Citation : Babikiana H. Antibacterial and Anti-Inflammatory Properties of Natural Herbal Oil Formulation (NHOF). Jacobs J Exp Cardiol Res 2019; 6(1): 025.

Research Article

Antibacterial and Anti-Inflammatory Properties of Natural Herbal Oil Formulation (NHOF)

Haig Babikian*

Pt. Central Proteina Prima Tbk, Pharmaceuticals Scineses Pte. Ltd, Gedung Pri Matari 2, 3rd Floor Jl. H.R, Rasuna Said Kav, H 1-2 Jakarta 12920, Indonesia

*Corresponding author: Haig Babikian, Pt. Central Proteina Prima Tbk, Pharmaceuticals Scineses Pte. Ltd, Gedung Pri Matari 2, 3rd Floor Jl. H.R, Rasuna Said Kav, H 1-2 Jakarta 12920, Indonesia; E-Mail: haig.babikian@cpp.co.id

Received Date: 05-29-2019 Accepted Date: 06-15-2019 Published Date: 06-25-2019 Copyright: © 2019 Haig Babikiana

Galley Proof

Abstract

Previously we studied the effect of this formula against white spot syndrome virus which was published in Journal of Pharmacognosy and Natural Products (Jha et al., J Pharmacogn Nat Prod 2016 and 2:4) and that gave us the insight to investigate the other effects of this formula (NHOF).

Natural Herbal Oil Formulation (NHOF) produced in the form of secondary metabolites from plants is originally used in traditional medicine. In the present work, the anti- inflammatory, -microbial and -fungal potential of-13 formulation contained Lavandula latifolia, Pinus sylvestris, Jasminum officinale, Citrus limon, Prunus avium, Viola odorata, Gardenia jasminoides, Cocos nucifera, Rosa damascene and Eucalyptus globulus was evaluated. For this, PBMCs from healthy donors were treated with different concentrations of Natural Herbal Formulation and cytokine content (IL-1β, IL-6, IL-10, TNFα and IFNγ) was determined in culture supernatants. The antimicrobial activity of natural formulation was studied by disc diffusion and broth dilution methods with using six bacterial (Staphylococcus aureus ATCC-6538, Staphylococcus epi-dermidis ATCC-12228, Escherichia coli ATCC-8739, Salmonella enteric serovar Typhimurium ATCC-14028, Pseudomonas aeruginosa ATCC-9027, Micrococcus luteus ATCC-10240) and two fungal (Candida albicans ATCC-10231, Candida albicans NCTC-885-653) strains. We observed a prominent suppression of proinflammatory cytokines production by LPS-primed PBMCs in the presence of Natural Herbal Formulation. A Natural Herbal Oil Formulation mixture showed moderate anti-microbial effect. Therefore, our results suggest that Natural Herbal Formulation may serve as a promising pharmaceutical agent with combined anti-inflammatory, -microbial and –fungal action.

Keywords: Natural Herbal Oil Formulation; anti-inflammatory; antimicrobial; herbal; blend

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Introduction

Throughout our evolution, plants were used as a source of products for the treatment of different illnesses. In recent years, there has been renewed interest in usage of natural compounds due to reduced efficacy of synthetic drugs and the number of adverse effects Special attention was focused on numerous plant-derived products. [1, 2] Among the plant-derived extracts, Natural Herbal Oil For-mulation (NHOF) from aromatic medical plants has been attracted substantial research interest. Natural Herbal Oil Formulation is mixture of volatile compounds (main-ly mono- and sesquiterpenoids, benzenoids, amino acid- derivatives, phenypropanoids, etc.) with wide range of pharmacological actions, e.g. antimicrobial, antifungal, an-ti-inflammatory, antioxidant, hyperemic, anti-nociceptive, spasmolytic, diuretic, choleretic, and carminative effects [3, 4]. For centuries, natural products have been widely used for combating infection in various cultures. Accordingly World Health Organization, a large proportion of the hu-man population and livestock still rely on herbal medicinal products [5]. Nowadays, the use of natural antimicrobial agents is noticeably growing for wide variety of purposes including human and animal medication, food industry and environment protection. In our study, the Natural Herbal Oil Formulation obtained from Lavandula latifolia, Pinus sylvestris, Jasminum officinale, Citrus Limon, Prunus avi-um, Viola odorata, Gardenia jasminoides, Cocos nucifera, Rosa damascene and Eucalyptus globulus were selected to develop a drug with antimicrobial and immune modulato-ry properties. Despite the biological activities of essential volatile oil blend (Natural Herbal Formulation) compounds were separately evaluated, combination of Natural Herbal Oil Formulation constituents may represent a valid strategy to achieve better antimicrobial and anti-inflammatory ef-fects. Essential volatile oils were traditionally used to treat many pathological conditions and become popular nowa-days. Despite recent studies have reported the anti- inflam-matory effect of NHOF, there is a substantial lack in under-standing of how single and mixed violate oils balance the pro- and anti-inflammatory responses in culture of human cells. This study was undertaken with the intention of find-

ing out the efficacy of essential volatile oil blend Natural Herbal Formulation as antimicrobial agent with immune modulatory activity for commercial purposes.

Experimental

Natural Herbal Formulation preparation and composi-tion

Each single oil Lavandula latifolia, Pinus sylves-tris, Jasminum officinale, Citrus limon, Prunus avium, Viola odorata, Gardenia jasminoides, Cocos nucifera, Rosa dama-scene and Eucalyptus globulus NHOF were obtained from the vendors who comply to the most strict industry prac-tices: Demeter Agro Research and Improvements Pty Ltd, New Directions Australia Pty Ltd and Australian Botanical Products Pty Ltd. Each NHOF is obtained through steam distillation process and should undergo thorough checking for the quality and chemical compositions based on Euro-pean Pharmacopeia. After the Natural Herbal Oil Formula-tion are declared to pass the quality checking, the mixture of the NHOF is conducted with the following sequence and percentage: Lavender oil-15%, Pine oil-20%, Jasmine oil-5%, C. Limon oil-7%, Prunus avium-8%, Viola odorata-5%, Gardenia jasminoides-10%, Cocos nucifera-9%, Rosa dam-ascene- 6% and Eucalyptus globules-15%. The mixing pro-cess approximately takes 45-60 min under the temperature of 40° C. Tween 80 was added with the quantity of 35% of the total mixture and the mixture was settled for additional 72 h.

Oil composition analysis by GC-MS

The GC–MS analysis of the oil was performed on a DANI Master GC gas chromatograph, coupled with Time of flight mass spectrometer TOF MS. The GC was equipped with a fused silica capillary column Rtx Sil5 (30 m × 0.25 mm i.d., film thickness 0.250 µm, from Restek, USA Helium was used as carrier gas at flow rate of 0,4 mL/min (split ratio 1:20), Injector temperature were 300° C. The oven program started with an initial temperature of 40° C held for 4 min, then the oven temperature was heated at 5° C/min to 80° C Held for 5 min, and then the oven temperature was heat-ed at 5° C/min to 260° C and finally held isothermally for

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25 min. For Master TOF MS detection, an election ionization system, with ionization energy of 70 eV was used. A scan rate of 0.6 s (cycle time: 0.2 s) was applied, Transfer line temperature was 300° C Sampling rate: 5 Hz Mass range: 40- 1000amu Solvent delay: 60 sec Acquisition time: 67 min. The identification of the compounds was based on the comparison of retention indexes and mass spectra of most of the compounds with data generated under identical ex-perimental conditions based on two dimensional searches, [6, 7] GCMS software, namely Master Lab 3 software with deconvolution mode (Dani Instruments Italy) was used for processing and interpretation of mass spectra with several commercially available libraries included: Wiley Registry of Mass Spectral Data (4th Ed.), NIST/EPA/NIH Mass Spectral Library (2012). Components relative concentrations were obtained by peak area normalization.

Antibacterial assay

Following microorganisms were purchased from the American Type Culture Collection and used as test strains: Staphylococcus aureus ATCC-6538, Staphylococcus epidermidis ATCC-12228, Escherichia coli ATCC-8739, Sal-monella enteric serovar Typhimurium ATCC-14028, Pseu-domonas aeruginosa ATCC-9027, Micrococcus luteus ATCC-10240, yeasts Candida albicans ATCC-10231, and Candida albicans NCTC-885-653.Screening of Natural Herbal For-mulation for antibacterial activity was done by the disk diffusion method. Mueller-Hinton agar plates and Cetrim-ide agar plates (for Pseudomonas aeruginosa ATCC- 9027) evenly seeded throughout the plate with each of overnight cultures strains at concentration 1x108 cfu/ml and 1x106 cfu/ml for Candida albicans ATCC-10231; Candida albicans NCTC-885-653. Sterilized standard paper discs (6 mm di-ameter) were placed on the already inoculated culture plates and 10 µl of each undiluted and1:10 and 1:100 warm dH2O diluted oil samples. A standard discs contain-ing 30 µg tetracycline, 40 µg fluconazole for bacterial cultures and Candida albicans respectively were used as reference controls. Tween-80 ̶ 35%, tween-80 ̶ 35% 1:10 and dH2O were used as negative controls. Antibacterial/antifungal activities of the Natural Herbal Formulation against the test strains were determined after incubation

of the test plates for 24 h at 37° C by measuring the di-ameter of zone of growth inhibition (IZ). The assessment of antibacterial activity was based on calculation of the average diameter of the inhibition zones and classified as follows: the strains were termed not sensitive (0) for a diameter smaller than 8 mm, moderately sensitive (+) for an 8–14 mm diameter and sensitive (++) for a diameter larger than 20 mm [8, 9]. Antimicrobial activity screen-ing was conducted in triplicates and results presented as an average mean ± SD.

Broth micro dilution method

Micro dilution susceptibility assay was per-formed for the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concen-tration (MBC). The standard 96 well micro-titer plate method was used. The plates were prepared by dispens-ing 100 µl of TSB (Tryptic Soy Broth) and 20 µl of the inoculums into each well. Each well had a final volume of 120 µl. The plates were incubated at 37 ± 1 °C, for 24 h for bacteria and at 34 ± 1° C, 48 h for yeasts. The in-hibition of growth of bacteria was revealed by the addi-tion of resazurin sterile solution (20 µl, 0.02 % and w/v) and re-incubation for 3 h. MIC was determined by the permanence of blue coloration in the wells. A change of colour from blue to red indicated the bacterial growth. The MIC was calculated as the highest dilution showing complete inhibition of the tested strains. The wells that showed no apparent growth were selected to evaluate the MBC, which was determined by the absence of mi-crobial growth on plates containing TSB. The tests were carried out in duplicates.

Peripheral blood sampling

After signed informed consent, 30 ml of venous blood samples were obtained from 6 healthy subjects: 3 men (mean age of 25 ± 3, 1 year) and 3 women (mean age of 26 ± 3, 4 years). The experiment was approved by the Eth-ical Committee of the Institute of Molecular Biology of the NAS RA (IRB IORG0003427). Healthy volunteer’s selection was conducted based on reviews of medical history with the following exclusion criteria

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1) Studied subjects were not taking any medication at the time of the experiments;

2) None of the individuals suffered from acute/chronic diseases or abnormalities;

3) Smoking, consumption of alcohol, drugs and current or past use of any active substance;

4) Pregnancy or lactating mothers.

In vitro stimulation of PBMCs with plant extracts and LPS

Peripheral blood mononuclear cells (PBMCs) were isolated from freshly obtained, EDTA-treated samples by gradient centrifugation on a discontinuous density gradient (Histopaque 1077, Sigma). The mononuclear cell fraction was washed three times in phosphate- buffered saline and counted.

Freshly isolated PBMCs were cultured in a 24-well flat-bottom plate at 1x106cells/ml in RPMI-1640 medium (Sigma), supplemented 10% fetal bovine serum (FBS) and 2 mML- glutamine (Sigma) in duplicates for each sample in a humidified 5% CO2 incubator for 24 hours at 37° C. For testing the responses to extracts, cells were stimulated with LPS (100 ng/ml) in presence or absence of Natural Herbal Formulation (6*10-2 µg/µl, 6*10-3 µg/µl and 6*10-4 µg/µl) and tween-80 as a negative control. The culture superna-tants were harvested after incubation period, centrifuged to remove cell debris, and stored at −80 for cytokine assay.

Cytokine measurements

Measurement of cytokine levels in the culture su-pernatant was performed with specific immunoassays, ac-cording to the manufacturer’s instructions. Cytokine con-tent was determined with using Human IL-1β, IL-6, IL-10, and TNF-α and IFNg ELISA MAX Deluxe kits (Bio legend, UK). The optical density was determined at 450 nm in a 96-well plate reader (Huma Reader HS, Human Diagnostics Worldwide, and Germany). Results were calibrated with se-rial dilutions of known quantities of recombinant cytokines.

Statistics: Statistical analyses were performed using the statistical software Graph Pad Prism 5.01 (Graph Pad Soft-ware, USA). Data were compared using One-way Repeated Measure ANOVA test. Normal distribution was checked vi-sually from distributions and with Shapiro-Wilk’s W test. P values ≤ 0.05 were considered as significant. Results are ex-pressed as the mean and standard error of the mean.

Oil composition: The phytochemical constituents of Natu-ral Herbal Formulation oil were analyzed using gas chroma-tography–mass spectrometry (GC–MS) having stationary phase nonpolar columns which led to the identification of a total of 168 different compounds from n-hexane extract-ed oil samples. Twenty-six volatile constituents were iden-tified with the corresponding CAS numbers, and they are listed in Table 3. Neryl (nerol) acetate (15.92%), citronellol (9.56%), eucalyptol (cineole, 9.33%), curzerene (8.67%), p-tert-butyl cyclohexyl-acetate (8.23 %) and amyl cinnamal (jasminal, 6.27%) were themain components. The minor constituents included beta-elemene (5.09%), beta methyl ionone (4.47%), canescine (4.40%), 4,6-dimethyl-2,7-non-adien-5-one (3.42%), alpha-hexylcinnamaldehyde (2.57%), myrrh related compound 4,4’- dimethyl-2,2’-dimethylen-ebicyclohexyl-3,3’-diene (2.50%) and beta.

The rest 17% of constituents included highly bio-logically active compounds in various amounts included isopropyl myristate, coumarin, borneol, elemol, pen-tadecanone, alpha-pinene, p-cymen, guaiazulene etc (Table1). 22 constituents out of the total 26 identified compounds (84.6%) were well known antibacterial and antifungal agents and 13(50%) were potent anti- in-flammatory molecules which included NF-κB and MAPK pathway inhibitors (beta methyl ionone, beta ionone, alpha- hexylcinnamaldehyde, borneol, cinnamein and alpha-pinene), LPS-induced COX2 expression inhibitors (citronellol)and STAT3/TLR4 signalling pathway inhibi-tions (beta-element). The nearly all major constituents of the Natural Herbal Formulation oil expressed both potent antimicrobial and anti-inflammatory, activity published in the literature (Table 1).

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Table 1: Natural Herbal Formulation composition (%) ionone (2.47%).

Alteration of cytokines production by oil mix in PBMCs culture

In this study we examined dose-dependent effect of essential volatile oil blend on PBMCs activation state and particularly cytokines production (Figure 1). Production of

IFNg (p<0.05) was significantly increased in cells treated with NHOF at 6*10-3 µg/µl. In contrast, secretion of IL-6 (p<0.01) by PBMCs were reduced, following induction with NHOF (6*10-2 µg/µl).

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Figure 2: Cytokines production by PBMCs, isolated from 6 healthy donors, stimulated with LPS (100 ng/ml) and LPS+Natural Herbal Oil Formulation (concentrations: Natural Herbal Oil Formulation/1 (6*10-2 µg/µl), Natural Herbal Oil Formulation/2 (6*10-3 µg/µl) and Natural Herbal Oil Formulation/3 (6*10-4 µg/µl) for 24h. *p<0.05, **p<0.01, ***p<0.001.

Figure 1: Cytokine production by PBMCs, isolated from 6 healthy donors, cultured for 24h in the presence (concen-trations: Natural Herbal Oil Formulation/1(6*10-2 µg/µl), Natural Herbal Oil Formulation/2 (6*10-3 µg/µl) and Natu-ral Herbal Oil Formulation/3 (6*10-4 µg/µl)) or absence (Ctl) of formulation measured by ELISA. *p<0.05, **p<0.01, ***p<0.001.

7Modulation of LPS-induced cytokines production by Natural Herbal Oil Formulation

We observed prominent anti- inflammatory effect of Natural Herbal Oil Formulation on PBMCs primed with LPS. Particularly, the ability of Natural Herbal Oil Formula-tion to inhibit secretion of several pro-inflammatory cyto-kines was demonstrated (Figure 2). Namely, IFNg (p<0.01) was down-regulated by the highest concentration of Natu-ral Herbal Oil Formulation (6*10-2 µg/µl). Similarly, pro-duction of IL-1β was significantly decreased by all used concentrations of Natural Herbal Formulation (6*10-2 µg/µl, 6*10-3 µg/µl, 6*10-4 µg/µl). Supplementation of medi-um with two highest concentrations of Natural Herbal For-mulation (6*10-2 µg/µl, 6*10-3 µg/µl) reduced the level of IL-6 (p<0.05) and TNFα (p<0.01) compared with the cells incubated only with LPS. Production of anti-inflammatory

IL10 (p<0.01) was also significantly decreased in this group by Natural Herbal Formulation (6*10-2 µg/µl, 6*10-3 µg/µl, 6*10-4 µg/µl).

Screening for antimicrobial activity

The degree of inhibition was determined by the values of inhibition zone (IZ) diameter of Natural Herbal Formulation (Table 2). Natural Herbal Formulation showed a moderate (+) inhibitory activity against four from six tested bacterial strains. The inhibition has been recorded against Gramm positive Micrococcus luteus (IZ, 10.13 ± 0.8762 mm), Staphylococcus aureus (IZ, 10.50 ± 1.756 mm) and Staphylococci occusepidermidis (IZ, 8.3 ± 0.7 mm) and Gramm-negative Pseudomonasaeruginosa (IZ, 12.59 ± 0.4185 mm). Even at the 1:10 water dilution the oil con-tinued shown an antibacterial effect against Pseudomonas aeruginosa (IZ, 8.37 ± 0.29 mm) (data not shown).

Table 2: Antibacterial activity of Natural Herbal Formulation against bacterial and yeast strains expressed by inhibition diameter (mm ± SD). * represent statistical difference (p ≤ 0.05) in the size of inhibition zones formed under the paper disc by Natural Herbal Oil Formulation and the control.

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MIC determination by micro dilution in broth

The antibacterial potency of the Natural Herbal Oil Formulation blend against a panel of pathogenic microor-ganisms was evaluated by the measurement of minimum inhibitory concentration (MIC). The results are presented in Table 3. Natural Herbal Formulation elicited an antibac-terial and antifungal activity. Particularly, Candida albicans strains exhibited a higher sensitivity to the tested formula-tion (from 0.625 mg/ml to 1.25 mg/ml in terms of MIC).

The use of plant-derived agents for the treatment of infectious diseases has a long and successful tradition. In this study we demonstrated that the herbal formula Natural Herbal Formulation which was prepared from a mixture of Lavandula latifolia, Pinus sylvestris, Jasminum officinale, Citrus limon, Prunus avium, Viola odorata, Gar-

denia jasminoides, Cocos nucifera, Rosa damascene and Eucalyptus globules showed moderate antimicrobial ac-tivity against tested bacterial and fungal [10, 11]. Antimi-crobial activity of listed Natural Herbal Oil Formulation against diverse range of human pathogens has previous-ly been shown [12, 13]. Evidently, active compounds of Natural Herbal Oil Formulation such as eucalyptol [14], guaiazulene [15], and citronellal [11, 16] isopropyl myri-state [17] that possess significant antibacterial activity, determine the efficiency of the formulation (Table 3).

In our study, Natural Herbal Oil Formulation in-hibited the growth of several tested microorganisms, including gram- positive Staphylococcus aureus, Staphy-lococcus epidermis, Micrococcus luteus, and gram-nega-tive Pseudomonas aeruginosa. The most prominent anti-

Table 3: Antibacterial activity of Natural Herbal Oil Formulation against bacterial and yeast strains expressed by MIC.

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microbial activity of Natural Herbal Oil Formulation was observed against Staphylococcus epidermis and Pseudo-monas aeruginosa. Nowadays, treatment of opportunis-tic infections caused by skin commensal Staphylococcus epidermis and environmental Pseudomonas aeruginosa is highly complicated due to their extreme resistance to multiple classes of antibiotics and capacity to evade the host defines system by forming biofilms [18, 19]. The use of natural compounds in treatment of gram-nega-tive bacterial infection is challenged by the presence of an outer envelope with impermeable structure which makes them less susceptible to Natural Herbal Oil For-mulation [20, 21]. Still, a variety of polyphenols and ter-penoids contained in high amounts in Natural Herbal Oil Formulation of Pinus sylvestris, Citrus limon, Prunus avi-um, Viola odorata, Cocos nucifera, Rosa damascene and Eucalyptus globules possess a strong binding affinity to different molecular structures of gram-negative bacteri-um and exhibit high potential to permeate through bac-terial cell envelope [22] which probably mediate antimi-crobial action of Natural Herbal Oil Formulation.

Mixed bacterial and fungal infections, usually associated with a more severe diseases course, contin-ue to challenge existing treatment strategies. Candida albicans is the predominant fungal pathogen of humans [23] which closely interact with its microbial neighbours [24]. For instance, C. albicans is most frequently found in mixed infections with Staphylococcus or Pseudomo-nas species [25]. Environmental conditions within such bacterial-fungal consortia promote their growth and can modulate the action of antibiotics or antifungal agents. We suggest that significant sensitivity of C. albicans to Natural Herbal Oil Formulation, elicited by MIC test, along with its ability to inhibit the growth of S. epider-midis and P. aeruginosa may be beneficial for new pro-phylactic, preventative or therapeutic strategies against mixed infections.

Natural Herbal Oil Formulation extracted from Citrus Limon, Cocos nucifera, and Eucalyptus globules is of the most popular natural derivate with known an-tibacterial activities against gram-negative as well as

gram-positive bacteria including resistant strains [8, 9]. In fact, combinations of two or more Natural Herbal Oil Formulation considered to have more beneficial value, therefore, the therapeutic potent from synergistic effect of blended Natural Herbal Oil Formulation are greater than the sum of the individual oils. However, in this study we observed relatively lower antibacterial efficiency than expected. Considering this fact, further investigations are required to optimize the formulation to fully exploit the favourable properties expected of compounds and attain better anti -bacterial and -fungal activity. While inflam-matory response is required for an effective host defence during infections, exaggerated or uncontrolled inflamma-tion is the main cause of healthy tissue destruction and chronic pathologies. Natural Herbal Formulation was de-signed not solely as a formulation with an antimicrobial and antifungal potency but also as an anti-inflammatory agent, capable to limit pathogen- induced inflammation. Anti-inflammatory properties of certain selected oils were assessed in a number of in vivo and in vitro studies using different animal and human models. Particularly, it was shown that flavonoids and terpenes contained in Pi-nus sylvestris, Gardenia jasminoides and Citrus species ex-tracts, exert significant anti-inflammatory effect through the inhibition of NO and inducible NOS (iNOS) expres-sion [26, 27, 28]. Plant extracts from Pinus sylvestris and Citrus species were shown to suppress COX-2 mRNA ex-pression in the murine macrophage cell line J774A.1 [26] and decrease the release of TNF-α from LPS- stimulated macrophage RAW264.7 cells [28], respectively. Garde-nia jasminoides, which is very popular in oriental tradi-tional medicine, is acknowledged for its protective effect against oxidative damage and cytotoxic activity [29, 30]. Anti-inflammatory effect of Gardenia jasminoides was further investigated in mouse model of cerulein-induced acute pancreatitis which resulted in reduced production of pro inflammatory cytokines, such as TNF-α, IL-1β and IL-6 [31]. Citronellal [32], that is one the major active component of R. damascene Natural Herbal Oil Formu-lation, constitutes 9% of the Natural Herbal Oil Formu-lation composition. It was demonstrated previously that citronellal is responsible for prominent anti-inflammato-

10ry properties of R. damascene volatile oil [32, 33]. Nat-ural Herbal Oil Formulation demonstrated a prominent anti- inflammatory effect as reflected by the suppression of IL-1β, TNFα, IL-6 and IFNγ production in a dose-de-pendent manner by LPS- stimulated human mononucle-ar cells. Production of anti- inflammatory IL-10 by LPS-primed cells was also suppressed by Natural Herbal Oil Formulation in a dose-dependent manner. The observed phenomenon is rather a consequence of Natural Herbal Oil Formulation -mediated suppression of LPS-induced pro- inflammatory responses than inhibition of anti-in-flammatory reactions. IL-10 is the most potent anti-in-flammatory cytokine that able to counter-regulate the production of several pro- inflammatory mediators [34]. In our experiment, LPS-induced pro- inflammatory re-sponses were restrained by the administration of Natu-ral Herbal Oil Formulation. Therefore, the level of IL-10, which produced to reduce inflammation, was markedly higher in the absence of Natural Herbal Oil Formulation. This assumption is also supported by the unchanged IL-10 levels in the presence of Natural Herbal Oil Formula-tion alone. Suppression of pro-inflammatory responses and manipulation the activity of anti-inflammatory cy-tokines, via inhibiting of MAPK/NF-κB signalling path-ways, are potent anti-inflammatory mechanisms that underlie the action of certain non-steroidal anti- inflam-matory drugs [35]. However, the use of modern medica-tions is still unsatisfactory and limited especially for a long-time treatment. In this regard, the development of newer anti- inflammatory agents from natural sources as an alternative to conventional therapeutics becomes top-ical. Natural Herbal Oil Formulation from the leaves of Eucalyptus globules, chiefly composed of eucalyptol (cin-eol), has been shown to inhibit pro- inflammatory IL-1β and TNF-α production in cultured human lymphocytes and monocytes [36]. The inhibition of MAPK/NF-κB sig-nalling pathway by eucalyptol [37] as well as β-methyl ionone, β- ionone [38], borneol [39], cinnamon [40], and α-pinene [41], presented in our formulation, is evidenced by numerous studies (Table 3). Hence, given the promi-nent anti-inflammatory power that overall associated with fewer side effects, our formulation provides prom-ising potential for the development of novel therapeutic

options. Nowadays it is well accepted that immune cells not only involved in immune responses to pathogens or damaged tissues but also in the development of inflam-matory and neuropathic pain [42, 43, 44, and 45]. Per-sistent exposure of elevated pro-inflammatory cytokines results in the activation or sensitization of nociceptors and enhanced painful behaviour. Natural products are traditionally used for the pain management [46]. The classical example of plant-derived drug employed in pain treatment is morphine, an opioid extracted from the Pa-paver somniferum [47]. Anti-nociceptive and analgesic effects of Natural Herbal Oil Formulation mixed in our formulation were previously investigated using different pain models. A growing number of investigations have demonstrated that the compounds present in the Nat-ural Herbal Oil Formulation from Rosa damascene [48], Eucalyptus globules [49], Citrus limon [50], Cocos nucif-era [51], and Lavandula latifolia [52], may have direct or indirect anti-nociceptive activities. Thus, having anti-in-flammatory activity, one can speculate that our formu-lation may exert anti- nociceptive effect which should be further studied in vivo. In conclusion, Natural Herbal Oil Formulation used for our mix are thought to have a strong biological potency associated with a suppression of unwanted inflammation and bacterial growth. There-fore, it has been anticipated that overlapping action of different natural compounds will amplify antimicrobi-al potency and influence the magnitude of immune re-sponses. Our data suggests that natural oil blend devel-oped by our group may help in combating bacterial and fungal infections and restrict exaggerated production of cytokines, supporting the normal immune response to inflammatory stressors.

Acknowledgment

I would like to convey my gratitude to Central Proteina Prima for the continuous supportto our research we would especially like to thank Mr. Benjamin Jiaravanon Chairman of Central Proteina Prima, Prof. Yusef Babik-yan senior Scientist of Central Proteina Prima for their guidance and support. Also I want to thank Prof Tigran Davtyan, Gayane Manukyan and Dr. Rajeev Kumar Jha for their guidance and support in this work.

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