international journal of pharma and bio sciences issn 0975 … · · 2016-01-16p+ phloroglucinol:...

Int J Pharm Bio Sci 2015 April; 6(2): (P) 47 - 54

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Research Article Pharmacognosy

International Journal of Pharma and Bio Sciences ISSN

0975-6299

PHARMACOGNOSTIC AND PRELIMINARY PHYTOCHEMICAL STUDIES OF

COLD AND HOT EXTRACTS OF STEM OF TINOSPORA MALABARICA

MIERS. - AN IMPORTANT MEDICINAL PLANT

SACHET HEDGE*1, JAYARAJ. M2 AND A. V. BHANDARKAR3

1 & 2- Department of Botany, Karnatak University, Dharwad – 580003. Mobile No : 96110 05644

3- Goa College of Pharmacy, Panjim, Goa – 403001.

ABSTRACT

Plant medicine is the oldest system of medicine in India known as Ayurveda which is gaining greater importance in recent years. In this system crude drugs are used, which may be adulterated by similar kind of plants which may be harmful. To check this adulteration pharmacognostic analysis is more reliable compared to other methods. For identification and authentication of crude drugs pharmacognosy is the best and the easy method. In the present study pharmacognosy of stem of Tinospora malabarica Miers. a climber belongs to the family Menispermaceae has been studied. It is used traditionally by Indians to treat diabetes, neurological disorders jaundice and used in many Ayurvedic preparations are similar to Tinospora cordifolia (Willd.) Miers. In this study organoleptic, macroscopic, microscopic, fluorescence analysis, ash values and extractive values have been carried out. This study also includes preliminary phytochemical analysis of successive hot and cold extracts. The powder microscopic study showed the presence of oval or reiniform starch grains, pitted and spiral vessels and fibers. The preliminary phytochemical analysis showed steroids, glycosides, proteins, amino acids, carbohydrates, mucilage and oxalic acids in both the extracts and gum is present only in cold extract.

KEY WORDS: Hot and cold extract, Menispermaceae, Pharmacognosy, Phytochemical analysis, Tinospora malabarica Miers

*Corresponding author

SACHET HEDGE

Department of Botany, Karnatak University, Dharwad – 580003.

Mobile No : 96110 05644



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INTRODUCTION Tinospora malabarica Miers. belongs to the family Menispermaceae which is native to India1. It is commonly known as Gurch or Giloe in Hindi, Kandamrata and Padma guduchi in Sanskrit, Madhuparne and Sudarshana balli in Kannada2. It is a large climber and its young parts clothed with whitish hairs; stem is 1.2 to 1.3 cm in diameter, it is smooth and shining with light colored papery bark, more or less warty. Leaves are membranous; it measures upto 8.8 to 12.7 cm. in length. The leaves are 7 nerved, broadly ovate, cordate, acuminate, pubescent above, whitish tomentose beneath; petioles reaching upto 12- 13 cm. long, thickened and twisted at the base. Flowers are green, inflorescence are in racemes, 7.5 – 15.3 cm. long; pedicels slender and clustered. Perianth is 6 in number, the outer whorl is small, ovate to oblong, obtuse at the tip, the inner whorl is large, oblong or sub-orbicular, concave. Perianth in the male flowers is obovate, cuneate, rounded at the apex, not embracing the stamens. Fruits are Drupes, found 1-3 (usually 2) on thick stalks and it is ovoid in shape, smooth, red in color; endocarp marked externally with many sharp- pointed tubercles3,4. The mature stem has tonic and stomachic properties which is used medicinally in the same way as Tinospora cordifolia(Willd.) Miers.5 and it is used in Ayurveda to treat fever, jaundice, burning sensation, diabetes, piles, skin ailments, respiratory disorders, neurological diseases and for improving intellect, it is a constituent of many Ayurvedic preparations for the treatment of general debility, dyspepsia, fever, leucorrhea and urinary diseases2,6,7. This plant extract reported to show anti- inflammatory activity8, anti- leishmenial activity9, anti ulcer activity10 against different animal models. There are few reports on Pharmacognosy and preliminary phytochemistry on the stem of T. malabarica Miers. and lack of comparative preliminary phytochemical analysis of hot and cold extracts of stem. The stem of T.malabarica Miers. has been used by the folk healers instead for T.cordifolia (Willd.) Miers. for

treating several disorders and in many Ayurvedic preparations. Therefore this work on pharmacognosy and preliminary phytochemistry on the stem of T. malabarica Miers. has been carried out as the stem is known for great medicinal properties as mentioned above and similar to T. cordifolia in the traditional system of Indian medicine5.

MATERIALS AND METHODS Collection and authentication T. malabarica Miers. was collected from the Botanical Garden at the Karnatak University, Dharwad campus during the month of August, 2013. The plant was identified by one of the authors and a voucher specimen was kept in the department herbaria for the future references. Pharmacognostic studies The collected plant material was shade dried for about 8-10 weeks in the laboratory until the moisture in the material gets evaporated. The dried material was coarsely powdered mechanically with the help of a grinder, passed through 20 mesh sieve and stored in an air tight container for further use. Fresh plant material was used to study the anatomical features. Microscopical characters, physicochemical parameters and phytochemical analysis were carried out using the powder and for microscopical studies, fresh transverse sections of the stem were prepared and stained according to the standard methods11,12,13,44. Organoleptic analysis Various sensory parameters such as color, odor, taste, size, shape, texture were studied by the organoleptic evaluation. Microscopic analysis Microscopic characters were studied by taking hand sections from the fresh plant material of stem and stained according to the standard procedures11,12,13,44.



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Physicochemical analysis Physicochemical parameters such as percentage of total ash value, acid insoluble ash, water soluble ash, extractive values and total percentage of fibers were investigated11,12. Fluorescence analysis Stem powder was subjected to fluorescence behavior and tested after 24 hours, treating with freshly prepared chemical reagents which were exposed to visible light and UV light (short and long wavelength)14,15. Extraction and phytochemical analysis Powdered material was subjected to two types of extraction procedures such as hot extraction or Soxhlet extraction and cold extraction to study the differences. For Soxhlet extraction 15gm of powdered plant material were taken and extracted for 18 hours successively with different solvents like Petrolium ether,

Chloroform, Acetone, Ethanol and Water. In case of cold extraction 25gm of powdered plant material was taken with 300ml of different solvents (Petrolium ether, Chloroform, Acetone, Ethanol and Water) and each one was extracted successively for 48-72 hours. Preliminary phytochemical analysis was carried out according to the standard procedure11,12.

RESULTS Macroscopic characters T.malabarica Miers. has a large climbing stem with thin yellowish gray papery bark and is warty, measures 1.2 to 1.3 cm. in diameter with watery latex. The stem gives rise to a thread like white aerial roots and prominent scattered lenticels are found on an old stem while the young stem is green, pubescent and having no papery bark and lenticels (Figure- 1: Habit).

Figure 1

Wild plant in Habit.

Organoleptic analysis Organoleptic analysis is one of the parameters of identifying crude drug macroscopically by its color, odor, taste and texture as mentioned below (Table- 1).

Table 1 Organoleptic analysis

Color Yellow

Odor Pungent

Taste Bitter

Texture Pubescent and warty



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Microscopic characters T. S of stem The transverse section of the stem of T. malabarica is circular in outline, showing 1-2 layer of epidermis which is covered with thin papery bark and multicellular uniseriate trichomes. In an old stem there is 2-3 layers of rectangular dead cells are called as papery bark. In young stem the single layered epidermis is covered by multicellular uniseriate trichomes. Cortex is 12-13 layered and consists of paranchymatous cells and presence of starch grains throughout. The endodermis and

the pericyclic cells are tangentially elongated 6-8 rows of sclerenchymatous cells; in old stem it continues to form a ring around the vascular bundles and pith. Stele consists of collateral open type of vascular bundles. The vascular tissues are continuously adding to the growth of the plant, consisting of phloem and xylem separated by parenchyma and medullary ray (Figure 2, A). In between xylem and phloem there exists thin band of cambium. Metaxylem is present towards the pith (Figure 2, B). Pith is at the centre and consists of paranchymatous cells.

Figure 2

T. S of stem of T. malabarica Miers.

Figure 3 microscopic character of the powdered stem material of T. malabarica Miers.

Fig. 2: A- Transverse section of stem, Fig. 2: B- enlarged view of vascular bundle. Fig. 3: A- oval or reiniform starch grains, Fig. 3: B- pitted vessels, Fig. 3: C- crystal fibers, Fig. 3: D- parenchyma cells, Fig. 3: E & G- fibers, Fig. 3: F- spiral vessel.

Powder microscopy Powdered crude drug consists of multicellular uniseriate trichomes (Fig. 3, G), oval or reiniform starch grains (Fig. 3, A), pitted vessels (Fig. 3, B), annular and spiral vessels (Fig. 3, F) and different types of fibers (Fig. 3, E & G).



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Physicochemical analysis Moisture content, ash values, extractive values and determination of crude fibers by Dutch method are as mentioned below (Table- 2).

Table 2 Physicochemical analysis

* Average of three readings. Standard Deviation. Calculated based on dry weight of the sample.

Fluorescence analysis Powdered material was tested with different chemical reagents and its fluorescence behavior with different wavelength of light was mentioned below (Table- 3).

Table 3 Fluorescence analysis

Material with chemical Day light UV light (short) UV light (long)

Powder as such (P) Yellowish gray - -

P+ Phloroglucinol: HCl Brown Dark green Brown

P+ methanol Light yellow Yellowish green Light Ceylon red

P+ ethanol Light yellow Yellowish green Light Ceylon red

P+ Pet. Ether Yellow Yellowish green Ceylon red

P+ Chloroform Yellow Green Light Ceylon red

P+ Acetone Light yellow Dark green Light Ceylon red

P+ 50% H2SO4 Dark brown Black Brownish black

P+ 50% HNO3 Dark yellow Light green Dark yellow

P+ 50% HCl Tan Black Black

P+ 10% NaOH Yellowis orange Green Green

P+ Ammonia Light yellow Green Light green

P+ glacial Acetic acid Creamish yellow Green Light green

Preliminary Phytochemical analysis

Table 4 Preliminary Phytochemical analysis of Cold and Hot extract of stem

Tests Extacts

Pet. Ether Chloroform Acetone Ethanol Water

Cold Hot Cold Hot Cold Hot Cold Hot Cold Hot

Carbohydrates Molish’s test - - - - + + + + + +

Reducing sugar Fehling’s test - - - + + + + + + +

Benedict’s test - - + + + + + + + +

Monosaccharides - - + + + + + + + +

Hexose sugars Tollen’sPhloroglucinol test + + + + + + + + + +

Non-reducing sugars Iodine test - - - - - - - - - -

Gums Fehling’s test - - + - - - - - - -

Benedict’s test - - - - - - - - - -

Mucilage + + + + + + + + + +

Parameters Values (w/w %)

Moisture content 8.0± 0.15

Ash values* Total ash 6.27 ± 0.12

Sulphated ash 11.13 ± 0.24

Extractive values*

Ether soluble 7.44 ± 0.12

Alcohol soluble 15.76 ± 0.34

Water soluble 21.6 ± 0.41

Crude fiber (Dutch method) 49.5± 0.26



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Proteins Biuret test - + - + + + + + + -

Millon’s test - - - - - - - - - - Proteins containing sulfur Precipitation test - - - - - - - - - -

Amino acids Ninhydrin test - - - - - + + + + +

Test for tyrosine - - - - - - - - - -

Steroids Salkowski test - - + + + + - + - +

Glycosides Deoxy sugars - - + - + + + + - + Anthraquinone glycosides Borntrager’s test - - - - - - - - - -

Modified Borntrager’s test - - - - - - - - - -

Saponins Foam test + - - - + - - - + +

Coumarine glycosides Extract + NaOH

- + + + - + - - - -

Flavonoids Shinodow’s test - - - - - - - - - -

Alkaloids - - - - - - - - - -

Tannins and phenols 5% FeCl3 - - - - - - - - - -

Gelatin solution - - - - + - - - - -

Acetic acid solution - - - - - - - - - - Potassium di-chromate solution - - - - - - - - - -

Dil. Iodine solution + + + - - - - - + +

Dil. HNO3 - - - - - - - - - +

Organic acids Oxalic acid - - - - + + - - - -

Tartaric acid - - - - - - - - - - Citric acid - - - - - - - - - -

Malic acid - - - - - - - - - -

Confirmatory test Oxalic acid - - - - + + - - - -

DISCUSSION To measure the quality, purity and authenticity of the crude drug, standardization is essential. One of the simplest and cheapest methods to establish the correct identification of the medicinal plant is microscopic method. It is necessary to standardize the crude drug of any medicinal plant according to the parameters of WHO standards. Macroscopic and microscopic measures, total ash values, extractive values and fluorescence analysis are unique for each plant, hence it is a measure to identify and authenticate the crude drug. The preliminary phytochemical analysis reveals the important chemical constituents of the plant16. Present study has been undertaken to differentiate and to standardize T. malabarica Miers. with that of T. cordifolia (Willd.) Miers. In T. malabarica Miers. stem is pubescent at the young stage and warty, papery brown bark at the older stage with prominent scattered lenticels while in T. cordifolia (Willd.) Miers. stem is a warty, glabrous with round lenticels, corky grooved bark with varying in thickness and light brown in color. Young stem is green with smooth surfaces. Comparative anatomical characters showed that, T. cordifolia (Willd.) Miers. stem have no trichomes, odorless, bitter in taste; cortex is made of 2-3 layers of

chollenchyma cells followed by parenchyma cells. Narrow pericycle made from persistent sclerenchymatous cells where as T. malabarica Miers. has 6-8 layers of tangential sclerenchyma cells which continuously forming rings with growth. In both the species collateral open type of vascular bundles can be seen and in T. cordifolia (Willd.) Miers. these vascular bundles are restricted but in T. malabarica Miers. it is forming continuous rings with growth. Primary xylem is absent in T. cordifolia (Willd.) Miers.17,18. Comparative phytochemical study of T. cordifolia (Willd.) Miers. with T. malabarica Miers., which is having alkaloids, terpinoids, flavonoids, fixed oils and fats in additions to phenols, steroids, saponins, glycosides and carbohydrates as in T. malabarica Miers19,20.

CONCLUSION This study on the pharmacognostic and preliminary phytochemical evaluation of the stem of T. malabarica Miers. providing the useful information to authenticate and to substantiate the genuine crude drug along with its phytoconstituent nature. This study helps



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presenting monographs to identify this medicinally important plant and to know adulterants in the drug. Though these two species fall under the same genus and family there are some anatomical and phytochemicals differences showed by the study, taking it as a basic step towards the experimental study to prove that, T. malabarica Miers. Can be used in place of T. cordifolia (Willd.) Miers. or vice versa. The elaborated comparative phytochemical study and the effect of different phytochemicals require further more investigations in this field.

ACKNOWLEDGEMENT Authors acknowledge the UGC for providing UPE fellowship for one of the author and Chairman, P. G. Department of Botany, Karnatak University, Dharwad, for extending facilities. CONFLICTS OF INTEREST We declare that we have no conflict of interest.

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international journal of pharma and bio sciences issn 0975 … · · 2016-01-16p+ phloroglucinol:...

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