Anticandidal Activity of Punica granatum. Exhibited in Different Solvents

Abstract

Anticandidal activity of Punica granatum L.. extracts in ethanol, methanol, propanol, acetone, benzyl alcohol, 1,4-dioxan, and N,N.-dimethyl formamide (DMF) is reported. The selection of solvents was on the basis of their polarity. Growth inhibition using the agar ditch diffusion assay was determined on four different strains of Candida. species isolated from HIV-positive patients. Ethanolic extract showed activity against three Candida. species, whereas it was ineffective against Candida albicans. 1. The methanol, acetone, and propanol extracts were effective against all four Candida. species. The DMF extract was effective against Candida albicans. 1 and 2 and showed moderate activity against Candida albicans. 3, whereas it was ineffective against “Candida species. 1.” The 1,4-dioxan extract was effective against Candida albicans. 1, Candida albicans. 2, and Candida species. 1 and was ineffective against Candida albicans. 3. The benzyl alcoholic extract was ineffective in showing any kind of activity against any of the four candidal strains. These different extracts of P. granatum. were also compared with commercially available antibiotics such as nystatin, clotrimazole, and amphotericin B, and the natural plant extract proved to be better against some candidal species.

Keywords:

• Anticandidal activity
• medicinal plant
• Punicaceae
• Punica granatum

Introduction

The discovery and development of antibiotics are among the most powerful and successful achievements of modern science and technology for the control of infectious diseases. However, the increasing antimicrobial resistance emergence and its dissemination among bacterial strains reduces the efficiency of treatment success of many drugs. The effects of plant extracts on bacteria and fungi have been studied by a very large number of workers in different parts of the world (Jelager et al., 1998; Ates & Erdogrul, 2003).

The investigation of traditional remedies largely of botanical origin, on which a worldwide majority of the population still relies, have been targeted for new drugs and lead compounds to create a bank of bioactive compounds through which the discovery and development of new and innovative therapeutic agents could be accelerated. Recent advances in drug discovery have revealed that natural product chemistry plays a significant role in developing new lead compounds of better therapeutic efficiency.

With consumers becoming more conscious about the side effects of synthetic health care products, the increased use of natural products for treating many diseases has recorded a landmark in the field of pharmaceuticals. It is now well recognized that the exploration of traditional herbal remedies is a viable research initiative for new pharmaceuticals. Judicious use of medicinal herbs can even cure deadly diseases that have long defied synthetic drugs (Bhattacharjee, 1998).

Traditional medicine is an important source of potentially useful new compounds for development of chemotherapeutics (Farnsworth et al., 1985). India is rich in medicinal plant diversity. All known types of agroclimatic, ecological, and edaphic conditions are met within India. India is rich in all the three levels of biodiversity, such as species diversity, genetic diversity, and habitat diversity (Zafar et al., 1999).

Plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines. A wide range of herbs has healing properties. The effects of herbal compounds and phytochemicals on pathogenic and economically important bacteria have been well studied. Although some therapeutic benefits can be traced to specific plant compounds, many herbs contain dozens of active constituents that, together, combine to give the plant its therapeutic value. Because of its vast and wide variations in soil and climate, the Indian subcontinent is suitable for the cultivation of a large number of medicinal and aromatic plants that can be used as raw materials for pharmaceutical, perfumery, cosmetics, flavor and food, and agrochemical industries. Four Siddha drugs, viz., Nandhi mezhugh, Parangi pattai choornam, Erasa kenthi mezhugu, and Vaan mezhugu (in order of efficacy), were found to have significant antifungal activity when tested against 14 strains of Candida albicans. 167 (Suresh et al., 1994).

Punica granatum. L. is a shrub belonging to the unigeneric family Punicaceae, a native of semitropical Asia. The different parts commonly used are leaf, flower, fruit, fruit rind, seed, dried bark of stem, and root (Holetz et al. 2002). The root bark shows activity against tapeworms. Astringent properties of the fruit rind and fruit juice explain the antidiarrheal activity. The bark and seeds are useful in bronchitis. The flowers are used in epistaxis. The unripe fruit is a good appetizer, and it is useful in nausea and vomiting. The ripe fruit is tonic, astringent to the bowels, and relieves burning sensation of the body. The rind of the fruit is very useful in diarrhea and dysentery. The fresh juice is used in cooling and refrigerant mixtures of some medicines for dyspepsia. The root bark has been used as an anthelmintic.

The aim of this study is natural drug discovery. Various plants belonging to this region were investigated. In the current work, in vitro. anticandidal activity of P. granatum. leaf was investigated, being extracted in different solvents.

Materials and Methods

Plant material and preparation of extract

The plant part selected for antibacterial study was the leaf of P. granatum.. Fresh leaf material of P. granatum. was collected randomly by leads supplied by local healers in the month of August 2002 at Rajkot, Gujarat, India. They were washed thoroughly under running tap water for 2 h and then gently dried, and the required amount, usually 25 g, was macerated with the help of a homogenizer. The slurry was taken in a conical flask, and 100 ml of the solvent was added. It was then kept on a rotary shaker at 180–190 rpm for 24 h. After 24 h, it was filtered through 8 layers of muslin cloth and centrifuged at 5000 × g. for 15 min. The supernatant was collected, and the solvent was made to evaporate so to make the final volume one-fourth of the original volume. It was then stored in airtight bottles for further phytochemical and microbiological assays.

Solvents used for antimicrobial assay

Plant extracts are complex, and isolating bioactive compounds is challenging. Only a few extractants have generally been used for isolating antimicrobial compounds from plants, and Cowan (1999) concluded that many classes of compounds are commonly obtained in only one solvent. The aim of this study was to investigate whether different solvents would simplify extracts to facilitate the isolation of anticandidal compounds from the complex crude mixture. The solvents were selected to represent a wide range of polarities and selectivity groups (Snyder & Kirkland, 1979). The solvents used and their polarities are as follows:

Table

Solvents	Dipole moment
1,4-Dioxan	0
Benzyl alcohol	1.66 (20)
Propanol	1.657 (20)
Methanol	1.7
Ethanol	1.69 (20)
Acetone	2.88 (20)
N, N.-Dimethyl formamide (DMF)	3.86 (25)

Microorganisms used and growth conditions

The test organisms were four different strains of Candida. species isolated from HIV-positive patients and obtained locally from a clinical microbiological laboratory (courtesy of Surendranagar Civil Hospital and Medical College). They are numbered as Candida albicans. 1, “Candida. species 1,” Candida albicans. 2, and Candida albicans. 3. The Candida. were grown on Sabouraud broth at 37°C and maintained on Sabouraud agar slants at 4°C.

Activation of test candidal strain

A loop full of the test strain was inoculated in 25 ml of Sabouraud broth and incubated for 24 h on a rotary shaker to activate the given test candidal strain.

The Sabouraud dextrose agar plates were prepared for the study of in vitro. antimicrobial activity by agar diffusion method. Agar ditch diffusion method (Perez et al., 1990) was performed for the antimicrobial assay.

Microbiological assay

Inoculation of the test strain was done by the pour-plate technique. The activated strain (0.2 ml) was inoculated into the media when it reached temperatures of 40–45°C. Proper homogenization of the strain was done by shaking the sugar tube in which the media was autoclaved first by inoculating the strain into it and then by shaking the tube for a couple of seconds and then pouring it gently into the Petri plate. The complete procedure of the plate preparation was done in laminar airflow to maintain a strict sterile and aseptic condition. The solidification of the media took about 30 min. After the media solidified, a ditch was made in the plates with the help of a cup-borer (0.85 cm), and then it was filled with the test compound. The inhibitory activity of the compounds in various solvents was determined by comparing the sizes of inhibition zones of the different compounds in different solvents with those of the positive controls.

Results and Discussion

The inhibitory zones produced by P. granatum. leaf extract in various solvents like 1,4-dioxan, benzyl alcohol, propanol, ethanol, methanol, acetone, and DMF on Candida albicans. 1 and Candida. species 1 are shown in Figure 1. The compound extracted in ethanol and benzyl alcohol did not produce any inhibitory zone in Candida albicans. 1, while maximum inhibition was against the compound extracted in methanol followed by acetone and 1,4-dioxan, respectively, and minimum inhibition was in propanol-extracted compound.

Figure 1 Anticandidal activity against Candida albicans. 1 and Candida. species 1.

The effect of the compounds extracted in various solvents showed an entirely different trend on Candida. species 1 except benzyl alcohol, which did not inhibit the candidal strain; it was also resistant against polar solvent DMF. Maximum inhibitory zone was produced by the compound extracted in acetone followed by 1,4-dioxan, ethanol, and methanol, respectively. Here, also, compound extracted in propanol was least effective.

The inhibitory zones produced by compounds extracted in different solvents on Candida albicans. 2 and Candida albicans. 3 are shown in Figure 2. The effect of compounds in different solvents on Candida albicans. 2 was similar to that of Candida. species 1. Here, also, benzyl alcohol did not produce any inhibitory zone, but unlike Candida. species 1, the compound extracted in DMF could inhibit Candida albicans. 2. In fact, maximum inhibitory zone was produced by acetone extract and DMF extract followed by all the other solvents. An entirely new trend was observed when Candida albicans. 3 was considered. The compounds extracted in DMF, 1,4-dioxan, and benzyl alcohol did not produce any inhibitory zone, whereas all the other four solvent-extracted compounds inhibited Candida albicans. 3.

Figure 2 Anticandidal activity against Candida albicans. 2 and Candida albicans. 3.

Thus, from the above results, it can be seen that the ethanolic extract showed maximum anticandidal activity against Candida. species 1 and Candida albicans. 3. It showed moderate activity against Candida albicans. 2, whereas no activity was seen against Candida albicans. 1. The methanolic extract showed maximum activity against Candida albicans. 1 followed by Candida albicans. 3, and moderate activity was seen against Candida. species 1 and Candida albicans. 2. The acetone extract showed almost similar activity against all the candidal strains. The propanol extract showed moderate activity against Candida albicans. 1 and Candida. species 1, and more activity was observed against Candida albicans. 2 and Candida albicans. 3. The DMF extract showed significant activity against Candida albicans. 1 and Candida albicans. 2, whereas no activity was seen against Candida. species 1 and Candida albicans. 3. The 1,4-dioxan extract showed considerable activity against Candida albicans. 1 and Candida. species 1, moderate activity was seen against Candida albicans. 2, whereas no activity was observed against Candida albicans. 3. The benzyl alcohol extract showed no activity against any of the given Candidal strains.

Hence, we conclude that for Candida albicans. 1, methanolic extract showed maximum activity, whereas for Candida. species 1, Candida albicans. 2, and Candida albicans. 3, acetone extract showed maximum activity. In Candida albicans. 2, the DMF extracts also showed activity similar to acetone. Here, the acetone and DMF proved to be better solvents compared to other solvents. Thus, the results indicate that the compounds extracted in acetone, methanol, and DMF had the greatest anticandidal activity, whereas benzyl alcohol had no activity. This discriminatory effect against specific candidal strains suggests the presence of different chemical compounds, and the most effective is that compound that is extracted in acetone solvent. Hence, this extract of acetone is carried on for further phytochemical analysis, as it is believed that this extract might contain some biologically active molecule that can be used to formulate a better drug for treatment against different ailments caused by different Candida. species.

These compounds were also compared to commercially available antibiotics such as nystatin, clotrimazole, and amphotericin B. For Candida albicans. 1, the methanol, propanol, acetone, 1,4-dioxan, and DMF proved to show better activity than clotrimazole, while among them the best activity was observed in methanol. Methanol also showed better activity than amphotericin B against Candida albicans. 1. In Candida albicans. 2, all the solvents except benzyl alcohol showed better activity than clotrimazole, but among them acetone and DMF showed better results. In the same way for Candida albicans. 3, ethanol, methanol, acetone, and propanol showed better activity than clotrimazole, but among them acetone showed the maximum activity. Thus, it appears that P. granatum. leaf has some compound with promising results and is better than some of the commercially available antibiotics. Hence, this compound can be used as a lead molecule to combat the diseases caused by these candidal species.