PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITY ON THE FRUITS OF HUGONIA MYSTAX L. (LINACEAE)

PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITY ON THE FRUITS OF HUGONIA MYSTAX L. (LINACEAE)

A. Vimalavady*¹, K. Kadavul ¹ and A. C. Tangavelou ²

Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies ¹, Lawspet, Puducherry- 605008, India

Bio - Science Research Foundation ², 166/1 Gunda Salai, Moolakulam, Puducherry 605010, India

ABSTRACT

An ethnomedicinal plant, Hugonia mystax L., was examined for preliminary phytochemical screening and antimicrobial activity. Preliminary phytochemical screening showed the presence of various classes of secondary metabolites such as flavonoids, phenols, saponins, steroids, tannins and terpenoids.  Antimicrobial activity of petroleum ether, chloroform, ethanolic and aqueous fruits extracts showed significant activity against the human pathogens such as Streptococcus pneumoniae causing brain abscesses, pneumonia and septic arthritis; Proteus vulgaris, Pseudomonas aeruginosa causing urinary tract infections and septicaemia; Salmonella typhi causing typhoid fever, Vibrio species causing diarrheal infections and the fungus Candida albicans causes urinary tract infections. The antimicrobial activity of the petroleum ether, chloroform, ethanolic and aqueous fruits extracts showed concentration-dependent activity against all the tested bacteria at various concentrations. Thus, the present findings revealed the medicinal potential of H. mystax to develop a drug against various human ailments.

Drug development

INTRODUCTION:Medicinal plants have always playing a vital role in the therapeutic armoury of mankind ¹. WHO estimated that 80% of world populations in developing countries are totally dependent on medicinal plants for their primary health care ². Over 25% of prescribed medicines in industrialised countries derive directly or indirectly from plants ³. At least 25% of the prescription drugs issued in the USA and Canada contain bioactive compounds that are derived from or modeled from plant natural products ⁴. It is estimated that only 5-15% of the approximately 250,000 described high plant species have ever been in the focus of phytochemical and pharmacological investigations ⁵. Only 122 compounds were identified from 94 plant species based on ethnomedicinal usage ⁶. Plants have been a prime source of highly effective conventional drugs for the treatment of various diseases ⁷.

The genus Hugonia L., of family Linaceae comprise about 40 species in the world; of which two species namely Hugonia mystax L., and H. ferruginea Wight & Arn., were reported from India ^{8, 9}. This plant Hugonia mystax L. locally known as Modirakanni. Ethnobotanically the fruits used by the tribals of Kalakad Mundanthurai for the treatment of rheumatism ¹⁰. Review of literature revealed less work on this plant. Hence, in the present study, preliminary phytochemical screening and antimicrobial activity of various extracts of the fruits of Hugonia mystax were reported to provide a scientific evidence to prove the ethnobotanical usages.

MATERIALS AND METHODS:

Collection of Plant material: The fruits Hugonia mystax L. was collected on June 2010 from the Marakanam forest of Villupuram district, Tamil Nadu, India.  The collected plant material was botanically identified and confirmed by using  Flora of Tamil Nadu Vols. 1-3 ¹¹ and ^’An Excursion Flora of Central Tamil Nadu, India ¹².The species confirmation was done at French Institute Herbarium, Puducherry (FIHP). The herbarium specimen was prepared and deposited at the Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Puducherry, for future reference.

Preparation of the Extracts: The collected fruit materials were chopped into small pieces separately, shade-dried and coarsely powdered using a pulverizor.  The coarse powders were subjected to successive extraction with organic solvents of increasing polarity such as petroleum ether, chloroform and ethanol by Soxhlet method ¹³. The extracts were collected and distilled off on a water bath at atmospheric pressure and the last trace of the solvents was removed in vacuo and stored at 4^oC. The resulted extracts were used for preliminary phytochemical screening and antimicrobial investigation.

The fresh fruits were collected and 30 g was weighed for aqueous extraction. Then, the fruits was chopped and divided into 3 portions. Each portion was crushed by grinding with the help of mortar and pestle, transferred in to a suitable glass bottle and added 50 mL of distilled water in each of it. First glass bottle was autoclaved at 10 lbs. for 20 min. The second was boiled (100^oC) for 20 min. The third was mechanically shaken (200 rpm) in cold condition for two hours. The extracts were filtered off using cheese cloth and 0.45µ filter papers, transferred into sterile closed containers.  The crude extract was considered as 100% extract.  By adding sterile distilled water, 50% and 25% of the extract was prepared ¹⁴.

Preliminary phytochemical screening: All the extracts were subjected to preliminary phytochemical tests followed by the standard methods ^{13, 15}.

In vitro antimicrobial activity: All the three extracts (petroleum ether, chloroform and ethanol extracts) were prepared in various concentrations such as 100, 50 and 25 mg/mL respectively and used for antimicrobial activity.

Test Microorganisms: The following bacterial strains and fungal strains were used for the study of antimicrobial activity. The microbial strains of human pathogens used were procured from IMTECH, Chandigarh  which include six Gram-negative bacteria, viz. Escherichia coli (MTCC 724), Proteus vulgaris (MTCC 426), Pseudomonas aeruginosa (MTCC 741), Salmonella typhi (MTCC 733), Vibrio parahaemolyticus (MTCC 451) and V. vulnificus (MTCC 1145);  three Gram-positive bacteria, viz. Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 96) and Streptococcus pneumoniae (MTCC 655); and four fungi, viz. Aspergillus flavus, A. fumigatus, A. niger (MTCC 1344) and Candida albicans (MTCC 227).

Determination of antimicrobial activity: Agar well-diffusion method ¹⁶ (Perez et al., 1990) was followed to determine the antimicrobial activity. Nutrient agar (NA) and Potato Dextrose Agar (PDA) plates were swabbed (sterile cotton swabs) with 8 hour old - broth culture of respective bacteria and fungi. Four wells (10mm diameter) were made in each of these plates using sterile cork borer. About 0.3 ml of different concentrations of plant solvent extracts were added using sterilized dropping pipettes into the wells and allowed to diffuse at room temperature for 2 h. The plates in triplicates were incubated at 37°C for 18-24 hour for bacterial pathogens and at 28°C for fungal pathogens. Respective proper controls of solvent plant extracts were also maintained. The experiment was repeated thrice.  Diameter of the inhibition zones and the average values were recorded.

RESULTS: The results of preliminary phytochemical screening were given in Table 1. From the tested all three extracts revealed the presence of phenolic compound and terpenoids in all the three extracts. Carbohydrates and flavonoids present both in petroleum ether and ethanol extracts. Likewise tannins present both in chloroform and ethanol extract. Saponins and steroids present only in ethanol extract. Alkaloids, amino acids, anthraquinones, catechins, coumarins, gum, oil & resins, proteins and quinones were absent in all the three extracts.

TABLE 1: PRELIMINARY PHYTOCHEMICAL SCREENING OF VARIOUS EXTRACTS ON THE FRUITS OF HUGONIA MYSTAX L.

+ = present, - = absent

The results of antimicrobial activity of fruits of H. mystax were given in the Table 2. All the extracts showed concentration-dependent activity against all the tested microorganisms.

Petroleum ether extract, zone of inhibition was ranged between 13-18 against gram positive bacteria, 12-18 against gram negative bacteria, P. vulgaris and V. vulnificus did shows any activity against gram negative bacteria, 12-16 against fungi. For petroleum extract did not recorded as maximum zone of inhibition.

TABLE 2:  ANTIMICROBIAL ACTIVITY OF VARIOUS EXTRACTS ON THE FRUITS OF HUGONIA MYSTAX L., AGAINST VARIOUS MICRO ORGANISMS.

(Measurement indicates the zone of inhibition). A – Ampicillin; Cl – Clotrimazole; Cf – Ciprofloxacin; K – Kanamycin;   P - Penicillin

Chloroform extract did not shows any activity against tested microorganisms except A. flavus the maximum zone of inhibition was recorded as 20mm against 100mg/mL concentration.

For ethanol extract, the zone of inhibition recorded ranged between 22 - 31mm against gram-positive bacteria. Maximum zone of inhibition was recorded as 31mm against B. subtilis at 100mg/mL; 29mm against S. pneumoniae at 100mg/mL; 28mm each against B. subtilis & S. pneumoniae at 50mg/mL; 26mm against S. aureus at 100mg/mL; 25mm each against B. subtilis & S. pneumoniae at 25mg/mL; 24, 22mm against  S. aureus at 100, 50mg/mL concentration respectively.

In gram-negative bacteria, the zone of inhibition recorded ranged between 23-33mm. Maximum zone of inhibition was recorded as 33&32mm against P. vulgaris at 100&50mg/mL; 31mm each against V. parahaemolyticus at 100 mg/mL & P. vulgaris at 25mg/mL; 30 mm against V. parahaemolyticus at 50mg/mL; 29mm each against P. aeruginosa at 100mg/mL, V. parahaemolyticus at 25mg/mL; 28mm each against S. typhi & V. vulnificus at 100mg/mL, P. aeruginosa at 50mg/mL; 27mm each against S. typhi & V. vulnificus at 100mg/mL, P. aeruginosa at 25mg/mL; 26mm each against E. coli at 100mg/ml, V. vulnificus at 25mg/mL; 25mm each against E. coli at 50mg/mL, S. typhi at 25 mg/mL; 23mm against E. coli at 25mg/mL concentration. In fungi, zone of inhibition recorded ranged between 20 and 32mm. Maximum zone of inhibition was recorded as 32mm against C. albicans at 100mg/mL, 30mm against A. fumigatus at 100mg/mL, C. albicans at 50mg/mL; 29mm each against A. flavus at 100mg/mL, A. fumigatus at 50mg/mL, C. albicans at 25mg/mL; 28mm each against A. flavus at 50mg/mL, A. fumigatus at 25mg/mL;  27mm against A. flavus at 25mg/mL; 25, 23 & 20mm against A. niger at 100, 50 & 25mg/mL concentration respectively.

Autoclaved extract, the zone of inhibition recorded ranged from 18-29mm against gram-positive bacteria. Maximum zone of inhibition was recorded as 29 mm against B. subtilis at 100%;  28mm against S. pneumoniae at 100%; 25mm each against B. subtilis and S. pneumoniae at 50%; 23mm against B. subtilis at 25%; 21mm against S. pneumoniae at 25% and 20mm against S. aureus at 100%. In gram-negative bacteria, the zone of inhibition recorded ranged between 16-26mm. Maximum zone of inhibition was recorded as 26mm against P. aeruginosa at 100%; 25mm each against E. coli and P. vulgaris at 100%; 24mm each against E. coli, P. aeruginosa and P. vulgaris at 50%, 23mm each against V. parahaemolyticus at 100%, P. aeruginosa at 25%, 21mm each against S. typhi at 100%, V. parahaemolyticus at 50% and E. coli at 25% respectively, 20mm each against S. typhi at 50%, P. vulgaris and V. parahaemolyticus at 25% concentrations respectively. In fungi, zone of inhibition recorded was ranged from 15-25mm. Maximum zone of inhibition was recorded as 25, 24 & 23mm against A. fumigatus at 100, 50 & 25%; 22, 21mm against A. flavus at 100, 50%; 20mm each against C. albicans at 100% and A. flavus at 25% concentration respectively.

Boiled extract, the zone of inhibition recorded ranged 22-27mm against gram-positive bacteria. Maximum zone of inhibition was recorded as 27mm against B. subtilis at 100%; 25mm each against S. aureus and S. pneumoniae at 100%; 24mm each against B. subtilis and S. aureus at 50%; 23mm each against S. pneumoniae at 50%, B. subtilis at 25%; 22mm each against S. aureus and S. pneumoniae at 25% concentration respectively. In gram-negative bacteria, the zone of inhibition recorded ranged from 18-26mm. Maximum zone of inhibition was recorded as 26mm against P. aeruginosa 100%; 25mm against V. parahaemolyticus at 100%, 24mm each against S. typhi at 100%,  P. aeruginosa and V. parahaemolyticus at 50%, 23mm each against E. coli at 100%, P. aeruginosa and V. parahaemolyticus at 100%; 22mm each against V. vulnificus at 100%,  E. coli at 50%; 21mm S. typhi and V. vulnificus at 50%, E. coli at 25%; 20mm each against P. vulgaris at 100%, S. typhi and V. vulnificus at  25%; In fungi, zone of inhibition recorded ranged from 14 - 20mm. Maximum zone of inhibition was recorded as 20mm against A. flavus and C. albicans at 100% concentration respectively.

Cold extract, the zone of inhibition recorded ranged 17-22mm against gram-positive bacteria. Maximum zone of inhibition was recorded as 22&21mm against S. aureus at 100&50%; 20mm against S. pneumoniae at 100%. In gram-negative bacteria, the zone of inhibition recorded ranged from 15-25mm. Maximum zone of inhibition was recorded as 25&24mm against P. aeruginosa at 100&50%; 23mm each against P. vulgaris at 100% and P. aeruginosa at 25%; 22mm against P. vulgaris at 50%; 21mm each against E. coli and S. typhi at 100%; P. vulgaris at 25%; 20mm each against E. coli at 50%; S. typhi at 50, 25% respectively. In fungi, zone of inhibition recorded ranged from 16 - 25mm. Maximum zone of inhibition was recorded as 25mm against A. flavus at 100%; 24mm each against A. fumigatus and C. albicans at 100%, A. flavus at 50%; 23mm each against A. fumigatus at 50%, A. flavus at 25%; 21mm each against A. niger at 100%, C. albicans at 50%, A. fumigatus at 25% concentration respectively.

DISCUSSION:Phytochemical screening of the petroleum ether, chloroform and ethanol extracts of fruits of H. mystax showed the presence of various classes of secondary metabolites such as flavonoids, phenols, saponins, steroids, tannins and terpenoids.  The presence of secondary metabolites in plants produces some biological activity and the major source of pharmaceuticals, food additives, fragrances and pesticides ^17-20. A number of plant secondary metabolites have been found to be valuable pharmacological probes or biochemical tools to help target various receptors as well as to explain cellular processes and assist with the elucidation of different kinds of molecular targets ²¹.

From the results of antimicrobial activity, it was found that the petroleum ether and ethanol extracts exhibited maximum antimicrobial activity against the tested human pathogens.

In our study, the maximum zone of inhibition against gram negative bacteria such as E. coli, P. vulgaris, P. aeruginosa, S. typhi and V. parahaemolyticus and against the fungi such as A. flavus and C. albicans may be due to the presence of various classes of phytochemicals such as flavonoids, phenolic groups and steroids as suggested by previous reports ^22-24. The significant activity of the results against the fungi, A. flavus and Candida albicans provides additional confirmation to the phenolic compounds and steroidal compounds which are more effective in higher concentration inhibited the growth of all fungi ^{25, 26}. Even in hospitals, majority of disinfectants such as phenols, lysol, cresols used are belonging to phenolic groups.

Thus, recent findings of antimicrobial activity against P. aeruginosa, P. vulgaris, S. typhi, V. parahaemolyticus and V. vulnificus revealed the medicinal potential values. Evidently, the petroleum ether, ethanol and all the aqueous extracts proved to possess the antibiotic potential against various pathogens causes abdominal pain, diarrhea, fever, nausea, septicaemia, urinary tract infections and vomiting (E. coli), wound and septicaemia infections (P. vulgaris and P. aeruginosa), typhoid fever (S. typhi) and diarrheal infections (Vibrio species).

Further, inhibition zone on against the growth of the fungal pathogen causes skin related diseases (C. albicans) and aspergillosis and respiratory tract infections (A. flavus) respectively.

CONCLUSION: The phytochemical compounds which are responsible for the significant inhibitory activity against various human infections should be isolated, purified and identified to develop a new lead of therapeutic interest to cure various human ailments.

ACKNOWLEDGEMENTS: We the authors express our profound gratitude to our Director, Dr. P. Shyma and Dr. A. Pragasam Head of the department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, for providing necessary lab facilities in the department. The first author (A. Vimalavady) thanks Puducherry Adidravidar Development Corporation (PADCO), Puducherry, for the financial assistance to carry out this research.

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