IN VITRO ANTI HAEMOLYTIC, ANTI BACTERIAL AND PHYTO CONSTITUENTS INVESTIGATION OF ANDROGRAPHIS PANICULATA METHANOLIC LEAF EXTRACT
HTML Full TextIN-VITRO ANTI-HAEMOLYTIC, ANTI-BACTERIAL AND PHYTO-CONSTITUENTS INVESTIGATION OF ANDROGRAPHIS PANICULATA METHANOLIC LEAF EXTRACT
Venkata Narasimha Kadali * 1, Tadi Ramesh 2, Sudhakara Rao Pola 1 and B. V. Sandeep 1
Department of Biotechnology 1, Andhra University, Visakhapatnam - 530003, Andhra Pradesh, India.
Department of Biotechnology 2, S V K P & Dr. K.S. Raju Arts and Science College, Penugonda - 534320, Andhra Pradesh, India.
ABSTRACT: The bitter plant Andrographis paniculata has been selected to evaluate the anti haemolytic activity, antibacterial activity, and various phytochemicals present in the methanolic leaf extract. The leaf extract of Andrographis paniculata shown to have significant anti-haemolytic activity. The extract showed anti-haemolytic activity in the range from 42% to 80%. At varying concentration of plant extracts 20, 40, 60, 80 and 100 mg/ml the percent of inhibition of haemolysis recorded were 42%, 56%, 62%, 70%, 80% respectively. The methanolic leaf extract of Andrographis paniculata showed significant inhibitory effect on all of the five bacterial species such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Streptococcus mutans. The inhibition zones for S. aureus were 12 mm, 14 mm and 19 mm (including well 4 mm) at concentrations of 15 µl, 20 µl, and 25 µl respectively. The inhibition zones P. aeruginosa were 11 mm, 13 mm and 20 mm, 8 mm, 10 mm and 15 mm for Streptococcus mutans. The zones for E. coli were 15 mm, 16 mm and 18 mm and 12 mm, 13 mm and 19 mm for Bacillus subtilis. The phytochemical investigation of methanolic extract of Andrographis paniculata leaves showed the presence of carbohydrates, flavonoids, alkaloids, tannins, phenols, and saponins.
Keywords: Andrographis paniculata, Phytochemicals, Anti-haemolytic, Anti-bacterial
INTRODUCTION: Despite the remarkable progress in the preparation of synthetic drugs, over 25% of prescribed medicines in industrialized countries are derived directly from plants 1. The World Health Organization (WHO) also considered phytotherapy in its health programs and suggested basic procedures for validation of drugs from plant origin in developing countries 2.
Indigenous plants are reservoirs of various metabolites and provide an unlimited source of important chemicals that have diverse biological properties 3. Medicinal plants are gaining a lot of importance nowadays because of efficacy they have been showing in the traditional healing 4. Herbs are the source of magnificent inhibitors that could act on a wide variety of diseases. One of the great aspects of herbs is they show 100% results when it comes to the healing. Herbs have all sorts of answers against various diseases 5.
The best source of drugs without lethal effects to human systems could be the plant source, and this has been proved by the Traditional healing system and the recent studies conducted on the experimental animals 6. Even expansion of modern medicines throughout the India people in rural areas still uses this wonder herbal medications for various sorts of diseases 7. The use of medicinal plants in developing countries is increasing, which offer a new source of antibacterial, antifungal, and antioxidants agents 8. The development of drug resistance, as well as the appearance of undesirable side effects of certain drugs, has led to the search of new antimicrobial agents in particular from medicinal plants 8. This medicinal herb A. paniculata otherwise called as king of bitters because it has extremely bitter where it is used to treat various infections and diseases 9.
Occurs quite commonly in the entire India subcontinent, which accounts for its widespread use since ancient times against a variety of disorders, in both Ayurvedic and Chinese Medicine 10. It has blood purifying property, so it is recommended for use in leprosy, gonorrhea, scabies, boils, skin eruptions, and chronic and seasonal fevers 9. The leaf extract of Andrographis paniculata was used to evaluate the enzyme inhibiting the activity of protease, phosphomono-esterase, phosphodiesterase, acetylcholinesterase, phospholipase A2, hyaluronidase and L-amino acid oxidase toxic enzymes present in snake venom 11.
The purified active compounds through TLC and crude plant extract were subjected to analyze the anti-inflammatory activity and shows potent inhibitory activity against the adverse effects of the inflammation such as tissue damage and protein denaturation 10. A. paniculata shows excellent anticancer activities against different cancer cell lines; it is alternatives medicines for cancer would replace side effect causing chemotherapeutic agent12.
Andrographolide active component of A. paniculata shown analgesic, antipyretic, and anti-ulcerogenic effect 13. Anti-hyperglycemic effect in streptozotocin-induced diabetic rats 14, it has anti-tumor activity via activation of cytotoxic T lymphocyte and attenuation of tumor growth in- vivo 15. A. paniculata is known to possess anti-HIV 16, cardioprotective 17 and hepatoprotective 18, properties. In this present study, the bitter plant Andrographis paniculata has been selected to evaluate the anti haemolytic activity, antibacterial activity, and various phytochemicals present in the methanolic leaf extract.
MATERIALS AND METHODS:
Materials: Leaves of Andrographis paniculata were collected from the campus S V K P & Dr. K S Raju Arts and Science College, Penugonda. The plant was authenticated by Dr. Suryanayana Raju, Department of Botany, S V K P & Dr. K S Raju Arts and Science College, Penugonda. Leaves were dried in sunlight for a week and then powdered using a blender to get a coarse powder. Chicken blood was collected from the local market in EDTA bottle.
Test Microorganisms: The methanolic leaf extract of Andrographis paniculata was tested against five pathogenic bacteria. The test organisms include Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Streptococcus mutans.
Extraction Process: Preparation of extract was done according to the procedure done by Sharmila et al., (2011) 19. 25g of leaf powder was packed in a Soxhlet extraction unit and exhaustively extracted using 100 ml of methanol at 60 ºC for 12 h. The extract was completely dried in a water bath at 40 ºC and subsequently stored at 4 ºC.
Phyto-chemical Analysis: A preliminary phyto-chemical investigation was conducted for the detection of steroids, terpenoids, flavonoids, saponins, tannins, carbohydrates, and phenols by using standard procedures 20, 21.
Determination of Antibacterial Activity: Antibacterial activity was measured by well diffusion method 22. Nutrient agar (Hi-media) was prepared and poured into the Petri plates. After solidification of media, overnight bacterial cultures were inoculated on the surface of media. By using a sterile gel puncher, 4 mm of wells were made in each Petri plates.
Then 15 µl, 20 µl, 25 µl of methanolic leaf extract Andrographis paniculata were added into the three wells respectively. The plates were incubated in the incubator at 37°C for optimum bacterial growth. In the next day, the diameter of the zone of inhibition was measured.
Assessment of Anti-haemolytic Activity: Anti-haemolytic activity against H2O2 induced haemolysis in chicken RBC determined by in-vitro method described by Tavazzi et al., 2001, Thagriki Dluya et al., 2015 23, 24. The chicken erythrocytes were separated by centrifugation at 200 rpm and washed with saline or isotonic sodium phosphate buffer (pH 7.4) until the supernatant is colorless. The erythrocytes were then diluted with saline or phosphate buffer to give a 4% suspension. Varying amounts of the plant extracts (20, 40, 60, 80 and 100 mg /ml) with saline or buffer was added to 2 ml of the suspension of erythrocytes, and the volume was made up to 3.5 ml with saline or buffer. This mixture was pre-incubated for 120 min and then 0.5 ml H2O2 solutions of appropriate concentration in saline or buffer was added. The concentration of H2O2 in the reaction mixture was adjusted to bring 90% haemolysis of blood cells after 120 min incubation. After 120 min of incubation, tubes were centrifuged, and the amount of haemolysis was determined by measurement of the absorbance at 540 nm corresponding to haemoglobin liberation. The anti-haemolytic activity was expressed as the inhibition percentage and was calculated using the following formula:
Anti-haemolytic activity (%) = Control 540 nm – Sample 540 nm / Control 540 nm × 100
Where, Sample 540 nm was the absorbance of the sample and Control 540 nm was the absorbance of the control.
RESULTS:
Phytochemical Analysis: The phytochemical investigation of methanolic extract of Andrographis paniculata leaves showed the presence of carbohydrates, flavonoids, alkaloids, tannins, phenols, and saponins Table 1.
TABLE 1: PRESENCE OF PHYTOCHEMICALS IN METHANOLIC EXTRACT OF ANDROGRAPHIS PANICULATA LEAVES
S. no. | Phytoconstituent | Presence of phytochemical in methanolic extract |
1 | Carbohydrates | + |
2 | Flavonoids | + |
3 | Alkaloids | + |
4 | Tannins | + |
5 | Phenols | + |
6 | Proteins | - |
7 | Saponins | + |
8 | Sterols | - |
+ indicates presence; - indicates absence.
Antibacterial Activity: The methanolic leaf extract of Andrographis paniculata showed significant inhibitory effect on all of the five bacterial species such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Streptococcus mutans. The inhibition zones for S. aureus were 12 mm, 14 mm and 19 mm (including well 4 mm) at concentrations of 15 µl, 20 µl, and 25 µl respectively. The inhibition zones P. aeruginosa were 11 mm, 13 mm and 20 mm, 8 mm, 10 mm and 15 mm for Streptococcus mutans. The zones for E. coli were 15 mm, 16 mm and 18 mm and 12 mm, 13 mm and 19 mm for Bacillus subtilis. The antibacterial results of methanolic extract were given below Table 2 and Fig. 1-6.
TABLE 2: INHIBITION ZONES OF METHANOLIC EXTRACT OF ANDROGRAPHIS PANICULATA AGAINST TEST PATHOGENIC BACTERIA
Test bacterial species | Inhibition zones in mm (including well 4 mm) | |||
15 µl (methanolic extract) | 20 µl (methanolic extract) | 25 µl (methanolic extract) | ||
Staphylococcus aureus | 12 mm | 14 mm | 19 mm | |
Pseudomonas aeruginosa | 11 mm | 13 mm | 20 mm | |
Bacillus subtilis | 12 mm | 13 mm | 19mm | |
Escherichia coli | 15 mm | 16 mm | 18 mm | |
Streptococcus mutans | 8 mm | 10 mm | 15 mm | |
FIG. 6: ZONES OF INHIBITION OF METHANOLIC EXTRACT ON DIFFERENT BACTERIAL SPECIES
Anti-haemolytic Activity: The methanolic leaf extract of Andrographis paniculata shown to have significant anti-haemolytic activity. The methanolic extract showed anti-haemolytic activity in the range from 42% to 80%. At varying concentration of plant extracts 20, 40, 60, 80 and 100 mg/ml the percent of inhibition of haemolysis recorded were 42%, 56%, 62%, 70%, 80% respectively Fig. 7.
FIG. 7: ANTI-HAEMOLYTIC ACTIVITY OF METHANOLIC LEAF EXTRACT OF ANDROGRAPHIS PANICULATA
DISCUSSION: The preliminary phytochemical investigation of methanolic extract of Andrographis paniculata leaves showed the presence of carbohydrates, flavonoids, alkaloids, Tannins, phenols, and saponins. The methanolic leaf extract of Andrographis paniculata shown to have significant anti-haemolytic activity. In this study, the inhibition of haemolysis found to be increased with increase in the concentration of extract. When red blood cells were treated with H2O2 (toxicant), % haemolysis was found to be increased. This may be because of the oxidizing nature of H2O2 concerning cell membrane degradation and release of haemoglobin from the cell 25. H2O2 also cause mobilization of Fe2+ by Ca2+ via Fenton reduction stimulating the production of OH- radicals 26.
All these factors combinedly cause destabilization of the cell membrane, which is probably the key event of the lysis of the cell 25. Polyphenols possess many biological effects, mainly attributed to their antioxidant activities in scavenging free radicals, inhibition of peroxidation and chelation of transition metals 27, 28. Chidambaram et al., (2011) 29 proved that Andrographis paniculata has significant ability to stabilize the Red blood cell membrane. These findings are by them. The methanolic leaf extract of Andrographis paniculata showed inhibitory effect on all of the five bacterial species such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Streptococcus mutans. It is shown that methanol extract had a broad spectrum of activity. The extract has shown inhibitory zones ranging from 8 mm to 20 mm.
In all the bacteria used in this study, Pseudomonas aeruginosa proved to be more susceptible and exhibited maximum inhibitory zone 20 mm. The minimum inhibitory zone was observed for Streptococcus mutans 15 mm. As modern antibiotics have various anarchic toxic effects, these plant extracts could serve as an alternative antibacterial agent. In single plant many active secondary metabolites are present and medicinal effect can be attributed to either to a single compound or synergistic effect of many compounds and present antibacterial activity can either be due to the presence of some specific bioactive molecule or due to the synergistic effect of different phytoconstituents 30.
CONCLUSION: Through this present study, it can be concluded that the methanolic extract of A. paniculata leaves showed significant antibacterial activity and membrane stabilizing activity. These effects are due to the phyto inhibitors present in the leaf extract. The feasibility of making efficient drugs from plant sources is very much near. Further research should be done to isolate inhibitors and necessarily formulated.
ACKNOWLEDGEMENT: The authors wish to thank the secretary and correspondent of S.V.K.P & Dr. K. S. Raju Arts & Science College, Penugonda for providing the research laboratory and also chemicals which enabled us to complete this work. The authors are grateful to the expertise available in the campus that helped a lot in species identification.
CONFLICT OF INTEREST: Nil
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How to cite this article:
Kadali VN, Ramesh T, Pola SR and Sandeep BV: In-vitro anti-haemolytic, anti-bacterial and phytoconstituents investigation of Andrographis paniculata methanolic leaf extract. Int J Life Sci & Rev 2016; 2(5): 102-08. doi: 10.13040/IJPSR.0975-8232.IJLSR.2(5).102-08.
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Article Information
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102-108
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English
IJLSR
V. N. Kadali *, T. Ramesh, S. R. Pola and B. V. Sandee
Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India.
vnsimhakadalibiotech@gmail.com
15 April 2016
16 May 2016
19 May 2016
10.13040/IJPSR.0975-8232.IJLSR.2(5).102-08
31 May 2016