Vitex negundo Linn (Nirgundi): Potential etiquette of an Important Medicinal Plant

Sadhana Yadav^*1, Dr. Prashant K Deshmukh²

1. Research Scholar, Department of Pharmacy, Sunrise University, Alwar (Raj)
2. Research Supervisor, Department of Pharmacy, Sunrise University, Alwar (Raj)

Abstract: Vitex negundo Linn. belong to family Verbenaceae. It is an important medicinal plant. Literature survey of V. negundo revealed the presence of different classes of natural products including essential oil, triterpenes, diterpenes, sesquiterpenes, lignan, flavonoids, flavones glycosides, iridoid glycosides, and stilbene derivative. The plant is traditionally reported for its use for the treatment of cough, asthma, fever, eye disease, inflammation, intestinal worms, skin diseases, nervous disorders, leprosy and rheumatism. Roots are tonic, anodyne, febrifuge, bechic, expectorant and diuretic. This research is short research of last two years reporting the natural products isolated and biological potential of Vitex negundo Linn.

Introduction: Vitex negundo Linn. (Verbenaceae), locally known as ‘Nirgundi’ an important medicinal plant¹, Vitex negundo Linn. is a woody, aromatic deciduous shrub growing to a small tree. It is an erect, 2-5 m in height, slender tree with quadrangular branchlets. The leaves have five leaflets in a palmately arrangement, which are lanceolate, 4-10 cm long, hairy beneath and pointed at both ends ^2,3. It thrives in humid places or along water courses in wastelands and mixed open forests and has been reported to occur in Afghanistan, India, Pakistan, Sri Lanka, Thailand, Malaysia, eastern Africa and Madagascar. It is grown commercially as a crop in parts of Asia, Europe, North America and West Indies, also finds use as a food crop and a source of timber ⁴.

Plant Anatomy:

• Kingdom – Plantae – Plants
• Sub Kingdom – Tracheobionta – Vascular plants
• Super division – Spermatophyta – Seed plant
• Division – Magnoliophyta – Flowering plants
• Class – Magnoliopsida – Dicotyledons
• Subclass – Asteridea
• Order – Lamilales
• Family – Verbenaceae
• Genus – Vitex Linn.
• Species – Vitex negundo Linn. (Chaste tree) ⁵.

Medicinal Plants: Plants used in traditional medicine contain a vast array of substances that can be used to treat chronic and even infectious diseases. According to a report of World Health Organization, more than 80% of world’s populations depend on traditional medicine for their primary health care needs. The demand for more and more drugs from plant sources is continuously increasing. It is therefore essential for systematic evaluation of plants used in traditional medicine for various ailments. Hence, there is need to screen medicinal plants for promising biological activity^6-8.

Figure 1: Vitex negundo Linn

Literature survey of V. negundo revealed the presence of volatile oil, triterpenes, diterpenes, sesquiterpenes, lignan, flavonoids, flavones glycosides, iridoid glycosides, and stilbene derivative. Though almost all parts of V. negundo are used, the extract from leaves and the roots is the most important in the field of phytomedicine and is sold as drugs. The leaf extract is used in Ayurvedic and Unani system of medicine. Water extract of mature fresh leaves exhibited anti-inflammatory, analgesic and antihistamine properties. Lignans, one class of natural compounds present in V. negundo, showed anti-cholinesterase activity in-vitro. However no studies were conducted to explore the effect of V. negundo extract against memory impairment in-vivo ^{9, 10}.

The leaf extract of Vitex negundo are generally used as a grain preservating material to protect the pulses against insects³. The leaves are the most potent for medicinal use. It is used for treatment of eye-disease, toothache, inflammation, leucoderma, enlargement of the spleen, skin-ulcers, in catarrhal fever, rheumatoid arthritis, gonorrhoea, and bronchitis. They are also used as tonics, vermifuge, lactagogue, emmenagogue, antibacterial, antipyretic and antihistaminic agents. Oil prepared with it, is applied to sinuses and scrofulous sores. Its extract has also shown anticancer activity against Ehrlich ascites tumour cells ¹¹. The roots are used in rheumatism, dyspepsia, dysentery, piles and considered as tonic, febrifuge, expectorant, antihelmintic and diuretic. The flowers are astringent and are employed in fever, diarrhoea and liver complaints. The dried fruits are vermifuge and the bark is used in toothache. The chemical constituents of the essential oil of V. negundo leaves have been reported which indicated viridifloral to be its chief constituents ^{12, 16}.

The plant has been reported to exhibit medicinal properties including the curing of rheumatic pains and reducing swellings of the joints. In Chinese traditional medicine, it has been used for the treatment of chronic bronchitis. An infusion of the twigs is considered to be an effective therapy for headaches, dizziness, convulsions, coughs, mental unrest and is said to promote wakefulness ¹⁷.

Its leaves and seeds are widely used externally for rheumatism and inflammations of joints and are also reported to have insecticidal properties. Internally, decoction of its leaves is taken as diuretic, expectorant, vermifuge, tonic and febrifuge. The chemical components of the essential oil of leaf isolated from V. negundo and other Vitex species have been reported by several researchers in the past. It’s essential oil is found to be useful for sloughing wounds and ulcers. The leaves of V. negundo are reported to possess pesticidal, antifungal and antibacterial properties ^18-20.

Leaves of this plant have been shown mosquito repellent effects as well as antiulcerogenic, antiparasitic, antimicrobial and hepatoprotective potentials. The methanolic root extract possessed potent snake venom neutralizing capacity The acetone extract of V. negundo was found to possess insecticidal, ovicidal, growth inhibition and morphogenetic effects against various life stages of a noxious lepidoteron insect-pest ⁶.

Petroleum ether extract of Vitex negundo leaves has shown significant analgesic activity and the anticonvulsant activity against strychnine and leptazole. Dried leaves powder of Vitex negundo showed anti-arthritic activity in rats ²¹.

V. negundo have diverse medicinal uses in the folk medicinal system of Bangladesh. Along with the utilization in traditional medicine by local practitioners and healers, this plant also reportedly showed diverse pharmacological properties including analgesic, antinociceptive, anti-inflammatory, anti-fertility, anti-feedant, anti-antioxidant, anti-hyperglycemic effect, cytotoxicity for human cancer cell line, hepatoprotective activity against liver damage induced by d-galactosamine, commonly used tubercular drugs and carbon tetrachloride, laxative activity, immunomodulatory effect, and mosquito repellent effect.

The plant parts are reported to have anti-microfilarial, anti-viral, anti-bacterial, anti-fungal, insecticidal, larvicidal, as well as significant effect on antagonizing the Vipera russellii and Naja kaouthia venom induced lethal activity in both in vitro and in vivo. The plant is reported to contain potent and novel therapeutic agents for scavenging of NO and the regulation of pathological conditions caused by excessive generation of NO and its oxidation product, peroxynitrite. Administration of V. negundo extracts also potentiated the effect of commonly used anti-inflammatory drugs sedative-hypnotic drugs (and anti-convulsive agents. Inhibitory effect of V. negundo against active enzymes has also been observed for lipoxygenase and butyryl-cholinesterase α-chymotrypsin xanthine-oxidase and tyrosinase²².

Medicinal herb and various parts of the plant have been employed in the folklore systems of medicine in Asia including India, China and Malaysia for various diseases. Many ethno botanical and pharmacological activities of V. negundo have been reported such as: analgesic and anti-inflammatory activity, antioxidant activity, enzyme inhibitions, nitric oxide scavenging activity, antiradical and antilipoperoxidative activity, CNS activity, hepatoprotective activity, anti-bacterial activity, antifungal activity, larvicidal activity, antiandrogenic effects and mosquito repellent activity. V. negundo leaves were found to have NSAIDs like activity ²³.

The plant is traditionally reported for its use for the treatment of cough, asthma, fever, eye disease, inflammation, intestinal worms, skin diseases, nervous disorders, leprosy and rheumatism. Roots are tonic, anodyne, febrifuge, bechic, expectorant and diuretic. The decoction of leaves is given as a drink to reduce phlegm in coughs, chronic bronchitis and asthma. Drugs currently used to treat cough are among the most widely used over-the-counter drugs in the world, despite a recent analysis suggesting that there is a little evidence to suggest that such drugs produce any meaningful efficacy.

The primary action of currently available cough suppressants (opiates, dextromethorphan etc.) is on the central cough pathway. The significant side effects of these agents such as constipation, respiratory depression, dependence, drowsiness and death limit their uses in humans and thus highly unsatisfactory. No peripherally acting antitussives, apart from local anesthetics such as lignocaine and possibly benzonatate are currently established and available for use in patients. There is a current need for the development of safe and effective antitussive therapeutic options in the treatment of persistent cough as alternative to existing medications¹.

Biological Activities:

1. Anti-amnesic activity: Anti-amnesic effect of V. negundo aqueous extract on scopolamine administered at different stages of active avoidance learning in rats. An automatic reflex conditioner with two-way shuttle box (Ugo Basile, Italy). The rats were treated orally with the standard drug through an intragastric feeding tube. Similarly the plant extract were administered for 14 days. For this purpose each rat is placed in a compartment separated from the other one by a guillotine door in the shuttle box.

Exploration period of 2 min is given initially. Thereafter, the trial start, in each trial the animal is subjected to a light for 30 s followed by a sound stimulus for 10s. Immediately after the sound stimulus, the rat receives a single low intensity foot shock (0.5 mA; 3 s) from 10th day to 14th through the floor grid if it does not transfer to the other shock free compartment. Infrared sensors monitor the transfer time from one compartment to another, which is recorded as avoid (after the stimulus of either light alone or both light and sound) and escape (after the foot shock) response.

Each animal received a daily session of 15 trials with an inter-trial duration of 15 s for 5 days i.e., a maximum of 75 trials. The rats were evaluated on the basis of their performance in the last session i.e., in the 5th session for their decrease in amnesic activity and increased learning and memory. The criterion for improved cognitive activity was taken as significant increase in the avoidance response on 5th session (retention) compared to 1st session ²⁴.

• Antioxidant activity: Preliminary studies showed that V. negundo leaf exhibited antioxidant properties and contain natural antioxidants. Thus, the objective of this study was to analyze the antioxidant activity of methanol and hexane extract and essential oil from V. negundo leaf using different in vitro antioxidant assays. In addition, total phenolic contents, flavonoids, tocopherol and carotenoids content of leaf of V.negundo were also quantified using high performance liquid chromatography (HPLC).
• 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method: DPPH method measured the ability of antioxidant in scavenging free radicals present. Antioxidant activity of V. negundo leaf was expressed as the concentration that inhibits 50% DPPH free radical (IC50). Results obtained in the study showed that the IC50 of methanol extract of V. negundo (138±11.68 μg/ml) was significantly (p < 0.05) lower than that of both essential oil (432±12.65 μg/ml) and hexane extract (567±17.37 μg/ml), revealing its higher antioxidant activity than those of hexane extract and essential oil.
• FRAP method: The FRAP test measures the ability of samples to reduce ferric ion to the ferrous form of TPTZ (2, 4, 6-tripyridylstriazine). Arbitrarily, one FRAP unit is defined as the reduction of 1 mol of Fe³⁺ to Fe²⁺. Similarly, result of the study showed that the antioxidant capacity of methanol extract (44.6±7.8 μM TE/g) was significantly (p < 0.05) higher than that of hexane (11.30±1.3 μM TE/g) and essential oil (11.53±1.35 μM TE/g) of leaves of V. negundo (Figure 1). However, there was no significant (p < 0.05) difference on the antioxidant capacity between hexane extract and essential oil. The antioxidant capacity of methanol extract was noted to be four times higher than that of hexane extract and essential oil. It is interesting to note that the trend of antioxidant activity obtained from FRAP assay was similar to that obtained in DPPH assay ¹⁷.
• Antibacterial activity: The bacteria used for antibacterial tests were Gram (+) Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 0121) and Gram (−) Escherichia coli (MTCC 0051), Pseudomonas aeruginosa (MTCC 0741). All the strains used for these studies were procured from MTCC, IMTECH, Chandigarh, India. Antibacterial potential of all three samples of essential oils and successive extracts was evaluated by agar well diffusion method. Nutrient agar plates were swabbed with the broth culture of the respective microorganisms (diluted to 0.5 McFarland Standard) and were kept at room temperature for 15 min for absorption to take place.

Wells of 8 mm diameter were punched into the agar medium and filled with 100 μl each of the essential oils and extracts. DMSO, DMF and hexane were taken as solvent blank and Ciprofloxacin was used as positive control. The inoculated agar plates were incubated for 24 h at 37°C. All the tests were made in triplicate and diameter of the inhibition zones was calculated in mm. The average of diameter of the inhibition zones of each sample was taken called clearing zone (CZ) and the antimicrobial index (AI) was computed as the clearing zone (CZ) minus the diameter of the hole divided by the diameter of the hole.

All the extracts and essential oils were found to be highly effective in inhibiting the growth of bacteria at a minimum concentration of 30 and 60 μg/100 μl, respectively. Each of the essential oil and extracts were found to be active against B. subtilis and E. coli with antimicrobial index (AI) ranging from 0.3 to 1.8. Leaf essential oil inhibited S. aureus with maximum AI of 1.5 while fruit essential oil showed its inhibition against E. coli and B. subtilis with AI of 1.3 and 1.0, respectively. Flower oil did not show any activity against S. aureus while leaf and fruit oils were ineffective against P. aeruginosa. Ethyl acetate extract was found to be most potent among all the extracts tested.

Petroleum ether and aqueous extracts did not show any activity against P. aeruginosa while all the extracts were found potent against S. aureus. Ciprofloxacin was used as positive standard control and the results of tested samples were very promising in comparison to standard drug ciprofloxacin ¹⁸.

• From the study, the zones of inhibition produced by the methanol extract, petether and carbon tetrachloride fractions were found to be 07-16 mm, 07-11 mm and 06-11 mm respectively at a concentration of 200 g/disc in case of 09 bacterial strains and 02 fungal strains where standard kanamycin (30μg/disc) showed zone of inhibition of 08-19 mm. Prominent activity was found against Bacillus subtilis (13-16 mm) by all of the fractions. Methanol extract showed significant inhibition (09-10 mm) against Bacillus cereus, Pseudomonas aeruginosa, Salmonella typhi Pet-ether and carbon tetrachloride fractions showed most prominent inhibitory action (zone of inhibition 11-18 mm) against Bacillus megaterium, Bacillus subtilis, Salmonella typhi and Vibrio mimicus in comparison to standard antibiotic (kanamycin, 30μg/disc). All the fractions of Vitex negundo were also tested for antifungal activity against 03 fungi. The extracts had inhibitory effect against all the test pathogens in different degree. The methanol extract and pet-ether fraction showed profound activity against Aspergillus niger and Candida albicans respectively.

• Volatile oil of Vitex negundo is reported to contain β-carryophyllene, sabinene, linalool, terpinen-4-ol, α-guaiene and globulol as major constituents along with sesquiterpenes, monoterpenes, terpenoids and sterols. A wide variety of essential oils are known to possess the antimicrobial properties and in many cases this activity is due to the presence of monoterpene constituents which exerts membrane damaging effects and stimulate leakage of cellular potassium ions which provides evidence of lethal action related to cytoplasmic membrane damage. Presence of terpenoids in supercritical fluid extract as evident by TLC pattern explains its stronger antibacterial potential ²⁵.
• Phytopathogenic antibacterial activity: There is a worldwide interest in searching for the safe and effective novel antibacterial compounds of plant origin for the control of plant pathogenic bacteria which is responsible for the great impact on the growth and productivity of agriculture crops. In this study an attempt was made to determine the in vitro antibacterial activity of sequentially extracted different solvent (dichloromethane, ethyl acetate, ethanol, methanol and water) extracts of leaf, flower and fruit of Vitex negundo L. and bulb of Allium sativum L. (Garlic) against phytopathogens namely Pseudomonas solanacearum and Xanthomonas axonopodis pv. citri.

The preliminary antibacterial activity was performed by agar well diffusion method and the minimum inhibitory concentration (MIC) values were determined by agar dilution method. The test samples were also subjected to qualitative phytochemical analysis. One way analysis of variance (ANOVA) followed by least significant difference (LSD) test were done for the statistical analysis of the data. All the test samples showed inhibitory effect on both of the test pathogens and the diameter of inhibition zone ranged from 9.9 ± 0.5 mm to 48.5 ± 1.3 mm and the inhibitory effect differed significantly (P<0.05) among the samples. Ethyl acetate extract of flower of Vitex negundo L. showed significantly (P<0.05) higher inhibition on Pseudomonas solanacearum and Xanthomonas axonopodis pv. citri.

The MIC values of ethyl acetate extracts of fruit and flower of Vitex negundo L. and Allium sativum and ethanol extract of flower of Vitex negundo L. ranged from 2.5mg/ml to 40mg/ml. Phytochemical analysis of above extracts revealed the presence of alkaloids, flavonoids, tannins, cardiac glycosides and terpenoids. Further studies are being carried out to elucidate the active principles responsible for the inhibitory effect of these pathogens and to determine their activity in vivo. This is the first report that reveals the inhibitory effect of Vitex negundo L. on Pseudomonas solanacearum and Xanthomonas axonopodis pv. Citri ²⁶.

• Antifungal activity: Sathiamoorthy et al., (2007) isolated six compounds from the powdered leaf extracts of Vitex negundo. The isolated compounds were evaluated for antifungal and anti-bacterial activity. From the isolated compounds two possess potent anti-fungal activities and very active when compared to other isolated compounds. Significant antifungal activity in ethanolic extract against Cryptococcus neoformans and Trichophyton mentagrophytes was offered by two compounds isolated from the leaf extract of Vitex negundo ²⁸.
• Anti-inflammatory and analgesic activities of Vitex negundoi: Inflammation may start in every part of our body. Any time when the word describing a disease ends with, it’s an inflammatory disease. Dermatitis means an inflammation of the skin, arthritis an inflammation of joints, an othitis an inflammation of the ear. Thus anti-inflammatory activity of a compound is considered to be a valuable feature. The leaves of Vitex negundo possess anti-inflammatory activity. Experimental investigations revealed that the mature fresh leaf of Vitex negundo have dose-dependent activity against inflammation as revealed in the carrageenan and formaldehyde models. Mature fresh leaf extract of Vitex negundo also demonstrated a dose-dependent prostaglandin (PG) synthesis inhibition, membrane stabilising and antihistamine activities. The inverse dose–response relationship shown by acute anti-inflammatory, antihistamine, PG synthesis inhibition and membrane stabilising activities may be due to reduction of the effectiveness of the active principle at its high concentrations.

Sedatives and stress are responsible for producing analgesia. There was no sign of stress observed in the rats treated with the mature fresh leaves extract of Vitex negundo. Mature fresh leaves extract of Vitex negundo is effective against the establishment of chronic inflammation which happens at the later stage of acute inflammation. Moreover treatment with the mature leaf extracts of Vitex negundo in rats did not show a gastric lesion which is an advantage when compared with the use of modern NSAIDs. Treatment of Mature fresh leaves extract of Vitex negundo for 14 days in rats orally did not produce detectable toxic effect in terms of body weight, serum concentrations of urea, creatinine, glucose and serum activity of ALT.

This is a very important criterion that favours the use of this extract for medicinal purposes. The anti-inflammatory and analgesic activities of the leaves did not disappear after the flowering of the tree in contrast to Anisomeles indica which lost these activities after flowering of the plant. These studies provide evidence for the anti-inflammatory and analgesic properties of mature fresh leaves of Vitex negundo claimed in Ayurveda medicine ^28-35.

Enzyme-inhibitory activity: Root extracts of Vitex negundo showed inhibitory activity against enzymes such as lipoxygenase and butyryl-cholinesterase, α-chymotrypsin, xanthine-oxidase and tyrosinase. Woradulayapinij et al. reported the HIV type 1 reverse transcriptase inhibitor activity of the water extract of the aerial parts of Vitex negundo ⁴.

Essentional Oils: The chemical components of the essential oil from V. negundo have been reported. Its essential oil is found to be useful for sloughing wounds and ulcers. The essentional oils from fresh leaves, flowers and dried fruits were extracted and analysis by GC/MS which may be responsible for the various medicinal properties of the plant.

1. From leaves: The identified constituent- p-cymene, cis-ocimene, citronellal, β-curcumene, β-caryophyllene, α-guaiene, guaia-3,7-diene, δ-guaiene, valencene, caryophyllene epoxide, ethyl-9–hexadecenoate, palmitic acid, (E)-nerolidol, humulene epoxide 1, globulol, humulene epoxide 2, epi-α-cadinol, α-muurolol, α-cadinol and α-bisabolol acetate represented about 85.5% of total composition of the essential oil of leaf ¹⁸.
2. From flowers: Twelve identified constituent in flower essential oil were formic acid, n-heptane, p-cymene, β-caryophyllene, trans-α-bergamotene, valencene, α-selinene, β-selinene, germacren-4-ol, caryophyllene epoxide, (E)-nerolidol and P-(1,1-dimethylethyl) toluene represented about 65% of total composition of the oil, (Khokra et al., 2008) from the flower oil of V. negundo, the main constituents of the oil were sabinene, linalool, terpinen-4-ol, β-caryophyllene, α-guaiene and globulol constituting 61.8% of the oil as major constituents along with sesquiterpenes, monoterpenes, terpenoids and sterols ^{35, 25}.
3. From fruits: The thirteen constituents namely α-copaene, β-caryophyllene, α-cedrene, α-guaiene, guaia-3,7-diene, α-humulene, aristolene, germacrene D, β-selinene, caryophyllene oxide, n-hexadecanoic acid, palmitolic acid and traces of acetyl lactyl glycerate were identified in dried fruit oil ¹⁸.

Proximate Analysis of Vitex negundo Linn. ³⁵:

S.No Parameters Quantitative (%) 1 Ash 7.5-8.5 2 Moisture 15.00-18.70 3 Crude protein 12.22-15.23 4 Crude fiber 25.50-30.50 5 Fat 5.00-9.00 6 Carbohydrate 7.5-10.57 7 Alkaloids 0.5

 

REFERENCES:

1. Rizwan UH, Azhar-ul-Haq AS, Zahoor U, Habib U, Rafeeq AK: Antitussive and Toxicological Evaluation of Vitex negundo Linn. Natural product research 2012; 26(5): 484-488.
   1. Raghavendra H, Lakshmanashetty, Vijayananda B, Nagaraj MG, Hiremath, and Vadlapudi K: In vitro Antioxidant Activity of Vitex negundo L. Leaf Extracts. Chiang Mai J. Sci. 2010; 37(3): 489-497.
   1. Rahman MS, Bhattacharya GN: Effects of leaf extract of vitex negundo on Lathyrus Sativus Linn. Curr. Sci 1982; 51(8): 434-435.
   1. Vishwanathan AS, Basavaraju R: A research on Vitex negundo L.–A medicinally important plant. EJBS 2010; 3(1): 30-42.
   1. Mahalakshmi R, Rajesh P, Ramesh N, Balasubramanian V, Rajesh Kannan V: Hepatoprotective activity on Vitex negundo Linn. (Verbenaceae) by using Wistar Albino Rates in Ibuprofen Induced Model. International Journal of Pharmacology 2010; 6 (5): 658-663.
   1. Chowdhury JA, Islam MS, Asifuzzaman Sk, Islam MK: Antibacterial and cytotoxic activity screening of leaf extracts of Vitex negundo (Fam: Verbenaceae). J. Pharm. Sci. & Res 2009; 1(4); 103-108.
   1. Duraipandiyan V, Ayyanar M, Ignacimuthu S: BMC Comp. Alter. Med 2006; 6: 35-41.
   1. Gupta AK, Tandon N, Sharma M: Quality standards of Indian medicinal plants. Indian Council of Medical Research, New Delhi, India 2005.
   1. Azhar UH, Abdul M: Enzymes Inhibiting Lignans from Vitex Negundo. Chemical and Pharmaceutical Bulletin 2004; 52(11); 1269-1272.
   1. Dharmasiri MG, Jayakodym JR, Galhenam G, Liyanagem SS, Ratnasooriyam WD: Anti Inflammatory and Analgesic Activities of Mature Fresh Leaves of Vitex Negundo. Jounal of Ethnopharmacology 2003; 87(2-3): 199-206.
   1. Tiwari OP, Yamini BT: Antioxidant properties of different fractions of Vitex negundo Linn. Food Chem 2007; 100: 1170-1176.
   1. Vinay KV, Siddiqui NU, Mohd A: Phytochemical constituents from the bark of Vitex negundo LINN. International Journal of Pharmaceutical Sciences Research and Research 2011; 7(2).
   1. Dhakal RC, Rajbhandari M, Kalauni SK, Awale S, Gewali MB: Phytochemical constituents of the bark of Vitex negundo. J. Nepal Chem. Soc 2009); 23: 89-92.
   1. Rastogi T, Bhutda V, Moon K, Aswar PB, Khadabadi SS: Comparative studies on anthelmintic activity of Moringa oleifera and Vitex negundo. Asian J. Res. Chem 2009; 2(2): 181-182.
   1. Subramani J, Damodaran A, Kanniappan M, Mathuram LN: Anti-inflammatory effect of petroleum ether extract of Vitex negundo leaves in rat models of acute and subacute inflammation. Pharmaceutical Biology 2009; 47(4): 335-339.
   1. Mahmud S, Shareef H, Farrukh U, Kamil A, Rizwani GH: Antifungal activity of Vitex negundo. Pak. J. Bot 2009; 41(4): 1941-1943.
   1. Zargar M, Azizah AH, Roheeyati AM, Fatimah AB, Jahanshiri F, Pak-Dek MS: Bioactive compounds and antioxidant activity of different extracts from Vitex negundo leaf. Journal of Medicinal Plants Research 2011; 5(12): 2525-2532.
   1. Khokra SL, Prakash O, Jain S, Aneja KR, Yogita D: Essential Oil Composition and Antibacterial Studies of Vitex negundo Linn Extracts. Indian J. Pharm Sci 2008; 70(4): 522–526.
   1. Leopold J, Gerhard B, Christiane P, Mohamed PS, Nambiar MK: Geetha Analysis of the essential oils of the leaves of the medicinal plants Vitex negundo var. negundo and Vitex negundo var. purpurescens from India. Acta Pharm 1998; 179 (48).
   1. Rasaga CY, Morales E, Rideout JA: Antimicrobial compounds from Vitex negundo. Philipp J. Sci 1999; 128(21).
   1. Adnaik RS, Pai PT, Sapakal VD, Naikwade NS, Magdum CS: Anxiolytic activity of Vitex negundo Linn in experimental models of anxiety in mice. International journal of green pharmacy 2009; 3(3): 243-247.
   1. Farzana BC, Safiul Azam FM, Maruf Hassan Md, Farhana  IJ, Anita RC, Syeda S, Zubaida K, Mohammed R: Studies with Callus Induction of Vitex Negundo an Aromatic Medicinal Plant. American-Eurasian Journal of Sustainable Agriculture 2011; 5(1): 6-14.
   1. Thangaraj V, Kaliya PK, Samuthirapandian R, Devadasan V: Active compound from the leaves of Vitex negundo L. shows anti-inflammatory activity with evidence of inhibition for secretory Phospholipase A2 through molecular docking. Biomedical Informatics 2011; 7(4): 199-206.
   1. Abhinav K, Jogender M, Madhusudana K, Vegi Ganga MN, Yogendra KG, Ramakrishna S: Anti-Amnesic Activity of Vitex Negundo in Scopolamine Induced Amnesia in Rats. Pharmacology & Pharmacy 2010; 1: 1-8.
   1. Nagarsekar KS, Nagarsenker MS, Kulkarni SR: Evaluation of composition and antimicrobial activity of supercritical fluid extract of leaves of Vitex negundo. Indian Journal of pharmaceutical sciences 2010; 72(5): 641-643.
   1. Jeyaseelan EC, Pathmanathan MK, Jeyadevan JP: Inhibitory Effect of Different Solvent Extracts of Vitex negundo L. and Allium sativum L. on Phytopathogenic Bacteria. Archives of Applied Science Research 2011; 3(1): 1-8.
   1. Rameshbabu AP, Anand PR: Research on the therapeutic potential of Vitex negundo Linn. Journal of Pharmacy Research 2010; 3(8): 1920-1922.
   1. Vishal R T: Medicinal uses and biological activities of Vitex negundo. Natural product radiance 2005; 4(3).
   1. Raama MJ, Venkataraman S, Meera R, Kiran SD, Chidambaranathan N, and Devi P: Phytochemical investigation and Antipyretic activity of leaf extract of Vitex negundo Linn. International Journal of PharmTech Research 2010; 2(2): 1068-1073.
   1. Suresh A, Thein ZL, Feng L, Yasuhiro T, Aung M, Akihiro T, Takao Y, Hiroyasu E, Shigetoshi K: Identification of Chrysoplenetin from Vitex negundo as a Potential Cytotoxic Agent against PANC-1 and a Panel of 39 Human Cancer Cell Lines (JFCR-39). Phytotherapy research 2011.
   1. Amit S, Sapna T, Ruchi N, Arjit C and Nag TN: Antimicrobial activity and cellular toxicity of flavonoid extracts from pongamia pinnata and Vitex negundo. Romanian Biotechnological Letters 2011; 16(4).
   1. Alama MI, Gomes A: Snake venom neutralization by Indian medicinal plants (Vitex negundo and Emblica officinalis) root extracts. Journal of Ethnopharmacology 2003; 86, 75–80.
   1. Kamaraj C, Bagavan A, Abdul Rahuman A, Abduz ZA, Elango G, Pandiyan G: Larvicidal potential of medicinal plant extracts against Anopheles subpictus Grassi and Culex tritaeniorhynchus Giles (Diptera: Culicidae). Parasitol Res 2009; 104, 1163–1171.
   1. Trapti R, Vijay B, Komal M, Aswar PB, Khadabadi SS: Comparative Studies on Anthelmintic Activity of Moringa Oleifera and Vitex Negundo. Asian J. Research Chem 2009; 2(2).
   1. Kaul, Pran N, Rao, Bhaskaruni RR, Bhattacharya, Arun K, Singh, Kamla, et al: Essential Oil Composition of Vitex negundo L. Flowers. Journal of Essential Oil Research 2005.