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

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Introduction
Medicinal plants are naturally God-gifted with a lot of chemical compounds that are formed in diferent plant parts and used in the treatment of various ailments.Tese chemical compounds are also known as secondary metabolites.Tese secondary metabolites can be utilized in the formulations of novel medications and have been reported as very efective in curing serious health problems.Te 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 efectiveness and easy availability [1].Advancements in biotechnology have made it possible to manufacture therapeutic protein drugs in signifcant 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.Te medicinal properties of plants are due to the presence of bioactive compounds such as saponins, glycosides, quinones, alkaloids, and favonoids [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 signifcantly altering consciousness.Te use of synthetic analgesic drugs has several side efects including gastrointestinal disorders, bleeding, and ulcers [7].A physiological response that protects a body from tissue injury is called infammation.Tere are two types of infammation, i.e., acute infammation and chronic infammation [8].Te nonsteroidal anti-infammatory drugs are used to treat infammation but it has a lot of side efects, including gastrointestinal and cardiovascular complications.Terefore, the development of new drugs is necessary for the management of pain and infammation.Plant-based beverages typically contain advantageous bioactive compounds, including favonoids, phenolic acids, lignans, and phytosterols, known for their exceptional antioxidant, analgesic, and anti-infammatory properties, contributing signifcantly to health benefts [9,10].In the past twenty years, numerous research studies have underscored the pivotal role of the gut microbiome in infuencing health and disease.Alterations in the composition of the intestinal microbiome have been linked to diverse intestinal and metabolic disorders, including infammatory bowel disease, diabetes, and obesity [11].
An imbalance in the gut microbiota plays a crucial role in the pathogenesis of functional Dyspepsia (FD).Tis imbalance disrupts the intestinal environment, ultimately causing a decrease in benefcial probiotics and triggering a range of acute and chronic diseases [12].Te condition of increased stool frequency, liquidity, or volume is called diarrhea.It mainly afects neonates and babies and is a signifcant cause of illness and mortality in developing countries [13].Medicinal plants are reservoirs of essential antidiarrheal bioactive constituents without any side efects 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.Te seed oil of O. biennis is used for the treatment of several ailments such as asthma, eczema, breast problems, rheumatoid arthritis, menopausal syndrome [19] fstulas, and lung disease [20].Te present study will analyze the plant in terms of drug standardization and evaluating its pharmacological efect which can be used as a potential candidate for future drug developments.

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.Te identifcation 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).

Extraction of Plant Material and Sample Preparation.
Whole plants were washed with distilled water, shade dried in the air dryer, and ground into a fne 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 fltration through flter paper.Te extract was concentrated using a rotary evaporator (RE-100D Phoenix) supplied by MED Lab Services.Te 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].

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 Termo GC-Trace Ultra version 5.0 with Termo MS DSQ II which was supplied by MED Lab Services.Te 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).Te 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.Te splitless mode was used to introduce the particle-free, diluted sample (10 mL/min split fow and 1 min spitless period).Helium was used as the carrier gas at a constant fow 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 identifed [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 modifcations.Mice were divided into fve groups containing fve 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 fve mins of postinjection; the number of writhing was counted for the next 20 min.Te percent analgesic efect was calculated by the following formula: % analgesic effect � 100 − no of writhing in tested animals no of writhing in control animals X 100. (1) 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 diferent concentrations (100, 200, and 300 mg body weight) of ethanolic and ethyl acetate extracts, respectively.Te 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 posttreatment reaction time of each animal after the administration of plant extracts recorded at 30 min, 60 min, and 90 min.
Te sample was prepared by adding 10 mg crude extract in 25 mL of respective solvents.Animals were divided into 5 groups comprising fve animals per group.In all groups, acute infammation was produced by subplantar injection of 0.1 ml freshly prepared 1% suspension of carrageenan.Te 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.Te 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: where Vt � increase in paw volume in mice treated with test compounds and Vc � increase in paw volume in the control group of mice.

Antispasmodic Activity.
Te antispasmodic activity of Z. mauritiana and O. biennis ethanolic and ethyl acetate extracts was carried out by adopting the method of [27].Te sample was prepared by adding 10 mg crude extract in 25 mL of respective solvents.Te selected mice were divided into four groups of fve mice each.At frst, 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 sacrifced, 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.
Journal of Food Quality where D � distance covered by charcoal (in meters) and L � intestinal length (in meters).1).

Writhing Method.
Te ethanolic and ethyl acetate extract of Z. mauritiana and O. biennis exhibited signifcant (p < 0.001) analgesic activity when measured by acetic acidinduced writhing inhibition method at a dose of 300 mg/kg body (Table 6).Te 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%).Te ethanolic extract of Z. mauritiana was more efective than O. biennis extracts.

Hot-Plate Method.
Te ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis showed a signifcant (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 efects, and a signifcant result was obtained after 90 min at 300 mg/kg comparable with diclofenac sodium.Te 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.Te highest central analgesic efect was shown by the ethanolic extract of O. biennis which was higher than diclofenac sodium.

Charcoal Meal Test.
Te ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis signifcantly (p ≤ 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).Te 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.

Discussion
For peripherally acting drugs, the acetic acid-induced abdominal constriction test is used.Te pain induction occurs by releasing endogenous substances and other pain mediators such as prostaglandins [28].Te dosage of 300 mg/kg of ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis signifcantly (p < 0.001) inhibited the writhing's paralleled to diclofenac sodium (p < 0.001).Te ethanolic extract of Z. mauritiana was more efective 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).Te results indicated that the reduction in pain was dose-dependent; hence, the 300 mg/kg dose proved to be most efective.Tese results are in line with [29] who reported efective analgesic results from methanolic extracts of Phyllanthus seeds.Pain inhibitory activity of plant extracts may be interrelated to the inhibition of prostaglandin synthesis [30].Te analgesic activity of Z. mauritiana and O. biennis might be due to several bioactive constituents such as terpenoids, favonoids, 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].Te ethyl acetate and ethanolic extract of Z. mauritiana and O. biennis possibly showed analgesic action by inhibiting the synthesis of the arachidonic acid metabolite.Te hot-plate test has been used for the assessment of centrally mediated analgesic responses, which emphasizes mostly changes above the spinal cord level.Te fndings 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 signifcantly (p < 0.001) increased in the reaction time (Tables 7 and 8).Te efect increased with an increase in time and dose, and a greater efect was attained after 90 min at a higher dose.Te ethanolic extract of O. biennis was more efective 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.Tese results are in line with [32] who reported similar results from the Moroccan medicinal plants.Te Z. mauritiana and O. biennis showed antinociceptive activity in the hot-plate test by increasing the latency to discomfort.Tis action could be stimulating the periaqueductal gray matter to release endogenous peptides (endorphins or encephalins) [33].Tese 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.Te analgesic activity of Z. mauritiana and O. biennis might be due to several secondary metabolites such as tannins, favonoids, 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-infammatory drugs can be tested by the carrageenan-induced infammation model [36].Acute infammation 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.Te fndings indicated that ethyl acetate and ethanolic extract (100, 200, and 300 mg/kg) of Z. mauritiana and O. biennis at the 5th hour exhibited signifcant inhibition (p < 0.001) in the volume of paw but less than the diclofenac sodium (p < 0.001) (Tables 9 and 10).Te 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%).Te ethanolic extract signifcantly suppressed edema in 1st phase, which might primarily be credited to the drop in the release and synthesis of serotonin and histamine.Te anti-infammatory activity shown by both plants was dose-dependent and timedependent revealing similar dose-dependent and timedependent anti-infammatory activity from the ethanolic

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Journal of Food Quality     1).Flavonoids inhibit signifcant enzymes involved in the biosynthesis of tissue activators, especially prostaglandins and arachidonic acid [37].Te triterpenes possess anti-infammatory potentials and prevent the production of infammatory mediators [38].Te efect of drugs on peristaltic movement can be tested by charcoal meal test [21].Te irritation and infammation 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].Te 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 signifcant reduction in the distance travelled by charcoal meal when compared with atropine sulfate which also showed signifcant (p < 0.001) reduction in charcoal meal movement (Table 11).Te extract showed a dose-dependent inhibition of charcoal meal motility.Tus, 300 mg/kg doses of both plants' extracts had more antimotility efect.Te ethyl acetate extract of Z. mauritiana and O. biennis was more efective and showed higher activity than atropine sulfate.However, the maximum antimotility efect was shown by the ethyl acetate extract of O. biennis than the ethyl acetate extract of Z. mauritiana.Te 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].Te antispasmodic activity of Z. mauritiana and O. biennis might be due to the presence of secondary metabolites such as favonoids, 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].12 Journal of Food Quality Journal of Food Quality

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, favonoids, 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 signifcant anti-infammatory, analgesic, and antispasmodic activity.Hence, these plants can be used for alleviating pain and treating various infammatory and diarrhoeal disorders.Both plants can be used in the future for drug development and various herbal formulations having fewer side efects.
Approval.Te 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.Te 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 signifcant.
3.1.Phytochemical Screening.Te phytochemical screening of ethanolic and ethyl acetate extracts of Z. mauritiana and O. biennis revealed the existence of diferent bioactive compounds.Te ethanolic extract of Z. mauritiana showed the presence of alkaloids, favonoids, tannins, steroids, and triterpenoids whereas saponins were found absent.Similarly, the ethyl acetate extracts of Z. mauritiana detect alkaloids, favonoids, saponins, tannins, and triterpenoids while steroids were absent.Likewise, the ethanolic extract of O. biennis revealed the presence of alkaloids, favonoids, saponins, and steroids and the absence of tannins and triterpenoids.Te ethyl acetate extract of O. biennis unveiled the presence of alkaloids, favonoids, tannins, steroids, and triterpenoids and the absence of tannins (Table
S. NoName of the compound Compound formula Retention time (min) Peak area (%)

Table 4 :
GCMS analysis of ethanol extract of Oenothera biennis.

Table 5 :
GCMS analysis of ethyl acetate extract of Oenothera biennis.

Table 8 :
Analgesic activity of Oenothera biennis L. by hot-plate method.