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International Journal of Recent Scientific Research Vol. 4, Issue, 6, pp.1032 1035, July, 2013 ISSN: 0976-3031

International Journal of Recent Scientific Research

RESEARCH ARTICLE PHARMACOGNOSTIC STANDARDIZATION OF STEM AND LEAF OF BRYOPHYLLUM PINNATUM KURZ.
Ranjan, R. and S. S. Deokule*
Department of Botany, University of Pune, Pune-411007 (M.S) India ARTICLE INFO
Article History:
Received 16th, June, 2013 Received in revised form 28th, June, 2013 Accepted 17th, July, 2013 Published online 30th July, 2013

ABSTRACT
Bryophyllum pinnatum Kurz. is an important medicinal plant also known as Kalanchoe pinnata Pers. It is used as immunosuppressive effects hepatoprotective activity. The leaf extracts of this plant have been routinely used for ailments like bacterial, fungal and viral infections, asthma, kidney stones, and ulcers. The leaves of this plant have been reported to possess antimicrobial, antifungal, anti-ulcer, anti-inflammatory and analgesic, antihypertensive, antidiabetic and antimutagenic activities. The present paper reveals the botanical standardization on the stem and leaf of Bryophyllum pinnatum. The pharmacognostic studies include the phytochemistry, histochemistry, microscopic, macroscopic evaluation, percentage extractives, ash and acid insoluble ash, fluorescence analysis and estimation of Protein, Carbohydrate and Flavonoid. The phytochemical and histochemical test includes starch, proteins, saponins, alkaloids, glycosides, sugar and flavonoid. Copy Right, IJRSR, 2013, Academic Journals. All rights reserved.

Key words: Pharmacognostic study, Standardization, Bryophyllum pinnatum Kurz.

INTRODUCTION
Bryophyllum pinnatum Kurz. is an important medicinal plant also known as Kalanchoe pinnata Pers. belonging to family Crassulaceae. It is used as immunosuppressive effects (RossiBergmann et al. 1994), hepatoprotective activity (Yadav; Dixit, 2003). It is found throughout India and is employed in the indigenous system of medicines. Bryophyllum are naturalized in many parts of the tropics, and deliberately cultivated for their attractiveness or for their interesting reproduction. Some species are toxic, containing plant alkaloids, calcium oxalate etc, and have become noxious weeds in parts of the world. Identified active ingredients include bufadienolides, flavonoids, glycosides, steroids and organic acids (Marriage et al. (1971); Gaind and Gupta, (1972); Gaind & Gupta, (1974). The leaf extracts of this plant have been routinely used for ailments like bacterial, fungal and viral infections, asthma, kidney stones, and ulcers. In traditional medicine, the leaves of this plant have been reported to possess antimicrobial (Mehta and Bhat, (1952); Akinpelu, (2000); Oliver-Bever, (1983), antifungal (Misra and Dixit, 1979), anti ulcer (Pal and Nag, 1991), anti-inflammatory and analgesic (Pal and Nag, 1989; 1999), and antihypertensive (Ojewole, 2002) activities. Bryophyllin, potassium malate, ascorbic, malic and citric acids have been isolated from the leaves of B. pinnatum {McKenzie et al. (1985); Siddigiuient et al. (1983); Singh (1976); Oliver (1983)}.

identified with the help of Flora of The Presidency of Bombay (Cooke, 1958). Microscopic and macroscopic evaluation Stem and leaf was examined macroscopically according to (Wallis, 1967 and Trease and Evans 2002) which includes shape and size, colour (inner and outer), odour and taste. For microscopic evaluation thin (25) hand cut sections were taken from fresh stem and leaf, permanent double stained and finally mounted in Canada balsam as per the plant micro-techniques method of Johansen (1940) and Krishnamurthy (1988). Histochemical study The thin transverse sections of fresh stem and leaf were taken (about 25). It was treated with respective reagent for the detection and localization of active constituent present in the tissues as per the method of Krishnamurthy (1988). Phytochemical study Some material were dried under the shade so as to avoid the decomposition of chemical constituents, powdered in blender and finally stored in dry air tied containers for phytochemical screening. Ash, acid insoluble ash and percentage extractives were accomplished by following standard pharmacopoeia techniques of Anonymous (1955). Fluorescence analysis was carried out as per Chase and Pratt (1949). Qualitative phytochemical tests were carried out by standard method of Harborne (1973) and Trease and Evans (2002). Quantitative phytochemical analysis was determined for proteins, carbohydrates, Starch, and Flavonoid by the methods of Lowry et al. (1951), Nelson (1944) and Boham and Kocipai (1994) respectively. The phytochemical screening is also detected by the High Performance Thin Layer Chromatography (HPTLC). HPTLC study was carried out on

MATERIAL AND METHODS

Collection and Identification of Plant Material The plant material was collected from in and around Pune district of Maharashtra state. The plant was collected in flowering and fruiting condition for the correct botanical identification. It was

* Corresponding author: Tel: + 09761482925 E-mail: drashok_01@yahoo.in

International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1032 - 1035, July, 2013 instrument comprising of Linomat5 for application using Densitometer- TLC scanner 3 with WINCATS software (Camag, Switzerland). These studies were carried out on HPTLC precoated aluminum fluorescent plates (E. Merck). For HPTLC studies, an extract of methanol (25% GR) solvent system was used and after development, plate was scanned at 254 and 366nm (Wagner and Bladt, 1966; Reich and Schibii, 2007) Table 3 Percentage extractives of Bryophyllum pinnatum Stem and Leaf
Sr. No. 1 2 3 4 5 6 Solvent Used Acetone Benzene Chloroform Petroleum ether Abs. Alcohol Distilled water % extractive (Dry Wt.) Stem 5.5% 17.4% 9% 2.8% 11.2% 6.6% % extractive (Dry Wt.) Leaf 5.1% 14.4% 5.3% 1.8% 10.2% 6.3%

RESULTS AND DISCUSSION

Macroscopic evaluation Habit It is an erect, more or less branched, smooth, succulent herb, 0.4 to 1.4 meters in height. Leaves are simple or pinnately compound, with the leaflets elliptic, usually about 10 centimeters long, thick, succulent, and scalloped margins. Plantlets grow along the notches of the leaf margins which can develop while still attached to the plant or when detached also. Table 1 Histochemical study of Bryophyllum pinnatum Stem and Leaf
Reagents Stem I2KI ++ Millans reagent ++ Acidic FeCl3 ++ Conc. H2SO4 ++ Sudan III ++ 20% aq. NaOH ++ Guignards test ++ Mayers reagent -Wagners reagent ++ Dragendorffs ++ reagent Tannic acid ++ Hagers Reagent. ++ + + (Positive sign) = denotes the presence of chemicals. -- (Negative Sign) = denotes the absence of chemicals. Sr. No. 1. 2. 3. 4. 5. 6. 7. 8. Test Starch Protein Tannin Saponin Fat Sugar Glycoside Alkaloids Leaf ++ ++ ++ ++ ++ ++ ++ -++ ++ ++ ++

Fig.1: Habit of Bryophyllum pinnatum

Fig.2: B.pinnatum Kurz. (Leaf)

Fig.3: B. pinnatum Kurz. (Stem)

Fig.4. Anatomy of Stomata B.pinnatum Kurz. (Leaf)

Fig.6: B. pinnatum Kurz. (Stem)

Fig.5: B. pinnatum Kurz. (Leaf)

Graph VI - Qualitative analysis of Quercetin in the Stem and Leaf of B.pinnatum Kurz. Table 4 Fluorescence analysis of Bryophyllum pinnatum Stem and Leaf
Sr. No. 1 2 3 4 5 Test Powder as such Powder as such U. V. light Powder + Nitrocellulose Powder + 1N NaOH in Methanol Powder + 1N NaOH in Methanol dry it for 30 min. + Nitrocellulose Stem Greenish brown Grayish yellow Grayish black Grayish green Grayish black Leaf Greenish yellow Grayish yellow Grayish black Dark brown Grayish black

Leaf: Leaves are pale green, have bitter taste and characteristic odour. The leaf is ovate or elliptic in shape. Their length ranges from 6-10cm in length and 4cm in breadth. Stem: Stems are faint green, have bitter taste and pungent odour. The stem is cylindrical in shape. Their length ranges from 11.5cm in diameter. The stems are circular in cross-section. Table 2 Ash and Acid insoluble ash of Bryophyllum pinnatum Stem and Leaf
Sample Stem Leaf Total ash % 13.8% 3.2% Acid insoluble % 13.5% 13.1%

Microscopic evaluation Transverse section of stem: The transverse section of the stem is circular in outline. The cells of epidermis are uniform. Cortex is narrow, chlorenchymatous, with large cells and big vacuoles. Vessels are few, Pith is small contains parenchymatous tissues and it is encircled by vascular bundles. Transverse section of leaf: The transverse section of leaf shows dorsiventraly structure. The outermost epidermis layer is uniseriate with stomata at the same or slightly above the level of other epidermal cells. Epidermis covered with the cuticle. The cuticle is moderately thick, stomata of anisocytic type observed. Under the epidermal region some layer of collenchyma cells are present which is followed by chlorenchymatous layer of cells. Mesophyll cells thick with large cells, large vacuoles and small intercellular space. Vascular bundle is collateral

Inflorescence: The inflorescence is terminal panicle. Flower: Flowers are cylindrical and pendulous. Calyx is tubular, cylindrical, inflated, brownish or purplish, 3.5 to 4 centimeters long. Corolla is tubular, about 5 centimeters long, inflated at the base, and then constricted, the exserted parts being reddish or purplish and the lobes tapering to a point. Fruit: Fruit is follicle with many seeds. Distribution: It is distributed in open settled areas, thickets, dry second-growth forests, sometimes planted, and locally abundant, tropical Asia or Malaya.

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International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1032 - 1035, July, 2013 Table 5 Phytochemical study of Bryophyllum pinnatum Stem and Leaf
Sr. No. Alcohol extract Reagents Wagners Mayers Dragendorffs Hagers Conc. HCL + Mg turning Benzene Water extract I2KI Millans Conc H2SO4 Test for Alkaloid Alkaloid Alkaloid Alkaloid Flavonoid Glycoside Starch Protein Saponin Stem + ve - ve + ve + ve + ve + ve + ve + ve + ve Leaf + ve - ve + ve + ve + ve + ve + ve + ve + ve Stem Leaf

Histochemical screening Histochemical screening showed the presence of starch, protein, fat, saponins, tannin, sugar and alkaloids (Table 1). Table 6 Quantitative estimation of Bryophyllum pinnatum Stem and Leaf.
Quantitative estimation (mg/gm) Stem Leaf Protein Reducing sugar 0.39524 0.45621 Non reducing sugar 0.48621 0.73281 Starch Flavono id 0.0507 0.0698

1.83025 2.35625

0.04946 0.04538

Fig. 6 Estimation of Phytoconstituents by HPTLC Analysis

B1 & B2 - B. pinnatum Kurz. (Stem & Leaf) Q1 & Q2 - Std. Quercetin

Phytochemical study B. pinnatum Stem and Leaf contains the total ash 13.8% and 13.5% and acid insoluble ash 3.2 and 13.1% (Table 2). The value of percentage extractive is higher in stem by using solvent Benzene and Lower in Leaf by using Petroleum ether (Table 3).

The extract was filter and filtrate is used as an application for quantification of Flavonoid. For each application 10l extracts were used and loaded on instrument comprising of Linomat5 for application

Table 7 Showing the peak values for Flavonoid for 10l plant extract
Peak 1 2 3 4 5 6 7 Start Rf 0.01 0.16 0.37 0.55 0.71 0.93 1.02 Start Height 1.1 94.7 221.6 2.6 81.5 38.6 25.4 Max Rf 0.13 0.34 0.44 0.67 0.74 0.94 1.04 Max Height 100.3 228.1 276.4 92.0 92.0 44.5 26.9 Max% 11.65 26.52 32.14 10.70 10.70 5.17 3.12 End Rf 0.14 0.36 0.52 0.70 0.79 0.95 1.06 End Height 96.2 221.9 0.1 79.9 50.4 21.4 21.2 Area 4529.4 19651.4 17058.0 5537.4 3841.7 602.6 644.8 Area% 8.73 37.89 32.89 10.68 7.41 1.16 1.24

Table 8 Showing the value of peak for Quercetin in the leaf of Bryophyllum pinnatum Kurz.
Peak 1 2 3 4 Start Rf 0.02 0.27 0.57 0.72 Start Height 6.8 146.1 1.0 84.8 Max Rf 0.15 0.45 0.69 0.74 Max Height 102.7 268.0 88.6 92.0 Max% 17.92 46.78 15.47 16.05 End Rf 0.17 0.53 0.71 0.82 End Height 93.3 2.8 83.9 29.8 Area 5635.7 28804.2 5363.3 3885.0 Area% 12.85 65.68 12.23 8.86

Fluorescence analysis was carried out to check the purity and potency of the drugs. The powdered drug was then observed in ultraviolet light, it was treated with reagents like Nitrocellulose, 1N NaOH, 1N NaOH in methanol, 1N NaOH in methanol dry it for 30 min + Nitrocellulose and observed under UV light to emits the color as shown in (Table 4). Qualitative analysis of the stem drug indicated the presence of proteins, reducing and non reducing sugars, saponins, fats, tannin, glycoside and alkaloids in the plant (Table 5). The quantity of proteins, carbohydrate, starch and flavonoid are mentioned in Table 6. In HPTLC study, the methanolic extract is sonicated for 5 min and then centrifuged at 10,000 rpm for 5 min.

using densinometer-TLC scanner3 with WINCATS software (Camag, Switzerland). These studies were carried out on precoated aluminum fluorescent plates (E. Merck). The plates were scanned at 254 and at 366nm (Wagner and Bladt, 1996; Reich and Schibii, 2007).

CONCLUSIONS
The plant B. pinnatum showed the correct taxonomy which is helpful for the standardization of drug. Findings of the present investigation will be useful for the correct botanical identification and authentication of the drug.

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International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1032 - 1035, July, 2013

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