Phytochemical screening, antioxidant, immunomodulatory and analgesic potential of aqueous-ethanolic extracts of Euphorbia prostrata ait. and Crotalaria burhia Buch–Ham

Abstract

Euphorbia prostrata (E. prostrata) and Crotalaria burhia (C. burhia) are widely found in flora of the Cholistan Desert of Bahawalpur, Pakistan and are traditionally used to treat pain and chronic disease. The current study aimed to evaluate their phytochemical screening, antioxidant activity, in-vivo phagocytic activity, and analgesic activity. Both the plant extracts were investigated for phytochemical screening, Fourier Transform Infrared (FTIR) analysis, in-vitro antioxidant by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method, in-vivo immunomodulatory activity by macrophages phagocytosis using carbon clearance rate assay and analgesic activity by acetic acid produced writhing method. Phytochemical screening showed the presence of carbohydrates, saponins, tannins, phenols, quinines, proteins, terpenes, glycosides, and alkaloids. FTIR analysis revealed the existence of different functional groups in both extracts. The DPPH method showed that E. prostrata exhibited a high antioxidant potential with an IC₅₀ of 62.5 μg/ml whereas C. burhia showed a lower antioxidant potential. At the dose of 200 mg/kg body weight (b. wt), both the extracts showed a significant increase in the phagocytic index by 5.2 ± 0.2, and, 4.8 ± 0.1 (p < 0.001) respectively which was close to the 100 mg/kg b. wt of the standard drug (Levamisole) 5.4 ± 0.2. Both the extracts at the dose of 200 mg/kg b. wt also significantly reduced the writhing (abdominal contractions) count by 13.7 ± 0.3 and, 25.3 ± 1.5 (p < 0.001), showing 71.8% and 47.6% of reduced analgesic activity compared to the standard drug dicloran (diclofenac sodium), respectively. In conclusion, extracts of both plants indicate their role in the management of various disorders to relieve pain and modulate the immune system.

1. Introduction

Around the world, the ethnomedicinal knowledge of herbs runs from one generation to the next in the local population [1] and is used throughout life till death [2]. Despite the widespread use of synthetic medications globally, traditional remedies are indeed essential among developing nations [[3], [4], [5]]. Likewise, traditional medicine is still used by a large part of the population in Pakistan, Africa, and Chile [6,7]. Consequently, Medicinal plants are affordable, simple to use, and have fewer adverse effects than current synthetic drugs [8]. Numerous studies show the fascinating benefits of plant-based medicines used in complementary, alternative, and traditional medicine to cure human ailments [[9], [10], [11]]. A report from the World Health Organization (WHO) has also recently reported that nearly three-quarters of the global population is currently using herbs and herbal remedies for therapeutic purposes [12]. Specifically in Pakistan, Indigenous Traditional Medicines are considered important for the prevention of different ailments and 40–59% of the population uses these [13].

The Cholistan desert because of its high species endemism and biodiversity, might be regarded as a unique environment. The wild flora of the Cholistan Desert in Pakistan includes diverse species that are commonly utilized by the local inhabitants to cure several diseases. Various important chemical compounds have been identified and isolated including glycosides, flavonoids, phenols, terpenes and triterpenoids, saponins, quinones, gums and resins, sterols, steroids, and anthocyanidins, etc. that exhibit pharmacological properties [14]. However, as the chemical composition and structure of chemical constituents found in many medicinal plant species are unknown, more research needs to be done in this area [14].

Euphorbia prostrata Ait. (E. prostrata) belongs to the Euphorbiaceae family, commonly known as “Hazar dani” [15] and reportedly has been used to treat hemorrhoids [16], asthma, dysentery, and diabetes mellitus [17]. Previous reports have indicated that several plants in Euphorbiaceae family have displayed pain relieving effects [18,19]. Crotalaria burhia (C. burhia) Buch–Ham belongs to the family Fabaceae, locally called “Chagg” or “Bhata” and traditionally used as a remedy for various diseases including typhoid, swelling, pain, gout, hydrophobia, eczema, and tumor [[20], [21], [22]]. Previously, phytochemical profiling revealed the presence of major compounds in C. burhia were pyrrilizidine like alkaloids (corhurhine, crotalarine, mono crotalarine, crosemperine, quercetin) and β-sitosterol like steroids [20]. Furthermore, anthraquinones, flavonoids, phenols, phlobatannins, polysaccharides, saponins, tannins, and terpenoids [23] are also documented. On the other hand, ten major compounds have been isolated from E. prostrata and identified as gallic acid, corilagin, 1,2,3-tri-O- galloyl-d-glucose, geraniin, tellimagradin I, II, rugosin A, rugosin E, rugosin D and rugosin G on the basis of physicochemical and spectroscopic methods [24].

Previous reports scientifically explored the various pharmacological properties belongs to these medicinal plants. Such as, C. burhia reported to have antimicrobial [25], anti-inflammatory [26] and anti-tumor [27] properties while E. prostrata was reported to possess antimicrobial [28], anti-inflammatory [29], anti-hyperglycemic [30], gastroprotective [31], anti-tumor, and anti-angiogenic [32], anti-hemorrhoidal effects [16] and Antiurolithiatic [33]. Similarly, various plants belonging to the same family, are traditionally used for the management of painful swellings and other chronic diseases [13,14] suggesting that the medicinal plant investigated in this study may also possess immunomodulatory and pain reducing properties.

As it demonstrated that herbal products are important in the development of new medications, screening and exploration of plants for significant active components, can be seen as an initial stage toward developing more potent remedies to treat a variety of illnesses [34]. To develop new pharmaceutical and nutraceutical materials with herbal constituents, the market for herbal applications is now growing at a rapid pace.

Consequently, it is imperative to come up with plans to fulfill the growing demand for herbal remedies, from both domestic and foreign consumers. Since it is commonly recognized that plant account for 70 percent of allopathic medications, so a significant number of medications are still derived directly from plant-based materials [35].

The full investigation of the Cholistan desert's medicinal plants will contribute to maximizing the effectiveness of medications, especially those derived from plants whose medicinal uses are not yet known [14]. Thus, the proposed research work intends to study the efficacy of the above mentioned herbs for medicinal purpose and to fulfill need of the day.

We aimed to comparatively investigate the E. prostrata and C. burhia for the diverse functional group analysis, in-vitro antioxidant, in-vivo immunomodulatory and analgesic potential. This study supports scientific validation of above mentioned herbs for their use in the Southern region of Punjab, Pakistan as an alternative treatment option.

2. Material and methods

2.1. Plant collection

C. burhia and E. prostrata were obtained from the Cholistan desert of the Bahawalpur, Pakistan during January–March. Both the plants were identified and authenticated by the Prof. Dr. Ghazala H. Rizwani, Director Research, Hamdard University Karachi, Pakistan. Dried samples of both the plants were stored in the Pharmacognosy Herbarium research laboratory of the Faculty of Eastern Medicine, Hamdard.

University, Karachi (voucher numbers: A138, A142). Prior to the processing, both the plants were cleaned and dried under the shade.

2.2. Preparation of the plant extracts

The stems and roots from the C. burhia while entire plant of E. prostrata was taken and cut into many pieces. The raw material of plants was powdered in electronic machine (National, Japan) and sieved by using sieve no. 40. The 300 g pounded plant material of each plant was first dipped for 3 days in the 1 L solvent, consisting of water and ethanol with the ratio of 30:70³⁶ and stirred alternatively once a day.

Inevitably, soaked plant material was passed through the filtration process firstly from muslin fabric and later through Whatman filter paper. Solvent used for soaking replaced 3 times to obtain maximum yield.

After the filtration extricate was transferred to rotary evaporator under controlled temperature 30–40° °C and diminish pressure.

2.3. Chemicals

All the solvents of analytical grade were obtained from Merck, Sigma-Aldrich. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) purchased form Alfa Aesar, United States and black carbon ink was purchased from Merck, Sigma-Aldrich. Levamisole was purchased from Helicon Pharmaceutek PVT LTD. Ketamine was purchased from the Indus Pharma PVT LTD. Xylazine was bought from Mylab Pvt. Ltd. Diclofenac sodium was bbought from the Abbott Laboratories PVT LTD (PAKISTAN).

2.4. Experimental animals

Male Wistar albino rats (150–200 g) and male Wistar albino mice (18–22 g) were utilized in the present research work. The four months old mice and rats in healthy condition were included in the study. Animals which were unhealthy or showed unwell behavior were excluded from the study. All the animals were acquired from the animal facility center of the Faculty of Medicine and Allied Health Sciences, The Islamia University of Bahawalpur (IUB), Pakistan. Animals were kept in polycarbonate cages having size 47 × 34 × 18 cm³ with a limit of 6 for each enclosure. The standard humidity conditions (56 ± 5%) with temperature (25 ± 1 °C) alongside the 12/12-h light/dull cycle were kept up throughout the study duration. Animals were acclimatized to lab conditions for about two-week preceding investigation and were on normal chow and water ad libitum. The entire investigations were done as per the ARRIVE rules [[36], [37]]. The experimental procedures were affirmed by the animal ethics committee (Pharmacy Research Ethics Committee), IUB (Notification no.1460-B/UCCM/23-04-2019) and as per the rules of advisor group with the purposes of analyses on the animal model.

2.5. Experimental plan

Animals were randomly divided into 6 groups of 6 rats in each group for each in vivo activity.

Group I animals were served as control.

Group II was served as standard (Levamisole 100 mg/kg b. wt or Diclorfenic sodium 10 mg/kg b. wt).

Group III were treated with aqueous-ethanolic extract of E. prostrata at doses of 100 mg/kg b. wt.

Group VI were treated with aqueous-ethanolic extract of E. prostrata at doses of 200 mg/kg b. wt.

Group V were administered with aqueous-ethanolic extract of C. burhia at the doses of 100 mg/kg b. wt.

Group VI were treated with aqueous-ethanolic extract of C. burhia at the doses of 200 mg/kg b. wt.

2.6. Phytochemical screening

The preliminary phytochemical screening was performed to observe the presence of various phytochemicals in aqueous-ethanolic extracts of E. prostrata and C. burhia. The entire test performed according to the previously documented protocols [38].

2.7. Fourier Transform Infrared (FTIR) spectroscopy

The aqueous-ethanolic extract of E. prostrata and C. burhia specimens were powdered and analyzed for FTIR spectroscopy (Shimadzu, IR Affinity 1, Japan). The sample was scanned at a scanning range of 400 and 4000 cm⁻¹ with 4 cm⁻¹ resolution [39].

2.8. Antioxidant activity via DPPH free radical scavenging assay

Antioxidant potential was determined by DPPH free radical reducing method [40]. 100 μM concentration of.

DPPH was utilized in methanol. Each of test drug (plant extracts) 5 mg dissolved in 1 mL methanol and in order to possess out the IC50 value sequential dilutions (5, 2.5, 1.25. 0.625 and 0.3125 mg/ml) were prepared. After mixing the components, 90 μL of DPPH solution and 10 μL of drug testing mixes were added to a 100 μL total volume microplate. Subsequently, the 96-well microplate solution was incubated at 37 °C for 30 min, and the absorbance was measured at HT Biotech USA microplate reader at 517 nm.

Readings were compared with the standard utilized and taken in triplicate [40]. Ez-fit-5 Perrella Scientic Inc., Amherst USA programming was utilized to ascertain the IC50. Lessening in absorbance demonstrated expanded radical scavenging action which was obtained by underlying equation.

$$\text{Inhibitory}\text{percentage}(\%)=\frac{\left(\text{abs}.\text{of}\text{control}‐\text{abs}.\text{of}\text{test}\text{solution}\right)}{\text{Abs}.\text{of}\text{control}}\times 100$$

Where,

Absorbance of control = Total radical activity without inhibitor.

Absorbance of test = Activity in the presence of test compound.

2.9. Acute oral toxicity study of E. prostrata and C. burhia

Rules of Organization for Economic Co-Cooperation and Development (OECD) were followed to perform acute lethality studies [41]. Wistar albino mice n = 45 having their weights between 22 and 42 g were isolated into nine gatherings, comprising of 5 animals in each gathering. Typical baseline conduct behaviors of every animal were observed. Trial mice were fasted the whole night and just got water ad libitum. One mice group got saline solution 10 ml/kg b. wt and rest of the 8 gatherings got the aqueous-ethanolic extract of E. prostrata and C. burhia at the dosages of 1, 3, 5, and 10 g/kg b. wt separately. The changes in behavior and reaction parameters; for example, readiness, hyperactivity, spasm, perspiring, lacrimation, urinary output, rightening reflex, corneal reflex, torment reaction, contact reaction, grasping quality as well as mortality were watched and recorded at different time periods including 0.5, 1, 2, 4, 6, 12, 24 along with 48 h.

2.10. In-vivo immunomodulatory activity by using macrophages phagocytosis using carbon clearance

2.10.1. Rate assay

The carbon clearance rate assay was done as demonstrated previously with certain modifications [42]. Group.

I animals obtained saline (10 ml/kg b. wt per orally). Group II was treated with standard

Levamisole 100 mg/kg b. wt orally starting from the day 4 till day 7. Group III-VI animals were treated with

aqueous-ethanolic extract E. prostrata and C. burhia orally as mentioned above. All the animals n = 36 were treated for 7 days. Following 7th day of treatment, rats from the all groups injected with an of Indian ink suspension; the mixture consisted of black carbon ink 3 mL, 3% gelatin solution 4 ml and saline 4 mL via tail vein (0.1 mL/10 g body weight). Blood was collected by retr-orbital plexus by utilizing capillary tubes at 6 min and 16 min after the administration of ink mixture.

To lyse the erythrocytes, blood was immediately mixed into 4 ml of 0.1% sodium carbonate mixture. Afterwards, absorbance was taken by using UV spectrophotometer at 675 nm. In the end, animals were sacrificed following the last blood sampling, spleen along with the liver harvested and weight was measured immediately in wet state.

The phagocytic action is illustrated by the carbon clearance rate K which determines the entire reticuloendothelial system functioning associated with the blood circulation and by corrected

phagocytic index α. These are determined by methods of underlying formula:

$$\mathrm{K}=\frac{\log \text{OD}1‐\text{logOD}2}{{\mathrm{t}}_2‐{\mathrm{t}}_1}$$

$$\mathrm{\alpha }=\frac{\surd \mathrm{K}\mathrm{x}\text{total}\text{Body}\text{weight}}{\text{weight}\text{of}\text{liver}+\text{weight}\text{of}\text{spleen}}$$

OD1 optical density at time 1 and OD2 optical density at time 2.

2.11. Evaluation of the analgesic activity by using acetic acid induced pain test (writhing test)

The writhing test in rats n = 36 was carried out following Ouachrif, A. et al. [43] with minor adjustments. The control group received 0.9% saline orally and the standard group was treated with Diclofenac sodium at a dose level of 10 mg/kg orally. Test groups III-VI treated with aqueous-ethanolic extract of E. prostrata and C. burhia at two different doses (100 and 200 mg/kg B·W) and orally by gavage 30 min before intraperitoneal administration of 0.6% acetic acid at a dose of 10 ml/kg and Diclofenac sodium (Dicloran) was administered 15 min prior to injecting acetic acid. A time of 5 min/mice was given to make sure the acetic acid bioavailability. After the 5 min, total no. of writhes developed in the animals was observed for the period of 30 min. In order to score, a writhe is prescribed by stretching of the abdomen with immediate stretching of at least one hind limb.

The obtained results characterize the total numbers of writhes saw in 30 min considered as writhing numbers. The analgesic activity was determined as a percentage of reduction in writhing as per using underlying formula:

$$\text{Inhibition}(\%)=\frac{NumberofWrithes\left[Control\right]–NumberofWrithes\left[Treatment\right]}{NumberofWrithes\left[Control\right]}$$

2.12. Effect of plant extracts on the body weight and organs weight

The Wister albino mice n = 36 were indiscriminately assigned into 5 different groups having 6 mice in each group. Animals of Group-I considered as control while animals in Groups-II, III, IV and V were treated with 100 and 200 mg/kg b. w of aqueous-ethanolic extract of E. prostrata and C. burhia, respectively and followed by the oral administration of plant extracts for the consecutive seven days. At the end of the treatment duration followed by the last dosage, body weight along with the other essential organs for example kidney, liver, bladder, thymus and spleen, was noted which is expressed as relative organ weight [44].

2.13. Statistical analysis

The outcomes were exhibited as mean ± SEM and analyzed by Statistical package for social sciences.

(SPSS) utilizing One-Way analysis of variance (ANOVA) by using post hoc Tukey test. Impacts were

viewed as significant when compared with control p < 0.05.

3. Results

3.1. Preliminary phytochemical screening of aqueous-ethanolic extract of E. prostrata and C. burhia

3.1.1. Preliminary phytochemicals screening of the aqueous-ethanolic extract of E. prostrata and C. was

performed according to the standard protocols. Results of preliminary phytochemicals screening showed

that E. prostrata had carbohydrates, saponins, tannins, phenols, terpenes, quinines, alkaloids, glycosides,

fats and oils while flavonoids and proteins were not detected. On the other hand, C. burhia was found to

have saponins, phenols, terpenes, quinines, glycosides, alkaloids, fats and oils except tannins, proteins

and carbohydrates as shown in Table 1.

Table 1.

Preliminary phytochemical screening of aqueous-ethanolic extracts of E. prostrata and C. burhia.

Sr. No.	Phytoconstituents	Tests	Euphorbia prostrata	Crotalaria burhia
1	Saponins	Froath test	+	+
2	Fats and oils	Stain test Saponification test	+	+
3	Tannins	Sodium nitrate test Gelatin method	+	-
4	Phenols	FeCl3 test	+	+
5	Flavonoids	Alkali solution test Lead acetate test	-	+
6	Alkaloids	Wagner′s reagent Mayer′s reagent	+	+
7	Glycosides	Keller killani test Salkowski test	+	+
8	Quinones	Test with Con. H2SO4	+	+
9	Terpenes	Salkowski test Copper acetate test	+	+
10	Proteins	Xanthoproteic test Ninhidrin test	-	-
11	Carbohydrates	Fehling reagent test Molisch test	+	-

+ indicates the presence of compounds while – shows the absence of compounds.

3.2. Fourier transform infrared spectrophotometer (FTIR) analysis

FTIR analysis of both the crude aqueous-ethanolic extract of E. prostrata and C. burhia showed various

peak values at various regions that further explored the presence of diverse functional groups such as

alkenes, alkanes, fluorine, alcohol, ether, carboxalic acid, ester, nitro compounds and carbonyl groups in

both plants extracts as shown in Table 2 and Fig. 1.

Table 2.

FTIR peak values of aqueous-ethanolic extracts of E. prostrata and C. burhia.

E. prostrata			C. burhia
Peak (wavenumber cm- [1])	Bond	Functional groups	Peak (wavenumber cm- [1])	Bond	Functional groups
716.97	C–H	Alkenes	698.81	C–H	Alkenes
883.51	C–H	Alkenes	724.79	C–H	Alkenes
914.55	C–H	Alkenes	777.65	C–H	Alkenes
1020.83	C–F	Fluorine group	846.17	C–H	Alkenes
1249.08	C–O	Alcohol, ether, carboxalic acid, ester	1015.36	C–F	Fluorine group
1336.01	NO2	Nitro compounds	1257.76	C–O	Alcohol, ether, carboxylic acid, ester
1496.91	C–H	Alkanes	1366.21	C–H	Alkanes
1616.08	C=O	Carbonyl group	3341.29	O–H	carboxylic acid
2341.17	C–H	Alkanes	3683.91	O–H	carboxylic acid
2923.33	C–H	Alkanes
3365.30	OH	carboxylic acid
3392.36	OH	carboxylic acid

Fig. 1.

(a): Peak absorbance values of FTIR spectra of the aqueous-ethanolic extract of C. burhia

(b): Peak absorbance values of FTIR spectra of the aqueous-ethanolic extract of C. burhia.

3.3. Antioxidant effect

3.3.1. Antioxidant effect of E. prostrata and C. burhia by DPPH free radical reduction assay

Antioxidant potential of aqueous-ethanolic extract of E. prostrata and C. burhia was performed by

utilizing the DPPH free radical reduction method and ascorbic acid was used as standard control. On

comparison with standard ascorbic acid 92.2 ± 0.18 with an IC50 of 0.0041 mg/mL (p < 0.001), E. prostrata showed a highly significant antioxidative potential 92.0 ± 1.1 with an IC50 of 62.5 mg/mL (p < 0.001) whereas C. burhia found to possess significant antioxidant potential 48.2 ± 1.7 with an IC50 of 5 mg/mL (p < 0.05) at the higher concentration of 5 mg/mL (Table 3).

Table 3.

Antioxidant effect of E. prostrata and C. burhia by DPPH free radical reduction assay.

Treatment	Dose (mg/kg) B·W	No. of writhes (mean ± SEM)	% of inhibition
Control	(10 ml/kg vehicle)	45.7 ± 1.2	–
Dicloran Standard	(10 mg/kg)	12.3 ± 0.9 C	74.7
E. prostrata	(100 mg/kg)	23.0 ± 0.6 b	53.8
E. prostrata	(200 mg/kg)	13.7 ± 0.3 C	71.8
C. burhia	(100 mg/kg)	28.7 ± 0.7a	40.9
C. burhia	(200 mg/kg)	25.3 ± 1.5a	47.6

Values are expressed as mean ± SEM, n = 6, ^ap < 0.05, significant, ^bp < 0.01 more significant, ^cp < 0.001 considered as highly significant. Compared with control.

3.4. Acute oral lethality studies of E. prostrata and C. burhia

Aqueous-ethanolic extracts of E. prostrata and C. burhia were fed to the mice orally at various dosages.

(1, 3, 5 as well as 10 g/kg b. wt). Following observations up to the dose of 5 g/kg b. wt, no signs of toxicity effect was observed as well as no mortality whereas E. prostrata, at the dose of 10 g/kg b. wt, induced

abdominal cramps followed by the death of 55% of the animals.

3.5. Effect of extracts on immunomodulatory activity

In-vivo comparative analysis of phagocytic potential of aqueous-ethanolic extracts of E. prostrata and.

C. burhia was analyzed by using macrophages phagocytosis assay using carbon clearance rate test in rats. Experimental results showed that the plant extracts at different dosages of 100 and 200 mg/kg

considerably raised the phagocytic index (Table 4). The maximum effect of E. prostrata was with 200 mg/kg dose and was highly significant 5.2 ± 0.2 (p < 0.001) and more significant 4.2 ± 0.1 at 100 mg/kg (p < 0.01) compared with standard Levamisole i.e. 5.4 ± 0.2 at 100 mg/kg. Similarly, C. burhia showed more significant results 4.8 ± 0.1 (p < 0.01) at 200 mg/kg dose and significant at 100 mg/kg i.e. 3.9 ± 0.1 (p < 0.05) was equipotent with standard. The effects of both the plant extracts at the dose of 200 mg/kg b. w were in line with the standard drug (levamisole) and suggesting its role in regulation of immune response that could be via augmenting macrophage phagocytic activity.

Table 4.

Immunomodulatory potential of the aqueous-ethanolic extract of E. prostrata and C. burhia.

Aqueous-ethanolic Extracts	Concentration (mg/mL)	% of inhibition	IC50 mg/mL
E. prostrata	5	92.0 ± 1.1b	0.63
C. burhia	5	48.2 ± 1.7a	5
Ascorbic Acid	0.5 (mmol/mL)	92.2 ± 0.18 b	0.0041
Methanol	5	Nil	Nil

Values are expressed as mean ± SEM, n = 6, ^ap < 0.05, significant, ^bp < 0.001 considered as highly significant. Compared with control.

3.6. Effect of extracts on analgesic activity

Analgesic activity of the aqueous-ethanolic extract of E. prostrata and C. burhia was carried out by acetic

acid induced pain test (writhing test). Results showed that E. prostrata and C. burhia extracts at the

dosages of 100 and 200 mg/kg b. w. considerably reduced (p < 0.001) the writhing count and contractions

in comparison to control group and standard dicloran. Administration of extract of E. prostrata and C.

burhia extracts showed significant reduction in percentage of writhing count which was closer to that of

standard value (Table 5). The maximum effect of E. prostrata was with 200 mg/kg dose and was highly significant 13.7 ± 0.3 with inhibition 71.8 % (p < 0.001) and more significant 23.0 ± 0.6 with inhibition 53.8% at 100 mg/kg (p < 0.01) compared with standard Dicloran i.e. 12.3 ± 0.9 with inhibition 74.7% at 100 mg/kg. Similarly, C. burhia showed significant results 25.3 ± 1.5 with inhibition 40.9% (p < 0.05) at 200 mg/kg dose and significant at 100 mg/kg i.e. 28.7 ± 0.7 with inhibition 47.6 (p < 0.05) was equipotent with standard. These results suggest that plants have considerable pain reducing property.

Table 5.

Analgesic activity of the aqueous-ethanolic crude of E. prostrata and C. burhia.

Grouping of animals	Dose of treatment (mg/kg) B·W	Phagocytic index
Control	(10 ml/kg vehicle)	2.3 ± 0.1
Levamisole	(100 mg/kg)	5.4 ± 0.2 C
E. prostrata	(100 mg/kg)	4.2 ± 0.1b
E. prostrata	(200 mg/kg)	5.2 ± 0.2 C
C. burhia	(100 mg/kg)	3.9 ± 0.1a
C. burhia	(200 mg/kg)	4.8 ± 0.1 b

Values are expressed as mean ± SEM, n = 6, ^ap < 0.05, significant, ^bp < 0.01 more significant, ^cp < 0.001 considered as highly significant. Compared with control.

3.7. Effects of crude extracts on body weight and organs

Crude aqueous-ethanolic extracts of E. prostrata and C. burhia at the 100 along with 200 mg/kg b. wt dosages did not show any visible physiological and behavioral changes. No change in the b. wt of animals was observed during the treatment with extracts. Spleen and liver were also weighed to know any change in their weights. In the extract fed animals, we did not observe any significant alteration in the weights of spleen, kidney, thymus and pancreas noted however there was a little increase in the weight of

Liver was observed as compared to the control, suggesting that these plants extracts are safe to use in vivo.

4. Discussion

In the present study, E. prostrata and C. burhia were tested for the preliminary phytochemical screening, antioxidant, in-vivo immunomodulatory and analgesic potential of the aqueous-ethanolicextracts. Preliminary phytochemical screening revealed the abundance of various phytoconstituents including carbohydrates, saponins, tannins, phenols, quinines flavonoids, proteins, fats and oils, terpenes, glycosides as well as alkaloids. Furthermore, the FTIR analysis was performed to evaluate the existence of various functional groups in both plant extracts and revealed the existence of alkenes, alkanes, fluorine, alcohol, ether, carboxalic acid, ester, nitro compounds and carbonyl groups (Table 2) (Fig. 1a & b).

Plants are considered the fundamental source of natural antioxidants, producing a variety of chemical compounds with therapeutically potential antioxidant properties. Polyphenols are the plant base substances, mainly abundant oxidative components [45]. The capacity to reduce free radicals is usually via splitting the free radical chain by contributing a covalent bond or avoiding the production of peroxide.

Naturally, plants are generally rich in phenolic acids, flavonoids, coumarins, lignans, tannins as well as lignin which are associated with various biomedical properties of such chemical compounds, including antioxidant properties [46]. Antioxidant potential of aqueous-ethanolic extracts of E. prostrata and C. burhia was confirmed by the DPPH free radical reduction method. Experimental results revealed that E. prostrata showed maximum percentage of inhibition of 92.0 ± 1.1 with the IC50 of 62.5 μg/ml whereas C. burhia didn't show significant inhibition when compared with ascorbic acid (Table 3). Hence, from the results, it is suggested that E. prostrata can be utilized as a strong antioxidant agent to gain maximum antioxidant effect in medicine, food and minimize the harm of free radicals.

Antioxidant effect of E. prostrata extract was evaluated by performing the DPPH radical scavenging activity and was compared with the effect of the standard drug ascorbic acid. The effect of extract was highly significant (p < 0.001) and was more pronounced compared with standard drug. It showed that crude extract of E. prostrata have the ability to reduce the pain. Antioxidant effect of C. burhia was evaluated and the effect was less significant as compared with crude extract from E. prostrata. The effect of extract C. burhia showed significant effect (p < 0.05). Antioxidative effect of both plant extracts was significant in reducing oxidative stress at the higher concentration of 5 mg/mL (Table 3).

Overall, various pathological conditions are associated with phagocytic defects. Medicinal plants are reported to be abundant in a variety of biological active compounds including alkaloids, phenols, tannins, flavonoids, glycosides, saponins and steroids and are linked with several biological properties including modulation via phagocytic index contributes to the immune functioning [47]. Therefore, we sought to evaluate the effect of aqueous-ethanolic crude extracts of E. prostrata and C. burhia on macrophage phagocytic activity by using the carbon clearance rate in animal model and found that both the crude extracts augmented the phagocytic index in comparison with control. Both the extracts significantly increased phagocytic index the maximum effect of E. prostrata was with 200 mg/kg dose and was highly significant 5.2 ± 0.2 (p < 0.001) and more significant 4.2 ± 0.1 at 100 mg/kg (p < 0.01) compared with standard Levamisole i.e. 5.4 ± 0.2 at 100 mg/kg. Similarly, C. burhia showed more significant results 4.8 ± 0.1 (p < 0.01) at 200 mg/kg dose and significant at 100 mg/kg i.e. 3.9 ± 0.1 (p < 0.05) was equipotent with standard (Table 4). The effects of both the plant extracts at the dose of 200 mg/kg b. w were in line with the standard drug (levamisole). Hence, our experimental results proposed that both the plants may have contributory role to modulate the immune response by potentiating phagocytic index to counter various life-threatening disorders.

Painful conditions are progressive and one of the largest health problems around the globe in both developed and developing countries. Furthermore, it leads to patient disability and a large public health burden [48]. These pathological conditions are portrayed by muscular pain, spasms, limited mobility, redness, and living standards compromise. In fact, individuals and families may be exposed to a heavy socio-economic burden. The pervasiveness of muscular pain including other joint related diseases ranges from 14% to 36% in advanced nations [49].

Thus, taking in account pain, we thought to analyze the analgesic potential of the aqueous-ethanolic crude extract of E. prostrata and C. burhia via acetic-acid induced writhing method in mice model. Our data indicated that both the extracts at the dose of 100 and 200 mg/kg b. wt exhibited significant % of writhing inhibition 53.8%,71.8% 40.9% and 47.6% respectively, when compared to control and standard showed 74.7%. The results showed that E. prostrata at the dose of 200 mg/kg b. w. exhibited statistically significant analgesic effect The maximum effect of E. prostrata was with 200 mg/kg dose and was highly significant 13.7 ± 0.3 with inhibition 71.8 % (p < 0.001) and more significant 23.0 ± 0.6 with inhibition 53.8% at 100 mg/kg (p < 0.01) compared with standard Dicloran i.e. 12.3 ± 0.9 with inhibition 74.7% at 100 mg/kg. Similarly, C. burhia showed significant results of 25.3 ± 1.5 with inhibition 40.9% (p < 0.05) at 200 mg/kg dose and significant at 100 mg/kg i.e. 28.7 ± 0.7 with inhibition 47.6 (p < 0.05) and found in line with that of dicloran when compared with the control. (Table 5). The findings of our research are in line with other studies; the Hosseinzadeh group has shown that saffron stigma, as well as petal aqueous-ethanolic extracts, have antinociceptive action in pain caused by acetic-acid in mice, the analgesic action of such extricates might be because of their tannin, saponin, alkaloids and flavonoid contents [50] and afsar et al. who reported that methanolic extract of the Acacia hydaspia exhibited significant anti-nocicepetive effect against acetic acid caused writhing method. The analgesic effect of the medicinal plants may be modulated via inhibitory action on the local peritoneal receptors through the inhibition of cyclooxygenase [51]. Previously, Ribeiro et al. have shown that acetic-acid's nociceptive property might be attributable to the cytokines release, like interleukin (IL)-1, IL-8 along with TNFα through resident peritoneal mast cells and macrophages.

Therefore, the earlier studies and yet these outcomes described here might suggest that all of the extracts used within the acetic-acid induced writhing experiment showed antinociceptive activity that might be attributed to inhibition through resident peritoneal cells contributes to the release of IL-1, IL-8 and TNFα [52]. A recent report also evidenced that aqueous-ethanolic crude extract of E. prostrata reduced the inflammation as well as oedema by inhibition proinflammatory cytokines modulating IL-1 via NF-κB dependent pathway [53] supporting its effect in regulation of immune response via reducing pain and augmenting phagocytic index.

Further studies are warranted to identify molecular target of these plants at cellular level and identification and isolation of the compound responsible. Moreover, therapeutic application in managing pain and immune system associated diseases.

5. Conclusion

We concluded that the plant extract of E. prostrata and C. burhia possesses in-vitro antioxidant, in-vivo

immunomodulatory and analgesic effects. Our phytochemical screening analysis showed that these plants are rich source of various chemical constituents including carbohydrates, saponins, tannins, phenols, quinines flavonoids, proteins, fats and oils, terpenes, glycosides as well as alkaloids. Furthermore, FTIR analysis revealed the existence of various functional groups such as alkenes, alkanes, fluorine, alcohol, ether, carboxalic acid, ester, nitro compounds and carbonyl groups in both plant extracts. Both the extracts exhibited the augmented phagocytic by regulating immune system and analgesic effects that justify their use traditionally in the management of different chronic and autoimmune diseases to relieve the pain and stimulate the immune system.

Funding

The authors received no financial support for the research.

Data availability statement

Data associated with study has not been deposited into a publicly available repository.

CRediT authorship contribution statement

Muhammad Rahil Aslam: Investigation. Hafiz Muhammad Asif: Supervision. Abdulaziz Alamri: Funding acquisition. Adnan Ahmed Bhutto: Writing – review & editing. Muhammad Mukhtiar: Funding acquisition. Khalil Ahmad: Visualization. Mehak Muhammad Ashfaq: Software. Hafiz Abdul Sattar: Methodology. Abdul Hayee: Resources. Sana Jabbar: Visualization. Rabia Zahid: Writing – review & editing, Data curation. Allah Nawaz: Methodology.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.