Metabolomics-based pharmaceutical evaluation of different parts of Swertia chirayita (Roxb.) Buch.-Ham. ex C.B. Clarke from the western Himalayas.

Swertia species are common ingredients in numerous herbal remedies. It is also used to treat a wide range of illnesses and possess diverse therapeutic activities. The aim of the study is to elucidate the comprehensive metabolomics profile of Swertia chirayita and the role of various extraction methods in the phytochemical compositions of the extracts of S. chirayita, and their antioxidant and enzyme inhibitory activities. Extraction of the stems, leaves, and flowering tops of S. chirayita was performed by maceration, infusion, and soxhlation using methanol and water as solvent. Extracts were subjected to phytochemical profiling by a liquid-chromatographic system. Antioxidant and enzyme inhibitory activity was carried out. The metabolomics profiling showed that a diverse range of specialized metabolites were present in the stems and leaves & flowering tops of the plant. All the extracts showed substantial antioxidant and enzyme inhibitory activities further confirmed by molecular docking studies. This study appraised the use of S. chirayita aerial parts as a potential antioxidant and its therapeutic application in various chronic illnesses including Alzheimer's disease, diabetes, and other skin-related disorders.

Himalayan Citrus jambhiri juice reduced renal crystallization in nephrolithiasis by possible inhibition of glycolate oxidase and matrix metalloproteinases.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrus fruits are a very rich source of electrolytes and citric acid. They have been used traditionally for treating urinary ailments and renal stones. Citrus jambhiri is indigenously used as a diuretic. AIM OF THE STUDY: Present study aimed at establishing the antiurolithiatic potential of the juice of Citrus jambhiri fruits along with the elucidation of the mechanism involved in the urolithiasis disease defying activity. METHODS: The antiurolithiatic activity was established by means of nucleation, growth and aggregation assay in the in vitro settings and by means of ethylene glycol mediated calcium oxalate urolithiasis in the male Wistar rats. Docking studies were performed in an attempt to determine the mechanism of the antiurolithiatic action. RESULTS: Present study revealed the role of C. jambhiri fruit juice in reducing nucleation, growth and aggregation of calcium oxalate crystals by possible reduction in the urinary supersaturation relative to calcium oxalate and raising the zeta potential of the calcium oxalate crystals. C. jambhiri fruit juice treatment in experimental rats produced significant amelioration of hypercalciuria, hyperoxaluria, hyperphosphaturia, hyperproteinuria, hyperuricosuria, hypocitraturia and hypomagnesiuria and ion activity product of calcium oxalate. It exhibited nephroprotection against calcium oxalate crystals induced renal tubular dilation and renal tissue deterioration. Docking studies further revealed high binding potential of the phytoconstituents of C. jambhiri viz. narirutin, neohesperidin, hesperidin, rutin and citric acid with glycolate oxidase and matrix metalloproteinase-9. CONCLUSION: C. jambhiri fruit juice possesses excellent antiurolithiatic activity. The study reveals antiurolithiatic mechanism that involves restoration of equilibrium between the promoters and inhibitors of stone formation; and inhibition of matrix metalloproteinases and glycolate oxidase.

Withania somnifera (L.) Dunal: Phytochemistry, structure-activity relationship, and anticancer potential.

BACKGROUND: Ayurveda is a highly recognized, well-documented, and well-accepted traditional medicine system. This system utilizes many natural products in various forms for therapeutic purposes. Thousands of plants mentioned in the Ayurvedic system are useful in disease mitigation and health preservation. One potential plant of the Ayurvedic system is "Ashwagandha" [Withania somnifera (L.) Dunal], commonly regarded as Indian Ginseng. It possesses various therapeutic activities, such as neuroprotective, hypoglycemic, hepatoprotective, antiarthritic, and anticancer effects. PURPOSE: Here we present a comprehensive insight on the anticancer effects of W. somnifera and mechanistic attributes of its bioactive phytocompounds. This review also provides updated information on the clinical studies pertaining to cancer, safety evaluation and opportunities for chemical modifications of withanolides, a group of specialized phytochemicals of W. somnifera. METHODS: The present study was performed in accordance with the guidelines of the Preferred Reporting Items for Systemic Reviews and Meta-Analysis. Various scientific databases, such as PubMed, Science Direct, Scopus, Google Scholar, were explored for related studies published up to May 2021. RESULTS: An updated review on the anticancer potential and mechanisms of action of the major bioactive components of W. somnifera, including withanolides, withaferin A and withanone, is presented. Comprehensive information on clinical attributes of W. somnifera and its active components are presented with the structure-activity relationship (SAR) and toxicity evaluation. CONCLUSION: The outcome of the work clearly indicates that W. somnifera has a significant potential for cancer therapy. The SAR revealed that various withanolides in general and withaferin A in particular have binding energies against various proteins and tremendous potential to serve as the lead for new chemical entities. Nevertheless, additional studies, particularly well-designed clinical trials are required before therapeutic application of withanolides for cancer treatment.

Nuclear Receptors in Asthma: Empowering Classical Molecules Against a Contemporary Ailment.

The escalation in living standards and adoption of 'Western lifestyle' has an allied effect on the increased allergy and asthma burden in both developed and developing countries. Current scientific reports bespeak an association between allergic diseases and metabolic dysfunction; hinting toward the critical requirement of organized lifestyle and dietary habits. The ubiquitous nuclear receptors (NRs) translate metabolic stimuli into gene regulatory signals, integrating diet inflences to overall developmental and physiological processes. As a consequence of such promising attributes, nuclear receptors have historically been at the cutting edge of pharmacy world. This review discusses the recent findings that feature the cardinal importance of nuclear receptors and how they can be instrumental in modulating current asthma pharmacology. Further, it highlights a possible future employment of therapy involving dietary supplements and synthetic ligands that would engage NRs and aid in eliminating both asthma and linked comorbidities. Therefore, uncovering new and evolving roles through analysis of genomic changes would represent a feasible approach in both prevention and alleviation of asthma.

An Accord of Nuclear Receptor Expression in CD4+ T Cells in Rheumatoid Arthritis.

Chronically activated CD4+ T cells drive uncontrolled inflammation, leading to tissue damage in various autoimmune disorders, such as rheumatoid arthritis (RA). Investigation of the molecular mechanisms involved in RA and recent analysis of transcriptomic profiles has implicated members of the nuclear receptor (NR) superfamily in RA. NRs are required for the development, differentiation, and effector function of CD4+ T cells; therefore, it is thought that NRs are important in shaping the CD4+ T cell repertoire and associated inflammation in RA. Despite their relevance, the full potential of the NR superfamily in RA, either as biomarkers or disease targets, has not been harnessed. To gain insight on the NR members that are closely associated with RA disease activity, we generated an expression atlas for the NR superfamily in CD4+ T cells isolated either in a steady state or over the course of collagen-induced arthritis mouse model of RA. We observed discrete expression patterns among the NR superfamily during the disease stages. NRs that instigate anti-inflammatory programs underwent major downregulation during disease onset; however, during the fully developed disease stage we noticed that NRs that induce proinflammatory programs had reduced transcript levels. These animal findings corroborated well with the expression patterns of NRs in clinical samples obtained from RA patients. Furthermore, we observed that targeting NRs using synthetic ligands alleviates the progression of collagen-induced arthritis. Overall, our data demonstrates the potential of the NR superfamily as novel therapeutic targets for the treatment of autoimmune disorders.

PPARγ: Potential Therapeutic Target for Ailments Beyond Diabetes and its Natural Agonism.

Intense research interests have been observed in establishing PPAR gamma as a therapeutic target for diabetes. However, PPARγ is also emerging as an important therapeutic target for varied disease states other than type 2 diabetes like neurodegenerative disorders, cancer, spinal cord injury, asthma, and cardiovascular problems. Furthermore, glitazones, the synthetic thiazolidinediones, also known as insulin sensitizers, are the largely studied PPARγ agonists and the only ones approved for the treatment of type 2 diabetes. However, they are loaded with side effects like fluid retention, obesity, hepatic failure, bone fractures, and cardiac failure; which restrict their clinical application. Medicinal plants used traditionally are the sources of bioactive compounds to be used for the development of successful drugs and many structurally diverse natural molecules are already established as PPARγ agonists. These natural partial agonists when compared to full agonist synthetic thiazolidinediones led to weaker PPARγ activation with lesser side effects but are not thoroughly investigated. Their thorough characterization and elucidation of mechanistic activity might prove beneficial for counteracting diseases by modulating PPARγ activity through dietary changes. We aim to review the therapeutic significance of PPARγ for ailments other than diabetes and highlight natural molecules with potential PPARγ agonistic activity.

Citrus medica: nutritional, phytochemical composition and health benefits - a review.

Citrus medica (Citron) is an underutilized fruit plant having various bioactive components in all parts of the plant. The major bioactive compounds present are iso-limonene, citral, limonene, phenolics, flavonones, vitamin C, pectin, linalool, decanal, and nonanal, accounting for several health benefits. Pectin and heteropolysachharides also play a major role as dietary fibers. The potential impact of citron and its bioactive components to prevent or reverse destructive deregulated processes responsible for certain diseases has attracted different researchers' attention. The fruit has numerous nutraceutical benefits, proven by pharmacological studies; for example, anti-catarrhal, capillary protector, anti-hypertensive, diuretic, antibacterial, antifungal, anthelmintic, antimicrobial, analgesic, strong antioxidant, anticancerous, antidiabetic, estrogenic, antiulcer, cardioprotective, and antihyperglycemic. The present review explores new insights into the benefits of citron in various body parts. Throughout the world, citron has been used in making carbonated drinks, alcoholic beverages, syrup, candied peels, jams, marmalade, cordials, and many other value added products, which suggests it is an appropriate raw material to develop healthy processed food. In the present review, the fruit taxonomical classification, beneficial phytochemicals, antioxidant activities, and health benefits are discussed.

Novel mutations conferring resistance to kanamycin in Mycobacterium tuberculosis clinical isolates from Northern India.

Twenty-nine Kanamycin resistant clinical isolates of Mycobacterium tuberculosis from Northern India were screened to evaluate genetic mutations in rrs gene, eis gene with its promoter, and whiB7 gene along with its 5'UTR. 14 strains (~48.0%) collectively exhibited mutations in rrs, eis or whiB7 target regions. While the highest frequency of mutations was found in rrs gene, eis and whiB7 loci displayed novel mutations. The novel mutations displayed by eis and whiB7 loci were found to be associated specifically with the Kanamycin resistance as none of the twenty nine Kanamycin sensitive strains harbor them. The inclusion of novel mutations of eis and whiB7 loci will be useful in improving the specificity of future diagnostics.

Toxicophore exploration as a screening technology for drug design and discovery: techniques, scope and limitations.

Toxicity is a common drawback of newly designed chemotherapeutic agents. With the exception of pharmacophore-induced toxicity (lack of selectivity at higher concentrations of a drug), the toxicity due to chemotherapeutic agents is based on the toxicophore moiety present in the drug. To date, methodologies implemented to determine toxicophores may be broadly classified into biological, bioanalytical and computational approaches. The biological approach involves analysis of bioactivated metabolites, whereas the computational approach involves a QSAR-based method, mapping techniques, an inverse docking technique and a few toxicophore identification/estimation tools. Being one of the major steps in drug discovery process, toxicophore identification has proven to be an essential screening step in drug design and development. The paper is first of its kind, attempting to cover and compare different methodologies employed in predicting and determining toxicophores with an emphasis on their scope and limitations. Such information may prove vital in the appropriate selection of methodology and can be used as screening technology by researchers to discover the toxicophoric potentials of their designed and synthesized moieties. Additionally, it can be utilized in the manipulation of molecules containing toxicophores in such a manner that their toxicities might be eliminated or removed.

Stem bromelain-induced macrophage apoptosis and activation curtail Mycobacterium tuberculosis persistence.

BACKGROUND: Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, stem bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune systems, is important. METHODS: We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS: SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS: Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

Inhibition of adipogenesis and induction of apoptosis and lipolysis by stem bromelain in 3T3-L1 adipocytes.

The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPß gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein G(i)α(1), as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point.

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.

Specific molten globule conformation of stem bromelain at alkaline pH.

Stem bromelain (SBM), a therapeutic protein, is rapidly absorbed across the gut epithelium. Because SBM encounters an alkaline pH at its principal site of absorption, we investigated the alkaline-induced denaturation of SBM. From pH 7 to 10, the protein's secondary structure remained the same, although a slight loss of tertiary structure was observed. Above pH 10, there was a significant and irreversible loss of secondary and tertiary structure. At pH 10, SBM showed enhanced tryptophan fluorescence, however, the number of accessible tryptophans remained the same. The thermodynamics of temperature transition at pH 7 and 10 were strikingly different, with the former showing a two-phase transition endotherm, and the latter a broad non-two-state transition. At pH 10, SBM showed a significant increase in 8-anilino-1-naphthalene-sulfonate binding relative to the native state, suggestive of a specific molten globule (SMG) state. These studies suggest a distinct conformational rearrangement in SBM, at the protein's isoelectric point.

Hexafluoroisopropanol-induced helix-sheet transition of stem bromelain: correlation to function.

Stem bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10-30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an alpha-helix to a beta-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in stem bromelain to a form with potentially useful therapeutic properties different from those of the native protein.

Extensively drug resistance (XDR) tb is not always fatal.

The chance of incidence of XDR TB is on the rise due to improper use of second line anti-tubercular drugs. XDR-TB is very difficult to treat successfully and is often referred to as "virtually untreatable form of TB". We herein report a case of XDR TB confirmed by bacteriological examination in a WHO recognised laboratory who after 12 months of regular treatment improved both clinically and radiologically with sputum smear conversion. To the best of our knowledge, there has been no previous report of any similar case in literature.

Regulation of retinal dehydrogenases and retinoic acid synthesis by cholesterol metabolites.

Retinoic acid (RA) constitutes the major active ingredient of vitamin A and is required for various biological processes. The tissue RA level is maintained through a cascade of metabolic reactions where retinal dehydrogenases (RALDHs) catalyze the terminal reaction of RA biosynthesis from retinal, a rate-limiting step. We showed that dietary supplement of cholesterol enhanced the expression of RALDH1 and 2 genes and the cellular RA content in vital organs such as brain, kidney, liver and heart. Consistently, the cholesterol-lowering agent (pravastatin sodium) downregulated the expression of RALDH1 and 2 genes in several organs especially the liver and in cultured liver cells. Further, cholesterol metabolites, predominantly the oxysterols, the natural ligands for liver X receptor (LXR), induced these genes via upregulation of sterol regulatory element binding protein-1c (SREBP-1c) that bound to the regulatory regions of these genes. Knockdown of LXRalpha/beta or SREBP-1c downregulated the expression of RALDH genes, which could be rescued by re-expressing SREBP-1c, suggesting SREBP-1c as a direct positive regulator for these genes. This study uncovered a novel crosstalk between cholesterol and RA biosynthesis.

Bioaffinity based oriented immobilization of stem bromelain.

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 degrees C for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K (m) values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V (max) values remained unaffected on immobilization.

Induction of 'molten globule' like state in acid-denatured state of unmodified preparation of stem bromelain: implications of disulfides in protein folding.

A denatured state of unmodified preparation of stem bromelain representing a structureless form has been characterized at pH 2.0 and the effect of increasing concentration of TFE on the acid-denatured state has been investigated by circular dichroism (CD), fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show considerable accumulation of secondary structure when the acid-denatured bromelain is subjected to 70% (v/v) TFE and exhibited close resemblance to spectral features of those of pH 7.0 preparation. Interestingly, the acid-denatured state also regained some tertiary structure/interactions, with increasing concentration of TFE and at 60% (v/v) TFE, these approached almost those of the native like state. However, further increase to 70% (v/v) TFE resulted in complete loss of tertiary structure/interactions. Tryptophan fluorescence emission studies also suggested the induction of significant compact structure at 60% (v/v) concentration of TFE. In addition the acid-denatured state showed enhanced binding of ANS in presence of 60% (v/v) TFE. Taken together these observations suggest the existence of a molten globule state in acid-denatured bromelain between 60 and 70% (v/v) TFE. A similar molten globule state under identical conditions has been identified in reduced and carboxymethylated preparation of stem bromelain as reported in our earlier communication [Arch. Biochem. Biophys. 413 (2003) 199]. Comparison suggests unfolding/folding behavior of the bromelain to be independent of the intactness of the disulfide bonds.

Trifluoroethanol-induced "molten globule" state in stem bromelain.

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.

Binding of antibromelain monomeric Fab' improves the stability of stem bromelain against inactivation.

Antienzyme polyclonal antibodies against stem bromelain were raised in male albino rabbits and the Fab' monomers isolated from the IgG of the immune sera. Incubation of bromelain with the Fab' resulted in binding and gel filtration of the resulting complex suggested a 1:1 stoichiometry. Complexing with the Fab' resulted in significant stabilization of bromelain against thermal inactivation and alkaline pH.