Phytochemical Profiling of Medicinal Plants Extracts and Their Antioxidant and Anticancer Potentialities Against Human Liver Cancer (Hep G2) Cell Lines

Although Pakistan is stacked with enormous varieties of medicinal plants, only a little proportion of these plants has been evaluated for their medicinal and therapeutic properties. Herein, four indigenous medicinal plants Citrus sinenesis, Citrus paradiasii, Moringa olifera, and Hibiscus rosa-sinenesis were collected and subjected to phytochemical analyses to scrutinize the presence of secondary metabolites. Qualitative analysis showed the presence of an array of secondary metabolites in the selected plants, which were further corroborated by high-performance liquid chromatography. Results revealed the presence of 33.24, 21.04, 15.2 ppm gallic acid in methanol, ethyl acetate and nhexane fraction of C. sinensis peels extract, respectively. C. paradaissi peels consist of 24.06, and 18.24 ppm of gallic acid and caffeic acid, respectively, in methanol and chloroform fractions, whereas its methanolic seeds extract contain caffeic acid as a major component (10.63 ppm). H. rosa-sinenesis has shown p-coumaric acid, caffeic acid, and gallic acid at 35.26, 15.04, and 11.4 ppm, respectively. M. olifera contained 3.24 ppm gallic acid in pods extract while stems and leaves extract contain a very low amount. Anticancer profile evinced that Citrus sinensis extract showed the highest percent inhibition (142.746%) of human liver cancer (Hep G2) cell lines followed by H. rosa-sinensis (132.49%), C. paradaisii (82.39%) and M. olifera (68.0%). The determined IC50 values for antioxidant activity were C. sinenesis (IC50=0.49 mM), C. paradaisii (IC50=0.43 mM), M. olifera (IC50=0.42 mM) and H. rosasinensis (IC50=0.41 mM). Conclusively, the selected plants could be an effective alternative and deliverable chemical therapeutic to the pharmaceutical industry due to their excellent biological effects.


Introduction
Fruits and vegetables are important for human life because their persistent utilization reduces the risk of many incurable diseases. With the advancements in technologies making life easier and facile, there is an increasing trend in the risk of stress-related diseases. Therefore, it is essential to take a healthy and natural diet. To cover this need, worldwide, intake of fruits, vegetables, plant-based foods, and nutrientrich plant products has been focused [1][2][3][4][5]. Nevertheless, the utilization of proper diet (a complex of vegetable, fruits, and nutritional juices) is not enough to prevent chronic diseases because a specific diet only provides more than 25,000 bioactive constituents. Among these, many are related to modification causing in processes of these chronic diseases. Thus, it is of paramount significance to develop biodrugs based on food and dietary products for the betterment of human health, which is only possible through a complete understanding of the complex relationship of food, vegetables, fruits, plant-products https://doi.org/10.37358/RC. 21.1.8407 to cure diseases. According to the recommendation of nutritionist and biologist, food-based drugs have synergic and additive effects, which are necessary to cover health benefits and maintain human health. Therefore, it is a better approach by researchers and pharmacists around the world to design and develop food-based strategies to derive bio-drugs for health improvement. Mostly, food constitutes, and bioactive reagents are obtained from plants by extraction, known as phytoconstituents. These phytochemicals are reduced products of plants acting as antioxidants and produced as defense tools in response to the environmental stress and conditions in which the plants exist ( Figure S1). As anti-oxidants, these bioactive constituents act as an inhibitor of free radicals like reactive oxygen and nitrogen species, which accelerate the chronic diseases [6]. From the beginning of life, natural products have been serving as excellent sources of novel drugs, which have proven as an inspiration and lead compounds for the synthesis of natural as well as non-natural compounds. It is important to introduce new pharmacological sources to scientific and social communityy [7]. Natural products are always acting as a supplier of bioactive scaffolds reservoir that has demonstrated significant avenues for the treatment of human diseasess [8]. Increasing interest in the use of medicinal plants and plant-derived drugs is due to their emerging effects in the maintenance of human health. It has been observed that the drugs derived from natural products are less toxic and free from adverse effects [9]. Keeping all beneficial properties in mind, the local flora of Pakistan was explored to identify their constituents to derive drugs and provide cost-effective, cheap, and promising remedies to treat diseases. Citrus belongs to the family Rutaceae and is the most admired and favored fruit crop. The undeveloped peels of Citrus sinensis are capable of acting as a chemotherapeutic mean. Hesperidin, a C. sinensis flavonoid, exhibits potential stability on the bone condition and their metabolism. It is demonstrated that many types of cancers like colorectal, stomach, and esophageal can be retarded by C. sinensis fruit. Citrus sinensis fruit also enhances the blood lipid profile and anti-stroke action [10]. Moringa olifera, locally called as drumstick-tree or horseradish tree, and its different parts are used as food since ancient times. Traditional medicine systems used this plant to cure different diseases like ulcers, wound information, heart disorders, cancer, obesity, liver problems [11]. Different parts of the plant have been evaluated for their potential as therapeutics and observed to exhibit anti-hypertensive, anti-tumor, anti-inflammatory, anti-ulcer, anti-spasmodic, cholesterol-lowering, anti-diabetic, anti- bacterial, and anti-fungal activities [12]. Citrus paradisi (commonly called grapefruit) is the second most worldwide used citrus crop [13]. Natural bioactive flavonoids reveal the antioxidant action and these flavonoids are present in higher amounts in the peels of C. sinensis. It has been utilized in various therapies as weight loss and immune system enhancement [14]. Traditionally, it has used as an antibacterial, anti-fungal, anti-inflammatory, antioxidant, and anticancer agent in the folk medicine system. Moreover, with health awareness and rising demand for anti-aging products, grapefruit has been used to clear oil from skin and acne issues [13]. A plant belongs to the genus Hibiscus is famous for its use in traditional medicines known as Hiboscus sabdariffa L. It has been used as food and herbal medicines, as well as, in herbal drinks, hot and cold drinks. Its extracts find use as antibacterial, antidiabetic, anti-oxidant, and antihypertensive agents ( Figure 1) [15].
A large variety of phytochemicals is present in nature, and many of these phytochemicals possess medicinal and health beneficial properties. Many components are present in plant extract that manifest the physiological and medicinal actions. Many biological properties are also exhibited by these components such as anti-fungal, anti-apoptosis, anti-inflammation, anti-microbial, anti-urease, anticarcinogen, anti-bacterial, anti-oxidant, and anti-aging etc. [16]. Parasitic infections, which generated due to viruses, fungi, bacteria, and others, were treated for thousands of years by medicinal plants and their products. Recently, a number of synthetic and semi-synthetic drugs have been synthesized using these plants [16]. Different parts of these plants like flowers, seeds, leaves, roots, barks, and fruits have been exploited to cure various diseases and disorders. The oils extracted from medicinal plants have also been tested for their activity against pests, bacteria, fungi, insects and other microorganisms [17]. Therefore, in this study, indigenous medicinal plants C. sinenesis, C. paradiasii, M. olifera, and H. rosasinenesis were subjected to phytochemical analyses to scrutinize their bioactive constituents. Moreover, anticancer and antioxidant activities were also investigated to appraise their therapeutic potentialities.

Plant materials and preparation of extracts
All selected plants C. sinenesis, C. paradaisii, H. rosa-sinensis, and M. olifera were collected locally from different regions of Faisalabad. The selected plants were identified by Dr. Fouzia Shakoor, Department of Botany, The Government College Women University, Faisalabad. The collected plants were dried under shade and ground to a fine powder. The powdered material of the plant was extracted with water, chloroform, methanol, petroleum, and ethyl acetate. For each plant extract, powdered plant material was soaked in respective solvents for 24 h to obtain all polar and non-polar compounds. The extracts were evaporated under reduced pressure by a rotary evaporator.  [18,19] to confirm the presence of secondary metabolites in the selected plants (Table 1). For quantitative analysis, the prepared extracts of selected plant species were subjected to HPLC to estimate the number of different phytochemicals. To perform a quantitative analysis of different solvent fractions of selected plants, their solutions were prepared. About 0.1 g of each extract (petroleum, chlororform, ethyl acetate, water, and methanol) was taken in a conical flask (100 mL). Then about 5 mL HCl and 10 mL distilled water was added, followed by the addition of 15 mL methanol. After mixing all, the mixture was put into an oven for 1.5 h and then subjected to HPLC for analysis. For HPLC analysis, sample preparation was done according to the method described in literature. The separation of plant samples on gradient HPLC was performed using shim-pack CLC-ODS (C18), 25 cm 4.6 mm, 5-µm column. The chromatographic separation was carried out using a mobile phase gradient: A (H2O : Acetic acid 94:6, pH = 6), B (Acetonitrile 100%) 0-15 min = 15% B, 15-30 https://doi.org/10.37358/RC.21.1.8407 min = 45% B, 30-45 min= 100% B with 1 mL/min flow rate using UV-visible detector at 280 nm wavelength at room temperature. The identification of each compound was established by comparing the retention time and UV-Vis spectra of the peaks with that obtained by the injection of standards. The quantification was performed by external calibration with standards. Precipitates of green color [29][30] 2. Flavonoids 1 mL of solution of (10%) lead tetra-acetate was mixed to the 1 mL of plant extract.

Qualitative and phytochemical analysis by HPLC
Yellow color 30 From 2% NaOH solution just 2mL was added to the crude extract Deep Yellow coloration, which disappears on acidification 30 In the crude extract, some or little fragments of magnesium ribbon were mixed and then drop wise concentrated hydrochloric acid was mixed.
Pink vermilion color 30 To the plant extract add alcohol and then filter paper strips were dipped in it and then the solution was ammoniated Change of color of strips to yellow 31 0.15 mL of 5% NaNO2 and 2 mL of distill water was merged to the plant extract. 0.15 mL of AlCl3 solution (10%) was added after 5 min and then for 5 min allows it to stand. Then mix 2 mL of NaOH solution (4%) to the mixture. To make the total volume up to 5 mL instantly water was mixed, stir and then for 15 min allow it to stand.

Phenols
The Crude extract was merged with 2 mL of FeCl3 solution (2%).
Black color 29 To 1 mL of plant extract, 4 mL of Na2CO3 and 5 mL of Folinciocalteu reagent were added.
Blue color 32 Few drops of lead acetate solution (10%) were merged to the solution.
White precipitates 33 4 Terpenoids To 2 mL of plant extract, 2 mL of acetic anhydride and 2-3 drops of concentrated H2SO4 was merged.
Deep red coloration 34 2 mL of chloroform was dissolved in the crude extract and evaporated to dryness. Afterwards this was heated for about 2 min with the addition of 2 mL of concentrated H2SO4.
Grayish color 30 5 Saponins 5 mL of distill water was merged to 5 mL of plant extract. Heat froth appearance 34 Some drops of olive oil were merged to the 5 mL extract of plant.
Formation of the emulsions 34 Some drops of sodium bicarbonate were merged to 1 mL extract of plant.
Formation of honey comb like structures 35-36 6 Steroids To the 2 mL of extract merged 2 mL of chloroform and 2 mL of H2SO4 (conc.).
Reddish brown ring at the junction indicated 30 7 Phytosterols 5 mL chloroform, 3 mL acetic anhydride, few drops of dil. Acetic acid and some of the drops of concentrated H2SO4 were mixed to the 1 mL extract of plant Bluish green coloration 37 8 Phlobatannins 1% solution of HCl (2 mL) was merged to 2 mL of the plant extract and then heat.
Reddish violet ring at the junction 30 Fehling B and Fehling A reagents were taken in same quantities then merged together and 2 mL from this mixture was merged to unrefined extract and boiled mildly.
Brick red precipitates 30 Unrefined extract was merged to 2 mL of Benedict's reagent and then boiled.
Reddish brown precipitates 30 Unrefined extract was merged to 2 mL of Benedict's reagent and then boiled.
Dark blue or purple coloration 30 10 Starch 1 mL of iodine solution is merged in extract of 1 mL. Blue coloration 30 11 Glycosides To 2 mL of the plant extract, 2mL of acetic acid and 2mL of chloroform was merged.
Color change violet to blue To 1 mL of plant extract, 1 mL of 5% FeCl3 solution and same quantity of acetic acid is used, and then some drops of H2SO4 were added to the mixture.
Greenish blue color 38 12 Cardiac glycosides 1-2 drops of FeCl3 (2%) were added to 2 mL of glacial acetic acid and then this mixture was mixed with the unrefined extract. Take another test tube having 2 mL of concentrated H2SO4 and then add the above-prepared mixture to this test tube.
Brown ring at the inter phase 30 13 Coumarins To 2 mL of the extract, mix 3 mL of 10% solution of sodium hydroxide.
Yellow color 34 14 Alkaloids To 2 mL of extract of plant, mix some drops of Hager's reagent.
Yellow precipitates 34 To 1 mL extract of the plant mix some drops of Dragandrof reagent Orange brown precipitates 36 2 mL of HCl (1%) was merged to the unrefined extract and heated lightly. Then the reagents added to the mixture were Wagner's and Mayer's reagents Opaqueness of the end precipitates 30 15 Proteins To 1 mL of extract of the plant, 1 mL of concentrated H2SO4 is mixed.
White precipitates 34 To 3 mL of the plant extract, 1 mL of 4% NaOH solution and 1 mL of 1% copper sulphates were mixed.
Violet or pink color 30 Precipitates of white color occurs when the unrefined was merged with 2 mL of Millon's reagent, and these precipitates were lightly heated Red Precipitates 30 A violet color occurred when 2 mL of Ninhydrin (0.2%) was boiled with unrefined extract Violet color 30 16 Emodin 2 mL of NH4OH and 3 mL benzene was mixed to 2 mL extract of the plant.
Red color 34 17 Anthraquinon 3 mL of benzene, 5 mL of 10% solution of NH3 was mixed to 3 mL of the plant extract.
Different colors in ammonical layer like pink, violet or red 36 18 Anthocyanins 2 mL of HCl (2N) and NH3 was mixed to 2 mL of extract. Pinkish red to bluish violet coloration To 1 mL extract of the plant, 1 mL of NaOH (2 N) was mixed and then heated.
Bluish-green colour 34 19 Betacyanin To 1 mL extract of the plant, 1 mL of NaOH (2 N) was mixed and then heated.
Yellow colour 34 20 Leucoanthocya nins 5 mL of isoamyl alcohol was mixed to 5 mL of plant extract. Red color in organic layer 34

21
Fixed oils Two filter papers were taken and then individually the extracts were compressed between these filter papers and let it dry.
Oil strain on filter paper 36 22

Gums and mucilage
To 0.1 mL of plant extract, 10 mL of distilled water and 2 mL of pure alcohol were mixed with perpetual stirring.

Biological evaluation 2.3.1 Cytotoxicity and cell viability analysis by MTT assay
The human HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin and maintained at 37°C with 5% CO2 in a humidified atmosphere. Cells were treated with extracts/compounds dissolved in DMSO with a final DMSO concentration of 0.05%. DMSO-treated cells were used as a control in all the experiments [20]. Cell viability was determined by MTT assay as described previously. Briefly, HepG2 cells were treated with different concentrations of compounds for 48 h. Following treatment, the MTT reagent was added (500 μg/mL) and cells were further incubated at 37°C for 4 h. Subsequently 150 μL DMSO was added to dissolve formazan crystals and absorbance was measured at 490 nm in a microplate reader (Thermo Scientific). The percentage of cell viability was calculated [21]. Different extracts of the selected plants were evaluated for their effects against human https://doi.org/10.37358/RC.21.1.8407 liver cancer lines. It was found that the extract of H. rosa sinensis flowers exhibits a strong activity as compared to the other four (Table 3).

Antioxidant (free radical scavenging) activity using DPPH assay
In order to perform the antioxidant activity, the stock solution of plant extracts having a concentration of 200 mM (0.022 g) was prepared. This solution was diluted to 250 µg, 200 µg, 150 µg, 100 µg, 50 µg, and 25 µg. DPPH solution of 4% was prepared by mixing 0.004 g of DPPH in 100 mL of methanol. Ascorbic acid was used as a control, which was prepared by mixing 0.0017612 g into 100 mL of water having 100-µg concentration. In test tubes, 1 mL of the sample from each diluted solution added and followed by the inclusion of 2 mL of DPPH solution. In the same way, 1 mL of the ascorbic acid was added along with 2 mL of DPPH as control and allowed to stay overnight. The next day, the readings were taken at 517 nm in triplicate by using a Hitachi U-2900 spectrophotometer and calculated their inhibitory concentration by using formula [22].

Results and discussions
Plants have been widely used for therapeutic purposes to cure diseases at the beginning of human history. According to the WHO report, about 80% population from rural areas depends upon natural products, herbs, and plants for their necessary health care. Unfortunately, not much attention has been given to investigate the medicinal plants for their useful effects, and constituents with medicinal properties as well as nutraceuticals to discover their health benefits. Therefore, some selected plants were considered to explore their phytochemical, pharmaceutical, and biological characteristics. For that, selected plants were analyzed qualitatively to discover the presence of compounds responsible for their health effects. In addition to qualitative analysis by HPLC to discover the percentage of these compounds, antioxidant and anticancer potential were also investigated.

Preliminary qualitative phytochemical analysis
Preliminary analysis showed the presence of an array of secondary metabolites in the selected plants. Results of qualitative analysis (Table 2) revealed that the targeted plants were rich sources of phytochemicals, particularly flavonoids, phenolic acids, alkaloids, steroids that were responsible for biological activities of plants helping in curing disease, increasing human body immune level and proved as a wound healer. Methanolic extract of C. paradaisii seeds has been observed to contain cardiac glycosides, which were not present in peels extract. Moreover, coumarins were found in seed extract while absent in peels extract of C. paradaisii. The results of the analysis of M. olifera showed that the phenols, phytosterols, and reducing sugars were present in leave extract while absent in pods and stem extract. Whereas extract of pods and stem (M. olifera) contain alkaloids, absent in leave extract as observed from Table 2. In comparison, H. rosa-sinenesus extracts lack of terpenoids, carbohydrates, cardiac glycosides, proteins, emodines, anthraquinones, fixed oils, and gums while others all present ( Table 2). Different fractions (Methanol, chloroform, n-hexane, ethyl acetate) of selected plant species (C. sinenesis peels, C. paradaisii peels, H. rosa sinenesis, M. olifera) were found to contain phytoconstituents in different amounts, which were further evaluated by HPLC analysis (quantitative analysis).

Quantitative phytochemical analysis by HPLC
Quantitative analysis by HPLC was performed to confirm the percentage of flavonoids and phenolic acids and results are shown in Table 3. Previously, a study of HPLC analysis has been done for the estimation of phenolics and flavonoids in the root and stem of C. sinenesis [23] but we selected its peels for HPLC analysis. Table 3 results showed that the methanolic extract of C. sinensis peels consists of quercetin, gallic acid, caffeic acid in high quantities than other fractions but C. sinensis peels lack of vanillic acid in all fractions. The m-coumaric acid and syringic acid were absent in ethyl acetate fraction, while synaptic acid, caffeic acid, m-coumaric acid, p-coumaric acid, chlorogenic acid, and syringic acid were not detected in n-hexane fraction. A previous report on C. sinenesis determined chlorogenic acid, quercetin, rutin, and hydroxybenzoic acid in both stem and root extract, whereas mandalic acid in root extract and phloroglucinol was detected only in stem extract [23]. In M. olifera, pod, stem, and leave extract to contain quercetin and gallic acid but previously Shervington et al. [24] reported that the M. olifera species were influenced by geographical changes strongly and resultantly, its chemical constituents (presence, percentage of occurrence) also varies [24]. HPLC results of H. rosa-sinensis showed the detection of quercetin, gallic acid, synaptic acid, caffeic acid, p-coumaric acid, and chlorogenic acid in good percentage. In comparison to the previous report [25] that showed the presence of only flavonoids quercetin, rutin, kaempferol, and myricetin in methanolic extract. From Table 3, it was found that vanillic acid was absent in all plant species but m-coumaric acid and syringic acid were absent in all plants except the methanolic fraction of C. sinensis peels. Gallic acid and quercetin were present in all fractions of selected plants except the n-hexane fraction of C. paradaisii peels, n-hexane fraction of C. sinenesis peels have only quercetin (0.1 ppm) and gallic acid (15.2 ppm). By observing HPLC results (Table 3), it was found that n-hexane fraction of C. paradaisii peels lack of any flavonoids and phenolic acids. Table 3. Results of quantitative phytochemical analysis by HPLC

Anticancer and antioxidant activities
Anticancer activity results (  [26]. Another report showed that M. olifera leaf extract has a significant inhibitory (80%) impact on Hep G2 cells [27]. H. rosa-sinenesis showed a cell viability of 132.49%, while a previous report showed its excellent apoptosis induction in breast cancer cell lines [28]. C. sinensis was found to exhibit significant cell viability of 142.74% as compared to other plant extracts due to the presence of phenolics and flavonoids, whereas C. paradaisii showed a cell viability of 82.39% ( Figure 2). Notably, there is no report on the selected citrus species (C. paradaisii and C. sinensis). Similarly, the selected plant extracts were evaluated for their antioxidant activity by using DPPH as a free radical using ascorbic acid as a standard. Table 5 showed the percentage of inhibition of each selected plant extracts. Among all extracts, H. rosa-sinenesis extract has the lowest IC50= 0.41 mM value, which makes it a good scavenger followed by M. olifera (IC50=0.42 mM), C. paradaisii (IC50=0.43 mM) and C. sinenesis (IC50=0.49 mM). This excellent scavenging activity confirms the presence of flavonoids and phenolic acid in an elevated concentration. In comparison to Ayyakkannu, [25] reports, the major factor responsible for excellent antioxidant potential was rutin [25] but in our findings, the main percentage was of p-coumaric acid (Table 3). Antioxidant potential results were in close agreement with HPLC findings, which confirmed that the excellent free radical scavenging potential of H. rosa-sinensis was due to a higher concentration of p-coumaric acid and caffeic acid in addition to other flavonoids and phenolic acids.

Conclusions
From the above study, it has been concluded that Citrus plants (sinensis and paradasii) are of enormous importance in pharmaceuticals and medicines due to the presence of many important phytoconstituents. The phytochemical analysis of C. sinensis peels proves to be very beneficial because of the presence of phytochemicals or bioactive compounds that are useful in various ways. These healing properties were observed due to the presence of tannins, flavonoids, terpenoids, saponins, reducing sugars. H. rosa sinensis has been used in various ways like tea, extract to weight loss, its flower water extract used to cure stomach disorders, skin irritations as anti-aging. Due to its much use as nutraceuticals, when it was phytochemically analyzed, it showed the presence of flavonoids, phenolics, alkaloids. Biological evaluation of the selected plants in different concentrations showed good results. C. sinensis showed the highest IC50 at 0.49 mM, while H. rosa-sinenesis plant species showed the lowest IC50 at 0.41 mM. These results showed that H. rosa sinensis has more activity, C. sinenesis has lower activity, while the C. paradaisii and M. olifera have medium activities.