MENTHA PIPERITA LINN: PHYTOCHEMICAL, ANTIBACTERIAL AND DIPTERIAN ADULTICIDAL APPROACH

SACHINKUMAR R. PATIL1, RAHUL SHIVAJI PATIL*2, A. G. GODGHATE3

1 Department of Zoology, Jaysingpur College, Jaysingpur 416101 (M. S.), India, 2Department of Microbiology, 3Department of Chemistry, Dr. Ghali College, Gadhinglaj 416502 (M. S.), India
Email: [email protected] 
 

Received: 17 Dec 2015 Revised and Accepted: 03 Feb 2016


ABSTRACT

Objective: There is an immense need to develop an alternative antimicrobial source for the treatment of several infectious diseases and restrict its vectors. So, evaluation of phytochemicals from Mentha piperita, its antibacterial and adulticidal potential was undertaken in the present study.

Methods: The organic solvent of ethanol and water were used for the preparation of extract for the study. These extracts were analyzed for the qualitative and quantitative phytochemicals. Antimicrobial activity was determined by using agar well diffusion method, and Bioassay was carried out by "Sugar Bait" Method to check adulticidal potential.

Results: The Mentha piperita were found a rich source of phytochemical compounds like diterpenes, steroids, tannin, flavonoids, cardial glycosides, alkaloids, phenols, coumarin, and saponin. Both the extracts possesses active antibacterial compounds which shown antibacterial activity. The aqueous (5% of 50 µl) and ethanolic (5% of 100 µl) extract (EA) were found as effective concentration. Salmonella typhimurium showed resistance against both the extracts. The aqueous extract (AE) has found with effective adulticidal potential. Hence it can be used to control and repel most popular disease spreading Dipterian vector, Musca domestica.

Conclusion: These results confirm the presence of antibacterial, adulticidal compounds in Mentha piperita which gives an opportunity to explore the possible usage of Mentha piperita in the development of antibiotics and insecticides.

Keywords: Mentha piperita L., Phytochemical analysis, Antibacterial, Adulticidal potential


INTRODUCTION

The plant kingdom is a treasure house of potential drugs, and there has been an increasing awareness about their importance as medicinal plants. They are used locally in the treatment of infections caused by fungi, bacteria, viruses and parasites. Different plants have been used as a source of inspiration in the development of novel drugs.

Phytochemicals are the chemical compounds that occur naturally in plants are responsible for color and organoleptic properties, such as the deep purple of blueberries and smell of garlic. Peppermint (Mentha piperita) is a hybrid mint, a cross between watermint and spearmint. The plant, indigenous to Europe and the Middle East, is now widespread in cultivation in many regions of the world. It is found wild occasionally with its parent species. Peppermint has a long tradition of use in folk medicine and aromatherapy. Peppermint is commonly thought to soothe or treat symptoms such as nausea, vomiting, abdominal pain, indigestion, irritable bowel, and bloating, although most of these effects have not been adequately demonstrated in human research.

Mentha piperita L. is a medicinally important plant which comes under the family Labiatae [1]. It is a non-native herbaceous plant, perennial, which can reach up to 40 inches in height and has a four-sided stem while leaves are stalked opposite and toothed. The flowers are irregular in shape with pinkish or purplish color [2].

Antibiotics are the main basis used in the therapy of various microbial infections. On the basis of evidence of the rapid global spread of drug-resistant in microbes, the need to find new antimicrobial agents have great importance [3]. Wide varieties of antibiotics are commonly used in the treatment of serious infections caused by some aerobic Gram-negative bacteria [4]. The increased use of antibiotics has resulted in the development of antibiotics resistant bacteria [5]. From recent years, misuse of antibiotics impacting in multi-drug resistance among bacteria which accelerated the search for drugs effective against such bacteria. In 1996, it has been reported that sales of botanical medicines peaked by 37% than that of 1995 [6]. In this relation, various parts of plants, herbs and spices have been used for many years to prevent infections. These parts are easily available and can be used in a domestic setting with self-medication. Most of their properties are due to the oils produced by their secondary metabolites [7].

So, as there is a need to develop an alternative antimicrobial source for the treatment of several infectious diseases from other sources like plants; however a large number of literature are available on the medicinal properties of essential oils present in Mentha;but, no much study has been directed towards its other useful properties which are locally available. Therefore, the present study was undertaken to evaluate antibacterial potential and qualitative phytochemical analysis with adulticidal effect from leaves of Mentha piperita.

MATERIALS AND METHODS

Collection of plant material

The fresh and healthy leaves of Mentha piperita L. was collectedfrom the various localities of Chandgad Tahsil, Maharashtra, India; during October 2014. Leaves were washedwith distilled water so as to remove dirt and soil particles. Theleaves were crushed and dried in a shadedarea at room temperature for a period of a week. Then thedried leaves were grinded with an ordinary grinderand then sieved through server.

Identification

Mentha piperita L.was authenticated by Mr. R. S. Sawant, Associate Professor, Department of Botany, Dr. Ghali College, Gadhinglaj, Kolhapur district, Maharashtra, India and Mr. Vinayak Sardesai, Assistant Professor, Department of Botany, R. B. M. College, Chandgad, Kolhapur district, Maharashtra, India and the voucher specimen (RSP 001) was deposited in Shivaji University, Kolhapur.

Preparation of test extract for antibacterial activity

The aqueous and ethanolic extract of leaves of Mentha piperita were prepared by addition of 0.5 gm of powder and 1 gm of powder into 10 ml of respective solvents for getting 5% and 10% concentration respectively. These extracts were kept at room temperature for overnight. Sample further used after centrifugation.

Preparation of extracts for phytochemical analysis

The collected leaves of Mentha piperita Linn were washed and then dried under shade. The coarse powder was soaked in 500 ml of distilled water and extracted in the cold for 3 d with occasional shaking. The solvent from the total extract was filtered & concentrated on a water bath for 8 h. The remaining was used for the analysis of phytochemicals test; same procedure was followed for ethanolic extraction using ethanol.

Identification tests for phytochemical analysis

The aqueous and ethanolic extracts of the plant were analyzed for the qualitative and quantitative phytochemicals analysis as shown in table 1 using standard methods [8-11].

Test. organisms

The standard test microorganisms were used in the present study as: Staphylococcus aureus NCIM 2654, Salmonella typhimurium NCIM 2501, Bacillus cereus NCIM 2703 and Proteus vulgaris NCIM 2813 and were obtained from National Collection of Industrial Microorganisms, Pune, (M. S.) India.

Antibacterial activity

Antimicrobial activity of the aqueous and ethanolic extract of Mentha piperita against various microorganisms (table 2) was determined by using agar well diffusion method by using Nutrient agar medium and respective solvent used as a control.

Preparation of bacterial suspension

A loop full suspension of the test organisms was aseptically streaked onto nutrient agar slants and incubated at 37 °C for 24 h. The obtained bacterial growth was harvested from the respective slant and suspension was prepared using sterile 1 ml normal saline (0.85 gm NaCl in 100 ml of distilled water). Then suspensions were stored in the refrigerator at 4 °C until used [12].

Adulticidal potential

Test insect

Musca domestica, house flies, was reared in the insect rearing cage in the laboratory at room temperature and 55-60% relative humidity. A standard rearing method [13] was adapted to provide adult flies of 0-24 h old for running bioassay tests. The adults of Musca domestica were collected from various poultry farms of Ajara Tahsil and kept under the starvation condition for a day.

Extraction

The method of Freedman et al. [14] was adapted with minor modification. The 20 gm of dried leaf powder was kept in 80 ml of distilled water for overnight to obtain 25 X 104 ppm. The extract obtained was double filtered and used as stock solution. The various concentrations (10%, 20%, 40% and 50% of 25 X 104 ppm) were prepared from the stock solution by diluting with distilled water and used as working extracts and kept in a deep freezer (-18 °C) until used.

Bioassay by "Sugar Bait" method

The Sugar Bait method of Mansour et al. [15] was adapted with minor modification. Quantities of 2 gm sugar with 10 ml of respective working solution was mixed to prepare bait and placed in petri dishes. Control sugar bait was prepared by dissolving 2 gm of sugar in 10 ml of distilled water. Each baited petri dish was placed in a rearing cage containing 50 adults of Musca domestica and maintained at room temperature for 24 h to estimate potency of the various concentrations. Usually, three replications were carried out for each tested concentration alongside with control tests.

RESULTS AND DISCUSSION

Phytochemicals analysis

According to Harborne and Willians [16], flavonoids exhibit anti-inflammatory, antimicrobial, antioxidant, vascular activities along with other medicinal properties. There are several reports on the antimicrobial activity of flavonoids [17-19]. The crude extracts with flavonoids, steroids, and triterpenes have significant activity against various strains of Staphylococcus aureus, Escherichia coli and Streptococcus faecalis [20]. Tannin may be toxic to the organisms like bacteria, yeast and filamentous fungi [21] having a potential antiviral [22] and antibacterial activity [23, 24]. Alkaloids are used as anesthetic agents [25]. Terpenoids exhibit various important pharmacological activities, i.e., anti-inflammatory, anticancer, antimalarial, inhibition of cholesterol synthesis, antiviral and antibacterial activities [26].

The results (table 1) of chemical test with aqueous extract of Mentha piperita contain phytochemicals like diterpenes, steroids, tannin, flavonoids, Carbohydrates, alkaloids, phenols, coumarin, and saponin while the ethanolic extract also contains the same chemicals except tannin, cardiac glycosides and saponin; whereas the aqueous extract found rich one.


Table 1: Phytochemicals present in aqueous and ethanolic extract of Mentha piperita

S. No.

Phytochemical

Results

Aqueous extract

Ethanolic extract

1

Anthocyanin

-

-

2

Diterpenes: Copper acetate test

+

++

3

Steroids

+

++

4

Tannin:

Lead acetate test

++

-

FeCl3

+

-

5

Cardial Glycosides: Keller-Killani test

+++

-

6

Flavonoid:

Alkaline Reagent Test

+++

-

NH4OH

++

+

Mg turning test

-

-

Zn dust test

-

-

7

Phlobatannins

-

-

8

Phytosterol: Salkowski’s test

+

+

9

Alkaloids

Wagner’s reagent

+++

+

Hager’s reagent

++

+

10

Phenols: FeCl3 test

+++

++

11

Leucoanthocyanin

-

-

12

Coumarin

++

+

13

Saponin: Foam test

+++

-

Key: (+) Positive test, (-) Negative test, n=2


Antibacterial activity

The aqueous extract of Mentha piperita showed an inhibitory effect against Proteus vulgaris and Staphylococcus aureus and its 5% of 50 µl solutions is most effective while Bacillus cereus and Salmonella typhimurium don’t shown a zone of inhibition (table 2). The ethanolic extract is more effective and shown a significant zone of inhibition against Proteus vulgaris and Bacillus cereus and its 5% of 100 µl solutions is most effective concentration. Salmonella typhimurium shown resistance against both the extract (table 2). These results confirm the presence of antibacterial compounds in Mentha piperita and it may be used to fulfill the need of alternative antibiotics.


Table 2: Antibacterial activity of active components present in the leaf extract of Mentha piperita

Organism used

Zone of inhibition (in mm)

Aqueous extract (AE)

Ethanolic extract (EE)

5%

10%

5%

10%

50 µl

100 µl

50 µl

100 µl

50 µl

100 µl

50 µl

100 µl

Staphylococcus aureus NCIM 2654

15.0±1.0

16.0±1.0

12.0±2.0

15.0±1.0

-

-

-

-

Salmonella typhimurium NCIM 2501

-

-

-

-

-

-

-

-

Proteus vulgaris NCIM 2813

13.0±1.0

11.0±2.0

13.0±1.0

13.0±1.0

14.0±2.0

16.0±1.0

20.0±1.0

21.0±1.0

Bacillus cereus NCIM 2703

-

-

-

-

16.0±1.0

19.0±1.0

18.0±2.0

17.0±1.0

Note: Each value is the mean of three readings±SD. n=3


Adulticidal potential

The adulticidal potential of Mentha piperita on Musca domestica was evaluated. The survival rate and Mortality rate are represented in table 3 and table 4 respectively. The present study revealed that 40% of working extract was effective as an adulticide. This proportion exhibited 100% Mortality. It might be due to various phytochemicals present in the leaf of Mentha piperita. Lower proportions have shown a comparatively low rate of Mortality ultimately due to the lower quantity of secondary metabolites. On the other hand, the rate of Mortality was expected higher in 50% but, it was found quietly similar to that of 10% of the extract.

This might be due to repellent properties of secondary metabolites. During the study, although Mortality rate at 10% was noticed low, the activity of adult flies got diminished. It indicates that higher quantity of secondary metabolites acts as repellent due to which files didn’t show any interest in sugar bait along with the extract.


Table 3: Survival rate of adult Musca domestica at various concentrations of Mentha piperita with respect to time

Proportion of working solution

No. of adults introduced

No. of adults survived after 6 h

No. of adults survived after 12 h

No. of adults survived after 18 h

No. of adults survived after 24 h

Control

50

50±0.0

50±0.0

49±1.0

48±1.0

10 %

50

47±1.0

42±1.0

37±1.0

25±1.0

20 %

50

42±1.0

31±2.0

19±1.0

10±0.0

40 %

50

39±1.0

16±1.0

03±1.0

01±1.0

50 %

50

48±1.0

39±1.0

28±2.0

20±1.0

Note: Each value is the mean of three readings±SD. n=3


Table 4: Mortality rate of adult Musca domestica at various concentrations of Mentha piperita after 24 h

Proportion of working solution

No. of adults survived after 24 h

Percent mortality

Control

48±1.0

04±2.0%

10 %

25±1.0

50±2.0%

20 %

10±0.0

80±0.0%

40 %

01±1.0

98±2.0%

50 %

20±1.0

60±2.0%

Note: Each value is the mean of three readings±SD. n=3


CONCLUSION

The Mentha piperita were found a rich source of secondary metabolites. The aqueous and ethanolic extract of Mentha piperita possesses antibacterial activity. These results confirm the presence of antibacterial compounds in Mentha piperita. The aqueous extract was effective against Staphylococcus aureus might be used as a source for finding the antibiotics effective against MRSA. Aqueous extract of Mentha piperita has effective adulticidal potential, hence it can be used to control and repel most popular disease spreading Dipterian vector, Musca domestica.

CONFLICT OF INTERESTS

Declare none

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