Comprehensive GC-MS Profiling and Multi-Modal Pharmacological Evaluations of Haloxylon griffithii: In Vitro and In Vivo Approaches
by
, , , , ,
Iram Iqbal
1,2
,
Mohamed A. M. Ali
3,
Fatima Saqib
1,*,
Kinza Alamgir
4,
Mohammad S. Mubarak
5
,
Anis Ahmad Chaudhary
3,
Mohamed El-Shazly
6 and 
Heba A. S. El-Nashar
6,* 1
Department of Pharmacology, Faculty of Pharmacy, Bahauddin Zakariya University, Multan 60800, Pakistan
2
Health & Population Department, Government of the Punjab, Lahore 54000, Pakistan
3
Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh 11623, Saudi Arabia
4
Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad 38000, Pakistan
5
Department of Chemistry, The University of Jordan, Amman 11942, Jordan
6
Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2025, 18(6), 770; https://doi.org/10.3390/ph18060770
Submission received: 4 May 2025
/
Revised: 15 May 2025
/
Accepted: 20 May 2025
/
Published: 22 May 2025
(This article belongs to the Special Issue Promising Natural Products in New Drug Design and Therapy)
Abstract
:Background/Objectives: Haloxylon griffithii is a medicinal plant possessing therapeutic effects in disorders associated with the gastrointestinal (GIT) system. This research aims to study the pharmacological activity of Haloxylon griffithii in a multidimensional manner, involving phytochemistry screening and in vitro and in vivo experiments. Methods: The whole dried plant was extracted with 80% methanol and further fractionation using solvents of increasing polarity. GC-MS analysis was performed on the crude extract to discover volatile compounds. The spasmolytic/spasmogenic effect was assessed in isolated rabbit jejunum using spontaneous and K⁺-induced contractions, as well as contractions induced by increasing concentrations of calcium ions in depolarized tissue. Antidiarrheal activity was evaluated in Swiss albino rats/mice (n = 6/group) using castor oil-induced diarrhea and peristaltic index models. In silico ADMET screening was conducted via SwissADME and pkCSM. Results: The GC-MS profiling of H. griffithii revealed the presence of 59 phytochemicals and a rare azulene derivative and constituents, including α-santonin and hexadecanoic acid esters, with favorable pharmacokinetic profiles, as predicted using SwissADME and pkCSM computational tools. The in vitro and in vivo experiments revealed the significant calcium channel blocking activity in non-polar fractions (n-hexane and ethyl acetate), while the polar extracts (ethanolic, aqueous) exhibited cholinergic effects, indicating a dual mode of action. Conclusions: This was a first-time demonstration of both antidiarrheal and smooth muscle-relaxant activity in H. griffithii, supported by GC-MS profiling and pharmacological assay. The findings lend scientific credibility to the traditional use of the plant in community healthcare, while also reinforcing the need for further pharmacological and clinical studies to explore its potential in drug development.
1. Introduction
Natural products have long been a cornerstone in traditional healing practices and modern therapeutic approaches. Scientists today are increasingly focused on understanding how plant-based medicines exert their effects at the molecular level. Nature remains a rich source of powerful and diverse chemical compounds, offering immense value to drug discovery. However, defining the specific actions of these substances remains difficult, mainly due to their complex activity and tendency to influence multiple biological targets [1].
Recent technological advancements have greatly facilitated the study and utilization of medicinal plants [2,3,4,5]. These methods enable rapid screening and modeling of functional compounds, accelerating the identification of potential therapeutic agents by predicting interactions between plant-derived compounds and biological targets [6,7].
Pakistan’s diverse and hospitable climate makes it a rich reservoir of medicinal plants, positioning the country as a major player in the global herbal and pharmaceutical industries. Out of the 6000 known medicinal plant species worldwide, Pakistan is home to approximately 400 to 600, highlighting its significant role in offering natural remedies for various health conditions [8]. Haloxylon griffithii has drawn particular attention for its pharmacological potential among Pakistan’s medicinal flora. Predominantly found in Northern Baluchistan, this plant is rich in secondary metabolites, contributing to its antioxidant, antimicrobial, and anti-inflammatory properties [9,10]. Its bioactive compounds, including flavonoids, alkaloids, and terpenoids, are crucial for developing therapeutic agents while enhancing plant resilience against pathogens and environmental stressors. Traditionally, H. griffithii has been used to treat gastrointestinal disorders, with studies supporting its role in modulating gut microbiota and reducing gastrointestinal motility [9,10]. Additionally, published research has demonstrated its antimicrobial activity against pathogenic bacteria like Escherichia coli and Staphylococcus aureus, both responsible for gastrointestinal infections. Additionally, phenolic constituents enhance its antioxidant potential, further validating its use in inhibiting oxidative stress and inflammation in the GIT. Scientific evidence also indicates that different extracts of H. griffithii possess anti-urease action, which is beneficial for controlling ailments such as peptic ulcers caused by Helicobacter pylori infection [11].
Despite the widespread use of medicinal plants, a significant gap remains in the scientific evaluation of their biological and pharmacological properties [8,12,13]. It is estimated that fewer than 20% of medicinal plants have been rigorously studied, highlighting an urgent need for comprehensive research [5,12,13,14,15]. Validating the efficacy and safety of these plants through pre-clinical and clinical studies is essential for integrating traditional remedies into modern medicine [16,17]. Expanding research efforts will allow underexplored plants like Haloxylon griffithii to be fully utilized, paving the way for novel drug discoveries and improved global health outcomes. Based on the previous discussion, the present work aimed to identify H. griffith’s medicinal relevance and how it can be applied in pharmaceutical formulations. As little information is available about this plant, our research will focus on screening it for its effect on the GIT and the mechanisms involved.
2. Results
2.1. GC-MS Analysis
The prime phytochemical components of the hydroethanolic extract were evaluated using GC-MS. The chromatogram shows the presence of 59 compounds in a given run time (Figure 1). Based on a NIST.20L and DEMO.L database comparative analysis of the compounds with a hit quality above 60, 23 components, listed in Table 1 and Table A2, were identified as major components. The complete list of all 59 identified compounds, including their retention times, peak areas, and spectral match scores, is provided in Table A1 for transparency and reference. Several compounds identified in this study have previously been unreported in H. griffithii, representing a novel contribution to its phytochemical profile. Notably, the detection of o-cymene and 2,4-di-tert-butylphenol, a potent antioxidant commonly found in other medicinal plants [6], represents a novel addition to the phytochemical repertoire of H. griffithii. Furthermore, the identification of a rare azulene derivative, ((3aR,4R,7R)-1,4,9,9-tetramethyl-3,4,5,6,7,8-hexahydro-2H-3a,7-methanoazulen-2-one) was also worth mentioning. The presence of complex aromatic hydrocarbons such as benzene, 1,2,4,5-tetrakis(1-methylethyl)- and benzene, hexaethyl- further emphasizes the chemical richness of the extract. This is the first documentation of these specific compounds in H. griffithii, underscoring the plant’s untapped potential and supporting its ethnomedicinal use in gastrointestinal disorders.
2.2. In Silico Models
ADME Profiling of GC-MS-Identified Compounds of H. griffithii
The in silico ADMET assessment of the phytochemicals derived from a GC-MS analysis of Haloxylon griffithii revealed a diverse array of pharmacokinetic and toxicity characteristics. Only compounds that satisfied essential criteria such as good GIT absorption, non-inhibition of major metabolic enzymes, and compliance with drug-likeness rules (e.g., Lipinski, Veber) were retained for detailed profiling (Table 2). Some compounds like hexadecanoic acid ethyl ester, its methyl counterpart, and n-hexadecanoic acid showed strong GIT absorption, lacked P-glycoprotein interactions, and favorable clearance parameters, highlighting their potential for effective oral delivery. In contrast, more lipophilic substances, such as (E)-9-octadecenoic acid ethyl ester and bis(2-ethylhexyl) phthalate, were obvious from the elevated total clearance rates (log mL/min/kg > 1.8). However, some were limited by poor GI absorption and multiple violations across the drug-likeness filters like Lipinski and Veber. In this context, α-santonin and o-cymene appeared as the most favorable in terms of drug-likeness, meeting all criteria set by Lipinski, Veber, and Egan rules and exhibiting favorable blood–brain barrier permeability, thus suggesting possible CNS-targeted activity. Meanwhile, benzene and hexamethyl combined good lipophilicity and broad tissue distribution but labeled potential hepatotoxicity, demanding more in-depth toxicological profiling. Positively, many of these compounds did not interfere with key CYP450 enzymes, indicating a low risk of metabolic interactions.
2.3. In Vitro Experiments
Effects of H. griffithii Extracts on Isolated Tissues of Rabbit Jejunum
The effects of crude (Hg.Cr), n-hexane (Hg.Hex), ethyl acetate (Hg.EA), ethanol (Hg.OH), and aqueous (Hg.Aq) extracts of H. griffithii were tested in spontaneously contracting jejunum preparations. Hg.Cr, Hg.Hex, and Hg.EA showed spasmolytic action by inhibiting the spontaneous rhythmic contraction in a dose-dependent way, with EC50 values of 0.65 mg/mL (0.29 to 1.61; 95% CI), 0.094 mg/mL (0.040 to 0.25; 95% CI), and 1.83 mg/mL (0.52 to 18.1; 95% CI), respectively. The results are comparable to verapamil, which has a spasmolytic action with an EC50 value of 0.43 mg/mL (0.30 to 0.65; 95% CI). On the contrary, when exposed to rhythmic or periodic jejunal contractions, Hg.OH and Hg.Aq exhibited dose-dependent contractility (Figure 2). The spasmogenic effects of Hg.OH and Hg.Aq were diminished, and a mild dose-dependent spasmolytic effect was seen in an atropine (1 µM)-pretreated jejunum preparation. In addition, the Hg.Cr. extract exhibited a dose-dependent inhibition to the sustained contractions of K+ (80 mM), with an EC50 of 0.47 mg/mL (0.30 to 0.77; 95% CI). Hg.Hex also inhibited the K+ (80 mM)-induced contraction in a dose-dependent manner, with an EC50 of 0.14 mg/mL (0.091 to 0.20; 95% CI), and Hg.EA also caused inhibition of K+ (80 mM)-induced contraction, with an EC50 of 0.41 mg/mL (0.18 to 0.96; 95% CI). These results are comparable to verapamil, which caused relaxation of K+ (80 mM)-induced contraction, with an EC50 of 0.038 mg/mL (0.024 to 0.060; 95% CI). Both Hg.OH and Hg.Aq did not cause relaxation in high K-induced contractions (Figure 2).
Concentration response curves (CRCs) of calcium in the absence and presence of extract were constructed to further confirm the calcium channel antagonism response of the extracts Hg.Cr, Hg.Hex, and Hg.EA. All these extracts caused the rightward shifting with suppression of calcium CRCs at 0.1 and 3 mg/mL (Figure 3).
2.4. In Vivo Experiments
2.4.1. Antidiarrheal Activity
Results from this investigation showed that Hg.Cr causes a significant decrease in the number of wet feces drops in mice (Table 3, Figure 4). At higher doses, Hg.Cr extract in mice caused significant anti-diarrheal effects compared to the disease control group. Hg.Cr 100 mg/kg, 200 mg/kg, and 400 mg/kg showed 34.4, 38.1, and 68.7% protection, respectively (Table 4, Figure 5).
2.4.2. Effect of H. griffithii on Charcoal Meal GIT Transit Time
The control group establishes the baseline with a 100% peristaltic index, representing normal GI motility. Loperamide caused a significant drop (p < 0.01) in the peristaltic index. Verapamil also significantly (p < 0.01) reduced the peristaltic index compared to the control. Hg.Cr doses of 200 and 400 mg/kg significantly (p < 0.01 to p < 0.001) reduced the peristaltic index, suggesting significant activity of the crude extract. In contrast, Hg.Hex caused significant drops (p < 0.05 to p < 0.01), especially at the higher dose (400 mg/kg). This suggests potent activity due to nonpolar bioactive compounds in the n-hexane fraction. In the Hg.EA, significant effects were noted at 200 mg/kg (p < 0.01). However, no significant reduction was observed at 400 mg/kg. This may indicate a plateau effect or reduced bioavailability at higher doses. On the other hand, Hg.EtOH caused significant reductions at 200 mg/kg (p < 0.01) but became nonsignificant (NS) at 400 mg/kg, similar to Hg.EA. Finally, Hg.Aq showed no significant effect at 200 mg/kg, while a mild reduction (p < 0.05) was observed at 400 mg/kg (Figure 6).
3. Discussion
The genus Haloxylon was traditionally used as medicine among local healers [9]. Many plant-derived compounds are potential medicinal agents based on their pharmacokinetic properties. These compounds can be screened through ADMET analysis [18]. Haloxylon griffithii extract contains several bioactive phytochemicals, identified through GC-MS analysis (Table 1). Numerous investigations revealed that the action of medicinal herbs is predominantly due to their Ca++ antagonistic action, and multiple pathologies can be treated through specific interactions between bioactive molecules and their respective target proteins [19,20].
To validate our hypothesis regarding the antidiarrheal potential of H. griffithii via calcium channel blockade, we conducted a broad series of both in vitro and in vivo experiments. The GIT motor tone is regulated by various physiological mediators responsible for the gut’s stimulatory action. Among them, an increase in the cytosolic calcium levels, either by the extracellular influx of calcium or the release of stored calcium in the cytosol of the endoplasmic reticulum, induces membrane depolarization and spontaneous contractions of the Jejunum preparation. Any substance interrupting this process is a confirmed antispasmodic [21,22] and therapeutically valuable for managing hyperactive gastrointestinal conditions [19]. Interestingly, in our in vitro experiments, the results for polar (ethanolic and aqueous) and non-polar (n-hexane and ethyl acetate) extracts of H. griffithii were opposite.
The crude H. griffithii extract and non-polar extracts (n-hexane and ethyl acetate) showed significant spasmolytic activity, supporting calcium antagonism, as evidenced by inhibition of high-K⁺-induced jejunal contractions (Figure 2) and a rightward shift in calcium CRCs (Figure 3). Findings showed that high doses of K+ activate voltage-dependent Ca++ channels, causing a flooding of free Ca++ in the cytosol and a profound depolarization of the action potential of the membrane, causing a protracted contraction of a long duration [23]. Reversal of this depolarization causes the relaxation of smooth muscles [24], and substances that inhibit smooth muscle contractions caused by K+ (80 mM) levels appear to impede Ca++ influx into the cytosol [23]. In this context, previous research has established that all Ca++ channel blockers exhibit the characteristic of blocking calcium’s slow entry, which can be reversed when Ca++ is introduced [25]. In our study, pretreatment with the extracts followed by the formation of calcium CRCs led to inhibition at doses of 1 and 3 mg/mL, producing a rightward parallel shift in the jejunum tissue preparation, like the effect of verapamil at doses of 0.1 and 0.3 μM [19] (Figure 3). These effects were twinned with those of verapamil, a standard L-type calcium channel blocker. In contrast, polar extracts (ethanolic and aqueous) produced dose-dependent spasmogenic effects. Interestingly, the spasmogenic properties of both ethanolic and aqueous extracts were blocked entirely by atropine (1 µM), pointing to cholinergic compounds likely acting through muscarinic receptor pathways (Figure 2) [26]. This duality highlights the presence of both excitatory and inhibitory phytoconstituents within H. griffithii—a notable strength of the study, offering a mechanistic explanation for its traditional use in both diarrheal and cramping conditions.
In an in vivo antidiarrheal model, the extract reduced the frequency and severity of diarrhea, delayed its onset, and exhibited a dose-dependent inhibition of wet stool production at 100, 200, and 400 mg/kg doses compared to the saline-treated group, where protection was absent (Figure 4). Castor oil, upon hydrolysis, forms ricinoleic acid, altering the transportation of electrolytes and water. This interruption leads to contractions in the gastrointestinal tract [27]. Studies show that a potential anti-diarrheal candidate may show its potential by inhibiting bowel contraction by acting on μ-opioid receptors [28] or reducing smooth-muscle contraction [29]. The antidiarrheal effect of the H. griffithii extract can be credited to multiple components present in the plant, which have an inhibitory action. In this regard, bioactive compounds such as flavonoids, alkaloids, and saponins inhibit the secretion and motility induced by ricinoleic acid [30,31].
The peristaltic index, used to quantify gut motility [32], further validated these effects. Non-polar fractions significantly reduced intestinal transit, similar to loperamide and verapamil. The crude extract (Hg.Cr) exhibited the most significant decrease in peristaltic index, indicating a synergistic effect of multiple phytochemicals (Figure 6A). Among all fractions, hexane extract (Hg.Hex) demonstrated notable activity (Figure 6B), possibly due to nonpolar compounds substantially contributing to the antidiarrheal effect. The ethyl acetate (Hg.EA) and ethanolic (Hg.EtOH) extracts demonstrated significant reductions in GI motility at lower doses. However, at higher concentrations, their reduced efficacy (Figure 6C,D) suggests possible complex pharmacokinetic interactions requiring further investigation. In contrast, the aqueous extract (Hg.Aq) demonstrated the least activity, indicating that water-soluble compounds in Haloxylon griffithii may be less prominent in influencing gastrointestinal motility. These results are correlated with in vitro studies (Figure 2), where atropine-pretreated jejunal tissues did not produce any relaxation after administration of Hg.Aq. The observed reductions in the peristaltic index across various Haloxylon griffithii extracts suggest potential antidiarrheal properties, likely due to bioactive compounds such as alkaloids, flavonoids, and tannins. Phytochemical analyses have confirmed the presence of these constituents in H. griffithii, supporting their role in modulating gastrointestinal motility [33].
These findings agree with studies highlighting the pharmacological potential of the Haloxylon genus, which includes species traditionally used to treat gastrointestinal disorders [34]. Our findings align with previous research on other medicinal plants whose bioactivity is associated with fatty acid esters, azulene derivatives, and monoterpenes, such as those identified in our GC-MS analysis. For instance, α-santonin and linoleic acid esters have previously been reported to exert antispasmodic and anti-inflammatory actions [35], consistent with our data. Hexadecanoic acid methyl ester has been previously reported for its anti-inflammatory, antioxidant, and antimicrobial activities [36]. Similarly, p-cymene has been reported to exhibit gastroprotective effects by enhancing gastric mucus production and reducing oxidative stress [37,38]. It has also demonstrated mild spasmolytic effects, likely mediated through calcium channel modulation and smooth muscle relaxation, supporting its traditional use in relieving intestinal cramps, colic, and spasms [39]. The identification of a rare azulene derivative, ((3aR,4R,7R)-1,4,9,9-tetramethyl-3,4,5,6,7,8-hexahydro-2H-3a,7-methanoazulen-2-one), is new for this species. Azulene derivatives, a structural class to which this compound belongs, are well-known for their spasmolytic and anti-inflammatory properties, often utilized in gastrointestinal and dermatological formulations. This aligns with the findings in pharmacological research on azulenes and related sesquiterpenes, such as bakkenolide and guaiazulene, which exhibit similar bioactivities [40], suggesting potential relevance to the plant’s traditional gastrointestinal applications.
One of the strengths of this study is the integration of multiple in vitro and in vivo experimental models: isolated jejunum relaxation, calcium concentration–response curves, castor oil-induced diarrhea, and peristaltic index assessment. These models collectively confirmed the spasmolytic and antidiarrheal actions of specific extract fractions. Nonetheless, our study has certain limitations. GC-MS profiling is confined to detecting volatile compounds, so it may have missed critical non-volatile phytochemicals, such as alkaloids or glycosides. The bioactivity results are likely due to the combined or synergistic effects of both volatile and non-volatile constituents present in the extract.
4. Materials and Methods
4.1. Identification and Collection of Haloxylon griffithii
A whole specimen of a Haloxylon griffithii plant was obtained in August 2021 in Quetta, Pakistan, and was identified and confirmed by Prof. Dr. Muhammad Zafar, Quaid-e-Azam University, Islamabad. A voucher specimen (215123) was deposited at the Pakistan Herbarium at Quaid-e-Azam University, Islamabad. Identification of the plant was alternatively validated through http://www.theplantlist.org, accessed on 31 August 2021.
4.2. Preparation of Crude Extract and Fractionations
The whole plant of Haloxylon griffithii was shade-dried for 10–12 days, crushed into a coarse powder with a mortar and pestle, and put through a sieve with a 0.3 mm aperture size. For crude extraction, approximately 500 g of the powder was subjected to Soxhlet extraction (65–85 °C) using 80% ethanol as the solvent. The extraction was carried out for 6–8 h until the solvent in the siphon tube appeared colorless, indicating complete extraction. The ethanolic extract was then concentrated under reduced pressure using a rotary evaporator at 40 °C to remove the solvent and obtain the crude extract. The resulting extract was stored at 4 °C in airtight containers for further analysis. A fresh portion of the powdered plant material was subjected to Soxhlet extraction with increasing polarity with n-hexane, ethyl acetate, ethanol, and water. The n-hexane, ethyl acetate, ethanol, and aqueous extracts were concentrated under reduced pressure with a rotary evaporator (BUCHI r-200, Büchi Labortechnik AG, Flawil, Switzerland) at 37 ± 2 °C and then kept in a refrigerator at 4 °C for later use.
4.3. Drugs and Chemicals
Chemicals, reagents, and standards were obtained from commercial sources (in parentheses) and were used as received without further purification. The following chemicals and standards were purchased from the Sigma Chemical Company (St. Louis, MO, USA): NaCl, KH2PO4, C6H12O6, CaCl2, NaH2PO4, KCl, MgSO4, MgCl2, NaHCO3, EDTA, acetylcholine chloride, atropine sulfate, loperamide hydrochloride, verapamil hydrochloride, charcoal, starch, and gum acacia. Castor oil was purchased from the local market.
4.4. Animals
Balb/c mice of either sex (18–25 g), Sprague–Dawley (SD) rats (200–250 g), and locally bred healthy rabbits (1–1.5 kg) of either sex were used throughout this investigation. These animals were kept in 12 h light/dark cycle at ambient room temperature and given free access to food and water at the Animal House of the Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan. The animals were kept under standard conditions and rules of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council [41] after the approval of the Ethical Committee of the Department of Pharmacy, Bahuaddin Zakariya University, Multan, Pakistan.
4.5. GC-MS Analysis
For GC-MS profiling, the crude hydroethanolic extract of Haloxylon griffithii was subjected to 1% dilution (w/v) with HPLC-grade methanol. The resulting extract was filtered with a 0.4-micron syringe filter and subjected to GC-MS analysis without further fractionation. GC-MS analysis of H. griffithii extracts was performed using the Agilent 7890B gas chromatograph (GC) (Agilent Technologies Inc., Santa Clara, CA, USA) coupled with mass selective detector 5977A (MSD), equipped with a DB5 (30 m × 250 µm × 0.25 µm, Agilent J&W, Santa Clara, CA, USA) capillary standard non-polar column. The oven temperature was set at 40 °C for 1 min, then increased to 10 °C per minute until it reached 260 °C, which was maintained for 28 min. The carrier gas (helium) flow rate was 1.2 mL/min, and the injection port temperature was 250 °C. The mass spectral (MS) source and quad temperatures were 230 °C and 150 °C, respectively. A split mode ratio of 10:1 was used for injecting the samples. The 40 to 650 (m/z) MS scan range was chosen for analysis [42]. Compounds were identified by comparing their retention times and mass spectra with those of authentic reference compounds stored in the DEMO.L and NIST20.L Library MS database and tabulated based on the CAS number (https://webbook.nist.gov/, analyzed 27 July 2024).
4.6. In-Silico Models
ADME Profiling of GC-MS-Identified Compounds
Selective bioactive compounds found from GC-MS analysis of H. griffithii were evaluated for absorption, distribution, metabolism, excretion, and toxicity (ADMET) in the “SwissADME” (http://www.swissadme.ch/, accessed on 28 January 2025) [43] and “PkCSM” (http://biosig.unimelb.edu.au/pkcsm/prediction, accessed on 28 January 2025) [44] to evaluate the ADMET and drug-likeness parameters. The ADMET properties, like intestinal absorption, volume of distribution, total renal clearance, ability to inhibit the P-glycoprotein, and hepatotoxicity, were also calculated using “PkCSM”.
4.7. In Vitro Investigation
Effect on Isolated Rabbit Jejunum
Experiments on the rabbit jejunum were carried out according to published procedures [22,24]. The animals were sacrificed and dissected to open the abdomen. After dissecting their abdomens, the jejunum tissues were carefully removed from the mesentery. Approximately 2 to 3 cm-long sections were cut and kept in freshly prepared Tyrode’s solution, which was aerated with carbogen gas (95% oxygen–5% carbon dioxide mixture) to keep them alive and ready for use. Each jejunum preparation was fixed in a 15 mL tissue organ bath maintained at 37 ± 0.5 °C filled with Tyrode’s solution and aerated with carbogen gas. Before test drug administration, a preload tensile strength of 1 ± 0.1 g was applied to each jejunal tissue preparation. Spontaneous recurrent contractions were observed after the jejunal tissue preparation was equilibrated for 15 ± 5 min by replacing Tyrode’s solution every 10 min with fresh Tyrode solution. For tissue response, we used an isotonic transducer (MLT0015, ADInstruments, Sydney, NSW, Australia) in conjunction with a data-accumulating system, Power Lab®® (4/25), and recorded in Lab Chart Pro (Version 8, ADInstruments). The response was calculated based on the percentage of the contraction taken just before adding the test substance to determine whether the extracts exhibited spasmogenic or spasmolytic activity [45].
The antispasmodic effect of the H. griffithii extracts was measured by inducing smooth muscle contractions by a calcium ion channel blockade with high K+ (80 mM KCl). The extracts were then added cumulatively to get a dose-dependent inhibitory response. High K+ (80 mM KCl) causes an influx of calcium ions into the cell, thus changing the cell’s polarity and producing persistent contractions in isolated tissue. The inhibitory effect of the extract on this induced contraction was calculated and designated as the calcium channel-blocking activity of the extract. Concentration-based response curves (CRCs) of calcium were constructed in the presence and absence of the extracts to further confirm the mechanism of action. To establish these curves, first, jejunal tissues were thrice incubated with K+ (80 mM) and then stabilized in EDTA-containing calcium-free Tyrode solution for about 30 ± 5 min, followed by 50 ± 5 min incubation in potassium-rich and calcium-free Tyrode solution, to eventually diminish the intracellular calcium stores. After the equilibration of tissues, calcium was then added to the tissue organ bath cumulatively to construct control CRCs. After the control curves, the tissues were washed and incubated for 50±5 min with varying doses of extracts, the curves were reconstructed, and the results were compared with the control CRCs [19].
4.8. In Vivo Investigations
4.8.1. Castor Oil-Induced Diarrhea Model
Blab/C mice (20–30 g) of either sex were randomly divided into seven groups of five mice each (n = 5). The animals were starved overnight (16 h) and given free access to water before the experiments. The following treatments were given orally:
Group 1: (Negative Control group) received saline solution (10 mL/kg);
Group 2: (Disease control group) received saline solution (10 mL/kg);
Group 3 (standard group) received 10 mg/kg of loperamide and was designated as the standard control group;
Group 4 (standard group) received verapamil 10 mg/kg and was designated as the standard control group 2;
Groups 5, 6, and 7 received various concentrations of Hg.Cr (100, 200, and 400 mg/kg, respectively).
Furthermore, the animals were given access to water before the administration of castor oil. One hour later, all groups except group 1 received castor oil via oral gavage at 10 mL/kg. Each animal was caged individually with flooring lined with blotting paper replaced every hour. The number of diarrheal stools was recorded over 4 h. The stools from the groups treated with Hg.Cr were compared to those of the positive control group, which received only castor oil. Data were reported as the number of defecations and their percentage representation [46]. The percentage protection from diarrhea was calculated according to the following equation:
where T1 represents the average number of defecations induced by the administration of castor oil, and E signifies the average number of defecations caused by the administration of the extract.
Percent (%) protection = [(T1 − E)/ T1] × 100;
4.8.2. Charcoal Meal GIT Transit Time
This test was performed according to the procedure outlined by Wahid et al. (2022) [25], with slight modifications to assess gastrointestinal motility. In this test, rats were randomly assigned to groups (n = 5 per group), starved for 14 h before the experiment, and allowed free access to drinking water. The groups received one of the following treatments orally:
Group 1 (Control): 0.9% normal saline (10 mL/kg);
Group 2 (Standard Drug): loperamide (10 mg/kg);
Group 3 (Standard Drug): verapamil (10 mg/kg);
Groups 4 and 5 (Hg.Cr) Hg.Cr at 200 and 400 mg/kg, respectively;
Groups 6 and 7 (Hg.Hex) Hg.Hex at 200 and 400 mg/kg, respectively;
Groups 8 and 9 (Hg.EA) Hg.EA at 200 and 400 mg/kg, respectively;
Groups 10 and 11 (Hg.OH): Hg.OH at 200 and 400 mg/kg, respectively;
Group 12 (Hg.Aq): Hg.Aq at 200 and 400 mg/kg, respectively.
Fifteen minutes after administering the treatment, each rat received a 10 mL/kg charcoal meal suspension. This suspension was made using distilled water and contained 10% gum acacia, 20% starch, and 10% charcoal. After thirty minutes of charcoal meal administration, the rats were euthanized by cervical dislocation, and their small intestines were removed after dissecting their abdomens. The distance the charcoal meal traveled was measured, starting 3 cm from the pylorus to the furthest point reached by the charcoal. The results were reported as the charcoal meal transport ratio, representing the percentage of gastrointestinal motility.
The following equation was used to calculate the percentage of gastrointestinal motility:
where D1 = distance covered by charcoal and D2 = total intestinal length
Charcoal Meal Transport Ratio (%) = [D1/D2] × 100;
4.9. Statistical Analysis
All determinations were conducted in triplicate, and the data were subjected to a one-way analysis of variance (ANOVA) and a two-way ANOVA, which were additionally analyzed using Dunnett’s test. The results are expressed as the mean ± standard error of the mean (S.E.M). The half maximal effective concentration (EC50) values are given as a geometric mean with 95% confidence intervals (CI). Statistical analysis was performed using GraphPad Prism 8.0.1. (GraphPad Software, San Diego, CA, USA, http://www.graphpad.com); the differences were significant at p ≤ 0.05 [47].
5. Conclusions
The present study contributes to the existing knowledge on Haloxylon griffithii by providing preliminary experimental support for its traditional use in gastrointestinal disorders. While the findings are promising, further in-depth phytochemical and mechanistic investigations are warranted to fully validate its therapeutic potential. A comprehensive pharmacological investigation demonstrated the plant’s significant antispasmodic and antidiarrheal properties, particularly in non-polar fractions. These effects were mechanistically linked to calcium channel-blocking activity, supported by both in vitro jejunum assays and calcium response studies. The lead molecules, including fatty acid esters, monoterpenes, and a rare azulene derivative, exhibit therapeutic effects that can be utilized as natural alternatives to synthetic drugs with fewer side effects. Even though such results provide promising prospects, more studies must be carried out to isolate individual bioactive constituents, ascertain their mechanisms of action, and verify their efficacy in a clinical setup.
Author Contributions
Conceptualization, I.I. and F.S.; Methodology, I.I.; Software, I.I. and K.A.; Validation, I.I. and F.S.; Formal analysis, M.S.M.; Investigation, I.I. and F.S.; Resources, F.S.; Data curation, I.I. and K.A.; Writing—original draft preparation, I.I., F.S. and H.A.S.E.-N.; Writing—review and editing, M.S.M. and H.A.S.E.-N.; Visualization, I.I., H.A.S.E.-N., M.A.M.A. and A.A.C.; Funding acquisition, M.A.M.A. and A.A.C.; Supervision, F.S., H.A.S.E.-N. and M.E.-S.; Project administration, I.I., F.S. and H.A.S.E.-N. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2501).
Institutional Review Board Statement
The animal study protocol was approved by the Institutional Ethics Committee of the Department of Pharmacy, Bahauddin Zakariya University, Multan. The approval code is EC/02PHDP6-21, and the approval was granted on 31 May 2022.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data is contained within the article.
Conflicts of Interest
The authors declare no conflicts of interest.
Abbreviations
The following abbreviations are used in this manuscript:
| H. griffithii | Haloxylon griffithii |
| CRCs | Concentration response curves |
| Hg.Cr | Haloxylon griffithii crude extract |
| Hg.Hex | Haloxylon griffithii n-hexane fraction |
| Hg.EA | Haloxylon griffithii ethyl acetate fraction |
| Hg.OH | Haloxylon griffithii ethanolic fraction |
| Hg.Aq | Haloxylon griffithii Aqueous fraction |
| GIT | Gastrointestinal tract |
| VGCC | Voltage-gated calcium channels |
Appendix A
Table A1.
List of all volatile and semi-volatile compounds identified in the crude hydroethanolic extract of Haloxylon griffithii using GC-MS analysis.
Table A1.
List of all volatile and semi-volatile compounds identified in the crude hydroethanolic extract of Haloxylon griffithii using GC-MS analysis.
| Peak no. | RT | % Area | Compound Name | Hit Quality |
|---|---|---|---|---|
| 1 | 3.466 | 0.21 | 2-Butanone, 4-hydroxy-3-methyl- | 52 |
| 2 | 7.703 | 0.10 | o-Cymene | 95 |
| 3 | 10.480 | 0.14 | 4-Hydroxy-.beta.-thujone | 72 |
| 4 | 11.750 | 0.18 | Cyclohexasiloxane, dodecamethyl- | 93 |
| 5 | 13.215 | 0.12 | 1-(1-Butyny)cyclopentanol | 50 |
| 6 | 13.677 | 0.26 | Hexanoic acid, 3,7-dimethyl-6-octenyl ester | 49 |
| 7 | 13.958 | 0.41 | Cycloheptasiloxane, tetradecamethyl- | 93 |
| 8 | 14.126 | 0.15 | Carane, 4,5-epoxy-, trans | 46 |
| 9 | 14.29 | 0.15 | Pentacosane | 74 |
| 10 | 14.38 | 0.06 | Ledol | 38 |
| 11 | 14.541 | 0.04 | 2,4-Di-tert-butylphenol | 78 |
| 12 | 15.930 | 0.25 | Cyclooctasiloxane, hexadecamethyl- | 94 |
| 13 | 16.174 | 0.03 | Bicyclo [2.2.1]heptane, 1,3,3-trimethyl | 46 |
| 14 | 16.217 | 0.04 | 2-Heptenoic acid, methyl ester, (E)- | 44 |
| 15 | 16.499 | 0.11 | 2-Propenal, 3-(2,6,6-trimethyl-1-cyclohexen-1-yl)- | 59 |
| 16 | 16.808 | 0.17 | 2-Hexen-1-ol, 2-ethyl | 89 |
| 17 | 16.979 | 0.23 | (S,E)-6-Hydroxy-6-methyl-2-((2S,5R)-5-methyl-5-vinyltetrahydrofuran-2-yl) | 68 |
| 18 | 17.315 | 0.14 | 1H-Indene, 1-ethylideneoctahydro-7a-methyl-, (1Z,3a.alpha.,7a.beta.) | 87 |
| 19 | 17.631 | 0.11 | Cyclononasiloxane, octadecamethyl | 90 |
| 20 | 18.154 | 0.12 | Isopropyl myristate | 35 |
| 21 | 18.536 | 0.14 | 7,10,10-Trimethyl-4-oxa-3,5-diazatricyclo [5.2.1.0(2,6)]deca-2,5-dien-3-one | 35 |
| 22 | 18.959 | 0.14 | 2,6-Pyrazinedicarbonitrile, 3-amino-5-(diethylamino)- | 41 |
| 23 | 19.045 | 0.15 | 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione | 99 |
| 24 | 19.146 | 0.18 | Cyclodecasiloxane, eicosamethyl- | 52 |
| 25 | 19.197 | 0.13 | Hexadecanoic acid, methyl ester | 96 |
| 26 | 19.419 | 0.10 | 5-(6-Methyl-7-oxooctyl)-2(5H)-furanone (isomer 2) | 47 |
| 27 | 19.533 | 0.34 | n-Hexadecanoic acid | 96 |
| 28 | 19.873 | 0.48 | Hexadecanoic acid, ethyl ester | 99 |
| 29 | 20.019 | 0.13 | (3aR,4R,7R)-1,4,9,9-Tetramethyl-3,4,5,6,7,8-hexahydro-2H-3a,7-methanoazulen-2-one | 78 |
| 30 | 20.216 | 1.89 | Lumisantonin | 95 |
| 31 | 20.326 | 0.92 | Propionic acid, 3-(2-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]diazepin-1-yl)- | 44 |
| 32 | 20.542 | 0.82 | Benzene, 1,2,4,5-tetrakis(1-methylethyl)- | 89 |
| 33 | 20.651 | 0.12 | 6-Hydroxy-5a,9-dimethyl-3-methylidene-4,5,6,7,9a,9b-hexahydro-3aH-benzo[g][1]benzofuran-2-one | 66 |
| 34 | 20.760 | 0.20 | 2-Butanone, 4-(2,6,6-trimethyl-2-cyclohexen-1-ylidene)- | 80 |
| 35 | 20.838 | 0.11 | 9,12-Octadecadienoic acid (Z,Z)-, methyl ester | 91 |
| 36 | 20.929 | 0.44 | Benzene, hexaethyl- | 89 |
| 37 | 20.990 | 0.33 | Cyclohexanol, 5-methyl-2-(1-methylethyl)-, (1.alpha.,2.alpha.,5.alpha | 43 |
| 38 | 21.182 | 0.36 | 9,12-Octadecadienoic acid (Z,Z)- | 95 |
| 39 | 21.235 | 1.55 | 4(1H)-Quinolinone,7-amino-3-carboxy-6-fluoro-1-ethyl | 44 |
| 40 | 21.460 | 0.90 | Linoleic acid ethyl ester | 99 |
| 41 | 21.515 | 0.67 | (E)-9-Octadecenoic acid ethyl ester | 95 |
| 42 | 21.707 | 0.18 | 3,6-Nonadien-5-one, 2,2,8,8-tetramethyl | 46 |
| 43 | 21.778 | 0.25 | 3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane | 27 |
| 44 | 22.024 | 33.57 | α-Santonin | 99 |
| 45 | 22.123 | 1.22 | N-[1-Ethylpyridinylidene]-1,3,2-dioxaphospholan-2-amine 2-sulfide | 42 |
| 46 | 22.260 | 3.11 | Phenanthrene, 3,6-dimethoxy-9,10-dimethyl | 59 |
| 47 | 22.305 | 2.30 | Alantolactone, 4.alpha.,4A.alpha.-epoxy | 64 |
| 48 | 22.615 | 0.30 | (-)-Neoclovene-(II), dihydro | 90 |
| 49 | 22.668 | 0.34 | Verrucarol | 62 |
| 50 | 22.941 | 0.31 | Oxazepam, 2TMS derivative | 56 |
| 51 | 23.039 | 0.23 | 2H-3,9a-Methano-1-benzoxepin, octahydro-2,2,5a,9-tetramethyl-, [3R-(3.alpha.,5a.alpha.,9.alpha.,9a.alpha.)]- | 56 |
| 52 | 23.203 | 0.13 | Spiro[2.5]octane, 5,5-dimethyl-4-(3-oxobutyl)- | 30 |
| 53 | 23.351 | 0.17 | Ethanone, 1-(1,2,3,4,7,7a-hexahydro-1,4,4,5-tetramethyl-1,3a-ethano-3aH-inden-6-yl)- | 47 |
| 54 | 23.545 | 0.26 | Benzothiophene-3-carboxylic acid, 4,5,6,7-tetrahydro-2-amino-6-ethyl-, ethyl ester | 38 |
| 55 | 23.850 | 0.11 | 2-Pyridinamine, N-(4,5-dihydro-5-methyl-2-thiazolyl)-3-methyl | 47 |
| 56 | 24.383 | 0.19 | Bis(2-ethylhexyl) phthalate | 68 |
| 57 | 24.563 | 0.19 | 2-N,2-N,4-N,4-N,8-Pentamethylquinoline-2,4,5-triamine | 70 |
| 58 | 24.963 | 43.93 | Bis(2-ethylhexyl) phthalate | 90 |
| 59 | 27.919 | 0.09 | 1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester | 94 |
Table A2.
2D-Structures of chief components identified in Haloxylon griffithii by GCMS analysis.
| S. No. | Peak no. | Compound Name | Mol. Weightg/mol | Mol. Formula | Structure |
|---|---|---|---|---|---|
| 1 | 2 | o-Cymene | 134.2182 | C10H14 |  |
| 2 | 4 | Cyclohexasiloxane, dodecamethyl- | 444.9236 | C12H36O6Si6 |  |
| 3 | 7 | Cycloheptasiloxane, tetradecamethyl- | 519.0776 | C14H42O7Si7 |  |
| 4 | 9 | Pentacosane | 352.6804 | C25H52 |  |
| 5 | 11 | 2,4-Di-tert-butylphenol | 206.3239 | C14H22O |  |
| 6 | 12 | Cyclooctasiloxane, hexadecamethyl- | 593.2315 | C16H48O8Si8 |  |
| 7 | 15 | 2-Propenal, 3-(2,6,6-trimethyl-1-cyclohexen-1-yl)- | 178.2707 | C12H18O |  |
| 8 | 16 | 2-Hexen-1-ol, 2-ethyl | 128.2120 | C8H16O |  |
| 9 | 19 | Cyclononasiloxane, octadecamethyl | 667.3855 | C18H54O9Si9 |  |
| 10 | 23 | 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione | 276.3707 | C17H24O3 |  |
| 11 | 24 | Cyclodecasiloxane, eicosamethyl- | 741.5394 | C20H60O10Si10 |  |
| 12 | 25 | Hexadecanoic acid, methyl ester | 270.4507 | C17H34O2 |  |
| 13 | 27 | n-Hexadecanoic acid | 256.4241 | C16H32O2 |  |
| 14 | 28 | Hexadecanoic acid, ethyl ester | 284.4772 | C18H36O2 |  |
| 15 | 29 | (3aR,4R,7R)-1,4,9,9-Tetramethyl-3,4,5,6,7,8-hexahydro-2H-3a,7-methanoazulen-2-one | 218.3346 | C15H22O |  |
| 16 | 32 | Benzene, 1,2,4,5-tetrakis(1-methylethyl)- | 246.4308 | C18H30 |  |
| 17 | 35 | 9,12-Octadecadienoic acid (Z,Z)-, methyl ester | 294.48 | C19H34O2 |  |
| 18 | 36 | Benzene, hexaethyl- | 246.4308 | C18H30 |  |
| 19 | 38 | 9,12-Octadecadienoic acid (Z,Z)- | 280.4455 | C18H32O2 |  |
| 20 | 40 | Linoleic acid ethyl ester | 308.4986 | C20H36O2 |  |
| 21 | 41 | (E)-9-Octadecenoic acid ethyl ester | 310.5145 | C20H38O2 |  |
| 22 | 44 | α-Santonin | 246.3016 | C15H18O3 |  |
| 23 | 58 | Bis(2-ethylhexyl) phthalate | 390.5561 | C24H38O4 |  |
References
- Temml, V.; Schuster, D. Molecular docking for natural product investigations: Pitfalls and ways to overcome them. In Molecular Docking for Computer-Aided Drug Design; Elsevier: Amsterdam, The Netherlands, 2021; pp. 391–405. [Google Scholar]
- Singh, H.; Bharadvaja, N. Treasuring the computational approach in medicinal plant research. Prog. Biophys. Mol. Biol. 2021, 164, 19–32. [Google Scholar] [CrossRef] [PubMed]
- Aly, S.H.; Elbadry, A.M.M.; Doghish, A.S.; El-Nashar, H.A.S. Unveiling the pharmacological potential of plant triterpenoids in breast cancer management: An updated review. Naunyn Schmiedebergs Arch. Pharmacology 2024, 397, 5571–5596. [Google Scholar]
- Askar, M.A.; El-Nashar, H.A.; Al-Azzawi, M.A.; Rahman, S.S.A.; Elshawi, O.E. Synergistic Effect of Quercetin Magnetite Nanoparticles and Targeted Radiotherapy in Treatment of Breast Cancer. Breast Cancer 2022, 16, 11782234221086728. [Google Scholar] [CrossRef] [PubMed]
- El-Nashar, H.A.S.; Adel, M.; El-Shazly, M.; Yahia, I.S.; El Sheshtawy, H.S.; Almalki, A.A.; Ibrahim, N. Chemical Composition, Antiaging Activities and Molecular Docking Studies of Essential Oils from Acca sellowiana (Feijoa). Chem. Biodivers 2022, 19, e202200272. [Google Scholar]
- Rahman, M.M.; Islam, M.R.; Rahman, F.; Rahaman, M.S.; Khan, M.S.; Abrar, S.; Ray, T.K.; Uddin, M.B.; Kali, M.S.K.; Dua, K. Emerging promise of computational techniques in anti-cancer research: At a glance. Bioengineering 2022, 9, 335. [Google Scholar] [CrossRef]
- Saeed, K.; Rahkama, V.; Eldfors, S.; Bychkov, D.; Mpindi, J.P.; Yadav, B.; Paavolainen, L.; Aittokallio, T.; Heckman, C.; Wennerberg, K. Comprehensive drug testing of patient-derived conditionally reprogrammed cells from castration-resistant prostate cancer. Eur. Urol. 2017, 71, 319–327. [Google Scholar] [CrossRef]
- Bappi, M.H.; Mia, M.N.; Ansari, S.A.; Ansari, I.A.; Prottay, A.A.S.; Akbor, M.S.; El-Nashar, H.A.S.; El-Shazly, M.; Mubarak, M.S.; Torequl Islam, M. Quercetin increases the antidepressant-like effects of sclareol and antagonizes diazepam in thiopental sodium-induced sleeping mice: A possible GABAergic transmission intervention. Phytother. Res. 2024, 38, 2198–2214. [Google Scholar] [CrossRef] [PubMed]
- Asif, R.; Ali, M.; Mazhar, M.; Mustafa, M.; Yasmin, R. Evaluation of Phytochemical Contents, Antimicrobial, and antioxidant Potential of Haloxylon Griffithii Collected From Northern Region of Balochistan, Pakistan. Dose-Response 2023, 21, 15593258231163389. [Google Scholar] [CrossRef]
- Kamal, S.; Bibi, I.; Rehman, K.; Zahoor, A.F.; Kamal, A.; Aslam, F.; Alasmary, F.A.; Almutairi, T.M.; Alhajri, H.M.; Alissa, S.A. Biological activities of in-house developed Haloxylon griffithii plant extract formulations. Plants 2021, 10, 1427. [Google Scholar] [CrossRef]
- Mengal, A.; Samiullah, N.K.; Baqi, A. Antibacterial and antifungal activities of extracts of Convolvulus leiocalycinus and Haloxylon griffithii of Balochistan, Pakistan. Pure Appl. Bio. 2019, 8, 2286–2294. [Google Scholar] [CrossRef]
- El-Nashar, H.A.S.; Al-Azzawi, M.A.; Al-Kazzaz, H.H.; Alghanimi, Y.K.; Kocaebli, S.M.; Alhmammi, M.; Asad, A.; Salam, T.; El-Shazly, M.; Ali, M.A.M. HPLC-ESI/MS-MS metabolic profiling of white pitaya fruit and cytotoxic potential against cervical cancer: Comparative studies, synergistic effects, and molecular mechanistic approaches. J. Pharm. Biomed. Anal. 2024, 244, 116–121. [Google Scholar] [CrossRef] [PubMed]
- Farasati Far, B.; Gouranmohit, G.; Naimi-Jamal, M.R.; Neysani, E.; El-Nashar, H.A.S.; El-Shazly, M.; Khoshnevisan, K. The potential role of Hypericum perforatum in wound healing: A literature review on the phytochemicals, pharmacological approaches, and mechanistic perspectives. Phytother. Res. 2024, 38, 3271–3295. [Google Scholar] [CrossRef] [PubMed]
- Ahmad, M.; Khan, M.P.Z.; Mukhtar, A.; Zafar, M.; Sultana, S.; Jahan, S. Ethnopharmacological survey on medicinal plants used in herbal drinks among the traditional communities of Pakistan. J. Ethnopharmacol. 2016, 184, 154–186. [Google Scholar] [CrossRef] [PubMed]
- El-Nashar, H.A.S.; Eldahshan, O.A.; Fattah, N.F.A.; Loutfy, S.A.; Abdel-Salam, I.M. HPLC-ESI/MS-MS characterization of compounds in Dolomiaea costus extract and evaluation of cytotoxic and antiviral properties: Molecular mechanisms underlying apoptosis-inducing effect on breast cancer. BMC Complement. Med. Ther. 2023, 23, 354. [Google Scholar] [CrossRef]
- Rosenblum, D.; Joshi, N.; Tao, W.; Karp, J.M.; Peer, D. Progress and challenges towards targeted delivery of cancer therapeutics. Nat. Commun. 2018, 9, 1410. [Google Scholar] [CrossRef]
- Waitkus, M.S.; Diplas, B.H.; Yan, H. Biological role and therapeutic potential of IDH mutations in cancer. Cancer Cell 2018, 34, 186–195. [Google Scholar] [CrossRef]
- Hochman, J.H. Adapting ADME and pharmacokinetic analysis to the next generation of therapeutic modalities. J. Pharm. Sci. 2021, 110, 35–41. [Google Scholar] [CrossRef]
- Saqib, F.; Janbaz, K.H. Ethnopharmacological basis for folkloric claims of Anagallis arvensis Linn. (Scarlet Pimpernel) as prokinetic, spasmolytic and hypotensive in province of Punjab, Pakistan. J. Ethnopharmacol. 2021, 267, 113634. [Google Scholar] [CrossRef]
- Zia-Ul-Haq, M. Past, present and future of Carotenoids Research. In Carotenoids: Structure and Function in the Human Body; Springer: Berlin/Heidelberg, Germany, 2021; pp. 827–854. [Google Scholar]
- Gilani, A.H.; Khan, A.-U.; Ghayur, M.N. Ca2+ antagonist and cholinergic activities explain the medicinal use of olive in gut disorders. Nutr. Res. 2006, 26, 277–283. [Google Scholar] [CrossRef]
- Wahid, M.; Saqib, F.; Ahmedah, H.T.; Gavris, C.M.; De Feo, V.; Hogea, M.; Moga, M.; Chicea, R. Cucumis sativus L. seeds ameliorate muscular spasm-induced gastrointestinal and respiratory disorders by simultaneously inhibiting calcium mediated signaling pathway. Pharmaceuticals 2021, 14, 1197. [Google Scholar] [CrossRef]
- Iqbal, I.; Saqib, F.; Latif, M.F.; Shahzad, H.; Dima, L.; Sajer, B.; Manea, R.; Pojala, C.; Necula, R. Pharmacological Basis for Antispasmodic, Bronchodilator, and Antidiarrheal Potential of Dryopteris ramosa (Hope) C. via In Vitro, In Vivo, and In Silico Studies. ACS Omega 2023, 8, 26982–27001. [Google Scholar] [CrossRef] [PubMed]
- Saqib, F.; Janbaz, K.H. Rationalizing ethnopharmacological uses of Alternanthera sessilis: A folk medicinal plant of Pakistan to manage diarrhea, asthma and hypertension. J. Ethnopharm. 2016, 182, 110–121. [Google Scholar] [CrossRef] [PubMed]
- Wahid, M.; Saqib, F.; Akhtar, S.; Ali, A.; Wilairatana, P.; Mubarak, M.S. Possible mechanisms underlying the antispasmodic, bronchodilator, and antidiarrheal activities of polarity–based extracts of cucumis sativus L. Seeds in in silico, in vitro, and in vivo studies. Pharmaceuticals 2022, 15, 641. [Google Scholar] [CrossRef] [PubMed]
- Gilani, A.H.; Ghayur, M.N.; Saify, Z.S.; Ahmed, S.P.; Choudhary, M.I.; Khalid, A. Presence of cholinomimetic and acetylcholinesterase inhibitory constituents in betel nut. Life Sci. 2004, 75, 2377–2389. [Google Scholar] [CrossRef] [PubMed]
- Rehman, N.U.; Ansari, M.N.; Ahmad, W.; Ali, A. Calcium Channel Inhibitory Effect of Marjoram (Origanum majorana L.): Its Med. Use Diarrhea Gut Hyperactivity. Front. Biosci. 2024, 29, 47. [Google Scholar] [CrossRef]
- Reynolds, I.J.; Gould, R.J.; Snyder, S.H. Loperamide: Blockade of calcium channels as a mechanism for antidiarrheal effects. J. Pharmacol. Exper. Therap. 1984, 231, 628–632. [Google Scholar] [CrossRef]
- Lu, Y.; Huang, J.; Zhang, Y.; Huang, Z.; Yan, W.; Zhou, T.; Wang, Z.; Liao, L.; Cao, H.; Tan, B. Therapeutic effects of berberine hydrochloride on stress-induced diarrhea-predominant irritable bowel syndrome rats by inhibiting neurotransmission in colonic smooth muscle. Front. Pharmacol. 2021, 12, 596686. [Google Scholar] [CrossRef]
- Na’Allah, A.; Bashir, S.; Alabi, M.; Kareem, F.A.; Olaifa, G.O.; Afolabi, F.J.; Aransiola, S.A.; Seth, C.S.; Maddela, N.R.; Prasad, R. Anti-diarrhoea properties of ethanol extract of Citrullus colocynthis fruit pulp: In vivo and in silico studies. Adv. Tradit. Med. 2025, 25, 269–285. [Google Scholar] [CrossRef]
- Woldeyohannes, M.G.; Eshete, G.T.; Abiye, A.A.; Hailu, A.E.; Huluka, S.A.; Tadesse, W.T. Antidiarrheal and antisecretory effect of 80% hydromethanolic leaf extract of Moringa stenopetala baker f. in mice. Biochem. Res. Int. 2022, 2022, 5768805. [Google Scholar] [CrossRef]
- Gilani, A.U.H.; Shah, A.J.; Ahmad, M.; Shaheen, F. Antispasmodic effect of Acorus calamus Linn. is mediated through calcium channel blockade. Phytother. Res. 2006, 20, 1080–1084. [Google Scholar] [CrossRef]
- Munir Ahmad, M.A.; Attiq-ur-Rehman, A.-u.-R.; Samiullah, S.; Rasool Bakhsh Tareen, R.B.T.; Naqeebullah Khan, N.K.; Abdul Baqi, A.B.; Abdul Manan, A.M. Qualitative and quantitative determination of phytochemicals in Convolvulus leiocalycinus and Haloxylon griffithii. Pure Appl. Bio. 2019, 8, 733–741. [Google Scholar]
- Amtaghri, S.; Eddouks, M. Comprehensive Review on the Genus Haloxylon: Pharmacological and Phytochemical Properties. Endocr. Metab. Immune Disord. Drug Targets 2024, 24, 1146–1160. [Google Scholar] [CrossRef] [PubMed]
- Gao, S.; He, Y.; Zhang, L.; Liu, L.; Qu, C.; Zheng, Z.; Miao, J. Conjugated linoleic acid ameliorates hepatic steatosis by modulating intestinal permeability and gut microbiota in ob/ob mice. Food Nutr. Res. 2022, 66, 29210–29219. [Google Scholar] [CrossRef] [PubMed]
- Abubakar, M.N.; Majinda, R.R.T. GC-MS analysis and preliminary antimicrobial activity of Albizia adianthifolia (Schumach) and Pterocarpus angolensis (DC). Medicines 2016, 3, 3. [Google Scholar] [CrossRef]
- Marchese, A.; Arciola, C.R.; Barbieri, R.; Silva, A.S.; Nabavi, S.F.; Tsetegho Sokeng, A.J.; Izadi, M.; Jafari, N.J.; Suntar, I.; Daglia, M. Update on monoterpenes as antimicrobial agents: A particular focus on p-cymene. Materials 2017, 10, 947. [Google Scholar] [CrossRef]
- Shareef, S.H.; Al-Medhtiy, M.H.; Ibrahim, I.A.A.; Alzahrani, A.R.; Jabbar, A.A.; Galali, Y.; Agha, N.F.S.; Aziz, P.Y.; Thabit, M.A.; Agha, D.N.F. Gastroprophylactic effects of p-cymene in ethanol-induced gastric ulcer in rats. Processeses 2022, 10, 1314. [Google Scholar] [CrossRef]
- Micucci, M.; Protti, M.; Aldini, R.; Frosini, M.; Corazza, I.; Marzetti, C.; Mattioli, L.B.; Tocci, G.; Chiarini, A.; Mercolini, L. Thymus vulgaris L. essential oil solid formulation: Chem. Profile Spasmolytic Antimicrob. effects. Biomolecules 2020, 10, 860. [Google Scholar] [CrossRef]
- Ammar, S.; Edziri, H.; Mahjoub, M.A.; Chatter, R.; Bouraoui, A.; Mighri, Z. Spasmolytic and anti-inflammatory effects of constituents from Hertia cheirifolia. Phytomedicine 2009, 16, 1156–1161. [Google Scholar] [CrossRef]
- Council, N. Committee for the Update of the Guide for the, C. & Use of Laboratory, A. Guide for the Care and Use of Laboratory Animals; National Academies Press: Washington, DC, USA, 2011. [Google Scholar]
- Vijh, D.; Gupta, P. GC-MS analysis, molecular docking, and pharmacokinetic studies on Dalbergia sissoo barks extracts for compounds with anti-diabetic potential. Sci. Rep. 2024, 14, 24936. [Google Scholar] [CrossRef]
- Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [Google Scholar] [CrossRef]
- Pires, D.E.; Blundell, T.L.; Ascher, D.B. pkCSM: Predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. J. Med. Chem. 2015, 58, 4066–4072. [Google Scholar] [CrossRef] [PubMed]
- Janbaz, K.H.; Zaeem Ahsan, M.; Saqib, F.; Imran, I.; Zia-Ul-Haq, M.; Abid Rashid, M.; Jaafar, H.Z.; Moga, M. Scientific basis for use of Pyrus pashia Buch.-Ham. ex D. Don. fruit in gastrointestinal, respiratory and cardiovascular ailments. PLoS ONE 2015, 10, e0118605. [Google Scholar] [CrossRef] [PubMed]
- Saqib, F.; Usman, F.; Malik, S.; Bano, N.; Ur-Rahman, N.; Riaz, M.; Mureşan, C.C. Antidiarrheal and Cardio-Depressant Effects of Himalaiella heteromalla (D. Don) Raab-Straube: In Vitro, In Vivo, and In Silico Studies. Plants 2022, 11, 78. [Google Scholar] [CrossRef]
- Zarei, M.; Mohammadi, S.; Komaki, A. Antinociceptive activity of Inula britannica L. and patuletin: Vivo Possible Mech. studies. J. Ethnopharmacol. 2018, 219, 351–358. [Google Scholar] [CrossRef] [PubMed]
Figure 1.
GC-MS chromatogram of volatile oil extracted from Haloxylon griffithii.
Figure 2.
Effect of Hg.Cr, Hg.Hex, Hg.EA, and verapamil on jejunal spontaneous and K+ (80 mM)- induced spastic contractions Hg.OH and HG.Aq on spontaneous and atropine-treated contractions. (Data expressed as the mean ± SEM, n = 5; data were analyzed by sigmoidal dose–response curve).
Figure 2.
Effect of Hg.Cr, Hg.Hex, Hg.EA, and verapamil on jejunal spontaneous and K+ (80 mM)- induced spastic contractions Hg.OH and HG.Aq on spontaneous and atropine-treated contractions. (Data expressed as the mean ± SEM, n = 5; data were analyzed by sigmoidal dose–response curve).
Figure 3.
Effect of Hg.Cr, Hg.Hex, Hg.EA, and verapamil on concentration response curves of calcium (CRCs) (data are expressed as the mean ± SEM, n = 5; data were analyzed by sigmoidal dose–response curve).
Figure 3.
Effect of Hg.Cr, Hg.Hex, Hg.EA, and verapamil on concentration response curves of calcium (CRCs) (data are expressed as the mean ± SEM, n = 5; data were analyzed by sigmoidal dose–response curve).
Figure 4.
Hg.Cr and loperamide impact on diarrhea brought on by castor oil. Data are expressed as the mean ± SEM (n = 5). For analysis, two-way ANOVA with multiple comparison testing was used. While **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns = non-significant in comparison with positive control.
Figure 4.
Hg.Cr and loperamide impact on diarrhea brought on by castor oil. Data are expressed as the mean ± SEM (n = 5). For analysis, two-way ANOVA with multiple comparison testing was used. While **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns = non-significant in comparison with positive control.
Figure 5.
The effect of Dr.Cr on caster oil-induced diarrhea. Data are expressed as the mean ± SEM (n = 5). The standard one-way ANOVA testing with multiple comparison tests was used for analysis. While **** p < 0.0001, * p < 0.05, ns = non-significant compared with negative control.
Figure 5.
The effect of Dr.Cr on caster oil-induced diarrhea. Data are expressed as the mean ± SEM (n = 5). The standard one-way ANOVA testing with multiple comparison tests was used for analysis. While **** p < 0.0001, * p < 0.05, ns = non-significant compared with negative control.
Figure 6.
Effect of Haloxylon griffithii Extracts (A). Hg.Cr (B) Hg.Hex, (C) Hg.EA, (D) Hg.OH, (E) Hg.Aq on gastrointestinal motility in comparison to standard antidiarrheal agents. Values are expressed as the mean ± SEM (n = 3) *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns: Not significant.
Figure 6.
Effect of Haloxylon griffithii Extracts (A). Hg.Cr (B) Hg.Hex, (C) Hg.EA, (D) Hg.OH, (E) Hg.Aq on gastrointestinal motility in comparison to standard antidiarrheal agents. Values are expressed as the mean ± SEM (n = 3) *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns: Not significant.
Table 1.
Chief components identified in Haloxylon griffithii by GC-MS analysis.
| Sr. No. | Peak no. | RT | % Area | Compound Name | Mol. Weight g/mol | Mol. Formula |
|---|---|---|---|---|---|---|
| 1 | 2 | 7.703 | 0.10 | o-Cymene | 134.2182 | C10H14 |
| 2 | 4 | 11.750 | 0.18 | Cyclohexasiloxane, dodecamethyl- | 444.9236 | C12H36O6Si6 |
| 3 | 7 | 13.958 | 0.41 | Cycloheptasiloxane, tetradecamethyl- | 519.0776 | C14H42O7Si7 |
| 4 | 9 | 14.29 | 0.15 | Pentacosane | 352.6804 | C25H52 |
| 5 | 11 | 14.541 | 0.04 | 2,4-Di-tert-butylphenol | 206.3239 | C14H22O |
| 6 | 12 | 15.930 | 0.25 | Cyclooctasiloxane, hexadecamethyl- | 593.2315 | C16H48O8Si8 |
| 7 | 15 | 16.499 | 0.11 | 2-Propenal, 3-(2,6,6-trimethyl-1-cyclohexen-1-yl)- | 178.2707 | C12H18O |
| 8 | 16 | 16.808 | 0.17 | 2-Hexen-1-ol, 2-ethyl | 128.2120 | C8H16O |
| 9 | 19 | 17.631 | 0.11 | Cyclononasiloxane, octadecamethyl | 667.3855 | C18H54O9Si9 |
| 10 | 23 | 19.045 | 0.15 | 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione | 276.3707 | C17H24O3 |
| 11 | 24 | 19.146 | 0.18 | Cyclodecasiloxane, eicosamethyl- | 741.5394 | C20H60O10Si10 |
| 12 | 25 | 19.197 | 0.13 | Hexadecanoic acid, methyl ester | 270.4507 | C17H34O2 |
| 13 | 27 | 19.533 | 0.34 | n-Hexadecanoic acid | 256.4241 | C16H32O2 |
| 14 | 28 | 19.873 | 0.48 | Hexadecanoic acid, ethyl ester | 284.4772 | C18H36O2 |
| 15 | 29 | 20.019 | 0.13 | (3aR,4R,7R)-1,4,9,9-Tetramethyl-3,4,5,6,7,8-hexahydro-2H-3a,7-methanoazulen-2-one | 218.3346 | C15H22O |
| 16 | 32 | 20.542 | 0.82 | Benzene, 1,2,4,5-tetrakis(1-methylethyl)- | 246.4308 | C18H30 |
| 17 | 35 | 20.838 | 0.11 | 9,12-Octadecadienoic acid (Z,Z)-, methyl ester | 294.48 | C19H34O2 |
| 18 | 36 | 20.929 | 0.44 | Benzene, hexaethyl- | 246.4308 | C18H30 |
| 19 | 38 | 21.182 | 0.36 | 9,12-Octadecadienoic acid (Z,Z)- | 280.4455 | C18H32O2 |
| 20 | 40 | 21.460 | 0.90 | Linoleic acid ethyl ester | 308.4986 | C20H36O2 |
| 21 | 41 | 21.515 | 0.67 | (E)-9-Octadecenoic acid ethyl ester | 310.5145 | C20H38O2 |
| 22 | 44 | 22.024 | 33.57 | α-Santonin | 246.3016 | C15H18O3 |
| 23 | 58 | 24.963 | 43.93 | Bis(2-ethylhexyl) phthalate | 390.5561 | C24H38O4 |
Table 2.
Comparative ADME profiling of compounds identified from GC-MS analysis of H. griffithii crude extract and Verapamil.
Table 2.
Comparative ADME profiling of compounds identified from GC-MS analysis of H. griffithii crude extract and Verapamil.
| Molecule | Verapamil | Alpha Santonin | O-Cymene | Hexadecanoic Acid, Ethyl Ester | Hexadecanoic Acid, Methyl Ester | n-Hexadecanoic Acid | (E)-9-Octadecenoic Acid Ethyl Ester | Bis(2-ethylhexyl) Phthalate | Benzene, Hexaethyl- | Cyclodecasiloxane, Eicosamethyl- | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Basic properties | Formula | C27H38N2O4 | C15H18O3 | C10H14 | C18H36O2 | C17H34O2 | C16H32O2 | C20H38O2 | C24H38O4 | C18H30 | C20H60O10Si10 |
| MW (g/mol) | 454.6 | 246.3 | 134.22 | 284.48 | 270.45 | 256.42 | 310.51 | 390.56 | 246.43 | 741.54 | |
| iLOGP | 4.5 | 2.25 | 2.43 | 4.65 | 4.41 | 3.85 | 5.03 | 4.77 | 3.85 | 6.55 | |
| XLOGP3 | 3.79 | 2.29 | 4.38 | 7.88 | 7.38 | 7.17 | 8.03 | 7.45 | 6.7 | 10.06 | |
| WLOGP | 5.09 | 2.42 | 3.12 | 6.03 | 5.64 | 5.55 | 6.59 | 6.43 | 5.06 | 7.18 | |
| MLOGP | 2.96 | 2.38 | 4.47 | 4.67 | 4.44 | 4.19 | 5.03 | 5.24 | 6.57 | –2.43 | |
| Absorption | GI absorption | High | High | Low | High | High | High | Low | High | Low | Low |
| Pgp substrate | Yes | No | No | No | No | No | No | Yes | No | Yes | |
| Bioavailability Score | 0.55 | 0.55 | 0.55 | 0.55 | 0.55 | 0.85 | 0.55 | 0.55 | 0.55 | 0.55 | |
| Skin Permeability log Kp (cm/s) | –6.38 | –6.18 | –4.01 | –2.44 | –2.71 | –2.77 | –2.49 | –3.39 | –3.05 | –3.68 | |
| Distribution | BBB permeant | Yes | Yes | Yes | No | Yes | Yes | No | No | No | No |
| BBB permeability log BB | –0.647 | 0.347 | 0.48 | 0.759 | 0.749 | –0.111 | 0.786 | –0.175 | 0.741 | –2.905 | |
| VDss (human) log L/kg | 0.931 | 0.205 | 0.744 | 0.373 | 0.334 | –0.543 | 0.332 | 0.36 | 1.334 | –0.106 | |
| Metabolism | CYP1A2 inhibitor | No | No | No | Yes | Yes | Yes | Yes | No | No | No |
| CYP2C19 inhibitor | No | No | No | No | No | No | No | No | No | No | |
| CYP2C9 inhibitor | No | No | No | No | No | Yes | No | Yes | No | No | |
| CYP2D6 inhibitor | Yes | No | Yes | No | No | No | No | No | No | No | |
| CYP3A4 inhibitor | Yes | No | No | No | No | No | No | Yes | No | No | |
| Excretion | Total Clearance logml/min/kg | 1.072 | 0.197 | 0.259 | 1.912 | 1.861 | 1.763 | 2.03 | 1.898 | 2.03 | −0.484 |
| Toxicity | Hepatotoxicity | No | No | No | No | No | No | No | No | Yes | No |
| Skin Sensitisation | No | No | Yes | Yes | Yes | Yes | yes | No | yes | No | |
| Drug Likeness | Lipinski #violations | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Ghose #violations | 2 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 4 | |
| Veber #violations | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | |
| Egan #violations | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 1 | |
| Muegge #violations | 0 | 0 | 2 | 2 | 1 | 1 | 2 | 2 | 2 | 2 |
The physicochemical and pharmacokinetic properties listed in this table were primarily obtained through computational prediction using online tools such as SwissADME (http://www.swissadme.ch, accessed on 28 January 2025) and pkCSM (http://biosig.unimelb.edu.au/pkcsm, accessed on 28 January 2025). Compound identification and basic molecular data (e.g., molecular formula, retention time) were confirmed using the NIST Chemistry WebBook and PubChem.
Table 3.
Effect of the hydroethanolic extract of Hg.Cr on castor oil-induced diarrhea in mice (n = 5).
Table 3.
Effect of the hydroethanolic extract of Hg.Cr on castor oil-induced diarrhea in mice (n = 5).
| Groups | Dose | No. of Wet Feces | ||||
|---|---|---|---|---|---|---|
| 0–0.5 h | 0–1 h | 0–2 h | 0–3 h | 0–4 h | ||
| Group I (Negative control) | 10 mL/kg (NS) | 0.2 | 1.0 | 1.0 | 2.0 | 4.0 |
| Group II (Disease control) | 10 mL/kg (NS) | 5.0 | 11.0 | 18.0 | 29.0 | 32.0 |
| Group III (standard) | 10 mg/kg (Loperamide) | 0.4 | 2.0 | 3.0 | 6.0 | 7.0 |
| Group IV (standard) | 10 mg/kg (verapamil) | 2.0 | 3.0 | 4.0 | 4.8 | 5.0 |
| Group V (Treatment) | 100 mg/kg (Hg.Cr) | 5.4 | 11.0 | 18.0 | 19.6 | 21.0 |
| 0Group VI (Treatment) | 200 mg/kg (Hg.Cr) | 5.2 | 9.0 | 13.0 | 17.0 | 19.8 |
| Group VII (Treatment) | 400 mg/kg (Hg.Cr) | 3.0 | 7.0 | 8.0 | 8.6 | 10.0 |
Table 4.
Percentage protection against castor oil-induced diarrhea by hydroethanolic plant extract (Hg.Cr) (n = 5; data are presented as the mean ± SEM).
Table 4.
Percentage protection against castor oil-induced diarrhea by hydroethanolic plant extract (Hg.Cr) (n = 5; data are presented as the mean ± SEM).
| Groups | Dose | Total Number of Feces | %Age Protection |
|---|---|---|---|
| Group I (Negative control) | 10 mL/kg (NS) | 4.0 ± 0.45 | 87.5 |
| Group II (Disease control) | 10 mL/kg (NS) | 32.0 ± 1.05 | 00.0 |
| Group III (standard) | 10 mg/kg (Loperamide) | 7.0 ± 0.71 | 78.1 |
| Group IV (standard) | 10 mg/kg (Verapamil) | 5.0 ± 0.45 | 84.4 |
| Group V (Treatment) | 100 mg/kg (Hg.Cr) | 21.0 ± 0.63 | 34.4 |
| Group VI (Treatment) | 200 mg/kg (Hg.Cr) | 19.8 ± 0.86 | 38.1 |
| Group VII (Treatment) | 400 mg/kg (Hg.Cr) | 10.0 ± 0.95 | 68.7 |
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Iqbal, I.; Ali, M.A.M.; Saqib, F.; Alamgir, K.; Mubarak, M.S.; Chaudhary, A.A.; El-Shazly, M.; El-Nashar, H.A.S. Comprehensive GC-MS Profiling and Multi-Modal Pharmacological Evaluations of Haloxylon griffithii: In Vitro and In Vivo Approaches. Pharmaceuticals 2025, 18, 770. https://doi.org/10.3390/ph18060770
AMA Style
Iqbal I, Ali MAM, Saqib F, Alamgir K, Mubarak MS, Chaudhary AA, El-Shazly M, El-Nashar HAS. Comprehensive GC-MS Profiling and Multi-Modal Pharmacological Evaluations of Haloxylon griffithii: In Vitro and In Vivo Approaches. Pharmaceuticals. 2025; 18(6):770. https://doi.org/10.3390/ph18060770
Chicago/Turabian StyleIqbal, Iram, Mohamed A. M. Ali, Fatima Saqib, Kinza Alamgir, Mohammad S. Mubarak, Anis Ahmad Chaudhary, Mohamed El-Shazly, and Heba A. S. El-Nashar. 2025. "Comprehensive GC-MS Profiling and Multi-Modal Pharmacological Evaluations of Haloxylon griffithii: In Vitro and In Vivo Approaches" Pharmaceuticals 18, no. 6: 770. https://doi.org/10.3390/ph18060770
APA StyleIqbal, I., Ali, M. A. M., Saqib, F., Alamgir, K., Mubarak, M. S., Chaudhary, A. A., El-Shazly, M., & El-Nashar, H. A. S. (2025). Comprehensive GC-MS Profiling and Multi-Modal Pharmacological Evaluations of Haloxylon griffithii: In Vitro and In Vivo Approaches. Pharmaceuticals, 18(6), 770. https://doi.org/10.3390/ph18060770
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