Detection of Adulteration in Rubia cordifolia –

A Chromatographic Approach

Ghanshyam Kamani, Rohan Sanghani, Vaibhavi Savalia*, Devang Pandya

School of Pharmacy, RK University, Kasturbadham, Rajkot, Gujarat - 360020 India.

*Corresponding Author E-mail: vaibhavi.savalia@rku.ac.in

ABSTRACT:

According to traditional medicinal texts as well as modern research, Rubia cordifolia (Desi Manjistha) stem cure various diseases of blood, skin diseases, inflammations, kidney stones, fever and various Kapha disorders. However, stems of Rubia cordifolia (Desi Manjistha) are often adulterated with stems of Rubia tinctorum (Irani Manjistha) due to morphological similarity. This adulteration is extremely harmful as R. tinctorum has Lucidin as one of the chemical constituents, which has severe genotoxic effects. The present work focuses on developing a comparative HPTLC fingerprint and GC-MS analysis of R. cordifolia and R. tinctorum, so as to detect the adulteration by R. tinctorum in R. cordifolia raw material form and in formulations. Methanol extracts of the individually powdered stems were used to develop a suitable mobile phase, such that a clear spot was obtained in TLC of R. tinctorum but not in TLC of R. cordifolia using the same mobile phase. This mobile phase was further used to develop a comparative HPTLC fingerprint of the two species. Methanol extracts of R. cordifolia and R. tinctorum were used for investigation of phytoconstituents by GC-MS analysis. The mobile phase n-butanol: ethanol (6:4) was the one which gave a clear single spot at R_f 0.89 in TLC of methanol extract of R. tinctorum but not in TLC of methanol extract of R. cordifolia, at 254nm and 366nm. Further, HPTLC analysis gave results clearly differentiating the two species using the same mobile phase. Further, GC-MS analysis of R. cordioflia revealed the presence of 9 phytoconstituents while R. tinctorum revealed the presence of 6 phytoconstituents, which are different from each other. Thus, these simple yet sophisticated techniques will be very useful for herbal industry in standardization and detection of adulteration of R. tinctorum in R. cordifolia formulations, thereby benefitting the patients who consume Manjistha formulations.

KEYWORDS: Adulteration, GC-MS, HPTLC, Manjistha, Quality Control, Rubia tinctorum.

INTRODUCTION:

Rubia cordifolia Family: Rubiaceae (Indian Manjistha/ Indian Maddar) stem have been prescribed in ayurveda to improve the color and complexion of the skin. It is well known as a rakta shodhak (blood purifier). It is also described for curing Kapha disorders, and in various diseases of blood, skin and urinogenital tract. Indian Manjistha can be used both internally and externally. Rubia cordifolia is also mentioned in Charak Samhita, Sushrut Samhita and in Ashtanga Hridaya, also official in Ayurvedic Pharmacopoeia of India and in Indian Pharmacopoeia^1,2,3,4.

Many of its formulations are available in the market, including Manjisthadi Taila and Manjisthadi Kwatha. Not only conventional formulations of Rubia cordifolia like tablets are available in market but wound healing formulation, gel and novel drug delivery system such as sustain release Rubia cordifolia matrix tablet have also been developed and evaluated^5,6,7.

However, stems of Rubia cordifolia are often adulterated with stems of Rubia tinctorum (Irani Manjistha) in crude drug or in its formulations due to morphological similarity. This adulteration is very harmful as R. tinctorum stem contains a genotoxic compound Lucidin which causes birth defects and miscarriages^1,8. It is difficult to differentiate powders or extracts of the two species on the basis of microscopy or phytochemical screening¹. Hyphenated techniques like GC-MS is a sophisticated technique which have potential to identify volatile phytoconstituents and which has effectiveness in determining molecular weight and structures of the unknown organic compounds in complex mixtures^9,10. High performance thin layer chromatography (HPTLC) is also a valuable tool for reliable identification plant, their extracts and formulations. It can provide chromatographic fingerprints that can be visualized and stored as electronic images^11,12,13. A RP-HPLC Method was developed for the determination of anthraquinone marker from the roots of Rubia cordifolia¹⁴. Thus, chromatographic approach can be used to detect adulteration and to identify original species of herbs by differentiating GC-MS chemo-profiling and HPTLC fingerprint both together¹⁵. But, to identify and detect adulteration of Rubia tinctorum in Rubia cordifolia stem chromatographic approach has not been developed as per our literature review. So, our aim of present work is TLC mobile phase development, HPTLC fingerprinting and GC-MS chemoprofiling of methanol extract to detect the adulteration of R. tinctorum in R. cordifolia.

Fig. 1: Marketed samples of Rubia cordifolia (Desi Manjisth) and Rubia tinctorum (Irani Manjistha)

A. Rubia cordifolia -Desi Manjistha stem, B. Rubia tinctorum – Irani Manjistha market samples

MATERIAL AND METHODS:

Chemicals and Reagents:

Methanol used were of analytical grade (Molychem chemicals, Mumbai, India). All other chemicals and reagents used were analytical grade unless otherwise indicated. Ethanol used for TLC and HPTLC was 99.5% ethyl alcohol.

Collection and Authentication of samples:

Dried stem of Rubia cordifolia and Rubia tinctorum species were Purchased from Yucca enterprise, Mumbai, India. (Fig. 1) The samples were authenticated by Dr. Kunjal Soni, Botanist, School of Science, RK University, Rajkot.

Preparation of Extract:

10g stem of each species were powdered separately using electric grinder and powder was passed through sieve #50. 50g dry powder of each species was macerated separately with 100ml methanol for 24h at room temperature. Methanol extracts were filtered and evaporated on water bath at 40°C to obtain the dried extracts. The collected samples were stored in air tight container and used for further study.

Thin Layer Chromatography:

Methanol extracts of the individually powdered stems of Rubia cordifolia and Rubia tinctorum were used to develop a suitable mobile phase, such that a clear spot was obtained in TLC of R. tinctorum but not in TLC of R. cordifolia using the identical mobile phase. For this, pilot TLC were developed using various mobile phases prepared using solvents like toluene, chloroform, methanol, ethyl acetate, etc., in varying ratios. Upon developing hundreds of pilot TLC, the mobile phase n-Butanol: Ethanol (6:4) was the one which gave a clear single spot at R_f 0.89 in TLC of methanolic extract of R. tinctorum but not in TLC of methanolic extract of R. cordifolia, at both 254nm and 366nm. This served as a guide for further comparative HPTLC analysis.

HPTLC:

HPTLC fingerprinting of methanolic extract of each species was performed in Dept. of Pharmaceutical Sciences, Saurashtra University, using the mobile phase n-Butanol: Ethanol (6:4) under the following conditions.

Stationary phase: Silica gel 60 F 254 (E. Merck KGaA)

Sample application: CAMAG Linomat 5

Detection: CAMAG TLC Scanner 3

Lamp: D2 & W

Measurement type: Remission

Measurement mode: Absorption

Optical filter: Second order

Data filtering: Savitsky-Golay 7

Scanning wavelength (UV): 254nm and 366nm.

GC-MS Analysis:

Comparative GC-MS of methanolic extracts was done at Bioresearch and Characterization Centre (BRCC) of RK University, Rajkot. Identification of compound on basis of GC-MS was done using the GC-MS database of National Institute Standard and Technology (NIST) library.

RESULTS AND DISCUSSION:

Thin Layer Chromatography:

A clear spot was observed in TLC of Rubia tinctorum stem methanol extract at 0.89 Rf value but not in Rubia cordifolia methanol stem extract with the mobile phase n-Butanol: Ethanol (6:4) (Fig. 2). Since, Manjistha formulations are widely available in the market and used for curing various disorders of blood and skin. This simple TLC technique will also serve as cheap and quick identification of adulteration of R. tinctorum in R. cordifolia powder as well as alcoholic extracts.

Fig. 2: Thin layer chromatography of Rubia cordifolia and Rubia tinctorum at UV 366nm

Where, sample A. Methanol extract of stem of Rubia tinctorum, B. Methanol extract of stem of Rubia cordifolia

HPTLC Fingerprinting of Manjistha Species:

For comparative HPTLC fingerprinting methanol extracts of R. cordifolia and R. tinctorum were applied on total 4 tracks, two each. n-Butanol: Ethanol (6:4) was used to develop HPTLC plate. Air dried plate were scanned at 254nm, and 366nm. Total of three and six peaks were detected at 254nm for methanol stem extract of R. cordifolia and R. tinctorum, respectively. Peaks were found at different R_f values for both the species (Table 1, Figure 3). Total of six and three peaks were detected at 366nm for methanol stem extract of R. cordifolia and R. tinctorum, respectively. Peaks were found at different R_f values for both the species (Table 2, Figure 4). The comparative HPTLC fingerprint analysis of methanolic extracts of stems of R. cordifolia and R. tinctorum reveals presence of peak at 0.89 Rf which confirm the thin layer chromatography with mobile phase n-Butanol: Ethanol (6:4). Thereby, present study serves a dual purpose. First purpose, it clearly distinguishes the two species from each other, and makes it simple to detect the adulteration of R. tinctorum in R. cordifolia by detecting presence of spot at 0.89 R_f value in sample HPTLC or TLC with mobile phase n-Butanol: Ethanol (6:4), confirms the adulteration of Rubia tinctorum in Rubia cordifolia. Second, it also serves as a quality control parameter of R. cordifolia itself by HPTLC fingerprinting of methanol extract with mobile phase n-Butanol: Ethanol (6:4).

Table 1: Comparative HPTLC of Manjistha species at 254nm (n-Butanol: Ethanol 6:4)

Peak	*Rubia cordifolia*		*Rubia tinctorum*
Peak	R_f	Area Under Curve	R_f	Area Under Curve
1	0.03	7892.3	0.02	11331.9
2	0.17	3702.6	0.21	20192.9
3	0.80	6348.2	0.42	1225.7
4	--	--	0.53	575.9
5	--	--	0.79	182.1
6	--	--	0.89	1182.7

Table 2: Comparative HPTLC of Manjistha species at 366nm (n-Butanol: thanol 6:4)

Peak	*Rubia cordifolia*		*Rubia tinctorum*
Peak	R_f	Area Under Curve	R_f	Area Under Curve
1	0.03	6380.3	0.02	9358.6
2	0.12	2192.6	0.21	10373.0
3	0.21	992.0	0.89	769.2
4	0.57	238.9	--	--
5	0.75	869.9	--	--
6	0.80	2698.8	--	--

Fig. 4: Comparative HPTLC Fingerprint of methanol extract of Manjistha species at 366nm (n-Butanol: Ethanol 6:4) (Where, A-3D

chromatogram; RC: Rubia cordifolia; RT: Rubia tinctorum, B- HPTLC plate scanned at 254nm; C- 2D chromatogram of Rubia cordifolia; D- 2D chromatogram of Rubia tinctorum)

GC-MS Analysis of Manjistha Species:

GC-MS chromatogram of methanol extract of stem of Rubia cordifolia and Rubia tinctorum are depicted in Fig. 5. Phytoconstituents identified are by GC-MS analysis of methanol extract of R. cordifolia and R. tinctorum are described in Table 3 and Table 4, respectively. Total of nine phytoconstituents were identified from methanol extract of R. cordifolia, while total of six phytoconstituents were identified from methanol extract of R. tinctorum. None of the phytoconstituents found similar in both species. Known phytoconstituents Caffeine was exclusively identified by GC-MS analysis in R. cordifolia. Simple chemoprofiling by GC-MS analysis of methanol extract of Rubia cordifolia will detect presence of adulteration and also confirm authenticity of raw material in powder form as well in alcoholic extracts. Thus, harmful adulteration of as R. tinctorum stem in R. cordifolia stem can be detected by simple HPTLC fingerprinting and GC-MS chemoprofiling in powder and alcohol extract and we can prevent genotoxic birth defects and miscarriages in females due to consumption of adulterated Manjistha formulations with Rubia tinctorum ⁵.

Fig.5: GC-MS chromatogram of methanol extract of stem of Manjistha species. (where, A- Rubia cordifolia, B- Rubia tinctorum)

Table 3: Integration Peak List of Phytochemicals Identified in methanol extract of stem of R. cordifolia by GC-MS analysis

Peak	Name of phytoconstituents	RT	Area	Area%	Structure of phytoconstituent
1	Pyridine, 2-nitro	2.15	87441.6	21.08
2	Propane, 2-fluoro-2-methyl	4.104	414875.83	100
3	Methane, isocyanato	4.291	77511.05	18.68
4	Sulfide, isobutyl o-tolyl	18.995	37145.85	8.95
5	Caffeine	22.483	116928.48	28.18
6	Benzene, 1-(1,1-dimethylethyl)-4-phenoxy	28.8	35758.68	8.62
7	Benzo[b]thiophene, 3-phenyl	32.663	290693.37	70.07
8	2-Naphthalene carboxylic acid, 1,4-dihydro-1,4-dioxo-3-(3-methyl-2-butenyl)-, methyl ester	56.378	159077.18	38.34
9	2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl-9-(phenylsulfonyl)-, (E,E)	58.546	49687.78	11.98

Table 4: Integration Peak List of Phytochemicals Identified in methanol extract of stem of R. tinctorum by GC-MS analysis

Peak	Name of phytoconstituents	RT	Area	Area%	Structure of phytoconstituent
1	2(1H)-Pyrimidinone	2.328	110752.64	100
2	1-Penten-3-one	2.756	64538.01	58.27
3	Butoxy acetic acid	3.72	36687.91	33.13
4	Ethanethiol, 2-phenoxy-	4.13	3194.18	2.88
5	2,4(1H,3H)-Pyrimidinedione, 6-iodo-5-methyl-	13.721	51423.66	46.43
6	2-Hydroxy-3-(thiophen-2-yl) methyl-5-methoxy-1,4-benzoquinone	22.483	15389.7	13.9

CONCLUSION:

A simple yet effective HPTLC fingerprint helps in the detection of adulteration by R. tinctorum in the stems of R. cordifolia. Also, a simple yet sophisticated chemoprofiling of R. cordifolia by GC-MS analysis also gives clear identification of Rubia cordifolia or adulteration of R. tinctorum in R. cordifolia. Moreover, the present work will be helpful to ensure the safety of patient by preventing adulteration of Manjistha formulation and improve the reliability of the products. Present work can be valuable for researchers, since it provides information which further opens up the scope of isolating phytoconstituents on the basis of the comparative HPTLC.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Department of Pharmaceutical Sciences, Saurashtra University, and Bioresearch and Characterization Centre (BRCC) of RK University, Rajkot, Gujarat, India for providing the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 08.05.2020 Modified on 19.07.2020

Research J. Pharm. and Tech. 2021; 14(8):4013-4018.

DOI: 10.52711/0974-360X.2021.00695