Sex-specific variations in phytochemicals and antimicrobial potentiality of Dioscorea

The monocotyledonous herbaceous crop Dioscorea is native to tropical and temperate regions of the world. Dioscorea tubers are dioecious in nature, have colossal therapeutic potentiality, and are frequently used in traditional medical practices throughout the world. Most of the research works are aimed to determine the medicinal property, nutritions, antinutrients, and biological activities of Dioscorea spp. without specifying the sexes of Dioscorea which promoted us to carry out this present research work. Sex-specific variation of phytoconstituents, antioxidants, and antimicrobial efficiency in tubers was appraised. The results obtained from this study divulge existence of significant quantitative variation between the male and female tubers. The female tubers are superior in acquiring phytochemicals compared to male counterparts and acquired maximum antioxidant and antimicrobial potentiality. This study will offer an apposite baseline for further sex-specific assessment which can be directed towards both qualitative and quantitative amelioration of medicinally important noble compounds by exploiting modern scientific strategies leading to their active participation in nutraceutical industries.


Background
The genus Dioscorea earlier positioned under order Liliales [1] but later included under Dioscoreales [2]. The highly medicinal dioecious Dioscorea of Dioscoreaceae contains more than 600 species globally [3][4][5]. The Dioscorea tubers are renowned for their ethnobotanical, nutritive, antioxidant, and biological potentiality that ensure the quality of daily nourishment of the indigenous people [4,6,7]. Dioscorea (Yam) is a staple food for the people of tropical countries of Africa, Asia, Caribbean, and the Pacific region [8]. The long-term storage potentiality of these tubers ensures seasonal food security in developing countries [9]. Diosgenin is a phytosteroidal saponin and a major bioactive compound found in the roots of wild yam [10]. It is the main precursor in the manufacture of synthetic steroids in the pharmaceutical industries [11].
Dioecy is attributed to seven percent of total plant taxa although most of the medicinal plants are monoecious [12]. Knowledge of the dioecious nature of plants has existed since Babylonian times but their consequences in the traditional medical system are not recognized appropriately [13]. Insinuation of dioecy in chemical and pharmacological properties has been pointed out [14][15][16]. The sex-specific biological activities of Piper betle, Carica papaya, and Tinospora cordifolia was recorded [17][18][19]. Sex determination in Dioscorea has not yet been fully elucidated although [20][21][22] have favored male as the heterogametic sex; Smith [22] and Meurman [23] emphasized the occurrence of an extra chromosome for the male expression, while [23][24][25] have reported absence of sex chromosomes. Literature survey concerning the biological potentiality and phytoconstituents' availability underpin that evaluation of sex-specific variation concerning phytoconstituents and biological efficacy of Dioscorea spp. has remained unexplored although reports are available [7].
Hence, this present study has entrained to evaluate the variation in the phytoconstituents and antimicrobial efficiency based on the male and female tubers of Dioscorea.

Sample collection
Matured male and female tubers of five edible Dioscorea were collected by using the shrivel and auger and packed into marked zipped sterile polythene bags from the forest bed of Tripura (Figs. 1, 2, 3, and 4). The collected plant samples were identified by using the Flora of Tripura [26], and two of them are a new addition to the Flora of the state [27,28]. Flowers, micro-and macromorphological characters were considered during the identification of the male and female plants and further authenticated with taking help from the expertise from Botanical Survey of India (Eastern Regional Centre, Shillong). The herbarium prepared for the selected Dioscorea spp. with their respective voucher numbers were deposited in the departmental herbarium and depicted ( Table 1). Analysis of International Union for Conservation of Nature (IUCN) status pointed out that among five of the selected Dioscorea spp., only Dioscorea wallichii is included in least concern category.

Plant samples
The male and female tubers of five Dioscorea spp. were collected during the flowering phase, and photographs were taken as reference for the identification from the forest bed of three different districts of Tripura (Table  1). Care was taken during the collection of the tubers that the tubers of both male and female plants were available at each of the selected study sites.

Sample extraction
Collected tubers were cleaned in running tap water, shade dried, and pulverized to powder in a mechanical grinder. Twenty grams tuber powder of each of the Dioscorea species was extracted separately with methanol (200 mL) in a shaker at room temperature. After, overnight extracts were filtered through Whatman No. 1 filter paper. The filtrates were subjected to analysis for total phenolic, flavonoid contents, and DPPH radical scavenging activities.

Determination of moisture content
Tubers samples were chopped into small pieces by using sterilized blades. Ten grams of the chopped samples were taken in the previously weighed Petri plates. Then, the sample was kept in a hot-air oven for overnight at 100 ± 2°C. The dried samples were cooled at room temperature and weighed to a constant weight. The loss in weight was considered as the moisture percentage and was calculated by using the following formula: Percentage of moisture content ð%Þ ¼ W1-W2Â100

W1
where W 1 = weight of the sample (leaf and rhizome) taken and W 2 = weight of the oven-dried samples.

Determination of carbohydrate
Carbohydrate was determined [29] from the dried tuber samples. One hundred milligrams of the sample was taken into boiling tubes and hydrolyzed with 5 ml of 2.5 N-HCl for 3 h and cooled at room temperature flowed by the neutralization with sodium carbonate pellets. The volume is made up to 10 ml and centrifuged at 5000 rpm for 15 min. The supernatant was collected and 1 ml aliquots were taken for analysis. Then, 4 ml of 2% anthrone (w/v in concentrated H 2 SO 4 ) reagent was added and heated in a boiling water bath for 10 min. The absorbance was taken at 630 nm using a spectrophotometer. Glucose was used as a standard.

Determination of protein
The protein content was determined [30]. One hundred milligrams of the sample was ground well with a pestle and mortar in 10 ml of the potassium phosphate buffer (0.1 M, pH 7.5) and centrifuged at 5000 rpm for 15 min. The pellet was discarded and the supernatant was used for protein estimation. From the supernatant, 1 ml of sample was taken in dried test tubes and 5 ml of reagent "C" was added. The reagent "C" was prepared by mixing reagent "A" and reagent "B" in a ratio 50:1 v/v. Reagent "A" is the mixture of 2% Na 2 CO 3 and 0.1 N NaOH and reagent "B" is a mixture of 0.5% CuSO 4 and 1% Na-K tartrate. The solution was shaken vigorously and allowed to stand for 20 min. After that, 0.5 ml of Folin-Ciocalteu reagent was added and incubated at room temperature for 30 min. Absorbance was measured at 660 nm using a spectrophotometer. Bovine serum albumin (BSA) was used as a standard.

Estimation of total free amino acids
The amount of total free amino acid was estimated [31]. For this, 100 mg of dried tubers sample was homogenized in 10 ml of 50% aqueous ethanol with a pinch of activated charcoal. The slurry was centrifuged at 5000 rpm for 10 min, and the free amino acid was extracted in the form of a clear supernatant which was used for spectrophotometric estimation. The volume of supernatant was raised to 10 ml with aqueous 50% ethanol. To 1 ml of the supernatant, 2 ml of 2% Ninhydrin (w/v in dehydrated alcohol) was added. The mixture was kept on a water bath at 75 + 2 o C for 10 min, and after cooling, aqueous alcohol (1:1) was added to make up the volume to 3 ml. The absorbance was measured at 570 nm on a spectrophotometer. Glycine was used as a standard.

Determination of fat content
The fat content was determined [32]. Two grams of the sample was taken in dried test tubes, and petroleum ether was added on that and allowed to stand for 16 h. After 16 h, the petroleum ether was evaporated to dryness and weights the flask before after for fat.

Estimation of total crude fiber
Crude fiber of the tuber samples was estimated [32]. One gram of dried leaf sample was subjected to acid and subsequent alkali digestion for degradation of native cellulose and lignin. The residue obtained after final filtration was weighed, incinerated, cooled, and weighed again. The loss in weight gives the crude fiber contents.

Determination of ascorbic acid
For determination of ascorbic acid content in the tubers, [33] method was employed. Five grams of the sample was weighed into a bottle containing 100 ml of ethylenediaminetetraacetic acid (EDTA)/tricarboxylic acid (TCA) The mixture was shaken vigorously for 30 min. The solution was transferred into a centrifuge tube, and centrifugation was done at 3000 rpm for 20 min. Then, the preparation was transferred to a 100-ml volumetric flask and 1% starch indicator was added followed by titration with 20% copper sulfate (CuSO 4 ) and waited until the dark color was developed.

Determination of riboflavin
For the determination of tubers riboflavin content, 5 g of the sample was extracted with 100 ml of 50% ethanol and shaken for 1 h followed by the filtration into 100 ml flask. From this preparation, 10 ml of the extract was pipetted into 50 ml volumetric flask and 10 ml of each 5% potassium permanganate, and 30% H 2 O 2 was added subsequently. This preparation was taken to a hot water bath for 30 min. This was followed by the addition of 2 ml of 40% sodium sulfate. The volume was made up to 50 ml and the absorbance measured at 510 nm [34].

Determination of thiamine
Five grams of the tuber sample was homogenized with 50 ml 10% ethanolic sodium hydroxide and filtered into a 100-ml conical flask. Ten milliliters of the filtrate was pipetted, and the color was developed by the addition of 10 ml 1% potassium dichromate; the absorbance was measured at 360 nm [34].

Determination of alkaloids
The alkaloid content was determined [35]. For this, 5 g of the sample was weighed and taken into a 250-ml beaker containing 200 ml of 10% acetic acid in ethanol and allowed to stand for 4 h. This preparation was concentrated on a water bath to one quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was completed. The precipitate was collected and washed with dilute ammonium hydroxide followed by filtration. The residue was dried and weighed.

Determination of total phenols
Total phenol was determined [36]. 200 mg of sample was crushed in 10% methanol and centrifuged for 20 min at 5000 rpm.1 ml supernatant was taken and 1 ml Folin Ciocalteu reagent was added flowed by the incubation for 3 min at room temperature. Then, 1 ml of saturated 20 % Na 2 CO 3 was added and kept in a water bath for 1 min. The absorbance was measured at 725 nm. Gallic acid was used as a standard.

Determination of flavonoids
The flavonoid content was estimated [37]. 0.5 ml of test sample solution in methanol (5 mg/100 ml) was mixed with 2 ml of distilled water and 150 μl of 5% sodium nitrate. After 6 min, 150 μl of 10% aluminum chloride and 2 ml of 1 M sodium hydroxide was added and left at room temperature for 15 min. The absorbance of the mixtures was measured at 510 nm. Catechin was used as a standard.

Determination of saponin
Saponin was determined [38]. Twenty grams of tuber samples were put into conical flasks containing 100 ml of 20% aqueous ethanol and heated at 55°C for 4 h. The mixture was then filtered and re-extracted with another 200 ml 20% ethanol. The combined extracts were reduced to 40 ml in a water bath. The concentrate was transferred into a 250-ml separating funnel containing 20 ml of diethyl ether and shaken vigorously. The aqueous layer was recovered and further purification was done in 60 ml of n-butanol. The preparation was washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath followed by the evaporation of the samples. The residue was dried and weighed.

Determination of tannin
The tannin content was estimated [39]. Five hundred milligrams of powdered sample was dissolved in 50 ml of distilled water and shaken for about 1 h in a mechanical shaker. This was filtered through cheese clothes into a 50-ml volumetric flask and made up to the mark. Then, 5 ml of the filtered was pipette out into a test tube and mixed with 2 ml of 0.1 M FeCl 3 in 0.1 N HCl and 0.008 M potassium ferrocyanide. The absorbance was measured at 760 nm within 10 min. Tannic acid was used as a standard.

DPPH radical scavenging activity
The free radical scavenging activities of methanol extract of all the samples were evaluated by 1,1-diphenyl-2picryl-hydrazyl (DPPH) method [40]. Different concentrations of methanol extracts (30, 60, 120, 240, 480, 600, 720, 840, 960 μg/ml of the sample) were mixed with 300 μl DPPH (0.02 mM). The absorbance was measured at 517 nm using a UV-VIS double beam spectrophotometer (Dynamica, DB-20and SL. No. -6622065) after 30 min of incubation at dark. Ascorbic acid was used as the reference sample. Scavenging of DPPH was calculated by using the formula: where A 0 is the absorbance of the control reaction and A 1 is the absorbance of the sample.

Evaluation of antimicrobial activity
The antibacterial potentiality of the tubers of male and female tubers was determined by the agar well diffusion method. Streptomycin and dimethyl sulfoxide (DMSO) were used as positive and negative controls for antibacterial study. The results were recorded by using a ruler with a sliding caliper [41], and the inhibition zone was expressed in millimeters. The anti-fungal activity of the compounds was determined by the agar well diffusion method.

Data analysis
Each of the analysis was performed in triplicate and expressed as mean ± SD. Antioxidant activity was determined and inhibition concentration (IC 50

Nutritional aspects
Moisture content

Total protein
The total protein (TP) content of the tubers ranged between 3.15 ± 0.05 and 13.25 ± 0.22 mg/gm fresh weight. Maximum and minimum TP content was recorded in the tuber of the male plant of D. pubera and D. oppositifolia, respectively. The total protein content differs significantly among the tuber of male and female plant of D. oppositifolia and D. pubera (P < 0.001), D. alata (P < 0.01), D. hamiltonii (P < 0.05).

Total carbohydrate
Maximum and minimum total carbohydrate (TC) content was observed in female tubers of D. hamiltonii and male tubers of D. wallichii, respectively. Significant differences were observed in the TC content of male and female tuber of D. alata, D. oppositifolia, D. pubera (P < 0.05), and D. wallichii (P < 0.01).

Total soluble sugar
Maximum and minimum total soluble sugar (TSS) content was recorded in female tubers of D. glabra and D. oppositifolia, respectively. No significant differences existed between the male and female tuber of D. hamiltonii, D. pubera, D. oppositifolia, and D. glabra while only D. alata showed significant difference (P < 0.01).

Total free amino acid
Total free amino acids (TFA) do not differ significantly among the male and female Dioscorea tubers. Only the male and female tubers of D. wallichii showed a significant (P < 0.05) difference. Maximum and minimum TFA was recorded in the tubers of the female plant of D. hamiltonii and D. pubera, respectively.

Total crude fiber
The total crude fiber (TCF) content significantly differed (P < 0.05) among the tuber of male and female plant of D. hamiltonii and D. wallichii. However, no significant variation was observed in the male and female plant tuber of D. alata, D. oppositifolia, and D. pubera. The maximum amount of TCF was recorded in the female tuber of D. alata and minimum in the male tuber of D. glabra.

Total fat
The total fat (TF) content varied significantly among the male and female tuber of D. oppositifolia (P < 0.01) and D. wallichii (P < 0.05). No significant variation was observed in the tuber of male and female plant of D. alata, D. pubera, and D. hamiltonii. Maximum and minimum TF was recorded in female tuber of D. alata and male tuber of D. hamiltonii, respectively.

Vitamins
Among the studied Dioscorea species, the maximum ascorbic acid (Aa) content was observed in the female tuber of D. oppositifolia while minimum in male tuber of D.

Anti-nutritional aspects Total alkaloid
Total alkaloid (TA) content in both male and female tubers of D. alata and D. pubera was significant (P < 0.05), whereas no significant difference was observed between the male and female tubers of other species. Maximum and minimum TA content was recorded from the tubers of female D. hamiltonii and D. oppositifolia, respectively.

Total phenol
The total phenol (TPH) content of male and female tubers of two species viz. D. wallichii and D. oppositifolia showed highly significant (P < 0.001) variation. Maximum phenol content was recorded in D. oppositifolia female tuber while the female plant of D. wallichii showed the least.

Total flavonoid
The

Total saponin
Tubers of male and female plants of D. alata showed a significant (P < 0.05) difference in total saponin (TS) content. Maximum and minimum TS were recorded from tubers of female D. hamiltonii and male tuber of D. oppositifolia, respectively.

DPPH radical scavenging activity
The methanolic extracts of tubers male and female Dioscorea plants possessed potent DPPH radical scavenging activity in terms of percentage of inhibition. Antioxidant activity varies significantly among the male and female tuber of the same species. Maximum and minimum antioxidant activity was recorded in the tubers of female D. oppositifolia and male D. hamiltonii, respectively. This study also revealed that tubers of the female plant tubers exhibit potent antioxidant activity compared to male counterpart. No significant difference was observed in the tubers of male and female D. alata.

Antimicrobial activity
Antimicrobial activity of methanol extracts of male and female tuber of five Dioscorea species were screened against four pathogenic bacteria and two pathogenic fungi. The antimicrobial activity was determined in terms of the inhibition zone around the respective microbial colonies. Maximum microbial activity against all the pathogenic bacteria and fungi was recorded in the methanolic extracts of both male and female tubers of D. pubera. Male and female tubers D. oppositifolia showed proficient activity against the selected fungal strains and bacterium Klebsiella pneumoniae. D. hamiltonii male and female tubers exhibited noticeable antibacterial activity against Streptococcus pneumoniae. The female tubers of D. wallichii showed strong activity than their male counterpart.

Discussion
This present study has been carried out to evaluate the sex-specific variation of nutrients and antinutrient aspects along with their antioxidant and antimicrobial potentiality of Dioscorea tubers (Tables 2, 3, 4, 5 and 6). A total of five edible species were selected for this present study out of which all are dioecious, i.e., male and female plants are developed separately. The tubers of superior crop yam [42] assist nutrients three times more than the most important food crops like cassava and sweet potato [43]. A considerable amount of research work has been carried out throughout the world but none of them are emphasizing the sex-specific evaluation of phytochemical constituents and biological efficacy of Dioscorea spp. The results obtained from the study revealed that significant differences existed between the most of the male and female tubers in terms of nutrient, vitamins, antinutrient, antioxidant, and antimicrobial efficacy. The possible reasons for these variations may be attributable to different factors such as genetic, climate, and environmental conditions [44][45][46][47]. In line with the earlier findings [48,49], experimental results showed that a significant and positive correlation exists between the phenolic contents and antioxidant potentiality. The high antioxidant potentiality of Dioscorea tubers may be one of the key determinants of their inclusion in traditional folkloric medicine. Mittelstrass et al. [50] proclaimed that male and female plants differ in their metabotypes which may be attributable to herbivore preferences for gender [51][52][53][54] which in turn are correlated secondary metabolite contents. Higher amounts of phenols and antioxidants are connected with the defense strategies of plants [55,56]. The pinpointing findings of this present study are the sexspecific study of the nutrient, antinutrient, antioxidant, and antimicrobial activity which is lacking in earlier findings. The methanolic extracts of the female tubers have proficient antibacterial potentiality which is in congruence with the earlier finding [57]. Among the tested bacterial strains, gram-positive bacterium (Streptococcus pneumoniae) is more susceptible compared to the gram-negative bacteria which reaffirmed the earlier findings [58][59][60]. Moreover, among all the selected Dioscorea species, most of the tubers of female plants are superior compared to the male counterparts although they are residing at the same climatic conditions or ecosystems which were the reconfirmation of earlier findings although their studied samples are different [17][18][19]. To consign, a specific reason for the findings of this present study is may be due to the different physiological and reproductive adaptive responses of both the sexes irrespective of their occurrences in the same microclimate.

Conclusion
This study is a precursive effort to estimate the sexspecific variations in the phytochemical constituents and biological activities of Dioscorea tubers. Further researches are needed to be carried out by incorporating modern scientific tools to underpin the actual physiological mechanism associated with these variations which may be helpful in the early delimitation of sexes. This study also ensures that female Dioscorea tubers are the reservoir of biological compounds compared to the male counterpart which may be able to draw the attention of nutraceutical industries leading to the discovery of noble drugs.