Metabolic Profiling and In Vitro Assessment of the Biological Activities of the Ethyl Acetate Extract of Penicillium chrysogenum “Endozoic of Cliona sp. Marine Sponge” from the Red Sea (Egypt)

Marine sponge-derived endozoic fungi have been gaining increasing importance as promising sources of numerous and unique bioactive compounds. This study investigates the phytochemical profile and biological activities of the ethyl acetate extract of Penicillium chrysogenum derived from Cliona sp. sponge. Thirty-six compounds were tentatively identified from P. chrysogenum ethyl acetate extract along with the kojic acid (KA) isolation. The UPLC-ESI-MS/MS positive ionization mode was used to analyze and identify the extract constituents while 1D and 2D NMR spectroscopy were used for kojic acid (KA) structure confirmation. The antimicrobial, antioxidant, and cytotoxic activities were assessed in vitro. Both the extract and kojic acid showed potent antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa with MIC 250 ± 0.82 µg/mL. Interestingly, the extract showed strong antifungal activity against Candida albicans and Cryptococcus neoformans with MIC 93.75 ± 0.55 and 19.53 ± 0.48 µg/mL, respectively. Furthermore, KA showed the same potency against Fusarium oxysporum and Cryptococcus neoformans with MIC 39.06 ± 0.85 and 39.06 ± 0.98 µg/mL, respectively. Ultimately, KA showed strong antioxidant activity with IC50 33.7 ± 0.8 µg/mL. Moreover, the extract and KA showed strong cytotoxic activity against colon carcinoma (with IC50 22.6 ± 0.8 and 23.4 ± 1.4 µg/mL, respectively) and human larynx carcinoma (with equal IC50 30.8 ± 1.3 and ± 2.1 µg/mL, respectively), respectively. The current study represents the first insights into the phytochemical profile and biological properties of P. chrysoenum ethyl acetate extract, which could be a promising source of valuable secondary metabolites with potent biological potentials.


Introduction
The oceans are home to a tremendous diversity of species and their inhabitants produce a wealth of natural products [1] that are intertwined in intra-specific and interspecific chemical communication [2]. Marine sponges, which are also known as microbial fermenters, are outstanding sources of highly diverse microbial communities, including new fungal species. The true producers of natural compounds from marine invertebrates are symbiotic microorganisms, which account for approximately 40 percent of the sponge's

Isolation and Screening of Endozoic Fungi with Potent Antimicrobial Activity from the Medicinal Marine Sponge
Two marine sponges, namely, Cliona sp. and Hymedesmia sp., were used as the source of endozoic fungi. In total, twelve fungal isolates were recovered from the marine sponges; four isolates were derived from Cliona sp. (Aspergillus orchaceous, Aspergillus terreus, Aspergillus niger, and Penicillium Chrysogenum), and eight isolates were recovered from Hymedesmia sp. (Aspergillus terreus, Aspergillus awamori, Aspergillus niger, Aspergillus oryzae, Alternaria alternata, Trichoderma viridae, Penicillium lilacinum, and Aspergillus astus) using malt yeast agar and malt extract agar media (Table 1). These fungal isolates were identified based on their species level according to the universal morphological keys [15][16][17]. The frequency of the genus Aspergillus is about 54 percent, whereas the frequency of the genus Penicillium is about 30 percent among the recovered endozoic fungi. The recovered isolates were assessed for their antimicrobial activities by inducing their growth on Wickerham's broth medium, incubating them at standard conditions, and extracting with ethyl acetate. The agar dilution method was used to evaluate the antimicrobial activity based on the inhibition zone and minimum inhibitory concentration (MIC) determination. De Oliveira et al. [18] and Sartoratto et al. [19] classified the antimicrobial activity of the extract based on the MIC values as follows: 50-500 µg/mL = strong activity; 600-1500 µg/mL = moderate activity; and >1500 µg/mL = weak activity or inactivity. Consequently, from the screening profile as illustrated in Table 1, the highest antimicrobial activity was reported for the Penicillium chrysogenum (C6) extract that showed a strong antibacterial activity against Staphylococcus aureus, with MIC value equivalent to 250 ± 0.82 µg/mL, and superior fungicidal activity against Candida albicans, with MIC 93.75 ± 0.78 µg/mL. Furthermore, Aspergillus terreus (H2) from Hymedesmia sp. is the second fungal isolate, which revealed potent antibacterial activity against Staphylococcus aureus and strong antifungal activity against Candida albicans, with MIC 250 ± 1.5 µg/mL and 187.5 ± 0.95 µg/mL, respectively. Interestingly, Aspergillus oryzae (H5) from Hymedesmia sp. exhibited strong antibacterial activity against highly resistant Gram-negative bacteria Pseudomonas aeruginosa and moderate fungicidal activity against Candida albicans, with MIC values equivalent to 250 ± 1.3 µg/mL and 750 ± 0.88 µg/mL, respectively. The remaining fungal isolates from both tested marine sponges (Table 1) displayed weak to negative antimicrobial activities against the tested organisms. IZ: Inhibition zone (mm) diameter; MIC: Minimum Inhibitory Concentration (µg/mL). MIC: 50-500 µg/mL (strong activity), 600-1500 µg/mL (moderate activity), >1500 µg/mL (weak activity) [18,19].

Characterization of Penicillium Chrysogenum Ethyl Acetate Extract (PC) and the Isolated Kojic Acid (KA)
Recently, marine-derived endophytic fungi investigation has drawn the attention of many researchers; as such, fungi are considered to comprise an unexplored repertoire of unprecedented bioactive compounds. The rationale behind the future use of endophytic fungi to produce biologically active secondary metabolites arises from their rapid growth on culture medium, bulk biomass production accessibility, and independence from environmental variables. Penicillium is one of the most isolated fungi from the marine environment. It has been proven that the Penicillium species comprise valuable biological resources, as they produce important enzymes and secondary metabolites. Penicillium is a decomposer of various marine organisms and play an essential role in pollutant degradation and nutrient recycling [20]. The recovered P. chrysogenum fungal isolate from Cliona sp. (GenBank accession no. OL597937.1) was screened for its phytochemical constituents, using UPLC-ESI-MS/MS analysis, and its biological activities.  Figure S1 [21]. The IR υ max (KBr) spectrum showed absorption bands at 3142, 2923, and 1656 cm −1 , thereby indicating the presence of hydroxyl, alkene, and carbonyl groups, respectively ( Figure S2). The 1 HNMR (400 MHz, DMSO-d 6 ) spectrum showed two protons at δ H 6.33 (1H, s, H-3) and 8.02 (1H, s, H-6) ppm, corresponding to vinylic protons [22] ( Figure S3 and Table S1). The 13 CNMR (100 MHz, DMSO-d 6 ) spectrum ( Figure S4) showed three quaternary carbons at δ c 173.9, 168.1, and 139.3, which corresponded to C-4, C-2, and C-5, respectively, in addition to two olefinic carbons (C-6 and C-3) at δ c 145.7 and 109.8, respectively, and one methylene carbon (C-7) (δc 59.5). The 1 H and 13 C NMR data in combination with the 1 H-13 C HSQC and 1 H-13 C HMBC analyses confirmed the presence of a pyrone ring [22] (Figures S5-S7). All these data are in good agreement with those previously reported [22,23].

Structural Identification of Constituents of P. chrysogenum Ethyl Acetate Extract by UPLC-ESI-MS/MS
In the current study, UPLC-ESI-MS/MS in the positive ion mode was used to analyze the ethyl acetate extract of P. chrysogenum (PC) for the first time. The compounds' identification was based on their MS 2 data, which was provided by the precursor ion mass and their fragments, neutral mass loss, and comparison with the available literature. The compounds were arranged based on their retention time (R t ), as shown in Table 2 and Figure 1. When using the positive mode in LC-MS analyses, the pseudo molecular ion [M + H] + was not the common MS signal, water losses were very common ([M + H-H 2 O] + ), and alkali adducts (mainly [M + Na] + ) also regularly existed. In total, 36 compounds were tentatively identified in the P. chrysogenum extract. Table 2 and Figure 1 indicate the tentatively identified compounds along with their retention times, experimental m/z in positive ionization mode, and MS/MS fragments. decomposer of various marine organisms and play an essential role in pollutant degradation and nutrient recycling [20]. The recovered P. chrysogenum fungal isolate from Cliona sp. (GenBank accession no. OL597937.1) was screened for its phytochemical constituents, using UPLC-ESI-MS/MS analysis, and its biological activities.  Figure S1 [21]. The IR υmax (KBr) spectrum showed absorption bands at 3142, 2923, and 1656 cm −1 , thereby indicating the presence of hydroxyl, alkene, and carbonyl groups, respectively ( Figure S2). The 1 HNMR (400 MHz, DMSO-d6) spectrum showed two protons at δH 6.33 (1H, s, H-3) and 8.02 (1H, s, H-6) ppm, corresponding to vinylic protons [22] ( Figure S3 and Table  S1). The 13 CNMR (100 MHz, DMSO-d6) spectrum ( Figure S4) showed three quaternary carbons at δc 173.9, 168.1, and 139.3, which corresponded to C-4, C-2, and C-5, respectively, in addition to two olefinic carbons (C-6 and C-3) at δc 145.7 and 109.8, respectively, and one methylene carbon (C-7) (δc 59.5). The 1 H and 13 C NMR data in combination with the 1 H-13 C HSQC and 1 H-13 C HMBC analyses confirmed the presence of a pyrone ring [22] ( Figures  S5-S7). All these data are in good agreement with those previously reported [22,23].

Structural Identification of Constituents of P. chrysogenum Ethyl Acetate Extract by UPLC-ESI-MS/MS
In the current study, UPLC-ESI-MS/MS in the positive ion mode was used to analyze the ethyl acetate extract of P. chrysogenum (PC) for the first time. The compounds' identification was based on their MS 2 data, which was provided by the precursor ion mass and their fragments, neutral mass loss, and comparison with the available literature. The compounds were arranged based on their retention time (Rt), as shown in Table 2 and Figure  1. When using the positive mode in LC-MS analyses, the pseudo molecular ion [M + H] + was not the common MS signal, water losses were very common ([M + H-H2O] + ), and alkali adducts (mainly [M + Na] + ) also regularly existed. In total, 36 compounds were tentatively identified in the P. chrysogenum extract. Table 2 and Figure 1 indicate the tentatively identified compounds along with their retention times, experimental m/z in positive ionization mode, and MS/MS fragments.  Compound 1 (R t . 0.79 min) was tentatively assigned as amphetamine. The ESI-MS spectrum showed a protonated molecular ion peak at m/z 136.0710 [M + H] + in addition to a daughter ion at m/z 119 [M + H-NH 3 ] + with loss of NH 3 moiety, and a fragment ion at m/z 92, which showed the loss of C 2 H 6 N leaving the benzyl moiety. The loss of the fragment C 2 H 6 N was confirmed by the presence of the fragment ion at m/z 54 (Figure 2A). From this fragmentation pattern and through comparison with the literature, compound 1 was concluded to be amphetamine [24]. Amphetamine is a psychostimulant drug of phenyl alanine class that is present in two enantiomers, d-and l-enantiomers. The d-enantiomer is more active than the l-form and is used for treating narcolepsy and as diet pills. It is noteworthy to say that amphetamine was used to promote wakefulness in the soldiers during the Second World War [25].
Compound 1 (Rt. 0.79 min) was tentatively assigned as amphetamine. The ESI-MS spectrum showed a protonated molecular ion peak at m/z 136.0710 [M + H] + in addition to a daughter ion at m/z 119 [M + H-NH3] + with loss of NH3 moiety, and a fragment ion at m/z 92, which showed the loss of C2H6N leaving the benzyl moiety. The loss of the fragment C2H6N was confirmed by the presence of the fragment ion at m/z 54 ( Figure 2A). From this fragmentation pattern and through comparison with the literature, compound 1 was concluded to be amphetamine [24]. Amphetamine is a psychostimulant drug of phenyl alanine class that is present in two enantiomers, d-and l-enantiomers. The d-enantiomer is more active than the l-form and is used for treating narcolepsy and as diet pills. It is noteworthy to say that amphetamine was used to promote wakefulness in the soldiers during the Second World War [25].   were tentatively identified as pyrroline carboxylic acid and its isomer [26]. Notably, it was previously identified from P. citrinum [27].
Compound 4 (R t . 1.13 min) showed a protonated molecular ion peak [M + H] + at m/z 143.0706, and was tentatively identified as kojic acid [21,22]. Kojic acid, a pyrone derivative, is a major secondary metabolite produced by a limited range of microorganisms, including Aspergillus oryzae, Aspergillus flavus, and Aspergillus tamarii, as well as Penicillium species and certain bacteria in the stationary phase of growth. The most important biological functions of KA are antibacterial, fungicidal, and insecticidal activities, and it has been approved as a food and cosmetic additive. Furthermore, KA can act as a tyrosinase inhibitor (inhibition of melanin formation) by chelating copper atoms at the tyrosinase active site. For clinical trials, the 1 percent KA-containing formulation was shown to be effective for treating hyperpigmentation disorders, such as melisma, post-inflammatory hyperpigmentation, age spots, and freckles. KA is now accepted to be safe as a cosmetic ingredient and continues to be used as a skin lightening product [29,30]. Notably, KA was previously isolated from P. chrysogenum endophyte of Strychnos toxifera, which is a poisonous plant [31].
Compound 5 (R t . 1.20 min) analysis generated ESI-MS at m/z 185.1388 [M + H] + and MS 2 fragmentation at m/z 167, which was produced by the loss of a water molecule. The daughter ion at m/z 139 showed subsequent loss of the carbonyl group. Furthermore, the fragment ions at m/z 125, 95, and 80, which showed the loss of CH 2 , CH 2 O, and CH 3 , respectively, were also observed in the MS 2 spectrum. Thus, compound 5 was identified as aspyrone according to the previously reported data [21]. It was reported that aspyrone showed nematocidal activity toward Pratylenchus penetrans [32].
Compound 6 (R t . 1.35 min) generated ESI-MS at m/z 143.0601 [M + H] + and MS 2 fragmentation at m/z 125, which was produced by the loss of a water molecule. On the other hand, the daughter ion at m/z 113 showed subsequent loss of the CH 2 OH group. Furthermore, the fragment ion at m/z 96 [113-OH] + was observed in the MS 2 spectrum with the base peak fragment at m/z 69 [97-CO]. Thus, compound 6 was tentatively identified as fulfuran [7].
Compound 8 (R t . 2.15 min) ESI-MS 1 showed a molecular ion peak at m/z 208.0558 [M + H] + . The MS 2 fragmentation of 8 showed loss of the methyl amine group leaving the fragment ion at m/z 177. Furthermore, the loss of C 4 H 8 O was evidenced from the fragment ion at m/z 136 in addition to the fragment ion at m/z 85, which corresponded to C 5 H 11 N. Compound 8 was tentatively identified as N-methyl benzodioxazoyl butan amine (MBDB) [24] from this fragmentation pattern and through comparison with the literature. This showed losses of two molecules of the carbonyl group from the protonated ion. Compound 11 was identified as KA aldehyde by comparison with literature [33].
Compounds 12 and 14 (R t . 6.11 and 7.87 min) were tentatively identified as penicillin G and its stereoisomer from ESI-MS 1 and MS 2 spectral data ( Table 2). The MS 2 spectrum ( Figure 2E which showed the loss of the thiazolidine ring. The mass data of these compounds are in good agreement with those in the literature of penicillin G along with its stereoisomers [34]. Penicillin G was also isolated from P. chrysogenum endophytic fungus on the mangrove plant Porteresia coarctata (Roxb.) [35].  [36].Consequently, this compound was identified to be anserine [26,36]. were also detected. According to the literature, compound 15 was tentatively identified as N-acetyl methionine [37].
Compound 16 (R t . 8.03 min) was tentatively identified as sorbicillin from ESI-MS ( Figure 2G), which showed a protonated molecule ion peak at m/z 233.1105 [M + H] + with an MS 2 base peak fragment ion at m/z 215 [M-H 2 O] + . In addition, the fragment ions at m/z 200 and 173 exhibited subsequent losses of the CH 3 and C 2 H 3 groups, respectively. Notably, a prominent fragment peak was observed at m/z 119 that corresponded to [M + H-H 2 O] + with the loss of the side chain [C 6 H 7 O] + . From this fragmentation pattern and through comparison with literature, the compound was concluded to be sorbicillin, which was previously isolated in addition to dihydrosorbicillin from Penicillin chrysogenum [13]. Sorbicillin was also isolated from Penicillium sp. endophyte of Chinese mangrove Bruguiera gymnorrhiza [38].
Compound 17 (R t 8.55 min) showed a molecular ion peak at m/z 335.1497 [M + H] + and a daughter ion at m/z 155 [M + 3H-C 9 H 10 O 4 ] + , which corresponded to the loss of [tropylium cation + 2COOH] + . According to the available literature, compound 17 was tentatively assigned as penillic acid. To the best of our knowledge, this compound was determined to be a degradation product of penicillin G [39].  Figure 2I). According to this fragmentation and through comparison with literature, these compounds were tentatively identified as L-saccharopine and its isomer [26].
Compound 19 (R t . 9.09 min) was identified as quinolactacide from ESI-MS 1 and MS 2 spectral data ( Figure 2B), as shown in Table 2. It facilitated a protonated molecular ion peak at m/z 237.1015 [M + H] + [100 percent relative abundance] and diagnostic fragment ions at m/z 209 [M + H-CO] + and 181 [209-CO] + , which showed the loss of two carbonyl groups. By comparison with the literature, it was confirmed that this compound could be named quinolactacide. Notably, quinolactacide has an excellent insecticidal activity against peach aphids. It is noteworthy to say that quinolactacide was previously isolated from Penicillium citrinum [40].  Figure 2H). These fragment ions, in addition to other fragment ions at m/z 160, 147, 133, 106, and 94, indicated that this compound is related to sorbicillin and, in turn, this compound was concluded to be dihydrosorbicillin [13]. Notably, dihydrosorbicillin was previously identified by P. chrysogenum [43]. The next fragment ion at m/z 181 showed loss of CH=CH moiety. The fragment ion at m/z 153 detected the loss of side chain moiety [CH 3 -CH=CH-CH=CH-C=O] + . This fact was confirmed from the base peak fragment at m/z 94, which corresponded to the side chain. Therefore, compound 24 was identified as sohirnone B, as previously published [38]. Sohirnone B was reported to have weak antibacterial activity on Staphylococcus aureus and Bacillus subtilis [44]. Sohirnone B was previously identified from P. chrysogenum [45].
Compound 25 (R t . 12.97 min) was tentatively identified as kynurenine along with its isomers, as the ESI/MS 1 shows a protonated molecular ion peak at m/z 209.2372 [M + H] + . The ESI-MS 2 fragment ions at m/z 164 and 148 showed the loss of the COOH and NH 2 groups, respectively. The base peak fragment ion at m/z 136 showed the loss of C 2 H 3 NO 2, and this loss was followed by the loss of one molecule of water to facilitate a fragment ion at m/z 118. This fragmentation pattern along with the other fragments shown in Figure 2F were in a good agreement with that of kynurenine [26,46]. To the best of our knowledge, kynurenine is found in the human lens, and it is thought to play an important role in protecting the lens and retina from UV-induced photodamage, reducing chromatic aberration, and sharpening the image on the retina [46].
Compound 26 (R t 14.42 min) ESI-MS showed a molecular ion peak [M + H] + at m/z 309.1964. Furthermore, the daughter ion was observed at m/z 217, and was produced after the loss of benzyl moiety, followed by the loss of the carbonyl group to facilitate the fragment ion at m/z 189. Subsequently, this resulted in the loss of cyanide moiety to facilitate a major fragment ion at m/z 159. Moreover, the loss of C 6 H 8 O 2 NS facilitated a fragment ion at m/z 148. From this ESI-MS/MS fragmentation and according to the available literature, the compound 26 was tentatively assigned as penilloic acid [47]. It is noteworthy to say that this compound could be produced by the exposure of penicillin G to an alkaline medium [39].
Compound 27 (R t . 15.17 min) gave a molecular ion peak [M + H] + at m/z 475.2501 and MS 2 fragment ions at m/z 248 [sohirnone B + H] + . Furthermore, daughter ions at m/z 207 and 180 (as previously mentioned in sohirnone B) were also detected. Thus, compound 27 was tentatively identified to be a sohirnone B derivative [38].
Compounds 28 and 29 (R t 16.08 and 17.02 min) were identified as sorrentenone and its isomer from ESI-MS 1 and MS 2 spectral data ( Table 2) Figure 2D). The mass information about this compound is in good agreement with the previously published data [21]. Fulvic acid is a phenolic acid and a fungal metabolite that was originally isolated from the Penicillium species. It acts as an immune modulator, reduces oxidative stress, induces apoptosis in hepatic cancer cell lines, influences the microbiome, and potentially affects gut health. Also, fulvic acid decreases proinflammatory markers and activates the immune system to kill bacteria [48]. Fulvic acid was previously identified from Penicillium sp. [49].  [50,51]. Citrinin is a polyketide mycotoxin with the molecular formula C 13 H 14 O 5 . It was first isolated as a pure compound from a culture of Penicillium citrinum [52]. It is mainly produced by Aspergillus, Penicillium, and Monascus fungi. Citrinin is a mycotoxin that contaminates various commodities of plant origin and it is nephrotoxic, genotoxic, and carcinogenic in nature [51,53]. Citrinin was previously isolated from P. chrysogenum [54].
Compound 32 (R t 21.98 min) was identified as zearalenone from MS 1 and MS 2 spectral data (  [55]. It was also produced by Penicillium sp. [56]. Furthermore, other fragments at m/z at 189, 156, and 134 were detected. This fragmentation helps in tentative identification of compound 33 as dehydroergosterol, as previously published [57]. Dehydroergosterol might be helpful in slowing down the deterioration of brain function by inducing a suitable microglial phenotype. However, the permeability of DHE through the blood-brain barrier was not assessed. DHE is considered safe to consume, and it is present in various dairy products, such as camembert cheese. Thus, DHE might be a valuable preventive tool for dementia [58]. It was also isolated from Penicillium roqueforti [59]. Compound 34 was tentatively identified as stigmasterol from these data and with the aid of the present literature [60,61]. Stigmasterol is an efficient antifungal and broad-spectrum antibacterial agent. As such, it may be used as a valuable compound in the development of novel antimicrobial drugs [62]. Stigmasterol was previously identified from Penicillium crusosum Thom [63]. Interestingly, this is the first report on the presence of amphetamine (1), 3-hydroxy kojic acid (3), aspyrone (5), fulfuran (6), asperlactone (9), acetyl kojic acid (10), kojic acid aldehyde (11), anserine (13), N-acetyl methionine (15), L-saccharopine (18), quinolactacide (19), camptothecine (21), kynurenine (25), penilloic acid (26), and brassicasterol palmitate (36) from penicillium sp., while other compounds were previously reported either from P. chrysogenum or from other Penicillium sp.  The discovery of novel classes of antimicrobial therapies has increased global concern over the span of the last few years. Many harmful microbes are now becoming resistant to many available antibacterial and antifungal drugs [66,67]. In the last few decades, interesting bioactive secondary metabolites from marine endophytic fungi have been reported with relevant clinical aims, some of which are matchless. The Red Sea, Egypt represents a diverse source of marine sponge-derived endophytic fungi. These organisms could be a potential host to a unique natural product [68]. The antibacterial and antifungal activity against different microorganisms by the well diffusion technique were expressed as the diameter of the inhibition zone and the percentage of activity, as shown in Tables 3 and 4. Table 3. Antibacterial activity of Penicillium chrysogenum (PC) total extract and kojic acid (KA) by agar diffusion method. MIC: 50-500 µg/mL (strong activity), 600-1500 µg/mL (moderate activity), >1500 µg/mL (weak activity) [18,19]. Table 4. Antifungal activity of Penicillium chrysogenum (PC) total extract and kojic acid (KA) by agar diffusion method.

Candida albicans ATCC 10231
Aspergillus fumigatus RCMB 002008  Figure 3). Furthermore, the extract showed weak activity against Aspergillus fumigatus and Aspergillus flavus, with no activity against Fusarium oxysporum. Interestingly, KA also showed strong antibacterial activity against highly resistant gram-negative bacteria Pseudomonas aeruginosa with an MIC value equal to 250 µg/mL, and moderate fungicidal activity against Candida albicans with an MIC value equal to 750 µg/mL (Figure 3). The antimicrobial activity of the extract, based on the MIC values, was classified as follows: 50-500 µg/mL = strong activity; 600-1500 µg/mL = moderate activity; and >1500 µg/mL = weak activity or inactive [18].

Antioxidant Activity
The series of concentrations ranged from 10 to 320 µg/mL in methanol of DPPH P. chrysogenum (PC) ethyl acetate extract, KA, and ascorbic acid were used in the cu study. The DPPH scavenging percentage of PC extract and KA, as well as ascorbic and IC50 values, are presented in Figure 4A, B, respectively. All of them showed a co tration-dependent antioxidant activity, as demonstrated by the increase in their D radical scavenging activity. . DPPH in methanol (without the tested sample) was used as a negativ trol. Data were analyzed by using one-way ANOVA and statistical significance was calculated Dunnett's multiple comparisons test. * p-value < 0.05 was considered as statistically significan data display the mean ±SD of three biological replicas. Asterisk indicates a significant diffe compared to the control (p < 0.01).

Antioxidant Activity
The series of concentrations ranged from 10 to 320 µg/mL in methanol of DPPH, and P. chrysogenum (PC) ethyl acetate extract, KA, and ascorbic acid were used in the current study. The DPPH scavenging percentage of PC extract and KA, as well as ascorbic acid and IC 50 values, are presented in Figure 4A,B, respectively. All of them showed a concentration-dependent antioxidant activity, as demonstrated by the increase in their DPPH radical scavenging activity.

Antioxidant Activity
The series of concentrations ranged from 10 to 320 µg/mL in methanol of DPPH, and P. chrysogenum (PC) ethyl acetate extract, KA, and ascorbic acid were used in the current study. The DPPH scavenging percentage of PC extract and KA, as well as ascorbic acid and IC50 values, are presented in Figure 4A, B, respectively. All of them showed a concentration-dependent antioxidant activity, as demonstrated by the increase in their DPPH radical scavenging activity. . DPPH in methanol (without the tested sample) was used as a negative control. Data were analyzed by using one-way ANOVA and statistical significance was calculated with Dunnett's multiple comparisons test. * p-value < 0.05 was considered as statistically significant. The data display the mean ±SD of three biological replicas. Asterisk indicates a significant difference compared to the control (p < 0.01). . DPPH in methanol (without the tested sample) was used as a negative control. Data were analyzed by using one-way ANOVA and statistical significance was calculated with Dunnett's multiple comparisons test. * p-value < 0.05 was considered as statistically significant. The data display the mean ±SD of three biological replicas. Asterisk indicates a significant difference compared to the control (p < 0.01).
The tested fraction and/or compound is considered as a very strong antioxidant when IC 50 values are <50 and is considered strong (50-100), moderate (100-150), or weak (151-200) µg/mL [69]. According to the previous range of IC 50 values, the total extract exhibited very weak antioxidant activity with IC 50 1086.2 ± 34.6, whereas pure KA showed a strong activity, with IC 50 33.7 ± 0.8 µg/mL, when compared to ascorbic acid, 14.2 ± 0.5 µg/mL. KA is used as a preservative and flavor enhancer because of its powerful antioxidizing and antimicrobial activities [70,71].

Cytotoxic Activity
An increasing worldwide interest has been directed toward exploring new economic and safe anticancer drugs of natural origin, which might help in chemotherapy and/or chemoprevention of different types of cancer [72]. There has been a significant increase in the number of anticancer compounds isolated from endophytic fungi following the first report of the production of paclitaxel by a fungus [73]. An MTT assay was used to assess the cytotoxic impact on two tumor cell lines in the concentration range between 0 and 500 µg/mL.
All tested fractions showed considerable cytotoxic activity against the cell lines under investigation (HCT-116 and HEP-2). In HCT-116 colon carcinoma cells ( Figure 5A), both ethyl acetate extract of P. chrysogenum and KA showed strong cytotoxic activity with IC 50 22.6 ± 0.8 and 23.4 ± 1.4 µg/mL, respectively, as represented in Table 5. With respect to HEP-2 larynx carcinoma, as shown in Figure 5B, the total extract and KA possessed strong activity and similar cytotoxic profile against HEP-2 cell lines with equal IC 50 30.8 ± 1.3 and 30.8 ± 1.2 µg/mL, respectively, as shown in Table 5. These significant antimicrobial and cytotoxic activities might be attributed to the presence of penicillin G, KA, sohirnone B, and camptothecin as major constituents in the ethyl acetate extract of P. chrysogenum. It is noteworthy that penicillin G was used in the treatment of a variety of skin, urinary tract, and respiratory infections [74], and that both KA and camptothecin exhibit antibiotic and antineoplastic activities [75]. The tested fraction and/or compound is considered as a very strong antioxidant when IC50 values are <50 and is considered strong (50-100), moderate (100-150), or weak (151-200) µg/mL [69]. According to the previous range of IC50 values, the total extract exhibited very weak antioxidant activity with IC50 1086.2 ± 34.6, whereas pure KA showed a strong activity, with IC50 33.7 ± 0.8 µg/mL, when compared to ascorbic acid, 14.2 ± 0.5 µg/mL. KA is used as a preservative and flavor enhancer because of its powerful antioxidizing and antimicrobial activities [70,71].

Cytotoxic Activity
An increasing worldwide interest has been directed toward exploring new economic and safe anticancer drugs of natural origin, which might help in chemotherapy and/or chemoprevention of different types of cancer [72]. There has been a significant increase in the number of anticancer compounds isolated from endophytic fungi following the first report of the production of paclitaxel by a fungus [73]. An MTT assay was used to assess the cytotoxic impact on two tumor cell lines in the concentration range between 0 and 500 µg/mL.
All tested fractions showed considerable cytotoxic activity against the cell lines under investigation (HCT-116 and HEP-2). In HCT-116 colon carcinoma cells ( Figure 5A), both ethyl acetate extract of P. chrysogenum and KA showed strong cytotoxic activity with IC50 22.6 ± 0.8 and 23.4 ± 1.4 µg/mL, respectively, as represented in Table 5. With respect to HEP-2 larynx carcinoma, as shown in Figure 5B, the total extract and KA possessed strong activity and similar cytotoxic profile against HEP-2 cell lines with equal IC50 30.8 ± 1.3 and 30.8 ± 1.2 µg/mL, respectively, as shown in Table 5. These significant antimicrobial and cytotoxic activities might be attributed to the presence of penicillin G, KA, sohirnone B, and camptothecin as major constituents in the ethyl acetate extract of P. chrysogenum. It is noteworthy that penicillin G was used in the treatment of a variety of skin, urinary tract, and respiratory infections [74], and that both KA and camptothecin exhibit antibiotic and antineoplastic activities [75].  were analyzed by using one-way ANOVA and statistical significance was calculated with Dunnett's multiple comparisons test. p-value < 0.05 was considered as statistically significant. The data display the mean ±SD of three biological replicas.

General Materials and Methods
The evaporation of solvents was achieved using a Buchi rotary evaporator (BUCHI R-100, Büchi Labortechnik AG, Flawil, Switzerland). A UV lamp λ max 254/366 nm (Model UV GL-58, Upland, CA, USA) was used for thin layer chromatography (TLC) visualization. A circulating hot-air oven, WT-binder 7200 (BINDER GmbH, Tuttlingen, Germany), was used for heating and drying in this study. Infrared (IR) spectral analysis was recorded in potassium bromide disks on a Pye Unicam SP 3000 and an IR spectrophotometer (FT/IR-460 plus; JASCO International Co., Ltd., Easton, MD, USA).
Nuclear magnetic resonance (NMR) analysis 1D and 2D experiments were performed on a Bruker AMX 400 MHZ (Billerica, MA, USA) for 1 HNMR and with standard pulse sequences operating at 100 MHz for 13 CNMR. 1 H-13 C one-bond connectivity was detected with the HSQC gradient pulse factor selection. Two-bond and three-bond connectivity was identified by the HMBC experiment. Chemical shifts are reported in δ (ppm) and coupling constants (J) are reported in Hz. Unless otherwise mentioned, tetramethylsilane was the internal standard. In addition, DMSO_d 6 (solvent at room temperature) was of a spectroscopic grade for spectral analysis (Sigma-Aldrich, St. Louis, MO, USA). Furthermore, a pH meter (Hanna instruments 85/9, Haryana, India), Autoclave (Medexport BK-75, Frankfurt, Germany), and Laminar air flow cabinet (Jinan, Shandong, China) were also used.

Collection of Marine Sponge Samples, Isolation, and Morphological Identification of Endophytic Fungi
Marine sponge, namely, Cliona sp. and Hymedesmia sp. class Demospongiae, were collected from the Red Sea, specifically, 20 km away from Sharm el-Sheikh [27 • 45 57.8 N 34 • 22 10.8 E], by scuba diving at a depth of 8:10 m off, during Nov-Dec/2018 Figure 6  (1 and 2). The collected material was immediately frozen and maintained at −20 • C until investigation, which was performed as soon as possible, to avoid the growth of ambient bacteria. The sponge biomass was identified by Prof. Saad Zakaria, Marine Science De ment, Faculty of Science, Suez Canal University. The sponges were brought to the l a sterile ice box, washed thoroughly with sterile sea water to eliminate adherent su debris prior to isolation of their endophytic fungi, and then sectioned into small segm of approximately 1 cm × 1 cm. The parts were surface-sterilized with ethanol 70 pe (v/v) for 60-120 s, followed by 2.5 percent sodium hypochlorite for 2 min, and then ri with sterile sea water to avoid any epiphytic microbes [76,77].
The surface-sterilized sponge parts were placed on the surface of malt yeast (MYA) and malt extract agar (MA) media [76,78] containing chloramphenicol (0.2 g shown in Figure 6 (3) and incubated for 15 days at 30 °C. Three biological replicat each fungal isolate were conducted. The developed fungal hyphal tips were collected purified by subculturing on the same media. Meanwhile, the purified fungal isolates maintained as slope cultures at 4 °C until further screening analyses. The recovered fu isolates were grown on MA media for 15 days at 30 °C and the developed colonie shown in Figure 6 (4,6), were examined every day based on their morphological feat Furthermore, they were identified on the basis of their species level according to the versal keys as colony diameter, extracellular exudates, pigmentation, mycelium colo nidial heads, fruiting bodies, and sporulation [15][16][17].

Preparation of P. chrysogenum Crude Ethyl Acetate Extract
The recovered P. chrysogenum fungal isolate from the selected medicinal m sponge Cliona sp. and Hymedesmia sp. (two agar plugs of 5 mm each) of a six-day-old ture of malt agar medium [Bacto agar 15 g/L, malt extract 15 g/L, pH 7.4-7.8 (adju with NaOH/HCl), sea water ad 1000 g] was screened for its biological activity by gro it on Wickerham's broth medium [yeast extract 3 g/L, malt extract 3 g/L, peptone 5 glucose 10 g/L, pH 7.2-7.4 (adjusted with NaOH/HCl), sea water ad 1000 g] for weeks, as depicted in Figure 6 (5). An equal volume of ethyl acetate (300 mL) was a to each flask for 24 h. The flask content was added thoroughly into an Ultra Turrax basic, IKA) at 4000 µ min −1 for 10 min for cell destruction and extraction. The mixture The sponge biomass was identified by Prof. Saad Zakaria, Marine Science Department, Faculty of Science, Suez Canal University. The sponges were brought to the lab in a sterile ice box, washed thoroughly with sterile sea water to eliminate adherent surface debris prior to isolation of their endophytic fungi, and then sectioned into small segments of approximately 1 cm × 1 cm. The parts were surface-sterilized with ethanol 70 percent (v/v) for 60-120 s, followed by 2.5 percent sodium hypochlorite for 2 min, and then rinsed with sterile sea water to avoid any epiphytic microbes [76,77].
The surface-sterilized sponge parts were placed on the surface of malt yeast agar (MYA) and malt extract agar (MA) media [76,78] containing chloramphenicol (0.2 g), as shown in Figure 6 (3) and incubated for 15 days at 30 • C. Three biological replicates of each fungal isolate were conducted. The developed fungal hyphal tips were collected and purified by subculturing on the same media. Meanwhile, the purified fungal isolates were maintained as slope cultures at 4 • C until further screening analyses. The recovered fungal isolates were grown on MA media for 15 days at 30 • C and the developed colonies, as shown in Figure 6 (4,6), were examined every day based on their morphological features. Furthermore, they were identified on the basis of their species level according to the universal keys as colony diameter, extracellular exudates, pigmentation, mycelium color, conidial heads, fruiting bodies, and sporulation [15][16][17].

Preparation of P. chrysogenum Crude Ethyl Acetate Extract
The recovered P. chrysogenum fungal isolate from the selected medicinal marine sponge Cliona sp. and Hymedesmia sp. (two agar plugs of 5 mm each) of a six-day-old culture of malt agar medium [Bacto agar 15 g/L, malt extract 15 g/L, pH 7.4-7.8 (adjusted with NaOH/HCl), sea water ad 1000 g] was screened for its biological activity by growing it on Wickerham's broth medium [yeast extract 3 g/L, malt extract 3 g/L, peptone 5 g/L, glucose 10 g/L, pH 7.2-7.4 (adjusted with NaOH/HCl), sea water ad 1000 g] for three weeks, as depicted in Figure 6 (5). An equal volume of ethyl acetate (300 mL) was added to each flask for 24 h. The flask content was added thoroughly into an Ultra Turrax (T18 basic, IKA) at 4000 µ min −1 for 10 min for cell destruction and extraction. The mixture was filtered, and the mycelium residue was discarded. The culture filtrate was transferred to a separating funnel and the ethyl acetate layer was isolated. The aqueous layer was extracted twice with 300 mL ethyl acetate. The combined ethyl acetate extract was washed with 100 mL demineralized water to eliminate any remaining sea salt and concentrated using a rotary evaporator at 50 • C to give (3 gm) solid residue.

Isolation of Kojic Acid from the Ethyl Acetate Extract of P. chrysogenum
Kojic acid was precipitated from the ethyl acetate extract of broth media after fermentation and concentration of the extract. The KA precipitate was washed several times with methanol to dispose of any impurities, redissolved in CH 2 Cl 2 , and recrystallized using acetone. It gave 168 mg of white needle crystals, with a melting point (m.p.) of 154 • C. It also gave a white spot with R ƒ value 0.74 in solvent system CH 2 Cl 2 -MeOH (9.5 − 0.5) after spraying with vanillin or H 2 SO 4 and heating. It also showed a protonated molecular ion peak at m/z 143.0706 [M + H] + using UPLC-ESI-MS/MS (positive mode).

UPLC-ESI-MS/MS Profiling for Fungal Strain P. chrysogenum
Ultra-performance liquid chromatography with electrospray ionization quadrupole linear ion trap tandem mass spectrometry analysis was performed in the ESI-MS positive ion acquisition mode with a XEVO TQD triple quadruple instrument. The method, in a multiple-reaction monitoring (MRM) mode, was employed for the determination of phytochemicals. The ethyl acetate extract of the fungal strain P. chrysogenum was analyzed by UPLC to obtain the chemical profile. The samples were dissolved in HPLC grade methanol and then filtered through a 0.2 µm membrane disc filter. The resultant solution concentrations were in the range between 0.2 and 0.5 mg/mL, based on the crude extract. The Waters Corporation, Milford, MA 01757, USA mass spectrometer was the UPLC system.
Reverse-phase separations were performed [ACQUITY UPLC-BEH C 18 1.7 µm-2.1 × 50 mm Column; 50 mm × 1.2 mm (inner diameter) and 1.7 µm particle size] at a 0.2 m/mL flow rate. The gradient program applied for the analysis was previously used by [79]. The mobile phase consists of acidified water containing 0. Electrospray Ionization (ESI) was carried out in a positive ion mode. The parameters for analysis were carried out using the positive ion mode as follows: source temperature 150 • C; cone voltage 30 eV; capillary voltage 3 kV; desolvation temperature 440 • C; cone gas flow 50 L/h; and desolvation gas flow 900 L/h. Mass spectra were detected in the ESI between m/z 100-1000 atomic mass units. The chemical constituents were identified by their ESI-QqQLIT-MS/MS spectra and fragmentation patterns. The peaks and spectra were processed using the Maslynx 4.1 software(Waters Corporation, Milford, MA, USA) and tentatively identified by comparing the retention time (Rt) and mass fragmentation with the reported data. Most analytes were detected as single-charged ions, specifically, either as protonated [M + H] + or sodium adducts [M + Na] + , in positive mode.

Antioxidant Activity
The antioxidant activity of the extract was determined at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University by the DPPH free radical scavenging assay, wherein triplicate and average values were considered.

DPPH Radical Scavenging Activity
A freshly prepared (0.1 mM) solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and different tested extracts prepared at 5, 10, 20, 40, 80, 160, and 320 µg/mL in methanol were vigorously mixed and allowed to stand for 30 min at room temperature in the dark [80]. The absorbances of the resultant solution were recorded with a UV-visible spectrophotometer (Milton Roy, Spectronic 1201, Houston, TX, USA) at λ max 517 nm. A DPPH radical in methanol with the reference antioxidant compound (ascorbic acid), or without the antioxidant sample, was used as positive and negative controls. The reference compound, ascorbic acid, was used at different concentrations (5,10,20,40,80,160, and 320 µg/mL). All the determinations were performed in three replicates and averaged. The percentage inhibition of the DPPH radical was calculated according to the following formula: where PI is the percentage inhibition of the DPPH radical, AC is the absorbance of the control solution, and AS is the absorbance of the sample in DPPH solution. The percentage of DPPH radical scavenging was plotted against each extract concentration and ascorbic acid (µg/mL) to determine scavenging capacity (IC 50 ), which is the concentration required to scavenge DPPH by 50 percent (that is, the concentration facilitating 50 percent reduction in the absorbance of a DPPH solution from its initial absorbance).

Cytotoxic Activity
The cytotoxic impact of the ethyl acetate extract of P. chrysogenum on HEP-2 (Human larynx carcinoma) and HCT-116 (Colon carcinoma) was investigated using 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) against DMSO and vinblastine sulphate as negative and positive controls, respectively. HEP-2 and HCT-116 cell lines were obtained from the VACSERA Tissue Culture Unit. Dimethyl sulfoxide (DMSO), crystal violet, and trypan blue dye were purchased from Sigma (St. Louis, MO, USA). Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin, and 0.25% Trypsin-EDTA were purchased from Lonza (Basel, Switzerland). The crystal violet stain (1 percent) is composed of 0.5 percent (w/v) crystal violet and 50 percent methanol, then made up to volume with ddH 2 O, and filtered through a Whatmann No. 1 filter paper. The cells were propagated on Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 percent heat-inactivated fetal bovine serum, 1 percent L-glutamine, HEPES buffer, and 50 µg/mL gentamycin. The cells were maintained at 37 • C in a humidified atmosphere with 5 percent CO 2 , and they were sub-cultured two to three times a week.
In the case of antitumor assays, the cells were seeded in 96-well plate at a cell concentration of 1 × 10 4 cells per well in 100 µL of growth medium. Fresh medium containing different concentrations of the test compound and the extract were added after 24 h of seeding. Serial two-fold dilutions of the tested compound and the extract were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. The microtiter plates were incubated at 37 • C in a humidified incubator with 5 percent CO 2 for a period of 24 h. Three wells were used for each concentration of the test sample. Control cells were incubated without the test sample and with or without DMSO. The small percentage of DMSO present in the wells (maximal 0.1 percent) was found to not affect the experiment. After incubation of the cells at 37 • C for 24 h, the viable cells' yield was determined by a colorimetric method.
Briefly, after the end of the incubation period, media were aspirated, and the crystal violet solution (1 percent) was added to each well for at least 30 min. The stain was removed, and the plates were rinsed using tap water until all excess stains were removed. Glacial acetic acid (30 percent) was then added to all wells and mixed thoroughly. Subsequently, the absorbance of the plates was measured after gently shaking the same on a microplate reader (TECAN, Inc., Morrisville, NC, USA), using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stains. The treated samples were compared with the cell control in the absence of the tested compounds. All experiments were carried out in triplicate. The cell cytotoxic effect of each tested compound was calculated.
The optical density was measured with the microplate reader (SunRise, TECAN, Inc., Morrisville, NC, USA) to determine the number of viable cells, and the percentage of viability was calculated, as the tumor cell lines were suspended in medium at a concentration of 5 × 10 4 cell/well in Corning ® 96-well tissue culture plates, and then incubated for 24 h. The tested extracts were then added into 96-well plates (six replicates) to achieve eight concentrations for each extract. Six vehicle controls with media or 0.5 percent DMSO were run for each 96-well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by the MTT test.
Cell viability percentage = [1 − (OD t /OD c )] × 100 percent, where OD t is the mean optical density of wells treated with the tested sample, and ODc is the mean optical density of untreated cells. The relation between surviving cells and each extract concentration (1-500 µg/mL) is plotted to find the survival curve of each tumor cell line after treatment with the tested fraction. The 50 percent inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50 percent of intact cells, was estimated from graphic plots of the dose response curve for each concentration using non-linear regression analysis of GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) [81,82]. All experiments were repeated at least three times. Results are reported as mean ± SD.

Antimicrobial Activity (Well Diffusion Method)
The antibacterial activity of P. chrysogenum ethyl acetate extract and kojic was evaluated by using the well diffusion method [83] against Staphylococcus aureus (S. aureus, ATCC 5368) as gram-positive bacteria. Escherichia coli (E. coli, ATCC 10536) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) as gram-negative bacteria, along with Candida albicans (C. albicans, ATCC 10231), Aspergillus fumigatus (A. fumigatus, RCMB 002008), Aspergillus flavus (A. flavus RCMB 002002), Cryptococcus neoformans (C. neoformans RCMB 0049001), and Fusarium oxysporum (F. oxysporum RCMB 001004) as fungi. All the tested strains were obtained from the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University, Egypt. The assay was performed on Muller Hinton Agar (MHA) medium for bacterial strains and potato dextrose agar for fungi. The tested samples were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 µg/mL. Ciprofloxacin (100 µg/mL) was used as a standard antibacterial drug, while Fluconazole (100 µg/mL) was used as a positive control for fungi. The wells were filled with 100 µL from a stock solution of each sample, with the standards and DMSO as a negative control. Cultures were incubated at 37 • C for 14-18 h for bacteria, and for 24-48 h for fungi. All the assays were conducted in triplicate. The activity was determined by measuring the diameter of the inhibition zone (mm) from three independent experiments, and the values were averaged. [84,85].

Determination of Minimum Inhibitory Concentration (MIC)
The MIC values of the tested compounds were determined using the agar dilution method. The tested compounds were dissolved and serially diluted in dimethyl sulfoxide (DMSO). A series of agar plates containing the assigned serial dilution of each compound was prepared. Each plate was prepared by mixing the indicated volume of the dissolved compound with molten Mueller-Hinton agar (MHA) medium to obtain agar with thickness between 3 and 4 mm, with final concentrations of DMSO not exceeding 5 percent and 2.5 percent for bacteria and fungi, respectively. The tested bacterial strains were grown overnight on MHA, and purified colonies were suspended in 0.9 percent saline. The turbidity of the bacterial inoculum was adjusted to equal 0.5 McFarland (2.5 × 10 8 cfu/mL) and then diluted to one-tenth of the solution with saline. Prepared MHA plates were inoculated by delivering 2 µL of the prepared inoculum on their surfaces to obtain a final concentration of 10 4 cfu per spot [86]. The tested fungal strains were streaked on potato dextrose agar, and purified colonies were suspended in saline. The turbidity of the prepared inoculum was adjusted to be equal to the turbidity of 0.5 Mcfarland (5 × 10 6 cfu/mL), then diluted to 1:10 with saline. Prepared potato dextrose agar containing the serially prepared concentration of each compound was inoculated by delivering 2 µL of the prepared inoculum, so the final concentration of the inoculum was 10 3 per the resultant spot. Inoculated plates were incubated at 30 • C for 24-48 h and were examined for the presence of fungal growth [87,88].

Fungal Deposition
The Internal Transcribed Spacer (ITS) sequence of the isolate Penicillium chrysogenum EFBL, an endozoic of Cliona sp., was deposited to the GenBank with accession No. OL597937.1.

Statistical Analysis
The data were collected and plotted using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The data were analyzed by using one-way ANOVA and statistical significance was calculated with Dunnett's multiple comparisons test. A p-value < 0.05 was considered statistically significant. The data display the mean ± SD of three biological replicas.

Conclusions
In the present study, Penicillium chrysogenum, an endozoic fungus recovered from Cliona sp. marine sponge, was investigated for the first time. The UPLC-ESI-MS/MS analysis revealed the tentative identification of 36 compounds, including amphetamine, pyrroline carboxylic acid, 3-hydroxy KA, KA, penicillin G, camptothecin, dihydrosorbicillin, sohirnone B, kynurenine, quinolactiocide, and sorrentanone as major compounds. KA was also identified from Penicillium chrysogenum ethyl acetate extract, and its structure was confirmed by IR, ESI-MS/MS, 1D and 2D NMR spectroscopy. The antioxidant, cytotoxic, and antimicrobial activities of the P. chrysogenum extract and KA were also investigated. Both the extract and KA showed significant cytotoxic activity against HCT-116 and HEP-2 cell lines. However, only KA showed a potent antioxidant activity compared to the P. chrysogenum extract, which is nearly inactive. Additionally, the PC extract and KA showed potent antibacterial activity against S. aureus and P. aeruginosa. Interestingly, the P. chrysogenum extract showed significant antifungal activity against C. albicans, C. neoformans, and F. oxysporum. In conclusion, based on the previous results, Penicillium chrysogenum isolated from Cliona sp. is a promising natural source of potent biologically active compounds for multi-drug resistant infections. Further studies of such endozoic fungi may result in the isolation of new natural drugs with variable therapeutic activities.