The Pharmacognostical, Phytochemical and Antimicrobial studies of leaves Cassia auriculata Linn.

Gharge Varsha G.*, Shelar Pournima A., Ghadge Dhairysheel M., Patil Anup A.,

Bhandwalkar Omkar S., Dr. Yadav Adhikrao V.

Gourishankar Institute of Pharmaceutical Education & Research, Limb, Satara, India-415001.

*Corresponding Author E-mail: varshagharge25993@gmail.com

ABSTRACT:

Purpose: Cassia auriculata Linn (Caesalpinaceae) popularly known, as Tarval. Cassia auriculata leaf extracts have shown hepatoprotective activity against alcohol induced liver injury and antimicrobial activity phytochemical analysis carried out revealed the presence of coumarins, flavanoids, glycosides, phenols, tannins,saponins and steroids. The results provide justification for the use of the leaves extract antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi were used for the study. Methods: Pharmacognostic characterization: In the present study pulverized dried Cassia auriculata Linn leaves were investigated for its physical constant (LOD, ash value, fluorescence analysis). Microscopic study: The fresh leaf of Cassia auriculata Linn was studied for microscopical characterization which shows different parts like upper and lower epidermis, palisade cells, spongy parenchyma in lamina region while Collenchyma and vascular bundles were observed in midrib region. The pulverized dried Cassia auriculata Linn leaves were extracted with ethanol using soxhlet apparatus. The powder of Cassia auriculata Linn leaves were also macerated with chloroform water. The antimicrobial activity of the concentrated extracts were evaluated by determination of the diameter of zone of inhibition against both gram negative and gram positive bacteria using agar disc diffusion and minimum inhibitory concentration (MIC). Results: Results of the phytochemical studies revealed the presence of alkaloids, glycosides, steroids, flavonoids, tannins and the extracts were active against both gram positive and gram negative bacteria. Conclusions: The ethanolic and aqueous extracts of Cassia auriculata Linn leaves were studied for antimicrobial activity, of which ethanolic extract shows significant results.

KEYWORDS: Cassia auriculata Linn, Physical constants, Microscopy, Antimicrobials activity.

INTRODUCTION:

Cassia auriculata Linn (Caesalpinaceae) popularly known, as Tarval in Hindi is a fast growing profusely branched, tall evergreen shrub, generally 1.2 - 3.0 m in height sometimes reaching a height of 6.0 m^1,2

Traditionally it is used for curing many diseases. Many therapeutic uses and medicinal properties are reported in whole plant. They are known for anti hyperglycemic activity³, hepatoprotective activity⁴, antihelmintic activity⁵, anti-microbial activity⁶ and antioxidant activity⁷. The dried flowers and flower buds are used as substitute for tea in case of diabetic patients. It is also believed to improve complexion in women. The powdered seed is also used in the treatment of leprosy, skin and liver diseases⁸. The flowers are used widely used in Ayurveda tradition system used as a varaipanchanga chooranum and the main constituents of Kalpa herbal tea.⁹The purpose of the study is to determine the antimicrobial and antioxidant potential of flavanoid rich fraction of leaf of Cassia auriculata.¹⁰ The cassia auriculata plant contain preliminary phytochemical constituents such as alkaloids, phenols, glycosides, flavonoids, tannins, saponins, proteins, carbohydrates and anthraquinone derivativesare responsible for the pharmacology activity. The plant has been widely used in traditional system of medicine as a cure for rheumatism¹¹.The plant has been reported to possess antipyretic¹²hepatoprotective¹³, antidiabetic, antiperoxidative and antihyperglyceamic¹⁴ and microbicidal activity.¹⁵The potential of plants as a source of drugs is largely uncharted. The medicinal properties of the plants are mainly due to the antioxidant, antimicrobial and antipyretic effect of the phytochemicals present in them.¹⁶the plants are rich source of antimicrobial agents. Thus it is significant to carry out a study on medicinal plants for their antimicrobial potential. Studies revealed that traditional medicines provides essential oils and other plant extracts that provoke the interest as sources of natural products for its potentiality as an alternate remedy for various infectious diseases.¹⁷A detailed investigation about the antimicrobial properties of the plants that are used local traditions can leads to the development of invaluable plant drugs for dreadful diseases.¹⁸The present study attempts to study the antimicrobial activity of separate extraction using ethanol and aqueous extracts from leaves of Cassia auriculata which is investigated against a few bacterial pathogens using agar well diffusion method and to report the phytoconstituents present in different extracts of leaves of Cassia auriculata¹⁹. Bacterial strains such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi were used for the study.^{20, 21}

Figure 1: Whole Plant of Cassia auriculata Linn

MATERIAL AND METHOD:

Collection of Plant Material:

The plant material Cassia auriculata Linn were collected from the Satara district, Maharashtra, during the month of July in the year 2016 and authenticated by Dept. of Botany, Y. C. I. S. Satara, Maharashtra, India. specimen voucher was deposited in the college hebarium for future reference. Fresh drug obtained were shade dried and coarsely powdered and passed through sieve 100 mesh sizes and stored in air - tight containers for further use.

Preparation of Extract²²

The pulverized dried Cassia auriculata Linn leaves were extracted with ethanol using soxhlet apparatus. The powder of Cassia auriculata Linn leaves were also macerated with chloroform water. Ethanol and water extracts were filtered and evaporated to dryness.

Macroscopic Characteristic:^{23, 24}

The macroscopy of fresh leaves were studied according to standard methods.

Microscopic characteristics:²⁵

For microscopy hand section of leaf was taken, stained and mounted following usual micro-techniques.

Physical Evaluation:^{26, 27, 28}

The ash values, extractive values and loss on drying were performed according to the officinal methods prescribed in Indian Pharmacopeia.

Fluorescence Analysis:

Many drugs shows fluorescence when their powder is exposed to ultraviolet radiation. It is important to observe all materials on reaction with different chemical reagents under U.V. light. The fluorescence characteristics of powdered drug were studied under U.V. light after treating with different chemical reagents is reported. Fluorescence analysis was carried out according to the method of Chase and Pratt²⁸ and Kokoski.²⁹

Phytochemical Screening:

The dried leaves were extracted with ethanol and water. The behavior of powder with various chemical reagent and preliminary chemical tests for various extracts were also carried out according to the standard procedures described by Kokate³⁰ and Horborne.³¹

Antimicrobeal Study:^32-37

Collection of microbes:

Bacterial strains such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi were used for the study. The collected microbes were maintained in Nutrient agar broth and cultured in Nutrient Agar media. (Hi Media (P) Ltd Mumbai).

Preparation of the Medium:

Nutrient agar medium was prepared by dissolving 2.8 g of nutrient agar in 100 ml of distilled water. The solution was sterilized in an autoclave at 121°C for 15 min. It was cooled and poured into sterile Petri dishes to solidify. The agar depth of the medium was measured (4 cm).

Determination of Antimicrobial Activity:

Agar well-diffusion method was followed to determine the antimicrobial activity. Nutrient agar (NA) plates were swabbed (sterile cotton plug) with 8 hour old-broth culture of respective bacteria. Three wells (10mm diameter) were made in each of the plates using sterile cork borer. About 0.3 ml of different concentration of plant solvent extract were added using sterilized dropping pipettes in to the wells and allowed to diffuse at room temperature for two hours. The plates were incubated at 37⁰C for 18-24 hr for bacterial pathogen. Respective proper controls of solvent plant extracts were also maintained. Diameter of the inhibition zones and the values were recorded.

Chromatographic Studies:^38-40

Thin Layer Chromatography studies were carried out for extracts to confirm the presence of different phytoconstituents in these extracts. TLC is a mode of liquid chromatography, in which the extract is applied as a small spot at the origin of thin sorbent layer supported on a glass plate. The mobile phase migrates through the stationary phase by capillary action. The separation of solutes takes place due to their differential absorption/ partition coefficient with respect to both mobile and stationary phases. Each separated component has same migration time but different migration distance. The mobile phase consists of a single solvent or a mixture of solvents. Although, a number of sorbent like silica gel, cellulose, polyamide, alumina, chemically modified silica gel etc. are used, silica gel (type 60) is most commonly used sorbent. Handmade plates are prepared by using techniques like pouring. The retardation factor (R_f) is calculated using following formula,

Distance traveled by sample from base line

R_f= ----------------------------------------------------------

Distance traveled by solvent from base line

Thin Layer Chromatography:^41-43

The extracts were subjected to thin layer chromatography for the presence of phytoconstituents. In this technique, the Silica gel-GF254 (for TLC) was used as an adsorbent and plates were prepared by pouring technique, then air dried for an over-night and activated for one hour at 110°C and used.

Preparative Thin Layer Chromatography:

A thick layer of silica gel GF-254 was coated on the square shaped plate and activated at 110⁰C for one hour. The broad spot of extracted sample was applied on the plate.

Characterization of Isolated Compound:

From the separated bands, the substance of interest was scrapped from the plate and it dissolved in methanol. The mixture was filtered and the filtrate was evaporated to dryness. The isolated compound was then subjected for further studies. (1mg/ml concentrations of the extracts were used).

IR of isolated compound:^44-46

IR spectrum was recorded in IR- spectrometer in 400-4000 frequency in cm^-1 for isolated moiety. IR spectrum of compound was carried in KBR pellet and reproduced on fig.No.6 and fig.No.7. The important absorption can be correlated.

RESULT:

Macroscopic Characteristic Leaves of Cassia uriculata Linn:

The shrub is especially famous for its attractive yellow flowers which are used in the treatment of skin disorders and body odour. It is widely used in traditional medicine for rheumatism, conjunctivitis and diabetes. It has many medicinal properties. Its bark is used as an astringent, leaves and fruits anthelminthic, seeds used to treat in eye troubles and root employed in skin diseases.

Microscopic Characteristics Leaves of Cassia auriculata Linn:

The Transverse Section of leaf is dorsiventral consists of Midrib and Lamina.

Midrib:

It consist of single layered epidermis, on either side, upper epidermis composed of single layer closely arranged elongated cells externally covered with striated cuticle. Leaf surface contains simple, multicellular covering trichomes and anomocytic type of stomata. Below the upper epidermis 3-4 layers of well developed more or less isodiametric collenchymatous tissue were observed. Midrib contains centrally located vascular bundle which is collateral surrounded by some parenchymatous cells filled with dark content. Xylem is well developed and the phloem consists of strands of sieve tubes and small celled parenchyma. Lower epidermis consisted of single layer elongated cells with cuticle and just above the lower epidermis 2-3 layers of parenchymatous cells followed by the layers of collenchymatous cells were present. Calcium-oxalate crystals were found in spongy parenchyma. Lower epidermis contains more number of covering trichomes as compared to upper epidermis.

Lamina:

Dorsi-ventral structure with single layered upper and lower epidermis with a layer of elongated closely arranged cells externally covered with cuticle. Epidermal cells show anomocytic stomata in surface view; below upper epidermis single layered palisade cells followed by 5-7 layers of masophyll parenchyma which are rounded in shape and are devoid of intracellular spaces.

Figure 2: Microscopy of leaves of Cassia auriculata Linn

Physical Evaluation:

The Loss on Drying, Ash Values likes (Total Ash, Acid insoluble ash and Water soluble ash), Ethanol soluble extractive and aqueous soluble extractive of leaf powder are given in table-1.

Fluorescence Analysis:

The fluorescence characteristics of powdered drug were studied under U.V. light after treating with different chemical reagents is reported.

Table 1: Physical Constants for leaves of Cassia auriculata Linn

Sr. No.	Physical Constants	Result
1.	Ash Value (% w/w) · Total Ash · Acid Insoluble Ash · Water Soluble Ash	11.5 4.35 1.35
2.	Loss on Drying (% w/w)	80.3
3.	Extractive Values (% w/w) Ø Ethanol soluble extractive. Ø Aqueous soluble extractive	0.420 0.290

Table 2: Result of Fluorescence Analysis of leaves of Cassia auriculata Linn

Reagents	Fluorescence Observed
	Leaf		Leaf Powder		Stem Powder
	At 254nm	At 366nm	At 254nm	At 366nm	At 254nm	At 366nm
Powder + 1N NaoH In Methanol	Green	Green	Light Green	Light Green	Dark brown	Dark brown
Powder + 1N NaoH In Water	Green	Green	Dark brown	Light Green	Dark brown	Light brown
Powder + 50% Hcl	Blue	Yellow	Yellowish Green	Green	Dark brown	Yellowish Green
Powder + 50% H₂SO₄	Light Green	Light Green	Dark Green	Light Green	Light brown	Light brown
Powder + 50% HNO₃	Dark Yellow	Greenish Yellow	Dark Yellowish	Dark Yellowish	Dark brown	Light Yellowish
Powder + Petroleum Ether	Faint Yellowish Green	Faint Yellowish Green	Dark Yellow	Faint Green	Dark brown	Light brown
Powder +Chloroform	Faint Green	Faint Green	Dark Green	Faint Green	Faint Green	Faint Green
Powder +Picric Acid	Faint Green	Faint Green	Yellow	Dark Green	Dark brown	Faint Green
Powder +5% Fecl₃	Yellowish Green	Yellowish Green	Dark brown	Faint Green	Yellowish	Yellowish Green
Powder +5% Iodine	Green	Faint Green	Dark brown	Dark Green	Dark brown	Black Green
Powder +Methanol	Black	Dark Green	Black	Dark Green	Light brown	Dark Green
Powder + (HNO₃+NH₃)	Faint Green	Yellowish Green	Faint brown	Light Green	Dark brown	Yellowish Green

Table 3: Phytochemical investigation of Ethanolic and Aqueous extracts of Cassia auriculata Linn

Sr. No.	Name of the test	Leaf
Sr. No.	Name of the test	Ethanolic extract	Aqueous extract
1.	Test for sterols	-	+
2.	Test for Triterpenoids	+	-
3.	Test for glycosides	+	+
4.	Test for carbohydrates	-	+
5.	Test for alkaloids	-	-
6.	Test for flavonoids	+	+
7.	Test for tannins	+	+
8.	Tests for proteins	+	-
9.	Test for amino acids	+	+
10.	Test for phenols	+	+
11	Test for Volatile oils	-	-

Table 4: Antimicrobial activity of Ethanolic extracts of Cassia auriculata Linn

Organism	Diameter of inhibition zone in cm
	Ethanolic extract		Streptomycin 100(µ/ml)
	200(µ/ml)	300(µ/ml)	Streptomycin 100(µ/ml)
Staphylococcus aureus	2.3	4	2.53
Escherichia coli	3.1	3.2	3.4
Salmonella typhi	2.5	3	3.5
Pseudomonas aeruginosa	2.4	2.6	3.2

Figure 3: Antimicrobial activity of Ethanolic extracts

Figure 4: Antimicrobial activity of Aqueous extracts Thin layer chromatography

Table 5: Antimicrobial activity of Aqueous extracts of Cassia auriculata Linn

Organisms	Diameter of inhibition zone in cm
	Aqueous Extract		Streptomycin 100(µ/ml)
	200(µ/ml)	300(µ/ml)	Streptomycin 100(µ/ml)
Staphylococcus aureus	2.3	3.2	3
Escherichia coli	2.9	3	3.2
Pseudomonas aeruginosa	3.5	3.2	3.5
Salmonella typhi	3	3.3	3.4

Thin layer chromatography of Ethanolic extract

Stationary phase: Silica gel GF-254

Mobile Phase: Toulene: ethyl acetate: Formic Acid (8.5:1:0.5).

Detection: UV-366

Solvent front: 5.2

Spot detection: 2.5

Thin layer chromatography of Aqueous extract

Stationary phase: Silica gel GF-254

Mobile Phase: Toulene: ethyl acetate: Formic Acid (8.5:1:0.5).

Detection: UV-366

Solvent front: 5

Spot detection: 2.3

Table 6: TLC Profile of steroids

Extract	Observation		R_f values
Extract	No. of spots	Colour of spots	R_f values
Ethanolic	1	Yellow	0.48
Aqueous	1	Yellow	0.46

Table 7: IR Spectral peaks of Ethanolic extract of Cassia auriculata Linn

Peak Observed	Assignment	Absorption Expected (cm^-1)
719.45	Alkanes	600-1500
1228.66	Amines	1180-1360
1373.32	Nitro compound	1330-1540
1635.34	Alkenes	1620-1680
2924.09	Hydrogen bonded Acid	2500-3000
3061.09	Aromatic Ring	3000-3100
3224.98	Amines	3300-3500

Figure 5: Thin layer chromatography of A) Ethanolic extracts B) Aqueous extracts

Figure 6: IR Spectra of Cassia auriculata Linn A) Ethanolic extract and B) Aqueous extract

Table 8: IR Spectral peaks of Aqueous extract of Cassia auriculata Linn

Peak Observed	Assignment	Absorption Expected (cm^-1)
792.84	Alkanes	600-1500
1188.15	Alcohol, Ether, Esters	1000-1300
1255.66	-F	1400-1000
2918.30	Hydrogen bonded Acid	2500-3000
3140.11	-CH	3150-3050
3219.19	-OH	3400-3200

DISCUSSION:

1. The shrub were collected from Satara district, Maharashtra region and authenticated. The shrub were subjected for Pharmacognostic investigation which includes determination of physical constants such as ash value, extractive value determination and fluorescence analysis. The powder of shrub shows fluorescence at 254 and 366 nm.

2. Macroscopic and microscopic characteristics of the leaf were studied. The microscopic study shows that it contains midrib and lamina portion. The lamina shows upper and lower epidermis, spongy parenchyma, palisade cell layer while midrib portion shows upper and lower epidermis, collenchyma, vascular bundles, etc., Powder characteristics shows presence of anomocytic stomata and covering trichomes.

3. The leaves of shrub were subjected to successive extraction by using Ethanol and Aqueous and these extracts were subjected to phytochemical investigation.

4. Phytochemical investigation of extracts of Cassia auriculata, shows that Aqueous extract contains sterols, glycosides, carbohydrates, alkaloids. While Ethanolic extract shows presence of sterols, flavonoids, glycosides, carbohydrate, alkaloids and tannins.

5. Chromatographic study of the extracts was carried out. Where Thin layer chromatography were carried out by using mobile phase Toulene: Ethyl acetate: Formic Acid (8.5:1:0.5) which shows R_fvalue 0.44 and 0.34 for steroids for Ethanolic and Aqueous extract respectively.

6. For these isolated compounds of Ethanolic extract infra red spectroscopy was carried out which shows that Cassia auriculata contains hydroxyl group, fluorine, alkenes, aldehydes, bromine, etc. While aqueous extract shows alkenes, amines, nitro-group, Chlorine, etc.

CONCLUSION:

Cassia auriculata Linn is widely found in India during rainy season. As there is less information available on pharmacognostical work on leaves hence the morphological study, microscopical studies, physico-chemical parameters, fluorescence analysis and chemical tests performed will guide in the proper identification of the plant species as well as help in authentication of the purity of the plant. All these parameters also help to build up a suitable plant profile.

ACKNOWLEDGEMENT:-

I sincerely thanks to the Gourishankar Institute of Pharmaceutical Education and Research, Limb, Satara, India for providing the facilities to complete this research work. I solicit my deep sense of appreciation and loves to my wonderful mother and father consider my self-privilege to have seen an entity of almighty in them. I consider myself as luckiest person being my brothers and sister Rupali always there besides me during my ups and downs in my life.

REFERENCES:

1. Kajal N. Chauhan, Hepatoprotective activity of flowers of Cassia auriculata R. Br. against paracetamol induced liver injury, Journal of Natural Remedies. 2009; 9(1):85 – 90

2. Nascimento, G.C.F., J. Locatelli, P.G. Freitas and G.L. Silva,. Antimicrobial activity of plant extracts and phytochemicals on antibiotics resistant bacteria. Braz. J. Microbiol. 2000;31: 247-256.

3. Gupta S, Sharma SB, Prabhu KM and Bansal SK, Protective role of Cassia auriculata leaf extract on hyperglycemia-induced oxidative stress and its safety evaluation, Ind. J. Biochem. Biophys. 2009 46: 371-377.

4. Das S, Sarma G and Barman S, Hepatoprotective activity of aqueous extract of fruit pulp of Cassia fistula (AFCF) against carbon tetrachloride (CCL4) induced liver damage in albino rats, J. Clinic Diagnostic Res . 2008; 2: 1133-1138.

5. Deore SL et al. In vitro Anthelmintic activity of Cassia tora, Int. J. Chem. Tech. Res. 2009; 1: 177-179.

6. Duraipandiyan V and Ignacimuthu S, Antibacterial and antifungal activity of Cassia fistula L.: An ethnomedicinal plant, J. Ethnopharmacol. 2007; 112: 590-594.

7. Kaur G, Alam MS, Jabbar Z, Javed, K and Athar M, Evaluation of antioxidant activity of Cassia siamea flowers, J. Ethnopharmacol. 2006; 108: 340-348.

8. Sankaranarayanan S, Medical Taxonomy of Angiosperms: Recent trends in Medicinal uses and Chemical Constituents. Harishi Publications, Chennai 73, 2009.

9. Ayyanar M and Ignacimuthu S. Pharmacological Actions of Cassiaauriculata L. and Cissusquadrangularis Wall. A short review. Journal of Pharmacology and Toxicology 2008; 3 (3): 213-32.

10. Sumathy R, Antibacterial and Antioxidant Activity of Flavanoid Rich Fraction From The Petals Of Cassia Auriculata – An In-Vitro Study, Int J Pharm Pharm Sci. 2013; 5( 3): 492-497.

11. Kirtikar KR and Basu BD. Indian Medicinal Plants. Oriental Enterprises, Dehradun 1984; 4:1216-9.

12. Vedavathy S and Rao KN. Antipyretic activity of six indigenous medicinal plants of Tirumala Hills J Ethnopharmacol. 1991; 33:1936.

13. Kumar RS et al. Effect of Cassia auriculata leaf extract on lipids in rats with alcoholic liver injury. Asia Pac J ClinNutr. 2002; 11(2):157–63.

14. Latha M and Pari L. Antihyperglycaemic effect of Cassia auriculata in experimental diabetes and its effects on key metabolic enzymes involved in carbohydrate metabolism. ClinExpPharmacol Physiol 2003; 30:38–43.

15. Maneemegalai S and Naveen N. Evaluation of Antibacterial Activity of Flower Extracts of Cassia auriculata Ethnobotanical Leaflets 2010;14: 8-20.

16. A.A. Adesokan, M.T. Yakubu, B.V. Owoyele, M.A. Akanji, A. Soladoye and O.K. Lawal, Effect of administration of aqueous and ethanolic extracts of Enantia chlorantha stem bark on brewer’s yeastinduced pyresis in rats, Afri. J. Biochem. Res. 2008 165-169.

17. C.K. Hindumathy, Invitro Study of Antibacterial Activity of Cymbopogon Citratus, World Academy of Science. Engineering and Technology 2011;50: 189-193.

18. P. Bharathi, Alex Thomas, Ansa Thomas, S. Krishnan and T. K. Ravi, Anti bacterial activity of leaf extracts of Calotropis gigantea Linn. Against certain gram negative and gram positive bacteria,Int. J. Chem. Sci. 2011;9: 919-923.

19. M. Kanthimathi, Phytochemical screening and Invitro antibacterial Potential of Cassia auriculata Linn. Flowers against Pathogenic Bacteria, IRJPBS. 2014; 1 (1): 45-56

20. M. Ayyanar, and Ignacimuthu, Pharmacologica Actions of Cassia quadrangular are wall: A short Review, J. Pharmacol. Toxicol. 3(2008): 213-221.

21. H. Mohamed Sham Shihabudeen, D. Hansi Priscilla, S.S. Kavitha and Thirumurugan, Antimicrobial activity and phytochemical analysis of selected Indian folk medical plants, International Journal of Pharma science and research IJPSR. 2010; 1: 430-434.

22. Chatwal GR, Anand SK. Instrumental methods of chemical analysis, Himalaya Publishing House. 1992.

23. Mukherjee PK, Quality control of herbal drugs, Bussiness Horizon’s, Pharmaceutical publisher, New Delhi, 2002, 138-141.

24. Evans WC, Trease and evans, Pharmacognosy, W.B. Saunders, Edinburgh London, New York 2002.

25. Johansen DA, Plant microtechniques, McGraw-hill Book Company, New York and London 1940.

26. Indian Pharmacopoeia, Government of India, Ministry of Health and Family Welfare, Controller of Publication, 4th ed, New Delhi, 1996. 4(II), A53-A54.

27. Pradeep Kumar Sharma, Mohammad Ali and Dinesh Kumar Yadav, Physicochemical and Phytochemical evaluation of different black tea brands. Journal of Applied Pharmaceutical Science. 2011; 01 (03):121-124.

28. Rinku Mathappan1, and Sanjay P Umachigi, Antioxidant Activity of the Methanolic and Aqueous Extracts of Urena lobata (Linn.) by DPPH Method RRJPP 2013,1(1): 6-9.

29. Chase, C.R and Pratt, R.S., Fluorescence of Powdered Vegetable drugs with particular reference to Development of a System of Identification. J. Am. Pharmacol. Assoc. 1949: 32-38

30. Kokoshi, C.J., Kokoshi, R.J and Sharma, F.T., Fluorescence of powdered vegetable drugsunder Ultraviolet radiation. J. Pharm. Asses.1958; 47: 715-717.

31. Kokate, C.K., Practical Pharmacognosy, Vallabh Prakashan, New Delhi 1986.

32. Harborne, J.B., Methods of extraction and isolation. In: Phytochemical methods, Chapman and Hall, London 1998.

33. Sayyad Sipai Babu at al. Pharmacological Review of Urena Lobata Plant, Asian J Pharm Clin Res, . 2016; 9(2) 20-22.

34. Perez C, Paul M, BazerqueP, Antibiotic assay by Agar well diffusion method, Acta Boil Med Ex, 1990; 15: 113-115.

35. Md. Sekendar Ali et al. Antioxidant and Cytotoxic Activities of Methanol Extract of Urena lobata (L) Leaves, hepharmajournal. 2013; 2(2): 9-14.

36. Son Radu and Cheah Yoke Kqueen, Preliminary Screening Of Endophytic Fungi Frommedicinal Plants In Malaysia For Antimicrobial And Antitumor Activity, Malaysian Journal of Medical Sciences. 2002; 9(2): 23-33.

37. I.Y. Mshelia, B.M. Dalori, L.L. Hamman and S.H. Garba, Effect of the Aqueous Root Extract of Urena lobata (Linn) on the Liver of Albino Rat, Res. J. Appl. Sci., Engine. Technol. 2013; 5(1): 01-06.

38. William Kemp. Organic Spectroscopy, Palgrave Macmillan. 1991.

39. Chatwal GR, Anand SK. Spectroscopy (Atomic ad Molecular), Himalaya Publishing House. 2001.

40. Sharma BK. Instrumental method of chemical analysis, Goel Publishing House, Merrut. 2002.

41. Ergon Stahl. Thin Layer Chromatography, A Laboratory Hand Book, New York 1990.

42. Khamkar S.P. et al. hsysicochemical And Phytochemical Investigation of Herbs of Kas Pathar, International Journal of Universal Pharmacy and Bio Sciences. 2014; 3(6): 155-176.

43. Khamkar S.P, Raskar,S, Preliminary Phytochemical Investigation and Microbial Activity. International Journal of Current Pharmaceutical Sciences. 2015; 3(6): 216-226.

44. P.A. Shelar, B.V. Jawlikar M.S. Mohite, Comparative study of isolated moiety from Senna leaves, Pods and Powder, Current Pharma Research2013; 3(4): 1019-1022.

45. Chatwal G R, Anand SK. Instrumental methods of chemical analysis, Himalaya Publishing House. 1992.

46. Chatwal G R, Anand SK. Spectroscopy (Atomic ad Molecular), Himalaya Publishing House 2001.

Received on 26.03.2017 Modified on 10.04.2017

Res. J. Pharmacognosy and Phytochem. 2017; 9(2): 87-94.

DOI: 10.5958/0975-4385.2017.00016.4