RESEARCH ARTICLE

Vasa (Justicia adhatoda L.) Leaves Best Procurement

Time with Special Reference to Assay of
Vasicine through HPLC and its Comparative
Pharmacognosy
Neelima Sharma1, Venkata N Cheemalapati2, Anupam K Mangal3, Prabhu Rekha4, Narayana Srikanth5

A b s t r ac t
Aim: Vasa (Justicia adhatoda L.) is a well-known plant for its medicinal properties, e.g., ailments of asthma, bronchitis, cough, dyspnea, external
bleeding, etc. Different preparations of vasa are described in the Ayurvedic texts to pacify these disorders. In Ayurveda, the season of collection
of its useful parts, i.e., leaves are not mentioned anywhere in the ancient texts. Since some research claim higher concentration of its active
metabolite in winter and rainy season, therefore, it has been planned to evaluate the suitable time of collection of plant parts.
Materials and methods: The leaves of vasa (Justicia adhatoda L.) were collected from the same habitat in all six seasons described in Ayurveda,
i.e., shishir (Jan to Feb), vasant (Mar to Apr), grishm (May to June), varsha (Jul to Aug), sharad (Sept to Oct), and hemant (Nov to Dec). The source of
the collected plant materials was authenticated at Regional Ayurveda Research Institute, RARI Jhansi, India, through the herbarium (accession no.
23595). Identification, comparative macroscopic, and microscopic along with powder microscopy of the leaves of the plant in each season were
carried out besides extraction of various solvents such as alcohol, hydroalcohol, and methanol though Soxhlet and its comparative quantitative
analysis for the extracted material in all six seasons through high-performance liquid chromatography (HPLC) for the different seasons.
Results: Pharmacognostical characteristics remain the same in all seasons, while the selected marker compound vasicine varied in quantity in
each season. Extractive value was found maximum, i.e., 1.3456 g in vasant ritu sample and also evident in the quantitative HPLC estimation for
the abundant vasicine in vasant ritu sample, i.e., 0.7330 to 1.0744 which is the clear indication for the best time to collect leaves in vasant ritu.
Conclusion: In vasant ritu, vasa (Justicia adhatoda L.) is in flowering stage, and at this stage the abundance of the secondary metabolites and
the extractive values clearly indicate to conclude that the suitable procurement time of collection of vasa leaves (Justicia adhatoda L.) is vasant
ritu (Mar to Apr).
Keywords: Best procurement time, High-performance liquid chromatography, Phytochemical studies.
Journal of Drug Research in Ayurvedic Sciences (2019): 10.5005/jdras-10059-0082

Introduction 1
Regional Ayurveda Research Institute, Gwalior Road, Jhansi, Uttar
Vasa (Justicia adhatoda L. syn. Adhatoda vasica Nees.) is an evergreen Pradesh, India
shrub found throughout the plains of India. It is a well-known drug 2,4
Captain Srinivasa Murthy Reginonal, Ayurveda Drug Development
in Ayurvedic system of medicine and is indicated for a variety Institute, Chennai, Tamil Nadu, India
of disorders such as bronchitis, asthma, fever, and jaundice. The 3,5
Central Council for Research in Ayurvedic Sciences, New Delhi, India
leaves are effective in curing cough, i.e., as an expectorant mild Corresponding Author: Neelima Sharma, Regional Ayurveda Research
bronchial antispasmodic.1 The leaves contain very small amount Institute, Gwalior Road, Jhansi, Uttar Pradesh, India, Phone: +91
of a crystalline alkaloid vasicine.2 The pharmacological properties 8004947960, e-mail: drneelimasharmayu@gmail.com
of A. vasica are attributed to vasicine. It has a slight but persistent How to cite this article: Sharma N, Cheemalapati VN, Mangal AK, et al.
bronchodilator effect and also reduces the blood pressure slightly. Vasa (Justicia adhatoda L.) Leaves Best Procurement Time with Special
Liquid extract of leaves is a very useful remedy in asthma. Reference to Assay of Vasicine through HPLC and its Comparative
Insecticidal and antiseptic properties are also attributed to Pharmacognosy. J Drug Res Ayurvedic Sci 2019;4(4):168–174.
vasicine. In Ayurvedic texts, the whole plant including branches, Source of support: Central Council for Research in Ayurvedic Sciences
flowers, and roots is indicated to check external bleeding or blood Conflict of interest: None
disorders. Leaves are useful in curing dyspnea and cough3 and the
root is used as a tonic.4
Vasa flowers are described to be useful in bleeding disorders,
emaciation, and bronchitis. according to Ayurvedic Pharmacopoeia of India, leaves are the
Leaf decoction (Siddhabheshajmanimala) is a useful wormicide.5 medicinally and economically useful part of the plant and are,
Leaf juice is very useful in curing vomiting, bronchitis, and external therefore, collected throughout the year. Hence, the establishment
bleeding.6 Although in Ayurvedic text, different parts of the plant of the best procurement time on the basis of the bioactive
are described for alignment of diseases, in common practice and secondary metabolites will definitely be useful in all aspects.

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.
org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to
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 Vasa (Justicia adhatoda L.) Leaves Best Procurement Time with Special Reference to Assay of Vasicine

Previous research suggests that Adhatoda contains a high flask, filtered through 0.22 μ membrane filter, and used for high-
percentage of vasicine content in March (3%) when it is in full bloom performance liquid chromatography (HPLC) analysis.
and 1.4% in September when it is in partial flowering. During the
vegetative stage, the plant contains very low concentration of Standard solution: Vasicine of 1.2 mg was dissolved in HPLC-grade
vasicine content (Raja et al.). During rainy and winter seasons, the methanol in a 10 mL volumetric flask and made up the volume
nonvolatile components, vasicine and deoxyvasicine, in A. vasica (Figs 2 to 5).
are found in optimum quantity.7
Buffer solution: Phosphate buffer is prepared by dissolving 0.14 g
M at e r ia l s and Methods of potassium dihydrogen orthophosphate in 500 mL of water and
adjusting the pH to 2.8 by using orthophosphoric acid (Figs 6 to 9).
Pharmacognostical Study
Purity of vasicine: 80%
Collection of Plant Materials and Authentication
The leaves of Justicia adhatoda L. were collected from the same Chromatographic conditions:
habitat, i.e., Regional Ayurveda Research Institute (RARI) Jhansi Instruments–Agilent 1200 series with manual sampler
garden and authenticated at RARI Jhansi. The collected plant ColumncC18 Eclipse, XBD, 4.6 mm × 150 mm
samples were cleaned by washing under running fresh tap water. Detection—VWD Detector at 300 nm
Triplicate plant samples were collected. Useful plant parts for Mobile phase—Methanol-buffer (40:60)
preparing respective drugs were preserved in 70% alcohol for Flow rate—0.8 mL/minute
histological studies. This process was repeated for each season. Injection volume—10 μL
Retention time—1.890
Drying of Plant Materials Purity—80%
The materials were subjected to shade drying for about 4 weeks.
The shade-dried materials were further crushed to powder and Calibration curve: Vasicine of 1.2 mg was accurately weighed
the powder was passed through the 85 no. mesh and stored in and added to a 10-mL volumetric flask, dissolved in HPLC-grade
airtight container for further analysis. This process was repeated methanol, and the volume was made up to 10 mL to obtain 0.12
for each season. mg/mL. This solution was appropriately diluted further to get
a concentration of 0.12, 0.06, and 0.03 mg/mL of vasicine. Each
Macroscopic and Microscopic Analyses of the standard solution was run through the HPLC and the
Macroscopic study: Macroscopic features were studied for each respective peak areas were recorded. Calibration was carried out by
season and photographs were also taken using Nikon DSLR establishing peak area vs concentration of vasicine applied (Fig. 10).
Camera. Macroscopic characters were studied with the help of
floras.8 Comparison of macroscopic characters has been done for Estimation of Vasicine in the Drug
different seasons. Ten microliters of each of the test solutions were injected through
manual injector for the analysis. The chromatogram was recorded
Morphology: Collected plants were observed carefully for its and the area of the peak of the test solution was determined,
identical morphological characters. Morphological characters like corresponding to that of vasicine from the above developed
color, shape, and size were noted down. Characters of plant parts calibration curve. The amount of vasicine present in the residues
were studied as per taxonomy and measurement was taken by extracted in various solvents such as hydroalcohol, ethanol, and
scale through naked eye. methanol for each of the test samples was obtained from the various
seasons of Justicia adhatoda L. (Table 4).
Microscopic analysis: Microscopic examination of the plant was
performed according to the standard method.9–11 Transverse
sections of plant parts were prepared and stained with safranin and
R e s u lts and Discussion
fast green as per the procedure. The same procedure was followed Salient Microscopic Features of Identification in
for powder microscopy. The microphotographs were taken by Powder Drug Analysis
bright-field microscope with digital camera (Tables 1 and 2). Stomata
The leaf was amphistomatic. The stomata of both the surfaces were
The HPLC Methodology/Quantitative Analysis of Vasa
diacytic, and the guard cells were surrounded by two subsidiaries.
(Justicia adhatoda L.)12–14 The subsidiary cells are parallel to the long axis of the pore
Extraction and guard cell. Stomata were found in abundance in the lower
The 5 g of dried powdered material was extracted with 200 mL epidermis, while they are very few in the upper epidermis.
of methanol by using soxhlet for 24 hours. The extracts were
evaporated to dryness under reduced pressure. In the same way, The Trichomes
the same procedure was followed for ethanol and hydroalcoholic Glandular and nonglandular trichomes are found, which are present
(5% aqueous) extraction with 5 g of sample separately. The obtained on both the adaxial and abaxial leaf surfaces. The trichomes of
residue weights for the above extractions given in Table 3. upper surface are unicellular with cytoplasmic content. The foot
was embedded into the epidermal cell and the tip of the trichome
The HPLC Estimation of Vasicine is pointed. The trichomes of lower surface were unicellular with
Test solution: The residues obtained from methanol, ethanol, and cytoplasmic content. Foot was embedded into the epidermal cell.
hydroalcohol extracts of each of the six seasons were weighed The length of trichome of upper surface was more than the lower
in triplicate and dissolved in methanol using 10 mL volumetric surface.

Journal of Drug Research in Ayurvedic Sciences, Volume 4 Issue 4 (October–December 2019) 169
 Vasa (Justicia adhatoda L.) Leaves Best Procurement Time with Special Reference to Assay of Vasicine

Table 1: Macro- and microscopical features of Vasa (Justicia adhatoda L.) in different seasons
Shishir (Jan–Feb) Vasant (Mar–Apr) Grishm (May–June) Varsha (July–Aug) Sharad (Sep–Oct) Hemant (Nov–Dec)s
Flowering (Fig. 1) Flowering (Fig. 1) Vegetative stage Vegetative stage Vegetative stage Flowering, fruiting
Macroscopic: The leaf is simple, entire, Same as the Same as the Same as the Same as the Same as the
wavy, ovate lanceolate, attenuate at the shishir ritu. shishir ritu. shishir ritu. shishir ritu. shishir ritu.
base, apex acuminate, 6–14 cm long,
3–4.5 mm broad, midrib prominent at the
lower surface, slightly grooved on the
upper surface, lateral veins 6–10 pairs
arising at an angle of 45 to 60°, running
parallel to each other, somewhat glabrous
but minutely puberulous on the veins.
Odor not characteristic, taste slightly bitter
Microscopic: TS passing through the Same as the Same as the Same as the Same as the Same as the
midrib is strongly convex on the lower shishir ritu. shishir ritu. shishir ritu. shishir ritu. shishir ritu.
side, slightly centrally depressed on the
upper side, containing a centrally located
arc of meristeles and few other second-
ary meristeles on its either side. Upper
and lower epidermii are composed of
rectangular to squarish, radially arranged
cells covered with thin cuticle, travers-
ing with stomata more on lower side;
in surface view, the cell wall of upper
epidermis is more wavy than that of lower
epidermis; it bears 2- to 3-celled, straight
or bent, thick-walled, knee shaped, simple
uniseriate covering trichomes and sessile
4-celled glandular trichomes, both being
more on the lower side; underneath the
upper epidermis lie two layers of palisade
cells containing oil globules and elon-
gated warty cystoliths extending from
the epidermis to the lower cells of the
palisade, discontinuous over the midrib,
the place being occupied by 5–10 rows of
collenchymatous tissues on both the sides;
spongy parenchyma 5–6 rows traversed
with circular to oval masses of cystolith.
Midrib contains 4–5 conjoint, collateral
meristeles, the centrally located being the
biggest one and in the form of an arc; the
parenchymatous and collenchymatous
tissues of the midrib contain spherical
masses of the cystolith
Powder: The leaf powder shows fragments Same as the Same as the Same as the Same as the Same as the
of lower and upper epidermii in surface shishir ritu. shishir ritu. shishir ritu. shishir ritu. shishir ritu.
view showing epidermal cells with
sinuous walls; spherical palisade cells and
spongy parenchymatous cells underneath
the upper and the lower epidermii,
respectively, showing circular to oval
masses of cystolith; diacytic stomata;
sessile glandular trichomes with 4-celled
head more on lower epidermis and
simple covering 2- to 3-celled trichomes;
fragments of transversely cut pieces of
lamina show sessile glandular trichomes
and 2-layered palisade cells containing oil
globules, embedded with cigar-shaped
cystolith

170 Journal of Drug Research in Ayurvedic Sciences, Volume 4 Issue 4 (October–December 2019)
 Vasa (Justicia adhatoda L.) Leaves Best Procurement Time with Special Reference to Assay of Vasicine

Fig. 1: Vasa in flowering vasant ritu photograph Fig. 2: Dried drug in vasant ritu

Fig. 4: Microscopic features of leaf in 10x. col, collenchyma; cys, cystolith;

Fig. 3: Microscopic features of leaf in 4x through midrib. col, collenchyma;
pal, palisade; vb, vascular bundle; xy, xylem; ph, phloem; per, pericycle;
pal, palisade; vb, vascular bundle; Pa, parenchyma
glt, glandular trichome

Table 3: Extractive value

Residue weight
Season name Methanol Ethanol Hydroalcohol
S. no. (5 g) extract (g) extract (g) extract (g)
1 Shishir ritu S1 0.8286 0.6575 0.8545
2 Vasant ritu S2 1.3456 0.8875 1.1448
3 Grishma ritu S3 1.1824 0.6647 0.9952
4 Varsha ritu S4 1.2455 0.5584 0.9963
5 Sharad ritu S5 1.2993 0.7459 1.1195
6 Hemant ritu S6 1.1452 0.6606 0.9335

Fig. 5: Microscopic features of leaf lamina. ue, upper epidermis; pal,

palisade; cys, cystolith; og, oil globules; le, lower epidermis; spp, spongy Vessels
parenchyma
Vessels are of both pitted and spiral types.
Table 2: Organoleptic character of Justicia adhatoda L.
S.no. Test Observation Inference Cystolith
1. Color Green* Leaf drug Starch grains as cigar-shaped cystoliths are present in spongy and
2 Odor Aromatic Crude drug palisade mesophyll of the leaf.
3 Taste Astringent Drug contain tannins
*The organoleptic characters remain same in shishir, vasant, grishm, varsha
Oil Globules
sharad, and hemant; but in varsha Ritu it become dark green and heavy in Oil globules of volatile nature are present mainly in the palisade
consistency due to the moisture content. and spongy mesophyll of the leaf.

Journal of Drug Research in Ayurvedic Sciences, Volume 4 Issue 4 (October–December 2019) 171
 Vasa (Justicia adhatoda L.) Leaves Best Procurement Time with Special Reference to Assay of Vasicine

The HPLC estimation of Vasicine

Figs 6A to D: Powder microscopy of Justicia adhatoda L. leaf. (A)

Fragments of upper epidermis in surface view; (B) Fragments of lower
epidermis in surface view; (C) Fragment of transversely cut piece of
lamina; (D), Cystolith; cys, cystolith; glt, glandular trichome; og, oil glands;
pal, palisade; ue upper epidermis
Figs 9A and B: The high-performance liquid chromatography of
Justicia adhatoda L. leaves. (A) Test solution; (B) Vasicine standard
Critical Analysis of the Drug
As far as the seasonal variation of microscopical features of leaf
is concerned, no remarkable difference has been noted in the
comparative pharmacognosy of the leaf.

Results of Chemical Analysis of Vasa (Justicia

adhatoda L.) Leaves

Fig. 10: Calibration curve

Table 4: The high-performance liquid chromatography estimation of

vasicine in different seasons
S. no. Seasonal name Vasicine (% w/w)*
Fig. 7: Plant profile Justicia adhatoda L. (leaves)
1 Shishir ritu 0.2133–0.3409
Selected chemical constituent: 2 Basant ritu 0.7330–1.0744
3 Grishm ritu 0.2152–0.4629
4 Varsha ritu 0.1470–0.2532
5 Sharad ritu 0.2771–0.3364
6 Hemant ritu 0.2278–0.4307
*Range of results was given from the means of triplicates of optimized
three solvents of hydroalcohol, ethanol, and methanol for all seasons
From the above table, it was observed Vasicine is abundant in vasant ritu
samples.

D i s c u s s i o n​
The leaves of Justicia adhatoda L. have high medicinal value and
are used in various Ayurvedic formulations.1–6,14–23 To increase
the efficacy of these formulations, it is very important to procure
Fig. 8: Vasicine the useful plant parts in suitable season, when secondary

172 Journal of Drug Research in Ayurvedic Sciences, Volume 4 Issue 4 (October–December 2019)
 Vasa (Justicia adhatoda L.) Leaves Best Procurement Time with Special Reference to Assay of Vasicine

metabolite is in highest concentration. This study was aimed to 5. Shah BG, Nighantu Adarsha; Uttarardh Vasadi verga, Chaukhambha
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