IJBPAS, June, 2016, 5(6): 1230-1243 ISSN: 2277–4998

ISOLATION AND CHARACTERIZATION OF VALERENIC ACID FROM wallichii ()

SAHU V, DHONGADE HJ*, SAWARKAR HA, SAHU P, SAHU R, PATEL D AND KASHYAP P Department of Phytopharmacognosy, Shri Rawatpura Sarkar Institute of Pharmacy, Kumhari, Durg, C.G, (India) *Corresponding Author: Hemant Dhongade: E Mail: [email protected]; 918827884865 ABSTRACT DC is commonly known as valerian belongs to Valerianaceae family. It is a hairy perennial herb, growing in temperate Himalaya from Kashmir hills up to an altitude of 3,000 m. In the present study soxhlet extraction was used to extract phytochemicals from collected roots using 95 % ethanol. After phytochemical screening, TLC, UV, and FTIR of Valeriana wallichii root extract was carried out. The result of TLC of an alcoholic extract of Valeriana wallichii roots and its extract was done in order to separate out as many compounds as possible. The result of UV is to be detecting absorbance maxima of the phytochemical component present in drug extract. The result of FTIR shows the identification of various functional groups present in plant extract. The extract obtained was dried and the dried was used for qualitative analysis of different constituents like alkaloids, tannins, flavonoids, cardiac glycosides, proteins, carbohydrates, reducing sugar, anthraquinone, Steroids in ethanolic extract of Valeriana wallichii. Keywords: Valeriana wallichii, Phytochemical Screening, TLC, UV, and FTIR 1. INTRODUCTION Valeriana wallichii DC is commonly known It is a hairy perennial herb, growing in as valerian belongs to Valerianaceae family. temperate Himalaya from Kashmir hills up to

1230 IJBPAS, June, 2016, 5(6) Dhongade HJ et al Research Article an altitude of 3,000 m. [1] The roots & 2. MATERIALS AND METHODS rhizome part of herb is used as medicine in 2.1 Plant material:- the Ayurvedic system of medicine for The roots of Valeriana wallichii were centuries. Traditionally it is also known as collected from local market of Raipur in 10 Tagara, Nata, Kutila, Nahusha, Vinamra, August 2015. The roots were cleaned and Nrpam, Sata, Bahram, Parthiva, Rajharshana, dried. The root was authenticated by Dr. Chatra, Deen, Jiwh, Munindhudha,[2] Rakshapal Gupta, Head of Department Kalanusarika, Kalanusari, Akrahavam Dravyaguna Vigyan, Govt. Ayurvedic Cheen, Katu, Mahoraga,[3] Karhaat, Swasna, College, Raipur (C.G.) Vishpushka. [4] Acharya Charaka mentioned 2.2 Preparation of Plant Extracts:- this plant under Sheeta and Tiktaskhandha [5] The roots of Valeriana wallichii were while Susruta described in Eladigana. [6] It is cleaned and the dust particles were removed. a perennial Leafy slightly hairy, tufted herb, It was the dried and made into a coarse up to 45 cm in height. Root stalk is thick, powder with the help of electronic grinder. horizontal, long petioled, deeply cordate and The powder was passed through sieve No. ovate, usually toothed or sinuate, small 40; about 150gm of grinded plant material pinnate and few are cauline leaves. was subjected to soxhlet extraction (50-60°C) [7,8]Flowers of valerian are often deciduous, employing ethanol (95%) as solvent. After white to ting with a pink, in terminal completion of extraction the extract was corymbs and unisexual, male and female in filtered and the solvent was removed by different .[9,10] There are two classical evaporation to dryness on a water bath. varieties of Valerian, they are Tagara and Within 4-5 days brown colour residue was Pindatagara.[11,12] The roots of valerian obtained and it was stored in a dessicator. contain valerianic acid, valerosidatum (iso- Coloured extract was found, and % yield was valery) glycoside, valepotriate (a derivative calculated. of iridoids or monoterpene), which is used as Yield estimation:- sedative. Essential oil is sweet smelling and Extract obtained was taken into a pre is extracted from the root and rhizomes of weighed china dish. The extract was then plant. [13,14] The oil contain valeriac acid, dried over water bath and subsequently terpene alcohol, bornyl ester of formic acid, weighed. The difference in weight was camphene and terineoland. [15, 16] measured and the extract was kept in the

1231 IJBPAS, June, 2016, 5(6) Dhongade HJ et al Research Article dessicator for 7 days. The weight difference plate was taken from the chamber and let the was used to calculate percentage yield. The solvent be vaporized. percentage yield of the extract was calculated 5. Detection of spot:- For the detection of using the following formula spot iodine chamber was used. The plate was

% yield= × 100 kept in the beaker containing iodine crystal and the color of the spot was analyzed. 2.3 Phytochemical Screening of Plant 6. Calculation of Rf value:-Rf value was Extracts:- calculated by using formula: Phytochemical screening of the extract was Rf∶= performed according to standard methods. The extract obtained was dried and the dried 2.4.2 Detection of absorbance maxima by was used for qualitative analysis of different UV method:- constituents like alkaloids, tannins, A UV spectrum was recorded on model UV- flavonoids, cardiac glycosides, proteins, 1800, a UV-Visible spectrophotometer carbohydrates, reducing sugar, (Shimadzu) between wavelengths 400 to anthraquinone, Steroids in ethanolic extract 200nm.Firstly, drug extract (10 mg) of of Valeriana wallichii. Valeriana wallichii ethanolic extract 2.4 Characterization of Valerenic acid in dissolved in about 10-20 ml of the ethanol extract:- make up the volume up to 50 ml with 2.4.1 TLC identification test and ethanol. 2.5 ml of filtrate was taken and Procedure:- diluted up to 25 ml of ethanol. 1ml of the 1. Developing container preparation:- resultant in solution was taken in 10 ml Solvent which was finalized was added to volumetric flask and the volume was made the container and was saturated for 1hr. up to the mark with ethanol. Prepared 2. TLC plate was prepared by using silica gel dilutions were scanned at detection G as a stationary phase. wavelength 400-200 nm observation were 3. Spotting TLC plates:- Spotting on TLC noted in terms of λ- max. plate was done using capillary tube 1-2 cm 2.4.3 Detection of functional group by FT- from the bottom of the plate. IR method:- 4. Developing the plate:- After spotting, the The FT-IR of Valeriana wallichii extract was plates were kept inside the chamber in recorded on Model- FT-IR 8400S. Fourier ascendant position. After developing the Transform Infrared Spectrophotometer

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(Shimadzu). The pellets were prepared on maximum separation. The developed KBr press using mixture of extract and KBr solvent system of TLC (chloroform: in about 1:10 ratio. The spectra were scanned methanol) 9:1) was used. at the range of 4000 to 400cm-1. . Detection: The developed plates 2.4.4 High performance thin layer were first observed under visible light chromatography (HPTLC):- and UV light (356 and 254 nm) for HPTLC is a modern chromatographic the detection of constituents. technique in which the principle of TLC is Densitometry scanning: The developed sophisticated and automated by which the plates were scanned at a suitable wavelength samples are accurately and precisely for the qualitative analysis. Peak areas and estimated which can be utilized for both peak heights were recorded from which the qualitative and quantitative purpose. unknown concentration and RF value of the . Preparation of sample: 500 mg of samples have been determined. extract was solubilised in the 2.5 Isolation of active constituent by respective solvent. Sonicator was Preparative TLC:- used to make the ethanolic extract Conventional preparative TLC: - Silica gel completely soluble. After G used for Thin Layer Chromatography solubilisation samples were filtered (TLC) was activated in hot air oven at 110°C using whatman filter paper to avoid for 1hour. residual impurities in samples. Preparation of thin layer plates and . Application and development of loading of samples:-The plate was plates: Commercially available developed in the solvent, Chloroform: precoated HPTLC plates (Merck) Methanol (45:5) to separate the compound. were used for the study.10µl of Chromatogram was detected with iodine sample solutions were applied on the chamber for 5 minutes. The active HPTLC plate using Linomat V fractions/pure compounds were scraped from applicator. The plates were then dried the silica gel plate and were dissolved in after application. mobile phase (↑).The active compound was . Solvent system: Solvent system was filtered through Millipore filters (0.45µm and used for each extract and solvent 0.22µm) to remove the silica gel and to yield system was developed to get

1233 IJBPAS, June, 2016, 5(6) Dhongade HJ et al Research Article compound fraction. The mobile phase was 3.1 TLC Profile:- evaporated to obtain the compound. On the basis of experiment chloroform: 3. RESULTS AND DISCUSSION methanol in the ratio of 9:1 was selected as a

The extraction was achieved using ethanol as solvent system for preparative TLC. The Rf solvent and % yield was found to be8.6 value of the given spot was found to be of %(w/w). Preliminary phytochemical 0.16, 0.48, 0.65 and 0.84, respectively which screening reveals the presence of: The results comes under the range of the Rfvalue of showed that alkaloids, tannins, flavonoids, Valerenic acid. Hence Valerenic acid is cardiac glycosides, proteins, carbohydrates, present in the extract. The reported Rf value reducing sugar, anthraquinone, Steroids in for Valerenic acid is 0.48 (Figure 1). ethanolic extract of the plant. The results are shown in Table 1. Table 1: Phytochemical screening of Valeriana wallichii roots extract S. No. Phytochemical Constituents Result 1 Alkaloids + 2 Tannins + 3 Flavonoids + 4 Terpenoids _ 5 Cardiac glycosides + 6 Proteins + 7 Carbohydrates + 8 Reducing Sugars + 9 Anthraquinone + 10. Steroids + + - Present - -Absent, all the determinants were carried out in triplicate

Figure 1: Developed TLC plate Table No. 2 Solvent system of TLC was analyzed by using various ratio of the solvent system S.No. Solvent System Ratio No. of spots Rf Values 1 Ethanol: Toluene: 7:2.5:0.5 No spot Negative GAA 5:3:2 Visualization of spot Negative 4:3:3 No spot Negative 2 Hexane: Methanol: 7:2:1 No spot Negative Water 4:2:4 Resolution of spot Negative 3 Chloroform: Methanol: 5:3:2 No spot Negative

1234 IJBPAS, June, 2016, 5(6) Dhongade HJ et al Research Article

Ethyl acetate 7:2:1 No spot Negative 2:5:3 Light spot Negative 4 Chloroform: Methanol 9:1 4 0.16, 0.48, 0.65 0.84 3.2 Detection of absorbance maxima by UV Method:- At sixth peak the absorbance maxima (λmax) was found to be = 254nm, which shows that Valerenic acid is present in extract.

Graph 1: UV spectrum of Valeriana wallichii root ethanolic extracts 3.3 Detection of functional groups by FT-IR method:-

Graph 2: FT-IR Spectrum of Valeriana wallichii ethanolic extract

Table No. 3 FT-IR absorption frequency of Valeriana wallichii root ethanolic extract S.No. Functional Group Wave Number (Cm-1) 1 C=S Stretching 1153.43 2 C-O Stretching (Alcohols) 1261.45

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3 C-O Stretching (Phenols) 1373.32 4 C=C Stretching (Aromatic) 1458.18 5 N-H Bending 1604.77 6 C=N Stretching 1654.92 3.4 HPTLC chromatogram of ethanolic extract:-

(a) (b) (c) Figure 2: (a) HPTLC plate at 254nm, (b) HPTLC plate at 365nm (c) HPTLC plate after derivatization with anisaldehyde H2SO4

Figure 3: 3D Spectrum of Valeriana wallichii extract

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Fig. No. 4 HPTLC chromatogram of Valeriana wallichii extract

Fig. No. 5 Densitometry scans of Valeriana wallichii extract

S. Start Start Max End End Assigned Max Position Max% Area Area% No. Position Height Height Position Height substance 4078.3 1. 0.09 Rf 232.5 AU 0.10 Rf 340.7 AU 17.89% 0.11 Rf 1.5 AU 7.82% unknown * AU 2. 0.13 Rf 0.1 AU 0.14 Rf 22.2 AU 1.17% 0.16 Rf 0.2 AU 244.1 AU 0.47% unknown * 5895.5 3. 0.19 Rf 0.0 AU 0.23 Rf 247.2 AU 12.98% 0.25 Rf 56.3 AU 11.30% unknown * AU 1986.2 4. 0.25 Rf 58.2 AU 0.26 Rf 88.6 AU 4.65% 0.30 Rf 0.1 AU 3.81% unknown * AU 5. 0.35 Rf 0.5 AU 0.37 Rf 19.9 AU 1.05% 0.39 Rf 8.3 AU 373.8 AU 0.72% unknown * 1017.7 6. 0.39 Rf 8.3 AU 0.41 Rf 54.6 AU 2.87% 0.42 Rf 44.3 AU 1.95% unknown * AU 2493.6 7. 0.43 Rf 43.9 AU 0.47 Rf 91.3 AU 4.79% 0.48 Rf 49.9 AU 4.78% unknown * AU

1237 IJBPAS, June, 2016, 5(6) Dhongade HJ et al Research Article

Figuew 6: HPTLC with Rf value

3.5 Result of preparative TLC:-

With the help of preparative TLC method 3 compounds were isolated have Rf value o.48, 0.65, 0.84.

Figure 7: Developed Preparative TLC

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4. CONCLUSION Stretching (Aromatic), (N-H This study deals with the extraction, Bending), C=N Stretching) phytochemical screening, TLC, UV, and . Isolation was done by using FTIR of Valeriana wallichii root extract. The preparation TLC & 3compounds result of TLC of an alcoholic extract of were isolated. Valeriana wallichii roots and its extract was 5. ACKNOWLEDGEMENT done in order to separate out as many I wish to express my deep sense of gratitude compounds as possible. The result of UV is to my esteemed guide Dr. H J Dhongade and to be detecting absorbance maxima of the also thankful to Dr. Mrs Pranita Kashyap for phytochemical component present in drug providing necessary facilities to carry out my extract. The result of FTIR shows the work. identification of various functional groups REFERENCE present in plant extract. This report [1] Sharma PV. Priya Nighuntu. 1st ed. documents for the first time, the Varanasi. Chaukhambha Bharati identification and quantification of an Academy; 2001:64- 65. alcoholic extract of Valeriana wallichii roots. [2] Indradev Tripathi. Raja Nighuntu. 4th . The % yield was found to be ed. Varanasi: Chaukhambha Krishnadas 8.6%w/w. Academy; 2006:41-42 . TLC- solvent system (chloroform: [3] Sri Khenraj. Madanapala Nighuntu. 1st methanol)shows 4spot. ed. Mumbai: Krishandas Prakashan; . UV At sixth peak the absorbance 2004:97. maxima (ʎmax) was found to be [4] Gyanendra Panday. Sodhala Nighuntu. 254nm, which shows that 1st ed. Varanasi: Chaukhambha Valerenic acid is present in Krishnadas Academy; 2009: 76. extract. [5] Charaka. Charakasamhita, Part-1 . FT-IR-Functional group were (Vidyotini Hindi commentary). present:- Absorption frequency of Kashinath Shastry, Valeriana wallichii root ethanolic GorakhnathChaturvedi, editors. 1st ed. extract [ (C=S Stretching),C-O Varanasi: Chaukhambha Bharati Stretching (Alcohols), C-O Academy; 1996. Sutra sthana 4/42; 94: Stretching (Phenols), C=C Vimanasthana 8/13; 740.

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