Extracted Trans-Resveratrol from Arachis hypogaea Enhances Expression of Sirtuin Gene and Replicative Life Span in Saccharomyces cerevisiae

Aims: Biotic stress given by Aspergillus niger enhances trans-resveratrol production in Arachis hypogaea plant. This plant extract increases sir2 gene expression and Replicative Life Span in Saccharomyces cerevisiae. Design of Study: Peanut plant was grown in aseptic environment, infected by Aspergillus niger. Plant extract used for quantification of trans-resveratrol by RP-HPLC. Yeast culture was grown in Potato dextrose media along with plant extract. Sir2 gene expression fold calculated by real time pcr. Replicative Life Span of yeast was measured by spectrophotometer. Place and Duration of Study: Allele Life Sciences Pvt. Ltd., Department of Biotechnology between February 2017 to March 2020. Methodology: Biotic stress in Arachis hypogaea plant was induced by wounding the leaves and introducing Aspergillus niger to enhance trans-resveratrol production. Tran-resveratrol was quantified by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC). Two methods Original Research Article Singh and Deval; JPRI, 32(29): 48-59, 2020; Article no.JPRI.62367 49 conducted to check reverse ageing, first one epigenetic based, when extracted trans-resveratrol from infected Arachis hypogaea plant extract added to Saccharomyces cerevisiae culture, it enhanced expression of Sir2 gene in Saccharomyces cerevisiae measured by qPCR, ABI applied biosystem. Process included RNA isolation, cDNA synthesis and thereafter qPCR. Enhanced expression of sirtuin responsible for gene silencing as sirtuin (Sir2 gene product) is a class of Histone deacetylase transferase enzyme. Second method, Replicative Life Span of Saccharomyces cerevisiae culture increased when Aspergillus niger infected peanut plant extract added to yeast culture which was measured through spectrophotometer at 600nm and showed high absorbance value. Results: Tran-resveratrol was quantified by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) and yield was 2.24 mg/g. Sir2 gene expression increased by 1.56 fold in yeast grown in infected peanut plant extract. Absorbance of yeast culture grown in infected peanut plant extract was 0.522±0.008 which was higher than control. Conclusion: Sir2 gene expression enhances along with replicative life span in yeast in presence of peanut plant extract.


INTRODUCTION
As we age older, our body deteriotes and thus it looses the ability to work. Aged body also adds extra pressure to a family, society and to country. If an aged body works normally to earn livelihood and takes care itself, then no burden on family to take care. A time comes when an aged group becomes worthless and looks quite heavy burden not to family but for a country also. At the same time, no one can ignore idea to remain healthy with all organ intact functioning properly and feel enthusiatic till last breathe in this world. Although deterioation of any living body is the process of life but understanding how living body deterioates and how the body can be brought back in state where body deterioation is possibly less is quite worthy. At juvenile age, we feel very energetic and our body has well worth for doing ethlete work but as we grow older we loss body's such ability. Understanding what changes have taken up in our body is the first challange to confirm and finding accurate pathways of those changes. Visiting history, we find that our ancestors followed some healthy practices in life style when there were no scientific evidences, will also be verified by this reverse ageing theory whether it comes food, exercise or religious customs to keep body calorie restricted (fasting). But lone these practices are having their own grey area such as regular food intake will also results into many free radicals accumulation in body which further damages cell [1], if some opts for regular extreme physical exercises then this also produces free radicals [2] and body will remain in demand of more energy whole day and if we may keep body nutrients restricted by fasting then this state may cause lack of key elements in body which can reduce body's immunity [3]. Above stated problems are not today's problem, but it has become part of curiosity for human being since we understood life.
When multifaceted decline of cellular and organismal function occurs over time, is described as ageing, and actually causes major risk factor for disease susceptibility [4]. Therefore, present researches are targeting to deaccelerate ageing process, and this is how lifespan or healthspan can be prolonged by therapeutics of such diseases. Molecular biology and epigenetics together give solution of above stated problems by reversing age. Gene expression is controlled by environmental factors which brings body in such condition where gene can be made silence or can enhance expression to produce desirable amino acids [5]. Naturally, peanut plant contains many polyphenol compounds, resveratrol is one of them. Chemically resveratrol is found in two forms, cis-resveratrol and trans-resveratrol. Trans-resveratrol has gained enough popularity as antiageing compound [6] in recent times, but its bioavailability is very low. Therefore, when peanut plant is infected with fungus (here Aspergillus niger), it is found that transresveratrol production increases in peanut plant.

Importance of Tran-resveratrol
In many religious tradition, fasting has been in regular practice. Scientific data says fasting is one of the reason of reverse-ageing which rejuvenates our cells to grow young. Religious Hindu books and Roza in muslims give same sense. If body is made somehow calorie restricted, cells do not reach to sensences or apoptosis. But practically, it is not possible to be calorie restricted as body demands energy molecule to do our daily routine work, therefore, we need to see other way to rejuvenate the cells. Groundnuts (Arachis hypogaea L.) has two very important anti-ageing nutrient compounds one is niacin and other one is trans-resveratrol [7][8]. Conceptually, if genes which switched on due to stress gained in life of organism, are made to switched off then, non stressed cell gain capacity to increase number of cell division. Biologically, Trans-resveratrol activates SIRTUIN enzyme which is NAD+ dependent. Sirtuin is Histone Deacetylases enzyme which removes acetyl group from lysine present at Histone to facilitate DNA binding thus causing gene silencing [9]. Thus amount of NAD+ is a marker of ageing in different animals.

Resveratrol is Sirtuin Activator
Resveratrol has been actively investigated to enhance sirtuin activity with consequent beneficial effects on ageing [10]. Sirtuin delays cellular senescence and extends the organismal lifespan through the regulation of diverse cellular processes.
Sirtuin suppresses cellular senescence and it is done mainly mediated through delaying the age-related telomere attrition, sustaining genome integrity and promotion of DNA damage repair. Sirtuin, additionally modulates lifespan by interacting with several lifespan regulating signaling pathways including insulin/IGF-1 signaling pathway, forkhead box O and AMP-activated protein kinase. Since Sirtuin mediates the prolongevity effect of calorie restriction. Many activators of Sirtuin have attracted the attention of researchers to develop therapeutics for agerelated diseases [11]. Sirtuins are proteins found in all domains of life. In Baker Yeast, the first known sirtuin, Sir2 (silent information regulator 2), from which the family derives its name, regulates ribosomal DNA recombination, gene silencing, DNA repair, chromosomal stability and longevity. Sir2 homologues have been studied to see its role in modulating lifespan in worms and flies, and may underlie the beneficial effects of caloric restriction, the only regimen whose effect we have seen in fasting that slows ageing and extends lifespan in organism. Considerable attention gained by sirtuins for their impact on mammalian physiology, since they may provide novel targets for treating diseases associated with ageing and perhaps extend human lifespan [12].
Biological platforms are requied for the reseach in anti-ageing interventions that may furher lead to drug discovery. Improved survival during ageing can be well studied at cellular platforms which is highly compatible and unbiased phentypical screeens. Such screening platform should be economical, simple and reliable and one organism fulfills such requirement is Saccharomyces cerevisiae, which is commonly known as Yeast, a model for human ageing and age related diseases. Many pathways in yeast and humans are relevent for ageing and disease are well conserved [13]. Based on sequence similarity, 30% of the yeast genome is conserved to humans. Yeast carries approximately 6000 genes, out of which 90% have already been characterized [14].

Test of Reverse ageing 1.3.1 First approach by up regulation of Sir2 gene
Here in present study, budding yeast has been selected as model orgnaism. In this organism we found that SIR2 was regulated up when they are grown in presence of peanut extract which contains tran-resveratrol (Quantified by HPLC). Sir2 up regulation causes sirtuin enzyme availability in cell. As Sirtuin, a class of Histone Deacetyl transferase enzyme removes N-acetyl from histone causing (+) positive charge on lysine exposed to (-) negative charge of phosphate group of DNA, therefore, part of DNA get tightly packed and then no transcription region is found for expression. In this way, those genes which switched on due to stress over the years (which actually increased the burden of cell and reduced ability to divide), will switch off ultimately.

Second approach by replicative life span
Over several hours, ability to divide in fresh liquid media of an ageing culture declines with age which can be monitored by measuring the optical density of culture aliquot that has been diluted in fresh media. When, in media peanut extract added, number of yeast cells increase.

MATERIALS AND METHODS
HPLC-grade methanol and ethanol were purchased from Merck (India). Trans-resveratrol (98% purity) was purchased from Cayman Chemical (India). Potato dextrose agar, Hoagland Solution were prepared at Allele Life Sciences Pvt. Ltd., Noida, UP.

Fungal Culture
Aspergillus niger was isolated from waste water previously using potato dextrose agar. It was identified and characterised using18s rDNA sequence.

Arachis hypogaea L. growth
Peanut seeds (Om Agro Industry, Bikaner, India) were sown in 3.8 liter pots (16 cm diameter) containing autoclaved mix soil (Add Value Biotech, Gurgaon, India) supplemented with Hoagland Solution. Seeds germinated in 10 days were grown in an insect-free greenhouse with natural light. The greenhouse temperature was kept at 25-30°C. After one month, seedlings were distributed to one individual per pot. Two months old plants with fully developed leaves were used in all experiments.

Aspergillus Niger Infection
The plants were divided into two groups; control and infected, each containing four plants. The leaves were wounded aseptically using scissor and Aspergillus niger culture was spotted on the wounded area. Each plant was then individually covered with a 4 liter plastic storage bag to provide adequate humidity and temperature conditions for fungal growth and colonization of the plant's leaves. The plants were incubated for three days before the removal of bags. Subsequently plants were grown with regular exposure of sunlight and water sprinkled whenever soil found dry till 3.5 months.

Extraction of Resveratrol from Whole Plant
At the end of experiment, whole plant was dried in heat incubator for 5 days at 50 ˚C and crushed to fine powder in mortar and pestle. Each powdered sample was weighed (0.5 g) and dissolved in ethanol (50 ml). The mixture was stirred at 250 rpm at 50°C for 5 days. Thereafter it was filtered to remove undissolved particles of larger size followed by centrifugation at 7000 rpm for 10 minutes. The supernatant was collected and used for further analysis. All the extraction steps were performed in dark to prevent photochemical degradation of the constituents.

Arachis hypogaea L. growth
After sowing the seeds, emergence of seedlings occurred in 20 days. The plant that grew normally, after two months it was taken for further treatment.

Yeast Growth
Baker Yeast collected from shop. For activation, 1 g table sugar was dissolved in 100ml warm water in glass beaker. 0.5 g yeast added in vessel and kept for overnight. Next day in morning when observed, fluffy rising growth of yeast found with froth.

Composition of PD agar media
Potato extract 0.4 gm, Dextrose 2.0 gm and Agar 1.5 gm pH-5.6

No. of primers used: 1. Beta actin and 2. Sir2
Wild type Yeast cultures and Test samples (above mentioned in sample info) were grown overnight at 30°C in a tube rotator to ~2x10 7 cells/ml in 5 ml of YPD. Samples from centrifugal elutriation size selection were immediately used from elutriation. Cell size and concentration were measured with a Z2 Coulter Counter. Total RNA Yeast lifespan variation correlates with cell growth and SIR2 expression was harvested from wild type and test cells using Tri-extract RNA isolation method (GBiosciences). After checking the isolated RNA concentration and purity with spectrophotometer, the samples were diluted to 20 ng/ul.

Synthesis of cDNA
Added Total RNA 5 µl, Oligo(dT)16 2 µl, Rnase free dd water 7.5µl. Heat at 70°C for 5 minutes and place the tube immediately on ice for 2 min. Vortex briefly and then added the following components. Added 5xM-MLV Buffer 4µl, dNTPs 1µl, RNAsin 0.5µl, M-MLV 1µl incubate at 42°C for 60 min. Heat the sample to 95°C to inactivate enzyme. Cool the sample on ice for downstream experiments or store at -20°C.
Primer Sequences of Sir2 forward-ggcagtgtcagcagcttcag, Sir2 reverse-gggcuencegtctctggtttcaaaa, primer sequence ACT1 forwardtcgttccaatttacgctggtt, ACT1 reverse-cggccaaatcgattctcaa. Normal PCR reaction carried out separately to ensure reaction prior to real time PCR with following cocktail and program. PCR master mix 25 µl, Primer F (10 µM) 1 µl, Primer R 1 µl cDNA 5 µl, nuclease free water 18 µl. PCR Program:95°C for 3 minutes, 95°C for 20 seconds, 50°C (Act1) / 52°C (Sir2) for 20 seconds,72°C for 40 seconds for 40x, 72°C for 3 minutes, 4°C for 10 minutes Real time PCR reactions were carried out using Kappa SYBR Fast Master Mix (2X) with ROX reference dye. Gene Expression primers for budding yeast SIR2 and ACT1 genes primers were obtained from GCC Biotech. Gene expression analysis of the real-time data was conducted with ABI Prism 7000 SDS software as well as Excel. Relative gene expression was calculated using the 2 -ΔΔCT comparative C T method.

Analysis of Trans-resveratrol through HPLC
The extracts were analysed by reversed phase high-performance liquid chromatography (HPLC) using mobile phase, methanol and phosphate buffer (63:37%, v/v). The mobile phase was chosen for its good baseline resolution and suitable analysis duration. The optimal detection wavelength was found to be at 307 nm, as determined by the maximum peak of resveratrol standard. This wavelength is reported in several studies and the retention time is ≈2.24 minutes. The limit of detection (LOD) is 1 µg/ml while the limit of quantification (LOQ) is 5 µg/ml.

Concentration of Trans-resveratrol
The values of resveratrol in control and infected plants is given in Table 1. The control plants exhibit 0.82±0.03 mg/g of resveratrol while the infected plants show 2.7 fold increase in resveratrol production as 2.24±0.3 mg/g of dried plant residue (Fig. 2), formula used in calculation was, Response Factor = (mAU of known std./ concentration of std), then concentration in sample = (mAU of sample/Response Factor).

Yeast Culture Preparation and Microscopic Examination
Yeast startup with sugar solution, single colony isolation of yeast in PDA media petri plate then yeast was examined through microscope at 100X

Agarose Gel Electrophoresis Image of RNA
Gel was prepared with 1% Agarose in 1X Tris Acetic Acid Ethylene Diamine Tetra Acetic Acid (TAE) buffer and run at 100 0 C. Gel was illuminated at 302 nm UV trans-illuminator to obtain image. (Fig. 4)

Amplification Curve
Real time pcr amplification plots for ACT1 gene of 16 reactions vials are shown in Fig. 5. and SIR2 gene in Fig. 6.
Delta Ct value of ACT1 gene and SIR2 gene are shown in table 2 and 3 respectively.   Table 2)     Table 4.
Relative gene expression of all 3 samples is in bar graph in Fig. 7.

Replicative Life Span
Pharmacological anti-ageing screens in yeast is very productive by Replicative Life Span method [15]. Result is in table 5 due to increased cells.

DISCUSSION AND CONCLUSION
In conclusion, biotic stress in the form of Aspergillus niger and abiotic stress in the form of wounding results in increase in resveratrol content of experimental peanut plants. Thus this study reports a combination of elicitation methods on peanut plant, an important agricultural crop in India and a promising functional food, to develop as a source of bioactive compound resveratrol.
Sir2 gene expression produces sirtuin enzyme. Sirtuin enzyme is group of Histone Deacetylate Transferase enzyme that removes acetyl (-) group from lysine (+) amino acid situated on Histone. Thus negative ion DNA shows affinity towards + charge and get wrapped along Histone. Due to wrapping, RNA polymerase will not find site for transcription, hence, this promotes gene silencing.
In all three test conditions sir2 gene expression increased in Saccharomyces cerevisiae. This result confirm the hypothesis that transresveratrol actually enhance expression of sirtuin enzyme which is a class of histone transferase enzyme. Due to over activation of histone transferase enzyme, process of gene silencing occurs. When organism aged accumulation of multiple genes or noise genes halt the process of cell and that effect rate of cell division also. Enhanced sirtuin enzyme (product of sir2 gene) accelerates gene silencing process of undesired gene and hence cell reverses its age.
Replicative Life Span of Yeast cell increased due to presence of enhance trans-resveratrol peanut plant extract. It related epigentics change in sir2 gene. All results indicated reverse ageing in yeast.
Indian groundnuts are available in Bold or Runner, Java or Spanish and Red Natal varieties. Peanut being good source of protein and fiber, also contains resveratrol which is slowly gaining attention in the Indian market as a health supplement.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.