Yassin et al. (2020a) evaluated the antibacterial efficiency of acetone, dichloromethane, ethanol and petroleum ether extracts of clove against Escherichia coli, Salmonella typhi, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA). The researchers reported that the dichloromethane extract exhibited the highest antibacterial potency against the pathogenic isolates.

A study by Radünz et al. (2019) showed that unencapsulated and encapsulated clove essential oil at 0.304 mg/ml inhibited S. aureus, E. coli, L. monocytogenes, and S. Typhimurium bacteria, independent of the microorganism membranes and in situ inhibition against S. aureus in meat products similar to burgers. The bactericidal action of clove essential oil is attributed to eugenol, which ruptures the bacterial cytoplasmic membrane, allowing leakage of ions and intracellular proteins, leading to cell death.

The high antibacterial effect of ethanol and methanol extracts of clove against Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae clinical isolates have been reported (Garba et al., 2019). Ethanolic extract of clove was shown to have broad spectrum (Gram-positive and Gram-negative) inhibition on five pathogens causing urinary tract infections, this inhibition was stronger than that shown by pure eugenol at the same concentration (Rosarior et al., 2021). Antibacterial activity of methanol extract was reported for Klebsiella pneumonia (Hemalatha et al., 2015).

Eugenol, the major component of clove oil was shown to have bactericidal action against Proteus mirabilis at the minimum inhibitory concentration of 0.125% (v/v) and at 0.25% (v/v) it reduced viability and completely inhibited P. mirabilis population within 30 min of exposure (Devi et al., 2013).

Another study showed that hexane extract of clove (Eugenia caryophylata), at 0.2% exhibits the highest inhibition against the pathogenic potato black stem and soft rot bacteria, Erwinia carotovora subsp. atroseptica when compared to ethanol extracts of cinnamon (Cinnamomum zelanicum) and datura (Datura metel) at the same concentration (Al-Jeboory et al., 2012).

Yassin et al. (2020b) showed that Syzygium aromaticum (clove) ethyl acetate extract exhibited the highest antifungal activity against C. tropicalis, C. albicans, C. glabrata, and with minimum inhibitory concentration of 250 μg/disc and 500 μg/disc respectively, while the minimum fungicidal concentration was 0.5 mg/disc against C. tropicalis and 1 mg/disc against the C. albicans and C. glabrata. The main bioactive compounds in the extract were eugenol (58.88%), eugenyl acetate (23.86%), trans-caryophyllene (14.44%) and α-humulene (1.88%).

Clove oil was reported to show maximum antifungal activity against C. tropicalis, C. albicans, and C. guilliermondii (Kumar et al., 2012). Encapsulated clove oil also exhibited strong antifungal action against Fusarium oxysporum (Estrada-Cano et al., 2017) and antiseptic effects on meat products at a concentration above 0.070% (Wang et al., 2018; Kaur and Kaushal, 2019).

Aqueous extract of clove was reported to exhibit a dose-dependent antiviral effect against Feline Calicivirus, used as a surrogate for Human Norovirus, a food-borne virus. The effect was attributed to the main bioactive component-eugenol which showed similar antiviral activity to clove extract, although at lower level, an indication that other components in clove worked synergistically with eugenol to inactivate Feline Calcivirus (Aboubakr et al., 2016).

Eugenol and Eugenin isolated from clove bud essential oil was also shown to have potent inhibitory effect against herpes simplex virus at a dose of 10 μg/ml (Milind and Deepa, 2011). The antiviral efficacy of aqueous extracts of clove against herpes simplex virus type 1 (HSV-1) and influenza A virus when combined with acyclovir has also been reported (Batiha et al., 2020).

According to the above mentioned studies, both the extracts and essential oil of clove possess antimicrobial activity. Most of these activities were dose dependent, broad spectrum and based on disruption of the microbial cytoplasmic membrane resulting in leakage of cytoplasmic content and subsequent cell death. However, most of the studies did not give the mechanism or type of inhibition, but all reported that clove can be used as a natural antibacterial, antifungal and antiviral agent in human/animal health, as well as the inhibition of pathogenic and spoilage microorganisms in foods and plants.

Anti-inflammatory activity of clove (Eugenia caryophyllata) essential oil was evaluated by simulating inflamed human skin cells in a dermal fibroblast system (HDF3CGF). Clove essential oil at a concentration of 0.001% showed strong anti-inflammatory action, demonstrated through resistance to the production of pro-inflammatory biomarkers like interferon-inducible T-cell α-chemoattractant (I-TAC), vascular cell adhesion molecule-1 (VCAM-1) and collagen III expression at gene and protein levels (Han and Parker, 2017).

Bachiega et al. (2012) reported that aqueous extract of clove and eugenol in non-cytotoxic concentrations exerted immunomodulatory or anti-inflammatory action on cytokine production by murine macrophages incubated with clove or eugenol (5, 10, 25, 50 or 100 µg/well) for 24 h. They showed that 100 µg/well of clove inhibited the production of IL-1β, IL-6, and IL-10, eugenol did not affect IL-1β production but inhibited IL-6 and IL-10. They concluded that the immunomodulatory/anti-inflammatory effects of clove was by inhibiting LPS action and the possible mechanism of action may be through suppression of nuclear factor-κB pathway by eugenol, because it was the major bioactive of clove extract.

Nikoui et al. (2017) investigated the anti-inflammatory and antipyretic effects of clove oil in healthy dogs after surgery. Animals surgically operated in the abdomen were grouped into four and administered 25 mg/kg of clove oil, 20 mg/kg betamethasone (anti-inflammatory), 15 mg/kg phenylbutazone (anti-pyretic), or nothing as control for 5 days consecutively. The researchers reported that clove oil-treated animals had significantly reduced edema, white blood cells, neutrophils, band neutrophils and rectal temperature compared to control. Also, histopathology assay showed that clove oil reduced inflammation significantly in dogs.

Also, the use of pure clove oil in the aromatherapy treatment of arthritis and rheumatism, in combination with honey for dermatitis and the paste (water and clove powder) for bites and cuts have been reported (Hussain et al., 2017).

Further, Naji et al. (2017) evaluated the anti-inflammatory capacity of Syzygium aromaticum extract (SAE) using formalin test and showed that mice fed with different doses of SAE exhibited visible increase in analgesia time and decrease in licking number (p < 0.05) compared to untreated animals.

Bachiega et al. (2012) showed that clove and eugenol exhibited immunomodulatory and anti-inflammatory action on cytokine production by murine macrophages when administered in non-toxic doses. In addition, clove oil at 0.05 and 0.20 ml/kg showed anti-inflammatory effect equal to that of anti-inflammatory drugs like etodolac and indomethacin at 0.025 and 0.1, and 0.05 and 0.2 ml/kg doses, respectively (Vicidomini et al., 2021).

The anti-inflammatory actions of clove extracts and essential oils though documented, were mostly performed using in vitro and in vivo assays using cell lines and animals respectively. No clinical study involving human subjects were reported. Many studies evaluated the anti-inflammatory potentials using eugenol the major bioactive component of clove. The studies report the potential of clove extracts and oil and provide support for its use in folk medicine. However, well planned studies across all levels (in vitro, in vivo and clinical/human) including dosages are urgently needed to validate the anti-inflammatory use of clove.

Azadi et al. (2020) using in silico mathematical modelling and 1H Nuclear Magnetic Resonance spectroscopy on clove oil treated Raji cells, used the Metaboanalyst software to predict that 50% inhibitory concentration of clove oil was 50 μg/ml and 74 genes with differentiating metabolites consisting of amino acids, cholesterol and fucose. They also predicted that clove oil mechanism of anti-cancer action was against novel enzymes, like 24-dehydrocholesterol reductase and 7-dehydrocholesterol reductase in cholesterol biosynthesis, dehydrofolate reductase in one carbon metabolism and serine palmitoyl-transferase long chain in sphingolipid biosynthesis.

Kubatka et al. (2017) showed that dietary administration (0.1 and 1%) of clove was effective against induced mammary carcinoma in rat model through significant decrease in tumor frequency but not latency. The antitumor effect was mediated through caspase three activation, reduction of protein (Bcl-2, Ki67, vascular endothelial growth factors A, MDA, CD24, CD44) expressions and increased methylation of RASSF1A promoter, as well as expressions of ALDH1A1, H4K16ac, and H4K20me3. Colon cancer was induced in mice by injecting them with naked HT-29 tumor cells, the mice were then treated with ethyl acetate extract of S. aromaticum once daily for 5 days. The authors reported that tumor growth was significantly reduced in the extract-treated group (Kubatka et al., 2017).

In vivo, the antitumor activity of ethyl acetate extract and oleanoic acid from clove were investigated using the HT-29 tumor xenograft model (Liu et al., 2014). The extract showed higher activity than oleanoic acid (one of its bioactive components) and 5-fluorouracil (a chemotherapeutic agent) at suppressing the growth of colon tumor xenografts. Most of the changes reported were at the mRNA level, suggesting transcriptional regulation by ethyl acetate extract. The authors concluded that clove extract and oleanoic acid, could be novel therapeutic agents for the treatment of colorectal cancer.

From the studies reviewed, clove extracts, essential oils and bioactive components exerted anti-cancer properties in vitro through cytotoxicity, apoptosis, chemopreventive, tumor formation, antiproliferative, modulation of gene expression and cancer signalling processes actions. In silico evaluation have also been used to predict anti-cancer potential of clove. In vivo, clove extracts inhibited tumor growth through caspase three activation, gene and protein expressions. Unfortunately, as with anti-inflammatory action of clove, no human studies have been documented so far. Thus it is pertinent that future studies be initiated to investigate the anticancer effect of clove in humans including the dosage. Presently though, since clove is a GRAS food, it can be used as an adjuvant in cancer treatment.

Bi et al. (2017) reported a study in which 36 people with T2D were given capsules containing 0, 1, 2, or 3 g of cloves per day for 30 days and a 10-days washout period. Results from the study showed that although no significance differences in responses to the dose was observed, diabetic patients who took clove supplement exhibited decrease in serum glucose, total cholesterol, triglycerides and LDL, compared to diabetic patients who did not.

An alcoholic extract of clove (containing majorly-eugenol (42.27%), acetyl eugenol (29.12%), caryophyllene (15.40%), and humulene (3.22%) was reported to inhibit S-phase DNA replication of HepG2 cells and adipocyte differentiation of OP9 cells (Ding et al., 2017). In vitro treatment using clove ethanol extract on 3T3-L1 cells showed that the extract efficiently inhibited the conversion of cells into adipocytes in a dose-dependent manner (Jung et al., 2012).

Ding et al. (2017) reported that an alcohol extract of clove [containing majorly-eugenol (42.27%), acetyl eugenol (29.12%), caryophyllene (15.40%), and humulene (3.22%)] prevents obesity in a mouse model by acting as a natural fatty acid inhibitor. The extract also reduced the development of high fat diet-induced obesity, body and abdominal adipose tissue weight, regulate total triglyceride, low-density lipoprotein cholesterol and lower lipid accumulation in the liver and epididymal adipose tissues.

In another study, Jin and Cho, (2011) showed that an aqueous extract of cinnamon and clove at 10 μg/ml administered to hyper-cholesterolaemic zebrafish exerted a potent dose-dependent cholesteryl ester transfer protein (CETP) inhibitory and hypolipidaemic activities; and that clove extract-fed group had the smallest increase in body weight and height and the strongest antioxidant activity after a 5-weeks high cholesterol diet.

Another study reported that clove ethanol extract reduced diet-induced obesity in mice fed high fat diet supplemented with 0.5% (w/w) for 9 weeks through down-regulation of adipogenic and lipogenic gene expression (Jung et al., 2012). Another study reported that simultaneous administration of clove and curcumin (Curcuma longa L.) extract on high fat diet-induced mice for 5 weeks led to reduced food intake, weight gain, adipose tissue, liver weight, and regulation of lipid profiles (Pérez Gutiérrez and Arrioja, 2021).

Regarding antidiabetic and anti-obesity potential of clove, most of the studies were on extracts and essential oils of clove and were mostly in vivo using mice or rat models and in vitro using cell lines and enzyme based assays. The activities were marked with reduction in fasting blood glucose, protein glycation, increased insulin production, improving glucose tolerance, and glucose and lipid metabolism-related enzyme activities and conversion of cells into adipocytes among other physiological actions. Only one clinical study in humans was reported for antidiabetic property of clove. These studies however, are not detailed enough, neither are they supported with human studies considering the GRAS status of clove.

Therefore, future studies should focus on the effect of clove on humans through randomized clinical trials, targeting specific biomarkers to confirm and validate the folkloric uses of clove as therapy for diabetes and obesity.

Paschoalinotto et al. (2021) showed that a tisane (T5) composed of lemon thyme, tutsan, cloves and cinnamon, exhibited health-promoting effects (lipid peroxidation inhibition, anti-inflammatory, and anti-diabetic activities) and potential for application in the food and nutraceutical industries.

In another study, Günes-Bayir et al. (2020) investigated the effect of adding clove (Syzygium aromaticum) in three different concentrations (0.1, 0.3, and 1%) and propolis (0.03%) to probiotic yoghurt as follows: 1) yoghurt without propolis or clove (control group); 2) yoghurt with propolis (0.03%); 3) yoghurt with propolis (0.03%) and clove (0.1%); 4) yoghurt with propolis (0.03%) and clove (0.3%); 5) yoghurt with propolis (0.03%) and clove (1.0%) respectively on the microbiological, chemical and sensory properties of probiotic yoghurts. They reported that whereas propolis exerted antibacterial effect on bacteria colonies except Streptococcus thermophiles, clove reduced the suppressive effect of propolis on bacteria colonies by supporting the number of B. animalis subsp. lactis growth, improving the microbial qualities and sensory acceptability of the probiotic yoghurts positively.

Nirmala et al. (2019) prepared nanoscale-based emulsions from essential oil of clove buds (Syzygium aromaticum) and varying concentrations of Tween 20 and Tween 80 surfactants. Cytotoxicity of the stable oil-based emulsion was evaluated using MTT colony formation, and Annexin V-FITC assays against thyroid cancer cell line (HTh-7). The authors reported that the product has potential as alternative cancer candidate drug because it showed apoptotic and reductive effect on cancer cell proliferation.

Cinnamon (Cinnamomum zeylanicum), fenugreek (Trigonella foenum-graecum), shallot (Allium hirtifolium Boiss) and clove (Syzygium aromaticum) were evaluated for their antidiabetic efficacy in rats, singly and as a polyherbal formulation (Tetraherbs) in streptozotocin (STZ)-induced diabetic rats with fasting blood sugar above 350 mg/dl. The rats were treated orally with 75 mg/kg dose ethanolic extracts of each specie separately, or in equal combination formulation (Tetraherbs) at 100–300 mg/kg once daily for 28 days. Tetraherbs gave a higher blood glucose lowering activity and pancreatic β cell regeneration than for each plant (Kiani et al., 2018).

Another study by Ramos et al. (2017) evaluated an herbal extract containing green mate (Ilex paraguariensis), clove (Syzygium aromaticum), and lemongrass (Cymbopogon citratus) optimized in fermented milk with or without sweet potato pulp. Addition of a lyophilized extract (1 g 100 g−1) of 87.5% clove and 12.5% green mate increased the antioxidant and total phenolic content, though fermented milk with added sweet potato pulp had the best sensory acceptance. Twenty-one-day storage revealed slight decrease in total phenolic acid and total reducing capacity. The authors proposed the addition and use of this extract for the development of new dairy foods with potential functional properties.

The safety and anti-ulcerogenic activity of a Clovinol, a novel polyphenol-rich extract of clove buds (Syzygium aromaticum L) was reported by Issac et al. (2015). Clovinol was derived as a water soluble free flowing powder from clove buds, without the characteristic pungency and aroma. The extract was characterized by electrospray ionization-time of flight mass spectrometry (ESI-TOF-MS) and evaluated for antioxidant, anti-inflammatory and anti-ulcerogenic activities in mice and rat models. The product exhibited significant antioxidant and anti-inflammatory effects measured by cellular antioxidant levels and inhibition of carrageenan-induced paw swelling in mice; while its anti-ulcerogenic activity was confirmed by greater than 97% inhibition of ethanol-induced stomach ulcers in Wistar rats administered at 100 mg per kg b w. orally and up regulation of in vivo antioxidants. Lipid peroxidation, oxidative stress and gastrointestinal health of ulcer induced rats were also improved by Clovinol, which was shown to be safe at 5 and 2.5 g per kg body weight for acute and 28 days of repeated dose toxicity studies respectively.

Babajide et al. (2013) blended equal portions of cucumber (50%) and pineapple (50%) juices with clove and ginger powder at 0.25% (CPCLG1), 0.5 (CPCLG2), 0.75% (CPCLG3), and 1% (CPCLG4) (w/v) respectively to develop a new fruit drink with health benefits. They reported the presence of alkaloids, flavonoids, saponins, steroids, tannins, terpenoids and phlobatannins. Various Quantities of isoquinoline, octanoic acid, metroprolol, fumaric acid, benzoquinone, and betaxolol among others.

Balaji et al. (2012) investigated the antidiabetic potentials of a novel polyherbal preparation containing Asparagus racemosus, Emblica officinalis, Salacia oblonga, Syzygium aromaticum, and Tinospora cordifolia in equal ratios. The authors showed that the product reverted the activities of glycolytic and gluconeogenic enzymes in diabetic rats and that lipid profile, antioxidant status and glycogen content were improved, with decrease in lipid peroxidation.

The studies reported here are some examples of the use of clove in preparation of functional foods and beverages, tailored towards the management of some chronic diseases. Some of these show that the therapeutic property of clove can withstand food processing/thermal changes and are carried through to the final products. However, more studies are needed to determine appropriate vehicles, doses, shelf-life and toxicity/adverse consequences in human trials to validate these therapeutic claims. These functional foods however, can be used co-adjuvantly in the prevention, treatments and management of chronic diseases.