Chemical Profiling and Biological Activities of Ziziphus Mauritiana var. spontanea (Edgew.) R.R. Stewart ex Qaiser & Nazim. and Oenothera Biennis L.

Abstract

Bioactive compounds of medicinal plants, including polyphenols, flavonoids, terpenoids, and alkaloids, are essential sources for developing analgesic, anti-inflammatory, and antidiarrheal drugs. In the current study, secondary metabolites were assessed through phytochemical screening and GC-MS analysis whereas analgesic activity was carried out through hot plate (HP) and acetic acid-induced method (AAI), anti-inflammatory through paw edema model (PEM), and antispasmodic activity via charcoal meal test (CMT) using ethyl acetate and ethanolic extract of Ziziphus mauritiana var. spontanea and Oenothera biennis. The phytochemical screening revealed that the ethyl acetate and ethanolic extracts of Z. mauritiana and O. biennis were rich in alkaloids, flavonoids, tannins, steroids, triterpenoids, and saponins. GC-MS analysis of Z. mauritiana and O. biennis of ethyl acetate and ethanolic extract showed the existence of many bioactive substances at various retention durations (min). These included pharmacologically active compounds such as heptadecane, 2-methoxy-4-vinylphenol, hexadecanoic acid, and tetradecanoic acid. The results of the HP method revealed that ethanolic and ethyl acetate extracts of Z. mauritiana and O. biennis at 300 mg/kg increased basal reaction time significantly ($p<0.001$) after 90 min. The results of the AAI method revealed that the ethanolic and ethyl acetate extract of Z. mauritiana and O. biennis showed significant ($p<0.001$) peripheral analgesic activity at the dose of 200 and 300 mg/kg body weight. The dosage of 100, 200, and 300 mg/kg body weight of the ethyl acetate and ethanolic extracts of Z. mauritiana and O. biennis showed significant ($p<0.001$) anti-inflammatory activity. According to PEM, the ethanolic extract of O. biennis showed the highest reduction in paw volume (73.3%) at 300 mg/kg. The results of CMT revealed that ethanolic and ethyl acetate extract of Z. mauritiana and O. biennis significantly ($p<0.001$) inhibited charcoal movement at 300 mg/kg. The maximum percent inhibition (67.2%) was shown by ethyl acetate of O. biennis at 300 mg/kg. From the present study, it can be concluded that ethanolic and ethyl acetate extracts of Z. mauritiana and O. biennis have the potential to manage inflammation, pain, and diarrhea-related problems mainly at a higher dose, i.e., 300 mg/kg. The presence of alkaloids, flavonoids, tannins, steroids, triterpenoids, and saponins might be among the responsible bioactive constituents. These plants showed significant medicinal and therapeutic efficacy which are novel. However, further studies are required to investigate the mechanism responsible for the activity.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Medicinal plants are naturally God-gifted with a lot of chemical compounds that are formed in different plant parts and used in the treatment of various ailments. These chemical compounds are also known as secondary metabolites. These secondary metabolites can be utilized in the formulations of novel medications and have been reported as very effective in curing serious health problems. The World Health Organization (WHO) reported that about 80% of the population in the world still depends on medicinal plants for their basic healthcare due to their effectiveness and easy availability [1]. Advancements in biotechnology have made it possible to manufacture therapeutic protein drugs in significant quantities [2]. Historically, fruits have served as a basis for various medicinal and liquor products [3]. Additionally, plant hydrocolloids have shown potential in mitigating cardiovascular disease risk, decreasing blood cholesterol levels, and enhancing immune function [4].

Aromatic medicinal plants are widely used by people for various purposes including the food industry, perfumery, textile industry, and pharmaceutical industry. The medicinal properties of plants are due to the presence of bioactive compounds such as saponins, glycosides, quinones, alkaloids, and flavonoids [5].

Pain is associated with potential or actual tissue damage. Pain is not only a symptom used to diagnose several diseases and conditions but also has a protective function [6]. Analgesic drugs have been used for the elimination of pain without significantly altering consciousness. The use of synthetic analgesic drugs has several side effects including gastrointestinal disorders, bleeding, and ulcers [7]. A physiological response that protects a body from tissue injury is called inflammation. There are two types of inflammation, i.e., acute inflammation and chronic inflammation [8]. The nonsteroidal anti-inflammatory drugs are used to treat inflammation but it has a lot of side effects, including gastrointestinal and cardiovascular complications. Therefore, the development of new drugs is necessary for the management of pain and inflammation. Plant-based beverages typically contain advantageous bioactive compounds, including flavonoids, phenolic acids, lignans, and phytosterols, known for their exceptional antioxidant, analgesic, and anti-inflammatory properties, contributing significantly to health benefits [9, 10]. In the past twenty years, numerous research studies have underscored the pivotal role of the gut microbiome in influencing health and disease. Alterations in the composition of the intestinal microbiome have been linked to diverse intestinal and metabolic disorders, including inflammatory bowel disease, diabetes, and obesity [11].

An imbalance in the gut microbiota plays a crucial role in the pathogenesis of functional Dyspepsia (FD). This imbalance disrupts the intestinal environment, ultimately causing a decrease in beneficial probiotics and triggering a range of acute and chronic diseases [12]. The condition of increased stool frequency, liquidity, or volume is called diarrhea. It mainly affects neonates and babies and is a significant cause of illness and mortality in developing countries [13]. Medicinal plants are reservoirs of essential antidiarrheal bioactive constituents without any side effects and can be used to treat gastrointestinal disorders, for example, constipation and diarrhea [14].

Ziziphus mauritiana belonging to the family Rhamnaceae Juss., locally called Ber, is a fruit tree that grows worldwide in tropical and subtropical regions [15]. Z. mauritiana is a medicinal plant used to cure several disorders such as ulcers, asthma, allergies, depression, digestive problems, weakness, obesity [16, 17], diabetes, urinary issues, and skin infections [18].

Oenothera biennis L. also known as evening primrose belongs to Onagraceae Juss. It is distributed in Peshawar Pakistan and eastern and central North America. The seed oil of O. biennis is used for the treatment of several ailments such as asthma, eczema, breast problems, rheumatoid arthritis, menopausal syndrome [19] fistulas, and lung disease [20]. The present study will analyze the plant in terms of drug standardization and evaluating its pharmacological effect which can be used as a potential candidate for future drug developments.

2. Materials and Methods

2.1. Plant Collection

Ziziphus mauritiana was collected from the area of Palosai Peshawar, and Oenothera biennis was collected from the Department of Botany, University of Peshawar KPK Pakistan which is located at 34.0011°N and 71.4874°E in August 2022. The identification of the plant was carried out with the help of Flora of Pakistan and Ghulam Jelani (plant taxonomist) and kept in the herbarium for future reference with Voucher Number Ambrin Bot. 33 (PUP) and Ambrin Bot. 34 (PUP).

2.2. Extraction of Plant Material and Sample Preparation

Whole plants were washed with distilled water, shade dried in the air dryer, and ground into a fine powder, and 50 g was soaked in 250 mL each in ethanol and ethyl acetate, respectively, which were supplied by U.M. enterprises. After 48 hours, the extract was passed through muslin cloth followed by filtration through filter paper. The extract was concentrated using a rotary evaporator (RE-100D Phoenix) supplied by MED Lab Services. The derived ethyl extracts (10.1 g) and ethanol extract (9.5 g) of Z. mauritiana and ethyl extracts (10.4 g) and ethanol extract (10.12 g) each were kept at 4°C in capped bottles before use [21].

2.3. Phytochemical Screening

Phytochemical analysis of the extracts was carried out to detect flavonoids, alkaloids, tannins, steroids, triterpenoids, and tannins following protocols [22].

2.4. Gas Chromatography-Mass Spectrometry (GC/MS)

A gas chromatography-mass spectrometry of the crude extract of Z. mauritiana and O. biennis was carried out by coupling Thermo GC-Trace Ultra version 5.0 with Thermo MS DSQ II which was supplied by MED Lab Services. The sample was prepared by adding 2 mg crude extract in 5 mL of respective solvents. To purify the samples, the mixtures were divided on a ZB 5-MS capillary regular nonpolar column (30 m 0.25 mm ID 0.25 lm FILM). The temperature of the column was kept at 70°C with an increasing rate of 2°C/minute. Finally, the increase in temperature was raised to 260°C at 6°C/minute with a holding time of about ten minutes. The splitless mode was used to introduce the particle-free, diluted sample (10 mL/min split flow and 1 min spitless period). Helium was used as the carrier gas at a constant flow rate of 1 mL/min, and 1 L of sample was injected. Peak area normalization was used to quantify the relative percentages of crude extract elements. In full scan mode, the mass spectral scan range was adjusted to 50 to 650 (m/z). By comparing the retention indices of the compounds with those of real samples stored on the Wiley and Main Lab computer library search software, the compounds were identified [23].

2.5. Analgesic Activity

2.5.1. Acetic Acid-Induced Writhing

Ziziphus mauritiana and Oenothera biennis ethanolic and ethyl acetate extracts (sample was prepared by adding 10 mg crude extract in 25 mL of respective solvents) were tested for their analgesic activity following the method of [24] with few modifications. Mice were divided into five groups containing five mice in each group. Group 1 served as control and administered only with normal saline (10 ml/kg i.p.) Group 2 was administered with standard diclofenac sodium (25 mg/kg), and groups 3–5 were administered with three doses of Z. mauritiana and O. biennis ethanolic and ethyl acetate extracts (100 mg/kg, 200 mg/kg, and 300 mg/kg), respectively, and these extracts were administered orally one hour before intraperitoneal injection of 0.6% v/v acetic acid and after five mins of postinjection; the number of writhing was counted for the next 20 min. The percent analgesic effect was calculated by the following formula:$$\begin{array}{}\text{(1)}& \%\text{\hspace{0.17em}analgesic} \text{effect}=100-\text{no} \text{of} \text{writhing} \text{in} \text{tested} \text{animals} \text{no} \text{of} \text{writhing} \text{in} \text{control} \text{animals} \mathrm{X} 100.\end{array}$$

2.5.2. Eddy’s Hot-Plate Method

Its requirements were similar to the previous method. To perform this activity, the method of [25] was followed, the albino mice (male) were divided randomly into 5 groups, and each group consisted of 5 mice. Group 1 served as control and administered only with normal saline (10 ml/kg ip), group 2 was treated with the standard drug diclofenac sodium (25 mg/kg), and groups 3–5 were orally administered with different concentrations (100, 200, and 300 mg body weight) of ethanolic and ethyl acetate extracts, respectively. The initial reaction time of control and test group animals was recorded by placing them on the hot plate (55 ± 0.5°c), and the licking of the paw or jumping was taken as the index of reaction of heat: the post-treatment reaction time of each animal after the administration of plant extracts recorded at 30 min, 60 min, and 90 min.$$\begin{array}{}\text{(2)}& \text{elongation\hspace{0.17em}}\%\text{\hspace{0.17em}latency\hspace{0.17em}}\text{test}-\text{latency\hspace{0.17em}}\text{control}\times 100\text{\hspace{0.17em}latency\hspace{0.17em}}\text{test}\text{\hspace{0.17em}statistical\hspace{0.17em}analysis}\end{array}$$

2.6. Anti-Inflammatory Activity

The anti-inflammatory activity of the Z. mauritiana and O. biennis ethanolic and ethyl acetate extracts was investigated on carrageenan-induced inflammation in mice paws following the procedure [26]. The sample was prepared by adding 10 mg crude extract in 25 mL of respective solvents. Animals were divided into 5 groups comprising five animals per group. In all groups, acute inflammation was produced by subplantar injection of 0.1 ml freshly prepared 1% suspension of carrageenan. The paw volume was measured plethysmometrically from 0 to 180 min after carrageenan injection. All the animals were orally premedicated with diclofenac sodium (10 mg/kg b.wt), two hours before the infection. The mean increase in paw volume was measured, and the percentage was calculated for all the extracts. Percentage inhibition of paw volume was calculated by the following formula:$$\begin{array}{}\text{(3)}& \text{Vc}-\text{Vt\hspace{0.17em}}\% \mathrm{i}\text{nhibition} \text{of} \text{paw} \text{edema}=\mathrm{x} 100 \text{Vc},\end{array}$$where Vt = increase in paw volume in mice treated with test compounds and Vc = increase in paw volume in the control group of mice.

2.7. Antispasmodic Activity

The antispasmodic activity of Z. mauritiana and O. biennis ethanolic and ethyl acetate extracts was carried out by adopting the method of [27]. The sample was prepared by adding 10 mg crude extract in 25 mL of respective solvents. The selected mice were divided into four groups of five mice each. At first, 1 ml of castor oil was given orally to every mouse in each group to produce diarrhea. After 1 hr, group I (control group) orally received saline (10 ml/kg). Group II received the standard drug (atropine sulfate 10 mg/kg b. wt in), and group it-V (the rest of the three groups) received ethanolic and ethyl acetate extracts of plants (100, 200, and 300 mg/kg b. wt ip, respectively). After 1 h, all animals orally revived of the charcoal meal (10 charcoal is a pension in 5% gum acacia). After one hour following the charcoal meal administration, all animals were sacrificed, and the distance covered by the charcoal meal in the intestine, from the pylorus to the caecum, was measured and expressed as a percentage of the distance moved intestinal transit.$$\begin{array}{}\text{(4)}& \%=\mathrm{D}/\mathrm{L}\times 100,\end{array}$$where D = distance covered by charcoal (in meters) and L = intestinal length (in meters).

2.8. Ethical Approval

The animal study was reviewed and approved by the Ethical Committee Pharmacy Lab, Qurtuba University of Science and Information Technology Peshawar, Pakistan, under permit no (148/VIEC/VRIP).

2.9. Statistical Analysis

The data were analyzed by Dunnett’s t-test statistical methods using SPSS Software 22.0. For the statistical tests, $p<0.001$, 0.01, and 0.05 was considered as significant.

3. Results

3.1. Phytochemical Screening

The phytochemical screening of ethanolic and ethyl acetate extracts of Z. mauritiana and O. biennis revealed the existence of different bioactive compounds. The ethanolic extract of Z. mauritiana showed the presence of alkaloids, flavonoids, tannins, steroids, and triterpenoids whereas saponins were found absent. Similarly, the ethyl acetate extracts of Z. mauritiana detect alkaloids, flavonoids, saponins, tannins, and triterpenoids while steroids were absent. Likewise, the ethanolic extract of O. biennis revealed the presence of alkaloids, flavonoids, saponins, and steroids and the absence of tannins and triterpenoids. The ethyl acetate extract of O. biennis unveiled the presence of alkaloids, flavonoids, tannins, steroids, and triterpenoids and the absence of tannins (Table 1).

Table 1

Phytochemical screening of Ziziphus mauritiana var. spontanea and Oenothera biennis.

Chemical constituents	Ziziphus mauritiana var. spontanea.		Oenothera biennis
	Ethyl acetate extract	Ethanol extract	Ethyl acetate extract	Ethanol extract
Alkaloids	+	+	+	+
Flavonoids	+	+	+	+
Saponins	−	+	−	+
Tannins	+	+	+	−
Steroids	+	−	+	+
Triterpenoids	+	+	+	−

3.2. Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry profiling identified the probable phytochemicals in the ethanolic and ethyl acetate extract of Z. mauritiana and O. biennis. In ethanolic and ethyl acetate extract of Z. mauritiana, fourteen phytoconstituents were detected by GC–MS (Tables 2 and 3; Figures 1 and 2). Similarly, eight phytoconstituents were detected in the ethanolic extract of O. biennis, and eight phytoconstituents were detected in its ethyl acetate extract (Tables 4 and 5; Figures 3 and 4). The results showed that phytoconstituents found in maximum concentration in ethanolic extract of Z. mauritiana was heptadecane (9.06%) followed by 2-methoxy-4-vinylphenol (6.54%) and dodecanoic acid (4.50%) (Table 2; Figure 1). In the ethyl acetate extract of Z. mauritiana, tetracosane (11.52%) was detected in the highest concentration followed by dodecane, 1,1-dimethoxy- (4.06%), and 2-methoxy-4-vinylphenol (6.36%) (Table 3; Figure 2). In an ethanolic extract of O. biennis, bioactive constituents detected in the maximum amount were phytol (7.45%), followed by furfural (6.53%) and 4-vinyl-2-methoxy-phenol (2.36%) (Table 4; Figure 3). Likewise, the phytoconstituents detected with maximum concentration in ethyl acetate extract of O. biennis include 9,12-octadecadienoic acid (Z, Z)- (7.33%), followed by hexathiane (1.06%) and ethyl hexadecane (4.37%) (Table 5; Figure 4).

Table 2

GCMS analysis of ethanol extract of Ziziphus mauritiana var. spontanea.

S. No	Name of the compound	Compound formula	Retention time (min)	Peak area (%)	Compound structure	Molecular weight
1	Heptadecane	C17H36	7.2	9.06		240.475 g·mol−1
2	2-Methoxy-4-vinyl phenol	C9H10O2	10.5	6.54		150.17 g/mol
3	Dodecanoic acid	C12H24O2	17.4	4.50		201.31 g/mol
4	2,3,6-Trimethyl decane	C11H24	25.3	8.43		156.31 g/mol
5	Octadecanoic acid	C18H34O2	30.2	5.36		282.5 g/mol
6	Methyl 14-methyl pentadecanoate	C17H34O2	39.5	3.40		270.5 g/mol
7	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	C19H38O4	42.6	2.53		330.5 g/mol

Table 3

GCMS analysis of ethyl acetate extract of Ziziphus mauritiana var. spontanea.

S. no	Name of the compound	Compound formula	Retention time (min)	Peak area (%)	Compound structure	Molecular weight
1	Dodecane, 1,1-dimethoxy-	C12H26O2	6.3	4.06		202.33 g/mol
2	2-Methoxy-4-vinyl phenol	C9H10O2	10.0	6.36		150.17 g/mol
3	9-Octadecene	C18H36	14.5	3.45		252.5 g/mol
4	Methyl 11-octadecenoate	C19H36O2	19.4	10.65		296.5 g/mol
5	Hexadecanoic acid	C16H32O2	24.4	7.06		287.61 g/mol
6	9,12-Octadecadienoic acid (Z, Z)-, methyl ester	C18H32O2	27.6	5.43		280.4 g/mol
7	Tetracosane	C24H50	30.6	11.52		389.0 g/mol

[figure(s) omitted; refer to PDF]

Table 4

GCMS analysis of ethanol extract of Oenothera biennis.

S. No	Name of the compound	Compound formula	Retention time (min)	Peak area (%)	Compound structure	Molecular weight
1	Furfural	C4H5O2	9.3	6.53		96.08 g/mol
2	4-Vinyl-2-methoxy-phenol	C9H10O2	14.2	2.36		150.17 g/mol
3	Hexadecanoic acid and methyl ester	C17H32O2	16.5	1.42		268.4 g/mol
4	7-Methyl hexadecane	C17H36	20.2	4.06		240.5 g/mol
5	2,3,6-Trimethyl decane	C11H24	22.4	3.40		156.31 g/mol
6	Squalene	C30H50	26.5	2.53		410.7 g/mol
7	Phytol	C20H40O	30.3	7.45		296.5 g/mol

Table 5

GCMS analysis of ethyl acetate extract of Oenothera biennis.

S. no	Name of the compound	Compound formula	Retention time (min)	Peak area (%)	Compound structure	Molecular weight
1	Hexathiane	C6H8	15.3	1.06		80.13 g/mol
2	Ethyl hexadecanoic	C98H184O10	18.6	4.37		1522.5 g/mol
3	2,3,6-Trimethyl decane	C11H24	22.4	3.16		156.31 g/mol
4	n-Eicosane	C41H86	25.3	6.05		579.1 g/mol
5	1,19-Eicosadiene	C8H14	28.5	5.32		110.20 g/mol
6	9,12-Octadecadienoic acid (Z, Z)-	C18H32O2	33.4	7.33		280.4 g/mol
7	Tetradecanoic acid	C14H28O2	45.5	1.56		228.37

[figure(s) omitted; refer to PDF]

3.3. Analgesic Activity

3.3.1. Writhing Method

The ethanolic and ethyl acetate extract of Z. mauritiana and O. biennis exhibited significant ($p<0.001$) analgesic activity when measured by acetic acid-induced writhing inhibition method at a dose of 300 mg/kg body (Table 6). The ethyl acetate and ethanolic extract of Z. mauritiana showed percent inhibition of 22.0%, 57.5%, 25.0%, and 72.6% at a dose of 100 mg/kg and 200 mg/kg body weight whereas the ethyl acetate and ethanolic extract of O. biennis showed percent inhibition of 24.0% and 64.1% and 29.1% and 59.4%, respectively, at a dose of 100 mg/kg and 200 mg/kg body weight which was comparable with the positive control diclofenac sodium (68.5%). The ethanolic extract of Z. mauritiana was more effective than O. biennis extracts.

Table 6

Analgesic activity of Ziziphus mauritiana. var spontanea and Oenothera biennis L. by writhing method.

Treatment	Dose (mg/kg)	Ziziphus mauritiana		Oenothera biennis
		Average no. of writhing	% Inhibition	Average no. of writhing	Inhibition %
Normal saline	5 ml/kg	70.6 ± 5.03	—	70.6 ± 5.03	—
Diclofenac sodium	10 mg/kg	22.3 ± 2.51${}^{\ast \ast \ast }$	68.5	22.3 ± 2.51${}^{\ast \ast \ast }$	68.5
Ethyl acetate	100 mg/kg	60.66 ± 9.45	14.1	58.66 ± 0.57	17.0
	200 mg/kg	55.00 ± 4.00${}^{\ast \ast }$	22.0	53.66 ± 3.21${}^{\ast \ast }$	24.0
	300 mg/kg	30.00 ± 5.00${}^{\ast \ast \ast }$	57.5	25.33 ± 5.50${}^{\ast \ast \ast }$	64.1
Ethanol	100 mg/kg	57.66 ± 6.65${}^{\ast }$	18.4	61.66 ± 6.65	12.7
	200 mg/kg	53.00 ± 4.35${}^{\ast \ast }$	25.0	50.00 ± 1.73${}^{\ast \ast \ast }$	29.1
	300 mg/kg	19.33 ± 1.15${}^{\ast \ast \ast }$	72.6	28.66 ± 7.02${}^{\ast \ast \ast }$	59.4

Values are expressed as mean ± standard deviation. Significance is shown as ${}^{\ast }$significant, ${}^{\ast \ast }$more significant, and ${}^{\ast \ast \ast }$highly significant.

3.3.2. Hot-Plate Method

The ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis showed a significant ($p<0.001$) increase in latency time after 90 min at a dose of 200 mg/kg and 300 mg/kg body weight (Tables 7 and 8). Both the plants showed dose-dependent effects, and a significant result was obtained after 90 min at 300 mg/kg comparable with diclofenac sodium. The inhibition percentage at 200 and 300 mg/kg of ethyl acetate and ethanolic extract of Z. mauritiana was 46.7%, 56.0% and 38.1%, 52.3% while that of O. biennis 40.3%, 53.7% and 48.5%, 59.5% after 90 min, respectively. The highest central analgesic effect was shown by the ethanolic extract of O. biennis which was higher than diclofenac sodium.

Table 7

Analgesic activity of Ziziphus mauritiana by hot-plate method.

Treatment	Dose (mg/kg)	After 0 min	After 30 min	After 60 min	After 90 min	Percent decrease in latency time
Normal saline	10 ml/kg	3.30 ± 0.62	4.20 ± 0.36	3.73 ± 0.41	4.53 ± 0.80	…
Diclofenac sodium	10 mg/kg	3.70 ± 0.17	8.10 ± 0.45${}^{\ast \ast \ast }$	9.23 ± 0.37${}^{\ast \ast \ast }$	10.60 ± 0.10${}^{\ast \ast \ast }$	57.2
Ethyl acetate	100 mg/kg	3.50 ± 0.30	4.56 ± 0.15	4.13 ± 0.55	6.40 ± 0.52${}^{\ast \ast }$	29.2
	200 mg/kg	3.53 ± 0.87	4.63 ± 0.45	5.23 ± 0.95${}^{\ast }$	8.50 ± 0.17${}^{\ast \ast \ast }$	46.7
	300 mg/kg	3.63 ± 0.20	4.73 ± 0.45	6.13 ± 0.50${}^{\ast \ast \ast }$	10.30 ± 0.45${}^{\ast \ast \ast }$	56.0
Ethanol	100 mg/kg	3.40 ± 0.79	4.26 ± 0.40	3.80 ± 0.36	5.56 ± 0.15	18.5
	200 mg/kg	3.30 ± 0.45	4.56 ± 0.11	4.70 ± 0.62	7.33 ± 1.05${}^{\ast \ast \ast }$	38.1
	300 mg/kg	3.46 ± 0.75	4.70 ± 0.10	5.80 ± 0.70${}^{\ast \ast \ast }$	9.50 ± 0.70${}^{\ast \ast \ast }$	52.3

Values are expressed as mean ± standard deviation. Significance is shown as ${}^{\ast }$significant, ${}^{\ast \ast }$more significant, and ${}^{\ast \ast \ast }$highly significant.

Table 8

Analgesic activity of Oenothera biennis L. by hot-plate method.

Treatment	Dose (mg/kg)	After 0 min	After 30 min	After 60 min	After 90 min	Percent decrease in latency time
Normal saline		3.30 ± 0.62	4.20 ± 0.36	3.73 ± 0.41	4.53 ± 0.80	…
Diclofenac sodium	10	3.70 ± 0.17	8.10 ± 0.45	9.23 ± 0.37	10.60 ± 0.10${}^{\ast \ast \ast }$	57.2
Ethyl acetate	100	3.40 ± 0.62	4.63 ± 0.41	3.96 ± 0.47	5.83 ± 0.41	22.2
	200	3.50 ± 0.60	4.73 ± 0.90	4.40 ± 0.20	7.60 ± 0.60${}^{\ast \ast \ast }$	40.3
	300	3.66 ± 0.90	4.86 ± 0.28	6.80 ± 0.62${}^{\ast \ast \ast }$	9.80 ± 0.55${}^{\ast \ast \ast }$	53.7
Ethanol	100	3.46 ± 0.90	4.23 ± 0.45	4.33 ± 0.05	7.23 ± 0.95${}^{\ast \ast \ast }$	37.3
	200	3.63 ± 0.45	4.30 ± 0.80	5.50 ± 0.26${}^{\ast \ast }$	8.80 ± 0.45${}^{\ast \ast \ast }$	48.5
	300	3.70 ± 0.85	4.50 ± 0.90	6.43 ± 0.25${}^{\ast \ast \ast }$	11.20 ± 0.40${}^{\ast \ast \ast }$	59.5

Values are expressed as mean ± standard deviation. Significance is shown as ${}^{\ast }$significant, ${}^{\ast \ast }$more significant, and ${}^{\ast \ast \ast }$highly significant.

3.4. Anti-Inflammatory Activity

3.4.1. Carrageenan-Induced Paw Edema

A significant reduction ($p<0.001$) in the paw volume was revealed by ethanolic and ethyl acetate extract (100, 200, and 300 mg/kg) of Z. mauritiana and O. biennis at the 5th hour dose-dependently. The diclofenac sodium caused a significant ($p<0.001$) reduction at the 4th and 5th hours in the volume of the paw (Tables 8 and 9). The inhibitory effect of the ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis at 100 mg/kg, 200 mg/kg, and 300 mg/kg was 64.6%, 68.6%, 70.0%, 57.3%, 60.6%, and 66.0% at 5th hour while the inhibitory effect of the ethyl acetate and ethanolic extract of O. biennis at 100 mg/kg, 200 mg/kg, and 300 mg/kg was 53.3%, 68.0%,71.3%, 66.6%, 69.3%, and 73.3%, respectively, at 5th hour. The percent inhibition of ethanolic extract of O. biennis was more than the Z. mauritiana but less than diclofenac sodium (10 mg/kg) which showed the highest 75.4% inhibition in paw volume at the 5th hour.

Table 9

Anti-inflammatory activity of Ziziphus mauritiana. var. spontanea.

Treatment	Dose (mg/kg)	Paw volume after drug administration (mean + SEM)
		1 hour	2 hour	3 hour	4 hour	5 hour
Normal saline	10 ml/kg	0.90 ± 0.02	0.77 ± 0.08	0.86 ± 0.10	0.95 ± 0.02	1.50 ± 0.27
Diclofenac sodium	10 mg/kg	0.80 ± 0.04 (11.11%)	0.54 ± 0.04${}^{\ast }$ (29.8%)	0.45 ± 0.03${}^{\ast }$ (47.67%)	0.52 ± 0.04${}^{\ast \ast \ast }$ (45.2%)	0.38 ± 0.08${}^{\ast \ast \ast }$ (75.4%)
Ethyl acetate extract	100 mg/kg	0.86 ± 0.06 (4.44%)	0.68 ± 0.08 (15.58%)	0.75 ± 0.08 (12.7%)	0.65 ± 0.04${}^{\ast \ast }$ (31.57%)	0.53 ± 0.06${}^{\ast \ast \ast }$ (64.6%)
	200 mg/kg	0.88 ± 0.07 (2.22%)	0.57 ± 0.08 (26.0%)	0.66 ± 0.06 (23.2%)	0.55 ± 0.08${}^{\ast \ast \ast }$ (42.1%)	0.47 ± 0.05${}^{\ast \ast \ast }$ (68.6%)
	300 mg/kg	0.76 ± 0.09 (15.5%)	0.59 ± 0.06 (23.3%)	0.64 ± 0.07 (34.3%)	0.53 ± 0.09${}^{\ast \ast \ast }$ (44.2%)	0.45 ± 0.04${}^{\ast \ast \ast }$ (70.0%)
Ethanolic extract	100 mg/kg	0.87 ± 0.03 (3.33%)	0.71 ± 0.04 (7.79%)	0.75 ± 0.18 (12.7%)	0.70 ± 0.14${}^{\ast }$ (26.3%)	0.64 ± 0.19${}^{\ast \ast \ast }$ (57.3%)
	200 mg/kg	0.86 ± 0.12 (4.44%)	0.66 ± 0.10 (23.2%)	0.70 ± 0.24 (26.3%)	0.64 ± 0.12${}^{\ast \ast }$ (57.3%)	0.59 ± 0.04${}^{\ast \ast \ast }$ (60.6%)
	300 mg/kg	0.84 ± 0.09 (6.66%)	0.63 ± 0.09 (18.1%)	0.67 ± 0.12 (22.0%)	0.73 ± 0.10 (23.1%)	0.51 ± 0.10${}^{\ast \ast \ast }$ (66.0%)

Values are expressed as mean ± standard deviation. Significance is shown as ${}^{\ast }$significant, ${}^{\ast \ast }$more significant, and ${}^{\ast \ast \ast }$highly significant.

3.5. Antispasmodic Activity

3.5.1. Charcoal Meal Test

The ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis significantly ($p\le 0.001$) decreased the distance travelled by a charcoal meal at a dose of 300 mg/kg body weight which was comparable to atropine sulfate (10 mg/kg) (Tables 10 and 11). The 300 mg/kg dose of ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis presented 64.4%, 56.8%, 67.2%, and 50.7% percent inhibition in the charcoal meal movement whereas the ethyl acetate extract of Z. mauritiana and O. biennis showed maximum percent inhibition of 64.4% and 67.2% in charcoal meal movement which was comparatively higher inhibition than the standard drug atropine sulfate. However, the ethyl acetate extract of O. biennis showed the highest percent inhibition (67.2%) than the ethyl acetate extract (64.4%) of Z. mauritiana.

Table 10

Anti-inflammatory activity Oenothera biennis L.

Treatment	Dose (mg/kg)	Paw volume after drug administration (Mean + SEM)
		After 1 hour	After 2 hour	After 3 hour	After 4 hour	After 5 hour
Normal saline		0.90 ± 0.02	0.77 ± 0.08	0.86 ± 0.10	0.95 ± 0.02	1.50 ± 0.27
Diclofenac sodium	10 mg/kg	0.80 ± 0.04 (11.11%)	0.54 ± 0.04${}^{\ast }$ (29.8%)	0.45 ± 0.03${}^{\ast }$ (47.67%)	0.52 ± 0.04${}^{\ast \ast \ast }$ (45.2%)	0.38 ± 0.08${}^{\ast \ast \ast }$ (75.4%)
Ethyl acetate extract	100 mg/kg	0.86 ± 0.01 (4.44%)	0.67 ± 0.07 (12.98%)	0.72 ± 0.15 (16.27%)	0.64 ± 0.10${}^{\ast \ast }$ (57.3%)	0.70 ± 0.11${}^{\ast \ast \ast }$ (53.3%)
	200 mg/kg	0.85 ± 0.02 (5.55%)	0.58 ± 0.08 (24.6%)	0.66 ± 0.05 (23.2%)	0.55 ± 0.11${}^{\ast \ast \ast }$ (42.1%)	0.48 ± 0.05${}^{\ast \ast \ast }$ (68.0%)
	300 mg/kg	0.83 ± 0.03 (7.77%)	0.61 ± 0.09 (20.7%)	0.68 ± 0.11 (20.9%)	0.49 ± 0.09${}^{\ast \ast \ast }$ (48.4%)	0.43 ± 0.02${}^{\ast \ast \ast }$ (71.3%)
Ethanolic extract	100 mg/kg	0.87 ± 0.07 (3.33%)	0.64 ± 0.11 (16.8%)	0.73 ± 0.15 (15.1%)	0.61 ± 0.07${}^{\ast \ast }$ (35.7%)	0.50 ± 0.06${}^{\ast \ast \ast }$ (66.6%)
	200 mg/kg	0.87 ± 0.06 (3.33%)	0.59 ± 0.11 (23.3%)	0.67 ± 0.100 (22.0%)	0.5733 ±0 .13051${}^{\ast \ast \ast }$ (26.0%)	0.4633 ±0 .06028${}^{\ast \ast \ast }$ (69.3%)
	300 mg/kg	0.84 ± 0.04 (6.66%)	0.57 ± 0.15 (26.0%)	0.63 ± 0.08 (18.1%)	0.56 ± 0.06${}^{\ast \ast \ast }$ (41.0%)	0.40 ± 0.02${}^{\ast \ast \ast }$ (73.3%)

Values are expressed as mean ± standard deviation. Significance is shown as ${}^{\ast }$significant, ${}^{\ast \ast }$more significant, and ${}^{\ast \ast \ast }$highly significant.

Table 11

Antispasmodic activity of Ziziphus mauritiana var. spontanea and Oenothera.

Treatment	Ziziphus mauritiana var. spontanea				Oenothera biennis L.
	Dose	The mean length of the intestine	Mean distance travelled by charcoal	Percent inhibition (%)	The mean length of the intestine	Mean distance travelled by charcoal	Percent inhibition (%)
Normal saline + castor oil	10 ml/kg	55.2	46.06 ± 2.51	16.6	55.2	46.06 ± 2.51	16.6
Atropine sulfate	10 mg/kg	54.3	20.60 ± 0.70${}^{\ast \ast \ast }$	62.0	54.3	20.60 ± 0.70${}^{\ast \ast \ast }$	62.0
Ethyl acetate extract	100 mg/kg	50.6	34.63 ± 3.61	31.6	49.5	36.63 ± 10.66	26.0
	200 mg/kg	51.3	29.13 ± 3.82${}^{\ast }$	43.2	54.3	30.80 ± 4.96${}^{\ast }$	43.2
	300 mg/kg	52.3	18.65 ± 0.21${}^{\ast \ast \ast }$	64.4	54.6	17.90 ± 2.26${}^{\ast \ast \ast }$	67.2
Ethanolic extract	100 mg/kg	52.5	37.3 ± 3.70	28.9	53.2	34.80 ± 15.39	34.5
	200 mg/kg	49.4	29.3 ± 2.72${}^{\ast }$	40.6	54.4	32.30 ± 9.83	40.6
	300 mg/kg	54.4	23.56 ± 1.85${}^{\ast \ast }$	56.8	52.2	25.70 ± 2.64${}^{\ast \ast }$	50.7

Values are expressed as mean ± standard deviation. Significance is shown as ${}^{\ast }$significant, ${}^{\ast \ast }$more significant, and ${}^{\ast \ast \ast }$highly significant.

4. Discussion

For peripherally acting drugs, the acetic acid-induced abdominal constriction test is used. The pain induction occurs by releasing endogenous substances and other pain mediators such as prostaglandins [28]. The dosage of 300 mg/kg of ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis significantly ($p<0.001$) inhibited the writhing’s paralleled to diclofenac sodium ($p<0.001$). The ethanolic extract of Z. mauritiana was more effective in inhibiting the writhing response which showed comparatively higher percent inhibition 72.6% in writhing than the standard drug diclofenac sodium (68.5%) (Table 6). The results indicated that the reduction in pain was dose-dependent; hence, the 300 mg/kg dose proved to be most effective. These results are in line with [29] who reported effective analgesic results from methanolic extracts of Phyllanthus seeds. Pain inhibitory activity of plant extracts may be interrelated to the inhibition of prostaglandin synthesis [30]. The analgesic activity of Z. mauritiana and O. biennis might be due to several bioactive constituents such as terpenoids, flavonoids, tannins, alkaloids, and steroids which were detected in plant extracts. Flavonoids showed analgesic action by increasing the endogenous serotonin level or its interaction with 5-HT2A and 5-HT3 receptors. Acetic acid-induced writhing has been connected with upgraded levels of prostaglandin (PGE2 and PGF2α) in the peritoneal liquids and the lipoxygenase items [31]. The ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis possibly showed analgesic action by inhibiting the synthesis of the arachidonic acid metabolite. The hot-plate test has been used for the assessment of centrally mediated analgesic responses, which emphasizes mostly changes above the spinal cord level. The findings revealed that after 90 min at 200 mg/kg and 300 mg/kg, the ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis significantly ($p<0.001$) increased in the reaction time (Tables 7 and 8). The effect increased with an increase in time and dose, and a greater effect was attained after 90 min at a higher dose. The ethanolic extract of O. biennis was more effective which showed a maximum percent increase (59.5%) in reaction time which was comparatively higher than the standard drug diclofenac sodium which showed a 57.2% percent increase in reaction time. These results are in line with [32] who reported similar results from the Moroccan medicinal plants. The Z. mauritiana and O. biennis showed antinociceptive activity in the hot-plate test by increasing the latency to discomfort. This action could be stimulating the periaqueductal gray matter to release endogenous peptides (endorphins or encephalins) [33]. These endogenous peptides run down the spinal cord and at the synapse in the dorsal horn and function as inhibitors of pain impulse transmission. Z. mauritiana and O. biennis showed central analgesic activity due to their action on the central opioid receptors or promoted release of endogenous opioid peptides. The analgesic activity of Z. mauritiana and O. biennis might be due to several secondary metabolites such as tannins, flavonoids, steroids, alkaloids, and terpenoids which were detected in plant extracts (Table 1). Flavonoids are involved in the management of pain by increasing the quantity of endogenous serotonin or by interacting with various receptors. Alkaloids have also been associated with the ability to inhibit pain perception [34, 35].

Anti-inflammatory drugs can be tested by the carrageenan-induced inflammation model [36]. Acute inflammation is produced in the rat paw by the subcutaneous injection of carrageenan, and certain mediators including histamine, prostaglandin, and serotonin are released causing fever and pain. The findings indicated that ethyl acetate and ethanolic extract (100, 200, and 300 mg/kg) of Z. mauritiana and O. biennis at the 5th hour exhibited significant inhibition ($p<0.001$) in the volume of paw but less than the diclofenac sodium ($p<0.001$) (Tables 9 and 10). The high percent inhibition (73.3%) in paw edema was caused by ethanolic extract of O. biennis at the 5th hour at 300 mg/kg when compared to Z. mauritiana but less than the standard drug diclofenac sodium (75.4%). The ethanolic extract significantly suppressed edema in 1st phase, which might primarily be credited to the drop in the release and synthesis of serotonin and histamine. The anti-inflammatory activity shown by both plants was dose-dependent and time-dependent revealing similar dose-dependent and time-dependent anti-inflammatory activity from the ethanolic extract and ethyl acetate of Albizia lebbeck (L.) Benth. and Senna sophera (L.) Roxb. Several secondary metabolites such as steroids, tannins, flavonoids, terpenoids, and alkaloids might be responsible for the anti-inflammatory activity of Z. mauritiana and O. biennis (Table 1). Flavonoids inhibit significant enzymes involved in the biosynthesis of tissue activators, especially prostaglandins and arachidonic acid [37]. The triterpenes possess anti-inflammatory potentials and prevent the production of inflammatory mediators [38].

The effect of drugs on peristaltic movement can be tested by charcoal meal test [21]. The irritation and inflammation of intestinal mucosa can result from the hydrolysis of castor oil into ricinoleic acid which results in diarrhea. It results in the release of prostaglandins which provoke gastrointestinal motility and result in secretion of water and electrolytes [22]. The result showed that ethyl acetate ($p<0.001$) and ethanolic extract ($p<0.01$) of Z. mauritiana and O. biennis at a dose of 300 mg/kg showed a significant reduction in the distance travelled by charcoal meal when compared with atropine sulfate which also showed significant ($p<0.001$) reduction in charcoal meal movement (Table 11). The extract showed a dose-dependent inhibition of charcoal meal motility. Thus, 300 mg/kg doses of both plants’ extracts had more antimotility effect. The ethyl acetate extract of Z. mauritiana and O. biennis was more effective and showed higher activity than atropine sulfate. However, the maximum antimotility effect was shown by the ethyl acetate extract of O. biennis than the ethyl acetate extract of Z. mauritiana. The inhibition in the peristaltic movement of the gastrointestinal tract might be due to the inhibition of acetylcholine by the plant extracts resulting in the absorption of water and electrolytes [39]. The antispasmodic activity of Z. mauritiana and O. biennis might be due to the presence of secondary metabolites such as flavonoids, alkaloids, steroids, phenol, terpenoids, phytosterol, and tannins which were detected in plant extracts (Table 1). Tannins present in the extract form protein tannates by precipitating the proteins in the intestinal mucosa which helps in the protection of intestinal mucosa by making it more resistant to certain chemicals [40]. Flavonoids and steroids help in absorbing electrolytes by inhibiting secretion induced by castor oil. Alkaloids and terpenoids are known to inhibit the secretion induced by castor oil by inhibiting the release of autocoids and prostaglandin [41, 42].

5. Conclusion

In the current research, both plants showed important phytochemicals and therapeutic potential. From the results, it can be concluded that Z. mauritiana and O. biennis contained important chemical constituents including alkaloids, tannins, saponins, flavonoids, steroids, and triterpenoids as determined by phytochemical screening. GCMS revealed the presence of pharmacologically active compounds such as hexadecanoic acid, 4-vinyl-2-methoxy-phenol, n-eicosane, and 2,3,6-trimethyldecane which may be responsible for the significant anti-inflammatory, analgesic, and antispasmodic activity. Hence, these plants can be used for alleviating pain and treating various inflammatory and diarrhoeal disorders. Both plants can be used in the future for drug development and various herbal formulations having fewer side effects.

Disclosure

The authors declare that they have no conflict of interest.

Authors’ Contributions

A.A. administrated the project and proposed methodology, M.A. wrote the original draft, and M.N. edited, supervised, and visualized the study. F.Z.F. developed software, collected data, and provided funding.

Acknowledgments

The authors are highly grateful for receiving support from the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Grant No. IFPRC-187-130-2020.

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