Research Journal of Pharmacognosy and Phytochemistry.

10(3): July- September, 2018

ISSN 0975-2331 (Print) Available online at

0975-4385 (Online) www.anvpublication.org
DOI: 10.5958/0975-4385.2018.00034.1
Research Journal of
Vol. 10| Issue-03|
July- September 2018 Pharmacognosy and Phytochemistry
Home page www.rjpponline.org

RESEARCH ARTICLE

Pharmacognostic Studies on Root-bark and fruit of Morinda tinctoria Roxb

Praveena. A1*, Sanjayan K. P2
1
Department of Biotechnology, Prathyusha Engineering College, Thiruvallur-602025, Tamilnadu, India.
2
Department of Zoology, Gurunanak College, Velachery, Chennai-600 042, Tamilnadu, India.
*Corresponding Author E-mail: praveena_bioinfo@yahoo.com

ABSTRACT:
Morinda tinctoria commonly known as Indian Mulberry is a species of flowering plant in the family Rubiaceae
which plays important role in traditional medicine. In the present study, the various anatomical characteristics
and proximate analysis of root-bark and fruit of Morinda tinctoria were investigated by the microscopic
sectioning using standard pharmacopoeia methods. Microscopic examination of the root-bark indicated the
presence of calcium oxalate crystals of raphide bundles in the axial parenchyma. Calcium oxalate crystals were
also present in the fruit, either as a 4-lobed druse type or as a spindle shaped Raphide type. Proximate analysis
was carried out to evaluate the plant as a potential source of active compounds which could be served as potent
drug or to develop novel insecticide against the major pest which involve in crop damage. A lower acid insoluble
ash content was recorded for the fruit than the root-bark. Acid insoluble ash value of M. tinctoria fruit (0.510%)
shows that small amount inorganic compound is insoluble in acid and therefore the fruit may be readily digested
and absorbed when consumed.

KEYWORDS: Morinda tinctoria, Microscopic sectioning, Calcium oxalate crystals, proximate analysis,
Raphide bundles.

INTRODUCTION: The Rubiaceae is one of the largest and most diverse

Medicinal plants are serving as source for the
development of medicines. Approximately 85-90% of
the world’s population consumes traditional herbal
medicines1. India has served as the base for the
development of many traditional medicine2. Accurate
characterization of plant material is a prime step to
ensure reproducible quality of herbal medicine which
will help us to justify its safety and efficacy3. The genus
Morinda, belonging to the family Rubiaceae, is
indigenous to tropical countries and is considered an
important traditional folk medicine.
Received on 29.11.2017 Modified on 02.01.2018
Accepted on 25.02.2018 ©A&V Publications All right reserved
Res. J. Pharmacognosy and Phytochem. 2018; 10(3): 211-215.
DOI: 10.5958/0975-4385.2018.00034.1
families of flowering plants, with approximately 650
genera and 13,000 species4, mostly of tropical
distribution. Historically fruit and seed characters have
been used to infer the classification of the family and in
several cases to define subfamilies5. In south India, 12
different species of Morinda are distributed throughout
Tamil Nadu and Kerala. Many species of Morinda are
available in India, of which Morinda tinctoria Roxb.
mainly grows in vacant agricultural land as weed tree.
M. tinctoria is a small tree, bark long fissured, leaves are
elliptic, white colour flowers in axillary globose heads
and the fruits are drupe, globose6. The leaves and roots
of M. tinctoria are used as astringent, deobstrent,
emmengogue and to relive pain in the gout in traditional
medicine7. There are many literatures available on the
anatomical and medicinal properties M. tinctoria leaves

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 Research Journal of Pharmacognosy and Phytochemistry. 10(3): July- September, 2018
8
. In the present study we have initiated the anatomical loss in the powdered material. The average percentage
and proximate characteristics of root-bark and fruit of M. weight loss, with reference to the air dried powdered
tinctoria. The pharmacognostical studies will be useful sample was determined for three replicates.
for the investigators to identify the plant in future.
Total ash determination:
MATERIAL AND METHODS: The crucibles were washed thoroughly, dried in hot oven
Collection of plant specimens: at 100°C, cooled in desiccator and weighed. A 2.0 g
The plant specimens (Root bark and fruit of Morinda portion of each of the sample was weighed into the
tinctoria) were collected from Kadambathur, Thiruvallur crucible and put in the furnace. Heating was started
district, Tamilnadu, India. Care was taken to select gradually until temperature of 600°C was reached. This
healthy and normal organs. The fruit and the bark of the temperature was maintained for 6 h. The crucible was
root were cut and removed from the plant and fixed in then put inside a desiccator and cooled. After cooling the
FAA (Formalin-5ml and Acetic acid-5ml+70% ethyl sample was reweighed and the percentage ash calculated.
alcohol-90ml). After 24hrs of fixing, the specimens were
dehydrated with graded series of tertiary-Butyl alcohol W-Z
(TBA) as per the schedule given by Sass, 1940. % Ash = ------ x 100
Infiltration of the specimens was carried by gradual N
addition of paraffin wax (Melting point 58-60°C) until Where,
TBA solution attained super saturation. The specimens W = weight of the crucible and ash;
were cast into paraffin blocks. Z = weight of empty crucible;
N = weight of the sample.
Sectioning:
The paraffin embedded specimens were sectioned with Water soluble ash value determination:
the help of Rotary Microtome. The thickness of the The crucible with the total ash was transferred into a
sections was 10-12 µm. dewaxing of the sections was by beaker containing 25 ml of distilled water. The beaker
customary procedure9. The sections were stained with and its contents were boiled for 5 min and filtered
Toluidine blue as per the method published by O’Brien through an ashless filter paper (Whatman). The filter
et al.,10 since Toluidine blue is a polychromatic stain. paper containing the residue was folded and placed in a
The staining results were remarkably good; and some weighed crucible. The crucible was then heated in the
cytochemical reactions were also obtained. The dye muffle furnace, until the filter paper was completely
rendered pink colour to the cellulose walls, blue to the ashed. The crucible and its contents were cooled and
lignified cells, dark green to suberin, violet to the weighed and the final weight noted. The weight of the
mucilage, blue to the protein bodies etc. Wherever residue was then calculated by subtracting the constant
necessary sections were also stained with safranin and weight of the second crucible and its ash. This is the
fast-green. water insoluble ash. The weight of the water soluble ash
was obtained by subtracting the weight of the water
Photomicrographs: insoluble ash from the total ash. The weight of the water
Microscopic descriptions of tissues are supplemented soluble ash divided by the initial weight of the crude
with micrographs whenever necessary. Photographs of sample was multiplied by 100 and was taken as the water
different magnifications were taken with Nikon lab soluble ash value.
photos 2 microscopic Unit. For normal observations
bright field was used. For the study of crystals, starch Acid insoluble ash value determination:
grains and lignified cells, polarized light was employed. The water insoluble ash obtained from the above
Since these structures have birefringent property, under experiment was transferred into a beaker containing 25
polarized light they appear bright against dark ml of diluted HCl. The beaker and its contents were
background. Magnifications of the figures are indicated boiled for 5 min and the boiled contents filtered through
by the scale-bars. an ashless filter paper (Whatmann). The washings were
then passed through the filter paper in a manner as to
PROXIMATE ANALYSIS: allow the collection of the residue at the tip of the cone
The following parameters were quantified using standard of the filter paper. The weight of the clean and heated
methods11-12. porcelain crucible was accurately determined. The filter
paper with the residue was folded with a small cone and
Moisture content/water loss on drying: transferred into the crucible. The crucible was gently
The powdered material of root-bark and fruit of M. heated until the filter paper was completely ashed, and
tinctoria (2.0 g) was weighed into a clean crucible of then heated strongly for a few minutes. The crucible and
known weight. After oven drying at 115°C for 5 hrs, the its contents were cooled, weighed and the final weight
crucible was cooled and weighed to determine weight was noted. The weight of the residue (ash) was then
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calculated. This was done by subtracting the constant
weight of the crucible and ash. The weight of the ash
divided by the initial weight of the sample and
multiplied by hundred was taken as the acid insoluble
ash value.
RESULTS AND DISCUSSION:
Microscopic features of root-bark:
The bark is brown or brownish-grey. It is rough and
deeply fissused; fissuses are longitudinal and parallel.
Old bark consists of thick chunky scales adhering on the
trunk. In a cut surface of the bark, three regions are
recognized. The inner most zone, measuring about 1mm
thick, represents the Cambial zone and noncollapsed
(intact) phloem; the middle zone is 7mm thick and is
brittle, granular in textures; narrow parallel white lines
representing the phloem rays, are visible in the middle
zone. The outer most zone is 3-4mm thick represents the
rhytidome which consists of brittle, granular substance
and exfoliates easily.
The root-bark consists of an outer most region of
rhytidome, which includes two or three layers of
rectangular, thick walled lignified phelloid cells
alternating with wider, thin walled suberised phellem
cells. The phellem and phelloid cells are arranged in
regular radial parallel rows. A narrow phelloderm zone
is seen inner to the periperm. In a bark of 12-13mm
thickness, the inner portion of secondary phloem
measures 7mm thick. The tissues from the inner border
of the rhytidome extending up to inner most end of the
bark represent the secondary phloem. The secondary
phloem can be divided into two region, namely outer
collapsed phloem and inner noncollapsed phloem.
The collapsed phloem (Fig 1) includes crushed sieve cells13.
elements which form dark, irregular tangential lives,
isolated clusters of brachysclereids and slightly diluted Microscopic features of fruit:
phloem rays. Calcium oxalate crystals in the form of The fruit is a drupe with membranous epicarp, fleshy
thick bundles consisting of thin pointed needles are mesocarp and thin endocarp. The fruit of M.tinctoria is
abundant in the collapsed phloem.
It is narrow zone of intact sieve elements companion
cells and narrow phloem rays. Brachysclereids and
raphide needle bundles are less or absent in the
noncollapsed phloem. The sieve tube members are
tangentially oblong and are arranged in radial parallel
lines. The sieve tubes are 18-34µm wide. The
companion cells are small and are located at the narrow
corner of the sieve tube. The phloem parenchyma cells
are comparatively smaller and are random in
distribution. Structure and arrangement of the phloem
rays and sieve-tubes can be viewed. The phloem rays are
nonstoried, i.e they occur at different levels. The rays are
uniseriate biseriate and multiseriate. Uniseriate rays are
less frequent. The rays are homocellular. The cells of the
ray are more or less uniform in shape and size. They are
angular in outline fairly thick walled and compact. The
height of the rays ranges from 40-110µm. The thickness
is 10µm (Uniseriate ray), 20µm (biseriate ray) or 50µm
(Multiseriate ray). Ray frequency is 13/1mm. Sieve
tubes are narrow and straight. They have simple sieve
plate, which is oblique. Axial parenchyma cells occur in
vertical, straight lines. The cells are vertically elongated
and thin walled.
Calcium oxalate crystals of raphide bundles in the axial
parenchyma were observed. The raphide consists of
hundreds of thin pointed needles tightly bundled into a
spindle shaped body. They are vertically oriented, so that
in transactional view, the raphides appear as circular
bundle and in LS view, they appear as spindle shaped
bodies. The raphide bundles are 210µm long and 50µm
thick. The bark of Rubiaceae plants Uncaria guianensis
Aubl. and U. tomentosa has been studied and reported
the presence of rhytidome with 2–4 periderms, with
phelloderm composed of sclereids and parenchymatic
multiple fruit, formed by fusion of several ovaries, so
that the fruit appears lobed and grooved. The surface of
the fruit consists of a thin epidermal layer of small
rectangular cells, which represents the epicarp. The
mesocarp is wide and parenchymatous. There are several
radially elliptical, wide chambers possessing abortive
seeds (Fig 2). In central part of the fruit is seen a ring of
discrete vascular strands, from the central circle of
strands several vascular traces deviate and enter into the
outer zone of the fruit.

Fig 1: Transverse section of the bark showing collapsed phloem

with raphides and Sclereid masses. (CPh- Collapsed phloem; DR-
Dilated ray; Ra- Raphides; Scl- Sclereid masses; NCPh- Non
collapsed phloem).

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 Research Journal of Pharmacognosy and Phytochemistry. 10(3): July- September, 2018
Fig 2. 1. Transverse section of fruit- outer epicarp and inner
mesocarp.
2. Transverse section of fruit- mesocarp with vascular
strand.
(EP- Epicarp; MC- Mesocarp; VS- Vascular Strand)
Calcium oxalate crystals are fairly abundant in the
ground tissue. There are two types of crystals: i) Small,
circular, 4-lobed druse type crystals is wide spread in the
cells. They are random in distribution (Fig 3) and ii)
Raphide type of crystals: These crystals are less in
frequency. They are spindle shaped cylinders comprising
numerous thin pointed needles. They are located within
wide cavities in the ground tissue. The raphides are
20µm thick and 80µm long.
Fig 3: Distribution of the druses and raphides. (Under polarized
light microscope)
1. Druses and raphides in the fruit mesocarp cells.
2. Druses enlarged.
3. Druses and raphide enlarged.
(Dr- Druses; Ra- Raphide)
214
The type of fruit in M. tinctoria is multiple fruit similar
to M. citrifolia. Calcium oxalate crystals occur in
different forms throughout the Plant Kingdom. They
perform various functions, including herbivory
deterrence14, calcium regulation15,16 and are associated
with heavy metal tolerance17. Calcium oxalate crystals
are produced and accumulated in over 215 plant familie
and are suggested functioning primarily in sequestering
excess calcium and acting as a defense against
herbivores18. Some investigations have shown a direct
correlation between Calcium oxalate and defense. In the
present study Calcium oxalate crystals was found in the
M. tinctoria fruit and root-bark which indicate the
defense property of the plant samples.
Proximate analysis:
Proximate analysis facilitates in evaluating the plant as a
potential source of insecticide. Data on the total ash
content of the plant material measures the total amount
of material remaining after ignition. This includes both
physiological ash, which is derived from the plant tissue
itself, and non-physiological ash, which is the residue of
the extraneous matter (e.g. sand and soil) adhering to the
plant surface. Results of the Proximate analysis of the
Morinda tinctoria fruit and Root-bark (bark of the roots)
are presented in the Table 1. In comparison to the fruit
the total ash content of the root-bark was higher.
However values of moisture Loss on drying (LOD) were
higher for the fruit than the root-bark. The total ash
content mainly is a measure of the presence of inorganic
compounds. A larger value indicates that the plant
material contains more of inorganic compounds.
Concentrated acid, when added to ash, reacts with the
calcium oxalate crystals. If the plant material contains a
large number of calcium oxalate crystals, the amount of
substance remaining after acid treatment will be quite
less. Thus, lower value of Acid insoluble ash content
indicates the presence of large number of calcium
oxalate crystals in the plant material and vice versa. In
the present study a lower acid insoluble ash content was
recorded for the fruit than the root-bark indicating a
higher content of calcium oxalate crystals in the fruit.
This high calcium oxalate crystals in the fruit may
account for defense property of the plant. Water soluble
ash content gives the crude estimate of the water soluble
extractable matter present in the ash. The relatively high
total ash values observed in the present study indicate a
high content of physiological ash. The organic
contaminants were negligible.
Table 1: Proximate parameters of Root-bark and fruit of Morinda
tinctoria
Parameters Root-Bark Fruit
Total Ash (%) 9.333 + 0.288* 7.166 + 0.288*
Water soluble Ash (%) 4.666 + 0.577* 0.833 + 0.288*
Acid insoluble Ash (%) 6.033 + 0.057* 0.510 + 0.01*
Loss on drying (%) 4.5 + 2.645* 14 + 2.179*
*Mean + standard deviation
 Research Journal of Pharmacognosy and Phytochemistry. 10(3): July- September, 2018

The moisture content of the stem bark powder of 11. African Pharmacopoeia. Vol. 2. 1st ed. OAU/ STRC Publications.
1986;128-144.
Triplochiton scleroxylon (0.68%) which fell below the
12. British Pharmacopoeia, BP. Vol.II. Her Majesty’s Stationary
pharmacopoeia limits of water content for vegetable Office, London. 1988; 1022.
drugs, which is between 8 to 14%19. Excessive water in 13. Helga Lindorf. Bark and Wood anatomy of Uncaria guianensis and
vegetable drugs, greater than the set limit will promote Uncaria tomentosa (“Cat’s claw”) IAWA Journal, 2005; 26 (2):
239–251.
the growth of microbes and fungi. This would also lead
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deterioration of the drug. Moisture content value members of the Pinaceae. Tree Physiology, 2003; 23: 361–371.
obtained in the present study was indicative that the 15. McNair JB. The interrelation between substances in plants:
essential oils and resins, cyanogen and oxalate. American Journal
material could be preserved over a long period of time
of Botany, 1932; 19: 225-271.
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bark (9.333%) and fruit (7.166%) of M.tinctoria, which VR. The role of druse and raphide calcium oxalate crystals in
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components and a rather low inorganic or mineral
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constituent. and function. Annual Review of Plant Biology, 2005; 56: 41–71.
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CONCLUSION: plants. Plant Cell, 1999; 11: 751-761.
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The anatomical characteristics of root-bark and fruit knowledge of chemical composition of stem bark of Triplochiton
were studied by the microscopic sectioning techniques. scleroxylon K. Schum. In traditional treatment of diabetes mellitus
The root-bark of M. tinctoria consists of rhytidome, in Nigeria. African Journal of Plant Science, 2011; 5(10): 552-556.
which includes lignified phelloid cells and phellem cells.
Microscopic examination of the root-bark indicated the
presence of calcium oxalate crystals of raphide bundles
in the axial parenchyma. The fruit was typical of any
other member of the family Rubiaceae, being a drupe
with membranous epicarp, fleshy mesocarp and thin
endocarp. Calcium oxalate crystals were also present in
the fruit, either as a 4-lobed druse type or as a spindle
shaped Raphide type. Presence of raphides showed the
defense mechanism against plant predators. Proximate
analysis of M. tinctoria in terms of the total ash content,
water soluble and acid insoluble ash and moisture loss of
drying was carried out to evaluate the plant as a potential
source of phytochemical. The moisture content value
obtained in the present study was indicative that the
material could be preserved over a long period of time
without deterioration.

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