In Vitro Assessment of The Bioactive Compounds and Anticancer Potential of Citrus medica Leaf Extract

Citrus medica is a horticultural crop grown in different parts of the world. The plant leaves have medicinal importance in traditional medicine for the treatment of various diseases. The leaves are an underutilised part of the plant, despite having various bioactive compounds with health benefits, with phytochemical analysis having revealed the presence of flavonoids, fatty acids, alkaloids, terpenoids, glycosides, carbohydrates and phytosterols. The biochemical constituents were identified using Fourier-transform infrared spectroscopy (FTIR) and gas chromatography–mass spectrometry (GC-MS), which confirmed the presence of terpenoids, alcohols, alkanes, phytosterols and fatty acids. Among these, methyl 8, 11, 14-heptadecatrienoate is a linolenic acid, and α-linolenic acid, trimethylsilyl ester and levulinic acid are the predominant compounds belonging to the omega-3 fatty acid group, which has known health benefits. Further, the antimicrobial activity of C. medica plant leaves were tested against certain food-borne pathogens and showed significant results. The minimum inhibitory concentrations ranged from 6.09 mg/mL to 390 mg/mL for bacterial organisms and 48.75 mg/mL to 390 mg/mL for fungal organisms. The antioxidant activity values were 300 μg/mL and 450 μg/mL by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, respectively. The methanolic extract from the C. medica leaves also showed anticancer activity against MCF7 breast cancer cell lines, with an IC50 value of material for developing a healthy processed food such as nutraceuticals and functional foods.

• The GC-MS analysis of leaf extract of Citrus medica revealed the presence of omega fatty acids which are considered to be as important as food supplements for boosting the immunity and also scavenging of free radicals.
• The active principles such as methyl 8, 11, 14-hepta decatrienoate which is a linolenic acid, α-linolenic acid trimethyl silyl ester and levulinic acid are the predominant compounds belongs to omega-3 fatty acids group with health benefits can be used in various food products that results in health benefits.

INTRODUCTION
Increased consumption of processed foods and exposure to stress have both been linked to the increased prevalence of several chronic diseases (Shridhar et al. 2015;Khan et al. 2010).Further, the human body harbours microorganisms that can cause disease (Caputo et al. 2018).Risk of these diseases can be reduced by consumption of natural ingredients such as fruits, leaves and vegetables that contain biochemical constituents, mineral components, dietary fibers and vitamins (Rafiq et al. 2018;Kausar et al. 2019).Natural foods available in the nature are functional to some extent (Varzakas et al. 2016).However, some foods are now being examined for its health benefits to reduce the risk of chronic diseases (Alissa & Ferns 2012).The biochemical constituents have the ability to treat various ailments such as cardiovascular diseases, cancer, gastrointestinal disorders and physiological functions like lowering triglycerides and glucose control in blood (Asif 2011;Eilat-Adar et al. 2013;McClements & Xiao 2017;Eswaraiah et al. 2020).Synthetic drugs used to treat these diseases can also affect healthy cells and cause adverse side effects (Karimi et al. 2015;Madigan & Karhu 2018).An alternative to these synthetic drugs is natural foods such as fruit and vegetables, which are natural sources of dietary fiber, vitamins and minerals (Bhaskarachary 2016).Compared with synthetic drugs, many fruits and vegetables deliver enhanced health benefits that exceed their nutritional value, termed functional foods, with biologically active substances such as antioxidants (Abuajah et al. 2015).Earlier studies also proved the relationship between functional ingredients of the food and health of wellbeing.The biochemical constituents of some functional foods positively impact health by reducing the risk of chronic diseases providing treatments for medical conditions such as cancer, cardiovascular disease and gastrointestinal disorders (Keservani et al. 2010;Das et al. 2012).
Developing functional foods which are supplements with added ingredients is currently a research priority in the developing world (Keservani et al. 2010).The natural food ingredients with antibacterial, antioxidant and anticancer properties have more importance in food processing industries (Cirmi et al. 2017;Adnan, Bibi, et al. 2014;Eswaraiah et al. 2019).Citrus plants have rich of bioactive molecules extracted from various parts of the plant used to treat a range of medical disorders (Lv et al. 2015;Chaudhari et al. 2016).Citrus medica plant extract has been used to treat arthritis, asthma, headaches, abdominal pain, intestinal parasites and psychological problems (Panara et al. 2012).C. medica leaves are rich in essential oils have huge demand in market and also the usage of these compounds increasing day by day.The C. medica plant is widely used in Chinese traditional medicine (Aliyah et al. 2017).This current study investigated the antimicrobial, antioxidant and anticancer properties of C. medica leaf extract.The antimicrobial property was tested against food borne pathogens, biochemical constituents by GC-MS analysis, antioxidant activity using DPPH and ABTS assay and anticancer activity against MCF7 breast cancer cell line.

Collection of Plant Sample
The leaves of Citrus medica (Citron-Dabbakaya), plant was collected from agricultural fields, Vijayawada, Krishna (Dt), Andhra Pradesh, India.Fresh leaves are collected during the winter season in the month of January 2018.The collected leaf samples were packed in plastic bags and transported to Biotechnology laboratory, Vignan's Foundation for Science Technology and research for further work.

Sample Preparation and Extraction of The Phytochemicals
Leaf samples were washed with sterile water to remove dust and foreign particles.The leaves were dried at 40°C in a hot-air oven for 3 days and then powdered.The Soxhlet extraction method was employed to extract the phytochemicals, using 100 g of powdered sample and 200 mL of methanol solvent at 12h intervals over three successive days at 65°C.For every hour, 3 cycles were run to extract the maximum number of compounds.After extraction, the methanol solvent was evaporated using a rotary evaporator under reduced pressure at 25°C for 1 h.The crude extract became a semi-solid mass and this was stored in Falcon tubes prior to further study (Adnan, Umer, et al. 2014).

Phytochemical Screening of Citrus Leaves Extracts
The crude extract was tested for presence of phytochemicals such as alkaloids, glycosides, saponins, phenolic compounds, steroids, flavonoids, tannins, anthraquinones, amino acids, carbohydrates, terpenoids and phytosterols in the leaf sample (Ali et al. 2018).

Fourier-Transform Infrared Spectroscopy
The functional groups of phytochemical compounds in the crude extract were identified using Fourier-transform infrared spectroscopy (FTIR) performed on a SHIMADZU FTIR-8400S.The crude extract 10 mg is mixed with 100 mg of KBr salt and compressed into thin pellet using mortar and pestle.The sample is loaded into spectroscope and the results were recorded at frequencies of 500 cm -1 to 4,000 cm -1 (Ashok Kumar & Ramaswamy 2014).

Gas Chromatography-Mass Spectrometry
The phytochemical constituents in the crude extract were identified using gas chromatography-mass spectrometry (GC-MS) analysis, performed on Agilent © 7890A-5975C equipment (Agilent Technologies, USA) using an HP 5 MS capillary 200 column (30 m × 0.25 mm).The injection port conditions were: sample injected = 1 µL; carrier gas = helium with a flow rate of 1.2 mL/min; and temperature = 250°C.The GC column temperature was programmed initially at 80°C for 1 min, then the temperature was increased to 200°C at a rate of 15°C/min, further increased to 300°C at a rate of 5°C, and then maintained at 300°C for 5 min.The MS conditions were: temperature of the ion source = 230°C; ionisation energy = 70 eV; and a scan range of 50 amu-800 amu.The sample is prepared using methanol solvent and filtered.50 µL of the sample is taken in 1.5 mL of autosampler and loaded into the injector port.The separated biochemical constituents were compared with the mass spectra in the NIST library (Rahman et al. 2019).

Agar Well Diffusion and Disk Diffusion Assay for Antimicrobial Activity
The selected indicator organisms were grown in a nutrient-broth medium and seeded for assay.Muller-Hinton agar plates (100 mm × 15 mm size) were prepared with 25 mL of medium and the inocula were seeded on the agar surface using the spread plate method.After lawn preparation, 4-mm-deep wells were created and loaded with 100 µL (1.6 × 10 8 CFU/mL) of crude citrus-leaf extract (methanol) before the plates were incubated at 37°C for 24 h.The zone of inhibition was then measured and compared to both the positive control--ampicillin antibiotic--and the negative control--dimethyl sulfoxide (Sah et al. 2011).The selected fungal indicator organisms were grown in a Potato Dextrose Broth medium for 5 days to 7 days at 28°C-30°C.Muller-Hinton agar plates were prepared and seeded with inocula using a sterile swab dipped in culture suspension.The inoculated plates were then dried before applying the disks.Sterile disks were placed in 10 µL of crude leaf extract for 30 min and were then placed on the surface of the agar and incubated at 28°C for 5 days.The zone of inhibition was measured and compared to the positive control--fluconazole antibiotic--and the negative control--dimethyl sulfoxide.The procedure was repeated for three replicates and the mean values are calculated (Agarwal et al. 2015).

Determination of Minimum Inhibitory Concentration
First, 10-mL measures of Mueller-Hinton broth medium were sterilised by autoclaving before being cooled and inoculated with 100 µL (1.6 × 10 8 CFU/ mL) of microbial cell suspension and 100 µL of plant extract (methanol) of known concentration.The concentrations of crude leaf extract used in this experiment were 0.76, 1. 52, 3.05, 6.09, 12.18, 24.38, 48.75, 97.50, 195.00 and 390.00 mg/mL.The contents of the test tubes were mixed well and incubated at 37°C for 24 h.The procedure was repeated for three replicates and the mean values are calculated.The lowest concentration of crude leaf extract to inhibit the growth of microorganisms was calculated according to the method of Mummed et al. (2018), with some modifications.

Antagonistic Activity Against Staphylococcus aureus and Candida albicans
The selected indicator organisms S. aureus and C. albicans were grown in nutrient broth and Sabouraud dextrose broth respectively at 35°C for 8 h (Li et al. 2019).After 8 h of incubation, the cells were separated by centrifugation at 5,000 rpm and suspended in phosphate-buffered saline (PBS) buffer with a pH of 7.4.The suspension (10 8 CFU/mL) was mixed with leaf extract and incubated for 4 h.The cells were separated by centrifugation and washed with PBS buffer, then fixed with glutaraldehyde (2.5%) and stored at 4°C for 30 min.A 50% to 100% graded series of ethanol was used to dehydrate the sample for 15 min per concentration before the cells were imaged using an S-3700N scanning electron microscope (SEM, Hitachi, Hitachi City, Japan).

DPPH assay
The antioxidant potential of the C. medica leaf extract was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay--a protocol suitable for the determination.Various concentrations of extract were prepared, in test tubes, ranging from 100 μg/mL to 1,000 µg/mL made up to a volume of 3 mL using 70% methanol.A 1-mL aliquot of 100-µM DPPH solution was added to each test tube, the mixture then shaken vigorously and incubated for 30 min.the absorbance was measured at 517 nm.Ascorbic acid was used as the control.All tests were performed in triplicate.The radical-scavenging activity was expressed as the inhibition percentage and was calculated using a standard formula.The antioxidant activity was also expressed as an IC 50 value (Sonboli et al. 2010).

ABTS assay
The antioxidant activity of C. medica leaf extract was measured by 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay.The reaction (1:1 v/v) was prepared via the oxidation of ABTS and potassium persulfate, with reaction allowed for 16 h under dark conditions.The mixture was then diluted with methanol until it achieved absorbance values of 1.0-1.5 at 734 nm.The leaf extract (0.1 mL) was mixed with 3.9 mL of ABTS solution and allowed to react for 2 h under dark conditions before the absorbance values were measured at 734 nm using a spectrophotometer.The results were expressed as a percentage of inhibition using the equation described for the DPPH method (Shah & Mehta 2018).

Antiproliferative Activity of Citrus medica
A recognised MCF-7 breast cancer cell line was procured from the National Centre for Cell Science, Pune, India.Using RPMI-1640 medium, the cancer cells were sub-cultured in culture flasks and passaged every three days.The cells were then seeded in 24 well plates for 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay.A series of crude leaf extract concentrations, ranging from 0 to 100 μg/mL, was prepared.First, the cells were trypsinzed and treated with trypan blue.Using a hemocytometer, the cells were counted and seeded at a density of 5.0 × 10 3 cells/well in 96 well plates, then incubated at 37°C overnight.The medium was then replaced with fresh medium and 100 μL of different concentrations of crude leaf extract were added.The cells were incubated for 48 h, after which fresh medium and MTT solution (0.5 mg/mL) were added to each well.The cells were then incubated for a further 3 h, after which the absorbance values were measured at 570 nm using a microplate reader.The percentage growth inhibition and corresponding IC 50 values were generated from the dose-response curve using Origin software (Stockert et al. 2018).

Phytochemical Screening
During the present study, the phytochemical constituents were identified by performing various tests and found the presence of alkaloids, glycosides, flavonoids, steroids, terpenoids, carbohydrates and phenolic compounds (Table 1).There is an absence of saponins, anthraquinones and tannins.

FTIR and GC-MS
The FTIR analysis revealed the presence of various functional groups and biochemical classes of compounds in the leaf extract (Table 2).Fig. 1 presents the GC-MS spectra, showing various peaks representing different bioactive compounds.The compounds were identified from the methanolic extract of the C. medica leaves, as indicated in Table 3 and Fig. 2.
All the results are written as mean of three individual observations ± SD.

Minimum Inhibitory Concentration
The minimum inhibitory concentration (MIC) of C. medica leaf extract was determined and the values found to range from 6.09 mg/mL to 390 mg/mL.High antibacterial activity was observed for the MIC value of 6.09 mg/mL against E. coli, S. aureus and E…aerogenes.For the fungal cultures, considerable antibacterial activity was observed at a MIC value of 48.75 mg/mL against C. albicans (Table 4).

Antagonistic Activity Against S. aureus and C. albicans
Antagonistic activity was evaluated against the food-borne pathogens S. aureus and C. albicans.The effect of C. medica leaf extract on the cell morphology of S. aureus and C. albicans was investigated using a scanning electron microscope (at 50,000× magnification) by comparing the morphological features of both treated and untreated cells.Untreated S. aureus cells presented as aggregations of rounded cells (Fig. 4a), whereas the treated cells varied in size and shape and demonstrated some cell shrinkage (Fig. 4b).The untreated cells of C. albicans were characterised by their regular round or oval shapes Fig. 4c), whereas the treated cells were characterised by ruptured hyphae and cell membranes (Fig. 4d).

Antioxidant Activity Assay
The methanolic extract of C. medica demonstrated strong antioxidant activity by reducing the formation of DPPH radicals by 50% at an IC 50 value of 300 μg.The mean IC 50 value of ascorbic acid was found to be 50 μg.In the case of the ABTS assay, the free-radical ABTS inhibition by methanolic extract was found to be 450 μg (Table 5).The methanolic extract of C. medica plant leaves was evaluated for its antiproliferative activity against the MCF7 breast cancer cell line by MTT assay.An inhibition of viable MCF7 cells counted after treatment with the extract was observed.The results revealed that the cell line viability was decreased gradually with an increase in sample concentration.The maximum reduction in cells was found at a concentration of 100 µg/mL, with the viability being 41.473%.The IC 50 value was 60.044 µg/mL at 48 h on the MCF7 cell line (Fig. 5).

DISCUSSION
Citrus leaves are rich in various biochemical compounds particularly phenolic compounds and flavonoids (Khettal et al. 2017).The phytochemicals screened in the citrus leaf extract are alkaloids, flavonoids, carbohydrates, glycosides, terpenoids and phenolic compounds.In recent study by Chhikara et al. (2018) also reported the presence of alkaloids, glycosides, flavonoids and steroids in the leaves of C. medica.In another study by Patil (2017) reported the presence of alkaloids, steroids, glycosides and phenols in the citrus plant.The FTIR analysis study revealed the presence of functional groups such as halogen compounds (C-I, C-Br and C-Cl stretching), primary and secondary alcohols (C=O stretch), aromatic amine (C-N stretch), aromatic compounds (C=C stretch, C-H bending), amines (N-H bending and N-H stretch), thiocyanate (S-C=N stretch), isothiocyanate (N=C=S stretching), isocyanate (N=C=O stretch), nitrile compounds (C=N stretch) and carboxylic acids (O-H stretching) represented in Table 2.In previous study, Onyeyirichi et al. (2014) reported that the Citrus medica Limonium leaf essential oil consists of alcohols, amines, alkyl halides and alkanes.
The antimicrobial activity was studied against the selected food borne pathogens.In support of the findings of this study, the inhibition of microbial growth by C. medica extract has previously been demonstrated for S. aureus (Aliyah et al. 2017;Sah et al. 2011;Li et al. 2019), E. coli (Li et al. 2019), B. subtilis (Li et al. 2019) and C. albicans (Aliyah et al. 2017).However, Sah et al. (2011) also failed to detect any inhibition of growth of P. aeruginosa, K. pneumoniae, E. coli, P. vulgaris, A. niger, A. flavus and C. albicans when exposed to C. medica extract.Further, previous studies have also found that C. medica extract inhibited the growth of some microorganisms not tested during this study, including E. faecalis (Sah et al. 2011), M. luteus (Li et al. 2019) and P. acne (Aliyah et al. 2017).In addition to this, the antagonistic activity was evaluated by selecting the S. aureus and C. albicans through Scanning electron microscopy and found changes in morphology, ruptured cell membranes and leakage of cellular constituents were observed.Similar type of study by Li et al. (2019) was reported for E. coli and S. aureus treated with citron essential oil.
In our body, the free radicals are produced and reacts with tissue causes oxidative damage.Our body has complex system of antioxidant defense mechanism of enzymes such as catalase, glutathione peroxidase and superoxide dismutase.However, under certain conditions, there is an imbalance occurs due to excessive production of free radicals results in oxidative stress (Khettal et al. 2017).Antioxidants are the chemicals that inhibits the oxidation and counteract the oxidative damage (Shah & Mehta 2018).Many studies illustrate the importance of natural antioxidants usage in the food processing industry and medical fields.The natural antioxidants have protective role against the reactive oxygen species due to the presence of bioactive compounds.In the present study, the methanolic extract was examined for in-vitro antioxidant activity through DPPH assay and ABTS assay.For DPPH assay and ABTS assay the IC 50 values are found to be 300 μg and 450 μg, respectively.There are no reports available related to anticancer activity of C. medica leaves.In this study, the anticancer activity was evaluated against MCF-7 breast cancer cell line.In previous study by Entezari et al. (2009) reported the anti-mutagenicity and anticancer effect of citrus fruit juice.

CONCLUSION
A GC-MS analysis of C. medica leaves confirmed the presence of various bioactive compounds with biological activity and food applications.The C. medica leaf extract showed good antioxidant, anticancer and antimicrobial activity against food-borne pathogens.The abundant compounds present in the extract were linolenic acid and α-linolenic acid trimethylsilyl ester compounds belonging to the omega-3 fatty acid group.Omega-3 fatty acids have a beneficial role in cancerrelated complications, as well as having antioxidant properties.Omega-6 fatty acid--eicosanoic acid plays a beneficial role in acting against inflammation and cardiovascular disease.This study indicates the potential for the development of C. medica extract as a natural functional food supplement for reducing the risk of chronic diseases.

Figure 2 .
Figure 2. Structures of compounds identified by GC-MS analysis.

Figure 3 .
Figure 3. (a) Antimicrobial activity of C. medica leaf extract; (b) Antifungal activity of C. medica leaf extract.

Figure 4 .
Figure 4. Antagonistic activity of Citrus medica leaf extract on S. aureus and C. albicans.(a) Untreated cells of S. aureus; (b) Treated cells of S. aureus; (c) Untreated cells of C. albicans; and (d) Treated cells of C. albicans.

Figure 5 .
Figure 5. Cytotoxic activity of methanolic extract of C. medica plant leaves against MCF7 cell lines.

Table 1 .
Phytochemical analysis of methanolic extract of C. medica.

Table 2 .
FTIR analysis of phytoconstituents of methanol leaf extract of C. medica.

Table 3 .
GC-MS analysis for leaf extract of C. medica.
Notes: RT = Retention time; RI = Retention index; SI = Similarity index

Table 4 .
MIC values of C. medica leaf extract versus selected indicator organisms.