Antimicrobial activity, antioxidant and chromatographic analysis of Varanus griseus oil extracts

doi:10.21203/rs.3.rs-1634893/v1

There is an urgent need to develop natural antimicrobials for the control of rapidly mutating drug-resistant bacteria and poultry viruses. Five extracts were prepared by using diethyl ether, ethyl acetate, methanol, 1-Butanol and n-Hexane from abdominal fats of Varanus griseus locally known as Indian desert monitor. Antibacterial, antioxidant and antiviral activities from oil extracts were done through disc-diffusion method, stable 1,1-diphenyl-2-picrylhydrazil (DPPH) free radical scavenging assay and In Ovo antiviral assay respectively. The gas chromatography mass spectrometry (GCMS) analyses were used to determine principal active compounds and chemical profile of each oil extract. n-Hexane extract showed clear zones of inhibition (ZOI) against Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae (12 ± 0.5 mm, 9 ± 0.5 mm, 9 ± 0.5 mm) while diethyl ether extract exhibited significant antibacterial activity (11 ± 0.5 mm) against Proteus vulgaris only. In case of drug resistant strains, methanol extract was active (6 ± 0.5 mm) against Staphylococcus aureus whereas n-Hexane extract has shown ZOI 11 ± 0.5 mm against P. aeruginosa. Range of percentage scavenging activity of V. griseus oil extracts from DPPH free radical assay was 34.9–70.7%. For antiviral potential, growth of new castle disease virus (NDV) was effectively inhibited by all five extracts (HA titer = 0–4). The highest antiviral activity against avian influenza virus (H9N2) was observed from methanol, diethyl ether and 1-Butanol oil extracts with HA titers of 2, 2 and 0, respectively. Methanol, diethyl ether, 1-Butanol and n-Hexane oil extracts produced best hemagglutination assay (HA) titer values (0, 0, 4 and 0) against infectious bronchitis virus (IBV). Ethyl acetate and 1-Butanol extract exhibited good antiviral potential against infectious bursal disease virus (IBDV) with indirect hemagglutination assay (IHA) titers of 8 and 4, respectively. Main classes of identified compounds through gas chromatography were aldehydes, fatty acids, phenols and esters. GC-MS identified 11 bioactive compounds in V. griseus oil extracts. It is summarized that V. griseus oil has strong antioxidant activity and good antimicrobial potential because of its bioactive compounds.

Hemagglutination

In Ovo screening

disc-diffusion technique

antiviral

zootherapy

Varanus griseus

In the history of folk medicine, reptiles have great significance. The significance of reptilian fauna was recognized by residents in magical and religious traditions, related to the fate and healing of chronic ailments (Riós-Orjuela et al. 2020). In Latin America, Brazil and Pakistan, although many animals are being used in traditional medicine (584, 354 and 81 species) but knowledge of their biological activities is rare (Alves and Alves 2011; Ferreira et al. 2018; Altaf et al. 2020). The tribes in Pakistan have used the fat and oil of V. bengalensis as a ointment for skin problems and rheumatic pain alleviation (Hashmi et al. 2013). The Rohi community in Cholistan used reptiles such as spiny-tailed lizard (Saara hardwickii Gray 1827), Indian cobra (Naja oxiana Eichwald, 1831), sand lizards (Lacerta agilis Linnaeus, 1758) and Indian desert monitor (Varanus griseus Daudin, 1803) including some other wild animals as traditional medication for the cure of a wide range of infections or medical settings (Ahmad et al. 2021).

Owing to their low cost and effectiveness with no or minimal side effects, about eighty percent of the world's population depends on conventional therapeutics for their primary healthcare requirements. Using remedies that are made or obtained from animals or from their products is known as zootherapy (Ahmad et al. 2021). In India, Sudan, and China, body parts of monitor lizards Varanus niloticus and Varanus bengalensis have been employed against different diseases. Various species of reptilian and amphibian fauna can be utilized to treat the same infection while a single animal often has various therapeutic applications and can be applied to cure multiple infections. In Brazil, lizard products Tupinambis merianae and T. teguixin are employed for curing different skin infections, respectively (Alves and Rosa 2007). In India, Bengal monitor products (V. bengalensis) are used to cure burns, body ache, hemorrhoids, and rheumatism, as well as snake and spider stings (Kakati et al. 2006).

Antimicrobial activities have been studied using natural resources available through ethno-guided knowledge. In these studies it was also found reptilian oil had antimicrobial activity against different microbes (Ferreira et al. 2009; Dias et al. 2013; Oliveira et al. 2014; Sales et al. 2015). Animal oil based extracts, powders, medicinal teas and oil-based emulsions have been used to treat viral infections for hundreds of years, but there is a strong urge for novel chemicals (Vijayan et al. 2004). Several ways of administrations for several inner and outer clinical signs are adapted to use medicinal products from the animal's oil and fat. The therapeutic activity of oil is mainly due to its fatty acid constituents. Animal-based medications are gaining importance through animal-based products (Mishra et al. 2020).

Brazilian lizard’s (Tupinambis merianae) body fat was widely exploited as zootherapeutic product. Skin infections, throat problems, inflammations, snake bites, tumors, asthma, deafness and respiratory problems are cured through these products (Ferreira et al. 2010). The evaluating procedures are related to antimicrobial sensitivity test for determination of the epidemiology, purpose of drug development and estimation of therapeutic features. In vitro investigation of drugs and extract to be considered as important microbicidal products was gauged through antimicrobial testing methods (Balouiri et al. 2016).

Oleic acid, palmitic acid, stearic acid, linoleic acid and linolenic acid are present in oil and play key roles as anti-microbial and anti-inflammatory agents. The zootherapeutic agents got from the fat of animal are acceptable for humans against various internal and external therapeutic indications. Because of a lack of scientific proof for traditional healing practices involving animal fats or oils, scientific studies to verify therapeutic action are being conducted, which may usher in a new age for the creation of a range of unique formations (Mishra et al. 2020). Free radicals are small diffusible molecules that are highly reactive because of the unpaired electron. Free radicals were initially thought to be oxygen centred radicals called reactive oxygen species (ROS) but also comprise a subgroup of reactive nitrogen species (RNS) and are all a product of normal cellular metabolism (Alkadi 2018). In normal metabolism levels of free radicals and antioxidants are balanced. However, the over-production of free radicals leads to oxidative damage, resulting to a range of chronic disease, such as inflammation, diabetes, and cancer (Cai et al. 2004). Antioxidant serves as singlet oxygen quencher, radical scavenger, enzyme inhibitor, electron donor, synergist, peroxide decomposer and metal-chelating agents (Lobo et al. 2010). Essential oils are rich sources of natural antioxidants, such as the phenolic compounds and due their high redox properties and chemical structure have the capacity to neutralize free radicals, chelate transitional metals, and quench singlet and triplet oxygen by delocalization or decomposition of peroxides both in humans and animals (Zheng and Wang 2001; Simitzis 2017). Lipid extracts or lipids taken from invertebrates or terrestrial vertebrates have shown to be effective against a wide range of bacteria (Alves et al. 2020). Several findings have examined the antibacterial action of fatty acids and their derivatives, such as monoglycerides. These are reviewed here, with an emphasis on the antibacterial capabilities that have been recognized as well as the therapeutic potential (Churchward et al. 2018). GC-MS identify as well as quantify the chemical constituents present in the compound studied, which makes GC-MS superior when compared with HPLC, only used for quantification purpose. Additionally, GC-MS is a fast, sensitive technique for minute sample quantity and reduces the use of organic solvents.he aims of the present research were to examine antimicrobial, antiviral, and antioxidant potential as well as GC-MS (Gas chromatography mass spectroscopy) profile of Varanus griseus oil extracts. To the best of our knowledge, there is no study on V. griseus oil extracts on these aspects.

Specimen collection:

Varanus griseus is a commonly occurring monitor lizard of Cholistan desert. Fats were obtained from ventral regions of body (Ferreira et al. 2010). Fats were extracted simply by heating small pieces of fat pads from the belly of lizard between 37-50 ˚C until complete dissolution and were kept at -20˚C for further use in aliquot form (Khunsap 2016). All the experiments were performed following the National Research Council’s Guidelines for the Care and Use of Laboratory Animals by approval of ethics committee of Bahauddin Zakariya University Multan.

The bacterial strains were obtained from Nishtar Medical University's Bacteriology Division and grown in the microbiology laboratory of Bahauddin Zakariya University in Multan. The bacterial species used in current study were Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumonia, Staphylococcus aureus and drug resistant S. aureus and drug resistant P. aeruginosa. Nutrient media was used to grow bacteria cultures.

LaSota vaccine strains of New Castle disease virus (NDV); Gallimune Flu H9 M.E (H9N2) of avian influenza virus (AIV) from Merial Labs of Italy; infectious bronchitis virus (IBV) H120 from IZO S.U.R.L 99/A-25124 Brassica of Italy and DS Gumboro Vac an infectious bursal disease virus (IBDV) from Dae Sung Microbiological Labs in Seoul, South Korea are four poultry viral strains used in current work.

Extract preparation:

The oil was mixed with five solvents separately on polarity basis to prepare the actual extract and were termed as n-Hexane, 1-Butanol, ethyl acetate, diethyl ether and methanol. Fractions of body oil of V. griseus through solvents were separated by using separating funnel and solvents were evaporated at room temperature from fractions. In Eppendorf tubes, we mixed one ml of each extract with one ml of the matching solvent, and the tubes were vortexed to mix the solvent (Tanhaeian et al. 2020).Antibacterial activity:

Determination of Zone of Inhibition:

Antibacterial activity of extracts of Varanus griseus oil against bacteria E. coli, S. aureus, P. aeruginosa, P. vulgaris, K. pneumonia, drug resistant S. aureus and P. aeruginosa species was recorded through disc diffusion method and the zone of inhibition was determined (Yaqeen et al. 2013).

Media preparation:

Nutrient Broth: By dissolving 1.3 g of it in 100 ml of distilled water, nutrient broth was formed and was autoclaved at 121 °C for 1 hour to sterilize (Hussain et al. 2015).

Nutrient agar: 2.8 g of nutrient agar was dissolved in distilled water for its preparation and was autoclaved at 121^oC for its sterilization (Hussain et al. 2015).

Evaluation of antibacterial activity through disc diffusion assay:

Agar plates preparation was done for disc diffusion technique to examine antibacterial activity of each oil extract. Nutrient broth media was used for the growth of bacterial cultures by inoculating strain from stock. Different aliquots were prepared by dispensing in the sterile Eppendorf tubes after refreshing culture (Cunha-Neto et al. 2020).

Principle of Disc diffusion method:

A medicine's or chemical's antibacterial activity is determined by putting a disc of filter paper containing the medication or compound on the surface of an agar plate containing a particular strain of bacteria. We recorded a distinct inhibition zone around the disc after overnight incubation. Clear lager inhibition zone showed the effectiveness of drug against bacteria (Fernando et al. 2020). The Ampicillin, the commercial antibiotic, was used as positive control.

Micro dilution method:

The minimum dose of antibiotic required to inhibit or kill the bacterium is determined using dilution susceptibility testing procedures. Antimicrobial agents are diluted in agar or broth medium to achieve this and are evaluated in two-fold log2 serial dilutions. The agar disc diffusion technique was used to determine antibacterial activity. The MIC of substances with antibacterial potential was determined using the broth dilution method (Jiang et al. 2013). After incubating the micro titer plates overnight at 37°C, a solution of p-iodonitro-phenyltetrazolium violet (INT) (2 mg/ml) was made. The MIC value shows the lowest concentration of extracts necessary for bacteriostatic action to be seen.

Antioxidant activity:

The antioxidant activity of the five produced extracts of Varanus griseus oil was determined using the extinction absorption technique of the radical 1,1-diphenyl-2-picrylhydrazil (DDPH). The DDPH technique was developed using Shimadzu model UV-1800 equipment (Shimadzu, Kyoto, Janpan) and spectrophotometry of molecule absorption in visible, ultraviolet light at 517 nm wavelength (Ak and Gülçin 2008). The 0.1mM solution of DPPH free radical was prepared in ethanol. Three hundred μl of each oil extract with 0.5 ml of 0.1 mM DPPH solution was incubated for 1hour in dark. The reading was recorded at 517 nm afterwards along with positive control of ascorbic acid and calculated DPPH scavenging percentage of each oil’s extract.

Antiviral study:

Each extract of Varanus griseus oil was combined with an equal quantity of a live virus and propagated for 7-11 days in embryonated eggs with adequate controls to determine antiviral activity. In Ovo antiviral assays were used to evaluate all species of viruses against different extracts (Rajbhandari et al. 2001). All of the selected viruses were injected in the chorioallantoic fluid of chick embryonated eggs of 9-11 days old. Before and after inoculations, the eggs were candled. Later the eggs were extracted 72 hours after vaccination (PI), then allantoic fluids were recovered and tested for HA or IHA (with IBDV) titers. We carried the entire process out in a category II biosafety cabinet. Before testing antiviral extracts of oil, we performed serial passages of viruses to improve titer (Bajpai and Chandra 1990).

Hemagglutination Test (HA):

Chicken blood was taken and centrifuged at 4000rpm for five minutes in freshly produced Alsevior solution. RBCs were washed in PBS buffer (Phosphate buffered saline, pH 7.2) after the supernatant was discarded, 10 μl packed RBCs were added to 1 ml PBS (pH 7.4) solution to make 1% suspension. The HA test was carried out using future cells (Danish et al. 2018). The antiviral impact was measured in HA titer, a greater HA titer value in the presence of oil extracts indicate a weaker antiviral activity (Akhtar et al. 2020). The medicine amantadine was employed as a positive control for H9N2 and NDV, while lithium chloride (LiCl) was used as a positive control for IBV.

Indirect Hemagglutination (IHA) Test:

IHA test was used to measure growth of infectious bursal disease virus (IBDV) and it employed human blood group "O". Three ml of blood was added in 4% sodium citrate solution and gently stirred. The supernatant was discarded by centrifugation at 4000 RPM for 5 minutes. The cells were suspended in 10 ml PBS (pH 7.4) before being centrifuged for 5 minutes at 4000 RPM. The process was carried out in triplicate. The cells were sensitized after washing with one volume of IBDV and two volumes of PBS (pH 7.4). The materials were carefully mixed and incubated at 37°C for 45 minutes. The cells were centrifugate at 4000 RPM after incubation and the supernatant was collected and rinsed again with PBS (pH 7.4) (Okwor et al. 2011). The sensitized cells were used to conduct the standard HA test (Hussain and Gorsi 2004). Because no approved drug was available therefore, we employed no positive control for IBDV. All injected eggs were retrieved for 72 hours after inoculation and tested for HA or IHA. Any differences in agglutination development compared to viral control were documented. By creating successive dilutions of extracts and challenging them against viruses, the IC₅₀ of each positive extract was measured. We performed all the experiments in triplicate.Gas chromatography mass spectrometry:

Gas chromatographic profile of five extracts of Varanus griseus oil was determined on Agilent Technologies 7890B Network Series GC System equipment and 5975C triple axis mass selective detector, with HP-5ms 19091S-433 capillary column (J&W Scientific, Folsom, CA, USA), 30 m × 250 µm × 0.25 µm following method Zamora-Gasga et al. 2015 with minor alterations. The injector temperature was set at 250°C. An oven temperature was kept at 50°C for 5 minutes and then increased to 200° C, with 5°C per minute, where it was kept for 2 minutes, and finally increased to 230°C, with 15°C per minute. This temperature was maintained for 15 min with a final run time was 53.5 minutes. The injection volume was 2 μL for samples by manual injection mode, flow control mode linear velocity, flow rate in column was 1.10 mL/min, and linear gas velocity was 27.458 cm/s. Helium was used as carrier gas, ion source temperature was set at 250 °C, start time was 3 min, and end time 53.5 min. A 50-550 m/z was used to calculate mass range. MS ChemStation and MS interpreter software were used for processing of chromatographic data.

Chemicals and reagents:

Methanol
Ethyl acetate
1-Butanol
n-Hexane
Diethyl ether
Ethanol
Amantadine
Lithium chloride
DMSO
DPPH
Ascorbic acid
Phosphate buffer saline
INT
EDTA
Alsever’s solution

These solvents and chemical reagents were supplied from Sigm-Aldrich (St. Louis, USA). These compounds were used in current study without further purification.

Statistical analysis:

ZOI, MIC and IC50 values were calculated in triplicate. Data was analyzed as mean ± standard deviation using MS (MS Excel 2016).

Antibacterial activity:

The antibacterial activity was analyzed through disc diffusion method. Zone of inhibition (ZOI) of pure solvents (methanol, ethyl acetate, diethyl ether, 1-Butanol and n-Hexane) were measured first. Only the methanol exhibited antibacterial activity against targeted bacteria, though it was not active against drug resistant bacterial strains. Later the ZOI procedure was repeated with five oil extracts. Final ZOI calculations were obtained after subtracting pure solvent activity from oil’s extracts activity. In current study antibacterial activity was determined against Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa, drug resistant S. aureus and drug-resistant P. aeruginosa. The disc diffusion method was used to calculate the zones of inhibition. n-Hexane extracts showed the highest antibacterial activity against S. aureus (Fig. 1a), P. aeruginosa, K. pneumoniae and P. vulgaris in the current study with (9 ± 0.5 mm; 12 ± 0.5 mm; 5 ± 0.5 mm; 9 ± 0.5 mm; 8 ± 0.5 mm) (Table 1). P. vulgaris was more susceptible to diethyl ether extract of V. griseus oil (11 ± 0.5 mm) as shown in Fig. 1b. Average inhibitory zone (6 ± 0.5 mm) was measured with methanol extract against drug resistant S. aureus (Fig. 1c) while n-Hexane extract showed strong antibacterial activity (11 ± 0.5 mm) against drug resistant P. aeruginosa. Positive oil extracts were subjected to MIC and values were shown (Table 1). The antibiotic drug ampicillin was employed as a positive control and it showed substantial inhibition zones against all tested bacteria: E. coli, S. aureus, P. aeruginosa, K. pneumoniae and P. vulgaris (5 ± 0.5 mm, 10 ± 0.5 mm, 9 ± 0.5 mm, 9 ± 0.5 mm and 11 ± 0.5 mm, respectively) (Table 1).

Table 1 MIC in microliters and inhibition zones of several bacterial strains against (V. griseus) oil (in millimeters).

Extract type	E. coli mm/ µg		S. aureus mm/ µg		Drug resist. S. aureus mm/ µg		P. aeruginosa mm/ µg		Drug resist. P. aeruginosa mm/µg		K. pneumonia mm/µg		P. vulgaris mm/µg
	ZOI	MIC	ZOI	MIC	ZOI	MIC	ZOI	MIC	ZOI	MIC	ZOI	MIC	ZOI	MIC
Methanol	3 ± 0.5	-	3 ± 0.5	-	6 ± 0.5	25	3 ± 0.5	-	5 ± 0.5	-	0	-	2	-
Ethyl acetate	1 ± 0.5	-	4 ± 0.5	-	1 ± 0.5	-	2 ± 0.5	-	0	-	6 ± 0.5	25	3 ± 0.5	-
Diethyl ether	0	-	6 ± 0.5	25	0	-	0	-	3 ± 0.5	-	0	-	11 ± 0.5	12.5
1-Butanol	0	-	1	-	2 ± 0.5	-	0	-	5 ± 0.5	-	4 ± 0.5	-	0	-
n-Hexane	9 ± 0.5	12.5	12 ± 0.5	12.5	0	-	5 ± 0.5	-	11 ± 0.5	12.5	9 ± 0.5	12.5	8 ± 0.5
Ampicillin	5 ± 0.5	12.5	10 ± 0.5	12.5		-	9 ± 0.5	25			9 ± 0.5	12.5	11 ± 0.5	25

Antioxidant activity using DPPH free radical assay:

The antioxidant capacity of five extracts of V. griseus oil was determined at 515 nm using UV spectrophotometer and found significant potential in DPPH free radical reaction system. Ethanol was used during DPPH radical preparation as it is miscible in majority of solvents. 1-Butanol oil extract displayed a significant (70.7%) inhibition of DPPH. Other oil extracts also showed antioxidant activities but comparatively less when compared with control as shown in Fig. 2.

Antiviral activity:

Activity against Newcastle disease virus (NDV)

Antiviral activity of oil extract was evaluated against NDV in terms hemagglutination test (HA). All five extracts of V. griseus oil were effective in controlling NDV growth. Three extracts viz. methanol, 1-Butanol and n-Hexane were fully efficient against NDV with HA titer 0. (Lane 1, Lane 4, Lane 5, Fig. 3). Diethyl ether extract was also effective with HA titer value of 2 as shown (Lane 3 Fig. 3). Ethyl acetate extract of V. griseus was moderately active against NDV with corresponding HA titer (04) shown (Lane 2 Fig. 3). NDV virus control displayed a HA titer of 1024 (Lane 6 Fig. 3).

Activity Against Avian Influenza Virus (H9n2):

1-Butanol extract was strongly active (HA titer 0) against H9N2 (Lane 4 Fig. 4). Methanol and diethyl ether extracts were also effective in controlling growth of H9N2; their HA titer was 2 (Lane 1; Lane 3 Fig. 4). Lowest HA titer 32 was recorded against H9N2 growth by ethyl acetate and n-Hexane extracts (Lane 3; Lane 5 Fig. 4). H9N2 virus control exhibited a HA titer of 2048 (Lane 6 Fig. 4).

Activity against Infectious Bronchitis Virus (IBV):

Three extracts viz methanol, diethyl ether and n-Hexane showed powerful antiviral activity with HA titer 0 over viral growth of IBV (Lane 1; Lane 3; Lane 5 Fig. 5). Similarly, 1-Butanol extract produced HA titer value 4 (Lane 4 Fig. 5). Overall, all five extracts were found effective against IBV varying in their antiviral potential (Fig. 5). IBV virus control showed a HA titer of 1024 (Lane 6 Fig. 5).

Activity against Infectious bursal disease virus (IBDV):

Antiviral activity of oil extract was evaluated against IBDV in terms indirect hemagglutination test (IHA). 1-Butanol extract was effective in limiting IBDV growth with IHA titer 4 as shown (Lane 4 Fig. 6). Ethyl acetate extract was moderately active against IBDV; HA titer produced was 8 (Lane 2 Fig. 6). Methanol, diethyl ether and n-Hexane extracts were equally but least effective against IBDV as shown (Lane 1; Lane 3; Lane 5 Fig. 6) and their IHA titer of 32. IBDV virus control revealed a HA titer of 1024 (Lane 6 Fig. 6). The IC₅₀ of each positive extract against all four viral strains was determined and reported in Table 2.

Table 2

Antiviral activity of five extracts with HA/IHA tests of four poultry viruses along with IC₅₀
Extract/viral strain	NDV		H9N2		IBV		IBDV
	HA titer	IC₅₀ (µg)	HA titer	IC₅₀ (µg)	HA titer	IC₅₀ (µg)	IHA titer	IC₅₀ (µg)
Methanol	0	256	2	-	0	64	32	-
Ethyl acetate	4	-	32	-	16	-	8	128
Diethyl ether	2	32	2	-	0	16	16	-
1-Butanol	0	8	0	64	4	-	4	32
n-Hexane	0	16	32	-	0	16	32	-
Positive control	0	10(mg)	0	10(mg)	0	10(mg)	-	-
Negative Control	1024		2048		1024		1024
Mean values of IC₅₀ were recorded. No drug is available which could be as positive control of IBDV.

Chemical profile of five extracts made from Varanus griseus oil:

GC-MS analysis was performed for all five extracts include diethyl ether, methanol, ethyl acetate, 1-Butanol and n-Hexane. The diethyl ether extract showed 34 peaks (each peak representing individual compound), 24 in methanol, 24 in 1-Butanol, 20 in ethyl acetate and 13 peaks in n-Hexane extracts. The diethyl ether extract was rich with 34 compounds consisted of 10 aldehydes, 14 unsaturated fatty acids, 3 saturated fatty acids, 4 alkanes and 3 alkenes (Fig. 7a). Methanol extract showed 24 peaks, which include 9 aldehydes, 7 unsaturated fatty acids, 3 saturated fatty acids, 3 alkanes and 1 alkene and 1 alkyne (Fig. 7b). 1-Butanol extract of 24 peaks comprised 10 aldehydes, 6 unsaturated fatty acids, 4 saturated fatty acids, 3 alkanes and 1 alkene (Fig. 7c). Ethyl acetate extract comprised of 20 compounds viz 7 aldehydes, 4 unsaturated fatty acids, 5 saturated fatty acids, 3 alkanes and 1 alkene (Fig. 7d). n-Hexane extract displayed 13 peaks including 2 aldehydes, 7 unsaturated fatty acids, 3 saturated fatty acids and 1 alkane were found (Fig. 7e).

Overall, eleven compounds in five extracts of Varanus griseus oil were biologically active against different microbes (Table 3). The biologically active compounds in diethyl ether contained 9,12-octadecadienoic (Z,Z)- [Peak 10], cis-p-mentha-1(7), 8-dien-2-ol [Peak 12] 2,4-decadienal [Peak 29] and 9-octadecenoic acid (Z) [Peak 34] (Fig. 7a). Methanol extract contained erucic acid [Peak 4], cis-11-eicosenoic acid [Peak 6], 9,12-octadecadienoic (Z,Z)- [Peak 10] and 2,4-decadienal [Peak 23] as bioactive compounds (Fig. 7b). 1-Butanol extract comprised of n-butyric acid 2-ethylhexyl ester [Peak 6], octanal [Peak 11], dodecanal [Peak16] and 2,4-decadienal [Peak 22] (Fig. 7c). Ethyl acetate extract comprised of n-butyric acid 2-ethylhexyl ester [Peak 5] and dodecanal [Peak 8] (Fig. 7d) while n-Hexane contained 9-octadecenoic acid (Z)- [Peak 2], eugenol [Peak 8], octadecanoic acid, 9,10-dichloro-, methyl ester [Peak 11] and 9,12-octadecadienoic acid (Z,Z)- [Peak 13] (Fig. 7e). Important peaks with chromatographic characters are described in Table 4. Complete chromatographic (GC-MS) analysis of five extracts of V. griseus body oil is provided in file of supported information.

Table 3

Compounds identified in five extracts of *V. griseus* body oil through GC-MS analyses.
Sr. No.	Tentative assignment	Methanol	Ethyl acetate	Diethyl ether	1-Butanol	n-Hexane
1	Erucic acid	+	-	+	-	-
2	cis-11-Eicosenoic acid	+	-	-	-	-
3	9,12-Octadecadienoic acid (Z,Z)-	+		+		+
4	n-Butyric acid 2-ethylhexyl ester	-	+	-	+	-
5	Dodecanal	+	+	-	+	-
6	trans-p-mentha-1(7),8-dien-2-ol/ cis-p-mentha-1(7),8-dien-2-ol	-	-	+	-	-
7	2,4-Decadienal	+	-	+	+	-
8	9-Octadecenoic acid (Z)-	-	-	+	-	+
9	Octanal	-	-	-	+	-
10	Eugenol	-	-	-	-	+
11	Octadecanoic acid, 9,10-dichloro-, methyl ester	-	-	-	-	+

Table 4

Important peaks with chromatographic characters.
Extract	Peak No.	Retention time (min)	m/z Experimental [M-H]-	m/z Calculated [M-H]-	Tentative assignment	Molecular formula	MS² Fragment ion
Methanol	4	8.562	338	338.31	Erucic acid	C22H42O2	55/181
	6	10.616	310	310.28	cis-11-Eicosenoic acid	C20H38O2	55/69
	10 ¹	11.772	280	280.24	9,12-Octadecadienoic acid (Z,Z)-	C18H32O2	67
	23¹	22.203	152	152.12	2,4-Decadienal	C10H16O	81
Ethyl acetate	5 ¹	8.477	200	200.17	n-Butyric acid 2-ethylhexyl ester	C12H24O2	71
Ethyl acetate	8 ¹	12.483	184	184.18	Dodecanal	C12H24O	55
Diethyl Ether	10²	11.601	280	280.24	9,12-Octadecadienoic acid (Z,Z)-	C18H32O2	67
	12	12.327	152	152.12	trans-p-mentha-1(7),8-dien-2-ol/ cis-p-mentha-1(7),8-dien-2-ol	C10H16O	109
	29 ¹	22.596	152	152.12	2,4-Decadienal	C10H16O	81
	34 ¹	52.679	282	282.25	9-Octadecenoic acid (Z)-	C18H34O2	55/97
1-Butanol	6 ²	8.136	200	200.17	n-Butyric acid 2-ethylhexyl ester	C12H24O2	71
	11	12.090	128	128.12	Octanal	C8H16O	57
	16 ²	15.752	184	184.18	Dodecanal	C12H24O	57
	22 ²	22.807	152	152.12	2,4-Decadienal	C10H16O	81
n-Hexane	2 ²	4.586	282	282.25	9-Octadecenoic acid (Z)-	C18H34O2	55/97
	8	24.258	164	164.08	Eugenol	C10H12O2	164
	11	46.551	366	366.20	Octadecanoic acid, 9,10-dichloro-, methyl ester	C19H36Cl2O2	74
	13 ³	52.342	280	280.24	9,12-Octadecadienoic acid (Z,Z)-	C18H32O2	67

According to an estimation about 80% of the world's population depends on traditional therapeutics for their primary healthcare requirements (Ahmad et al. 2021). Traditional therapeutic medicinal properties were reported from 584, 283 and 81 animal species from Latin America, Brazil and Pakistan including 8 reptilian species (Alves et al. 2007, 2011, Altaf et al. 2020).

The five extracts were used to analyze their antimicrobial efficacy and each exhibited different result. n-Hexane extract of V. griseus oil produced best inhibition zone against E. coli, S. aureus, drug resistant P. aeruginosa and K. pneumonia. The above findings are in line with the previous studies conducted by (Inguglia et al. 2020; Brazil et al. 2020) on animal oil products as antibacterial agent. The same extract showed an average antioxidant activity of 54.6% scavenging capacity of DPPH. The extract was efficiently active against growth of NDV and IBV and comparatively less active against H9N2 and IBDV viruses. Current findings are in accordance with earlier work carried on n-Hexane extract of Saara hardwickii body oil (Arshad et al. 2022). GC-MS analysis of n-Hexane extract displayed bioactive compounds viz. 9-octadecenoic acid (Z)- acid, 9,10-dichloro-, methyl ester and 9,12-octadecadienoic acid (Z,Z). Similar compounds were reported from n-Hexane extract of different medicinal plants (Rahman et al. 2014; Abubakar and Majinda 2016). Antiviral activity of n-Hexane extract was due to its eugenol component as also reported from (Schnitzler et al. 2011).

Diethyl ether extract inhibited growth of P. vulgaris while did not show good antioxidant activity in terms of DPPH scavenging. Best antiviral activities were displayed by diethyl ether extracts against NDV, H9N2 and IBV viruses with lowest HA titer values while lowest antiviral activity was observed against IBDV. GC-MS analysis of diethyl ether extract displayed bioactive compounds viz. 9,12-octadecadienoic (Z,Z)-9-octadecenoic acid (Z), cis-p-mentha-1(7),8-dien-2-ol and 2,4-decadienal. The bioactive compound 9,12-octadecadienoic (Z,Z)-9-octadecenoic acid (Z) was described as antibacterial by Rahman et al. 2014; Abubakar and Majinda 2016 and cis-p-mentha-1(7),8-dien-2-ol by Spencer et al. 2021 while 2,4-decadienal as antiviral component in nature (Setzer 2016).

Methanol extract of V. griseus oil showed growth inhibition of drug resistant S. aureus. The current findings are in accordance with studies on antimicrobial activity of body oils from Rhinella jimi and Podocnemis expansa (Sales et al. 2015; Brazil et al. 2020). The Methanol extract showed an average antioxidant activity of 57.7% scavenging capacity of DPPH. These results are in agreement with antioxidant potential by Emu oil as reported by Bennett et al. 2008. Best antiviral activities were exhibited against NDV, H9N2 and IBV viruses with lowest HA titer values when compared against IBDV. These results are consistent with antiviral study of essential oil and natural antimicrobials (Barbour et al. 2008; Balta et al. 2020). GC-MS analysis of methanol extract showed erucic acid, cis-11-eicosenoic acid, 9,12-octadecadienoic (Z,Z) and 2,4-decadienal. According to Kim et al. 2018 erucic acid, cis-11-eicosenoic acid, 9,12-octadecadienoic (Z,Z) have good antibacterial potential while 2,4-decadienal has excellent antiviral potential (Hayashi et al. 1995; Setzer 2016).

Ethyl acetate extract of V. griseus oil inhibited growth of K. pneumoniae. The results are in line with antimicrobial work on skin secretions of Bufo melanostictus (Thirupathi et al. 2018) while no antioxidant activity in terms of DPPH scavenging was observed. Ethyl acetate extract was moderately active against growth of NDV, IBV, H9N2 and IBDV viruses. GC-MS analysis of methanol extract showed 9,12-octadecadienoic (Z,Z)-, dodecanal and n-butyric acid 2-ethylhexyl ester. According to a study conducted by Hayashi et al. 1995 and Abubakar and Majinda 2016 the compound 9,12-octadecadienoic (Z,Z)- has best antibacterial while n-butyric acid 2-ethylhexyl ester and dodecanal have good anti-viral activity (Thormar et al. 1987; Hayashi et al. 1995).

Surprisingly, 1-Butanol extract of V. griseus oil did not exhibited any anti-bacterial strain though highest antioxidant activity of 70.7% scavenging capacity of DPPH. 1,1-diphenyl-2-picrylhydrazil (DPPH) consumption by the sample was considered proportional to its antioxidant activity, thus proved as an effective natural antioxidant. The V. griseus oil has potential to scavenge free radical DPPH when compared with body oils obtained from other reptiles like Naja kaouthia (Cobra) and Uromastyx harwickii (Khunsap 2016; Shams et al. 2019). Best antiviral activities were exhibited against NDV and H9N2 as compared against IBV and IBDV. Current study verifies with previous antimicrobial study on fixed oils harvested from ruminant fats (Dias et al. 2019). GC-MS analysis of methanol extract showed bioactive compounds trans-p-mentha-1(7),8-dien-2-ol, n-butyric acid 2-ethylhexyl ester, octanal, dodecanal and 2,4-decadienal. The bioactive compound trans-p-mentha-1(7),8-dien-2-ol and octanal was considered best antibacterial agent from the work conducted by Reichling et al. 2009 and Spencer et al. 2021 on different amphibian oils. The antiviral activity of 1-Butanol extract was due to presence of n-butyric acid 2-ethylhexyl ester, octanal, dodecanal and 2,4-decadienals reported from earlier research work showed by Thormar et al. 1987, Hayashi et al. 1995, Reichling et al. 2009, and Setzer, 2016.

Remarkable antimicrobial and antioxidant potential of essential oils was recorded due to the presence of higher concentration of phenolic compound eugenol in the oil in a previous investigation by Purkait et al. 2020. In present study n-Hexane extract of V. griseus body oil showed similar results due to presence of eugenol along with other bioactive molecules.Current study documented a remarkable antimicrobial potential of V. griseus oil traditionally used in treatment of different ailments faced by inhabitants of Cholistan desert (Ahmad et al. 2021). GC-MS analysis recorded 11 bioactive compounds in different V. griseus body oil extracts. Current antimicrobial study proved V. griseus oil extracts have specific bioactive natural compounds which proved useful against different microbial pathogens.

Current findings of antibacterial, antioxidant and antiviral activities of Varanus griseus body oil suggests its potential use in modern natural medicines to cure various microbial infections. The n-Hexane oil extract exhibited best antibacterial activity and effective antioxidant potential due to presence of phenolic and bioactive compounds while 1-Butanol oil extract showed the best antioxidant potential. All the five extracts used in current work demonstrated the antiviral potential. The following major classes of compounds were identified in all five extracts viz. Aldehydes, fatty acids, phenols, terpenoids and esters. Among them 1-Butanol extract found diverse in nature with 34 different chemical constituents. More antimicrobial studies could be done to explore the medicinal values of body fats from different animal species used in zootherapeutic practices.

DPPH: 1,1-diphenyl-2-picrylhydrazil
GC-MS: Gas chromatography mass spectrometry
NDV: NewCastle disease virus
AIV: Avian influenza virus
IBV: Infectious bronchitis virus
IBDV: Infectious bursal disease virus
HA: Hemagglutination test
IHA: Indirect hemagglutination test
ZOI: Zone of inhibition
INT: p-iodonitro-phenyltetrazolium violet
MIC: Minimum inhibition concentration
PBS: Phosphate buffer saline solution
RPM: Revolutions per minute
PI: Post infection
EDTA: Ethylenediaminetetraacetic acid

Acknowledgements: The authors would like to thank “Institute of Pure and Applied Biology” Bahauddin Zakariya University, Multan, Pakistan for facilitating to conduct this study and Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa, Mexico for GC-MS analysis.

Author contributions: AAK and TR designed the study and supervised the experiments, SA collected and verified animal specimens, SA and MIS performed laboratory experiments. GR and DVNC executed chromatographic analysis of oil, and SA corrected and finalized the manuscript.

Funding: None.

Conflict of interest: The authors declare no conflict of interest.

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No competing interests reported.

Antimicrobial activity, antioxidant and chromatographic analysis of Varanus griseus oil extracts

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Antibacterial activity:

Antioxidant activity using DPPH free radical assay:

Antiviral activity:

Activity Against Avian Influenza Virus (H9n2):

Activity against Infectious Bronchitis Virus (IBV):

Activity against Infectious bursal disease virus (IBDV):

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1