Phytochemical analysis, in silico study and toxicity profile of Cycas pectinata Buch.-Ham seed in mice

Abstract Fruit of Cycas pectinata Buch.-Ham has been used as medicine by the local community in some parts of the north eastern state of India. Despite its uses for different purposes, the safety assessment study has not been conducted. Therefore, we have evaluated the acute and the sub-acute toxicity of methanolic extract of C. pectinata fruit (CPFE) in a mice model via oral route of administration. Phytochemicals analysis was carried out by liquid chromatography-mass spectroscopy (LC-MS), nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR). The acute toxicity study was performed at a single dose of 1000, 3000 and 5000 mg/kg and the sub-acute toxicity study at a dose of 100, 300 and 500 mg/kg was administered daily for 28 days. The calculated Lethal dose 50 (LD50) of CPFE was found to be 4000 mg/kg. Both acute and sub-acute studies showed that 5000 mg/kg and 500 mg/kg dose was toxic to the mice. The results of acute toxicity showed CPFE could have a mild toxic effect on the kidney at a dose of 3000 and 5000 mg/kg, as some deteriorated changes in the kidney along with increase creatinine levels were observed. Acute toxicity also showed an increase in white blood cells (WBC) at a dose of 3000 mg/kg. However, sub-acute toxicity studies do not show any detrimental effects on liver, kidney and hematological parameters. Thus, it can be suggested that CPFE at a dose of 100 and 300 mg/kg would be safe for consumption. The phytochemicals analysis by LC-MS, NMR and FTIR showed the presence of 32 major chemical compounds with certain biological activity like anti-neoplastic, antioxidant, and possible modulator of steroid metabolism (cholesterol antagonist and agonist of testosterone 17β-dehydrogenase) as predicted by PASS analysis.


Introduction
Medicinal plants have been used since ancient times. Medicinal plants having curing properties played an important role in traditional medicines and also in home remedies. Due to its preventive and curative properties in several diseases, the use of medicinal plants is immensely increasing each day (Maity et al. 2009). Cycas pectinata Buch.-Ham belonging to the family Cycadaceae are tall, evergreen, palmlike trees. This acrogymnospermae has a branched stem arranged in a crown manner and the leaves range from greygreen to deep green. The different parts of C. pectinata are eaten as vegetables by some local communities. The large, ovoid male cone with elongated and narrow microsporophylls, with long apical spines distinguishes C. pectinata from other species (Khuraijam and Singh 2015). The seeds are round to oval with a thick fibrous layer and a smooth sclerotesta. It has long megasporophylls with glabrous and deeply pectinate with soft lateral spines (Khuraijam and Singh 2015). In addition, C. pectinata are famous for traditional medicines in many cultures. Traditionally it is used in hair swellings (Nair and Van Staden 2012), stomach aches, and ulcers while some local communities in Meghalaya and Assam eat to enhance sexual male potency (Khuraijam and Singh 2015).
It has been shown that the extract of plants either pure or crude has enormous potential for human use as a medicine and food (Cos et al. 2006). It has been reported by WHO that 80% of the world's population uses traditional medicine for primary healthcare needs. The isolation and characterization of a plant's important constituents are necessary to unravel its specific biological activity. The phytochemical analysis of C. pectinata fruit and leaves has been performed and it revealed the presence of flavonoids, alkaloids, terpenoids, glycosides, carbohydrates, saponins, coumarin, amino acids, resins, gums and mucilage, phytosterols, sterols, cardiac glycosides and steroids (Laishram et al. 2015, Tareq et al. 2020. Toxicological studies are very essential in finding a new drug for clinical use and it provides information on toxic doses, therapeutic indices of drugs and xenobiotics (Aneela et al. 2011). Pharmacology drugs and food supplements cannot be established without doing clinical trials and toxicity studies. Therefore, toxicity studies in animals can determine the safety of medicinal plants and further benefits for development into pharmacological products (Al-fifi et al. 2018). Acute toxicity studies are required to determine the immediate toxic effect of a drug, where single doses of drug are given in large quantities. Acute toxicity study of C. pectinata leaves has been reported recently. However, no work has been carried out on sub-acute toxicity of C. pectinata plant. A subacute toxicity study needs to be carried out to find the safest dose of the drug in long term treatment where the repeated dose of drugs are given for 28 days (Aneela et al. 2011).
It has been shown that various traditionally used plants for the treatment of cancer, diabetes, and infertility contains high radical scavenging activity due to the presence of various phytochemicals and help to counter oxidative stress (Greenwell and Rahman 2015, Nasri et al. 2015, Ngaha Njila et al. 2019. Reactive oxygen species (ROS) have the potential to cause a number of destructive effects on human health and increase oxidative stress due to increase ROS has been the core of pathogenesis. ROS like hydrogen peroxide; act as key signaling molecules, while others are harmful to biological systems (Hancock et al. 2001).
The great potential of C. pectinata as herbal medicine can be suggested by various phytochemicals present in the other species of Cycas (Prakash et al. 2021) and it has been shown that its phytochemicals can ameliorate radiation induced oxidative stress in the rat (Ismail et al. 2020). The knowledge we obtained from the local people and traditional healers about the C. pectinata also suggested that fruits of C. pectinata are consumed for medicinal uses and food supplements, thus, it is important to evaluate the safety of the plant and its constituents. However, there are insufficient data demonstrating the safety of the crude extract from the C. pectinata fruit. To the best of our knowledge, no work has reported on C. pectinata fruit extract and its pharmacological activities.
Therefore, we investigated the phytochemicals composition by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR) and we also investigated the toxicity of C. pectinata fruit extract using in vivo experimental models. To the best of our knowledge, this will be the first report on the detailed phytochemicals analysis by LC-MS, NMR, and FTIR of C. pectinata fruit extract along with its acute and sub-acute toxicity assessment in a mice model.

Plant material
The unripe fruit of C. pectinata was collected from Meghalaya, Garo Hills, India in August 2019. A voucher specimen (BSI/ ERC/Tech/2020/1309) was deposited at the herbarium in Botanical Survey of India (BSI), Shillong, Meghalaya and accession number 96583 was obtained from BSI, Shillong.

Preparation of plant extract
The fruit of C. pectinate was collected in a fresh condition and chopped into smaller pieces and dried for 2 weeks at room temperature. The dried fruit was grounded into a coarse powder using a mechanical grinder. About 100 g of C. pectinata fruit powder was soaked in 500 ml methanol on a beaker for three days by occasional shaking and stirring at room temperature. The crude part of the materials was removed by using Whatman filter paper (Grade 1 circles -150 mm). The obtained filtrate was evaporated to yield a viscous mass.
In vitro anti-oxidant 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was performed using the standard protocol by Leong and Shui (2002) with minor modifications. Serial dilutions of plant extract and ascorbic acid were assembled at concentrations of 2000, 1600, 800, 400, 200 and 100 lg/ml. The tests of all concentrations were performed in triplicate. Briefly, 0.5 ml of C. pectinata extracts (1-400 mg/ml) were mixed along with 1 ml of methanol solution of 0.1 M DPPH (1.97 mg in 50 ml of methanol) followed by 30 min incubation in the dark. The absorbance of the solution was achieved at 523 nm by using a UV-visible spectrophotometer. The extract antioxidant activity was expressed as half-maximal inhibitory concentration (IC50), the concentration (mg/ml) of extract that inhibits the formation of DPPH radicals by 50%. Ascorbic acid (ASA) was used as the standard. The percentage scavenging activity was calculated using the following formula: where Ao is the absorbance of the control (solution containing all the reagents except the extracts) and A sample is the absorbance of the solution containing the extract.
Functional groups were identified by Fourier transform infrared spectrophotometer (FTIR) analysis. The methanolic extract was used for FTIR analysis (Shimadzu, Kyoto, Japan, IR Affinity1), with a scan range from 400 to 4000 cm À1 with a resolution of 4 cm À1 .
1 H NMR (300 MHz) spectra of methanolic extract of fruit were recorded on JEOL AL300 FTNMR spectrometer (JEOL Ltd., Tokyo, Japan) using TMS as an internal reference, and chemical shift values are expressed in d, ppm units.

Prediction of activity spectra for substances (PASS)
PASS is online-based software that can be used to evaluate the possible binding affinity of organic drug-like molecules based on the structure of organic compounds. All phytochemicals were subjected to PASS analysis (Filimonov et al. 2014). It employs multilevel neighborhood of atom (MNA) descriptors and a Bayesian algorithm to predict the activity of organic compounds based on the structure-activity relationship (SAR) model of a large number of pharmaceutical compounds.

Experimental animals
Male adult Swiss albino mice (32.28 ± 0.52g) were used for the acute and the sub-acute oral toxicology test. Mice were kept in an aerated polypropylene cage with ad libitum access to food and water at 25 ± 3 C temperature under a 12/12 h light/dark cycle. All experimental procedures were conducted in compliance with the protocols approved by the Mizoram University Institutional Animal Ethical Committee (approval no. MZU/IAEC/2020/06).

Acute toxicity
Male adult Swiss albino mice were used to test for acute toxicity of C. pectinata fruit extract (CPFE) according to the method described by the Organization of Economic Cooperation and Development 423 guidelines (OECD). The animals were randomly divided into four groups (n ¼ 6 per group). Each group consist of six animals and a single dose of 1000, 3000, and 5000 mg/kg of CPFE that dissolved in 10% dimethyl sulfoxide (DMSO) was administered orally to the overnight fasted mice. For control animals, water and 10% dimethyl sulfoxide (DMSO) were mixed and administered orally as a vehicle. The animals were monitored individually for signs of acute toxicity for the first 4 h, and then 8 h and then once a day for 14 days for delayed toxicity signs. All morphological observations like changes in skin and fur color, movement patterns, convulsions, sleep, diarrhea, coma, salivation, Lethargy, and mortality were recorded. The initial body weight was taken before the administration of CPFE and at the end of the experiment.

Sub-acute toxicity
Male Swiss albino mice were randomly divided into four groups (n ¼ 6 per group) to test for sub-acute toxicity of CPFE. Three groups were orally administered with CPFE extract that dissolved in distilled water at a concentration of 100, 300, and 500 mg/kg which is one-tenth of the acute toxicity dose for 28 consecutive days. One group was administered with distilled water which served as a control for 28 consecutive days. All general toxicity signs of animals were monitored throughout the study. The initial body weight of all the experiment animals has been recorded before CPFE administration and at the end of each week.

Hematological analysis
Hematological analyses were performed at the end of the experiment in all surviving animals that is on the 15th day for acute toxicity and the 29th day for sub-acute toxicity. Animals were sacrificed and blood was collected in tubes. Hematological parameters including Red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were determined.

Serum biochemistry analysis
The biochemical assay was done in all surviving animals at the end of the experiment. The blood was collected in the tubes and centrifuge at 3500 rpm for 30 min. The serum from each sample was collected and stored at À80 C until assayed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, direct bilirubin, total protein, and creatinine activity were determined by using a diagnostic kit (Euro Diagnostic System).

Relative organs weight and histopathological observations
After sacrificing the animals on the 15th day (acute toxicity) and 29th day (sub-acute), the vital organs such as liver, kidney, lungs, heart, and spleen were collected for histological studies. The fats from collected organs were removed and blotted with clean tissue paper and then weighed. The relative organ's weight (ROW) was calculated and recorded in proportion to the bodyweight according to the following equation: ROW ¼ ðAbsolute organ weightÞ=ðBody weight at sacrificeÞ Â 100 The collected vital organs (liver, kidney, lungs, heart, and spleen) of both acute and sub-acute were subjected to histological evaluation. After collection of organs, they were put in Bouin's fixative for 24 h and then transferred into 70% ethanol until it was routinely processed. The organs were embedded in paraffin wax and sections of 7 lm thicknesses were cut in Leica rotary microtome (RM2125 RTS). The sections were spread on glass slides, deparaffinized with xylene, and stained with hematoxylin and eosin for general histology. Histopathology of organs was examined and evaluated under a Nikon microscope (Model E200, Nikon, Tokyo, Japan) and photographs were taken for analysis (Bancroft and Gamble 2002).

Statistical analysis
All results were expressed and presented as mean (±) standard error of the mean (SEM). Statistical significance was set at p < 0.05. All data were analyzed by using one-way ANOVA, to compare means and significant differences were further analyzed by Tukey's multiple comparisons using GraphPad prism (version 9, GraphPad Software, La Jolla, CA).
FTIR spectroscopy study of methanolic extract was done to ascertain the functional groups in extracted compounds ( Figure 1). The broad characteristic absorption band at the region 3394-3286 cm À1 can be attributed to the presence of -OH and -NH functional groups. Further, the intense absorption band at 2924 cm À1 and 2854 cm À1 corresponds to C-H stretching. The observed bands at 1712 cm À1 and 1643 cm À1 can be attributed to the presence of C ¼ O carbonyl groups or broadly the absorption band at 1720-1640 signifies the likely presence of ketonic, ester, and carboxylic groups. Moreover, the absorption band at 1040 cm À1 can be attributed to the C-O stretching of ether or ester groups. The medium absorption band at 1604 corresponds to C ¼ C stretching. The (-CH-O-CH 2 ) stretching and CH 2 O-CH bending frequencies at 3055 cm À1 and 941 cm À1 , respectively, also suggests the likely presence of epoxy-containing compounds.
The absorption band at 1296 cm À1 may be accounted for the stretching vibrations of P ¼ O of phosphodiester containing compounds. The crude product of methanolic extract was also subjected to 1 H NMR spectroscopy for structural analysis. It reveals that aromatic protons, carboxylic acids, and aldehydic hydrogens are either absent or present in negligible amounts. The NMR spectrum further indicates the presence of different alkyls, vinyl, alkynes, R-OH, R-NH 2 , -OCH 3 , etc. groups. The peaks at d 0.8-1.45, 1.2-1.47, 1.4-2.69, 3.2-4.8 suggest the presence of -CH 3 , -CH2-, -C ¼ C-CH 3 , -OCH 3 type hydrogens. Moreover, the presence of peaks at d 1.4-2.69 and 4.2-5.3 which corresponds to -C ¼ C-CH 3 and -C ¼ CH-, respectively, indicates the presence of polyisoprene groups. The peaks at d 1.0-5.2 also correspond to the presence of R-OH and R-NH 2 hydrogens.
Measurement of anti-radical activity by DPPH test 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is one of the first free radicals used to study antiradical activity, therefore, we have performed the DPPH scavenging of fruit extract of C. pectinata.
The methanolic extract of C. pectinata fruit (CPFE) exhibited DPPH scavenging activities from concentration 100 mg/ ml to 2000 mg/ml (Figure 2). The DPPH scavenging activities were observed by the discoloration of DPPH by the extract. CPFE showed a dose-dependent significant (p < 0.05) increase in DPPH radicals scavenging activity from the concentration of 400 mg/ml to 2000 mg/ml, whereas the concentration of 200 mg/ml and 400 mg/ml did not show significant (p > 0.05) change. The IC 50 of CPFE was found to be 1612.67 ± 52.44 mg/ml.

Acute toxicity test
The acute toxicity of C. pectinata fruit extract (CPFE) revealed that oral administration of the extract at a single dose of different concentrations (1000 mg/kg, 3000 mg/kg, and 5000 mg/kg), the highest concentration 5000 mg/kg showed signs of mortality in treated animals during the 14 days. For every group, six male mice were taken and in the 5000 mg/ kg groups only one male mouse survived and five mice died within 3 days of treatment. Therefore, data could not be collected from the 5000 mg/kg groups, however, the histopathological analysis of kidney and liver was performed.
The other doses of CPFE 1000 mg/kg and 3000 mg/kg did not show any sign of mortality. The biochemical parameters AST, ALT, ALP, total protein, total bilirubin, and direct bilirubin did not show any significant change compared to the control animals (Figure 3(A-F)). However, the creatinine levels were found to be higher in the 1000 mg/kg and 3000 mg/kg groups compared to the control, but it was not significant ( Figure 3G). ALT and ALP also showed a decrease in 1000 mg/kg and 3000 mg/kg groups compared to the control, but it was not significant (Figure 3(B,C)). The organ weight of liver, kidney, heart, lung, and spleen from 1000 mg/kg and 3000 mg/kg groups did not show significant change compared to the control (Table 2).
We have also measured various hematological parameters such as RBC, WBC, hemoglobin, hematocrit, MCV, MCH, and MCHC. These parameters also did not show any significant change compared to the control, except the WBC, which showed a significant increase in the 3000 mg/kg group compared to the control and 1000 mg/kg groups (Table 3).

Histopathology of liver and kidney after acute toxicity
The histopathological analysis of the liver from control, 1000 mg/kg, and 3000 mg/kg groups did not show the  (mg/dl); (E) total bilirubin (mg/dl); (F) direct bilirubin (mg/dl); (G) creatinine (lmol/l). All data are expressed in mean ± SEM (n ¼ 6). One-way ANOVA followed by Tukey's multiple comparison test, p > 0.05; not significantly different from control group was observed. Values are expressed as mean ± SEM, n ¼ 6. Ã p < 0.05 (one-way ANOVA followed by Tukey's multiple comparison). deteriorated change in the histology (Figure 4(A-C)). The liver of a single surviving animal in the 5000 mg/kg group also showed no pronounced changes (Figure 4(D)). The kidney histology revealed pathological changes in the form of diminished, distorted glomeruli, and mild necrosis, in 3000 mg/kg and 5000 mg/kg groups, however, the kidney from control animals and animals from 1000 mg/kg groups showed normal histology ( Figure 5(A-D)).

Sub-acute toxicity test
The three different doses of CPFE were selected for the subacute toxicity. The oral administration of CPFE at a dose of 500 mg/kg for 28 days caused the toxicity. Out of six mice, only one mouse survived. Therefore, biochemical parameters could not be performed. However, we have subjected the tissues for histopathological analysis. Oral doses of CPFE at 100 mg/kg and 300 mg/kg for 28 days did not cause any sign of adverse effects and no deaths were recorded. The biochemical parameters such as ALT, total protein, total bilirubin, and creatinine did not show any significant variation among the control, 100 mg/kg and 300 mg/kg (Figure 6(C,D,E and G)) in the result section of sub-acute toxicity test. However, ALP levels and direct bilirubin showed a significant decrease in the 100 mg/kg and 300 mg/kg compared to the control (Figure 6(A,F)). The AST levels were found to be significantly lower in the 300 mg/kg group compared to the control and 100 mg/kg groups (Figure 6(B)). The different organs showed no change in their weight after the treatment (Table 4).
The various hematological parameters such as RBC, WBC, hemoglobin, hematocrit, MCV, MCH, and MCHC also did not show the significant change compared to the control ( Table 5).

Histopathology of liver and kidney after subacute toxicity
The histopathological study of the liver (Figure 7(A-D)) and kidney (Figure 8(A-D)) exhibited the normal histoarchitecture without any sign of toxicity. Even the histology of a single survived mouse in 500 mg/kg groups showed normal histoarchitecture.

PASS prediction of the compounds identified in Cycas extract by LC-MS
The outputs can be interpreted based on the estimated probability of Pa and Pi for each type of activity, where p a is the 'probability to be active' and p i is the 'probability to be inactive.' In the present work, the compounds that have p a > 0.7 (70%) have been considered to possess potent biological activity. Probable activity (p a ) and inactivity (p i ) values of the compounds for the activities as antineoplastic, cytochrome P450 substrate, a b-adrenergic receptor kinase inhibitor, antioxidant, cholesterol antagonist, and testosterone 17b-dehydrogenase inhibitors are given in Table 6.

Discussion
The present study investigated the safety assessment of C. pectinata fruit in relation to acute and sub-acute toxicity in the mice model along with the phytochemical characterization by LC-MS, NMR, and FTIR analysis. There are two reports on the C. pectinata, which described its ethno medicinal uses (Laishram et al. 2015, Tareq et al. 2020. The study by Tareq et al. (2020) reported that methanolic extract of C. pectinata leaves showed antioxidant, anti-inflammatory, and analgesic properties. As the previous study on fruit extract of C. pectinata showed the antidiabetic property in the rat model, thus, it is evident that C. pectinata has great medicinal uses. Despite these two studies on the pharmacological effects of C. pectinata fruit and leaves, there is no detailed investigation on its possible toxic impact, fruit in particular. To the best of our knowledge, no study has been reported the acute and the sub-acute toxicity of C. pectinata in any animal model. Therefore, the present study focused on the evaluation of the acute and the sub-acute toxicity of CPFE on mice models.
Moreover, the phytochemicals constitutes of the C. pectinata fruit have also been reported and it was shown that anti-diabetic properties of C. pectinata fruit extract (CPFE) were attributed to amentoflavone and 2,3-dihydroamentoflavone (Laishram et al. 2015). The present study characterized major phytochemicals constituents of C. pectinata fruit in the methanolic extract by LC-MS showed the presence of 32 major phytochemicals (Table 1). FTIR analysis of the methanolic extract revealed the presence of -OH, -NH, C¼O carbonyl, ketonic, ester, alkene, alkyne, epoxy, and carboxylic groups in the extract. Furthermore, the NMR spectrum further indicated the presence of different alkyls, alkene, alkynes, R-OH, R-NH 2 , -OCH 3 , etc. groups and aromatic protons, carboxylic acids, and aldehydichydrogens were likely to be negligible in the extract. LC-MS, NMR, and FTIR data together confirm the presence of steroids, carotenoid, tetraterpenoids, fatty acid esters, triglycerides, carbohydrates, and alkaloids. A total of 25 phytochemicals belonging to these groups have also been reported in the leaves of C. pectinata by GC-MS analysis (Tareq et al. 2020). There are reports of phytochemicals constitution of different Cycas species. The GC-MS analysis of ethanol extract of the sarcotesta layer of Cycas revoluta revealed seven compounds like o-cymene,4hydroxy-4-methyl-2-pentanone, methoxy-phenyl-oxime, tetradecanoic acid,10,13-dimethyl-, methyl ester; 9,12-octadecadienoic acid, methyl ester; 9-octadecenoic acid, methyl ester; and methyl stearate (Prakash et al. 2021). On the other hand, the different parts of Cycas Sancti-lasallei have been shown to contain squalene b-sitosterol, stigmasterol, triglycerides, phytyl fatty acid esters, and b-sitosterol fatty acid esters (Ng et al. 2015). In the Cycas armstrongii, various compounds have also been reported, such as naringenin, dihydroamentoflavone, 2,3-dihydrohinokiflavone, amentoflavone, 2,3-dihydrobilobetin, isoginkgetin, pruning, naringin, vanillic acid, p-coumaric acid, b-sitosterol, b-sitosterol glucoside, 3,7,9,11-tetramethyl heptadecanoic acid, and these compounds ameliorated the radiation-induced oxidative stress in a rat model (Ismail et al. 2020). As the previous study by Laishram et al. (2015) showed the anti-diabetic potential of CPFE, and the two compounds, amentoflavone and 2,3-dihydroamentoflavone have been attributed to its activity in the ethyl acetate sub-fraction. The difference in the chemical composition of C. pectinata with other species of Cycas could be due to species-dependent variation of phytochemical compounds and it has been suggested that the chemical composition of plants is highly variable and same species may differ significantly in their chemical composition (Koutsoukis et al. 2019). The difference in our results from the findings of Laishram et al. (2015) may also be attributed to the method of extraction, as we have used crude methanolic extract for the phytochemicals analysis; however, in the previous study ethyl acetate fraction were used. Furthermore, it can be suggested that either fruit or leaves of C. pectinata contain a variety of phytochemical compounds with certain biological activities. To ascertain the possible biological activities in the CPFE extract, we have used the PASS prediction analysis. The results of PASS prediction showed that all the compounds except 7, 23-25, and 28-31 have 0.76<p a < 0.965 for antineoplastic indicating good anti-neoplastic activity of the extracted crude compounds. The compounds 1-3, 6, 7, 10-13, 15-23, and 26 exhibited 0.72<p a < 0.977 in cytochrome P450 substrates and compounds 1-3, 7, 11-13, 15-18, 22, 23, and 26, 28, 31 have 0.71<p a < 0.951 in b-adrenergic receptor kinase. The total protein (mg/dl); (E) total bilirubin (mg/dl); (F) direct bilirubin (mg/dl); (G) creatinine (lmol/L). All data are expressed in mean ± SEM (n ¼ 6). One-way ANOVA followed by Tukey's multiple comparison test. Ã p < 0.05; significantly different from control group. Values are expressed as mean ± SEM, n ¼ 6. p > 0.05 and data are not statistically significant, one way ANOVA followed by Tukey's multiple comparison. Values are expressed as mean ± SEM, n ¼ 6. p > 0.05 and data are not statistically significant, one way ANOVA followed by Tukey's multiple comparison.
compounds that have more than 70% p a for antioxidant activity are 1-3, 11, 13, 16, 18, 20, and 22 with 0.72<p a < 0.945. Our DPPH assay supported the antioxidant potential of CPFE like methanolic extract of C. pectinata (Tareq et al. 2020). However, only the steroidal alkaloids 6 and 7 and triterpenoid 8 exhibited more than 70% p a as cholesterol antagonists with 0.75<p a < 0.884. Moreover, it can be suggested that crude compounds are less active against testosterone 17b-dehydrogenase except for compounds 12, 13, 18, and 23 that displayed 0.74<p a < 0.90. However, the activities predicted are probable and only further experimental validation can confirm it. The predicted biological activities of various phytochemicals also open an avenue on the antineoplastic activity of C. pectinata fruit. However, the possible impact of phytochemicals on the antioxidant potential, as a cholesterol antagonists and as agonist on testosterone 17b-dehydrogenase might be involved in the regulation of male reproduction as it is claimed by local healers. As our aim was not to evaluate its effect on male reproduction, thus, further study is needed to support the claims of C. pectinata fruit as aphrodisiac drug. Since we aimed to find the safety assessment of C. pectinata fruit extract, in the mice model, therefore, we have evaluated the various parameters of the acute and the sub-acute toxicity in the present study. Moreover, the acute toxicity study of the present work showed the oral administration of a single dose of CPFE (from 1000 mg/kg to 3000 mg/kg) did not show any observable changes in terms of mortality, or behavioral or bodyweight changes, however, in the 5000 mg/ kg group, only one animal survived and thus it can be suggested that higher dose is very toxic to the mice. The OECD 423 suggests that if all the animals survived until their scheduled euthanasia, then extract has low toxicity and should be included in category 5 (Brondani et al. 2017), thus, the dose up to 3000 mg/kg was safe and exhibited very low toxicity. The LD50 of CPFE was found to be 4000 mg/kg, which also showed the feasibility of extract to fall in category 5. The previous studies also showed that the acute and the sub-acute toxicity evaluation of medicinal plants is prerequisite conditions for any medicinal for wider use in human (Brondani et al. 2017, Villas Boas et al. 2018, Porwal et al. 2020. Since the treatment of many conditions may require the longer use of plant extract and the information based on the local healer, it was revealed the multiple doses of fruit of C. pectinata can be given for improving male fertility. Moreover, as leaves of C. pectinata have been shown to exhibit antioxidant, anti-inflammatory and analgesic properties (Tareq et al. 2020), and fruit contain anti-diabetic properties, whether herbal or non-herbal formulation must require safety assessment (Zhang et al. 2015). Therefore, we have also evaluated the sub-acute toxicity of CPFE in a mice model. The subacute toxicity study on the CPFE at doses of 100 and 300 mg/kg did not exhibit any change in animal behavior, body weight and mortality. Thus 300 mg/kg can be considered no observed adverse effect level (NOAEL) because it is the highest dose at which the response is not statistically   Table 6. PASS prediction of the compounds identified in C. pectinata fruit extract by LC-MS, where p a refers to probability 'to be active' and p i refers to probability 'to be inactive'.

Antineoplastic
Cytochrome P450  Despite no change was observed in the various organs weight after acute and sub-acute toxicity, we have monitored the liver function test, because liver is one of the vital organs, which is a target of xenobiotics (Bariweni et al. 2018). The various parameters such as AST, ALT, ALP, and bilirubin levels were measured and data showed that ALT, ALP, and direct bilirubin decreased in the 1000 and 3000 mg/kg groups compared to the control, but it was statistically non-significant. For the sub-acute toxicity ALP, ALT, AST, and bilirubin were also statistically non-significant in the 100 mg/kg and 300 mg/kg compared to the control. Despite the non-significant in the ALT in the sub-acute toxicity at 300 mg/kg dose compared to the control, it was slightly more than the normal range, it may be due to very mild abnormal liver functions. The previous study also showed that AST, ALT, and bilirubin-sensitive markers of hepatocellular toxicity and its increased activity indicate liver damage (Ramaiah 2011). The histopathological examination of the sub-acute study showed that the extract did not cause any change in the liver histology, which suggest that CPFE may be safe and non-toxic for consumption. The use of this parameter is well reported for liver function in laboratory animals as well as humans The kidney histological analysis after acute toxicity showed the deteriorated changes along with increase serum creatinine levels (although it is a non-significant increase). Creatinine levels have been considered as important markers for kidney dysfunction (Mukinda andEagles 2010, Chebaibi et al. 2019). Thus, these results suggested possible kidney toxicity of CPFE at a single higher dose, whereas a dose of 100 mg/kg and 300 mg/kg for 28 days did not show any change in creatinine levels and kidney histology, therefore, it may be suggested that a small dose would be safer for chronic use of CPFE. It has also been reported that organs weight may be very important for toxicity assessment for any chemicals (Ugwah-Oguejiofor et al. 2019). The organs weight after the treatment either increase due to hypertrophy or decrease due to necrosis or degeneration (Teo et al. 2002). Our results showed the vital organs did not cause any change in their weight after acute and sub-acute toxicity assessment. Except for an increase in WBC count in 3000 mg/ kg of acute toxicity study, all other hematological parameters were also unaffected after an acute and sub-acute dose of CPFE. It has been suggested that an increase in WBC after treatment of herbal extract may show its immune modulation effect (Tousson et al. 2011). However, further study would be required to support this hypothesis in the case of CPFE.

Conclusion
The present study gathered worthy information on both the acute and the sub-acute toxicity of CPFE in a mice model. CPFE was found to be practically less toxic in both acute and sub-acute oral administration in mice in relation of liver, kidney, and hematological parameters. In addition, LC-MS, NMR, and FTIR analysis of methanolic fruit extract of C. pectinata have identified more than 30 major phytochemicals constituents. The PASS prediction also suggested some biological functions of extract such as antioxidant, anti-neoplastic activity and moreover, the cholesterol antagonist activity and agonist of testosterone 17b-dehydrogenase, may further suggest its role in steroid biosynthetic pathways. However, further study would be required to confirm this role of CPFE. Thus, these observations suggest that CPFE may be nontoxic to vital organs and can also be used at a dose of 100-400 mg/kg for curative purposes.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.