Physicochemical Analysis of Sumbul-al –Teeb (Nardostachys jatamansi D.C.) Rhizome along with its HPLC Profile

Rashid, Rahman, Ahmad, Tajuddin, and Mian: Physicochemical Analysis of Sumbul-al –Teeb (Nardostachys jatamansi D.C.) Rhizome along with its HPLC Profile

Authors

INTRODUCTION

Nowadays the Indian herbal industry is flourishing at an admirable pace with remarkable increase in the introduction of new herbal pharmaceutical and cosmetic products in the market. But due to ignorance and awful supply chain management of herbal medicines, quality and purity of herbs and their products is not assured. As the efficacy and safety of herbal products is strongly based on their good quality therefore, determination of identity, quality and purity of the herbal medicines is unavoidable. The traditional approach towards standardization for obtaining good quality herbal medicines seems to be insufficient for the current herbal market which invites the need for more advanced techniques. Standardization of the crude drugs involves passport data of the drugs viz., botanical identification, and macroscopic, microscopic and molecular examination, identification of Phytochemical constituent by various chromatographic techniques and biological activity of the whole plant.1 Due to the high commerce, traders have imperilled plants and their products to adulteration and substitution.

As the rhizome of N. jatamansi is often contaminated and adulterated with different plant materials such as Selinium vaginatu.2 Therefore, in this study Nardostachys jatamansi DC, family – Caprifoliaceae (Valirenaceae) was selected and standardized on their physico-chemical characteristics along with HPLC profile. Nardostachys jatamansi is native to the Himalayan regions of India and commonly known as muskroot.3 It is an excellent remedy popular to the Indian medicinal system and is used from centuries for its health benefits; it can also be used for treatment and other skin diseases. Medicinally it possesses anti-inflammatory, sedative/anodyne, detergent, sialagogue, desiccant, carminative, cardiac tonic, brain tonic, diuretic properties and can be used in cephalalgia, flatulence, ascites, jaundice, hepatitis, ureteralgia, cystitis etc.4 The rhizome is the source of spikenard oil.5 It has been reported that Spikenard oil contained p-maaline (18%) and calarene (65%).6 Monoterpenes are α-pinene, β-pinene, ∆3-carene; alcohols- β-eudesmol, elemol, oroselol; a semisolid long chain hydrocarbon with molecular formula C30H62. β-sitosterol; a terpenic coumarin - jatamansin, a polyoxygenated compound-angelicin and jamansinol have been identified by GLC.7,8 Nardostachnol 9-hydro aristolene, 1(10)-dehydroaristolene, 2β-maaline and 1, 2, 9, 10-tetrahydro aristolene have been isolated from roots.9 There are number of compounds reported, which have been isolated from its roots and rhizomes, e.g. sesquiterpene ketone –jatamansone.10 Liquid alcohols - nardol, calarenol and n-hexacosanol; a ketonevaleranone and diethenoid ketone - nardostachone, n-hexacosen, n-hexacosanylisovalerate, n-hexacosanylarachiedate, isovaleric acid,valeranal and β-sitosterol have been reported from powdered roots.11

Other sesquiterpenes include nardostachone, jatamansinol, jatamansic acid, jatamansinone, nardostachyin, nardosinone, jatamol A and B etc. A new sesquiterpene acid and new pyranocoumarin: 2’, 2’-dimethyl-3’-methoxy-3’, 4’-dihydropyranocoumarin were reported. Actinidine, an alkaloid was also reported.5

The present study was carried out in order to standardise the rhizome of Nardostachys jatamansi with a view to develop its quality parameters, and also to deliver referential information for the identification of the crude drug so as to check the substitution and adulteration and to ensure the effectiveness of a drug in treating different body ailments.12 Parameters include macroscopy, powder analysis, physicochemical parameters and preliminary phyto-chemical screening alongwith HPLC profile.

MATERIAL METHOD

Collection of sample

Dried rhizome of N. jatamansi was procured from local market of Aligarh and was properly recognized from the accessible literature and authenticated by Prof. S. H. Afaq. The sample with specimen voucher no. SAMU/NJ-R-0001/17 was deposited in the Department of Saidla, Faculty of Unani medicine, Aligarh Muslim University, Aligarh, for future reference. It was crushed and sieved to coarse powder mechanically and stored in air tight container for study.

Macroscopy and organoleptic characters

The organoleptic characters of the crude drug were observed with sensory organs and was analysed for its colour, odour and taste, size, shape, fracture and surface.

Physicochemical parameters

Ash values, alcohol and water soluble extractive values, volatile oil estimation and loss on drying of the test drug was determined as per the methods recommended by Ayurvedic Pharmacopeia of India (API).13 and British Pharmacopeia.14

The fluorescence analysis of the rhizome powder was done by treating with the different chemical reagents and observed under Ultra violet light and day light.15,16

TLC

Thin layer chromatographic analysis of the methanolic extract of Nardostachys jatamansi was carried out via chloroform: methanol (24:1) as mobile phase in percolated silica gel 60F254 TLC plates. Spotted TLC plates were exposed to iodine vapours in iodine chamber and were also visualized in day light and UV short and long wavelength. The Rf value of spots was determined by the given formulae.14,17

Rf value =Distance travelled by the SpotDistance travelled by the solvent

Preliminary phyto-chemical screening

The extracts were introduced to preliminary phyto-chemical analysis and investigated for the presence of various phyto-constituents like alkaloids, carbohydrates, glycosides, flavonoids, proteins, steroids, saponins, etc. with following parameters.18,19

HPLC profile of N. Jatamansi

HPLC profile of the methanolic extract of the N Jatamansi was done. For this Shimadzu Prominence Isocratic HPLC System equipped with LC-20 AD Solvent delivery unit, Rheodyne Injector, SPD-20A prominence Uv-vis detector system along with C18G120A column, 250x4.6 mm 5U with guard column was used. The methanolic extract of coarsely powdered drug was obtained with the help of soxhlet’s extraction method, extract was filtered and allowed to evaporate on water bath. This dried alcoholic extract was dissolved in HPLC grade methanol and used for study. The chromatographic analyses were carried out at room temperature using reversed phase and software driven peaks were obtained (Figure 2). The pressure and flow rate was 127 kgf and 1.0 ml/min, respectively. Detector for HPLC was UV and the wavelength was 254 nm. Mobile phase for HPLC profile of extract consisted of HPLC grade methanol (Merk life science Pvt. Ltd.) only.

RESULTS AND DISCUSSION

Modern system of medicine relies on sound experimental data, toxicity studies and human clinical studies. But there is a lack of pharmacopoeial standards on raw material / finished products. The insufficient quality standards have led to the occurrence of mild to serious adverse effects. Hence, the standardization of herbal ingredients is the basic requirement in order to establish the identity, purity and quality.20 Herbals are traditionally considered safe and are remarkably consumed by people without prescription. However, it is advocated that some can cause health problems, some are not effective and some may interact with other medicines. Standardization is crucial for the assessment of the quality, purity and authenticity of the drugs, based on the physicochemical parameters, TLC, HPLC and on the presence of active principles.21 A standardized and good quality drug is the assurance of its therapeutic effectiveness and global acceptance. Nardostachys jatamansi is a well-known drug of Unani System of Medicine used to treat various body ailments such as inflammatory conditions. Therefore, for this study Nardostachys jatamansi was selected and standardized on their physicochemical parameters such as organoleptic characters, ash values, extractive values, volatile oil estimation, fluorescence analysis, qualitative estimation, TLC along with HPLC profile.

Organoleptic characters of N. jatamansi

Organoleptic properties are the critical parameter for the rapid identification and consumer acceptance. Sensory evaluation-visual macroscopy, colour, odour, taste, fracture are the common features helped in identification of the crude drug. The organoleptic properties of rhizome of N. jatamansi have been mentioned in Table 1.

Physicochemical analysis of N. jatamansi

Ash values, alcohol and water soluble extractive values, loss of weight in powdered drug after drying at 105°C and moisture contents are the indicators of the purity, quality and authenticity of any crude drug. Therefore, to standardise a herbal drug these parameters have basic importance and unavoidable. Total ash values, acid insoluble and water soluble ash values reveals the information related to the adulteration of crude drug with inorganic matter. The water and alcohol soluble extractive values indicate the amount of the extract that the drug yields in a solvent.16 Less or more extractive value indicates addition of exhausted material, adulteration or incorrect processing during drying, or storage of plant products.22 Low or high moisture contents may affect the quality of the drug and hence, its efficacy. The excessive moisture is an ideal medium for the growth of the different types of microorganisms which subsequently damages the drug.23 This drug is also well known for its oil contents. The inappropriate method of extraction of oil or distillation and storage may spoil the quality of the drug and hence the oil.23 Therefore, to assess the quality of N. jatamansi it is also necessary to determine volatile oil percentage of the drug. All the values were determined in triplet and the results are depicted in Table 2.

Table 1

Organoleptic characters of N. jatamansi

Rhizome of N. jatamansiCharacters
ShapeElongated and cylindrical
SizeRhizomes are 2.5 to 7.5 cm in length
ColourDark grey rhizomes are crowned with reddish brown tufted fibers. Internally they are reddish brown in colour
FractureEasy and splintery
SurfaceHairy
OdourHighly agreeable, aromatic
TasteAcrid, slightly bitter and aromatic
Table 2

Physicochemical values of N. jatamansi

ParametersObserved Valuesin % (Mean ± SD)
Total ash7.08 ± 0.03
Acid insoluble ash3.71 ± 0.18
Water soluble ash0.58 ± 0.07
Alcohol soluble extract3.54 ± 0.72
Water soluble extract2.5 ± 0.06
Volatile oil02 ± 0.04
Loss on drying9.12 ± 0.12
Moisture content8.6 ± 0.2517
Table 3

Fluorescence analysis of N. jatamansi

ReagentsVisible lightUV light
Short 254 nmLong 366 nm
Powder as suchBrownGreenDark Brown
Powder+1N HClLight BrownGreenLight Brown
Powder+50% H2SO4BlackBlackIndigo
Powder+50% HNO3YellowLight greenBlack
Powder+Glacial acetic acid (GAA)BrownLight greenGrey
Powder+1N NaOH in waterDark BrownBlackBlack
Powder+1N NaOH in methanolYellowDark BrownIndigo
Powder+Wagner’s reagentBrownBrownIndigo
Powder+Drangendorff reagentYellowGreenIndigo
Powder+Benedict’s reagentBrownBrownIndigo
Powder+Fehling reagentBrownBrownIndigo
Powder+Lead Acetate (1%)BrownBrownIndigo

Fluorescence analysis of N. jatamansi

Some constituents in many natural products exhibit fluorescence in the daylight ultra violet light and if the substance itself is not fluorescent, it may often be converted into fluorescent through the application of different reagents. Hence, the qualitative assessment of the test drug is carried out in this manner also which serves as an important parameter for pharmacognostic evaluation of crude drugs.24 (Table 3).

Phytochemical analysis of N. jatamansi

The efficacy and pharmacological therapeutic effects of any herbal medicine is depends on their secondary metabolites i.e. phytoconstituents such as alkaloids and glycosides etc. The presence or absence of these phytoconstituents also indicates the quality of the crude drug.24 Therefore, it is also necessary to determine the presence of active secondary metabolites in the test drug, the results are shown in Table 4.

TLC of N jatamansi

TLC is one of the important parameter equips with the qualitative and semi-quantitative information of the drug. If the drug is adulterated or exhausted which in turn may increase or decreases the number of spots and change in the Rf values.16 The TLC profile alongwith images of TLC are illustrated in Table 5 and Figure 1 respectively.

Table 4

Phytochemical analysis of N. jatamansi

Chemical ConstituentsTest / ReagentsInference
AlkaloidsDragendroff’s reagentPresent
 Hager’s testPresent
 Wagner’s reagentPresent
 Tannic acid testPresent
 Mayer’s reagentPresent
CarbohydratesFehling’s testPresent
 Molish’s testPresent
 Barfoed’s testPresent
 Benedict’s testPresent
FlavonoidsShinoda testPresent
 Zinc hydrochloride testPresent
 Alkaline reagent testPresent
TanninsFerric chloride testPresent
 Gelatin testPresent
 Lead acetate testPresent
ProteinsWarming testPresent
 Biuret testPresent
SteroidsSalkowski testPresent
 Sulphur powder testPresent
 Hosse’s reactionPresent
 Moleschott’s reactionPresent
GlycosidesNaOH TestPresent
SaponinsFrothing with NaHCO3Present
Fats and fixed oilsCopper sulphate / Sodium hydroxidePresent
Table 5

TLC Profile of N. jatamansi

DrugExtractMobile PhaseObservation
Sumbul-ut-TeebMethanolicChloroform:Methanol 24:1In long wavelengthIn short wavelengthAfter exposure to iodine vapours
   Rf valueColour of BandsRf valueColour of BandsRf valueColour of Bands
   0.74Light blue0.10Light Violet0.03Brown
   0.81Light blue0.18Ash Colour0.10Light Brown
   0.92Light blue0.21Ash Colour0.34Yellowish
   0.95Pink0.46Light Violet0.42Yellowish
     0.54Light Violet0.50Yellowish
     0.71Ash Colour0.71Yellowish
     0.91Violet0.80Yellowish
     0.95Light Violet0.85Yellowish
       0.91Light Brown
       0.95Brown
Table 6

HPLC peak table of N. Jatamansi

Peak#Ret. TimeAreaHeightArea %Height %
12.80051022490399986225.97427.040
22.9581749873720351768.90813.758
33.1121713371214488478.7229.795
43.35822115659139839011.2589.453
53.63154040089127712.7516.171
63.760126806709678246.4556.543
74.05985071075993964.3314.052
84.39958866076325998829.96722.038
94.7144175791690.0210.062
104.8619461967920.0480.046
116.5204258234730.0220.023
127.137191120218660.0970.148
137.4584189317330.0210.012
147.96748407090.0020.005
158.88339384860.0020.003
169.615602923550.0030.016
1710.7154893034400.0250.023
1811.31412662197230.0640.066
1911.5419343979040.0480.053
2011.843159512114170.0810.077
2112.480243216145270.1240.098
2213.0619865058270.0500.039
2316.8002122412740.0110.009
2418.50892674420.0050.003
2519.2059832942790.0500.029
2620.984135857310.0070.005
2721.291215329200.0110.006
2821.961169374960.0090.003
2923.315253699520.0130.006
3023.93324711293340.1260.063
3125.3287433824350.0380.016
3226.6611442445490670.7340.332
3335.632105642490.0050.002
3437.411297025230.0150.004
Total 19643601114792377100.000100.000
Figure 1

TLC Profile of N. jatamansi rhizome.

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HPLC profile of methanolic extract of N jatamansi

The preparative and analytical HPLC has been widely employed for the analysis of herbal medicines in lieu of its high separation capacity. It can also be utilized to analyse almost all constituents of herbal products provided that an optimized procedure is developed which involves optimization of mobile phase and stationary phase along with other chromatographic parameters.25 The adulteration and impurities can also be determined by this technique. If there is any change in number of peaks or retention time or area of peaks from standard it indicates adulteration or deterioration in the drug. The HPLC pattern shows 34 peaks and the peak no. 01 and 08 are major peaks having area concentration and retention time as 25.974% at 2.8 min. and 29.967% at 4.399 min. respectively followed by peak no. 04, 02, 03, 06 and 07 with concentration of 11.258%, 8.908%, 8.722%, 6.455% and 4.331% respectively. The HPLC profile of the test drug was obtained and recorded for future reference. The details are depicted in Figure 2 and Table 6.

Figure 2

HPLC profile of alcoholic extract of N. Jatamansi rhizome.

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CONCLUSION

Present study shows that the methods of standardization and identification of N jatamansi i.e. organoleptic characters along with physico-chemical analysis are the basic and useful parameters to analyse the originality of the test drug. A good quality of drug is the assurance of its efficacy. The results of phytochemical analysis and HPLC fingerprinting also play a key role in identification and authentication of N jatamansi. Further these analytical parameters for quality assurance also indicating effectiveness of N jatamansi for treating various body ailments. The data obtained in the present work will be useful in identification, standardisation and quality assurance of different samples of N. jatamansi and will also be useful in the preparation of the drug’s monograph for inclusion in various pharmacopoeias.

GRAPHICAL ABSTRACT

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SUMMARY

  • The present study aims towards the evaluation of the parameters involved in the determination of the quality and purity of Nardostachys jatamansi rhizome and its standardization. The parameters used for the standardisation of the test drug includes organoleptic characters, extractive values, ash values, phyto-chemical analysis, TLC, fluorescence analysis and HPLC profile etc. The study will provide referential information for the good quality, purity and identification for the future batches of Nardostachys jatamansi.

ABOUT AUTHORS

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Dr. Aziz ur Rahman is working as Assistant Professor in Department of Saidla (Unani Pharmacy), Faculty of Unani Medicine, Aligarh Muslim University, Aligarh. His research area includes standardization, drug design & discovery, formulation development and experimental research.

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