Assessment of the Therapeutic Potential, Antioxidant, Antidiabetic, and Analgesic Properties of Vitex trifolia

Assessment of the Therapeutic Potential, Antioxidant, Antidiabetic, and Analgesic Properties of Vitex trifolia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of the Therapeutic Potential, Antioxidant, Antidiabetic, and Analgesic Properties of Vitex trifolia Taskia Azad Konika, Md Rahimul Hasan, Md Nazmul Hasan, Md Shamsuzzaman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5996186/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Vitex trifolia , a plant traditionally utilized for treating ailments such as chronic colds, coughs, dysentery, mastitis, and liver disorders, was evaluated for its antioxidant, antidiabetic, anti-inflammatory, and analgesic properties in this study. Phytochemical analysis of the leaf extracts revealed high levels of total phenolics (95.12 mg GAE/g) and flavonoids (42.50 mg QE/g). The in vitro assays demonstrated significant antioxidant activity, with 77.85% DPPH radical scavenging at 100 µg/mL and 73.33% nitric oxide radical scavenging at 1000 µg/mL. The extracts also exhibited potent antidiabetic effects, inhibiting α-amylase by 67.25% at 100 µg/mL, and strong anti-inflammatory activity, with 70.25% inhibition of albumin denaturation at 800 µg/mL. In vivo analgesic activity was confirmed through acetic acid-induced writhing and tail-flick assays in mice, where significant reductions in writhing responses were observed at doses of 50 and 100 mg/kg, comparable to diclofenac sodium. Additionally, the extract reduced glutamate-induced nociception by 40.91% and 55.64% at doses of 50 mg/kg and 100 mg/kg, respectively, in the tail-flick test. These findings suggest that V. trifolia has significant therapeutic potential, exhibiting strong antioxidant, antidiabetic, anti-inflammatory, and analgesic effects. Plant Molecular Biology and Genetics Medicinal Chemistry Vitex trifolia Antioxidant activity Analgesic properties α-amylase inhibition Anti-inflammatory activity Figures Figure 1 Figure 2 Figure 3 1. Introduction Vitex trifolia, a medicinal plant prevalent in tropical and subtropical regions, has been traditionally used to treat a variety of ailments such as colds, fevers, inflammation, and pain [1, 2]. Its longstanding use in ethnomedicine is increasingly supported by modern scientific research, which highlights the plant's therapeutic potential in addressing chronic diseases and other health concerns [3]. One of the primary attributes of V. trifolia is its strong antioxidant activity [4], which is attributed to its rich phenolic and flavonoid content [6]. Oxidative stress, caused by an imbalance between free radicals and antioxidants, plays a pivotal role in the onset of chronic diseases, including diabetes, cardiovascular conditions, and cancer [5]. Extracts from V. trifolia leaves have demonstrated significant free radical scavenging abilities, particularly against DPPH and nitric oxide radicals, which helps mitigate oxidative stress and related cellular damage [6, 7]. In addition to its antioxidant properties, V. trifolia exhibits considerable analgesic effects [8, 10], and has been used in traditional medicine for pain relief in conditions like inflammation and rheumatism [9]. Animal studies, including acetic acid-induced writhing and tail-flick tests, have shown that its analgesic activity is comparable to that of Diclofenac sodium, suggesting its potential as a natural pain reliever, particularly for inflammatory pain [10, 11]. Moreover, V. trifolia possesses significant anti-inflammatory properties [12, 14, 15]. It inhibits albumin denaturation, a key marker of anti-inflammatory activity [14], and although it may be less potent than aspirin, V. trifolia ’s effects are nonetheless substantial, aligning with its traditional use in treating inflammatory disorders [15]. Additionally, V. trifolia has been shown to have antidiabetic potential due to its α-amylase inhibitory activity [16, 17], a mechanism that is critical for managing postprandial blood glucose levels and is targeted by many antidiabetic medications [16]. Research has demonstrated that V. trifolia extracts can effectively inhibit α-amylase, showing efficacy comparable to that of sitagliptin, a commonly used antidiabetic drug [17, 18]. This highlights its promise as a natural antidiabetic agent. In conclusion, the broad spectrum of bioactive properties exhibited by V. trifolia , supported by both traditional use and contemporary scientific evidence, indicates its significant potential for managing chronic diseases and inflammatory conditions. Further research into its phytochemical composition and mechanisms of action could pave the way for the development of novel natural therapeutics. 2. Materials and Methods 2.1. Plant Collection and Extraction Vitex trifolia was collected from Mirpur-01, Dhaka, Bangladesh. The collected plant material was washed, shade-dried, and ground into a fine powder. To extract the active compounds, 300 g of powdered plant material was macerated in 5 L of methanol for 5 days at room temperature (303 ± 1 K), with daily solvent replacement. The extract was then filtered using Whatman grade 1 filter paper (11 µm) and concentrated using a rotary evaporator, yielding 112.5 g of crude extract for further analysis. 2.2. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) Analysis The total phenolic content (TPC) of the V. trifolia extract was determined using the Folin-Ciocalteu method [19]. In brief, 40 µL of the extract was mixed with 3.16 mL of distilled water, 200 µL of Folin-Ciocalteu reagent, and 600 µL of 20% sodium carbonate. The mixture was incubated at 303 ± 1 K for 2 hours, and the absorbance was measured at 765 nm using a Mecasys Optizen 2120 UV Plus UV-spectrophotometer. TPC was calculated from a gallic acid standard curve and expressed as mg gallic acid equivalents (GAE) per gram of extract.Total flavonoid content (TFC) was determined using the aluminum chloride colorimetric method [20]. A solution containing 0.5 mL of extract (100 mg/mL), 0.1 mL of 1 M potassium acetate, 0.1 mL of 10% aluminum chloride, and 4.3 mL of distilled water was incubated for 30 minutes at room temperature. Absorbance at 415 nm was measured, and TFC was expressed as mg quercetin equivalents (QE) per gram of extract. 2.3. Antioxidant Assays The antioxidant capacity of V. trifolia extract was evaluated using various assays, including DPPH radical scavenging, nitric oxide scavenging, and hydrogen peroxide scavenging assays, as well as reducing power and total antioxidant capacity assays [19]. Ascorbic acid was used as a reference standard in all assays. 2.3.1. DPPH Radical Scavenging Assay To assess DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, 0.2 mL of extract (10–100 µg/mL) was mixed with 2 mL of 0.5 mM DPPH in methanol. After 30 minutes of incubation in the dark, absorbance was measured at 517 nm against a methanol blank. The percentage inhibition was calculated as: Inhibition (%) = [(Acontrol - Asample) / Acontrol] x 100 where A control and A sample represent absorbance of DPPH without and with extract, respectively [21]. 2.3.2. Nitric Oxide Scavenging Assay To evaluate nitric oxide scavenging, 2 mL of 10 mM sodium nitroprusside in 0.5 mL PBS (pH 7.4) was mixed with 0.5 mL of extract (100–1000 µg/mL) and incubated at 298 ± 1 K for 2 hours. After incubation, 0.5 mL of Griess reagent (a mixture of 2% phosphoric acid, 1% sulfanilic acid, and 0.1% naphthyl ethylenediamine dichloride) was added and incubated for an additional 30 minutes at 303 ± 1 K. Absorbance was measured at 546 nm, and nitric oxide scavenging activity was calculated similarly to DPPH scavenging [19–22] 2.4. α -Amylase Inhibition Assay The α-amylase inhibitory activity of the extract was assessed by mixing 500 µL of extract (25–100 mg/mL) with 500 µL of α-amylase (0.5 mg/mL) in 0.02 M sodium phosphate buffer (pH 6.9) containing 0.006 M sodium chloride. After 10 minutes of pre-incubation at 25°C, 500 µL of a 1% starch solution was added and incubated for 10 minutes at the same temperature. The reaction was stopped by adding 1 mL of 3,5-dinitrosalicylic acid (DNSA) and boiling the mixture for 5 minutes. After cooling, 10 mL of distilled water was added, and the absorbance was measured at 540 nm. The percentage inhibition was calculated as: Inhibition (%) = [(Acontrol - Asample) / Acontrol] x 100 [23-24]. 2.5. Anti-inflammatory Assay The anti-inflammatory potential of Vitex trifolia extract was evaluated using the albumin denaturation method, adapted from Qamar et al. (2021) [25]. Various concentrations of the extract were mixed with a 1% aqueous bovine serum albumin solution. The pH was adjusted to optimal denaturation conditions using 1N HCl. The mixture was incubated at 37°C for 20 minutes, followed by heating at 57°C for 20 minutes to induce protein denaturation. After cooling to room temperature, absorbance was measured at 660 nm. The percentage inhibition of albumin denaturation was calculated using the following formula: Inhibition (%) = [(Acontrol - Asample) / Acontrol] x 100. 2.6. Analgesic Assays The analgesic effects of V. trifolia extract were evaluated using several animal models, including the acetic acid-induced writhing test, tail immersion test, hot plate test, and glutamate-induced nociception test. Swiss albino mice weighing 16–20 g and aged 3–4 weeks were used in the experiments. The mice were acclimated for one week prior to testing. In the acetic acid-induced writhing test, V. trifolia extract (50, 100, and 200 mg/kg) was administered intraperitoneally or orally. Distilled water and diclofenac (10 mg/kg, i.p.) served as negative and positive controls, respectively. For the tail immersion and hot plate tests, diclofenac (75 mg/kg) and morphine (5 mg/kg, i.p.) were used as positive controls. Additionally, the glutamate-induced nociception test was performed using V. trifolia extract at the same doses, with diclofenac (200 mg/kg) as the positive control [26-27]. 3. Result and Discussion 3.1. Phytochemical Analysis Total phenolic content (TPC) and total flavonoid content (TFC) are key indicators of a plant's antioxidant capacity and therapeutic potential [28]. The Vitex trifolia extract demonstrated a TPC of 95.12 mg gallic acid equivalents (GAE)/g and a TFC of 42.50 mg quercetin equivalents (QE)/g [Figure 1]. This high phenolic and flavonoid content underscores the plant's strong antioxidant, antidiabetic, and anti-inflammatory properties [29]. Elevated levels of phenolic and flavonoid compounds in plants are known to contribute to significant free radical scavenging activity, offering protection against oxidative stress-related diseases such as diabetes, neurodegenerative disorders, and osteoporosis [30]. These phytochemical constituents play a crucial role in mitigating oxidative damage and regulating inflammation, which are central to the pathogenesis of chronic conditions. 3.2. Antioxidant Activity The antioxidant potential of Vitex trifolia extract was assessed through various assays, including DPPH, nitric oxide, hydrogen peroxide scavenging, reducing power, and total antioxidant capacity assays [4]. In the DPPH radical scavenging assay, the extract exhibited concentration-dependent activity, ranging from 5.20% to 77.85% inhibition at concentrations of 10–100 µg/mL [Figure 2a]. This result is consistent with previous findings, suggesting that antioxidant efficacy is influenced by both the concentration and molecular structure of bioactive compounds [31]. Similarly, the nitric oxide scavenging assay [Figure 2b] demonstrated a concentration-dependent trend, with maximum inhibition of 73% at 1000 µg/mL and a minimum of 8% at 100 µg/mL. The ability of V. trifolia to scavenge nitric oxide, a free radical linked to inflammation and carcinogenesis, is likely due to its rich phenolic content, as these compounds are known to act as hydrogen donors and reducing agents, neutralizing reactive nitrogen species [7]. This antioxidant activity highlights the plant's potential in preventing oxidative stress and related pathological conditions. 3.3. α -Amylase Inhibition and Antidiabetic Potential α-Amylase inhibition is a crucial mechanism in the management of diabetes, as it reduces the breakdown of carbohydrates into glucose, thereby controlling postprandial blood sugar spikes. Vitex trifolia extract (25–100 mg/mL) demonstrated significant α-amylase inhibitory activity in vitro, achieving a maximum inhibition of 67% at 100 mg/mL [Figure 3a]. This inhibitory effect is important for mitigating hyperglycemia, a common issue in diabetes management [32]. The α-amylase inhibition by V. trifolia is likely due to interactions between the polyphenols and flavonoids present in the extract and the enzyme's active site, preventing the enzyme from breaking down carbohydrates effectively [33]. In addition to their role as enzyme inhibitors, plant-based compounds such as polyphenols and flavonoids often possess antioxidant properties, which can provide added therapeutic benefits by reducing oxidative stress, further supporting their use in antidiabetic therapies [34]. 3.4. Anti-inflammatory Activity The anti-inflammatory activity of Vitex trifolia extract was assessed using the albumin denaturation assay. At a concentration of 800 µg/mL, the extract exhibited significant inhibition of protein denaturation (85.92 ± 1.48%), surpassing the inhibition seen with aspirin at 200 µg/mL (75.89 ± 0.56%) [Figure 3b]. These results suggest that V. trifolia possesses strong anti-denaturation properties, which are indicative of its potential as an effective anti-inflammatory agent [35]. The extract's superior inhibition compared to aspirin, although at a higher concentration, highlights its promising anti-inflammatory potential and justifies further investigation into the mechanisms driving this effect, as well as its comparative efficacy in various inflammation-related conditions. 3.5. Analgesic Activity The analgesic properties of Vitex trifolia ethanol extract were evaluated using four established pain models in mice: the acetic acid-induced writhing test, tail immersion test, hot plate test, and glutamate-induced nociception test. These models allowed assessment of both peripheral and central analgesic mechanisms. Diclofenac sodium and morphine were used as positive controls. 3.5.1. Acetic Acid-Induced Writhing Test V. trifolia extract showed significant dose-dependent peripheral analgesia, with a maximum inhibition of 95.5% observed at 50 mg/kg, surpassing the analgesic effect of diclofenac sodium (68.5%) [Table 1]. Interestingly, higher doses (100 and 200 mg/kg) resulted in reduced efficacy, which may be due to receptor saturation or metabolic limitations [36]. The strong analgesic effect at 50 mg/kg suggests that V. trifolia may inhibit prostaglandin synthesis or reduce the release of inflammatory pain mediators, similar to nonsteroidal anti-inflammatory drugs (NSAIDs) like diclofenac [37]. This highlights the importance of dose optimization to maximize analgesic efficacy. 3.5.2. Tail Immersion and Hot Plate Tests Both the tail immersion and hot plate tests, which assess central analgesia, demonstrated dose-dependent increases in withdrawal latency and response latency, respectively, indicating the modulation of central pain pathways [Table 2 and S2]. While the effects were significant, they were less potent compared to diclofenac sodium and morphine, suggesting that V. trifolia may interact with endogenous opioid receptors or other central pain-modulating systems. Further investigation is required to elucidate the precise mechanisms involved. Similar to the acetic acid-induced writhing test, the reduced efficacy at higher doses in these tests suggests potential receptor saturation [27, 38]. 3.5.3. Formalin and Glutamate-Induced Nociception Tests In both the formalin and glutamate-induced nociception tests, V. trifolia extract (50–200 mg/kg) significantly inhibited both the early (0–5 min) and late (15–30 min) phases of formalin-induced paw licking in a dose-dependent manner (p < 0.001 vs. control; Table S3). At 50 mg/kg, the extract's efficacy was comparable to that of diclofenac sodium (10 mg/kg); however, higher doses (100 and 200 mg/kg) exhibited lower efficacy than diclofenac sodium. In the glutamate-induced nociception test, V. trifolia showed significant dose-dependent antinociception, with 55.64% and 51.44% inhibition at 100 and 200 mg/kg, respectively, though this was less potent than diclofenac sodium (87.18%) [Table 3]. Given that glutamate plays a key role in both peripheral and central pain transmission, especially in inflammatory and neuropathic pain [39], these results suggest that V. trifolia may modulate excitatory neurotransmitter pathways, possibly by interacting with glutamate receptors or inhibiting glutamate neurotransmission. The significant analgesic activity observed through both peripheral and central mechanisms indicates that V. trifolia may serve as a potential natural analgesic, particularly for treating inflammatory and neuropathic pain. However, further research into its mechanisms of action and optimal dosing is essential [40]. 4. Conclusion This study provides scientific validation for the traditional medicinal uses of Vitex trifolia , revealing its potent antioxidant, antidiabetic, anti-inflammatory, and analgesic properties. The antidiabetic effects were linked to α-amylase inhibition, a critical mechanism in glucose regulation. The extract demonstrated superior anti-inflammatory activity compared to aspirin in the albumin denaturation assay, and its analgesic effects were mediated through both peripheral and central mechanisms. The non-linear dose-response observed across assays highlights the need for further research to optimize dosing and better understand the mechanisms driving V. trifolia 's bioactivity. Future investigations should focus on identifying its key phytochemical constituents and molecular pathways, exploring potential synergistic effects with conventional treatments, and evaluating its clinical applications for the management of chronic diseases and inflammatory pain. 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Results of Hot Plate Test for Vitex trifolia Extract Test Group Dose 0 min 30 min 60 min 90 min 120min Control 0.1ml/kg 3.19±0.69 4.19±0.98 5.19±2.25 6.284±1.01 6.32±0.367 Positive Control 5 mg/kg 1.23±0.07 4.54±1.25 8.21±2.92 9.44±1.193 5.49±0.605 Group-I 50 mg/kg 2.12±0.46 7.15±1.12 6.23±0.36 7.32±1.494 5.13±0.82 Group-II 100 mg/kg 3.81±0.31 4.03±1.12 4.23±0.45 5.11±0.650 3.89±0.95 Group-III 200 mg/kg 4.88±0.33 5.12±0.97 4.9±0.22 6.2±0.98 4.99±0.71 Table 3 : Analgesic activity of extract of Vitex trifolia on Glutamate induced abdominal writhing test. Treatment Dose (mg/kg) Number of licking % Inhibition Control 0.1 ml/mouse 104.60±1.07 - Standard Diclofenac Sodium 10mg/kg 13.40±0.6*** 87.18% Group I 50 61.80±1.16*** 40.91% Group II 100 46.40±0.68*** 55.64% Group III 200 51.36±0.47*** 51.44% Values are expressed as Mean ±SEM (n=5);*the mean difference is significant at the 0.05 level ٭٭ the mean difference is significant at the 0.01 level; ٭٭٭ the mean difference is significant at the 0.001 level. Dunnett tests treat one group as control and compare all other group against it. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5996186","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":420574796,"identity":"7489baae-2e50-4b96-b08b-fd8a5f2b798e","order_by":0,"name":"Taskia Azad Konika","email":"","orcid":"","institution":"Department of Pharmacy, Primeasia University, 12 Kemal Ataturk Ave, Dhaka 1213, Bangladesh","correspondingAuthor":false,"prefix":"","firstName":"Taskia","middleName":"Azad","lastName":"Konika","suffix":""},{"id":420574797,"identity":"002b10ab-d2c5-429f-a8cb-42e06fe22dad","order_by":1,"name":"Md Rahimul Hasan","email":"","orcid":"","institution":"Department of pharmacy, Stamford University Bangladesh,51 Siddeswari Road (Ramna), Dhaka-1217","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Rahimul","lastName":"Hasan","suffix":""},{"id":420574798,"identity":"0185b6aa-6b5b-441b-8088-d5d24c2cfb02","order_by":2,"name":"Md Nazmul Hasan","email":"","orcid":"","institution":"Department of pharmacy, Stamford University Bangladesh,51 Siddeswari Road (Ramna), Dhaka-1217.","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Nazmul","lastName":"Hasan","suffix":""},{"id":420574799,"identity":"64e3abb7-3795-4293-8bbe-285eba1381ab","order_by":3,"name":"Md Shamsuzzaman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCQjFw8DA2MDAUAFkMjM3kKLlDEgLI3FaIICxDUzi12Iu3fzs0w2GbTLy/YcbHxfOq43mbwdq+VGxDacWyznHjGfnMNzmMbiR2Gw8c9vx3BmHGRsYe87cxqnF4EaCMTNYiwRjmzTvtmO5DUAtzIxt+LSkfwZrke8/CNQy51jufMJaciC2MBxIBGppqMndQEiL5YycYuYcA6hfeI4dyN0I1HIQn1/MJdI3M+dU3LaX7z/+8DFPTV3uvPOHDz74UYHHYUgkCBwGkwdwqkdRDAF1+BSPglEwCkbBCAUAOPpX6yVrDyQAAAAASUVORK5CYII=","orcid":"","institution":"Department of Biomedical Science, School of Medicine, Kyungpook National University, Daegu, Republic of Korea","correspondingAuthor":true,"prefix":"","firstName":"Md","middleName":"","lastName":"Shamsuzzaman","suffix":""}],"badges":[],"createdAt":"2025-02-10 06:33:37","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5996186/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5996186/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77669753,"identity":"dcab9804-8af3-4709-8513-f863ac5f0afa","added_by":"auto","created_at":"2025-03-04 06:45:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16835,"visible":true,"origin":"","legend":"\u003cp\u003eTPC and TFC of \u003cem\u003eC. Vitex trifolia \u003c/em\u003elevels\u003cem\u003e \u003c/em\u003eextract.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5996186/v1/5123153eeed57ca80b11fcc8.png"},{"id":77668427,"identity":"471fa70c-5d25-4f1a-aace-1bfb725c26d0","added_by":"auto","created_at":"2025-03-04 06:37:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93891,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activities of \u003cem\u003eVitex trifolia \u003c/em\u003elevels (\u003cstrong\u003ea\u003c/strong\u003e) DPPH free-radical scavenging and (\u003cstrong\u003eb\u003c/strong\u003e) nitric oxide inhibition.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5996186/v1/73add0bc9d44588c5087bd67.png"},{"id":77669757,"identity":"6cae50ec-4bf3-4759-9b99-c81780f0eef7","added_by":"auto","created_at":"2025-03-04 06:45:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110883,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of α-amylase activity and albumin denaturation by Vitex trifolia extract, demonstrating a dose-dependent protective effect. Data presented as mean ± SD (n=3), with statistical significance at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5996186/v1/343e856c5e1c7351539a8b05.png"},{"id":77671129,"identity":"bc2361dc-2e7a-4b51-84b1-943d1e3dd7cb","added_by":"auto","created_at":"2025-03-04 07:01:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1004077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5996186/v1/7374544f-c3d5-49e1-a46e-9524642ac3be.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAssessment of the Therapeutic Potential, Antioxidant, Antidiabetic, and Analgesic Properties of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVitex trifolia\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eVitex trifolia,\u003c/em\u003e a medicinal plant prevalent in tropical and subtropical regions, has been traditionally used to treat a variety of ailments such as colds, fevers, inflammation, and pain [1, 2]. Its longstanding use in ethnomedicine is increasingly supported by modern scientific research, which highlights the plant's therapeutic potential in addressing chronic diseases and other health concerns [3]. One of the primary attributes of \u003cem\u003eV. trifolia\u003c/em\u003e is its strong antioxidant activity [4], which is attributed to its rich phenolic and flavonoid content [6]. Oxidative stress, caused by an imbalance between free radicals and antioxidants, plays a pivotal role in the onset of chronic diseases, including diabetes, cardiovascular conditions, and cancer [5]. Extracts from \u003cem\u003eV. trifolia\u003c/em\u003e leaves have demonstrated significant free radical scavenging abilities, particularly against DPPH and nitric oxide radicals, which helps mitigate oxidative stress and related cellular damage [6, 7].\u003c/p\u003e\n\u003cp\u003eIn addition to its antioxidant properties, \u003cem\u003eV. trifolia\u003c/em\u003e exhibits considerable analgesic effects [8, 10], and has been used in traditional medicine for pain relief in conditions like inflammation and rheumatism [9]. Animal studies, including acetic acid-induced writhing and tail-flick tests, have shown that its analgesic activity is comparable to that of Diclofenac sodium, suggesting its potential as a natural pain reliever, particularly for inflammatory pain [10, 11].\u003c/p\u003e\n\u003cp\u003eMoreover, \u003cem\u003eV. trifolia\u003c/em\u003e possesses significant anti-inflammatory properties [12, 14, 15]. It inhibits albumin denaturation, a key marker of anti-inflammatory activity [14], and although it may be less potent than aspirin, \u003cem\u003eV. trifolia\u003c/em\u003e’s effects are nonetheless substantial, aligning with its traditional use in treating inflammatory disorders [15].\u003c/p\u003e\n\u003cp\u003eAdditionally, \u003cem\u003eV. trifolia\u003c/em\u003e has been shown to have antidiabetic potential due to its α-amylase inhibitory activity [16, 17], a mechanism that is critical for managing postprandial blood glucose levels and is targeted by many antidiabetic medications [16]. Research has demonstrated that \u003cem\u003eV. trifolia\u003c/em\u003e extracts can effectively inhibit α-amylase, showing efficacy comparable to that of sitagliptin, a commonly used antidiabetic drug [17, 18]. This highlights its promise as a natural antidiabetic agent.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the broad spectrum of bioactive properties exhibited by \u003cem\u003eV. trifolia\u003c/em\u003e, supported by both traditional use and contemporary scientific evidence, indicates its significant potential for managing chronic diseases and inflammatory conditions. Further research into its phytochemical composition and mechanisms of action could pave the way for the development of novel natural therapeutics.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Plant Collection and Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVitex trifolia\u003c/em\u003e was collected from Mirpur-01, Dhaka, Bangladesh. The collected plant material was washed, shade-dried, and ground into a fine powder. To extract the active compounds, 300 g of powdered plant material was macerated in 5 L of methanol for 5 days at room temperature (303 \u0026plusmn; 1 K), with daily solvent replacement. The extract was then filtered using Whatman grade 1 filter paper (11 \u0026micro;m) and concentrated using a rotary evaporator, yielding 112.5 g of crude extract for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total phenolic content (TPC) of the \u003cem\u003eV. trifolia\u003c/em\u003e extract was determined using the Folin-Ciocalteu method [19]. In brief, 40 \u0026micro;L of the extract was mixed with 3.16 mL of distilled water, 200 \u0026micro;L of Folin-Ciocalteu reagent, and 600 \u0026micro;L of 20% sodium carbonate. The mixture was incubated at 303 \u0026plusmn; 1 K for 2 hours, and the absorbance was measured at 765 nm using a Mecasys Optizen 2120 UV Plus UV-spectrophotometer. TPC was calculated from a gallic acid standard curve and expressed as mg gallic acid equivalents (GAE) per gram of extract.Total flavonoid content (TFC) was determined using the aluminum chloride colorimetric method [20]. A solution containing 0.5 mL of extract (100 mg/mL), 0.1 mL of 1 M potassium acetate, 0.1 mL of 10% aluminum chloride, and 4.3 mL of distilled water was incubated for 30 minutes at room temperature. Absorbance at 415 nm was measured, and TFC was expressed as mg quercetin equivalents (QE) per gram of extract.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Antioxidant Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant capacity of \u003cem\u003eV. trifolia\u003c/em\u003e extract was evaluated using various assays, including DPPH radical scavenging, nitric oxide scavenging, and hydrogen peroxide scavenging assays, as well as reducing power and total antioxidant capacity assays [19]. Ascorbic acid was used as a reference standard in all assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1. DPPH Radical Scavenging Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, 0.2 mL of extract (10\u0026ndash;100 \u0026micro;g/mL) was mixed with 2 mL of 0.5 mM DPPH in methanol. After 30 minutes of incubation in the dark, absorbance was measured at 517 nm against a methanol blank. The percentage inhibition was calculated as:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInhibition (%) \u0026nbsp;= [(Acontrol - Asample) / Acontrol] x 100\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ewhere A control and A sample represent absorbance of DPPH without and with extract, respectively [21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2. Nitric Oxide Scavenging Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate nitric oxide scavenging, 2 mL of 10 mM sodium nitroprusside in 0.5 mL PBS (pH 7.4) was mixed with 0.5 mL of extract (100\u0026ndash;1000 \u0026micro;g/mL) and incubated at 298 \u0026plusmn; 1 K for 2 hours. After incubation, 0.5 mL of Griess reagent (a mixture of 2% phosphoric acid, 1% sulfanilic acid, and 0.1% naphthyl ethylenediamine dichloride) was added and incubated for an additional 30 minutes at 303 \u0026plusmn; 1 K. Absorbance was measured at 546 nm, and nitric oxide scavenging activity was calculated similarly to DPPH scavenging [19\u0026ndash;22]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. \u003cem\u003e\u0026alpha;\u003c/em\u003e-Amylase Inhibition Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026alpha;-amylase inhibitory activity of the extract was assessed by mixing 500 \u0026micro;L of extract (25\u0026ndash;100 mg/mL) with 500 \u0026micro;L of \u0026alpha;-amylase (0.5 mg/mL) in 0.02 M sodium phosphate buffer (pH 6.9) containing 0.006 M sodium chloride. After 10 minutes of pre-incubation at 25\u0026deg;C, 500 \u0026micro;L of a 1% starch solution was added and incubated for 10 minutes at the same temperature. The reaction was stopped by adding 1 mL of 3,5-dinitrosalicylic acid (DNSA) and boiling the mixture for 5 minutes. After cooling, 10 mL of distilled water was added, and the absorbance was measured at 540 nm. The percentage inhibition was calculated as:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInhibition (%) = [(Acontrol - Asample) / Acontrol] x 100 [23-24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Anti-inflammatory Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe anti-inflammatory potential of \u003cem\u003eVitex trifolia\u003c/em\u003e extract was evaluated using the albumin denaturation method, adapted from Qamar et al. (2021) [25]. Various concentrations of the extract were mixed with a 1% aqueous bovine serum albumin solution. The pH was adjusted to optimal denaturation conditions using 1N HCl. The mixture was incubated at 37\u0026deg;C for 20 minutes, followed by heating at 57\u0026deg;C for 20 minutes to induce protein denaturation. After cooling to room temperature, absorbance was measured at 660 nm. The percentage inhibition of albumin denaturation was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003eInhibition (%) = [(Acontrol - Asample) / Acontrol] x 100.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Analgesic Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analgesic effects of \u003cem\u003eV. trifolia\u003c/em\u003e extract were evaluated using several animal models, including the acetic acid-induced writhing test, tail immersion test, hot plate test, and glutamate-induced nociception test. Swiss albino mice weighing 16\u0026ndash;20 g and aged 3\u0026ndash;4 weeks were used in the experiments. The mice were acclimated for one week prior to testing.\u003c/p\u003e\n\u003cp\u003eIn the acetic acid-induced writhing test, \u003cem\u003eV. trifolia\u003c/em\u003e extract (50, 100, and 200 mg/kg) was administered intraperitoneally or orally. Distilled water and diclofenac (10 mg/kg, i.p.) served as negative and positive controls, respectively. For the tail immersion and hot plate tests, diclofenac (75 mg/kg) and morphine (5 mg/kg, i.p.) were used as positive controls. Additionally, the glutamate-induced nociception test was performed using \u003cem\u003eV. trifolia\u003c/em\u003e extract at the same doses, with diclofenac (200 mg/kg) as the positive control [26-27].\u003c/p\u003e"},{"header":"3. Result and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. Phytochemical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal phenolic content (TPC) and total flavonoid content (TFC) are key indicators of a plant's antioxidant capacity and therapeutic potential [28]. The \u003cem\u003eVitex trifolia\u003c/em\u003e extract demonstrated a TPC of 95.12 mg gallic acid equivalents (GAE)/g and a TFC of 42.50 mg quercetin equivalents (QE)/g [Figure 1]. This high phenolic and flavonoid content underscores the plant's strong antioxidant, antidiabetic, and anti-inflammatory properties [29]. Elevated levels of phenolic and flavonoid compounds in plants are known to contribute to significant free radical scavenging activity, offering protection against oxidative stress-related diseases such as diabetes, neurodegenerative disorders, and osteoporosis [30]. These phytochemical constituents play a crucial role in mitigating oxidative damage and regulating inflammation, which are central to the pathogenesis of chronic conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Antioxidant Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant potential of \u003cem\u003eVitex trifolia\u003c/em\u003e extract was assessed through various assays, including DPPH, nitric oxide, hydrogen peroxide scavenging, reducing power, and total antioxidant capacity assays [4]. In the DPPH radical scavenging assay, the extract exhibited concentration-dependent activity, ranging from 5.20% to 77.85% inhibition at concentrations of 10–100 µg/mL [Figure 2a]. This result is consistent with previous findings, suggesting that antioxidant efficacy is influenced by both the concentration and molecular structure of bioactive compounds [31]. Similarly, the nitric oxide scavenging assay [Figure 2b] demonstrated a concentration-dependent trend, with maximum inhibition of 73% at 1000 µg/mL and a minimum of 8% at 100 µg/mL. The ability of \u003cem\u003eV. trifolia\u003c/em\u003e to scavenge nitric oxide, a free radical linked to inflammation and carcinogenesis, is likely due to its rich phenolic content, as these compounds are known to act as hydrogen donors and reducing agents, neutralizing reactive nitrogen species [7]. This antioxidant activity highlights the plant's potential in preventing oxidative stress and related pathological conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. \u003cem\u003eα\u003c/em\u003e-Amylase Inhibition and Antidiabetic Potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eα-Amylase inhibition is a crucial mechanism in the management of diabetes, as it reduces the breakdown of carbohydrates into glucose, thereby controlling postprandial blood sugar spikes. \u003cem\u003eVitex trifolia\u003c/em\u003e extract (25–100 mg/mL) demonstrated significant α-amylase inhibitory activity in vitro, achieving a maximum inhibition of 67% at 100 mg/mL [Figure 3a]. This inhibitory effect is important for mitigating hyperglycemia, a common issue in diabetes management [32]. The α-amylase inhibition by \u003cem\u003eV. trifolia\u003c/em\u003e is likely due to interactions between the polyphenols and flavonoids present in the extract and the enzyme's active site, preventing the enzyme from breaking down carbohydrates effectively [33]. In addition to their role as enzyme inhibitors, plant-based compounds such as polyphenols and flavonoids often possess antioxidant properties, which can provide added therapeutic benefits by reducing oxidative stress, further supporting their use in antidiabetic therapies [34].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Anti-inflammatory Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe anti-inflammatory activity of \u003cem\u003eVitex trifolia\u003c/em\u003e extract was assessed using the albumin denaturation assay. At a concentration of 800 µg/mL, the extract exhibited significant inhibition of protein denaturation (85.92 ± 1.48%), surpassing the inhibition seen with aspirin at 200 µg/mL (75.89 ± 0.56%) [Figure 3b]. These results suggest that \u003cem\u003eV. trifolia\u003c/em\u003e possesses strong anti-denaturation properties, which are indicative of its potential as an effective anti-inflammatory agent [35]. The extract's superior inhibition compared to aspirin, although at a higher concentration, highlights its promising anti-inflammatory potential and justifies further investigation into the mechanisms driving this effect, as well as its comparative efficacy in various inflammation-related conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Analgesic Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analgesic properties of \u003cem\u003eVitex trifolia\u003c/em\u003e ethanol extract were evaluated using four established pain models in mice: the acetic acid-induced writhing test, tail immersion test, hot plate test, and glutamate-induced nociception test. These models allowed assessment of both peripheral and central analgesic mechanisms. Diclofenac sodium and morphine were used as positive controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.1. Acetic Acid-Induced Writhing Test\u003c/strong\u003e\u003cbr\u003e\u003cem\u003eV. trifolia\u003c/em\u003e extract showed significant dose-dependent peripheral analgesia, with a maximum inhibition of 95.5% observed at 50 mg/kg, surpassing the analgesic effect of diclofenac sodium (68.5%) [Table 1]. Interestingly, higher doses (100 and 200 mg/kg) resulted in reduced efficacy, which may be due to receptor saturation or metabolic limitations [36]. The strong analgesic effect at 50 mg/kg suggests that \u003cem\u003eV. trifolia\u003c/em\u003e may inhibit prostaglandin synthesis or reduce the release of inflammatory pain mediators, similar to nonsteroidal anti-inflammatory drugs (NSAIDs) like diclofenac [37]. This highlights the importance of dose optimization to maximize analgesic efficacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.2. Tail Immersion and Hot Plate Tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth the tail immersion and hot plate tests, which assess central analgesia, demonstrated dose-dependent increases in withdrawal latency and response latency, respectively, indicating the modulation of central pain pathways [Table 2 and S2]. While the effects were significant, they were less potent compared to diclofenac sodium and morphine, suggesting that \u003cem\u003eV. trifolia\u003c/em\u003e may interact with endogenous opioid receptors or other central pain-modulating systems. Further investigation is required to elucidate the precise mechanisms involved. Similar to the acetic acid-induced writhing test, the reduced efficacy at higher doses in these tests suggests potential receptor saturation [27, 38].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.3. Formalin and Glutamate-Induced Nociception Tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn both the formalin and glutamate-induced nociception tests, \u003cem\u003eV. trifolia\u003c/em\u003e extract (50–200 mg/kg) significantly inhibited both the early (0–5 min) and late (15–30 min) phases of formalin-induced paw licking in a dose-dependent manner (p \u0026lt; 0.001 vs. control; Table S3). At 50 mg/kg, the extract's efficacy was comparable to that of diclofenac sodium (10 mg/kg); however, higher doses (100 and 200 mg/kg) exhibited lower efficacy than diclofenac sodium. In the glutamate-induced nociception test, \u003cem\u003eV. trifolia\u003c/em\u003e showed significant dose-dependent antinociception, with 55.64% and 51.44% inhibition at 100 and 200 mg/kg, respectively, though this was less potent than diclofenac sodium (87.18%) [Table 3].\u003c/p\u003e\n\u003cp\u003eGiven that glutamate plays a key role in both peripheral and central pain transmission, especially in inflammatory and neuropathic pain [39], these results suggest that \u003cem\u003eV. trifolia\u003c/em\u003e may modulate excitatory neurotransmitter pathways, possibly by interacting with glutamate receptors or inhibiting glutamate neurotransmission. The significant analgesic activity observed through both peripheral and central mechanisms indicates that \u003cem\u003eV. trifolia\u003c/em\u003e may serve as a potential natural analgesic, particularly for treating inflammatory and neuropathic pain. However, further research into its mechanisms of action and optimal dosing is essential [40].\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study provides scientific validation for the traditional medicinal uses of \u003cem\u003eVitex trifolia\u003c/em\u003e, revealing its potent antioxidant, antidiabetic, anti-inflammatory, and analgesic properties. The antidiabetic effects were linked to α-amylase inhibition, a critical mechanism in glucose regulation. The extract demonstrated superior anti-inflammatory activity compared to aspirin in the albumin denaturation assay, and its analgesic effects were mediated through both peripheral and central mechanisms. The non-linear dose-response observed across assays highlights the need for further research to optimize dosing and better understand the mechanisms driving \u003cem\u003eV. trifolia\u003c/em\u003e's bioactivity. Future investigations should focus on identifying its key phytochemical constituents and molecular pathways, exploring potential synergistic effects with conventional treatments, and evaluating its clinical applications for the management of chronic diseases and inflammatory pain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eall experiments involving non-human vertebrates were conducted according to the ethical principles and guidelines set by the Swiss Academy of Medical Sciences and were approved by the Ethics Committee of Stamford University Bangladesh (SUB/IAEC/23.05).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWee, Hai-Ning, et al. \u0026quot;Effects of Vitex trifolia L. leaf extracts and phytoconstituents on cytokine production in human U937 macrophages.\u0026quot; \u003cem\u003eBMC complementary medicine and therapies\u003c/em\u003e 20 (2020): 1-15.\u003c/li\u003e\n \u003cli\u003eYan, Chun-Xiao, et al. \u0026quot;Vitex rotundifolia L. f. and Vitex trifolia L.: A review on their traditional medicine, phytochemistry, pharmacology.\u0026quot; \u003cem\u003eJournal of Ethnopharmacology\u003c/em\u003e 308 (2023): 116273.\u003c/li\u003e\n \u003cli\u003eVenkateshwarlu, Goli, et al. \u0026quot;Vitex trifolia-An Important Medicinal Plant: A Review of Its Folklore Medicine and Traditional Uses.\u0026quot; \u003cem\u003eAsian Journal of Pharmaceutical Research\u003c/em\u003e 4.2 (2014): 70-71.\u003c/li\u003e\n \u003cli\u003eSaklani, Sarla, et al. \u0026quot;Comparative evaluation of polyphenol contents and antioxidant activities between ethanol extracts of Vitex negundo and Vitex trifolia L. leaves by different methods.\u0026quot; Plants 6.4 (2017): 45.\u003c/li\u003e\n \u003cli\u003eShamsuzzaman, Md, et al. \u0026quot;Genome insight and probiotic potential of three novel species of the genus Corynebacterium.\u0026quot; \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e 14 (2023): 1225282.\u003c/li\u003e\n \u003cli\u003eGoh, Wei Wen, Sharmin Sultana, and Azrina Azlan. \u0026quot;Antioxidant Properties of Lemuni Leaves (Vitex trifolia var. purpurea) in Different Concentrations of Ethanol-Water Solvent Extraction.\u0026quot; Indonesian Journal of Agricultural Research 7.2 (2024): 106-118.\u003c/li\u003e\n \u003cli\u003eAkbari, Behnaz, et al. \u0026quot;The role of plant‐derived natural antioxidants in reduction of oxidative stress.\u0026quot; BioFactors 48.3 (2022): 611-633.\u003c/li\u003e\n \u003cli\u003eGoverdhan, P., and Diwakar Bobbala. \u0026quot;Anti-nociceptive and anti-inflammatory effects of the leaf extract of Vitex trifolia Linn. in experimental animals.\u0026quot; Ethnobotanical Leaflets 2009.1 (2009): 8.\u003c/li\u003e\n \u003cli\u003eAnkalikar, Aryaa, Agadi Hiremath Viswanathswamy, and A. 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Kitts. \u0026quot;Antioxidant property of coffee components: assessment of methods that define mechanisms of action.\u0026quot; \u003cem\u003eMolecules\u003c/em\u003e 19.11 (2014): 19180-19208.\u003c/li\u003e\n \u003cli\u003eZinjarde, Smita S., Shobha Y. Bhargava, and Ameeta R. Kumar. \u0026quot;Potent \u0026alpha;-amylase inhibitory activity of Indian Ayurvedic medicinal plants.\u0026quot; \u003cem\u003eBMC complementary and alternative medicine\u003c/em\u003e 11.1 (2011): 1-10.\u003c/li\u003e\n \u003cli\u003eAli, Hasenah, P. J. Houghton, and Amala Soumyanath. \u0026quot;\u0026alpha;-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus.\u0026quot; \u003cem\u003eJournal of ethnopharmacology\u003c/em\u003e 107.3 (2006): 449-455.\u003c/li\u003e\n \u003cli\u003eQamar, Muhammad, et al. \u0026quot;Syzygium cumini (L.), Skeels fruit extracts: In vitro and in vivo anti-inflammatory properties.\u0026quot; \u003cem\u003eJournal of Ethnopharmacology\u003c/em\u003e 271 (2021): 113805.\u003c/li\u003e\n \u003cli\u003eHasan, Md Rahimul, et al. \u0026quot;Phytochemical Screening From Ficus religiosa Leaves and Determination of Sedative-Hypnotic Activity in Mice By Using Ethyl Alcohol Extract.\u0026quot; \u003cem\u003eInternational journal of research in pharmaceutical and nano sciences\u003c/em\u003e 8.3 (2019): 116-127.\u003c/li\u003e\n \u003cli\u003eAryal, Sushant, et al. \u0026quot;Total phenolic content, flavonoid content and antioxidant potential of wild vegetables from Western Nepal.\u0026quot; \u003cem\u003ePlants\u003c/em\u003e 8.4 (2019): 96.\u003c/li\u003e\n \u003cli\u003eAbdel-Aleem, Enas R., et al. \u0026quot;Total phenolic and flavonoid contents and antioxidant, anti-inflammatory, analgesic, antipyretic and antidiabetic activities of Cordia myxa L. leaves.\u0026quot; \u003cem\u003eClinical Phytoscience\u003c/em\u003e 5 (2019): 1-9.\u003c/li\u003e\n \u003cli\u003eMutha, Rakesh E., Anilkumar U. Tatiya, and Sanjay J. Surana. \u0026quot;Flavonoids as natural phenolic compounds and their role in therapeutics: An overview.\u0026quot; \u003cem\u003eFuture journal of pharmaceutical sciences\u003c/em\u003e 7 (2021): 1-13.\u003c/li\u003e\n \u003cli\u003eDa Silva Porto, Patrıcia Andr\u0026eacute;ia Leite, Joao Ant\u0026oacute;nio Nave Laranjinha, and Victor Armando Pereira de Freitas. \u0026quot;Antioxidant protection of low density lipoprotein by procyanidins: structure/activity relationships.\u0026quot; \u003cem\u003eBiochemical Pharmacology\u003c/em\u003e 66.6 (2003): 947-954.\u003c/li\u003e\n \u003cli\u003eHossain, Uday, et al. \u0026quot;An overview on the role of bioactive \u0026alpha;-glucosidase inhibitors in ameliorating diabetic complications.\u0026quot; \u003cem\u003eFood and chemical toxicology\u003c/em\u003e 145 (2020): 111738.\u003c/li\u003e\n \u003cli\u003eŞ\u0026ouml;hretoğlu, Didem, and Suat Sari. \u0026quot;Flavonoids as alpha-glucosidase inhibitors: Mechanistic approaches merged with enzyme kinetics and molecular modelling.\u0026quot; \u003cem\u003ePhytochemistry Reviews\u003c/em\u003e 19.5 (2020): 1081-1092.\u003c/li\u003e\n \u003cli\u003eDewanjee, Saikat, et al. \u0026quot;Plant-based antidiabetic nanoformulations: the emerging paradigm for effective therapy.\u0026quot; \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e 21.6 (2020): 2217.\u003c/li\u003e\n \u003cli\u003eBallo, Mahamadou, et al. \u0026quot;In vitro inhibition of cyclooxygenases, anti-denaturation and antioxidant activities of Malian medicinal plants.\u0026quot; \u003cem\u003eAfrican Journal of Pharmacy and Pharmacology\u003c/em\u003e 17.2 (2023): 34-42.\u003c/li\u003e\n \u003cli\u003eMeriema, Belghoul. \u0026quot;Anti-inflammatory and antioxidant effect of Oxalis cernuaareal part and root methanolic extracts.\u0026quot; (2021).\u003c/li\u003e\n \u003cli\u003eGan, Tong J. \u0026quot;Diclofenac: an update on its mechanism of action and safety profile.\u0026quot; \u003cem\u003eCurrent medical research and opinion\u003c/em\u003e 26.7 (2010): 1715-1731.\u003c/li\u003e\n \u003cli\u003eRamabadran, Krishnaswami, et al. \u0026quot;Tail immersion test for the evaluation of a nociceptive reaction in mice: Methodological considerations.\u0026quot; \u003cem\u003eJournal of pharmacological methods\u003c/em\u003e 21.1 (1989): 21-31.\u003c/li\u003e\n \u003cli\u003eOsikowicz, Maria, Joanna Mika, and Barbara Przewlocka. \u0026quot;The glutamatergic system as a target for neuropathic pain relief.\u0026quot; \u003cem\u003eExperimental physiology\u003c/em\u003e 98.2 (2013): 372-384.\u003c/li\u003e\n \u003cli\u003eQuintans, Jullyana SS, et al. \u0026quot;Natural products evaluated in neuropathic pain models‐a systematic review.\u0026quot; \u003cem\u003eBasic \u0026amp; clinical pharmacology \u0026amp; toxicology\u003c/em\u003e 114.6 (2014): 442-450.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. \u0026nbsp;Effect of the \u003cem\u003eVitex trifolia\u003c/em\u003e on acetic acid-induced writhing in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"612\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7124%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7255%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026plusmn; SEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.451%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% of writhing\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4444%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% of Inhibition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7124%;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7255%;\"\u003e\n \u003cp\u003e5ml/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e17\u0026plusmn;8.804\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.451%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4444%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7124%;\"\u003e\n \u003cp\u003ePositive control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7255%;\"\u003e\n \u003cp\u003e10mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e7.3\u0026plusmn;3.246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.451%;\"\u003e\n \u003cp\u003e31.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4444%;\"\u003e\n \u003cp\u003e68.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7124%;\"\u003e\n \u003cp\u003eGroup-I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7255%;\"\u003e\n \u003cp\u003e50mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e1.5\u0026plusmn;0.781\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.451%;\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4444%;\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7124%;\"\u003e\n \u003cp\u003eGroup-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7255%;\"\u003e\n \u003cp\u003e100mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e9.1\u0026plusmn;8.975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.451%;\"\u003e\n \u003cp\u003e65.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4444%;\"\u003e\n \u003cp\u003e34.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7124%;\"\u003e\n \u003cp\u003eGroup-III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7255%;\"\u003e\n \u003cp\u003e200mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e11.32\u0026plusmn;2.365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.451%;\"\u003e\n \u003cp\u003e67.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4444%;\"\u003e\n \u003cp\u003e32.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues are expressed as Mean \u0026plusmn; SEM (n=5); [Control (Water 0.1 ml/Mouse), Positive Control diclofenac-Na (10 mg/kg), Group I =\u003cem\u003e\u0026nbsp;Vitex trifolia\u003c/em\u003e (50 mg/kg), Group II = \u003cem\u003eVitex trifolia\u003c/em\u003e (100 mg/kg), Group III = \u003cem\u003eVitex trifolia\u0026nbsp;\u003c/em\u003e(200 mg)]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Results of Hot Plate Test for \u003cem\u003eVitex trifolia\u003c/em\u003e Extract\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"611\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0 min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e30 min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e60 min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.082%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e90 min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e120min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e0.1ml/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e3.19\u0026plusmn;0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e4.19\u0026plusmn;0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e5.19\u0026plusmn;2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.082%;\"\u003e\n \u003cp\u003e6.284\u0026plusmn;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e6.32\u0026plusmn;0.367\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.23\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.54\u0026plusmn;1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8.21\u0026plusmn;2.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.082%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9.44\u0026plusmn;1.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5.49\u0026plusmn;0.605\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup-I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e50 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e2.12\u0026plusmn;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e7.15\u0026plusmn;1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e6.23\u0026plusmn;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.082%;\"\u003e\n \u003cp\u003e7.32\u0026plusmn;1.494\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e5.13\u0026plusmn;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup-II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e100 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e3.81\u0026plusmn;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e4.03\u0026plusmn;1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e4.23\u0026plusmn;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.082%;\"\u003e\n \u003cp\u003e5.11\u0026plusmn;0.650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e3.89\u0026plusmn;0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup-III\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e200 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e4.88\u0026plusmn;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e5.12\u0026plusmn;0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6066%;\"\u003e\n \u003cp\u003e4.9\u0026plusmn;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.082%;\"\u003e\n \u003cp\u003e6.2\u0026plusmn;0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.2459%;\"\u003e\n \u003cp\u003e4.99\u0026plusmn;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAnalgesic activity of extract of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eVitex trifolia\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eon Glutamate induced abdominal writhing test.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"left\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.5918%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDose (mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5102%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of licking\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Inhibition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.5918%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e0.1 ml/mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5102%;\"\u003e\n \u003cp\u003e104.60\u0026plusmn;1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.5918%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDiclofenac Sodium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e10mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5102%;\"\u003e\n \u003cp\u003e13.40\u0026plusmn;0.6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e87.18%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.5918%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5102%;\"\u003e\n \u003cp\u003e61.80\u0026plusmn;1.16***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e40.91%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.5918%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5102%;\"\u003e\n \u003cp\u003e46.40\u0026plusmn;0.68***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e55.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.5918%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup III\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5102%;\"\u003e\n \u003cp\u003e51.36\u0026plusmn;0.47***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.449%;\"\u003e\n \u003cp\u003e51.44%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues are expressed as Mean \u0026plusmn;SEM (n=5);*the mean difference is significant at the 0.05 level\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e٭٭\u003c/span\u003e\u003c/strong\u003ethe mean difference is significant at the 0.01 level; \u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e٭٭٭\u003c/span\u003e\u003c/strong\u003ethe mean difference is significant at the 0.001 level. Dunnett tests treat one group as control and compare all other group against it.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Department of Biomedical Science, School of Medicine, Kyungpook National University, Daegu, Republic of Korea","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vitex trifolia, Antioxidant activity, Analgesic properties, α-amylase inhibition, Anti-inflammatory activity","lastPublishedDoi":"10.21203/rs.3.rs-5996186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5996186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eVitex trifolia\u003c/em\u003e, a plant traditionally utilized for treating ailments such as chronic colds, coughs, dysentery, mastitis, and liver disorders, was evaluated for its antioxidant, antidiabetic, anti-inflammatory, and analgesic properties in this study. Phytochemical analysis of the leaf extracts revealed high levels of total phenolics (95.12 mg GAE/g) and flavonoids (42.50 mg QE/g). The in vitro assays demonstrated significant antioxidant activity, with 77.85% DPPH radical scavenging at 100 \u0026micro;g/mL and 73.33% nitric oxide radical scavenging at 1000 \u0026micro;g/mL. The extracts also exhibited potent antidiabetic effects, inhibiting α-amylase by 67.25% at 100 \u0026micro;g/mL, and strong anti-inflammatory activity, with 70.25% inhibition of albumin denaturation at 800 \u0026micro;g/mL. In vivo analgesic activity was confirmed through acetic acid-induced writhing and tail-flick assays in mice, where significant reductions in writhing responses were observed at doses of 50 and 100 mg/kg, comparable to diclofenac sodium. Additionally, the extract reduced glutamate-induced nociception by 40.91% and 55.64% at doses of 50 mg/kg and 100 mg/kg, respectively, in the tail-flick test. These findings suggest that \u003cem\u003eV. trifolia\u003c/em\u003e has significant therapeutic potential, exhibiting strong antioxidant, antidiabetic, anti-inflammatory, and analgesic effects.\u003c/p\u003e","manuscriptTitle":"Assessment of the Therapeutic Potential, Antioxidant, Antidiabetic, and Analgesic Properties of Vitex trifolia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-04 06:37:16","doi":"10.21203/rs.3.rs-5996186/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60bd164a-416b-4e45-b0dc-0be0dab60e43","owner":[],"postedDate":"March 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44831335,"name":"Plant Molecular Biology and Genetics"},{"id":44831336,"name":"Medicinal Chemistry"}],"tags":[],"updatedAt":"2025-03-04T06:37:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-04 06:37:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5996186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5996186","identity":"rs-5996186","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}