INTRODUCTION:

Vasa is a sanskritized Ayurvedic name of a medicinal shrub that is botanically equated to Justicia adhatoda L. (Adhatoda zeylanica Medic.) belonging to the family Acanthaceae. The leaves, flowers, fruits and roots of Vasa are useful for treating cold, cough, chronic bronchitis and asthma, as a sedative-expectorant. It is also useful as an antispasmodic and anthelmintic¹. This shrub has been used in Ayurvedic medicinal formulations since times immemorial and has been mentioned in Ayurvedic classical texts. In addition to leaves, flowers also have medicinal uses both in Ayurveda, Unani and in Folklore. Vasa is useful to cure various ailments like malarial fever, intrinsic haemorrhage, cough and asthma, skin diseases, piles, leprosy, for retention of urine, diseases of mouth etc.².

Keeping in view about the different uses of flowers of vasa, there is a necessity to develop standardized identification parameters to aid quality control and to avoid adulteration with special focus on flowers. Although botanical identification studies and some chemical studies have been reported, comprehensive studies involving Pharmacognosy and phytochemical and TLC fingerprint studies are sparse. Hence this study is an attempt to develop and report comprehensive authentication parameters including macroscopical, microscopical, physico-chemical, preliminary phytochemical profile of the flowers of Vasa.

Ayurveda Therapeutic and Traditional uses of Vasa Flowers:

Shade dried Vasa flowers powder with honey is useful for intrinsic haemorrhage². Fresh flowers of Vasa are used in Opthalmia or the severe inflammation of the eye or conjunctiva, the decoction of the flowers is useful for blood purification, to improve the circulation, to check the painful and difficult urination and jaundice; flowers are antispasmodic³. The flowers of Vasa are helpful in the management of eye disorders caused due to ushna (heat) (pitta), as an external application⁴. The flowers of Vasa are useful, internally to combat bleeding⁵. Different formulations containing Vasa flowers, have been clinically evaluated on Tamaka shwasa (dyspnoea/ asthma) viz., Vasaghana vati (dry extract) and Vasa ghritha (ghee) with significant improvement when compared to formulations like Vasa Avaleha, vasarishta, Vasakasava, which don’t have Vasa flower as an ingredient⁶. Vasa flowers pacify Kapha and Pitta, they are bitter in taste, cold in potency and pungent after digestion⁷. Sushruta mentions that the flowers of vasa alleviate cough and Pthisis⁸. Vrunda advocates the use of juice of vasa flowers along with sugar and honey to combat fever due to vitiation of pitta and kapha and also jaundice and bleeding disorders⁹. A lehya prepared with shade dried vasa flowers with honey is useful as a treatment for cough, bleeding disorders and Pthisis¹⁰. The flowers and fruits are bitter and aromatic and their uses are similar to those of the leaves.

The Vasa flowers are a source of nectar for bees, children suck the flowers for nectar¹¹. The Vasa flowers are used for acute bronchitis, cough^12,13to enhance blood circulation and in jaundice¹⁴and as an antispasmodic¹⁵. The Vasa flowers and the fruits are bitter, aromatic and antispasmodic. The fresh flowers are used in Opthalmia. As Per the Unani system of Medicine flowers are useful to improve the blood circulation and lessen strangury and jaundice. In Assam a traditional preparation of the juice of young shoots and flowers of the vasa is administered with ghee or honey for curing asthma¹⁶. The leaves, flowers and roots, especially the flowers are considered antispasmodic, and are given in cases of asthma and intermittent fever, successfully employed in chronic bronchitis and in other pulmonary and catarrhal affections when attended with fever. The fresh flowers are bound over the eyes in cases of ophthalmia¹⁷. The flowers of vasa have Anti-oxidant and Anti-bacterial activity¹⁸. Kurumba tribes of Nilgiris, Tamilnadu are inhaling the smoke of dried flowers (burnt) to cure asthmatic problems, daily once before bed time for 3-7 days¹⁹. The tender leaves and flowers of are used as vegetable after cooking, by Khasi tribes of Meghalaya, India and Nepal^{20, 21}.

Habitat:

Vasa is an evergreen, gregarious, perennial shrub with a height up to 6.0 meters, distributed all over India, commonly as edge plant up to an altitude of 1,300 m. Leaves ellipptic-lanceolate or ovate-lanceolate, hairy, margin entire, light green above, dark below, leathery¹¹. Flowers are closely arranged in axillary pedunculate spikes. Bracts green, elliptic, subacute at apex, glabrous, reticulately veined. Bracteoles oblong-lanceolate, acute, with ciliolatemargins, 1-nerved. Calyx 5-lobed, glabrous or slightly pubescent; lobes imbricate, lanceolate, shortly connate. Corolla cream-white, pubescent outside; lobes 5, bilipped; upper lip erect, obtuse, shortly 2-fid; Lower lip 3-fid, crested with purple lines, middle lobe broad, lateral lobes recurved, obtuse. Stamens 2, attached at the throat of corolla tube, excerted; filaments thick, hairy at the very base; anther cells oblong, parallel, basally apiculate. Ovary pubescent, 4-valved, lower part of style pubescent. Capsules clavate, shortly and bluntly pointed, pubescent. suborbicular. Seeds orbicular-oblong, tubercular-verrucose, glabrous ¹⁶.

Regional Names:

Assamese: Titabahak, Bahak, Vachaka; Bengali: Baksa, Vasaka; English: Malabar nut, Vasaca; Gujrati: Aduso, Ardusi, Adulso; Hindi: Aduss, Arusa; Kannada: Adsale, Adusoge, Atarusha, Adsole, Adasale; Malayalam: Valiya adalotakam, Peradalotakam; Marathi: Vasa, Adulsa; Oriya: Basanga; Punjabi: Bhekar, Vansa, Arusa; Sanskrit: Brihat vasa, Brihat vasaka, Vrisha, Vrishaka, Simhasya, Adarushaka, Vajidanda; Tamil: Vasambu, Adathodai; Telugu: Addasaramu; Urdu: Adusa, Basa; Unani: Arusaa; Folk: Vasaca ²².

Ayurvedic Description:

The flowers of vasa are used in Ayurveda system of medicine. In terms of Rasa panchaka theory of Ayurveda, the properties of Vasa are as follows – Rasa (taste): Tikta, Kasaya (astringent); Guna (quality): Ruksa (Creates dryness) and laghu (light for digestion); Veerya (Potency): Sheeta (Conserves energy during digestion and metabolism); Vipaka (Digestive effect): Katu (pungent); Karma (action): Grahi (constipative); Doshagnata (effect on doshas): kaphapittashamaka (pacifies impaired kapha and pitta doshas). Vyadhiharatva/Rogaghnata (indications in diseases): Kasa (cough), Swasa (respiratory dysfunction like breathlessness) Rajayaksma (disorders characterized by diminution of tissues as seen in chronic systemic disorders with respiratory compromise), Rakta pitta (Bleeding disorders), Jwara (fever), Mutraghata (diseases characterized by urinary outlet obstruction symptoms), Gulma (Gastrointestinal Disorders characterized by pain abdomen), Kustha (Skin diorders), krimi (parasitic infestations like helminthes) etc¹.

Doses:

Flower juice: 10-20 ml; Leaf Juice: 10-20 ml; Root decoction: 40-80 ml (10-20 gm of dried drug)¹.

Chemical constituents:

An alkaloid Vasicinine, {2, 3-(α-hydroxy trimethylene) -4- quinazolene}, m.p. 208-209⁰C, isolated from young inflorescence²³. A flavonoid compound Luteolin has been isolated and identified from the flowers of A. vasica²⁴. A chalcone glycoside, 2’, 4-dihydroxy chalcone –4-glucoside (C₂₁H₂₂O₈, melting point 195-197⁰C) was isolated from the flowers²⁵. High Pressure Liquid Chromatography (HPLC) analysis of Methanolic extract of flowers showed the presence of Vanillic acid (10.5 mg), Tannic acid (1.33 mg), 3,4-dihydroxy benzoic acid (1.98 mg), Syringic acid (3.13 mg), p-hydroxy benzoic acid (6.84 mg) Gallic acid (2.89 mg) and p-coumaric acid (1.80 mg) in 100 g. of dry drug. The flowers also contains a very good amount of phenolic compounds and flavonoid contents¹⁸. The sample from Pakistan showed the presence of Vasicine (inflorescences, 0.64; flowers, 0.15% on dry basis). The inflorescences also contained β-sitosterol (up to 0.035%). The flowers yield a golden yellow essential oil. The colouring matter in the flowers includes Luteolin, Quercetin and Kaempferol (free, and as 3β-D-glucoside and 3-sophoroside). Other non-nitrogenous constituents include α-amyrin, tritriacontane (flowers, 0.07; flowering tops, 0.09% on dry basis), and β- sitosterol (both free and as 3β-D-glucoside) ¹¹.

Vasicine

MATERIALS AND METHODS:

Plant material:

The flowers of vasa (J. adathoda) were collected from the vicinity of Bangalore, Karnataka, authenticated from Survey of Medicinal Plant Unit (coll. no. RRCBI-8602), National Ayurveda Dietetics Research Institute, Ashoka pillar, Jayanagar, Bangalore-560011, India. The collected flowers were shade dried, coarsely powdered and stored in a closed vessel, to carryout microscopical, physico-chemical, preliminary phytochemical analysis.

Study methods:

The macroscopical characters of the flowers were observed. Then, for powder microscopical study, the powder was stained with phloroglucinol and concentrated HCl to study the lignified cells, trichomes, fibres, xylem vessels, etc. The powder was also stained with N/50 iodine solution to detect the presence of starch. A small portion of powder was mounted in water to identify calcium oxalate crystals. Microscopy of different parts of the flowers was carried out by the methods prescribed by Trease and Evans²⁷.

The drug (flower) powder is successively extracted with Petroleum ether (60-80⁰C), Chloroform and Alcohol by using soxhlet apparatus and water bath. The dried extractives were obtained after evaporation of solvent under reduced pressure by rotary evaporator. Preliminary Phytochemical analysis carried out according to standard procedures²⁸ and recorded in table-1. Physico-chemical parameters such as ash values, alcohol soluble and water soluble extractive values and loss on drying and pH, of flowers were determined as per the standard Ayurvedic Pharmacopoeial methods, ²⁹ and recorded in Table-2.

Macroscopy –

(Fig.1): Flower bracteate, sessile, complete, hermaphrodite, zygomorphic, pentamerous, hypogynous, whitish with pink streaks.

Calyx:

Sepals 5, gamosepalous, green, imbricate, inferior.

Corolla:

Petals 5, gamopetalous, personate (2/3), posterior lip with 2 lobes, imbricate, white with pink streaks, inferior.

Androecium:

Stamens 2, polyandrous, epipetalous, filaments long, hairy at the base, basifixed, dithecous, introrse, anther lobes at unequal height and spurred.

Gynoecium:

Bicarpellary, syncarpous, superior, bilocular, one ovule in each locules, axile placentation, style long, stigma bifid.

Microscopy:

Bract – (Fig. 2):

T.S. of the bract shows typical leaf structure. T.S. of the bract shows upper and lower epidermis covered with thick cuticle showing uniseriate to bi seriate trichomes and single headed glandular trichome followed by 4-5 layers of collenchymatous cells and 3-4 layers of parenchymatous cells on both upper and lower epidermis. Centrally well developed vascular bundle is located with metaxylem facing towards the lower region and prtoxylem towards upper epidermis. Phloem 5-8 layered. Laminar region shows typical leaf structure where both upper and lower epidermis covered with thick cuticle, single layered palisade tissue with abundant oilgloubles and spongy loosely arranged 1-2 layered. Both epidermal layers shows glandular and non glandular trichomes.

Calyx– (Fig. 3):

T.S. of the calyx shows shows deeply planoconvex in structure, towards the lower epidermis and towards the upper epidermis plain, both the epidermis covered with thick cuticle showing uniseriate to bi seriate trichomes and single headed glandular trichome followed by 2-3layers of collenchymatous cells and 1-3 layers of parenchymatous cells on both upper and lower epidermis. Collenchymatous cells show prominent cystolith. Centrally well developed vascular bundle is located with metaxylem facing towards the lower region and protoxylem towards upper epidermis. Phloem 5-8 layered. Below the phloem region well developed 5-6 layers of collenchymatous layers are present. Laminar region shows typical leaf structure where both upper and lower epidermis covered with thick cuticle, single layered palisade tissue with abundant oilgloubles and spongy parenchymatous loosely arranged which 1-3 are layered. Both epidermal layers showed glandular and non glandular trichomes. Small veins represent vascular bundles, where small xylem cells and phloem cells are present.

Corolla – (Fig. 4):

T.S. of the corolla shows slightly planoconvex in structure, towards the lower epidermis, and towards the upper epidermis plain, both the epidermis covered with thick cuticle showing uniseriate to bi seriate trichomes and single headed glandular trichome followed by 2-1, layers of collenchymatous cells and 1-3 layers of parenchymatous cells. Epidermal cells show prominent pappilae like outgrowth. Centrally well developed vascular bundle is located with metaxylem facing towards the lower region and protoxylem towards upper epidermis. Phloem 5-6 layered with well developed phloem fibers. Below the phloem region well developed 5-6 layers of collenchymatous layers are present. Laminar region shows typical leaf structure where both upper and lower epidermis covered with thick cuticle, single layered palisade tissue with abundant oilgloubles and spongy parenchymatous loosely arranged which 1-4 are layered. Both epidermal layers show glandular and non glandular trichomes. Small veins represent vascular bundles, where small xylem cells and phloem cells are present. Glandular trichomes show brown content of tannin and spongy parenchymatous cells also show brown content of tannin.

Androecium – (Fig. 5):

T.S. of anther is slightly cresent shaped in outline with a broad connective bearing 2 Pollen chambers 1 each on the lateral sides. The wall of the anther is composed of a layer of epidermis covered by thin cuticle. Underneath the epidermis lies a layer of column shaped cells of endothecium near the pollen chamber which on complete maturity disintegrates for the dispersal of pollen grains. Spores are enclosed in the cavity surrounded by sporogenous tissue which in turn is surrounded by tapetum. The connective tissue shows closely packed parenchymatous cells which are thin walled compactly arranged with abundant tannin contents and vascular bundles are scattered in 3-6 groups with xylem and phloem cells. In surface view endothecium cells are compactly arranged with many layers.

Gynoecium/ Ovary T.S. – (Fig. 6):

T.S. of the ovary is circular in outline and shows outer epidermis bearing abundant unicellular to multi cellular trichomes, centrally located bilocular ovary with 2 ovules in each locule encircled by compressed thin walled parenchymatous cells with vascular bundle in the centre with tannin content in the parenchymatous tissue.

Diagnostic Characters:

1) Presence of Glandular and non glandular trichomes in all most all parts of the flower.

2) Presence of cystolith in the calyx portion.

3) Presence of brown content in the glandular trichome and also in the spongy tissue of corolla region.

4) Presence of Papillae like outgrowth in the epidermal region of corolla.

5) Presence of abundant unicellular to bi, tricellular trichomes on the walls of ovary wall.

Powder Microscopy – (Fig. 7):

Powder light brown in colour, rough to touch, when it is treated with chloral hydrate and water and observed under the microscope, the following different fragments of tissues were observed/noted.

· Abundant epidermal cells, pollengrains, pappilose cells in surface view.

· Uni to biseriate trichomes.

· Parenchymatous cells.

· Helical to spiral xylem vessels.

· Lower Epidermal cells with stomata.

· Rounded Pollen grains.

Table-1: Preliminary Phytochemical tests for flower extracts of Justicia adhatoda

S. No.	Natural product group	Test for natural Products	Presence (+) /Absence (-)
1	Alkaloids	(a) Dragendorff’s test	+++
		(b) Hager’s test	+
		(c) Mayer’s test	+
		(d) Wagner’s test	+
2	Carbohydrates	(a) Anthrone test	++
		(b) Benedict’s test	++
		(c) Fehling’s test	+++
		(d) Molisch’s test	++
3	Flavonoids		+
4	Phenols	(a) Ferric chloride test	++
4	Phenols	(b) Libermann test	++
5	Proteins	(a) Biuret’s test	+
5	Proteins	(b) Millon’s test	++
6	Saponins		+++
8	Starch		+
9	Tannins	(a) Ferric chloride test	+
9	Tannins	(b) Lead acetate test	+

+++ = abundantly present, ++ = good amount present, + = slightly present.

Table-2: Physicochemical parameters

S. No.	Name of the parameter	Values (%) w/w
1	Description	Greenish brown
2	Foreign matter	Less than 1%
3	pH (5% w/v aq. solution)	6.20
4	Loss on drying at 105⁰C	13.11
5	Total ash	6.89
6	Acid-insoluble ash	1.11
7	Water-soluble extractive	11.96
8	Alcohol-soluble extractive	14.87
9	Petrolium ether 60⁰-40⁰C	1.66
10	Chloroform	0.85

Table-3: Thin layer Chromatography (TLC) studies of Justica adhatoda flower extracts.

DISCUSSION:

The World Health Organization (WHO) manual, The Use of Traditional Medicine in Primary Health Care, recommends Vasa as a non-toxic expectorant in treatment of cough, asthma and bleeding piles, and it can be used for both adults and children for a long period. ²⁹It is a most frequently mentioned medicinal shrub in various published hand books, encyclopaedias and research articles of various countries in the treatment of respiratory diseases including cough, colds, asthma, bronchitis and phthisis (a wasting of the body, tuberculosis). It is used as an expectorant, bronchodilator. The drug has been used for a long period of time with no serious adverse effects reported/documented. In traditional health practices of India, Pakistan, Sri Lanka and Nepal, flowers are used apart from leaves and roots to cure various ailments like opthalmia, fever, gonorrhoea, antiseptic, hectic heat of blood, to improve blood circulation, Jaundice, lessen strangury, abdominal tumour and in rheumatism since long time³⁰. This study therefore is an attempt to develop multidimensional identification characteristics of vasa flowers. The striking features in Macro and Microscopy, Powder Microscopy and Phytochemical constituents are listed here for authentic identification of the vasa flowers.

Macro and Microscopy:

This study therefore brings out comprehensive identity profile of the medicinal flowers of Vasa including macroscopical, microscopical, physico-chemical, preliminary phytochemical profile. In terms of Pharmacognosy, the following features are striking to the flowers of Vasa and can serve as principal parameters for authentication in fresh, dry form as well as in powder form also.

1) Presence of Glandular and non glandular trichomes in all most all parts of the flower.

2) Presence of cystolith in the calyx portion.

3) Presence of brown content in the glandular trichome and also in the spongy tissue of corolla region.

4) Presence of Papillae like outgrowth in the epidermal region of corolla.

5) Presence of abundant unicellular to bi, tri cellular trichomes on the walls of ovary wall.

Powder Microscopy:

Powder light brown in colour, rough to touch, when it is treated with chloral hydrate and water, following different fragments of tissues was observed.

· Abundant epidermal cells, pollen grains, pappilose cells in surface view.

· Uni to biseriate trichomes.

· Parenchymatous cells.

· Helical to spiral xylem vessels.

· Lower Epidermal cells with stomata.

· Rounded Pollen grains.

Phytochemical Constituents and Medicinal Properties:

The chief bitter quinazoline alkaloids Vasicine and Vasicinone are reported to have bronchodilatory activity in vitro and in vivo, respiratory stimulant, uterotonic activity, oxytocic, weak cardiac stimulant and antianaphylactic actions^31-33. Though the chief alkaloid vasicine is reported in all parts of the plant, highest percentage present in inflorescence, the flowers may also be used in view of its major constituent vasicine’s proved vasodilatory and bronchodilatory actions among the public for respiratory problems in addition to other parts of the plant.

These Pharmacognostical studies and physico-chemical data evolved from the present investigation may be utilized for the standardization of the drug in order to check and ensure the quality of the drug in quality control laboratories and also for laying down Pharmacopoeial standards for the flowers of Vasa.

ACKNOWLEDGEMENT:

Authors are thankful to the Director General, CCRAS, New Delhi for providing necessary facilities and Dr. Kishore Kumar, R.O. (Ay.), and Dr. Kayva N., S.R.F. (Ay.) for their suggestions to carry out the work successfully.

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Received on 09.03.2015 Modified on 15.03.2015

Res. J. Pharmacognosy & Phytochem. 7(2): April-June 2015; Page 73-90

DOI: 10.5958/0975-4385.2015.00014.X