2.1 Collection of sample and extract-preparation

Nepeta paulsenii Briq, was gathered from Skardu (Gilgit-Baltistan), Pakistan. The plant was identified by Prof. Dr. Mansoor Hameed, and voucher specimen number 1002 was banked at the science herbarium of Quaid-e-Azam University, Department of Botany, Islamabad, Pakistan. The plant sample was collected at mature stage and the identification was carried out on the basis of morphological characteristics of sample. Leaves of N. paulsenii plant were dehydrated under shadow at RT and grinded to a very fine powder. Leaves powder was placed in Soxhlet apparatus for methanolic extraction. Extracted material was dried under vacuum with rotary-evaporator.

2.2 Phytochemical-analysis

Various qualitative trials were performed to recognize the classes of phytochemicals that exists in crude methanolic extract of leaves.

2.3 Gas chromatograph coupled with mass spectrometre (GC–MS) analysis

N. paulsenii was scrutinized for the existence of active compounds for mass determination on the gas chromatograph“ Thermo GC-Trace Ultra Ver; 5.0 ” supporting with a“ Thermo MSSDSQ II. The elements were parted on a 60 m long 'ZB 5-MS Capillary Standard Non-polar Pole' with a film thickness of 0.25-μm.-The temp. was increased from 70 − 260 °C at a rate of 6 °C per min. throughout the experiment. The rate of flow of carrier gas was 1 mL per min. of helium, while the sample injection vol. was 1 μL. The-characterization of chemical components was based on analogy with those obtained from genuine sample and Wiley library spectra.

2.4 Experimental design

In the UAF animal house, male albino rats were separated for this trial. Tap water and food chaw ad libitum were given to rats. The rats of different groups were kept in separate cages. Eight-groups (n = 6/ group) of male albino rats were established to scrutinize the hepatoprotective role of N. paulsenii extract. Group-I was referred as control. Group-II was treated with olive oil and dimethyl-sulfoxide (1:1, v/v) at a dose of 1 mL per kg bw orally. Rats in group III were treated with Olive oil + CCl₄ (8:2, 1 mL per kg bw). CCl₄ + silymarin (50 mg per kg bw) in dimethyl sulfoxide was administered to group IV rats. Doses of 200 and 400 mg/kg bw of N. paulsenii extract were orally administered to group V and VI rats respectively with CCl₄. Dose of 200 and 400 mg/kg bw in N. paulsenii extract only were given to VII and VIII group rats respectively.

Trial was carried out for thirty days and after completion; all of the rats were examined then blood sample was stored in sterilized tubes and liver of every single rat was removed out. For removal of plasma, centrifugation of blood carried out for 15 min at 3000 revolution per minutes and kept at −20 °C for further analysis. Liver was washed with ice cold saline. A segment of each liver was packaged in zip lock bag and kept at −20 °C for evaluation of anti-oxidant enzymes and DNA damage studies. And other part of liver was used for histopathological studies. This protocol was certified by the ethical board of Agriculture University, Faisalabad-Pakistan.

2.5 Anti-oxidant enzymes assessment

The assessments of anti-oxidant enzymes were carried out according to the reported procedures (Ijaz et al., 2021, Rehman et al., 2022). Finally, the standard curve was plotted (Hayashi et al., 2007).

2.6 Biochemical studies of serum

Liver marker enzymes, i.e., ALT, ALP and AST were analyzed utilizing relevant commercially available diagnostic kits (Wiesbaden-Germany).

2.7 Comet assay

For the estimation of DNA-injury, process of (Dhawan et al., 2009) was followed. According to this method, a reasonable section of liver tissue was placed and crushed in a number of fragments on autoclaved slides and then immersed in 0.5 % of low melting agarose and 0.6 % of NMA (Normal melting agarose) and let it set at normal room temp. After solidification, these slides were bathed in cold lysis solution for 1 hr. at 4 ℃. For the DNA un-winding, these slides were kept in electrophoretic buffer for 20 min. and then processed for electrophoresis for 30 min. After that, Tris-hydrochloric acid buffer at 25 °C was used to bring pH 7.0 of each slide. Ethidium bromide (1 %) was used for staining of slides before studying under fluorescence microscope. For DNA assessment, injury level image software (TRITEK) was used. Parameters i.e., comet number, olive moment, tail moment, head length, tail length, comet length % DNA contents in head and tail were considered for DNA integrity.

2.8 Histopathological examination

Histopathological analysis was performed to observe hepatic injuries. Samples were fixed in fixative solution (glacial acetic acid10 %, absolute alcohol 70 %, formaldehyde 20 %). For the gradual dehydration process, the tissues were passed through various grades of ethanol (70 %, 90 % and 100 %). For embedding Paraffin wax was used. Thick slices of tissues were cut to the 4–5 μm using spencer rotatory microtome (model 820) was used for this purpose. Lastly stained using the stain (hematoxylin-eosin) which was dissolved in 70 % of alcohol. Light microscope (Nikon model 187–842, Japan) was used to observe these slides at × 40 and microphotography was done using Leica LB microscope was adapted to utilize the photographs.

2.9 Statistical analysis

Results are displayed as Mean ± SEM. The difference between the results of different groups was evaluated as reported earlier (Samad et al., 2020).

3.1 Qualitative phytochemical analysis

The phytochemical examination of crude extract is shown in Table 1. The existence of flavonoids, coumarins, saponins, terpenoids and betacyanins in methanol extract of N. paulsenii was confirmed by a qualitative study.

3.2 GC–MS analysis

3.2.1

3.2.1 Identification of components

N. paulsenii crude extract was designated for GC–MS study to reveal the compounds accountable for hepatoprotective activity. GC–MS exploration specified that N. paulsenii contains 10 chemical-components eluted between 2.0 and 10.0 min. (Fig. 1). The documentation of chemical components was based on a comparison of their mass spectra and relative-rt with those attained from reliable samples and Wiley libraries spectra (Table 2). In N. paulsenii, out of these 10 chemical constituents, there was 2-glycerin (4.37 % as per peak area and 2.32 min. R/T), geranic acid (4.87 % and 4.08 min.), 6-octadien-1-ol (2.32 % and 4.22 min.), 2-pentadecanone (3.74 % and 6.53 min.), benzenepropanoic acid (2.20 % and 6.95 min.), ascorbic acid (22.1 % and 7.05 min.), phytol (10.8 % and 7.67 min.), 9, 12, 15-octadecatrienoic acid (36.1 % and 7.80 min.), octadecanoic acid (7.78 % and 7.87 min.) and 1,2-benzenedicarboxylic acid (4.43 % and 9.71 min.) (Fig. 2).

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Fig. 1

GC–MS chromatogram of leaves methanol extract of N. paulsenii plant.

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Table 2 GC–MS analysis of N. paulsenii leaves extracts (methanol).

Sr#	Name of compound	Peak area %	Class	R/T (mins.)
1	glycerin	4.37	Alcohol	2.33
2	geranic acid	4.87	Polyunsaturated fatty acid	4.08
3	2,6-octadien-1-ol	2.34	Alcohol	4.22
4	2-pentadecanone	3.74	Ketone	6.53
5	benzenepropanoic acid	2.20	Carboxylic acid	6.95
6	1-(+)-ascorbic acid	22.1	Vitamin C	7.05
7	phytol	10.8	Diterpene alcohol	7.67
8	9,12,15-octadecatrienoic acid	36.1	Linolenic acid	7.80
9	octadecanoic acid	7.78	Stearic acid ester	7.87
10	1,2-benzenedicarboxylic acid	4.43	Plasticizer	9.71

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Fig. 2

(a) MS of glycerin (RT: 2.33) (b) MS of geranic acid (RT: 4.08) (c) MS of 2, 6-Octadien-1-ol (RT: 4.22) (d) MS of 2-Pentadecanone (RT: 6.53) (e) MS of Benzenepropanoic acid (RT: 6.95) (f) MS of 1-(+)-ascorbic acid (RT: 7.05) (g) MS of phytol (RT: 7.67) (h) MS of 9, 12, 15-octadecatrienoic acid (RT: 7.80) (i) MS of octadecanoic acid (RT: 7.87) (j) MS of 1, 2-benzenedicarboxylic acid (RT: 9.71).

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Fig. 2

(a) MS of glycerin (RT: 2.33) (b) MS of geranic acid (RT: 4.08) (c) MS of 2, 6-Octadien-1-ol (RT: 4.22) (d) MS of 2-Pentadecanone (RT: 6.53) (e) MS of Benzenepropanoic acid (RT: 6.95) (f) MS of 1-(+)-ascorbic acid (RT: 7.05) (g) MS of phytol (RT: 7.67) (h) MS of 9, 12, 15-octadecatrienoic acid (RT: 7.80) (i) MS of octadecanoic acid (RT: 7.87) (j) MS of 1, 2-benzenedicarboxylic acid (RT: 9.71).

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3.3 Effect of crude N. Paulsenii extract on antioxidant enzymes activity

Antioxidant enzymes protect the cell against toxic radical reactions and show defensive effect against cellular damage. Variation in activity of antioxidant enzymes was assessed after CCl₄ treatment to illustrate the protecting effect of N. paulsenii extract. Action of CAT, SOD, GST, POD, GSH & GSR were substantially (p < 0.05) reduced in comparison with control after CCl₄ administration. N. paulsenii extract reinstated the antioxidant enzymes activities in co-treated groups and it was statistically analogous to the group treated with silymarin at its higher dose. Oral administration of N.paulsenii extract alone demonstrated a marginal difference in the activities compared to control (Table 3).

3.4 Effect of N. Paulsenii extract on ROS and TBARS levels

Compared to control group, the rats treated with CCl₄ reported a significant (p < 0.05) eleviation in ROS and TBARS levels. The levels of TBARS and ROS reduced remarkably (p < 0.05) when co-treated with CCl₄ + silymarin as opposed to the CCl₄ group. As matched with CCl₄-intoxicated group, the N. paulsenii + CCl₄ groups showed significant decreases in ROS and TBARS levels (dose-dependent). Rats administered with N. paulsenii extract alone showed ROS and TBARS levels within standard range (Table 4).

3.5 Effect of N. Paulsenii extract on serum markers

Levels of ALP, AST and ALT are directly linked to oxidative stress in liver tissues (Table 5). Treatment of CCl₄ significantly (p < 0.05) enhanced the level of hepatic serum markers (ALP, AST and ALT) which was diminished significantly (p < 0.05) by co-treatment with N. paulsenii extract. However, in 200 and 400 mg/kg dose of N. paulsenii alone treated groups, the serum markers levels were almost comparable to control group values.

3.6 Effect of N. Paulsenii extract on DNA damage

CCl₄ intoxication remained significant (p < 0.05) in comet length, comet-numbers, tail length, tail and olive moment, whereas a major decrease was reported in head length and percentage DNA in head as compared with control rats. CCl₄ + silymarin treatment exhibited a notable (p < 0.05) reduction in comet number, comet and tail length, % DNA in tail, tail and olive moment and a substantial (p < 0.05) rise in percentage DNA in head and head length was observed as compared to CCl₄ intoxicated rats. In identical way, significant reductions in tail length, comet number, comet length, percent DNA in tail, tail moment and olive moment, and a significant increase in percent DNA in head and head length relative to CCl₄ administered rats were observed in N. paulsenii + CCl₄ administered groups dose dependently. Rats administered with N. paulsenii extract alone showed normal parameters as indicated in the control group (Table 6).

3.7 Effect of N. paulsenii extract on histomorphology of liver

Effect of N. paulsenii extract in various groups with different doses and CCl₄ treatment on histology of liver is presented in Fig. 3 (40X). Vehicle control and Control group indicated normal histomorphology and did not show any disruption in central venule and hepatocytes. CCl₄ administration resulted in severe variations that occur in histomorphology of liver tissues. The substantial variations noticed in CCl₄ intoxicated group were deterioration of lobule, fatty change, congested blood vessels and severe steatosis. Co-treatment of N. paulsenii extract with CCl₄ exhibited tissue recovery and abolition of chronic damages at different doses. N. paulsenii extract (200 mg/kg b.w.) treatment showed a visible level of tissue recovery.

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Fig. 3

Microscopic images of liver tissues were obtained from different groups (H&E 40X). (a) Normal histoarchitecture (control) (b) Vehicle control (normal histoarchitecture) (c) CCl₄ (1 mL/kg) Tissues show extensive and marked necrosis (d) CCl₄ + Silymarin (decrease in necrosis throughout the liver tissues and recovery of damaged tissues (e) N.P (200 mg/kg) + CCl₄ (decreased necrosis) (f) N.P (400 mg/kg) + CCl₄ (shows recovery in damaged tissues) (g) N.P (200 mg/kg) shows normal histoarchitecture (h) N.P (400 mg/kg)shows normal histomorphology: H; Hepatocytes, CV; Central venule, S; Sinusoids, KC; Kupffer cells, N; Nucleus.

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3.8 Discussion

Liver performs metabolic functions in the body. Hepatic diseases are a major problem even in advanced countries (Adewusi and Afolayan 2010). Hepatic injury is related with distortion of these metabolic functions. There is a lack of effective medicines which stimulate the functions of liver and protect the liver from damage and regenerate the hepatic cells so there is need to replace the current medicines with new effective medicines (Adewusi and Afolayan 2010). Mostly new drugs are being synthesized from natural products (secondary metabolites) and from those compounds that are obtained from natural products Genus Nepeta have medicinal properties because many species of genus Nepeta are used in folk medicines and components found in species of Nepeta genus have biological activities (Karaman 2007). Herbal medicines that are synthesized from plants have been used against hepatic diseases for a long time and some preparations are accessible in market. Many active components are present in plants from where herbal medicines are synthesized. Therefore, the present investigation emphasizes the hepatoprotective potential of N. paulsenii and its active ingredients, which can be further studied as a pharmacore for reducing the toxic effects of carbon tetrachloride.

Many phytocomponents identified in preliminary phytochemical analysis of extract like coumarins, terpenoids, flavonoids, saponins and betacyanins. Among these phytochemicals, flavonoids, coumarins, saponins and betacyanins have increased a certain importance because of their wide range antioxidant-activities (Sajid et al., 2016). Ten components identified in further analysis (GC–MS) which have medicinal properties these components are glycerin, geranic acid, phytol, octadecanoic acid, 2,6-octadien-1-ol, 2-pentadecanone, benzene propanoic acid, 1-(+)-pscorbic acid, 1,2-benzene-dicarboxylic acid and 9,12,15-octa-decatrienoic acid. benzenepropanoic and 1-(+)-ascorbic acids have anti-oxidant activity (Akpuaka et al., 2013). So benzenepropanoic acid and 1-(+)-ascorbic acid are foremost components providing to the antioxidant activity of the plant. Benzenepropanoic acid is used to extend the shelf life of foods as an antioxidant, and it is also used to protect the shelved foods from deterioration by microorganisms. Moreover, 9,12,15-octadecatrienoic acid has hepatoprotective effect and it is the one of major constituents of the plant (Akpuaka et al., 2013).

Antioxidant enzymes are considered to be the foremost species to defend the biological molecules. The metabolic function of the liver is to detoxify the effect of xenobiotics on ROS development, where CAT, POD, SOD, GSH, GSR and GST play a crucial role in oxidative stress deterrence in liver (Khan et al., 2013). CAT reacts with hydrogen peroxide to form molecular oxygen and water. Administration of CCl₄ influenced the activity of POD, CAT, SOD, GSH, GST and GSR while the levels of ROS and TBARS were elevated in CCl₄ administered group. Lipid peroxidation of hepatocytes caused when CCl₄ is metabolized into highly reactive metabolites by liver. The observed decline in the level of CAT, SOD, POD due to CCl₄ exposure is in consensus with former observation of (Sajid et al., 2016), who reported that treatment with CCl₄ reduced the level of these antioxidant enzymes. Reduction in the level of these antioxidant enzymes indicated the oxidative damage in liver (Cheng et al., 2013). Administration of plant extract instigated a noteworthy recovery in activities of enzymatic antioxidants CAT, SOD, POD, GSH, GST and GSR, however, ROS and TBARS levels were significantly reduced. This demonstrates that plant extract can scavenge free radicals that might cause reduction in oxidative damage to liver tissue and restore the antioxidant enzymes activities. The existence of bioactive phytochemicals in the methanolic extract of the N. paulsenii may be a major cause of its curative potential.

CCl₄-administration significantly increased the AST, ALP and ALT levels in treated rats. The biochemical damage and cellular function defects can be detected by the levels of these enzymes (Khan et al., 2011). Previous studies have shown that high levels of serum ALT, AST, and ALP have a strong linkage with oxidative liver damage (Sharifi-Rigi et al., 2019). Aminotransferases including ALT, ALP and AST are enzymes ked with liver parenchyma cells. These enzymes are normally present in the cytosol, but due to the damage in plasma membrane of hepatic cells, these markers are released into the bloodstream, which indicates liver damage. So, the level of these enzymes is considered as an important marker that is used in the diagnosis of liver diseases (Carobene et al., 2013). Deleterious effects of CCl₄ may be linked with its reactive metabolites which are formed intermediately during its metabolism in the body. Previous investigations showed that over production of ROS impairs the structural integrity of hepatic cells, as indicated by the unusual increase in hepatic serum markers. CCl₄ treatment caused noteworthy hepatic injury as evident by increased levels of ALP, ALT and AST that are indicator of disturbance in functional-integrity of liver. Treatment with N. paulsenii extract reduced the hepatic markers level which is the sign of stabilization in hepatic functions. It has been reported earlier, phytochemicals (flavonoids, coumarins, saponins and betacyanins) act as an antioxidant which acts as both free radical scavengers and antioxidant (Engwa 2018). This reduction in the level of hepatic serum markers can be accredited to antioxidant potential of N. paulsenii extract.

DNA can be damaged by several exogenous and endogenous factors. Common factors include ROS produced during the normal metabolic functions, spontaneous or enzymatic conversions and errors during DNA replication (Tiwari and Mishra 2017). It was previously reported that several cytotoxic agents pose genotoxic impacts through the generation of ROS, which results in single/double-stranded breakdowns and disrupt the DNA integrity (Srinivas et al., 2019). In the current investigation, the outcomes of the comet parameters show significant variations in comet parameters. CCl₄ administration induced a remarkable reduction % DNA in the head and in head length compared to the standards, on the other hand, increased the comet length, comet numbers, tail length, olive-tail moment, and % DNA in tail. It has been reported that CCl₄ potentially generates ROS, which leads toward DNA fragmentation (Alkreathy et al., 2014). However, N. paulsenii plant extract recovered the DNA injury provoked by CCl₄. Our study evaluated that N. paulsenii plant extract is a potent bioactive agent to avert CCl₄-induced DNA damage by decreasing OS in the liver tissues which is further supported by (Samad et al., 2020), who observed that N. paulsenii plant extract decreased oxidative stress produced due to CCl₄ administration in the testicular tissues of rats.

Histopathological study of liver showed the severe damages like hepatic damages, inflammatory cell infiltration, fatty changes, degeneration of lobular structure and degeneration of nuclei in CCl₄ treated group. Level of fatty degeneration and necrosis were obvious after treatment of CCl₄. CCl₄ induced hepatic tissue injury is due to the production of free radicals and lipid peroxidation that induces cellular damage. CCl₄ forms a CCl₃ and OOCCl₃ radicals then these radicals bind with polyunsaturated fatty acids (PUFA) and form peroxy and alkoxy radicals. These radicals produce injury in cell membrane, hepatic damage and altered the activity of different enzymes. These radicals bind with macromolecules and become source of oxidative degradation of cellular membrane leading to necrosis of hepatocytes (Ranawat et al., 2010). Administration of high and medium dose of N. paulsenii plant extract reduced the injury and fatty degeneration. This may be due to the presence of phytochemical components like coumarins, terpenoids, flavonoids, saponins and betacyanins. These components exhibited hepatoprotective effect and work individually or combined action against hepatotoxicity. Histological study exhibited a defensive effect of N. paulsenii plant extract on CCl₄ induced hepatoxicity which may be attributed to the active components of the plant extract.

3.9 Conclusion

The current study revealed that the hepatoprotective nature of N. paulsenii leaves extract is possibly due to the endogenous antioxidant-molecules and scavenging of free radicals. Natural antioxidants tend to promote this effect in N. paulsenii plant, which greatly decreased oxidative stress and contributed to normal liver functions. Further research is needed to expose the pathways concerning the hepatoprotective function of N. paulsenii at molecular level. Moreover, ethanolic extract of N. paulsenii needs to be studied in future as for the intake of oral drugs ethanolic extracts may be relatively safer compared to methanolic extracts. Also, LC-MS based studies needs to be carried out for N. paulsenii.