A rapid, efficient and cost-effective DNA isolation protocol for various herbal products which is appropriate for different downstream genomics applications

Medicinal plants have been used for the treatment of diseases and its demand is expanding in both developed and developing countries. The extensive consumption to meet the demand and supply ratio exerts a heavy strain on the existing resources. This subsequently led to adulteration and substitution. The DNA-based method is the most widely adopted for the determination of adulteration in herbal products. Quality DNA extraction is regarded as the prerequisite for the application of DNA-based techniques in herbal plant authentication. Various protocols for DNA isolation could only be applied to a small number of species and are often expensive and time-consuming. Furthermore, herbal products undergo various conditions and are mixed with different substances, these factors affect the integrity of the DNA making it difficult to isolate. In this study, a simple, cost-effective and rapid CTAB-based DNA extraction protocol was developed which successfully isolated quality DNA from different samples of medicinal plants and their respective adulterants without using liquid nitrogen. Quality DNA was also isolated from different herbal products of reputed companies. The protocol was also validated by the isolation of quality DNA from different herbarium samples. DNA quality of both plants and herbal drugs was measured by A260/A280 ratio, which was ranging between 1.3 and 1.8. Thus, the developed protocol can be adapted for medicinal plants and herbal drugs quality DNA isolation and authentication as well as herbarium samples for molecular identifications. The isolated DNA was found to be of good quality and sufficient quantity for ISSR-PCR, SCoT-PCR and restriction digestion reactions. The protocol has successfully demonstrated its suitability for different genomics downstream applications.


Introduction
World Health Organization (WHO) defined herbs or herbal preparations as herbal products that contain whole plants, parts of plants, or other plant materials, including leaves, bark, berries, flowers and roots or their extracts as active ingredients intended for human therapeutic use (World Health Organization 2001).Herbal products are used in the treatment of the spectrum of diseases and it is being used by almost 80% of the World's population (World Health Organization 2011; Khan and Ahmad 2019).The global market for herbal medicine is estimated at US$ 110.2 billion in the year 2020 and is expected to reach US$ 550 billion by 2030 (Raclariu et al. 2018).The increasing demand for herbals is stimulating the economically-motivated adulteration and substitution of medicinal plants with look-alike species or inferior taxa to meet the annual demand of the herbal industry (Mishra et al. 2016).Adulteration and substitution are currently a major concern with confirmed reports from several countries (Newmaster et al. 2013;Urumarudappa et al. 2016).A prior study determined the authenticity of 5957 commercial herbal products sold in 37 countries, distributed on all six continents, using DNA-based methods.The number of adulterated herbal products varies significantly among continents, being highest in Australia (79%), South America (67%), lower in Europe (47%), North America (33%), Africa (27%) and the lowest in Asia (23%) (Ichim 2019).In Australia, Japan, Taiwan and China, chronic use of Aristolochia fangchi adulterated products led to the death of patients (Yang et al. 2002;Hong et al. 2006).Several studies have reported the adulteration of Stephania tetrandra roots with roots of the toxic herb, Aristolochia fangchi (Guang-Fang-Ji) leading to major renal failure (Michl et al. 2013).
The adulteration in herbal drugs accentuates the necessity for proper identification, certification and toxicity assessment of herbal products (Dutta et al. 2018).Hence, identifying a comprehensive method for effective monitoring of raw herbal drugs is a prerequisite.Since raw drugs are available in extremely dried, shredded, or powdered form, species identification using traditional tools may not always be possible (Seethapathy et al. 2019).In recent years, several techniques have been developed to identify medicinal plants in trade including chemical fingerprinting and DNA-based approach.
Molecular markers of the DNA-based method are the most widely adopted for the determination and test for adulteration in herbal products (Joshi et al. 2004).Considering the wide application and the use of DNA-based methods for medicinal plant authentication in the herbal medicine sector, quality DNA extraction is regarded as the prerequisite for these techniques.An excellent DNA isolation protocol from medicinal plants must be rapid, simple, inexpensive and effective (Rao et al. 2007).Various protocols for plant DNA isolation were designed by scientists, some of these protocols include Dellaporta et al. 1983;Keim et al. 1988;Doyle and Doyle 1990;Khanuja et al. 1999;Kumar et al. 2003.Although many of the developed protocols could only be applied to a small number of species due to the chemical variability of the species of plants, occasionally closely related species fail to respond to the same protocol (Weishing et al. 1994).There are many secondary metabolites found in plants, particularly medicinal plants.These metabolites are the appealing features of medicinal plants thereby attracting molecular biology research.However, these compounds interfere with the process of DNA isolation (Ounzar et al. 1998).Some DNA isolation protocols are time-consuming and yield low-quality DNA that could not be suitable for further analysis such as PCR due to the presence of secondary metabolites.In this study, a simple and rapid genomic DNA isolation protocol was optimized and tested for DNA extraction from samples of medicinal plants and herbal products.Inter Simple Sequence Repeat (ISSR) and Start Codon Targeted (SCoT) polymorphism markers were used to confirm the quality of the isolated DNA.CTAB extraction buffer: [100 mM TRIS-Cl pH 8.0 (WV −1 ), 20 mM EDTA pH 8.0 (WV -1 ), 1.6 M NaCl (WV −1 ), 4% CTAB (WV −1 ), 2% β-Mercaptoethanol (VV −1 )].
The optimized DNA extraction protocol -This modified CTAB protocol is ideal for DNA isolation from both plant samples (leaves, roots and bark) and herbal drug samples (powder, capsules and tablets).The protocol does not require liquid nitrogen for grinding the sample.
1.For plant samples (leaves and roots), ground 300 mg of plant samples with 3 ml CTAB buffer using mortar and pestle.Transferred 1.2 ml of the mixture in a sterile 2 ml tube.2. For herbal drugs (powders, capsules, tablets), transferred 200 mg of the sample into a 2 ml tube containing 1.2 ml CTAB buffer.3. Mixed vigorously and incubated in a water bath for 40 min at 70 °C with shaking at 10 min intervals.4. To the incubated sample mixture, 500 µl Chloroform: Isoamyl alcohol (24:1) was added and mixed for 2 min then centrifuged for 10 min at 12,000g. 5.The supernatant was transferred into a clean tube, and an equal volume of Chloroform: Isoamyl alcohol was added; mixed thoroughly for 3 min and then centrifuged the samples at 13,000g for 10 min.6.The supernatant was collected into a sterilized tube, then 10 µl of RNase A (20 mg/ml) was added and incubated at 37 °C for 30 min.7. To the incubated sample mixture; an equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) was added and mixed for 5 min, followed by centrifugation at 12,000g for 15 min.8.The supernatant was transferred into a clean tube; an equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) was added and mixed for 5 min, followed by centrifugation at 12,000g for 10 min.9.The supernatant was collected into a clean tube and 1 ml of ice-cold absolute Isopropanol was added; mixed gently and centrifuged at 11,000g for 3 min.10.The Isopropanol was discarded and the DNA was washed twice with 70% Isopropanol with centrifugation at 12,000g for 2 min.11.Air-dried the DNA samples under a running ceiling fan for 2-5 min.12.The DNA was suspended in 150 µl TE buffer and stored at − 20 °C for further analysis.
Quantification of extracted DNA -DNA quality and quantity were determined by measuring optical density (O.D.) at A260 and A280 with Nanodrop Spectrophotometer (M/S TECAN Infinite ® 200 PRO NanoQuant, Switzerland).5 μL of the isolated DNA was loaded on agarose gel containing ethidium bromide (0.5 μg mL − 1 ) and were subjected to agarose gel (0.8%) electrophoresis in 1X TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA) at 70 V for 1 h.Known standard (λ-DNA, 100 ng μL − 1 , Bangalore Genei, India) was also run to check the DNA quality and quantity.The gels were photographed under a Molecular Imager Gel Doc XR+ (Biorad, California, USA).The result obtained is illustrated in Table 1 and Fig. 1.
Restriction enzymes digestion Restriction digestion.Total cellular DNA (1.5 μg) of three species was digested with EcoRI (Thermo scientific FastDigest).The reaction was carried out in buffered condition, incubated at 37 °C for 30 min following the manufacturer's instructions (Puregene, Genetix Biotech).The digested DNAs were run on 0.8% agarose gel electrophoresis along with standard (λ-DNA, 50 ng mL −1 ) and marker (λ-Hind III digested and 50 bp DNA ladder; MBI Ferment, USA).The gels were photographed under a Molecular Imager Gel Doc XR+ (Biorad, California, USA).Doyle and Doyle (1990) protocol has been widely used for DNA isolation, however, it fails in the isolation of quality DNA from various medicinal plants (Ajmal Iqbal et al. 2013).The low-quality DNA is a result of contaminations with polysaccharides, proteins and secondary metabolites (Puchooa and Venkatasamy 2005).The optimized protocol prescribed an increase in concentrations of CTAB, NaCl among other modifications that helped in the removal of polysaccharides and other DNA contaminants which corresponds with the findings of Paterson et al. (1993).Furthermore, this protocol does not require the use of liquid nitrogen thereby reducing the expenses of DNA extraction.Using the optimized protocol, DNA was successfully extracted from leaves of five different samples of medicinal plant namely; Withania somnifera WS (A) Asparagus racemosus AR (C) Saraca asoca SA (E) and Chlorophytum borivilianum CB (G) and their respective adulterants Trigonella foenum-graceum TR (B), Asparagus sarmentosus AS (D), Polyalthia longifolia PL (F), Chlorophytum arundinaceum CA (H).The clear DNA bands are illustrated in Fig. 1A-H.

Results and discussion
The purity of the isolated DNA samples from all medicinal plants and their respective adulterants ranged from 1.5 to 1.8 at A260/A280 except for samples A1, A4 and CB5 with the value of 1.3 and CB4 with value of 1.4.The concentration of isolated DNA samples of the plants varied between 60 and 210 ng µl −1 ; except for AR5, AS3 and SA5 which have concentrations of 54 ng µl −1 , 56 ng µl −1 and 53 ng µl −1 , respectively (Table 1).The explanation for the DNA Samples A1, A4, CB5 and CB4 having relatively low purity compared with other samples of the same species and AR5, AS3 and SA5 having lower concentration compared to other samples of the same specie despite using the same protocol could be attributed to an accidental increase or decrease in the quantity of sample.This could have affected the ratio of sample quantity to the volume of extraction buffer which influenced the quality and yield of DNA as proposed by Križman et al (2006).DNA was extracted from five different samples (obtained from different companies) of Ashwagandha ASH1-ASH5 (L), Shatavari STV1-STV5 (M) and Brahmi BRH1-BRH5 (N) herbal drugs using the optimized protocol.Quality DNA with good concentration was observed in most of the herbal drug samples (Fig. 1).The concentration ranged from 20 to 300 ng µl −1 and the quality range of 1.3-1.8 at A260/ A280 (Table 1).Medicinal plants experience diverse conditions and undergo treatment with various chemicals during the process of herbal drug production.These factors have a significant impact on the DNA of the processed medicinal plant.Different companies adopt distinct treatment methods and procedures for herbal drug formulation, which consequently influence the quality of DNA in the samples.This variability accounts for the variations observed in terms of purity and concentration levels among the isolated DNA.The reason for the lack of DNA in sample STV3 can be attributed to the high level of chemical composition and procedure followed by the company in processing the drug (Mohammed Abubakar et al. 2017).
There are several molecular markers used in the authentication process of medicinal plants and herbal drugs, some of the most commonly used molecular primers in this regard include ISSR and SCoT primers.To determine the application of isolated DNA using the protocol for the PCR technique; ISSR-PCR was performed with isolated DNA from Withania somnifera (WS) and its adulterant Trigonella foenum graceum (TR).Saraca asoca (SA) and its adulterant Polyalthia longifolia (PL) using [CA] 8 RC as a primer (Fig. 1I).Amplifications were observed in all four samples of various sizes with clear DNA bands that would be used to discriminate between the plants.Furthermore, SCoT-PCR technique was also carried out on extracted DNA samples of Asparagus racemosus (AR) with its adulterant Asparagus sarmentosus (AS) and Chlorophytum borivilianum (CB) with its adulterant Chlorophytum arundinaceum (CA).Clear DNA bands of Chlorophytum arundinaceum (CA) sample can be observed on the gel Fig. 1J, which can be applied in authenticating the plant CB from CA.This is also proof of the reliability of this protocol in such analysis.DNA of low purity that contains secondary metabolites or polysaccharides is not suitable for PCR amplification reaction as stated by Lodhi et al. (1994).Extracted DNA showed amplification in both ISSR-PCR and SCoT-PCR, thus the optimized protocol has proven to be ideal for PCR-based techniques and other downstream applications.
The inhibitory compounds found in low-quality DNA such as proteins, polyphenols and other secondary metabolites interfere with enzymatic reactions (Mishra et al 2008;Amani et al. 2011).To further determine the applicability of the optimized protocol in various molecular analyses such as genomic sequencing and gene cloning, isolated DNA from Withania somnifera (WS), Saraca asoca (SA) and Asparagus racemosus (AR) were digested with EcoRI (Thermo scientific FastDigest) as indicated in Fig. 1K.There was successful digestion of all the DNA samples dWS, dSA and dAR while the untreated DNA samples were intact WS, SA and AR.Such downstream molecular reactions are not possible without high-quality DNA.(Varma et al. 2007).This result further confirmed the quality of DNA isolated using  this protocol.The protocol is also suitable for herbal drug DNA isolation.Five different drug samples of Ashwagandha, Shatavari and Brahmi samples were taken; DNA isolation was carried out using the optimized protocol and PCR quality DNA has been obtained (Fig. 1L-N).
To further demonstrate the applicability of this protocol for the isolation of DNA from different samples.The protocol was used to isolate DNA from the herbarium of Vinca rosea L. (S1), the fresh bark of Polyalthia longifolia plant (S2) dried bark of Saraca asoca plant (S3).Successful DNA with good quality was isolated from all the samples with the purity of ratio of A260/A280 1.7, 1.8, 1.7 and concentrations of 137 ng µl −1 , 240 ng µl −1 and 189 ng µl −1 respectively as shown in Table 1.
The study developed a simple cost-effective DNA isolation protocol that successfully extracted DNA from various medicinal plants, herbal drugs and herbarium samples.Moreover, the protocol does not require liquid nitrogen for grinding the plant samples making the protocol more robust and universally acceptable which will help the researchers when liquid nitrogen is a limiting factor.The quality and quantity of isolated DNA were successfully validated in different PCR applications as well as restriction digestion systems.

Table 1
Illustrating optical density (O.D.) and concentration of DNA extracted from different herbal plants and products along with their adulterant plants.