytes. Further, it significantly elevated the expression levels of cytokines (IFN-γ and IL-2) over the control in murine thymocytes. Taken together, purified ACA induces a Th1-type immune response in vitro. Though present in low amounts, ACA may contribute to the immune-boosting potential of the popular spice onion since considerable amounts are consumed on a daily basis universally.

Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate.

D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI-TOF analyses, an increase in apparent molecular weight (SDS-PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P-BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P-BSA antiserum subjected to caprylic acid precipitation followed by hapten-affinity chromatography; its affinity constant is 7.1 × 10(8) M(-1). Rbt-5-P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to D-ribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P-specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P-protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P-specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide.

Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol.

D-Ribitol, a five-carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27-30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid-Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol-KLH-Sepharose CL-6B resulted in pure ribitol-specific antibodies (~45-50 µg/mL). The affinity constant of ribitol antibodies was found to be 2.9 × 10(7) M(-1) by non-competitive ELISA. Ribitol antibodies showed 100% specificity towards ribitol, ~800% cross-reactivity towards riboflavin, 10-15% cross-reactivity with sorbitol, xylitol and mannitol, and 5-7% cross-reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4%) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.

Identification and characterization of a basic thaumatin-like protein (TLP 2) as an allergen in sapodilla plum (Manilkara zapota).

SCOPE: Cases of oral allergy syndrome following the ingestion of sapodilla plum (Manilkara zapota) have been reported rarely. As the causative allergens are not known, the main objective of this study was to identify and characterize the important allergens in sapodilla. METHODS AND RESULTS: Allergy to sapodilla was diagnosed by case history, skin prick test, and serum allergen-specific IgE. The allergen was detected by IgE immunoblotting, purified on SP-Sepharose and characterized by native/SDS-PAGE, IEF, MS, and amino acid composition. Several cases of allergy to sapodilla fruit were identified; majority of the sapodilla-allergic subjects (6/7) experienced typical oral allergy syndrome symptoms, and allergen-specific IgE to the purified protein was positive. The allergen has a pI ≥9.5 and high contents of arginine, threonine, glycine, and cysteine. Circular dichroism revealed a secondary structure rich in beta sheets/turns. Based on its N-terminal sequence of A-T-F-D-I-Q-N-N-C-X-Y-, the allergen (21 578 Da) was identified as a thaumatin-like protein by homology. CONCLUSION: The causative allergen in sapodilla plum has been identified and characterized as a highly basic thaumatin-like protein belonging to the pathogenesis-related protein (PR-5) family, which has been recognized as a new family of conserved, cross-reactive plant allergens.

In silico analyses of structural and allergenicity features of sapodilla (Manilkara zapota) acidic thaumatin-like protein in comparison with allergenic plant TLPs.

Thaumatin-like proteins (TLPs) belong to the pathogenesis-related family (PR-5) of plant defense proteins. TLPs from only 32 plant genera have been identified as pollen or food allergens. IgE epitopes on allergens play a central role in food allergy by initiating cross-linking of specific IgE on basophils/mast cells. A comparative analysis of pollen- and food-allergenic TLPs is lacking. The main objective of this investigation was to study the structural and allergenicity features of sapodilla (Manilkara zapota) acidic TLP (TLP 1) by in silico methods. The allergenicity prediction of composite sequence of sapodilla TLP 1 (NCBI B3EWX8.1, G5DC91.1) was performed using FARRP, Allermatch and Evaller web tools. A homology model of the protein was generated using banana TLP template (1Z3Q) by HHPRED-MODELLER. B-cell linear epitope prediction was performed using BCpreds and BepiPred. Sapodilla TLP 1 matched significantly with allergenic TLPs from olive, kiwi, bell pepper and banana. IgE epitope prediction as performed using AlgPred indicated the presence of 2 epitopes (epitope 1: residues 36-48; epitope 2: residues 51-63), and a comprehensive analysis of all allergenic TLPs displayed up to 3 additional epitopes on other TLPs. It can be inferred from these analyses that plant allergenic TLPs generally carry 2-3 IgE epitopes. ClustalX alignments of allergenic TLPs indicate that IgE epitopes 1 and 2 are common in food allergenic TLPs, and IgE epitopes 2 and 3 are common in pollen allergenic TLPs; IgE epitope 2 overlaps with a portion of the thaumatin family signature. The secondary structural elements of TLPs vary markedly in regions 1 and 2 which harbor all the predicted IgE epitopes in all food and pollen TLPs in either of the region. Further, based on the number of IgE epitopes, food TLPs are grouped into rosid and non-rosid clades. The number and distribution of the predicted IgE epitopes among the allergenic TLPs may explain the specificity of food or pollen allergy as well as the varied degree of cross-reactivity among plant foods and/or pollens.

Characterization and gene cloning of an acidic thaumatin-like protein (TLP 1), an allergen from sapodilla fruit (Manilkara zapota).

BACKGROUND: Allergy to sapodilla (Manilkara zapota) fruit ingestion is rare. An independent study from our group has identified a basic thaumatin-like protein (TLP 2) as the major allergen. The present study was aimed at identifying and characterizing additional allergens from sapodilla. METHODS: Allergic subjects were identified by case history, skin prick test (SPT) and allergen-specific IgE. Sapodilla extract was fractionated using SP-Sepharose into 3 components (SP1, SP2 and SP3) which were analyzed by native/SDS-PAGE, IgE-immunoblot, isoelectric focusing (IEF) and N-terminal sequencing. The conserved regions of plant TLPs and the N-terminal sequence were used to design primers for PCR. RESULTS: SPT and ELISA confirmed a subject with oral allergy syndrome (OAS) to sapodilla and custard apple. Two proteins (26.9 and 24.5kDa; reducing conditions) were detected as allergens, of which the latter in SP2 has already been identified as basic TLP (TLP 2). The 26.9kDa protein present in SP1 was identified as an acidic TLP based on native PAGE, IEF and N-terminal sequencing. Presence of a basic ß-1,3-glucanase in SP3 was inferred by zymography. Sequence analysis of the genomic clone of the acidic TLP gene revealed that it is intronless and non-glycosylated. Evolutionary relatedness to olive, grape and kiwi fruit allergenic TLPs were inferred by phylogenetic analysis. CONCLUSIONS: An acidic TLP (TLP 1) was identified as a new allergen in sapodilla. TLP 1 is a single polypeptide (207 residues) belonging to the thaumatin family of the GH64-TLP-SF superfamily. Clinically, sapodilla should be considered in the list of fruits causing OAS.

Promoter analyses and transcriptional profiling of eggplant polyphenol oxidase 1 gene (SmePPO1) reveal differential response to exogenous methyl jasmonate and salicylic acid.

The transcriptional regulation of multigenic eggplant (Solanum melongena) polyphenol oxidase genes (SmePPO) is orchestrated by their corresponding promoters which mediate developmentally regulated expression in response to myriad biotic and abiotic factors. However, information on structural features of SmePPO promoters and modulation of their expression by plant defense signals are lacking. In the present study, SmePPOPROMOTERs were cloned by genome walking, and their transcription start sites (TSS) were determined by RLM-RACE. Extensive sequence analyses revealed the presence of evolutionarily conserved and over-represented putative cis-acting elements involved in light-regulated transcription, biosynthetic pathways (phenylpropanoid/flavonoid), hormone signaling (abscisic acid, gibberellic acid, jasmonate and salicylate), elicitor and stress responses (cold/dehydration responses), sugar metabolism and plant defense signaling (W-BOX/WRKY) that are common to SmePPOPROMOTER1 and 2. The TSS for SmePPO genes are located 9-15bp upstream of ATG with variable lengths of 5' untranslated regions. Transcriptional profiling of SmePPOs in eggplant seedlings has indicated differential response to methyl jasmonate (MeJA) or salicylic acid (SA) treatment. In planta, while MeJA elicited expression of all the six SmePPOs, SA was only able to induce the expression of SmePPO4-6. Interestingly, in dual treatment, SA considerably repressed the MeJA-induced expression of SmePPOs. Functional dissection of SmePPOPROMOTER1 by deletion analyses using Agrobacterium-mediated transient expression in tobacco leaves has shown that MeJA enhances the SmePPOPROMOTER1-ß-glucuronidase (GUS) expression in vivo, while SA does not. Histochemical and quantitative GUS assays have also indicated the negative effect of SA on MeJA-induced expression of SmePPOPROMOTER1. By combining in silico analyses, transcriptional profiling and expression of SmePPOPROMOTER1-GUS fusions, the role of SA on the modulation of MeJA-induced SmePPO1 expression has been elucidated. It is concluded that similar to the coding regions of multigenic SmePPOs, the regulatory elements are also evolutionarily conserved and fall into two distinct sub-classes based on their responses to MeJA and SA.

Fructans from aged garlic extract produce a delayed immunoadjuvant response to ovalbumin antigen in BALB/c mice.

Garlic (Allium sativum) is known for its innumerable biological activities including immunomodulation. Aged garlic extract (AGE), an odorless garlic preparation, has been shown to have superior immunomodulatory properties over raw garlic extract. Although garlic is a very rich source of fructans (17%, fresh weight basis), AGE contains only 0.22% of raw garlic fructans. Aged garlic fructans (AGF) have recently been shown to possess immunomodulatory activities in vitro. Natural adjuvants capable of eliciting better immune response of a model antigen are important in developing newer vaccines. In the present study, the adjuvant activity of AGF has been investigated in BALB/c mice using ovalbumin (OVA, 30 µg) as an experimental antigen. The body weights of animals did not change significantly indicating that the administration of garlic fructans is well-tolerated. AGF produce a significant humoral (serum IgG) response to OVA in BALB/c mice administered mucosally by either intranasal or oral route--a delayed response appearing on 50th day at a dose of 30 µg AGF by intranasal route. However, the serum IgG response was seen earlier on 35th day at a dose of 100 µg AGF by oral route. Higher concentrations of AGF (>50 µg) were inhibitory for adjuvant activity by intranasal administration. These observations indicate that AGF display immunoadjuvant activity for a test antigen though the humoral immune response is delayed.

Immunostimulatory properties of the major protein from the stem of the Ayurvedic medicinal herb, guduchi (Tinospora cordifolia).

ETHNOPHARMACOLOGICAL RELEVANCE: Guduchi (Tinospora cordifolia), a widely used plant in folk and Ayurvedic systems of medicine is well known for its immunomodulatory activity; however, the presence of an immunomodulatory protein (ImP) in guduchi has not been investigated. MATERIALS AND METHODS: Guduchi ImP was purified from dry stem powder extract by anion-exchange chromatography on Q-Sepharose. Characterization of guduchi ImP was performed by SDS-PAGE, periodic acid-Schiff staining, HPLC, and immunochemical analyses. Immunostimulatory activity was assessed by lymphocyte proliferation and macrophage activation assays. Fresh guduchi stem/leaf, guduchi satwa and guduchi capsules were also analyzed for the presence of guduchi ImP. RESULTS: Guduchi ImP was purified to homogeneity from dry stem powder extract (~150 mg protein per 100 g guduchi stem powder) as a single chain acidic protein (25 kDa) without glycans; it was noticeably absent in guduchi leaf. Guduchi satwa and guduchi capsule preparations also lacked this protein. Guduchi ImP showed ~3-fold mitogenic activity compared to untreated murine splenocytes in the 1-10 µg/mL concentration range; 5-7-fold increase in mitogenic activity was seen in the case of murine thymocytes vs. control. The purified protein also induced nitric oxide production from macrophages present in isolated murine peritoneal exudates cells. Guduchi ImP displays enhanced phagocytosis of yeast cells by macrophages. Guduchi ImP does not possess hemagglutination activity (towards rabbit and human erythrocytes of all blood groups) indicating that the immunomodulatory protein is not a lectin. CONCLUSIONS: The confirmation of an immunomodulatory protein in guduchi stem showing lymphoproliferative and macrophage-activating properties reinforces the rationale of the use of guduchi preparations in several Ayurvedic medicines for immunomodulation. To our knowledge, this is the first report of an immunomodulatory protein isolated from guduchi.

Eggplant polyphenol oxidase multigene family: cloning, phylogeny, expression analyses and immunolocalization in response to wounding.

Though polyphenol oxidase (PPO) genes from tomato and potato have been extensively studied, information about PPO genes in eggplant (Solanum melongena) is lacking. The main objective of this study is to understand the structural and functional aspects of eggplant PPO genes. Six eggplant PPO genes (SmePPO1-6) cloned by RACE and genome walking were found to be intronless and correspond to eight eggplant unigenes. Comprehensive sequence analyses indicated that the eggplant PPO genes exhibit considerable variation in the transit peptide regions, copper-binding domains and UTRs, and fall into two distinct structural classes. Further, PPO gene members appear to exist in clusters on eggplant chromosome 8 as seen in the case of tomato and potato PPOs. During normal growth and development, SmePPO1 and 2 are expressed in roots, whereas the transcript levels of all the eggplant PPO genes vary considerably in leaves, flowers and fruits. SmePPO1 was expressed in Escherichia coli as a GST fusion protein, and immunoblot using rabbit polyclonal antiserum to GST-SmePPO1 detected a major protein band (~70 kDa) and a minor band (~67 kDa) in eggplant fruit extract. Tissue printing indicated the predominant presence of PPO in the exocarp and the areas surrounding the seeds in the mesocarp of eggplant fruits. Immunolocalization of PPOs in eggplant infested with shoot-and-fruit borer revealed localization of the PPO at the site of infection in tender shoots and fruits, and further inside the mature tissues. The upregulation of eggplant PPO gene transcripts following mechanical injury shows that all the genes except SmePPO2 are induced in the fruit over 6h. On the contrary, the transcripts of SmePPO2 and PPO3 are not detectable in the stem, and expression seems to be prominent over a 2h period for SmePPO1 and SmePPO4-6. Our results show that eggplant PPO genes are structurally different, and are differentially expressed in various tissues of eggplant indicating their functional diversity.

Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract.

Traditionally, garlic (Allium sativum) is known to be a significant immune booster. Aged garlic extract (AGE) possesses superior immunomodulatory effects than raw garlic; these effects are attributed to the transformed organosulfur compounds. AGE is also known to contain fructans; the amount of fructans in AGE represents a small fraction (0.22%) of the total fructans in raw garlic. In order to evaluate the biological activity of fructans present in AGE, both high molecular weight (>3.5 kDa; HF) and low molecular weight (<3 kDa; LF) fructans were isolated. The structures of purified HF and LF from AGE determined by (1)H NMR and (13)C NMR spectroscopy revealed that both have (2→1) ß-D-fructofuranosyl bonds linked to a terminal glucose at the non-reducing end and ß-D-fructofuranosyl branching on its backbone. Biological activity of fructans was assessed by immunostimulatory activity using murine lymphocytes and peritoneal exudate cells (source of macrophages). Both HF and LF displayed mitogenic activity and activation of macrophages including phagocytosis. These activities were comparable to that of known polysaccharide immunomodulators such as zymosan and mannan. This study clearly demonstrates that garlic fructans also contribute to the immunomodulatory properties of AGE, and is the first such study on the biological effects of garlic fructans.

Dietary garlic (Allium sativum) lectins, ASA I and ASA II, are highly stable and immunogenic.

The immunomodulatory proteins present in garlic have recently been shown to be identical to the garlic lectins ASA I and ASA II [Clement F, Pramod SN, Venkatesh YP. Int. Immunopharmacol. 2010; 10: 316-324]. In this study, the stability of garlic lectins as a function of pH, temperature and denaturants has been examined in relation to biological activity (hemagglutination and hagocytosis). Stability of garlic lectins in simulated gastric fluid (SGF) was assessed by their hemagglutination activity, immunoreactivity, and intactness by SDS-PAGE. Garlic lectins were moderately stable in SGF for up to 30 min; while they retained hemagglutination activities, immunoreactivity with the respective rabbit antiserum decreased immediately (0.5 min) to 10-30%. ASA I retained ~80% hemagglutination activity in the pH range 2-12; however, ASA II retained only 40% in the pH ranges 2-4 and 10-12. Garlic lectins exposed to 60 °C (30 min) and pepsin (1 and 2 min) retained hemagglutination and phagocytic activities. Urea (4M) and Gdn.HCl (2M) did not affect hemagglutination. The immunogenicity of garlic lectins upon oral feeding in BALB/c mice was examined. A lectin-specific serum IgG response was seen in mice comparable to the oral immunogen, phytohemagglutinin. The recovered lectin in feces of mice administered with garlic lectins showed antigenicity identical to that of the administered proteins. The stabilities of the garlic lectins, their ability to withstand the gastrointestinal passage, and their recognition by the immune system upon oral feeding reinforce the reported presence of natural antibodies to garlic proteins in normal human sera.

Identity of the immunomodulatory proteins from garlic (Allium sativum) with the major garlic lectins or agglutinins.

Garlic (Allium sativum), an important medicinal spice, displays a plethora of biological effects including immunomodulation. Although some immunomodulatory proteins from garlic have been described, their identities are still unknown. The present study was envisaged to isolate immunomodulatory proteins from raw garlic, and examine their effects on certain cells of the immune system (lymphocytes, mast cells, and basophils) in relation to mitogenicity and hypersensitivity. Three protein components of approximately 13 kD (QR-1, QR-2, and QR-3 in the ratio 7:28:1) were separated by Q-Sepharose chromatography of 30 kD ultrafiltrate of raw garlic extract. All the 3 proteins exhibited mitogenic activity towards human peripheral blood lymphocytes, murine splenocytes and thymocytes. The mitogenicity of QR-2 was the highest among the three immunomodulatory proteins. QR-1 and QR-2 displayed hemagglutination and mannose-binding activities; QR-3 showed only mannose-binding activity. Immunoreactivity of rabbit anti-QR-1 and anti-QR-2 polyclonal antisera showed specificity for their respective antigens as well as mutual cross-reactivity; QR-3 was better recognized by anti-QR-2 (82%) than by anti-QR-1 (55%). QR-2 induced a 2-fold higher histamine release in vitro from leukocytes of atopic subjects compared to that of non-atopic subjects. In all functional studies, QR-2 was more potent compared to QR-1. Taken together, all these results indicate that the two major proteins QR-2 and QR-1 present in a ratio of 4:1 in raw garlic contribute to garlic's immunomodulatory activity, and their characteristics are markedly similar to the abundant Allium sativum agglutinins (ASA) I and II, respectively.