COMPARATIVE PHYTOCHEMICAL EVALUATION OF ETHNOBOTANICALLY IMPORTANT MEDICINAL PARASITIC HERB: ALECTRA CHITRAKUTENSIS
SHITAL K. SHARMA1*, ANITA S. PATIL1, R. L. S. SIKARWAR2
1Lab no. 106, Plant Secondary Metabolite Lab, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati 444602
(M. S.), India, 2Arogyadham, (J. R. D. Tata Foundation for Research in Ayurveda and Yoga Sciences), Deendayal Research Institute, Chitrakoot, Distt-Satna (M. P.), India
Email: shital_sharma24@yahoo.co.in
Received: 28 Jan 2016 Revised and Accepted: 01 Mar 2016
ABSTRACT
Objective: Alectra chitrakutensis (M. A. Rau) R. Prasad & R. D. Dixit is a critically endangered and ethnobotanically very important plant. Official drug i.e. dried rhizome of the plant has been reported to be used for treatment of leprosy, tuberculosis, paralysis, oedematous swelling, fevers, intestinal worms and constipation. Besides having high medicinal properties, detail studies on chemical constituents present in the rhizome of this particular species have not been done so far. Thus, in present study efforts were made for evaluation of phytochemical as well as physicochemical analysis of the rhizome collected from six different places of the Chitrakoot region of Madhya Pradesh (M. P.) and Uttar Pradesh (U. P.).
Methods: Phytochemical analysis of the rhizome was carried out as per standard protocol given in Ayurvedic Pharmacopoeia of India (API). Separation and qualitative phytochemical screening were done by using an advance technique of High-performance Thin Layer Chromatography (HPTLC).
Results: The study revealed the presence of alkaloids, steroids, triterpenoids, flavonoids, glycosides, carbohydrates, starch, saponin, tannins, etc. in the rhizome of the plants, and the study confirmed the chemotaxonomic resemblance among all the collected plant materials.
Conclusion: Comparative study of six rhizome samples of A. Chitrakutensis provides authenticity for establishing Pharmacopoeial standardization of drug and evidence of the study proves the chemotaxonomic similarities of official drug.
Keywords: Alectra chitrakutensis, Endangered plant, Quality control, Physicochemical, Phytochemical analysis, HPTLC
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
INTRODUCTION
A. chitrakutensis, family Scrophulariaceae is a critically endangered annual root parasitic herb. It is locally known as ‘Nirgundikand’ found in confined localities in the Chitrakoot region of Madhya Pradesh and also in Uttar Pradesh, India [1]. The species grows as a parasite on the threadlike roots of white-flowered Vitex negundo L. Chitrakoot is very rich in ethnomedicobotanical diversity. The tribal people use locally available plant species for the treatment of human as well as livestock ailments [2]. A. chitrakutensis is one of them having high-value medicinal properties. Ethnobotanically, the whole plant has been reported to be used for treatment of diverse deadened diseases. However, the rhizome is used to cure leprosy, tuberculosis, paralysis, oedematous swelling, fevers, intestinal worms and constipation. The previous study has reported various ethnobotanical uses of rhizomes, for example, paste of the rhizome mixed with cow urine is given to leprosy patients [3]; similarly, dried and powdered rhizome is used to cure other skin diseases. For instant relief from constipation rhizome paste is taken with milk. Besides, rhizome paste is given once a day for 2-3 d to expel intestinal worms. Likewise, for the treatment of malaria, fresh rhizome paste with cow urine is given to a patient twice a day. Rhizome powder is also given to cure paralysis and piles. The plant and rhizome extract with almond, cucumber, watermelon, long cucumber, cardamom and rose petal is taken as an invigorating tonic. In case of Spermatorrhoea, rhizome powder is given with milk [2]. Preliminary phytochemical investigations carried out and reported the presence of steroids, glycosides, flavonoids, carbohydrates, alkaloids, saponins and tannins in the whole plant extracts [4]. A carotinoid, an orange-yellow color pigment azafrin, and mannitol were also found in the rhizome [5].
Since, A. chitrakutensis is a critically endangered and endemic species of high medicinal value. Until now, the importance of this species was recorded only in few folklore literatures; furthermore, no records were found to establish pharmacopoeial evaluation of the rhizome which is the plant part of medicinal interest. Owing to the significant medicinal properties of the rhizome, there is an urgent need to identify and characterize the various primary as well as secondary metabolites present in the rhizome so as to prepare pharmacopoeial standards and a novel drug of therapeutic use. Therefore, the present study deals with the physicochemical and phytochemical evaluation of the rhizome of the plant A. chitrakutensis collected from six different places.
MATERIALS AND METHODS
Collection and identification of plant material
Authentic plant materials were collected from six different places in the Chitrakoot region of M. P. and U. P. in between the month of February-March 2012 under the supervision and guidance of R. L. S. Sikarwar; speciemen herbarium was prepared and submitted vide herbarium number 319 in the repository at “Arogyadham” (JRD Tata Foundation for Research in Ayurveda and Yoga Science), Deendayal Research Institute, Chitrakoot, (M. P.). The distinctive places from where the material was collected are Herbal Garden, Deendayal Research Institute, Chitrakoot (M. P.), Narayanpur Hills, Chitrakoot (U. P.), Bank of Mandakini river, Satna, Chitrakoot (M. P.), Bank of Bagey river, Badausa (U. P.), Bank of Bagey river, Banda (U. P.) and Lodwara Hill, Chitrakoot (U. P.).
Processing of plant material
Rhizomes were separated from aerial parts of the plant, thoroughly washed with tap water and dried in the hot air oven at 50C and powdered by using a grinder. After grinding all the samples were passed through sieve of 40-60 mesh size and kept in a container for further use.
Powder analysis
The fluorescence study of powdered drugs was performed using different chemicals under the UV light (254 nm and 366 nm) and in visible light according to the standard methods [6-7].
Physicochemical evaluation
Physico-chemical analysis such as moisture content, ash value, and extractive values were performed using standard protocols given in Ayurvedic Pharmacopoeia of India [8].
Table 1: Fluorescence study of rhizome powder of ‘A. chitrakutensis’ collected from six different places
Reagents/Samples |
Herbal Garden, Chitrakoot (M. P.) |
Bank of Bagey river, Banda (U. P). |
Lodwara Hill, Chitrakoot (U. P.) |
Narayanpur Hills, Chitrakoot (U. P.) |
Bank of Bagey river, Badausa (U. P.) |
Bank of Mandakini river, Satna, Chitrakoot (M. P.) |
||||||||||||
Day Light |
UV 254 nm |
UV 366 nm |
Day Light |
UV 254 nm |
UV 366 nm |
Day Light |
UV 254 nm |
UV 366 nm |
Day Light |
UV 254 nm |
UV 366 nm |
Day Light |
UV 254 nm |
UV 366 nm |
Day Light |
UV 254 nm |
UV 366 nm |
|
Powder (P) as such |
Brown |
Dark Brown |
Black |
Brown |
Dark Brown |
Black |
Brown |
Dark Brown |
Black |
Brown |
Dark Brown |
Black |
Brown |
Dark Brown |
Black |
Brown |
Dark Brown |
Black |
P+Nitrocellulose in amyl acetate |
Blackish Brown with Greenish Tinge |
Fluorescence Greenish brown |
Black with Yellow tinge |
Blackish Brown with Greenish Tinge |
Fluorescence Greenish brown |
Black with Yellow tinge |
Blackish Brown with Greenish Tinge |
Fluorescence Greenish brown |
Black with Yellow tinge |
Blackish Brown with Greenish Tinge |
Fluorescence Greenish brown |
Black |
Blackish Brown with Greenish Tinge |
Fluorescence Greenish brown |
Black |
Dark Brown with Greenish Tinge |
Fluorescence Greenish brown |
Black |
P+1N NaOH |
Chocolate Brown |
Dark Brown |
Black |
Chocolate Brown |
Dark Brown |
Black |
Chocolate Brown |
Dark Brown |
Black |
Chocolate Brown |
Dark Brown |
Black |
Chocolate Brown |
Dark Brown |
Black |
Chocolate Brown |
Dark Brown |
Black |
P+1N NaOH+Amyl acetate |
Dark Brown |
Dark Brownish Black |
Black |
Dark Brown |
Dark Brownish Black |
Black |
Dark Brown |
Dark Brownish Black |
Black |
Blackish Brown |
Black |
Black |
Blackish Brown |
Black |
Black |
Dark Brown |
Black |
Black |
P+1N HCl+Nitrocellulose in amyl acetate |
Brown with Greenish Tinge |
Dark Brown with fluorescence Green tinge |
Black |
Brown with Greenish Tinge |
Dark Brown with fluorescence Green tinge |
Black |
Brown with Greenish Tinge |
Dark Brown with fluorescence Green tinge |
Black |
Dark Brown |
Blackish Brown with fluorescence Green tinge |
Black |
Dark Brown |
Blackish Brown with fluorescence Green tinge |
Black |
Blackish Brown |
Brown |
Black |
P+1N NaOH in methanol |
Dark Green |
Dark Green |
Green |
Dark Green |
Dark Green |
Green |
Dark Green |
Dark Green |
Green |
Dark Green |
Blackish Brown |
Green |
Dark Green |
Blackish Brown |
Green |
Dark Green |
Dark Brown |
Green |
P+50% KOH |
Chocolate Brown |
Black |
Black |
Chocolate Brown |
Black |
Black |
Chocolate Brown |
Black |
Black |
Chocolate Brown |
Black |
Black |
Chocolate Brown |
Black |
Black |
Blackish Brown |
Fluorescence Green tinge |
Black |
P+1N HCl |
Dark Brown |
Dark Brown |
Black |
Dark Brown |
Dark Brown |
Black |
Dark Brown |
Dark Brown |
Black |
Brown |
Black |
Black |
Brown |
Black |
Black |
Brown |
Black |
Black |
P+50% H2SO4 |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Blackish Brown |
Black |
Black |
P+50% HNO3 |
Rusty Yellow |
Black with Green tinge |
Black with Green tinge |
Rusty Yellow |
Black with Green tinge |
Black with Green tinge |
Rusty Yellow |
Black with Green tinge |
Black with Green tinge |
Almond Brown |
Dark Brown |
Black |
Almond Brown |
Dark Brown |
Black |
Chocolate Brown |
Black |
Black |
P+Conc. HNO3 |
Rusty Yellow |
Black with Yellow tinge |
Black with Green tinge |
Rusty Yellow |
Black with Yellow tinge |
Black with Green tinge |
Rusty Yellow |
Black with Yellow tinge |
Black with Green tinge |
Turmeric dark/pale Yellow |
Rusty Yellow |
Black |
Turmeric dark/pale Yellow |
Rusty Yellow |
Black |
Pale Yellow |
Brown |
Black |
P+Acetic acid |
Dark Brown |
Black |
Black |
Dark Brown |
Black |
Black |
Dark Brown |
Black |
Black |
Blackish Brown |
Brown with fluorescence Green tinge |
Black |
Blackish Brown |
Brown with fluorescence Green tinge |
Black |
Dark Brown |
Rusty Yellow |
Black |
P+Conc. H2SO4 |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Black |
Dark Brown |
Black |
P+Iodine water |
Brown |
Dark Brown |
Black |
Brown |
Dark Brown |
Black |
Brown |
Dark Brown |
Black |
Dark Green |
Dark Brown |
Black |
Dark Green |
Dark Brown |
Black |
Dark Green |
Black |
Black |
Phytochemical screening
The preliminary phytochemical screenings for the chemical constituents like steroids, alkaloids, saponins, glycosides, flavonoids, starch, tannins, resins, etc. were determined by the standard methods [9].
HPTLC fingerprint analysis
HPTLC (CAMAG, Switzerland), an advance technique was used for qualitative screening of different phytoconstituents [10]. For this, 1 g powdered material of each sample was extracted using 10 ml methanol in water bath for 5 min. The extracts were filtered through Whatman No. 1 filter paper separately for each sample. Similarly, stock solution (1 mg/ml) of standard azafrin; a pure compound isolated from plant material by using standard protocol [11] was also prepared. Filtrated extracts and standard solution of azafrin (2 µl) were applied on 20x20 cm TLC plates of silica gel G F254 (Merck, Germany) with the help of Linomat 5 CAMAG TLC Applicator.
The plate was developed up to 18.5 cm by using the solvent system Toluene: Ethyl acetate: Glacial acetic acid (4:5:0.1 v/v/v) in a CAMAG Twin-through chamber previously saturated with mobile phase vapor for 30 min. at room temperature. After development, plates were air dried at room temperature and documented by using CAMAG visualizer documentation system. Anisaldehyde sulfuric acid reagent was used as derivetizing agent. After derivetization, plates were dried at 100 °C on a hot plate for 5 min and visualize under 254 nm, 366 nm and visible light.
RESULTS
Powder analysis
Fluorescence study of all six samples of rhizome powder under UV light, with or without chemical, treatment was done. The color developed by the powder with different chemical reagents under UV, and visible lights are summarized (table 1). Plant materials collected from different places showed somewhat different color characterization of rhizome powder. Similarities in results were obtained among the rhizome samples collected from Herbal garden, Arogyadham, Dindayal Research Institute, Chitrakoot (M. P.), Bank of Bagey river, Banda (U. P.) and Lodwara Hill, Chitrakoot (U. P.). While, Narayanpur Hills, Chitrakoot (U. P.) and Bank of Begey river, Badausa (U. P.) also produced similar results, whereas material from Bank of Mandakini river, Satna, Chitrkoot (M. P.) showed unlike color than others.
Quantitative physico-chemical analysis
The results of physicochemical analysis viz., foreign matter, moisture content, total ash, acid insoluble ash, alcohol, water extractive values and successive Soxhlet extractive values of different extractives like hexane, chloroform, methanol and water of the rhizome powder are presented in table 2 and 3. Plant material collected from different places throughout the Chitrakoot region showed not much significant difference. Thus, the values obtained from a physicochemical study are useful to establish the identity and strength on the rhizome part for further study.
Qualitative phytochemical screening of successive soxhlet extractives
The phytochemical analysis using different reagents such as n-hexane, chloroform, methanol and water extracts showed the presence of starch, steroids, terpenoids, alkaloids, tannins, flavonoids, glycosides, proteins, and amino acids in different successive extracts of all the collected samples have been presented in table 4. Glycoside was the major constituent found in chloroform, methanol and water extracts.
HPTLC study
In densitometry scan of Thin Layer Chromatography (TLC) profiles of the methanolic extracts of all six rhizome samples of ‘A. chitrakutensis’ showed an almost resembling band pattern which indicated that the six samples are having similar phytochemicals. Only the quantity of particular phytoconstituents may vary (fig. 1). For TLC, solvent system was developed to get high-quality resolution and separation of bands. Even if there were some variations in the profiles which might be due to some environmental factors. Rf values were calculated under 366 nm and are summarized in table 5.
Table 2: Comparative physicochemical values of the rhizome of ‘A. chitrakutensis’ collected from six different places
S. No. |
Physico-chemical parameters |
Herbal garden, chitrakoot (M. P.) |
Narayanpur hills, chitrakoot (U. P.) |
Bank of mandakini river, satna, chitrakoot (M. P.) |
Bank of bagey river, badausa (U. P.) |
Bank of bagey river, banda (U. P.) |
Lodwara hill, chitrakoot (M. P.) |
1. |
Foreign matter |
2.12 |
2.00 |
2.00 |
2.2 |
2.12 |
2.1 |
2. |
Moisture content (%) |
7.06 ±0.13 |
5.27 ±0.04 |
6.16 ±0.02 |
7.96 ±0.03 |
7.01 ±0.03 |
5.99 ±0.01 |
3. |
Total Ash (%) |
5.98 ±0.07 |
5.83 ±0.15 |
5.91 ±0.02 |
5.88 ±0.06 |
5.89 ±0.08 |
5.79 ±0.09 |
4. |
Acid Insoluble Ash (%) |
1.34 ±0.03 |
1.32 ±0.04 |
1.29 ±0.01 |
1.29 ±0.05 |
1.30 ±0.00 |
1.29 ±0.01 |
5. |
Alcohol soluble extractive value (%) |
7.66 ±0.74 |
7.81 ±0.44 |
7.52 ±0.24 |
8.26 ±0.56 |
8.02 ±0.15 |
7.55 ±0.25 |
6. |
Water soluble extractive value (%) |
27.93 ±1.43 |
27.92 ±0.06 |
27.00 ±0.05 |
27.91 ±0.09 |
27.77 ±0.05 |
27.75 ±0.06 |
Table 3: Comparative soxhlet extractive values of rhizome of ‘A. chitrakutensis’ collected from six different places
Solvent |
Herbal garden, chitrakoot (M. P.) |
Narayanpur hills, chitrakoot (U. P.) |
Bank of mandakini river, satna, chitrakoot (M. P.) |
Bank of bagey river, badausa (U. P.) |
Bank of bagey river, banda (U. P.) |
Lodwara hill, chitrakoot (M. P.) |
Hexane |
1.83±0.03 |
1.64±0.02 |
1.74±0.03 |
1.81±0.02 |
1.59±0.02 |
1.41±0.02 |
Chloroform |
5.29±0.05 |
5.24±0.02 |
5.28±0.02 |
5.34±0.03 |
5.32±0.02 |
5.13±0.01 |
Methanol |
32.99±0.45 |
31.24±0.23 |
30.18±0.42 |
33.26±0.43 |
29.83±0.31 |
32.23±0.26 |
Water |
20.73±0.12 |
20.71±0.09 |
20.74±0.07 |
20.79±0.07 |
20.79±0.11 |
20.72±0.13 |
Table 4: Qualitative phytochemical screening of successive soxhlet extractives ofrhizome samples of ‘A. chitrakutensis’ collected from six different places
Phytoconstituent (s) |
Hexane |
Chloroform |
Methanol |
Water |
Starch |
- |
- |
- |
+ |
Steroids |
+ |
+ |
- |
- |
Triterpenes |
+ |
- |
- |
- |
Flavonoids |
- |
- |
+ |
- |
Resins |
+ |
+ |
- |
- |
Alkaloids |
- |
- |
+ |
- |
Reducing sugars |
- |
- |
+ |
+ |
Glycosides |
- |
+ |
+ |
+ |
Tannins |
- |
- |
+ |
+ |
Saponins |
- |
- |
+ |
+ |
Carbohydrates |
- |
- |
- |
+ |
Lipids |
+ |
- |
- |
- |
Amino acids |
- |
- |
- |
+ |
Oils |
+ |
- |
- |
- |
Table 5: TLC analysis of six rhizome samples of ‘Alectra chitrakutensis’ collected from six different places at 366 nm
Rf values |
Azafrin |
Herbal Garden, Chitrakoot (M. P.) |
Narayanpur Hills, Chitrakoot (U. P.) |
Bank of Mandakini river, Satna, Chitrakoot (M. P.) |
Bank of Bagey river, Badusa (U. P.) |
Bank of Bagey river, Banda (U. P.) |
Lodwara Hill, Chitrakoot (U. P.) |
0.02 |
- |
+ |
+ |
+ |
+ |
+ |
+ |
0.04 |
- |
+ |
+ |
- |
- |
- |
- |
0.05 |
- |
- |
+ |
- |
+ |
- |
- |
0.24 |
- |
- |
- |
+ |
+ |
- |
- |
0.32 |
- |
+ |
+ |
+ |
+ |
+ |
+ |
0.41 |
- |
+ |
+ |
+ |
+ |
+ |
+ |
0.54 |
- |
+ |
+ |
+ |
+ |
+ |
+ |
0.59 |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
0.70 |
- |
+ |
+ |
+ |
+ |
+ |
+ |
0.75 |
- |
+ |
+ |
+ |
+ |
+ |
+ |
Fig. 1: HPTLC Fingerprint profile of methanolic extracts after derivetization. a. Azafrin standard b. Narayanpur Hills, Chitrakoot (U. P.), c. Bank of Mandakini river, Satna, Chitrakoot (M. P.), d. Bank of Bagey river, Badausa, (U. P.), e. Bank of Bagey river, Banda (U. P.), f. Herbal Garden, Deendayal Research Foundation, Chitrakoot (M. P.) g. Lodwara Hill, (U. P.) at A.254 nm, B.366 nm and C.422 nm respectively
DISCUSSIONThe presence of medicinal phytochemicals viz. alkaloids, steroids, flavonoids, glycosides, terpenoids, etc. provides authenticity to the high curative value of the plant. Only very few records are noted in the physicochemical or pharmacognostical studies which were done on whole plants and not confined to rhizome, which is the plant part of medicinal interest [4, 12]. Similarly, preliminary phytochemicals analysis of another species Alectra parasitica A. Rich collected from Akola district (Maharashtra) has been conducted by using whole plant powder in various extracts, and they found the analogous phytochemicals present on the plant [13]. On the other hand, as also indicated above no records were found in the pharmacognostical standardization/phytochemical analysis by using only rhizomatous part of the plant. Therefore, it is the first attempt to study pharmacognostical evaluation and screening of phytochemicals of plant A. chitrakutensis by using an advanced and sophisticated instrument HPTLC. The results obtained from the different tests performed for physicochemical, and phytochemical analysis was given in a comparable manner for all six rhizome samples to lay down quality standards using a wide range of data. Thus, the study indicates that there is no significant chemotaxonomic variation found among the plants collected from different places of Chitrakoot region.
CONCLUSION
The present manuscript will be proven as an important document for pharmacognostical standardization and quality control because a wide range of rhizome samples was compared on their physicochemical as well as the phytochemical basis. The study also revealed that the plants are found to be similar chemotaxonomically as well as in their phytochemical, compounds present under the form of primary as well as secondary metabolites, in particular, species of A. chitrakutensis and was confirmed by HPTLC finger print profile, which showed similar bands in all the samples. The present study will definitely open up the new avenues for further researches on isolation, identification and characterization of some new secondary metabolites of promising medicinal uses. Consequently, the present data will certainly provide the platform to make new drugs for mankind to cure threatened diseases.
CONFLICT OF INTERESTS
Declared none
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