ISOLATION AND SPECTROSCOPIC CHARACTERIZATION OF NARDOSINONE FROM THE ROOTS OF NARDOSTACHYS JATAMANSI

INTRODUCTION:

Natural products, also known as "natural sources deriving compounds," are found in plants, animals, and microorganisms. In particular, a wide range of species from the kingdom of plants are still utilized as medicine to treat a number of illnesses in various regions of the world, including South America (Bolzani et al., 2012), Asia (Durajpandiyan et al., 2006; Grover et al., 2002), Africa (Jurg et al., 1991; Nugueym et al., 2008; Brusotti et al., 2011; Khalid et al., 2012)."Natural product and/or natural product structures continued to play a highly significant role in the drug discovery and development process," according to Newman and Cragg's recent review (New man and Cragg, 2012).Biodiversity provides

numerous novel chemical entities (NCEs) for therapeutic purposes. These secondary metabolites, produced by plants as defense against pathogens and herbivores, belong to three primary chemical families: phenolic compounds, terpenoids, and alkaloids. Understanding plant parts used for treatments is crucial for ethno pharmacological methods. However, due to lack of information about the secondary metabolite's nature, extraction, purification, and separation procedures are conducted to isolate and identify the bioactive compounds (Brusotti et al., 2014). Nardostachys jatamansi (D.Don) DC. (Caprifoliaceae Juss.: Dipsacales Batsch) has a long history of use in ayurveda and traditional medicine systems (Sharma

et al., 2000; Chen and Mukherji, 2016). Phytochemical research demonstrated the presence of essential oils (coumarins and sesquiterpenes) in both the roots and rhizomes of N. jatamansi, which are directly related with several medicinal applications (Mishra et al., 1995; Nautiyal, 2013; Singh et al., 2015; Rekha et al., 2013; Liu and Liu, 2014). It is also one of the top 20 most traded plants in India and is heavily exploited (Rai et al., 2000; Olsen, 2005; Chauhan et al., 2011; Dhiman and Bhattacharyya, 2020). It is presently on the verge of extinction due to its vast range of uses in traditional medicine, severe over-exploitation, and small reproductive phase and poor germination rate (10- 20%) (Nautiyal et al., 2003).The tiny, perennial rhizomatous herb Nardostachys jatamansi DC grows from 2200 m to 5000 m above sea level on steep, damp, rocky, undisturbed grassy slopes in India, Nepal, China, Tibet, and Bhutan (Ghimire et al., 2005).The Ayurvedic classics Charaka Samhita, Nighantus Chikitsagranthas, and Sushruta Samhita all contain descriptions of jatamansi. It is explained in the Charaka Samhita as Sangyasthapa Mahakashaya is utilized in Kushtha as a dhumvarti for hikkashwasa. Used in Kasa, Hriveradighrita is used in Arsha. Used in unmade is Mahapaishachikaghrita. It is known as Kumararasayana in the Sushruta Samhita (Airi et al., 2000). It's also referred to as spikenard, Indian nard, jatamansi, and balchar. Several medical systems use its rhizomes in traditional remedies (Yang, 1996). It has been tested for depressing effects in herbal mixtures containing other plants. According to Ayurveda, Nardostachys jatamansi roots and rhizome have different effects on the doshas. In the end, trido shashamak, but kapha-pitta nashak in particular, has been used medicinally due to its anti-ischemic, antioxidant, neuroprotective, and anticonvulsant properties. Additionally, Nardostachys jatamansi improves memory. It is also intended to treat amnesia brought on by aging because mice age naturally. The rhizome of Nardostachys jatamansi DC has been shown to be an effective antistress and memory-restoring medication for dementia patients (Jadhav et al., 2009). For millennia, India has utilized jatamansi extensively in perfumes and medicine. Numerous therapeutic qualities are associated with it, including anti-lipid peroxidative, hypolipidemic, antioxidant, hepatoprotective, sedative, tranquilizing, antihypertensive, anti-inflammatory, antidepressant-like, anticonvulsant, hypotensive, anti-asthmatic, and anti-estrogenic activity (Rahman

et al., 2011). In addition, it is used to treat a number

of nervous problems, including excitement, epilepsy, neurosis, insomnia, Alzheimer's disease, and learning and memory impairments (Joshi and Parle, 2006; Rahman et al., 2011). It is also used to cure

hair loss, growth, and shine (Bagchi et al., 1991). Additionally, the antispasmodic and stimulating qualities of their extracts can be utilized to control

constipation, urine, menstruation, and digestion in addition to treating fits and heart palpitations (Anon, 1993).

MATERIAL AND METHOD :

2.1 Plant Material

The roots and rhizomes of Nardostachys jatamansi, were collected from Garhwal region in Uttarakhand in the month of September, 2021. The plant was authenticated by Dr. V.P Bhatt, Scientist, Herbal rese*arch and development Institute, Chamoli, Uttarakhand. A voucher specimen (Ref No 320/HRDI/21-1/2020-21) has been deposited in the herbarium of our institute, Herbal research and development Institute, for future reference.

2.2 Plant Extraction

Extraction of dried leaves powder was done with petroleum ether and by placing in a thimble of Soxhlet apparatus using Hydroalcoholic solvent system at 40-60oC temperature of the heating mantle for 8-10 hours. Further evaluation of that extract like isolation of phenols and characterization is carried out in this paper.

2.3 Preliminary Thin Layer Chromatography

Thin Layer Chromatography was performed utilizing a different solvent system that included standard sesquiterpeniod on TLC plates of silica gel 60 F254 precoated with a layer thickness of 0.2 mm. The capillary tube was used to manually apply the spots, the plates were allowed to air dry, and a solvent system was used to produce the TLC chamber at room temperature. Spots on TLC plates were seen using an iodine chamber in visible light and UV light

254 & 365nm respectively. Rf values were computed (Kagan et al., 2014).

Rf Value = (Distance traveled by solute)/ (Distance traveled by solvent)

A solvent system was created for the initial TLC analysis of the Nardostachys jatamansi, extract, wherein the standard sesquiterpeniod was observed in the Toluene: Ethyl acetate: Acetic acid (9:1:0.2) mobile phase. For column chromatography, toluene: ethyl acetate: acetic acid (9:1:0.2) solvent was used as the mobile phase.

2.4 Column chromatography

Sesquiterpeniod was separated from Nardostachys jatamansi extract using silica gel column chromatography using hydro alcoholic extract. Chromatography was performed using a vertical glass column composed of borosilicate material. Before packing, the column was thoroughly dried and cleaned with acetone. Wet packing was used to pack the column, with silica gel (60–120) serving as the adsorbent. Toluene was used to produce the slurry, which was then added to the column. One

gram of extract was applied to the column's top. Column chromatography was performed using the gradient elusion technique. Toluene was used to elude the column: Nineteen elutes of ethyl acetate: acetic acid (9:1:02) were obtained. To find a single compound, TLC was used to concentrate the fractions or elutes that had been collected (Srivastava et al., 2021).

2.5 Spectroscopic characterization: -

2.5.1 UV-visible Spectroscopy

The isolated fraction H of Nardostachys jatamansi extract was scanned from 200 to 800 nm wavelength using UV-Visible Spectrophotometer (Shimadzu UV- 1700) and the characteristic peaks were detected and recorded (Patel et al., 2022).

2.5.2 FT-IR

FT-IR spectroscopy was carried out using a Perkin Spectrum BX spectrophotometer to determine whether the functional groups were present in the separated fraction H of Nardostachys jatamansi extract. After being dried and crushed using KBr pellets, the samples were examined using a Thermo

Nicolet model 6700 spectrum analyzer. A mixture of 2% finely dried sample was used to make a disk containing 100 mg of KBr, which was subsequently analyzed using an IR spectrometer. Spectra of infrared light were obtained between 400 and 4,000 cm-1 (Luciene et al., 2008).

2.5.3 NMR Spectroscopy

NMR spectroscopy was performed for the isolated fraction H of Nardostachys jatamansi extract to identify the structure of the compound present in the isolated fraction. NMR spectroscopy for this purpose was Fourier Transform Nuclear Magnetic Resonance spectroscopy, Model AVNACENEO500 Ascend Bruker BioSpin International AG, Switzerland (Zia et al., 2019).

2.5.4 Mass Spectroscopy

Mass spectrometry converts molecules into ions and according to their mass and charge the ions can be separated and sorted. The mass spectrometer used for the identification of the molecular weight of the compound was mass spectrometer instrument micrOTOF-Q 228888.10348 (Wiley et al., 1995).

RESULTS:

3.1 Preliminary TLC preparation for the estimation of active constitutes –

TLC of Nardostachys jatamansi Hydro alcoholic extract For Sesquiterpeniod: -

Mobile Phase- Toluene: Ethyl acetate: Acetic acid (9: 1: 0.2)

Figure 1: TLC estimation by UV lamp for TLC of NJ with Std. Sesquiterpeniod (Std. = Standard, NJ = Nardostachys Jatamansi)

Table 1: TLC of Nardostachys Jatamansi Hydro alcoholic extract

No.

Solvent system

No. of spots

Colour of spots at Wavelength (254 & 365nm)

Rf value (Extract)

Rf value (Std.Sesquiterpeniod)

`1.

Toluene: Ethyl Acetate: Acetic acid (9:1:02)

Light Green (Std) Florescence Light Blue Florescence

Florescence(Green) Dark Florescence Florescence

- 0.17

0.20

0.26

0.53

0.62

0.67

0.53

TLC of Nardostachys jatamansi extract was performed on different solvent systems (solvent system was selected on the basis of literature survey). TLC performed in Toluene: Ethyl Acetate: Acetic acid (9:1:02) that were clearly visible bands of Nardostachys jatamansi extract with Std. Sesquiterpeniod. The Rf values of Nardostachys jatamansi extract with std. Sesquiterpeniod were found to be 0.53 and 0.53.

3.2 Column Chromatography

The fractions/elutes obtained from silica gel column chromatography of Nardostachys Jatamansi Hydro alcoholic extract were tested for the detection of various Phyto compounds using TLC. The collected fractions/elutes were taken properly and do the UV spectrum.

3.2.1 Column Chromatography of Nardostachys jatamansi Hydro alcoholic extract -

Table 2: Fraction collected from Column Chromatography of Nardostachys jatamansi Hydro alcoholic extract

Sr. No.	Eluent composition	Fraction collected	Remarks
1	Toluene: Ethyl Acetate: Acetic acid (9:1:02)	01 (A)	White coloured mixture of compound
2		02 (B)	White creamy coloured mixture of compound
3		03 (C)	White coloured mixture of compound
4		04 (D)	White creamy coloured mixture of compound
5		05 (E)	Light Yellowish coloured mixture of compound
6		06 (F)	White creamy coloured mixture of compound
7		07 (G)	Very Light Yellowish coloured mixture of compound
8		08 (H)	Yellowish coloured mixture of compound
9		09 (I)	Light Yellowish coloured mixture of compound
10		10 (J)	White Creamy coloured mixture of compound

3.2.2 TLC of all collected fractions-

A) TLC of all collected fractions of Nardostachys jatamansi Hydro alcoholic extract –

Figure 2: TLC estimation by UV lamp for NJ fractions after column chromatography with std. Std.

Sesquiterpeniod. a) Short-UV (254 nm), b) Long-UV (365 nm), c) visible light. (Std. = Standard, NJ = Nardostachys Jatamansi)

Sr. No.	Fraction	Solvent system	No. of spots	Colour of spots at Wavelength (254 & 365nm)	Rf value (Extract)	Rf value (std. Sesquiterpeniod)
1.	A	Toluene: Ethyl Acetate: Acetic acid (9:1:02)	-	-	-	0.54
2.	B		01	Light Fluorescence	0.46
3.	C		01	Light Blue	0.18
4.	D		01	Light Blue	0.18
5	E		01	Fluorescence	0.26
6	F		01	Light Green	0.29
7	G		01	Light Green	0.29
8	H		02	Green Dark Green	0.54 0.23
9	I		01	Dark Green	0.23
10	J		-	-	-

TLC of fractions (A, B, C, D, E, F, G, H, I & J) of Nardostachys jatamansi Hydro alcoholic extract - Table 3: Rf values of all collected fractions of Nardostachys jatamansi after column chromatography

Rf value resulted after performing the TLC estimation was also done for the confirmation of active constituent in fraction H of Nardostachys jatamansi Hydro alcoholic extract with mobile phase Toluene: Ethyl Acetate: Acetic acid (9:1:02) by comparing with std. Sesquiterpeniod.

3.3 Spectroscopic characterization: -

3.3.1 Active constitutes estimation by UV-Spectroscopy and FTIR – Spectroscopy

UV spectra of the isolated fraction H of Nardostachys jatamansi was recorded over a scanning range of 200-800 nm and λmax of isolated compound were determined. The Blank was Toluene: Ethyl acetate: Acetic acid (9:1:0.2). The wavelength of isolated fraction H of Nardostachys jatamansi Hydro alcoholic extract was found to be 251 nm.

Figure 3: Active constitutes estimation by UV- Spectra of H fraction of Nardostachys jatamansi Hydro alcoholic extract after column chromatography

Figure 4:IR spectra of the isolated Fraction H of Nardostachys jatamansi Hydro alcoholic extract

Table 4: FTIR- Spectrum Frequency Range of the isolated Fraction H of Nardostachys jatamansi Hydro alcoholic extract

Sr. No.	Fraction	Frequency Range	Group Absorption (cm^-1)	Appearance	Group	Compound Class
1	H	4000- 3000 (cm-1 )	3450.65	Strong, Broad	O-H stretching	Hydroxyl Group
		3000- 2500 (cm-1 )	2927.48	Medium	C-H stretching	Alkane
		2400-2000 (cm-1 )	2345.90	Strong	O=C=O stretching	Carbon dioxide
		2000- 1600 (cm-1 )	1653.88	Medium	C-O stretching	Carbonyl group
		1400- 1100 (cm-1 )	1103.70	Weak	C-C stretching	Alkane
		1600-1400 (cm-1 )	1438.24	Strong	C=C stretching	Benzene Ring
		840-790 (cm-1 )	796.91	Medium	C=C bending	Alkene

The IR Spectra of isolated fraction H of Nardostachys jatamansi Hydro alcoholic extract showed that -OH group broad absorption peak appeared at 3450.65 cm-1. The C-H stretching peak appeared at 2927.48 cm-1 for Alkane. O=C=O stretching peak appeared at 2345.90cm-1, Carbonyl group C-O stretching absorption peak appeared at 1653.88 cm-1 and C-C stretching peak at 1103.70 cm-1 for Alkane. The C=C stretching absorption peak at 1438.24 cm-1 for Benzene ring and C=C bending peak appeared at 796.91 cm-1.

3.3.3 1H NMR - Spectroscopy-

1H NMR spectra of isolated fraction H of Nardostachys jatamansi Hydro alcoholic extract was recorded on NMR Spectrometer. Tetramethylsilane used as an internal standard. The signals are denoted with the symbols s, d, t, and m for singlet, doublet, triplet, and multiplet, respectively.

(A) 1H NMR spectra of the isolated compound (Fraction H) of Nardostachys jatamansi –

In 1H NMR spectra of isolated fraction H of Nardostachys jatamansi Hydro alcoholic extract showed that lH-3 protons appeared at 0.93 (d) ppm, lH-9 protons appeared at 1.15-1.34 ppm (1.20 (s) ppm, 1.25 (s) ppm, 1.30 (s) ppm), lH-2 protons appeared at 1.55-1.75 ppm (1.60 (dddd) ppm, 1.62 (dddd) ppm), lH-2 protons appeared at 1.92-

2.15 ppm (1.99 (dqd) ppm, 2.05 (d) ppm), lH-2 protons appeared at 2.20-2.47 ppm (2.29 (dtd) ppm, 2.38 (dddd) ppm), lH-2 protons appeared at 2.70-3.00 ppm (2.85 (dd) ppm, 3.01 (dd) ppm), lH-1 proton appeared at 4.60 (ddd) ppm and lH-1 proton appeared at 5.68 (dd) ppm.

Figure 5: ^lH-NMR spectra of the isolated compound (Fraction H) of Nardostachys jatamansi Hydro alcoholic

extract

3.3.4 Mass – Spectroscopy-

A mass spectrum of isolated compound (Fraction H) of Nardostachys jatamansi Hydro alcoholic extract was recorded on Mass Spectroscopy. Mass spectra of isolated fraction (Fraction H) of Nardostachys jatamansi Hydro alcoholic extract showed molecular ion [M+] peaks at mlz 251.1615 which corresponds to the molecular formula C15H22O3 according to their fragments.

(A)Mass spectra of the isolated compound (Fraction H) of Nardostachys jatamansi Hydro alcoholic extract-

Figure 6: Mass spectra of the isolated compound (Fraction H) of Nardostachys jatamansi Hydro alcoholic extract

Nardosinone

IUPAC NAME: (3aR,9R,9aR,9bS)-1,1,9,9a-Tetramethyl-1,3a,4,7,8,9,9a,9b-octahydro-5H-naphtho [2,1- c ] [1,2] dioxol-5-one

DISCUSSION:

Different solvent systems were used for the preliminary TLC of Nardostachys Jatamansi Hydro alcoholic extract. TLC was done in Toluene: Ethyl acetate: Acetic acid (9:1:0.2) using standard sesquiterpeniod, which was observed in the bands of Nardostachys jatamansi Hydroalcoholic extract. The Nardostachys jatamansi and Std. Sesquiterpeniod Rf values were determined to be

0.53 and 0.53, respectively. For column chromatography, toluene: ethyl acetate: acetic acid (9:1:0.2) was chosen as the mobile phase. The mobile phase of Toluene: Ethyl acetate: Acetic acid (9:1:0.2) for Nardostachys jatamansi is used in column chromatography to isolate the active ingredients, yielding Fractions 01 (A), 02 (B), 03

(C), 04 (D), 05 (E), 06 (F), 07 (G), 08 (H), 09 (I),

and 10 (J) (Table 2). TLC estimation was performed for the validation of active ingredients in fractions H of Nardostachys jatamansi using mobile phase Toluene: Ethyl acetate: Acetic acid (9:1:0.2) by comparing with standard sesquiterpeniod (Fig 2, Table 3). The fractions were appropriately collected, and a UV spectrum study was carried out. The UV spectra of the isolated fractions H of Nardostachys jatamansi were recorded throughout a scanning range of 200-800 nm, and the maximum of fractions H was measured, and the wavelength of Nardostachys jatamansi, H fraction was found to be 251 nm (Fig 3). The separated fraction H of the Nardostachys jatamansi Hydro alcoholic extract's IR spectrum revealed that a wide absorption peak for the -OH group had emerged at 3450.65 cm-1. For alkane, the C-H stretching peak was seen at 2927.48 cm-1. For alkane, the O=C=O stretching peak was seen at 2345.90 cm-1, the C-O stretching absorption peak for the carbonyl group was at 1653.88 cm-1, and the C-C stretching peak was at 1103.70 cm-1. The benzene ring's C=C stretching absorption peak was observed at 1438.24 cm-1, whereas the C=C bending peak was observed at

796.91 cm-1(Table 4, Figure 4). In 1H NMR spectra of isolated fraction H of Nardostachys jatamansi Hydro alcoholic extract showed that lH-3 protons appeared at 0.93 (d) ppm, lH-9 protons appeared at 1.15-1.34 ppm (1.20 (s) ppm, 1.25 (s) ppm, 1.30 (s)

ppm), lH-2 protons appeared at 1.55-1.75 ppm (1.60 (dddd) ppm, 1.62 (dddd) ppm), lH-2 protons appeared at 1.92-2.15 ppm (1.99 (dqd) ppm, 2.05

(d) ppm), lH-2 protons appeared at 2.20-2.47 ppm (2.29 (dtd) ppm, 2.38 (dddd) ppm), lH-2 protons appeared at 2.70-3.00 ppm (2.85 (dd) ppm, 3.01 (dd) ppm), lH-1 proton appeared at 4.60 (ddd) ppm and lH-1 proton appeared at 5.68 (dd) ppm (Fig 5). Using mass spectroscopy, a mass spectrum of the isolated component (Fraction H) of the alcoholic extract from Nardostachys jatamansi was

captured. The isolated fraction (Fraction H) of the alcoholic extract from Nardostachys jatamansi displayed mass spectra with molecular ion [M+] peaks at mlz 251.1615, which, based on their fragmentation, corresponds to the chemical formula C15H22O3 (Fig 6). It was determined from this physical, chemical, and spectroscopic analysis that fraction H of the Nardostachys jatamansi Hydro alcoholic extract contained Nardosinone.

CONCLUSION:

Nardostachys jatamansi contains a number of bioactive chemicals, including crystalline acid, Jatamansic acid, hydrocarbons, a polyoxygenated crystalline solid together with A-endesmol, B- eudesmol, ethanol, angelicin, 4-hydroxythymol dimethyl ether. The range of examination of the Hydro alcoholic extracts of plant Nardostachys jatamansi belonging to the family Caprifoliaceae was effectively carried out. From these physically, chemically and spectral investigation were confirmed the presence of Nardosinone in fraction H Nardostachys jatamansi Hydro alcoholic extract. The identification and characterization of the bioactive molecule, achieved through advanced analytical techniques, have paved the way for a deeper understanding of the plant's pharmacological properties. The identified compound has antioxidant, anti-inflammatory, and neuroprotective effects. Nardosinone can suppress the generation and differentiation of OCs from mouse bone marrow macrophages (BMMs) through JNK, ERK, PLCγ2, c-Fos, and NFATc1 signaling pathways in association with scavenging the RANKL-induced ROS. These findings highlight the plant's medicinal significance and the need for further research to explore its therapeutic potential. The outcomes of this study lay the groundwork for further research and development, fostering a bridge between traditional knowledge and contemporary scientific advancements in the pursuit of effective and natural healthcare solutions.

FUNDING

NIL

CONFLICT OF INTEREST

None

REFERENCES:

1. Airi S, Rawal RS, Dhar U, Purohit AN. Assessment of availability and habitat preference of Jatamansi – a critically endangered medicinal plant of west Himalaya. Curr Sci. 2000; 78:1467–1471.

2. Anon. Medicinal plants of Nepal. Bulletin of the

Department of Medicinal Plants No. 3. Kathmandu: His Majesty's Government of Nepal, Ministry of Forests and Soil Conservation; 1993.

3. Bagchi A, Oshima Y, Hikino H. Neolignans and lignans of Nardostachys jatamansi roots. Planta Med. 1991;57(1):96–97.

4. Bolzani VDS, Valli M, Pivatto M, Viegas C Jr. Natural products from Brazilian biodiversity as a source of new models for medicinal chemistry. Pure Appl Chem. 2012;84(9):1837–1846.

5. Brusotti G, Cesari I, Dentamaro A, Caccialanza G, Massolini G. Isolation and characterization of bioactive compounds from plant resources: The role of analysis in the ethnopharmacological approach. J Pharm Biomed Anal. 2014; 87:218–

228. doi: 10.1016/j.jpba.2013.03.007

6. Brusotti G, Cesari I, Frassà G, Grisoli P, Dacarro C, Caccialanza G. Antimicrobial properties of stem bark extracts from Phyllanthus muellerianus ( Kuntze) Excell. J Ethnopharmacol. 2011;135(3):797–800.

7. Chauhan RS, Nautiyal MC, Kumar A. Analysis of variabilities in populations of Nardostachys jatamansi DC. in Garhwal Himalaya, India. J Plant Breed Crop Sci. 2011;3(9):190–194.

8. Chen KK, Mukerji B, editors. Pharmacology of oriental plants: Proceedings of the First International Pharmacological Meeting, Stockholm, 22–25 August 1961. Amsterdam:

Elsevier; 2016.

9. Dhiman N, Bhattacharya A. Nardostachys jatamansi (D. Don) DC. – Challenges and opportunities of harnessing the untapped medicinal plant from the Himalayas. J Ethnopharmacol. 2020; 246:112211.

10. Duraipandiyan V, Ayyanar M, Ignacimuthu S. Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC Complement Altern Med. 2006; 6:1–7.

11. Ghimire SK, McKey D, Aumeeruddy-Thomas

Y. Conservation of Himalayan medicinal plants: Harvesting patterns and ecology of two threatened species, Nardostachys grandiflora DC. and Neopicrorhiza scrophulariiflora (Pennell) Hong. Biol Conserv. 2005;124(4):463– 475.

12. Grover JK, Yadav S, Vats V. Medicinal plants of India with anti-diabetic potential. J Ethnopharmacol. 2002;81(1):81–100.

13. Jadhav VM, Thorat RM, Kadam VJ, Kamble

SS. Herbal anxiolytic: Nardostachys jatamansi. J Pharm Res. 2009;2(8):1208–1211.

14. Joshi H, Parle M. Nardostachys jatamansi improves learning and memory in mice. J Med Food. 2006;9(1):113–118.

15. Jurg A, Tomás T, Pividal J. Antimalarial activity of some plant remedies in use in Marracuene, southern Mozambique. J Ethnopharmacol.

1991;33(1–2):79–83.

16. Kagan IA, Flythe MD. Thin-layer chromatographic separations and bioassays of plant extracts to identify antimicrobial compounds. J Vis Exp. 2014;(85):51411.

17. Khalid H, Abdalla WE, Abdelgadir H, Opatz T, Efferth T. Gems from traditional North-African medicine: Medicinal and aromatic plants from Sudan. Nat Prod Bioprospect. 2012; 2:92–103.

18. Liu XC, Liu ZL. Evaluation of insecticidal activity of Nardostachys jatamansi essential oil against some grain storage insects. J Entomol Zool Stud. 2014;2(4):335–340.

19. Mishra D, Chaturvedi RV, Tripathi SC. The fungitoxic effect of the essential oil of the herb Nardostachys jatamansi DC. Trop Agric. 1995.

20. Moraes LGP, Rocha RSF, Menegazzo LM, Araújo EBD, Yukimitu K, Moraes JCS. Infrared spectroscopy: A tool for determination of the degree of conversion in dental composites. J Appl Oral Sci. 2008;16(2):145–149.

21. Nautiyal BP, Chauhan RS, Prakash V, Purohit H, Nautiyal MC. Population studies for the evaluation of germplasm and threat status of the alpine medicinal herb, Nardostachys jatamansi. Not Recur Fitogenet. 2003; 723:896.

22. Nautiyal OH. Anti-fungal activity of Nardostachys jatamansi essential oil beneficial for treating dermatophytosis (ringworm). J Nat Prod. 2013; 9:241.

23. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod. 2012;75(3):311–335.

24. Ngueyem TA, Brusotti G, Marrubini G, Grisoli P, Dacarro C, Vidari G, et al. Validation of use of a traditional remedy from Bridelia grandis stem bark against oral Streptococci. J Ethnopharmacol. 2008;120(1):13–16.

25. Olsen CS. Trade and conservation of Himalayan medicinal plants: Nardostachys grandiflora DC. and Neopicrorhiza scrophulariiflora (Pennell) Hong. Biol Conserv. 2005;125(4):505–514.

26. Patel D, Panchal D, Patel K, Dalwadi M, Upadhyay U. A review on UV-visible spectroscopy. Int J Creat Res Thoughts. 2022;2320–2882.

27. Rai LK, Prasad P, Sharma E. Conservation threats to some important medicinal plants of the Sikkim Himalaya. Biol Conserv. 2000;93(1):27– 33.

28. Rahman H, Shaik HA, Madhavi P, Eswaraiah MC. A review: Pharmacognostics and pharmacological profiles of Nardostachys jatamansi DC. Elixir Pharm. 2011; 39:5017–

5020.

29. Rekha K, Rao RR, Pandey R, Prasad KR, Babu KS, Vangala JR, et al. Two new sesquiterpenoids from the rhizomes of Nardostachys jatamansi. J Asian Nat Prod Res. 2013;15(2):111–116.

30. Sharma PC, Yelne MB, Dennis TJ, Joshi A, Billore KV. Database on medicinal plants used in Ayurveda. New Delhi: CCRAS; 2000.

31. Singh M, Khan MA, Khan MS, Ansari SH, Ahmad S. Standardization and in vitro antioxidant activity of Jatamansi rhizome. J Pharm Bioallied Sci. 2015;7(4):275–280.

32. Srivastava N, Singh A, Kumari P, Nishad JH, Gautam VS, Yadav M, et al. Advances in extraction technologies: Isolation and purification of bioactive compounds from biological materials. In: Natural Bioactive Compounds: Technological Advancements. Elsevier; 2021. p. 409–425.

33. Wiley WC, McLaren IH. Time-of-flight mass spectrometer with improved resolution. Rev Sci Instrum. 1955;26(12):1150–1157.

34. Yang YC, editor. Tibetan Materia Medica. Qinghai: Qinghai People's Press; 1996.

35. Zia K, Siddiqui T, Ali S, Farooq I, Sohail MZ, Khurshid Z. Nuclear magnetic resonance spectroscopy for medical and dental applications: A comprehensive review. Eur J Dent. 2019;13(1):124–128.