Introduction

Vanilla is arguably the world’s most popular flavour and is derived from mature pods of the orchid Vanilla planifolia. It constitutes one of the most preferred flavours and fragrance ingredients in ice-creams, confectioneries, milk products, perfumes, pharmaceuticals, liqueur and other cordial industries, thereby forming a whopping multimillion-dollar market (Gallage and Møller 2018). For centuries vanilla flavour remained classified for the rest of the world since it was ascribed as a flavour of nobility by Aztecs and pre-Columbian Mayas. It was in 1519 that vanilla was exposed to the world with the Spanish invasion of the Aztecs. It was transported to Europe and subsequent development of hand pollination techniques led to its expansion to other parts of the world (Teoh 2019). Today, Madagascar is the largest producer of natural vanilla with 75% of world production followed by Indonesia, China, Mexico, and Papua New Guinea. Vanilla is a mixture of ~ 200 compounds; however, it’s characteristic flavour and fragrance comes mainly from the molecule vanillin (Gallage and Møller 2018).

Vanillin is a specialized metabolite and the main ingredient of vanilla extract that occurs in concertation of 1.0–2.0% w/w in cured vanilla beans (Zhang and Mueller 2012). Vanillin has different functional groups, like aldehyde, hydroxyl and ether attached to an aromatic ring. The physicochemical properties of vanillin are described in Table 1. Vanillin is either isolated from vanilla extract or is chemically synthesized from guaiacol. Besides being known for flavour and fragrance, it has diverse bioactive properties, namely anticancer, neuroprotective, antibiotic potentiation, and anti-quorum sensing (Arya et al. 2019; Bezerra et al. 2016; Li et al. 2018). Moreover, the bioactivities of curcumin are now attributed to the constituent and stable degradation products, i.e. vanillin and ferulic acid (Iannuzzi et al. 2017). Though recent studies on vanillin have eluded to its bioactive potential, in comparison to curcumin the level of research activity is very limited. Therefore, due to the potential emerging reports of usage of vanillin as a therapeutic molecule and its inclusion in the food additive on generally regarded as safe (GRAS) list, it is an ideal candidate for health care applications (Tai et al. 2011b). Our focus here is to provide an in-depth look at the bioactive properties of vanillin (Table 2) as an attempt to identify it as a mainstream bioactive small molecule like curcumin. Readers are also directed to the reviews by Singletary (2020), Sharma et al. (2020), Anand et al. (2019) and Bezerra-Filho et al. (2019) that highlighted the therapeutic use of vanilla, vanillin, and vanillic acid.

Table 1 Physicochemicalcharacteristics of vanillin
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The literature for this review was extracted from the last three decades published in various research and review articles, book chapters, and conference proceedings. The search engines used to search this information includes PubMed, Google Scholar, Science direct and ScopeMed. The keywords or search terms, “vanillin”, “vanillin derivatives, bioactivities, anticancer, antioxidant, anti-inflammatory, neuroprotective, anti-sickling, anti-amyloid aggregation and inhibition of non-enzymatic glycation, antibacterial, anti-fungal, anti-quorum sensing, antibiotic potentiation, wound healing/tissue engineering, antiviral”, “toxicity”, “nanoparticles”, “nanocarriers” and their combination were used (Fig. 1).

Fig. 1
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Number of published articles on bioactivities of vanillin (Accessed on 07th September, 2020)

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Sources of vanillin

Typically, there are three sources of vanillin, i.e. natural, chemical/synthetic and biotechnological (Fig. 2). Depending on the source and the synthesis procedure, the vanillin is categorized as either natural or artificial flavour. Of these, the natural and biotechnologically produced vanillin (from ferulic acid as a substrate) is considered as food-grade additives by most food control authorities across the world.

Fig. 2
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Different sources and routes of vanillin synthesis

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Major sources

Natural

Vanillin is naturally extracted from vanilla pod extract of Vanilla planifolia, Vanilla tahitensis, and Vanilla pompona which are by far the main sources of vanillin (Bezerra et al. 2016). Commercial extraction methods for vanillin include Soxhlet, supercritical fluid extraction (SCEF), microwave and ultrasound-assisted extraction, enzymatic extraction, solid-phase extraction and biphasic sonoelectroanalysis (Hardcastle et al. 2001; Kun 2002; Sharma et al. 2006; Sostaric et al. 2000; Voisine et al. 1995; Waliszewski et al. 2007). Natural vanillin is the most expensive form at a cost of nearly US$ 1200/kg to more than US$ 4000/kg (Gallage and Møller 2018).

Chemical synthesis

Compared to the natural source, chemically synthesized vanillin it is considerably cheaper ($15/kg), however, is labeled as artificial vanillin which attracts negative consumer sentiments. Various substrates have been tried for the synthesis of synthetic vanillin, like lignin, guaiacol, 4- hydroxybenzaldeyde, 3-bromo-4-hydroxybenzaldeyde, 3-methoxy-4-hydroxybenzyl alcohol, cow dung and lignin-rich crop residual waste materials with varying success (Banerjee and Chattopadhyay 2019; Ciriminna et al. 2019).

Minor source

Biotechnological

Bioengineering is a modern route for the production of vanillin. Various proprietary bacterial and fungal strains are genetically engineered that use a spectrum of starting materials like ferulic acid, eugenol, iso-eugenol and glucose. Also, enzymatic synthesis of vanillin using proteins from Nocardia sp. and white-rot basidiomycetes have been reported (Banerjee and Chattopadhyay 2019). Furthermore, genetically engineered plants or plant cell cultures producing vanillin are proposed as a future alternative to produce vanillin and increase its commercial and medical applicability (Chee et al. 2017).

Bioactivities

As a popular flavour and fragrance compound, vanillin has received less attention for the bioactive properties it possesses. However, to be used as a pharmaceutical ingredient, it must have the desired bioactivity and should be bioavailable in humans and/or animals. In this regard, bioavailability studies have identified the rate and concentration at which vanillin is absorbed in the blood, plasma and also its target site (Beaudry et al. 2010). It is shown that vanillin has an LD50 (lethal dose to kill half of a tested population) of 4333 mg/kg for mice and 4730 mg/kg for rats (Makaruk 1980). Furthermore, toxicology studies on rats via oral and intraperitoneal administration of vanillin confirms that it is safe even at a high concentration of 300 mg/kg and did not exhibit any toxic effect on kidney, liver, blood cells, and also showed blood and neuroprotective properties (Ho et al. 2011). Owing to its non-toxicity in rats, it is worthwhile to consider vanillin as a candidate bioactive molecule and highlight its potential pharmacological applicability.

Anticancer activity

Reports that implicate vanillin in mediation of DNA damage and antimutagenic potential have encouraged researchers to evaluate the anticancer effects at cellular and molecular levels (Bezerra et al. 2016). Vanillin (1000 µg/mL) inhibited the proliferation of HT-29 cells (Colon cancer cells) where significant cell arrest occurred during the G0/G1 phase and an increase in apoptotic cells in sub-G0 phase was observed (Ramadoss and Sivalingam 2019). Further, a derivative of vanillin, 4-(1H-imidazo [4,5-f] [1,10]-phenanthrolin-2-yl)-2-methoxyphenol (IPM711) showed growth inhibition, invasion and migration of HT-29 and HCT116 cells by binding to a Wnt/β-catenin signalling receptor (Ma et al. 2019). In this study, vanillin down-regulated proteasome genes in colon tissues and significantly suppressed proteasome activity. Furthermore, at 10 mM it hindered the mitogen-activated protein kinase (MAPK) phosphorylation, reducing the number of granulocytes in colon tissue, proliferating cells and p65-positive cells. Amelioration of cancerous activity by vanillin might be associated with downregulation of the proteasome genes, MAPK pathway and nuclear factor-κB (Li et al. 2018). A vanillin derivative VND3207 has shown a strong radio-protective effect in radiation-induced intestinal injury in mice (Li et al. 2020). VND3207 was found to alleviate the radiation injury in human lymphoblastoid cells by enhancing the expression of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) which is an essential part of DNA double-strand break repair mechanism. Another in vitro study suggested that vanillin induces apoptosis in human hepatic carcinoma and neuroblastoma cells (Naz et al. 2018). Further molecular docking reveals binding of vanillin to CAMKIV enzyme associated with cancer and neurodegenerative diseases. Also, monodimer of vanillin was found to decrease the metastatic potential of HepG2 cells by inhibiting FAK/PI3K/Akt signalling pathway (Jantaree et al. 2017). With these leads, we can use a multi-omics and modelling approach to more precisely identify the potential molecular targets of vanillin.

Table 2 Summary of literature into the effects of vanillin and vanillin derivatives on potential cellular targets and associated molecular mechanisms
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Antioxidant and anti-inflammatory activity

Vanillin is reported to be a potent scavenger of ROS as observed in multiple antioxidant assays like ORAC (oxygen radical absorbance capacity), ABTS+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and oxidative haemolysis inhibition where it operates by self-dimerization contributing to high reaction stoichiometry (Tai et al. 2011b). Also, it is found to have anti-inflammatory activity, for instance, vanillin was found to inhibit nitric oxide in the lipopolysaccharide activated (LPA) RAW264.7 macrophages (Lim et al. 2008). Moreover, suppression of inducible nitric oxide synthase (iNOS) is closely related to anti-inflammatory activity, RT-PCR studies revealed that vanillin concentration-dependently reduced the induction of iNOS mRNA in LPA macrophages.

Neuroprotective activity

Experimental evidence in animals has shown that vanillin acts as a neuroprotective agent in Huntington’s disease (HD) and global ischemia (Gupta and Sharma 2014; Kim et al. 2007). Vanillin significantly affected the 3-nitropropionic acid (3-NPA) induced HD in rats by attenuating motor coordination, learning-memory, locomotory and biochemical impairments (Gupta and Sharma 2014). Moreover, vanillin (40 mg/kg) exhibited neuroprotection against neuronal cell damage in the hippocampal CA1 region (Kim et al. 2007). Vanillin is further reported to promote early neurofunctional development, ameliorates histomorphological damage, brain infarct volume and brain edema after hypoxic-ischemic damage in neonatal rats (Lan et al. 2019). In spinal cord injury rat model, vanillin exerted neuroprotective effect reducing apoptosis and downregulating the expression of HIF-α in spinal tissues (Chen et al. 2019). This neuroprotective effect of vanillin is proposed to be mediated by ROS scavenging, attenuating mitochondrial dysfunction, decreasing lipid peroxidation, and apoptosis (Dhanalakshmi et al. 2015). Recently, it was reported that vanillin and vanillic acid modulate antioxidant system via alleviation of metabolic complications linked to Fe2+-induced brain tissue damage (Salau et al. 2020). Thus, vanillin and its analogues can be further evaluated as a potential therapeutic agent for neuroprotection and stroke therapy.

Sickle cell anaemia

Vanillin was evaluated as an agent to treat sickle cell disease (SCD) by Abraham et al. in 1991. It showed dose-dependent inhibitory effect on deoxygenation (HbA) induced sickling and sickle haemoglobin (HbS) polymer formation with no adverse effect on cellular water or ionic content. Through X-ray crystallography, it is realized that binding of vanillin is near His 103α, Cys 104α and Gln 131β in central water cavity, with a secondary binding site at His 116β and His 117β (Abraham et al. 1991). o-vanillin also affects the membrane permeability of red blood cells stimulating the efflux of K+ ions which further ameliorated the complication of SCD (Hannemann et al. 2014). Moreover, numerous vanillin derivatives have been developed which exhibit enhanced in vitro allosteric inhibition and anti-sickling as compared to vanillin (Pagare et al. 2018). Thus, vanillin or its derivatives can be designed and tested for allosteric modulation in stereospecific inhibition of HbS polymerization and high-affinity HbS.

Amyloid aggregation and non-enzymatic glycation (NEG) of insulin

Advanced glycation end products (AGE) are formed as end products of glycation reaction and are associated with developing sever diabetic complications that include neuropathy, nephropathy, retinopathy, and further progress in amyloid based neurodegenerative diseases. Vanillin was found to restrain NEG and AGE of albumin by functioning like a chemical chaperone (Awasthi and Saraswathi 2016). This in vitro study provided preliminary evidence for vanillin mediated insulin glycation and amyloid aggregation and AGE formation by methyl-glyoxal was strongly reduced in the presence of vanillin. It is presumed that vanillin binds non-covalently to positively charged Arg22 of insulin B chain and hinder the glycation reaction (Iannuzzi et al. 2017). Furthermore, vanillin also showed cytoprotective and anti-oxidant effect against AGE induced ROS products. These studies open new avenues for vanillin in the treatment of NEG and AGE induced diabetes.

Antifungal activity

Fungal pathogens are well known to affect food, human health and agriculture. It is found that vanillin can impede the growth of such fungal pathogens. For instance, vanillin (250 mg/L) decreased the growth of Alternaria strains, suggesting its fungistatic behaviour where the lag time of fungal life cycle was increased from initial 50 h to 112 h and also inhibition of mycelial growth of up to 37.5% was observed (Romero-Cortes et al. 2019). Antifungal activity of vanillin and its 33 variants were tested against Cryptococcus neoformans which is the causative agent of cryoptococcal meningitis (Kim et al. 2014). RNA-seq of o-vanillin and o-ethyl vanillin treated C. neoformans showed that they caused mitochondrial dysfunction and triggered oxidative stress, significantly reducing their growth. Omics based analysis of vanillin treated fungus may further reveal the molecular targets of vanillin and pave a way for its use as an antifungal molecule in food, agriculture and the pharmaceutical industry.

Antibacterial activity

Vanillin was found to affect the growth of spoilage bacteria like Pantoea agglomerans, Aeromonas enteropelogenes, Micrococcus lylae and Sphingobacterium spiritovorun with the minimum inhibitory concentration (MIC) ranging from 10 to 13.3 mM (Ngarmsak et al. 2006). It was found that exposure to 10–40 mM vanillin inhibited respiration of E. coli and Listeria innocua and treatment with 50–100 mM resulted in complete dissipation of proton ion gradient with loss of pH homeostasis in Lactobacillus plantarum (Fitzgerald et al. 2004). In order to gain detailed insight into the cellular response to vanillin, the proteomics of vanillin treated E. coli showed that around 147 proteins exhibited a significant change in abundance in response to vanillin (Pattrick et al. 2019). The treatment caused accumulation of ROS invoking adaptations mediated by a MarA, OxyR, and SoxS regulatory network and increased in RpoS/DksA-dependent gene expression. Also, AcrD and AaeAB were identified as potential vanillin efflux systems (Pattrick et al. 2019). Further omics-based studies are required for other pathogenic bacteria specially listed as critical threats by world health organization in order to identify novel gene/protein targets of vanillin in bacteria.

Antibiotic potentiation activity

Vanillin at sub-inhibitory concentrations was found to modulate the activities of antibiotics. It was reported to regulate the activities of gentamycin, imipenem, norfloxacin and spectinomycin used against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli (Bezerra et al. 2017; Brochado et al. 2018). It also potentiated the activities of some commonly used and last line antibiotics like chloramphenicol, ciprofloxacin, levofloxacin, tigecycline, meropenem, trimethoprim and fosfomycin against extremely drug-resistant P. aeruginosa clinical isolates (Arya et al. 2019, 2020). These studies suggest that vanillin has the potential to be used as an antibiotic adjuvant in future.

Anti-quorum sensing activity

Bacteria either grow as planktonic cells or in films known as biofilms. These biofilms are highly resistant towards antibacterial agents and can be inhibited by anti-quorum sensing molecules that affect bacterial signalling. Reports on vanillin suggest that it can inhibit short-chain homoserine lactones and long-chain acyl-homoserine lactones in Aeromonas hydrophila (Ponnusamy et al. 2009). Recently, the in vitro analysis in P. aeruginosa and in silico docking studies revealed that vanillin binds to the active site of PqsR (PQS-binding response regulator) and inhibits pqs expression which is associated with pyocyanin (quorum sensing molecule) and the virulence thereafter (Mok et al. 2020). Vanillin can, therefore, be explored to evaluate its antibiofilm properties against other biofilm-forming bacteria which are usually found resistant to antibacterial agents.

Application in wound healing and tissue engineering

Vanillin is used as a natural crosslinker to fabricate chitosan hydrogel for wound healing. Self-healing chitosan-vanillin hydrogel is developed based on Schiff base and hydrogen bond hybrid linkages between chitosan and vanillin (Xu et al. 2018). At the atomic level, aldehyde moiety of vanillin reacts with amino group of one chitosan molecule through Schiff-base reaction and its hydroxyl moiety forms hydrogen bond with the hydroxyl or the amino groups in another chitosan molecule. The self-healing effect is generated by reconstruction of Schiff-base bond. Along with wound healing, rat skin samples treated with chitosan-vanillin membrane showed angiogenic stimulus, collagen deposition, re-epithelialization, and reduced levels of IL-1β and TNF-α as well as increased IL-10 and gene expression of TGF-β and VEGF (de Aragão Tavares et al. 2018). Various concentrations of vanillin/chitosan along with other metallic and organic components are used for wound healing and tissue engineering such as osteochondral tissues (Hunger et al. 2019). Although chitosan-vanillin hydrogels have promising outcomes for wound healing and tissue engineering, these studies are yet to be replicated in human and therefore clinical trials are needed to determine their applicability.

Antiviral activity

A novel vanillin derivative MY21 was designed, synthesized and evaluated for its anti-neuraminidase (NA) activity (Hariono et al. 2016). Vanillin with guanidino group (MY21) at the C3 position played a vital role in NA inhibition. Modelling studies suggested that these predicted activities might be due to the interaction with conserved and essential residues of NA with ∆Gbind (binding affinity of the ligand to the active site of the receptor) values comparable to those of oseltamivir and zanamivir, two commercially available NA inhibitors. Recently reports on SARS-CoV-2 suggests that vanillin has moderate affinity towards spike protein and main protease. Thus, further studies should be undertaken to enhance the inhibitory potential of vanillin and its derivative on SARS-CoV-2. Altogether, such findings suggest that vanillin and its derivatives can become suitable starting compounds for further lead optimization as NA inhibitors.

Vanillin as a cosmeceutical ingredient

Vanillin is used in many cosmeceuticals owing to its fragrance and antioxidant properties. At non-toxic concentrations, vanillin was found to up-regulate the stemness mediators Oct-4, pOct-4 and Nanog (transcription factors that control the stem cell signatures in humans) and it also increased the expression of epithelial adhesive protein (E-cadherin) (Taboonpong et al. 2017). Vanillin decreased the production of pro-inflammatory cytokines and reduced UV-B induced phosphorylation of ataxia telangiectasia mutated (ATM), serine-threonine kinase checkpoint kinase 2 (Chk2), tumor suppressor protein 53 (p53), p38/mitogen-activated protein kinase (p38), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), S6 ribosomal protein (S6RP), and histone 2A family member X (H2A.X) (Lee et al. 2014). All these factors play a central role in skin renewal and repair; therefore, using vanillin or its derivatives as cosmeceutical ingredients could also provide therapeutic benefit in addition to providing fragrant and antioxidant effects.

Clinical studies

So far, only a few clinical trials with vanilla or vanillin have been undertaken or completed. The details of these studies are summarized in Table 3. However, only one out of these clinical trials was directed to assess the therapeutic potential of vanilla, while others were aimed to study the calming effect of vanilla/vanillin fragrance on the distressed infants with neonatal hypoxia and temporary Apnoea. Although few in numbers, these trails suggest that it is time to work towards and realize the therapeutic potential of vanilla/vanillin. The increase in the number of reports on the cyto-, neuro, nephron-, cardio-, and hepatoprotective potential of vanillin may therefore enhance the chances of vanillin to be considered for clinical trials in the future.

Table 3 Clinical studies involving vanillin/vanilla
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Nanoparticles to deliver vanillin

The bioavailability and hydrophobicity limit the bioactive efficiency and pharmacokinetics of vanillin. Nanocarriers or nanoparticles (NPs) can potentiate the bioactive profile of vanillin (Fig. 3). Various reports are available were vanillin is either capped /functionalized onto the NPs or encapsulated into the NPs (Table 4). These NPs also allow controlled/sustained release to prolong the effect of vanillin. Apart from delivering vanillin using NPs, vanillin itself can be used to synthesize NPs for the delivery of other drug molecules (Table 4). It is an interesting development that a popular and one of the oldest flavoring molecule vanillin has found applications in the latest nanotechnology discipline as well. Due to these developments, it is essential to realize the potential of vanillin and consider it for therapeutic purposes.

Fig. 3
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Specific features of nanoparticles as delivery systems

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Table 4 Summary of nanoparticles with vanillin as cargo/component of nanoparticles
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Conclusions

To date, vanillin has been utilized primarily as a flavour and fragrance ingredient. As discussed in this review, vanillin has shown diverse bioactivities that can be harnessed for human, animal and agricultural benefits. As it exhibited non-toxic effects in rat models, it is likely that vanillin is efficiently assimilated and eliminated from their bodies. Future studies in nanocarrier systems for vanillin may increase its stability, bioavailability and bioactivity. Hence with some promising inroads in this area, it would be interesting to systematically investigate the possible effects of vanillin with the multi-omics approach at cellular and molecular levels. This will enable us to further assess its applicability as an active biopharmaceutical ingredient to tackle important issues like neurodegeneration, antibiotic resistance, sickle-cell anaemia, tissue engineering, viral infections and industrial applications such as food preservation.