Antifungal activity of Rhein isolated from Cassia fistula L. flower

15 Pages·2012·0.12 MB·English

Article ID: WMC00687 ISSN 2046-1690
Antifungal activity of Rhein isolated from Cassia
fistula L. flower
Corresponding Author:
Dr. S Ignacimuthu,
Director, Entomology Research Institute, Loyola College, Nungambakkam, Chennai, 600 034 - India
Submitting Author:
Dr. V Duraipandiyan,
Scientist, Division of Ethnopharmacology, Entomology Research Institute, Loyola College, 600 034 - India
Article ID: WMC00687
Article Type: Research articles
Submitted on:20-Sep-2010, 11:14:49 AM GMT Published on: 20-Sep-2010, 05:16:23 PM GMT
Article URL: http://www.webmedcentral.com/article_view/687
Subject Categories:PHARMACOLOGY
Keywords:Antifungal, Cassia fistula, MIC, Rhein
How to cite the article:Duraipandiyan V, Ignacimuthu S. Antifungal activity of Rhein isolated from Cassia fistula
L. flower . WebmedCentral PHARMACOLOGY 2010;1(9):WMC00687
Competing Interests:
We have no competing interest
Additional Files:
Full manuscript
Cover letter
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Antifungal activity of Rhein isolated from Cassia
fistula L. flower
Author(s): Duraipandiyan V, Ignacimuthu S
Abstract amount of alkaloids have also been reported in the
flowers; traces of triterpenes have been observed in
both flowers and fruits [8,9].
Our preliminary evaluation of ethyl acetate extract
Antifungal activity of rhein (1, 8-
from Cassia fistula flowers showed significant
dihydroxyanthraquinone- 3carboxylic acid) isolated
antifungal activity [10]. In the present work, we report
from the ethyl acetate extract of Cassia fistula flower
the separation and identification of rhein from C. fistula
was studied. Rhein inhibited the growth of many fungi
flowers and its antifungal effect.
such as Trichophyton mentagrophytes (MIC 31.25
Materials and Methods
µg/ml), Trichophyton simii (MIC 125 µg/ml),
Trichophyton rubrum (MIC 62.5 µg/ml) and
Epidermophyton floccosum (MIC 31.25 µg/ml).
Introduction Plant material
Cassia fistula flowers were collected from Loyola
College Campus, Chennai, India. It was authenticated
by Dr. S. Amerjotthy, Department of Botany,
Plants used in traditional medicine usually constitute
Presidency College, Chennai, India. A voucher
an important source of new biologically active
specimen (ERIC-D-73) is deposited at the herbarium
compounds. Numerous useful drugs have been
of Entomology Research Institute, Loyola College,
discovered from higher plants by following up
Chennai.
ethnomedical uses [1]. During the past several years,
Preparation of crude extract
there has been an increasing incidence of fungal
The extracts were taken using cold percolation method.
infections due to a growth in immunocompromised
Fresh flowers were collected (9kg) and shade dried at
population such as organ transplant recipients, cancer
room temperature and ground in a manual mill. The
and HIV/AIDS patients. This fact coupled with the
powder (1kg) was extracted with 3 ltr (1:3 w/v) of
resistance to antibiotics and toxicity during prolonged
hexane for 48 hours. The extract was filtered through
treatment with several antifungal drugs, has been the
a Buchner funnel with Whatman number 1 filter paper.
reason for an extended search for newer drugs to treat
The filtrate was evaporated to dryness under reduced
opportunistic fungal infections [2,3].
pressure using rotary evaporator at 40ºC. The
Cassia fistula L., (Caesalpiniaceae), a semi-wild Indian
remains of the plant material were extracted with
Labernum (also known as the Golden Shower), is
chloroform (11g), ethyl acetate (17g), methanol (20)
distributed in various countries including Asia, South
and water (13g) sequentially in a similar manner. The
Africa, Mexico, China, West Indies, East Africa and
crude extracts were stored at 4ºC until further use.
Brazil. It is an ornamental tree with beautiful bunches
Isolation of active compound
of yellow flowers. C. fistula exhibited significant
The crude ethyl acetate extract (10 g) was subjected
antifungal activity and showed properties that support
to column chromatography over silica gel (200
folkloric use in the treatment of some diseases as
g-acme’s 100–200 mesh) and eluted with hexane
broad-spectrum antifungal agent [4]. The whole plant
followed by the combination of hexane: ethyl acetate
is used to treat diarrhea; seeds, flowers and fruits are
ranging from 95:5 to 100. 117 fractions were collected
used to treat skin diseases, fever, abdominal pain and
in a 200ml conical flask. After checking TLC, the
leprosy by traditional people [5]. This plant has a
fractions were combined in to 24 fractions. Fraction 10
strong tendency to contain anthraquinone derivatives.
showed a crystal which was subjected to
From the genus Cassia many quinone derivatives
crystallographic analysis and identified reported.10
such as Kaempferol have also been isolated; a
Fraction 18 showed single spot on TLC (Rf = 0.36)
proanthocyanidin has been isolated from the acetone
and yielded 210 mg; this fraction was eluted using
extract of the flower [6]. A bianthraquinone glycoside,
hexane: ethyl acetate (10:9) as mobile phase solvent
fistulin, together with kaempferol and rhein have been
system. The spot turned pink on exposure to
isolated from ethanol extracts of C. fistula flowers [7].
ammonia vapor; it indicated the presence of
Besides phenolics and their derivatives, a certain
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anthraquinones. The compound was subjected to MHz, dimethyl sulfoxide (DMSO)] d ppm: 161.2 (C-1),
spectroscopic analysis. 124.2 (C-2), 165.6 (C-3), 119.0 (C-4), 124.7 (C-5),
Spectroscopic analysis 138.5 (C-6), 124.7 (C-7), 161.5 (C-8), 181.2 (C-9),
IR, 1H-NMR, 13C NMR and MASS were taken from 181.2 (C-10), 130.0 (C-4a), 118.9 (C-8a), 118.7
Nicholas Primal Pvt. Ltd. Ennore, India and used to (C-9a), 133.3 (C-10a), 191.4 (3-COOH) which
identify the isolated compound. corresponded to those reported [12]. The active
Fungi compound was identified as Rhein
Fungi, Trichophyton rubrum, T. rubrum 57/01, T. (1,8-dihydroxyanthraquinone-2-carboxylic acid) (Fig
Mentagrophytes, T. simii, Epidermophyton floccosum, 1). The same compound was previously reported from
Scopulariopsis sp., Aspergillus niger, Botyritis cinerea, some other plants. Sun et al. [14] chromatographed
Curvularia lunata and Candida albicans MTCC 227 and purified emodin, chrysophanol and rhein from
were used for the experiments. All cultures were Rheum officinale extract. Wang et al. [15] separated
obtained from the Department of Microbiology, and determined active anthraquinone components
Christian Medical College, Vellore, Tamil Nadu, India physcion, chrysophanol, aloe-emodin, emodin, and
Assay for antifungal activity rhein from the Chinese herb Polygonum multiflorum.
The antifungal activity of the isolated compound was The isolated compound (rhein) was tested for
determined using standard method [11]. The antifungal activity and results are shown in table 1.
compound was tested by micro broth two-fold serial Rhein showed activity against T. mentagrophytes (MIC
dilution technique. The crude extract and compound 31.25 µg/ml), T. simii (MIC 125 µg/ml), T. rubrum (62.5
were dissolved in water + 2% dimethyl sulfoxide µg/ml), T. rubrum 57(MIC 62.5 µg/ml), E. floccosum
(DMSO). The initial concentration of extract was 1mg (MIC 31.25 µg/ml), Scopulariopsis sp (MIC 250 µg/ml),
/ml; the initial concentration of the compound was C.lunata (MIC 250 µg/ml) and A. niger, M .grisea, B.
250µg/ml. The initial test concentrations were serially cinerea and C. albicans were not inhibited.
diluted two-fold. Each well was inoculated with 5 ml of
Discussion
suspension containing 104 spore/ml of fungi.
Fluconazole and Ketoconazole were included in the
assays as positive controls. The plates were incubated
for 24, 48 or 72 h at 27 ° C up to 9 days. MIC was Fungal diseases have increased dramatically in recent
defined as the lowest extract concentration showing years. The treatment of mycoses has lagged behind
no visible fungal growth after incubation time. and fewer antifungal than antibacterial substances are
available. Therefore, a search for new antifungal drugs
Results
is extremely necessary [16]. Many plants are now
used to treat various infectious diseases. In this study
we examined the antifungal activity of C.fistula flower
The present study deals with the antifungal activity of ethyl acetate extract and isolated compound rhein.
crude ethyl acetate extract and an isolated compound Traditional uses of this plant favor its use as antifungal
from C. fistula flower. The crude ethyl acetate extract drug. Ethyl acetate extract of C.fistula showed
inhibited the growth of fungi T. mentagrophytes (MIC promising antifungal activity. This indicated that there
250 µg/ml), T. simii (MIC 1000 µg/ml), T. rubrum (1000 may be some active compound involved. So we
µg/ml), T.rubrum 57 (MIC 500 µg/ml), E. floccosum selected ethyl acetate extract for isolation of active
(MIC 500 µg/ml), Scopulariopsis sp (MIC 500 µg/ml). compound. Isolated active compound was confirmed
The structural identification of compound was carried by comparing the IR, 1H NMR, 13C NMR, MASS and
out using IR, MS, 1H NMR and 13C NMR spectra as m.p., data with that of the known compound rhein
follows: the EI-MS: m/z 284, 267, 256, 239, 228, 211, Previously the same compound was reported against
183, 155, 142, 126. It showed the molecular ion at m/z Botrytis cinerea [17]; Candida albicans, Trichophyton
284, which was corresponding to the molecular mentagrophytes [18]. However, rhein was not yet
formula C15H8O6 of rhein; its melting point was at tested against T. rubrum, T. rubrum 57/01, T. simii, E.
321ºC [12]. The 1H NMR spectrum revealed five floccosum, Scopulariopsis sp., A. niger, C. lunata
aromatic protons of which two were broad singlets due and M. grisea. Rhein inhibited the growth of T.rubrum,
to meta coupling. 1H NMR (75 MHz, DMSO): 11.9 (1H, T. rubrum 57, T. simii, E.floccosum and Scopulariopsis
brs, C1–OH), 11.5 (1H, brs, C8–OH), 8.13 (1H,brs, sp. The anthraquinone derivative rhein is significantly
C2–H), 7.40 (1H, d, J = 8.5 Hz, C5–H), 7.73 (1H, m, antiseptic and is especially toxic to the pathogen [19].
C6–H), 7.75 (1H,brs, C4–H), 7.81(1H, d, J = 8.0 Hz, Trichophyton rubrum is the most prevalent pathogenic
C7–H) corresponded to rhein [13]. 13C NMR [(300 fungus; worldwide it represents 80% of clinical isolates
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[20]. In our findings rhein inhibited the growth of T. 7. Kumar A, Pande CS, Kaul RK. Chemical
rubrum at 62.5 µg/ml. examination of Cassia fistula flowers. Ind J Chem
Generally anthraquinone derivatives, which include 1966;4: 460.
emodin, chrysophanol, rhein, aloe-emodin, physcion, 8. Asseleih LMC, Hernandez OH, Sanchez JR.
and their glucosides, and important active components Seasonal variation in the content of Sennosides in
with various pharmacological actions such as leaves and pods of two Cassia fistula populations.
purgation, antibacterial, antifungal and antitumor Phytochem 1990; 29: 3095–3099.
activity [21]. Antifungal activities of anthraquinones 9. Guri-Fakim A, Gueho J, Sewraj MD, Dulloo E.
and napthoquinones isolated from natural sources Plantes Medicinales de lile Maurice, Editions de
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(3,4-dihydroxy-1-methoxyanthraquinone- 11. National Committee for Clinical Laboratory
2-carboxaldehyde) was isolated from ethanolic extract Standards. Reference method for broth dilution
of Saprosma fragrans and tested against T. antifungal susceptibility testing of filamentous fungi.
mentagrophytes (12.5 µg/ml) [27]. Our results Approved standard M38-A. Wayne, PA: National
showed that the isolated compound significantly Committee for Clinical Laboratory Standards, 2002.
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µg/ml and E. floccosum 31.25 µg/ml. rhein from Chinese traditional herb by repeated
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fungi. 15. Wang D, Yang G, Engelhardt H, Liu H, Zhao J.
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Illustrations
Illustration 1
Figure 1: Rhein (1, 8-dihydroxyanthraquinone-3-carboxylic acid) isolated from ethyl acetate extract
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Illustration 2
Table 1: Antifungal activity of Rhein (MIC µg/ml)
Tested Fungi EA C Fl Ket
Trichophyton mentagrophytes 66/01 250 31.2 25 <12.5
T. simii 110/02 1000 125 <12.5 <12.5
T. rubrum MTCC 296 1000 62.5 <12.5 <12.5
T. rubrum 57/01 500 62.5 25 <12.5
Epidermophyton floccosum 73/01 500 31.2 12.5 <12.5
Scopulariopsis sp. 101/01 500 250 <12.5 <12.5
Aspergillus niger MTCC 1344 >1000 >250 100 <12.5
Curvularia lunata 46/01 >1000 >250 <12.5 <12.5
Magnaporthe grisea >1000 >250 nt nt
Botrytis cinerea >1000 >250 nt nt
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Candida albicans MTCC 227 >1000 >250 >100 25
EA- Ethyl acetate (Crude extract) C- Rhein; Fl – Fluconazole (antifungal agent); Ket-
Ketoconazole (antifungal agent); nt- not test
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Reviews
Review 1
appropriate
Review Title:
Posted by Lead Faculty Dr. Gurpreet K Randhawa on 17 Dec 2011 06:11:43 AM GMT
1 Is the subject of the article within the scope of the subject category? Yes
2 Are the interpretations / conclusions sound and justified by the data? Yes
3 Is this a new and original contribution? Yes
4 Does this paper exemplify an awareness of other research on the topic? Yes
5 Are structure and length satisfactory? Yes
6 Can you suggest brief additions or amendments or an introductory statement that will increase Yes
the value of this paper for an international audience?
7 Can you suggest any reductions in the paper, or deletions of parts? No
8 Is the quality of the diction satisfactory? Yes
9 Are the illustrations and tables necessary and acceptable? Yes
10 Are the references adequate and are they all necessary? Yes
11 Are the keywords and abstract or summary informative? Yes
Rating: 6
Comment:
comparative comments on antifungal activity of rhein, ketoconzole and fluconazole can be given under result
heading.
Future prospects concerning further animal/ clinical studies while concluding the study can be added

a grapghical representation of above comaparison will be welcomed
Competing interests: no
Invited by the author to make a review on this article? : No
Experience and credentials in the specific area of science:
10 years
Publications in the same or a related area of science: No
How to cite: RU A.appropriate[Review of the article 'Antifungal activity of Rhein isolated from Cassia fistula L.
flower ' by ].WebmedCentral 1970;2(12):WMCRW001278
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Review 2
Comments on Antifungal activity of Rhein isolated from Cassia
Review Title:
fistula L. flower published in Webmed Central
Posted by Dr. M Ayyanar on 14 Feb 2011 04:13:42 PM GMT
1 Is the subject of the article within the scope of the subject category? Yes
2 Are the interpretations / conclusions sound and justified by the data? Yes
3 Is this a new and original contribution? Yes
4 Does this paper exemplify an awareness of other research on the topic? Yes
5 Are structure and length satisfactory? Yes
6 Can you suggest brief additions or amendments or an introductory statement that will increase Yes
the value of this paper for an international audience?
7 Can you suggest any reductions in the paper, or deletions of parts? Yes
8 Is the quality of the diction satisfactory? Yes
9 Are the illustrations and tables necessary and acceptable? Yes
10 Are the references adequate and are they all necessary? Yes
11 Are the keywords and abstract or summary informative? Yes
Rating: 7
Comment:
7. No, there is a no need for any reduction or deletion in any parts of the paper.
Invited by the author to make a review on this article? : No
Experience and credentials in the specific area of science:
I am working in the field of complementary and Alternative medicine in the last seven years. In addition, I have
adequate knowledge in the areas of Plant Taxonomy and Plant Tissue Culture.
Publications in the same or a related area of science: Yes
References: 1. Ayyanar, M. and Ignacimuthu, S., 2005. Traditional Knowledge of Kani tribals in Kouthalai of
Tirunelveli hills, Tamil Nadu, India. Journal of Ethnopharmacology 102 (2): 246 ? 255. 2. Ayyanar, M. and
Ignacimuthu, S. 2011. Ethnobotanical survey of medicinal plants commonly used by Kani tribals in Tirunelveli
hills of Western Ghats, India. Journal of Ethnopharmacology (In press, doi:10.1016/j.jep.2011.01.029), 3.
Ignacimuthu, S., Ayyanar, M. and Sankarasivaraman, K., 2008. Ethno-botanical study of medicinal plants used
by Paliyar tribals in Theni district of Tamil Nadu, India. Fitoterapia 79, 562 ? 568. 4.Ignacimuthu, S., Ayyanar, M.
and Sankarasivaraman, K., 2006. Ethno-botanical investigations among tribes in Madurai District of Tamil Nadu,
India. Journal of Ethnobiology and Ethnomedicine; http://www. ethnobiomed.com /content/2/1/25. 5. Muthu, C.,
Ayyanar, M., Raja, N. and Ignacimuthu, S., 2006. Medicinal plants used by traditional healers of Kancheepuram
district of Tamil Nadu, India. Journal of Ethnobiology & Ethnomedicine;
http://www.ethnobiomed.com/content/2/1/43. 6. Ayyanar, M. and Ignacimuthu S. 2009. Herbal medicines for
wound healing: Ethnobotanical and Scientific evidence from south Indian traditional medicine. International
Journal of Natural Products in Applied Research 2(3): 29-42.
How to cite: Ayyanar M.Comments on Antifungal activity of Rhein isolated from Cassia fistula L. flower
published in Webmed Central[Review of the article 'Antifungal activity of Rhein isolated from Cassia fistula L.
flower ' by ].WebmedCentral 1970;2(2):WMCRW00470
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