Nirgundi (Vitex negundo)

Recent Research on Nirgundi (Vitex negundo)

• Tiwari, Ashutosh. (2014). Analgesic and anti-inflammatory activities of Vitex Negundo (leaves) extract. International Journal of Pharmacognosy. 1. 301- 306. 10. 13040/ IJPSR. 0975- 8232. 1 (5). 301- 06. Aims: Analgesic and anti-inflammatory effects of Vitex negundo leaves extract (VNE) were evaluated in the animal models. The VNE was evaluated for phytochemical analysis.  Settings and Design: Factorial design was used for experimental models.  Methods and Materials: The analgesic effects at graded doses of VNE (40- 320 mg/ kg, p.o.) were evaluated in mice against acetic acid-induced writhing (chemically induced pain) and a hot-plate method (thermally induced pain). The analgesia produced by VNE was compared with the standard analgesic’s diclofenac sodium (DIS, 5 mg/ kg, p.o.) and pentazocin (PTZ, 5 mg/ kg, p.o.). Acute anti-inflammatory activity of VNE was also analysed using a carrageenan-induced rat paw edema model at doses 40, 80, and 160 mg/ kg i.p., using diclofenac sodium (5 mg/ kg, i.p.) as standard.  Statistical analysis used: ANOVA was applied, followed by Tukey’s post hoc test for comparison between the groups. Results: In comparison to the control group, VNE showed highly significant, dose-dependent analgesic activity against chemical as well as thermally induced pain models (P < 0.05). In comparison to the control, VNE at the employed doses produced marked acute anti-inflammatory activity in rats (P < 0.05).  Conclusions: The results suggest that the aqueous extract of Vitex negundo has significant analgesic and anti-inflammatory potential as reflected by the parameters investigated. Further investigations are, however, necessary to explore the mechanism(s) of action involved in these pharmacological activities.
• Adnaik, Rahul & Pai, P. & Mule, S. & Naikwade, N. & Magdum, Chandrakant. (2008). Laxative Activity of Vitex negundo Linn. Leaves. Crude aqueous extract of Vitex negundo Linn. leaves (family: verbenaceae) at doses of 100 and 200 mg/kg was investigated for laxative activity according to Cappaso et al. in albino rats that were compared with the standard drug agar-agar (300mg/kg, p.o.) in normal saline. The rats were fasted for 12 hours before the experiment. After 8 hours of drug administration, the faeces were collected and weighed. The extract was found to produce significant laxative activity in a dose-dependent manner. The activity may be attributed to the phytoconstituents present.
• Sarvankumar, Guguloth & Vivekanandan, Lalitha & Sengottuvelu, Singaravel & Sharif, Sheik & Sivakumar, T. (2011). In vitro antioxidant activity of Vitex negundo Linn bark. International Research Journal of Pharmacy. 2. 105- 109. The present study shows the in vitro antioxidant activity of methanolic extracts of Vitex negundo Linn bark (VBE). The methanolic extracts of Vitex negundo Linn bark result in significant in vitro antioxidant activity in a concentration-dependent manner. Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and our metabolism. They are continuously produced by the body’s normal usage of oxygen, such as respiration and some cell-mediated immune functions. The oxygen consumption inherent in cell growth leads to the generation of reactive oxygen species (ROS) (Gulcin et al., 2007). Most harmful effects are produced by the reactive oxygen species (ROS) in our body. ROS act as oxidants. Antioxidants are substances that scavenge the oxidation process. Antioxidants are a type of complex compounds found in our diet that act as a protective shield for our body against certain disastrous diseases such as arterial and cardiac diseases, arthritis, cataracts, and also premature ageing, along with several chronic diseases.
• Kulkarni, Roshan & Virkar, AD & D’mello, Priscilla. (2008). Antioxidant and Antiinflammatory Activity of Vitex negundo. Indian journal of pharmaceutical sciences. 70. 838- 40. 10. 4103/ 0250- 474X. 49140. Reactive oxygen species are implicated in various inflammatory disorders. Vitex negundo is mentioned in Ayurveda as useful in treating arthritic disorders. The present work was undertaken to evaluate the antioxidant potential and anti-inflammatory activity of the plant. The total methanol extract of the plant was standardized in terms of total polyphenols. The standardized extract in a dose of 100 mg/kg caused a comparable reduction in edema with that of diclofenac sodium (25 mg/kg) when evaluated for anti-inflammatory activity by the carrageenan-induced rat paw edema method. The extract also exhibited a strong free radical scavenging activity by 1, 1- diphenyl- 2-picrylhydrazyl method and caused a significant reduction in the formation of thiobarbituric acid reacting substances when evaluated for its lipid peroxidation inhibitory activity. The results strongly suggest that radical quenching may be one of the mechanisms responsible for its anti-inflammatory activity.
• Tandon, Vishal & Gupta, Rajesh. (2006). Anti-Inflammatory Activity and Mechanism of Action of Vitex negundo Linn. International Journal of Pharmacology. 2. 10. 3923/ ijp. 2006. 303. 308. To evaluate the anti-inflammatory activity of Vitex negundo (VN) leaf extract and its mechanism of action in experimental animals. Carrageenin-induced hind paw edema and cotton pellet granuloma test in albino rats were employed to study the anti-inflammatory activity of Vitex negundo (VN) leaf extract. The mechanism of anti-inflammatory action was explored by observing its effect on oxytocin-induced contractions in the rat uterus and oxidative stress. VN extract was administered orally in graded doses (100, 250, and 500 mg kg-1) as single-dose therapy and twice daily for 7 days in respective inflammatory experimental models. The effects were compared with phenylbutazone (100 mg kg-1) orally in the carrageenin-induced hind paw edema method and ibuprofen (200 mg kg-1, B.DX 7 days) orally in the cotton pellet granuloma test as standard controls, respectively. The test drug showed significant anti-inflammatory activity in a dose-dependent manner in both experimental models. VN inhibited oxytocin-induced contractions of the rat uterus and plasma MDA (malondialdehyde) levels significantly. These observations suggest that VN possesses anti-inflammatory activity against acute as well as sub-acute inflammation, which appears to be due to prostaglandin inhibition and reduction of oxidative stress, respectively, which needs to be substantiated by further study.
• Patil, Rajeshwari & M., Naveen Kumar & Lamani, Babu & Sharma, S. (2015). Anti-inflammatory activity of Vitex negundo, Boswellia serrata, and Aegle marmelos leaf extracts in LPS-treated A549 cells. International Journal of Phytopharmacy. 5. 12- 20. 10. 7439/ ijpp. v5i2.2009. The present study aims to evaluate the therapeutic properties of the medicinal plants (Vitex negundo, Aegle marmelos, and Boswellia serrata) for their potential anti-inflammatory activity in LPS-treated human lung adenocarcinoma A549 cells. A549 cells were treated with or without LPS and methanolic leaf extracts of the above medicinal plants, and the cell viability and nitric oxide production were measured by MTT assay and Griess reaction, respectively. The expression of pro-inflammatory cytokines mRNAs (IL-8 and TNF-α) was measured by semi-quantitative RT-PCR. The methanolic leaf extracts of all three 3 plants significantly decreased LPS-induced NO production and pro-inflammatory cytokines expression. Out of the 3 plants tested, Vitex negundo and Aegle marmelos leaf extracts at 50 and 100 µg/ ml show potent anti-inflammatory activity. The novel pharmacological action provides a new avenue for the isolation and purification of phytochemicals from Vitex negundo and Aegle marmelos that may be of therapeutic modality for the treatment of inflammation.
• R Valarmathi & K Sundari & Puratchikody, Ayarivan & Jayakar, Balasundaram. (2000). Anti-anti-inflammatory Activity of Dried Root Extracts of Vitex negundo Linn. Indian Drugs. 37. 42- 43. Extracts of Vitex Negundo Linn dried root obtained by continuous hot percolation were given orally to study its effects on the exudative and proliferative phases of the inflammatory reaction, using the technique of carrageenan-induced paw edema in male albino rats. The anti-inflammatory activity was compared with that of diclofenac sodium. In carrageenan-induced paw edema, 45 mg/ kg of the dried extracts, such as chloroform, acetone, and alcohol, showed anti-inflammatory activity comparable to that of diclofenac sodium.
• Petchi, R. & Kathiresan, Vijaya & Parasuraman, Subramani & Natchiappan, A., & Devika, G. S. (2011). Anti-arthritic effect of ethanolic extract of leaves of Vitex negundo Linn., (Verbenaceae) in male albino Wistar rats. International Journal of Research in Pharmaceutical Sciences. 2. 213- 218. To determine the anti-arthritic effect of ethanolic extract of leaves of Vitex negundo Linn. (Verbenaceae) in male albino Wistar rats using Freund’s complete adjuvant model. The plant was collected in the agricultural college, Madurai, and preliminary phytoconstituents were identified by chemical analysis. The fatty materials from the leaves were extracted with petroleum ether by the hot percolation method. The dried leaves were extracted with ethanol and used for pharmacological experiments. The male Wistar rats were used for the chronopharmacological and anti-arthritic study. The arthritis was induced by Freund’s complete adjuvant (FCA), and the anti-arthritic effect of the ethanolic extract of the leaves of Vitex negundo Linn (VNE) was studied at 100, 250, and 500 mg/ kg dose levels. At the end of the study, the liver function test and radiological examination were carried out to assess the anti-arthritic effect of the VNE. VNE at 250 and 500 mg/kg showed significant inhibition of FCA-induced arthritis in the evening time drug-administered group. The animals that received VNE in the evening showed a significant anti-arthritic effect than morning-treated animals. VNE-treated animals showed a significant (p< 0.05) increase in body weight. The present study concluded that the leaf extracts of Vitex negundo showed significant anti-arthritic activity against Freund’s complete adjuvant-induced arthritis in male Wistar rats.
• Kumar Bs, Ashok. (2014). Evaluation of In-Vitro Anti-Arthritic Activity of Vitex negundo Against the Denaturation of Protein. Journal of Traditional Medicine. 1. 1-3. Introduction: Vitex negundo (Verbenaceae) is traditionally used as an arthritic anti-inflammatory for arthritis. The main aim of the study is to screen anti- arthritic activity of aqueous extract of leaves of Vitex negundo by protein denaturation. Methods: The extract at different concentrations was incubated with egg albumin and bovine albumin in controlled experimental conditions and subjected to the determination of absorbance to assess the anti-inflammatory property. Diclofenac sodium was used as the reference drug. Results: The present findings exhibited a concentration-dependent inhibition of protein (albumin) denaturation by the Vitex negundo. The effect of diclofenac sodium was found to be less when compared with that of Vitex negundo (1000 μg/ ml). Conclusion: From the present study, it can be concluded that Vitex negundo showed marked in vitro anti- arthritic effect against the denaturation of protein.
• Ladda, Padma & Magdum, Chandrakant. (2018). Antitubercular Activity and Isolation of Chemical Constituents from the Plant Vitex negundo Linn. Iranian Journal of Pharmaceutical Research: IJPR. 17. 1353- 1360. Tuberculosis remains a leading cause of death in the world. Currently, there is considerable interest in natural products and their derivatives in the area of drug research for multidrug-resistant tuberculosis (MDR-TB). The present investigation focused on the identification, isolation, and characterization of lead constituents and determined the antitubercular activity of their enriched fractions and isolated compounds by the Nitrate reductase assay (NRA) method. Leaves extracted with ethanol by Soxhlet extraction and ethanol extract separated in petroleum ether, chloroform, and methanol by separating funnel and fractionated by column chromatography. Ethanol extract, petroleum ether, and chloroform fraction showed antitubercular activity at 150 µg/ mL. Isolated HEA- 2, CM- 20, and CM- 24 showed MIC at 100, while PE- 34 showed MIC at 50 and 100 µg/mL. β-sitosterol content in chloroform and petroleum ether fractions was calculated by using HPTLC. Pet. ether and chloroform fractions of ethanol extract, which contain betulinic acid, ursolic acid, and β -sitosterol, show anti-TB activity. HPTLC, IR, 1H NMR, and GC-MS study of isolated PE-34 gave satisfactory results for confirmation of the structure as ursolic acid with significant antitubercular potential.
• Chowhdury, Jakir Ahmed & Islam, M.S. & Asifuzzaman, Sk & Islam, Mohammad Kaisarul. (2009). Antibacterial and cytotoxic activity screening of leaf extracts of Vitex negundo (Fam: Verbenaceae). Journal of Pharmaceutical Sciences and Research. 1. The work described in this paper details the biological investigation on Vitex negundo, a species of Verbenaceae. The methanol crude extract of Vitex negundo was fractionated with the Kupchan method, and petroleum ether and carbon tetrachloride were used for screening the antimicrobial and antitumor potentials using the disc diffusion method and brine shrimp lethality bioassay, respectively. An established antibiotic (Kanamycin, 30 μg/ disc) and cytotoxic agent (Vincristine sulfate) were used to compare the results. From the graphs, the LC50 (50% mortality) values were found as 12.5 g/ ml, 1.55 g/ ml, and 1.56 g/ ml for methanolic crude extract, pet-ether, and carbon tetrachloride fractions, respectively. LC90 was also determined from the graph to establish the therapeutic index, and the values were found to be 150.0 g/ ml, 50 g/ ml, and 50 g/ ml for methanolic crude extract, pet-ether, and carbon tetrachloride fractions, respectively. The four fractions were also assayed for antimicrobial screening, and all the fractions showed the most prominent zone of inhibition against a number of bacterial and fungal strains. Especially in comparison to the standard kanamycin, all fractions gave a prominent zone of inhibition against Bacillus subtilis, Bacillus megaterium, Salmonella typhi, Vibrio mimicus, and a fungal strain, Aspergillus niger.
• Dewade, D.R. & Christina, A.J.M. & Bhajipale, N.S. & Tekade, N. P. (2010). Antitumor activity of Vitex Negundo Linn. Against Dalton’s Ascitic Lymphoma. International Journal of PharmTech Research. 2. 1101- 1104. The present study aims to evaluate the effect of ethanolic (EEVN) and aqueous (AEVN) extract of leaves of Vitex Negundo Linn. Against Dalton’s Ascitic Lymphoma (DAL) in Swiss Albino mice. DAL cells were injected intraperitoneally (1* 106 cells) into each mouse. Two days after cell injection, the animals were treated with 200 mg/ kg of ethanolic extract of Vitex negundo (EEVN) and aqueous extract of Vitex negundo (AEVN) for 14 days. 5 -Fluorouracil (20 mg/ kg) was used as the standard drug. The entire animals were evaluated for Median Survival Time (MST), and Cancer cell count, packed cell volume, and Hematological parameters were compared with the same parameters in standard conditions by collecting blood from the retro-orbital blood vessel of mice. All the parameters were normalized in tumor-induced mice. These observations suggest that both the extract (EEVN and AEVN) possess antitumor effect against Dalton’s Ascitic Lymphoma (DAL).
• Islam, Md & Akhtar, Most Mauluda & Parvez, Md & Alam, Jahangir. (2013). Antitumor and antibacterial activity of crude methanol leaf extract of Vitex negundo L. Archives of Biological Sciences. 65. 229- 238. 10. 2298/ ABS- 1301229I. In this study, we evaluated a methanol leaf extract of Vitex negundo L. (Verbenaceae) for antitumor and antibacterial activities using the potato disc bioassay and the agar disc diffusion method, respectively. Taking ≤ 20 % tumor inhibition as significant, we found significant crown gall inhibition (24- 48.39 %) with 1 and 10 mg/ ml extracts, while 0.1 mg/ml of the extract was ineffective (14.67 % to 18.28 %). Maximal tumor inhibition was observed with 10 mg/ml extract against Agrobacterium tumefaciens strain AtS-l0105 (48.39 %), followed by AtTa-0112 (45.9 %) and AtAc-0114 (44 %). The methanol leaf extract showed growth inhibitory potency against all of the studied bacteria (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi). The minimum inhibitory concentrations ranged from 0.312 mg/ ml to 1.25 mg/ml. The least MIC value was recorded against S. aureus and P. aeruginosa. The presented findings indicate that the methanol leaf extract could be considered as a source of novel antitumor and antibacterial compounds.
• Kothandan, Sangeetha & Swaminathan, Rajarajan. (2014). Evaluation of in vitro antiviral activity of Vitex Negundo L., Hyptis suaveolens (L) Poit., Decalepis hamiltonii Wight & Arn., against Chikungunya virus. Asian Pacific Journal of Tropical Disease. 4. S111– S115. 10. 1016/ S2222- 1808 (14) 60424- 2. Objective: To screen the three Indian plants for the antiviral activity against the chikungunya virus since chikungunya infections are treated symptomatically without specific drugs to date. Methods: In vitro cytotoxicity assay of the lyophilized extracts was assessed in Vero cells for the determination of maximum non-toxic concentration, and in vitro antiviral assay was evaluated by the inhibition of virus-induced cytopathic effect. Results: Aqueous and aqueous ethanolic extracts of Hyptis suaveolens exhibited partial inhibition to the Asian strain of the Chikungunya virus. Conclusion: Of all three plants tested for antiviral activity against both lineages of chikungunya virus, Hyptis suaveolens was found to be effective against the Asian strain of chikungunya virus.
• Ganapathy, Sujan. (2017). IN VITRO AND IN SILICO ANTICANCER ACTIVITY OF NEGUNDOSIDE ISOLATED FROM LEAVES OF VITEX NEGUNDO LINN. European Journal of Biomedical and Pharmaceutical Sciences. 4. 349- 354. Vitex negundo Linn. [Verbenaceae], commonly known as Five-leaved Chaste tree or Monk’s Pepper (Hindi —Sambhalu, Nirgundi) is used as medicine fairly throughout the greater part of India. The present study aims to isolate negundoside and evaluate anticancer activity by in vitro and in silico methods. Negundoside was isolated by column chromatography from ethyl acetate fractionation of the methanol extract of leaves of V.negundo. Negundoside was characterized by UV, IR, 1H-NMR, 13C-NMR, and Mass spectrum. Standardization of negundoside was done by HPTLC fingerprinting. In vitro anticancer activity was done using HeLa cell lines by MTT assay at different concentrations ranging from 20 to 100 μg/ml, and in silico docking studies using the enzyme EGFR tyrosine kinase. Fingerprinting of isolated negundoside was done by the HPTLC method. The IC50 value was found to be 62.69 μg/ ml in in vitro anticancer activity in HeLa Cell lines. Negundoside was subjected to molecular docking studies for the inhibition of the enzyme EGFR tyrosine kinase, which is one of the targets for the inhibition of cancer cells. It has shown -7.32 kJ mol-1 binding and -11.32 kJ mol-1 docking energy with five hydrogen bonds. Negundoside has been shown to possess anticancer activity both in vitro and in silico studies.
• Audipudi, Amrutha Valli & Reddy, P. & Vajha, Madhuri & Bhaskar, Chitturi. (2014). Evaluation of anti-arthritic and immunostimulatory activities of chloroform extracts of leaves of Vitex negundo L. International Journal of Pharmacognosy and Phytochemical Research. 6. 194- 199. To search for anti-arthritic bioactive compounds with immunostimulatory activities, chloroform extracts of Vitex negundo L. (VCE) were screened for the first time. Chloroform extracts of V.negundo L. leaves were screened for various photochemical constituents and in-vitro anti-arthritic activity, along with other Immunostimulatory activities in terms of DPPH assay, %- 5-Lipoxigenase assay, α-amylase assay, and Brine Shrimp Lethality assay. Levels of Flavonoids and alkaloids of VCE were found to be significantly high. VCE showed 100 % anti-arthritic activity at the concentration of 100 μg/ ml. Antioxidant activity was found to be significantly high (IC50 value of >100 μg/ ml) when compared to Vitamin- C. Crude extracts showed greater value of anti-inflammatory activity (IC50 19.26 μg/ ml) and. α- amylase inhibitory activity (IC50 > 100 μg/ ml) when compared to respective standards curcumin (IC50 9.38μg/ml) and Acarbose (IC50 9.88 μg/ ml). VCE showed moderate cytotoxicity activity (ED50 76.77 μg/ ml) when compared with standard Podophyllotoxin (ED50 2.50 μg/ ml). Results of the current investigation revealed that phytochemical constituents of VCE have strong anti-arthritic potential, and further investigation may be advocated for the identification of a lead molecule with pharmacological significance.
• Garbi, Mohammed & E. Osman, Elbadri & Kabbashi, Ahmed & Saleh, Mahmoud & Yuosof, Yuosof & Mahmoud, Sara & Abdel Salam, Hamza. (2015). Cytotoxicity of Vitex trifolia leaf extracts on MCF-7 and Vero cell lines. Journal of Scientific and Innovative Research. 4. 89- 93. 10. 31254/ jsir. 2015. 4208. The pharmacological and preventive properties of Vitex trifolia leaf extracts are well known, but the anticancer activity of methanol and petroleum ether extracts of Vitex trifolia leaves on human breast cancer cells has not been explored so far. Therefore, the present study was designed to investigate the cytotoxic activities of these extracts against the MCF-7 and Vero cell lines. Cells were exposed to 125 to 500 μg/ ml of the extracts of Vitex trifolia for 72 h. Post-treatment, percent cell viability was studied by 3- (4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide (MTT) assays. The results showed that petroleum ether and methanol extracts significantly reduced the cell viability of MCF-7 cells in a concentration-dependent manner. Concentrations of 125 μg/ ml and above of petroleum ether and 500 μg/ ml of methanol extract were found to be cytotoxic in MCF-7 cells. Cell mortality at 125, 250, and 500 μg/ ml of petroleum ether extract was recorded as 98.80 %, 93.40 % and 87.75 % respectively, whereas at 125, 250, and 500 μg/ml of methanol extract, values were 79.98 %, 75.70 % and 70.25 %, respectively, by MTT assay. MCF- 7 and Vero cells were exposed to 125, 250, and 500 μg/ ml. The data revealed that the treatment with petroleum ether and methanol of Vitex trifolia leaf extract induced cell death in MCF-7 cells. Meanwhile, the same extract showed a moderate inhibition against Vero cell lines. It may be concluded that Vitex trifolia can cause cell death in MCF-7 cancer cells, which can be considered a promising chemotherapeutic agent in breast cancer treatment.
• Matsui, Mariko & Kumar-Roine, Shilpa & DARIUS, Hélène & Chinain, Mireille & Laurent, Dominique & Pauillac, Serge. (2009). Characterisation of the anti-inflammatory potential of Vitex trifolia L. (Labiatae), a multipurpose plant of the Pacific traditional medicine. Journal of Ethnopharmacology. 126. 427-33. 10.1016/j.jep.2009.09.020. Vitex trifolia L. (Labiatae) is a plant commonly employed against Ciguatera Fish Poisoning (CFP) in the Pacific region. Here, the anti-inflammatory potential of an aqueous extract of Vitex trifolia leaves was evaluated by monitoring its effects on the modulation of cytokines, the mediators of inflammation, as well as on the expression profiles of inducible nitric oxide synthase (iNOS), which produces the free radical nitric oxide (NO). We prepared an aqueous extract from Vitex trifolia leaves and evaluated its anti-inflammatory potency by monitoring its effect on the lipopolysaccharide (LPS)-induced cytokines and iNOS mRNA over-production in RAW 264.7 macrophages using quantitative Polymerase Chain Reaction (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) methods. Aqueous extract of Vitex trifolia leaves showed significant dose- and time-dependent inhibitory activity on interleukin (IL)- 1 beta, IL- 6, and iNOS mRNA synthesis, but the slight effect on tumor necrosis factor (TNF)-alpha, all of which are involved in the inflammatory response. Moreover, the plant extract seemed to induce the LPS-dependent IL-10 anti-inflammatory cytokine. These results were further confirmed by ELISA using specific antibodies to mouse IL-6, IL-10, and TNF-alpha. The anti-inflammatory effects of Vitex trifolia could validate its utilization as a traditional remedy against CFP and emphasize its potential therapeutic value against other inflammatory diseases. Therefore, this plant is a promising candidate for further screening of its active compounds through activity-guided fractionation.
• Kadir, Farkaad & Kassim, Normadiah & Abdulla, Mahmood & Yehye, Wageeh. (2013). PASS-predicted Vitex negundo activity: Antioxidant and antiproliferative properties on human hepatoma cells- an in vitro study. BMC complementary and alternative medicine. 13. 343. 10. 1186/ 1472- 6882- 13- 343. Hepatocellular carcinoma is a common type of tumour worldwide with a high mortality rate and with low response to current cytotoxic and chemotherapeutic drugs. The Prediction of Activity Spectra for Substances (PASS) software, which predicts more than 300 pharmacological effects, biological, and biochemical mechanisms based on the structural formula of the substance, was efficiently used in this study to reveal new multifaceted properties of Vitex negundo (VN) constituents. Experimental studies based on antioxidant and antiproliferative assays verified the predictions obtained by the PASS-predicted design strategy. Antioxidant activity of VN extract was studied using 1, 1- diphenyl- 2-picrylhydrazyl (DPPH) and Ferric reducing or antioxidant power (FRAP) assays. The antiproliferative activity of VN extract against WRL68 and HepG2 was investigated based on methylthiazol tetrazolium (MTT) spectrophotometric assay. VN extract showed 79.43% inhibition of the DPPH stable radical with an IC50 of 13.31 ± 0.18 μg/mL. This inhibition was too close to butylated hydroxytoluene (BHT) 82.53 % (IC5013.8 +/- 0.14) and gallic acid 89.51 % (IC50 3.1 +/- 0.08). VN extract exhibited the strongest free radical scavenging power compared with two commercial antioxidants, BHT and ascorbic acid. VN increased the activities of antioxidant enzymes in normal embryonic liver cells (WRL68), including superoxide dismutase (SOD) and glutathione peroxidase (GPX), compared with the H2O2 group. The ethanolic extract of VN showed cytotoxicity to HepG2 cells in a dose and time-dependent manner, with IC50 66.46 μg/ ml, 57.36 μg/ ml, and 65.12 μg/ ml at 24, 48, and 72 hours of incubation, respectively, with no sensitivity in WRL68 cells. This was associated with a significant elevation in lactate dehydrogenase (LDH) release in HepG2 cells. In addition, the activation of caspase-3 3 enzyme suggests that the observed cytotoxicity was mediated via an intrinsic apoptosis pathway. PASS-predicted plant activity could efficiently help in selecting promising pharmaceutical leads with high accuracy and the required antioxidant and antiproliferative properties. This is the first report on PASS-predicted VN activity.
• Kanwal, Abhinav & Mehla, Jogender & Kuncha, Madhusudana & Naidu, Vgm & Gupta, Yogendra & Sistla, Ramakrishna. (2010). Anti-Amnesic Activity of Vitex Negundo in Scopolamine-Induced Amnesia in Rats. Pharmacology & Pharmacy. 1. 1-8. In the present study, we investigated the anti-amnesic activity of Vitex negundo in scopolamine-induced amnesia in rats. Wistar rats (180- 200 g) were trained on active avoidance tasks. Each animal received a session of 15 trials with an inter-trial duration of 15 s for 5 days. Scopolamine (3 mg/ kg, i.p) was administered at different time periods on the basis of stages of memory, i.e., acquisition, consolidation, and retention in different groups (n = 6). The effect of Vitex negundo extract was evaluated and compared to a standard drug, Donepezil. A significant (p < 0.05) increase in the avoidance response on the 5th session has been observed as compared to the 1st session in the control group. Scopolamine treatment significantly (p < 0.05) reduced the avoidance response compared to control. Extract-treated groups showed a significant (p < 0.05) increase in the number of avoidance responses as compared to scopolamine-treated groups. Increased oxidative stress in the brain after scopolamine treatment, as observed by an increase in MDA & decrease in GSH & SOD, was lowered in the groups treated with extracts. AChE activity was also improved after V. negundo treatment. Results of the study have shown that V. negundo treated groups decrease the phenomenon of amnesia by increasing learning and memory through antioxidant effects and decreasing AChE activity.
• M Kannan & P, Rajendran & V Vedha & Ashok Gohil & S Anushka & Chandran Pratap. (2012). HIV-1 reverse transcriptase inhibition by Vitex negundo L. leaf extract and quantification of flavonoids in relation to anti-HIV activity. Journal of Cell and Molecular Biology. 10. 53-59.. This study aimed to determine the activity of the ethanolic leaf extract of Vitex negundo L. against HIV-1 Reverse Transcriptase (RT) and to identify and quantify the flavonoids present. The effects of ethanolic (85 %) leaf extract of Vitex negundo L. on RT activity in vitro were evaluated with recombinant HIV-1 enzyme, using a non-radioactive HIV-RT colorimetric ELISA kit. In addition, the identification and quantification of flavonoids such as Rutin, Luteolin, Myricetin, Quercetin, Kaempherol, Isorhamnetin, and Quercetagetin were analysed using HPLC. The plant Vitex negundo L. ethanolic leaf extract exhibited the most notable activity of 92.8 % against HIV-1 RT at 200 μg/ ml concentration. Phytochemical analysis revealed the presence of steroids, triterpenes, alkaloids, flavonoids, antroquinone glycosides, and amino acids. Among the 7 flavonoids tested, 6 were identified in the decreasing order of quantity as Kaempherol, Myricetin, Quercetin, Quercetagetin, Isorhamnetin, and Luteolin. The study revealed that the plant Vitex negundo L. leaf possesses anti-RT substances, and probably the flavonoids act as anti-virus agents.
• Tandon, Vishal & Khajuria, Vijay & Kapoor, Birender & Kour, D & Gupta, Shravan. (2008). Hepatoprotective activity of Vitex negundo leaf extract against anti-tubercular drugs-induced hepatotoxicity. Fitoterapia. 79. 533- 8. 10. 1016/ j. fitote. 2008. 05. 005. Hepatoprotective (HP) activity of Vitex negundo (VN) leaf ethanolic extract was investigated against hepatotoxicity (HT) produced by administering a combination of three anti-tubercular drugs: isoniazid (INH)- 7.5 mg/ kg, rifampin (RMP)- 10 mg/ kg, and pyrazinamide (PZA)- 35 mg/ kg for 35 days by oral route in rats. V. negundo leaf ethanolic extract was administered in three graded doses of 100, 250, and 500 mg/ kg orally, 45 min before anti-tubercular challenge for 35 days. The HP effect of V. negundo leaf ethanolic extract was evident in the doses of 250 and 500 mg/ kg, as there was a significant decrease in TB, AST, ALT, and ALP levels in comparison to the control. Histology of the liver section of the animals treated with the V. negundo leaf ethanolic extract in doses of 250 and 500 mg/kg further confirms the HP activity.
• Cheng, Zheng & Zhao, Xiang-Xiang & Ai, Hong-Wei & Lin, Bing & Han, Ting & Jiang, Yi-Ping & Xing, Xin & Qin, Lu-Ping. (2014). Therapeutic effects of standardized Vitex negundo seeds extract on complete Freund’s adjuvant-induced arthritis in rats. Phytomedicine. 21. 10. 1016/ j. phy- med. 2014. 02. 003. The seeds of Vitex negundo L. (Verbenaceae) have been commonly used as a folk remedy for the treatment of rheumatism and joint inflammation in Traditional Chinese Medicine. This study aimed to evaluate the anti-arthritic activity of the extract of V. negundo seeds (EVNS) using Freund’s complete adjuvant (CFA) induced arthritis (AA) in a rat model. As a result, EVNS, with abundant phenyl-naphthalene-type lignans, significantly inhibited the paw edema, decreased the arthritis score and spleen index, and reversed the weight loss of CFA-injected rats. Histopathological studies showed a marked decrease in synovial inflammatory infiltration and synovial lining hyperplasia in the joints of EVNS-treated animals. The remarkable decrement of serum inflammatory factors (TNF-alpha, IL-1 beta, and IL-6) was observed in EVNS-treated rats, whereas IL-10, an anti-inflammatory cytokine, was found to be significantly increased by EVNS. The expressions of COX-2 and 5-LOX in PBMC were also inhibited by the administration of EVNS. Our results demonstrated that V. negundo seeds possessed a potential therapeutic effect on adjuvant-induced arthritis in rats by decreasing the levels of TNF-alpha, IL-1 beta, and IL-6 and increasing those of IL-10 in serum, as well as down-regulating the levels of COX-2 and 5-LOX, and therefore may be an effective cure for the treatment of human rheumatoid arthritis.
• Rahmath, Abdulrahiman & Rajan, Nisha & Edakkot, Sreekumaran. (2015). Neuroprotective effect of Vitex negundo against scopolamine-induced cognitive impairment and oxidative stress in Wistar albino rats. 12. 301- 307. 10. 13005/ bbra/ 2040. The present study was undertaken to investigate the neuroprotective effect of Vitex negundo on cognitive impairment induced by scopolamine, a muscarinic antagonist, in Wistar albino rats. Memory impairment was induced by the administration of scopolamine intraperitoneally. Aqueous extract of Vitex negundo was fed to the rats at a dosage of 1000 mg per kilogram body weight of the animal for a period of 15 days. Learning and memory were evaluated using the classical T-maze test. Oxidative stress parameters like Malondialdehyde, glutathione-S-transferase, and superoxide dismutase were assessed, and acetylcholinesterase activity was estimated. We conclude that the aqueous extract of Vitex negundo leaf has potential therapeutic effects on improving cognitive impairment in Wistar albino rats through inhibiting lipid peroxidation, augmenting endogenous antioxidant enzymes, and decreasing acetylcholinesterase activity in the brain. We suggest a further detailed study in this area to separate the exact compound in the extract responsible for the effect for therapeutic purposes.
• Adnaik, Rahul & Pai, P. & Sapakal, V. & Naikwade, N. & Magdum, Chandrakant. (2009). Anxiolytic activity of Vitex Negundo Linn. In experimental models of anxiety in mice. International Journal of Green Pharmacy. 3. 10. 4103/ 0973- 8258. 56284. The purpose of this study was to characterize the putative anxiolytic-like activity of an ethanolic extract prepared from the roots of Vitex negundo (VN) using the elevated plus maze (EPM) and light-dark exploration test in mice. Male mice were either treated orally with the VN extract or the positive control diazepam, respectively, 1 hour before behavioral evaluation. Oral administration of 100 and 200 mg/kg of VN extract significantly (P > 0.01) increased the percentage time spent on and the number of entries into the open arms of the EPM. The effect was comparable to that of the benzodiazepine diazepam (2 mg/ kg p.o.). In the light-dark exploration test, diazepam-treated rats significantly increased the time spent in the light arena and decreased the duration of immobility, while VN treated rats also showed a significant (P > 0.01) increase in the time spent (100 and 200 mg/ kg) in the light arena. Diazepam and the VN extracts did not produce any overt motor dysfunction. These results indicate that VN is an effective anxiolytic agent. In conclusion, the action of extract upon the anxiety models tested is in accord with the traditional use of VN L. and could be useful in primary medical care.
• Mohd Salleh, Rabeta & Dashti, Neda & Ong, Ming Thong. (2014). Proximate Analysis and Anti-Proliferative Properties of Vitex negundo L. Sains Malaysiana. 43. 1543- 1547. The study of the proximate analysis on the leaves of Vitex negundo L. (VN) was done to gain preliminary data on the anti-proliferative properties of cancer cell lines. Aqueous and organic extracts of the leaves of VN were used to identify its cytotoxic effect on six types of cancer-origin and normal cells, namely hormone-dependent breast cancer cell line (MCF-  7), non-hormone-dependent breast cancer cell line (MDA-MB-231), ovarian cancer cell line (Caov-3), cervical cancer cell line (HeLa), liver cancer cell line (Hep- G2) and human foreskin fibroblast cell line (Hs27). The anti-proliferation activities of these extracts were investigated by employing colorimetric MTT (3- (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide) assay through time periods of 24, 48, and 72 h. Preliminary results showed that the methanol extracts had significant effects (p< 0.05) on MDA- MB- 231 with IC50 values of 65.38 μg/ mL. The nutritional composition of the leaves provides a strong basis for emphasizing the nutritional value of Vitex negundo L.
• Anusha, D., & Sharanya, S., & Ramya, & David Darling. (2019). Anticancer Screening of the Phytochemicals Present in the Medicinal Plant Vitex Negundo Against Mutant Anaplastic Lymphoma Kinase (ALK) Protein: An In-Silico Approach. Biomedical and Pharmacology Journal. 12. 993- 1000. 10. 13005/ bpj/ 1727. The lymphomas are a heterogeneous group of cancers of the lymphocytes and the lymphatic system, and account for up to 3% of all malignancies.1 Most of the drugs currently used for the treatment of lymphoma produce various side effects; hence, in this study, we focus on natural compounds obtained from the medicinal plant Vitex negundo, which exhibits selective toxicity against cancer cells. The objective of this research was to formulate the binding energies and interaction of selected phytochemicals present in the medicinal plant Vitex negundo2 against anaplastic lymphoma kinase protein, which is overexpressed in an anaplastic large cell lymphoma. 3, 4, 5 The structure of mutant human anaplastic lymphoma kinase protein was retrieved from the Protein Data Bank (PDB ID: 4ANL ), and the 3D chemical structure of the phytochemicals present in the medicinal plant Vitex negundo was obtained from the PubChem database. A molecular docking study was performed for these natural compounds to evaluate and analyze their anti-lymphoma-cancer activity. A total of 16 compounds present in Vitex negundo, based on a comprehensive literature survey, were selected for this molecular screening. Molecular docking analysis was carried out by Molegro Virtual Docker software to screen the 16 chosen compounds and rank them according to their binding affinity towards the site of interaction of the oncoprotein, anaplastic lymphoma kinase. Out of the 16 screened phytocompounds, only 4 compounds showed promising interactions against the oncoprotein ALK (4ANL). 6′-p-hydroxybenzoyl mussaenosidic acid exhibited a very good binding with a molecular docking score of -127.723 kcal/ mol, ranking first among the compounds screened. This was followed by Betulinic acid, Viridiflorol, and protocatechuic acid with molecular docking scores of -95.596 kcal/ mol, -76.1648 kcal/ mol, and -63.0854 kcal/ mol, respectively. The docking scores from the above study show that the phytocompounds present in Vitex negundo extract exhibit an effective inhibitory effect against anaplastic lymphoma kinase protein that is overexpressed in lymphoma.
• Cheng, Zheng & Zhao, Xiang-Xiang & Ai, Hong-Wei & Lin, Bing & Han, Ting & Jiang, Yi-Ping & Xing, Xin & Qin, Lu-Ping. (2014). Therapeutic effects of standardized Vitex negundo seeds extract on complete Freund’s adjuvant-induced arthritis in rats. Phytomedicine. 21. 10. 1016/ j. phymed. 2014. 02. 003. The seeds of Vitex negundo L. (Verbenaceae) have been commonly used as a folk remedy for the treatment of rheumatism and joint inflammation in Traditional Chinese Medicine. This study aimed to evaluate the anti-arthritic activity of the extract of V. negundo seeds (EVNS) using Freund’s complete adjuvant (CFA) induced arthritis (AA) in the rat model. As a result, EVNS, with abundant phenyl- naphthalene- type lignans, significantly inhibited the paw edema, decreased the arthritis score and spleen index, and reversed the weight loss of CFA-injected rats. Histopathological studies showed a marked decrease in synovial inflammatory infiltration and synovial lining hyperplasia in the joints of EVNS-treated animals. The remarkable decrement of serum inflammatory factors (TNF-alpha, IL-1 beta, and IL-6) was observed in EVNS-treated rats, whereas IL-10, an anti-inflammatory cytokine, was found to be significantly increased by EVNS. The expressions of COX-2 and 5-LOX in PBMC were also inhibited by the administration of EVNS. Our results demonstrated that V. negundo seeds possessed a potential therapeutic effect on adjuvant-induced arthritis in rats by decreasing the levels of TNF- alpha, IL- 1 beta and IL- 6 and increasing that of IL- 10 in serum as well as down-regulating the levels of COX- 2 and 5- LOX, and therefore may be an effective cure for the treatment of human rheumatoid arthritis.
• Prakash, Ved & Rana, Shelly & Sagar, Anand. (2017). Studies on the Analysis of Antioxidant and Enzyme Inhibitory Activity of Vitex negundo Linn. International Journal of Pharmacognosy and Phytochemical Research. 9. 10. 25258/ phyto. v9i6. 8187. The current study was designed to investigate the leaf extracts of Vitex negundo Linn. for their antioxidant and enzyme inhibitory (α-amylase and urease) activity. The antioxidant capacity of the different extracts (methanol, acetone and aqueous) of this plant was evaluated by DPPH (1, 1- diphenyl- 2- picrylhydrazyl) and reduced power tests. The plant exhibited good DPPH radical scavenging activity and moderate reducing power potential. Further, all the extracts of V. negundo were reported to possess good anti-alpha amylase and anti-urease activity of greater than 50 % in all the solvents used at a concentration of 1 mg/ mL. Thus, the study provided scientific evidence for the traditional uses of this plant in the treatment of obesity, diabetes, ulcers, kidney stones, etc. Therefore, the leaf extracts of this plant can be selected for further investigation to determine their therapeutic potential.
• To evaluate the possible antifilarial effect of ethyl acetate extract of Vitex negundo (Verbenaceae) leaves against Setaria cervi filarial parasite in vitro. In vitro screening was done by the method of motility inhibition and MTT reduction assay with a concentration of 0.03 to 1.00 mg/ mL for 2 to 24 h incubation periods, respectively, for possible antifilarial effect by comparing with the control. The present study recorded a significant antifilarial effect of Vitex negundo plant extract and contributed toward the development of a database for novel drug candidates for lymphatic filariasis. Sahare KN, Singh V. Antifilarial activity of ethyl acetate extract of Vitex negundo leaves in vitro. Asian Pac J. Trop Med. 2013 Sep; 6 (9): 689- 92. Doi: 10. 1016/ S1995- 7645 (13) 60119- 4.
• The hepatoprotective activity of ethanolic extract from the leaves of Vitex negundo (VN) was studied against thioacetamide (TAA) induced hepatic injury in Spurge Dawley rats. Rats were administered with VN extract at two different doses, 100 mg/kg and 300 mg/kg body weight. After 12 weeks, the rats administered with VN should have a significantly lower liver-to-body weight ratio. Their abnormal levels of biochemical parameters and liver malondialdehyde were restored closer to the normal levels and were comparable to the levels in animals treated with the standard drug, SY. Kadir FA, Kassim NM, Abdulla MA, Yehye WA. Hepatoprotective Role of Ethanolic Extract of Vitex negundo in Thioacetamide-Induced Liver Fibrosis in Male Rats. Evid Based Complement Alternate Med. 2013; 2013:  739850. doi:10.1155/ 2013/ 739850. Epub 2013 May 19.
• Two new glycosides, 2-methyl pyromeconic acid 3- 0- B-d-glucopyranoside-6 6′-(O-4″- hydroxybenzoate) (1), 6′- O-p-hydroxybenzoly-gardoside (2), and four known iridoid glycosides (3- 6) were isolated from the whole plant of Vitex negundo. Huang J, Wang GC, Wang CH, Huang XJ, Ye WC. Two new glycosides from Vitex negundo. Nat Prod Res. 2013 Jan 29.
• Chemical investigations of the seed extract afforded a new labdane diterpenoid, named negundol (la+1b), as an inseparable mixture of two diastereoisomers in a 5:4 ratio. Zheng CJ, Lan XP, Wang Y, Huang BK, Han T, Zhang QY, Qin LP. A new labdane diterpene from Vitex negundo. Pharm Biol. 2012 Jun; 50 (6): 687- 90. doi: 10. 3109/ 13880209. 2011. 597410. Epub 2012 Apr 10.
• This study was carried out to investigate its cough-relieving potential. The antitussive effect of the butanolic extract of V. negundo (Vn) on sulphur dioxide (SO(2))-induced cough was examined in mice. In toxicity tests, no signs of neural impairment and acute behavioral toxicity were observed at antitussive doses, and the extract has been well-tolerated at higher doses. The results indicate that V. negundo exhibits an anti-tussive effect, and it was found to be devoid of toxicity. Hag RU, Shah AU, Khan AU, Ullah Z, Khan HU, Khan RA, Malik A. Antitussive and toxicological evaluation of Vitex negundo. Nat Prod Res. 2012; 26 (5): 484- 8. doi: 10. 1080/ 14786419. 2010. 534472. Epub 2011 Aug 2.

Recent Research on Nirgundi (Vitex trifolia Linn.)

• Rak, Aweng & Hanisah, Nur & M.A, Mohd & Yunos, Nurhanan & Muhamad, Shamsul. (2012). Antioxidant activity and phenolic compounds of Vitex trifolia var. Simplicifolia is associated with cancer.  ISCA Journal of Biological Sciences. 1. 65- 68. The Vitex trifolia var simplicifolia plant was found growing wildly at the beach, especially at Pantai Sri Tujuh, Tumpat, Kelantan, Malaysia. The extracted leaves were commonly used to prepare a traditional dessert among Siamese communities in Kelantan called “Khanom Bai Kunthi” for a long time. Water was used as an extraction agent by the people in this area. These extracted leaves were used by the Siamese communities in the Tumpat area because it has natural color, flavor, and fragrance properties. The Siamese communities who consumed “Khnom Bai Kunthi” were found to have a healthy life and live longer. This is the starting point of the study. For this study, Vitex trifolia var simplicifolia leaves were extracted by using 80% methanol in the laboratory. The extracted V. trifolia var. simplicifolia was analyzed and found to be high in antioxidant activity of phenolic compounds. The result shows that the extracted V. trifolia var. simplicifolia had a high amount of total phenolic compounds (44.66μg of GAE/ mg of fresh weight). The plant’s secondary metabolites normally play a role in cancer treatment. Cytotoxicity activity of the extracted V. trifolia var. simplicifolia was evaluated on MCF- 7 (Breast cancer cell line), HT- 29 (colorectal cancer cell line), and WRL- 68 (normal liver cell line), and the results show that, cytotoxicity activity (IC50 values 78.87 μg/ ml, 77.50 μg/ ml, and 78.29 μg/ ml, respectively).
• Bao, Fengyan & Tang, Ruotian & Cheng, Li & Zhang, Cuiyun & Qiu, Chongyue & Yuan, Ting & Zhu, Lihan & Li, Hua & Chen, Lixia. (2018). Terpenoids from Vitex trifolia and their anti-inflammatory activities. Journal of Natural Medicines. 72. 10. 1007/ s11418- 018- 1178- x. A new diterpenoid glucoside, (3S, 5S, 6S, 8R, 9R, 10S)- 3, 6, 9- trihydroxy-13 (14)- labdean- 16, 15- olide 3- O- β- D- glucopyranoside (1), and a new iridoid glucoside, (1S, 5S, 6R, 9R)- 10- O- p- hydroxybenzoyl- 5, 6β-dihydroxy iridoid 1- O- β- D- glucopyranoside (2), along with six known compounds (3- 8) were isolated from Vitex trifolia L. Their structures were elucidated by extensive spectroscopic analysis. All these isolated compounds were evaluated for their inhibitory effects on nitric oxide production in LPS-induced RAW 264.7 macrophages. Compounds 2, 4, 5, and 7 showed moderate inhibitory activities with IC50 values of 90.05, 88.51, 87.26, and 76.06 μM, respectively.
• E. Osman, Elbadri. (2015). Antigiardial, antiamoebic, and cytotoxic activity of the leaf extracts of Vitex trifolia. The World Health Organization ranks diarrheal disease as the second (after acute respiratory infections) most common cause of morbidity and mortality in children in the developing world. Entamoeba histolytica and Giardia lamblia are two of the most important and most widespread diarrhea-related parasitic protozoa in the world. Metronidazole is used as a drug of choice against amoebiasis and giardiasis. However, like a lot of other chemical agents, this drug has its own side effects. The present study was carried out to evaluate anti-giardial, anti-amoebic activity and explore the cytotoxicity of Vitex trifolia (leaves) variety supreme court leaves, petroleum ether, and methanolic extracts in vitro. The highest activity against Giardia lamblia, with respect to time, was obtained from petroleum ether extract, which exhibited 75.25 % mortality within 72 h with a concentration of 1000 ppm, followed by the same extract, which exhibited 72.07 % mortality within 72 h with a concentration of 500 ppm. On the other hand, the lowest anti- giardial activity was recorded by petroleum ether extract, 58.92 % mortality with 125 ppm concentration in 72 h. The highest activity against Entamoeba histolytica, with respect to time, was obtained from the methanolic extract, which exhibited 61.64 % mortality within 72 h with a concentration of 1000 ppm. On the other hand, the lowest anti-amoebic activity was recorded by petroleum ether extract, 53.62% mortality with 125 ppm concentration within 72 h. The cytotoxicity of methanol and petroleum ether extract had varying degrees of toxicity to Vero cell lines, with IC 50 349.07 μg/ml for the methanol extract and 369.77 μg/ml for the petroleum ether extract.
• Winarno, Ermin & Susanto, Susanto & Winarno, Hendig. (2020). Antiproliferative activity against cancer cell lines of gamma irradiated “Legundi” (Vitex Trifolia L.) leaves and its chromatogram profiles. AIP Conference Proceedings. 2296. 020068. 10.1063/5.0030628. Legundi (Vitex trifolia L.) is a plant of the Vitex genus that has efficacy as an anti-inflammatory, for bronchitis, fever, and as an anti-cancer because it has high antioxidant activity. The main content of secondary metabolites present in the leaves of Vitek trifolia is terpenoids, flavonoids, and phenolic acid. This research was conducted to study the effect of gamma irradiation dose of 7.5 kGy treatment on dried legundi leaves coarse powder on its potential as an anti-cancer and its chromatographic profile. Gamma irradiation technology (source ⁶⁰Co) is used to extend the shelf life of herbal medicinal ingredients. Maceration of legundi leaves was done in ethanol. The activity of ethanol extracts from legundi leaves, which were not irradiated and which were irradiated with a gamma dose of 7.5 kG, was carried out against cell lines (MCF, HeLa, and K-562). The cytotoxic activity of ethanol extract from legundi leaves showed that the irradiation dose of 7.5 kGy on legundi leaves powder did not damage its anti-cancer potential. The IC50 value of legundi leaves extract before and after an irradiated dose of 7.5 kGy against MCF cancer cells was 8.2 µg/mL, increased to 12.1 µg/ mL; HeLa from 7.6 to 16.9 µg/ mL, and K-562 from 19.7 to 22.4 µg/  mL. The efficacy of unirradiated and irradiated legundi leaves was still in the active category as a potential anticancer extract, because the IC50 value of the ethanol extract was less than 30 µg/ mL. The TLC profile of ethanol extract from irradiated legundi leaves was the same as the unirradiated leaves’ profile. This was a good result, because no irradiation effect on the TLC profile of the ethanol extract.
• Fang, Shi-Ming & Liu, Rui & Li, Lin & Yao, Jin-Long & Liu, Er-Wei & Fan, Guan-Wei & Han, Zhang & Gao, Xiu-Mei. (2018). Anti-inflammatory diterpenes from the fruits of Vitex trifolia L. var. simplicifolia Cham. Journal of Asian Natural Products Research. 21. 1-7. 10.1080/10286020.2018.1482881. Two new labdane-type diterpenes, named viterotulin C (1) and vitexilactone D (2), together with five known diterpenes (3-7), were isolated from the fruits of Vitex trifolia L. var. simplicifolia Cham. Their structures were elucidated by a detailed analysis of spectroscopic data. All the compounds were evaluated for their inhibitory effects on the nuclear factor-kappa B (NF-κB) pathway in the HEK 293 cell line. These compounds presented inhibition on TNF-α-induced NF-κB activation, with inhibition rates ranging from 42.52 ± 10.69% to 68.86 ± 10.76% at the concentration of 50 μM.
• Vasanthi, V.J. & Radhjeyalakshmi, R., & Nasrin, Fatema. (2014). Evaluation of anticancer activity using hexanic extract of Vitex trifolia on two different cancer cell lines. International Journal of Pharmacognosy and Phytochemical Research. 6. 449- 453. The present study was designed to evaluate the cytotoxicity of n-Hexane fractions of Vitex trifolia in two cancer cell lines, viz., Hep G2 and HeLa. Different concentrations, such as 20 and 40. 80, 160, 320, and 640 µg/ ml of extract were tested for cytotoxicity using the MTT Assay. Based on our previous research findings on the qualitative and quantitative analysis of phytochemicals in Vitex trifolia, it was evident that phytochemicals in Vitex trifolia had strong antibacterial activity. Hence, in the current study, anticancer activity was tested using Vitex trifolia extracts. Concentration required for a 50 % inhibition of viability (IC50) was determined graphically. MTT Assays showed that hexanic extract of Vitex trifolia was highly effective against both Hep G2 and HeLa cancer cell lines at a concentration of 80 µg/ml. This concludes that Vitex trifolia can be a potent drug for cancer treatment and an alternative medicine. International Journal of Pharmacognosy and Phytochemical Research. All rights reserved.
• Hossain, M & Paul, N & Sohrab, Mohammad & Kabir, Eva, & Rashid, Mohammad. (2001). Antibacterial activity of Vitex trifolia. Fitoterapia. 72. 695-7. 10.1016/S0367-326X(01)00304-5. The petroleum ether and ethanol extracts of Vitex trifolia leaves exhibited moderate inhibiting activity against both gram-positive and gram-negative bacteria.
• Li, Wen-Xin & Cui, Cheng-Bin & Cai, Bing & Wang, Hai-Yan & Yao, Xin-Sheng. (2005). Flavonoids from Vitex trifolia L. inhibit cell cycle progression at G (2)/ M phase and induce apoptosis in mammalian cancer cells. Journal of Asian Natural Products Research. 7. 615- 26. 10. 1080/ 10286020310001625085. Six flavonoids, persicogenin (1), artemetin (2), luteolin (3), penduletin (4), vitexicarpin (5), and chrysosplenol-D- D (6), have been isolated for the first time as new cell cycle inhibitors from Vitex trifolia L., a Chinese folk medicine used to treat cancers, through a bioassay-guided separation procedure. They were identified by spectroscopic methods. The inhibitory effects of 1- 6 on the proliferation of mammalian cancer cells have been evaluated by the SRB (sulforhodamine B) method, and their effects on cell cycle and apoptosis were investigated by flow cytometry with the morphological observation under a light microscope and by agarose gel electrophoresis to detect internucleosomal DNA fragmentation. Compounds 1- 6 inhibited the proliferation of mouse tsFT210 cancer cells with the IC50s (μg ml-1) > 100 (inhibition rate at 100 μg ml-1, 47.9 %) for 1, > 100 (inhibition rate at 100 μg ml-1, 49.6  %) for 2, 10.7 for 3, 19.8 for 4, 0.3 for 5, and 3.5 for 6. Flow cytometric investigations for 1-6 demonstrated that 1-5 mainly inhibited cell cycle at the G2/ M phase in a dose-dependent manner with a weak induction of apoptosis on the tsFT210 cells, while 6 induced mainly apoptosis of the same tsFT210 cells also in a dose-dependent manner together with a weak inhibition of the cell cycle at the G0/ G1 and G2/ M phases, demonstrating that 1- 6 exert their anti-proliferative effect on tsFT210 cells through inhibiting cell cycle and inducing apoptosis. In contrast to the cell cycle G2/ M phase inhibitory main effect on tsFT210 cells, 5 induced mainly apoptosis on human myeloid leukemia K562 cells with a weak inhibition of the cell cycle at the G2/M phase. The present result provides flavonoids 1- 6 as new cell cycle inhibitors and 1 and 4 as new anticancer flavonoids, which not only provide the first example of cell cycle G2/ M phase inhibitory and apoptosis-inducing constituents of V. trifolia L. but also explain the use of Vitex trifolia L. by Chinese people to treat cancers. Garbi, Mohammed & E. Osman, Elbadri & Kabbashi, Ahmed & Saleh, Mahmoud & Yuosof, Yuosof & Mahmoud, Sara & Abdel Salam, Hamza. (2015). Cytotoxicity of Vitex trifolia leaf extracts on MCF-7 and Vero cell lines. Journal of Scientific and Innovative Research. 4. 89- 93. 10. 31254/ jsir. 2015. 4208. The pharmacological and preventive properties of Vitex trifolia leaf extracts are well known, but the anticancer activity of methanol and petroleum ether extracts of Vitex trifolia leaves on human breast cancer cells has not been explored so far. Therefore, the present study was designed to investigate the cytotoxic activities of these extracts against the MCF-7 and Vero cell lines. Cells were exposed to 125 to 500 μg/ ml of the extracts of Vitex trifolia for 72 h. post-treatment, percent cell viability was studied by 3- (4, 5 dimethylthiazol- 2yl)- 2, 5 5-biphenyl tetrazolium bromide (MTT) assays. The results showed that petroleum ether and methanol extracts significantly reduced cell viability of MCF-7 cells in a concentration-dependent manner. Concentrations of 125 μg/ ml and above of petroleum ether and 500 μg/ ml of methanol extract were found to be cytotoxic in MCF-7 cells. Cell mortality at 125, 250, and 500 μg/ ml of petroleum ether extract was recorded as 98.80 %, 93.40 % and 87.75 % respectively, whereas at 125, 250, and 500 μg/ml of methanol extract, values were 79.98 %, 75.70 % and 70.25 %, respectively, by MTT assay. MCF- 7 and Vero cells were exposed to 125, 250, and 500 μg/ ml. The data revealed that the treatment with petroleum ether and methanol of Vitex trifolia leaf extract induced cell death in MCF-7 cells. Meanwhile, the same extract showed a moderate inhibition against Vero cell lines. It may be concluded that Vitex trifolia can cause cell death in MCF-7 cancer cells, which can be considered as a promising chemotherapeutic agent in breast cancer treatment.
• Matsui, Mariko & Kumar-Roine, Shilpa & DARIUS, Hélène & Chinain, Mireille & Laurent, Dominique & Pauillac, Serge. (2009). Characterisation of the anti-inflammatory potential of Vitex trifolia L. (Labiatae), a multipurpose plant of the Pacific traditional medicine. Journal of Ethnopharmacology. 126. 427-33. 10.1016/j.jep.2009.09.020. Vitex trifolia L. (Labiatae) is a plant commonly employed against Ciguatera Fish Poisoning (CFP) in the Pacific region. Here, the anti-inflammatory potential of an aqueous extract of Vitex trifolia leaves was evaluated by monitoring its effects on the modulation of cytokines, the mediators of inflammation, as well as on the expression profiles of inducible nitric oxide synthase (iNOS), which produces the free radical nitric oxide (NO). We prepared an aqueous extract from Vitex trifolia leaves and evaluated its anti-inflammatory potency by monitoring its effect on the lipopolysaccharide (LPS)-induced cytokines and iNOS mRNA over-production in RAW 264.7 macrophages using quantitative Polymerase Chain Reaction (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) methods. Aqueous extract of Vitex trifolia leaves showed significant dose- and time-dependent inhibitory activity on interleukin (IL)- 1 beta, IL- 6, and iNOS mRNA synthesis, but the slight effect on tumor necrosis factor (TNF)-alpha, all of which are involved in the inflammatory response. Moreover, the plant extract seemed to induce the LPS-dependent IL-10 anti-inflammatory cytokine. These results were further confirmed by ELISA using specific antibodies to mouse IL-6, IL-10, and TNF-alpha. The anti-inflammatory effects of Vitex trifolia could validate its utilization as a traditional remedy against CFP and emphasize its potential therapeutic value against other inflammatory diseases. Therefore, this plant is a promising candidate for further screening of its active compounds through activity-guided fractionation.