1. Introduction
Diseases of the gastrointestinal tract are a common medical problem in modern society. A number of mechanism-different diseases of the gastrointestinal tract are as follows: typical feed or chemical poisoning [
1,
2], chronical [
3] or situational digestive disorders, old age related disorders [
4], bacterial infections [
5,
6,
7,
8] and rotavirus infections that lead to gastritis gastroduodenitis, peptic ulcer of the stomach and duodenum, gastroesophageal reflux disease, pancreatitis, functional diseases of the gastrointestinal tract [
9]. These infections lead to inflammation and disruption of the gastrointestinal tract with more or less pronounced symptoms.
Helicobacter pylori is the leading etiological factor of many diseases of the stomach and duodenum.
H. pylori can cause gastroesophageal reflux disease and chronic atrophic gastritis [
10]. The European Group for the study of
Helicobacter pylori has adopted a number of treatment regimens for the disease related to this bacterial infection (
https://www.ehmsg.org/publications). The treatment strategy is that it is necessary to use a combination of 2 antibiotics with different mechanism that prevents the development of the resistance. Thus, the first line: 1) proton pump inhibitor (omeprazole or analogues), 2) clarithromycin, 3) amoxicillin or metronidazole (MN) – at least 7-10 days. Second line: 1) proton pump inhibitor (omeprazole or analogues), 2) bismuth-based drug, 3) MN and 4) tetracycline. However, these drugs have an adverse effect on the organism due to the high dosage. In particular, MN is well absorbed (up to 80%) and metabolized in liver, causing hepatotoxicity, and in the case of gastrointestinal diseases caused by
H. pylori, it would be better if it remained longer and acted on the mucous shell.
Currently, the direction of bio-friendly medicine is actively developing, in other words, the use of safe natural extracts and essential oils [
11,
12,
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24,
25,
26], which have antiinflammatory, antitumor, antioxidant and regenerating properties. Earlier we showed that adjuvants (allylmethoxybenzenes, terpenoids, etc.) have an enhancing effect on antibacterial drugs, including LF and MF [
15,
17,
18,
27].
Plant extracts exhibiting an anti-
H.pylori effect are the main source of the most important biologically active compounds, in particular such as polyphenols, flavonoids, terpenoids, etc. Flavonoids (for example, baicalein) have multiple biological effects, including antiviral, antithrombotic, anti-ischemic, anti-inflammatory, antihistamine, antioxidant, and block the activity of free radicals [
28,
29]. The gastroprotective effect of flavonoids is associated with an increase in endogenous prostaglandins, a decrease in histamine secretion, absorption of free radicals, oxygen derivatives, and even stimulation of mucus formation by stomach cells. Catechins – the main component of green tea, inhibit
H. pylori urease [
30].
For a number of biologically active components, anti-H.pylori activity was shown: Eugenol (EG) and cinnamaldehyde inhibited the growth of H. pylori strains tested, at a concentration of 2 µg/ml, while in an acidic environment (gastric juice model) activity increases, and bacterial resistance does not develop. These data indicate the potential use of adjuvants in combination with the main components (antibiotics LF or MN) for the treatment of gastrointestinal diseases, in particular H.pylori- or E.coli-associated infections.
To improve the effectiveness of drugs, it is necessary to increase solubility, bioavailability and prolongation, as well as to give the composition mucoadhesive, enveloping and wound healing properties which will promote treatment. It is very appropriate to use chitosan (a natural polymer of D-glucosamine) for this purpose [
10,
31,
32]: since it is biocompatible, biodegradable, mucoadhesive and capable of enhancing the effect of drugs and accelerating tissue regeneration [
33,
34,
35,
36]. Since there is a pH gradient in the gastrointestinal tract, medicinal formulations based on chitosan will “intelligently” release the drug, as well as adhere to the stomach wall and cause local healing of ulcers [
36]. Chitosan and its derivatives enhance the antibacterial effect on Gram-negative bacteria and gram-positive bacteria of various drugs due to adsorption on the cell surface and increased influx [
10,
37]. It is known that one of the important mechanisms of
H. pylori infection is the interaction of bacterial oligosaccharides located on the outer cell wall with glycoproteins and epithelial mucins of the mucous membrane [
38,
39]. In this regard, an important role in suppressing this activity of adhesives belongs to natural polysaccharides, such as chitosan. Polysaccharides can interact with bacterial patterns and prevent infection of the gastrointestinal mucosa with
H. pylori [
40].
Inflammatory diseases of the gastrointestinal tract can also be caused by other bacteria:
Clostridioides difficile in ulcerative colitis and invasive adhesive
E. coli in Crohn's disease [
41].
E. coli can be used as a model of
H. pylori available for research – as a primary screening of the effectiveness of the formulations being developed.
E. coli is a gram-negative bacterium, a common inhabitant of the human gastrointestinal tract and pathogenic
E. coli can cause gastrointestinal diseases.
Here we have attempted to develop a gut-restricted drug formulation for potentially more safe and efficacious treatment of gastrointestinal tract infections: as a combination of levofloxacin (LF) [
42,
43], a powerful fluoroquinolone, less toxic and less prone to the development of bacterial resistance, in the complex with adjuvants and included in chitosan-based systems for mucoadhesiveness and prolongation of action on the gastral- (gut-)mucosa. Этo пoзвoлит избежать всасывания и high systemic exposure, and hence, unfavourable side effects, whereas their exposure in stomach mucus, the predominant location of the bacteria, будет усилена.
Thus, this paper presents experimental bases for creating a combined formulation for the treatment of gastrointestinal diseases based on several components: main drug LF/MN or miramistin (MM) enhanced with salicylhydroxamic acid (SHA, a powerful and irreversible inhibitor of the urease enzyme of various bacteria) and plant extracts (EG, menthol, baicalein, limonene, linalool as efflux inhibitors, membrane-penetrating enhancing agent, antioxidant, tissue regenerating agent).
2. Materials and Methods
2.1. Reagents
Chitosan oligosaccharide lactate 5 kDa (Chit5), methyl-β-cyclodextrin (MCD), baicalein, (+)-limonene were purchased from Sigma Aldrich (St. Louis, MI, USA). Menthol and linalool were purchased from Rotichrom GC (Germany). Eugenol (EG) and menthol at the highest commercial quality were purchased from Acros Organics (Belgium). Levofloxacin (LF) from Zhejiang Kangyu Pharm Co Ltd. (China). The rest of the reagents were kindly provided by the Institute of Organic Chemistry of the Russian Academy of Sciences (Krylov S.S.). Organic solvents, salts and acids – production Reakhim (Russia). Components for LB medium were bactotrypton, agarose and yeast extract (Helicon, Russia), NaCl (Sigma Aldrich, USA).
2.2. Preparation of β-Cyclodextrin Inclusion Complexes, Chitosan Nanogels
Preparation of methyl-β-cyclodextrin (MCD) inclusion complexes was performed as earlier described [
18].
Using CD spectroscopy (Jasco J-815 CD Spectrometer, Japan), the degree of deacylation of Chit5 was determined by the peak at 215 nm corresponding to the absorption of the amide bond and it was 92–95%.
2.3. Nanoparticles Obtaining and Characterization
Chit5 nanoparticles was obtained after 1 h incubation of 5 mg of Chit5 and drug-MCD inclusion complex (5 mg on drug for 20-35% of mass content in final formulation) followed by extrusion (200 or 400 nm membrane, Avanti Polar Lipids)
Chit5-genipin (Chit5-gen) nanoparticles was obtained after 24 h incubation of Chit5 nanoparticles with 0.1 mg of genipin (dissolved in 10 µL of EtOH).
Particles hydrodynamic diameter sizes and ζ-potentials were measured using Zetasizer Nano S «Malvern» (Malvern, UK) as earlier described [
44]. Topography, phase and magnitude signal images of the nanogels deposited onto freshly cleaved surface of mica were obtained by atomic force microscopy (AFM) using a scanning probe microscope NTEGRA Prima (NT-MDT, Russia) [
44].
2.4. FTIR Spectroscopy
FTIR spectra of samples were recorded using a Bruker Tensor27 spectrometer equipped with a liquid nitrogen-cooled MCT (mercury cadmium telluride) detector, as described earlier [
15,
37,
45,
46].
2.5. UV Spectroscopy
UV spectra were recorded on the AmerSham Biosciences UltraSpec 2100 pro device (USA) three times in the range of 200–700 nm in a quartz cell Hellma 100–QS with an optical path of 1 cm.
2.6. Drug Capacity of Nanogels
Drug capacity of nanogels was determined using UV and FTIR spectroscopy using calibration spectra. Mass content of drug was varied from 20 to 35%.
2.7. Study of the Mucoadhesive Properties of Nanogels
To study the mucoadhesive properties, a film of mucin of 1 mg per cell of a culture tablet was used, then 200 µl of nanogel (0.5 mg/mL) was added. UV and FTIR spectra of the solution over the mucin film were recorded at certain intervals and the amount of adsorbed substance was determined.
2.8. Antioxidant Activity Using ABTS Assay
The antioxidant activity of drug formulations was determined by using ABTS assay as described in papers [
29,
47,
48]: registering an absorbance at 734 nm of ABTS cation-radical mixed with drugs.
2.9. Antibacterial Activity Studies: FTIR Spectroscopy, Microbiology
The strains used in this study were Escherichia coli (ATCC 25922) from National Resource Center Russian collection of industrial microorganisms SIC "Kurchatov Institute") and Lactobacillus (Lactobacillus plantarum 8P-АЗ, Lactobacillus fermentum 90TC-4, Lactobacillus casei) from commercially available drug Lactobacterin. The culture was cultivated for 18-20 h at 37 °C to CFU/mL ≈ 4×107 (determined by A600) in liquid nutrient medium Luria-Bertani (pH 7.2) with stirring 100 rpm.
FTIR spectra of cells samples suspension were recorded using followed procedure: overnight cell suspensions were washed twice with sterile PBS (pH 7.4) from the culture medium by centrifuging (Eppendorf centrifuge 5415C, 5 min, 5,000×g). The cells are precipitated by centrifugation and separated from the supernatant, washed twice and resuspended in PBS (2×109 CFU/mL) to register FTIR spectra. Cell suspensions were incubated with drug samples and FTIR spectra were registered at 37 °C online.
Microbiologic studies. The culture was cultivated for 18–20 h at 37 °C to CFU ≈ 0.3 × 108 (colony-forming unit) in liquid nutrient medium Luria–Bertani (pH 7.2). The experiments in liquid media were conducted by adding 50 μL of the samples to the 5000 μL of cell culture. The specimens were incubated at 37 °C for 8 days. At the specific time, 300 μL of each sample was taken, diluted with PBS, and the absorbance was measured at 600 nm (with CFU control on Petri dishes). For quantitative analysis of the dependences of CFU (cell viability) on the concentration of MF 50 μL of each sample was diluted 106–108 times and seeded on the Petri dish. Dishes were placed in the incubator at 37 °C for 24 h. Then the number of the colonies (CFU) was counted. The number of living cells was additionally determined by the fluorescence of the DAPI dye relative to obviously dead cells and living ones after 10 minutes incubation of 200 µL of a cell suspension sample with 1 µg/mL of DAPI.
4. Conclusions
Gastrointestinal diseases are a serious problem in the modern world, pathogenic microorganisms resistant to antibiotics pose a particular threat. An actual direction in modern science is the use of safe, biocompatible components of essential oils (such as terpenoids, flavonoids, allylbenzenes, etc.), which have a number of important biological properties: antibacterial, anti-inflammatory, antioxidant, regenerating activity. The second promising direction in the field of drug development is the use of polymers of natural origin (chitosan, mannan, heparin, pectin, etc.) as drug delivery systems in form of nanogels. In this paper, we have combined these two approaches to achieve a powerful effect in the aspects of creating medicinal formulations for the treatment of infectious diseases of the gastrointestinal tract. In this work, we have developed a promising drug formulation based on following components: the main drug (antibiotic), its adjuvant in the complex with cyclodextrin (MCD) (for solubility) and chitosan for the formation of nanogels that demonstrate improved properties compared to a simple polymer. E. coli cells (H. pylori model) as target and Lactobacilli (“good” cells) were considered to study the selectivity of the combined formulation against target bacteria. We selected 4 candidates for the main component and 10 as an additional one and optimized the composition of the drug formulation in terms of strength and selectivity to antibacterial action. The use of natural extracts and essential oils is a promising direction for the creation of non-toxic drugs, besides having antibacterial, antioxidant, wound-healing activity. Adjuvants are able to inhibit efflux in bacteria due to which it is possible to overcome drug resistance. The genipin stitched chitosan-based nanogels used in the present work are well sorbed on the surface of the mucous membrane, thereby increasing the bioavailability of the drug and at the same time acting as an antiadhesive agent for pathogenic bacteria, preventing infection of the body. Free drugs are absorbed into the gastrointestinal tract, and this should be avoided at the expense of chitosan nanogels, if we want it to act specifically on infection in the gastrointestinal tract, and not get into the system bloodstream and metabolized in the liver. Thus, the presented combined formulation is due to the joint action of each component by different mechanisms, which potentially significantly strengthens existing methods of treating infectious diseases of the gastrointestinal tract and, in principle, claims the right to be an independent therapy strategy.
Figure 1.
FTIR spectra of drugs in free form, in form of MCD-inclusion complexes, including wrapped in a Chit5 polymer globule or cross-linked Chit5 with genipin: (a) linalool, (b) zephirol, (c) quercetin, (d) dihydroquercetin, (e) EG, (f) baicalein, (g) myristicin, (h) limonene, (i) azaron, (j) LF, (k) MN, (l) MM, (m) SHA. PBS (0.01 M, pH 7.4). T = 22 °C.
Figure 1.
FTIR spectra of drugs in free form, in form of MCD-inclusion complexes, including wrapped in a Chit5 polymer globule or cross-linked Chit5 with genipin: (a) linalool, (b) zephirol, (c) quercetin, (d) dihydroquercetin, (e) EG, (f) baicalein, (g) myristicin, (h) limonene, (i) azaron, (j) LF, (k) MN, (l) MM, (m) SHA. PBS (0.01 M, pH 7.4). T = 22 °C.
Figure 2.
(a) FTIR spectra of limonene-MCD in Chit5-genipin particles depending on temperature – phase transition of chitosan nanogels. (b) Corresponding dependences of the position of the characteristic peaks on temperature. (c), (d) Flow cytometry diagrams of Chit5-genipin nanogel with loaded FITC (CFITC = 1 µg/mL). SSC – side scattering, FSC – front scattering, FITC – fluorescence channel.
Figure 2.
(a) FTIR spectra of limonene-MCD in Chit5-genipin particles depending on temperature – phase transition of chitosan nanogels. (b) Corresponding dependences of the position of the characteristic peaks on temperature. (c), (d) Flow cytometry diagrams of Chit5-genipin nanogel with loaded FITC (CFITC = 1 µg/mL). SSC – side scattering, FSC – front scattering, FITC – fluorescence channel.
Figure 3.
The dependences of E. coli and Lactobacillus colony-forming units on the incubation time of cells with antibacterial drugs. For E. coli: С(LF) = 1 μg/mL, C(MN) = 0.1 mg/mL, C(zephirol) = 0.1 mg/mL, C(EG) = 0.1 mg/mL. For Lactobacillus: С(LF) = 10 μg/mL, C(MN) = 1 mg/mL, C(zephirol) = 0.1 mg/mL, C(EG) = 1 mg/mL. LB medium (pH 7.2). 37 °C.
Figure 3.
The dependences of E. coli and Lactobacillus colony-forming units on the incubation time of cells with antibacterial drugs. For E. coli: С(LF) = 1 μg/mL, C(MN) = 0.1 mg/mL, C(zephirol) = 0.1 mg/mL, C(EG) = 0.1 mg/mL. For Lactobacillus: С(LF) = 10 μg/mL, C(MN) = 1 mg/mL, C(zephirol) = 0.1 mg/mL, C(EG) = 1 mg/mL. LB medium (pH 7.2). 37 °C.
Figure 4.
(a) FTIR spectra of suspension of E. coli cells (109 CFU) after a day of incubation with drug formulations in Chit5 particles. С(LF) = 1 μg/mL, C(MN, MM, SHA) = 0.1 mg/mL, C(other substances) = 1 mg/mL. LB medium (pH 7.2). (b) FTIR spectra of suspension of E. coli cells (109 CFU) during incubation (online) with linalool-MCD in Chit5 particles. 37 °C. (c), (d) Flow cytometry diagrams of E. coli cells incubated with FITC-labelled Chit5-genipin nanogel 15 min (CFITC = 1 µg/mL). (e), (f) Flow cytometry diagrams of E. coli cells (control). Green indicates a population with a high intensity of FITC fluorescence. SSC – side scattering, FSC – front scattering, FITC – fluorescence channel.
Figure 4.
(a) FTIR spectra of suspension of E. coli cells (109 CFU) after a day of incubation with drug formulations in Chit5 particles. С(LF) = 1 μg/mL, C(MN, MM, SHA) = 0.1 mg/mL, C(other substances) = 1 mg/mL. LB medium (pH 7.2). (b) FTIR spectra of suspension of E. coli cells (109 CFU) during incubation (online) with linalool-MCD in Chit5 particles. 37 °C. (c), (d) Flow cytometry diagrams of E. coli cells incubated with FITC-labelled Chit5-genipin nanogel 15 min (CFITC = 1 µg/mL). (e), (f) Flow cytometry diagrams of E. coli cells (control). Green indicates a population with a high intensity of FITC fluorescence. SSC – side scattering, FSC – front scattering, FITC – fluorescence channel.
Figure 5.
Free radical-scavenging activity of adjuvants and main drugs (LF, MM, MN, SHA) examined by using ABTS assay.
Figure 5.
Free radical-scavenging activity of adjuvants and main drugs (LF, MM, MN, SHA) examined by using ABTS assay.
Figure 6.
(a) FTIR spectra of pre-incubated mucin with limonene-containing formulations and chitosan. pH 2. T = 37 °C. (b) Adsorption curves of LF (0.1 mg/mL), baicalein (0.1 mg/mL) and zephirol (0.1 mg/mL) in polymeric particles on a mucin (1 mg) substrate. 0.01M HCl or 0.01M Na-phosphate buffer (pH 7.4).
Figure 6.
(a) FTIR spectra of pre-incubated mucin with limonene-containing formulations and chitosan. pH 2. T = 37 °C. (b) Adsorption curves of LF (0.1 mg/mL), baicalein (0.1 mg/mL) and zephirol (0.1 mg/mL) in polymeric particles on a mucin (1 mg) substrate. 0.01M HCl or 0.01M Na-phosphate buffer (pH 7.4).
Table 1.
Cell viability (% relative to control) of E. coli cells after two days of incubation with samples in LB medium. Selectivity coefficients (the ratio of activities against different strains) of antibacterial formulations (E. coli vs Lactobacillus). T = 37 °C.
Table 1.
Cell viability (% relative to control) of E. coli cells after two days of incubation with samples in LB medium. Selectivity coefficients (the ratio of activities against different strains) of antibacterial formulations (E. coli vs Lactobacillus). T = 37 °C.
|
in MCD |
in MCD-Chit5 |
in MCD-Chit5-gen |
1 mg/mL |
0.1 mg/mL |
Selectivity (E. coli vs Lactobacillus) |
1 mg/mL |
0.1 mg/mL |
Selectivity (E. coli vs Lactobacillus) |
1 mg/mL |
0.1 mg/mL |
Selectivity (E. coli vs Lactobacillus) |
Linalool |
78±6 |
85±8 |
0.6±0.1 |
73±4 |
82±5 |
0.20±0.05 |
58±3 |
76±7 |
0.30±0.06 |
Menthol |
83±6 |
90±4 |
1.1±0.1 |
81±5 |
90±3 |
0.4±0.1 |
79±6 |
84±5 |
0.4±0.1 |
Zephirol |
12±2 |
34±3 |
0.8±0.1 |
10±1 |
33±2 |
1.2±0.2 |
10±1 |
23±2 |
1.6±0.2 |
Quercetin |
85±5 |
92±4 |
1.1±0.1 |
87±4 |
92±3 |
0.9±0.1 |
82±6 |
89±5 |
0.9±0.1 |
Dihydroquercetin |
86±3 |
91±4 |
1.1±0.1 |
87±5 |
95±2 |
1.0±0.1 |
83±4 |
87±6 |
0.24±0.02 |
Eugenol |
79±3 |
90±4 |
0.9±0.1 |
70±2 |
85±3 |
1.1±0.1 |
67±5 |
82±4 |
1.2±0.1 |
Baicalein |
26±3 |
72±7 |
1.8±0.2 |
49±4 |
68±6 |
1.6±0.2 |
66±3 |
77±5 |
0.8±0.1 |
Myristicin |
80±5 |
87±5 |
1.0±0.1 |
86±4 |
86±7 |
1.2±0.1 |
90±2 |
89±3 |
1.0±0.1 |
Limonene |
83±4 |
90±2 |
1.0±0.1 |
87±3 |
88±5 |
1.0±0.1 |
87±6 |
89±4 |
0.9±0.1 |
Azaron |
81±5 |
87±4 |
0.7±0.1 |
86±6 |
91±3 |
0.8±0.1 |
64±3 |
87±2 |
1.0±0.2 |
|
1 μg/mL |
0.1 μg/mL |
Selectivity (E. coli vs Lactobacillus) |
1 μg/mL |
0.1 μg/mL |
Selectivity (E. coli vs Lactobacillus) |
1 μg/mL |
0.1 μg/mL |
Selectivity (E. coli vs Lactobacillus) |
LF |
9±1 |
16±3 |
2.3±0.2 |
7±1 |
14±2 |
3.6±0.3 |
7±1 |
13±3 |
4.0±0.3 |
|
10 μg/mL |
1 μg/mL |
Selectivity (E. coli vs Lactobacillus) |
10 μg/mL |
1 μg/mL |
Selectivity (E. coli vs Lactobacillus) |
10 μg/mL |
1 μg/mL |
Selectivity (E. coli vs Lactobacillus) |
MN |
41±4 |
77±5 |
0.8±0.1 |
46±7 |
82±8 |
0.7±0.1 |
20±2 |
69±3 |
1.4±0.1 |
MM |
10±2 |
26±4 |
0.7±0.1 |
9±1 |
18±3 |
2.3±0.3 |
10±1 |
15±1 |
2.4±0.1 |
SHA |
20±3 |
84±5 |
0.5±0.1 |
20±2 |
85±3 |
0.6±0.1 |
17±2 |
76±8 |
0.7±0.1 |
Table 2.
Synergy coefficients (SYC) of antibacterial activity of LF+X in MCD-Chit5 in comparison with alone LF and X in MCD-Chit5 against E. coli and Lactobacillus cells. The SYC was interpreted as: synergism (> 1.2), additivity (0.8-1.2), or antagonism (< 0.8). Selectivity coefficients (SEC, the ratio of activities against different strains) of antibacterial formulations (E. coli vs Lactobacillus). The SEC was interpreted as: highly specific against E. coli (>2), highly specific against Lactobacillus (<0.5), specific against E. coli (> 1.3), specific against Lactobacillus (<0.85), indifference (0.85<SEC<1.3). T = 37 °C.
Table 2.
Synergy coefficients (SYC) of antibacterial activity of LF+X in MCD-Chit5 in comparison with alone LF and X in MCD-Chit5 against E. coli and Lactobacillus cells. The SYC was interpreted as: synergism (> 1.2), additivity (0.8-1.2), or antagonism (< 0.8). Selectivity coefficients (SEC, the ratio of activities against different strains) of antibacterial formulations (E. coli vs Lactobacillus). The SEC was interpreted as: highly specific against E. coli (>2), highly specific against Lactobacillus (<0.5), specific against E. coli (> 1.3), specific against Lactobacillus (<0.85), indifference (0.85<SEC<1.3). T = 37 °C.
Compound X
|
Linalool |
Menthol |
Zephirol |
Quercetin |
Dihydroquercetin |
Eugenol |
Baicalein |
Myristicin |
Limonene |
Azaron |
MN |
MM |
SHA |
E. coli |
1.06±0.13 |
1.49±0.20 |
1.14±0.05 |
1.3±0.1 |
1.3±0.2 |
1.16±0.07 |
1.15±0.05 |
1.08±0.09 |
1.27±0.08 |
1.0±0.1 |
0.23±0.02 |
1.23±0.05 |
1.73±0.24 |
Lactobacillus |
0.76±0.03 |
1.11±0.05 |
0.26±0.02 |
1.15±0.06 |
1.06±0.08 |
0.91±0.05 |
0.83±0.03 |
1.24±0.12 |
1.02±0.06 |
0.91±0.09 |
0.69±0.05 |
1.06±0.07 |
0.91±0.08 |
Selectivity E. coli vs Lactobacillus
|
1.4±0.1 |
1.3±0.1 |
4.4±0.4 |
1.1±0.1 |
1.2±0.1 |
1.3±0.1 |
1.4±0.1 |
0.9±0.1 |
1.2±0.1 |
1.1±0.1 |
0.33±0.04 |
1.2±0.1 |
1.9±0.2 |
Table 3.
IC50 values of free radical scavenging of the studied substances in nanogels (Chit5-genipin) examined by using ABTS assay.
Table 3.
IC50 values of free radical scavenging of the studied substances in nanogels (Chit5-genipin) examined by using ABTS assay.
Compound |
IC50, mg/mL |
Linalool |
0.46±0.07 |
Menthol |
>3 |
Zephirol |
0.27±0.05 |
Quercetin |
<0.01 |
Dihydroquercetin |
Eugenol |
Baicalein |
Myristicin |
Limonene |
1.5±0.2 |
Azaron |
~0.01 |
LF |
0.015±0.005 |
MN |
0.04±0.01 |
MM |
0.008±0.002 |
SHA |
<0.01 |