Antimicrobial, anti-inflammatory and antioxidant activities of natural organic matter extracted from cretaceous shales in district Nowshera-Pakistan

2.4.1 Humic acid

50 g of powdered sediment was treated with sodium hydroxide solution (0.4 L; 0.1 M) in distilled water. The extraction was carried out for 24 h with occasional stirring. After 24 h, the supernatant was collected and centrifuged at 10,000 × g for 15 min. The supernatant was then acidified up to pH 1 with hydrochloric acid solution (4.0 M) and left overnight to precipitate the humic acid. The precipitate was then obtained by centrifugation at 10,000 × g for 15 min (Laurynas et al., 2021).

2.4.2 Fulvic acid

The supernatant (Crude fulvic acid) was filtered through 0.2 µm cellulose nitrate membrane filter and neutralized with sodium hydroxide solution (0.1 M; pH 4 to 7). Solutions were left overnight to precipitate fulvic acid. Precipitates of fulvic acid was then obtained by centrifugation at 6000 × g for 15 min (Laurynas et al., 2021).

2.4.3 Characterization of humic and fulvic acid

2.5.1 Bacterial strains

The following strains of bacteria were used; Bacillus subtilisin (ATCC 6633), Staphylococcus aureus (ATCC 25924), Salmonella typhi (19430), Pseudomonas aeruginosa (ATCC 27853), and Escherichia coli (ATCC 25922). The bacterial suspension was standardized to a turbidity equivalent to 0.5 on the McFarland scale (CLSI, 2012).

2.5.2 Agar-well diffusion method

Microbial cultures were prepared using a validated method (Fazli et al., 2021). Different strains were added to Muller Hinton Agar (MHA) and poured into sterile petri dishes. Well (6 mm) were made using a sterile borer into the agar plate for both control and test samples. 3 mg of NOM was dissolved in DMSO (1 ml; 3%) and about 100 µl of it was transferred into each well. The petri dishes were then incubated for 48 h (37℃) and finally, zone of inhibition was measured (n = 3). Imipenem and DMSO were used as positive and negative control, respectively.

2.5.3 Fungal strains

Fungal strains include Aspergillus niger (ATCC 10568), Alternaria alternata (ATCC 96255), Fusarium solani (ATCC 11213) and Aspergillus terreus (ATCC 20445).

2.5.4 Agar slanting method

The selected fungal strains were grown on Sabouraud dextrose agar (SDA) and subsequently incubated for 24 h at 37℃ 4 ml of SDA along with fungal inoculum was poured into sterilized test tubes. Test sample was prepared by dissolving 23 mg in 1 ml of DMSO (3%) and 67.5 µl of it was added into each test tube. The cultured media was solidified in tilting position and then incubated for seven days at 37℃. Miconazole was used as positive control. Finally, the linear growth inhibition was measured and percent inhibition was calculated using Eq. (1). (Fazli et al., 2021)

2.5.5 Minimum inhibitory concentration (MIC)

The MIC was determined by the broth microdilution method, using 96 well microtiter plates (CLSI, 2012). Initially, NOM was dissolved in DMO and serially diluted with water (sterile) to get various dilutions (i.e. 0.1–30 mg/ml), in microtiter plates. To each well, susceptible strains of growing cultures (1.5108 cfu/ml) were added and subsequently incubated at 37℃ for 72 and 24 hr for fungi and bacteria, respectively. Tetrazolium salt was used as an indicator. It was added to each well and the appearance of violet color in culture media indicated the growth of microorganisms. Imipenem and miconazole were used as positive, while DMSO was used as a negative control.

2.5.6 Cyclooxygenase (COX-2) assay

The method of Olomola et al. was used for the determination of in vitro enzyme inhibition activity (Olomola et al., 2020). For 10 min, the enzyme (300 U/ml) and co-factor (50 µl) were preincubated on ice. In addition 50 µl of co-factor solution (1 mM Hematin, 0.9 mM glutathione and 0.24 mM N,N,N,N-tetramethyl-pphenylenediamine dihydrochloride in 0.1 M Tris HCl buffer, pH 8.0) was mixed with the enzyme solution. Subsequently, the enzyme solution and NOM (25–1000 µg/ml) were mixed and allowed to stand for 5 min at 25℃. In order to initiate the reaction, arachidonic acid (20 ml, 30 mM) was added to this mixture and incubated for 4 to 5 min. Finally, maximum absorbance was determined at 570 nm.

2.5.7 5-lipoxygenase (5-LOX) assay

Initially, solutions of montelukast, NOM (25–1000 µg/ml), phosphate buffer (50 mM, pH 6.3) and 5-LOX enzyme (10,000 U/ml) were prepared. Test samples were dissolved in buffer solution (250 µl) and the enzyme solution (250 µl) was added to it before incubation at 25℃ for 5 min. In addition, the enzyme solutions and linoleic acid (0.6 mM, 1 ml) were mixed and maximum absorbance (234 nm) was determined with a UV–vis spectrophotometer (Okur et al., 2021). Percent inhibition values were determined using Eq. (2).

2.5.8 Antioxidant activity

2.6.1 Animals

Healthy BALB/c mice (25–30 g) of either sex were used for the in vivo anti-inflammatory activity. Standard protocol was adopted for conducting the experiments. The experiment was approved by the “Ethical Committee of the Department of Pharmacy, University of Peshawar” (Application No. 03/EC-18/Phar).

2.6.2 Carrageenan-induced paw edema

The in vivo anti-inflammatory activity was determined using carrageenan-induced paw edema model (Lingadurai et al., 2007). The animals were divided into five groups (n = 5). They were treated with NOM (50, 100 and 200 mg/kg, i.p), indomethacin (10 mg/kg, i.p), and normal saline. Paw edema was induced by injecting carrageenan solution (0.05 ml; 1%), 1 hr after the administration of NOM, into the left hind paw’s subplantar region. The standard and control groups received indomethacin and normal saline, respectively. The linear paw circumference was measured before and 1, 2, 3, 4 and 5 h after the injection of carrageenan, using a digital plethysmometer (model 7150, Ugo Basile, Italy). Percent inhibition was measured using Eq. (4).

3.2.1 pH determination

The pH of fulvic and humic acid solution (2%) was 3.2 and 3.8, respectively.

3.2.2 UV–Vis spectral analysis

UV–Vis spectrums of fulvic and humic acid was in the range of 200 to 600 nm and 200 to 550 nm, respectively (Fig. S2). Both the acids exhibit wide uncharacteristic spectra consistent with earlier reported studies conducted on structural elucidation of natural organic matter (Kyoichi, 1987).

3.3.1 Agar-well diffusion method

The cretaceous period was the warmest period in earth history. During that period, the temperature raised three times than the current global temperature (Ohkouchi et al., 2015). As a result, the polar glaciers melted and all water was added to oceans. The high global temperature and water on continents and ocean might have activated different strains of pathogens because they are sensitive to high temperature and water. The diverse pathogens from continents were shredded into oceans which might have further added to the diversity of oceanic pathogens. In response, the immunity of phytoplankton and zooplankton might have enhanced by producing different chemicals. The same algae and cyanobacteria are now preserved in rock record. Therefore, this study was designed to preliminary investigate the antimicrobial potential of NOM which may lead to the discovery of new lead compounds.

In present study, the NOM was tested for antimicrobial activity against some well-known pathogens, including P. aeruginosa, S. typhi, S. aureus, B. subtilis and E. coli. Literature review showed that typhoid fever, skin and other frequent infections are caused by these bacteria (Eng et al., 2015). E. coli is the most common facultative anaerobe in humans, causing illnesses such as gastroenteritis, urinary tract infection, meningitis and peritonitis (Arwyn-Jones and Surjo, 2019).

NOM exhibited significant activity against S. typhi, P. aeruginosa, E. coli and B. subtilis with MIC values 0.82, 0.87, 0.79 and 0.93 mg/ml, respectively. In addition, it showed considerable activity against S. aureus with MIC values 1.12 mg/ml (Table 2).

The antibacterial activity shown by NOM may involve several mechanisms including, but not limited to, pore formation, nucleic acid synthesis, DNA gyrase inhibition, and change in physiochemical properties of the plasma membrane (JJose and Cesar, 2016). It has been previously reported that humic and fulvic acids possess significant antibacterial activity particularly against methicillin-sensitive S. aureus, S. thyphi, MRSA, E. coli, and P. aeruginosa (Maguey-Gonzalez et al., 2018; Winkler and Ghosh, 2018). According to literature review, both fulvic and humic acids are the prominent constituents of NOM. Therefore, the antibacterial activity of NOM in this study against the mentioned pathogens may be partially due to these compounds.

3.3.2 Agar slanting method

Despite the fact that there are few studies in the literature regarding the antifungal activity of NOM. Here, it proved to be active against all the tested fungi especially against A. alternata and F. solani with MIC values 0.60 and 0.68 mg/ml, respectively, whereas it showed considerable activity against A. niger and A. terreus with MIC values 0.82 and 1.10 mg/ml, respectively (Table 3). Fusarium and Alternaria are well-known plant pathogens responsible for significant productivity losses in agricultural crops globally. These fungi causes early blight and wilt of tomato (Haytham et al., 2021). It has been documented that synthetic fungicides are extensively used to control plant pathogens; however, in addition to its toxicity to consumers they are considered the most lethal pollutants for the environment. Contrary to that, natural pesticides are environment friendly with minimal or no health hazards or pollution (Asmaa et al., 2020). Based on our results NOM may be a potential source for developing an eco-friendly phytofungicides.

3.3.3 Cyclooxygenase and lipoxygenase assays

Both COX-2 and 5-LOX are considered as pro-inflammatory enzymes, involved in arachidonic acid metabolism (Violette and Ebtisam, 2018). They are also involved in the production of eicosanoids, including thromboxane, leukotrienes and prostaglandins that primarily cause inflammation. Therefore, these enzymes are the rate-limiting factors in the production of mediators of inflammation and commonly used for screening anti-inflammatory drugs. It is pertinent to mention that co-inhibition of COX/5-LOX potentially reduce side effects preferably on the GIT and cardiovascular system, while retaining the activity of COX-1/2 inhibitors (Martel-Pelletier et al., 2003).

Based on its traditional uses, we investigated the anti-inflammatory effect of NOM using in vitro models. In this study, COX-2 and 5-LOX enzymes were inhibited by NOM in a dose-dependent manner. The COX-2 inhibition results showed that NOM had significant activity with IC₅₀ value of 31.34 µg/ml, compared to reference drugs Celecoxib (18.23 µg/ml). Concerning 5-LOX inhibition capacity, the sample showed considerable activity with IC₅₀ value of 38.45 µg/ml, compared to Montelukast (25.34 µg/ml) (Fig. 3).

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Fig.3

The relative inhibition of COX-2 and 5-LOX enzymes by NOM and standard.

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Inflammatory reactions are commonly triggered by higher level of prostaglandins and leukotrienes, which are produced by 5-LOX and COX-1/2 enzymes, respectively. Prostaglandins sensitize nociceptive fibres and facilitates the passage of pain stimuli, while leukotrienes stimulates cell adhesion to vascular endothelium (Felipe et al., 2017). Therefore, inhibition of COX-1/2, reduce prostaglandin production, which subsequently reduce pain and inflammation. However, inhibition of prostaglandins may lead to activation of 5-LOX pathway, which in turn raise gastrotoxic leukotrienes. The use of dual inhibitors of COX-2/5-LOX, help to reduce these problems (Afshin and Sara, 2011). The results from the current study clearly exhibited that NOM had a high potency against COX and LOX and thus may be considered in anti-inflammatory drug discovery and development.

3.3.4 Antioxidant activity

According to WHO, nearly 80% of the world population rely on herbal traditional medicines for their primary health care needs and most of these therapies involves the use of herbal extracts (Nisa et al., 2013). It has been documented that our body produces more reactive oxygen species (ROS) than enzymatic antioxidants under stress condition. These includes superoxide anion radical (O₂·^–), perhydroxyl radical (HO₂·) and hydroxyl radical (·OH) as well as some non-free radicals i.e·H₂O₂ (Duduku et al., 2011). In the absence of antioxidants, these free radicals facilitates the development of several diseases which includes but not limited to cancer, liver cirrhosis, diabetes, atherosclerosis, cardiovascular diseases, inflammation, and a variety of neurodegenerative disorders (Nisa et al., 2013; Sajjad et al., 2015). Synthetic antioxidants such as butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) have been widely used in food industry but these agents are reported to be associated with carcinogenesis and liver cirrhosis (Duduku et al., 2011). Therefore, in last few decades the use of natural antioxidants have got much attention.

3.3.4.3

3.3.4.3 Hydroxyl scavenging activity

Hydroxyl radicals are the primary active oxygen species responsible for lipid peroxidation and massive biological injuries. These radicals are generated from the reaction of transition metals with various hydroperoxides. It damages unsaturated fatty acids in membrane, proteins, DNA and other biological molecules (Anju et al., 2019). In this study, NOM depicted concentration dependent activity against hydroxyl radicals produced in the Fenton reaction. As shown in Fig. 4, the sample and standard exhibited 73 and 92% hydroxyl scavenging activity at 1000 µg/ml, respectively.

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Fig. 4

Hydroxyl scavenging activity of NOM and standard at different concentrations. Each value represents mean ± SD (n = 3).

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3.3.4.4

3.3.4.4 Hydrogen peroxide scavenging

This is a weak oxidizing agent that directly inactivates few enzymes, typically by oxidation of essential thiol (-SH) groups. It has the ability to rapidly cross cell membranes; once inside the cell, it is likely to react with Fe²⁺ and possibly Cu²⁺ ions to form ^–OH radicals, which may be the source of many of its toxic effects (Ergul, 2016). NOM showed weak hydrogen peroxide scavenging activity i.e. 40 % at the highest tested dose, compared to standard, which depicted 73% activity at the same dose (Fig. 5).

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Fig. 5

Hydrogen peroxide scavenging activity of NOM and standard at different concentrations. Each value represents mean ± SD (n = 3).

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These in vitro tests show that NOM contains a significant amount of natural antioxidants, which may aid in the prevention of a variety of oxidative stresses. However, the compounds responsible for the antioxidant activity are unknown at this time. As a result, additional research is required to isolate and identify the antioxidant compounds present in NOM. Furthermore, the antioxidant activity of NOM in vivo must be determined before it can be used in clinical trials.

3.4.1 Carrageenan-induced paw edema

The anti-inflammatory activity of NOM against carrageenan-induced paw edema is shown in Table 5. The NOM at all doses significantly (P < 0.001) decreased the edema volume against control and maximum activity (33, 47 and 54% at 50, 100 and 200 mg/kg dose, respectively) was observed at fifth hr. of treatment. The standard indomethacin showed maximum inhibition (58%) at fourth hr. of administration.

It is evident from the above results that NOM has significant anti-inflammatory potential by controlling biphasic inflammatory events. Carrageenan-induced inflammation in animals occur in two phases. The early phase (90–180 min) is characterized by the release of histamine, serotonin and similar substance whereas; the later phase (270–360 min) is associated with the release of prostaglandins, lysosome and proteases. In this study, NOM showed significant inhibitory effect particularly in the later phase of inflammation. This suggests that NOM possibly acts by inhibiting the release and/or actions of vasoactive substances (serotonin, histamine) and prostaglandins. The present results corroborate the importance for screening cretaceous shales as a potential source for bioactive compounds. Phytochemical composition of NOM showed the presence of humic substances including fulvic acid and some complex organic matters mainly produced by cyanobacteria (Ohkouchi et al., 2015). Literature review showed that humic substances possess significant anti-inflammatory activity (De-Melo et al,. 2016). In light of the abovementioned facts, this preliminary study could lead to the discovery of novel anti-inflammatory drugs derived from cretaceous shales that can be used for the rapid and effective management of infectious diseases.