names bium dulce (Roxb.) Benth. chili Perr. Willd. olata Blanco lcis Roxb.

guis-cuti Blanco cis Roxb.

Kamatsile Pithecellobium dulce (Roxb.) Benth. SWEET TAMARIND Niu ti dou Common names Camachile (Pamp.) Chamultis (Ig.) Damortis (Ilk.) Damulkis (Bon.) Kamachili (Tag., Bik.) Kamachilis (Tag.) Kamanchilis (P. Bis., Mag.) Kamansile (Tag.) Kamantilis (Pang.) Kamarsiles (Tag.) Kamatsile (Tag.) Kamatsele (Tag.)

Kamonsiles (Tag.) Kamunsil (P. Bis.) Karamansili (Ibn.) Komonsili (P. Bis.) Komontos (Ting.) Komontres (Ting.) Madras thorn (Engl.) Monkeypod (Engl.) Sweet tamarind (Engl.) Manila tamarind (Engl.) Niu ti dou (Chin.)

Gen info / Etymology Referred to as manila tamarind because of the sweet-sour tamarind-like taste. Genus Pithecellobiumderives from from the Greek words 'pithekos' (ape) and 'lobos' (pod), and the species name 'dulce' from the Latin 'dulcis' meaning sweet. Botany Kamatsile is a tree 5 to 18 meters high, with pendulous branches, with short, sharp stipular spines. The leaves are evenly 2-pinnate, 4 to 8 centimeters long. The flowers are white, in dense heads, 1 centimeter in diameter. Pods are turgid, twisted, and spiral, 10 to 18 centimeters long, 1 centimeter wide, and dehiscent along the lower suture. Seeds are 6 to 8, with an edible, whitish, pulpy aril. The arillus is sweet when the fruit is ripe. Distribution Found throughout the Philippines at low or medium altitudes. Constituents - Tannin, 25.36%; fixed oil, 18.22%, olein. - A glycoside quercitin has been isolated. - Seeds have been reported to contain steroids, saponins, lipids, phospholipids, glycosides, glycolipids and polysaccharides. - Bark yields 37% tannins of the catechol type. - Leaves yield quercitin, kaemferol, dulcitol and afezilin. Properties - Considered abortifacient, anodyne, astringent, larvicidal, antibacterial, antiinflammatory, febrifuge, antidiabetic. - Bark considered astringent.

- Leaves considered astringent, emollient, antidiabetic, and abortifacient. - Roots reported to be estrogenic.

Parts used and preparation Bark, leaves. Uses Edibility Pulp around the seed is edible. Folkloric Frequent bowel movements: Decoction of bark taken as tea. The leaves, when applied as plasters, used for pain, venereal sores. Salted decoction of leaves, for indigestion; also used as abortifacient. Bark used in dysentery, dermatitis and eye inflammation. In Brazil, P. avaremotem, used as a cancer elixir. In Mexico, decoction of leaves for earaches, leprosy, toothaches and larvicide. In India, bark of the plant used as astringent in dysentery, febrifuge. Also used for dermatitis and eye inflammations. Leaves used as abortifacient. In Guiana, root bark used for dysentery and as febrifuge. Studies Anti-Inflammatory / Antibacterial:Study of the fresh flowers of Pithecellobium dulce yielded a glycoside quercitin. The activity of the flavonol glycoside confirmed its antiinflammatory and antibacterial properties. Phenolics / Antioxidant: Free Radical Scavenging Activity of Folklore: Pithecellobium dulce Benth. Leaves:Study of the aqueous extract of Pithecellobium dulce leaves revealed phenolics including flavonoids and showed potent free radical scavenging activity.. Anti-inflammatory Triterpene: Anti-inflammatory triterpene saponins of Pithecellobium dulce: characterization of an echinocystic acid bisdesmoside. A new bisdesmodic triterpenoid saponin, dulcin, was isolated from the seeds of PD Genotoxicity: Mutagenic and Antimutagenic Activities in Philippine Medicinal and Food Plants: In a study of 138 medicinal plants for genotoxicity, Pithecellobium dulce was one of 12 that exhibited detectable genotoxicity in any system. Anti-tuberculosis / Antimicrobial: Hexane, chloroform and alcoholic leaf extracts were studied for activity against Mycobacterium tuberculosis strains. The alcoholic extract showed good inhibitory activity and antimicrobial activity against secondary pathogens. Anti-Diabetic: Study of ethanolic and aqueous leaf extract of P dulce in STZ-induced diabetic model in rats showed sigificant activity, aqueous more than the alcoholic extract, comparable to glibenclamide. Anti-Ulcer / Free Radical Scavenging: Study of the hydroalcoholic extract of PD was found to possess good antioxidant activity and suggests possible antiulcer activity with its free-radical scavenging and inhibition of H, KATPase activities comparable to omeprazole. Phytochemical screening yielded flavonoids - quercetin, rutin, kaempferol, naringin, daidzein. Hepatoprotective: Study of an aqueous extract of P. dulce in a murine model showed hepatoprotection against CCl4induced oxidative impairments probably through its antioxidative property. Results were supported by histological findings. CNS Depressant: Study evaluating the locomotor activity of aqueous and alcoholic extracts of PD in albino mice

showed significant CNS depression, the alcoholic extract exhibiting greater effect when compared to chlorpromazine. The activity was attributed to an increase in the concentration of GABA in the brain. Analgesic / Anti-Inflammatory: Study of methanol extract showed significant anti-inflammatory and analgesic effects comparable to standard drugs. Availability Wild crafted. Last Update September 2011 Additional Sources and Suggested Readings (1) Phytochemical Investigation and Pharmocological Studies of the Flowers of Pithecellobium dulce / P G R Chandran and S Balaji / Ethnobotanical Leaflets 12: 245-253. 2008. (2) Free Radical Scavenging Activity of Folklore: Pithecellobium dulce Benth. Leaves / M Sugumara et al / Ethnobotanical Leaflets 12: 446-451. 2008. (3) Anti-inflammatory triterpene saponins of Pithecellobium dulce: characterization of an echinocystic acid bisdesmoside / N P Sahu, S B Mahato / Phytochemistry. 1994 Nov; 37(5): 1425-7 (4) Mutagenic and Antimutagenic Activities in Philippine Medicinal and Food Plants / Clara Y Lim-Sylianco and W Thomas Shier / Toxin Reviews, Volume 4, Issue 1 1985 , pages 71 - 105 / DOI: 10.3109/15569548509014414 (5) Pithecellobium Dulce Benth - A Review / M Sugumaran (6) Manila Tamarind could help treat tuberculosis / S D Shanmugakumar / Natural News Today (7) Antidiabetic potential of aqueous and alcoholic leaf extracts of Pithecellobium dulce / M Sugumaran et al / Asian J. Research Chem.. 2(1): Jan..-Mar. 2009 (8) Free radical-scavenging and H+, K+-ATPase inhibition activities of Pithecellobium dulce / J Megala and A Geetha / Food Chemistry, Vol 121, Issue 4, 15 August 2010, Pages 1120-1128 / doi:10.1016/j.foodchem.2010.01.059 (9) Phytomedicinal Role of Pithecellobium dulce against CCl4-mediated Hepatic Oxidative Impairments and Necrotic Cell Death / Prasenjit Manna, Sudip Bahttacharyya, Joydeep Das et al / Evidence-Based Complementary and Alternative Medicine, Volume 2011 (2011) / doi:10.1093/ecam/neq065 (10) Locomotor Activity of Leaf extracts of Pithecellobium dulce Benth. / M. Sugumaran, T.Vetrichelvan, S. Darline Quine / Ethnobotanical Leaflets 12: 490-493. 2008. (11) Analgesic and anti-inflammatory activities of leaf extract of Pithecellobium dulce Benth. / S.Arul Selvan, P.Muthukumaran / International Journal of PharmTech Research, Vol.3, No.1, pp 337-341, Jan-Mar 2011

Ethnobotanical Leaflets 12: 245-253. 2008.

Phytochemical Investigation and Pharmocological Studies of the Flowers of Pithecellobiumdulce P.G.R. Chandran1 and S. Balaji2 1 Lecturer in Chemistry, School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamilnadu-613402, India 2 Lecturer in Bioinformatics, Department of Biotechnology, Manipal Institute of Technology, Manipal University, Manipal-576104, India
2

Corresponding Author: Tel: mobile-9895592347 E-mail: blast_balaji@rediffmail.com Issued 24 May 2008

ABSTRACT To evaluate the effects from the fresh flowers of Pithecellobium dulce (Roxb.) Benth, belonging to the family ofLeguminosae subfamily Mimosoideae, a glycoside quercitin has been isolated. The ethyl acetate soluble of P. dulcecontaining the above glycoside was studied both in silico and in vitro for the anti-inflammatory and antibacterial properties. The concatenation of the in silico and in vivo has been done. Results indicated the activity of this flavonolglycoside in the protection of HRBC lysis and against the gram positive micro organisms, thus confirming its anti-inflammatory and anti-bacterial properties. INTRODUCTION Pithecellobium dulce (Roxb.) Benth, belonging to the family of Leguminosae, subfamily Mimosoideae, is a small evergreen thorny tree1. The tree is reported to be active against veneral diseases2. The decoction is given as enema3. The seeds contain saponin4. As there is no work on the flowers of P. dulce, the flowers of same have now been examined for their poly-phenolic constituents. The crude extract of the flowers has also been investigated in silico and in vitro for their anti-inflammatory and antibacterial properties and our results are presented in this communication. METHODOLOGY

From the fresh flowers (300g) of P. dulce collected from Thanjavur district of Tamilnadu, India, during February, were dried in shade and extracted with 80% ethanol in cold. The combined alcoholic extract was concentrated in vacuum and the aqueous concentrate was successfully fractionated with benzene, peroxide free Et2O and EtOAc. The benzene fraction did not yield any crystalline solid. The residue from the EtOAc fraction was taken up in minimum quantity of Me2CO and left in an ice chest for about a week when yellow solid separated. On re-crystallization from aqueous methanol, it came out as pale yellow needles, m.p 313-315C (yield 0.01 %). 255,269sh, 370 ;(MeOH+NaOMe):262sh,321,420(dec.);(MeOH+AlCl3): 267,303,458 MeOH+AlCl3+HCl):267,303,351,428;(MeOH+NaOAc): 275,328,390 ;( MeOH+NaOAc+H3BO3):262,303sh, 386; red color with Mg-HCl5, Olive green color with alc.Fe3+, golden yellow color fluorescence with conc.H2SO4, yellow under UV/NH3, responded to HorhammerHansel 19, Wilsons boric acid test 20 and Gibbs test21 test indicative of a flavonol 3-O glycoside and the identity was confirmed by co- and mixed PC with an authentic sample of quercetin. The 13C- NMR data of the isolated compound is given as follows: with NH3 and NaOH, yellow solution with pale green ;(

13

C-NMR (125MHz,DMSO-D6,TMS) ppm:157.3(C-2),134.2(C-

3),178.1(C-4).162.1(C-5),99.5(C-6),165.0(C-7),94.3(C-8),157.0(C-

9),104.8(C-10),121.8(C-11),116.0(C-21),145.6(C-31),148.9(C-41),116.0(C51) and 122.0(C-61)

The UV and 13C- NMR spectral data were in agreement with the flavonol quercetin. A solution of the glycoside was hydrolyzed (7%H2SO4, 100 C, 2 hr.) and the aglycone was characterized as quercetin(m.m.p, uv data, Rf, acetate and methyl ether) and the sugar was identified as rhamnose. A quantitative hydrolysis of the same by the Folin-wus micro method 22 revealed to be a monoside. The glycoside was thus characterized as quercetin 3O rhamnoside (quercitrin) and the identity was confirmed by direct comparison with an authentic sample of quercitin. Based on the UV and 13CNMR spectral data, it is crystal clear that the isolate is flavonol quercetin. The structure of thequercitrin was drawn by using ACD-3D ChemSketch.5 Then we performed the conversion of the drawn chemical structure into SMILES 7 notation, so as to predict biological activity and to find similar chemical compounds. The SMILES notation used for screening similar compounds and biological activity is given below. C5(O)C(O)C(O)C2[O+]([H]C1C(O)C(O)CCC1C3C(O2)C(O)C4C(O)CC(O)CC4O3)C5 To find similar chemical compounds we have screened the compounds from NCI. 8 For predicting biological activity we used PASS. 6 RESULTS AND DISCUSSION Quercetin 3-O-rhamnoside (quercitrin) see figure 1, has been isolated from the fresh flowers of P. dulce. The UV spectrum of the glycoside exhibited two major absorption peaks at 350 nm (band I) and 256 nm (band II). The band I absorption of the glycoside is reminiscent of a flavonol skeleton. A comparison of band I absorption of the glycoside and that of the aglycone revealed that there may be 3-glycosilation in the flavonol. A bathochromic shift of 43 nm (band I) inNaOMe confirmed the presence of

a free OH at C-41 .The AlCl3 spectra (with and without HCl) showed four absorption peaks to reveal the presence of a free 5-OH group. It was confirmed by the bathochromic shift of 50 nm on the addition of AlCl3-HCl in the glycoside. The presence of a free OH group at C-7 was evident from the +16 nm (band II) shift on the addition of NaOAc. The band I absorption in AlCl3 spectrum is 30 nm more than that noticed on addition of AlCl3-HCl.This is indicative of the existence of an O- dihydroxyl group in the B-ring. In the 1H-NMR spectrum (400MHz,DMSO-D6,TMS) the signal at 6.47 and 6.42 ppm corresponds to the A-ring protons at C-8 and C-6.The 5_OH protons resonates at 12.56 ppm.The proton C-51 appears at 6.86 ppm as a doublet. The signal 10.88 ppmcan be traced to the-OH at C-7 and C-21.The C-61 protons show up at 7.30 ppm.The methyl protons of rhamnosidemoiety resonate at 1.18 nppm and the H-1 of rhamnoside resonates at 5.31 ppm.The remaining sugar protons appear in the range of 3.37-4.0 ppm Supporting evidence for the structure of the glycoside was provided by the analysis of 13C-NMR (100MHz, DMSOD6, TMS) data. Due to glycosilation at 3-position, the C-2 and C-4 carbons absorb at 157.3 and 178.1 ppm respectively.

Figure 1: Quercitrin structure. The stick model shown in this picture (color by CPK) is based on the actual chemical structure provided by the analysis of 13C-NMR (100MHz, DMSOD6, and TMS) data.

Thus on the basis of the above mentioned Physical and Chemical evidences the glycoside obtained from P. dulce has been characterized as quercetrin. Biological activity prediction is pivotal in any structure prediction. Based on the UV and 13C- NMR spectral data, it is crystal clear that the isolate is flavonol quercitrin. We were much interested to predict its biological activity. So as to predict structure activity relationship, for that the structure of the quercitrin was drawn by using ACD-3DChemSketch v 5.12 5. We have performed in silico studies like chemical structure similarity search and the Prediction of Activity Spectra for Substances: PASS 6 prediction was done by converting the quercetin structure into the Simplified Molecular Input Line Entry System (SMILES notation) 7 proposed by Dave Weininger (Weininger, 1988). Finally we have found some similar structures in enhanced NCI database browser release 2. 8 The obtained quercitrin structure was submitted for Pass prediction and we obtained the 41 substructure chemical descriptors. We have taken Pa >0.7 and found that the highest hit had predicted membrane integrity agonist and anti-inflammatory activities. Because if the Pa >0.7, the substance is very likely to exhibit the activity in experiment. The list of predicted properties by PASS Pa >0.7 is given in the Table 1. The reliable effects and mechanisms are listed in Table 2, and 3.

Pa 0,969 0,891 0,885 0,849 0,751 0,731 0,728 0,725

Table 1: PREDICTED ACTIVITY Pi Activity 0,004 0,004 0,002 0,008 0,007 0,006 0,005 Membrane integrity agonist Membrane permeability inhibitor Capillary fragility treatment Topoisomerase II inhibitor Emetic Sweetener Osmotic diuretic

0,006 Vascular (periferal) disease treatment

Table: 2. PREDICTED RELIABLE EFFECTS. Pa Pi Activity:

0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969 0.969

0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004

Membrane integrity agonist Antiinflammatory Antibacterial Psychotropic Antiepileptic Immunostimulant Antiviral (HIV) Antiviral (herpes) Antineoplastic Antiprotozoal Dermatologic Antieczematic Antiseborrheic Antiischemic Antiischemic renal

Table: 3. PREDICTED MECHANISMS Pa 0.969 0.969 0.969 0.969 0.969 0.969 0.969 Pi 0.004 0.004 0.004 0.004 0.004 0.004 0.004 Activity Membrane integrity agonist Antiinflammatory Antineoplastic Antifungal Antiviral Antiseborrheic Antiviral (HIV)

So from the in silico predicted information, we have decided to test the activity in vitro especially for the membrane permeability inhibitor (Table-1), membrane integrity agonist (Table 1, 2, 3), anti-bacterial effects(Table-2) and anti-inflammatory mechanisms (Table-2 & 3). ANTI-INFLAMMATORY STUDIES

Lysosomal enzymes play an important role in the development of acute and chronic inflammation.9 Increased enzyme activity has been reported in certain types of experimental inflammation.10 The inhibitory effects of nonsteroidal antiinflammatory drugs on lysosomal enzymes have been proposed as an explanation for one of their many mechanisms of actions in vitro.11 Acidic anti-inflammatory compounds such as phenyl butazone, Mefenamic acid and indomethacin have been shown to exert their beneficial effect by inhibiting the activities of either released lysosomal enzyme or by stabilizing thelysosomal membrane12-14. It has been reported that the structure of RBC is similar to that of lysosomal membrane components.15 Since lysosomal membranes resemble human RBC (HRBC) membranes, the lysosomal membrane effects have been studied using HRBC. When the RBC is subjected to hypotonic stress, the release of haemoglobin from RBC is prevented by anti-inflammatory drugs because of the membrane stabilization. Hence the HRBC membrane stabilization by drugs against hypotonicity induced haemolysis serves as a very useful in vitro method for assessing the anti-inflammatory activity of compounds. In this present investigation, an in vitro study of the EtOAc isolates of P.dulce by finding the stabilization of the HRBC membrane against hypotonicity induced haemolysis has been made,16 the results are indicated in the Table 4 and the Graph-I. From the results the in silico predicted membrane permeability inhibitor (Table-1), membrane integrity agonist (Table 1, 2, 3), and anti-inflammatory mechanisms (Table-2 & 3) have been understood in vitro. Table: 4: Stabilization Effect of isolates of P.dulce on the HRBC membrane stabilization against hypotonicity induced haemolysis.

Sl. No 1 2 3

Concentration of the drug in mg 10 50 100

Percentage protection 40 58 64

4 5

250 500

70 90

Based on the in vitro Stabilization Effect of isolates of P.dulce on the HRBC membrane stabilization against hypotonicityinduced haemolysis, the tabular values are plotted as a graphical representation. (Graph-I) Graph-I: The in vitro Stabilization Effect of isolates of P.dulce on the HRBC membrane stabilization against hypotonicityinduced haemolysis. The concentration of the drug (in mg) used in the protection of HRBC membrane is plotted in the graph.

The crude extract was observed to be effective in stabilizing the HRBC membrane against hypotonicity induced haemolysisand hence would be effective as non steroidal anti-inflammatory compounds in the control of

inflammation. With in the experimental range of dosages of (10 to 250 mg /ml) the flavonoid drug exhibited 70% protection at 250 mg dose and at subsequent doses, the protection increases and reached a maximum with a sharp increase at 500 mg. At higher concentrations the activity climbs up showing the anti-inflammatory activity of this flavonoid drug under in vitro experimental conditions dependent upon the concentration of the drug. The membrane is stabilized by the flavonoidal drug at a concentration of 500 mg.

ANTI-MICROBIAL STUDIES In this investigation , the anti- bacterial activity of the residue of the EtOAc fraction containing the flavonoid glycoside isolated from the flowers of P.dulce have been studied in gram negative vitro by Petri-dish Escherichia organisms. method coli Anti-

using Staphylococcus aureus a and Salmonella typhii two gram

positive, as test

bacterials produce their effect by interfering with one or more vital metabolic pathways in the organism. The object of the treatment with an anti-microbial drug which is higher than the minimal effective concentration and which is maintained at that level until the organisms have been eliminated 17.The extracts of various medicinal plants containing flavonoids have been reported to possess anti- bacterial activity 18.A standard volume (2.5mL) of MuellerHinton agar medium that would support the growth of the test organisms was added to sterile Petridishes. Solutions of the test compound (EtOAc residue) at six different concentrations viz., 25, 50,100,200,300 and 400 mg/mL in sterile water were prepared. Standards containing streptomycin at concentration of 50,100 and 200 mg/mL and a control containing no drug were prepared. A standard inoculum of a suspension of turbidity equal to a McFarland standard 0.5 of the test organism was added to all Petridishes. After inoculation, the plates were incubated at 37C and minimum inhibitory concentration (MIC) is found out after 48 hours of incubation. The number of colonies that grow on this subculture is then counted and compared to the

number of CFU/mL(Colony Forming Units) in the original inoculum. In the anti-microbial studies only traces of the growth has been observed at a lower concentration of the drug. The growth of the organism is inhibited with higher concentration. ACKNOWLEDGEMENTS The authors express their thanks to their Dean, Dr. K .N. Somesekharan, and Dr. D. Venkappayya, Professor, School of Chemical & Bio Technology, SASTRA Deemed University, Thanjavur, Tamilnadu for their keen interest and constant encouragement .They are also grateful to Dr.K.M.Matthew, Rapinat herbarium, St.Josephs college, Tiruchiraapalli f or his help in the identification of the plant. REFERENCES 1. Allen, O. N. and Allen, E. K, The Leguminosae, A source book of characteristics, uses and modulation, Wisconsin press, Wisconsin, 1981, 812. 2. Duke, J. A. and Wain, K. K., Medicinal plant of the world, Computer index with more than 85,000 entries, 3vols, 1981. 3. 4. 5. 6. Chopra, R. N.; Nayar, S. L.; Chopra, I. L., The Glossary of Indian Medicinal Plants, C.S.I.R, New Delhi, 1956, 196. Anonymous. The Wealth of India, Raw materials, C.S.I.R, NewDelhi, 1969, vol.140, P8. Spessard, G. O., ACD labs/logP dB 3.5 and Chemsketch 3.5. J. Chem. Inf. Comput. Sci. 1998, 38, 218 230. Lagunin, A.; Stepanchikova, A.; Filimonov, D. and Poroikov, V., PASS: prediction of activity spectra for biologically active subsatances. Bioinformatics. 2000, Vol 16, (No. 8), P 747748, Oxford University Press. Weininger, D., "SMILES .3. depict: graphical depiction of chemical structures". J. Chem. Inf. Comp. Sci., 1990, 30(No.3), 237243. "Frederick/Bethesda Data and Online Services" and NCI website references: http://cactvs.cit.nih.gov/about3.html and

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