Pharmacognostic Study and Establishment of Quality Parameters of ...

Journal of Medicinal Plants Studies Year : 2013, Volume : 1, Issue : 1 First page : (38) Last page : (46) ISSN: 2320-3862 Online Available at www.plantsjournal.com

Journal of Medicinal Plants Studies Pharmacognostic Study and Establishment of Quality Parameters of Leaves, Root and Bark of Ficus retusa. Linn Shivani Chauhan*1, Lubhan Singh1, Ashish Kr Sharma2 1. 2.

Department of Pharmaceutical Technology, M.I.E.T., Meerut, U.P., India- 250005 [E-mail: [email protected]] Adarsh Vijendra Institute of pharmaceutical Sciences, Gangoh, Saharanpur, U.P., India- 247341

Ficus retusa. Linn (Family- Moraceae) is found commonly in India. Ficus species are rich source of polyphenolic compounds, flavonoids which are responsible for strong antioxidant properties that help in prevention & therapy of various oxidative stress related diseases. In present investigation, the detailed comparative pharmacognostic study of Ficus retusa (leaf, root and bark) was carried out to lay down the standards which could be useful in future experimental studies. However, no conclusive pharmacognostic study of its leaves, bark and roots has been performed yet. The study includes macroscopy, microscopy, preliminary phytochemical screening, physicochemical evaluation, fluorescence analysis and chromatographic evaluation. Highest extractive values were found with water and ethanol. The fluorescence analysis under visible light and under UV light by treatment with different chemical reagents showed different colour changes. The presences of alkaloids, steroids, flavonoids, saponins were confirmed during preliminary phytochemical screening. Conclusion: Such a study would serve as a useful gauge in standardization of the plant material, isolation of medicinally important phytoconstituents, performing pharmacological investigations and ensuring quality formulations Keyword: : Ficus retusa, Pharmacognostic, Phytochemical, Fluorescence Analysis.

1. Introduction The Ficus retusa, also known as Chinese banyan, plaksha, pakur and kamarup plant belongs to Moraceae family mainly distributed throughout Western Peninsula and also found in chhota Nagpur, Bihar, Central india, Andamans, Sundribuns, Malaya islands and Australia. It is a huge tropical, decidous, evergreen tree with more than 800 species. It has an evergreen rosette with oval, dark green, shiny leaves, & a chunky trunk with exposed aerial roots (Ajasa A M, 2004 and Arya V, 1997). Leaves are ellipitic, ovate or obovate, 8 to 14 cm long with tapering points, narrowing into a petiole 6 to 12 mm long. The barks are grey to dark brown, fruity in young in odor, taste in bitter, cylindrical shape, Vol. 1 No. 1 2013

and rough irregularly cracked bark in surface characteristics (Kollipillai B et al, 2010 and Jaya R et al, 2011). It mainly contains sterols, terpenoids, glycoside, flavonoids, polyphenols, proteins and carbohydrates. Leaves and bark used as poultice for rheumatism. Juice of leaves used for abdominal colic and flatulence. Bark juice used for liver diseases. In china, rootlets are used for toothaches (Ravichandra V D et al, 2011 and shrisha N et al, 2011) 2. Materials and Method 2.1 Plant Material The plant material (leaves, roots and barks) were collected from (Gangoh) distt. Saharanpur U.P. India. The plant was identified and authenticated

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Journal of Medicinal Plants Studies

by Dr. Anjula Pandey, Principal Scientist, National Bureau of Plant Genetic Resources (Indian council of Agricultural Research), New Delhi. Leaves, Roots and Barks were then washed to remove adhering material, shade dried and powdered (#60) using a blender. The powders were stored in an airtight container. An exhaustive pharmacognosy was carried out using standard methodology. 2.2 Pharmacognostical Studies 2.2.1 Macroscopy Morphological studies of leaf, root and bark such as color, size, odor, taste, surface characteristic and fracture were examined using the terms and outlined given in (Evans, 1989). 2.2.2 Microscopy Free hand transverse sections of fresh leaf, root and bark were taken, cleaned in chloral hydrate solution with gentle warming. A drop of concentrated hydrochloric acid and phloroglucinol were used to detect the lignified cells in the cross sections and in the powder drugs. They were mounted on slide in glycerin and studied under microscope according to the standard method given in the textbook of pharmacognosy by T E.Wallis 1967. Photomicrographic images were taken by using Trino CXR camera. 2.2.3 Chemo-Microscopical Examinations The transverse sections were treated with various chemical reagents to detect the presence or absence of chemical constituents such as starch grains, lignin, mucilage and calcium oxalate crystal. The fluorescence behaviors of the powdered drugs in different chemical solutions towards the ordinary and ultra violate light was also carried out as per method described by Lal, V.K.1980. 2.2.4 Quantitative Evaluation of the Powdered Leaves, Bark and Roots The moisture content of the powdered material was determined by the loss on drying method.

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The ash value and the acid insoluble ash value were determined using methods described in the British Pharmacopoeia (British Pharmacopoeia, 1980). The extractive (water and alcohol) values were determined using methods described in Brain and Turner, 1975. Average of three determinations for each procedure was calculated. The preliminary phytochemical screenings of Ficus retusa leaves bark and roots extracts were carried out according to Harbone J.B, 1976. 2.2.5 Fluorescence Characteristics Of Powdered Drug Under Ultra-Violet Light Powdered drug were screened for fluorescence characteristic with and without chemical treatment. The observations pertaining to their color in day light and under ultra-violet light (short and long) were noticed (Kokashi V J et al, 1958). 2.3 Chromatographic Evaluation The chromatographic studies were performed using various solvent systems to confirm the phytochemical studies. Prepared silica gel TLC plates were used for the chromatographic evaluation. Finally Rf values were calculated and shown in table 6, 7 and 8. (Mukherjee P K, 2008, Stahl E, 1985, Wagner and Bladt 1996 and Wallis TE 1984). 3. Result 3.1 Macroscopic And Sensory Characters Leaves: The leaves are pale green, fruity in young in odor, taste in bitter, oblong shape, 8.0 to 14.2 cm in length, 4 to 5 cm in width in size, glabrous in surface characteristics. Root: The root is light brown, fruity odor, taste in bitter, cylindrical shape, and rough irregularly cracked in surface characteristics. Bark: The barks are grey to dark brown, fruity in odor, taste in bitter, cylindrical shape, and rough irregularly cracked bark in surface characteristics as shown in table1.


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Table1: Morphology of Leaves, Root and Bark of Ficus retusa Characters

Description (Leaves)

Description (Root)

Description (Bark)

Color

Pale Green

Light brown

Grey to dark brown

Odor

Fruity in young leaves

Fruity

Fruity

Taste

Bitter

Bitter

Bitter

Size

8.0 to 14.2 in length, 4 to5 in width

-

-

(mature leaves)

Shape

Oblong

Cylindrical

Cylindrical

Surface

Glabrous, shiny appearance

Rough irregularly cracked

Rough irregularly cracked

roots

bark

characteristics

Table 2: Physicochemical Parameters of Ficus Retusa.Linn (Mean)n %w/w Leaf)

(Mean)n %w/w (Root)

(Mean)n %w/w (Bark)

Total ash value

10.25

4.75

6.25

Acid insoluble ash

3.0

2.0

2.5

Water soluble ash

6.5

4.0

4.5

Loss on Drying

8.9

9.2

9.7

Parameter

* Where n=3 on dry weight basis

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Table3. Extractive values of Ficus retusa. Linn (Mean)n %w/w (Leaves)

(Mean)n %w/w (Root)

(Mean)n %w/w (Bark)

Water soluble extractive values

8.8

4.8

3.2

Alcohol soluble extractive values

13.6

1.6

6.4

Parameter

* Where n=3 on dry weight basis

3.2 Phytochemical Investigation: Preliminary phytochemical screenings were performed to find out the phytoconstituent present in the leaves, roots and barks of Ficus retusa. Linn (Table 4) Table4. Preliminary phytochemical screening of leaves, root and bark of Ficus retusa.Linn Leaves

Root

Bark

Alkaloids

+

-

+

Glycoside

-

-

-

Tannin

+

-

+

Saponin

-

-

-

Steroids

-

-

-

Flavonoids

+

+

+

Carbohydrates

+

+

-

Amino acids

-

-

-

Terpenoids

+

+

-

Description

(+) = Present;

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(-) = Absent;

3.3 Microscopic Characters 3.3.1 Leaves: The transverse section of leaf showed palisade, epidermis, spongy mesophyll, phloem, xylem and abaxial surface as shown in Fig 1a, 1b and1c. 3.3.2 Root: The transverse section of root shows, vascular bundles, medullary rays, calcium oxalate crystal, cortex region and pith as shown in Fig 2a, 2b, 2c and 2d and powdered sample of root of Ficus retusa showed Trichomes, Starch grain, Lignified cell, fibres and oil globules in Fig 3a. 3.3.3 Bark: The transverse section of barks show fibers showing thick cells of the parenchymatous cells, cork cells, cortex, pigment, vascular bundles and starch grain as shown in Fig 4a

3.4 Physicochemical studies The moisture content was 8.9% (leaves), 9.2% (root) and 9.7% (bark) which were not so high as to facilitate bacterial growth. The other physicochemical parameters which ascertain the quality, purity and also help in evaluating the crude drug, and the total ash value, acid insoluble ash value and water soluble ash value which were determined to be not more than 10.25 %w/w, 3.0 %w/w, 6.5 %w/w (leaves), 4.75 %w/w, 2.0 %w/w, 4.0 %w/w (root), and 6.25 %w/w, 2.5 %w/w, 4.5% w/w (bark) respectively which indicated the presence of the total foreign inorganic matter. While study of extractive values can serve as a valuable source of information and provide suitable standards to determine the quality of plant material in future investigations or application. (Table 2 & 3) 3.5 Fluorescence Analysis: The results are summarized in Table 5.


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Table 5: Fluorescence Analysis of Powdered Ficus Retusa. Linn Leaves Roots and Bark. Leaves

Root

Bark

Sr No

Powdered crude drug + reagent

Day Light

UV (Short) 254nm

UV (Long)366nm

Day Light

UV (Short)254 nm

UV (Long) 366 nm

Day Light

UV (Short) 254 nm

UV (Long) 366 nm

1

Powder Crude Drug as such

Green

Dark green

Dark green

Yellowish green

Dark green

Light green

Light yellow green

Dark green

Light yellow green

2

Drug + 1M NaOH

Dark green

Dark green

Blackish green

Dark brown

Blackish green

Dark green

Dark brown

Blackish green

Dark green

3

Drug + 1M NaOH +Methanol

Dark green

Blackish green

Dark green

Yellowish brown

Blackish green

Yellowish green

Dark brown

Blackish green

Dark brown

4

Drug + 1M NaOH + Water

Green

Dark green

Green

Yellowish brown

Blackish green

Yellowish green

Dark brown

Blackish green

Yellowish brown

5

Drug + 1M HCl

Yellowish brown

Dark brown

Yellowish brown

Yellowish green

Dark green

Dark green

Light yellow green

Dark green

Yellowish green

6

Drug + dil. HNO3

Yellowish brown

Light brown

Yellowish brown

Yellowish green

Blackish green

Dark green

Light yellow green

Dark green

Dark yellowish green

7

Drug + 5% Iodine

Blackish green

Blackish green

Dark green

Dark green

Blackish green

Blackish green

Dark green

Blackish green

Blackish green

8

Drug + 5% FeCl3

Blackish green

Blackish green

Dark green

Blackish green

Blackish green

Blackish green

Dark green

Blackish green

Blackish green

9

Drug + dil. Ammonia

Green

Dark green

Green

Yellowish green

Dark green

Green

Yellowish green

Dark green

Light green

10

Drug + Methanol

Pale green

Pale green

Yellowish green

Light green

Dark green

Light yellowish green

Light green

Dark green


11

Drug + dilute HCl

Brownish green

Brownish green

Dark green

Light brown

Dark green

Yellowish green

Light yellow green

Blackish green

Dark green

12

Drug + 1M H2SO4

Brownish green

Brownish green

Brownish green

Brownish green

Blackish green

Dark green

Yellowish green

Dark green

Yellowish green

13

Drug + Conc. HNO3

Reddish brown

Brown

Brown

Reddish brown

Blackish brown

Blackish brown

Reddish brown

Dark brown

Blackish brown

14

Drug + K2Cr2O7

Dark green

Blackish brown

Dark green

Dark brown

Dark green

Blackish brown

Dark brown

Blackish brown

Blackish brown

15

Drug + Ethanol

Green

Green

Yellowish green

Yellowish green

Yellowish green

Green

Yellowish green

Dark green


16

Drug + Toluene

Light green

Dark green

Yellowish green

Light green

Dark green


Light green

Dark green


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3.6 Chromatographic Evaluation: Thin layer chromatography was performed using various solvent systems to confirm the phytochemical studies. The Rf values were calculated for the optimum solvent system. 

TLC analysis of leaves: The observations are shown in Table 6. Table 6: TLC analysis of methanol extract of leaves Ratio

No. of spots

8:2

9

Ethyl acetate : Benzene

9 : 11

Flavonids

Chloroform : Acetone : Formic acid

Alkaloids Triterpenoids

Category

Solvent system

General

Hexane : Ethyl acetate

Tannins



Spraying reagent

Rf

Iodine Chamber/U.V Chamber

0.21,0.23, 0.26, 0.25, 0.28, 0.41, 0.45, 0.8, 0.85

3

Vanillin-Sulphuric Acid Reagent

0.72, 0.69, 0.96

7.5 : 16.5 : 13.5

5

Iodine Chamber

0.37, 0.39, 0.43, 0.54, 0.87

Chloroform : Diethyl amine

9:1

5

Dragendroff’s reagent

0.50, 0.64, 0.79, 0.92, 0.90

Chloroform: Methanol

10:1

4

Iodine Chamber

0.16,0.70,0.32, 0.61,

TLC analysis of root: The observations are shown in Table 7. Table7. TLC analysis of methanol extract of root

Category

Solvent system

Ratio

No. of spots

Spraying reagent

Rf

Tannins


9 : 11

4


0.21, 0.22, 0.55, 0.51

Triterpenoids

Chloroform : Methanol

10 : 1

2


0.73, 0.78

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

TLC analysis of bark: The observations are shown in Table 8. Table8. TLC analysis of methanol extract of bark. Ratio

No. of spots

8:2

6

Category

Solvent system

General

Hexane : Ethyl acetate

Tannins


9 : 11

3


0.78, 0.79, 0.74

Triterpenoids

Chloroform: Methanol

10:1

3

Iodine Chamber

0.81, 0.75, 0.83

4. Discussion The leaves of Ficus retusa was pale green in color, with fruity odour and Bitter taste. The size was 8.0 to 14.2 cm in length and 4 to 5 cm in width. The shape of leaves is oblong with glabrous, shiny surface characteristics.The bark of Ficus retusa was dark brown in color, with fruity odour and bitter taste. The shape was cylindrical with rough irregularly cracked surface. The root of Ficus retusa was light brown in color with fruity odour and bitter taste. The leaves, roots & bark of Ficus retusa were subjected to microscopical studies using transverse section and powder microscopy. The transverse section of bark of Ficus retusa showed cork cells, cork cambium, medullary rays, starch grain and oil globules. Medullary rays were straight. The transverse section of root of Ficus retusa showed cork cells, vascular bundles, medullary rays, calcium oxalate crystals and oil globules. The transverse section of leaves of Ficus retusa showed fibres, starch grain, oil globules. The powder of root of Ficus retusa showed starch grain, Trichomes, fibers and oil globules. The powder of leaves of Ficus retusa showed Fibro-vascular, starch grain and oil globules. Physiochemical parameters and extractive value of leaves, bark & root of Ficus retusa were studied and results were shown in

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Spraying reagent

Rf


0.16, 0.15, 0.14, 0.39, 0.37, 0.35

Table 2 and 3 respectively. Preliminary phytochemical studies on the plants revealed the presence of alkaloids, glycoside, tannins, saponin, steroids, tri-terpenoid and flavonoids. The preliminary phytochemical test results were rationalized by the Thin Layer chromatographic studies. 5. Conclusion The comparatives pharmacognostic standards for the leaves, bark & root of Ficus retusa are laid down for the first time in this study. Morphological and anatomical studies of plant parts will enable to identify the crude drug. The information obtained from preliminary phytochemical screening will be useful in finding out the genuity of the drug. Ash values, extractive values can be used as reliable aid for detecting adulteration. These simple but reliable standards will be useful to a person in using the drug as a home remedy. Also the manufacturers can utilize them for identification and selection of the raw material for drug production. So further study should be carried out in future to isolate the specific chemical constituents as well as detailed pharmacological activity will be carried out in proper scientific way.


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Figure 1a: T.S of leaf of Ficus retusa. Figure 2a: T.S of root of Ficus retusa. Linn shows vascular bundles, epidermis, medullary rays and cortex region.

(

Figure 1b: Magnified T.S of leaf of Ficus retusa. Figure 2b: T.S of root of Ficus retusa. Linn showing vascular bundles and pith region

Figure 1c: T.S of petiole of Ficus retusa

Figure 2c: L.S of root of Fcus retusa showing fusiform cells

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3.

4. 5. 6.

Figure 2d: L.S of root of Ficus retusa showing epidermis, cortex region and fusiform cells. 7.

8. 9. 10.

11.

Figure 3a: Powdered sample of root of Ficus retusa. Linn showed lignified cells, trichomes, fibres, oil globules and starch grains.

12.

13.

14.

Figure 4a: T.S of bark of Ficus retusa showed vascular bundles, parenchymatous cells, starch grains and pigment.

15.

16.

3. Reference: 1.

2.

Ajasa A.M.O. (2004) ‘Heavy trace metals and macronutrients status in herbal plants of Nigeria’ Food Chemistry, Vol. 85 No. 1, pp. 6771. Brain K.R. and Turner T.D. (1975) The Practical Evaluation of Phytopharmaceuticals (Wright Scientecnica, Bristol):81-82.

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17. 18.

British Pharmacopoeia. (1980). Vol.II.Ash Value, Acid Insoluble Ash, WaterSoluble Extractive, (Her Majestry’s Stationary Office, London), Appendix XI.A108-113. Evans W.C, Trease and Evans- Pharmacognosy, 15 edition, (Elsevier Science limited. 2002) 538-539. Evans, W.C., In; Trease and Evans’ Pharmacognosy 13th Edn., (2004), Bailliere Tindall & Cansell Ltd., London, 538-546. Harborne J.B. (1998) ‘Phytochemical methodsA guide to modern techniques of plant analysis’ Third edition, published by Springer (India) Pvt. Ltd., pp. 49-129. Harbone J.B. (1976) Phytochemical Methods. A guide to modern techniques of plant analysis(Charpan and Hall). 78. Indian Medicinal Plants, (1997) Arya Vaidya Sala, Orieny Longman, pp.91. Jaya Raju et al (2011) “Investigation of Hepatoprotective activity of Ficusretusa (Moraceae)” IJRAP, 2(1), 166-169. Koilpillai.Babu, Sabesan Gokul Shankar, SadanandRai, (2010), “Comparative Phrmacognostic Studies on the bark of four Ficus species” Turk J Bot, 34, 215-224. Kokashi CJ, Kokashi RJ, Sharma M. (1958). Fluorescence of powdered vegetable drugs in ultra-violet radiation. J American Pharm Assoc; 47:715–717. Lal,V.K., Khosa,R.L.,Wahi,A.K.,(1980).Pharmacognostical studies on Haebennaria edgeworthii., Indian.J.Bot. 1:18. Mukherjee P. K. (2008) ‘Quality Control of Herbal Drugs’ Third reprint, published by Business horizons, New Delhi, pp. 247-378, 562-563. Ravichandra VD, Padmaa M Paarakh(2011) “Pharmacognostic& Phytochemical Investigation on leaves of FicusHispida” IJPPS, Vol 3, Issue 2. Sirisha.N, Sreenivasulu.M, Sangeeta.K, Chetty.C.M (2010) “Antioxidant properties of Ficus species-A Review” International Journal of Pharm Tech Research, Vol.2, No.4, pp 21742182. Stahl E., “Thin layer chromatography- A laboratory handbook” second edition, Published by Springer Verlag Berlin Heidelberg, pp. 241-247, 427-431. Wallis TE: Practical Pharmacognosy. J & A Churchill Ltd., London, EditionV, 1984. Wagner H, Bladt S, Plant drug analysis- A thin layer chromatography atlas, II edition, (Thomson Press Ltd. India, 1996) 275-278.


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