8 V May 2020

http://doi.org/10.22214/ijraset.2020.5146
 International Journal for Research in Applied Science & Engineering Technology (IJRASET)
ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429
Volume 8 Issue V May 2020- Available at www.ijraset.com

Phytochemical and Antibacterial Activity

Screening of Acalypha Indica and Ocimum
Tenuiflorum
Monica C1, Sheela2
1, 2
Department of Plant Biology and Biotechnology, Loyola College

Abstract: The aim of this study was to assess the secondary metabolities of Ocimum tenuiflorum and Acalypha indica. The
ethanol and methanol extracts were screened phytochemically to check for the presence of various bioactive compounds.
Furthermore, the extracts were qualitatively analysed by high-performance liquid chromatography, which showed the presence
of flavonoids (keampferol and quercetin) and tannins (tannic acid, gallic acid and resorcinol). The extracts at various
concentrations were used against common pathogens isolated from street food. It was seen that as the concentration increases,
the activity exhibited also increased. It also showed less activity against the natural beneficial bacteria present in the human
body. Methanol extracts, of both the plants, showed more activity when compared with the ethanol extract. This can be due to the
ability of the methanol solvent to extract low-molecular-weight bioactive compounds, which results in the accumulation of more
bioactive compounds.
Keywords: Methanol extract; Ethanol extract; Flavonoids; Tannins; High-performance liquid chromatography

I. INTRODUCTION
Plants are the naturally available medicines that are used as a potential material for maintaining good health and conditions. The
reasons for considering herbal plant sources are as follows: (1) herbs can act in pathways similar to those of pharmaceutical
medications, (2) the bioactive compounds find their way into the arsenal of the antimicrobial drug easily, and (3) the public is
becoming aware of the use of chemical antibiotics, their side effects, and starts looking for alternative sources. Many potential drugs
have been derived from various plants that have interdependent pathways for synthesizing effective metabolites. The higher the
concentration of phytochemical in a plant, the greater therapeutic potency or medicinal importance of that plant. Research shows
that plants exhibit antimalarial, antibacterial, antioxidant, antitumour, antifungal, anticancerous, and antiviral properties, which have
been exploited extensively for developing new drugs against a wide range of ailments. The potential of higher plants as a source of
drugs is still unexplored (Verma et al., 2008).

A. Herbal Extracts as Antimicrobial Agents

Antibiotics are effective treatments for the microbial diseases. But, in recent years, bacteria under continuous stress due to various
environmental factors result in developing resistance, which is an adaptation of bacteria to the changing environment (Schlundt et
al., 2004). Owing to the lack of new antimicrobial agents to combat them, there is a rapid emergence of resistance pathogens. The
safe and effective compounds that fight against these drug resistant pathogens, especially the pathogens associated with the food
borne illness, could be plant-derived antimicrobial agents (Gyawali R et al., 2017). Plant extracts in combination with the antibiotics
can restore the efficacy of the already existing drugs against resistant pathogenic bacteria (Abreu et al., 2012).
Plant extracts, organic acids, essential oils, and bacteriocins are natural antimicrobial agents that could be used as a good alternative
to ensure food safety.
Food antimicrobial agents are substances that cause microbial death or delay the microbial growth in food. The major target for such
antimicrobial agents is food-poisoning microorganisms and spoilage organisms whose metabolic end products cause off-odours, off-
flavours and discoloration (Davidson, 2001).
Antimicrobial agents from plants are mostly used in the form of biofilms and edible coatings in food systems. These antimicrobial
films and coatings slowly diffuse inside the food package, thus extending the duration of the antimicrobial activity. The direct usage
of antimicrobial agents in food system is avoided as these agents could diffuse into food bulks, and the concentration of
antimicrobial agents at food surface could reduce and eventually results in microbial growth and spoilage at surface. The
antimicrobial films are used in meat industry, beverage industry, and pharmaceuticals industry as per the FDA-prescribed format.

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ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429
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B. Acalypha indica
A. indica, commonly known as kuppaimeni in Tamil, Indian copperleaf, or three-seeded mercury, is widely used in traditional
medicinal systems such as Ayurveda, unani, siddha, and so on. The whole plant has been extensively used as a medicinal plant
owing to the presence of phytochemicals such as alkaloids, flavonoids, phenolic compounds, saponins, and sterols (Chitravadivu et
al., 2009). This plant is an emetic, expectorant, laxative and diuretic, which is used to treat bronchitis, pneumonia, asthma, and
pulmonary tuberculosis. Leaves have laxative and antiparasiticide properties. Powdered dry leaves are used to expel intestinal
worms. Fresh leaf juice is used to treat arthritis and scabies.

C. Ocimum Tenuiflorum
O. tenuiflorum, popularly known as holy basil, tulasi in tamil, is native to the Indian subcontinent widely used in the traditional
medicinal system. O. tenuiflorum is a very important herb in the Ayurvedic tradition. It is a pungently aromatic, warming, antiseptic
herb; Some of its applications are that it induces perspiration, lowers fevers, relaxes spasms, eases pain, clears bacterial infections,
strengthens the immune and nervous systems, reduces inflammations, and benefits the digestive system. This herb is used externally
as antiseptic to treat skin infections. The seeds are used as a tonic. The herb has been suggested to possess antifertility, anticancer,
antidiabetic, antifungal, hepato protective, cardio protective, antiemetic, antispasmodic, analgesic, adaptogenic and diaphoretic
actions.

II. MATERIALS AND METHODS

A. Plant Collection
O. tenuiflorum (Tulasi) was purchased from a flower shop in Chennai, and A. indica (Kuppaimeni) was collected from the Loyola
College campus. The leaves of these plants were washed, shade-dried for 24 hours, powdered, and stored in air tight containers for
further analysis.

B. Plant Extract
Extracts of A. indica and O. tenuiflorum were prepared by dissolving 20 gms of the leaf powder in 200 ml of ethanol and methanol
separately. This mixture was kept in a shaker for 24 hours, filtered, and allowed to evaporate. The extract was stored at -20°C for
further use.

C. Phytochemical Screening
1) Test for Flavonoids: To 1 ml of the extract, 2 ml of NaOH was added, and the appearance of intense yellow colour occurs,
which on addition of dilute acids, appearance of pale yellow colour indicated the presence of flavonoids.
2) Test for Saponins: Few amount of the extract was dissolved in a test tube containing 3 ml of hot distilled water. Then, the
mixture was shaken vigorously for 1 minute, and the foam was observed.
3) Test for Terpenoids: To 2 ml of the extract, 2 ml of chloroform and 3 ml of concentrated sulphuric acid was added, and the
appearance of reddish brown colour indicates the presence of terpenoids.
4) Test for Tannins: To the extract, 2 ml of bromine water was added. Disappearance of yellow colour indicates the presence of
tannins.
5) Test for Glycosides: To the extract, 2 ml of sulphuric acid was added, and the red colouration indicates the presence of
glycosides.
6) Test for Steroids: To the extract, 2 ml of chloroform and concentrated sulphuric acid was added. The red layer appears at the
lower level of chloroform.

D. High-Performance Liquid Chromatography

The specifications of HPLC for the analyses of the samples are as follows:
Equipment: HPLC, Waters make, pump-515
Column: RP, C-18; (5µm particle size)
Injection volume : 20µl
Temperature: 28°C
Mobile phase:
Solvent A, phosphate buffer (pH 5.8)
Solvent B, acetonitrile
Flow rate: 1 ml/min

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 International Journal for Research in Applied Science & Engineering Technology (IJRASET)
ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429
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Detector: UV–VIS detector (model 2487 Waters make)

Wavelength: 254 nm
The analyzed samples were compared with the standards from the literature to identify the compounds present in the analyzed
samples.

E. Antibacterial Assay
The antibacterial activity for the leaf extract of A. indica and O. tenuiflorum was evaluated by agar well-diffusion method on
Muller–Hinton Agar. The leaf extracts were prepared at different concentrations (100 mg to 600 mg) with dimethylsulfoxide as a
solvent. The bacterial cultures isolated from the food samples (previous work by the authors) were used to examine the antibacterial
activity of the leaf extract. Fresh inoculum was prepared by inoculating the bacterial culture in nutrient broth and incubated for 2
hours. The Muller–Hinton agar plates were prepared, and wells were punched. The cultures were spread evenly by the swabbing
method. The wells were filled with 25μl of the extract with streptomycin antibiotic discs as a positive control. The plates were
incubated at 37°C for 24 hours. Using the standard scale (mm in measurement), the zone of clearance was measured.

III. RESULTS
A. Phytochemical Screening
Phytochemical analyses revealed that A.indica and O.tenuiflorum contained alkaloids, tannins, saponins, terpenoids, flavonoids, and
phenolic compounds in the extracts which are shown in the Table 1.
TABLE 1: PHYTOCHEMCIAL SCREENING
Phytochemical test Acalypha indica Ocimum tenuiflorum
Methanol extract Ethanol extract Methanol extract Ethanol extract
Alkaloids + + + +
Flavonoids + + + +
Tannins + - + +
Saponins + + + -
Carbohydrates + - - -
Terpenoids + - - +
Glycosides - - - -
Note(s): +, Presence; -, Absence.

B. High-Performance Liquid Chromatography

High-performance liquid chromatography was performed for the methanol extract of both the plants. This is a qualitative analysis
carried out to determine the presence of various bioactive compounds. The chromatogram shows various peaks at different retention
period of time. The chromatographs are shown in Fig 1 and 2. The bioactive compounds that were identified are given in the tables
2 & 3. The compounds kaempherol and quercetin belongs to the group of flavonoids, whereas gallic acid, tannic acid, and resorcinol
belong to the group of tannins.
TABLE 2: HPLC ANALYSIS OF METHANOL EXTRACT OF A. INDICA
Peak Retention time (min) Area (mAU*s) Amount/area Compound
1 5.899 1819.96753 5.4946 Kaempherol

2 29.298 144.19958 6.9434 Quercetin

TABLE 3: HPLC ANALYSIS OF METHANOL EXTRACT OF O.TENUIFLORUM

Peak Retention time Width (min) Area (mAU*s) Height (mAU) Area % Compound
(min)
1 1.579 0.2128 3633.00146 219.80565 17.3301 Gallic acid
2 3.451 0.2999 1446.57849 67.52967 6.9005 Tannic acid
3 7.502 0.2862 336.06171 16.7901 1.6031 Resorcinol
4 29.194 0.7351 998.53998 17.51589 4.7632 Quercetin

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C. Antibacterial Activity of the Extracts

A total of 7 isolates were used against the prepared extracts. These isolates were isolated from a common street food, pani puri
water, and were biochemically characterized. As per the test results, possible genus of the isolates were Bacillus, Escherichia,
Streptococcus, Pseudomonas, and Klebsiella. When these organisms were tested against the extracts of the plant, zone of clearance
was observed. The efficiency of the plant extracts on the bacterial cultures is shown in Table 4 and 5.
TABLE 4: EFFICIENCY OF A.INDICA EXTRACT AGAINST BACTERIAL CULTURES
Acalypha indica

Isolates Ethanol extract Methanol extract concentration (mg)

concentration (mg)
100 200 300 400 500 600 100 200 300 400 500 600
1 - - 10 10 11 11 - - 10 10 11 11
2 - - - - 10 11 - 10 11 11 12 12
3 - 8 10 11 11 12 - - 10 12 14 15
4 - 8 10 10 12 12 - 10 10 10 11 11
5 - 6 11 11 12 12 - 12 12 13 14 14
6 - 9 10 11 11 12 - 10 10 11 12 13
7 - 6 10 10 11 12 - 10 11 11 12 12
TABLE 5: EFFICIENCY OF O.TENUIFLORUM EXTRACT AGAINST BACTERIAL CULTURES
Ocimum tenuiflorum

Isolates Ethanol extract Methanol extract concentration (mg)

concentration (mg)
100 200 300 400 500 600 100 200 300 400 500 600
1 - 9 10 11 12 12 - 10 11 12 12 13
2 - 9 10 11 11 12 - 10 10 11 11 12
3 - 8 10 10 11 11 - - 10 11 11 12
4 - 8 10 11 11 12 - 9 10 10 11 11
5 - - 10 11 11 12 - 10 11 12 13 13
6 - 6 10 11 12 12 - 10 11 11 12 12
7 - 8 10 11 12 12 - 10 11 12 13 13

IV. DISCUSSION
A. Phytochemical Screening
Phytochemical screening showed the presence of various bioactive compounds such as alkaloids, flavonoids, tannins, and saponins.
These compounds are non-nutritive chemicals, which have enormous significance due to their beneficial effects on human health.
HPLC analyses showed the presence of bioactive compounds: flavonoids and tannins. Flavonoids, hydroxylated phenolic substances,
are synthesized by the plants in response to the microbial infections. They exhibit antifungal and antibacterial activity against some
human pathogenic fungi and bacteria (Owoyale JA et al., 2005).
Their antibacterial activity is due to the formation of complex with extracellular and soluble proteins thus inactivating the proteins.
This leads to the disruption of microbial membranes (Tsuchiya et al., 1996). Kaempferol is an important flavonoid found abundantly
in tea, broccoli, apples, strawberries, and beans (Somerset & Johannot, 2008). It is a strong antioxidant and helps to prevent
oxidative damage of cells, lipids and DNA. Kaempferol seems to prevent arteriosclerosis by inhibiting the oxidation of low density
lipoprotein and the formation of platelets in the blood (Kowalski et al., 2005). Studies have also confirmed that kaempferol acts as a
chemopreventive agent as they exhibit less toxicity to normal cells in comparison to chemotherapy drugs (Zhang et al., 2008). Fig. 1
shows the molecular structure of kaempferol.

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Fig. 1: Structure of kaempferol

Quercetin is categorized as a pigmented flavonol, a subclass of flavonoid compounds that are abundant in variety of foods including
apples, berries, grapes, onions, shallots, and tomatoes. It possesses anti-inflammatory potential that can be expressed on different
types of cells, both in human and animal models (Chirumbolo S, 2010). They also possess antiproliferation and gene expression–
changing capacities in vitro (Boots et al., 2008). Fig. 2 shows the structure of quercetin

Fig. 2: Structure of quercetin

Tannins are polyphenols present in medicinal plants. They have astringent properties which hasten the healing of wounds and
inflamed mucous membrane due to their physiological activities such as antioxidant, antimicrobial, and anti-inflammatory
properties (Sule et al., 2010). Tannic acid (Fig. 3), gallic acid (Fig. 4), and resorcinol (Fig. 5) were the tannins present in the
analysed samples, which provide the antibacterial property to the plant. The antimicrobial mechanisms of tannins can be
summarized as follows. (i) The astringent property of the tannin may induce complexation with enzymes or substrates. Many
microbial enzymes in raw culture filtrates or in purified forms are inhibited when mixed with tannins. (ii) A tannin's toxicity may be
related to its action on the membranes of the microorganisms. (iii) Complexation of metal ions by tannins may account for tannin
toxicity (Chung et al., 1998).

Fig. 3: Structure of tannic acid

Fig. 4: Structure of gallic acid

Fig. 5: Structure of resorcinol

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 International Journal for Research in Applied Science & Engineering Technology (IJRASET)
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B. Antibacterial Assay
The antibacterial assay by disc diffusion method showed no activity against the pathogens. This negative result could be due to the
difference in the phytochemicals present or the composition of the extracts and the methodology of the antimicrobial tests
(Pochapski et al., 2011). The plant extracts were inoculated directly into the wells of the culture swabbed agar to diffuse. After
incubation, the extracts showed antibacterial activity against the inoculated organism. The methanol extracts of both the plants A.
indica and O. tenuiflorum showed higher effect when compared with ethanol extracts. Ethanol and methanol are polar solvents used
frequently to recover polyphenols from the plants. Ethanol has been commonly used for polyphenol extraction and is safe for human
consumption. Methanol was found to be more efficient in the extraction of lower molecular weight polyphenols (Quy Diem Do et
al., 2014). Hence, the methanol extract may contain maximum amount of alkaloids and flavonoids compared with ethanol extract
which could be reason for higher antibacterial activity.

REFERENCE
[1] Abreu, A. C., McBain, A. J., & Simoes, M. (2012). Plants as sources of new antimicrobials and resistance-modifying agents. Natural Product Reports, 29(9),
1007e1021.
[2] Boots, Agnes W., Guido RMM Haenen, and Aalt Bast. "Health effects of quercetin: from antioxidant to nutraceutical." European journal of
pharmacology 585.2-3 (2008): 325-337.
[3] Chirumbolo, Salvatore. "The role of quercetin, flavonols and flavones in modulating inflammatory cell function." Inflammation & Allergy-Drug Targets
(Formerly Current Drug Targets-Inflammation & Allergy) 9.4 (2010): 263-285.

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ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429
Volume 8 Issue V May 2020- Available at www.ijraset.com

[4] Chitravadivu, C., Manian, S., & Kalaichelvi, K. (2009). Qualitative analysis of selected medicinal plants, Tamilnadu, India. Middle East Journal of Scientific
Research, 4(3), 144-146.
[5] Choi, Jae Sue, et al. "The structure–activity relationship of flavonoids as scavengers of peroxynitrite." Phytotherapy Research 16.3 (2002): 232-235.
[6] Davidson, P. M., & Naidu, A. S. (2000). Phyto-phenols. In A. S. Naidu (Ed.), Natural food antimicrobial systems (pp. 265e294). Boca Raton, FL: CRC Press.
[7] Do, Quy Diem, et al. "Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica." Journal of
food and drug analysis 22.3 (2014): 296-302.
[8] Gyawali, R., Hayek, S. A., & Ibrahim, S. A. (2015). Plant extracts as antimicrobials in food products: Mechanisms of action, extraction methods, and
applications. Handbook of natural antimicrobials for food safety and quality, 49-68.
[9] Kowalski, J., Samojedny, A., Paul, M., Pietsz, G., & Wilczok, T. (2005). Effect of apigenin, kaempferol and resveratrol on the expression of interleukin-1beta
and tumor necrosis factor-alpha genes in J774. 2 macrophages. Pharmacological reports: PR, 57(3), 390-394.
[10] Owoyale JA, Olatunji GA, Oguntoye SO. Antifungal and antibacterial activities of an ethanolic extract of Senna alata leaves. J Appl Sci Environ Mgt.
2005;9(3):105-7.
[11] Pochapski, Márcia Thaís, et al. "Phytochemical screening, antioxidant, and antimicrobial activities of the crude leaves’ extract from Ipomoea batatas (L.)
Lam." Pharmacognosy magazine 7.26 (2011): 165.
[12] Schlundt, J., Toyofuku, H., Jansen, J., & Herbst, S. A. (2004). Emerging food-borne zoonoses. Revue Scientifique et Technique-Office International des
Epizooties, 23(2), 513-534.
[13] Somerset SM, Johannot L. Dietary flavonoid sources in Australian adults. Nutr Cancer. 2008; 60:442–449.
[14] Sule, I. O., and G. P. Oyeyiola. "Bacteriological assessment of some swimming pools within Ilorin Metropolis, Kwara, Nigeria." Bioresearch Bulletin 1 (2010):
13-7.
[15] Tsuchiya, H., M. Sato, T. Miyazaki, S. Fujiwara, S. Tanigaki, M. Ohyama, T. Tanaka, and M. Iinuma. 1996. Comparative study on the antibacterial activity of
phytochemical flavanones against methicillin-resistant Staphylococcus aureus. J. Ethnopharmacol. 50:27–34.
[16] Verma, Sheetal, and S. P. Singh. "Current and future status of herbal medicines." Veterinary world Vol. I (11), pp. 347-350 (2008).
[17] Zhang Y, Chen AY, Li M, Chen C, Yao Q. Ginkgo Biloba Extract Kaempferol Inhibits Cell Proliferation and Induces Apoptosis in Pancreatic Cancer Cells. J
Surg Res. 2008;148(1):17–23.

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