Noni fruit was harvested by hand in French Polynesia and allowed to fully ripen. The fruit was then processed into a puree by mechanical removal of the seeds and skin with a micro-mesh screen in a commercial fruit pulper. This puree was then pasteurized at 87˚C for 3 seconds at a good manufacturing certified fruit processing facility in Mataiea, Tahiti. This is the same source of noni fruit puree that was approved by the European Commission in 2003 as a safe novel food for use in pasteurized beverages [29]. The puree was then filtered through a 0.45 μm PTFE filter to produce a clarified noni juice for testing.

Previously, we identified iridoids as the major phytochemical constituents of noni fruit. We confirmed the presence of these iridoids (deacetylasperulosidic acid, asperulosidic acid and scandoside) according to our validated method [30]. The filtered puree was diluted with methanol-H₂O (1:1), and chromatographic separation was performed using a C-18 column (4.6 mm × 250 mm; 5 μm, Waters Corporation, Milford, MA, USA) and two mobile phases: A; acetonitrile (MeCN), and B; 0.1% formic acid in H₂O (v/v). A linear gradient of 100% B for 0 - 5 minutes was followed by 70% B and 30% A for 40 minutes at an elution rate of 0.8 mL/min. Eluted compounds were detected by photodiode array (PDA) within 210 - 400 nm. Pure deacetyl asperulosidic acid, asperulosidic acid and scandoside were also obtained for use in the spike protein binding experiments.

The ability of noni juice and iridoid samples to inhibit the binding of SARS-CoV-2 spike protein to ACE2 was measured by an enzyme-linked immunosorbent assay kit (Cayman Chemical Company, Ann Arbor, Michigan, USA) involving recombinant rabbit immunoglobulin (Fc)-tagged SARS-CoV-2 spike S1 receptor binding domain (RBD) and His-tagged ACE2. In this assay, a polystyrene 96-microwell plate is precoated with a mouse anti-rabbit monoclonal antibody attached to the rabbit Fc-tagged spike S1 RBD. In the absence of an inhibitor, a recombinant histidine (His)-tagged ACE2 protein binds the spike S1 RBD. The resulting complex is detected with a horseradish peroxidase (HRP)-conjugated anti-His antibody which is quantified by measuring absorbance at 450 nm in a microplate reader (Synergy™ HT, BioTek Instruments, Winooski, Vermont, USA). The absorbance measured without any inhibitor served as the initial activity control, representing 100% spike protein binding. Prior to absorbance measurements, the experiment was performed as follows. A buffer composed of 0.14 M NaCl, 10 mM dibasic sodium phosphate, 0.1% bovine albumin, 1.76 mM monobasic potassium phosphate and 2.68 mM KCl was prepared. 100 µL of this buffer was added to the precoated microwells. Iridoid samples were prepared by dissolving them in the buffer. Next, 50 µL of the dissolved sample solutions, noni juice or buffer (initial activity control) were added to the microwells. Lyophilized His-tagged ACE2 protein (Cayman Chemical Company) was dissolved in the buffer, and 50 µL of this solution was added to each microwell. The Lyophilized SARS-CoV-2 spike RBD (Cayman Chemical Company) was also dissolved in buffer, with the 50 µL of the resulting solution being added to each microwell. The microwell plate was incubated 60 minutes at room temperature on an orbital shaker. Following this initial incubation, the wells were washed three times with 300 µL of a wash buffer composed of 7.61 mM dibasic potassium phosphate and 2.37 mM monobasic potassium phosphate and Polysorbate 20 (500 µL/L). After washing, 150 µL of HRP conjugated anti-His-antibody solution, prepared with a new buffer (0.4 M NaCl, 75.78 mM dibasic potassium phosphate, 23.51 mM monobasic potassium phosphate, 1.1% bovine albumin and 0.038% ETDA tetrasodium salt), was added to each well. The wells were then incubated a second time at room temperature for 30 minutes on an orbital shaker. Next, the wells were emptied and washed three times again followed by the addition of 175 µL of 3,3',5,5'-tetramethylbenzidine (TMB) solution, a substrate of HRP and incubation for 10 to 20 minutes at room temperature. 75 µL of 2.8% sulfuric acid solution was added to each well to stop the enzymatic reaction. Finally, absorbance was read with the microplate reader.

Inhibition percentage was calculated from the difference between control and sample absorbance divided by the absorbance of the control. Replicate samples of the iridoids were prepared and analyzed at 500 µg/mL. A mixture of the three iridoids, at 167 µg/mL each in the final assay, was also evaluated. The expected percent inhibition of the mixture was calculated from the result of each replicate test and the ratio of the concentration of each iridoid in the mixture to its concentration in the individual test. Replicate assays were performed with the noni juice.

Chromatographic analysis confirmed the presence of iridoids in the noni juice samples (Figure 1). Their concentrations were within the ranges that we had observed previously in pasteurized noni fruit puree [31]. The average (± standard deviation) deacetylasperulosidic acid, asperulosidic acid and scandoside contents of the samples were, respectively, 1.40 ± 0.12, 0.41 ± 0.06 and 0.04 ± 0.01 mg/g.

The results of the SARS-CoV-2 spike protein binding inhibition experiments are summarized in Table 1. Noni juice exhibited significant anti-spike protein potential by reducing average spike protein-ACE2 binding by 69.14%. Deacetylasperulosidic acid did not exhibit significant activity, only reducing binding by approximately 8%. Asperulosidic acid was somewhat more active, reducing binding by almost 15%. Scandoside, however, displayed a high degree of biological activity. On average, it prevented 79.25% of spike protein-ACE2 binding.

*The expected % inhibition of the mixture, determined from individual iridoid results and concentration ratios, is 32.60 ± 1.89.

The mixture of these iridoids, with each at lower concentrations, was expected to reduce spike protein-ACE2 interaction by 32.60%. But the mixture was much more active than expected, preventing binding by an average of 55.48%. Deacetylasperulosidic acid is the major phytochemical constituent of noni juice. It has been found to be responsible, to a large degree, for the antioxidant, immunomodulatory and DNA protective properties of noni juice. However, our results suggest that it has a minor role in the ability of noni juice to prevent SARS-CoV-2 binding of the ACE2 receptor. Scandoside displayed very high activity in preventing SARS-CoV-2 binding. But asperulosidic acid and scandoside are present in smaller concentrations in noni juice than those evaluated in our assay. Although it appears that all three iridoids act synergistically when combined, with an additional 22.88% reduction in spike protein binding beyond the expected levels, they do not appear to be responsible for all the inhibitory activity of noni juice.

Other phytochemicals in noni juice may also be contributing to its anti-spike protein binding activity. Scopoletin, rutin, quercetin and kaempferol are present in noni fruit and juice [32] [33]. In silico studies (computer modeling or simulation) have revealed that scopoletin and related compounds have structural properties that allow it to bind to and inhibit SARS-CoV-2 main protease (Mpro) as well as interact with ACE2, thereby interfering with spike protein-ACE2 recognition [34] [35] [36]. Rutin exhibited potent spike protein binding and anti-Mpro activity in molecular docking studies [37] [38]. Quercetin and kaempferol demonstrated high binding affinity for the spike protein-ACE2 complex, potentially perturbing effective binding [39]. Thus, it is likely that the combination of multiple phytochemicals in noni juice act synergistically to prevent the binding of SARS-CoV-2 to ACE2.

Our current experiment focused solely on binding interactions between SARS-CoV-2 spike S1 RBD and ACE2 and did not evaluate inhibition of cellular proteases. As host cell proteolytic priming of S1 RBD enhances binding affinity, inhibition of furin and serine proteases by noni juice and its phytochemical constituents may further impede the ability of SARS-CoV-2 to infect cells. Scopoletin is reported to be a potent furin inhibitor [40]. Molecular docking studies found that quercetin also inhibits furin [41]. Geniposide, an iridoid with a chemical structure very similar to deacetylasperulosidic acid and scandoside, displayed a high degree of interaction with TMPRSS2 in silico [42]. This suggests that phytochemicals in iridoids in noni juice may also limit spike protein priming.