PHYTOCHEMICAL AND BIOLOGICAL EVALUATION OF VITEX NEGUNDO LINN: A REVIEW

PHYTOCHEMICAL AND BIOLOGICAL EVALUATION OF VITEX NEGUNDO LINN: A REVIEW

Zahoor Ullah*¹,Riaz Ullah ^{1, 2}, Azhar-ul-Haq Ali Shah², Ijaz Ahmad² and Sajjad Haider¹

Department of Chemical Engineering, College of Engineering, King Saud University ¹, P.O BOX 800, Riyadh 11421, Saudi Arabia

Department of Chemistry, Kohat University of Science and Technology ², Kohat, Pakistan

ABSTRAC

Vitex negundo Linn. belong to family Verbenaceae. It is an important medicinal plant.Literaturesurvey 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 review is short review of last two years reporting the natural products isolated and biological potential of Vitex negundo Linn.

Biological activities

INTRODUCTION:Vitex negundo Linn. (Verbenaceae), locally known as ‘Nirgundi’ an important medicinal plant¹, Vitex negundo Linn. is a woody, aromaticdeciduous shrub growing to a small tree. It is an erect, 2-5 m in height, slender treewith quadrangular branchlets. The leaves have five leafletsin a palmately arrangement, which arelanceolate, 4-10 cm long, hairy beneath andpointed 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 Americaand 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.

PHOTOGRAPHS OF VITEX NEGUNDO LINN.

Medicinal Plants: Plants usedin traditional medicine contain a vast arrayof substances that can be used to treatchronic and even infectious diseases.According to a report of World HealthOrganization, more than 80% of world’spopulations depend on traditionalmedicine for their primary health careneeds. The demand for more and moredrugs from plant sources is continuouslyincreasing. It is therefore essential forsystematic evaluation of plants used intraditional medicine for various ailments.Hence, there is need to screen medicinalplants for promising biological activity^{6, 8}.

Literaturesurvey of V. negundo revealed the presence of volatile oil,triterpenes, diterpenes, sesquiterpenes, lignan, flavonoids,flavones glycosides, iridoid glycosides, and stilbene derivative. Though almost allparts ofV. negundo are used, the extract from leaves andthe roots is the most important in the field of phytomedicineand is sold as drugs. The leaf extract is used in Ayurvedicand Unani system of medicine. Water extractof mature fresh leaves exhibited anti-inflammatory,analgesic and antihistamine properties. Lignans, one class of natural compoundspresent in V. negundo, showed anti-cholinesterase activityin-vitro. However no studies were conductedto explore the effect of V. negundo extract against memoryimpairment in-vivo^{9, 10}.

The leaf extract of Vitexnegundo are generally used as a grainpreservating material to protect the pulsesagainst insects ³.The leaves are the mostpotent for medicinal use. It is used fortreatment of eye-disease, toothache, inflammation,leucoderma, enlargement of the spleen,skin-ulcers, in catarrhal fever, rheumatoidarthritis, gonorrhoea, and bronchitis. They arealso used as tonics, vermifuge, lactagogue,emmenagogue,antibacterial, antipyretic andantihistaminic agents. Oil prepared with it, isapplied to sinuses and scrofulous sores. Itsextract has also shown anticancer activityagainst Ehrlich ascites tumour cells ¹¹.

The roots are usedin rheumatism, dyspepsia, dysentery, piles andconsidered as tonic, febrifuge, expectorant, antihelminticand diuretic. The flowers are astringent and are employedin fever, diarrhoea and liver complaints. The dried fruitsare vermifuge and the bark is used in toothache. Thechemicalconstituents of the essential oil of V. negundoleaves have been reported which indicated viridifloral tobe its chief constituents^{12, 16}.

The plant hasbeen reported to exhibit medicinal properties includingthe curing of rheumatic pains and reducing swellings ofthe joints. In Chinese traditionalmedicine, it has been used for the treatment of chronicbronchitis. An infusion of the twigs is considered to be aneffective therapy for headaches, dizziness, convulsions,coughs, mental unrest and is said to promotewakefulness¹⁷.

Its leaves and seedsare 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 specieshave 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 shownmosquito repellent effects as well asantiulcerogenic, antiparasitic,antimicrobial and hepatoprotective potentials. The methanolic root extractpossessed potent snake venom neutralizingcapacity The acetone extract of V.negundo was found to possess insecticidal,ovicidal, growth inhibition andmorphogenetic effects against various lifestages of a noxious lepidoteron insect-pest⁶.

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 negundoshowed anti-arthritic activity in rats²¹.

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 alsoreportedly showed diverse pharmacological properties including analgesic, antinociceptive, anti-inflammatory, anti-fertility,anti-feedant, anti- antioxidant, antihyperglycemic 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 onantagonizing 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 forscavenging of NO and the regulation of pathological conditions caused by excessive generation of NO and itsoxidation product, peroxynitrite. Administration of V. negundo extracts alsopotentiated the effect of commonly used antiinflammatory drugs sedative-hypnotic drugs (and anticonvulsive agents. Inhibitory effect of V. negundo against active enzymes has also been observed forlipoxygenase and butyryl-cholinesterase α-chymotrypsin xanthine-oxidase and tyrosinase²².

Medicinal herb and various parts of the plant have beenemployed in the folklore systems of medicine in Asia includingIndia, China and Malaysia for various diseases. Manyethnobotanical and pharmacological activities of V. negundohave been reported such as: analgesic and anti-inflammatoryactivity, antioxidant activity, enzyme inhibitions,nitric oxide scavengingactivity, antiradical andantilipoperoxidative activity, CNS activity,hepatoprotective activity, anti-bacterial activity, antifungalactivity, larvicidal activity, antiandrogeniceffects  and mosquito repellent activity. V. negundoleaves were found to have NSAIDs like activity²³.

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. negundoaqueous extract on scopolamine administered atdifferent 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 thestandard drug through an intragastric feeding tube. Similarlythe plant extract were administered for 14 days. Forthis purpose each rat is placed in a compartment separatedfrom the other one by a guillotine door in the shuttlebox.

Exploration period of 2 min is given initially.Thereafter, the trial start,in each trial the animal is subjectedto a light for 30 s followed by a sound stimulus for10s. Immediately after the sound stimulus, the rat receivesa single low intensity foot shock (0.5 mA; 3 s)from 10th day to 14th through the floor grid if it does nottransfer to the other shock free compartment. Infraredsensors monitor the transfer time from one compartmentto another, which is recorded as avoid (after the stimulusof either light alone or both light and sound) and escape(after the foot shock) response. Each animal received adaily session of 15 trials with an inter-trial duration of 15s for 5 days i.e., a maximum of 75 trials. The rats wereevaluated on the basis of their performance in the lastsession i.e., in the 5th session for their decrease in amnesicactivity and increased learning and memory. The criterionfor improved cognitive activity was taken as significantincrease in the avoidance response on 5th session(retention) compared to 1st session²⁴.

2. Antioxidant activity: Preliminary studies showed that V. negundo leafexhibited antioxidant properties and contain naturalantioxidants. Thus, the objective of this study was toanalyze the antioxidant activity of methanol and hexaneextract and essential oil from V. negundo leaf usingdifferent in vitro antioxidant assays. In addition, totalphenolic contents, flavonoids, tocopherol and carotenoids content of leaf of V.negundo were also quantified usinghigh performance liquidchromatography (HPLC).
3. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method: DPPH method measured the ability of antioxidant inscavenging free radicals present. Antioxidant activity ofV. negundo leaf was expressed as the concentration thatinhibits 50% DPPH free radical (IC50). Results obtained inthe study showed that the IC50 of methanol extract ofV. negundo (138±11.68 μg/ml) was significantly (p <0.05) lower than that of both essential oil (432±12.65μg/ml) and hexane extract (567±17.37 μg/ml), revealingits higher antioxidant activity than those of hexane extractand essential oil.
4. FRAP method: The FRAP test measures the ability of samples to reduceferric ion to the ferrous form of TPTZ (2, 4, 6-tripyridylstriazine).Arbitrarily, one FRAP unit is defined as thereduction of 1 mol of Fe³⁺ to Fe²⁺. Similarly, result of thestudy showed that the antioxidant capacity of methanolextract (44.6±7.8 μM TE/g) was significantly (p < 0.05)higher than that of hexane (11.30±1.3 μM TE/g) andessential oil (11.53±1.35 μM TE/g) of leaves of V.negundo (Figure 2). However, there was no significant (p< 0.05) difference on the antioxidant capacity betweenhexane extract and essential oil. The antioxidant capacityof methanol extract was noted to be four times higherthan that of hexane extract and essential oil. It is interesting to note that the trend of antioxidant activityobtained from FRAP assay was similar to that obtained inDPPH assay¹⁷.
5. 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°. 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 ciprofloxacin¹⁸.

• From the study, the zones of inhibitionproduced by the methanol extract, petetherand carbon tetrachloride fractionswere found to be 07-16 mm, 07-11 mmand 06-11 mm respectively at aconcentration of 200g/disc in case of 09bacterial strains and 02 fungal strainswhere standard kanamycin (30μg/disc)showed zone of inhibition of 08-19 mm.Prominent activity was found againstBacillus subtilis (13-16 mm) by all of thefractions. Methanol extract showed  significant inhibition (09-10 mm) againstBacillus cereus, Pseudomonas aeruginosa,Salmonella typhi Pet-ether and carbontetrachloride fractions showed mostprominent inhibitory action (zone ofinhibition 11-18 mm) against Bacillusmegaterium, Bacillus subtilis, Salmonellatyphi and Vibrio mimicus in comparison tostandard antibiotic (kanamycin,30μg/disc).All the fractions of Vitex negundo werealso tested for antifungal activity against03 fungi. The extracts had inhibitory effectagainst all the test pathogens in differentdegree. The methanol extract and pet-etherfraction showed profound activity againstAspergillus niger and Candida albicansrespectively⁶.
• 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 posses 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 explainsitsstrongerantibacterialpotential²⁵.

6. 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 forthe great impact on the growth and productivity of agriculture crops. In this study an attemptwas made to determine the in vitro antibacterial activity of sequentially extracted differentsolvent (dichloromethane, ethyl acetate, ethanol, methanol and water) extracts of leaf, flowerand fruit of Vitex negundo L. and bulb of Allium sativum L. (Garlic) against phytopathogensnamely Pseudomonas solanacearum and Xanthomonas axonopodis pv. citri.

The preliminaryantibacterial activity was performed by agar well diffusion method and the minimum inhibitoryconcentration (MIC) values were determined by agar dilution method. The test samples werealso 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 thedata. All the test samples showed inhibitory effect on both of the test pathogens and the diameterof inhibition zone ranged from 9.9 ± 0.5 mm to 48.5 ± 1.3 mm and the inhibitory effect differedsignificantly (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 andXanthomonas axonopodis pv. citri.

The MIC values of ethyl acetate extracts of fruit and flower ofVitex negundo L. and Allium sativum and ethanol extract of flower of Vitex negundo L. rangedfrom 2.5mg/ml to 40mg/ml. Phytochemical analysis of above extracts revealed the presenceof alkaloids, flavonoids, tannins, cardiac glycosides and terpenoids. Further studies are beingcarried out to elucidate the active principles responsible for the inhibitory effect of thesepathogens and to determine their activity in vivo. This is the first report that reveals theinhibitory effect of Vitex negundo L. on Pseudomonas solanacearum and Xanthomonasaxonopodis pv. Citri²⁶.

7. Antibacterial effect of silver nanoparticles using Vitex negundo Linn.: Different biological methods are gaining recognition for the production of silver nanoparticles (Ag-NPs) due to their multiple applications. One of the most important applications of Ag-NPs is their use as an anti-bacterial agent. The use of plants in thesynthesis of nanoparticles emerges as a cost effective and eco-friendly approach. In thisstudy the biosynthesis of silver nanoparticles using Vitex negundo L. extract and itsantimicrobial properties has been reported. The resulting silver particles are characterizedusing transmission electron microscopy (TEM), X-ray diffraction (XRD) and UV–Visible(UV-Vis) spectroscopic techniques.

The TEM study showed the formation of silvernanoparticles in the 10–30 nm range and average 18.2 nm in size. The XRD study showed that the particles are crystalline in nature, with a face centered cubic (fcc) structure. Thesilver nanoparticles showed the antimicrobial activity against Gram positive and Gramnegative bacteria. Vitex negundo L. was found to display strong potential for the synthesisof silver nanoparticles as antimicrobial agents by rapid reduction of silver ions (Ag+ to Ag0)²⁷.

8. Antifungal activity: Sathiamoorthy et al., (2007) isolated six compounds from the powdered leafextracts of Vitex negundo. The isolated compounds were evaluated for antifungaland anti-bacterial activity. From the isolated compounds two possess potent anti-fungal activities and very active when compared to other isolated compounds.Significant antifungalactivity in ethanolic extract against Cryptococcus neoformans and Trichophytonmentagrophytes was offered by two compounds isolated from the leaf extract ofVitex negundo²⁸.
9. Anti-inflammatory and analgesic activities of Vitex negundoi:Inflammation may start in every part of our body. Any time whenthe word describing a disease ends with - it is, it’s an inflammatory disease.Dermatitis means an inflammation of the skin, arthritis an inflammation ofjoints, an othitis an inflammation of the ear. Thus anti-inflammatory activityof a compound is considered to be a valuable feature. The leaves of Vitex negundopossess anti-inflammatory activity. Experimentalinvestigations revealed that the mature fresh leaf of Vitex negundohave dose-dependent activity against inflammation as revealed in the carrageenanand formaldehyde models. Mature fresh leaf extract of Vitex negundoalso demonstrated a dose-dependent prostaglandin (PG) synthesis inhibition,membrane stabilising and antihistamine activities. The inverse dose–response relationship shown by acute anti-inflammatory, antihistamine, PGsynthesis inhibition and membrane stabilising activities may be due to reductionof the effectiveness of the active principle at its high concentrations.

Sedatives and stress are responsible for producing analgesia. Therewas no sign of stress observed in the rats treated with the mature fresh leavesextract of Vitex negundo. Mature fresh leaves extract of Vitex negundois effective against the establishment of chronic inflammation which happensat the later stage of acute inflammation. Moreover treatment with themature leaf extracts of Vitex negundoin rats did not show a gastric lesionwhich is an advantage when compared with the use of modern NSAIDs.Treatment of Mature fresh leaves extract of Vitex negundofor 14 days inrats 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 formedicinal purposes. The anti-inflammatory and analgesic activities of the leaves did not disappearafter the flowering of the tree in contrast to Anisomeles indicawhichlost these activities after flowering of the plant. These studies provideevidence for the anti-inflammatory and analgesic properties of maturefresh leaves of Vitex negundoclaimed in Ayurveda medicine²⁸.

1. Enzyme-inhibitory activity: Root extracts of Vitex negundoshowed 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.
2. Effect on Reproductive Potential: The flavonoid rich fraction of seeds of Vitex negundocaused disruption of the latter stages of spermatogenesis in dogs and interfered with male reproductive function in rats. It must however be noted that these findings are in sharp contrast with the traditional use of Vitex negundoas aphrodisiac. Hu et al. determined that ethanolic extracts of Vitex negundo showed estrogen-like activity and propounded its use in hormone replacement therapy⁴.
3. Histomorphological and Cytotoxic Effects:  Tandon and Gupta studied the histomorphological effect of Vitex negundo extracts in rats and found the stomach tissue to be unaffected even by toxic doses; while dose-dependent changes were observed in the heart, liver and lung tissues. Cytotoxic effect of leaf extracts of Vitex negundowas tested and affirmed using COLO-320 tumour cells. On one hand, Diaz et al., found the chloroform extracts of Vitex negundoleaves to be toxic to a human cancer cell line panel while on the other; Yunos et al. reported that Vitex negundoextracts were non-cytotoxic on mammary and genito-urinary cells of mice⁴.
4. Anticonvulsant Activity: The plant has been studied for its anticonvulsant activity. The petroleum and butanol leaf extracts have shownprotection, whereas, none of root extract has shown protection against maximal electro shock (MES) seizures. Petroleum root extract could only provide protection against Leptazole induced convulsios whereas methanolic leaf extract showed significant protection against Strychnine and Leptazole induced convulsions. Gupta and Tandon (2002) not only suggested anticonvulsant activity of ethanolic leaf extract of this plant but also indicated that it can potentiate the effects of standard anticonvulsanta, which may help to reduce dose and dose related side effects of standard anticonvulsants ²⁹.
5. Anxiolytic Activity: Anxiety is an exaggerated feeling of apprehension, uncertainty, and fear. It is an unpleasant state of tension with an anticipation of imminent danger.Itmay be regarded as a particular form of behavioral inhibition that occurs in response to environmental events that are novel. Anxiety affects one-eighth of the total population worldwide and has become a very important area of research interest in psychopharmacology during this decade.There are various ways of explaining the mechanisms of action of anti-anxiety agents because of the involvement of many CNS chemical mediators.

The effect of most of the anxiolytic agents is to enhance the response to GABA, by facilitating the opening of GABA-activated chloride channels. GABAA receptors were involved in anxiety and their direct activation would have an anxiolytic effect.Anti-anxiety drugs have also been shown to act on limbic system, hypothalamus,and the brain stem reticularsystem.Benzodiazepines are still the most frequently used drugs for the treatment of generalized anxiety disorder despite their undesirable side effects such as muscle relaxation, sedation, physical dependence, memory disturbance, and interaction with other drugs.

However, the realization that benzodiazepines present a narrow safety margin between the anxiolytic effect and those causing unwanted side effects has prompted many researchers to evaluate new compounds in the hope that other anxiolytic drugs will have less undesirable effects.In recent years, the development of new anxiolytics has been an area of interest.

It has been established that there are lot of secondary plant metabolites being employed in the treatment of psychotic disorders especially for anxiety in traditional medicine practice, most of which directly or indirectly affect the central nervous system such as noradrenaline, serotonin, gamma-aminobutyric acid (GABA) and benzo-diazepine (BDZ) neurotransmitters' activities. On the basis of these considerations, it was the purpose of this study to characterize the anxiolytic-like activity of an ethanolic extract prepared from the roots of Vitex negundo Linn (VN)²¹.

5. Antipyretic Activity: The literature reveals that the leaf extract of Vitexnegundo causes a significant antipyretic effect in yeastprovoked elevation of body temperature. In the cases,the methanol extract caused a significant lowering ofbody temperature, with the effect being comparable tothat of paracetamol. Thus the present pharmacologicalevidence provides support for the folklore claim as anantipyretic agent. Flavonoids are known to targetprostaglandins which are involved in the late phase ofacute inflammation, pyrexia and pain perception.Flavonoids reduce lipid peroxidation by preventing orslowing the onset of cell necrosis and by increasing thevascularity. Hence the presence of flavonoids in themethanol extract of Vitex negundo may be contributoryto its antipyretic activity³⁰.
6. Cytotoxic agent against PANC-1 and a Panel of 39 Human Cancer Cell: Recently it was observed that the methanol extract of Vitex negundo from Myanmar displayed potent cytotoxic activity preferentially in the nutrient starvation condition with 100% cell death at a concentration of 10μg/mL. Therefore, it was subjected to phytochemical analysis to identify active constituents. The active constituent was further tested against PANC-1 cells as well as a panel of 39 human cancer cell lines (JFCR-39) at the Cancer Chemotherapy Center, Japanese Foundation for Cancer Research.

The ethanol extract of V. negundo showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells. Bioassay-guided fractionation and purification of Vitex negundo led to the isolation of chrysoplenetin (1) and chrysosplenol D (2) as the most active constituents with a PC₅₀ value of 3.4μg/mL and 4.6μg/mL, respectively, against the PANC-1 human pancreatic cancer cell line. They showed characteristic apoptosis-like morphological changes of PANC-1 cells in NDM.

The most active compound, chrysoplenetin was further evaluated against a panel of 39 human cancer cell lines at the Japanese Foundation for Cancer Research. Chrysoplenetin exhibited differential cell growth inhibition at submicromolar concentration against most of the cell lines. Vitex negundo and its active constituents might have possible beneficial effects for patients suffering from cancer in a real clinical situation³¹.

7. Testing for Cellular Toxicity: The cellular toxicity of extracts was determined against human erythrocytes adopting the procedure of He and Ursula. Eight fold serial dilutions of the extract were made in phosphate buffered saline and a total volume of 0.9 ml for each dilution was placed in an eppendorf tube. Fresh human erythrocytes were added to each tube to give a final volume of 1ml.

A negative control (containing saline only) and a positive control (containing test extracts 5mg/ml) were also included in the analysis. Solutions were incubated at 37°C for 30 min and alltubes were centrifuged at 3000 rpm for 5-10 min and then observed for hemolysis. Completehemolysis was indicated by a clear red solution without any deposits of erythrocytes.Hemolysis was also checked microscopically by presence or absence of intact RBCs.leaf and seeds extracts of Vitexnegundo were also assayed for cellular toxicityto fresh human erythrocytes and found to have no cellular toxicity³².

8. Antitussive and Toxicological Evaluation of Vitex negundo Linn: Antitussive effects of the n-butanol fraction of Vitex negundo on sulphur dioxide-induced cough have been examined in mice.Cough is the most common respiratory symptom that has been experienced by every human. It is an essential protective and defensive act whose action secures the removal of mucus, noxious substances and infections from the larynx, trachea and larger bronchi. On the other hand, a number of patients have non-productive cough, which is not associated with mucus clearance and may have a different stimulation.

It may be first overt sign of disease of airways or lungs and may significantly contribute to the spread of airborne infections and in some instances, may result in severe functional and structural damage to the organism. There is an acute current need for the development of safe, effective antitussive therapeutic options for the treatment of persistent cough as alternative to the existing medications. Vitex negundo reflects potency to inhibit chemically induced cough.

From the experimental results, it was observed that extract of Vitex negundo demonstrated a significant inhibition of cough reflex in a dose dependent manner.

For instance, the activity possessed by 250 mg/kg at 30 min was lesser than other increased doses in all the experimental duration. The highest inhibition of cough reflex was achieved by the extract at the dose level 1000 mg/kg at 60 min of experiment. These results provide pharmacological evidence in support of folklore claims as an antitussive agent.The  available antitussives have been derived from various compounds that have different pharmacological actions, such as analgesic, anticholinergic and antihistaminic.

Plant extracts tend to exhibit numerous pharmacological actions with multiple mechanisms, which are activated by several different types of compounds. There is considerable ethnomedical and pharmacological evidence that Vitex negundo possesses analgesic, antipyretic, antihistaminic, antiallergic, antibacterial, antioxidant and antiphlegmatic, potential.

This not only suggests that extract of Vitex negundo in improved formulation may inhibit coughing more effectively, but it may also provide interesting and meaningful efficacy in respiratory conditions such as asthma, bronchitis, tuberculosis and other lower respiratory tract infections which are accompanied with combined symptoms of fever, inflammation, phlegm and cough.The data received indicates that Vitex negundo possesses obvious antitussive activity against SO₂ induced cough in mice. The antitussive activity of this plant correlates with its various biological and ethnopharmacological properties which may justify its widespread use in various respiratory conditions in folk medicine. It was found a safer drug in the different toxicity assays performed¹.

9. Hepatoprotective activity: The study was intended to evaluate the hepatoprotective activity on ethanolic extract of leaves  of Vintex negundo was determined by using Wistar Albino rats in male sex. The V.negundo Linn is a natural plant product, in its the leaves are used with the added advantage to revert Ibuprofen induced hepatotoxicity. Oral administration of ethanol extract of V.negundo (100 300 mg) produced a significant and dose dependent inhibition to the acute hepatotoxic induced rats and various parameters were analysed, when compared with negative control V.negundo showed that the significant activity in 300 mg/kg/b.wt. They exhibited a significant inhibition of hepatic toxicity by using various marker enzymes and the histopathalogical analysis.

The inhibitory effect of the V.negundo on hepatotoxicity was compared to hat of positive control group. The various parameters such as glucose, protein, triglycerides, bilirubin, urea, creatinine, ALP, ACP, SGPT, SGOT and histo-pathalogical parameters was measured by dissectionthe rats. A significant index and values were observed in the acute assays; concluded that the V.negundo having the potentional effectiveness at the dose of 300 mg/kg/b.wt, (p<0.01) significance in a dose dependent manner. These results suggest that leaves of V.negundo having the hepatoprotective activity, which support the hepatic cells protection ⁵.

1. Antisnake VenomActivity: The methanolic root extracts of Vitex negundo Linn and Emblica officinalis Gaertn were explored for the first time for antisnake venomactivity. The plant (V. negundo and E. officinalis) extracts significantly antagonized the Vipera russellii and Naja kaouthia venom inducedlethal activity both in in vitro and in vivo studies. V. russellii venom-induced haemorrhage, coagulant, defibrinogenating and inflammatoryactivity was significantly neutralized by both plant extracts. No precipitating bands were observed between the plant extract and snake venom.

The above observations confirmed that the plant extracts possess potent snake venom neutralizing capacity and need further investigation³³.

1. Larvicidal Activity: Mosquito control is facing a threat due to theemergence of resistance to synthetic insecticides. Insecticidesof botanical origin may serve as suitable alternativebiocontrol techniques in the future. The acetone, chloroform,ethyl acetate, hexane, methanol and petroleum etherextracts of leaf, flower and seed of Cassia auriculata L.,Leucas aspera (Willd.), Rhinacanthus nasutus KURZ.,Solanum torvum Swartz and Vitex negundo Linn. Weretested against fourth instar larvae of malaria vector,Anopheles subpictus Grassi and Japanese encephalitisvector, Culex tritaeniorhynchus Giles (Diptera: Culicidae).The larval mortality was observed after 24 h of exposure.

All extracts showed moderate larvicidal effects; however,the highest mortality was found in leaf petroleum ether,flower methanol extracts of C. auriculata, flower methanolextracts of L. aspera and R. nasutus, leaf and seed methanolextracts of S. torvum and leaf hexane extract of V. negundoagainst the larvae of A. subpictus (LC₅₀=44.21, 44.69,53.16, 41.07, 35.32, 28.90 and 44.40 ppm; LC₉₀=187.31,188.29, 233.18, 142.66, 151.60, 121.05 and 192.11 ppm,respectively) and against the larvae of C. tritaeniorhynchus(LC₅₀= 69.83, 51.29, 81.24, 71.79, 44.42, 84.47 and65.35 ppm; LC90=335.26, 245.63, 300.45, 361.83,185.09, 351.41 and 302.42 ppm, respectively).

These results suggest that the leaf petroleum ether, flowermethanol extracts of C. auriculata, leaf and seed methanolextracts of S. torvum and leaf hexane extract of V. negundohave the potential to be used as an ideal eco-friendlyapproach for the control of the A. subpictus and C.tritaeniorhynchus. This is the first report on the mosquitolarvicidal activity of the medicinal plant extracts³⁴.

2. Antihelmintic activity: Ethanolic extracts of Moringa oleifera and Vitex negundo were taken for anthelmintic activity against Indian earthwormPheritima posthuma. Various concentrations of both extracts were tested and results were expressed in terms of time forparalysis and time for death of worms. Piperazine citrate (10 mg/ml) was used as a reference standard and distilled wateras a control group. Dose dependent activity was observed in both plant extracts but Moringa oleifera shows more activityas compared to Vitex negundo³⁵.

Essentional Oils: The chemical components of the essential oilfrom V. negundohave 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/MSwhich 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 leaf¹⁸.
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 oilas major constituents along with sesquiterpenes, monoterpenes, terpenoids and sterols^{36, 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 oil¹⁸.

Proximate Analysis of Vitex negundo Linn. ³⁷:

ACKNOWLEDGEMENT: The authors wish to thanks the Deanship of scientific research King Saud University Saudi Arabia for their support and interest to enhance the research activities in this university.

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    How to cite this article:

    Ullah Z, Ullah R, Shah AHA, Ahmad I and Haider S. Phytochemical and Biological Evaluation of Vitex negundo Linn: A Review. Int J Pharm Sci Res, 2012; Vol. 3(8): 2421-2431.

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