07-Physicochemical and biological properties assessment of Pituranthos chloranthus from Algerian - Enfermagem (2024)

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Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=tcyt20CyTA - Journal of FoodISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/tcyt20Physicochemical and biological propertiesassessment of Pituranthos chloranthus fromAlgerian SaharaMekhadmi Nour Elhouda, Mlik Randa, Ben Amor Safia, Boussebaa Walid,Segueni Bentahar Assia, Ramdani Messaoud, Ghemam Amara Djilani,Ayomide Victor Atoki, Mohammed Messaoudi & Adekunle Ismahil AdeniyiTo cite this article: Mekhadmi Nour Elhouda, Mlik Randa, Ben Amor Safia, Boussebaa Walid,Segueni Bentahar Assia, Ramdani Messaoud, Ghemam Amara Djilani, Ayomide Victor Atoki,Mohammed Messaoudi & Adekunle Ismahil Adeniyi (2024) Physicochemical and biologicalproperties assessment of Pituranthos chloranthus from Algerian Sahara, CyTA - Journal of Food,22:1, 2337001, DOI: 10.1080/19476337.2024.2337001To link to this article: https://doi.org/10.1080/19476337.2024.2337001© 2024 The Author(s). Published withlicense by Taylor & Francis Group, LLC.Published online: 18 Apr 2024.Submit your article to this journal Article views: 242View related articles View Crossmark datahttps://www.tandfonline.com/action/journalInformation?journalCode=tcyt20https://www.tandfonline.com/journals/tcyt20?src=pdfhttps://www.tandfonline.com/action/showCitFormats?doi=10.1080/19476337.2024.2337001https://doi.org/10.1080/19476337.2024.2337001https://www.tandfonline.com/action/authorSubmission?journalCode=tcyt20&show=instructions&src=pdfhttps://www.tandfonline.com/action/authorSubmission?journalCode=tcyt20&show=instructions&src=pdfhttps://www.tandfonline.com/doi/mlt/10.1080/19476337.2024.2337001?src=pdfhttps://www.tandfonline.com/doi/mlt/10.1080/19476337.2024.2337001?src=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1080/19476337.2024.2337001&domain=pdf&date_stamp=18 Apr 2024http://crossmark.crossref.org/dialog/?doi=10.1080/19476337.2024.2337001&domain=pdf&date_stamp=18 Apr 2024Physicochemical and biological properties assessment of Pituranthos chloranthus from Algerian Sahara Mekhadmi Nour Elhoudaa,b, Mlik Randac, Ben Amor Safiad,e, Boussebaa Walidf,g, Segueni Bentahar Assia h, Ramdani Messaoud i, Ghemam Amara Djilania, Ayomide Victor Atoki j, Mohammed Messaoudik and Adekunle Ismahil AdeniyilaDepartment of Biology, SNV Faculty, University of Chahid Hamma Lakhdar, El-Oued, Algeria; bLaboratory of Biodiversity and Applications of Biotechnology in the Agriculture Field, University of Chahid Hamma Lakhdar, El-Oued, Algeria; cNational Institute of Agronomic Research of Algeria, Adrar, Algeria; dLaboratory for Research on Medicinal and Aromatic Plants, Faculty of Nature Sciences and Life, University of Saad Dahlab, Blida, Algeria; eFaculty of nature sciences and life, University of Saad Dahlab, Blida, Algeria; fHigher School of Saharan Agronomy, El Oued, Algeria; gScientific and Technical Research Center in Physico-Chemical Analysis (CRAPC), Tipaza, Algeria; hLaboratory of Phytotherapy Applied to Chronic Diseases SNV Faculty, University of Setif 1, Setif, Algeria; iLaboratory of Natural Resources Valorization, SNV Faculty, University of Setif 1, Setif, Algeria; jDepartment of Biochemistry, Kampala International University, Ishaka, Uganda; kNuclear Research Centre of Birine, Ain Oussera, Algeria; lDepartment of Physiology, Kampala International Universit, Ishaka, UgandaABSTRACTMany plants are well-known for their use in traditional medicine to treat diseases associated with inflammations and oxidative stress. This study aimed to investigate a medicinal plant from the Saharan region of Algeria, Pituranthos chloranthus. Phytochemical analysis showed that it is rich in polyphenols (8.93 ± 0.736 mg GAE/g) and flavonoids (0.78 ± 0.023 mg EQ/g). The treatment of hypo-glycemic individuals with a P. chloranthus extract showed effectiveness when compared to control diabetic rats not treated with this extract. The latter showed a high average glucose at 3.11 ± 0.769 g/L when compared to the two treated rat groups, 0.8 ± 023 g/L and 0.54 ± 0.08 g/L when fed 500 and 250 mg/Kg body weight of P. chloranthus extract, respectively. The in vitro antioxidant evaluation, using the DPPH (IC50 = 0.156 ± 0.23 mg/mL) and FRAP (IC50 = 0.718 ± 0.09 mg/mL) tests and ratio to ascorbic acid 0.0037 ± 0.505 mg/mL showed an important antioxidant potential.ARTICLE HISTORYReceived 22 November 2023 Accepted 26 March 2024 KEYWORDSP. chloranthus; aqueous extract; HPLC; UPLC-ISI/MS; antidiabetic; antioxidant; anti-inflammatoryIntroductionHistorically, plants have served not only as nutritional sources but also as integral components of traditional reme-dies for various health issues, a fact documented in ancient Arabic, Chinese, Egyptian, Hindu, Greek, and Roman litera-ture (Kamatou et al., 2017). 70 to 95% of the population living in developing countries uses medicinal plants for pri-mary treatments due to the lack of access to prescribed medications. The biological activities of various chemical compounds, particularly secondary metabolites like alka-loids, flavonoids, polyphenols, polyterpenes, saponifies, ster-ols, and tannins, which are used in treatments as enzyme inhibitors, antioxidants, antidiabetics, antibacterials, antican-cer, antifungals, diuretics, etc., are responsible for these therapeutic effects (Khacheba et al., 2014; N’Guessan et al., 2009). Traditional medicine is based on the use of medicinal plants for the treatment of many diseases, including dia-betes which is a metabolic disease characterized by a disorder in the regulation of carbohydrate metabolism leading to hyperglycemia. Statistical results from 2017 showed that 451 million people were living with diabetes worldwide which will increase to 693 million by 2045 . Whereas, 7 million diabetics have been estimated in Africa (Bouchenak et al., 2018). Furthermore, diabetes is also char-acterized by hyperglycemia and insufficient secretion or action of endogenous insulin (Derouiche et al., 2017). Diabetes is generally accompanied by an increase in the production of free radicals or impaired antioxidant defenses (Ceriello, 2000) which induces a state of oxidative stress (Tiwari et al., 2013). Secondary metabolites are also known to function as anti-inflammatories, facilitating the body’s defensive reactions to diverse assaults, including physical, chemical, or biological origins (immune response) or infec-tious agents (Dièye et al., 2008). Inflammation can some-times be detrimental. The detrimental effect stems from the interplay of various factors, including the virulence of the pathogen, its prolonged presence, and the specific loca-tion of the inflammatory response. Pituranthos chloranthus, locally known as “Guezzah,” belongs to the Apiaceae family and is among the most utilized medicinal plants with sig-nificant antidiabetic and hypoglycemic properties in Algeria, as well as neighboring countries such as Tunisia and Morocco. It is recommended for the treatment of several diseases (Dahia et al., 2007; Yangui et al., 2009). The primary objective of our study is to evaluate the antidiabetic, anti-oxidant, and anti-inflammatory properties of the aqueous extract of Pituranthos chloranthus through in vitro and in vivo experiments.Material and methodsPlant materialThe plant species examined in this study is frequently employed in traditional medicine for its anti-diabetic proper-ties (Hammiche & Maiza, 2006), antioxidant effects, and anti- inflammatory characteristics. Pituranthos chloranthus (Coss. CONTACT Ayomide Victor Atoki atokiav@kiu.ac.ug Department of Biochemistry, Kampala International University, Ishaka, Uganda© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.CYTA – JOURNAL OF FOOD 2024, VOL. 22, NO. 1, 2337001https://doi.org/10.1080/19476337.2024.2337001http://orcid.org/0000-0002-7609-0386http://orcid.org/0000-0002-3536-6358http://orcid.org/0000-0003-1914-973Xhttp://www.tandfonline.comhttps://crossmark.crossref.org/dialog/?doi=10.1080/19476337.2024.2337001&domain=pdf&date_stamp=2024-04-16and Dur.) (Figure 1) was harvested in March 2022 from the province of El Menia (Algerian Sahara). The samples were dried at room temperature and protected from light. After drying, the specimens were cut and partially crushed using an electric mill, specifically the IKA/MF 10 basic Microfine grinder (Germany/Deutschland). They were then stored in tightly closed jars in a dry location until needed (Gao et al., 2015).Preparation of the aqueous extract by macerationTraditionally, water extraction commonly employed in tradi-tional medicine was carried out as described by Majhenic et al. (2007), and Bougandoura and Bendimerad (2012), with some modifications. P. chloranthus powder (10 g) was added to 100 mL of distilled water, then shaken for 21 h, and finally allowed to macerate for an additional 3 hours at 25°C. Subsequently, it was filtered using Whatman No. 1 filter paper. After filtration, the extract was dried in an oven at a temperature not exceeding 50°C. The resulting extract obtained in the form of a thin solid film was scraped off using a flat spatula, stored in closed glass vials covered with aluminum foil, and kept in a 4°C refrigerator until ready for use within 30 days. Qualitative phytochemical tests were conducted on the aqueous P. chloranthus extract to detect polyphenols (flavonoids, tannins), reducing sugars, alkaloids, and saponins.High-performance liquid chromatography (HPLC)In the current work, we used a High-Performance Liquid Chromatography (HPLC) system, type Shimadzu LC 20 AL equipped with a universal injector (Hamilton 25 µL). The analytical column used was a Shim-pack VP-ODS C18 (4.6 mm × 250 mm, 5 µm), type Shimadzu. A UV-VIS detector SPD 20A (Shimadzu) was used. The mobile phase was a mixture of acetonitrile and acetic acid 0.1%. The contents of the mobile phase were filtered before use through a 0.45 μm membrane filter, sonicated, and pumped from the sol-vent reservoir to the column at a flow rate of 1 mL/min. According to Chouikh et al. (2018), and 20 µL of plant extract solution was injected into the flow of the mobile phase. We adjusted the high pressure that drives the mobile phase by using a pump. The separated compounds shall be deter-mined using the column for 50 min with the mobile phase in the effluent detected at λ = 268 nm and to the computer which records the results as chromatographic curves. In this study, the quantification of some peaks was compared by calibration of standards as mentioned below.Ultra-performance liquid chromatography-mass spectrometry (UPLC/MS-MS)For polyphenol standards optimization, we employed direct injection of 5 µL without a column (Restek Ultra C18 3 µm 150 × 4.6 mm) on a SHIMADZU 8040 Ultra-High Sensitivity system with UFMS technology, equipped with a binary pump Nexera XR LC-20AD. Gradient elution was performed using a mobile phase composed of solvent A (water with 0.1% formic acid) and solvent B (methanol), with a total flow rate of 0.4 ml/min. The ESI conditions were as follows: CID gas, 230 KPs; conversion dynode, −6.00 Kv; DL temperature, 250 ◦C; nebulizing gas flow, 3.00 L/min; heat block, 400 ◦C; drying gas flow, 15.00 L/min. A summary of MS/MS detection parameters is presented in Table 1.Animal materialOur study was conducted on female Wistar Albino rats aged between 9 and 11 weeks, with weights ranging from 150 g to 180 g. These animals were brought from the Pasteur Institute in Algiers, and raised in an animal facility at the Faculty of Natural and Life Sciences at the University of El-Oued. The rats were acclimatized and housed for two weeks prior to the com-mencement of the experiment under controlled conditions of light and temperature (12-hour light cycle, temperature main-tained at 24°C). They were kept in plastic cages filled with sawdust, which was changed three times a week throughout the duration of the experiment. All procedures involving the treatment and handling of rats were conducted in accordance with the guidelines outlined in the manuals for the care and use of experimental animals, as per the standards established by the Canadian Council on Animal Care (CCAC) in 1984. In addition, these experiments received approval from the ethical committee, as evidenced by the report under code 35 EC/ DCMB/FNSL/EU2024. The female rats weight change was mon-itored twice a week using an electronic scale.Study of the toxicity of P. chloranthus extract on ratsThe acute toxicity of P. chloranthus extract was assessed using 15 female Wistar rats, randomly allocated into three groups, each comprising 5 rats. The groups were designated as follows: G1 (untreated), G2 (treated with 500 mg/kg of Figure 1. Pituranthos chloranthus.2 M. N. ELHOUDA ET AL.body weight), and G3 (treated with 1000 mg/kg of body weight). Prior to administration, the rats underwent a 16- hour fasting period, with access to water provided ad libi-tum. Each group received a single daily dose of the aqueous extract orally using a gavage syringe, with all rats being maintained under consistent conditions throughout the study. The signs of toxicity and the weight of the rats were recorded during the duration of the experiment (7 days) according to the method of Pissang et al. (2022) with some modifications.All individuals were monitored at regular intervals during the initial 24 hours post-administration, with particular atten-tion given to the first 4 hours. Subsequently, they were monitored daily for the duration of the experiment following the administration of the solution. Weight variations were calculated and recorded (El Kabbaoui, 2019).Evaluation of the antidiabetic activity of P. chloranthus in vivoAfter the toxicity test, another group of 15 adult Wistar Albino rats (between 150 g and 190 g) were injected with alloxan (Sigma, UK) after fasting for 12 h, intraperitoneal injection, prepared freshly in a volume of 5 mL of physiological water solution at a dose of 150 mg/kg b.w., was induced (Sabu et al., 2002). After the injection, the water bottles were replaced with those containing 5% glucose solution for 24 h to overcome any normal or fatal hypoglycemia that could occur. Alloxan- induced destruction of pancreatic β cells and the massive release of insulin (Chahlia, 2009).Induced diabetes was confirmed in each of the rats to be studied after 48 h by measuring fasting blood sugar (Owoyele et al., 2005). Treatment with P. chloranthus extract was initiated 48 h after diabetes induction (El Kabbaoui, 2019), they were administered orally (gastric gavage) daily for a total treatment duration (21 days). All rats were divided into four groups, 5 rats for each. Normal group on a standard diet (G1); diabetic group subjected to a standard diet (G2); diabetic group subjected to a standard diet and treated daily with 500 mg/Kg b.w. of P. chloranthus extract (G3); diabetic group subjected to a standard diet and treated daily with 250 mg/Kg b.w. of P. chloranthus extract. During the treat-ment period, two parameters were monitored at regular intervals, namely: weight and blood sugar. In addition, bio-chemical parameters were taken into consideration i.e. glu-cose, triglycerides,total cholesterol, serum urea, creatinine, aspartate aminotransferase (ASAT) and alanine aminotrans-ferase (ALT). Moreover, histological sections of the liver, heart, kidneys and spleen were made according to the tech-nique described by Houlot (1984).Evaluation of the in vitro antioxidant activity of P. chloranthusThe capacity of an extract or a plant compound to eliminate free radicals was estimated in several ways.Free radical scavenging test (DPPH)This method involves completely dissolving 4 mg of DPPH in methanol. 12 mg of the extract was dissolved in 2 mL of methanol. 200 μL of the extract dissolved in methanol was added to 800 μL of the DPPH solution (3 repetitions), after which the solution was hom*ogenized and incubated in the dark for 30 minutes. Finally, the absorbance of the prepared solutions were measured at 517 nm by a spectrophotometer (Azouaou et al., 2020; Dziri et al., 2012). Ascorbic acid was used as a standard.Table 1. LC/MS detection parameters for analysed compounds in P. chloranthus (negative and positive ionization mode) (n = 3).ID# NameMolecular FormulaMolecular WeightESI Charge (±) m/zRet. Time Height Area1 Hydroxy quinolin C9H7NO 145.16 + 146.0500>101.0000 5.101 3541 320412 Thymol C10H14O 150.22 + 151.7500>88.1000 0.768 7156 430273 2-Methoxybenzoic Acid C8H8O3 152.15 + 153.0500>135.0500 5.370 61958 14841264 4-mythoxybenzoic Acid C8H8O3 152.15 + 153.0500>70.7500 3.423 20446 2060355 Coumaric acid C9H8O3 164.16 (+) 165.1000>69.1500 0.777 3436371 237851066 Kojic acid C6H6O4 142.11 (+) 143.0000>69.0500 3.231 45890 2937747 Ferulic acid C10H10O4 194.18 (+) 194.9000>177.1500 7.206 25396 1513128 Naringenin C15H12O5 272.25 (+) 272.9500>177.0000 5.321 21123 2806749 Beta carotene C40H56 536.87 (+) 537.2000>23.1000 23.551 1150 477510 Keampferol C15H10O6 286.24 (+) 287.1000>255.2500 1.121 6479 4902211 Quercetin C15H10O7 302.23 (+) 303.1000>225.1500 9.032 8628 8371412 Vanillin C8H8O3 152.15 (+) 153.1000>65.1500 5.672 28976 29226413 Chrysin C15H10O4 254.24 (+) 255.0500>69.0500 22.275 2329 1004114 Myricetin C15H10O8 318.23 (+) 336.2500>238.4000 22.370 1924 534515 Folic acid C19H19N7O6 441.14 (+) No peak is found in Window/Band range.0.000 0 016 Rutin C27H30O16 610.5 (+) 611.2000>303.2000 6.689 16125 9121717 Catechin hydrate C15H16O7 308.28 (-) 306.9500>93.0500 1.092 2692 1180918 Sinapic acid C11H12O5 224.21 (-) 223.0000>193.1000 0.867 2612 745819 4-hydroxy coumarin C27H30O16 162.14 (-) No peak is found in Window/Band range.0.000 0 020 3,5-Dihydroxybenzoic acidC7H6O4 154.12 (-) 153.1000>109.1000 3.232 4235679 2987793821 Caffeic acid C9H8O4 180.16 (-) 179.1500>135.0500 5.415 65027 45725822 Cis-p.coumaric acid C9H8O3 164.16 (-) 163.1500>119.1500 20.118 11731 19000823 Syringic acid C9H10O5 198.17 (-) 196.9500>182.0000 6.616 3081 1763024 Salysilic acid C7H6O3 138.12 (-) 137.1000>93.1500 7.238 707376 407931325 Gallic acid C4H4O4 170.12 (-) 169.1000>125.0500 21.070 538 216226 Luteonil C15H10O6 286.24 (-) 284.9500>151.0000 21.886 543 97227 Hespertin C16H14O6 302.28 (-) 300.9000>255.2500 16.770 7876 5689728 Chlorogenique acid C16H18O9 354.31 (-) 352.9000>177.1500 0.870 3928 15031CYTA - JOURNAL OF FOOD 3Ferric reducing-antioxidant power (FRAP) iron reduction testThe activity of the reducing power was determined according to the method of Oyaizu (1986), Benzie and Strain (1996). 0.25 mL of the aqueous extract of the plant, at different concentrations (between 0.0625 and 1 mg/mL) was added to 0.625 mL of a buffer solution (sodium phosphate: 0.2 M, pH = 6.6). The mixture was added to 0.625 mL of a 1% solution of potassium ferricya-nide [K3Fe (CN)6]. Test tubes containing the reaction mix-ture were incubated in a water bath at 50°C for 20 min. To stop the reaction, 2.5 mL of 10% trichloroacetic acid (TCA) was added. All tubes were centrifuged at 3000 rpm for 10 min. 0.625 mL of the supernatants were transferred to separate test tubes, into which 0.625 mL of distilled water and 0.125 mL of a freshly prepared solution of ferric chloride FeCl3 0.1% were already present. The absorbance reading of the reaction mixture was measured at 700 nm against a similarly prepared blank. The positive control was represented by ascorbic acid.Evaluation of anti-inflammatory activity in vitroTo evaluate the anti-inflammatory activity, we used egg whites, according to the protein denaturation method described by Chandra et al. (2012) with some modifica-tions. We mixed 200 µL of fresh egg albumin with 2.8 mL of phosphate buffer saline (PBS, pH 6.4) and 2 mL of aqueous extract at different concentrations (0.0625 mg/ mL). For the control, it consists of 2 mL of distilled water instead of the aqueous extract. Acetylsalicylic acid was used as the reference drug. Then, the mixtures were left at room temperature for 15 min, and then heated in a water bath at 70°C for 5 min. After cooling, the absor-bance was measured at 660 nm. The percentage inhibition (PI%) of protein denaturation was calculated according to the formula of Bouhlali et al. (2018):Evaluation of anti-inflammatory activity in vivoThis activity was evaluated according to the method of Amezouar et al. (2013) and Harchaoui (2019) with some modifications. The rats were starved for 16 h before admin-istering the product intragastrically. The rats were divided into four groups with five individuals for each. The first group was a negative control, the second was a positive control (reference), the third was treated with 500 mg/Kg b. w. and the last was treated with 250 mg/Kg b.w. The percentage of inhibition of edema was calculated according to the following formula:- Dn: the diameter of the leg at time t after the injection of carrageenan.- D0: the initial diameter of the paw before causing edema.Statistical analysisStatistical analysis was carried out using the statistical soft-ware R 4.2.3. The analysis included Principal Component Analysis (PCA) and ANOVA.ResultsIn the present study, we obtained a dry extract weighing 1.012 g, corresponding to a yield of 10.12% but displayed low levels of alkaloids, terpenes, and tannins. Saponins and reducing sugars were found to be absent (Table 2).Based on the qualitative results obtained showing the significant presence of polyphenols and flavonoids, we selected these last two metabolites to carry out quantita-tive tests. According to the results, we saw that our extract contains polyphenols at a rate of 8.93 ± 0.74 mg GAE/g and also flavonoids estimated at 0.78 ± 0.02 mg EQ/g (Figure 2).The present experiment revealed the presence of poly-phenols and flavonoids in the aqueous extract of P. chloranthus. According to Chabane et al. (2013), flavo-noids and tannins play a crucial role in the hypoglycemic effect of this plant. Additionally, they contribute to the body’s defense against free radical damage through their antioxidant and anti-enzymatic properties, as well as in limiting inflammatory reactions in various tissues (Hertel, 2003; Stoclet & Schini-Kerth, 2011). In addition, they are responsible for hepato-protective activity (Sangare et al., 2012). It also contains biological properties such as anti- allergic, antiviral, antibacterial and anti-tumor (Middleton, 1998). The antioxidant property of polyphenols is likely to prevent molecular and cellular oxidative damage inducing various pathologies (Amiot et al., 2009). In our study, the quantification of polyphenols revealed higher concentra-tions compared to previous research. Seladji (2013) reported 4.089 ± 0.38 mg EAG/g in the aqueous extract of P. chloranthus, while Ben Nasr et al. (2020) found 1.36 ± 0.02 mg EAG/g. In contrast, reported a higher quan-tity of polyphenols in this plant, amounting to 11.22 ± 0.98 mg EAG/g. The variation in phenol values across dif-ferent extracts can be attributed to the presence of Table 2. Phytochemical test of aqueous extract of P. chloranthus.Secondarymetabolites Reactions ObservationPolyphenol +++ Greenish-blueFlavonoids ++ YellowTannins + Greenish-brownAlkaloids + White precipitation (Mayer’s reagent)+ Reddish-brown precipitation (Drajendorf’s reagent)Saponins - No foamReducing sugars - Absence of brick red precipitationTerpenes + Appearance of a red-purple ring(+): Weakly positive test, (++): Positive test, (+++): Strongly positive test, (-): Negative test. 4 M. N. ELHOUDA ET AL.diverse phenolic compounds and their respective propor-tions. Their characteristics varies depending on their che-mical structure and the environment in which they are situated, including factors such as acidity and basicity (Hayouni et al., 2007; Mellouk, 2013). Regarding the quan-titative results of flavonoids, our results were close to those found by Khacheba et al. (2014).High-performance liquid chromatography (HPLC)Phenolic compounds detected in the P. chloranthus fraction by HPLC are listed in Table 3.The HPLC analyzes have identified seven phenolic compounds in the aqueous extract of P. chloranthus, e.g. gallic acid, chlorogenic acid, vanillic acid, caffeic acid, p-coumaric acid, rutin, and quercetin (Table 3, Figures 2 and 3).Ultra-performance liquid chromatography-mass spectrometry (UPLC/MS-MS)UPLC/MS chromatography profiles detailing the detection and quantification of the 21 separated phenols are shown in Figure 4. The detected compounds in the aqueous extract of P. chloranthus are kojic acid, catechin hydrate, synaptic acid, chlorogenic acid, kaempferol, 2-methoxybenzoic acid, 4-methoxybenzoic acid, thymol, salicylic acid, coumaric acid, vanillin, naringenin, syringic acid, ferulic acid, 3,5 dihydrox-ybenzoic acid, p-coumaric acid, caffeic acid, quercetin, hespetin, hydroxyquenolin, and rutin.Toxicity of P. chloranthusThe oral administration of the aqueous extract of P. chloranthus at doses of 500 and 1000 mg/kg body weight to rats did not elicit any signs of acute toxicity throughout the 14-day observa-tion period. There were no observable effects on the skin, hair, eyes, behavior, somato-motor activity, sleep, or mortality. A normal course of body growth was observed (Figure 5).The oral administration of P. chloranthus extract via gavage resulted in a natural increase in the growth rate for the two treated batches compared to the control group. Importantly, no signs of toxicity were observed during the study period. This observation indicates the non-toxic nature of these doses. Consequently, we proceeded to investigate its antidiabetic and anti-inflammatory activity at these doses in vivo. 0 10 20 30 40 min0255075100mVDetector A:268nm Figure 3. HPLC profile of the ethyl acetate fraction of P. chloranthus.Table 3. The concentration (µg/mg extract) of major phenolic compounds identified by HPLC in extracts of P. chloranthus (n = 3).Compounds (µg/ml) Retention time ExtractGallic acid 5.29 4.84Chlorogenic acid 13.39 11.45Vanillic acid 15.53 0.47Caffeic acid 16.28 0.5P-Comaric acid 23.82 1.20Rutin 28.37 20.82Quercetin 45.05 7.93y = 0.0928x + 0.0797R² = 0.993900.050.10.150.20.250.30.350.40 1 2 3 4Abserbance (765 nm)Gallic acid oncentration (mg/mL)y = 0.455x + 0.146R² = 0.99600.20.40.60.811.21.41.60 1 2 3 4Abserbance (420 nm) Quercetin concentration (mg/mL)Figure 2. Calibration curve for gallic acid (total phenols) and quercetin (flavonoids).CYTA - JOURNAL OF FOOD 5Effect of diabetes induction in ratsIn this study, rats made diabetic by intraperitoneal injection with 150 mg/kg of Alloxan (Sabu et al., 2002) showed hyper-glycemia after 48 hours after the injection compared to con-trol rats (Figure 6). This was consistent with the results of other previous studies. This effect was explained by the cytotoxicity of Alloxan to pancreatic Langerhans cells, which can cause severe necrosis of the latter and thus causes a significant drop in insulinemia, which induces type 1 dia-betes (Lenzen, 2008; Szkudelski, 2001).After 48 h of diabetes induction, the hypoglycemic activ-ity of the aqueous extract of P. chloranthus was studied by following the evolution of body weight (Figure 7). During the first days of treatment, severe weight loss was recorded in the three groups of diabetic rats (control and treated), while the healthy control exhibited normal growth develop-ment. Auroba (2010) indicated that the loss of body weight in diabetic rats was caused by alloxan. From the second week, an increase in body weight was observed in all treated and control groups.Figure 4. UPLC-ISI/MS profiles for the phenolic compounds detected in the aqueous extract of P. chloranthus.6 M. N. ELHOUDA ET AL.On the 16th day of the experiment, the treatments admi-nistered with P. chloranthus extract resulted in a reduction in blood sugar levels compared to the untreated diabetic group. This explains the biological potential of the extract to reduce hyperglycemia induced by alloxan.The decrease in body weight was generally attributed to the stimulation of gluconeogenesis. The acceleration of pro-tein and fat catabolism, due to the carbohydrate deficit. This deficit was due to continuous lipolysis by a lack of insulin because of the onset of diabetes leading to a significant loss of body weight after an increase in muscle atrophy and loss of tissue proteins (Daisy et al., 2012; Sathishsekar & Subramanian, 2005). The increase and improvement in body weight was perhaps due to the protective effect of our plant against weight loss associated with diabetes disease and ensuring normal growth through the activation of structural protein synthesis (Gandhi et al., 2012). In addi-tion, this plant can stimulate pancreatic secretion of insulin, which promotes the storage of lipids and triglycerides (Babu et al., 2007; Kim et al., 2006) to control hyperglycemia.After the injection of alloxan, the results showed an increase in blood sugar levels in control diabetic rats and rats treated with the extract, this effect is due to the administration of alloxan leading to the selective destruction of β cells of the islets of Langerhans, which leads to permanent hyperglycemia (Etuk, 2010). Alloxan is a glucose analog that preferentially accumulates in pancreatic β cells via the GLUT2 transporter and thus acts to destroy pancreatic cells (Pinheiro et al., 2011).The effect of P. chloranthus extract represented a reduction in blood sugar levels, which was evident from 0501001502002501st 4th 7thBody weight (g)DaysControl 1000 mg/kg b.w. 500 mg/kg b.w.Figure 5. Evolution of weight (g) of rats treated with P. chloranthus extract.140145150155160165170175180185190D1 D4 D7 D10 D13 D16 D19 D21Evolution of body weight (g)DaysControl Diabetic D500 mg/kg D250 mg/kgFigure 6. Evolution of body weight (g) of treated and control rats during the experiment.0123456D1 D4 D7 D10 D13 D16 D19 D21Blood suger (g/L)DaysControl Diabetic D500 mg/kg D250 mg/kgFigure 7. Evolution of blood sugar (g/l) for all batches of rats during the experiment.CYTA - JOURNAL OF FOOD 7the 16th day of the experiment in the group treated with this extract. Thus, the molecules included in the composition of this plant can influence the stimulation of insulin secre-tion at the level of pancreatic β cells by activating the SIRTUIN 1 enzyme, which stimulates the signaling chain of insulin secretion (Jang et al., 2000; Kilic et al., 1998). In addition, they can influence the absorption of glucose and its use by different tissues.Variations in organ weightIn the present study, a significant increase in the weight of organs including the liver, kidneys, and heart was observed in untreated diabetic rats compared to the other groups (Figure 8). While the weight of the spleen did not present a differencebetween the treated (G3, G4) and control (G1) groups, except the treated group (G2).According to studies carried out on the diabetogenic effect of alloxan, a loading of the liver is marked by the increase in the hepatic concentration of free TG and AG, which is due to their mobilization of adipose tissues towards the liver and the reduction of their degradation. This increase is caused by the absence of the hormone insulin, which leads to the breakdown of fats and glycogen under the influence of the hormone glucagon (Mihaela et al., 2006). Concerning the relative weight of the kidneys, its increase in diabetic rats is due to the proliferation of glomerular cells (Kumar et al., 2007).In our study, we found that the treatment of diabetic rats with P. chloranthus extract attenuated the diabeto-genic effect of alloxan and reduced the degree of dia-betes as well as improving the lipo-distribution mechanisms controlling overloads lipids in non-adipose tissues such as the liver and heart (Unger, 2002). In addi-tion, it contains chemical compounds that eliminate free radicals from pancreatic β cells, which leads to improving their efficiency in secreting insulin, which contributes to the conversion of glucose into glycogen (Ghorbani et al., 2019). It is attributed to the fact that diabetes causes a deficiency of hemoglobin (accompanied by high crea-tine levels (Babu et al., 2007), which leads to anemia, that causes the spleen to absorb iron and maintain a constant blood flow in the capillaries (Ghorbani et al., 2019). This decrease in hemoglobin levels can be explained by the increase in antibodies released during the onset of dia-betes, resulting from the destruction of β cells in islets of Langerhans under the effect of autoimmunity (Beam, 2001).Analysis of biochemical parametersThe results illustrated in the Table 4 below showed the antidiabetic potential of the aqueous extract of P. chloranthus on rats treated with it (250-500 mg/Kg b.w.).The application of P. chloranthus extract on hypoglyce-mic individuals showed the existence of a very significant effectiveness compared to controls whose diabetic rats not treated with this extract recorded a very high average glucose (3.11 ± 0.77 g/L) compared to treated individuals (D500 = 0.8 ± 023; D250 = 0.54 ± 0.08) (Table 4). Idem, all the other biochemical parameters tested were very high in untreated diabetic individuals whose other diabetic sub-jects treated with the P. chloranthus extract recorded values similar to or close to the control individuals (Table 4).Analysis of the correlation of doses and biochemical parametersThe analysis of the matrix revealed high correlation coeffi-cients, 65.31% of which their variables were significantly correlated (Figure 9). The eigenvalues representing the dosage of biochemical parameters of the groups studied on the axes are average, 82.9% for the first axis and 9.7% for axis 2, thus giving a good contribution to the total variance. The total information explained by the first three axes from the PCA was 92.6%.00.511.522.533.544.5G1 G2 G3 G4Relative weight % LiverKidneyHeartSpleenFigure 8. Variation in the weight of internal organs of female rats after sacrifice.Table 4. Biochemical parameters.Lots Control Diabetic D500 D250Glucose (g/L) 0.75*** ± 0.28 3.11 ± 0.77 0.8*** ± 0.23 0.54 ± 0.08Cholesterol (g/L) 0.45 ± 0.05 0.89 ± 0.10 0.47 ± 0.06 0.51 ± 0.02TG (g/L) 0.48 ± 0.05 1.097 ± 0.10 0.55 ± 0.07 0.84 ± 0.01Urea (g/L) 0.38 ± 0.47 0.86 ± 0.14 0.46 ± 0.03 0.79 ± 0.12Creatinine (mg/L) 6.022 ± 0.47 8.70 ± 1.21 6.53 ± 0.56 7.92 ± 0.35TGO (UI/L) 47.22 ± 5.01 96.97 ± 16.67 61.45 ± 10.43 74.35 ± 2.5TGP (UI/L) 102.38 ± 6.34 171.7 ± 4.41 110.27 ± 20.10 137.5 ± 33.79***Very highly significant difference. 8 M. N. ELHOUDA ET AL.The positive part of axis 1 was explained by triglyceride, GTP and GOT, while the positive part of axis 2 was explained by glucose. On the other hand, the negative part was explained by urea, creatinine and total cholesterol. The pro-jection of the groups indicated the presence of a negative correlation between G1 and G3 with a disturbance of bio-chemical parameters. While G2 and G4 are positively corre-lated with these (Figure 10).According to the analysis of ANOVA and PCA, the results indicated a significant relationship between groups 1 and 2 in stabilizing biochemical parameters, while the other groups exhibited a notable association with the disruption of these parameters (Figure 10).In our study, a very highly significant increase in the serum concentration of total cholesterol was recorded in rats rendered diabetic by alloxan because insulin inhibited the 3-hydroxy-3-methyl-glutaryl coenzyme. Insulin deficiency results in the inability to activate lipoprotein lipase, thereby causing hypertriglyceridemia and hypercho-lesterolemia (Shirwaikar et al., 2005). The differences in glu-cose levels between treated and non-treated rats may be explained by decreasing the expression of genes that control gluconeogenesis, increasing the storage of glucose in the liver and reducing the degradation of glycogen, inhibiting glucose transporters in the intestines (Sarkhail et al., 2007), and the conversion into fatty acids in the liver (Palsamy & Subramanian, 2008). Additionally, the reduction in uric acid levels may be due to the reduction in lipid peroxidation of triglycerides and cholesterol, while the elevation of these metabolites may increase uric acid synthesis (Derouiche, 2020). As for the increase in creatinine being explained by the effect of diabetes on the kidneys, it has been found that high doses of alloxan cause necrosis of the renal tubules (Nicolas, 2010). This result was explained by the Figure 9. Circle of correlations, projection of variables on the plane (1x2).Figure 10. Combination analysis between PCA and ANOVA of the biochemical parameters of the studied groups.CYTA - JOURNAL OF FOOD 9accumulation of amino acids (alanine) in the serum coming from the degradation of protein compounds in the body (Bouiddouh, 2012). As a result, these amino acids can be transformed under the action of serum transaminases into carboxylic compounds such as α ketoglutarate and pyruvate. Which then implies a strong enzymatic activity of TGO and TGP. This can also be explained by the hepatotoxic effect of alloxan (Bouiddouh, 2012).Histological sectionsHistological sections made from treated and untreated indi-viduals showed congestion of blood and vacuolation of cytoplasm in the liver of untreated diabetic rats; unlike the diabetic individuals treated with P. chloranthus extract pre-sented just one small congestion of the blood (Figure 11). Regarding the kidneys, the untreated batches showed some histopathological changes represented by a clear dilation of Bowman’s capsule, congestion of the blood vessels, conges-tion within the glomeruli and dilation of the tubules. Furthermore, the observation of an improvement in renal tissue following treatment with P. chloranthus extract was noted. This improvement was characterized by the restora-tion of glomerular size with reduced inflammation. Additionally, slight blood congestion was observed in certain areas with the administration of a higher dose (500 mg/kg) (Figure 11), while examination of the heart revealed conges-tion of cardiac muscles in untreated diabetic individuals. However, both treated groups exhibited slight histological changes, manifested by the divergence of cardiac muscle fibers, particularly at lower doses. These changes could potentially be attributed to the effects of Alloxan (Figure 11). Concerning the spleen, normal morphology was observed in the control batches and the treated diabetic batches, as for the untreated diabetic batch, the white pulp was densely compared to the red pulp, because it contains cells, which participate in the immune responseresulting an inflammation in the spleen (Figure 11).In this study, we noted a complete absence of fatty agglomerations in the liver of treated groups, whilst the presence of blood congestion was observed in the diabetic group. According to Mir et al. (2008), all these signs are caused by poor blood drainage following hepatic venous obstruction, causing cessation or disruption of blood flow through hepatic visceral cells. On the other hand, a vacuolation of cytoplasm in diabetic and untreated rats was observed. It was caused by damage to liver cells result-ing from immunological causes or from the toxic effect of alloxan (oxidative stress) resulting from the accumulation of radicals which cause the destruction of liver cells by lipid peroxidation of cell membrane or mitochondrial mem-branes, causing the emergence of an inflammatory and immune response, leading to severe blood congestion, hepatic hemorrhage, and the destruction of hepatocytes, as described by Majumdar et al. (2008).The untreated group exhibited some histopathological changes represented by a clear dilation of Bowman’s cap-sule, congestion of the blood vessels, congestion within the glomeruli and dilation of the tubules. It has been observed that kidney necrosis is among the changes induced by dia-betes, as highlighted by Teoh et al. (2010). Oxidative stress plays a significant role in the development of diabetic nephropathy, as emphasized by Abo-Salem et al. (2009).The notable improvement observed can be attributed to the active terpenoid and flavonoid components present in the tested plant. These compounds contribute to a reduction in lipid peroxidation and an increase in antiox-idants in the rats. This leads to a decrease in accumulated fat and hypoglycemia, ultimately resulting in the enhancement of renal tissues (Abd-Alla et al., 2014). In the untreated Figure 11. Histological section of the different organs of treated and untreated individuals (G: 40x10) (BC: Blood congestion; VC: Vacuolization of the cytoplasm).10 M. N. ELHOUDA ET AL.diabetic group, the white pulp exhibited denser character-istics compared to the red pulp. This denser appearance may be attributed to the presence of cells involved in the immune response, leading to inflammation. It is plausible that alloxan toxicity contributed to the occurrence of this reaction (Tennenbaum et al., 2023).Evaluation of the antioxidant activity of P. ChloranthusIn this study, the utilization of free radical trapping test (DPPH), revealed an antioxidant potential of our extract, demonstrating an inhibitory concentration of 0.156 ± 0.23 mg/mL. Comparatively, ascorbic acid exhibited exceptionally strong anti-radical activity, with an IC50 value of 0.0037 ± 0.505 mg/mL. The lower the IC50 value, the more the extract is considered a powerful antioxidant. On the other hand, the iron reduction test (FRAP) revealed that our extract has a lower reducing activity (0.718 ± 0.17 mg/mL) when com-pared with the standard ascorbic acid (0.064 ± 0.08 mg/mL). It was clear that our extract has considerable modest reduc-tive power. This discrepancy could be explained by the variation in the phenolic compounds present in our extract.Our extract showed lower antioxidant activity than the aqueous extract of Kadouri and Tabaa (2019) as well as Ben Nasr et al. (2020) who found an inhibitory concentration equal to 0.54 ± 0.011 mg/mL, and 0.44 ± 0.01 mg/mL, respec-tively. On the other hand, Seladji (2013) recorded a higher antioxidant activity with an IC50 = 501.7 mg/mL. In another study, the methanolic extract of P. chloranthus was 2.01 ± 0.34 µg/ml (Yangui et al., 2009). The presence of inhibitory capacity in the extract was attributed to the presence of active substances capable of scavenging or capturing the free radicals. Mohammedi (2013) confirmed the existence of a direct correlation between flavonoid content and antiox-idant capacity.Regarding the effectiveness of iron reduction in our plant, it exceeded the values reported by Seladji (2013), who found 7.32 ± 0.69 mg/mL. Additionally, Dridi (2018) reported values of 0.0389 mg/mL and 0.0659 mg/mL for extracts obtained before and after the flowering period, respectively, using a methanolic extract of the same species. The functional groups present in phenolic compounds play an important role in the adsorption and neutralization of free radicals, quenching of singular and triplet oxygen or decomposition of peroxides (Amarowicz et al., 2004; Zheng & Wang, 2001). In addition, the antioxidant activity is linked to the structure and nature of the compounds contained in the extract (Baghiani et al., 2010). Phenolic compounds can be free radical acceptors or hydrogen donors or by readily donating an electron or proton (Yordi et al., 2012).Anti-inflammatory activity in vitro and in vivoThe results of the anti-inflammatory activity in vitro indicated an inhibition percentage equal to 82.32% with concentra-tions of 1 mg/mL of the extract, while acetylsalicylic acid presented an inhibition rate of 89.64% for the same concen-tration. On the other hand, the inhibitory concentration of the extract and acetylsalicylic acid was almost identical, i.e. 0.305 mg/mL and 0.203 mg/mL, respectively.Regarding the in vivo test, we observed that the inflam-mation caused by carrageenan increased as a function of time, reaching a maximum of 6.96 ± 0.02 mm at the third hour. In addition, we have noted that acetylsalicylic acid (reference) gradually reduced the edema.Furthermore, the P. chloranthus extract exhibited a highly significant inhibition against inflammation, with the dia-meter reduced to 5.74 ± 0.01 mm for the extract dose of 250 mg/kg body weight and 4.81 ± 0.02 mm for the extract dose of 500 mg/kg body weight. The progression of the kinetics of edema inhibition showed that the two extracts have a considerable anti-inflammatory effect, at 5 hours, with a very similar effect of Diclofinac. We can deduce the order of this inhibition as follows: Aspirin ˃ Diclofenac ˃ Extract (500 mg/Kg b.w.) ˃ Extract (250 mg/Kg b.w.) (Figure 12).Given the limited research on our species as an anti- inflammatory substance, we compared our results to those reported by Bouhlali et al. (2018) on seeds of Cuminum cyminum, which belongs to the same family. They reported an IC50 value of 234.87 µg/mL. In addition, Derrouiche et al. (2020) found that the IC50 equal to 247.12 µg/mL. The anti- inflammatory potential increased with increasing concentra-tions of P. chloranthus extract. These results, when compared to those of acetylsalicylic acid, the reference anti- inflammatory, indicated that the inhibitory effect of the tested extract varied depending on the dose. Consequently, the extract demonstrated potent anti- inflammatory action, particularly in preventing the thermal denaturation of egg white proteins. This effect can be attrib-uted to the presence of the B vitamins group, notably vita-min B6, which possesses anti-inflammatory properties, along with vitamin B12 (Hosseinzadeh et al., 2012; Paez-Hurtado et al., 2023). Several studies report the anti-inflammatory potential of plants belonging to the Apiaceae family. After 3 h of carrageenan injection, the methanolic extract of 0 20 40 60 80 100ControlDiclofenacAspirineD500 mg/kgD250 mg/kgInhibition (%) Figure 12. Inhibition rate (%) of different standards in comparison with P. chloranthus extract.CYTA - JOURNAL OF FOOD 11Thapsia garganica at a dose of 500 mg/Kg caused an inhibi-tion (48.7%) lower than that of our plant.The inhibition of plantar edema in rats suggests the pre-sence of bioactive molecules in the plant extract, particularly phenolic compounds such as flavonoids and tannins. These compounds potentially function as non-steroidal anti- inflammatories by inhibiting the enzymatic activity of cyclooxygenase (COX) and lipoxygenase (LOX). Consequently, theyhelp reduce the production of pro- inflammatory mediators during the immune process. Additionally, flavonoids, as a subgroup of polyphenols, are known to modulate prostaglandin biosynthesis (Adebayo et al., 2015).ConclusionWith the use of in vitro and in vivo tests, the current study enabled us to determine the biological characteristics of the Algerian Saharan medicinal plant. Its chemical compo-sition, which is particularly rich in polyphenols, confers both therapeutic and commercial significance. According to the findings of the current study, the compounds pre-sent in P. chloranthus exhibit promising pharmacological properties that could be valuable in traditional medicine. These include anti-diabetic, antioxidant, and anti- inflammatory properties.Disclosure statementNo potential conflict of interest was reported by the author(s).FundingThis research was not funded.Ethical approval and consent to participateThe experimental procedures adhered to ethical guidelines and fol-lowed an approved protocol. In addition, these experiments were approved according to the Algerian ethical committee report under a code 35 EC/DCMB/FNSL/EU2024.ORCIDSegueni Bentahar Assia http://orcid.org/0000-0002-7609-0386Ramdani Messaoud http://orcid.org/0000-0002-3536-6358Ayomide Victor Atoki http://orcid.org/0000-0003-1914-973XReferencesAbd-Alla, H. I., Aly, H. F., Shalaby, N. M., Albalawy, M. A., & Aboutabl, E. A. (2014). Hunting for renal protective phytoconstituents in Artemisia judaica L. and Chrysanthemum coronarium L. (Asteraceae). Egyptian Pharmaceutical Journal, 13(1), 46–57. https://doi.org/10.4103/1687- 4315.135597Abo-Salem, O. M., El-Edel, R. H., Harisa, G. 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ELHOUDA ET AL.https://doi.org/https://doi.org/10.1016/S0378-8741(02)00217-9https://doi.org/https://doi.org/10.1016/S0378-8741(02)00217-9https://doi.org/https://doi.org/10.1016/j.phrs.2007.07.003https://doi.org/https://doi.org/10.1016/j.phrs.2007.07.003https://doi.org/https://doi.org/10.1016/j.jep.2004.11.034https://doi.org/https://doi.org/10.1016/j.jep.2004.11.034https://doi.org/https://doi.org/10.1016/j.pharma.2010.11.004https://doi.org/https://doi.org/10.1016/j.pharma.2010.11.004https://doi.org/https://doi.org/10.1016/j.revmed.2023.01.005https://doi.org/https://doi.org/10.1016/j.revmed.2023.01.005https://doi.org/https://doi.org/10.1155/2013/378790https://doi.org/https://doi.org/10.1146/annurev.med.53.082901.104057https://doi.org/https://doi.org/10.1111/j.1472-765X.2008.02499.xhttps://doi.org/https://doi.org/10.1111/j.1472-765X.2008.02499.xhttps://doi.org/https://doi.org/10.5772/29471https://doi.org/https://doi.org/10.1021/jf010697nhttps://doi.org/https://doi.org/10.1021/jf010697nAbstractIntroductionMaterial and methodsPlant materialPreparation of the aqueous extract by macerationHigh-performance liquid chromatography (HPLC)Ultra-performance liquid chromatography-mass spectrometry (UPLC/MS-MS)Animal materialStudy of the toxicity of P.chloranthus extract on ratsEvaluation of the antidiabetic activity of P.chloranthus invivoEvaluation of the invitro antioxidant activity of P.chloranthusFree radical scavenging test (DPPH)Ferric reducing-antioxidant power (FRAP) iron reduction testEvaluation of anti-inflammatory activity invitroEvaluation of anti-inflammatory activity invivoStatistical analysisResultsHigh-performance liquid chromatography (HPLC)Ultra-performance liquid chromatography-mass spectrometry (UPLC/MS-MS)Toxicity of P.chloranthusEffect of diabetes induction in ratsVariations in organ weightAnalysis of biochemical parametersAnalysis of the correlation of doses and biochemical parametersHistological sectionsEvaluation of the antioxidant activity of P.ChloranthusAnti-inflammatory activity invitro and invivoConclusionDisclosure statementFundingEthical approval and consent to participateReferences