Praveena. A1*, Sanjayan K. P2
1Department of Biotechnology, Prathyusha Engineering College, Thiruvallur-602025, Tamilnadu, India.
2Department of Zoology, Gurunanak College, Velachery, Chennai-600 042, Tamilnadu, India.
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.
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.
The Rubiaceae is one of the largest and most diverse 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 8. In the present study we have initiated the anatomical and proximate characteristics of root-bark and fruit of M. tinctoria. The pharmacognostical studies will be useful for the investigators to identify the plant in future.
MATERIAL AND METHODS:
Collection of plant specimens:
The plant specimens (Root bark and fruit of Morinda tinctoria) were collected from Kadambathur, Thiruvallur district, Tamilnadu, India. Care was taken to select healthy and normal organs. The fruit and the bark of the root were cut and removed from the plant and fixed in FAA (Formalin-5ml and Acetic acid-5ml+70% ethyl alcohol-90ml). After 24hrs of fixing, the specimens were dehydrated with graded series of tertiary-Butyl alcohol (TBA) as per the schedule given by Sass, 1940. Infiltration of the specimens was carried by gradual addition of paraffin wax (Melting point 58-60°C) until TBA solution attained super saturation. The specimens were cast into paraffin blocks.
The paraffin embedded specimens were sectioned with the help of Rotary Microtome. The thickness of the sections was 10-12 µm. dewaxing of the sections was by customary procedure9. The sections were stained with Toluidine blue as per the method published by O’Brien et al.,10 since Toluidine blue is a polychromatic stain. The staining results were remarkably good; and some cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc. Wherever necessary sections were also stained with safranin and fast-green.
Microscopic descriptions of tissues are supplemented with micrographs whenever necessary. Photographs of different magnifications were taken with Nikon lab photos 2 microscopic Unit. For normal observations bright field was used. For the study of crystals, starch grains and lignified cells, polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against dark background. Magnifications of the figures are indicated by the scale-bars.
The following parameters were quantified using standard methods11-12.
Moisture content/water loss on drying:
The powdered material of root-bark and fruit of M. tinctoria (2.0 g) was weighed into a clean crucible of known weight. After oven drying at 115°C for 5 hrs, the crucible was cooled and weighed to determine weight loss in the powdered material. The average percentage weight loss, with reference to the air dried powdered sample was determined for three replicates.
Total ash determination:
The crucibles were washed thoroughly, dried in hot oven at 100°C, cooled in desiccator and weighed. A 2.0 g portion of each of the sample was weighed into the crucible and put in the furnace. Heating was started gradually until temperature of 600°C was reached. This temperature was maintained for 6 h. The crucible was then put inside a desiccator and cooled. After cooling the sample was reweighed and the percentage ash calculated.
% Ash = ------ x 100
W = weight of the crucible and ash;
Z = weight of empty crucible;
N = weight of the sample.
Water soluble ash value determination:
The crucible with the total ash was transferred into a beaker containing 25 ml of distilled water. The beaker and its contents were boiled for 5 min and filtered through an ashless filter paper (Whatman). The filter paper containing the residue was folded and placed in a weighed crucible. The crucible was then heated in the muffle furnace, until the filter paper was completely ashed. The crucible and its contents were cooled and weighed and the final weight noted. The weight of the residue was then calculated by subtracting the constant weight of the second crucible and its ash. This is the water insoluble ash. The weight of the water soluble ash was obtained by subtracting the weight of the water insoluble ash from the total ash. The weight of the water soluble ash divided by the initial weight of the crude sample was multiplied by 100 and was taken as the water soluble ash value.
Acid insoluble ash value determination:
The water insoluble ash obtained from the above experiment was transferred into a beaker containing 25 ml of diluted HCl. The beaker and its contents were boiled for 5 min and the boiled contents filtered through an ashless filter paper (Whatmann). The washings were then passed through the filter paper in a manner as to allow the collection of the residue at the tip of the cone of the filter paper. The weight of the clean and heated porcelain crucible was accurately determined. The filter paper with the residue was folded with a small cone and transferred into the crucible. The crucible was gently heated until the filter paper was completely ashed, and then heated strongly for a few minutes. The crucible and its contents were cooled, weighed and the final weight was noted. The weight of the residue (ash) was then 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 elements which form dark, irregular tangential lives, isolated clusters of brachysclereids and slightly diluted phloem rays. Calcium oxalate crystals in the form of thick bundles consisting of thin pointed needles are abundant in the collapsed phloem.
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).
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 cells13.
Microscopic features of fruit:
The fruit is a drupe with membranous epicarp, fleshy mesocarp and thin endocarp. The fruit of M.tinctoria is 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 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)
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 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
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
The moisture content of the stem bark powder of Triplochiton scleroxylon (0.68%) which fell below the pharmacopoeia limits of water content for vegetable drugs, which is between 8 to 14%19. Excessive water in vegetable drugs, greater than the set limit will promote the growth of microbes and fungi. This would also lead to hydrolysis of constituents resulting ultimately to deterioration of the drug. Moisture content value obtained in the present study was indicative that the material could be preserved over a long period of time without deterioration of the drug. Total ash value of root-bark (9.333%) and fruit (7.166%) of M.tinctoria, which is low, implies that the plant has good or high organic components and a rather low inorganic or mineral constituent.
The anatomical characteristics of root-bark and fruit were studied by the microscopic sectioning techniques. The root-bark of M. tinctoria consists of rhytidome, 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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Received on 29.11.2017 Modified on 02.01.2018
Accepted on 25.02.2018 ©A&V Publications All right reserved