A Comparative Study of the Quality Assessment of Vitex negundo Linn Leaves Collected from Three Different Geographical Locations

Ajay Kumar Meena¹*, Uttam Singh², Arjun Singh³, Sudeep Mishra³, M. M. Rao¹, M. M. Padhi³, Ramesh Babu³ and A. Gaurav²

¹National Institute of Ayurvedic Pharmaceutical Research, Patiala- 147001, Punjab, (India)

²School of Pharmaceutical Sciences, Shobhit University, Meerut, UP,(India)

³Central Council for Research in Ayurveda and Siddha (CCRAS), Janakpuri, Delhi-110058.

*Corresponding Author E-mail: ajaysheera@gmail.com

ABSTRACT:

Vitex negundo Linn (Five-leaved chaste tree), Hindi-Nirgundi, Sanskrit-Sindhuvara, Tamil and Malyalam-Vennochi, Telugu-Nalla vavili and Punjabi-Marwan (Family: Verbenaceae), is an important medicinal plant found throughout India. All parts of this plant especially the leaf and root extracts are being used in various formulations in Ayurveda and Unani systems of medicine. Vitex negundo Linn is pungent, bitter and astringent in taste and according to Ayurveda , it alleviates vata and kapha doshas, but aggravates pitta dosha. It possesses light and dry attributes. Its leaves and seedsare widely used externally for rheumatism and inflammations of joints and 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 the plant are used to treat colds and coughing spells. Vitex negundo Linn is used in Classical Ayurvedic Preparations like Nirgundi kalka, Nirgundi ghrta, Nirgundi kvatha, Vranasodhana taila, Visagarbha taila etc. The evaluation studies of Pharmacopoeial standards, phytochemical parameters suggested that the observed physiochemical parameters are of great value in quality control and formulation development of Vitex negundo Linn. Physicochemical parameters like preliminary characteristics, toxic heavy metals, and aflatoxin analysis were carried out. The study revealed that the results on different parameters of the crude drug will be useful in identification and control of adulterations.

KEYWORDS: Ayurveda, Vitex negundo Linn, Toxic metals, Aflatoxin and Quality control.

INTRODUCTION:

Vitex negundo Linn is pungent, bitter and astringent in taste and according to Ayurveda , it alleviates vata and kapha doshas, but aggravates pitta dosha. It possesses light and dry attributes. It has antipyretic, anti-arthritic and anti-inflammatory properties and is used in diseases like fever, worms, dermatoses, adenitis and splenic diseases etc⁵

Phytochemical studies on Vitex negundo Linn have afforded several types of compounds, such as volatile oils^6-9, lignans^10,11, Flavonoids ^12-14, iridoids^15-17, terpenes (triterpenes, diterpenes, sesquiterpenes)^18-19, and steroids.²⁰The most Flavanoid, glycoside from Leaves of Vitex negundo Linn of ethanolic extract is 5-hydroxy-3, 6,7-trimethoxy-2-(3,4-dimtoxypheny)-4H-chrome-4-on and 5, 7-dihydroxy-2- (3,4- dihydroxyphenyl) -4H-chromen-4-one.²¹Methanolic extract also contains, Negundoside, Agnuside, Vitegnoside²². Bark of Vitex negundo Linn. p-Hydroxybenzoic acid and β-sitosterol were isolated, and identified from the methanol and hexane extracts of Vitex negundo.

From the seed of Vitex negundo Linn the following compound Isolated are -acetoacetate fraction, two phenylnaphtha- lene-typelignans and identified as 6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxy-methyl-7-methoxy-3,4-dihydro-2-naphthaldehyde and vitedoamine A, both of which have been previously reported and isolated from the seeds of Vitex negundo.Powdered roots are used for piles and as a demulcent for dysentery. It also used in dyspepsia, colic, rheumatism, worms, boils and leprosy.²³The roots are used as an antidote to snake venom which contain a furanoeremophilane, Tyrosinase inhibitory lignin’s from the methanol extract.²⁴

Hepatoprotective activity of Vitex negundo Linn was investigated against hepatotoxicity produced by administering a combination of three anti-tubercular drugs isoniazid -7.5 mg/kg, rifampin-10 mg/kg and pyrazinamide-35 mg/kg from the ethanolic extract of leaves. Vitex negundo Linn contains many polyphenolic compounds, terpenoids, glycosidic iridoids and alkaloids. Since polyphenolic compounds have high antioxidant potential, the antioxidant potency of Vitex negundo was investigated by employing various established in vitro systems, such as 2, 20-azino-bis 3-ethyl benzothiazoline- 6-sulfuric acid /Lipid Peroxide /Superoxide/Hydroxyl radical scavenging and iron ion chelation. The anti-inflammatory, analgesic and antihistamine properties of mature fresh leaves of Vitex negundo Linn claimed in the Ayurvedic medicine were established in Rats by orally treating water extract of the leaves to rats. The carrageenan-induced rat paw oedema was significantly suppressed in inversely dose-dependent manner. anti-hyperglycemic activities.²⁵ Vitex negundo exhibited significant activity against E. coli, K. aerogenes, P. vulgaris and P. aerogenes at all dosages. Extract of leaves of V. negundo showed activity against bacteria.²⁶ The antiasthmatic activity was evaluated by various experimental models like mast cell degranulation. ²⁷ Anti-implantation activity was found in the methanolic extract of leaves of Vitex negundo Linn, ²⁸ Xanthine oxidase inhibitory activity was assayed from Vitex negundo.

The methanolic root extracts of Vitex negundo Linn. and Emblica officinalis Gaertn. were explored for the first time for anti snake venom activity. The plant (Vitex negundo and Emblica officinalis) extracts significantly antagonized the Vipera russellii and Naja kaouthia venom induced lethal activity both in vitro and in vivo studies.²⁹Petroleum ether extracts of the leaves of Vitex negundo were evaluated for larvicidal activity against larval stages of Culex tritaeniorhynchus in the laboratory.³⁰Crude aqueous extract of Vitex negundo Linn leaves are investigated for laxative activity.³¹Ethanolic extracts of Vitex negundo were taken for anathematic activity against Indian earthworm Pheritima posthuma.

MATERIALS AND METHODS:

Plant material:

The Leaves of Vitex negundo Linn Were collected in September, 2009 from Gwalior M.P. (Sample S1), National Institute of Ayurvedic Pharmaceutical research, Patiala, Punjab (Sample S2) and Chennai (Sample S3) specimen were identified and authenticated at the National Institute of Ayurvedic Pharmaceutical Research (NIAPR), Patiala (Punjab). Leaves were washed in running water and air-dried. The fresh leaves were then studied for physiochemical evaluation. Powder of the samples was used for chemical analysis. Physicochemical studies like total ash, water soluble ash, and loss on drying at 105°C, TLC and extractive values were carried out as per the WHO/AOAC guidelines.^32,33

RESULTS AND DISCUSSIONS:

Preliminary phytochemical study:

Preliminary phytochemical results showed the presence or absence of certain phytochemical constituents in the Vitex negundo Linn sample. Hexane extract showed the presence of Fixed oils and fats, alcohol extracts gave positive results for Carbohydrate, Alkaloid, Flavanoid, steroid, tannins, Phenolic compounds, Saponins and gave negative results for Anthraquinones, Fixed oils and fats. Water extract gave positive results for Alkaloid, Flavanoid, steroid, tannins, Phenolic compounds and gave negative results for Carbohydrate, Saponins, Fixed oils and fats shown in Table 1.

Physico-chemical parameters of the leaves of Vitex negundo Linn are tabulated in Table 2. Deterioration time of the plant material depends upon the amount of water present in plant material. If the water content is high, the plant can easily be deteriorated due to fungus. The loss on drying at 105 °C in samples S1, S2 and S3 were found to be 9.47, 8.5 and 8.0 % w/w respectively. Total ash value of plant material indicated the amount of minerals and earthy materials attached to the plant material. Analytical results showed total ash value samples S1, S2 and S3 content were 8.0, 6.4 and 8.2 % w/w respectively. The amount of acid insoluble siliceous matter present in the plant samples were 0.31, 0.81 and 0.87 % w/w respectively. The water-soluble extractive value was indicating the presence of sugar, acids and inorganic compounds. The water soluble extractive value in the drug sample were 28.0 (S1), 28.34(S2) and 28.76(S3) % w/w and alcohol soluble extractive value were 16.44 (S1), 17.19 (S2) and 16.87 (S3) % w/w. The alcohol soluble extractive values indicated the presence of polar constituents like phenols, alkaloids, steroids, glycosides, flavonoids and secondary metabolites present in the plant sample. Other parameters, like pH, are given in Table 2. Aflatoxin B1, B2, G1, G2 is also not detected in the drug sample Heavy metals and microbial contaminations are below permissible limit in the samples.

Table 1. Preliminary phytochemical tests of various solvent extract of Vitex negundo Linn.

S. No.	Phytoconstitunts	Successive extraction of Sample, S1, S2, S3
S. No.	Phytoconstitunts	n-Hexane	Alcohol	Water
1.	Carbohydrate	-ve	+ve	-ve
2.	Alkaloid	-ve	+ve	+ve
3.	Flavanoid	-ve	+ve	+ve
4.	Steroid	-ve	+ve	+ve
5.	Tannin	-ve	+ve	+ve
6.	Phenolic compounds	-ve	+ve	+ve
7.	Saponins	-ve	+ve	-ve
8.	Anthraquinones	-ve	-ve	+ve
9.	Fixed oils and fats	+ve	-ve	-ve

Table 2. Analysis of physico chemical parameters of Vitex negundo Linn

Parameters	*S1 ( Gwalior)*	*S2 (Patiala)*	*S3 ( Chennai)*
pH (10% w/v aqueous solution)	4.8	5.0	4.8
Total Ash (%w/w)	8.0	6.4	8.2
Acid insoluble ash (%w/w)	0.31	0.81	0.87
Water soluble extract (%w/w)	28.0	28.34	28.76
Alcohol soluble extract (%w/w)	16.44	17.19	16.87
Loss on drying at 105°C (%w/w)	9.47	8.5	8.0

2gm of sample was soaked overnight in 20 ml of 90% ethanol. The solutions were continuously stirred for 6 hrs and kept for next 18 hrs., next day filtered, dried and made 10% solution. The solution was applied on Merck Aluminium pre-coated plate with silica gel 60 F₂₅₄ of 0.2 mm thickness. The plate was developed in Toluene: Ethyl acetate (9: 2 v/v). After air dry the plates were visualized in UV 254 and 366 nm. The plates were dried and then dipped in Vanillin- Sulphuric acid and heated at 105 ° C till the spots appeared.

TLC of the ethanol extract developed in the mobile phase of Toluene: Ethyl acetate (9: 2 v/v) and observed under UV 254 nm 3 spots at R_fvalue 0.24,0.35 (Pale yellow) and 0.47 (Brown); under UV 366 nm showed 3 spots at R_f 0.24, 0.35 and 0.47 (Pale red), and after derivatization with Vanillin- Sulphuric acid, showed 5 spots at R_f 0.37 (Pale green), 0.43 (Pale blue), 0.50 (Pale green), 0.62 (Blue) and 0.68 (Pale blue). Heavy metals contamination is below permissible limit in the samples and Aflatoxin B_1,B_2,G_1,G₂ are also not detected in the samples.

CONCLUSION:

Physiochemical parameters that are loss on drying at 105ºC, Total ash, Acid insoluble ash and extractive values on comparison revealed that extractive value is highest in sample S3. Highest extractive values show the highest active component in sample S2, S3 comparison with sample S1. It was observed that there is variation in the values obtained on analysis of the various samples of Vitex negundo Linn collected from different regions. It has been concluded from this study that estimation of physico-chemical parameters, heavy metals and aflatoxin is highly essential for raw drugs or plant parts used for the preparation of compound formulation. The periodic assessment is essential for quality assurance and safer use of herbal drugs, So that study can be considered as the Pharmacopoeia standard and helpful to confirm the genuineness of the single plant drug to be used for the preparation of effective Ayurvedic formulation.

ACKNOWLEDGEMENT:

The authors wish to thank the Director, Shobhit University Meerut for providing encouragement to complete the work successfully.

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Received on 22.02.2011 Modified on 07.03.2011

Asian J. Research Chem. 4(6): June, 2011; Page 986-989