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Comparison of Antioxidant Properties and Flavonoid of Natural and in vitro Cultivated Nardostachys jatamansi

Hem Chandra Pant1, Indra Rautela2, Harsh Vardhan Pant3, Arun Kumar1, Pankaj Kumar4, Kaneez Fatima5, Naveen Gaurav1,*
1Department of Biotechnology, Shri Guru Ram Rai University, Dehradun, Dehradun-248 001, Uttarakhand, India.
2Department of Biotechnology, Uttaranchal University, Dehradun-248 001, Uttarakhand, India.
3Shri Guru Ram Rai P.G. College, Dehradun-248 001, Uttarakhand, India.
4Department of Microbiology, Dolphin (PG) Institute of Biomedical and Natural Sciences, Dehradun- 248 007, Uttarakhand, India.
5Department of Microbiology, Galgotias University, Greater Noida-201 310, Uttar Pradesh, India.
Background: India has very rich diversity of medicinal plants. Medicinal plants are thought to be a rich source of ingredients that can be used in the development of pharmaceutical or synthetic drugs. Aside from that, these plants play an important role in the development of human drug all over the world. Whether in modern or traditional medicine, medicinal plants are used to maintain health, to treat a specific condition, or both. Nardostachys is one of the most important medicinal plant having several therapeutic properties. It is threatened in its natural habitat due to over exploitation for therapeutic purposes and high demand in the traditional medicine system. Keeping these points in mind, we attempted to investigate alternative uses of in vitro grown plants in place of wild plants without disrupting plant-based therapeutics and market demand. And then compare the root extract of the Nardostachys jatamansi plant’s antioxidant and flavonoid levels under in vitro and natural growth conditions.

Methods: Nardostachys jatamansi is a plant that is widely used in traditional medicine systems. Because of its wide spread use in traditional medicine, this plant is considered endemic. In our study, we compare of antioxidant quality and Flavonoid amount of Natural and in vitro propagated Nardostachys jatamansi. Firstly we cultivated in vitro plants from Nardostachys jatamansi nodal explants for comparative analysis. The methanol extract of in vitro grown and wild-type plant root extract was then prepared using the maceration method. The extract was subjected to a comparative DPPH method to determine the presence of antioxidant potential in natural and in vitro grown plants. Furthermore, the HPLC analysis was used to detect and quantify the amount of Quercetin in both natural and in vitro propagated plants. 

Result: When grown in vitro at a higher concentration, the roots of Nardostachys jatamansi have greater antioxidant potential than when they are grown naturally. They demonstrated antioxidant DPPH radical scavenging activity, with an IC50 value of 29.55 µg/ml for in vitro generated plants and 24.18 µg/ml for naturally grown plants. The concentration of Quercetin (mg/ml) for natural plant species is 1.95 and for in vitro propagate plant is 1.83. The HPLC analysis presents distinct peaks, with the main peaks having retention time for standard Quercetin (10.38) in the natural plant (10.34) and in vitro grown plants (10.32). In the end, natural-type species that had been produced in vitro were used to obtain the potential of micro propagated plants. The DPPH test and flavonoid were tested on the root extract of natural and in vitro plants. Both plants displayed promising antioxidant activity and an HPLC study identified the Quercetin component.
In the region of 3000 to 5000 m high altitude plant is found, Nardostachys jatamansi is a fragmented, perennial, flower-patterned plant with a height of 10 to 60 cm. The plant’s short, dark grey, woody and thickened rhizome is a modified stem. The herb has rosette-shaped, whole, lanceolate leaves. According to Kumar et al., (2011)  the plant’s flowers have a pale purple color. From the plant’s rhizome, essential oil was extracted using the hydro-distillation method. According to Pradhan and Paudel (2014) the oil has a smell that is comparable to musk pod and is greenish in hue. In addition to alpine meadows, juniper scrub, dwarf rhododendron forests and open pine forests, rocky outcrops are the usual habitat type, according to (Ghimire et al., 2005). In the Kumaon, Garhwal and Nepal Himalayan regions, the Nardostachys jatamansi plant is quite plentiful, but because local herb collectors and traders have taken advantage of it, it is now scarce. When Nardostachys jatamansi was first mentioned as being heavily exploited in the Indian Himalayas, it was suggested that its systematic cultivation was required to preserve the plants for the Himalayan region. According to Airi et al., (2000) it is a plant species that is critically endangered in the Kumaon region of Uttarakhand. The study of Nardostachys jatamansi includes a thorough description of the plant, its development patterns, applications and other relevant information that may be useful in establishing the species’ long-term cultivation. It is intriguing to notice that the chemical compositions of the essential oils from Nardostachys jatamansi and Indian valerian have been compared in published studies. State that India, Nepal and Bhutan have banned the export of Nardostachys jatamansi (Bhattarai et al., 2002).This plant reproduces vegetative and by its seeds, which are presumably pollinated by tiny insects like flies. Nautiyal et al., (2003) review that seed germination rates between 10 and 20%. According to a study from Dolpo, Nepal, adult plants have higher survival rates (88-100%) than seedlings do (68-90%). The plant species may also take 3-4 years before the boom of seedlings to reproductive size. Nepal has determined survival prices of adults to be high (88-100%), at the same time decreasing for juveniles and seedlings (68-90% and 46-78%) (Ghimire et al., 2008). According to Sharma et al., (2016) and Malik et al., (2018) the plant roots contain a variety of phytochemicals, including sesquiterpenes, jatamansic acid, jatamansinone, nardostachone, jatamansinol, jatamol, nardostachyin, nardosinone, angelicin, jatamansin, calarene, atchoulense. Singh et al., (2009) and Rahman et al., (2011) describe that the plant’s rhizomes contain a variety of substances, including gum, sugar, starch, ketone, corrosive, lupelol, malliene, calarenol, coumarin, propionate and cyclohexanal.

The month of October sees the ripening of seeds that may later be sown following the winter. Jatamansi rhizomes can therefore be harvested around October, typically following Nepal’s Dashain festival. Even in November, if green plants are still visible, the rhizome can be taken. However, after November, the plants dry out and become hidden by snow, making it impossible to harvest the rhizome. So, according to Pant et al., 2021 October is the ideal month for collecting Jatamansi rhizomes. Both conventional and contemporary therapies have employed Nardostachys jatamansi. This study’s goal is to gather naturally growing Nardostachys jatamansi plants from various Himalayan regions and compare their antioxidant activity and flavonoid (Quercetin) content to that of in vitro cultivated plants.
Selection of the plant
 
The Nardostachys jatamansi plant was obtained from Pithoragarh and Baghwesher districts of Kumaun region of Uttarakhand, India. Plants authenticated by the Botanical survey of India, Dehradun which will provide a certificate with the number 702 on it.

In vitro cultivation of the mother plant
 
The plant hormones BAP (6-benzyl amino purine) and IBA (Indole butyric acid) were utilized in MS (Murashige and Skoog) media at various concentrations to initiate shoot growth in the Nardostachys jatamansi plant. Before inoculation, the explants were surface sterilized to completely eradicate fungus, bacteria and spores from the plant. When a shoot was first noticed in the plant, the plant hormones cytokinin, BAP (6-benzyl amino purine) and kinetin (6-furfural amino purine) were used to promote the growth of the shoot. After that, half-strength MS Medium with various concentrations of NAA and BAP was used to promote rooting in the plant. Sucrose was employed as a carbon and energy source at 0.8% (w/v), while agar was used as a freeze to MS Medium at 0.5% (w/v). Explants injected with MS medium were then moved into a tissue culture facility at 25°C for a 16-hour photoperiod of light and dark after the MS Medium’s pH was fixed at 5.8. After two weeks of incubation, routine data collection was started, including the number of plant shoots and root length.
 
Antioxidant activity
 
Because of their calming and relaxing effects on the central nervous system, Nardostachys jatamansi’s roots and rhizomes have been employed in Ayurvedic medicine. This investigation focused on the methanolic extract of Nardostachys jatamansi’s anti-stress abilities. By using a DPPH scavenging assay with an IC50 value of 10 g/ml, the antioxidant activities of Nardostachys jatamansi were examined.
 
DPPH radical scavenging activity
 
The free radical scavenging capacity of phytochemicals was assessed by adding 0.1 mM DPPH to 1.0 ml of standard/extract solution in methanol at various concentrations. After that, let the combination sit for 20 minutes before measuring it at 517 nm. The log dose inhibition curve was used to determine the sample’s IC 50 value, or the concentration of the sample needed to inhibit the DPPH free radical 50% of the time. Higher levels of free radical activity were indicated by the reaction mixture’s lower absorbance. Blosis et al., (1958) used the following equation to compute the percentage of DPPH that was scavenged.
A positive control was ascorbic acid. The DPPH radical scavenging activity was calculated using the formula,
 
 
 
Where,
At = The absorbance of the sample.
Ao = Absorbance of the control (without the extract).
The tests were run three times for a total of 30 minutes.
 
Preparation of DPPH
 
4 mg of 2, 2-diphenyl-1-picryl hydrazyl (DPPH) was dissolved in 100 ml of 95% methanol in the absence of light to create a 0.004% (w/v) DPPH solution. Three milliliters of various extracts in methanol were combined with one milliliter of this solution at concentrations of 10, 15, 20, 25, 30 and 35µg/ml (Fig 2). Here, only those extracts that could be dissolved in methanol were used and different quantities of those extracts were made using the dilution approach. After shaking, the mixture was left to sit at room temperature for 30 minutes. The sample’s absorbance was then measured at 517 nm with a UV-VIS spectrophotometer. Ascorbic acid was employed as the reference standard chemical and three copies of the experiment were run.
 
HPLC outline of  Nardostachys jatamansi
 
For the HPLC analysis of the plant, Nardostachys  jatamansi’s  methanolic extract was used. Using an extract of Nardostachys jatamansi, the flavonoid Quercetin was produced; a straight forward HPLC method was developed and verified for this. Due to the quick chromatographic run time, the new method can be applied to in-vivo research as well as routine quantification and quality control of Quercetin. The previously described HPLC method for Quercetin was outperformed by this technique. As a mobile phase, the experiment used acetonitrile, water and methanol. A UV-VIS detector with a variable wavelength of 242 nm was used for the detection. The amount of Quercetin in the plant material ranged from 1 to 2.0 mg/ml with a correlation coefficient. The method of standard addition was used to test both the HPLC method’s accuracy and the correctness of the complete extraction process. The Quercetin isolated from various extracts and preparations can be standardized using this method. The limit of quantification was determined to be ten times this ratio (LOQ = 10•SD/slope) and the limit of detection to be three times the ratio between the SD and the slope of the low concentration curve (LOD = 3•SD/slope) (Stefova et al., 2001).
 
Nardostachys jatamansi is a threatened plant species and necessary for many types of medicine (Bhatt et al., 2015). In this research study firstly a tissue culture technique can be utilized to grow Nardostachys jatamansi plant in in vitro condition without damaging the plant species.
 
In vitro propagation of Nardostachys jatamansi
 
According to Pant et al., 2021 MS (Murashige and skoog) medium containing phytohormone BAP + IBA (2.0 mg/l +1.5 mg/1) appear to have the maximum shoot growth. The largest shoots are seen in the Cytokines Carry MS medium (2 mg/l BAP and 1.5 mg/l KN) for the plant’s post-subculture shoot proliferation. Rooting branches that were between 1.0 and 5.5 cm long were cut from the culture plant and transferred to a rooting media containing various concentrations of NAA combined with BAP. For inducing roots in plants, NAA and BAP (1.5 mg/l+1.0 mg/1) in the 1/2 Strength of MS medium works very well (Fig 1).

Fig 1: Different stage of In vitro clonal propagation of Nardostachys jatamansi from nodal ex plants. A. Mother plant of N. jatamansi where explant taken for further propagation process; B. Shoot Initiation in the explants; C. Shoot multiplication occur in the plant after subculture of in vitro raised plant; D and E. Rooting is obtained inthe plant after the multiplication; F. Hardening of the plantsin the mixture of different soil.



Then, after being carefully taken from the culture flask and being well cleaned with distilled water, the better-developed rooted plants were transplanted to a small pot filled with a unique soil mixture.

Nardostachys jatamansi plant is over bleeding from the Himalayan region of India Purohit et al., (2012). In vitro clonal propagation, which produces genetically stable and disease-resistant clones of plants while limiting cultural variation, is the more powerful tool for plant propagation. The current work provides a possible in vitro propagation method for Nardostachys jatamansi that is large-scale, prolific, fast and affordable through proliferation shoot growth. So by the in vitro method the Nardostachys jatamansi plant was multiplied in a lab. In order to create shoots and multiple shoots in plants, a combination of BAP, IBA and kinetin appears to be the best response. Next, a concentration of BAP, NAA and half-strength MS medium demonstrated the most successful rooting, after two weeks of the culture, healthy plants were collected for future research study, according to (Dhiman et al., 2021).This study can be applied to the cost-effective in vitro production and preservation of this medicinal plant that is in grave danger of extinction.
 
DPPH Activity in Nardostachys jatamansi
 
After the in vitro propagation of the plant both natural and in vitro cultivated plant extracts are used to compare for the DPPH test. In the DPPH test, varied time durations between 10 and 60 min were needed for the plant extracts to reach chemical equilibrium (Sarbhov et al., 1978). This is a characteristic predicted because the reactivity of the antioxidant molecules is not constant (Berghe et al., 2012). The investigation began with the top phases or unambiguous solutions (Rahman et al., 2007). The final DPPH level was expressed  in mole per  cent and plotted against the mass of dried plant (i.e., the theoretical mass of dried plant needed to make the volume of extract utilized for a measurement) in the cuvette when equilibrium was attained at various doses of the extracts. The roots of methanol extract of naturally and in vitro showed better antioxidant potential when compared to standard ascorbic acid by DPPH scavenging assay method. The absorbance at 517 nm by UV visible spectrophotometer were found to be as 0.174±0.075 and % free radical inhibition 56.25±11.34 for the wild extract and for 0.063±0.022 in vitro methanolic extract and % free radical inhibition 66.83±6.81, IC 50 value obtained were as 24.18 for naturally species and 29.55 µg/ml for in vitro propagated species (Choudhary et al., 2007; Hasan et al., 2008).

Fig 2: Preparation of the DPPH and various different concentrations of wild and in vitro Propagated Extract of N. jatamansi. A. Preapration of the free radical DPPH; B. Different concentration of wild species of N. Jatamansi; C. Different concentration of in vitro propagated N. jatamansi.



The inhibition values of all the different plant extracts both naturally and in vitro have been provided in Table 1 and Table 2 and Fig 3 Fig 4.

Fig 3: N. jatamansi (wild) root extract with standard ascorbic acid.



Fig 4: N. jatamansi root extract (in vitro) with standard ascorbic acid.



Table 1: 517 nm UV visible spectrometer study of N. jatamansi (wild) root extract with standard ascorbic acid (DPPH scavenging assay method).



Table 2: 517 nm UV visible spectrometer study of N. jatamansi root extract (in vitro) with standard ascorbic acid (DPPH scavenging assay method).



It determined that methanolic extract of plants at higher concentration captured more free radicals formed by DPPH resulting in decrease in absorbance and increase in IC 50 Value (Sharma et al., 2020).

This study discovered that Nardostachys jatamansi species methanolic extract shown superior antioxidant capability by DPPH radical scavenging method, indicating that this plant has antioxidant activity (Kinsella et al., 1993; Mishra et al., 1995). This determining the possible antioxidant activity is found all plant extracts, both natural and in vitroand by the comparison with naturally plant in vitro propagated plant show better results (Sharma et al., 2008).
 
HPLC detection of phytochemicals of N. jatamansi
 
The phytochemical constituents i.e. flavonoid (Quercetin) was evaluated using HPLC (Yu-ling et al., 2017). By this technique identification and other types of impurities was detected. Any form of deviation from the norm in the peaks number, peaks area, or retention time indicates that the medication is impure or failing (Rahman et al., 2013). The Nardostachys jatamansi HPLC examination produced a number of peaks, however the most important ones with high area concentration were recorded as 29619399 for the naturally species with a retention time of 10.34 2.8 min and 27876969 and 10.32 min for the in vitro cultivated plant. UV detector used to study the concentration of Quercetin in the natural type and in vitro grown plants for the identification of Quercetin, liquid phase acetonitrile, methanol and water used and absorbance of Quercetin measured at 242 nm by using UV detector. Concentration of Quercetin in mg/ml is 1.95 and 1.83 for natural and in vitro grown plant species Fig 5 and Table 3.

Fig 5: HPLC detection of phytochemical of N. jatamansi. A. HPLC Chromatogram of Standard profile of Quercetin; B. HPLC Chromatogram of natural species profile of Nardostachys jatamansi; C. HPLC Chromatogram of In vitro propagated profile of Nardostachys jatamansi.



Table 3: Flavonoid (Quercetin) Concentration of Natural and in vitro propagated Nardostachys jatamansi (mg/ml).



For the identification, standardization of methanolic extract of plant roots, HPLC is an impressible and authentic tool that widely used for the standard estimation of plant extract and it obtain formulation (Mahendra et al., 2011). Results of HPLC analysis of Nardostachys jatamansi methanolic extract of natural species at 242 nm mobile phase: acetonitrile, water and methanol in ratio (40:30:30) various constituents were recorded as evidenced by the chromatogram obtained at various retention times (10.341, 11.002, 14.058, 16.074, 17.347, 23.507 and 25.183) are the constituents found in Nardostachys jatamansi roots mainly. The methanolic extract of Nardostachys jatamansi root extract of in vitro chromatogram shows different constituents at various retention times (10.321, 10.999, 14.034, 16.027, 17.303, 25.867 and 28.773) (Rashid et al., 2018). According to Marquez et al., (2005) these peaks indicate the chemical components found in plant roots.

Currently, research is being done on the isolation and characterization of bioactive compounds. Quantitative results show that HPLC analysis is crucial for determining the quantity, legitimacy and identification. The effectiveness of Nardostachys jatamansi in treating various bodily ails is also demonstrated by this analytical methodology for quality assurance. To enhance quality, clarity and identification of the upcoming herb Nardostachys jatamansi, this study will offer preliminary information about it. The Quercetin chemical was found in Nardostachys jatamansi and measured the amount of Quercetin in both naturally occurring and in vitro cultivated plant species (Garg et al., 2021). Le et al., 2017 explain that the chromatography method gives us a chromatogram of the several substances found in the methanolic extract of the plant’s roots.
Firstly, the Nardostachys jatamansi plant was grown utilizing the in vitro micropropagation technique. The best potential and activity of both naturally occurring and in vitro cultivated plants are compared utilizing antioxidant tests and HPLC analysis. According to the results of the current study’s DPPH test, antioxidant levels were found to be higher in in vitro propagated plants than in naturally growing plants. The analytical techniques described here can easily separate the natural and in vitro cultured Nardostachys jatamansi species when it comes to Quercetin identification and quantification. According to this compression study, plants cultivated in vitro can also be employed as a source of medicine.
The authors are thankful to the Management and Staff members of Shri Guru Ram Rai University Patel Nagar, Dehradun for their support and providing necessary facilities.
Develop thought work, Conducted the research, experimentation and data analyses, Prepared and written the initial draft.
 The research content of manuscript is original and has not been published elsewhere.
Not applicable.
Not applicable.
All authors agree to publish the paper in Agricultural Science Digest.
The authors declare that there is no conflict of interest.

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