Introduction

Medicinal plants are known for their acceptability by human and animal system. According to a report from World Health Organization, about 80% of the population of developing countries depend on traditional plant-based medicines, for their primary health care needs (Hassan 2009) due to their antioxidant, antibacterial, antifungal and antiviral activity (Barbour et al. 2004; Yasunaka et al. 2005). Indigenous systems such as Siddha, Ayurveda and Unani depend upon several plants species to treat different ailments (Rabe and Staden 1997).

The present study has been carried out on Psidium guajava which is a fruit tree with medicinal importance and is a native of South America. It is commonly known as guava, lemon guava, mubera, mupeera etc. Every part of this tree, whether its fruit, leaves, bark or roots, have been used for treating stomach-ache and diarrhoea in many countries. Ethanolic extracts of stem have a high anti-diabetic activity (Mukhtar et al. 2006; Rai et al. 2007). Guava contains a large number of antioxidants and phytochemicals including essential oils, polysaccharides, minerals, vitamins, enzymes, and triterpenoid acid alkaloids, steroids, glycosides, tannins, flavonoids and saponins (Smith and Siwatibau 1975). Guava contains a higher content of vitamin C and vitamin A. Guava is also a very good source of the pectin which is an important dietary fiber. It has high content of flavonoids (Das 2011), fructose sugar (Khan and Ahmad 1985) and carotenoids (Naseer 2018). Its leaves, pulp and seeds have been used for treatment of gastrointestinal and respiratory disorders (Barbalho et.al. 2012). The leaf extract was found to possess anticestodal (Tangpu and Yadav 2006), analgesic, anti-inflammatory properties (Ojewole 2006), antimicrobial (Nair and Chanda 2007), hepatoprotective (Roy et al. 2006) and antioxidant activities (Chen and Yen 2007). In addition, the leaf extract is used in many pharmaceutical preparations as a cough sedative.

Guava leaf extract contains flavonoids, mainly quercetin derivatives, which are hydrolyzed in the body to give the aglycone quercetin which is responsible for the spasmolytic activity of the leaves (Lozoya et al. 1994). Quercetin has several pharmacologic actions; it inhibits the intestinal movement, reduces capillary permeability in the abdominal cavity (Zhang et al. 2003) and possesses dose-dependent antioxidant properties (Nakamura et al. 2000), anti-inflammatory activity (Middleton et al. 1985; Amella et al. 1985), antiviral and antitumor activities (Kaneuchi et al. 2003). It also inhibits the aldose reductase enzyme (Chaundry et al. 1983). Several flavonoids have been reported from guava leaf such as quercetin, avicularin, guaijaverin, isoquercetin, hyperin, quercitrin, quercetin 3-O-gentiobioside, quercetin 4′-glucuronoide (Lozoya et al. 1994; Kandil et al.1997; Arima and Danno 2002). Keeping in mind the importance of P. guajava, the present study was conducted to analyze the phytochemical analysis, antimicrobial activity, antioxidant activity and cytotoxicity activity of different extract of P. guajava leaves.

Materials and methods

Plant Sample and chemicals

Fresh leaves of P. guajava were collected from different regions of Ambala District of Haryana, India. The solvents such as methanol, ethanol, chloroform, n-butanol, n-hexane, ethyl acetate, petroleum ether, acetone used were obtained from Loba Chemie Pvt. Ltd., Mumbai India. Antibiotics such as Gentamycin and Fluconazole, DPPH and MTT were obtained from Sigma-Aldrich Co. LLC, Mumbai.

Media and microbial strains

Two Bacterial strains Staphylococcus aureus (MTCC096) and Pseudomonas aeruginosa (MTCC741) and a fungal strain i.e. Candida albicans (MTCC227) were used to analyze the inhibitory effect of various solvent extract of leaves of P. guajava. Both bacterial and fungal strains were procured from Institute of Microbial Technology, Chandigarh and maintained on Muller-Hinton agar (MHA) and Potato Dextrose (PDA) agar slants at 37 ºC and 30 ºC respectively. The antibacterial and antifungal assays were performed on Muller-Hinton agar (MHA) and Potato Dextrose (PDA) agar medium using agar well diffusion method (Perez et al.1990).

Preparation of leaves extracts using different solvents

The collected leaves (1 kg) were thoroughly washed with running tap water followed by rinsing in distilled water. The leaves were completely dried for two weeks in shady area, and grounded to powder using an electric grinder. Extracts of different solvents (Methanol, ethanol, chloroform, n-butanol, n-hexane, ethyl acetate, petroleum ether, acetone) were prepared using a cold maceration method, whereas decoction method was used to prepare aqueous extract and filtrate was allowed to dry and stored at 4 °C till use (Kumar et al. 2016).

Qualitative analysis of phytochemicals in various solvent extracts

The various solvent extracts were subjected to preliminary phytochemical analysis for the presence or absence of different bioactive components such as phenolics, flavonoids, tannins, saponins, alkaloids, glycosides, and carbohydrate using standard methods (Khandelwal 2008; Guleria et al. 2016).

Determination of total phenolic and flavonoid content in various in various solvent extracts

The total phenolic content (TPC) was determined using Folin–ciocalteau reagent method (Singleton et al. 1999; Kumar et al. 2018, 2020); whereas total flavonoid content (TFC) of different solvent extracts of leaves of T. foliolosum were quantified by using aluminum chloride (AlCl3) method (Zhishen et al. 1999; Kumar et al. 2018, 2020). Gallic acid and Rutin was used as standard for calculation of TPC and TFC respectively.

Agar well diffusion method for antimicrobial activity

Antimicrobial activity in various solvent extracts of leaves of P. guajava was evaluated in terms of zone of inhibition using agar well diffusion method (Perez et al. 1990). Gentamycin (for antibacterial activity) and Fluconazole (for antifungal activity) were used as a positive control, and Dimethyl sulfoxide (DMSO) was used as a negative control. In agar well diffusion method, the bacterial/fungal culture of 0.5 McFarland Standard (∼2 × 108 colony forming units (CFU/mL) was uniformly spread on the surface of MHA/ PDA plates using sterile cotton swabs. The wells were punched with the cork borer (6 mm) in the agar. Approximately 50 μL of the prepared extract (50 mg/mL in DMSO) of P. guajava were added into the wells, allowed to stand at room temperature for about 1 h and incubated at 37 °C for bacteria and 30 °C for fungal growth. After 24 h (bacteria)/48 h (fungus) of incubation, the zone of inhibition was measured using a HiAntibiotic Zone scale-C (Himedia Biosciences, Mumbai (India).

DPPH radical scavenging activity of solvent extracts of P. guajava

DPPH radical scavenging activity of the extract was measured by the method described by Barros et al. (2007) with modifications. For performing DPPH assay, 10 test tubes were covered with aluminium foil to mark as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In each test tube, 200 µL of each extract (1 mg/mL), 800µL of 0.1 M Tris–HCl buffer (pH-7.4) and 1 mL of DPPH solution (0.004%) was added, mixed thoroughly and incubated in dark for about 30 min. After incubation, the absorbance was taken at 517 nm. For blank, 1.2 mL of ethanol and 800µL of Tris–HCl was used. For control, 1 mL of DPPH solution, 200 µL of ethanol and 800 µL of 0.1 M Tris–HCl was taken. Ascorbic acid (10 mg/10 mL) was used as standard antioxidant and standard curve was prepared using various dilutions of ascorbic acid (10–1 to 10–8). To 200µL of ascorbic acid sample from each dilution, 800µL of Tris–HCL buffer and 1 mL of DPPH solution was added and incubated in dark for 30 min and the absorbance was taken at 517 nm.

Percentage scavenging activity was calculated using following formula-

$$\% {\text{ DPPH radical scavenging activity }} = \, \{ {\text{A}}_{{\left( {{\text{control}}} \right) - }} {\text{A}}_{{({\text{sample}})]}} /{\text{ A}}_{{({\text{control}})}} \} \times { 1}00$$

In vitro blood lymphocyte proliferation response by MTT assay

Lymphocytes from bovine blood were extracted using Histapaque 1077 and suspended in PMI1640 media supplemented with 10% fetal calf serum (FCS), 10 mM HEPES buffer, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Lymphocyte suspension was diluted to 5 × 106 live cells/mL using culture media (RPMI 1640). To 100 μL of diluted cells, add 50μL of each extracts and mitogen (concanavalin A, 25 µg/mL in RPMI), and then final volume was adjusted to 200μL using RPMI media. The reaction mixture was incubated at 37 °C in a humidified CO2 incubator (95% air and 5% CO2) for 48 h. After incubation, supernatant was discarded without disturbing the adhered cells and then 25 μL of MTT (5 μg/mL of PBS) solution to each well and again allowed to react for 4 h in a humidified CO2 incubator. Then 100 μL of DMSO was added to each mixture in order to dissolve crystals formed. The amount of MTT formazan produced during the incubation was measured by an ELISA reader at a test wavelength of 550 nm and a reference wavelength of 630 nm. The results were based on the optical density at the wavelength of 550 nm (OD550) and expressed as a stimulation index (SI), which was calculated as follows: (Soper et al.1978; Mosmann 1983; Norian et al. 2015):

Stimulation index (SI) = OD550 of stimulated cells-OD550 of blank/ OD550 of unstimulated cells.

Results and discussions

Phytochemical analysis

All the extracts were tested for the presence of different phytochemicals and the results was summarized in Table 1. All the extracts also varied in number of phytocompounds present and comparison of number of phytocompounds showed higher number of phytocompounds were present in extracts of methanol, ethanol, acetone and Aqueous extract prepared through decoction method. Least number of bioactive compounds were reported from n-hexane and ethyl acetate extract (Fig. 1). Several studies have reported the presence of alkaloids, flavonoids, glycosides, polyphenols, reducing compounds, saponins and tannins in the different solvent extract of P. guajava leaves (Uboh et al. 2010; Biswas et al. 2013; Gayathri and Kiruba 2014).

Table 1 Qualitative screening of phytocompounds in various extracts of P. guajava
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Fig. 1
figure 1

Comparison of number of phytocompounds present in various extracts of P. guajava leaves

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Quantitative analysis of phytocompounds in various extracts of P. guajava leaves

The amount of total phenolic (TPC) and flavonoid content (TFC) was calculated using spectrophotometric methods and the results were showed in Fig. 2. Among all extracts, acetone extract of P. guajava leaves showed higher amount of TPC (256.32 ± 4.56 mg/g GAE) followed by n-butanol extract (210.32 ± 4.3 mg/g GAE), while TFC was higher in n-butanol extract (198.64 ± 1.23 mg/g RE) followed by acetone extract (165.43 ± 2.44 mg/g RE). Amount of TPC and TFC was also quantified by various researchers (Nantitanon et al. 2010; Ashraf et al. 2016; Camarena-Tello et al. 2018) in different solvents. However, there may be variation in TPC and TPC in solvents of different polarities, which can be explained due to difference in the chemical nature of solvents, variation in agro-climatic conditions, maturity at harvest, difference in extraction method and polarity of extracting solvent (Bae et al. 2012). Díaz-de-Cerio et al. (2015) quantified different category of phenolic compounds in Guava leaves infusions and ultrasound aqueous extract using HPLC–ESI–MS method. Four different classes of phenolic compound such as Gallic and ellagic acid derivatives, flavonols, flavanones, and flavan-3-ols and some benzophenones were quantified.

Fig. 2
figure 2

Comparison of total phenolic content (TPC) and flavonoid content (TFC) in various extracts of P. guajava leaves. TPC was expressed as mg/g Gallic acid equivalents; while TFC was expressed as mg/g Rutin equivalents. Values are expressed as mean ± S.D. of two readings

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Antimicrobial activity of various extracts of P. guajava

Antimicrobial activity of various extracts of P. guajava was evaluated against bacterial and fungal strains and expressed in terms of diameter of zone of inhibition. The data of zone of inhibition was given in Table 2. Acetone (13.5 ± 2.12 mm), ethanol (13 ± 1.41 mm) and methanol (12.5 ± 3.53 mm) extract showed maximum zone of inhibition against P. aeruginosa; while acetone (14 ± 0.71 mm) and ethanol (14.5 ± 2.12 mm) showed maximum zone of inhibition against S. aureus (Fig. 3). Chloroform (10.2 ± 1.13 mm) and ethanol extract (8.75 ± 0.50 mm) showed maximum inhibition to the growth of C. albicans (Fig. 4). The higher antibacterial activity of guava against gram positive bacteria and moderate against the gram-negative bacterial strains was also reported in previous study (Nair and Chanda 2007). Essential oil from guava also showed activity against Salmonella and S. aureus (Gonçalves et al. 2008). Aqueous and methanol extract of the guava leaves reported to inhibit the growth of bacteria by producing a remarkable zone of inhibition (Puntawong et al. 2012). Antifungal activity of leaves of P. guajava showed in the present study was in accordance with the reports of Morais-Braga et al. (2017) and Shinde et al. (2018).

Table 2 Diameter of Zone of Inhibition against tested bacterial and fungal strains
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Fig. 3
figure 3

Agar well diffusion method of antibacterial activity of various extracts of P. guajava against P. aeruginosa (a–d), where plate A-methanolic extract; plate B-n-hexane extract; plate C-n-Butanol extract; plate D-Ethyl acetate extract and against Staphylococcus aureus (e–h) where plate A-Methanolic extract; plate B-Pet ether extract; plate C-n-Butanol extract; plate D-Ethyl acetate extract

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Fig. 4
figure 4

Agar well diffusion method of antifungal activity of various extracts of P. guajava against C. albicans. Plate A-Methanolic extract; Plate B-Ethanolic extract

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Antioxidant activity in terms of DPPH radical scavenging

Antioxidant activity followed concentration-dependence, as radical scavenging activity of ascorbic acid increase with increase in concentration (Fig. 5). Comparison of % DPPH radical scavenging activity of various solvent extracts of P. guajava was done at same concentration (100 µg/mL) and found that acetone extract (41%) showed highest radical scavenging followed by chloroform (40%) and n-butanol extract (35.9%). Pet ether and n-hexane extract showed least radical scavenging activity (Table 3). Ascorbic acid an important antioxidant compound is present in leaves in excess (He and Venant 2004; Thaipong et al. 2005) . The other phytocompounds which are present in guava are protocatechuic acid, quercetin, ferulic acid, quercetin, gallic acid and caffeic acid and are also important antioxidants. Study from Fernandes et al. (2014), Seo et al. (2014) and Santhoshkumar et al. (2014) also reported the maximum antioxidant activity of methanol and aqueous extract of guava leaves. Ethanolic extract of guava showed low activity in all antioxidant assays like DPPH and FRAP assay (Vijayakumar et al. 2015). Quercetin isolated from leaves of guava was also reported to possess most active and strong antioxidant activity (Soman et al. 2010; Nantitanon and Okonogi 2012).

Fig. 5
figure 5

Concentration dependent DPPH radical scavenging activity of ascorbic acid

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Table 3 Comparative % DPPH radical scavenging activity of various extracts of P. guajava at same concentration
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Cytotoxic activity assay

Stimulation Index of different solvent extracts of P. guajava showed that highest stimulation index was found with methanol extract (1.432 ± 0.193) followed by ethanol (1.09 ± 0.045), butanol (1.005 ± 0.103) and acetone extract (0.88 ± 0.026) (Fig. 6). Aqueous leaf extract of P. guajava was reported to possess anticancer activity against MCF-7 cell lines using MTT assay (Sukanya et al. 2017). Ashraf et al. (2016) also evaluated in vivo cytotoxic against Artemia salina nauplii using brine shrimp lethality assay and in vitro anticancer activity against KBM5 (human chronic myelogenous leukaemia), SCC4 (human tongue squamous carcinoma cells) and U266 (human multiple myeloma cells) of methanol, hexane and chloroform extracts of P. guajava. Among all tested extracts, hexane extract showed better cytotoxicity and anticancer effect which may be attributed to presence of tetracosane, α-copaene, γ-sitosterol, vitamin E and squalene.

Fig. 6
figure 6

Stimulation index of guava leaves in different solvent extracts

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Conclusion

The present study reveals that the plant P. guajava is rich in many bioactive compounds which are of medicinal importance. The results also showed that the most effective solvent used for the extraction of phytochemicals are methanol, ethanol and acetone and highest antimicrobial, antioxidant and cytotoxicity activity was reported with acetone extract. As the leaves have been reported for biological importance, therefore, it can be further used to treat many acute and even lethal diseases in future. Also, we know that, there is a rapid emergence of drug resistant organisms which remains ineffective from the present medicines and antibiotics used by us. Therefore, the necessity of continuous searching for new antimicrobial substances and natural products may act as alternative for antibiotic and chemotherapeutic agents. Through phytochemical analysis, the presence of many bioactive compounds such as terpenoids, tannins, steroids, flavonoids, saponins, alkaloids have been observed. However, results indicate that the extraction was best in methanol and ethanol. Therefore, by keeping in mind the benefits and availability of the guava leaves, we should look forward the isolation of these bioactive compounds for the development of versatile drugs from a versatile tree, P. guajava.