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Chemical and Biological Activity Profiling of Hedyosmum strigosum Todzia Essential Oil, an Aromatic Native Shrub from Southern Ecuador

The present study aimed to determine the chemical composition, enantiomeric distribution and the biological profile of Hedyosmum strigosum essential oil (EO). The antioxidant properties and anticholinesterase effect were measured by spectroscopic methods and antimicrobial potency assessed against 8 bacteria and two fungi. H. strigosum is a native shrub, particularly found in Ecuador and Colombia at 2000 to 3500 m a.s.l. Chemical composition was determined by GC-MS and GC-FID. A total of 44 compounds were detected, representing more than 92% of the EO composition. The main compounds were thymol (24.35, 22.48%), α-phellandrene (12.15, 13.93%), thymol acetate (6.59, 9.39%) and linalool (8.73, 5.82%), accounting for more than 51% of the EO. The enantioselective analysis revealed the presence of 5 pure enantiomers and 3 more as a racemic mixture. The EO exerted a strong antioxidant capacity, determined by ABTS assay, with a SC50 of 25.53 µg/mL and a strong and specific antimicrobial effect against Campylobacter jejuni with a MIC value of 125 µg/mL. A moderate acetylcholinesterase inhibitory effect was also observed with an IC50 value of 137.6 µg/mL. To the best of our knowledge this is the first report of the chemical composition and biological profile of H. strigosum EO.

Introduction
Chloranthaceae is the smallest family of vascular plants and it is the only family in the Chloranthales order. This family is composed mainly of lowering plants (angiosperms) and is considered one of the most primitive of its type [1]. The Chloranthaceae family consists of herbs, shrubs and trees, the species are characterized by the presence of secretory cells in the stems and leaves [2]. The family comprises 80 species belonging to the Ascarina (12 accepted species), Chloranthus (20 accepted species), Hedyosmum (44 accepted species) and Sarcandra genera (4 accepted species) [3]. Species of Chloranthaceae are naturally occurring in tropical and subtropical areas of Southeast Asia, Pacific Islands, New Zealand, Madagascar, Central and South America, and the West Indies [4]. In Ecuador, the Chloranthaceae family is represented only by the genus Hedyosmum [5].
Pharmacological investigations have revealed that the extracts of Chloranthaceae species are highly bioactive. Phytochemical studies showed the presence of terpenoids, flavonoids, coumarins, organic acids, amides, and sterols can be obtained from plants of this family [4]. Some species of the family Chloranthaceae have been used as folk medicine, especially in eastern Asia. A common use of these species includes: invigorating blood circulation and eliminating blood-stasis. The sarcandrae herb (Sarcandra glabra) is a folk medicine with a long history in China, widely used for its medicinal properties and it has been has been recorded in the Pharmacopoeia of the People's Republic of China [6]. The genus Hedyosmum consists of 44 species of shrubs or trees, with branches hinged at the height of the nodes or bulging. Its leaves are opposite, petiolate, simple, toothed and with pinnate veins [7]. Most of the species of this genus are aromatic, for this reason, the genus name finds its origin in the Greek words, hedy (pleasant) and osmum (smell) [8]. This genus is represented mainly in America, and only one species is registered in Southeast Asia [9]. Hedyosmum species are widespread in low and high mountain rain forests at altitudes of 500 to 3000 m a.s.l. [10]. The species of this genus are distributed mainly in the mountains from the state of Veracruz (Mexico) to Brazil (through southern Mexico, Central America and the Andes of South America) [11][12][13].
In Ecuador, this genus is represented by at least 15  H. purpurascens is the only endemic species occurring in the high Andean forest in the south of the country. All the reported species for Ecuador grown mainly in cloud forests between 600 and 3000 m a.s.l. [14,15].
Previous studies have reported non-volatile and volatile secondary metabolites (essential oil) in species of the genus Hedyosmum. Among the identified non-volatile compounds are flavonoids, hydroxycinnamic acid derivatives, neolignans, sesquiterpenes and sesterterpenes. The essential oils (EO) have been extracted from leaves, flowers, and fruits from plants of this genus [12,13,16]. Traditional uses assigned to Hedyosmum genus include the use as sources of firewood, construction materials and food (fruits). The leaves of some species are used in infusion as a medicinal preparations or aromatic beverages [7]. The pharmacological effects associated with species of this genus include analgesic, anxiolytic, antibacterial, anticancer, antidepressant, antinociceptive, antiplasmodial neuroprotective and sedative properties [17]. Some members of this genus have been reported to possess antispasmodic and digestive effects, and also, they were reported for the treatment of kidney problems and anxious stomach. In addition, folk medicine report traditional uses such as, headaches, snake bites, rheumatic joint pain, fever, and cold symptoms [2,18].
Hedyosmum strigosum Todzia (class: Equisetopsida C. Agardh; subclass: Magnoliidae Novák ex Takht.; superorder: Austrobaileyanae Doweld ex M.W. Chase & Reveal; order: Chloranthales R. Br.; family: Chloranthaceae R. Br. ex Sims; genus: Hedyosmum Sw.) is a native shrub or tree of Ecuador, also found in Colombia. In Ecuador, this species is distributed in the Andean and Amazon region between 2000 to 3500 m a.s.l., especially in the northern andean provinces of Cotopaxi, Imbabura, Napo, Pichincha and Tungurahua, and the eastern province of Sucumbíos [5]. The plant is popularly known as "Quinillo" or "Olloco," and traditionally, the infusion of the leaves is drunk as an aromatic beverage, its fruits are food for birds, its branches and stems are used as fuel, and the stem is timber and is used for the construction of fences [18].
In south America, Ecuador is considered a megadiverse country because has many species per unit surface area. Currently, this country occupies the sixth position worldwide as a biodiversity hotspot [19]. However, there are few studies of its plant species, especially of the aromatic species of the Hedyosmum genus. The importance of the family and the genus, and the fact that only four species of this genus have been studied in Ecuador (H. luteynii [14], H. racemosum [2], H. scabrum [17] and H. sprucei [13]) encouraged us to determine a complete chemical profile of H. strigosum and validate its biological properties. To the best of our knowledge, this is the first report made for this specie, for that reason, the aim of this research was to determine the chemical composition, enantiomeric distribution, antimicrobial, antioxidant and anticholinesterase effect of the H. strigosum essential oil.

Results
The EO from Hedyosmum strigosum obtained by hydrodistillation exhibited a paleyellow color and a yield of 0.66% (4.62 g from 700 g of plant material). The relative density was 0.9823 ± 0.004 g/mL, refractive index of 1.4893 ± 0.001 and a specific rotation of α 20 D of +21.21 ± 0.035 (c 10.25, CH 2 Cl 2 ).

Enantiomeric Analysis
The results obtained for the enantiomeric analysis from H. strigosum essential oil are depicted in Table 2.

Antimicrobial Activity
According to the results displayed in Table 3, the most relevant outcome was obtained for Campylobacter jejuni, and the two dermatophytes with a MIC value of 125 µg/mL. The remaining microorganisms exhibited weak or null inhibitory effects at concentrations higher than 1000 µg/mL.

Antioxidant Capacity
The results obtained for the antioxidant capacity of the EO from H. strigosum are depicted in Table 4. Trolox was used as reference control with its corresponding IC 50 value.

Anticholinesterase Activity
This is the first study of the inhibitory potency of H. strigosum, EO, against acetylcholinesterase, measured by the modified Ellman's method. Results showed a moderate inhibitory effect with an IC 50 value of 137.6 ± 1.02 µg/mL (Figure 1). Donepezil hydrochloride was used as positive control with an IC 50 of 13.6 ± 1.02 µM.

Antioxidant Capacity
The results obtained for the antioxidant capacity of the EO from H. strigosum are depicted in Table 4. Trolox was used as reference control with its corresponding IC50 value.

Anticholinesterase Activity
This is the first study of the inhibitory potency of H. strigosum, EO, against acetylcholinesterase, measured by the modified Ellman's method. Results showed a moderate inhibitory effect with an IC50 value of 137.6 ± 1.02 µg/mL (Figure 1). Donepezil hydrochloride was used as positive control with an IC50 of 13.6 ± 1.02 µM.

Discussion
There are no previous reports about the extraction of EO from H. strigosum, however, a study from a related species, H. luteynii, from Chimborazo-Ecuador, reported a very low yield of the EO of ca. 0.07% by using the same extraction method [14]. In contrast, by means of microwave radiation-assisted hydrodistillation from H. translucidum, the EO yield obtained was 1.2% [20], approximately twice the yield obtained in our study.
According to Radice et al. [8] the main chemical compounds occurring in essential oils from representative species of the genus Hedyosmum (≥10%) corresponds to There are no reports about the antimicrobial activity of H. strigosum, however, H. brasiliense, a related species, showed a good profile of inhibition against several pathogenic bacteria such as Bacillus subtillis, Staphylococcus aureus, Staphylococcus saprofiticus and dermatophytic fungi such as Microsporus canis, Trichophyton rubrum and Trichophyton mentagrophytes with MIC values ranging from 0.31 to 0.12% v/v (3.12 to 1.25 mg/mL ca.) [9]. There are no extended criteria to value the potency of MIC values, however, Van Vuuren and Holl [21] suggested a detailed classification for extracts and essential oils and found that a MIC value between 101 to 500 µg/mL can be considered as a strong activity and according to our report, the activity for C. jejuni and the two dermatophytes lies between this range.
The H. strigosum EO as shown in Table 4, reveals a strong scavenging effect for ABTS radical, meanwhile a weak or null scavenging capacity for DPPH radical. This can be due to the mechanistic differences of both assays. ABTS cation radical is stabilized by electron transfer mechanism from antioxidants present in samples and for DPPH assays, this radical can be mainly stabilized by hydrogen atom transfer mechanism [22]. The occurrence of the aliphatic monoterpene α-phellandrene in concentrations higher to 28% in the species H. racemosum, another Ecuadorian related species, and its null antioxidant activity [2] could suggest at least in our research that, the antioxidant capacity could be related to the main compound, thymol, which demonstrated a strong antioxidant effect as described by Torres-Martínez et al. [23] against ABTS assay with a scavenging effect higher than 96% at a 0.1 mg/mL dose. However, according to Beena et al., thymol exerted a moderate antioxidant effect in the DPPH assay with a SC 50 value of 167.57 µg/mL [24] which can agree with our results.
Enantiomeric analysis showed the presence of 5 pure enantiomers and 3 pairs found as a racemic mixture, other equally interesting studies showed enantiomeric compositions of EO from the Hedyosmun genus, such as; H. scabrum (Ruiz & Pavon) leaves collected in Ecuador and Bolivia, and H. angustifolium collected in Bolivia the presence of similar enantiomers such as β-pinene, sabinene, limonene and linalool [17,25].
Regarding to the anticholinesterase effect, the H. strigosum EO exerted a moderate inhibitory potency with an IC 50 value of 137.6 µg/mL, much higher than the reported effect of H. barisiliense EO which exhibited an inhibition percentage of 69.82% at a dose of 1 mg/mL. Many species of Hedyosmum genus have been explored for their antibacterial or antioxidant potential and their chemical profile but little or nothing is said about their inhibitory potential against acetylcholinesterase, however, information related to chemical entities isolated from the EO of this genus and their inhibitory potential can be found in literature, such as thymol which is the characteristic occurring compound in Thymus vulgaris but, it was found as the majority compound in our EO. As reported by Jukic et al. [26], thymol presented an IC 50 of 0.74 mg/mL and linalool was inactive at a dose higher than 1 mg/mL, surprisingly, carvacrol, the structural isomer of thymol, exerted a ten times higher potency with an IC 50 of 0.063 mg/mL against acetylcholinesterase, suggesting that the hydroxyl functionality in -orto position, like occurring in carvacrol, plays an important role for the AChE inhibitory effect.
Despite its low concentration in the EO of H. strigosum, terpenes like terpinolene, terpinen-4-ol and E-caryophyllene (< to 1%) can be collaborating for the moderate AChE inhibitory effect observed. As reported by Bonesi  156.4 µg/mL [27], meanwhile, terpinen-4-ol and E-caryophyllene exhibited a moderate effect with inhibition percentages of 21.4% at 1.2 mM and 32% at 0.06 mM doses, respectively. There is no report about the AchE inhibitory effect of α-phellandrene, the second major compound occurring for H. strigosum EO, however, in the same study of Bonesi, the AChE inhibitory effect of β-phelandrene is reported with an IC 50 value of 120.2 µg/mL. highlighting the importance of terpenes containing EO and their AChE inhibitory capacity.

Plant Material
The aerial parts of Hedyosmum strigosum were collecteced in Villonaco hill, Loja, province at 2720 m a.s.l. at 3 • 59 42.62 S and 79 • 16 02.60 W coordinates. The identified specimen was located at the herbarium of the Universidad Técnica Particular de Loja with voucher code HUTPL14299. The aerial parts were cleaned and dried at 34 • C for 6 days until the plant material was used for distillation.

Essential Oil Distillation
The aerial plant material was submitted to hydrodistillation in a Clevenger-type apparatus for a period of 3 h. Anhydrous sodium sulfate was added to the flask containing the EO with moisture to remove it and then, the EO properly labeled was stored at −4 • C until was used in the biological and chemical assays.

Physical Properties Determination
Three physical properties were determined for the EO. Relative density (RD 20 ), according to the AFNOR NFT75-111 standard, with an 1 mL pycnometer and an analytical balance (Mettler AC100 model), refractive index (RD 20 ), according to the ANFOR NF 75-112 25 standard in an ABBE refractometer (Boeco, Germany) and specific rotation [∝ 20 514 ], according to the ISO 592-1998 standard in a Hanon P 810 automatic polarimeter, were carried out. All measurements were performed by triplicate.

GC-MS
Chemical analysis of H. strigosum EO was carried out using a gas chromatographymass spectrometry (GC-MS) model Trace1310 gas chromatograph, coupled to a simple quadrupole mass spectrometry detector model ISQ 7000 (Thermo Fisher Scientific, Walthan, MA, USA). The mass spectrometer was operated in SCAN mode (scan range 35-350 m/z), with the electron ionization (EI)source 70 eV.
A non-polar column, based on 5%-phenyl-methylpolysiloxane, DB-5 ms (30 m long, 0.25 mm internal diameter, and 0.25 µm film thickness), was used for the qualitative and quantitative analysis of EO. The thermal program was as follow: initial temperature 60 • C for 5 min, followed by a gradient of 2 • C/min until 100 • C, then at 3 • C/min until 150 • C, and at 5 • C/min until 200 • C. Finally, a new gradient of 15 • C/min until 250 • C was applied. The final temperature was maintained for 5 min. GC purity grade helium 5.0 grade ultra-pure, was used as carrier gas, set at the constant flow of 1 mL/min [28].

GC-FID
Quantitative analysis was performed on an Agilent Gas Chromatograph (6890 series) with a flame ionization detector (CG-FID) the chromatographic conditions were the same as in the GC-MS analysis. The average percentage of each component EO was calculated from the area of the corresponding CG-FID peak with respect to the total area of peaks without applying any correction factor. The values and standard deviations were calculated from the results of the three injections [29].

Enantioselective Analysis
Enantioselective analysis was carried out through an enantioselective from Mega, MI, Italy capillary column, based on 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin (25 m × 250 µm internal diameter × 0.25 µm phase thickness). GC method was as follows: initial temperature was 60 • C for 2 min, followed at 2 • C/min until 220 • C, that was maintained for 2 min. The homologous series of n-alkanes (C9-C25) was also injected, in order to calculate the linear retention indices. The enantiomers were identified determined by injection of enantiomerically pure standards [30].

Antimicrobial Activity
The antimicrobial activity and Minimal inhibitory concentration values were determined by means of the broth microdilution technique with four gram positive bacteria (Enterococcus faecalis ATCC ® 19433, Enterococcus faecium ATCC ® 27270, Staphylococcus aureus ATCC ® 25923, Listeria monocytogenes ATCC ® 19115), four gram negative bateria (Escherichia coli (O157:H7) ATCC ® 43888, Pseudomonas aeruginosa ATCC ® 10145, Salmonella enterica serovar Thypimurium ATCC ® 14028) and two fungi (Trichophyton rubrum ATCC ® 28188, Trichophyton interdigitale ATCC ® 9533) as microbial assay models. Two-fold serial dilution method was used to achieve concentrations ranging from 4000 to 31,25 µg/mL and a final inoculum concentration of 5 × 10 5 cfu/mL for bacteria, 2.5 × 10 5 cfu/mL for yeast and 5 × 10 4 spores/mL for sporulated fungi were used. Meller Hinton II (MH II) for bacteria and Sabouraud broth for fungi as assay media were used. The method followed was previously described for our research group. The culture of Campylobacter jejuni, was activated by adding horse serum at 5% in Tioglycolate medium and incubated at anaerobic conditions provided by a Campygen sachet (Thermo, scientific) at 37 • C for 48 h. The broth microdilution test was performed in MH II supplemented with 5% horse serum (Thermo) at the same conditions previously described as oil sample dilution and bacterial inoculum, and anaerobic conditions as described fully by our research group [31].
Antimicrobial commercial agents were used as positive control as follows: Ampicilin 1 mg/mL solution for S.aureus, E. faecalis and E. faecium. Ciprofloxacin 1 mg/mL solution for P.aeruginosa, S. enterica, E. coli. and L. monocytogenes, finally, Itraconazol 1 mg/mL for the two fungi. The procedure was adapted from the methodology suggested by Thaipong et al. [32] and fully developed for our research group in previous reported jobs [31]. Briefly, a stable solution of DPPH in MeOH was prepared at a concentration of 625 µM. Working solution was prepared by adjusting an aliquot of the stable solution in methanol until an absorbance of 1.1 ± 0.02 was reached. An EPOCH microplate reader was used for absorbance reading at 515 nm and 96 microplates were used for the test. 270 µL of the working solution were allowed to react with 30 µL of the sample solution for 1 h at room temperature in darkness. The same procedure was followed for the blanks and positive control (Trolox). A solution representing 100% of the DPPH radical was prepared with 270 µL of working solution and 30 µL of MeOH. Sample solutions of essential oil were prepared by dissolving 10 mg of EO in MeOH. Three more 10 × dilutions were included to calculate the SC50 (half scavenging capacity). 4.6.2. The 2,2-Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic acid) Radical Scavenging Assay The procedure was adapted from the methodology described by Thaipong et al., [32] and Arnao et al. [33] and fully developed for our research group in previous reported jobs [32] Briefly, a stable solution of ABTS* radical was prepared by reacting in darkness for 24 h, equal volumes of a 7.4 µM aqueous ABTS solution and 2.6 µM potassium peroxydisulfate. Working solution was prepared by adjusting an aliquot of the stable solution in methanol until an absorbance of 1.1 ± 0.02 was reached. An EPOCH microplate reader was used for absorbance reading at 734 nm and 96 microplates were used for the test. The same procedure as described for DPPH was followed for ABTS assay.
Half Scavenging capacity defined as the concentration of the EO required to scavenge 50% of the radical was calculated from the corresponding curve fitting of data (Radical scavenging percentage vs. EO concentrations). Radical scavenging percentages were calculated according to Equation (1): where, Ac. Absorbance of the 100% radical adjusted solution.
As. Absorbance of the radical adjusted solution with the sample at different concentrations.

Anticholinesterase Assay
Acetylcholinesterase inhibitory effect of the essential oil was measured with a variation of the method described originally by Ellman et al. and reported in our previous jobs [28,33] and more recently in Andrade et al., [34]. Briefly, 20 µL of a 15 mM solution of acetylthiocoline in PBS pH 7.4 were placed along with 40 µL of Tris buffer pH 8.0 (containing 0.1 M NaCl and 0.02 M of MgCl 2 .6H 2 O), 100 µL of Ellman's reagent solution in Tris buffer (DNTB, 3 mM) and 20 µL of EO sample solution. The reaction was placed in an EPOCH 2 microplate reader and preincubated for 3 min at 25 • C. Finally, the addition of 20 µL of the enzyme solution (0.5 U/mL) started the reaction. The amount of product released was monitored at 405 nm for 60 min. EO sample solutions were made by dissolving 10 mg of EO in MeOH. Three more 10 × factor dilutions and two intermediate log based doses were included to obtain final concentrations of 1000, 500, 100, 50 and 10 µg/mL and the experiments were performed by triplicate.
IC 50 value was calculated from the corresponding curve fitting of data obtained from the calculated rate of reactions with Graph pad Prism 8.0.1 (San Diego, CA. USA) Donepezil hydrochloride was used as positive control.

Conclusions
The chemical composition and enantiomeric distribution of the EO of H. strigosum, as well as, a biological activity profile, were determined for the first time. This research contributes to our knowledge about the native aromatic species from Ecuador, particularly of the Hedyosum genus which is the only genus of the Chlorantaceae family, represented in Ecuador. The importance of aromatic species is well supported with this study where, an important antibacterial and antifungal effect was observed. Also, the antioxidant and anticholinesterase effect observed for this species encourage us to continue exploring our native flora, searching for novel candidates for the pharmaceutical or cosmetic industry.


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

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Phytotoxic, Antifungal Activities and Phytochemical Screening of Medicago Denticulata

Introduction: The plants are an alternative source for the management of a number of human disorders because of diverse metabolites. Aims: Current study targets to probe underexplored therapeutic potentials of Medicago denticulata (M. denticulata) through phytochemical and biological assays. Methods: The dried whole plant (aerial and root parts) was extracted in succession with dichloromethane (DCM) and methanol by simple maceration process and designated with codes MDWD and MDWM respectively. Results: The phytochemical investigation uncovered the occurrence of saponins, terpenoids, tannins, phenols and flavonoids in the extracts. MDWD fraction unveiled the highest total phenolic contents 54.47 μg GAE / mg dry weight (DW), The total reducing potential 58.125 μg AAE / mg DW and total antioxidant capacity 81.71 μg AAE / mg, while MDWM fraction revealed maximum flavonoids content 16.72 μg QE / mg DW. The phytotoxicity of methanol and DCM extracts exhibited 73% and 63% growth regulation respectively at concentration of 1000 μg/mL. Extracts revealed antidiabetic activity through α-amylase inhibition. The maximum inhibition i.e. 116.91% was detected at the concentration of 1000 mg / mL by DCM extract. Conclusion: Dichloromethane and methanolic extracts of M. denticulata are well thought-out as the potential source of antioxidants and could provide immunomodulatory, antifungal, phytotoxic and a-amylase inhibition activities.

INTRODUCTION
The assorted plant kingdom of the world is generally acknowledged for its medicinal significance. Potential medicinal constituents of natural plants have significantly contributed in the development of many herbal therapies used for several diseases around the world. 1 The herbs have been utilized by all cultures for quite a long time. Each area of the world uses herbs indigenous to that area. 2 The remedies derived from plants play very important role in the traditional medicine system, like Ayurveda, Egyptian and Chinese, which represents their recurrent usage till today. 3 Eastern area of Mediterranean have been recognized throughout generations with a rich record of the natural medicinal herbs. 4 A striking number of new drugs had been developed from terrestrial plants being utilized as medicines in China, Egypt, India and Greece from antiquated time. There is a prediction that plants can give potential bioactive compounds for development of new 'leads' for example, vinblastine and vincristine used in therapy of cancer. 5,6 verse flora. Generally in deserts, western and northern seashore and mountain areas of Pakistan approximately 6000 taxa comprising the flowering plants were accounted. 11 To best of our knowledge, the most of traditional plants in the vicinity have not been characterized up till now to demonstrate their biological and phytochemical potentials. Thus, current study has been concentrated predominantly on phytochemical and pharmacological profiling of M. denticulata.

Genus Medicago
The genus Medicago L. belonging to family Fabaceae (subfamily Papilionoideae, tribe Trifolieae) comprises of fifty species, of which Medicago sativa L. (alfalfa) is renowned. These species are perennial or annual herbs and rarely shrubs. 12 Medicago polymorpha (Burr medic) is a common, polymorphic annual legume of Mediterranean origin. It has important value in many dry land farming systems as a selfreseeding and very effective nitrogen fixer. 13 Fresh leaves of Medicago polymorpha cooked in water are taken orally to treat indigestion and constipation. 14

Extraction of plant material
For effective extraction, the whole plant material was dried at a room temperature for a period of 3 to 4 weeks before it was comminuted to coarse powder and weighed. Extraction of that powdered material was done through simple maceration procedure. Weighed quantity of that powdered plant was soaked in a measured volume of the dichloromethane (DCM) solvent for 24 hours in an extraction bottle before filtration was carried out. Above process was repeated 3 times with dichloromethane solvent. Then extraction of this marc was completed in a similar way by using methanol solvent. To attain crude extract, Dichloromethane and methanol extracts were subjected to dryness in a rotary evaporator (Buchi, Switzerland) under the reduced pressure. The dichloromethane (12 g) and methanol (23.6 g) extracts collected in the separate sample bottles were labeled with codes as MDWD and MDWM respectively.

Qualitative Phytochemical Analysis
Preliminary phytochemical examination for extracts was completed as described by. 15

Quantitative Phytochemical Analysis
Total phenolic along with flavonoid contents were measured by following described procedures.

Estimation of total phenolics
Total phenolic contents (TPC) of M. denticulata fractions were determined using a method based on the study of 16 with a slight change. Briefly, 20 µL of the each fraction was put in the 96-well plate, followed through adding 90 µL of diluted FolinCiocalteu reagent in the each well plate. After incubating that mixture for five minutes at a room temperature 6% sodium carbonate solution (90 µL) was added and then incubated again for 1 hour at a room temperature. The absorbance was documented on microplate reader at 630 nm wavelength. Value of total phenolic content was determined as gallic acid equivalent (GAE). 17

Estimation of total flavonoids
Aluminum chloride based colorimetric technique was utilized for quantification of total flavonoid content (TFC), using quercetin as a standard. 18 Concisely, the reaction mixture comprising 20 µL of the sample solution (20 mg/mL DMSO), 10% aluminum chloride (10 µL), 1M potassium acetate (10 µL), and 160 µL of distilled water was incubated for half an hour at room temperature. Then, spectrophotometric absorbance of this mixture was determined at 415 nm wavelength and total phenolic content was communicated µg QE/mg DW. 3

Total Antioxidant Capacity (Phosphomolybdate Assay)
For determination of antioxidant capacity of samples Phosphomolybdate technique was utilized. 19 The assay mixture (1000 microliter) containing sodium phosphate (0.028 M), H 2 SO 4 (0.6 M) and 0.004 M ammonium molybdate was transferred to the sample tubes holding 100 µL test sample. The mixture samples were cooled after heating for 1.5 hours at 95 ˚C hot water bath. The absorbance of reaction mixture was recorded at 765 nm. As reference standard ascorbic acid was utilized. 20

Total Reducing Power (Potassium Ferricyanide Colorimetric Assay)
Reducing powers of the extracts were evaluated as per process described previously by. 21,22 Briefly, the mixture containing 200 µL of test extract (4 mg/mL DMSO), 0.2 mol/L phosphate buffer having pH value 6.6 (400 µL and) and 1% potassium ferricyanide [K 3 Fe(CN) 6 ] were incubated at temperature 50˚C for twenty minutes. About 400 µL trichloroacetic acid (10%) was added to above mixture and then centrifuged at 3000 rpm for ten minutes at a room temperature. A 500 µL solution from upper layer, distilled water (500 µL) and 100 µL FeCl 3 (0.1%) were mixed. The absorbance was note down at 700 nm wavelength and an increased value of absorbance of the mixture is related with increased reducing power. For the preparation of blank instead of the extract a 200 µL of DMSO was added to above mentioned reaction mixture. Reducing powers of those samples were stated as per µg ascorbic acid equivalent per mg plant dry weight (µg AAE/mg DW). 23

BIOLOGICAL EVALUATION α-amylase inhibitory assay
The assay was performed with the help of modified procedure described by 24 An amount 250 µL of the extract solution was placed in the tube followed by the addition of equal volume of sodium phosphate buffer (0.02M) having pH=6.9 comprising 0.5 mg/mL of -amylase solution.
After pre-incubation of this solution at 25 ∘ C for ten minutes, an amount 250 µL of the starch solution (1%) in a sodium phosphate buffer (0.02M) with pH=6.9 was added in different intervals of time and after that incubation was done for 10 minutes at 25 ∘ C. Reaction was ended through addition of 500 µL of dinitrosalicylic acid (DNS) reagent. Tubes were incubated in the boiling water for five minutes and then cooled at room temperature. After diluting with distilled water (5 mL), the absorbance of the reaction mixture was measured by the use of spectrophotometer at 540 nm wavelength. By the use of same procedure control was set through substituting extract by distilled water. Then -amylase inhibitory potential was determined as %inhibition by the following given formula. 25 Abs control Abs extracts % Inhibition 100 Abs control

Antifungal assay
Agar tube dilution assay was performed for screening of extracts about their in vitro antifungal action against Aspergillus flavus, Candida albicans, Fusarium solani and Microsporum canis. Test samples were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solution. Sabouraud dextrose agar (4%) was prepared in the distilled water. After dissolving the contents with the help of magnetic stirrer, known quantity was distributed into the screw capped tubes. These tubes holding media were then autoclaved for 15 minutes at 121˚C. Then after cooling these tubes to 50˚C, test samples were taken from the stock solution into non-solidified Sabouraud agar medium. Then at room temperature the tubes were permitted to solidify. Inoculation of each tube was done using piece of the inoculum (4mm diameter) detached from 7 days old fungi growth culture. Incubation of the tubes containing culture was done for growth at optimal temperature 28-30˚C for seven days. Humidity (40-50%)

Phytotoxicity assay
In this assay, E-Medium was prepared by mixing a number of constituents in 1L distilled water, the adjustment of pH 6-7 was done by addition of potassium hydroxide pellets (stock solution). The working E-medium was prepared via mixing 900 mL distilled water and 100 mL stock solution. 30 mg of the extract sample was dissolved into 1.5 mL of methanol to make a stock solution of sample. Extract sample was pipetted from stock solution into 3 flasks. The solvent of 3 flasks containing 10, 100 and 1000 μg/mL concentration of extract was evaporated over a night. Working E-medium (20 mL) along with plant Lemna minor (each having rosette of 2 -3 fronds) were added to every flask (total = 20 fronds). The standard drug (paraquat) was present in other flask supplemented with E-medium. The number of fronds was note down in each flask at 7th day after keeping the flasks in the growth cabinet. The results were evaluated as percentage growth regulation by comparing with negative control. 28 Ahmad et al: Natural plant with biological potential and phytochemical constituents Number of fronds in test samples % Regulation 1 00 1 00 number of fronds in negative control = − ×

Preliminary phytochemicals screening
The extracts were preliminarily evaluated for presence of different phytochemical groups with the help of precise chemical-based methods. As indicated by qualitative screening, presence of the terpenoids, tannins, saponins, phenols, and flavonoids was declared in these extracts. The results indicating preliminary phytochemical screening of the extracts are presented in Table 1.

Phytotoxicity bioassay
The phytotoxic capability of dichloromethane and methanol extracts of whole plant was examined with the help of Lemna minor phytotoxicity bioassay. Growth regulation (%) revealed by crude extracts is presented in Table 2. Results exposed that sample MDWM indicated % growth regulation 26% and 36% at the concentration of 10 and 100µg/mL respectively, but at concentration of 1000µg/mL, it presented 73% growth regulation. While sample MDWD indicated % growth regulation 21% and 26% at a concentration of a 10 and 100µg/mL respectively, however at concentration 1000 µg/mL, it revealed 63% growth regulation. In this way, the extracts exposed low growth regulation at concentrations 10 and 100µg/mL however appeared to have moderate % growth regulation at high concentration value (1000 µg/mL) by comparing with a standard drug.

Immunomodulatory activity
Luminol-enhanced chemiluminescence assay showed that sample MDWD had moderate activity against ROS (reactive oxygen species) with 55.9% inhibition while sample MDWM showed insignificant immunomodulatory activity with 38.9% inhibition. The results of immunomodulatory activity are given in Table 3. Ibuprofen was used as standard for this assay with % inhibition at 25 µg/mL = 73.2%

α-amylase inhibitory activity
The extracts were also assessed for α-amylase inhibition activity through standard in-vitro α-amylase inhibition assay. Extracts showed highest inhibitory action against -amylase. The %age inhibition results of M. denticulata extracts on activity of α-amylase are given in Table 4.
The %age inhibition of -amylase increases by increasing concentration of extract as presented in the graph (Figure 1).

Antifungal activity
Antifungal activity of M. denticulata extracts against 4 strains of fungi namelyAspergillus flavus, Fusarium solani, Candida albicans and Microsporum canis is mentioned in Table 5. The information obtained indicates a moderate antifungal action shown by extracts contrary to tested strains. Extracts indicated varying degree of activity against more or less all tested fungi. The maximum inhibition zone was revealed by MDWM extract against Microsporum canis. The effects were compared using standard drugs Amphotericin B and Miconazole. (Table 5)

Total phenolic and flavonoid contents
The recorded values of TFC and TPC in the extracts of M. denticulata are given in

Total antioxidant capacity (TAC) and total reducing power (TRP)
Phenolic components in several medicinal plants have been demonstrated to be promising contributor towards the antioxidant activity. It is shown that among plant extracts tested, MDWD extract was found to reveal highest antioxidant and reducing potential 81.716µg AAE /mg DW and 58.125 μg AAE/mg DW respectively. The phosphomolybdenum based antioxidant capacity and reductive power outcomes of Medicago denticulata extracts are given in Table 6.

DISCUSSION
Considering the results of the qualitative screening, these plant extracts are assumed to contain some health-promoting phytochemicals like terpenoids, tannins, saponins, phenols and flavonoids. The presence of these secondary metabolites in various medicinal plants belonging to different families was also reported by. 29 The past investigations confirm that Medicago genus is appreciated source of the saponins. Triterpene saponins were recognized in Medicago polymorpha as glycosides of hederagenin, echinocystic acid, bayogenin, caulophyllogenin and soyasapogenol B. The Echinocystic acid, (detected 67.3 ± 0.9% of the total sapogenins), was a main aglycone in Medicago species. [30][31][32] A moderate immunomodulatory activity of dichloromethane extract with 55.9% inhibition was exhibited by the plant. The literature about the reported immunomodulatory activities of medicinal plants supports the current findings. Several plants native to Iran were examined for immunomodulatory properties and at higher concentrations these extracts exhibited peripheral blood lymphocytes (isolated from the healthy individual) inhibitory potential. These inhibitory properties of some plants may be due to the induction of apoptosis. 33 The inhibition of ROS generation in whole blood phagocytes by herbal plants has already been reported. 34 Methanolic extract of Enicostema axillare acts on cell-mediated and humoral immune functions, reducing release of inflammatory cytokines in peritoneal macrophages. 35 An alcoholic extract from the bark of Mangifera indica Linn (having 2.6% mangiferin), was explored for its potential on the humoral and cell-mediated components of mice immune system. It revealed promising immunostimulatory effects. 36 M. denticulata has antifungal potential like other Medicago species. The maximum inhibition zone was revealed by MDWM extract against Microsporum canis. Antifungal effect of triterpenoidal glycosides from Medicago sativa has also been previously reported. 37 Among plant extracts tested, maximum phenolic contents were quantified into MDWD extract. This extract also revealed highest antioxidant and reducing potential 81.716 µg AAE /mg DW and 58.125 μg AAE/mg DW respectively. A significant correlation among reducing power, antioxidant capacity, and the total phenolic contents were shown in the investigation, signifying that the phenolic compounds may be main contributor towards antioxidant potential of that plant. Numerous classes of polyphenols, including phenolic acids, flavonoids, lignans, and stilbenes present in the medicinal plants had been linked to their antioxidant potentials which are helpful in preventing age-related diseases, mainly produced via oxidative stress. 38,39 Thus, currently phenols and flavonoids assurance in these extracts propose this medicinal plant to be a source of valuable natural antioxidants. The previously reported antioxidant activity of Medicago polymorpha extract against free radicals for example DPPH, ABTS and significant reducing power 40, 41 support the above findings.
Phytotoxicity is an important factor to consider when determining a plant's allelopathic potential. The determination of a plant's phytotoxicity aids in the development of biological herbicides or naturally occuring plant growth regulators. The necessity for phytotoxic compounds cannot be overstated, as the majority of the agricultural products are affected by weeds. Human health is negatively impacted by synthetic herbicides. Researchers are battling to find an effective, safe and human health-friendly phytotoxic compounds. Due to the occurence of natural phytotoxic components in the current studied plant make it valuable to isolate important agro-chemicals. Phytotoxic effects of crude methanolic extracts of various parts of some medicinal plants on the germination of radish seeds were investigated. 42 The phytotoxicity of medicinal plants was also described by. 43 The extracts showed a concentration dependent significant α-amylase inhibitory activity. α-amylase catalyses hydrolysis of 1,4-glycosidic bonds of carbohydrates into monosaccharide single sugar units that can be absorbed more easily from intestine. Inhibition of α-amylase enzyme in human digestive tract is thought to be useful in treating diabetes by reducing of glucose absorption. 44 It is indicated from the studies that Azadirachta indica, Mangifera indica and Murraya koenigii are beneficial to manage postprandial hyperglycemia. 45 The pancreatic α-amylase inhibitory action of roselle (Hibiscus sabdariffa Linn.) tea extract was described by. 46 α-amylase enzyme was significantly inhibited by Phyllanthus amarus suggesting that the plant contains potential α-amylase inhibitor compounds that may contribute to its antidiabetic activity. 47

CONCLUSION
The current study concludes Medicago denticulata as a potential source of phytochemicals. The dichloromethane extract (MDWD) and methanolic extract (MDWM) have potential phenolic and flavonoid contents with important biological activities. Maximum gallic acid equivalent (GAE) total phenolic contents [54.47 and 41.35 μg GAE/mg dry weight] and quercetin equivalent (QE) total flavonoid contents (13.49 and 16.72 μg QE /mg DW) were documented in DCM and methanolic extracts of M. denticulata respectively. Maximum antioxidant capacity represented as ascorbic acid equivalent in DCM and methanol extracts was 81.71 and 72.01 01μg AAE / mg DW respectively, however reducing power noted as ascorbic acid equivalent in DCM and methanol extracts was 58.12 & 54.51 01μg AAE /mg DW. Screened plant extracts are found to have adequate antioxidant potential which may be credited to the existence of flavonoid and phenolic content. The effects of immunomodulatory activity conclude that sample MDWD revealed moderate activity against ROS. The findings also confirm the antifungal, phytotoxic and -amylase inhibitory potential of these extracts. Inhibition of enzyme α-amylase (responsible to hydrolyze carbohydrates) is a significant tactic in lowering postprandial blood glucose level. Obtained results demand further pharmacological description and the bioactivity guided isolation of those components that are responsible for these observed activities.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
α-Tocopherol Prevents Sperm Apoptosis and Necrosis in Rats Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant that induces overproduction of reactive oxygen species (ROS). Studies on avoiding the adverse effects of dioxin pollution exposure are needed in all aspects, including reproductive health. This study aimed to determine the effect of α-tocopherol on superoxide dismutase (SOD) and malondialdehyde (MDA) levels, live spermatozoa, apoptosis, and necrosis in male rats exposed to dioxin as a model. Thirty healthy 12-week-old male rats were randomly divided into five groups. Rats in the control group were given corn oil twice daily at 4-hour intervals. The remaining rats were given TCDD 700 mg/kg BW daily, followed by administration of corn oil and α-tocopherol at doses of 77, 140, and 259 mg/kg BW/d for T0, T1, T2, and T3 groups, respectively. The treatments were conducted for 45 days; all rats were euthanized to collect blood and testicular samples on day 46. The results showed that exposure of TCDD resulted in a decrease in SOD activity and live spermatozoa and increased MDA level and death, apoptosis, and necrosis of spermatozoa (T0) compared to the control (C) group (p < 0.05). The administration of α-tocopherol, starting from the doses of 77 (T1), 149 (T2), and 259 mg (T3) per kg BW, was sequentially followed by returning MDA levels, recovering SOD activities, and restoration in the percentage of living, dead, apoptotic, and necrotic spermatozoa, similar (p > 0.05) to those of the control group. It could be concluded that the administration of α-tocopherol resolves the harmful effects of TCDD on the viability of spermatozoa in rats as a model.

Introduction
e formation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was as a by-product in the burning processes of organic materials or a side product in organic synthesis. It was a persistent organic pollutant and a colorless solid with no distinguishable odor at room temperature [1]. TCDD induced premature senescence in neuronal cells by promoting intracellular ROS production that accelerates the onset of neuronal senescence to induce neurotoxic effects [2]. e neuroendocrine activity along the hypothalamicpituitary-testicular axis regulated spermatogenesis [3]. A reverse set of changes in the AhR protein and mRNA response to TCDD suggested that AhR functions as a physiological regulator [4]. Our previous study indicated that TCDD depressed spermatogenic staging, number of Leydig cells, spermatozoa, seminiferous tubule diameter, and thickness of tubule epithelium [5]. TCDD caused mitochondrial dysfunction and the accumulation of cellular reactive oxygen species (ROS) [2]. Polyunsaturated fatty acids in the cell membrane, including the membrane of mitochondria, are sensitive to ROS exposure, causing lipid peroxidation.
e end product of lipid peroxidation was MDA; therefore, MDA was a biomarker for oxidative stress [6].
In living beings, there are several endogenous antioxidants, including SOD. SOD is essential for protecting cells against oxidative stress [7]. e antioxidant matter can scavenge free radicals due to TCDD [8]. Meanwhile, α-tocopherol is an exogenous antioxidant, a fat-soluble nonenzymatic antioxidant. e antioxidant α-tocopherol can break the TCDD chain bond with AhR (aryl hydrocarbon receptor) found in the cell cytoplasm, thus protecting cells from lipid peroxidation [7]. α-tocopherol was a fertility triggering agent that can normalize seminiferous tubular epithelium [9] and had a protective effect on oxidative stress and sperm apoptosis in diabetic mice [10]. e administration of α-tocopherol proved to repair the damage of testicle histology in rats exposed to dioxin [5]. e decreasing spermatogenic staging due to TCDD exposure may be caused by the death of sperm due to apoptosis and necrosis. ere is no report about the use of α-tocopherol to avoid apoptosis and necrosis of spermatozoa on rats exposed to TCDD. erefore, this study aimed to determine the effect of α-tocopherol for maintaining sperm viability, decreasing sperm apoptosis and necrosis through maintaining SOD, and decreasing MDA levels on male rats exposed to dioxin.

Ethical Approval.
e experimental protocol of this study was approved by the Animal Care and Use Committee, Airlangga University, Surabaya, Indonesia (no. 267/ HRECC.FORM/VI/2020). Experiments have been conducted with minimum pain or discomfort, following the guidelines established by the Institutional Animal Ethics Committee.

TCDD and α-Tocopherol Dosages.
e dose determination of TCDD and α-tocopherol (both from Sigma-Aldrich, Singapore) was based on our earlier study [5]. is study used a single dose of 700 ng/kg BW/day TCDD. Meanwhile, the doses of α-tocopherol were 77, 140, and 259 mg/kg/day. Corn oil (Mazola ® , Codaa Switzerland AG) was used as a solvent for TCDD and α-tocopherol.

Treatment of Experimental Animals.
irty healthy male rats (Rattus norvegigus, Wistar strain) aged 12 weeks, weighing about 200 grams, were randomly divided into five groups equally for control (C), T1, T2, T3, and T4 group. e control group rats were administered corn oil twice a day in four-hour intervals. e remaining rats in the treatment group were given TCDD 700 mg/kg BW/day, and four hours later, they were given corn oil, α-tocopherol 77, 140, and 259 mg/kg BW/day for the T0, T1, T2, and T3 groups, respectively. TCDD and α-tocopherol were administered orally in 1 mL volume daily for 45 days, and on day 46, all rats were euthanized for blood and testicular samples. Euthanasia was performed via decapitation by trained personnel.

SOD Activity Measurements.
e tunica albuginea of the testes was removed; then, the testes were homogenized at a ratio of one gram tissue per ml of 0.05 M Tris-HCl buffer pH 7.5. e homogenate was centrifuged at 12000 × g for 15 minutes to remove most of the tubules and other large sediment tissue fragments. e supernatant was centrifuged for 20 minutes at 50,000 × g and 150,000 × g for 60 minutes. e lipid layer on the surface of the supernatant was removed after each centrifugation, and the final supernatant was used for SOD activity measurement using a colorimetric superoxide dismutase activity assay kit (CS0009, Sigma-Aldrich, Singapore) at a wavelength of 550 nm [11].

MDA Level Measurements.
Blood samples were collected from below the aortic arch for measuring serum MDA levels using the thiobarbituric acid-reactive substance (Sigma-Aldrich, Singapore) with a UV-1601 spectrophotometer at a maximum wavelength of 535 nm [12,13].
2.6. Live and Dead Spermatozoa Count. Semen was collected from the cauda epididymis and diluted 200 times with 0.9% NaCl. A drop of the semen sample and a drop of eosinnigrosin were mixed homogeneously, smeared, and quickly dried over the flame. e viability of the sperm was examined under a light microscope Nikon E200 (Nikon Corporation, Tokyo, Japan) at 400× magnification. Live spermatozoa were white and transparent on the head since they did not absorb color, while the dead spermatozoa absorbed a red-purple color [14]. e percentages of live and dead spermatozoa were determined out of the total of 300 spermatozoa.

Apoptosis and Necrosis Spermatozoa Count.
Dry smeared semen samples were fixed with absolute methanol and glacial acid for 15 min, stained with acridine orange, and then observed with a fluorescence microscope at 100× (Nikon Eclipse E800, Tokyo, Japan). Apoptotic spermatozoa were yellow to reddish, and the necrotic ones were brownish-orange. Meanwhile, live spermatozoa were greenstained [15,16]. e percentages of apoptosis and necrosis spermatozoa were determined out of the total of 300 spermatozoa.

Statistical Analysis.
e data were analyzed using a oneway ANOVA followed by Tukey's honestly significant difference test at a 95% confidence level (SPSS version 23, IBM, New York, United States).

Results
TCDD exposure was followed by a decrease in SOD activity by half and an increase in MDA levels twice (T0) compared to the control group (C) (p < 0.05). Administration of α-tocopherol starting at a dose of 140 mg per kg BW restored the same SOD activity as in the normal mice (group C) ( Table 1).
Different superscripts in the same column show significant differences (p < 0.05). Rats in the control group (C) were administered 0.5 mL of corn oil twice daily at 4 h intervals for 45 d. Rats in groups T0, T1, T2, and T3 were administered TCDD 700 ng/kg BW daily and 4 h later were administered corn oil, 77, 140, and 259 mg/kg BW daily α-tocopherol, respectively, for 45 d.
3.1. Live, Dead, Apoptotic, and Necrotic Spermatozoa. Rats exposed to TCDD (T0 group) showed decreases of live spermatozoa (leaving only about 40%) and 4-5-fold increases, respectively, in dead, apoptotic, and necrotic spermatozoa compared to normal rats (C group) (p < 0.05). Administration of α-tocopherol resulted in increased live spermatozoa. e dose of 259 mg α-tocopherol per kg BW recovered all of those parameters to those of the control group (p > 0.05) ( Table 2 and Figures 1 and 2).
Different superscripts in the same column show significant differences (p < 0.05). Rats in the control group (C) were administered 0.5 mL of corn oil twice daily at 4 h intervals for 45 d. Rats in groups T0, T1, T2, and T3 were administered TCDD 700 ng/kg BW daily and 4 h later were administered corn oil, 77, 140, and 259 mg/kg BW daily α-tocopherol, respectively, for 45 d.

Discussion
TCDD is a toxic substance that induces oxidative stress, which is an imbalance between free radicals and antioxidants [8]. Free radicals react with polyunsaturated fatty acids, resulting in lipid peroxidation [17]. MDA was a marker of lipid peroxidation [18]. Lipid peroxidation is a degenerative process that plays a crucial role in the pathogenesis of many diseases by decomposing hydroperoxide products into cytotoxic derivatives [19]. Actually, there are many antioxidants in the living cell as a defense mechanism against the oxidant, such as superoxide dismutase (SOD), catalase (CAT), and seleno-dependent glutathione peroxidase (GSH-Px) [20]. However, those antioxidant levels decrease when there is excessive free radical production [21].

SOD and MDA.
e SOD activities were lower in rats exposed to TCDD (T0) than those of normal rats. TCDD activated the aryl hydrocarbon receptor (AhR) and led to dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) and transcriptional activation of several xenobiotic-metabolizing enzymes, such as SOD. SOD was essential for protecting cells against the harmful superoxide radical derived from mitochondrial respiratory chain leakage. SOD causes the dismutation of the superoxide radicals to molecular oxygen and hydrogen peroxide [7,13].
SOD inactivation was resulted from oxidative stress due to excessive reactive oxygen species (ROS) [22]. Administration of α-tocopherol, starting from 140 mg per kg BW, was sufficient to restore the SOD activity level to that of the normal rats. is may be due to the antioxidant of α-tocopherol scavenges excess free radicals in cells, preventing SOD inactivation [20]. e MDA level of rats exposed to TCDD were higher than those of normal rats. Polyunsaturated fatty acids of the cell membrane easily accepted unpaired electrons from ROS, causing lipid peroxidation [6]. e end product of lipid peroxidation was MDA; therefore, MDA was a biomarker for oxidative stress [23]. e increased MDA levels in rats exposed to TCDD reflect the level of damage to the plasma membrane attacked by free radicals. When ROS levels were higher than endogenous antioxidants (among others is SOD), membrane integrity was lost, and MDA levels increased. Administration of α-tocopherol starting from 77 mg per kg BW was sufficient to return MDA levels like those of normal rats. e result of this study was following the earlier report of the higher antioxidant level, followed by the lower MDA levels [24].

Live Spermatozoa.
In the control group, about 14-15% of the spermatozoa population died. e normal metabolic activity could also cause death in the spermatozoa itself because the lactic acid produced lowered semen pH [25]. e rat sperm viability decreased along with increasing the dead, apoptotic, and necrotic spermatozoa due to the TCDD exposure. A substantial decrease in the quality of spermatozoa occurred due to a large number of dead spermatozoa [26]. Short-term exposure to TCDD has been shown to affect live spermatozoa [27]. TCDD elicits its effects by inducing AhR degradation and requires ubiquitination of the protein as a mechanism of mediated gene induction [28]. Sperm DNA fragmentation due to increased ROS affected live spermatozoa [29]. TCDD also inhibited the action of the 17β-hydroxysteroid oxidoreductase enzyme in testosterone synthesis, causing a decrease in testosterone levels. Testosterone was essential in maintaining the viability of spermatozoa as long as they are stored in the epididymis [30][31][32].
α-tocopherol could protect the health of the mitochondrial plasma membrane to maintain the viability of spermatozoa [33]. In this study, a dose of 259 mg/kg BW/d α-tocopherol completely covered the effects of TCDD on sperm viability. e antioxidant effects of α-tocopherol worked not only at the spermatogenesis stage but also during endocrine regulation that maintains spermatozoa's viability [33]. α-tocopherol is a fat-soluble antioxidant, so it is more effective as a protector against oxidative stress [34], and it scavenges ROS and stops the chain propagation reaction in the lipid peroxidation process [9,10] due to TCDD is also fat-soluble. erefore, α-tocopherol is essential in maintaining the physiological integrity of the testicular cells, epididymis, and accessory glands that play essential roles in spermatogenesis and the maturation and viability of spermatozoa [35,36].
ese results were consistent with the findings of Ghafarizadeh et al. [37] that α-tocopherol increases sperm viability in vitro.

Necrosis and Apoptosis.
Cell death could be caused by apoptosis and necrosis; both could be induced by toxic substances [38], including overproduction of ROS [39]. Staining with ethidium bromide and acridine orange in necrotic cells made all the cells appear brown [16,40]. In apoptotic cells, not all dyes could enter the cell because the cell membrane was still intact, but the DNA was fragmented [41,42] and stained so that the nucleus is yellow with brownish-red cytoplasm [16,40]. Necrosis was an acute and irreversible cell death due to damage to the plasma membrane that could not maintain homeostasis to allow the entry of water and extracellular ions [43]. Cell necrosis may occur due to the activity of the lysozyme that digests the cell membrane's organelles, such as the mitochondrial membrane, ribosomes, and other cell apparatus, including intracytoplasmic fluid, followed by cell lysis [44]. However, the cell damage was reversible if the cause was immediately removed [45]. Apoptosis was a programmed cell death that was not preceded by cell swelling or inflammation. Apoptotic cells would shrink due to a breakup of the cell nucleus and chromosomes, which all form apoptotic bodies [46]. e mitochondrial membrane was rich in lipids sensitive to free radical attack [47]. In the mitochondrial membrane, there were Bcl-2 or Bcl-XL proteins that bound to Bax proteins that regulate the mechanism of apoptosis [48]. e apoptosis is triggered by lipid peroxidation, which leads to ROS activation in a continuous redox cycle. is cascade also includes the increased activity of a p53 protein, which will activate the Bax protein and then stimulate mitochondria to produce excessive cytochrome c that will cause apoptosis [44]. Cytochrome c is made up of apoptogenic proteins, such as the apoptosis-inducing factor and the endonuclease G [35], followed by caspase activation and exposure to phosphatidylserine on the surface of spermatozoa a few hours later [49]. e increasing ROS affected the damage of DNA, especially the integrity of DNA as a cause of cell death [50], and caused the limited cytoplasmic capacity for DNA repair. e higher DNA fragmentation correlated with the activation of caspase-3 [51] as the executioner of apoptosis [52]. Antioxidant effect of α-tocopherol at 259 mg/kg BW recovers live, dead, necrosis, and apoptosis of spermatozoa which returns to like those of normal rats.
An increase in MDA levels and a decrease in SOD indicated oxidative stress followed by cell death mechanisms, such as apoptosis or necrosis [53]. Administration of α-tocopherol started from a dose of 77 and 140 mg per kg BW, returning MDA levels and SOD activity to those of normal rats. However, a dose of 259 mg/kg BW α-tocopherol was needed to recover the percentage of living, dead, apoptosis, and necrosis of spermatozoa. is may be due to the effect of TCDD on sperm viability, and its recovery by α-tocopherol requires a more complicated mechanism and extended pathway than are needed to recover MDA levels and SOD activity. e maintaining of testicle health involves the dynamics of complex upregulation of the endocrine. e explanation of α-tocopherol role for recovery testicular damage due to TCDD exposure in this study was limited based on SOD and MDA levels. erefore, further studies were needed to determine changes in GnRH, FSH, LH, and testosterone levels, including the role of α-tocopherol to prevent apoptosis of testicular tissue at gene level such as AhR, Bax, and Bcl casp3.
Necrosis is acute and irreversible cell death due to damage to the plasma membrane that cannot maintain homeostasis to allow the entry of water and extracellular ions [43]. Cell necrosis may occur due to the activity of the lysozyme that digests the cell membrane's organelles, such as the mitochondrial membrane, ribosomes, and other cell apparatus, including intracytoplasmic fluid; damage is caused to the cells, followed by cell lysis [44]; however, the cell damage is reversible if the cause is immediately removed [45]. Administration of α-tocopherol starts from a dose of 77 and 140 mg per kg BW, returning MDA levels and SOD activity to those of normal rats. MDA levels and SOD activity of rats exposed to TCDD and treated with α-tocopherol at dose 140 or 259 mg/kg BW daily were not statistically significant (p > 0.05) than those of normal rats (control group). Table 1: SOD activity (%) and MDA level (nm/mL) of rats exposed to TCDD and treated with α-tocopherol.  Table 2: e percentage of live, dead, apoptotic, and necrotic spermatozoa (%) of rats exposed to TCDD and treated with α-tocopherol.  Figure 1: Live and dead sperm of rat exposed to TCDD and treated with α-tocopherol. Rats in the control group (C) were administered 0.5 mL of corn oil twice daily at 4 h intervals for 45 d Rats in groups T0, T1, T2, and T3 were administered TCDD 700 ng/kg BW daily and 4 h later were administered corn oil, 77, 140, and 259 mg/kg BW daily α-tocopherol, respectively, for 45 d Live spermatozoa do not absorb color on the head, whereas dead spermatozoa absorb a red-purple color from the stain (eosin-nigrosin staining), at 400× magnification under a light microscope Nikon E200 (Tokyo, Japan).

Group
Veterinary Medicine International C T0 T3 T4 T1 Figure 2: Apoptotic and necrotic spermatozoa of rats exposed to TCDD and treated with α-tocopherol. Rats in the control group (C) were administered 0.5 mL of corn oil twice daily at 4 h intervals for 45 d. Rats in groups T0, T1, T2, and T3 were administered TCDD 700 ng/kg BW daily and 4 h later were administered corn oil, 77, 140, and 259 mg/kg BW daily α-tocopherol, respectively, for 45 d. e membrane of apoptotic spermatozoa will be green with yellowish cytoplasm and nucleus, and necrotic spermatozoa will be brown. Meanwhile, live spermatozoa were green (acridine orange staining), at 100× magnification under a fluorescence microscope (Nikon Eclipse E800, Tokyo, Japan).

Conclusion
α-tocopherol rebalances SOD and MDA levels to recover spermatozoa's viability from necrosis and apoptosis death of male rats exposed to TCDD.

Data Availability
e data used to support the findings of this study are available from the first author.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
DKM, IM, and WW compiled ideas, designed the research work, and provided the draft manuscript. KR, ISH, and WW performed treatment of rats. NS and SU measured SOD activity and MDA levels. DKM, DKSCP, and KR evaluated viability, apoptosis, and necrosis of spermatozoa. DKM and IM conducted the statistical analysis and conceived the manuscript. ISH, NS, DKSCP, and SU critically read and revised the manuscript for intellectual content. All authors read and approved the final manuscript.


text:
Biological characteristics of Russian black pied cattle

The biological characteristics of farm animals depend on their species and breed differences, but at the same time they are subject to certain variability due to individual heredity. In this regard, from the number of first-calf cows (daughters of different bulls) eight groups of the same age cows were organized and examined. In addition, a group of 65 animals was formed to assess the exterior by the method of random sampling without taking into account their heredity. The current research is aimed to evaluate the first-calf cows in the following options: milk productivity, functional properties of the udder, body type and the main disadvantages of the exterior. As a result of the analysis, Alta Genetics cows showed the best milk productivity among other breeds. The cows of Alta Gost and Alta Samuari breeds turned out to have the best indicators for a number of economically useful traits. The AltaGost cows were characterized by the best milk yield. When assessing the exterior features and constitution, it was found that the greatest number of conformation defects is associated with the limbs: close hocks (35%); wide interdigital cleft (71%); narrow hoof (22%). Nevertheless, a visual assessment of a complex of features allows to refer all the examined cows to a good body type.

Relevance
One of the current trends in the development of cattle breeding is the use of intensive milk production technologies. At the same time, the requirements for the dairy cattle as the main mean of production are increasing. Nowadays the cows used in a high-technological agricultural complex should have high productivity, strong exterior and excellent reproductive qualities. [2,[5][6][7][8]

Methods and Materials
In regard to the aim of the current research and the formulated tasks in the breeding economy of LLC "Konstantinovo" (Penza region, Russia), 8 groups of cows were formed from the number of first-calf cows (daughters of different proven bulls). The scheme of the experiment is given below (Figure 1).
The selection of data for analysis was taken from zootechnical and breeding records of the SELEX program. The level of milk productivity of cows was assessed by milk yield for 305 days of lactation or the entire shortened lactation. In addition, the quality indicators of milk from the experimental animals were assessed. The milk flow rate and the milk yield index were calculated according to the generally accepted method. Thus, a group of 65 animals was formed to assess the exterior by the method of random sampling without taking into account their heredity. A comprehensive description of the exterior and constitution of cows was organized according to a 100-point system with 5 indexes: general exterior -20%, milk characteristics -15%, body size -10%, legs -15%, udder -40%. According to the obtained results for 5 indexes in a complex assessment, a general complex score for  When determining the selection and genetic parameters of the studied indexes in all groups, the following options were calculated: the arithmetic mean (M) and its error (m), variability expressed by the standard deviation (σ) and the coefficient of variation (Cv) according to generally accepted methods. The significance of differences between the characteristics was assessed by comparison with the Student's t-test. At the same time, the probability of a prediction error was established according to three reliability thresholds (* -Р<0.05; ** -Р<0.01; *** -Р<0.001). Statistical data processing was carried out on a personal computer using the Excel program. All indicators of milk productivity of the daughters of each bull were compared with the average data of all first-calf heifers of the herd.

Results
As a result of assessing the milk productivity of first-calf cows, taking into account their heredity (Table 1, Table 2), it could be seen that in terms of milk yield, a significant superiority of the daughters of Alta Samuari was revealed. The difference was 252 kg (P <0.05). The daughters of Alta Gost showed higher milk yields, but the difference was not significant. It should be noted that all Alta Genetics daughters tend to have superiority over the daughters other bulls' offspring. The daughters of the Benedict, Carl-A Appleton, Titanium and Eubrox daughters differed in milk yield below the average of all first-calf cows in the herd, relatively, by 394 kg. (P <0.001); 59 kg; 708 kg (P <0.01), and 515 kg (P <0.01). There were no significant differences in the mass fraction of fat and protein in milk. However, due to the high milk yield of the Alta Gost's daughters, they surpassed in milk fat yield by 18.6 kg (P <0.05), and the daughters of Alta Samuari -by 9 kg (P <0.05). The latter also differed in protein yield, their superiority was 7 kg (P <0.05). The fat and protein yield was significantly lower than the average for first-calf heifers in the Benedict's daughters by 15.3 kg and 11.5 kg (P <0.001), and the Eubrox's daughters -by 17.6 and 14.8 kg (P <0.01). When using cows in modern dairy complexes, much attention is paid to the milking time of the cow. The milk flow rate and the udder capacity are the main features characterizing its functional properties ( Table 2).
The daughters of AltaGost were characterized by the best milk yield at the highest daily milk yield with a difference of 0.6 kg/min by the first index and 3.0 kg by the second one (P <0.001). The daughters of Benedikt, Carl-A Appleton, Titanium and Eubrox had significantly lower indicators for both studied characteristics.
Characteristics of cows in terms of weight and milk productivity ratio are given below (Table 3). There were no differences in weight of first-calf cows. This index is significantly more dependent on the level of feeding the young herd during rearing and is determined by the plan of rearing the young herd, as evidenced by the low variability of the index. At the same time, the daughters of Alta Samuari showed the best milk yield -16.7 kg, that shows the superiority over the other cows of 0.6 kg (P <0.05). The daughters of Benedict, Titanium and Eurbrox, on the other hand, were inferior in terms of milk productivity by 0.8; 1.3 (P <0.001) and 0.8 (P <0.05), relatively.
In improving the productive qualities of animals, breeding of individuals of the desired body type is of great importance. In countries with developed dairy cattle breeding, and more recently in Russia, for the selection of animals of the desired type, a linear assessment of the exterior is used, which makes it possible to evaluate the exterior of the daughters from different bulls to carry out selection in order to correct constitutional deficiencies. [1,3,4,9] Visual assessment for a complex of indexes showed that animals scored more than 80 points for all its components, which allows us to classify them as "good with a positive tendency" in terms of body type.  At the same time, first-calf heifers received the lowest score of 77 points for the complex of indexes characterizing the limbs, despite their correct formulation, revealed during the linear assessment (Table 4). To detail the reasons for the decrease in the score of the body type index, it is necessary to take into account the shortcomings of the exterior (Figure 2).   Figure 2 shows the disadvantages that frequently occur than 10%. These include the disadvantages of the udder: inclined bottom (17%); asymmetry (16%); close nipples (40%). The greatest number of deficiencies is associated with the limbs: close hocks (35%); wide interdigital cleft (71%); narrow hoof (22%). The last listed disadvantages, among other reasons, increase the risk of limb disease, and could lead to abnormal growth of the hoof horn, injury to the sole or interdigital space.

Conclusion
As a result, the following results were obtained. Since the daughters of the bulls Alta Gost and Alta Samuari have a significant superiority over the other examined cows, it could be recommended their preferred use as the bulls-improvers at dairy complexes. When selecting bulls, it is advised to assign herds of bulls to cows, assessed by the exterior of their daughters. Also it is important to pay special attention to the structure of the limbs and the characteristics of the udder.


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Population genetic structure analysis and identification of backfat thickness loci of Chinese synthetic Yunan pigs

Yunan is a crossed lean meat pig breed in China. Backfat thickness is the gold standard for carcass quality grading. However, over 14 years after breed registration, the backfat of Yunan thickened and the consistency of backfat thickness decreased. Meanwhile, no genetic study has been ever performed on Yunan population. So, in this study we collected all the 120 nucleus individuals of Yunan and recorded six backfat traits of them, carried out population genetic structure analysis, selection signals analysis and genome-wide association study of Yunan pigs with the help of their founder population Duroc and Chinese native Huainan pigs, to determine the genomic loci on backfat of Yunan. Genetic diversity indexes suggested Yunan pigs had no inbreeding risk while population genetic structure showed they had few molecular pedigrees and were stratified. A total of 71 common selection signals affecting growth and fat deposition were detected by F ST and XP-CLR methods. 34 significant loci associated with six backfat traits were detected, among which a 1.40 Mb region on SSC4 (20.03–21.43 Mb) were outstanding as the strong region underlying backfat. This region was common with the results of selection signature analysis, former reported QTLs for backfat and was common for different kinds of backfat traits at different development stage. ENPP2, EXT1 and SLC30A8 genes around were fat deposition related genes and were of Huainan pig’s origin, among which Type 2 diabetes related gene SLC30A8 was the most reasonable for being in a 193.21 Kb haplotype block of the 1.40 Mb region. Our results had application value for conservation, mating and breeding improvement of backfat thickness of Yunan pigs and provided evidence for a human function gene might be reproduced in pigs.

Introduction
Huainan is one of the oldest northern China native pig breeds. It is native to south of the upper reaches of Huai River and North of Dabie Mountains in Henan Province. Henan is located in the middle and lower reaches of the Yellow River and is one of the earliest pig domestication areas in China (Zhang et al., 2007). Huainan is an all-black pig breed with large body size, large ears, short mouth, strong and robust limbs, high fertility, strong adaptability and delicious meat quality (Wang et al., 2005). People in and around Huainan pig producing areas have the habit of eating black pigs. Black pig is an essential ingredient of the famous local cuisine there. However, the poor growth rate of Huainan pigs limited its development.
Since 1980s, a large number of foreign commercial pigs with fast growth rate and high lean meat rate have been introduced to China to improve growth performance of Chinese native pig breeds (Xu, 2004). Among them, the American Duroc with golden coat color was widely used because of its high growth rate and high feed conversion rate ( Figure 1A). So, in 1996, we started to use Huainan and American Duroc pigs as the base herd to intercross to develop a new high-performance black pig, Yunan pig (Figures 1B,C) (Zhu et al., 2009).
Yunan pig is a black crossbred pig breed obtained by nine generations crossbreeding between Huainan and American Duroc ( Figure 2). In 2008, Yunan was registered as a new pig breed in China by National Livestock and Poultry Genetic Resources Committee. Theoretically, it contains 37.50% of Huainan's lineage and 62.50% of Duroc lineage. Yunan exhibits good growth performances from Duroc with an average daily gain of 648 g during the stage of body weight at 30 kg-90 kg and lean meat rate of 56%, and has good meat quality from Huainan with an excellent meat quality (intramuscular fat content as 4.11%). So, Yunan becomes a popular black pig breed in and around Henan.
Since the year of 2008, the systematic breeding of Yunan pigs have been no longer carried out which resulted in a decline of backfat consistency. Backfat thickness now is still the only gold standard for black pig carcass grading as same as the commercial pigs in China. Therefore, the decline of backfat performance of Yunan brings great economic loss to farmers. Meanwhile, with the emergence of the African swine fever in China in 2018, the population of Yunan pigs reduced seriously.
However, there has never been a genomic study on Yunan pigs. Only a few correlation analyses of several genes and comparative analysis of production traits of Yunan were reported (Li et al., 2016;Wang et al., 2021). Herein, in this study, we recorded six backfat thickness traits of the core group of Yunan population, and used SNP array genotyping data of Yunan, Huainan and Duroc with the following objectives: 1) determine the genetic diversity of Yunan by calculating the observed heterozygosity and the expected heterozygosity; 2) access the inbreeding state by detecting runs of homozygosity (ROH); 3) detect selection signals of Yunan by pairwise F ST and XP-CLR methods; 4) investigate genomic evidence on the population structure of Yunan by phylogenetic tree, principal component and admixture analysis; 5) identify the genomic region that controlled backfat thickness of Yunan.
We used PLINK v.1.9 (Purcell et al., 2007) to perform the quality control (QC) of the total 193 individuals genotype data. SNPs without positions on pig reference genome (Sus scrofa 11.1), or with genotyping rate less than 90%, or with minor allele Frontiers in Genetics frontiersin.org 02 frequency (MAF) lower than 1% or on Y chromosome were excluded. Individuals with genotyping rate less than 90% were excluded. Genotypic data of the same SNP in Yunan, Huainan and Duroc populations were extracted for genetic diversity, genetic differentiation and population genetic structure analyses.

Genetic diversity and selection signal analysis
Genetic indicators including the observed heterozygosity (Ho), the expected heterozygosity (He) and MAF were calculated for Yunan, Huainan and Duroc by PLINK v.1.9 to compare the genetic diversity of these populations, command set is "-hardy". In addition, SNePv1.1 (Barbato et al., 2015) software was used to calculate the effective population size (Ne).
The length and frequency of ROH can reflect the group history. A long ROH indicates recent inbreeding, while a short ROH indicates ancient inbreeding. Genomic homozygous fragments of each individual were detected using runs of homozygosity of PLINK v.1.9. The following parameters were used of define ROHs: 1) the minimum length of ROHs was 500 Kb; 2) a sliding window of 50 SNPs across the genome; 3) each window allowed one heterozygous genotype and five missing SNPs  to avoid false negatives caused by occasional genotyping errors and missing genotypes. Then the ratio of the total length of the ROH fragment to the total length of autosomal genome was calculated to get the coefficient of inbreeding (Silió et al., 2013) using the following formula: where L ROH is the sum of the lengths of the autosomal ROH fragments and L auto is the total size of 18 autosomes of pigs covered by SNPs, which is 2.265 Gb (https://www.ncbi.nlm.nih.gov). We used SNP data from three breeds, using genetic differentiation coefficient (F ST ) and cross-population compound likelihood ratio test (XP-CLR) for genome-wide selective detection. The genetic differentiation coefficient (F ST ) between populations was calculated using Vcftools v.0.1.13 (Sun et al., 2020), The command set was as follows: 1) window size was set to 500 Kb; 2) step length was set to 40 Kb. The F ST values ranging from 0 to 0.05, 0.05 to 0.15, and 0.15 to 0.25 indicate that there are no genetic, moderate, and large differentiations among populations, respectively. XP-CLR was calculated using XP-CLR (Chen et al., 2010) software. XP-CLR models the difference in frequency of multiple alleles between the two populations. Further, we used the parameters ("-w1 0.005 200 2000 -p0 0.95") to calculate the XP-CLR score for each chromosome. The empirical cutoffs for the genomic windows with top 1% F ST and XP-CLR values across the whole genome were considered as selective sweeps.

Population genetic structure analysis
To assess the individual genetic distances between populations to illustrate the relationship between populations, we carried out principal components analysis (PCA) of the SNP dataset using PLINK v.1.9 and selected the first two principal components for visualization by ggplot package in R v.4.1.3 (Team, 2014). To gain more insight into the Yunan pig population, we performed t-distributed stochastic neighbour embedding (T-SNE) analysis on the dataset using the R package "Rtsne" (Krijthe, 2015). The phylogenetic tree was built by neighbor-joining (NJ) method and visualized using R v.4.1.3, the genetic distance matrix was constructed by PLINK v.1.9. To explore the population structure of Yunan, the rapid model of ADMIXTURE v.1.3 (Alexander et al., 2015) software was used to cluster all Yunan samples in a context of some related populations and the results were visialized using the "barplot" package of R v.4.1.3 software.

Genome-wide association analysis of backfat thickness
Genome-wide association analysis for backfat traits of Yunan was performed using a univariate mixed linear model of GEMMA v.0.98.5 (Zhou et al., 2012) as follows.

FIGURE 2
Schematic representation of the setup to generate Yunan using Huainan and Duroc.
Frontiers in Genetics frontiersin.org where y is a vector of phenotypic values for the trait; W is a matrix of fixed effects including the first three PCs; α is a vector of corresponding coefficients including the intercept; x is a vector of genotypes for the marker; β is an effect size for the marker; u is an n-vector of random effects; and ε is an n-vector of errors.
The results of genome-wide association analysis were visualized using the rMVP (Yin et al., 2021) package in R software. The suggestive significance threshold was 1/N, where N is the number of SNPs used for the analysis. We used 1 Mb upstream and downstream significant SNPs region as traits related candidate region.

Genotyping
There are 57,466 SNPs sites in the original data. After quality control, SNPs without positions on pig reference genome (Sus scrofa 11.1), or with genotyping rate less than 90%, or with minor allele frequency lower than 1% or on Y chromosome were excluded. Individuals with genotyping rate less than 90% were excluded. Finally, 50,717 SNPs were used for the following analysis.

Genetic diversity analysis
To assess genetic diversity of Yunan, we analyzed MAF, Ho, He and Ne of Yunan, Huainan and Duroc. The results were shown in Table 1. By comparison, the Ne of Huainan was lower than that of Yunan and Duroc. Indexes MAF, Ho and He of Yunan were higher than those of Huainan and Duroc indicating that Yunan was rich in genetic diversity.
ROH analysis results of Yunan, Huainan and Duroc were shown in Table 2. A total of 3,317 homozygous fragments were detected in the three populations. Duroc had the highest average number of ROH per animal (48.8 ± 6.55 with a range of 2-80 Mb) while Huainan had the lowest (7.21 ± 8.14 with a range of 1-105 Mb). Mean length of ROH (MGL ROH ) was maximum in Yunan (10.01 ± 4.28 Mb) and minimum in Duroc (8.91 ± 1.14 Mb). Duroc revealed the highest ROH based inbreeding (F ROH = 0.1925 ± 0.037). F ROH of Huainan (F ROH = 0.0324 ± 0.043) and Yunan (F ROH = 0.043 ± 0.033) were lower than Duroc. Yunan did not have severe inbreeding.

Population genetic structure analysis of yunan population
The first and second principal components of Yunan, Huainan and Duroc explained 29.68% and 9.99% of the total variance. Yunan, Huainan, and Duroc were clearly separated ( Figure 4A). The first PC clearly separated Yunan, Huainan and duroc. Yunan was located between Huainan and Duroc. We used t-SNE to best classify the populations to perform dimensionality reduction clustering analysis on all the breeds. From Figure 4B, these results indicate that different subpopulations exist in Yunan pig population. From the result of admixture analysis in Figure 4C, When K = 2, Yunan, Huainan and Duroc could be clearly distinguished. Yunan contained 33.78% of Huainan and 66.22% of Duroc blood. When K = 3, a new bloodline in red was appeared in Yunan. When K = 4-5, different degrees of differentiation appeared in Yunan population. To investigate genetic structure of Yunan pigs, we constructed the NJ tree of the 193 individuals, which indicated the existing Yunan pigs could be divided into four main branches, each of which was divided into two or more subbranches ( Figure 4D).

Selection signal screening among yunan, Huainan and Duroc
To screen the selection signals among Yunan, Huainan and Duroc, and analyse the possibly origin of the signals, we divided Yunan, Huainan and Duroc into three groups (Yunan-Duroc Vs. Huainan, Yunan-Huainan Vs. Duroc and Yunan Vs. Huainan-Duroc) and used two methods of F ST and XP-CLR. Top 1% regions of F ST and XP-CLR were considered as salient loci under selected. F ST screening results were shown in Figure 5A (Yunan-Duroc Vs. Huainan), 5C (Yunan-Huainan Vs. Duroc) and 5E (Yunan Vs. Huainan-Duroc). We detected several significant loci on SSC1-12, SSC14-17 and SSCX in Yunan-Duroc Vs. Yunan group, SSC1-16 and SSCX in Yunan-Huainan Vs. Duroc group, and SSC1-10, SSC12-18 and SSCX in Yunan-Duroc Vs. Yunan group. XP-CLR analysis results of the above three groups were shown in Figure 5B (Yunan-Duroc Vs. Huainan), 5D (Yunan-Huainan Vs. Duroc) and 5F (Yunan Vs. Huainan-Duroc). We obtained 71 significant loci on each of the 18 autosomes in each of the three groups. Then we combined the outputs of F ST and XP-CLR analysis. In Yunan-Duroc Vs. Huainan group, we found 37 significant regions that over threshold under selected were overlapped in F ST and XP-CLR results. A total of 104 genes were annotated in these regions in the Ensembl (http://ensemble.org) database, 17 out of which were involved in fat production and metabolism, nine out of which were related to growth and development. These 26 genes under selected were thought to be of Duroc origin.
Similarly, in Yunan-Huainan Vs. Duroc group, we got 21 overlapping regions containing 103 annotated genes, 14 genes out of which were related to fat regulation while eight genes were related to growth development. These 22 genes were thought to be of Huainan origin. Also, 13 overlapping regions covering 116 genes were found in Yunan Vs. Huainan-Duroc. Only eight genes were involved in fat metabolism and adipose production, three genes were involved in skeletal muscle development. These outstanding 11 genes may have been selected by artificial selection in Yunan population.
To further look into former reported economic characters underlying these genomic regions, we extracted the common regions between these results and PigQTLdb (www. animalgenome.org/cgi-bin/QTLdb/index).

Genome-wide association study on backfat traits in yunan pigs
To understand genetic background of backfat thickness variation in Yunan, Yunan pigs were divided into gilt (n = 48), 1 st parity (n = 48) and 2 nd parity (n = 24) according to the age. Descriptive statistics of the six backfat traits (P2BF, SBF, SSRBF, LRBF, LBF and ABF) of this three groups were shown in Table 4. From Table 4, Yunan was much fatter from the ideal body shape. For example, the average P2BF thickness of gilt, 1 st parity and 2 nd parity in Yunan was 36.90, 35.28 and 35.93 mm. This was much thicker than the commercial sows (usually 16-22 mm). Moreover, the coefficient variation (CV) of the backfat traits was larger, ranged from 12.80% to 34.96%.
With the goal of pinpointing genomic region associated with backfat phenotypes of Yunan, we performed genome-wide association studies of six backfat traits using a linear mixed model. We identified 34 suggestive significant SNPs associated with six backfat traits in total. Thereinto, nine significant SNPs on SSC1-2, SSC4, SSC11-12, SSC14 and SSC16 were detected in 1 st parity pigs. 14 significant SNPs on a 1.4 Mb segment of SSC4  Frontiers in Genetics frontiersin.org (20.03-21.43 Mb) were detected in 2 nd parity pigs while 11 significant SNPs on SSC1-2, SSC8, SSC13 and SSCX were obtained in gilts. 202 genes were annotated in the 34 genomic regions in the Ensembl database, five, six and three genes of which were involved in fat metabolism or growth in gilt, 1 st and 2 nd parity pigs ( Figure 6). The genomic regions where these significant loci were located partially overlapped. The most significant associated SNP was the same for P2BF and LRBF on SSC8 (10.43 Mb) in gilts, for P2BF and ABF on SSC16 (21.97 Mb) in 1 st parity, for P2BF, LRBF and ABF on SSC4 (20.03-21.43 Mb) and for SSRBF and ABF on SSC4 (106.29 Mb) in 2 nd parity pigs. Then we combined these 34 regions with PigQTLdb database (www.animalgenome.org/ cgi-bin/QTLdb/index) and found that significant loci on SSC4 (20.03-21.43 Mb) were overlapped with a reported average backfat thickness QTL (20,366,121-21,945,045 bp) in large white pigs (Bink et al., 2000) (Table 5).
In view of the existence of selective signals related to fat deposition and growth, and to investigate whether the genomic regions associated with backfat traits mapped by GWAS might have been selected in the population, we analyzed the regions common to the results of F ST , XP-CLR and GWAS. The selected region on SSC4 (21.24-21.83 Mb) in Yunan-Huainan Vs. Duroc group which was thought to be of Huainan origin and the P2BF, LRBF and ABF associated region on SSC4 (20.03-21.43 Mb) in 2nd parity stood out. These two regions were overlapped, and ENPP2 and EXT1 genes (SSC4:19.02-21.06 Mb) in this region were functionally related to fatty acid production (Crespo-Piazuelo et al., 2020). Therefore, the genome region of 20.03-21.83 Mb on SSC4 was considered as an important candidate region for backfat thickness in Yunan pig population.

Discussion
The genetic diversity of a population was a key factor in ensuring the survival and evolution of a species. Commercial pigs have been subjected to high-intensity artificial selection for a time, resulting in a decrease in genetic diversity, while local varieties were mainly selected by natural conditions such as environmental factors and geographical factors, thus maintaining a high level of genetic diversity. China's native pig breeds has a better genetically diverse (SanCristobal et al., 2006;Ai et al., 2013). The genetic diversity of Chinese hybrid pigs was higher than that of Chinese local pig breeds (Yang et al., 2003;Huang et al., 2020) and commercial pig breeds (Wang et al., 2021). There were many statistical methods to evaluate population genetic diversity, such as allele frequency and population heterozygosity. The higher the heterozygosity, the higher the genetic diversity. In this study, the MAF, Ho and He of the hybrid pig breed Yunan were higher than those of its founders Duroc and Huainan, indicating Yunan had a richer genetic diversity. Effective population size (Ne) is also an important indicator of diversity and species conservation. If the Ne was below 65, the breed might have a population crisis Frontiers in Genetics frontiersin.org (Simon, 1999). The Ne of Yunan was 67. This suggested that although having experienced the threat of African swine fever, Yunan did not have a group crisis now. ROH segments contains information about population inbreeding, and its length and frequency can reflect the population history. Compared with pedigree inbreeding number, the calculation of genomic inbreeding number based on ROH was more accurate and can better reflect the real inbreeding number of an individual (Purfield et al., 2012;Zanella et al., 2016;Deniskova et al., 2019;Bhati et al., 2020). A longer ROH usually indicates a closer genetic relationship while a shorter ROH indicates an ancient inbreeding. A larger number of ROHs and fragments means a higher probability of inbreeding (Kirin et al., 2010). Here, the average total length of ROH of Yunan was between Huainan and Duroc. Duroc had the highest average number of ROH per animal while Huainan had the lowest. Mean length of ROH was maximum in Yunan and minimum in Duroc. Duroc had the highest ROH based on inbreeding. F ROH of Huainan and Yunan were lower than Duroc. Compared with some other local breed pigs (Mashen and Chun'an) in China (Cai et al., 2021;Dai et al., 2021) and European commercial breeds (Zhan et al., 2020), the average inbreeding coefficient of Yunan was relatively lower, and Yunan black pigs did not show obvious inbreeding.
From few large branches in NJ trees, population stratification in PCA, and having no one main lineage when K = 3 to 5 of ADMIXTURE implied much more breeding work should be done in Yunan. In addition, different subsets of Yunan may exist according to PCA, which was also indicated by t-SNE analysis. So, even if Yunan pigs had no risk of inbreeding and recession according to the population diversity and inbreeding analysis, but this diversity may partly be due to stratification within groups. Therefore, in the future breeding work of Yunan pigs, we should carry out mating among lineages according to the NJ molecular pedigree to avoid multiple invalid matings, otherwise the consistency of the population would not be improved. Selection signals can reflect loci and genes that have been strongly selected in a population during long-term domestication. The selective signals we detected in Yunan black pigs were not only related to fat deposition, but also to growth. Both directions had both Duroc origin (Yunan-Duroc VS. Huainan) and Huainan origin (Yunan-Huainan VS. Duroc). For example, GPRC6A (Mukai et al., 2021) and MBD5 (Du et al., 2012) for fatness and growth of Duroc while ANGPTL3 (Jiang et al., 2018) and DOCK3 (Reid et al., 2020) of Huainan. These results indicated that both Duroc and Huainan pigs had genetic variants affecting growth and fat deposition. In addition, some genes, such as CDH13 (Göddeke et al., 2018) and RAB23 (Hasan et al., 2020), were found to be involved in fat deposition and growth in the Yunan VS. Huainan-Duroc group. These loci might be derived from the founder effect of the initial breeding group of Yunan or might be the result of the 14 years of breeding process of Yunan.
Genomic loci associated with backfat thickness partially overlapped between different parities and different traits. This suggested the influence of some genes on backfat was a long-term process, there's no time and space specificity. When it came to the nonoverlapped genes or regions, we could not come to a conclusion of time or space specificity. Because although the population used in this study covers the core population of Yunan black pigs, the number of populations was too small. This reminded us that under the epidemic environment, it was necessary to adopt multi-site conservation for species, especially for local genetic resources.
Three genes related to fat deposition, ENPP2, EXT1 and SLC30A8, were found in the upper and lower 1 Mb of SSC4 (20.03-21.43 Mb) (Du et al., 2009;Hutley et al., 2011;Nishimura et al., 2014). Among the four linkage disequilibrium blocks in the 1.40 Mb interval of SSC4, only one gene, SLC30A8, was present in the fourth block (193.21 Kb). This gene is the star gene of type 2 diabetes. In 2009, there was a study that attempted to find the causal mutation for human type 2 diabetes by analyzing the correlation between the mutation of this gene with fat deposition in pigs (Du et al., 2009) but failed. Further analysis of this gene in Yunan might be needed.
In addition, QTL has been widely used in the study of important economic traits in pigs. The hunt for QTL in pigs has been ongoing for nearly 2 decades, beginning with the first publication of a QTL for fatness on pig chromosome 4 in 1994 (Andersson et al., 1994). In crossbreeding analysis, SNP markers Frontiers in Genetics frontiersin.org

FIGURE 6
Manhattan plot of genome-wide association analysis of P2-backfat, shoulder backfat, sixth and seventh ribs backfat, last rib backfat, lumbar joint backfat and average backfat at different parity. The annotated genes represent the relevant candidate genes.
Frontiers in Genetics frontiersin.org 10 and QTL linkage analysis can be used to identify genomic regions with complementary effects from potential resource groups, so as to increase the degree of genetic complementarity between varieties or lines in a planned way, and thus improve the economic traits of hybrids and the genetic diversity of varieties.
In conclusion, we analyzed genetic structure and mapped genomic regions affecting backfat thickness of Yunan black pigs. Significance of SNP.
Frontiers in Genetics frontiersin.org Although there was no risk of inbreeding depression, the stratification phenomenon existed in Yunan population. The increasing backfat thickness and decreasing consistency had a particular genetic basis. The 1.40 Mb interval of SSC4 (20.03-21.43 Mb) was a strong candidate region associated with backfat thickness. ENPP2, EXT1, and SLC30A8, particular SLC30A8, were strong candidate genes. Our findings are helpful for the subsequent breeding and conservation, as well as genomic improvement of backfat thickness of Yunan black pigs and suggested the importance of multi-site breeding conservation method.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://bigd.big.ac. cn, GVM000386.