Int J Biol Med Res.2019 ;10(1):6631-6634 Int J Biol Med Res www.biomedscidirect.com Volume 10, Issue 1, Jan 2019

Contents lists available at BioMedSciDirect Publications International Journal of Biological & Medical Research

Journal homepage: www.biomedscidirect.com BioMedSciDirect International Journal of Publications BIOLOGICAL AND MEDICAL RESEARCH

Original article Therapeutic properties of extracts of Leucas aspera and Anisomeles malabarica Rajeev Ramchandra Kolgia,c, Shivakumar S La, Sajeeda Niketha,c, Shivaraj Y3and Chandrakant S. Karigarb* aDepartment of P.G. Studies and Research in Biochemistry, Tumkur University, Tumkur, 572103, India. bDepartment of P.G. Studies and Research in Biochemistry, Bangalore University, Bengaluru, 560056, India. cDepartment of Chemistry and Biochemistry, Government Science College, Bengaluru, 560001, India.

A R T I C L E I N F O A B S T R A C T

Keywords: Leaves of two plant extracts from Leucas aspera and Anisomeles malabarica are studied for Phytochemical analysis their therapeutic activity. These plants have been evaluated for antibacterial property against Leucas aspera Anisomeles malabarica Bacillus cereus and Pseudomonas aeruginosa. The plant extracts showed low antibacterial antibacterial effects on the selected microbes. However, the methanol leaf extracts showed significant anticancer antibacterial activity. The determination of anticancer activity showed that the plants are poor zone of inhibition. in substances with appreciable therapeutic potential. c Copyright 2010 BioMedSciDirect Publications IJBMR - ISSN: 0976:6685. All rights reserved.

1. Introduction Leaves of plants with varied therapeutic potential have known to The plant leaves are found active against convulsions, colic, for benefit humans. These plants are selected as they are known to dyspepsia in intermittent fevers, boils, tetanus, inflammation, possess useful properties like anti-inflammatory, anti-cytotoxic, cough, cold, stomachache, itches and in uterine affections. hepato-protective effects, etc. Inflorescence and leaves extracts have been found to control mosquitoes. This plant extract have known to possess antibacterial Leucas aspera (Wild.) Linn. belonging to the famiy of Lamiaceae and anticancer properties. is popularly known to grow all over India. This beneficial herb rises to an altitude of 15-60 cm with stout and hispid acutely quadrangular branches and stems [1]. Its leaves are found beneficial in treatment of psoriasis, chronic rheumatism and other chronic skin eruptions. L. aspera plant is found to be useful for cure of respiratory tract disorders, gastrointestinal disorders, edema, and used as an antidote to poison [2]. Antiplasmodial activity of the leaf extract of plant against chloroquine-sensitive (3D7) strain of Plasmodium falciparum has been discovered [3].

The methanol extract of L. aspera flowers and its fractions has been found to show the good antibacterial activity. The expressed flower juice and alkaloidal residue also exhibited good antibacterial activity [4]. The L. aspera root ethanolic extract is found to inhibit acetic acid induced writhing in mice at the doses of Figure 1: The herb Leucas aspera. 250 mg/kg and 500 mg/kg. The extract revealed an important free radical scavenging activity with an IC50 of 8 μg/ml [5].

Anisomeles malabarica is a highly scented plant belonging to the family Lamiaceae (Labiatae) and is commonly well-known as Malabar catmint. It is a perennial plant which has woolly stems and grows 50 – 150 cm tall. The leaves and roots are useful as an astringent, a carminative, febrifuge and a tonic. It is found to be used or the treatment of amentia, colic, fever, dyspepsia, etc.

* Corresponding Author : E-mail: [email protected] Figure 2: The herb Anisomeles malabarica.

c Copyright 2011. CurrentSciDirect Publications. IJBMR - All rights reserved. Rajeev Ramchandra Kolg et.al/Int J Biol Med Res.10(1):6631-6634 6632

Antibacterial property Determination of Anti Bacterial Activity

An antimicrobial agent is a substance which kills or prevents the The antibacterial activity was determined by agar well diffusion development of microorganisms. Due to continuous use of method [11] against Bacillus cereus, Pseudomonas aeruginosa. antibiotics, the microorganisms have developed resistance. Sometimes industrial antibiotics are associated with side effects Petri plates containing 20 ml Nutrient Agar Medium were seeded such as low immunity and allergic reactions [6]. Hence, plant with 24 hr culture of bacterial strains B. cereus and P. aeruginosa. materials remain an important resource to fight severe infections in Wells were cut and 50 µl of the plant extracts were added with the world. A scientific study has be conducted on plants to regulate different concentrations (1 mg/ml, 5 mg/ml, 10 mg/ml, standard- their antimicrobial activity in a compounds, is a reasonably new field Ciprofloxacin 10 µg/ml). The plates were then incubated at 37 °C for approach. 24 hours. The antibacterial activity was recorded by measuring the diameter of the inhibition zone formed around the well (NCCLS, Anticancer Activity 1993) [9, 10, 11].

Cancer is a devastating disease which occurs with no specific Anticancer activity symptoms and no specific age. There is a constant demand for new therapies to treat and prevent this life-threatening disease. The MTT cytotoxicity assay for in vitro anticancer study disease is characterised by cells in the human body continually multiplying with the inability to be controlled or stopped. The cells The cytotoxicity assay was performed according to the consequently form malignant tumours with the potential to be microculture MTT method with slight modifications [12]. The cancer metastatic [7]. Current treatments include chemotherapy, cells MCF-7 obtained from Merck, which are cultured and harvested radiotherapy and chemically derived drugs. Treatments such as (1.5 × 104 cells/well) and inoculated in 96-well microtiter plates. chemotherapy can put patients under a lot of strain and further They were washed with phosphate-buffered saline (PBS) and the deteriorate their health. Therefore, there is a focus on using cultured cells were then inoculated with and without the extract. alternative treatments and therapies against cancer particularly After 72 h of incubation, the medium was aspirated. Ten microliters from plant origin [8]. of MTT solution (5 mg/ml in PBS, pH 7.2) was added to each well and the plates were incubated for 4 h at 37 °C. After incubation, 100 μl of In the present study, Leucas aspera and Anisomeles malabarica dimethyl sulfoxide (DMSO) was added to the wells followed by leaves extract are tested for their potentials as antibacterial and gentle shaking to solubilize the formazan dye for 15 min. Absorbance anticancer agents. Antibacterial property is evaluated by agar well was read at 570 nm and the surviving cell fraction was calculated. diffusion method (Mixed culture method) and the anticancer Camptothecin was used as the reference standard for anticancer potential is estimated by MTT Assay. activity,

MATERIALS AND METHODS The inhibition of cell viability calculated using the formula: [12]

Plants Material Inhibition activity (%) = 1-T/C X 100 Where T= Absorbance of the test sample, C= Absorbance of the The plants, Leucas aspera and Anisomeles malabarica were control sample collected from Devarayana Durga Hills,Tumkur District. The samples Results were authenticated at Regional Ayurveda Research Institute for Metabolic Disorders (Central Council for Research in Ayurvedic Antimicrobial Activity Sciences, Ministry of AYUSH, Govt. of India) Bengaluru, Karnataka. The zone of inhibition was measured after treatment of bacteria Preparation of plant extracts with plant extracts

Leaves of plants are collected separately, washed thoroughly and dried under the shade. These leaves are coarsely powdered and packed in Soxhlet apparatus and is continuously extracted individually using solvents such as ethanol, methanol, water, chloroform till all the constituents are extracted. Later, rotary vacuum evaporator was employed to concentrate the extracts.

Phytochemical Analysis of the Extracts

he leaves extracts of Leucas aspera and Anisomeles malabarica were subjected to phytochemical examinations as per the standard Figure 3: Petri dishes showing the zone of inhibition of extracts methods adopted as per Brain and Turner (1975) and Evans (1996). (crude). Rajeev Ramchandra Kolg et.al/Int J Biol Med Res.10(1):6631-6634 6633

The cytotoxicity assay was performed according to the microculture MTT method with slight modifications. The results are tabulated (data not shown). R1M alkaloid fraction showed an IC50 of 647.54, and R2M alkaloid fraction showed IC50 462.90. R1M flavonoid fraction showed an IC50 of 564.42, and R2M flavonoid fraction showed IC50 623.74. The effects of plant extracts alkaloid Table 1: Antibacterial activity of methanolic extracts of fraction on cancer cells is represented graphically (Fig.3 & 4). The Leucas aspera and R2 is Anisomeles malabarica. R1; Leucas influence of plant extracts flavanoid fraction on cancer cells is aspera, R2; Anisomeles malabarica, M; methanolic extract. represented graphically (Fig.5 & 6). Then Agar well diffusion method was carried out for R1M (alkaloids), R1M (flavonoids), and R2M (alkaloids) R2M (flavonoids), the concentration of the extracts used for both alkaloids and flavonoids were 1 mg/ml, 5 mg/ml, 10mg/ml and that of standard Ciprofloxacin (Standard antibacterial agent) 10 µg/ml. The experiment was carried out in triplicates.

Fig. 4. Effect of alkaloid extract (R2M) on MCF cell viability.

Fig.5. Effect of flavanoid extract (R1M) on MCF cell viability

Figure 4: Antibacterial activity of methanolic extracts of Leucas aspera (R1) and Anisomeles malabarica (R2). Fla; flavanoids; Alk; alkaloids. Anticancer activity

Fig.6. Effect of flavanoid extract (R2M) on MCF cell viability.

Fig.3. Effect of alkaloid extract (R1M) on MCF cell viability. Rajeev Ramchandra Kolg et.al/Int J Biol Med Res.10(1):6631-6634 6634

Conclusion [11] Mounyr Balouiri, Moulay Sadiki, Saad Koraichi Ibnsouda, Methods for in vitro evaluating antimicrobial activity: A review, J Pharmaceutical Herbal medicine is known as botanical medicine or Analysis, 2016; 6(2), 71-79. phytomedicine. It refers to use of plant seeds, roots, berries, leaves, [12] Rafik Shaikh, Mahesh Pund, Ashwini Dawane, and Sayyed Iliyas. bark and flowers for medicinal purposes. Recently, the World Health Evaluation of Anticancer, Antioxidant, and Possible Anti-inflammatory Properties of Selected Medicinal Plants Used in Indian Traditional Organization estimated that 80% of people worldwide relay on Medication. J Tradit Complement Med. 2014; 4: 253–257. herbal medicines for some part of their primary health care. Herbal medicines have been the oldest forms of health care. In this study Leucas aspera and Anisomeles malabarica plant extracts have been investigated for antibacterial and anticancer properties. These plant extracts showed poor antibacterial and anticancer properties when treated as crude extracts. However, alkaloid and flavanoid fractions obtained after methanol extraction showed appreciable antibacterial and anticancer activities.

Conflict of Interest

Authors declare no conflicts of interest.

Acknowledgements:

Authors are grateful to Government Science College (Autonomous), Bengaluru for the instrumental facility which are received under DST-FIST programme. REFERENCES

[1] Srinivasan R, Ravali, B, Suvarchala A, Honey A, Tejaswini A, Neeraja P. Leucas aspera - Medicial plant: A Review. Int J Pharma Biosci 2011; 2:153- 159.

[2] Kirtikar KR, Basu BD In: Indian Medicinal Plants, Vol. III, Deharadun: Lalit Mohan Basu, 2019.

[3] Bagavan A, Rahuman AA, Kamaraj C, Geetha K. Larvicidal activity of saponin from Achyranthes aspera against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitology Res 2008; 103:223- 229.

[4] Mangathayaru K, Lakshmikant J, Shyam Sundar N, Swapna R, Grace XF, Vasantha J. Antimicrobial activity of Leucas aspera flowers. Fitoterapia. 2005; 76:752–4.

[5] Rahman MS, Sadhu SK, Hasan CM. Preliminary antinociceptive, antioxidant and cytotoxic activities of Leucas aspera root. Fitoterapia. 2007; 78:552–5.

[6] Lopez A, Hudson JB, Towers GHN. Antiviral and antimicrobial activities of Colombian medicinal paints. Journal of Ethnopharmacology, 2001;77: 189-96.

[7] Ochwang'I DO, Kimwele CN, Oduma JA, Gathumbi PK, Mbaria JM, Kiama SG. Medicinal plants used in treatment and management of cancer in Kakamega County Kenya. J Ethnopharmacology. 2014; 151:1040–1055.

[8] M. Greenwell and P.K.S.M. Rahman. Medicinal Plants: Their Use in Anticancer Treatment. Int J Pharm Sci Res. Int J Pharm Sci Res. 2015; 1: 4103–4112.

[9] Osuntokun OT, Olajubu FA. Antibacterial and phytochemical properties of some Nigerian medicinal plant on Salmonella typhi and Salmonella paratyphi isolated from Infected Human Stool in Owo local Government. J Scientific Research & Reports. 2015; 4: 441- 449.

[10] NCCLS Performance standards for antimicrobial disc suspectibility tests. c Copyright 2019 BioMedSciDirect Publications IJBMR - ISSN: 0976:6685. Approved Standard NCCLS Publication M2-A5, Villanova, PA, USA, 1993. All rights reserved.