Evaluation of the effect of Cassia surattensis Burm. f., flower methanolic extract on the growth and morphology of Aspergillus niger

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European Review for Medical and Pharmacological Sciences

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Abstract. – BACKGROUND AND AIM: Cassia(C.) surattensis Burm. f. (Leguminosae), a medici-nal herb native to tropical equatorial Asia, wascommonly used in folk medicine to treat variousdiseases.The aim of the present study is to inves-tigate the effects of methanolic flower extract of C.

surattensis against Aspergillus (A.) niger .MATERIALS AND METHODS: Antifungal ac-

tivity of C. surattensis flower extract was studiedby using agar disc diffusion method, broth dilu-tion method, percentage of hyphal growth inhibi-tion and scanning electron microscopy (SEM)observation.

RESULTS:The extract exhibited good antifun-gal activity with zone of inhibition 15 mm and

minimum inhibitory concentration (MIC) 6.25mg/ml. The flower extract exhibited considerableantifungal activity against A. niger with a IC50 of2.49 mg/ml on the hyphal growth. In scanningelectron microscopy (SEM) squashed, collapsed,empty and deformation of hyphae were the ma-jor changes observed. Shrunken conidiophoreswere the obvious alteration on the spores. Mor-phological alterations observed on A. niger caused by the flower extract could be the contri-bution of chemical compounds present in theCassia flower. Phytochemical screening revealsthe presence of carbohydrate, tannins, saponinsand phenols in the extract.The amount of tannin,total phenolics and flavonoids were estimated tobe 55.14 ± 3.11 mg/g, 349.87 ± 5.41 mg/g gallicacid equivalent and 89.64 ± 3.21 mg/g catechinequivalent respectively.

CONCLUSIONS: C. surattensis flower extractpotently inhibited the growth of A. niger and are,therefore, excellent candidates for use as thelead compounds for the development of novelantifungal agents.

Key Words: Cassia surattensis , Aspergillus niger , Antifungal,

Phytochemical screening.

Evaluation of the effect of Cassia surattensis Burm. f., flower methanolic extract on the

growth and morphology of Aspergillus niger

V. SUMATHY, Z. ZAKARIA, Y. CHEN1, L.Y. LATHA 2, S.L. JOTHY 2,S. VIJAYARATHNA 2, S. SASIDHARAN2

Biological Program, School of Distance Education, Universiti Sains Malaysia, Minden, Penang, Malaysia1Dental Research and Training Unit, and Oral Cancer Research and Coordinating Centre (OCRCC),Faculty of Dentistry, Universiti of Malaya, Kuala Lumpur, Malaysia2Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Pulau Pinang,Malaysia

Corresponding Author: S. Sasidharan, Ph.D.; e-mail: [email protected]

Introduction

In most of the Asian tropical countries likeMalaysia and Indonesia floral diversity has beenan important asset to mankind to be explored forvarious therapeutic purposes. Since ancient days,these medicnal plants and herbs were used fortherapeutic purposes due to the presence of phy-tochemical with various medicinal properties.Literature has proven 65% of the world’s popula-tion depended on medicines derived from natural

product such as herbs and edible plants

1

. Naturalresources are always favorable especially thosewhich are edible because they are rich with phy-tochemicals, vitamins and minerals. On the con-trary, it is not deniable that some medicinalplants can be toxic and poisonous to human if proper dose is not consumed.

Cassia surattensis Burm. f., shrub belongs tothe family of Leguminosae and locally called asbushy cassia. It is commonly found along theroadside in Malaysia. The height of this shrub isnormally about 6 m tall with bipinnate and broadleaves. C. surattensis is a very popular herb

amongst practitioners of traditional medicine,widely used as a decoction or infusion to treatvarious ailments. In Chinese traditions, leaves areused to treat constipation problem, sore throatand cough by consuming the infusion of boiledleaves orally. A study on antioxidant assessmenton C. surattensis flowers proved the flowers havegood antioxidant property2. Bark and leaves of this shrub is believed to be antihemorrhagic. It isalso believed that Balinese rub the leaves of C.surattensis into both external and internal cool-ing medicine3. Until today very little literature

has been reported on the medicinal properties of

2013; 17: 1648-1654



C. surattensis. Therefore, the present study wasconducted to evaluate the antifungal properties of C. surattensis flower against human pathogenicmicroorganism A. niger .

Materials and Methods

Plant Collection and Extraction C. surattensis flowers were collected in Uni-

versiti Science Malaysia, Penang. The flowerswere washed under the running water and driedin the oven at 50°C for 24 h. Dried flowers weremilled to a fine powder using an electronicblender1. 200 g of the powdered plant materialwere macerated and percolated with 600 mL of

methanol in a beaker for 4 days. The solvent wasthan filtered using No. 1 Whatman filter paperand evaporated using rotary evaporator. Crudeextract obtained in a paste form was stored atroom temperature for further use.

Bacterial and Fungal Test Organism Aspergillus niger used in this study was ob-

tained from the School of Biological Sciences,USM Penang. The fungus was maintained inPotato Dextrose Agar (PDA) at 4°C until fur-ther use.

Antimicrobial Assay Agar disc diffusion method was used for anti-

fungal susceptibility test. Inoculums of spore sus-pension spread uniformly on the plate. Sterile 6mm discs were placed on the plate and loadedwith 25 µl of methanolic extract (100 mg/ml).Similarly 25 µl of 100% methanol loaded fornegative control and miconazole nitrate (30µg/ml) for fungus positive control. The fungusplate was incubated at 28°C for 48 h. Zone of in-hibition was measured and recorded. This experi-ment was performed in triplicates.

Minimum Inhibitory Concentration (MIC) Minimum inhibitory concentration of the plant

extract was determined using broth dilutionmethod. The test extract was dissolved inmethanol to obtain 1000 mg/ml of stock solu-tions. 1 ml of the stock solution was pipetted intopotato dextrose broth for fungus to obtain theconcentration as 100 mg/ml. Two-fold serial di-lution was performed and 500 µl of test culturewere added into each tube. Turbidity of the testculture was adjusted using the 0.5 McFarland

standard. Control tubes contained organisms,

broth and extract individually. Tubes with funguswere incubated at 28°C for 48 h. MIC value indi-cate the tubes with the lowest concentration with-out any growth of the microorganisms4.

Minimum Fungicidal Concentration (MFC)

This test was performed to determine the anti-fungal activity of C. surattensis flower extractagainst A. niger as percentage of hyphal growthinhibition. Serial dilutions were performed withthe test solution and PDA to give the final con-centrations as 100, 50, 25, 12.5, 6.25, 3.125,1.562, 0.78, 0.39 and 0.195 mg/ml. The mixturewas than mixed well and poured onto Petriplates. 1 mm plugs of fungal mycelium were cut

from the actively growing culture and placed onthe centre of each Petri plate after solidificationof the PDA medium. Methanol was used as nega-tive control and Miconazole nitrate as positivecontrol. The plates were incubated at 28°C for 48h. Diameter of the A. niger growth was measuredand the results were expressed as percentage of hyphal growth inhibition. IC50 was calculated forthe % inhibition of hyphal growth against con-centration graph.

Sample Preparation for Scanning

Electron Microscopy (SEM) Scanning Electron Microscopy (FESEM LEOSupra 50 VP, Carl Zeiss, Germany) was per-formed on A. niger which was treated with andwithout C. surattensis flower extract. The treatedsample was taken at various incubation periods[0 (control), 3, and 7 days] for SEM examina-tions. Small plugs of 5-10 mm samples were cutand placed on a planchette. The samples werethan fixed with 2% osmium tetraoxide for 1 hand followed by freeze drying process for 10 h.Finally the specimens were coated with gold be-fore viewing the sample at different magnifica-

tions5.

Phytochemical Screening Standard procedures for phytochemical

screening described by Evans6, Parekh and Chan-da7 and Mishra et al8 were adopted to screen themethanolic extract of C. surattensis flower.

Determinations of Total Phenolic Contents Total phenolic content of C. surattensis flower

extract was determined using the Folin-Ciocal-teau assay according to the previously described

method9

. Total phenolic content was calculated

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Evaluation of the effect of Cassia surattensis Burm. f., flower methanolic extract



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Hyphal growthZone of inhibition Concentration inhibition atselected

(mm)a,b tested for concentration (%)b

hyphal growthCassia surattensis MICc MFCd inhibition assay Cassia surattensis

Mircoorganisms flower extract M ME (mg/ml) (mg/ml) (mg/ml) flower extract M

Aspergillus niger 15 22 0 6.25 12.5 0.195 0 NDe

0.39 18 ND

0.78 24.7 ND

1.562 42 ND

3.125 55.3 ND

6.25 62 100

12.5 100 100

25 100 100

50 100 100

100 100 100

IC50 2.49 < 0.5

Table I. Antimicrobial activity of Cassia surattensis flower.

aDisk diffusion technique; bThe values (average of triplicate) are diameter of zone of inhibition at 100 mg/ml crude extract, 30µg/ml Chloramphenicol and 30 µg/ml Micanozole nitrate; cBroth dilution method; dAgar dilution method; eND, not detected.The MIC values for Escherichia coli, Bacillus subtilis, Micrococcus sp, Proteus mirabilis, Staphylococcus aureus, Candida al-bicans and Rhizopus sp also determined and the values were 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, 3.125 mg/ml, 1.562

mg/ml, 6.25 mg/ml and 3.125 mg/ml respectively. M = Micanozole nitrate; ME = methanol.

from the calibration curve of a gallic acid stan-dard solution. Results were expressed as gallicacid equivalents, in mg/g dry extract.

Determination of Total Flavonoid Content The amount of total flavonoid in the C. suratten-

sis flower extract was measured spectrophotometri-cally following method described by Djeridane etal10. Catechin was used as standard to construct acalibration curve. Various concentrations of C.surattensis flower extract (1.5 mL) were mixedwith of 2% methanolic AlCl3 solution (1.5 mL). Af-ter incubation at room temperature for 15 min, theabsorbance of the reaction mixture was measuredat 430 nm (UV-9100; Ruili Co., Beijing, China).The flavonoids content was expressed as mg cate-

chin equivalents (CE) in 1 g extract (mg CE/g).

Tannin Estimation Tannins were estimated spectrophotometrical-

ly11, with minimal modifications. 0.5 ml of suit-ably diluted extract was taken in a test tube andvolume was made up to 2.5 ml with distilled wa-ter. 0.25 ml of 1:19 diluted Folin Ciocalteaureagent and 0.5 ml of 20% sodium carbonate so-lution were added. The solution was kept for 30minutes at room temperature. Subsequently, ab-sorbance was measured at 775 nm (UV-9100;

Ruili Co., Beijing, China) and concentration wasestimated with respect to tannic acid as standard.Total tannin in the extract was expressed asequivalent to tannic acid (mg TE/g extract).

Results

Antimicrobial Activity Methanol extract of C. surattensis flower

showed significant inhibition zone against A.niger . The diameter of the inhibition zone mea-sured for A. niger was 15 mm. The extract showedMIC value of 6.25 mg/ml against A. niger (TableI). The fungicidal activity against A. niger was de-termined by the agar dilution method. Percentage

of hyphal growth inhibition of this fungus was de-termined at concentrations 0.195, 0.39, 0.78,1.562, 3.125, 6.25, 12.5, 25, 50 and 100 mg/ml.100% inhibitions were observed at 12.5 mg/mL.The percentage reduced from 62% at 6.25 mg/mlto 0 at 0.195 mg/ml (Table I). IC50 value as 2.49was obtained from the graph plotted.

SEM Observations of Aspergillus niger Morphology

The efficiency of C. surattensis flower crudeextract on the ultrastructure of A. niger (myceli-

Evaluation of the effect of Cassia surattensis Burm. f., flower methanolic extract on the growth



um and conidiophores) treated with and withoutthe extract is shown in Figure 1a-f at the samemagnification. Untreated (control) A. niger ap-peared as thick tube like hyphae and with spheri-cal conidiophores attached together as a network in a chain form (Figure 1a and b). When A. niger treated with C. surattensis flower extract a majoralteration on the morphology of hyphae was ob-served as the incubation period increased. Thereis obvious changes observed on the hyphae onDay 3 (Figure 1c) where the smooth hyphae cellwall was flattened and shrunken compared to thecontrol cells. Folding, empty and collapsed hy-phae were visible on Day 7 (Figure 1e). Treatedconidiophores on Day 3 (Figure 1d) did notshowed many changes on the microscopic obser-

vations. The activity of the extract was observedon the spores on Day 7 where the spore arrange-ment was altered, outer layer were distorted andshrunken spores were observed at the end of thetreatment period (Figure 1f).

Phytochemical Analysis Phytochemical screening on the methanolic

extract revealed the presence of active com-pounds such as tannins, saponins, phenols, carbo-hydrate and reducing sugar.

Total Phenolic and Flavonoid Content The total phenolic content of C. surattensisflower extract was determined to be 349.87 ±5.41 mg/g gallic acid equivalent. In addition, thetotal flavonoid content of C. surattensis flowerextract was 89.64 ± 3.21 mg/g catechin equiva-lent.

Tannin Estimation Estimation of total tannin is based on oxida-

tion of molecules which contain phenolic hy-droxyl groups. For C. surattensis flower extract,total tannin was found to be 55.14 ± 3.11 mg/g of

samples.

Discussion

Out of the 350,000 plant species identified sofar, about 35,000 (some estimate up to 70,000)are used worldwide for therapeutic reasons andless than about 0.5% of these have been studiedfor their phytochemical and pharmacological po-tential12. This medicinal plants and herbs, thus,embody a huge pool of putative lead bioactive

compounds to be exposed for the development of

various therapeutic agents for human diseases. C.surattensis alleged to have many medicinal prop-erties. Voon et al13 reported that methanol extractof C. surattensis flower exhibited good antibacte-rial and antifungal activity against Proteusmirabilis, Staphylococcus aureus , Escherichiacoli, Salmonella typhi, Micrococcus spp., Enter-obacter aerogenes, Bacillus subtilis, S. sonnei, A.lipoferum, Klebsiella pneumoniae, Pseudomonaaeruginosa, Candida albicans and Aspergillusniger . Moreover, Sangetha et al14 reported that C.surattensis flower exhibited good antioxidant ac-tivity. They reported that the C. surattensis flow-ers extract revealed the best antioxidant property,presenting much lower IC50 values (423.32 µg/mlfor DPPH assay and 11.1 µg/ml for xanthine oxi-

dase assay). Furthermore, the highest antioxidantcontents (polyphenols) were found for these ex-tracts (657.2392 ± 2.0321 mg GAEs/g extract).Therefore, in this research C. surattensis flowerswere used to evaluate the antimicrobial activityagainst A. niger . Furthermore, we mainly focuson A. niger because it may cause allergic bron-chopulmonary disease, invasive aspergillosis, ormay be a colonizer of natural or preformed cavi-ties of the human15,16.

From the antimicrobial screening favorablezone of inhibition was obtained for A. niger . For

MFC the flower extract showed only moderatereduction (42%) at 1.562 mg/ml of hyphalgrowth, while inhibition was substantial at 12.5mg/ml with 100%. At 6.25 mg/ml of extract inbroth medium fungal growth was completely in-hibited but for MFC at 12.5 mg/ml. Earlier anti-fungal investigations on the aqueous andmethanolic extracts of Roman chamomile ( An-themis nobilis) dried leaves and Matricaria (M.)chamomilla flower heads was done by Magro etal17 and Rauha et al18. Magro et al17 reported thatthe aqueous extract of Roman chamomile driedleaves was able to inhibit A. niger growth at a

concentration of 920 mg/ml. Meanwhile, Rauhaet al18 also shown that the methanolic extract of

M. chamomilla flower heads also possessed in-hibitory effect against the fungus. The flower ex-tract significantly reduced the growth of A. niger in a dosage response manner. Antifungal activityby the MIC and MFC methods were further veri-fied by SEM observations. Flower extract com-pletely squashed and severely collapsed hyphaewere observed from SEM study, which results inflattening and ultimately death of hyphae. Thehyphae showed lack of cytoplasm, damage and

loss of integrity and rigidity of the cell wall.

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V. Sumathy, Z. Zakariah, Y. Chen, L.Y. Latha, S. L. Jothy, S. Vijayarathna, S. Sasidharan

Figure 1. Scanning electron micrographs of A. niger conidiophores grown on PDA with and without C. surattensis flower ex-tract during 7 days of incubation at 28°C. A and B , control mycelium and conidiophores. C and E , flattened and squashedmycelium; and (D and F ) shrunken conidiophores treated with 6.25 mg/ml of extract.

A B

CD

E

F



Treated conidiophores did not demonstrate manyalterations on the SEM microscopic observa-tions. These findings indicate that the mode of antifungal activity of flower extract of C. surat-tensis is an outcome of attack of extract on theplasma membrane and retraction of cytoplasm inthe hyphae and ultimately death of the A. niger mycelium. Alternatively, Tolouee et al19 reportedsuch alteration induced by extract may be relatedto the interference of bioactive components withenzymatic reactions of wall synthesis, which af-fects morphogenesis and growth of A. niger . Toconfirm this, we will investigate through trans-mission electron microscopy (TEM) in our futurestudies. Similar results of ultrastructural changesof fungal morphology have been reported for A.

niger hyphae treated with Cympobogon nardus20,Citrus sinensis21, Thymus eriocalyx and Thymus

x-prolock 22 and Cymbopogon citratus23.The objective of performing phytochemical

screening is to preliminarily characterize the ac-tive principles that are responsible for observedbioactivity by the extract when treated againstmicroorganism24. In this study the presence of various secondary metabolites in C. surattensisflower extract may contributed to the observedantifungal activity. Previous study on Terminaliaspecies reported that the antifungal activity ob-

served in their study could be due to the presenceof tannins and saponins25. Tannins at high con-centrations can act as antifungal agents by coagu-lating the protoplasm of the microorganism26.Saponins is commonly stored in plant cells as in-active precursor but easily can be converted intobiologically active antibiotics by enzyme activi-ties immediately after infection. This compoundwill act by disrupting the membrane integrity of fungal cells. Phenolic and flavonoid compoundsthat were presence in the methanolic extract of C.surattensis flower is also reported to possessedantimicrobial activity27. It is possible that these

compounds were mainly responsible for the ob-served antibacterial and antifungal effects in thisstudy. However, further phytochemical studiesare needed to isolate the active compound(s) re-sponsible for this pharmacological activity.

Conclusions

At present, there is growing interest in usingnatural products such as medicinal plant extractsfor the development of antifungal agents to re-

duce the infection of pathogenic microorganism.

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Therefore, C. surattensis flower extract is ap-pears to have promise as a safe alternative naturalproduct. The present study clearly shows themode of action of extract against the testpathogen A. niger . In conclusion, C. surattensisflower extract is an excellent candidate for use asthe lead compounds for the development of novelantifungal agents.

–––––––––––––––––––– Acknowledgements

Vello Sumathy was supported by the Universiti SainsMalaysia Fellowship Scheme from Institute for Postgradu-ate Studies (IPS) of Universiti Sains Malaysia.

––––––––––––––––––––

Conflict of InterestNone to declare.

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Evaluation of the effect of Cassia surattensis Burm. f., flower methanolic extract on the growth and morphology of Aspergillus niger