Indian Journal of Experimental Biology Vol. 42. June 2004, pp. 589-594

Antibacterial and antidiarrhoeal effects of alkaloids of Hola rrhena Gnticiysenterica WALL

D Kav itha, P N Sh ilpa & S Niranjali Devaraj*

Department of Biochemistry, Uni versity of Madras, Gu indy Campus, Chennai 600 025 , Indi a

Received 14 Septell/ber 2003; revised 12 Decell/ber 2003

The alkaloids from the ethanolic extrac t o f H. alltidyselllerica seeds were evaluated for their antibacterial acti vity against clinical isolates o f enteropathogenic Escherichia coli (EPEC) ill vitro, and their antidiarrlloeal acti vity on castor o il-induced diarrhoea in rats, ill vivo. The pl asmid DN A, whole cell lysate and outer membrane protein profil e o f a clinical isolate of EPEC was determ ined in presence of alkaloids o f H. alllidysellIerica. T he disc di ffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy. The alkaloids showed strong antibacterial acti vity against EPEC straitls. In castor oil -induced diarrhoea, alkaloids reduced the diarrhoea with decrease in the number o f wet faeces in pretreated rats at a dose of 200-800 mg/kg. T he loss o f plasmid DNA and suppression o f high molecular weight proteins were observed on alkaloids treatment. Taking into account the mUltiple antibiotic resistance of EPEC, the results suggest usefulness of alkaloids o f H. aHtidyselllerica seeds as ant ibacterial and antidi arrhoeal agents.

Keywor ds: Holarrhella mllidysell terica , alka loids, EPEC, antidi arrhoeal.

IPCCode: Int C I7 A6 1K

Dian-hoea is a common symptom of intestinal di sorders and it is a global threat to human health 1.2. It is a leading cause of morbidity and n10ttality , with over 1000 million episodes and over 4 million deaths annually in children under five year of age3

.

Enteropathogenic Escherichia coli (EPEe) is a member of the attaching and effaci ng (NE) family of pathogens that induce di arrhoeal disease in a wide range of mammalian species4

. The AlE les ion is characterized by localized destruction (effacement) of brush border mi crovill i, intimate attachment of the bacillus to host ce ll membrane and the formation of an underlying pedestal-like structure on the host cell consisting of polymerized actin , a -actinin , ezrin , talin and myosins-7 . The pathogenesis of EPEe infec tion encompasses 3 di stinct stages (i) Initi al adherence: type IV fimbliae-is assoc iated with a high molecular weight plasmid (90 kb), present in most EPEe strains8

, (ii) signal transduction: the second stage consists of signaling in the host ce ll s by the bacteri a, their signaling requires type III secreted proteins (EspA, Esp8, EspD, EspF and Tir)9, and (iii) intimate attachment: T ir functions as a receptor on the host cell membrane for initimate attachment of the outer

*Con'espondent author: Phone: 9 1-44-2235 1269 Fax: 9 1-44-22352494 E-mail :niranjali @yahoo.com

membrane protein, inti min 10. The type III secretion system and type IV fimbri ae in EPEe play principal role in the interaction between the bacteri a and host epithelial cells, and can thus be exploited for therapy .

The WHO has constituted a di arrhoeal disease control program , which includes traditional medi cinal practices together with the evaluati on of health education and prevention approaches II . Holarrhena anlidysenlerica WALL (Apocyanaceae) commonl y known as Tellicherry bark (Engli sh), Kurchi (Hindi), is a small deciduous tree wi th white flowers. Thi s plant has many sanskrit names, the better known being Kutaja and Kalinga. It grows in the tropical Himalayas and is distributed throughout [ndi a I2

.13

.

Almost all the parts of the plant, viz bark, root, stem and seeds, are known to have various medici nal properties l 4

• The main ingredient of Ku tajarishta. an antidi atThoeal formulation, is H. anlidysenlerica, wh ich has been reported to exhibit antiamoebic activity. The antimicrobial activity of H. antidysenterica bark extract has been reported against enteropathogens like enteroinvasive Escherichia coli, EPEe, Salmonella typhimurium, Salmonella enteritidis, Shigella .fiexlleri, Sh.boydii and Vibrio cholerae l 5

• In addition, the plant has been reported to possess antihelminthic, appetisti ng and astringent properties l6

.

The seeds of H. antidysenterica are used in the treatment of dysentery , diarrhoea and fever l7

. The'

590 INDIAN J EXP BIOL, JUNE 2004

seeds contain many alkaloids but at low concentrations (1.82%)18. The multiple antibiotic resistance of EPEC I9.20 h as promoted the search for new antibacterial agents from traditional medicinal plants. The present study has been undertaken to justify the antibacterial and castor oil-induced antidiarrhoeal activity of the alkaloids of H. alltidysellferica.

Materials and Methods Plant material-The seeds of H. alltidysenterica

were collected during August 2001, from trees growing in Tamil Nadu, India. Identification of the plant was established by the Centre for Advanced Studies in Botany, University of Madras, Chennai. After collection, the seeds were shade-dried and ground into coarse powder and stored in air-tight container for further use.

Isolafioll of alkaloids from the seeds of H. alltidysenterica--The dried pulverized powder of the seeds of H. alltidysellferica (1000 g) were defatted

with petroleum ether (40°-60°C) in Soxhlet extraction apparatus for 2 hr. The plant material was dried under vacuum and mixed with 100 g calcium hydroxide and made into a slurry. The mixture was heated in a water bath for 30 min. Then the material was extracted with alcohol + 10% acetic acid mixture in a Soxhlet unit for 5 hr. After that the alcoholic extract was acidified with I N HCI (pH 3.2). After the acidification process, added 250 ml of ethyl acetate and mixed using a magnetic stirrer for 10 min. The aqueous solution was separated and the alkaloids precipitated by dropwise addition of concentrated ammonium hydroxide solution (pH 9). The precipitate was collected by centrifugation and the precipitated alkaloids was washed with I % ammonium hydroxide solution. The precipitate was dissolved in 200 ml ethyl acetate and the ethyl acetate portion was separated and concentrated under vacuum. A yellowish brown residue was obtained which was re-crystallized with chloroform (Fig. I ) The isolated alkaloid was confirmed by its melting point and by TLC.

The melting point of the isolated alkaloid was 123°-

124°C. TLC was canied out on silica gel G pre-coated sheets using methanol:chloroform (3: 17) as solvent system. The alkaloids were detected with Dragen dOlffs reagent (Rr = 3.0).

Test Microorganisms-The in vitro antibacterial study was carried out against enteropathogenic Escherichia coli (EPEC 1402fc; EPEC 136Ifc). The clinical isolates were obtained from All India Institute

of Medical Sciences, New Delhi, India. The bacteria were grown on nutrient agar plates supplemented with

ampicillin (100 Ilg/ml) for 24 hr at 37°±2°C. An isolated colony was suspended in sterile saline and was used for further studies.

Antibaderial activity Disc diffusion method (European pharmacopo­

eia/3-Paper discs (<p = 9 mm) were impregnated with 1.25, 2 .5 and 3.5 mg of the sample and allowed to air dry . Over the agar plate, the diluted bacterial culture was spread with a sterile swab. The discs impregnated with extract were placed over the agar plate and incubated at 37° ± 2°C for 24 hr. The antibactelial activity was measured as a diameter of inhibitory zone on the agar plate.

Agar well dijfusiolllllethod (Hugo alld Russel/ 4-In this technique, bacterial culture was added aseptically

to the agar medium at 45°C, mixed well and poured immediately onto sterile petri dishes. After solidification, wells of about 6 mm in diameter were cut into agar and 1.25, 2.5 and 3.5 mg of the alkaloid preparation were placed in the wells. The plates were incubated at 37°C and observations were made after 24 to 72 hr.

The experiments were repeated thrice and the results were expressed as the average of 3 experiments. Cefotaxine, chloramphenicol and ciproflaxin (Ranbaxy) were used as reference antibiotics for comparison.

Microdilutioll lIlethod-The method of Makane and Kandal25

, was followed to determine the minimum inhibitory concentration (MIC). The total alkaloids were di luted to obtain concentrations ranging from 1.6-5 mg/ml. The test tubes containing 3 ml LB broth, 0.1 ml bacterial suspensions and different concentration of

samples were incubated at 37°C for 24 hr. Bacterial turbidity was measured at 560 nm to determine bacterial inhibition . Chloramphenicol (100 ~lg/ml) was used as a reference antibiotic and tubes containing only the growth medium and organism were used as control.

Isolatioll of whole cell lysate (WCL) and outer membrane proteills (OMP) of EPEC 1402fc-The method of Carlene et a/.,26 was followed for OMP and for SDS-PAGE the method of Laemmli27 was followed. OMP and WCL were isolated from an overnight LB broth culture of EPEC 1402fc and LB broth culture containing alkaloids (1 and 1.5 mg/ml) . The isolated proteins were subjected to 12% SDS­PAGE and stained with Coomassie brilliant blue.

KA VITHA el af. : ANTIBACfERIAL & ANTlDIARRHOEAL EFFECfS OF HOLARRH ENA ALKALOIDS 59 1

Dried pulverized H.antidysenterica seeds (1000 g)

~ Defatted with petroleum ether (40° - 60°C) in

Soxhlet apparatus (2hr)

~ Dried under vacuum

~ Mixed with Ca(OH)2 (100g)

~ Slurry

~ Heated in a water bath (30 min)

~ Material extracted with alcohol + 10% acetic acid

in Soxhlet apparatus (5hr)

-+

~ Alcoholic extract Concentrated

~ Acidified (1 N HCI - pH 3.2)

~ Added ethyl acetate (250 m I)

and stir (10m in)

t • Aqueous Phase Organic Phase

+ Aqueous solution ppted

with dropwise addition of

NH40H (pH 9)

I t

+ evaporated (40°C)

~ Neutral & acidic material

ppt collected by centrifugation (3000 rpm/15 min)

+ ppt washed 1% NH

40H and dissolved 200 ml ethyl acetate

~ Concentrated - vacuum

+ Yellowish brown residue (alkaloid) crystallized with chloroform

• Alkaloid - Melting point (123°-124°C) - identified by TLC using Dragendroff's reagent

Fi g. I---Outline of the extraction procedure for a lka loids carriedout fo r H. all lidysell lerica.

592 INDI AN J EXP BIOL, JUN E 2004

Isolation of plaslllid DNA-The method of Sambrook et al., 28 was foll owed. Plasmid DNA was isolated from an overni ght LB broth culture of EPEC 1402fc and a culture containing the alkaloids (O.S, l.6 and 3.2 mg/ml) and was subj ected to O.S% agarose gel electrophoresis.

Castor oil-induced diarrhoea ill rats-The method followed was that of Awouter et al., 29 with modification by Das et al. 30. Rats were fasted for IS hr and were housed in steel cages containing six rats in each group. Group I received normal saline as negative control , group II received d iphenoxy late hydrochloride (Iomotil ; 5 mg/kg) as positi ve control. Group III , IV , V and VI were admini stered, orally ,with alkaloids at a dose of (200, 400, 600, SOO mg/kg). After one hour treatment all the rats received I ml castor oi l, orally by gavage, and were observed for defecation for 4 hr after the castor o il challenge. The presence of characteristic di arrhoeal droppings was noted in transparent plas ti c dishes placed beneath the individual cages.

Results

Antibacterial activity

Disc diffusion method-The zone of inhibition exhibited by the alkalo ids was observed for different concentrations (1.25 , 2.5 and 3.5 mg) using di sc diffusion method. It was fo und to be 12, 16 and 20 mm for the isolate 1402fc and I I , 12 and 17 mm fo r the isolate 136 1 fc (Table I ).

Agar well diffusion method-The effect of the alkalo ids of H. antidysellterica was eval uated against EPEC ( 1402fc and 136 1fc) us ing this method. Inhibition zones were 15, 16 and 24 mm fo r 1402 fc and IS, 19 and 2 1 mm for 136lfc (Table 1).

Microdilutioll method-In thi s tech nique, with increasing concentrat ion o f alkalo ids, the op tical density (00 ) grad uall y decreased. Thi s was compared with the standard antibi ot ic, chl oramphenicol ( 100 /-Ig/ml) (da ta not shown).

The ant ibacterial properties of H. antidysenterica alkalo ids were clearl y ev ident and compared well with the antibacteri al ac tivity of the standard an ti bioti cs (Tables I).

Proteill profile of OMP and WCL of 1402fc-The protein profiles of OMP and WCL of EPEC 1402fc culture grown in the presence and absence of alka lo ids were compared. Suppression of high molecular weight proteins was revealed in alkaloids­treated culture (Fig. 2).

Plasmid profile of EPEC 1402fc-In thi s method, the plasmid DNA of EPEC 1402fc, which was grown in presence and absence of alkaloids were compared . Fig. 3 shows the loss of plasmjd DNA bands in a concentration-dependent manner.

Castor Oil-induced diarrhoea-The alka lo ids of H. alltidysel1lerica, like the standard an tidiarrhoeal drug diphenoxylate, signi ficantly inhibited the defecation when compared to untreated control rats in dose­dependent manner (Table 2).

Discussion Research on natural sources has been act ively

encouraged by the WH03). The present results clearly

indicate that the ethanolic ex tract of H. an.tidysel1terica seed alkaloids strong ly inhi bit the

Table I-Determinat ion of antibacterial act ivi ty of <Ilkaloids of H. alllidysell lerica seeds and reference antibiotics by (A) di sc

diffu sio n and (B) agar well diffusion methods

Samples

Alka loids

Cefotax ine

Chloramphen icol

Ciproflax in

kDo

97 • 66 • 43 • 29 • 2 0 • 14 •

Concentration

1.25 mg

2.5 Illg

3.5 mg

100 pg

I ()() pg

100 pg

2 3

Inhi bition zone diameter (Ill m) (mean value, n=3)

EPEC 1402fc EPEC 136 1fc

A 12 II B 15 18 A 16 12 B 16 19 A 20 17 B 24 2 1 A 23.0 18 B 11.4 12.4 A 20 15 B 19.8 18.4 A 29 28 B 27 27

4 5 6 7

Fig.2-Protein profi le of EPEC 1402fc o n 12% SDs-pAGE [aMP (Lane 2 - EPEC 1402 fc; Lane 3 and 4, EPEC grown in presence of alkaloids of H. alllidyselllerica. 0.8 mg/mi and 1.6 rng/ml respecti ve ly] WCL (Lane 5-EPEC 1402fc; Lane 6 and 7 EpEC grown in presence of a lkaloids of H. antidysell ierica. 0.8 rng/1ll1 <Ind 1.6 mg/rn l respec ti vely] Lane I ind ica tes mo lecular weight (kDa)]

KAVITHA et al.: ANTIBACTERIAL & ANTIDIARRHOEAL EFFECTS OF HOLARRHENA ALKALOIDS 593

growth of EPEC strains. Most of the alkaloids having antibacterial 32

, antidiarrhoeal33, antimicrobial 34

,

anticancer35, antimalarial 36 activity and cure several

infectious di seases . The present results are in agreement with the findings of Ballal et ai.,15 and Chakraborty et ai., 14. A total 29 different alkaloids are present in the crude extract of H. anlidysenlerica, conessine may be the component that inhibits the growth of bacteria l5

.

The antidiarrhoeal effect of the alkaloids from H. alllidyselllerica was similar to the standard drug, diphenoxylate, by inhibiting the production of watery stool s or fluid . The above observation suggests that the alkaloids of H. alllidysenterica (in graded doses) reduced diarrhoea in castor oil induced rats. Tripathi 37

reported that astringent properties of tannins of PUllica granatum, are responsible for protein denaturation producing protein tannate, which reduces secretion from intestinal mucosa. Similarly, H. alltidysellterica also contains the astringent

Table 2-Effect of alkaloids of H. alltidysellterica on castor o il-induced diarrhoea in rats

(Values are mean ± SO from 6 rats in each group)

Group

II 1lI IV V VI

*P < O.ool

Pretreatment

Saline 5 ml /kg (Control) Diphenoxylate 5 mg/kg Alkaloid 200 mg/kg

400 mg/kg 600 mg/kg 800 mg/kg

2 3

Defecati ona per group drops

2.632 ± 0.01 0.557 ± 0.20 2.021 ± 0.023 1.257 ± 0.020 0.810 ± 0.026 0.565 ±0.35

4

Fig.3-Plasmid profile of EPEC 1402fc on 0.8% agarose ge l. [Lane I-EPEC 1402 fc, Lane 2, 3 and 4 treated with alkalo ids of H. alltidysellterica. 0.8mg 1.6mg and 3.2 mg/ml respective ly]

properties of alkaloids which may produce . . • 12

anti secretory activity . Abbanat et ai., 38 reported that the new antibacterial

agents, Hongoquericins damage the membrane of E. coli imp strains, as the primary mode of their bactericidal action39

. Fukao et ai. ,40 reported that the combination of hop resins sodium hexametaphosphate damage the cell membrane of the E. coli strains. The present observations, suggest that the suppression of high molecular weight outer membrane proteins (Fig. 2) may be due to the alkaloids of H. antidysenlerica. The loss of plasmid DNA bands (Fig. 3) may be due to the involvemet of alkaloids.

Further studies are necessary to confirm the role of alkaloids on plasmid and outer membrane proteins of EPEe. These findings have initiated search for a new drug target for the antidiarrhoeal activity of alkaloids. However, the antibacterial and antidiarrhoeal activity of alkaloids of H. aillidysenterica look promising .

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