INTRODUCTION:

Hemidesmus indicus commonly known as Indian sarsaparilla (Anantamul), a member of the family Asclepiadaceae is a twining shrub commonly found in India. It is a slender laticiferous, twining, sometimes prostrate or semi erect shrub, occurring over the greater part of India. Roots are woody and aromatic; stems numerous, slender, terete, thickened at the nodes; leaves opposite, short-petioled, very variable, elliptic-oblong to linear - lanceolate often variegated with white above, sometimes silvery white and pubescent beneath; flowers are greenish outside, purplish inside, crowded in subsessile axillary cymes; follicles are slender, four inches long, cylindrical, sometimes curved, divaricate; seeds numerous, black, flattened, with a silvery white coma¹. The woody roots of the plant have a strong fragrance and sweet taste with cooling effect. Because of this typical aroma, the roots are used as a flavoring agent in ‘sherbets’ (sweet drinks).

The roots are also used to treat a variety of ailments or diseases and are a well-known drug in the Ayurvedic system of medicine². It is used as a blood purifier and also for curing fever, leprosy, rheumatism and liver disorders^3,4. Moreover, root extracts of H. indicus have also proved to be an effective snake venom antidote ⁵.

It is a common medicinal plant widely used in Indian Systems of Medicine⁶. It is also an official drug in Indian Pharmacopoeia⁷ and British Pharmacopoeia⁸. Various market samples are available in the name, identified as H. indicus, Decalepis hamiltoni, Cryptolepis buchananii, Ichnocarpus frutescens and Vallaris solanacea¹.

The main chemical constituents are sarsaponin, smilacin, p-methoxy salicylic aldehyde, beta-sitosterol, sarsapogenin, smilgenin, sitosterol, stigmasterol fatty acids and tannins ⁹. The roots are used as antipyretic, anti-diarrhoeal, astringent, blood purifier, diaphoretic, diuretic, refrigerant and tonic^{10, 11}. Roots are also found useful in biliousness, dysentery, respiratory disorders, skin diseases, syphilis, fever, leprosy, leucoderma, leucorrhoea, itching, bronchitis, asthma, eye diseases, epileptic fits in children, loss of appetite, burning sensation and rheumatism^{12, 13}. Stem of H. indicus is used as diaphoretic, diuretic, laxative and for treating brain, liver and kidney diseases, syphilis, gleet, urinary discharges, uterine complaints, leucoderma, cough and asthma¹.

Many phytochemical studies have been carried out on H. indicus. From the roots, hemidesmol and glucoside, tannin and resin¹⁴, lupeol, α and β amyrins, β-sitosterol¹⁵, lupeol acetate, β-amyrin acetate, hexa triconate acid and lupeol octacosonate¹⁶, 2-hydroxy-4-methoxy benzenoid¹ and a coumarino liganoid¹⁷ like hemsidesmin-2 were isolated. The constituents of oil obtained from the roots of H. indicus contains, 80% crystalline matter, glucose, hemidesmol, hemidestrol, 2-hydroxy-4-methoxy benzaldehyde, resin acid, glucoside, sterol and tannins. Recently, 2-hydroxy-4-methoxybenzaldehyde which is also present in several African medicinal plants, has been identified as a potent tyrosinase inhibitor¹⁸ and is thus being used as an ingredient in cosmetic¹⁹ and other medicinal products, primarily to treat hyperpigmentation^20,21. The compound is also known to have antimicrobial²² and insecticidal²³ properties. Literature survey revealed HPLC method for quantification of 2-hydroxy-4-methoxybenzaldehyde in the root of Hemidesmus indicus²⁴and for simultaneous analysis of 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid in root tissues of this plant²⁵, and a GC–MS method for determination of the chemical composition of volatile oil of H. indicus²⁶.

In any herbal medicine and its extract there are numerous unknown components and many of them are in low amount²⁷. Secondary metabolites, as markers have been extensively used in quality control and standardization of botanical drugs²⁸.

In recent years, development of ﬁngerprints has been widely used to evaluate the quality of herbal raw material and preparation. Fingerprint analysis also controls the stability of quality because it represents the entire composition of the chemical components and their relative concentration²⁹. Therefore, development and validation of HPTLC method for estimation of ferulic acid in the root of H. indicus was undertaken for the first time.

MATERIALS AND METHOD:

Chemicals and Plant Materials:

All chemicals used were of AR grade and were purchased from Sisco Research Laboratories (Mumbai, India). Ferulic acid purity more than 99.5% was used for the analysis. Roots of Hemidesmus indicus were purchased from Khari Baoli, New Delhi, India and identified by the Dr. D. K. Agrawala, Research Officer, CCRAS, New Delhi.

Extraction Method:

H. indicus root (100 g) was accurately weighed and crushed in disintegration machine. After disintegration, powder was mixed with 500 ml of hydro-alcoholic (60% water and 40% ethanol) solution. Hot extraction (by refluxing) of this mixture was done for 4 hours at 80⁰C±5⁰C. The mixture was left overnight at room temperature and later filtered through Whatmann no.1 filter paper. The filtrate was evaporated on a water bath to furnish a solid mass of extract. The hydroalcoholic solid extract (18.22 g) so obtained indicated the presence of tannins. For the removal of tannins, 5 g extract was washed 2-3 times with 50 ml ethyl acetate in a separating funnel. Ethyl acetate fractions were collected and pooled and passed over dry anhydrous sodium sulphate for removal of water molecules. The filtrate was evaporated to dryness to furnish a solid mass of extract (516.12 mg).

Sample Preparation:

Accurately weighed 10 mg dry extract of H. indicus roots obtained from ethyl acetate fraction was taken in a 10 mL volumetric flask. Added 5 mL of ethyl acetate followed by sonnication to achieve a clear and homogenous solution. Solution volume was made up to 10 mL with ethyl acetate to get sample solution of concentration, 1 mg mL^-1.

Standard Preparation:

Standard stock solution containing 10 mg mL^-1 of ferulic acid was prepared by dissolving 10 mg ferulic acid in 10 mL methanol. The stock solution was further diluted to attain concentration of 25 μg mL^-1 for HPTLC analysis.

Chromatography:

Chromatography was performed on 20 cm × 10 cm TLC plates precoated with 250 μm layers of silica gel 60 F₂₅₄(E. Merck, Germany). Samples were applied to the plates as bands 7 mm wide by use of a CAMAG (Switzerland) Linomat 5 applicator fitted with a 100 μL syringe (Hamilton, Switzerland). The application positions X and Y were 15 mm and 10 mm, respectively, to avoid edge effects. Linear ascending development to a distance of 80 mm with Toluene: Ethyl acetate: Formic acid; 5: 5: 0.2, (v/v/v) as mobile phase was performed in a twin-trough glass chamber (20 cm × 10 cm) previously saturated with mobile phase vapour for 20 min, Figure 1. The plates were dried in air and densitometric scanning at λ 366 nm was performed with a CAMAG TLC scanner III, in absorbance/reflection mode, operated by WinCATs software (V 1.4.2; CAMAG). The slit dimensions were 5 mm × 0.45 mm and the scanning speed 100 nm s^-1.

Figure 1: TLC of ethyl acetate extract of Hemidesmus indicus root (I) and ferulic acid (II), using Toluene: Ethyl acetate: Formic acid; 5:5:0.2 (v/v/v) as mobile phase; 1A - visualized at 254 nm; 1B – visualized at 366 nm.

Method Validation for ferulic acid analysis:

The method was validated for linearity, accuracy, precision, limits of detection and quantitation, robustness, and specificity in accordance with ICH guidelines³⁰ reported methods^31-32.

Linearity and Calibration curve for the method:

Linearity was assessed by construction of six point calibration plot, Figure 2. From the standard stock solution (25 μg mL^-1) 2, 4, 6, 8, 10 and 12 μL of solution were applied to TLC plate furnish amounts in the range 50-300 ng spot^-1. Linear equation was obtained from the plot between peak areas and concentrations. Least-squares regression analysis was performed to obtain r-square (r²), multiple r (r) and standard deviation (SD).

Figure 2: Calibration plot of peak area Vs concentration of ferulic acid

Precision study of the method:

Repeatability and intermediate precision were assessed by measurement of intra-day variation. For the intra-day study, 4 µL of standard stock solution was applied (n=6 times) and measured on the same day. Mean, SD and RSD (%) were calculated for peak area and R_f.

Accuracy and Recovery study:

Accuracy was assessed by measurement of recovery by the method of standard additions; the method was applied to previously analyzed sample to which known amounts of standard had been added at three levels (100, 200 and 300). Analyses were performed in triplicates for each level, and the results obtained are expressed in terms of % recovery.

Limit of Detection (LOD) and Limit of Quantitation (LOQ) :

LOD of an analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantified accurately. This was calculated by using the formula:

LOD = (3.3 × standard deviation of the y-intercept) / (slope of the calibration plot).

LOQ is defined as the lowest amount of analyte in a sample which can be quantified accurately. This was calculated by using the formula:

LOQ = (10 × standard deviation of the y-intercept) / (slope of the calibration plot).

RESULTS AND DISCUSSION:

Optimization of Extraction method:

The root powder of H. indicus was extracted by hot extraction technique using hydro-alcohol solution (60% water and 40% ethanol) as extracting solvent. The dried hydro-alcoholic extract was rich in tannins, which interfered in separation and resolution of the bands in chromatogram. Therefore, to remove excess of tannins from hydro-alcoholic extract, fractionation of the extract was carried out repeatedly between ethyl acetate and water. Aqueous fractions were high in tannin content due to higher polarity and therefore, discarded. The ethyl acetate fractions were collected and used for HPTLC analysis.

Optimization of the Chromatographic Conditions:

Chromatographic separation studies were conducted on the ethyl acetate extract of H. indicus roots using ferulic acid as standard. Initially, plates were developed in mono-phase solvents as mobile phase like n-hexane, toluene, dichloromethane, ethyl acetate, chloroform, methanol and ethanol alongwith chamber saturation. To achieve good separation and optimum resolution of the developed bands, binary and ternary mixtures of solvents as mobile phase were investigated. Use of toluene: ethyl acetate: formic acid 5:5:0.2 (v/v/v) as mobile phase at room temperature (28±2⁰C) led to good resolution of ferulic acid with R_f0.59 ± 0.01. Wavelength for detection of ferulic acid was selected after evaluation of complete UV spectrum of ferulic acid. Quantitative analysis of the chromatogram was performed in the remission /absorbance mode at λ 366 nm for ferulic acid, Figure 3.

Figure 3: HPTLC chromatogram of standard ferulic acid (3A) and ethyl acetate extract of H. indicus root (3B).

Validation of HPLC Developed Method:

Validation parameters like linearity, precision, specificity, recovery and accuracy were evaluated for the developed chromatography method.

A six point calibration curve was constructed for linearity study. Correlation coefficient (r²) was found to be 0.999, indicating a good linear relation between peak area (peak response) and concentration applied in the range 50-300 ng spot^-1. The results from linear regression analysis are summarized in Table 1. The method was found to be precise, as indicated by RSD (≤ 1.95%) determined after intra-day precision, Table 2. The recovery at three different levels of ferulic acid was found in the range 91.43% - 105.21% indicating good accuracy and recovery as evident in Table 3. The developed method was found to be appropriate and suitable for the quantitative analysis of ferulic acid as indicated by limit of detection and limits of quantification which were 1.40 ng spot^-1and 4.25 ng spot^-1, respectively as shown in Table 1.

Table 1: Linearity, LOD and LOQ of Ferulic acid

S. No	Conc.(ng spot^-1)	Average peak Area
1.	50	931.5
2.	100	1450.7
3.	150	1929.95
4.	200	2396
5.	250	2932.2
6.	300	3454.2
Slope (m)		10.01
Intercept (c)		430.0
R- square (r²)		0.999
Multiple R (r)		0.999
SD		2.660
LOD		1.40 ng spot^-1
LOQ		4.25 ng spot^-1

Table 2: Precision study (intraday) of ferulic acid

Repeatability Study of Ferulic Acid (100 µg spot ^-1)
Spot no.	R_f	Peak area
1	0.59	1489
2	0.57	1412
3	0.58	1469
4	0.59	1475
5	0.59	1446
6	0.59	1436
Mean	0.59	1454.50
SD	0.01	28.43
% RSD	1.43	1.95

Table 3: Recovery study of ferulic acid

S. No.	Track	Area	Conc. (ng spot^-1)	% Recovery
S. No.	Sample	769.2	33.88
Level I	Std (100 ng)	1450.56	101.95	91.43
Level I	Spike	1760.7	132.93	91.43
Level II	Std (200 ng)	2397.06	196.50	98.47
Level II	Spike	2731.1	229.88	98.47
Level III	Std (300 ng)	3454.62	302.15	105.21
Level III	Spike	3811.5	337.81	105.21

Quantitative determination of Ferulic acid in H. indicus root:

The amount of the ferulic acid present in the H. indicus was estimated by the developed and validated HPTLC method. Content of ferulic acid in ethyl acetate extract of root powder was 1.694 %, w/w, Figure 3. Further, identification and quantitation of the other secondary metabolites present in whole plant of H. indicus by the developed method or other new method is currently under progress.

CONCLUSION:

Ferulic acid is an important secondary metabolite present in Hemidesmus indicus roots. A densitometric HPTLC method for quantitation of this compound in ethyl acetate extract of the dry root powder has been established and validated. The method proved to be simple, rapid, accurate, precise, and sensitive, and thus can be used for routine quality control and fingerprint analysis of H. indicus root powder using ferulic acid as chemical marker.

ACKNOWLEDGMENTS:

The authors are grateful to Arbo Laboratories Pvt. Ltd., New Delhi, for providing necessary facilities to carryout the research work.

REFERENCES:

1. Austin A. A review on Indian Sarsaparilla, Hemidesmus indicus (L.) R. Br. Journal of Biological Sciences. 8 (1) ; 2008 : 1-12.

2. Kirthikar KR and Basu BD. Indian Medicinal Plants. Bishen Singh Mahendra Pal Singh, Deharadun, India. 1980.

3. Prabakan M, Anandan R and Devaki T. Protective effect of Hemidesmus indicus against Rifampicin and Isoniazid-induced hepatotoxicity in rats. Fitoterapia. 71 (1) ; 2000: 55–59.

4. Gupta PN. Antileprotic action of an extract from Anantmul. (Hemidesmus indicus R.Br.). Lepr. India. 53 (3) ; 1981: 354-359.

5. Alam AI, Auddy B and Gomes A. Isolation, purification and partial characterization of viper venom inhibiting factor from the root extract of the Indian medicinal plant sarsaparilla (Hemidesmus indicus R.Br.). Toxicon, 32 (12); 1994 : 1551–1557.

6. Anonymous. The Wealth of India. Raw materials. Vol. III, V and X. CSIR, New Delhi, India. 1997.

7. Anonymous. Indian Pharmacopoeia. Indian Pharmacopoeia Committee, Ministry of Health and Family Welfare, Government of India, New Delhi. 1996.

8. Anonymous. British Pharmacopoeia. British Pharmacopoeia Commission, London. 2003.

9. Available from URL http://www.motherherbs.com/sarsaparilla.html

10. Anonymous. The useful plants of India. CSIR, New Delhi, India. 1986.

11. Nadkarni AN. Indian Material Medica. Vol. I. Popular Book Depot, Bombay, India. 1989.

12. Mukherjee B. The Indian Pharmaceutical Codex. Vol. II. Indigenous drugs. CSIR, New Delhi, India. 1953.

13. Chopra RN, Nayar SL and Chopra IC. Glossary of Indian Medicinal Plants, CSIR, New Delhi, India. 1956.

14. Murthi BR and Seshadri TR. A study of the chemical components of the roots of Decalepis hamiltoni, Part III. Comparison with Hemidesmus indicus (Indian Sarasaparilla). Proceeding Indian. Acad. Sci. 13 ; 1941: 3999-4003.

15. Chatterjee I. Chakravarty AK and Gomes A. Daboia russellii and Naja kaouthia venom neutralization by lupeol acetate isolated from the root extract of Indian sarasaparilla Hemidesmus indicus R. Br., J. Ethnoparmacol. 106 (1) ; 2006 : 38-43.

16. Padhy SN, Mahato SB and Dutta NL. Asclepiadaceae terpenoides from the roots of Hemidesmus indicus. Phytochemistry. 12 (1) ; 1973 : 217-218.

17. Das PC, Joshi PC, Mandal S, Das A and Chatterjee A. New Coumarinoliganoids from Hemidesmus indicus R. Br. Indian J. Chem. 31B ; 1992 : 342-345.

18. Isao K and Ikuyo KH. 2-Hydroxy-4-methoxybenzaldehyde: A Potent Tyrosinase Inhibitor from African Medicinal Plants. Planta Med. 65 (1) ; 1999 : 19–22.

19. Maeda K and Fukuda M. In vitro effectiveness of several whitening cosmetic components in human melanocytes. J. Soc. Cosmet. Chem. 42 (6) ; 1991 : 361–368.

20. Fitzpatrick TB, Seiji M and McGugan AD. Melanin pigmentation. New Eng. J. Med. 265 (Aug 24) ; 1961: 374–378.

21. Prota G. Melanins and Melanogenesis. Academia Press, San Diego. 1992.

22. Phadke NY, Gholap AS, Ramakrishnan K and Subbulakshmi G. Essential oil of Decalepis hamiltonii as an antimicrobial agent. J. Food Sci. Technol. 31 (6) ; 1994 : 472–475.

23. George J, Pereira J, Ravishankar GA and Divakar S. Bioinsecticide by swallowroot (D. hamiltonii) Wight and Arn. Protects food grains against insect infestation. Curr. Sci. 77 (4) ; 1999 : 501–502.

24. Mandal SK, Haty S, Paul A, Panja D and Deb S. Standardization of herbal drugs part III. A High Performance Liquid Chromatography (HPLC) method for the quantitative estimation of 2-hydroxy-4-methoxybenzaldehyde from Hemidesmus indicus (L.) AIT. (Anantamul). Indian Drug, 44 (4) ; 2007 : 257–260.

25. Sircar D, Dey G and Mitra A. A Validated HPLC Method for Simultaneous Determination of 2-Hydroxy-4-methoxybenzaldehyde and 2-Hydroxy-4-methoxybenzoic Acid in Root Organs of Hemidesmus indicus. Chromatographia 65 (5-6) ; 2007 : 349–353.

26. Nagarajan S, Rao LJM and Gurudutt KN. Chemical composition of the volatiles of Hemidesmus indicus R. Br. Flavour Fragr. J. 16 (3) ; 2001 : 212–214.

27. Raghavendra HL, Yogesh HS, Gopalakrishna B, Chandrashekhar VM, Sathish BP and Vadlapudi K. An overview of herbal medicine. Int. J. Ph. Sci. 1(1) ; 2009 : 1-20.

28. Sahoo N, Padmavati M and Dey S. Herbal drugs: Standards and regulation. Fitoterapia. 81 (6) ; 2010 : 462-471.

29. Xin-miao Liang, Yu Jin, Yan-ping Wang, Gao-wa Jin, Qing Fu and Yuan-sheng Xiao. Qualitative and quantitative analysis in quality control of traditional Chinese medicines. J. Chromatogr. A. 1216 (11) ; 2009 : 2033–2044.

30. International Conference on Harmonization Q2B: Validation of Analysis Procedures, US FDA Federal Register .1997. 62: 27463-27467.

31. Verma SC, Nigam S, Jain CL, Pant P, Padhi MM and Devalla RB. Solvent polarity based microwave assisted extraction of ferulic acid from whole plant of Cynodon dactylon L. and it’s quantitative determination by developed and validated HPTLC Method. Asian J. Research Chem. 4 (9) ; 2011 : 1460-1465.

32. Verma SC, Nigam S, Jain CL, Pant P and Padhi MM. Microwave-assisted extraction of gallic acid in leaves of Eucalyptus x hybrida Maiden and its quantitative determination by HPTLC, Der Chemica Sinica. 2 (2) ; 2011 : 268-277.

Received on 19.09.2011 Modified on 05.10.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1747-1751