Phytochemical analysis, GC–MS profile and determination of antibacterial, antifungal, anti-inflammatory, antioxidant activities of peel and seeds extracts (chloroform and ethyl acetate) of Tamarindus indica L

Tamarindus indica L., is widely used tree in ayurvedic medicine. Here, we aimed to understand the presence of important constituents in seeds and peel of Tamarind fruits and their biological activities. Hence, seeds and peel of Tamarind fruits are used for further extraction process by soxhlet method (chloroform and ethyl acetate solvents). Results suggest that the ethyl acetate extract (seeds) consists of terpenoids (72.29 ± 0.513 mg/g), phenolic content (68.67 ± 2.11 mg/g) and flavonoids (26.36 ± 2.03 mg/g) whereas chloroform extract (seeds) has terpenoids (42.29 ± 0.98 mg/g). Similarly, chloroform extract (peel) has terpenoids (25.96 ± 3.20 mg/g) and flavonoids (46.36 ± 2.03 mg/g) whereas ethyl acetate extract (peel) has terpenoids (62.93 ± 0.987 mg/g). Furthermore, anti-inflammation activity results revealed that the chloroform extract of peel was found to be more effective with IC50 of 226.14 µg/ml by protein denaturation analysis and with IC50 of 245.5 µg/ml on lipoxygenase inhibition activity. Chloroform extract (peel and seeds) shown better antioxidant activity using DPPH than ethyl acetate extract (peel and seeds). Ethyl acetate extract of seeds showed impressive potency by inhibiting the growth of fungus, Candida albicans. Additionally, ethyl acetate extract of seeds showed impressive potency inhibiting the growth of Escherichia coli than Bacillus cereus. GC–MS analysis shown the existence of diverse set of phytochemicals in each extract. Overall, comparative studies highlight the effectiveness of seeds extracts than peel extracts. Moreover, GC–MS results suggest that the seeds and peel extracts (chloroform and ethyl acetate) contains a wide range of compounds (including flavonoids, isovanillic acid, fatty acids and phenolic compounds) which can be utilized for therapeutic purpose.

Tamarindus indica L., is widely used tree in ayurvedic medicine.Here, we aimed to understand the presence of important constituents in seeds and peel of Tamarind fruits and their biological activities.Hence, seeds and peel of Tamarind fruits are used for further extraction process by soxhlet method (chloroform and ethyl acetate solvents).Results suggest that the ethyl acetate extract (seeds) consists of terpenoids (72.29 ± 0.513 mg/g), phenolic content (68.67 ± 2.11 mg/g) and flavonoids (26.36 ± 2.03 mg/g) whereas chloroform extract (seeds) has terpenoids (42.29 ± 0.98 mg/g).Similarly, chloroform extract (peel) has terpenoids (25.96 ± 3.20 mg/g) and flavonoids (46.36 ± 2.03 mg/g) whereas ethyl acetate extract (peel) has terpenoids (62.93 ± 0.987 mg/g).Furthermore, anti-inflammation activity results revealed that the chloroform extract of peel was found to be more effective with IC 50 of 226.14 µg/ml by protein denaturation analysis and with IC 50 of 245.5 µg/ml on lipoxygenase inhibition activity.Chloroform extract (peel and seeds) shown better antioxidant activity using DPPH than ethyl acetate extract (peel and seeds).Ethyl acetate extract of seeds showed impressive potency by inhibiting the growth of fungus, Candida albicans.Additionally, ethyl acetate extract of seeds showed impressive potency inhibiting the growth of Escherichia coli than Bacillus cereus.GC-MS analysis shown the existence of diverse set of phytochemicals in each extract.Overall, comparative studies highlight the effectiveness of seeds extracts than peel extracts.Moreover, GC-MS results suggest that the seeds and peel extracts (chloroform and ethyl acetate) contains a wide range of compounds (including flavonoids, isovanillic acid, fatty acids and phenolic compounds) which can be utilized for therapeutic purpose.
Similarly, Tamarindus indica tree source is also utilized for medicinal purpose which is a long lived evergreen hardwood tree found in different countries/region, for example, Egypt, Africa (tropical region) and Asia (including India) (Aly et al., 2023;Bhadoriya et al., 2011;Ghaly et al., 2023).Tamarindus indica tree yields high amount of green fruits.Once green fruit completely ripens in the tree, the fruit becomes a paste like texture (brown/reddish brown color).And the taste of the fruit will get sweet and sour (Yahia, 2011).Over the years, every part of the tree is traditionally used in ayurvedic medicinal system with nutritional value (Morton, 1958).For example, pulp of tree was used as ayurvedic ingredient for detoxifying skin as well as the body polishing purpose (Morton, 1987).Additionally, paste of Tamarind has anti-microbial properties, because of this reason, the Tamarind paste was frequently used to treat wounds (Gupta et al., 2014).The fruits of Tamarind have most of the essential amino acids as per the report of World Health Organization (Glew et al., 2005;Kuru, 2014).
From the Tamarind tree and its parts, many active phytochemicals have been isolated (Ghaly et al., 2023;Usman et al., 2023) namely some phenolic compounds like tartaric acid, l-(-)mallic acid; triterpenes such as lupanone and lupeol; oils components such as n-hexacosane, eicosanoic acid, b-sitosterol, octacosanyl ferulate and so on including fatty acids and proteins (Rana et al., 2018).However, not much information available on seeds and peel extracts of Tamarind fruits.
Hence, in the current study, we collected Tamarind fruits and separated seeds and peel.Further, these seeds and peel are used for extraction by soxhlet method using ethyl acetate and chloroform solvents, respectively.The extracts of seeds and peel of Tamarind fruits are used for the determination of qualitative and quantitative analysis of phytochemicals.Gas chromatography-mass spectrometry (GC-MS) study was conducted to detect the bioactive molecules presence in both seeds and peel extracts.Furthermore, we studied antibacterial, antifungal, antiinflammatory and antioxidant activities of peel and seeds extracts (ethyl acetate and chloroform).Here, our research study will further insight in seeds and peel extracts (ethyl acetate and chloroform) components which will eventually help in additional information to current knowledge of Tamarind fruits.That can be further utilized for treatment purpose.

Tamarindus indica seeds collection and extraction
Tamarindus indica fruits collected from agriculture field in Yatanhalli village (Lat: 15.0066082 and Lon: 75.1448748),Shiggaon taluk, Karnataka, India.The collected fruits are further used to separate seeds.The separated seeds are further washed.Seeds and peel are separated and shade dried.Dried seeds and peels are powdered thoroughly using stone mortal and pestle and were used for further extraction.
Approximately 10 g of fine powdered samples of each seeds and peel are used for extraction by soxhlet method using 200 ml of chloroform and ethyl acetate solvents, respectively (Bagewadi et al., 2019).Rotary evaporator was used to dry the supernatants (containing bioactive molecules) collected from seeds and peel samples and dried samples were stored at 4 • C, till further use.

Biochemical methods
Seeds and peel extracts (chloroform and ethyl acetate) of Tamarindus indica fruits were evaluated by standard protocols to detect existence of various phyto-constituents like flavonoidss, alkaloids, saponins, phenols, and so on (Bagewadi et al., 2019;Harborne, 1984).

Total phenolic content
Folin-Ciocalteu reagent was used to quantify the total phenolics content in seeds and peel extracts (chloroform and ethyl acetate) with little modification (Bagewadi et al., 2019;Blainski et al., 2013).Here, we have taken 0.5 ml of each extracts (seeds and peel) in clean and dried test tubes, to this 0.5 ml of Folin-Ciocalteu reagent was added and the reaction mixture was kept for 5 to 8 min (at 25 • C).After that, 2 ml of 7.5 % sodium carbonate solution was added and the solution was makeup to 8 ml with double distilled water and kept for further 2 h.The absorbance was noted at 750 nm.Here, to get a calibration curve, standard compound, gallic acid was used and the total phenolic content was determined as described previously as per following equation, Gallic acid (mg) equivalents/gram of sample (mg GAE /g E) (Bagewadi et al., 2019).

Total flavonoids content
The total flavonoids content in each seeds and peel extracts (chloroform and ethyl acetate) were measured by colorimetric assay with little modification (Bagewadi et al., 2019;Fattahi et al., 2014).In a clean and dried test tubes, 0.5 ml of each extracts (seeds and peel) were added.To this solution, 4 ml of double distilled water was added.The mixed solution was further mixed with 0.4 ml 5 % of sodium nitrite and incubated up to 5 min.After specified time, the solution was mixed with 0.3 ml of 10 % aluminium chloride.At 6 min, the solution was mixed with 2 ml of sodium hydroxide (1 M) solution and the final volume was further diluted with 3.3 ml double distilled water and mixed meticulously without delaying the reaction.At 510 nm, the absorbance was noted.Here, to get calibration curve, standard compound quercetin was used.Total flavonoids content of the extract was determined using an equation, Quercetin (mg) equivalents/gram of sample (mg QuE /g E).

Total terpenoids content
100 mg of each seeds and peel extracts (wi) (chloroform and ethyl acetate) were taken separately and soaked in approximately 9 ml of ethanol for a day and filtered with whatman filter paper.The filtrate was further fractionated with 10 ml of petroleum ether.The separated ether extract sample (s) was pre-weighed glass vials and dried completely (wf).The yield (%) of total terpenoids contents was determined as per the procedure described in Malik (2017).

Protein denaturation assay
In brief, in a clean 1.5 ml of centrifuge tubes, the reaction mixture consisted of 1 ml of PBS as well as 50 µl of BSA were added, to this, different concentration of each seeds and peel extracts (chloroform and ethyl acetate) were added individually and also performed similar way with standard solution.All the tubes are incubated for 15 min (at 35 • C).Later, denaturation was induced by keeping at 60 • C in hot water bath for 15 min.At 660 nm, the absorbance was noted in UV-Vis spectrophotometer (Company, Labman UV-Visible Spectrophotometer).Aspirin was used as standard (Leelaprakash and Dass, 2011).The percent of inhibition of protein denaturation was evaluated as described A. N. Tavanappanavar et al. in Revankar et al., (2023).

Lipoxygenase assay
The lipoxygenase activities of seeds and peel extracts (chloroform and ethyl acetate) are determined as described previously (Chen et al., 2009) with little modification.134 μM of linoleic acid as a substrate and 165 U/ml of enzyme was used final concentration.Test extracts of seeds and peel were dissolved in DMSO (1.6 %).At 234 nm, the absorbance was noted in UV-Vis spectrophotometer (Company, Labman UV-Visible Spectrophotometer).Here, inhibition of enzyme was determined in percentage (%) as per the following equation.
Where X = absorbance measured at 234 nm (without a test sample), and Y = absorbance measured at 234 nm with a test sample.

Antioxidant assay
Seeds and peel extracts (chloroform and ethyl acetate) are used to determine antioxidant activity as described previously with minor modification (Shettar et al., 2023).In a test tube, 1.0 ml of α, α-diphenylβ-picrylhydrazyl (DPPH) (0.2 mM) was taken.To this, 0.5 ml of different concentrations test samples (Seeds and peel extracts) were added and the also standard solutions having concentrations between 100 and 500 µg/ml were also added.The reaction mixture of each test samples and standards are kept individually at room temperature under dark condition for half an hour.At 517 nm, the absorbance was determined in UV-Vis spectrophotometer (Company, Labman UV-Visible Spectrophotometer).Here, standard compound ascorbic acid was used as specified previously (Shettar et al., 2023;Baliyan et al., 2022).The antioxidant or radical scavenging activity was determined in %.The following equation was used to calculation.
Where, X and Y are the absorbance of control and test samples, respectively.

Anti-fungal assay
Potato dextrose agar (PDA) containing Petri plates were used to grow a fungal pathogen, Candida albicans.Freshly grown fungal strain (20 h) was inoculated on PDA.Wells were made using sterile well-borer.Here, for positive control, Itracanozole (10 mg/ml) was used as standard drug and for negative control, DMSO was used.Individual extract of different concentrations like 30 µg, 60 µg and 90 µg per ml are added to the well.All plates were kept at 37 • C for incubation (24 h).The antifungal activity was confirmed by determining the diameter of the inhibition zone formed around the well (Biemer, 1973).

Antibacterial assay
The antibacterial effect was evaluated on gram positive [Bacillus cereus (MTCC-1369)] and gram negative [Escherichia coli (MTCC-739)] pathogens as described previously (Rajagopalachar et al., 2022) with little modifications.Nutrient agar was sterilized and poured into sterile petri plates.After solidification, the agar plate surface is inoculated by spreading 0.1 ml of overnight grown each pure microbial inoculum.Then well made (5 mm diameter) aseptically at specific distance in the petri plates with a sterile cork-borer.Here, for positive control Ampicillin (100 μg/ml) was used standard drug and for negative control DMSO was used to determine the sensitivity of bacterial culture.Various concentrations of extracts (chloroform and ethyl acetate) of seeds and peel samples are used to test antibacterial activity.

Molecular characterization of seeds and peel extracts (chloroform and ethyl acetate) are carried out using GC-MS method
For the determinations of seeds and peel extract samples, GC-MS analysis was performed with little modifications (Massada, 1976;Mulla et al., 2016a).Here, fused silica used as a closed column and Helium gas was used as mobile phase for separation of active components present in the test samples.An aliquot of each extracts (1 µl) were injected into the GC-MS apparatus.The initial temperature of column programmed from 60 • C. The injector temperature was set at 250 • C and during the process temperature flow was set at the speed of rising 10 • C/min with standard specifications.Final temperature was set to 300 • C. Once it reaches the final temperature, it was holding for 6 min.The identification of components was based on the comparison of their mass spectra with those of NIST mass spectral library (Massada, 1976;Mulla et al., 2016a).

Statistical analysis
All the experimental studies are carried out in triplicates and the results were determined as mean ± SEM.Statistics were performed using GraphPad Prism 8.0 and Microsoft excel 2013.

Phytochemical analysis
The phytocompound analysis of extracts (chloroform and ethyl acetate solvents) of Tamarind fruit seeds showed the presence of different groups of phyto-components.Ethyl acetate extract (seeds) tested positive for terpenoids, saponins, phenolic compounds and flavonoids.Chloroform extract (seeds) tested positive only for terpenoids (Table 1).Additionally, phytochemical analysis of solvent extract of peel indicated the existence of various types of phytocomponents.Chloroform extract of peel sample tested positive for terpenoids and flavonoids.Ethyl acetate extract of peel sample tested positive only for terpenoids (Table 1).

Estimation of total phytochemical content
Based on the presence of phytochemicals, their contents were estimated according to their respective standard linear curve.The results of ethyl acetate extract of seeds in accordance with the respective standards shown the presence of high content of terpenoid (72.29 ± 0.513 mg/g), phenolic content (68.67 ± 2.11 mg/g) and flavonoids (26.36 ± 2.03 mg/g) whereas 42.29 ± 0.98 mg/g of terpenoid found in (+ve = Present and -ve = Absent).
Similarly, the results of ethyl acetate extract of peel in accordance with the respective standards shown the presence of high content of terpenoid (62.93 ± 0.987 mg/g) whereas 46.36 ± 2.03 mg/g of Flavonoids content and 25.96 ± 3.20 mg/g of total terpenoids content found in chloroform extract of peel (Fig. 1b).

Anti-inflammatory
Anti-inflammatory activity of extracts (chloroform and ethyl acetate) of seeds and peel were analyzed by very effective protein denaturation protective method and enzyme inhibition lipoxygenase assay.

Protein denaturation
Both seeds and peel extracts showed the concentration dependent anti-inflammatory activity.At increasing concentration, it was observed that ≥ 40 % of anti-inflammatory activity in extracts (chloroform and ethyl acetate) of seeds and peel sample.IC 50 was calculated accordingly and compared with the standard drug Aspirin (IC 50 = 83.64µg/ml) (Table 2a).
On comparison of IC 50 concentrations of seeds and peel extracts, all the extracts showed IC 50 of > 200 µg/ml concentration which was very much distinguishable from the standard drug (Table 2A).

Lipoxygenase assay
Both seeds and peel extracts (chloroform and ethyl acetate) showed anti-inflammatory potency against lipoxygenase, an inflammatory enzyme.Results suggest that the increasing concentrations of extracts were increases the inhibition level.Chloroform and ethyl acetate extracts of both seeds and peel sample showed more than 50 % inhibition of lipoxygenase enzyme.IC 50 concentrations of chloroform extract of seeds showed 318.6 µg/ml whereas ethyl acetate extract of seeds showed 297.1 µg/ml.Similarly, IC 50 concentrations of chloroform extract of peel showed 245.5 µg/ml whereas ethyl acetate extract of peel showed 258.2 µg/ml.All the extracts showed IC 50 > 200 µg/ml concentration which was very much distinguishable from the standard drug of IC 50 = 96.11µg/ml (Table 2B).

Anti-oxidant activity
On assessment of the radical scavenging capacity of extracts (chloroform and ethyl acetate) of seeds as well as peel samples by DPPH method.Both the samples showed a significant radical scavenging ability on comparison with the standard Vitamin-C (Table 3).The inhibitory effect was concentration dependent.The results of DPPH activities are follows, IC 50 concentrations of chloroform extract of seeds showed 277.4 µg/ml whereas ethyl acetate extract of seeds was 290.2 µg/ml.Similarly, IC 50 concentrations of chloroform extract of peel showed 283.8 µg/ml whereas ethyl acetate extract of peel showed 306.9 µg/ml (Table 3).

Antifungal activity
Antifungal activity was studied using 3 different concentrations of each extracts (chloroform and ethyl acetate) of seeds and peel.As compared with the standard drug, only the ethyl acetate solvent extract of seeds showed inhibition against fungus Candida albicans with linear increase of concentration (Fig. 2).The zone of inhibition was measured as 14 ± 0.3 mm (30 µg/ml), 17 ± 0.5 mm (60 µg/ml) and 18 ± 0.5 mm (90 µg/mm) with respect to ethyl acetate extract of seeds.However, both solvent extracts of peel are not showed inhibition against Candida albicans (data was not shown).

Antibacterial assay
Antibacterial potency of three different concentrations of each extracts (chloroform and ethyl acetate) of seeds and peel were assessed against gram positive (Bacillus cereus) and gram negative (E. coli) pathogens.Positive control, ampicillin showed zone of inhibition measuring about 30 ± 0.7 mm against Bacillus cereus and 35 ± 0.2 mm against E. coli (Fig. 3) and negative control was not shown inhibition   against both pathogens.The seeds extract was very potent against both the bacteria's.Against gram positive Bacillus cereus bacteria, chloroform extract of seeds showed a zone of inhibition only at 90 µg/ml whereas the ethyl acetate extract of seeds showed a zone of inhibition at all the treated concentrations.Apparently, against the E. coli, only the ethyl acetate extract of seeds showed a zone of inhibition parallel to increased concentration (Fig. 3).Extracts (Chloroform and ethyl acetate) of peel has not shown inhibition against Bacillus cereus and E. coli (data was not shown).

Discussions
Presently, reports suggest that the several groups of microorganisms are shown resistant towards antibiotics and antimicrobial agents and also some of the organisms have capacity to degrade such medicines (Chibwe et al., 2023;Mulla et al., 2016a and b;Mula et al., 2018;Wang et al., 2023a and b;Zarzecka et al., 2022).It is because of their presence in the environment either through excretion by animals as well as human beings and/or through incomplete removal from the wastewater treatment systems (Golgeri et al., 2022;Mulla et al., 2016c and2020).For example, antimicrobial agents like triclosan and triclocarban are degraded by microorganisms (Mulla et al., 2016a and b;Mulla et al., 2020).Similarly, sulphonamide drugs like sulfadiazine, sulfamethazine and sulfamethoxazole are also among the antibiotics and were degraded individually by various types of microorganisms (Mulla et al., 2016c(Mulla et al., , 2018(Mulla et al., and 2023;;Wang et al., 2023a).Moreover, there are reports on medicines like ciprofloxacin, tetracycline, levofloxacin    (Fluoroquinolone), carbamazepine and diclofenac were degraded/ removed by microorganisms (Ben Ayed et al., 2022;Chen et al., 2022b;Shah et al., 2022;Wang et al., 2023b;Wojcieszyńska et al., 2023).Hence, various researchers are looking alternative ways like plant based molecules to control such organisms' growth.Additionally, it was observed that biomolecules extract from plant sources are useful in industrial purpose.
Tamarind fruits is a household fruit of Asian countries (including India).Maximum south Indian cuisines involve the usage of tamarind fruit and plant young leaves (Rao and Mathew, 2012).Even the Indian Ayurveda uses all the major parts of the plant to cure various human diseases.Based on these data, in the current study we have selected the seeds and peel of Tamarind fruits plant parts to look their biological activities.The selected seeds and its peel are extracted using solvent systems (chloroform and ethyl acetate).The extracts were analyzed for the presence of diverse phytochemicals.In both the samples extracts, presence of phenolic compounds, flavonoids and terpenoids were observed.Further total phenolic, flavonoids and terpenoid contents were determined based on the presence of the phytochemicals according to their solvent extracts.Subsequently, in the seeds ethyl acetate extract, terpenoid content was found highest (72.20 ± 0.51 mg/g) on comparison with the seeds chloroform extract and peel solvent extracts.Whereas total phenolic content was estimated only in seeds ethyl acetate extract based on its presence and was found to be 68.67 ± 2.11.However, total flavonoids content was found to be significantly high (46.36 ± 2.03) in peel chloroform extract than the seeds ethyl acetate extract.Similarly, previous studies on screening of phytochemicals in the different parts of the tamarind tree also have shown specific results (Abdelrahman and Mariod, 2019;Roy et al., 2020;Sookying et al., 2022).
With the hint of vast content of major phytochemicals presence, seeds and peel samples were analyzed for its bioactivities.Primarily they were tested against inflammation using protein denaturation and lipoxygenase assay.Here, protein denaturation was done using heat induction.Heat-denatured proteins exhibit antigens linked to type III hypersensitivity reactions (Johansen et al., 2005).Denatured proteins are one of the main reasons of inflammation (Silvestrini and Silvestrini, 2022).Because of this sever inflammation in the human system, all the major interactions of the protein abruption lead to protein denaturation.Many non-steroidal anti-inflammatory drugs show protection towards protein denaturation (Kpemissi et al., 2023).Hence, this parameter was considered to evaluate the anti-inflammatory effect of the two samples.On analysis, the anti-inflammatory activity was found to be dependent on increasing concentration among all the concentration (Table 2).The data was compared for its effectiveness using the standard antiinflammatory drug, aspirin.In association with the IC 50 in protein denaturation assay of the extracts (chloroform) of peel was found to be effective as it showed a prominent inhibition of protein denaturation in its lower concentration.Even though it was distinguishable from the standard drug.Many other plant extracts have also shown the similar kind of anti-inflammatory effect (Anokwah et al., 2022;Dharmadeva et al., 2018).
Lipoxygenase in the human body is mainly involved in stimulation of the inflammation.It's been linked to many prominent inflammatory disorders (Mashima and Okuyama, 2015).Prostaglandins and leukotrienes are synthesized by lipoxygenase (Cook et al., 1993), and so they are linked to disease development, and their inhibition is regarded as an important step in disease prevention (Hu and Ma, 2018).Hence, inhibition of inflammatory enzyme lipoxygenase is considered for the study.On analysis, extracts (chloroform and ethyl acetate) of both seeds and peel samples showed prominent inhibition of lipoxygenase enzyme in concentration dependent manner.On consideration of IC 50 values of the solvents of two samples highlight the potency of peel than seeds sample.The results from these samples which was found to be evident with the previous similar work (Chung et al., 2009;Lončarić et al., 2021;Muleya et al., 2017).
As the inflammation in interlinked with oxidative stress and damage, the extracts were explored for its anti-oxidant activity (Biswas, 2016).A stable radical was generated using DPPH, and the radical scavenging ability was accessed.On analysis, extracts (chloroform and ethyl acetate) of seeds and the peel samples depicted very significant anti-oxidant activity.Results suggest that the seeds extracts have better antioxidant activity than peel extracts.There are reports on different parts of the plant source extracts are used to study antioxidant activity and found good results (Natukunda et al., 2015;Sandesh et al., 2014).Hence, plant based extracted molecules have advantage over chemically synthesized molecules due their non-toxicity.Anti-fungal activity was tested mainly against, Candida albicans.It is a most common human fungal pathogen.Hence, in our present study, extracts of both seeds and peel samples were tested against C. albicans growth.Among the seeds and peel samples, ethyl acetate extract of seeds showed better zone of inhibition (18 mm ± 0.5) at higher concentration of (90 µg/ml).On the other hand, there is report on silver nanoparticles synthesis carried out using root extract of Furcraea foetida showed a zone of inhibition (16 mm ± 2) at 100 µg/100 µl against C. albicans (Sitrarasi et al., 2022).Similar kind of results also observed previously (Essghaier et al., 2022;38 Liu et al., 2017).However, extracts of peel had no effect on inhibition of Candida albicans.It's been evident from the results that only the seeds had anti-fungal effect against C. albicans.
Furthermore, on screening for anti-bacterial potency between the seeds and peel extract against gram positive bacteria Bacillus cereus and gram negative bacteria E. coli.Majority of the pathogenic and drug resistant bacteria are gram negative because of its unique structure (Breijyeh et al., 2020).Hence, structurally and pathologically distinguishable bacterial species were considered for the inhibition study using plant source extracts as well as nanoparticles (Bagewadi et al., 2019;Essghaier et al., 2022;Rajagopalachar et al., 2022) and compared with the standard drugs.Here, both solvent extracts of peel sample had zero effect on both organisms.Contradictorily, seeds chloroform extract at 90 µg/ml concentration and seeds ethyl acetate extract at all three concentration range showed inhibition of Bacillus cereus growth.Additionally, against gram negative E. coli bacteria only the seeds ethyl acetate extract showed an impressive inhibition whereas the seeds chloroform extract had no effect on the bacteria at all three concentrations.On a note, it was definite that only the seeds ethyl acetate had the significant antibacterial effect.
As the extracts presented impressive bioactivities, the potent extracts from both the samples were explored for their specific phytochemicals using analytical technique like GC-MS.Various researcher studied plant source extraction using different solvents and were further analyzed using chromatographic techniques like GC-MS, UPLC-ESI-Orbitrap-MS, UPLC-ESI-Q-TOF-MS E , HPLC etc., to observe the presence of important biomolecules that can be utilized to treat inflammatory, wound healing, antioxidants, antimicrobial activity, hepatoprotective activity and so on (Aly et al., 2023;Chen et al., 2022a;Djebari et al., 2023;Razali et al., 2012;Wang et al., 2022;Youssef et al., 2023).On analysis with GC-MS, it revealed the presence of versatile group of phytocompounds among all the solvent extracts from both the samples.On keen observation of the GC-MS reports, mainly flavonoids, isovanillic acid, fatty acids, fatty acid methyl esters and phenolic compounds are high among all the extracts.The phytochemicals were differed from solvent to solvent showcasing the unique phytoconstituents presence.

Conclusions
In the current study, the extracts (chloroform and ethyl acetate extract) of seeds and peel samples showed biological activities.Results further revealed that the bioactivities of the samples with different extracts differ based on their phytochemical compositions.Furthermore, peel extracts effectiveness against inflammation was significant.Results are also suggesting that the seeds extracts have significant effectiveness on antioxidant activity.Additionally, on anti-fungal and anti-bacterial potency, seeds extract showed significant inhibition than peel extract.

Fig. 1 .
Fig. 1.Terpenoid, flavonoids and total phenolic contents of the seeds extracts (A) and peel extracts (B) with respect to the standards.Results mentioned are triplicate study and data expressed in mean ± SEM.

Fig. 2 .
Fig. 2. Antifungal activity of seeds extracts (chloroform and ethyl acetate).A) Positive control, B) Seeds chloroform extract, C) Seeds ethyl acetate extract.D) Zone of inhibition of seeds extract and controls.Results mentioned are triplicate study and data expressed in mean ± SEM.

Fig. 3 .
Fig. 3. Antibacterial activity of extracts (chloroform and ethyl acetate) of seeds.A) Seeds ethyl acetate extract (Gram + ve), B) seeds chloroform extract (Gram + ve), C) Seeds ethyl acetate extract (Gram -ve).D) Zone of inhibition of seeds extract and controls.In graph* (B) = Bacillus cereus and (E) = E. coli.Results mentioned are triplicate study and data expressed in mean ± SEM.

Table 1
Phytochemical analysis of extracts (chloroform and ethyl acetate) of seed and peel samples.

Table 3
Anti-oxidant activity of extracts (chloroform and ethyl acetate) of seed and peel samples.

Table 4
GC-MS compounds profile of peel chloroform solvent extract.

Table 5
GC-MS compounds profile of seed chloroform solvent extract.

Table 6
GC-MS compounds profile of peel ethyl acetate solvent extract.

Table 7
GC-MS compounds profile of seed ethyl acetate solvent extract.