G. Parimala Devi et al. / Journal of Pharmacy Research 2012,5(4),1942-1945 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Pharmacognostical standardisation of Antidesma ghaesembhilla Gaertn, family: G. Parimala Devi * 1, C.Pooja Chowdary, D.Prasanna,T.Damodar,Balreddy,Satish,Ramamohan Gupta 1Department of Pharmacognosy, Pullareddy Institute of Pharmacy, Annaram, Medak Dist. 2 Pullareddy Institute of Pharmacy, Annaram, Medak Dist.,Department of Pharmacognosy, Andhra Pradesh, India Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012

ABSTRACT Gaertn, Phyllanthaceae , is the small tree which is widely distributed in the Tirumala hills, Indomalaysia to in the western Ghats-south central and Maharashtra sahyadris Vijayanagaram and the Mahaboobnagar districts. The parts of the have the various uses as herbal medicine for headache; stems used to facilitate menorrhea; used as purgative. The chemical constituents of Antidesma Ghaesembilla Gaertn, include fats, alkaloids, phenol compounds, flavonoids and carbohydrates. Though the plant has pharmacological potentials no standardization has been done pharmacognostically, hence form the basis for performing this work. The transverse section of stem and numerous unicellular, uniserrate covering trichomes are abundant, pointed towards apex and broader at base measures about 260-450 microns in length. The powder microscopy showed the presence of fibers, phloem and xylem vessels and the absence of calcium oxalate crystals. The proximate analysis, fluorescence analysis and preliminary phytochemi- cal tests were performed and reported. These data would help in the development of the profile of Antidesma Ghaesaembilla.

Key words: Antidesma Ghaesaembilla Gaertn, endarch vascular bundles, physicochemical, Fluorescence analysis. 1.INTRODUCTION: Antidesma Ghaesaembilla Gaertn is the small tree which is widely distrib- muffle furnace (3003137), Rotary vacuum evaporator, Stage micrometer, uted in the Tirumala hills, Vijayanagaram, Mahaboobnagar district, etc. This Eyepiece micrometer. is the dicot plant A small to medium tree upto 20 m tall; young twigs pubes- cent. Leaves oblong, more rarely ovate or obovate, (2-)3-7(-16) × (2-)3-5(- 2.1.1Chemicals and reagents: 9) cm, papery to thinly leathery, pubescent to glabrous especially adaxially, All the chemicals and reagents like chloral hydrate, phloroglucinol, hydro- often only major veins and margin pubescent, dull or shiny adaxially, dull chloric acid, nitric acid, potassium hydroxide, picricacid, lead acetate, alco- abaxially, drying olive green to reddish green, base rounded to cordate, rarely hol, acetone, chloroform, petroleum ether, etc. Used were of analytical grade. obtuse, apex rounded, more rarely obtuse or acute, sometimes mucronate or retuse; domatia sometimes present; midvein flat adaxially, lateral veins 5-7 2.2. Microscopical studies: pairs, tertiary veins reticulate to weakly percurrent. Synonyms of the plant include black currant tree. It is not indigenous to this country but is rarely 2.2.1Transverse section of stem: cultivated. It is also found along hills and valleys. The leaves are used as a Microtome sectioning was done for fresh stem to obtain a thin section. medicine for headaches; the stem is used as a medicine to stimulate the Phuloroglucinol and hydrochloric acid in the ratio 1:1 was used as a stain and menstrual flow. Propagation is usually done by means of seeds, especially mounted on a glass slide and focused under a microscope. A thin transverse when the seeds are its final position. The flowering and fruiting time of the section of Antidesma Ghaesaembilla Gaertn stem was taken and studied plant is usually from September – October. The leaves are mainly used for 4.The descriptions are given as per standard anatomical references. (Figure1) medicinal purposes. (1, 2, 3) 2.2.2Transverse section of leaf: 2. MATERIALS AND METHODS: Microtome sectioning was done for fresh stem to obtain a thin section. The plant Antidesma Ghaesaembilla Gaertn was collected in regions of Phuloroglucinol and hydrochloric acid in the ratio 1:1 was used as a stain and Karnataka, in the months of November to December. The plant material was mounted on a glass slide and focused under a microscope. A thin transverse authenticated by Dr.Madhava chetty, Dept. of Botany and was certified section of Antidesma Ghaesaembilla Gaertn stem was taken and studied 4 under the Voucher No -892, deposited in the Department of pharmacognosy, .The descriptions are given as per standard anatomical references. (Figure 2 Pullareddy institute of pharmacy,medak. and 3)

2.1Instruments used: 2.3 Powder microscopy: Micro senior precision rotary microtome (latest Spencer 820 types), Sisco Shade dried whole plant were powdered with the help of an electric grinder till a fine powder was obtained5, 6, 7 .This fine powder was subjected to powder microscopy, as per standard procedures mentioned. (Figure 3) *Corresponding author. G. Parimala Devi 2.4 Determination of Physicochemical properties: Department of Pharmacognosy, Total ash, acid insoluble ash and water soluble ash, sulphated ash of Antidesma Pullareddy Institute of Pharmacy, Ghaesaembilla Gaertn were determined by standard methods and the results Annaram, Medak Dist. are tabulated in table. The crude fiber content, moisture content, alcohol Andhra Pradesh,India soluble extractive value, water soluble extractive value, chloroform soluble extractive value and the petroleum ether soluble extractive values Antidesma

Journal of Pharmacy Research Vol.5 Issue 4.April 2012 1942-1945 G. Parimala Devi et al. / Journal of Pharmacy Research 2012,5(4),1942-1945 Ghaesaembilla Gaertn were determined by standard methods and the results c)Endodermis: This is the innermost layer of cortex, and is single layered obtained were tabulated in the table 5, 6, 7. with laterally stretched barrel shaped cells arranged in wavy manner. Cells of this layer contains numerous dense starch granules. 2.4.1 Measurement of cell structure and content: The length and width of phloem fibers and the diameter of the starch grains PERICYCLE: were measured using a stage micrometer and the eyepiece micrometer by It occurs in the form of alternating patches of parenchyma and Scleren- standard procedures5, 6, and 7. chyma.

2.5 Extraction: VASCULAR BUNDLES: The collected plant was washed and dried under the shade. It was then Many in number and all are of small size and are arranged in the form of a ring. coarsely powder using an electric grinder. 50 g of the coarsely powdered stem Each vascular bundle is conjoint, collateral and open with endarch xylem. and leaf was packed separately in soxhlet apparatus and extracted with, Cambium is present between xylem and phloem. chloroform, acetone, alcohol, and water after defatting with petroleum ether at 60 -800c for 72 hours. The extract obtained was concentrated under vacuum Medulla (or pith): using a rotary vacuum evaporator 8, 9. It is most conspicuous region and it contains Parenchymatous cells with profuse intercellular spaces. 2.5.1 Preliminary chemical screening: The extract obtained was subjected to various chemical tests as per the Medullary Rays: procedure mentioned in the standard reference books8, 9. Parenchyma cells of the pith extend radialltty between the vascular bundles and connect the cortex reaching upto the endodermis. These Parenchyma- 2.6 Determination of Fluorescence analysis: tous rays are called medullary rays. The powdered mass was subjected to analysis under ultraviolet light after treatment with various chemical and organic reagents 10, 11.

3. RESULTS AND DISCUSSION:

3.1Microscopical studies

3.1.1: Transverse section of stem:

3.1.2: Transverse section of leaf: Figure 3 Figure 2

Epidermis: I t occurs on both upper and lower sides and are respectively called as,

a)Upper epidermis: Composed of uniseriate, compactly arranged more or less rectangular cells without chloroplasts. The outer exposed tangential wall is covered by a waxy substance called cutin, and is called cuticle. Stomata are usually absent on the upper epidermis: when present these are very few in number.

b)Lower epidermis: Similar to the upper epidermis, but with numerous stomata distributed throughout its surface. A few multicellular hairs are present on the lower epidermis. Figure1: Mesophyll tissue: EPIDERMIS: The green tissue of leaf in between the two epidermal layers is known as This is the outermost bordering tissue made up of a single layer of tubular mesophyll tissue. And is made-up of parenchyma cells, which are distinctly cells covered externally by a cuticle. On the surface of the epidermis multiple differentiated into upper palisade parenchyma and lower spongy paren- hairs are present and are called as trichomes. chyma.

CORTEX: a) Palisade parenchyma: It is composed of columnar, elongated compactly It is a multilayered and is differentiated into three regions: arranged two or more rows of cells below the upper epidermis. It possesses a)Hypodermis: It is collenchymatous and is present below the epidermis in numerous chloroplasts around their radial walls. 2-3 layers of cells. b)General cortex: This is made up of parenchyma cells. All the cells of b) Spongy parenchyma: This is found below the lower epidermis and is parenchyma are cholenchymatous.

Journal of Pharmacy Research Vol.5 Issue 4.April 2012 1942-1945 G. Parimala Devi et al. / Journal of Pharmacy Research 2012,5(4),1942-1945 composed of loosely arranged oval or spherical parenchyma cells with abun- Table 2: Leaf constants dant intercellular spaces. Few chloroplast cells are present. Leaf constants Measurements

3)Vascular bundles: Upper epidermis: 8 – 4.8 The midrib and veins which forms a network all through the lamina repre- Lower epidermis : 10 - 15 sents the vascular system of the leaf. The midrib represents the large mid The Stomatal index for lower surface 8.5 The Stomatal index for upper surface 6.6 vein. Each vascular bundle is composed of xylem facing upper epidermis and Vein islet number 10-11mm2 phloem facing lower epidermis. Cambium is absent if present, functionless. Vein termination number 9-10mm2 Thus the vascular bundle is conjoint, collateral and closed. Parenchyma as Table 3: Cell structure and content well as collenchyma cells extend radially and connect the upper and lower epidermis forming bundle sheath extensions. Parameters Length Width Fibers 2.25 to 3mm 6µm

3.2. Powder microscopy: Phloem vessels 170 to 300µm 40-50µm Figure 4: Figure 5 Table 4: Measurement of starch grains Parameter Diameter

Starch grains 5µm 3.4. Preliminary chemical screening: The extracts obtained after successive solvent extraction with petroleum ether, chloroform, acetone, ethanol, and water were analyzed for the color, odor and percentage yield. These extracts were subjected to qualitative chemi- cal test for the identification of various chemical constituents. The chemical Figure 6: Figure 7 test helps in the confirmation of the chemical nature of the active principles present in the plant extract .The results of the chemical tests are tabulated in table 5, 6.

Table 5: Total extract

Extracts Color Consistency %yield

Petroleum ether Yellow Gummy mass. 0.9% Alcohol Green Oily mass. 4.1% Water Brown Powdery mass. 3.1%

Table 6: Preliminary Phytochemical screening of total extract

Fibers of phloem were found to lignified with lumen in it. Compounds Pet ether Alcohol Water Calcium oxalates are absent. Diacytic stomata are present. Saponins -ve -ve +ve Carbohydrates +ve +ve +ve Trichome is present. Glycosides +ve +ve -ve Spiral vessels are present. Steroids +ve -ve +ve Proteins -ve -ve -ve Phenolic compounds -ve +ve +ve 3.3. Determination of physico chemical properties: Flavonoids -ve +ve +ve The physico chemical properties will help to estimate the amount of impu- Alkaloids +ve -ve -ve rities like soil and particles present in the drug. It also helps to assess the Fats and oils -ve +ve +ve calcium salts present in the drug sample. The results obtained for the ash 3.5. Determination of Fluorescence analysis: values, extractive values, moisture content and crude fiber content, leaf con- Fluorescence analysis is a tool to determine the kind of the chemical nature of stants, length and width of the fibers, and phloem vessels, diameter of starch the drug. The fluorescence obtained in short wavelength, long wave length grains, yield and physical characters are tabulated in Table 1, 2,3,4. and daylight after treatment with different chemicals and reagents are tabu- lated in table 7. Table 1: Physicochemical properties

Parameter Stem (%w/w) Leaf (%w/w ) Table 7: Fluorescence analysis of total extract:

Total ash 0.247%w/w 0.495%w/w Chemicals Daylight Short wavelength Long wavelength Acid insoluble ash 0.231%w/w 0.218%w/w

Water soluble ash 4.2%w/w 3.1%w/w Drug+50%H2SO4 Dark green Green Black Moisture content 0.356%w/w 0.940%w/w Drug +50%HNO3 Reddish brown Green Black Crude fiber content 0.58%w/w 0.67%w/w Drug+1N methanolic NAOH white Green Black. Alcohol soluble extract value 4.1%w/w 9.4%w/w Drug +1N KOH Greenish yellow Green black Black Water soluble extract value 6.03%w/w 9.3%w/w Drug +5%KOH yellow Green Black

Chloroform extract value 6%w/w 4.2%w/w Drug +5%FECL3 Blackish green Greenish brown Yellowish red Petroleum ether extract value 1.3%w/w 2.2%w/w Drug + methanol Green Green Red

Sulphated ash 1.56%w/w 1.36%w/w Drug+conc H2SO4 Black Greenish black Reddish black Loss of drying at 105°C, % w/w 9.8% 1.9%w/w Drug+ ammonia Blackish brown Greenish black Black

Drug+conc HNO3 Brown Greenish black Black

Journal of Pharmacy Research Vol.5 Issue 4.April 2012 1942-1945 G. Parimala Devi et al. / Journal of Pharmacy Research 2012,5(4),1942-1945

4. CONCLUSION: Botany ,Telugu Akademi Publication; Hyderabad, 2004. The macro and microscopical characters along with physicochemical and fluo- 5. Khandelwal KR. Practical Pharmacognosy Techniques and Ex- rescence characters of powder of Antidesma Ghaesaembilla Gaertn, is used to periments. Published by D.K Furia, Nirali prakashan, Pune, establish the Pharmacognostical standards and qualitative parameters as per 2002, 9th Ed, pgno220-222. pharmacopoeia and WHO guidelines. Preliminary phytochemical screening of 6. The Indian pharmacopoeia Ghaziabad, volume 1, 2007, , Page no different plant extracts revealed the presence of phenols, flavonoids, saponins, glycosides and carbohydrates. 78. 7. Kokate CK, Practical Pharmacognosy, Vallabh Vrakashan; Delhi, 5. ACKNOWLEDGEMENT: 2008.149-156. The authors are highly grateful to the management of pullareddy institute of 8. Deshmukh Hafsa, Pradnya J Prabhu, Standardization Of Stem- pharmacy for providing necessary help and laboratory facilities in performing Bark Of Dendrophthoe Falcate Linn,International Journal of Phar- this experiment. They express their gratitude to for Dr.Madhava chetty authen- macy and Pharmaceutical Sciences, Vol 3, 2011, ISSN- 0975- ticating the plant specimen. 1491, Suppl 4,.. 9. Sathis Kumar. D, Veena Mandarapu, David Banji, Rao Knv, 6. REFERENCES: Chandrashekar, Sudhakar.et al, Pharmacognostical Study On Piper 1. http://www.fruitipedia.com/black_currant_ tree% 20aantidessma_ Trioicum Roxb, International Journal of Pharmacy and Pharma- ghaesembilla.htm ceutical Sciences, Vol 3. 2011,ISSN- 0975-1491 Suppl 3. 2. Pullaiah T, D. Ali Moulali Flora of Andhra Pradesh (India). Jodh- 10. Chase CR, Pratt R. Fluorescence of powdered vegetable drugs pur: Scientific Publishers, Vol-II 1997, pg no-910 . with particular reference to the development of a system of iden- 3. Madhava KC, Sivaji K, Tulasi KR. Flowering of Chitoor tification, J Am Pharmacology Assoc 38: 1949, 324-331. Dist A.P. India, Students Offset Printers, Tirupati,2008, First 11. Kokoski CJ, Kokoski RJ, Slama FJ. Fluorescence of powdered edition pg.no.328. vegetable drugs under ultraviolet radiation. J Am Pharm Assoc 4. Kailasnath Sarma T,.Rama Krishna KN,.Sai Siva Ramakrishna V, 47: 1958, 715-717. Gourinath A,Satyanarayana Reddy P,Intermediate First Year

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 4.April 2012 1942-1945