P a g e | 2130

eISSN: 2231-0541 CAS CODEN: PHARN8 An EMBASE, EMCare Covered Journal PHARMANEST An International Journal of Advances in Pharmaceutical Sciences

Volume 5|Issue 3|May-June 2014|Pages 2130-2141

Review Article

A REVIEW ON PHARMACOGNISTIC, PHYTOCHEMICAL, CHEMICAL PROFILE AND APOPTOSIS INDUCTION IN YEAST CELLS OF GUAZUMA ULMIFOLIA

aUV RAMAKRISHNA, aSUKESH NARAYAN SINHA*, bNEELAM KUMARI, cVIJAY BHATNAGAR aResearch Scholar, Food and Drug Toxicology Research Centre, National Institute of Nutrition, Jamai-Osmania, Tarnaka, Hyderabad – 500007, INDIA a*Scientist-D, Food and Drug Toxicology Research Centre, National Institute of Nutrition, Jamai-Osmania, Tarnaka, Hyderabad – 500007, INDIA bNarma Niketan, Kameshwar lane, Kalambagh road, Muzaffarpur - 842002, INDIA cDepartment of Environmental Engineering, Swami Vivekanand Subharti University, Meerut – 250005, INDIA

Author for Correspondence: [email protected]

Received: 04-06-2014 Revised: 21-06-2014 Accepted: 26-06-2014 Available online: 01-07-2014

ABSTRACT

Guazuma ulmifolia Lam. belonging to the family Sterculiaceae and commonly called Pigeon wood or Bastard cedar, in Telugu known as Bhadraksh or Rudraksha. It is Native to Tropical America and widely distributed in tropical America from Mexico to northern part of Argentina & the middle part of Brazil. The bark and leaves have been traditionally used antibacterial, antidysenteric, antifungal, anti-inflammatory, antimicrobial, astringent, depurative, diaphoretic, emollient, febrifuge, hepatoprotective, pectoral, refrigerant, stomachic, styptic and sudorific. Phytochemically plant contains fatty acids, steroids, flavanoids, tannins, glycosides, carbohydrates, mucilage. Preclinical study on plant performed till date Anti-diabetic action, Anti-Hypertensive, Anti-microbial action, Anti-Oxidant, Antiulcer Action, Hair growth promoter, Weight loss agent. Key Words: Guazuma ulmifolia, Pharmacognistic, Phytochemical, Clinical, Essential Oils.

INTRODUCTION of fodder for livestock, especially at the end of Medicinal Plants, as a source of remedies, are the dry season when there are no grasses. widely used as alternative therapeutic tool for In many places farmers feed the leaves and the prevention or treatment of many diseases1. fruits to cattle. Taxonomy of plant as Kingdom: Essential oils from aromatic and medicinal Plantae, Subkingdom: Tracheobionta, Order: plants have been known to possess biological Malvales, Superdivision: Spermatophyta, activity, notably antibacterial, antifungal and Division: Magnoliophyta, Class: Magnoliopsida, antioxidant activities2,3. Biological activity of Subclass: Dilleniidae, Family: Sterculiaceae, essential oils depends on their chemical Genus: guazuma, Species: Guazuma ulmifolia composition determined by genotype and Lam 9. It is commonly known as Bastard cedar influenced by environmental and agronomic but in different languages it has different conditions 4,5. common or vernacular names in Guazuma ulmifolia Lam. belonging to the Bengali- Nipaltunth family Sterculiaceae and commonly called English- Bastard cedar, Honey fruit tree, Bastard cedar is native to tropical American Musket tree countries. It was introduced into India more Kannada- Bhadrakshi mara, Bucha, Rudrakshi than 100 years ago 6. It has naturalized to the Malayalam- Rudraksham, Uttharasham, local climatic conditions. This species has high Oriya- Debodaru economic importance. Its wood is used as fuel Tamil-Rudrasam, Tenbachai, Thenmaram, wood and charcoal, and its leaves are used as tubakki fodder for livestock. A beverage prepared from Sanskrit (Ayurveda)-Pundraaksha, Rudraakshi crushed seeds soaked in water is used to treat (fake rudraaksha) ailments like diarrhoea, dysentery, cold, cough Telugu- Rudraksha, Thene-chettu, Udrikpatta, and venereal disease. It is also used as a Siddha system- Rudraksham, diuretic and astringent 7. Rope and twine are Trade name -guazima firewood 10-12. made from the tough, fibrous bark and young Sterculiaceae family is widespread in the stems 8.In dry areas it is an important source tropics of the world, especially in America and Africa, with about 68 classes and 430 species.

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2131 Guazuma ulmifolia Lam. known as “chico- are in a branched pattern around 2.5–5 cm in magro” or “mutamba”occurs in all of Latin. It is length and are found at the bottom of the popularly used for the treatment of dandruff, leaves. The flowers come in many, are short hypercholesterolemic and to reduce13. stalked, small in size, have a brown-yellow Antimicrobial properties have been described color, five parted, 1 cm in length and have a previously from G. ulmifolia leaves and stem small fragrance to them. The calyx contains bark 14 species also showed antiulcer activity are lobed (2-3), have hairs that are brown or that is related to the presence of several light grey in color, as well as greenish. They anthocyanidins isolated from stem bark of G. have 5 petals with a yellow-like stamen, ulmifolia ethyl acetate fraction15. In addition, 15anthers per pistil, 5 stigmas (combined), this species showed promising antioxidant ovary lighter green in color with hairs, and also capacity, being related to the presence of contains a style. The fruit which have capsules phenolic compounds and flavonoids 15. The that are round to elliptical in shape are 15- literature search did not reveal any report on 25mm in length. They have many seeds which the essential oil composition of G. ulmifolia. are shaped like eggs and are 3mm in length, However, in a study conducted by Reid et al. 16 grey in color18. with five species of Sterculiaceae family, was Pharmacognistic and Phytochemical verified the presence of essential oils in three Profile species.Chemical composition of essential oil Guazuma ulmifolia is wood Tree. It is of Helicteres guazumifolia leaves commonly found in deciduous forest. It is 30 (Sterculiaceae) was investigated, and the main m in height leaves are alternate with two rows components essential oil this species are non- distributed in alternate pattern. Leaves are terpenoids volatile secondary metabolites ovate to lanceolate with toothed margin (30.28%) 17. usually texture is rough. Leaves are covered Description with small star shaped hairs. The panicles are Guazuma ulmifolia grows to 30 m in height and in branched pattern around 2.5 to 5 cm in 30–40 cm in diameter and comes with a length flowers many and are short stalked rounded crown. Leaves are distributed in an small in size, brown to yellow in colour. Seeds alternate pattern with 2 rows in assembled are brown in colour19. flatly. The leaves are ovate to lance-shaped, T.S OF LEAF finely saw toothed margin, usually have a Leaf anatomy shows upper and lower rough texture and are 6–13 cm in length and epidermis i9s composed of rectangular cells 2.5–6 cm in diameter.3-5 main veins arise with thin visible cuticle.The mesophyll tissue from the base (rounded or notched, unequal differentiated in to palisade parenchyma and sided) of the leaf which has a darker green spongy parenchyma.Trichomes are present on upper surface and a fairer green color leaves and stem they are unicellular, underneath. They are virtually hairless and on-glandular and stellate. Anisocytic stomata thin. The leaf stalks of this species are lean, are confine to abaxial surface(figure 1-2) 19. approximately 6-12mm long, and are covered with small “star-shaped” hairs18. The panicles (indeterminate flower clusters)

Fig.1.Stomata Fig.2.Trichome

T.S STEM Stem is circular in out line, differentiated in to The endodermis surrounds the vascular tissue. epidermis, cortex and conjoint, collateral open Vascular bundles are conjoint, collateral,open vascular bundles. The epidermis is single and arranged in ring. Secondary xylem is layered with in the parenchyma cells covered present. At the center thin walled by thick cuiticle.Just beneath it 2 to 3 layered parenchymatous pith is present. vessels are collenchymatous hypodermis and multilayered long with scalariform thickening on lateral parenchymatous genral cortex is present. walls and both the end wall plates are oblique(figure 3) 19.

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2132

PHYTOCHEMICAL SCREENING Photochemical screening of G.Ulmifolia fruit and leaf showed the presence of different groups of secondary metabolites viz.Alkaloids, Tannins, Sapponins, Flavonoids,Terpenoids, cardiac Glycosides,Steroids which are of medicinal importance. The aqueous extract showed positive result for most of the test compounds (Table 1) 19.

Fig.3.T.S Stem

Table.1. Photochemical screening results of G.Ulmifolia fruit and leaf

Plant Cardiac Tannins Saponins Flavonoids Terpenoids Alkaloids Steroids Parts Glycoside Fruit + + + + + + -

Leaf + + + + + + +

Leaves: Contain octacosanol, taraxerol-oac,  Dysentery, friedelin-3-á-oac, â-sitosterol, and friedelinol-  Elephantiasis, 3-acetate.  Fevers, Heartwood: Kaempferol.  Fractures, Fruit: Sweet edible mucilage. When eaten in  Gastrointestinal Pain, excessive quantities it is reported to cause  Gonorrhea, diarrhoea.  Grippe, Bark: Contains friedelin, betulin, and  Hemorrhage, â-sitosterol 19.  Hemorrhoids, ETHNOPHARMACOLOGICAL USE  Hypertension, The bark and leaves have been traditionally  Infections, used by herbal medicine practitioners for their  Influenza, following properties:  Kidney Problems,  Antibacterial,  Leprosy,  Antidysenteric,  Liver Problems,  Antifungal,  Malaria,  Anti-Inflammatory,  Nephritis,  Antimicrobial,  Pneumonia,  Astringent,  Prostate Problems,  Depurative,  Pulmonosis,  Diaphoretic,  Skin Conditions,  Emollient,  Stomach Inflammation,  Febrifuge,  Stomachache,  Hepatoprotective,  Syphilis,  Pectoral,  Ulcers,  Refrigerant,  Uterine Pain and Wounds.  Stomachic, Fruit: Diarrhea, hemorrhage, infection, uterine  Styptic, pain. Leaves: Alopecia, asthma, bruises,  Sudorific and Vulnerary 20. dermatitis, dysentery, erysipelas, fevers, Bark: inflammation, kidney diseases, liver diseases,  Alopecia, skin eruptions, skin diseases, sores, ulcers,  Asthma, wounds.  Bronchitis, Root: Childbirth, Stem bark: Diarrhea. Wood :  Bruises, furniture, panels of coaches, packing cases and  Burns, slack cooperage. It is also used as fuel and for  Childbirth, making charcoal 21.  Constipation, Bark: Demulcent, sudorific, tonic, In West  Coughs, Indies, inner bark remedy for elephantiasis,  Dematosis, decoction of inner bark used in clarifying sugar  Dermatitis, juice, disease of chest and cutaneous 22,23.  Diarrhea,

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2133 Leaf extract: An extract of the leaves used to byhydrodistillation for 3 h. The obtained reduce corpulence. essential oil was separated, dried over Seed: Astringent, carminative, antidiarrhoeal, anhydrous sodium sulphate, and stored under In Java, stomach troubles 24. argon in a sealed vial, at -20 °C before usage Extraction of the Essential oil 25. The yield in terms of percentage of the One hundred grams of plant material and 500 fresh weight of the leaves was determined. ml water were placed in a Clevenger type apparatus. The essential oil was isolated Table.2.Chemical compounds present in Guazuma ulmifolia essential oil 26

Rt (min) Compounds (%) RIa RIb Mol. Formula

3.261 α- Pinene 1.07 939 939 C10 H16 3.880 β – Pinene 0.35 981 980 C10 H16 50174 Sabinene 5.18 990 990 C10 H16 10.625 ρ – Cymene 1.56 1025 1026 C10 H14 10.851 α- Phellandrene 0.43 1028 1027 C10 H16 11.381 1,8 – Cineol 2.68 1029 1033 C10 H18 O 13.054 Y – Terpinene 3.27 1279 1251 C10 H16 16.153 Thymol 20.97 1288 1290 C10 H14 O 16.947 Carvacrol 13.76 1298 1298 C10 H14 O 18.042 Eugenol 10.13 1357 1356 C10 H12O2 19.511 Thymol acetate 0.21 1357 1355 C10 H16O2 23.760 α- Copaene 3.17 1378 1376 C15 H24 25.934 β - Cubebene 0.26 1400 1390 C15 H24 28.486 β - Elemene 2.04 1390 1391 C15 H24 28.627 Methyl eugenol 0.51 1400 1401 C11 H14O2 28.951 Bicycloelemene 1.93 1439 1435 C15 H24 28.975 β - Caryophyllene 6.74 1443 1451 C15 H24 29.002 α - Humulene 1.21 1454 1454 C15 H24 30.165 Germacrene D 2.81 1477 1480 C15 H24 31.931 Butylated hydroxytoluene 0.39 1509 1512 C15 H24 O 32.116 Camphor 0.96 1526 1521 C10 H16 O 32.471 Eugenol acetate 1.55 1537 1536 C12 H16O3 32.381 Linalool 2.12 1563 1557 C10 H18 O 33.502 Spathulenol 7.09 1577 1576 C15 H24 O 35.099 Globulol 3.56 1583 1583 C15 H26 O 35.710 Humulene epoxide 0.18 1598 1606 C15 H24 O 36.420 Y- Eudesmol 1.59 1630 1630 C15 H26 O 40.015 Cubenol 0.78 1643 1642 C15 H26 O 43.127 α - Eudesmol 0.34 1651 1652 C15 H26 O 5.613 n-Hexanol 2.73 867 867 C6 H14 O 34.701 Hexadecanol 0.28 1871 1879 C16 H34 O Total identified (%) 99.85 Relative proportions of the essential oil constituents were expressed as percentages. Rt = Retention time according their order on MS. a = Retention indices experimental (based on homologous series of n-alkane C7-C30). b = Retention indices from literature (Adams, 1995).

Table.3.Minimal inhibitory concentrations (MIC) of essential oil of the G. ulmifolia leaves26

Microorganisms Essential oil (µg/mL) P. aeruginosa 62.50 S. aureus 125.00 E. coli 500.00 S. epidermidis 750.00 C. albicans 1000.00 C. neoformans 1000.00 K. pneumoniae > 1000.00 E. faecalis > 1000.00 P. mirabilis > 1000.00 Malassezia sp. > 1000.00 Aspergillus sp. > 1000.00 Aeromonas sp. > 1000.00

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2134

Gas Chromatography–Mass Spectrometry shows maximum absorption. A solution of (GC-MS) 26 DPPH (1 mL; 0.3 mM) in ethanol (2.5 mL) was The analyses of the volatile compounds were used as a negative control and ascorbic acid in run on a Agilent Technologies AutoSystem XL the same concentrations used for the essential GC-MS system operating in the EI mode at 70 oil provided the positive control. Ethanol was eV, equipped with a split/splitless injector used to calibrate the spectrophotometer. The (250°C). Two columns were used: a HP 5MS test was performed in triplicate and the (30m x 0.25 mm; film thickness 0.25 mm) and calculation of the antioxidant activity followed an HP Innowax (30m x 0.32mm i.d., film the equation: thickness 0.50 mm). Oven temperature was % Inhibition = [(A0 - A1)/A0] x 100 programmed as following: isothermal at 70 °C Where, for 4 min, then increased to 180 °C, at a rate A0 was the absorbance of the control sample of 4 °C/min and subsequently held isothermal (without essential oil) and for 15 min (for Innowax column); isothermal at A1 was the absorbance in the presence of the 70 °C for 2 min, then increased to 200 °C, at a sample 30. rate of 3 °C/min and held isothermal for 15 Antioxidant activity of Flavonoids from min (for 5MS column). The carrier gas was leaves and apoptosis Induction in Yeast helium (1.3 ml/min). The injection port Cells temperature was 250 ºC and the detector Flavonoid fractionation of Guazuma temperature was 280 °C. Ionization of the ulmifolia Lamk. Leaf: sample components was performed in the EI Guazuma leaves were extracted by using mode (70 eV). Injected volume was 1 μl. Mabry method (Mabry et al.,1970). Leaf Identification of the Components powder was macerated in methanol/H2O 1:1 Identification of the constituents was for 24 hrs to produce 50% methanol filtrate. performed on the basis of retention index (RI), Similarly, another 100g of leaf powder was determined with reference of the homologous macerated with methanol/H2O 9:1 for 24 hrs series of n-alkanes, C7-C30, under identical to produce 90% methanol filtrate. Each was experimental conditions, comparing with the repeated 3 times. Both filtrates (50 and 90%) mass spectra library search (NIST and Wiley), were mixed and then concentrated to obtain a and with the mass spectra literature date less aqueous methanol/H2O extract. Adams 27,28 The relative amounts of individual Subsequently, the extract was partitioned components were calculated based on the CG using n-Hexane that gave n-hexane fractions peak area (FID response). and residue. Then, the residue was Antioxidant Activity fractionated with chloroform to obtain Qualitative Analysis flavonoid extract. This flavonoid extract will be Ten microlitres of 1:50 dilution of the essential diluted in 50% ethanol. oil in hexane was applied to TLC plates (silica Malondialdehyde (MDA) concentration gel 60 GF254), quercetin and ascorbic acid analysis by TBA method: (Sigma-Aldrich, ≥ 98% HPLC) standards also Standard curve was made by diluting 1,1,3,3- were used. The TLC plate was sprayed with a tetramethoxypropane (TMP) 6 M to various 0.2% 1,1-diphenyl-2-picrylhydrazyl (DPPH) concentrations series. Sample mixtures which solution in methanol and left at room consisted of 2 mL phosphate buffer 0.1 M pH temperature for 30 minutes. Active compounds 7.2mL linoleic acid 50mM in 99.8% ethanol appear as yellow spots against a purple and 1mL of extract were prepared similar to background, indicating possible antioxidant sample mixture, except that the extract was activity 29. substituted with deionized water. Control for Quantitative Analysis antioxidant activity was prepared by mixing The antioxidant activity of the essential oil was 2mL of phosphate buffer 0.1 M pH 7 with 2mL evaluated by monitoring their ability in of linoleic ahanol 99.8% which contained α – quenching the stable free radical DPPH, tocopherol in final concentration of 200ppm according to a slightly modified method and 1mL of deionized water. All mixtures previously described by Boligon et al. except the standards were incubated in the Spectrophotometric analysis was used to dark using water-bath at 40̊C for 8 days prior measure the free radical-scavenging capacity to MDA determination. The samples were and to determine the scavenging concentration incubated in the dark using water-bath at 40ºC or inhibitory concentration (IC50). The DPPH for 8 days prior to MDA determination. The quenching ability was expressed as IC50 (the samples were incubated in a water-bath at essential oil concentration (μg/mL) required to 40ºC for 8 days. One milliliter of each sample inhibit 50% of the DPPH in the assay medium). was taken and was added with 2mL Six different ethanol dilutions of essential oil at trichloroacetic acid (TCA) 20% and 2mL 250, 125, 62.5, 31.25, 15.62 and 7.81 μg/mL thiobarbituric acid )TBA) 1% in 50% acetic acid were mixed with 1.0 mL of DPPH 0.3 mM in and mixed thoroughly. Sample mixtures and ethanol solution. After 30 min, absorption was standards were placed in a hot water bath at measured at 518 nm, where the radical DPPH 100ºC for 10min. After cooling and

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2135 centrifugation at 3000rpm for 15 min, the absorbance of the samples was measured using spectrophotometer at wavelength of 532 nm.

Fig.5.No. of petite cells yeast cells on various treatments

Fig.4.AntiOxidant Activity of Guazuma ulmifolia various concentrations The same letter on the bars indicated statistical non-significance (p.0.05)

Role of falconoid extract in apoptosis induction of yeast cells31 Cells regeneration and maintenance: yeast cells were obtained from IPB culture collection (IPBCC), biology department, Bogor Agriculture University. For cell regeneration, two colonies of cells were grown on solid medium of yeast extract peptones dextrose (YEPD) which composed of 1% yeast extract, 2%pepton,2%gilcose and1.8%bactoagar. All culture media were processed form difco laboratories(Becton,Becenson and Company,Newjersey,USA) cells were incubated at 28cg for 2days. After that cells were ready to be used or stored in a refrigerator (8c)(Campbell and duff us,1988) 31. Yeast cells apoptosis induction: An ose of yeast cells which had been rejuvenated was transferred into 10ml YEPD liquid medium and placed in water bath shaker (121 RPM)for 24h at room temperature(28c).from this 200mul of cell cultures were transferred into 20ml YEPD Liquid medium .cell culture were incubated feather at 28c for 48 hours or and till the reading of optical density (OD)at wave length off 600nm measured between 1.355- 1.458.subseqently, cell cultures were centrifuged hettich centrifuge D-7200 Tuttlingen 1200,germany)at 3000RPM for 15min at low temperature 4c(and washed twice by 15ml sterile distil water. Group of cell pellets were then incubated with (1)flavonosed in ethanol, (2)glucose 4%,(3)ethanol (final concentration 1%)and (4)YEPD medium (normal cell control).except normal control cells which were incubated at 28c,other cell cultures were incubated at 37c for period off 24h(grantor et al.,2003) 31.

Fig.6-8.Morphology of Yeast cell by SEM (20 kV, 10.000x)

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2136

Viable cells and ‘petite cell’ frequency Quantitative analysis of antioxidant test: after 24 hours incubation, treated cells activity were diluted to 106 and 200µl aliquots were distributed on normal YEPED medium or petit The antioxidant activity of the essential oil was YEPD medium the composition off petit evaluated by monitoring their ability in medium consisted of 1% yeast extract,2% quenching the stable free radical DPPH, pepton,0.1%glucose and 1.8% bactoager. according to a slightly modified method 16 After incubation at 28 c for 24 h, cells which previously described by Boligon et al. . Spectrophotometric analysis was used to were growing on each Petridis were classified measure the free radical-scavenging capacity as petit cells for smaller cells and smaller and and to determine the scavenging concentration larger cells as normal cells. Petit cells or inhibitory concentration (IC50). The DPPH frequencies were calculated use in the quenching ability was expressed as IC50 (the equation: essential oil concentration (μg/mL) required to inhibit 50% of the DPPH in the assay medium). Six different ethanol dilutions of essential oil at The total colonies of yeast grown were counted 250, 125, 62.5, 31.25, 15.62 and 7.81μg/mL as viable cells. were mixed with 1.0 mL of DPPH 0.3 mM in Cells morphological examination by ethanol solution. After 30 min, absorption was scanning electron microscope (SEM): measured at 518 nm, where the radical DPPH samples were fixed by 2% guttural dehyde, shows maximum absorption. A solution of centrifuged and the supernatant was DPPH (1 mL; 0.3 mM) in ethanol (2.5 mL) was discarded. Tannic acid 2% was added to used as a negative control and ascorbic acid in samples and soaked for 12 h. after the same concentrations used for the essential centrifugation, the fixating solvent was oil provided the positive control. Ethanol was discarded and pellets were added with used to calibrate the spectrophotometer. The cacodylic buffer after soaking for 2×10min,the test was performed in triplicate and the samples were centrifuged and the buffer were calculation of the antioxidant activity followed discarded.The samples were added with the equation: % Inhibition=[(A0-A1)/A0]×100, 1%OSMUM tetroxide and soaked for 1 h, where A0 was the absorbance of the control followed by centrifugation and the supernatant sample (without essential oil) and A1 was the were discarded. the remaining samples were absorbance in the presence of the sample 30. soaked twice in 50% alcohol for 10min felled Antimicrobial Assay Determination by alcohol 70.80and 95%washing,every The essential oil was evaluated against 10min.finally,absolute alcohol was added twice Candida albicans ATCC 28967, Cryptococcus for 10min.samples were centrifuged and the neoformans ATCC 2857, Klebsiella pneumoniae solvent were discarded . Samples were soaked ATCC 700603, Pseudomonas aeruginosa ATCC twice in t-butanol for 10min, followed by 27853, Enterococcus faecalis ATCC 51299, centrifugation. After discarding the solvent, Proteus mirabilis ATCC 7002, Staphylococcus sample were resuspended in butanol and aureus ATCC 29213, Staphylococcus applied on to the frozen coverslip, dried and epidermidis, Malassezia sp., Aspergillus sp., Scand with sem (Jevol JSM 5310-lv, Tokyo, Aeromonas sp. and Escherichia coli (clinical japan) 31. isolates). The minimal inhibitory concentration (MIC) of the oil against the test microorganisms were determined by the broth micro dilution method M27-A2 32. The experiments were repeated twice and the results were determined as an average value. Six different dilutions (1000, 750, 500, 250, 125, and 62.5 μg/mL) were prepared in DMSO. Bacterial strains were cultured overnight at 37 °C in Mueller-Hinton agar. Yeasts were cultured overnight at 30 °C in Potato dextrose Fig.10.Petite colonies agar. The first column of the plate was reserved for negative control wells (without inoculants) and the last column, for the positive growth control wells (without antimicrobial agents). The MIC was considered as the lowest concentration of the essential oil inhibiting the total growth of microorganisms. MIC was detected by lack of visual turbidity (matching the negative growth control).

Fig.9.Culture of S.Cerevisiae colonies

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2137 Anti-diabetic activity work carried out on this plant, has focused on Alonso-Castro et al 33 has studied used in the bark because of its high conc. of treatment of type 2 diabetes in Mexico. Non- antioxidant proanthocyanidins. Study was to toxic concentrations of aqueous extracts (GAE) assess gastroprotective effects of aqueous were assayed on adipogenesis & 2-NBD- suspension of ethanolic extract from leaves glucose uptake in murine 3T3-F442A and flowers in model of acute gastric ulcer preadipose cell line. GAE added to adipogenic induced by diclofenac as ulcerogenic agent medium (AM) did not affect adipogenesis at using proton pump inhibitor Omeprazole as any of the tested concentrations (1-70 μg/ml), protection reference. Extract administered 2 whereas in AM lacking insulin GAE 70 μg/ml times orally to 3 groups of wistar rats at doses induced triglyceride accumulation by 23 %. of 500, 250 &125 mg/kg, with a 24-h interval On,other hand, GAE 70 μg/ml stimulated 2- between doses. Diclofenac (100 mg/kg) has NBDG uptake by 40% in insulin-sensitive 3T3- given1 h after last F442A adipocytes & by 24% in insulin-resistant administration of extract. Pretreatment with adipocytes, with respect to incorporation Guazuma ulmifolia Lam. or Omeprazole showed by insulin-sensitive adipocytes decreased ulcerated area in a dose-dependent stimulated with hormone. GAE exerts way. Myeloperoxidase activity as a marker of antidiabetic effects by stimulating glucose neutrophile in filtration slightly reduced in vivo. uptake in both model adipocytes without In vitro anti-inflammatory action clearly inducing adipogenesis. Alarcon-Aguilara et al 16 inhibited in dose dependent way. Lowest doses has reported anti-hyperglycemic effect on 28 of extract significantly decreased levels of medicinal plants. Each plant processed in lipoperoxides & superoxide dismutase action traditional way & intragastrically administered increased similar extent as with Omeprazole. to temporarily hyperglycemic rabbits. Conclude Aerial parts Guazuma ulmifolia Lam. protect eight out of 28 studied plants significantly gastric mucosa against injurious effect of decrease hyperglycemic peak and/or area NSAIDs by anti-inflammatry & radical under glucose tolerance curve. Concluded scavenging mechanisms. validity of Guazuma ulmifolia Lam their clinical Hair growth promoter use in diabetes mellitus, after their Tsutsumi et al 36 has studied depilatory toxicological investigation. inhibitor which inhibits epilation & exerts hair Anti-Hypertension activity growing and restoring effects has no injury to Magos et al 34 has studied in vivo & in vitro scalp and does not generate foul smell to cardiovascular activity of procyanidin fraction scalp. Depilatory inhibitor, hair growing & (PCF) obtained from acetone extract of restoring promoter composed of Pilocarpus Guazuma ulmifolia.Lam bark which traditionally jaborandi (Jaborandi) as a major component used as antihypertensive. 10 mg/kg PCF doses method for preventing epilation & promoting orally administered to sugar-fed hypertensive hair growth or restoring hair are implemented rats decreased both systolic arterial pressure by pilocarpine extract of leaf, trunk, branch of and the heart rate, whereas same doses i.v Pilocarpus jaborandi. Additional, plant administered induced arterial hypotension materials used in combination with other which attenuated by NG-nitro-L-arginine plants and its formulations. methylester (L-NAME 31 mg/kg) pretreatment. Nakaguchi et al 37 has studied for promoting In these experiments employed carbachol as hair growth contains extract of plant selected positive control test. PCF reduced the from including rhizomes of Curcuma contraction induced by norepinephrine (1 x 10 xanthorrhiza Roxb.(Zingiberaceae), fruits of to power-7 M) in isolated aortic rings of Helicteres isora L.(Sterculiaceae). Stems of normotensive (IC50 -35.3+12.4 ng/mL) & Tinospora crispa L. (Menispermaceae), roots of sugar-fed hypertensive (IC50-101.3 + 57.2 Vetiveria zizanioide (Poaceae), Elephantopus ng/ml) rats. Relaxant activity inhibited by scaber L (Compositae). Vitex trifolia L. either vascular endothelium removal or LNAME (Verbenaceae). Fruits of Elaeocarpus (30 μM) pretreatment, while indomethacin (10 grandiflorus J.Smith (Elaeocarpaceae), Ficus μM) or atropine (10 μM) had no effect. septica Burm(Moraceae). Fruits of Anacardium Preliminary analysis of PCF by HPLC-DAD-MS & occidentale L. (Anacardiaceae). Rhizomes of FAB-MS allowed detection of complex of Amorphophallus campanulatus Roxb.(Araceae), procyanidin oligomers consisting tetramers & rhizomes of Dioscorea hispida Roxb. trimers. Guazuma ulmifolia.Lam bark having (Dioscorea), leaves of Hibiscus tiliaceus L. long-lasting antihypertensive & vasorelaxing (Malvaceae), leaves of Guazuma ulmifolia.Lam properties linked to endothelium factors, where (Sterculiaceae), roots of Merremia mammosa nitric oxide involved. Hall. (Convolvulaceae), fruits of Sapindus rarak Anti-Ulcer Activity DC of (Sapindaceae) and leaves of Erythrina Berenguer et al 35 has studied Guazuma lithospermae folia (Leguminosae). ulmifolia Lam., member of Sterculiaceae family Weight loss agent issued in folk medicine because of its Galustyan et al 38 has studied potential of plant antioxidant, antimicrobial and anti- dry extract plant leaves, murraya leaves, hypertensive properties. Most of the research Curcuma aeruginoea Rhizoma, rhubard

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2138 rootstock, Curcuma xanthorrhizae rootstock, George et al 44 has studied the activity guided- Orthosiphonis folium rootstock, maltodextrin bioassay fractionation of 70% acetone extract and natural instant lyophilized coffee in specific of bark of Guazuma ulmifolia Lam. inhibition of component ratio effect, effective complex angiotensin II binding to AT 1 receptor led to weight loss agent. isolation & identification of bioactive oligomeric CHEMICAL REVIEW & polymeric proanthocyanidins consisting Seigler et al 39 has studied cyanogenic mainly of (-)-epicatechin units. Displacement glycoside Guazuma ulmifolia (2R)- taxiphyllin of [3H]- angiotensin II binding was dose- (>90 %), high occurs with (2S")-dhurrinin. The dependent and correlated with the degree of cyanogenic glycosides of report of identification polymerization of different fractions containing of cyanogenic compounds from the Betulaceae proanthocyanidins. Strong displacement is based on NMR spectroscopic and TLC data seen for residual fraction suggesting that most cyanogenic glucoside of Tiquilia plicata is active substances corresponding to highly dhurrin, whereas major cyanide-releasing polymerized proanthocyanidins. Angiotensin II compound of Tiquilia canescens is nitrile AT 1 receptor binding might be considered as a glucoside, menisdaurin. NMR and TLC data potentially interesting biological activity of indicate that both compounds are present in proanthocyanidins contributing to very broad each of these species. The spectrum was spectrum of biological activities of the examined by CI-MS, 1H and 13C NMR, COSY, condensed tannins. 1D selective TOCSY, NOESY, and 1J/2,3J Hoer M et al 45 studied antisecretory activity of HETCOR experiments; all carbons and protons Guazuma ulmifolia.Lam examined in rabbit are assigned. The probable absolute distal colon mounted in using chamber. configuration of (2R)-dhurrin is established by Chloride secretion stimulated by cholera toxin an Xray crystal structure. and prostaglandin E2 (PGE2). Guazuma Jastrezebski et al 40 has studied bioactive ulmifolia Lam. extract (GUE) completely compounds & antioxidant action of Prolipid, an inhibited cholera toxin-induced secretion if the herbal mixture, containing extracts of extract added to the mucosal bath prior to the Guazuma ulmifolia Lam. Muraya paniculata toxin. Adding extract after administration of (Rutaceae) and sonchus arvensis (Asteraceae). toxin had no effect on secretion. GUE did not The contents of polyphenols and flavonoids inhibit PGE2-induced chloride secretion. These were 19.87 + 2.09 and 3.09 + 0.31 mg gallic results indicate an indirect antisecretory acid equivalent GAE/g DW and 2.09 + 0.24 mechanism. SDS-PAGE analysis of cholera and 0.57 + 0.05 mg catechin equivalent CE/g toxin treated with GUE confirmed this DW in water and methanolfractions, presumption. GUE specifically interacted with respectively. Anthocyanins & flavanols were subunit of the toxin. Preliminary phytochemical found only in water fraction. Antioxidant action examinations showed that most active fraction of Prolipid as determined by 4 different contains procyanidins with a degree of antioxidant assays higher in water than in polymerization higher than 8. methanol fraction. Correlation coefficients David et al 46 has isolated cyanogenic glycoside between polyphenols, flavonoid and of Guazuma ulmifolia.Lam (2R)-taxiphyllin antiioxidant activities of Prolipid water extracts (>90%), with (2S)-dhurrin. with TEAC 0.97 & 0.90 respectively. Concluded To date, cyanogenic compounds have not been that high content of polyphenol compounds are characterized from the Sterculiaceae. The prolipidis contributor to antioxidant activity. cyanogenic glycosides of Ostrya virginiana Shekhawat et al 41 has studied primary (Betulaceae) and (2S)-dhurrin and (2R)- metabolites such as protein, lipid, starch, taxiphyllin in an approximate 2:1 ratio. This phenol, and carbohydrate in parts which are marks the first report of the identification of present in different proportion in plant species cyanogenic compounds from the Betulaceae. Guazuma ulmifolia Lam. The highest amt. of Based on NMR spectroscopic and TLC data, the sol. sugar observed. 129.0 mg/gdw, protein major cyanogenic glucoside of Tiquilia plicata is 67.0 mg/gw in leaf, lipid 42.0 in root mg/gdw. dhurrin, whereas the major cyanide-releasing Arriaga et al 42 has suggested oil from leaves compound of Tiquilia canescens is the nitrile of G. ulmifolia Lam was analyzed by GC and glucoside, menisdaurin. NMR and TLC data GC/MS. The major constituents were indicate that both compounds are present in precocene I (56.0%), caryophyllene (13.7%) each of these species. The spectrum was and (2Z,6E)-farnesol (6.6%). examined by CI-MS, 1H and 13C NMR, COSY, Arriaga et al 43 has evaluated the constituents 1D selective TOCSY, NOESY, and 1J/2,3J of Guazuma ulmifolia Lam., Sterculiaceae, a HETCOR experiments; all carbons and protons tropical american tree, were studied. Twenty- are assigned. The probable absolute four constituents (fattyacids, hydrocarbons and configuration of (2R)-dhurrin is established by a diterpene, ent-kaur-16-en-19-oic acid) were an X-ray crystal structure. The 1H NMR identified from hexane ext. of its leaves by spectrum of menisdaurin is more complex than inspection of their MS spectra and Kovats might be anticipated, containing a planar indexes. The fatty oil compound has not been conjugated system in which most elements are previously reported. coupled to several other atoms in the

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2139 molecule. The coupling of one vinyl proton to 9. http://luirig.altervista.org/schedenam/fna the protons on the opposite side of the ring m.php?taxon=Guazuma+ulmifolia involves a 6Jand a 5/7J-coupling pathway. A 10. Kirtikar KR, Basu BD: Indian Medicinal biogenetic pathway for the origin of nitrile Plants. International book distributors glucosides is proposed. Dehradun 1999; 1: 381-382. CONCLUSION 11. Anonyms: The wealth of India- A Guazuma ulmifolia Lam. widely distributed dictionary of Indian raw materials and throughout India. The plant appears to have a industrial product.NISCAIR, New Delhi broad spectrum of activity on several ailments. 2005, vol-4(f-g), 268-269. Various parts of plant have been explored for 12. Parrotta JA: Healing Plants of Peninsular Anti-diabetic, Anti-Hypertensive, Anti-microbial India. CABF publishing- international, new action, Anti-Oxidant, Antiulcer Action, Hair Delhi 2001; 685-686. growth promoter, Weight loss agent. 13. Feltrin AC, Boligon AA, Janovik V, Athayde Phytochemically plant contains fatty acids, ML (2012) Antioxidant potential, total steroids, flavanoids, tannins, glycosides, phenolics and flavonoid cantents from the carbohydrates, mucilage, and essential oil. The stem bark of Gazumaulmifolia Lam. Asian pharmacological studies reported in this review J Biol Sci 5: 268-272 confirm therapeutic value of G. ulmifolia Lam. 14. Camporese A, Balick MJ, Arvigo R, The research suggested that the flavonoid from Esposito RG, Morsellino N, et al. (2003) Guazuma ulmifolia Lamk. possess active Screening of anti-bacterial activity of compound(s) which may include apoptotic medicinal plants from Belize (Central events on yeast cells. The antioxidant activity America). J Ethnopharmacol 87: 103-107. of the flavonoid extracts positively correlated 15. Hor M, Heinrich M, Rimpler H (1996) with their ability to induce petite colonies. The Proanthocyanidin polymers with anti present review is the study to demonstrate secretory activity and proanthocyanidin that flavonoid extract from Guazumz's leaves oligomers from Guazuma ulmifolia bark. have inhibitory effect on cell proliferation, Phytochem 42: 109-119. indicating the effect of flavonoid on other 16. Reid KA, Jager AK, Ligt ME, Mulholland mechanisms in cellular activities. DA, Van Staden J (2005) Phytochemical CONFLICT OF INTEREST and pharmacological screening of Authors declare no conflict of interest. Sterculiaceae species and isolation of REFERENCES compounds. J Etnopharmacol 97:285-291. 1. Ramakrishna uv, Qualitative Assessment 17. Ordaz G, DÁrmas H, Yáñez D, Moreno S Of Antioxidant Potential In Various Dosage (2011) Chemical composition of essential Forms Of Terminalia arjuna, International oils from leaves of Helicteres guazumifolia Journal of Phytotherapy Research, Earth (Sterculiaceae), Piper tuberculatum Journals, Volume 3, Issue 3, 2013. ISSN (Piperaceae), Scoparia dulcis (Arecaceae) 2278 – 5701. and Solanum subinerme (Solanaceae) 2. Boligon AA, Schwanz TG, Piana M, from Sucre, Venezuela. Revista de Bandeira RV, Frohlich JK, Brum TF, Zadra Biologia Tropical 59: 585-595. M, Athayde ML. Chemical composition and 18. http://en.wikipedia.org/wiki/Guazuma_ul antioxidant activity of the essential oil of mifolia. Tabernaemontana catharinensis A. DC. 19. Jayshree U. Patil, S.D. Biradar, leaves. Natural Product Research 2013; Pharmacognostic study of Guazuma 27:68-71. ulmifolia, International Research journal 3. Carovic-Stanko K, Orlic S, Politeo O, of Pharmacy,2013,4(4),ISSN2230 - 8407. Strikic F, Kolak I, Milos M, Satovic Z. 20. www.rain-tree.com/Mutamba- Composition and antibacterial activities of Monograph.pdf essential oils of seven Ocimum taxa. Food 21. Sammbamurthy AVSS: Dictionary of Chemistry 2010; 119:196-201. Medicinal Plants. CBS Publishers and 4. Edris AE. Pharmaceutical and therapeutic Distributors, new delhi, 1st edition, 2005, potentials of essential oils and their 149. individual volatile constituents: A review. 22. Prajapati and Kumar: Agro’s Dictionary of Phytotherapy Res 2007; 21:308-323. Medicinal Plants. Agrobiosis, India, 2003, 5. Zouari N. Essential Oils Chemotypes: A 150. Less Known Side. Med. Aromat. Plants. 23. Kirtikar KR and Basu BD: Indian Medicinal 2013; 1:e145. Plants. International book distributors, 6. www.ias.ac.in/currsci/oct252009/1112.pdf Dehradun, 1999; 1: 381-382. 7. Vallejo MA and Oveido FJ: Arboles 24. Khare CP: Indian Medicinal Plants - An yarbustos forrajeros en América Central Illustrated Dictionary. Springer (India) 1994; 2: 676–677. Pvt. Ltd., New Delhi, India, 1st Indian 8. Little E L and Wadsworth FH: Agricultural Reprint 2007, 296-297. Handbook 1964: 338–340. 25. Carovic-Stanko K, Orlic S, Politeo O, Strikic F, Kolak I, et al. (2010)

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2140 Composition and antibacterial activities of ulmifolia bark in normotensive and essential oils of seven Ocimum taxa. Food hypertensive rats. Journal of Chem 119: 196-201. Ethnopharmacology 2008; 117(1): 58-68. 26. Aline Augusti Boligon, Andrieli 36. Felipe AMM, Rincao VP, Benati FJ, CasselFeltrin, Amanda Leitao Gindri and Linhares REC ,Galina KJ, de Toledo CEM, Margareth Linde Athayde, Essential Oil Lopes GC, deMello JCP, Nozawa C: composition, Antioxidant and Antiviral effect of Guazuma ulmifolia and Antimicrobial Activities of Guazuma Stryphnodendron adstringens on ulmifolia from Brazil, Medicinal & Aromatic poliovirus and bovineherpesvirus. Plants, 2013, 2:3, ISSN: 2167-0412. Biological & Pharmaceutical Bulletin 2006; 27. Adams RP (1995) Identification of 29(6): 1092-1095. essential oil components by Gas 37. Kaneria M, Baravalia Y, Vaghasiya Y, Chromatography/Mass spectroscopy. Chanda S: Determination of antibacterial Allured Publishing Corporation: Illinois and antioxidant potential of some USA 456. medicinal plants from saurashtra region, 28. Mahlke JD, Boligon AA, Machado MM, India. Indian Journal Pharmaceutical Athayde ML (2013) Composition of the Science 2009; 71(4):406-12. essential oil of Buddleja thyrsoides Lam. 38. Navarro MC, Montilla MP, Cabo MM, leaves from Brazil. Nat Prod Res. Galisteo M, Cáceres A, Morales C, Berger 29. Boligon AA, Schwanz TG, Brum TF, I: Antibacterial, antiprotozoal and Frohlich JK, Nunes L, et al. (2012) antioxidant activity of five plants used in Chemical composition, antioxidant and Izabal for infectious diseases. antimicrobial activities of the essential oil Phytotherapy Research 2003; 17 (4): of Scutiabuxifolia Reissek leaves. 325-9. Pharmaceut Anal Acta 3: 199-202. 39. Yoshitani S, Yoshimi F, Tabata T, 30. de Brum TF, Boligon AA, Frohlich JK, Haraguchi H: Inhibitor of lipid peroxide Schwanz TG, Zadra M, et al. (2013) production and composition containing the Composition and antioxidant capacity of same/A lipid peroxide production inhibitor the essential oil of leaves of Vitex containing Guazuma ulmifolia extract. Jpn megapotamica (Sprengel) Moldenke. Nat Kokai Tokkyo Koho 2000. Prod Res 27: 767-770. 40. Berenguer B , Trabadela C, Sanchaz- 31. Syaefudin, W.T Wahyuni, I.M. Artika & Fidalgo S , Quilez A, Mino P, De la Puerta Sulistiyani, Antioxidant activity of R, Martin Calero MJ: The aerial parts of Flavonoid from Guazuma ulmifolia Lamk. Guazuma ulmifolia Lam. protect against Leaves and Apoptosis Induction in Yeast NSAID-induced gastric lesions.Journal of Cells, Journal of Biological Sciences 14 Ethnopharmacology 2007 ;114(2): 153- (4): 305-310, 2014. ISSN 1727 - 3048. 160. 32. National Committee for Clinical Laboratory 41. Tsutsumi, Shigetoshi: Depilatory inhibitor, Standards (2002) Reference method for hair growing and restoring promoter, broth dilution antifungal susceptibility depilatory preventive and hair growing testing of yeast: approved standard, and restoring promotion/Pilocarpus document M27-A2 Methods for dilution jaborandi and its uses in inhibiting and antimicrobial susceptibility tests for preventing depilation and promoting hair bacteria that grow aerobically: document growth. Jpn. Kokai Tokkyo Koho 1999. M7-A5, Wayne, PA. National Comittee for 42. Nakaguchi O, Okamoto H, Matsuyama Y, Clinical Laboratory Standards. Hashigaki T, Sakano T, Katsata, Masanori: 33. Alonso-Castro AJ, Salazar-Olivo LA:The Hair-growing agent composition/novel use anti-diabetic properties of Guazuma of plant extract as hair growth promoter. ulmifolia Lam are mediated by the Jpn. Kokai Tokkyo Koho; 2001. stimulation of glucose uptake in normal 43. Galustyan VA: Weight loss agent. Russ and diabetic adipocytes without inducing 2007. adipogenesis. Journal of 44. Seigler DS, Pauli GF, Frohlich R,Wegelius Ethnopharmacology 2008 ;118(2): 252- E, Nahrstedt A, Glander KE, Ebinger JE: 256. Cyanogenic glycosides and menisdaurin 34. Alarcon-Aguilara FJ, Roman-Ramos R, from Guazuma ulmifolia, Ostrya Perez-Gutierrez S, Aguilar-Contreras A, virginiana, Tiquilia plicata, and Tiquilia Contreras-Weber CC, Flores-Saenz J L: canescens. Phytochemistry 2005; 66(13): Study of the anti-hyperglycemic effect of 1567-1580. plants used as antidiabetic. Journal of 45. Jastrezebski Z, Tashma Z, Katrich E, ethnopharmacology1998;61(2):101-10. Gorinstein S: Biochemicalcharacteristics of 35. Magos GA, Mateos JC, Paez E, Fernandez the herb mixture prolipid as a plant G, Lobato C, Marquez C, Enriquez RG: foodsupplement and medicinal remedy. Hypotensive and vasorelaxant effects of Plant Foods for Human Nutrition 2007; the procyanidin fraction from Guazuma 64(4): 145-150.

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net

P a g e | 2141 46. Shekhawat N, Vijayvergia R: Comparative study of primary metabolites in different plant parts of Clitoria ternatea (L.), Guazuma ulmifolia (Lam.) & Madhuca indica (Gmel). Journal of Chemical and Pharmaceutical Research 2010; 2(2): 168-171.

HOW TO CITE THIS ARTICLE aUV Ramakrishna, aSukesh Narayan Sinha*, bNeelam Kumari, cVijay Bhatnagar.(2014 July1). A Review on Pharmacognistic, Phytochemical, Chemical Profile and Apoptosis Induction in Yeast Cells of Guazuma Ulmifolia. PHARMANEST,5(3),2130-2141. http://www.pharmanest.net

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 3| May-June 2014 Available online: http://www.pharmanest.net