Ananain

Ananain: A Comprehensive Reference

1. Identity, Nomenclature, and Natural Source

Ananain is a plant-derived cysteine endopeptidase first characterized in 1988. A previously unknown cysteine proteinase, named ananain, was isolated from crude commercial pineapple stem bromelain. It is officially catalogued in the Enzyme Commission system as EC 3.4.22.31, placing it within the hydrolase class of enzymes that cleave peptide bonds.

The Ananas comosus stem extract is a complex mixture containing various cysteine proteases of the C1A subfamily, such as bromelain and ananain. It has been shown that pineapple tissue contains at least four cysteine endopeptidases. The most prominent are stem bromelain (EC 3.4.22.32) and fruit bromelain (EC 3.4.22.33), followed by ananain (EC 3.4.22.31) and comosain.

Ananain belongs to the papain superfamily (CA clan, C1 family) of cysteine proteases. The Ananas comosus stem extract (often improperly described as stem bromelain) is a complex extract containing various cysteine proteases (iso)forms of the papain family (CA clan, C1 family) and other partially characterized non-proteolytic compounds.

Despite its presence in the same raw material as stem bromelain, ananain is a biochemically and immunologically distinct enzyme. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain.

Regarding its quantitative presence in the pineapple stem extract, ananain is a minor but disproportionately active component. Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. In the broader context of proteases identified in bromelain preparations, proteases constitute the major components of bromelain and include stem bromelain (80%), fruit bromelain (10%), and ananain (5%).

1.1 Common Forms and Preparations

Ananain is not commercially available as a standalone isolated supplement. In practice, it reaches end users as a constituent within preparations described broadly as bromelain — specifically pineapple stem bromelain. Bromelain is a mixture of different thiol endopeptidases and other components like phosphatase, glucosidase, peroxidase, cellulase, escharase, and several protease inhibitors.

The most clinically significant preparation containing ananain is the pharmaceutical product NexoBrid® (anacaulase-bcdb, MediWound Ltd., Yavne, Israel). The proteolytic enzyme mixture obtained from bromelain comprises at least two of the cysteine proteases present in bromelain: stem bromelain (EC 3.4.22.32) and ananain (EC 3.4.22.31). NexoBrid is presented as lyophilised bromelain powder and gel vehicle for preparation of a gel for cutaneous use, including concentrate of proteolytic enzymes enriched in bromelain as the active component. Following mixing of the powder with the gel vehicle, each gram of the prepared product contains 0.09 g partially purified bromelain. Partially purified bromelain is a mixture of enzymes extracted from the stem of Ananas comosus (pineapple plant).

The lyophilized formulation used in NexoBrid is notable for its stability. As the last step of the preparation, the proteolytic mixture is lyophilized and stored as a lyophilized powder until use. The proteolytic enzyme mixture is highly stable as a lyophilized or dried powder and can be stored at 2–8°C for 3 years. After this period of time, the proteolytic enzyme mixture maintains at least 90% of the original enzymatic activity as determined at the end of the production process.

2. Traditional and Historical Use

Ananain, as a specific molecular entity, was unknown before 1988 and therefore has no tradition of use as an isolated compound. Its traditional history is subsumed within the broader ethnobotanical use of the pineapple plant and its preparations.

Pineapple was traditionally used for medicinal purposes in South and Central America. Bromelain has a long history of traditional medicinal use in various cultures, particularly in Central and South America, where pineapple is native. The history of bromelain dates back to the ancient civilizations of South America, where the pineapple plant (Ananas comosus) is native.

The Ananas comosus stem extract is a complex mixture containing various cysteine proteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine has several potential therapeutic applications as an anti-cancer, anti-inflammatory, and ecchymosis degradation agent.

In the context of wound debridement specifically, the use of plant-based products for burn treatment dates back to 1600 BC. Historically, various debridement methods have been used for burns, with plant-derived products tracing back to 1600 BC. Bromelain, the active ingredient in NexoBrid, is a proteolytic enzyme derived from pineapple fruit, initially described by Heinicke et al.

The scientific interest in the pineapple stem extract as a debriding agent extends into the mid-twentieth century. In the late 19th century, bromelain, a proteolytic enzyme extracted from the stems of pineapples, began to attract attention, with reports indicating its ability to promote wound healing by selectively dissolving necrotic tissue.

3. Key Constituents, Molecular Characteristics, and Mechanisms of Action

3.1 Primary Structure and Molecular Properties

Ananain is comprised of 216 residues with a theoretical mass of 23,464 Da. Ananain was originally purified from the bromelain extract as a 25 kDa protein with an overall charge more positive than that of stem bromelain, the most abundant cysteine protease derived from pineapple stem. The small difference between the theoretical mass (23.4 kDa) and the apparent molecular mass (~25 kDa) observed in early characterizations is consistent with the behavior of such proteins under different analytical techniques.

This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain, and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain.

Unlike stem bromelain, ananain is not a glycoprotein. The inhibition profiles of ananain and bromelain (e.g., by cystatin and iodoacetate) are also clearly different, and only bromelain is glycosylated. The most common feature between ananain and bromelain remains their 77% sequence identity.

Ananain exists as a proenzyme (zymogen) that must be processed to its active form. Pro-ananain is formed by an inhibitory pro-sequence of 98 amino acids and the cysteine protease portion of 223 residues. Pro-ananain most likely uses a self-activation process to form the mature form.

3.2 Active-Site Mechanism and Substrate Specificity

As a canonical cysteine protease of the C1A subfamily, ananain catalyzes the hydrolysis of peptide bonds through a nucleophilic cysteine residue in its active site. The enzyme's catalytic activity is dependent on the presence of a free thiol group. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. Mass spectrometry provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.

Crystal structural analyses published in 2019 have provided the most detailed picture yet of ananain's binding sites. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high k_cat/K_m value of 1.7 × 10⁶ M⁻¹s⁻¹, with cleavage confirmed to occur after the Gln residue.

Crystal structures of unbound ananain and an inhibitory complex of ananain and E−64, solved at 1.73 and 1.98 Å, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E-64 and ananain explains the high inhibitory efficiency of E-64 toward ananain.

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference, and inhibitory profile compared to stem bromelain.

Regarding the broad substrate hydrolysis profile, ananain is characterized by hydrolysis of proteins with broad specificity for peptide bonds, with the best reported small molecule substrate being Bz-Phe-Val-Arg-NHMec, but with broader specificity than fruit bromelain.

3.3 Structural Comparison with Related Plant Proteases

The amino acid sequences of ananain (EC 3.4.22.31) and stem bromelain (EC 3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. The complete amino acid sequence of ananain has been compared with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya, and actinidin from kiwifruit.

A related enzyme in the Bromeliaceae family further illustrates evolutionary conservation. Fastuosain is a 25 kDa cysteine endopeptidase from the unripe fruit of Bromelia fastuosa and has been shown to have high homology to the pineapple proteinases ananain (74%) and stem bromelain (66%).

4. Scientific Evidence by Area of Use

4.1 Enzymatic Debridement of Burns

The most robustly evidenced clinical application of ananain-containing preparations is the non-surgical enzymatic debridement of deep thermal burns. Ananain is a defined active component, alongside stem bromelain, of the pharmaceutical product NexoBrid®.

Preclinical research (animal models): The earliest in vivo work on ananain's debriding potential used animal burn models. A limited in vivo study using 12 rats with full-thickness skin burn injuries was carried out. The animals were treated 24 hours post-burn with two newly discovered enzyme fractions derived from the stem of the pineapple (Ananas comosus). The results indicated that even debridement of the injury could be effected rapidly (within 4 hours). Although the details of enzyme formulation and clinical application had yet to be established, these findings clearly suggested that two enzyme fractions from pineapple stem have potential as non-surgical debriding agents. One of the subsequent references from that body of work specifically cited a porcine study using an ananain-based cysteine protease preparation, demonstrating refinement of the research toward isolating ananain's specific contribution.

An important historical barrier to clinical translation was batch inconsistency. The most important reason for inconsistent findings in earlier studies was the use of commercially available bromelain that was not standardized, and there was no measurable enzyme composition. The presence of at least four distinct cysteine proteinases, namely ananain1, ananain2, stem bromelain, and comosain, has been described. Therefore, each patient within a trial was treated with an agent with different proteolytic enzyme composition with different debriding properties, leading to variations in debriding intensity.

Regulatory approval and clinical evidence (NexoBrid®): In 2012, the European Medicines Agency approved a pineapple stem-derived bromelain-based debridement concentrate of proteolytic enzymes (NexoBrid®, MediWound Ltd, Yavne, Israel) for adult deep burns. The clinical evidence base for this approval was substantial. The safety and efficacy of NexoBrid (for burn eschar debridement) has been extensively investigated in 7 clinical studies, including a phase 3 pivotal trial. Over 10,000 patients have been successfully treated with NexoBrid® globally, including in the US.

A 2023 systematic review synthesized the body of clinical evidence. Investigators identified 103 relevant studies, of which 34 were found eligible. The included studies report the positive effects of NexoBrid® on burn debridement, functional and cosmetic outcomes, scarring, and quality of life. They also validated high patient satisfaction thanks to enhanced protocols of analgosedation and/or locoregional anaesthesia during bromelain-based debridement. Two studies investigating potential risks (coagulopathy, burn wound infection) concluded there is no strong evidence of these adverse events.

Specific clinical benefits documented in the NexoBrid program include: effective eschar removal by NexoBrid, which removes necrotic tissue without surgery. Non-surgical effective debridement facilitates debridement in children, the aged, and difficult skin areas (hands, feet, joints etc.). NexoBrid removes the eschar in a minimally invasive manner that leaves surrounding uninjured and viable tissues unharmed and thus allows maximization of spontaneous healing and reduces the incidence and extent of skin grafting.

The debridement preparation has also been studied in hands specifically. Findings suggest that enzymatic debridement can significantly reduce the number of procedures required for hand burns initially classified as deep by clinicians, which might otherwise have been deemed for early surgical debridement with a standard-of-care approach.

Dosage in debridement studies: Two grams or five grams of NexoBrid sterile powder are mixed in 20 grams or 50 grams of sterile gel vehicle (ratio of 1:10), respectively, to obtain sterile NexoBrid Gel. NexoBrid Gel is applied to the burn wound at a dose of 2 g NexoBrid sterile powder mixed with 20 g sterile gel vehicle per 1% of total body surface area (~surface of an adult palm) for four hours, or 5 g NexoBrid sterile powder mixed with 50 g sterile gel vehicle per 2.5% of total body surface area.

Evidence strength: The evidence for ananain-containing preparations (as NexoBrid®) in enzymatic burn debridement is strong, supported by multiple randomized controlled trials, a systematic review of 34 studies, and regulatory approval by the EMA and other agencies. However, it must be noted that the clinical evidence pertains to the multicomponent NexoBrid® formulation (containing both stem bromelain and ananain), not to ananain as an isolated agent. The specific independent contribution of ananain versus stem bromelain to clinical outcomes has not been formally separated in human trials.

4.2 Anti-Inflammatory Activity

Ananain has been studied in the context of inflammation, largely through its membership in the pineapple cysteine protease family. Cysteine proteases in pineapple (Ananas comosus) plants are phytotherapeutical agents that demonstrate anti-edematous, anti-inflammatory, anti-thrombotic, and fibrinolytic activities.

However, the research specifically attributed to ananain as an isolated compound in anti-inflammatory contexts is limited. The anti-inflammatory properties extensively documented in the literature largely apply to the bromelain mixture as a whole. In vitro and in vivo studies demonstrate that bromelain exhibits various fibrinolytic, anti-edematous, antithrombotic, and anti-inflammatory activities. Because ananain is a major active component within this mixture, it is understood to contribute to these activities, but direct human clinical evidence attributing anti-inflammatory effects specifically to isolated ananain does not exist in the peer-reviewed literature reviewed here.

Evidence strength: Preliminary and indirect. In vitro and animal studies suggest ananain contributes to the anti-inflammatory activity of pineapple stem preparations, but no human clinical trials have isolated ananain's anti-inflammatory effects from those of the full bromelain complex.

4.3 Anti-Cancer and Cytotoxic Activity

A 2020 structural and functional study published in Scientific Reports examined ananain's cytotoxicity against cancer cell lines directly. Results with purified and fully active bromelain, ananain, and papain showed a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 μM, with control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.

The finding that papain (an enzyme with comparable proteolytic activity to ananain but divergent sequence) lacked this effect on non-adherent cell lines implies that something structurally specific to ananain and bromelain — beyond mere nonspecific protease activity — accounts for the anti-proliferative effect on those cell lines. This is a mechanistically interesting observation but remains at the in vitro level.

More broadly, studies have shown that bromelain has the capacity to modulate key pathways that support malignancy. It is now possible to suggest that the anti-cancer activity of bromelain consists in the direct impact on cancer cells and their micro-environment, as well as in the modulation of immune, inflammatory, and haemostatic systems.

Evidence strength: Preliminary; in vitro only. The cytotoxic effects of purified ananain against multiple cancer cell lines have been demonstrated in cell culture, but no animal studies or human clinical trials have been conducted with isolated ananain for anti-cancer purposes. This area remains investigational.

4.4 Proteolytic / Digestive Activity

Ananain hydrolyzes proteins with broad specificity for peptide bonds. As a component of pineapple stem extracts and preparations described commercially as bromelain, ananain contributes to the well-known protein-digesting capacity of these preparations. Bromelain is considerably absorbable in the body without losing its proteolytic activity and without producing any major side effects. Bromelain accounts for many therapeutic benefits like the treatment of angina pectoris, bronchitis, sinusitis, surgical trauma, and thrombophlebitis, debridement of wounds, and enhanced absorption of drugs, particularly antibiotics. Again, these properties are attributed to the mixture; the isolated contribution of ananain in human digestive or therapeutic studies has not been separately quantified.

5. Body Systems and Health Areas Associated with Ananain

• Integumentary system (skin): The primary documented clinical role — as a constituent of NexoBrid® for the enzymatic debridement of deep partial-thickness and full-thickness thermal burns. NexoBrid is indicated for the removal of eschar in burn patients with deep partial and full-thickness burns.
• Oncology (investigational): In vitro evidence of anti-proliferative activity against breast cancer (MDA-MB231), melanoma (A2058), lymphoma (OCI-LY19), and leukemia (HL-60) cell lines, as described above.
• Inflammation and immune modulation: As part of pineapple stem cysteine protease preparations, ananain is associated with the anti-inflammatory and anti-edematous activity ascribed to bromelain mixtures.
• Digestive system: Broad-spectrum proteolytic activity relevant to protein digestion, as a component of pineapple-derived enzyme preparations used in food technology and as dietary supplements.

6. Dosage Forms Reported in Studies

Because ananain is not marketed or studied as a standalone supplement, dosage information is available only in the context of multi-enzyme preparations containing it:

• Topical pharmaceutical (NexoBrid®): Each bottle contains 5 g of NexoBrid and is applied to a burn wound area covering 2.5% of the total body surface area. Following mixing of the powder with the gel vehicle, each gram of the prepared product contains 0.09 g partially purified bromelain.
• Application duration: As reported in clinical protocols, NexoBrid Gel is applied to the burn wound at a dose of 2 g NexoBrid sterile powder mixed with 20 g sterile gel vehicle per 1% of total body surface area (~surface of an adult palm) for four hours.
• In vitro cytotoxicity studies: Results with purified and fully active bromelain, ananain, and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 μM.

No human oral dosing studies with isolated ananain have been identified in the peer-reviewed literature.

7. Safety Considerations and Known Interactions

7.1 Allergy and Hypersensitivity

Given that ananain is a pineapple-derived cysteine protease sharing 77% sequence identity with stem bromelain, the well-characterized allergy and hypersensitivity profile of bromelain is directly relevant. Consumption of pineapple has been associated with systemic immunoglobulin E (IgE)-mediated hypersensitivity reactions (both delayed and immediate phase reactions) along with respiratory and gastrointestinal symptoms, and can even result in anaphylaxis. This may be attributed to the presence of bromelain in pineapple.

Cross-reactivity studies with bromelain indicate an immunoglobulin E (IgE)-mediated reaction, and cross-reaction with honeybee venom, olive tree pollen, celery, cypress pollen, and papain has been reported. Sensitization to enzymes may follow inhalation (through occupational or other exposure; some reviews show up to 50% occurrence) or ingestion (rare).

Occupational exposure to pineapple-derived enzymes carries specific risks. Repeated exposure of pineapple cutters to bromelain can result in the obliteration of fingerprints.

7.2 Inhibitor Profile Relevant to Safety

The distinct inhibitor profile of ananain relative to bromelain has mechanistic significance for safety. Ananain behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. This means that endogenous cystatin-family inhibitors present in human tissues may modulate ananain activity differently than they do stem bromelain, a factor relevant to understanding its selectivity in clinical debridement and its behavior in biological contexts.

7.3 Thiol Oxidation and Inactivation

Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. This has practical implications: ananain's enzymatic activity is sensitive to oxidizing conditions, which can irreversibly inactivate the enzyme at its catalytic cysteine. This property is also exploited in formulation strategies that use thiol-modifying reagents for chromatographic separation.

7.4 NexoBrid®-Specific Safety Data

For the ananain-containing pharmaceutical NexoBrid®, clinical safety data across 34 eligible studies was reviewed in the 2023 systematic review. Two studies investigated potential risks (coagulopathy, burn wound infection) and concluded there is no strong evidence of these adverse events. The product is applied topically and under controlled medical conditions.

7.5 Drug Interactions Relevant to Pineapple Protease Preparations

The bromelain complex, of which ananain is a component, has documented pharmacological interactions when taken orally. These are attributed to the protease mixture as a whole and are directly applicable to any ananain-containing preparation. Bromelain ingestion is associated with a low incidence of adverse reactions, including diarrhea, menorrhagia, nausea, skin rash, and vomiting. No effects in human clinical tests were documented with bromelain supplementation up to 460 mg; however, doses of up to 1,840 mg increased heart rate.

One cause of inconsistent clinical findings in early debridement studies was the inactivation of bromelain by the commonly used silver sulfadiazine. This chemical incompatibility is specifically relevant when ananain-containing preparations are used in clinical wound care settings where silver-containing antimicrobials are co-administered.

7.6 Batch Variability in Crude Preparations

Crude bromelain preparations do not deliver standardized amounts of ananain. As noted above, early debridement clinical trials yielded inconsistent results partly because the use of commercially available bromelain that was not standardized meant there was no measurable enzyme composition. This has safety and efficacy implications for any therapeutic application: the amount of ananain in a dietary supplement marketed as "bromelain" is not defined and may vary substantially between products and batches.

8. Research Gaps and Current Status

Despite ananain's notable biochemical properties — including its disproportionate contribution to the proteolytic activity of pineapple stem extract and its distinct structural features — it remains one of the least independently studied enzymes of the pineapple protease complex. Key research gaps include:

• No human clinical trials have assessed ananain as an isolated agent for any indication.
• The anti-proliferative activity observed in cancer cell lines exists only at the in vitro level; no animal or human studies have been performed.
• The relative contribution of ananain versus stem bromelain to the clinical efficacy of NexoBrid® in burn debridement has not been formally dissected in controlled studies.
• These data reveal the first in-depth structural and functional data for ananain, with the first crystal structures only solved in 2019 — underscoring how recently foundational structural research has been completed.
• Pharmacokinetics and bioavailability of ananain after oral ingestion have not been characterized in human subjects.

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