Abstract

Aim: To study the antimycotic, antibacterial and phytochemical properties of inherent in leaves, roots, stems, seeds and seed pods of Gynandropsis pentaphylla DC. Procedure: Fresh leaves, roots, stems, seeds and seed pods of G. pentaphylla were used to extract with methanol, acetone and water. Fresh extracts of were tested against 2 bacteria and also against 3 fungi using well diffusion method of susceptibility testing on sensitivity testing agar medium. Extracts of roots, stems, leaves, seeds and seed pods were screened phytochemically for the presence of secondary metabolites such as alkaloid, flavones, sugar, phenolic group, essential oil, amino acids and saponin. Results: Bacterial cultures of Escherichia coli and Staphylococcus aureus were highly susceptible to the acetone extracts of all the five parts of G. pentaphylla. Aspergillus niger, Aspergillus flavus and Metarhizium anisopliae were highly susceptible to the methanol extracts of all the parts of G. pentaphylla. The seed showed highest activity among all the plant parts. Highest activity index was observed for all extracts against the entomopathogenic fungi M. anisopliae. Both M. anisopliae and G. pentaphylla are active pest control agent, since both are incompatible to each other, it is difficult to use the both in integrated pest management (IPM) combinedly to manage the pests. Conclusion: The active antimicrobial ingredients in G. pentaphylla should be identified while its medicinal value to humans properly investigated in this regard.

Introduction

The cat's whiskers 11 , 5 (Gynandropsis pentaphylla DC.) is one vegetable, which grows as a weed in most tropical countries. The leaves and seeds of cat's whiskers are used in indigenous medicine in many countries 11 . In India the common names include kurhur and karaila 11 . Gynandropsis pentaphylla belongs to the plant family Capparidaceae. Gynandropsis pentaphylla DC syn Gynandropsis gynandra L. (Briq.) and Cleome gynandra L. (Briq) 11 . It is a herb indigenous to the tropical and sub tropical regions. The herb is edible and grows up to 60cm high 12 , 10 , 14 , 1 .

Kurhur has been used for several years in Indian traditional medical practices. G. pentaphylla leaves with a high percentage of vitamin C is taken as a pot herb in soups, fresh or dried 11 . The leaves are used as disinfectants. Inhalation of the leaves also relieves headaches; leaf juice and oil, for earache and eye wash 11 . Seeds have been reputed to have antihelmintic properties and oil is used as fish poison 25 , 26 , 11 . Sterols are also found in in these plants; with lupeol, campesterol and epi-lupeol having been isolated from this palnt 19 , 20 . In previous studies, the anthelmintic and antimicrobial properties of Capparidaceae plants have been reported 4 , 6 , 5 from different countries and in continuation of these objectives on this plant family, the phytochemical, antibacterial and antifungal properties of Gynandropsis pentaphylla are presented in the current study for the Indian G. pentaphylla.

G. pentaphylla plants have been observed to have insecticidal, antifeedant and repellent characteristics 7 , 21 , 22 , 11 . The leaves have anti-tick properties. They also have repellent and acaricidal properties for larvae, nymphs and adult Rhipicephalus appendiculatus and Amblyomma variegatum ticks. Ticks may not be found for a distance of 2-5 m from the plant 11 . The ethanol extract is toxic to insect pests, such as the painted bug (Bagrada cruciferarum Kirk) and the diamond back moth (Plutella xylostella L.) of cruciferous vegetables. The volatile oils permanently repel the diamond back moth larvae from treated cabbage leaves. The plant has an anti-feedant action against the tobacco caterpillar (Spodoptera litura F.). The extract from the mature seeds is toxic to brinjal aphid (Aphis gossypii Glov.), and the larvae of Helicoverpa armigera (Hubner). The seeds contain phenolic compounds, which are natural products 15 , 11 . Lipids from seeds could be used in soap manufacture 13 , 11 . The biocontrol agent, Metarhizium anisopliae (Metsch.) Sorokin (Deuteromycotina: Hyphomycetes) is an extensively studied cosmopolitan filamentous fungus, which is a key regulatory organism of insect populations 8 . No reports were available on the activity of G. pentaphylla against this entomopathogenic fungi. With this views in mind the present is designed to carried out the activity of leaves, roots, stems, seeds and seed pods extracts of indigenous G. pentaphylla against two bacterial and three fungal species such as Escherichia coli and Staphylococcus aureus and Aspergillus niger, Aspergillus flavus and M. anisopliae.

Materials And Methods

Determination of antimicrobial activity

Sterile cottons swap was taken and dipped in 24 hours old S. aureus culture. The entire agar surface of LA plate was seeded first in horizontal direction and then in vertical direction to ensure the even distribution of organism over the agar surface using the above swap. The seeded agar surface was allowed to dry for five min. Tip of well cutter was sterilized by heating on the blue flame of Bunsen burner and used for the well preparation in the seeded LA plates. Three wells were prepared in each LA plate. As soon as the wells were prepared all plant part extracts (10µl) were poured in each well separately using sterile micro-tips. The same procedure was followed for E. coli in LA plate. The above protocol was adapted for A niger, A. flavus and M. anisopliae in PDA plates using 48 hours old PDB cultures. Antimicrobial agents [Cloroamphenicol (10mg/ml) and fluconozole (10mg/ml)] were pored as positive control and respective solvents were used as negative control. All LA plates and PDA plates were incubated at 37±2 ^o C for 24 hrs and 27±2 ^o C for 72 hrs. After incubation the zone of inhibition was measured using HiAntibiotic Zone Scale (HiMedia, Mumbai, India).

Activity Index

The zone of inhibition in extract and the standard antimicrobial agents were used to calculate the activity index.

Proportion index

Number of positive results obtained for water, methanol and acetone extract of one plant part was against all the microbials and total number tests carried out were used to evaluate the proportion index.

Results and Discussion

The phytochemical screening of Gynandropsis pentaphylla revealed the secondary metabolites which are of medicinal interest as presented in table 1. G. pentaphylla is rich in sugar, amino acid and phenolic group. However, in G. pentaphylla seeds extract (both water and other solvents) contained alkaloids, flavones, sugar, phenolic group, saponin, amino acid and essential oil in plenty of amounts (table 1). Saponins was comparatively less in this species. The variance in the quantitative composition of the secondary metabolite establishes the fact that different plant parts are not likely to have the same medicinal potential. These secondary metabolites are known to exhibit medicinal activity as well as physiological activity 23 .

Table 1: Phytochemical screening of Gynandropsis pentaphylla and yields of extracts

The results of the antimicrobial activity presented in table 2 shows that all extracts exhibited appreciable antimicrobial properties, inhibiting the growth of all microorganisms. While stem aqueous extracts not inhibit the growth of Escherichia coli, Staphylococcus aureus, Aspergillus niger and Aspergillus flavus. This is interesting as water is one of the medium through which traditional healers utilize medicinal plants to their clients 11 . Two clinical strains of human pathogenic fungi like A. niger and A. flavus and one entomopathogenic fungi, Metarhizium anisopliae were used to find out the antimycotic activity of G. pentaphylla. M. anisopliae was the most sensitive, this was closely followed by A. flavus and A. niger was the least sensitve in the assay.

Table 2: Antimicrobial activities of G. pentaphylla extracts

Generally, proportion index of antimicrobial activities of G. pentaphylla extracts shows the highest activity in seeds extract as presented in figure 1.

Figure 1: Proportion index of antimicrobial activities of G. pentaphylla extracts

The relationship between the sensitivity pattern of different microbials against various extracts used for the antimicrobial assay had been studied using STATISTICA/w 5.0. software (table 3).

Table 3: Correlation matrix of sensitivity of different microorganism by G. pentaphylla extracts

A high correlation coefficient (near 1 or -1) means a good relationship between two variables and its value around zero means no relationship between them at a significant level of p < 0.05. More precisely, it can be said that parameters showing r > 0.7 are considered to be strongly correlated whereas r between 0.5 and 0.7 shows moderate correlation. In the present study, the resultant matrix reveals strong positive correlation among different bacteria (table 3). The relationship between the antimicrobial activity pattern of different extracts against various microbials used for the assay had been studied. Highest correlation (0.99) was observed in between aqueous extract of root and acetone extract of seeds. Higher correlation (0.97) was observed in between acetone extract of leaves and roots; acetone extract of stems and standard antibiotic. 0.96 correlation was observed in between aqueous extracts of leaves and seed pods.

A data matrix of five organisms and 19 variables (different extracts with one aqueous and two solvents) were used for this cluster analysis, the results are presented in figure 2.

Figure 2: Cluster analysis of sensitivity of different microorganism by G. pentaphylla extracts

The microbes were separated into two main groups I and II based on their sensitivity: the bacteria, S. aureus with the highest resistant was clustered alone in group II and all the remaining organism with moderate and low resistant were grouped into I (figure 2). In the group I, only one fungi, A. flavus was sub clustered into IB, which is the second resistant organism (figure 2). G. pentaphylla extracts were more active against fungi than that of bacteria as presented in figure 3.

Figure 3: Activity index of G. pentaphylla extracts

Presence of alkaloids and reducing sugars in leaves and stems were reported by Ajaiyeoba 5 . In the present study also we observed the same results in stem and leaves. Apart from both leaves and stems we additionally observed the presence of alkaloids and reducing sugars in seeds, roots and seed pods extracts of G. pentaphylla. This is not suprising for plants of the Capparidaceae family 18 , 20 , 5 .

From the result of the antibacterial studies as shown in Table 2, all the extracts exhibited appreciable antibacterial properties, inhibiting the growth of all the bacteria and fungi. The same results were repotted by Ajaiyeoba 5 , but the zone of inhibition was comparatively less in methanol extracts of stems and leaves. However, in the present study we noted the highest activity (28.4±0.0) against E. coli in acetone extract of leaves. In most instances, activities were greater than the standard therapeutic agent, chloramphenicol as presented in figure 3. The current study was correlated with the previous report 5 . However, in the current investigation we haven't observed any insensitive bacteria to chloramphenicol.

In the antimycotic assay, G. pentaphylla have again displayed high antifungal activities. Methanol extract of G. pentaphylla stem showed the highest activities, inhibiting the growth of A. niger up to 20.2±0.2mm. Ajaiyeoba 5 observed less activity (13.0±0.5) against this fungi in his study. This difference in activity is may be because of the difference in geographical origin 11 . Highest activity index was observed generally against the entomopathogenic fungi M. anisopliae. M. anisopliae 8 and G. pentaphylla 11 , 24 are active pest control agents, since both are incompatible to each other, it is difficult to use the both pest control agents simultaneously in integrated pest management (IPM).

Conclusively, all extracts have displayed antimicrobial activities from present studies. The leaves and seeds of cat's whiskers are used in indigenous medicine in many countries 11 . This has further confirmed the use of this plants in Indian ethnopharmacology for treatment of bronchitis, boils, earache, eye wash, disinfectant and nasal congestion, analgesic, headaches, epileptic fits, facilitate childbirth in pregnant women, stomach-ache, constipation, conjunctivitis, thread-worm infection, chest pains, arthritis, inflammation, neuralgia, rheumatism, localized pains, pus, anaemia, uterine complaints, malaria, pneumonia, head lice and reduce coughing 10 , 3 , 17 , 14 , 11 , 16 , 5 . There is need for the development of new antibiotics due to acquired resistance, more importantly from natural sources as this delays resistance. G. pentaphylla used for the study provide good opportunities for drug development in this area. Further studies are needed to find out the exact compound responsible for the antimicrobial activity using thin layer chromatography, column chromatography and HPLC. Further purification and bioactivity is in progress in our laboratory and will be communicated in future.

Correspondence to

J. Francis Borgio Genecity Laboratories Pvt. Ltd. 128 / 1B, First Floor, Chhatrapati House, Paud Road, Kothrud, Pune – 411038, India. E.mail: borgiomicro@gmail.com Mobile: 0996005686

References