Content uploaded by Rakesh Eshwarlal Mutha
Author content
All content in this area was uploaded by Rakesh Eshwarlal Mutha on Jan 25, 2022
Content may be subject to copyright.
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 232
Pharmacognostic Studies on Anisomeles Heyneana Benth. (Labiatae)
Rakesh E. Mutha
1
, Kundan J. Tiwari
2
, Dashan M. Kokate
3
, Yogesh V. Ushir
2
*
1
Department of Pharmacognosy, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur,
Dist. Dhule 425405, India
2*
SMBT Institute of D. Pharmacy, Dhamangaon, Dist. Nashik 422401, India
3
Department of Botany, MVPS's K. S. K. W. Arts, Science & Commerce College, Nashik 422008, India
Corresponding authors email: ushir29@gmail.com
ABSTRACT
Adulteration is considered to be the main cause for the production of inferior and sometimes unsafe and toxic drug.
So correct identification and selection of crude drugs becomes utmost important to avoid further health
complications. Out of the various techniques available, morphological and microscopic evaluation is found to be
most suitable and promising for the plant drug authentication. The present work deals with the study of
morphological and microscopical characteristics along with phytochemical, physicochemical and fluorescence
analysis of leaf, petiole and stem of Anisomeles heyneana Benth. Along with morphological study, detail
microscopical study gives more authenticity in the identification of medicinal plant. The microscopy of
dorsiventral lamina of the leaf shows adaxial and abaxial epidermal cells, palisade and spongy parenchyma. The
petiole consists of a central and two lateral vascular bundles. The stem of the herb is quadrangular with very
narrow phloem and collenchymatous hypodermis. Microchemical tests of leaf revealed occurrence of phenolic
compounds, lignin, starch, etc. Phytochemical analysis revealed occurrence of phytosterols, triterpenes, phenolic
compounds, flavonoids, tannins, saponins, etc when studied using different extracts. Physicochemical evaluation of
the herb was done using parameters such as extractive values, ash values, foreign matter, moisture content, water
insoluble matter and volatile oil content. Variation in the fluorescence colour response was detected in fluorescence
analysis using different solvents. This finding may be useful as identifiers of herb parts and serve as a guiding path
for the development of official monograph and detailed standardization of Anisomeles heyneana.
Keywords: Anisomeles heyneana, Leaf, Petiole, Stem, Micrometry.
INTRODUCTION
Anisomeles Linn. R. Br. is thought to be important genera of family Lamiaceae, This genus comprises herbs or
under-shrubs, scattered from Africa to India, South East Asia to North East Australia and east from China to
Taiwan, Japan and Philippines. In India, Anisomeles genus is represented by three species viz. A. indica, A.
malabarica and A. heyneana. Anisomeles heyneana commonly called as Western Hill Catmint in English or Gopali
in Marathi. It's a tall, upright herb with slim stems and quadrangular branches that may reach 1 to 1.5 meters in
height. Leaves are 5 to 12 cm long, ovate lance-like, and oppositely oriented. Flowers are tiny, white with pink
tinges, and 2-lipped, and appear in lengthy cymes in October-November. The flowers resemble cow's earlobes [1].
Till date A. indica and A. malabarica have been investigated for various pharmacognostical and pharmacological
studies. These species and essential oil obtained from them was already explored for their antibacterial activity
[2,3]. With this, these two herbs are also investigated for the presence of various fatty acids from their fixed oil [4].
Whereas, A. heyneana has been investigated for the presence of various phyllocladane diterpenes and evaluated for
inhibition of Mycobacterium tuberculosis [5]. The pharmacognostical study (morphological and microscopical) of
A. heyneana is not done yet so far. The rationale made here to row up the most valuable medicinal plant A.
heyneana like A. indica and A. malabarica. So that this species undertaken to study and make it familiar
throughout the world in view of at least morphological, microscopic, phytochemical, physicochemical and
fluorescence analysis. The objective of the present investigation is to figure out an overall pharmacognostic
assessment of the A. heyneana.
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 233
MATERIAL AND METHODS
Instruments, chemicals and other supplies
The study's basic equipment included a simple microscope, a compound microscope, a projection microscope, hot
air oven, UV cabinet and other ordinary glasswares. OLYMPUS photomicroscope provided with Magnus
MagVision image analysis software with Magcam DC 5MP camera was employed to get HD images. All the
genuine chemicals and organic solvents used in this study were procured from Pallav Chemicals, Mumbai, India.
Collection and processing
A. heyneana herb was collected in flowering stage from Western Ghats of India from ‘Waghere Valley’ (Latitude:
20° 6' 53" N; Longitude: 73° 27' 7" E), of Tribakeshwar Tehsil, District Nashik, Maharashtra (India) on 30th
October, 2020. A herbarium is prepared and authenticated by Prof. D. M. Kokate, Dept. of Botany, M.V.P’s
K.S.S.W College, Nashik. The plant voucher specimen has been preserved at the institute level
(SMBTIODP/HERB/16- 2020). Entire herb was cleaned off with water to remove adhering dust and unwanted
plant material was removed. It was further dried in shade, cut and pulverized. The pulverized powder was sieved
using sieve #44 and stored in airtight polythene containers for future use according to research requirements.
Morphological examination
Fresh stem and leaf were utilized for morphological observations. Under a simple microscope, the macro-
morphological characteristics of the herb components (leaves and stem) were examined.
Microscopic examination
Fresh stem, leaves and petiole of the herb were examined by taking a transverse section (TS) using surface
preparations and sections. The longitudinal section (LS) of the stem was also taken for study. The lamina and
midrib of the leaf were investigated using techniques of Brain and Turner [6]. Cross sections of these plant parts
were produced and stained according to K. R. Khandelwal's method for microchemical investigations [7]. The
micrometric study of leaves and stem and pulverized A. heyneana aerial parts (AHA) powder was done using
Magnus MagVision image analysis software with Magcam DC 5MP camera.
Chemo-microscopical examination
Chemo-microscopical examination of the leaf and stem was carried separately. The TS of these specimens were
treated individually with solutions of dilute ferric chloride, dilute iodine, Dragendroffs reagent, Phloroglucinol +
Hydrochloric acid (1:1). These TS were further observed under a compound microscope to detect the occurrence of
chemical/phytoconstituents in it.
Phytochemical Analysis
The proximate chemical analysis of the A. heyneana aerial parts (AHA) powder was proceeded for the
identification of different phytoconstituents. The phytochemical investigation for detection of alkaloids, phenols,
flavonoids, saponins, tannins, cardiac glycosides, steroids, anthraquinone glycosides, phlobatanins, triterpenes,
anthocyanins, etc were carried out. The standard experimental procedures were used to conduct this investigation
[7].
Physicochemical Analysis
The physicochemical/quantitative parameters like total ash, acid-insoluble ash, water-soluble ash, sulfated ash,
alcohol, water and ether soluble extractive, loss on drying (moisture content), volatile oil in drug and water
insoluble matter were determined as per Ayurvedic Pharmacopoeia of India [8].
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 234
Fluorescence Analysis
Fluorescence analysis of A. heyneana, aerial parts (AHA) powder was done by using the method of Chase and
Pratt [9]. A small amount of the crude powder was placed on a clean, grease free microscopic slide and 1-2 drops
of freshly prepared various chemicals/reagents viz. 50% Hydrochloric acid, 50% Sulfuric acid, 50% Nitric acid,
1N (ex.) Sodium hydroxide, 1N (alco.) Sodium hydroxide, 50% Potassium hydroxide, Ammonia, etc. were added.
Then chemicals and crude powder mixed by the gentle movement of the glass slide and hold for a 1-2 minutes.
After this, one by one these slides were kept for observation in an ultraviolet chamber in visible light, 254 nm
(short) and 365 nm (long) ultra violet radiations. The colors noticed in different radiations by the addition of above
chemicals/reagents were documented.
RESULTS
Morphological Examination
The leaves are orienting themselves at an angle to the main axis and perpendicular to the direction of sunlight. The
stem is quadrangular and shows internodes with a length of 9 to 11 cm (Fig. 1). The detail description of aerial
part’s morphological appearance of the herb A. heyneana is described in Table 1.
Fig. 1 Anisomeles heyneana herb
1a and 1b-Herb; 1c-Stem & Leaf; 1d-Fflowers; 1e-Macroscopy
Table 1: Macroscopy of Anisomeles heyneana
Part Characteristic
Stem Green in color, woody, annual and slender stems. Up to 50 cm to 70 cm height
bearing numerous branches which are quadrangular. Internodes 9 cm to 11 cm
long, pith white, fracture is fibrous.
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 235
Leaves The petiole being 1 cm to 3 cm in length. Acute apex, crenate margin,
asymmetric base, reticulate venation and oppositely arranged ovate -lanceolate
like leaves are 5-
12 cm long. Color is green to dark green, taste is mild
astringent with aromatic odor
Flowers Flowers occur in cymes which are 10- 30 cm long. Small 2 cm flowers are white,
tinged with pink, and 2-lipped. Upper lip is 5 mm which is entire, hooded. The
lower lip is 3-lobed. The median lobe large, reflexed.
Seeds Black-brown color, small, sub quadrate and numerous
Microscopical Examination
Leaf
While studying A. heyneana leaf transverse section it has been observed as a dorsiventral leaf (Fig. 2). Lamina
portion shows epidermis and mesophyll. The compact epidermis present on adaxial and abaxial surfaces. At both
surfaces, epidermal cells are compactly arranged with no intercellular spaces except stomata. Epidermis is straight
walled with a polygonal shape present at both surfaces. The outermost wall of the epidermal cells is cutinized (Fig.
2c). The cuticle present on adaxial epidermis is comparatively thicker than the cuticle present on abaxial epidermis.
The ventral or adaxial epidermal cells (0.94 X 0.76 µm) shows the existence of chloroplast whereas, it is absent in
dorsal or abaxial epidermal cells (0.74 X 0.51 µm). The caryophyllaceous stomatal number is more on the abaxial
epidermis. Beneath the stomata, air chamber also observed. Uniseriate, multicellular straight, blunt tip non-
glandular covering trichomes along with unicellular stalk with unicellular head glandular trichomes were present in
both the epidermis (Fig. 2b). Epidermis also contains mucilage.
Mesophyll in A. heyneana differentiated into the palisade parenchyma (13.41 X 2.77 µm) and spongy parenchyma
(2.46 µm). Palisade parenchyma is single layered elongated compactly arranged, narrow columnar cells with right
angle to the upper epidermis. Uniseriate layer has been continuous over the midrib region. Palisade parenchyma
cells were filled with numerous chloroplasts, which are close to the cell wall. Spongy parenchyma contains loosely
arranged, 2-4 layered cells with varying size and shape in between palisade layer and lower epidermis. Many broad
calcium oxalate and starch containing intracellular spaces were observed in this region (Fig. 2c).
Midrib is present on both surfaces with different degree of concavity (Fig. 2a). The dorsal surface is more convex
than ventral surface. Lower palisade is replaced by patch of 2-4 layered collenchymatous cells, which is about 0.45
µm in diameter. The midrib with its middle part found to be occupied by a significant conjoint and collateral
vascular bundle. Broader and narrower vascular bundles are observed at the base of the leaf lamina and leaf apex
respectively. A bundle sheath surrounds each vascular bundle. Xylem vessels are covered by xylem fibers. Xylem
parenchyma (1 µm) is made up from semi rectangular, lignified cells. The phloem cell (0.52 µm) is non-lignified
(Fig. 2c). The micrometric analysis is tabulated in Table 2.
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 236
Fig. 2 Cross section of Anisomeles heyneana leaf
Ctl-Cuticle; uEpi-Upper Epidermis; PlSd-Palisade cells; lEpi-Lower Epidermis; Pchym- Parenchyma; uCol-
Upper collenchyma; lCol-Lower collenchyma; ngTrch-Non glandular Trichomes; gTrch-Glandular
Trichomes; Xylm-Xylem; Phlm-Phloem
Table 2: Micrometry of cellular elements of Anisomeles heyneana leaf
Cellular element Measurement (µm)
Adaxial epidermal cell L: 0.17-
0.94
-1.28;W: 0.45-
0.76
-1.06
Abaxial epidermal cell L: 0.14-
0.74
-1.01;W: 0.34-
0.51
-0.77
Palisade cell L: 10.32-
13.41
-16.50;
W: 1.93-2.77-3.57
Adaxial collenchymatous cell 0.34-
0.90
-1.21
Abaxial collenchymatous cell 0.14-
0.45
-0.77
Parenchymatous cell 0.80-
2.46
-3.81
Phloem cell 0.14-
0.52
-0.96
Xylem parenchyma 0.01-
1.00
-1.93
L- Length; W- Width
Petiole
Adaxial epidermis present iso-diametric cells (0.68 X 0.53 µm) with straight walls with some unicellular, glandular
trichomes followed by 4-5 layers of collenchyma (0.45 µm) and parenchyma (2.13 µm) cells. Epidermis covered
by a very characteristic cuticle (Fig. 3a). The central vascular bundle is mixed, collateral closed, and U-shaped.
Two lateral vascular bundles (Fig. 3b), circularly arranged and are collaterally closed. The phloem region is formed
of primary and secondary phloem, followed by the xylem composed of secondary xylem vessels separated by
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 237
xylem sclerenchyma and xylem parenchyma separates a few primary xylem vessels (0.84 µm). The abaxial
epidermis is the main site for the presence of unicellular or multicellular (2-3 celled) trichomes with pointed tip
type and sessile glandular type (Fig. 3c). The micrometric analysis is tabulated in Table 3.
Fig. 3 Cross section of Anisomeles heyneana petiole
Ctl- Cuticle; Epi- Epidermis; Pchym- Parenchyma; Col- Collenchyma; ngTrch- Non glandular Trichomes;
gTrch- Glandular Trichomes; VscrBdl- Vascular Bundle; Xylm- Xylem; Phlm- Phloem
Table 3: Micrometry of cellular elements of Anisomeles heyneana petiole
Cellular element Measurement (µm)
Abaxial epidermal cell L: 0.16-
0.68
-1.13;W: 0.36-
0.53
-0.73
Abaxial epidermal cell L: 0.25-
0.73
-1.28;W: 0.41-
0.62
-0.85
Collenchymatous cell 0.14-
0.45
-0.91
Parenchymatous cell 0.75-
2.13
-3.35
Phloem cell 0.09-
0.46
-0.73
Xylem parenchyma 0.01-
0.84
-1.77
L- Length; W- Width
Stem
Stem’s cross section (Fig. 4a; 4b) is quadrangular with four pubescent ridges evenly spaced, a central broad
parenchymatous four-angled pith bordered by a ring of xylem and extremely thin phloem (0.49 µm) with
collenchymatous hypodermis. The epidermis (1.06 X 0.66 µm) made up of one layered rectangular cells, which is
covered by a cuticle layer along with very limited stomata. It also bears simple, multicellular (4-5 celled) covering
trichomes and blunt tipped glandular trichomes. The cortex (Fig. 4c) is a 2 to 4 layered collenchymatous (0.68 µm)
structure that can extend up to 10 layers under the primary ridges.
Distinct endodermis and cambium is present in the given structure. Between the cortex and the vascular tissue are
lignified sclerenchyma fibres (Fig. 4d). The parenchymatous central broad pith (3.71 µm) contains crystals of
calcium oxalate and simple starch granules. The micrometric analysis is tabulated in Table 4.
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 238
Fig. 4: Transverse section of Anisomeles heyneana stem
Bldg- Buldge; Ctl- Cuticle; Epi- Epidermis; ngTrch- Non glandular Trichomes; gTrch- Glandular
Trichomes; CrTx- Cortex; Col- Collenchyma; SclRch- Sclerenchyma; Xylm- Xylem; XylmP- Xylem
Parenchyma; Phlm- Phloem; Pth- Pith
Table 4: Micrometry of cellular elements of Anisomeles heyneana stem
Cellular element Measurement (µm)
Epidermal cell L: 0.15-
1.06
-1.88; W: 0.37-
0.66
-1.53
Collenchymatous cell 0.14-
0.68
-0.97
Sclerenchyma cell 0.64-
1.70
-2.01
Parechymatous cell 1.79-
3.71
-6.52
Phloem cell 0.13-
0.49
-1.03
Xylem parenchyma 0.01-
1.30
-2.16
L- Length; W- Width
Powder characteristics
The AHA powder microscopic characteristics are shown in Fig. 5. The specific characteristics of powder
determined by microscopic investigation showed following as;
Lignified characteristics
When AHA powder treated with phloroglucinol+Conc. HCl (1:1) then slide observed under 10X the distinguishing
characteristics with pink color found. These characteristics are lignified fibers (Fig. 5a), lignified xylem vessels
(Fig. 5b), lignified spiral xylem fiber (Fig. 5c; Fig. 5d), lignified bordered pitted xylem fiber (Fig. 5e), and lignified
tissues (Fig. 5f).
Calcium oxalate crystals
In without any chemical treated slide, the acicular and prismatic, calcium oxalate crystals were observed (Fig. 5h).
Journal of the Maharaja Sayajirao University of Baroda
Volume-55, No.1(X) 2021
Lamina portion
The palisade cells with epidermal cells of lamin
Trichomes
The AHA powder was shown abundant various type of covering trichomes in 10X. They are uniseriate
multicellular 3 celled blunt tip (Fig. 5k), 5 celled thick walled, pointed (Fig. 5l) cover
walled, blunt tip covering trichomes (Fig. 5m), and, 4 celled blunt tip (Fig. 5n) covering trichomes.
Parenchyma cells
The spongy parenchyma cells (Fig. 5o) with cellular content observed during observation of powder slide under
10x of microscope.
Oil globules
When AHA powder treated with Sudan red III, red colored oil globules were found (Fig. 5p, Fig. 5q & Fig. 5r).
Stone cells
The non-
lignified, rectangular and oval shaped stone cells observed (Fig. 5s & Fig. 5t).
Starch grains
The abundant blue colored simple starch grains (Fig. 5u, Fig. 2v & Fig. 5w) were found when AHA powder treated
with dilute iodine solution.
Periderm
The periderm portion (Fig. 5x) with 2
without any cellular content and 4-
6 layered of thin walled rectangular phelloderm cells without any cellular
content observed.
Cork cells
The thin walled with yellowish to brown matter cork cell (Fig. 5y) are also observed during microscopic study
AHA powder.
Fig. 5 Microscopy of
5a- Lignified fibre; 5b-
Xylem vessels,
fiber; 5f- Lignified tissue; 5g-
Fibers & xylem vessels;
Journal of the Maharaja Sayajirao University of Baroda
The palisade cells with epidermal cells of lamin
a (Fig. 5i) and stomatal opening (Fig. 5j) also observed.
The AHA powder was shown abundant various type of covering trichomes in 10X. They are uniseriate
multicellular 3 celled blunt tip (Fig. 5k), 5 celled thick walled, pointed (Fig. 5l) cover
walled, blunt tip covering trichomes (Fig. 5m), and, 4 celled blunt tip (Fig. 5n) covering trichomes.
The spongy parenchyma cells (Fig. 5o) with cellular content observed during observation of powder slide under
When AHA powder treated with Sudan red III, red colored oil globules were found (Fig. 5p, Fig. 5q & Fig. 5r).
lignified, rectangular and oval shaped stone cells observed (Fig. 5s & Fig. 5t).
The abundant blue colored simple starch grains (Fig. 5u, Fig. 2v & Fig. 5w) were found when AHA powder treated
The periderm portion (Fig. 5x) with 2
-
3 layer of thin walled, flat, polygonal cork cells, 3
6 layered of thin walled rectangular phelloderm cells without any cellular
The thin walled with yellowish to brown matter cork cell (Fig. 5y) are also observed during microscopic study
Fig. 5 Microscopy of
Anisomeles heyneana
aerial parts (AHA) powder
Xylem vessels,
5c,5d- Lignified spiral xylem fiber; 5e-
Lignified bordered pitted xylem
Fibers & xylem vessels;
5h- Calcium oxalate crystals-
ISSN :0025-0422
239
a (Fig. 5i) and stomatal opening (Fig. 5j) also observed.
The AHA powder was shown abundant various type of covering trichomes in 10X. They are uniseriate
multicellular 3 celled blunt tip (Fig. 5k), 5 celled thick walled, pointed (Fig. 5l) cover
ing collapsed cell thick
walled, blunt tip covering trichomes (Fig. 5m), and, 4 celled blunt tip (Fig. 5n) covering trichomes.
The spongy parenchyma cells (Fig. 5o) with cellular content observed during observation of powder slide under
When AHA powder treated with Sudan red III, red colored oil globules were found (Fig. 5p, Fig. 5q & Fig. 5r).
The abundant blue colored simple starch grains (Fig. 5u, Fig. 2v & Fig. 5w) were found when AHA powder treated
3 layer of thin walled, flat, polygonal cork cells, 3
-5 layer of phellogen
6 layered of thin walled rectangular phelloderm cells without any cellular
The thin walled with yellowish to brown matter cork cell (Fig. 5y) are also observed during microscopic study
of
aerial parts (AHA) powder
Lignified bordered pitted xylem
acicular & prismatic; 5i-
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 240
Lamina portion- palisade with epidermal cells; 5j- Stomatal opening; 5k- Uniseriate multicellular (3 celled) blunt
tip covering trichomes; 5l- Uniseriate multicellular (5 celled) thick walled, pointed covering trichomes; 5m-
Uniseriate, multicellular (collapsed cell) thick walled, blunt tip covering trichomes; 5n- Uniseriate, multicellular (4
celled) blunt tip covering trichomes; 5o- Parenchyma cells, 5p,5q,5r- Oil globules; 5s,5t- Stone cells; 5u,5v,5w-
Starch grains; 5x- Periderm:cork cells, phellogen and Phelloderm; 5y- Cork cells: thin walled with yellowish to
brown matter; 5z- Fibre
Chemo-microscopical examination
The counter idea about the occurrence of primary and secondary metabolites is obtained through stated
examination. Metabolites like phenolic compounds, starch, proteins, lignin were identified with various laboratory
chemicals and staining reagents. When a ferric chloride solution treatment is given to leaf’s cross section, a blue
black stain is developed which directs occurrence of phenolic compounds in the palisade zone. Whereas, when
stem’s cross section treatment using Phloroglucinol and HCl (1:1) shows pink color for lignin, present between the
cortex and vascular tissue and xylem tissue (Table 5).
Table 5: Micro-chemical tests of Anisomeles heyneana leaf and stem
Reagent Color Chemical Histological Zone
Leaf Stem
Phloroglucinol + HCl
Pink
Lignin
Vascular bundle
Between the cortex and
Vascular tissue; Xylem
tissue.
Iodine solution
Blue
Starch
Mesophyll region
Below Vascular tissue
FeCl
3
solution
Bluish black
Phenolic
compound Palisade cells
Beneath to primary
ridges; Hypodermis
Libermann-Burchardlt
Reagent Greenish
Steroids
Mesophyll region
Between Vascular
tissue and pith
Million’s reagent
Blue Proteins
Mid rib
Pith
Phytochemical Analysis
The qualitative phytochemical investigation of the A. heyneana aerial parts (AHA) powder in different solvent
extracts is presented in Table 6. The phytochemicals were soluble in the solvent depending on its polarity. The
solvent choices were made in this protocol as per polarity index (PI) from none polar to semi-polar to polar [10].
Solvent used for extraction as petroleum ether (PI - 0.1), benzene (PI - 2.7), chloroform (PI - 4.1), Propanone (PI -
5.1), ethanol (PI - 5.2), and, water (PI - 10.1). In petroleum ether and benzene extract, phytosteroids and triterpenes
were present. In chloroform extract, chemical tests for the detection of flavonoid, phytosteroids, and triterpenoids
were found positive. In propanone extract, tests for phenolic compounds, tannins and triterpenoids were found
positive. In ethanol extract, flavonoids, mucilage, phenolic, saponins, and triterpenes were present. Whereas, in
water extract, carbohydrates, protein, mucilage, phenolics, saponins were present.
Table 6: Phytochemical analysis of AHA extracts
Phytoconstituents Pet. Ether
(40-60
o
) Benzene Chloroform Propanone Ethanol
(95%) Water
Alkaloids
- - - - - -
Amino acid - - - - - -
Carbohydrates
- - - - - +
Flavonoid
- - + - + -
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 241
Mucilage - - - - + +
Phenolic
- - - + + +
Phytosterols
+ + + - - -
Proteins - - - - - +
Saponins
- - - - + +
Tannins
- - - + + -
Triterpenoids + + + + + -
‘+’ presence; ‘-’ absence
Physicochemical Analysis
Physicochemical parameters are a vital for the standardization and quality control of any crude drug. The results of
physicochemical factors such as extractive values, ash values, foreign matter, etc. are presented in Table 7.
Table 7: Physicochemical analysis of AHA powder
Parameter Values % (w/w)* ± SD
Ash Values
Total Ash 6.99 ± 0.636
Acid-Insoluble Ash 0.49 ± 0.030
Water-Soluble Ash 2.28 ± 0.208
Sulphated Ash 10.32 ± 0.741
Extractive values
Alcohol Soluble 5.54 ± 0.051
Water Soluble 16.01 ± 0.074
Ether Soluble (Fixed Oil Content) 1.33 ± 0.741
Foreign Matter 4.16 ± 0.753
Moisture Content (Loss on Drying) 5.36 ± 0.314
Water Insoluble Matter 79.40 + 1.501
Volatile Oil in Drug 0.08 + 0.021
* Values are expressed as mean of three readings; SD-Standard Deviation
Ash values
Determination of ash values is used to control the quality and purity of crude drugs. The total ash value (6.99 ±
0.636% w/w), acid-insoluble ash value (0.49 ± 0.03% w/w) and water-soluble ash value (2.28 ± 0.208% w/w)
carried out for AHA powder. The sulphated ash value also determined which to be found as 10.32 ± 0.741% w/w.
A total ash value indicates presence of various inorganic impurities like phosphates, carbonate, oxalate and
silicates of potassium, sodium, magnesium, calcium. The amount of inorganic components contained in given drug
is estimated using water soluble ash. The acid insoluble ash is mostly composed of silica and shows that it has been
contaminated with earthy material [11].
Extractive values
The tentative idea about the plant material’s possible active chemical components when extracted with a specific
solvent is determined by extractive values. The chemical constituents' compositions are influenced by the drug's
nature and the solvent utilized. It also indicates whether or not the crude drug has been exhausted [12]. The
maximum soluble extractive value of AHA powder was found in water (16.01 ± 0.074% w/w) and minimum
soluble extractive value was found in petroleum ether (1.33 ± 0.741% w/w). The alcohol soluble extractive value
was 5.54 ± 0.051% w/w.
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 242
Foreign matter
When analyzed, medicinal plant materials must be free of obvious mold growth, rodent excrement, insects,
sliminess, stones or any other toxic foreign matter [8]. The foreign matter for AHA powder was observed as 4.16
± 0.753% w/w.
Moisture content
Moisture content (loss on drying) is commonly used test procedure for determination of moisture content in a
powdered sample. Moisture content of drugs should be at minimal levels to discourage the growth of bacteria,
yeast, or fungi during storage. The moisture content of AHA powder was found to be 5.36 ± 0.314% w/w.
Water insoluble matter
The water insoluble matter is a residue remains after boiling with water mainly sugar free residue, it was confirmed
by negative Molisch test. The water insoluble matter for AHA powder was found to be 79.40 ± 1.501% w/w.
Volatile oil in drug
The AHA powder gives strong aroma during its morphological examination; it indicates the presence of volatile oil
in it. To get an exact idea about content of volatile oil in drug is determined by Clevenger’s apparatus. The AHA
powder shows the presence of 0.08 ± 0.021% w/w volatile oil content on dry weight basis.
Fluorescence Analysis
The medicinal plant material has different chemical constituents which exhibit fluorescence. The A. heyneana,
aerial parts (AHA) powder fluorescence analysis showed greenish blue (UV light- 254 nm) and waxy yellow (UV
light- 366 nm) color when treated with 1 N aqueous NaOH. Under visible light, a yellowish green colour was
noticed for the same using 1% Iodine solution. Various colors and some extended fluorescence like greenish black,
violet brown, bluish black and dark green were also observed under different light conditions when treated with
different laboratory chemicals. Table 8 shows all the observations of fluorescence analysis.
Table 8: Fluorescence analysis of AHA powder
Treatment Observation
Visible light UV light- 254 nm UV light- 365 nm
Powder Light Green Brown Dark brown
Powder + 1 N (alc.) NaOH Green Reddish orange
*
Dark violet
Powder + 1 N (aqs.) NaOH Green Greenish blue* Wax yellow
*
Powder + 1% Iodine Yellowish green Red
*
Dull violet
Powder + 50% H
2
SO
4
Green Black Light green
Powder + 50% HCl Dark green Violet brown* Dark green
Powder + 50% HNO
3
Brown Green
*
Reddish violet
Powder + 50% KOH Brown Black Dark green
Powder + Acetic acid Dark green Violet brown* Brown
Powder + Ammonia Light green Greenish blue
*
Dull violet
Powder + Ethyl acetate Green Brown Green
Powder + 95% Ethyl alcohol Green Yellowish green* Light green
Powder + 5% FeCl
3
Blackish green Greenish grey
*
Dark violet
Powder + Methanol Green Bluish red
*
Yellow
Powder + Petroleum ether Green Green Yellowish green
*
Presence of fluorescence
DISCUSSION
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 243
As the proof of identity and authentication are the important prerequisites to start any research activity relevant to
the plant material. Considering the same and to promote the research activities of A. heyneana, the above stated
data were generated. The macro and microscopic characteristics that characterize the authentic herb A. heyneana
were determined by the findings of botanical research described here. These morphological features are useful to
quickly identify a medication, which is especially valuable when dealing with powdered drug. The detailed
macroscopic and microscopic study of this herb with respect to leaf, petiole and stem will helpful for the correct
identification and differentiating this herb from the other species of Anisomeles. The microscopic study included
the micrometric inclusion of cellular components, which will give more precise insight of the herb. Microchemical
tests of a leaf revealed the presence of phenolic compounds, lignin, starch, etc. Phytochemical analysis revealed
presence of triterpenes, phytosterols, tannins, phenolic compounds, flavonoids, saponins, etc when studied using
different extracts. In the standardization and quality evaluation, investigation of its physicochemical parameters is
extremely essential. The extractive value of water, alcohol and petroleum ether were found to be 16.01 ± 0.074%
w/w, 5.54 ± 0.051% w/w and 01.33 ± 0.741% w/w respectively. The total of minerals and earthy debris associated
with the herb is expressed by the total ash (6.99 ± 0.636% w/w). The acid insoluble siliceous material is expressed
by the acid insoluble ash (0.49 ± 0.03% w/w). The lower value of loss on drying of the AHA powder (5.36 ±
0.314% w/w) represents higher stability and further prevention of degradation due to bacteria, fungi and yeast.
With this, results of fluorescence study of the herb could also serve as a standard for the authentication and
identification and further investigation of adulteration, if any. The more detail investigation needs to be performed
using advance techniques like scanning electron microscopy, random amplified DNA (RAPD) analysis including
other physicochemical parameters in near future.
CONCLUSION
Morphological and cross sectional study helps to ensure proper authentication, identification and quality of
traditional plant. The pharmacognostical study of A. heyneana has been evaluated with respect to its morphology,
microscopy, chemo-microscopy, phytochemical, physicochemical and fluorescence analysis.. These
pharmacognostical results of A. heyneana can be employed in its identification, preparation of official monographs
and standardization. These findings will undoubtedly aid in the identification, assessment and development of a
monograph for this herb in the future.
REFERENCES
1. K. S. R. Murthy, M. C. Reddy, T. Pullaiah, Ethnobotany, chemistry and pharmacology of an aromatic genus
Anisomeles linn. in India, Int. J. Life Sci. Pharma. Res., 5, L-34-L-47, 2015.
2. Y. V. Ushir, A. U. Tatiya, S. J. Surana, U. K. Patil, Gas chromatography−Mass spectrometry analysis and
antibacterial activity of essential oil from aerial parts and roots of Anisomeles indica Linn., Int. J. Green
Pharm., 4, 98-101, 2010. https://doi.org/10.4103/0973-8258.63884
3. R. R. Kulkarni, K. Shurpali, R. L. Gawde, D. Sarkar, V. G. Puranik, S. P. Joshi, Phyllocladane diterpenes
from Anisomeles heyneana, J. Asian Nat. Prod. Res., 14, 1162-1168, 2012.
https://doi.org/10.1080/10286020.2012.738672
4. Y. Ushir, K. Patel, Chemical composition and antibacterial activity of essential oil from Anisomeles species
grown in India, Phcog. J., 2, 55-59, 2011. https://doi.org/10.1016/S0975-3575(11)80026-X
5. Y. Ushir, K. Patel, N. Sheth, Analysis of fatty Acid in Anisomeles Species by Gas Chromatography-Mass
Spectrometry, Phcog. J., 3, 44-47, 2011. https://doi.org/10.5530/pj.2011.22.9
6. K. R. Brain, T. D. Turner, “The practical evaluation of phytopharmaceuticals”, Wright-Scientechnica, Bristol,
81 -86, 1975.
7. K. R. Khandelwal, “Practical Pharmacognosy techniques and experiments”, 13th Ed., Nirali Prakashan, Pune,
130-149, 2005.
Journal of the Maharaja Sayajirao University of Baroda ISSN :0025-0422
Volume-55, No.1(X) 2021 244
8. The Ayurvedic Pharmacopoeia of India, Part-I, 1st Ed., Volume-VI, Government of India, Ministry of Ayush,
New Delhi, 242-245, 2008.
9. C. R. Chase, R. J. Pratt, Fluorescence of powdered vegetable drugs with particular reference to development
of a system of identification, J. Am. Pharm. Assoc., 38, 324-31, 1949.
https://doi.org/10.1002/jps.3030380612
10. K. Ramluckan, K. G. Moodley, F. Bux, An evaluation of the efficacy of using selected solvents for the
extraction of lipids from algal biomass by the soxhlet extraction method. Fuel, 116, 103-108, 2014.
https://doi.org/10.1016/j.fuel.2013.07.118
11. S. Chanda, Importance of pharmacognostic study of medicinal plants: an overview, J. Pharmacognosy
Phytochem., 2, 69–73, 2014.
12. A. Tatiya, S. Surana, S. Bhavsar, D. Patil, Y. Patil, Pharmacognostic and preliminary phytochemical
investigation of Eulophia herbacea Lindl. Tubers (Orchidaceae), Asian Pac. J. Trop. Dis. 2 (Suppl 1), S50–
S55, 2012. https://doi.org/10.1016/S2222-1808(12)60123-6
