Eugenol May Be Highly Effective Against Covid / "Spike Protein"

[Limon9]

A common covid/vaccine-preventative approach discussed on the forum is using ACE2-binding substances to "inhibit viral entry", as has been advocated with vitamin D and Losartan (an early recommendation by Dr. Peat). The function of ACE2 is to degrade angiotensin II into angiotensin I, the former being ostensibly inflammatory and the latter ostensibly anti-inflammatory or at least vasodilatory. Therefore these authors argued, somewhat unusually, that it would be preferable to have substances which directly bind and inactivate the [recombinant - disclaimer - which means it's not real, guys] spike protein without interfering with ACE2...

In any case, they did screening and found eugenol to be one such, verifying it with several in-vitro assays and a mouse covid test. It normalized the abnormal inflammatory mediators, fever, ECG and activity levels consequent of recombinant spike protein exposure. They seemed to find powerful inhibitory activity from upwards of 5 micromoles; I'm not sure about the kinetics of eugenol but it seems like a pretty low dose. The mouse dose mentioned was 25mg/kg, allometrically scaled to 2.5mg/kg, which should be attainable with a few drops of clove oil. Perhaps @haidut can chime in, as he has been an advocate of this substance in the past and even toyed with the idea of a designated eugenol product.

Eugenol, a Component of Holy Basil (Tulsi) and Common Spice Clove, Inhibits the Interaction Between SARS-CoV-2 Spike S1 and ACE2 to Induce Therapeutic Responses​

J Neuroimmune Pharmacol. 2021 Dec;16(4):743-755. Paidi RK et al.

". . . we screened different components of Tulsi leaf and found that eugenol, but not other major components (e.g. ursolic acid, oleanolic acid and β-caryophylline), inhibited the interaction between spike S1 and ACE2 in an AlphaScreen-based assay. By in silico analysis and thermal shift assay, we also observed that eugenol associated with spike S1, but not ACE2. Accordingly, eugenol strongly suppressed the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus (VSV), into human ACE2-expressing HEK293 cells. Eugenol also reduced SARS-CoV-2 spike S1-induced activation of NF-κB and the expression of IL-6, IL-1β and TNFα in human A549 lung cells. Moreover, oral treatment with eugenol reduced lung inflammation, decreased fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice."

"Angiotensin-converting enzyme 2 (ACE2) is a beneficial molecule as it converts angiotensin II (AngII), a vasoconstrictor, to Ang1-7, a vasodilator (Vickers et al. 2002; Zaman et al. 2002). Since the spike protein on the surface of SARS-CoV-2 binds to ACE2 (Machhi et al. 2020; Stower 2020) to enter into human cells and the spike S1 subunit harbors the receptor-binding domain (RBD), we screened different components of Tulsi leaf and found that eugenol was capable of inhibiting the interaction between spike S1 and ACE2. In addition, eugenol inhibited the entry of pseudotyped SARS-CoV-2, but not VSV, into human ACE2-expressing HEK293 cells and suppressed spike S1-induced activation of NF-κB and expression of proinflammatory cytokines in human lungs cells. Oral administration of eugenol also decreased lung inflammation, reduced fever, inhibited arrhythmias, and enhanced locomotor activities in an animal model of COVID-19, indicating that naturally available eugenol may be beneficial for COVID-19."

"As described recently (Paidi et al. 2021), we employed a chemiluminescence-based ACE2:SARS-CoV-2 spike S1 binding assay (catalog# 79,936; BPS Bioscience). We noticed that SARS-CoV-2 spike S1 binding to immobilized ACE2 was inhibited by different doses of eugenol (Fig. 1A). However, ursolic acid, oleanolic acid and β-caryophylline remained unable to inhibit the association between SARS-CoV-2 spike S1 and ACE2 (Fig. 1A), indicating the specificity of the effect."

"In an effort to understand whether eugenol binds to SARS-CoV-2 spike S1 or ACE2, we performed an in silico analysis. We applied a rigid-body protein-protein interaction tool to model the interaction between ACE2 and receptor-binding domain (RBD) of spike S1 in the absence or presence of eugenol. As expected, in the absence of eugenol, various residues (Lys417, Tyr449, Gly496, Asn501, and Tyr505) of spike S1 interacted with Asp30, Asp38, Gln42, and Lys353 residues of ACE2 (Fig. 1B). However, eugenol showed association with spike S1, not ACE2 (Fig. 1C). Interestingly, eugenol interacted with Lys417 residue of spike S1 and due to this binding, the ionic bond (salt bridge) that bound with Asp30 of ACE2 was broken and Lys417 exhibited a different rotameric pose (Fig. 1C)."

"In order to understand whether the complex of spike S1 and ACE2 was conformationally stable and whether eugenol was capable of influencing the preexisting complex, we examined the melting profile of the combination of spike S1 and ACE2 in the absence and presence of eugenol. As evident from sigmoidal melting curve, the complex of spike S1 and ACE2 was conformationally stable (Fig. 1F). However, the presence of eugenol led to a shift of the melting curve of the spike S1:ACE2 complex by 3.27 °C from 59.66 °C to 62.93 °C (Fig. 1F). Together, these results indicate that eugenol binds to SARS-CoV-2 spike S1, but not ACE2, and that eugenol is also capable of associating with the established spike S1:ACE2 complex"

"Since eugenol suppressed the binding of SARS-CoV-2 spike S1 with ACE2, next, we investigated whether eugenol inhibited viral entry. Pseudoviruses are suitable for virus entry assays, as they permit viral entry to be distinguished from other virus life-cycle stages. Therefore, we used lentiviral particles pseudotyped with the SARS-CoV-2 Spike S1 protein. Since human embryonic kidney 293 (HEK293) cells do not have any detectable ACE2 receptors, we used HEK293 cells expressing human ACE2 for entry assay. In pseudovirus luciferase assay, viral entry into cells correlates to the levels of luciferase signals in the cells. While lenti-naked infection did not increase luciferase signals in hACE2-expressing HEK293 cells, marked increase in luciferase activity was seen in pseudo-SARS-CoV-2-infected cells (Fig. 2A), indicating the entry of pseudo-SARS-CoV-2 into hACE2-HEK293 cells. However, eugenol at 5, 10 and 20 µM concentrations strongly inhibited pseudo-SARS-CoV-2-induced luciferase activity (Fig. 2A), suggesting that eugenol inhibits the entry of pseudo-SARS-CoV-2 into hACE2-HEK293 cells. Similar to pseudo-SARS-CoV-2, infection with pseudo-VSV also led to marked increase in luciferase activity in hACE2-HEK293 cells (Fig. 2B). However, in contrast to that seen with pseudo-SARS-CoV-2, eugenol did not inhibit pseudo-VSV-induced luciferase activity in hACE2-HEK293 cells (Fig. 2B)."

"Some COVID-19 patients present a severe symptom of acute respiratory distress syndrome (ARDS) with high mortality. This high severity is dependent on pulmonary inflammation induced by a cytokine storm (Pia 2020), which is most likely mediated by interleukin-6 (IL-6) and other proinflammatory cytokines. NF-κB is a proinflammatory transcription factor (Vallabhapurapu and Karin 2009) and recently we have demonstrated that recombinant SARS-CoV-2 spike S1 induces the activation of NF-κB and the expression of IL-6 in human A549 lung cells (Paidi et al. 2021)."

"As evident by EMSA, recombinant spike S1 induced the DNA-binding activity of NF-κB in A549 cells (Fig. 3A). However, eugenol inhibited spike S1-induced activation of NF-κB (Fig. 3A). To confirm these results, we monitored the expression of TNFα, IL-1β and IL-6, proinflammatory cytokines that are driven by activated NF-κB. Spike S1 increased the expression of TNFα (Fig. 3B), IL-1β (Fig. 3C) and IL-6 (Fig. 3D) in A549 cells. However, eugenol dose-dependently inhibited SARS-CoV-2 spike S1-induced mRNA expression of TNFα (Fig. 3B), IL-1β (Fig. 3C) and IL-6 (Fig. 3D) in A549 cells."

"Although SARS-CoV-2 does not easily bind to ACE2 and infect normal mice, we have observed that intranasal intoxication [?] of SARS-CoV-2 spike S1 induces fever and important cardiac and respiratory symptoms of COVID-19 in normal C57/BL6 mice (Paidi et al. 2021). Therefore, we studied whether eugenol could reduce these symptoms in mice"

"Parallel to that observed in human lung cells, intranasal exposure with recombinant SARS-CoV-2 spike S1 (Fig. 4A) led to the expression of TNFα (Fig. 4C), IL-1β (Fig. 4D) and IL-6 (Fig. 4E) in vivo in the lung of C57/BL6 mice. However, oral administration of eugenol strongly inhibited the mRNA expression of TNFα (Fig. 4C), IL-1β (Fig. 4D) and IL-6 (Fig. 4E) in the lungs of SARS-CoV-2 spike S1-insulted mice. Similarly, eugenol treatment also suppressed the level of TNFα in serum of SARS-CoV-2 spike S1-insulted mice (Fig. 4F). Fever is probably one of the prominent symptoms of COVID-19 (Machhi et al. 2020; Pahan and Pahan 2020) and oral administration of eugenol also led to the normalization of body temperature of SARS-CoV-2 spike S1-intoxicated mice (Fig. 4G)."

"Non-invasive ECG demonstrated cardiac arrhythmias in SARS-CoV-2 spike S1-intoxicated mice [no way!] as compared to control untreated mice (Fig. 5A, B). However, eugenol treatment normalized electrical activity of the heart as evident from ECG (Fig. 5A−C). Similarly, eugenol also steadied heart rate (Fig. 5D), heart rate variability (Fig. 5E), JT interval (Fig. 5F), ORS interval (Fig. 5G), QT interval (Fig. 5H), and RR interval (Fig. 5I) in SARS-CoV-2 spike S1-intoxicated mice. As expected, the level of LDH was also markedly higher in serum of SARS-CoV-2 spike S1-intoxicated mice than normal mice (Fig. 5J). However, eugenol treatment normalized serum LDH in spike S1-intoxicated mice (Fig. 5J)."

"... we found a decrease in overall locomotor activities in SARS-CoV-2 spike S1-intoxicated mice (Fig. 6A–G). Therefore, we investigated whether oral eugenol could improve such behavioral deficits. Interestingly, eugenol treatment increased overall locomotor activities as evident by heat map (Fig. 6A), distance travelled (Fig. 6B), velocity (Fig. 6C), cumulative duration (Fig. 6D), center zone frequency (Fig. 6E), center zone cumulative duration (Fig. 6F), and rotorod performance (Fig. 6G). We did not notice any drug-related side effect (e.g. hair loss, appetite loss, weight loss, untoward infection, irritation, etc.) in any mouse upon treatment with oral eugenol at a dose of 25 mg/kg body weight/d."

"... our chemiluminescence-based binding assay, in silico structural analysis and thermal shift assay clearly delineated that eugenol binds to spike S1, but not ACE2, to inhibit the association between ACE2 and spike S1. Therefore, in this case, without affecting ACE2 function, eugenol should not pose any risk for COVID-19 patients with preexisting cardiovascular, pulmonary and kidney problems. Recently, in a review, Vicidomini et al. (Vicidomini et al. 2021) have described the potential role of clove (a rich source of eugenol) in the frame of anti-COVID-19 therapy, focusing on the antiviral, anti-inflammatory, and antithrombotic effects of clove."

"Several studies have described antioxidant potential of eugenol (Gulcin 2011; Barboza et al. 2018). It has been shown that the antioxidant behavior of eugenol is greater than most of the known or standard antioxidants such as Trolox (Gulcin 2011). According to Jin and Cho (Jin and Cho 2011), eugenol decreases lipidemia in hyperlipidemic zebra fish model, where eugenol causes a great reduction in triglyceride and cholesterol levels in serum samples. Diabetes is an important risk factor for COVID-19 (Kumar et al. 2020) and eugenol has been shown to enhance the activities of carbohydrate metabolism enzymes such as glucose-6-phosphate dehydrogenase and instance hexokinase (Srinivasan et al. 2014). Moreover, in a number of studies (Jaganathan and Supriyanto 2012; Fujisawa and Murakami 2016), eugenol has shown efficacy against a number of diseases such as reproductive disorders, nervous system disorders, microbial infections, tumorigenesis, hypertension, inflammations, and digestive complications. Here, we delineate a new property of eugenol in inhibiting the binding of SARS-CoV-2 spike S1 with ACE2 and suppressing viral entry into host cells. Reduction of lung inflammation, normalization of heart functions, reduction of fever, decrease in serum markers, and improvement in locomotor activities in SARS-CoV-2 spike S1-intoxicated mice by oral administration of eugenol suggest that oral eugenol may be beneficial for COVID-19."

 

[Motorneuron]

A common covid/vaccine-preventative approach discussed on the forum is using ACE2-binding substances to "inhibit viral entry", as has been advocated with vitamin D and Losartan (an early recommendation by Dr. Peat). The function of ACE2 is to degrade angiotensin II into angiotensin I, the former being ostensibly inflammatory and the latter ostensibly anti-inflammatory or at least vasodilatory. Therefore these authors argued, somewhat unusually, that it would be preferable to have substances which directly bind and inactivate the [recombinant - disclaimer - which means it's not real, guys] spike protein without interfering with ACE2...

In any case, they did screening and found eugenol to be one such, verifying it with several in-vitro assays and a mouse covid test. It normalized the abnormal inflammatory mediators, fever, ECG and activity levels consequent of recombinant spike protein exposure. They seemed to find powerful inhibitory activity from upwards of 5 micromoles; I'm not sure about the kinetics of eugenol but it seems like a pretty low dose. The mouse dose mentioned was 25mg/kg, allometrically scaled to 2.5mg/kg, which should be attainable with a few drops of clove oil. Perhaps @haidut can chime in, as he has been an advocate of this substance in the past and even toyed with the idea of a designated eugenol product.

Eugenol, a Component of Holy Basil (Tulsi) and Common Spice Clove, Inhibits the Interaction Between SARS-CoV-2 Spike S1 and ACE2 to Induce Therapeutic Responses​

J Neuroimmune Pharmacol. 2021 Dec;16(4):743-755. Paidi RK et al.

Very interesting, it should also be very promising for degenerative diseases but honestly I have still found very little in the literature.

 

[J.R.K]

A common covid/vaccine-preventative approach discussed on the forum is using ACE2-binding substances to "inhibit viral entry", as has been advocated with vitamin D and Losartan (an early recommendation by Dr. Peat). The function of ACE2 is to degrade angiotensin II into angiotensin I, the former being ostensibly inflammatory and the latter ostensibly anti-inflammatory or at least vasodilatory. Therefore these authors argued, somewhat unusually, that it would be preferable to have substances which directly bind and inactivate the [recombinant - disclaimer - which means it's not real, guys] spike protein without interfering with ACE2...

In any case, they did screening and found eugenol to be one such, verifying it with several in-vitro assays and a mouse covid test. It normalized the abnormal inflammatory mediators, fever, ECG and activity levels consequent of recombinant spike protein exposure. They seemed to find powerful inhibitory activity from upwards of 5 micromoles; I'm not sure about the kinetics of eugenol but it seems like a pretty low dose. The mouse dose mentioned was 25mg/kg, allometrically scaled to 2.5mg/kg, which should be attainable with a few drops of clove oil. Perhaps @haidut can chime in, as he has been an advocate of this substance in the past and even toyed with the idea of a designated eugenol product.

Eugenol, a Component of Holy Basil (Tulsi) and Common Spice Clove, Inhibits the Interaction Between SARS-CoV-2 Spike S1 and ACE2 to Induce Therapeutic Responses​

J Neuroimmune Pharmacol. 2021 Dec;16(4):743-755. Paidi RK et al.

I am wondering if eugenol has all of these effects on various maladies if perhaps the method of action on achieving these results is an anti inflammatory ability to quiet the RAAS in general.
Dr Peat has talked about how it is best to not have the system activated at all as it only causes inflammation and problems.
It was noticed in Italy that people with hypertension that were controlled (presumably by ACE inhibitors as that is the preferred go to) had higher mortality.
Just a layman’s thoughts.

 

[Limon9]

I am wondering if eugenol has all of these effects on various maladies if perhaps the method of action on achieving these results is an anti inflammatory ability to quiet the RAAS in general.
Dr Peat has talked about how it is best to not have the system activated at all as it only causes inflammation and problems.
It was noticed in Italy that people with hypertension that were controlled (presumably by ACE inhibitors as that is the preferred go to) had higher mortality.
Just a layman’s thoughts.

When something has broadly therapeutic effects it's almost never doing just one thing, but it could be involved. At the very least, eugenol blocks lipoxygenase and cyclooxygenase enymes, and is an MAO-B inhibitor.

 

[J.R.K]

When something has broadly therapeutic effects it's almost never doing just one thing, but it could be involved. At the very least, eugenol blocks lipoxygenase and cyclooxygenase enymes, and is an MAO-B inhibitor.

Thank you for this @Limon9 I was unaware that eugenol was a COX and LOX inhibitor like Aspirin. As well as MAO-B inhibitor. Whether it would be therapeutic or not including some ginger cloves or Ceylon cinnamon into the daily diet might be helpful to keep metabolism on the right track.
What are your thoughts respectfully @Limon9 ?

 

[Limon9]

Thank you for this @Limon9 I was unaware that eugenol was a COX and LOX inhibitor like Aspirin. As well as MAO-B inhibitor. Whether it would be therapeutic or not including some ginger cloves or Ceylon cinnamon into the daily diet might be helpful to keep metabolism on the right track.
What are your thoughts respectfully @Limon9 ?

I think eugenol is going to be reasonably safe in any dose below 200mg if someone's liver is not highly compromised or burdened by a dozen other supplements (because nobody does that...) Discounting suspicious cigarettes, clove oil hasn't too many toxicity reports, with cases of liver failure occurring most in children who ingested the entire bottle (5-20ml) at once. With your typical dropper bottle dispensing drops of weight 20-50mg (0.02-0.05ml) at ~80% eugenol content, nobody should enter such a range. 150mg eugenol with a light breakfast is cleared pretty much completely within 24 hours in healthy humans:

​

The metabolism of eugenol in man​

Xenobiotica. 1990 Feb;20(2):209-22. Fischer IU, Unruh GE, Dengler HJ.

"1. The metabolism of eugenol (4-hydroxy-3-methoxy-allylbenzene) was investigated in male and female healthy volunteers. It was rapidly absorbed and metabolized after oral administration and was almost completely excreted in the urine within 24 h. Unmetabolized eugenol excreted in urine amounted to less than 0.1% of the dose. 2. The urine contained conjugates of eugenol and of nine metabolites. The structures of these metabolites, elucidated using g.l.c.-mass spectrometry, and by comparison with synthetic reference compounds, were identified as: eugenol, 4-hydroxy-3-methoxyphenyl-propane, cis- and trans-isoeugenol, 3-(4-hydroxy-3-methoxyphenyl)-propylene-1,2-oxide, 3-(4-hydroxy-3-methoxyphenyl)-propane-1,2-diol, and 3-(4-hydroxy-3-methoxyphenyl)-propionic acid. 3. The structures of the following metabolites were tentatively deduced from mass spectra only, as reference compounds were not available: 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-allylbenzene, 3-(6?-mercapto-4-hydroxy-3-methoxyphenyl)-propane, and 2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-propionic acid. 4. The amounts of the individual metabolites excreted were determined by g.l.c. Some 95% of the dose was recovered in the urine, most of which (greater than 99%) consisted of phenolic conjugates; 50% of the conjugated metabolites were eugenol-glucuronide and sulphate. Other metabolic routes observed were the epoxide-diol pathway, synthesis of a thiophenol and of a substituted propionic acid, allylic oxidation, and migration of the double bond."

"LaVoie et al. (1986) determined the LD50 of eugenol administered by intratracheal instillation, which in rats was: 11 mg/kg body wt, and in hamsters was: 17 mg/kg body wt. These results have shown that eugenol was nearly 250 times more toxic for rats when administered by inhalation than when administered orally (LD50 = 2.68 g/kg (US FDA 1978))."

 

[haidut]

Perhaps @haidut can chime in, as he has been an advocate of this substance in the past and even toyed with the idea of a designated eugenol product.

Great find, thanks for posting.
It looks like eugenol works quite well to prevent the binding of SARS-CoV-2 with ACE2, at concentrations achievable with human doses in the range of 100mg-150mg. Since preventing the binding/deactivation of ACE2 (which can be done by many other substances, not only SARS-CoV-2) has anti-inflammatory and anti-hypertensive effects, this would explain the anti-hypertensive and anti-inflammatory effects eugenol has shown in many studies, even at low doses. I guess combining eugenol with vitamin A/D or K (though not taken at the same time as K, as they can interact in a negative way) would be a great way to handle not only SARS-CoV-2 but also all of its future mutations, as long as they contain the spike protein. Same with the vaccines against COVID-19 and its future incarnations.

Vitamin K, D and A bind to the SARS‐CoV‐2 spike protein

This study shows that Vitamin K, D, and A very effectively bind to a certain part of the SARS‐CoV‐2 spike protein (fatty acid binding site) . Through this mechanism it inhibits viral entry, because it blocks binding to the endogenous ACE2 enzyme. At least this is how I'm reading it, correct me...

raypeatforum.com

 

[J.R.K]

Great find, thanks for posting.
It looks like eugenol works quite well to prevent the binding of SARS-CoV-2 with ACE2, at concentrations achievable with human doses in the range of 100mg-150mg. Since preventing the binding/deactivation of ACE2 (which can be done by many other substances, not only SARS-CoV-2) has anti-inflammatory and anti-hypertensive effects, this would explain the anti-hypertensive and anti-inflammatory effects eugenol has shown in many studies, even at low doses. I guess combining eugenol with vitamin A/D or K (though not taken at the same time as K, as they can interact in a negative way) would be a great way to handle not only SARS-CoV-2 but also all of its future mutations, as long as they contain the spike protein. Same with the vaccines against COVID-19 and its future incarnations.

Vitamin K, D and A bind to the SARS‐CoV‐2 spike protein

This study shows that Vitamin K, D, and A very effectively bind to a certain part of the SARS‐CoV‐2 spike protein (fatty acid binding site) . Through this mechanism it inhibits viral entry, because it blocks binding to the endogenous ACE2 enzyme. At least this is how I'm reading it, correct me...

raypeatforum.com

Great information as always @haidut! Do you think the levels in ginger, cloves and Ceylon cinnamon would be at levels that would provide the same benefits against SARS COV-2 viruses or all other coronavirus for that matter?

 

[J.R.K]

Great find, thanks for posting.
It looks like eugenol works quite well to prevent the binding of SARS-CoV-2 with ACE2, at concentrations achievable with human doses in the range of 100mg-150mg. Since preventing the binding/deactivation of ACE2 (which can be done by many other substances, not only SARS-CoV-2) has anti-inflammatory and anti-hypertensive effects, this would explain the anti-hypertensive and anti-inflammatory effects eugenol has shown in many studies, even at low doses. I guess combining eugenol with vitamin A/D or K (though not taken at the same time as K, as they can interact in a negative way) would be a great way to handle not only SARS-CoV-2 but also all of its future mutations, as long as they contain the spike protein. Same with the vaccines against COVID-19 and its future incarnations.

Vitamin K, D and A bind to the SARS‐CoV‐2 spike protein

This study shows that Vitamin K, D, and A very effectively bind to a certain part of the SARS‐CoV‐2 spike protein (fatty acid binding site) . Through this mechanism it inhibits viral entry, because it blocks binding to the endogenous ACE2 enzyme. At least this is how I'm reading it, correct me...

raypeatforum.com

Are there any indications that eugenol would have a similar property to Aspirin and vitamin E lowering PUFA levels. There are similar characteristics here if I am not mistaken.
eugenol has shown efficacy against a number of diseases such as reproductive disorders, nervous system disorders, microbial infections, tumorigenesis, hypertension, inflammations, and digestive complications. Here, we delineate a new property of eugenol in inhibiting the binding of SARS-CoV-2 spike S1 with ACE2 and suppressing viral entry into host cells. Reduction of lung inflammation, normalization of heart functions, reduction of fever, decrease in serum markers, and improvement in locomotor activities in SARS-CoV-2 spike S1-intoxicated mice by oral administration of eugenol suggest that oral eugenol may be beneficial for COVID-19."