Assessment of Preclinical Antioxidative and Anti‐Inflammatory Activities of Cornus macrophylla Wall. Bark

TL;DR

This study shows that the ethyl acetate fraction of Cornus macrophylla bark has strong anti-inflammatory and neuroprotective effects in mice.

Contribution

The study identifies the ethyl acetate fraction of C. macrophylla as a potent antioxidant and anti-inflammatory agent through in vivo and ex vivo experiments.

Findings

The ethyl acetate fraction of C. macrophylla significantly reduced inflammation and oxidative stress in mice.

It restored antioxidant levels like glutathione and catalase in a dose-dependent manner.

The fraction improved locomotor activity in LPS-treated mice.

Abstract

Cornus macrophylla
Wall. Bark has been traditionally utilized for its therapeutic properties; however, limited scientific evidence is available that supports its role as having anti‐inflammatory and neuroprotective properties. Exploring the neuroprotective and anti‐inflammatory properties of
C. macrophylla
Wall. Bark may help in validating the therapeutic properties, presenting insights into its use as a potent natural component for modulating inflammation and oxidative stress‐related neurological ailments. In this study, crude extract along with different fractions of
C. macrophylla
Wall. Bark have been investigated for anti‐inflammatory and neuroprotective effects. In the in vivo anti‐inflammatory assay, the ethyl acetate (EA) fraction showed activity of 68.3%, 79.7%, and 83.7% at dosages of 50, 100, and 200 mg/kg B.W. (Body weight) of all the mice decreased when treated with LPS while it increased when treated with aspirin, crude extract, dichloromethane (DCM), and EA, respectively. Rectal temperature increased when treated with lipopolysaccharide (LPS) and gradually decreased with aspirin, crude, DCM, and EA fraction. Moreover, locomotor activity was weak when treated with LPS and enhanced when treated with aspirin, crude, DCM, and EA, respectively. Neuroprotective ex vivo results showed that the EA fraction decreased oxidative stress by restoring the glutathione (GSH) level by 21.10, 18.20, and 14.04 μM/mg proteins at doses of 100, 200, and 300 mg/kg body weight, respectively. The EA fraction decreased oxidative stress by restoring the superoxide dismutase (SOD) level by 0.17, 0.15, and 0.13 units/mg proteins at doses of 100, 200, and 300 mg/kg BW, respectively. The EA fraction decreased oxidative stress by restoring the malondialdehyde (MDA) level by 0.17, 0.15, and 0.13 units/mg proteins at dosages of 100, 200, and 300 mg/kg body weight, respectively. The EA fraction markedly increases the level of catalase (CAT) by 46.26, 41.12, and 39.37 units/mg proteins at doses of 100, 200, and 300 mg/kg body weight, respectively. In vivo results validated the in vitro results of different fractions of
C. macrophylla
.

C. macrophylla
, belonging to the Cornaceae family, exhibits significant anti‐inflammatory and neuroprotective properties. The ethyl acetate fraction showed the highest efficacy in reducing oxidative stress, restoring antioxidant levels (GSH, SOD, TBARS, and CAT), and improving locomotor activity in LPS‐treated mice, validating its therapeutic potential.

Figures

C. macrophylla bark, different extracts effects on weight alteration by LPS administration. Values are represented by mean ± SEM. p < 0.001 compared to control mice. p < 0.001 compared to LPS‐treated mice.

Effect of different extracts of C. macrophylla on the LPS‐induced variation in rectal temperature. Values are represented by mean ± SEM. p < 0.001 compared with control mice. p < 0.001 compared with LPS‐treated mice. p < 0.001 compared to aspirin‐treated mice.

Effect of different extracts of C. macrophylla on LPS‐induced variation in locomotor activity using actophotometer. Values are represented by mean ± SEM, p < 0.001 when compared with control mice and p < 0.001 compared to the LPS‐treated mice.

Tables

TABLE 1. The ferric reducing antioxidant power (FRAP) assay of crude extract and different fractions of C. macrophylla .

Item	FRAP (μM TE/g)
Dichloromethane	21.65 ± 3.14
Ethyl acetate	21.94 ± 2.20
Crude	8.46 ± 0.60
n‐Hexane	11.24 ± 1.56
Aqueous	7.59 ± 0.76
p	< 0.001

TABLE 2. H2O2 inhibitory assay of the crude extract and different fractions of C. macrophylla .

Samples names	Concentration (μg/mL)	% H2O2 activity	IC50 (μg/mL)
Crude	1000 500 250 125 62.5	85.15 ± 0.72*** 81.70 ± 0.40*** 75.64 ± 0.62*** 71.22 ± 0.20*** 64.34 ± 0.94***	13.56
n‐Hexane	1000 500 250 125 62.5	76.62 ± 0.66*** 64.34 ± 0.94*** 70.38 ± 0.12*** 63.16 ± 0.44*** 57.42 ± 0.54***	31.54
Dichloromethane	1000 500 250 125 62.5	89.78 ± 1.42*** 83.22 ± 0.78*** 75.56 ± 0.42*** 70.34 ± 0.44*** 66.42 ± 0.64***	16.38
Ethyl acetate	1000 500 250 125 62.5	87.77 ± 0.73*** 82.22 ± 1.44*** 77.54 ± 0.52*** 73.49 ± 0.45*** 65.10 ± 0.34***	12.68
Aqueous	1000 500 250 125 62.5	71.51 ± 0.77*** 66.34 ± 0.92*** 58.57 ± 0.51*** 52.22 ± 0.34*** 46.79 ± 0.47***	93.41
Ascorbic acid	1000 500 250 125 62.5	96.54 ± 0.66 91.46 ± 0.34 86.62 ± 0.46 82.80 ± 0.84 77.37 ± 0.78	7.94

TABLE 3. In vitro albumin denaturation and protease inhibition activities of C. macrophylla .

Tested fractions	Albumin denaturation IC50 (μg)	Protease inhibition IC50 (μg)
Crude	27.10	21.88
n‐Hexane	45.40	40.32
Dichloromethane	25.30	15.80
Ethyl acetate	22.15	14.20
Aqueous	60.10	90.88
Diclofenac sodium	20.10	13.67

TABLE 4. Acute toxicity result of crude drug extract of C. macrophylla bark.

Group	Animals	Tested crude drug toxicity (mg/kg b. wt)
1	5	500
2	5	1000
3	5	1500
4	5	2000
5	5	3000

TABLE 5. Percent inhibition of various extracts of C. macrophylla bark in carrageenan‐induced paw edema.

Test sample/drug	Dose mg/Kg	1st hour	2nd hour	3rd hour	4th hour	5th hour
Vehicle	10 ml	70.0 ± 0.03	71.5 ± 0.07	71.0 ± 0.03	71.2 ± 0.07	71.3 ± 0.03
Diclofenac sodium	10 mg	71.8 ± 0.80	74.2 ± 0.06	80.8 ± 0.80	77.7 ± 0.70	73.8 ± 0.04
Crude	50 100 200	42 ± 0.02ns 51 ± 0.05ns 60 ± 0.04***	56 ± 0.04ns 57.6 ± 0.02ns 62 ± 0.02***	58.75 ± 0.03ns 63 ± 0.03ns 65.8 ± 0.08***	54.4 ± 0.20ns 51.6 ± 0.04ns 54.4 ± 0.40***	51.6 ± 0.08ns 51.6 ± 0.60ns 55.8 ± 0.06***
EA	50 100 200	61.9 ± 0.20ns 67.5 ± 0.30ns 73.4 ± 0.40***	63.4 ± 0.40ns 72.3 ± 0.30ns 76.1 ± 0.50**	66.6 ± 0.20 ns 76.2 ± 0.20 ns 81.2 ± 0.40***	68.3 ± 0.15ns 79.7 ± 0.31ns 83.7 ± 0.03ns	59.4 ± 0.20ns 62.7 ± 0.50ns 68.3 ± 0.03**
DCM	50 100 200	58.7 ± 0.30ns 60.9 ± 0.50ns 68.4 ± 0.08***	58.8 ± 0.02ns 66 ± 0.06ns 72.6 ± 0.04***	63 ± 0.50ns 70.8 ± 0.08ns 75 ± 0.50***	57 ± 0.30ns 73.8 ± 0.02ns 77.2 ± 0.08***	52.2 ± 0.04ns 67.2 ± 0.02ns 72.2 ± 0.06***
NH	50 100 200	44 ± 0.08ns 51 ± 0.11ns 57.5 ± 0.50***	46 ± 0.06ns 53 ± 0.30ns 58.8 ± 0.06***	51.6 ± 0.04ns 54.1 ± 0.30ns 61.2 ± 0.22***	42.2 ± 0.06 ns 43.4 ± 0.08 ns 51.6 ± 0.02***	40 ± 0.02ns 41.1 ± 0.11ns 47.7 ± 0.07***
AQ	50 100 200	39.3 ± 0.11ns 45 ± 0.30ns 53.7 ± 0.07***	44.2 ± 0.02ns 47.3 ± 0.50ns 54.6 ± 0.08***	46.2 ± 0.02ns 49.5 ± 0.30ns 57.9 ± 0.07***	37.2 ± 0.08ns 41.1 ± 0.50ns 53.3 ± 0.30***	35.4 ± 0.04ns 40 ± 0.06ns 43.4 ± 0.08***

TABLE 6. Effect of various fractions of C. macrophylla on LPS‐induced alterations in GSH levels.

Treatment		GSH (μmoles/mg protein)
Control		34.00 ± 0.60
LPS + aspirin		15.00 ± 0.92***
EA	(100 mg/kg)	21.10 ± 0.80***
	(200 mg/kg)	18.20 ± 0.62***
	(300 mg/kg)	14.04 ± 1.02***
CHF	(100 mg/kg)	27.08 ± 1.10n.s.
	(200 mg/kg)	25.40 ± 1.20*
	(300 mg/kg)	22.00 ± 1.10***
Crude	(100 mg/kg)	31.50 ± 1.20n.s.
	(200 mg/kg)	27.00 ± 1.40*
	(300 mg/kg)	24.00 ± 0.90***

TABLE 7. Effect of various fractions of C. macrophylla on LPS‐induced alterations in SOD levels.

Treatment		SOD (units/mg protein)
Control		0.28 ± 0.004
LPS + Aspirin		0.15 ± 0.008***
EA	(100 mg/kg)	0.17 ± 0.007**
	(200 mg/kg)	0.15 ± 0.006***
	(300 mg/kg)	0.13 ± 0.007***
CHF	(100 mg/kg)	0.19 ± 0.005*
	(200 mg/kg)	0.17 ± 0.008**
	(300 mg/kg)	0.15 ± 0.009***
Crude	(100 mg/kg)	0.20 ± 0.004n.s.
	(200 mg/kg)	0.18 ± 0.007**
	(300 mg/kg)	0.16 ± 0.009***

TABLE 8. Effect of various fractions of C. macrophylla on LPS‐induced alterations in MDA levels.

Treatment		MDA (nmoles/mg protein)
Control		3.20 ± 0.08
LPS + Aspirin		2.40 ± 0.07***
EA	(100 mg/kg)	2.90 ± 0.07*
	(200 mg/kg)	2.42 ± 0.08***
	(300 mg/kg)	2.20 ± 0.08***
CHF	(100 mg/kg)	3.30 ± 0.09n.s.
	(200 mg/kg)	3.10 ± 0.09*
	(300 mg/kg)	2.50 ± 0.09***
Crude	(100 mg/kg)	3.20 ± 0.09n.s.
	(200 mg/kg)	3.10 ± 0.08*
	(300 mg/kg)	2.70 ± 0.08**

TABLE 9. Effect of various fractions of C. macrophylla on LPS‐induced alterations in CAT levels.

Treatment		CAT (unit/mg protein)
Control		58.10 ± 0.92
LPS + Aspirin		38.10 ± 1.20***
EA	(100 mg/kg)	46.24 ± 0.82**
	(200 mg/kg)	41.12 ± 0.80***
	(300 mg/kg)	39.37 ± 0.93***
CHF	(100 mg/kg)	51.52 ± 1.52n.s.
	(200 mg/kg)	47.10 ± 1.20*
	(300 mg/kg)	42.22 ± 1.20***
Crude	(100 mg/kg)	54.60 ± 1.60n.s.
	(200 mg/kg)	46.10 ± 1.10**
	(300 mg/kg)	43.43 ± 1.53***

Full text

Introduction

1

Cornus macrophylla Wall. (family Cornaceae), also known by its indigenous name “large leaf dogwood”, has been widely consumed as a potent therapy for treating various ailments. Moreover, C. macrophylla is known among folk practitioners as diuretic, analgesic, and for preserving foods. However, the fruits of C. macrophylla are being used as a remedy against varied diseases such as allergies, malaria, inflammation, cancer, diabetes, and oxidative stress (Noshiro and Baas 2000; Shah et al. 2015).

Inflammation is a degenerative process resulting from the accumulation of low‐molecular‐weight catabolic entities generated from damaged cells, causing alteration of osmotic pressure. Swelling during inflammation occurs due to elevated osmotic pressure, which leads to the exudation of fluid from the cell into the surrounding tissue, with or without liberation of heat, depending upon the metabolic activity at the inflamed area. Numerous pathological conditions are linked to chronic inflammation, such as cancer, soreness, redness, increased tissue temperature, and organ failure (Pan et al. 2014). Neuro‐inflammation is a syndrome that occurs through the hallmarks associated with the loss of neuronal structure and function in the central nervous system, resulting from a viral insult, but not limited to it. Neurodegeneration is profusely noticed in old‐aged people, resulting in different neurological diseases. Parkinson's disease, multiple sclerosis, and Alzheimer's disease are caused due to neurodegeneration and pose detrimental effects on mental and physical functionalities. It is still important to classify the causative agents of neurodegeneration. The inflammatory mechanism, however, has been found to be among the main reasons leading to multiple neurodegenerative pathways that are associated with depression. In the pathophysiology of neurodegeneration, pro‐inflammatory cytokines are found to play intriguing roles and therefore, are considered essential in managing neurodegenerative disorders.

Clinically, administration of carrageenan manifests weight loss, diarrhea, and visible blood in feces due to intestinal inflammation and mucosal injury induced by carrageenan exposure (Estakhr and Javdan 2011). Histamine, serotonin, and bradykinin are the first detectable mediators in the early phase of carrageenan‐induced inflammation; prostaglandins are involved in the increased vascular permeability and are detectable in the late phase of inflammation. Local and/or systemic inflammation is associated with enhanced levels of the pro‐inflammatory cytokines TNF‐α, IL‐1, and IL‐6 (Wu et al. 2022). Previous studies have highlighted the role of superoxide dismutases (SODs), metalloenzymes present across various prokaryotes and eukaryotes, as major components of cellular antioxidant defense mechanisms (Landis and Tower 2005). These enzymes aid the conversion of •O_2_ ^−^ (superoxide anion free radical) to H_2_O_2_ (hydrogen peroxide) and O_2_ (molecular oxygen). Afterward, hydrogen peroxide in the presence of CAT (catalase), GPx (glutathione peroxidase), and/or Trx (thioredoxin)‐dependent Prx (peroxiredoxin) is reduced to water (Lesser 2005).

Catalase enzyme is an oxidoreductase enzyme as it plays a crucial role in quenching the reactive oxygen species (ROS), that is, hydrogen peroxide, often produced as a by‐product of aerobic respiration (Kaushal et al. 2018). The basic mechanism of the working of this enzyme involves the breakdown and subsequent breakdown of the reactive oxygen species, that is, hydrogen peroxide (H_2_O_2_), into oxygen and water, thus relieving the oxidative stress caused by this substrate as depicted in the following reaction (Vasiliev et al. 2001). Lipid peroxidation is one of the main consequences of oxidative stress, a process in which reactive oxygen species degrade polyunsaturated fatty acids in cell membranes. This degradation forms different toxic byproducts, among which malondialdehyde (MDA) is a well‐recognized marker of inflammation. Increased content of MDA indicates the extent of oxidative damage, contributes to cellular dysfunction, and contributes to the progression of inflammation‐related disorders. Hence, in biological systems, MDA is considered an authentic indicator of oxidative stress and inflammatory conditions (Ayala et al. 2014). Malondialdehyde (MDA) is a solid compound (melting point, 72°C–74°C) that is soluble in water, methanol, ethanol, and moderately soluble in methylene chloride, while insoluble in diethyl ether (Wencel‐Delord et al. 2001), with a pK _ a _ value of 4.46 (Tsikas 2017). MDA level is commonly known as a marker of oxidative stress and the antioxidant status in cancerous patients (Gaweł et al. 2004). MDA is the prototype of the so‐called thiobarbituric acid reactive substances (TBARS). Glutathione reductase (GR) plays an essential central role in cell defense against reactive oxygen metabolites by efficiently maintaining the cellular reduced GSH pool through catalyzing the reduction of GSSG to GSH with the accompanying oxidation of NADPH (Anjum et al. 2010; Trivedi et al. 2013).

It is evident from the literature that medicinal plants and natural products have potential in curtailing inflammation and achieving optimal health (Calder et al. 2017; Pan et al. 2010). Herbal and medicinal plants provide an alternative and safe approach to potentially address chronic inflammatory conditions that are directly linked to several ailments such as diabetes, cardiovascular complications, and other metabolic disorders (Agarwal and Shanmugam 2022; Tasneem et al. 2019). It is the need of the time to investigate the potential of medicinal plants and their constituents in managing inflammation. Overall health and wellness can be achieved through dietary modifications and interventions. The present study aimed to highlight the effectiveness of C. macrophylla Wall. bark extracts in modulating inflammation, hence providing insights for their integration as dietary intervention and therapeutic strategies. Keeping in view the medicinal properties of C. macrophylla Wall. bark, this study was designed to evaluate the crude extract along with different fractions of C. macrophylla Wall. bark for in vivo anti‐inflammatory potential via carrageenan‐induced paw edema in mice. The experimental fractions were further subjected to lipo‐polysaccharide (LPS) induced neuro‐protective effect by performing various ex vivo activities.

Materials and Methods

2

Plant Materials

2.1

Bark of C. macrophylla Wall. was procured from the local market of Malakand District, KPK (Khyber Pakhtunkhwa), Pakistan in April. Prof. Dr. Nisar Ahmad (Department of Botany), University of Malakand, authenticated the plant's bark, and the specimen was stored in the herbarium of the department against voucher # H.B.UOM‐102.

Extraction and Fractionation

2.2

The bark (4.5 kg) was washed and shade‐dried, followed by grinding in a commercial grinder to collect respective bark powder. The bark powder was macerated in methanol for 15 days with continuous stirring. After maceration, the crude extract was initially filtered using muslin cloth to separate coarse solid particles, followed by filtration through filter paper. The filtered extract was concentrated at 40°C by the application of a rotary evaporator. After concentration, the final weight of the extract was 510 g with a yield of 11.3% which was measured using the standard formula ((Weight of extract after concentration/Weight of dried plant) * 100). For pharmacological analysis, out of 510, 260 g of crude methanolic extract was separated and stored till further analysis. Meanwhile, the remaining 250 g were subjected to fractionation through n‐hexane (NH), ethyl acetate (EA), and dichloromethane (DCM). Initially, the methanolic extract (250 g) was mixed with distilled water (500 mL), which was later mixed with n‐hexane (500 mL) for the preparation of a reaction mixture in a separating funnel, which was mixed thoroughly. The resultant reaction mixture was held for 5 min to ensure the formation of two immiscible layers. To obtain the n‐hexane fraction, the top layer was removed and concentrated using a rotary concentrator. To ensure effective removal of the n‐hexane fraction, this procedure was performed thrice. To collect the DCM fraction, the DCM was mixed with the aqueous layer of the crude methanolic extract solution. Again, the solution was held for a few minutes to ensure the formation of two immiscible layers of dissimilar solvents. To obtain the DCM fraction, the bottom layer was concentrated in a rotary concentrator. To ensure effective removal of the DCM fraction, this procedure was performed thrice. Similarly, in a separating funnel, ethyl acetate was added to the top aqueous layer and mixed thoroughly. Finally, this solution was again held for a few minutes to ensure the formation of two immiscible layers. The ethyl acetate fraction was removed from the solution and concentrated using a rotary concentrator. To ensure effective removal of the EA fraction, this procedure was performed thrice. The fraction left at the end of the procedure was an aqueous fraction (Ngo et al. 2018; Sadiq et al. 2019).

In Vitro Antioxidant Activity

2.3

Ferric Reducing Antioxidant Power (FRAP) Assay

2.3.1

To evaluate the in vitro antioxidant potential of extracts and fractions, in this study the FRAP assay was conducted by following the protocols adopted by Du et al. (2019). Initially, 10 times diluted extract (1 mL) was added to PBS (0.2 mL) and potassium ferricyanide (1.5 mL: 0.3%), followed by incubation for 20 min at 50°C. Afterward, 1 mL trichloroacetic acid (10%) was mixed with the incubated solution, followed by centrifugation (3000 rpm) for 10 min. Later, the resultant supernatant (2 mL) was mixed with distilled water (3 mL) and 0.5 mL of ferric trichloride (3%). In the end, the absorbance of the final mixture was noted at 700 nm. The results were presented as μM TE/g fw.

Hydrogen Peroxide Free Radical Assay

2.3.2

To evaluate the in vitro antioxidant potential through hydrogen peroxide free radical assay of extracts and fractions, in this study, the protocols described by Ruch et al. (1989) were adopted. Purposely, hydrogen peroxide solution (2 mmol) was made in phosphate buffer (50 mmol) at 7.4 pH. 0.1 mL of extracts and fractions were mixed in phosphate buffer (50 mmol) to raise the final volume to 0.4 mL. Before checking the final absorbance with spectrophotometer at 230 nm, 0.6 mL of hydrogen peroxide was mixed in each test tube containing extracts and fractions, respectively. Following formula was used for determination of percent hydrogen peroxide scavenging activity;

$$[eqn]$$

In Vitro Anti‐Inflammatory Activity

2.4

Inhibition of Albumin Denaturation

2.4.1

Inhibition of albumin denaturation method was used for assessing the anti‐inflammatory properties of extracts and fractions. For this purpose, 0.05 mL of extracts and fractions in varied concentrations (31.25–1000 mg mL^−1^) were mixed with 0.5 mL of albumin aqueous solution (5%). 1 N hydrochloric acid was added to the prepared solution for adjustment of pH to 6.3. The resultant solution was given a stay time of 20 min at 37°C and heated afterward for 3 min at 51°C. Later, the reaction solution was cooled, and phosphate buffer (2.5 mL) was mixed in it, followed by measurement of absorbance at 660 nm (Jan et al. 2020). The following formula was used for measuring the percent inhibition of protein denaturation;

$$[eqn]$$

Protease Inhibitory Assay

2.4.2

In this assay, each 2 mL of reaction mixture contained trypsin (0.06 mg mL^−1^), Tris‐HCl buffer (1 mL of 20 mM), and extracts/fractions (1 mL) at varied concentrations ranging from 31.25 to 1000 mg mL^−1^. This mixture was given a stay time of 5 min at 37°C, followed by the addition of casein (1 mL of 0.8%) before again incubating for 20 min and adding perchloric acid (2 mL of 70%). Finally, before measuring the absorbance at 217 nm, the cloudy suspension was subjected to centrifugation (3000 rpm) for 5 min (Jan et al. 2020). The following formula was used for measuring the percent inhibition of protease;

$$[eqn]$$

Acute Toxicity Testing

2.5

For acute toxicity studies, BALB/C mice (male) were divided into five groups (n = 6). The first group was considered as control, while the other four groups were treated with different doses/concentrations of extract and its fractions. The animals were observed for 1–2 h for any gross effects, and the mortality of mice was checked after 24 h.

Assay for Carrageenan‐Induced Paw Edema

2.6

The crude extract of C. macrophylla Wall. bark and its several fractions as NH, DCM, EA, and AQ were screened for their anti‐inflammatory effect. Albino mice weighing 25–30 g of both sexes were selected. Six different groups were made of selected animals. Each group consisted of six mice (n = 6). For negative and positive controls, Group I and Group II were selected. At a 10 mL/kg body weight dose, normal saline was administered to Group I as a negative control, and at a 10 mg/kg body weight dose, diclofenac sodium was administered to Group II as a positive control group. To the remaining groups (IV, V, and VI), different extracts were administered at various doses (50, 100, and 200 on body weight), respectively. Carrageenan 1% was injected into the subplanter right‐hand paw after 30 min of above treatment to each mouse, and the anti‐inflammatory effect was measured with the help of a plethysmometer for 5 h at 0, 1, 2, 3, 4 h, and 5 h) after administration of carrageenan (Khan et al. 2009). The paw edema percent inhibition will be calculated using the standard formula as follows.

$$[eqn]$$

Where an A is the negative control edema volume, while B represents tested group paw edema.

Assay for LPS‐Induced Inflammatory Activity

2.7

C. macrophylla bark crude extract and its fractions DCM and EA for the presence of neuroprotective effects were screened. Albino mice for this purpose, having an average weight of 25–30 g body weight were selected. The mice were divided into five groups, each having six mice. Control group I first received normal saline for 14 days orally once daily. The second group II was treated with normal saline orally for 14 days, followed by intraperitoneal LPS (Beijing Solarbio Technology Co. Ltd. CAS No: 111C039) administration at 100 μg/kg body weight. The third group III of mice was treated with aspirin at a dose of 200 mg/kg body weight orally for 14 days, followed by LPS administration intraperitoneally at a dose of 100 μg/kg body. IV, V, and VI groups of mice were treated with above‐mentioned three extracts of C. macrophylla bark orally at a dose of 100, 200, and 300 mg/kg body weight, followed by LPS intraperitoneally at a dose of 100 μg/kg body weight. After 14 days of treatment, several behavioral assessments were carried out, like body weight, rectal temperature, and locomotor activity (Prakash et al. 2017).

Dissection and Homogenization

2.8

The mice were sacrificed on the 15th day with mild anesthesia. Their brains were removed, and the forebrain was dissected out after the completion of the behavioral assessment posttreatment period. Brains dissected were placed on ice and rinsed with isotonic saline for the removal of blood. A 0.1 M phosphate buffer was added to make the resultant homogenate (Prakash et al. 2017).

Estimation of Glutathione Reductase

2.9

For determining the activity of glutathione reductase, a reaction mixture of NADPH, EDTA, oxidized glutathione, and phosphate buffer was formed by using distilled water. Tissue homogenate was mixed with the reaction mixture, and absorbance was measured at 340 nm for 2 min at each 30 s interval. Results were presented as mol NADPH oxidized min^−1^ mg^−1^ protein (Davidson and Hird 1964).

Estimation of Superoxide Dismutase

2.10

For determining the activity of glutathione reductase, the absorbance of the reaction mixture comprising epinephrine and carbonate buffer was noted at 480 nm for 2 min at a regular interval of 15 s (Misra and Fridovich 1976).

Assay for TBARS (Thio‐Barbituric Acid Reactive Substances)‐Malonaldehyde Level

2.11

In order to assess the lipid peroxidation, the reaction of TBARS was measured using an indirect determination method. Homogenates were mixed with trichloroacetic acid along with TBA and HCl (0.25 M) in test tubes. The solutions were mixed vigorously and placed in an ice bath to lower the temperature. Afterward, the solution was subjected to centrifugation for 10 min. The top layer was separated, and absorbance was measured at 532 nm (Ohkawa et al. 1979).

Determination of Catalase Level

2.12

The measurement of catalase level was assessed by the procedure of Aebi (1984). The reaction mixture consisted of phosphate buffer, tissue homogenate, and hydrogen peroxide. The absorbance (240 nm) was noted for 30 s at intervals of 15 s.

Determination of Body Weight

2.13

Digital weighing balance was used for determination of body weights, before and after the administration of extracts/fractions and drug control.

Determination of Rectal Temperature

2.14

A thermostat probe was used for measuring rectal temperature. For this purpose, the probe (1.3 cm) was inserted into the rectum and taped to the base of the tail.

Assessing Locomotor Activity

2.15

Actophotometer digital apparatus was used to monitor locomotor activity. The apparatus was placed in a light and sound‐attenuated, darkened testing room. There was a digital counter that presented any pause of the beam on the x‐axis or y‐axis and created a subsequent electric impulse. On Day 14th, activities were performed on animals followed by LPS administration, and at counts per 5 min, values were expressed respectively (Reddy and Kulkarni 1998).

Results

3

In Vitro Antioxidant Assay

3.1

The in vitro antioxidant assay investigated the free radical scavenging potential of methanolic extract of C. macrophylla along with its prepared fractions. Results revealed that DCM and EA fractions possessed the most potential FRAP activities, that is, 21.65 and 21.94 μM TE/g, respectively. The other fractions, like crude, n‐Hexane, and aqueous, also displayed good to moderate activity, causing 8.46, 11.24, and 7.59 μM TE/g FRAP inhibition, as shown in Table 1.

TABLE 1: The ferric reducing antioxidant power (FRAP) assay of crude extract and different fractions of C. macrophylla .

Hydrogen Peroxide Free Radical Assay

3.1.1

In the H_2_O_2_ free radicals scavenging activity, DCM and EA again showed the highest activities causing 89.78% ± 1.42%, 83.22% ± 0.78%, 75.56% ± 0.42%, 70.34% ± 0.44%, 66.42% ± 0.64% and 87.77% ± 0.73%, 82.22% ± 1.44%, 77.54% ± 0.52%, 73.49% ± 0.45%, 65.10% ± 0.34% inhibitions, respectively at concentrations ranging from 1000 to 62.5 μg/mL with IC_50_ values of 16.38 and 12.68 μg/mL. Similarly, the other fractions, like crude, hexane, and aqueous, displayed 85.15% ± 0.72%, 76.62% ± 0.66%, and 71.51% ± 0.77% inhibition at 1 mg/mL concentration (Table 2). The calculated IC_50_ values were 13.56, 31.54, and 93.41 μg/mL, respectively. Ascorbic acid inhibition was 96.54% ± 0.66%, 91.46% ± 0.34%, 86.62% ± 0.46%, 82.80% ± 0.84%, and 77.37% ± 0.78% at the same tested concentration, with an IC_50_ of 7.94 μg/mL.

TABLE 2: H2O2 inhibitory assay of the crude extract and different fractions of C. macrophylla .

In Vitro Anti‐Inflammatory Activity

3.2

Inhibition of Albumin Denaturation and Protease

3.2.1

Among our tested samples, it was noticed that EA fraction showed good albumin denaturation (IC_50_: 22.15 μg) and protease inhibition (IC_50_: 14.20 μg) potentials. The other fractions showed moderate activity in these assays (Table 3).

TABLE 3: In vitro albumin denaturation and protease inhibition activities of C. macrophylla .

Acute Toxicity

3.3

The results for acute toxicity displayed no associated mortalities in mice administered a single dosage of 3000 mg/kg of any of the investigated crude extracts. 3000 mg/kg is the lethal dose (LD_50_) for crude drug extract. No signs of illness or behavioral changes were noticed in mice during this study trial (Table 4).

TABLE 4: Acute toxicity result of crude drug extract of C. macrophylla bark.

In Vivo Anti‐Inflammatory Activity

3.4

In carrageenan induced paw edema assay, the EA fraction of Cornus mass L. demonstrated excellent activity against carrageenan induced inflammation at dose of 200 mg/kg which is the highest dose of that extract, and reached its maximum value at the 4th hour, remaining significant (***p < 0.001) until the 5th hour of sample administration. At the 4th hour, the EA fraction showed an activity of 83.7 ± 0.03 at a dose of 200 mg/kg body weight, while the standard drug, that is, diclofenac sodium, showed marked activity of 80.8 ± 0.80 and remained significant up to the 5th hour. Similarly, the dichloro methane fraction also exhibited marked activity of 77.2 ± 0.08 at a dose of 200 mg/kg body weight at the 4th hour. The remaining fractions also exhibited good activity, but the EA fraction remained the most active fraction among all. The n‐hexane fraction (61.2 ± 0.22) is more active than the aqueous fraction, which had an activity of 57.9 ± 0.07 at a dose of 200 mg/kg body weight, as shown in Table 5.

TABLE 5: Percent inhibition of various extracts of C. macrophylla bark in carrageenan‐induced paw edema.

Effect of

C. macrophylla Bark on LPS‐Induced Changes in Body Weight

3.4.1

LPS‐treated mice at a dose of 100 μg kg^−1^ body weight momentously (p < 0.001) reduced the body weight in comparison to the control group. The mice treated group with aspirin at a dose of 200 mg/kg body weight showed increased significantly (p < 0.001) in body weight as compared to the LPS‐treated mice. Different C. macrophylla bark extracts, like crude extract, DCM, and ethyl acetate, showed a significant (p < 0.001) increase in body weight in comparison with the LPS‐treated mice (Figure 1).

C. macrophylla bark, different extracts effects on weight alteration by LPS administration. Values are represented by mean ± SEM. p < 0.001 compared to control mice. p < 0.001 compared to LPS‐treated mice.

Effect of

C. macrophylla Bark on LPS‐Induced Changes in Rectal Body Temperature

3.4.2

LPS‐induced mice, when treated with aspirin at a dose of 200 mg/kg body weight, showed a significant reduction in rectal temperature as compared to the LPS‐treated group. Different C. macrophylla extract‐treated groups, like crude, DCM, and EA, revealed a reduction (p < 0.001) in rectal temperature when compared with the LPS‐treated mice (Figure 2).

Effect of different extracts of C. macrophylla on the LPS‐induced variation in rectal temperature. Values are represented by mean ± SEM. p < 0.001 compared with control mice. p < 0.001 compared with LPS‐treated mice. p < 0.001 compared to aspirin‐treated mice.

Effect of

C. macrophylla Bark on LPS‐Induced Alteration in Locomotor Activity

3.4.3

LPS‐treated mice, when subjected to aspirin treatment (200 mg/kg body weight), the locomotor activity showed significant reduction as compared to the untreated control group. Pretreatment with different C. macrophylla extracts like crude, DCM, and EA momentously (p < 0.001) elevated the locomotor activity as compared to LPS‐treated mice (Figure 3).

Effect of different extracts of C. macrophylla on LPS‐induced variation in locomotor activity using actophotometer. Values are represented by mean ± SEM, p < 0.001 when compared with control mice and p < 0.001 compared to the LPS‐treated mice.

Effect of

C. macrophylla on Antioxidant Level

3.5

Antioxidant activities include the below‐mentioned ex vivo activities.

Effect of

C. macrophylla on LPS‐Induced Changes in GSH Level

3.5.1

LPS induction reduced the GSH level in mice, which was elevated on administration of aspirin as compared to LPS‐treated mice. Similarly, the mice treated with C. macrophylla in doses of 100, 200, and 300 mg/kg body weight also enhanced the GSH levels significantly (p < 0.001) as compared to LPS‐treated mice (Table 6).

TABLE 6: Effect of various fractions of C. macrophylla on LPS‐induced alterations in GSH levels.

Effect of

C. macrophylla on LPS‐Induced Alterations in SOD Level

3.5.2

Mice group treated with LPS significantly (p < 0.001) decreased the levels of superoxide dismutase when compared with the control group. Whereas the SOD levels were enhanced in the aspirin‐treated mice group. In the experimented groups, C macrophylla at doses of 100, 200, and 300 mg/kg body weight restored SOD level momentously (p < 0.001). However, the antioxidant effect showed at 300 mg/kg was much better than that of C. macrophylla (200 and 100 mg/kg) (Table 7).

TABLE 7: Effect of various fractions of C. macrophylla on LPS‐induced alterations in SOD levels.

Effect of

C. macrophylla on LPS‐Induced Alterations in MDA Level

3.5.3

The MDA levels in LPS‐treated mice were elevated compared to the control group. The MDA levels were reduced in the aspirin‐treated mice group as compared to the LPS‐treated mice group. Similarly, the MDA levels in pretreated C. macrophylla at doses of 100, 200, and 300 mg/kg body weight decreased significantly (p < 0.001) as compared to the LPS‐treated mice (Table 8).

TABLE 8: Effect of various fractions of C. macrophylla on LPS‐induced alterations in MDA levels.

Effect of

C. macrophylla on LPS‐Induced Changes in CAT Level

3.5.4

CAT levels were diminished in the LPS‐treated mice group as compared to the control group of mice. Meanwhile, the aspirin‐treated mice group significantly (p < 0.001) enhanced the level of the CAT enzyme compared to LPS‐treated mice. Moreover, the level of CAT increased in the pretreated C. macrophylla (100, 200, and 300 mg/kg body weight) group as compared to LPS‐treated mice (Table 9).

TABLE 9: Effect of various fractions of C. macrophylla on LPS‐induced alterations in CAT levels.

Discussion

4

Literature shows that species in genus Cornus possess therapeutic potential against various ailments. Traditionally, different parts of C. macrophylla were used as a potential medicinal plant for treatment of various diseases. The bark of this plant is commonly utilized in the form of powder for treatment of stomach ulcer, jaundice, and backache (Khan et al. 2009). C. macrophylla inhibits the activity of aldose reductase and hence can be a potent candidate for treating diabetic retinopathy (Prakash et al. 2017). Previously, diverse bioactivities such as the antifungal (Davidson and Hird 1964), antibacterial (Misra and Fridovich 1976), and antioxidant (Ohkawa et al. 1979) properties of an array of phytoconstituents isolated from Cornus species have also been investigated. In the current investigation, we explored the antioxidant capacity of using H_2_O_2_ and FRAP assays for the first time. In this assay, the DCM and EA fractions showed excellent inhibition, as shown in Tables 1 and 2. Furthermore, Cornus mas

s (C. mass) fruit extract oral administration for 14 days to rats showed hepatoprotection by decreasing serum enzyme levels, albumin, total serum protein, and lipid peroxidation content (Alavian et al. 2014). At all concentrations, it was clearly shown that aqueous extract of Cornus mas

s L. has a significant antioxidant effect on various in vitro antioxidant assays (Gülçin et al. 2005). C. mass (Cornelian cherry) is a valuable source of substances with a high antioxidant activity (Gąstoł et al. 2013). C. mass L. various genotypes showed highest antioxidant activity as genotype C24 82.37%, followed by genotype C27 77.6% and genotype C15 76.32% (Hassanpour et al. 2011). C. mass leaf and fruit extracts showed significant radical scavenging activity (Milenković‐Anđelković et al. 2015). In murine RAW 264.7 macrophage cells, aqueous extract of Corni fructus inhibited the synthesis of PGE (2) and production of NO through suppression of LPS‐induced upregulation of iNOS (inducible NO synthase) and COX‐2 (cyclooxygenase‐2) (Sung et al. 2009). Cornus walteri extracts have in vivo and in vitro activity significantly more sensitive to lipopolysaccharide‐induced lethality and inflammatory cytokines (Lee et al. 2011). Water and ethanolic extracts of Cornus walteri show potential antioxidant activity due to the presence of polyphenol and flavonoid contents (Kim et al. 2013). C. walteri hot water extract showed a significantly protective effect against tert‐butyl hydroperoxide‐induced oxidative stress in HepG2 cells, and also antioxidant enzymes modulation by C. walteri extract has important antioxidant activity against tert‐butyl hydroperoxide‐induced oxidative insult in HepG2 cells (Yeon et al. 2013). C. walteri ethanolic leaf extracts showed in vitro chemical and cellular antioxidant activity by treating with enzymes in human dermal fibroblast irradiated by type B ultraviolet (UVB) phototherapy (Park et al. 2014). C. walteri stem bark extracts showed a positive effect in lipid‐lowering, anti‐inflammatory, and antioxidative effects in rats fed a high‐fat diet (Park and Cha 2009). Fractions of ethyl acetate and aqueous from adventitious roots of C. capitata showed antioxidant activity against 1,1‐diphenyl‐2‐pycrylhydrazyl (DPPH) and superoxide anion radicals (Tanaka et al. 2003). Cornus kousa was evaluated for its antioxidant, antidiabetic, and tyrosinase inhibitory activity and exhibited a significant inhibitory effect (Lee et al. 2015). Cornus alba , locally known as Siberian dogwood, possesses marked anti‐inflammatory effect (Park et al. 2019). C. macrophylla, locally khadang, showed marked antioxidant activity (Shah et al. 2015).

In our previous investigation regarding the phytochemical investigation of C. macrophylla extract, results showed the occurrence of terpenes, flavonoids, alkaloids, and tannins. The cache of phytochemicals present in the extract of C. macrophylla may be responsible for various bioactivities. Diverse nutraceutical properties, such as anti‐inflammatory and analgesic characteristics, are associated with the presence of diverse flavonoids in the extracts (Aebi 1984; Ohkawa et al. 1979; Reddy and Kulkarni 1998). Evidently, flavonoids may have a significant effect on inflammatory biomarkers (Alavian et al. 2014). Likewise, terpenes have also been shown to possess analgesic and anti‐inflammatory activities (Gąstoł et al. 2013; Gülçin et al. 2005). These activities are ascribed to the inhibitory potential against phospholipase A2 and hence result in the obstruction of arachidonic acid metabolism (Hassanpour et al. 2011). Moreover, the presence of alkaloids is also thought to be a reason for preventing inflammation via their inhibitory effects on arachidonic acid metabolism (Milenković‐Anđelković et al. 2015; Sung et al. 2009).

The most commonly used model to assess the anti‐inflammatory potential of natural compounds in animal models is the carrageenan‐induced inflammatory mouse model (Park et al. 2019). In this model, induced inflammation occurs in two phases. In the first phase, the release of kinins, serotonin, and histamine is observed in the first hour, whereas, in 2nd phase, lysosomal enzymes and prostaglandins are released in the next 2–4 h (Gad 2018). The 2nd phase is known to be the most sensitive to anti‐inflammatory compounds and/or drugs (Ong et al. 2007). Outcomes of this study show that the administered extracts and fractions had significant inhibitory effects on carrageenan‐induced acute inflammation in the 3rd hour of the study, when compared with the standard anti‐inflammatory drug, as shown in Table 5. In the anti‐denaturation assay, heat treatment results in the denaturation of egg albumin. Likewise, native proteins and heat‐induced denatured proteins are also considered to effectively delay hypersensitivity. Furthermore, it has already been established that NSAIDs like indomethacin and phenylbutazone not only inhibit the production of endogenous prostaglandins through suppression of the COX enzyme but also through prevention of denatured proteins (Ullah et al. 2014). The current study showed that the crude extract and fractions possessed substantial anti‐inflammatory properties. Both crude extract and fractions controlled autoantigen production and hence inhibited protein denaturation as compared to the standard drug. Moreover, the secondary constituents, including tannins, flavonoids, and phenolics that were identified in our previous study, may also be responsible for this anti‐inflammatory characteristic (Khan et al. 2022).

Conclusion

5

It was concluded from our results that the in vivo anti‐inflammatory activity EA fraction showed strong inhibition of paw edema, while in LPS‐induced neuroprotective effect, the EA fraction showed the most potent antioxidant activity by increasing antioxidant level of GSH, SOD, and CAT in brain tissues (Tables 6, 7 and 9) and decreasing the level of MDA (Table 8). Moreover, the body weight of all the mice decreased when treated with LPS, while it increased when treated with aspirin, crude extract, DCM, and EA, respectively. Rectal temperature increased when treated with LPS and gradually decreased with aspirin, crude, DCM, and EA fraction. More research is required to evaluate the specific compounds present in C. macrophylla that are responsible for its neuroprotective effect.

Author Contributions

Ali Khan: conceptualization (lead), data curation (lead), formal analysis (lead), investigation (lead), methodology (equal), supervision (equal), writing – original draft (lead), writing – review and editing (equal). Aini Pervaiz: conceptualization (supporting), data curation (supporting), investigation (lead), methodology (supporting), validation (supporting), visualization (equal), writing – original draft (equal), writing – review and editing (equal). Muhammad Saeed Jan: data curation (equal), investigation (supporting), methodology (supporting), visualization (supporting), writing – original draft (supporting), writing – review and editing (equal). Bushra Ansari: data curation (supporting), formal analysis (supporting), investigation (supporting), methodology (equal), writing – original draft (equal), writing – review and editing (equal). Imad Ahmad: formal analysis (supporting), investigation (supporting), methodology (supporting), validation (supporting), visualization (supporting), writing – original draft (equal), writing – review and editing (equal). Syed Muhammad Mukarram Shah: formal analysis (equal), investigation (supporting), methodology (supporting), validation (supporting), visualization (equal), writing – original draft (supporting), writing – review and editing (supporting). Abdur Rauf: conceptualization (lead), investigation (lead), methodology (lead), project administration (equal), validation (lead), visualization (supporting), writing – original draft (equal), writing – review and editing (equal). Ahood Khalid: methodology (supporting), validation (supporting), visualization (equal), writing – original draft (supporting), writing – review and editing (equal). Anees Ahmed Khalil: data curation (lead), data curation (lead), investigation (lead), methodology (equal), validation (lead), visualization (equal), writing – original draft (lead), writing – review and editing (lead). Hassan A. Hemeg: formal analysis (supporting), investigation (supporting), methodology (supporting), validation (equal), visualization (equal), writing – original draft (supporting), writing – review and editing (supporting). Yahya S. Al‐Awthan: investigation (supporting), methodology (supporting), validation (equal), visualization (equal), writing – original draft (supporting), writing – review and editing (supporting). Omar S. Bahattab: investigation (supporting), methodology (supporting), validation (equal), visualization (equal), writing – original draft (supporting), writing – review and editing (supporting). Mohammed Mansour Quradha: conceptualization (lead), data curation (supporting), investigation (supporting), methodology (lead), validation (equal), visualization (equal), writing – original draft (lead), writing – review and editing (lead).

Conflicts of Interest

The authors declare no conflicts of interest.